Progress in Opioid Research

Proceedings of the 1986

International Narcotics

Research Conference

Editors:

John W. Holaday, Ph.D.

Ping-Yee Law, Ph.D.

Albert Herz, M.D.

NIDA Research Monograph 75

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

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NIDA Research Monographs are prepared by the research divisions of the

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Editorial Advisors

MARTIN W. ADLER, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic Institute

Troy, New York

RICHARD E. BELLEVILLE, Ph.D.

NB Associates, Health Sciences

Rockvllle, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

Washington. D.C.

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph.D.

The Johns Hopkins Umversity School of

Medicine

Baltimore, Maryland

THEODORE J. CICERO, Ph.D.

Washington University School of

Medicine

St Louis, Missouri

SIDNEY COHEN, M.D.

Los Angeles, California

MARY L. JACOBSON

National Federation of Parents for

Drug-Free Youth

Omaha, Nebraska

REESE T. JONES, M.D.

Langley Porter Neuropsychiatric Institute

San Francisco, California

DENISE KANDEL, Ph.D.

College of Physicians and Surgeons of

Columbia University

New York, New York

HERBERT KLEBER, M.D.

Yale University School of Medicine

New Haven, Connecticut

RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

CHARLES R. SCHUSTER, Ph.D.

Director, NIDA

JEAN PAUL SMITH, Ph.D.

Acting Associate Director for Science, NIDA

Acting Editor

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Progress in Opioid Research
ACKNOWLEDGMENT

This monograph is based upon papers from the 1986 International

Narcotics Research Conference, held on July 6-11, 1986, in San

Francisco, California. In the interest of rapid dissemination, it

is published in the NIDA Research Monograph series as reviewed and

submitted by the editors, whose names and affiliations follow.

JOHN W. HOLADAY, Ph.D.

Chief, Neuropharmacology Branch

Department of Medical Neurosciences

Division of Neuropsychiatry

Walter Reed Army Institute of Research

Washington, D.C. 20307-5100, U.S.A.

PING-YEE LAW, Ph.D.

Associate Research Pharmacologist

Department of Psychiatry

Langley Porter Psychiatric Institute

University of California

San Francisco, California 94143, U.S.A.

ALBERT HERZ, M.D.

Director, Department of Neuropharmacology

Max Planck Institut für Psychiatrie

D-8033 Martinsried, F.R.G.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the

National Institute on Drug Abuse or any other part of the U.S.

Department of Health and Human Services.

The U. S. Government does not endorse or favor any specific

commercial product or company. Trade, proprietary, or company names

appearing in this publication are used only because they are

considered essential in the context of the studies reported herein.

DHHS publication number (ADM)87-1507

Printed 1986

NIDA Research Monographs are indexed in the Index Medicus. They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
FOREWORD

The National Institute on Drug Abuse (NIDA) is pleased to publish

Progress in Opioid Research, 1986, the proceedings of the 17th annual

scientific meeting of the International Narcotic Research Conference

(INRC). This meeting was held in San Francisco in July 1986.

The INRC is a unique international organization seeking to develop a

fundamental understanding of the basic mechanisms of action of

opioids, opioid peptides, and their receptors, research supported in

large part by NIDA. This group and its meetings are different from

others in the field because a definite effort is made to have young

as well as senior investigators attend and present data and results

of current, ongoing studies. For instance, in recent years, 75 per

cent of the travel funds provided by the Division of Preclinical

Research of NIDA for attendance at the meeting have been allocated

to young investigators involved in laboratory research on opioids or

related subjects. As is true for many other fields, excellence

attracts more participants every year and this publication is the

largest of any INRC Proceedings, with 155 papers and 113 abstracts

included. The trio of editors worked swiftly and vigorously to

review and assemble this compendium for publication by NIDA.

We are sure that this significant "state-of-the-art" volume of the

latest research on the basic mechanisms of drug abuse will be of

great value to members of the scientific community and other readers

interested in the prevention and treatment of substance abuse.

Monique C. Braude, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

v

PREFACE

From our "bay view" in South San Francisco, we witnessed the recent

developments in opioid research and reflected on the many notable scientific

revelations from prior meetings of the INRC. Historically, the thrust of

opioid research has progressed from classic in vivo and in vitro pharmacology

toward a molecular description of events at a cellular and sub-cellular level.

The 1966 Meeting of the INRC has provided us with an opportunity to view the

cutting edge of research defining opioid ligands, receptors, and their

functional consequences.

In many ways, the opiates have been at the forefront of scientific

advances which were subsequently applied to other areas of biochemical,

pharmacological, physiological and behavioral research. For example, the

early definition of opiate receptors in 1973 has resulted in experimental

strategies which have been directly applied to many other endogenous

receptor systems. Clearly, the initial description of endogenous peptide

ligands for opioid receptors in 1975 provided the major impetus for research in

the neurobiology of many other opioid and non-opioid peptides. Likewise, the

current emphasis on molecular biology as a means by which to define the

biosynthesis and metabolism of endogenous opioids has obvious pertinence to

other biologically active peptides. The complex biochemical procedures that

have been developed to isolate and purify opiate receptors are already being

applied to other receptor systems. Finally, the experimental strategies used

to define the functional effects of endogenous opioids have resulted in a

further understanding of the interactions between opioid and non-opioid

systems.

Once again, this year’s International Narcotics Research Conference

provided a stimulating environment for the exchange of the most recent

advances in opioid research. The program not only reflected many

outstanding voluntary papers, but also included four invitational symposia and

plenary lectures by international luminaries in pharmacology and the

neurosciences. It was the intent of the program committee, under the

leadership of Drs. Horace Loh and Nancy Lee, to utilize the plenary lectures

as a means by which to acquaint opioid researchers with recent advances in

related non-opioid fields. The first plenary lecturer, Dr. Daniel Koshland,

discussed bacterial chemotaxis as a model sensory system. Dr. Julius Axelrod

presented an update on the transduction of receptor mediated signals and the

release of adrenocorticotropin. CTP binding proteins in signal transduction

was the topic of the lecture by Dr. John Northrup, and Dr. 5. Numa provided

the final plenary lecture on a molecular approach to the function of ionic

channels. These outstanding presentations provided a refreshing break from

the tachyphylactic effects of continued "opioid exposure", and also fostered

new ideas and perspectives with pertinence to opioid research.

Unfortunately, despite the tireless efforts of many of our participants,

the structures of isolated, purified opiate receptors were not available for

presentation at this conference. Additionally, the fundamental problems of

tolerance and physical dependence were only minimally addressed. Hopefully,

future meetings of the INRC will provide a forum to reveal these advances as

prior meetings have served to define the many advances described above.

vii
Immediately following the INRC Meetings, the editorial board met for

two days to review the papers and organize the monograph. In organizing this

volume, it was our intention to provide a flowing framework, with related

areas presented in logical groupings. We have attempted to arrange the

papers into chapters which progress sequentially from biochemical studies of

receptors and ligands through morphological descriptions of their localization

to the final chapters defining the functional effects of opioid systems.

Following the manuscripts, we have included the abstracts that were not

submitted as papers.

As editors of this volume and participants in this year’s meetings, we

feel that the outstanding efforts of Dr. Horace Loh, Dr. Nancy Lee, Barbara

Halperin and their colleagues in San Francisco, and Jean Paige in Washington,

D.C., ensured both an outstanding scientific and social agenda for the

meetings and the timely review and assemblage into this monograph of the

many manuscripts based on those meetings. Their work was complemented by

the tireless efforts of Dr. Eddie Way in his capacity as secretary to the

IN RC. This meeting was generously supported by the National Institute on

Drug Abuse, the U.S. Army Medical Research and Development Command,

and several sponsors and contributors as listed in the program. Finally, Dr.

Monique Braude, of the National Institute on Drug Abuse, provided guidance in

our preparation of Progress in Opioid Research for publication in the NIDA

Research Monograph series.

JOHN W. HOLADAY

PING-YEE LAW

ALBERT HERZ

viii
CONTENTS

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface ............................................................ vii

OPIOID RECEPTOR ISOLATION

Purification of Kappa-opioid Receptor Subtype to Homogeneity

from Frog Brain.

A. Borsodi, A. Khan, J. Simon, S. Benyhe, J. Hepp,

M. Wollemann and K. Medzihradsky . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Characterization, Solubilization and Purification of an Opioid

Receptor from the Brain of the Frog Rana ridibunda.

A. Pascaud, C. Mollereau, A. Puget, G. Baillat,

M. Mazarguil and J.C.Meunier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

The Direct Demonstration of Binding of Mu, Delta and Kappa Agonists

to Digitonin-solubilized Opioid Receptors from Bovine Striatum.

G. Crema, T.L. Cioannini, J.M. Hiller and E.J. Simon . . . . . . . . . . . . . 9

Identification of Rat Brain Opioid (Enkephalin) Receptor by

Photoaffinity Labeling.

Clement W.T. Yeung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Photoaffinity Labeling of Opiate Receptors with 3H-etorphine:

Possible Species Differences in Clycosylation.

W.D. Bowen and G. Kooper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Binding to Mu and Delta Opioid Sites but not Kappa Sites is Inhibited

by FAB Fragments from a Monoclonal Antibody Directed Against the

Opioid Receptor.

J.M. Bidlack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Monoclonal Antiidiotypic Antibodies which Recognized the Binding Site

of Delta Receptor: Fine Specificity of the Antiidiotypic Antibodies.

A. Cupo and P. Kaldy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Anti-idiotypic Antibodies Raised Against Anti-metenkephalin

Antibodies in Rabbits.

Min-Feng Jen, Liu Yan-Xin and Chin Yin-Chang . . . . . . . . . . . . . . . . 29

Development of a Biotin-avidin Probe for Detecting Opioid Receptors.

G.Hochhaus, B.W.Gibson and W.Sadee . . . . . . . . . . . . . . . . . . . . ...33

MULTIPLE OPIOID RECEPTORS AND THEIR LIGANDS

Considerations on the Nature of Mu, Kappa, and Delta Receptors.

A. Gero . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

A Novel Comprehensive Opiate-receptor Model

V.M. Kolb and J.P. Snyder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

ix
Stereochemistry of Novel Delta Selective Opiate Antagonists.

V.M. Kolb, D.H. Hua and W.L. Duax . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Structure Activity Studies of a Series of 4-(m-OH Phenyl) Piperidines.

G. Loew, J. Lawson, L. Toll, E. Uyeno, G. Frenking

and W. Polgar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49

Theoretical Structure-activity Studies of Methyl Substituted

4-(m-OH Phenyl) Piperidines.

G. Frenking, C.H. Loew and J. Lawson . . . . . . . . . . . . . . . . . . . . . . . . 53

Conformational Studies and Receptor Binding of Delta Selective

Opioid Peptides.

C. Keys, P. Amsterdam, P. Payne, L. Toll and G.. Loew . . . . . . . . . . 57

Site-dependent Effects of Ions on μ-, - and -opioid

Binding in Suspensions of Guinea-pig Brain Membranes.

L.E. Robson, S.J. Paterson and H.W. Kosterlitz.. . . . . . . . . . . . . . . .61

Binding Characteristics of [D-Ala2-MePhe4-Clyol5] Fnkephalin

and [D-Pen2,D-Pen5] Enkephalin in Rat Brain: Inhibition

Studies with Fentanyl-like Drugs.

M. Yeadon and I. Kitchen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Development of -opiois Receptors in Rat Brain Characterised

by [

3

H]-[D-Pen2, D-Pen5] Fnkephalin Binding.

I. Kitchen and J. McDowell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Comparison of the Effects of Calcium Concentration on Mu and

Kappa Agonist Actions in the Guinea Pig Ileum.

M.A. Johnson, R.C. Hill and J. Hughes . . . . . . . . . . . . . . . . . . . . . . . . 69

Kappa Opioid Binding Sites in Rat Spinal Cord.

J.R. Traynor, M. Stuart Wood, J.A. Carroll

and M.J. Rance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Opioid Receptors of Bovine Posterior Pituitary Neurosecretosomes

are Exclusively Kappa.

G.O. Pesce, M.A. Lang, R.A. Cruciani, J.T. Russell, H. Gainer

and D. Rodbard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

The Pharmacological Profile of BAM 18.

D.E. Hurlbut, C.J. Evans, J.D. Barchas

and F.M. Leslie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Cross-protection of μ and Opioid Receptors in the Mouse Vas

Deferens.

M.J. Sheehan, A.C. Hayes and M.B. Tyers . . . . . . . . . . . . . . . . . . . . . . 85

Affinity Crosslinking of 125I-Human beta-endorphin to Cell Lines

Possessing either mu or delta Type Opioid Binding Sites.

O. Keren, T.L. Cioannini, J.M. Hiller and E.J. Simon . . . . . . . . . . ..89

x
Upregulation of the Mu-noncompetitive Delta Binding Site by

Chronic Morphine Administration: Effect of Preincubating

Membranes in 400 mM Sodium Chloride.

J.A. Danks, F.C. Tortella, V. Bykov, A.E. Jacobson,

K.C. Rice, J.W. Holaday and R.B. Rothman . . . . . . . . . . . . 9 3

Interaction between Highly Selective Mu and Delta Opioids

in vivo at the Rat Spinal Cord.

R.D. Russell, J.B. Leslie, Y.-F. Su, W.D. Watkins

and K.-J. Chang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Opioid Receptor Activity in the Dentate Region of the Rat Hippocampus.

J.F. Neumaier and C. Chavkin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Delta and Kappa Opioid Receptors in the Rabbit Ear Artery.

I.P. Berzetei, S.P. Duckles, and A. Fong . . . . . . . . . . . . . . . . . . . . 105

POST-RECEPTOR EVENTS

A Comparsion of Microsomal and Synaptic Delta Site Binding Kinetics.

J.W. Spain, D.E. Petta and C.J. Coscia . . . . . . . . . . . . . . . . . . 109

Multiple Sites of Divalent Cation Modulation of Delta Opioid

Receptor Binding.

M. Szucs, G.M. Oetting and C.J. Coscia . . . . . . . . . . 113

Effects of Morphine and beta-endorphin on Ca2+-ATPase Activity of

Synaptic Plasma Membranes.

Shih-chia C. Lin and E. Leong Way . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Opioids and Rat Erythrocyte Deformability.

D.L. Rhoads, L. Wei, E.T. Lin, A. Rezvani

and E.L. Way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121

Calmodulin Content in Rabbit Reticulocyte and the Influence

of Opioid Peptides on Calmodulin Activity in its Membrane.

L. Jingsheng, L. Ye, J. Haiyan and C. Yinchang. . . . . . . . . . . . . . . . 125

Adrenal Medullary Opioid Receptors are Linked to CTP-binding

Proteins, Pertussis Toxin Substrates.

K. Kamikubo, H. Murase, M. Niwa, K. Miura, M. Nozaki

and K. Turumi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Varying Expression of Opioid Receptors and CTP-binding

Proteinsin Human Pheochromocytomas.

K. Kamikubo, H. Murase, M. Niwa, K. Miura, Y. Miura,

T. Satoh, A. Tomita, S. Nakamura, T. Miyashita, T. Imai,

Y. Kondo, M. Nozaki and K. Tsurumi . . . . . . . . . . . . . . . . . . .133

Pertussis Toxin Treatment Results in Tolerance to the Depressant

Effects of Opioid, Monoaminergic, and Muscarinic Agonists on

Dorsal-horn Network Responses in Mouse Spinal Cord-ganglion

Cultures.

S.M. Crain, B. Crain and M.H. Makman . . . . . . . . . . 137

xi
Inhibition of Adenylate Cyclase and Induction of Heterologous

Desensitization by Kappa Agonists in Rat Spinal Cord.

B. Attali and Z. Vogel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Spare Ligand Binding Sites in the Coupling of Opioid Receptor

to Brain GTPase.

M.J. Clark and F. Medzihradsky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Comparative Properties of SH-groups at Mu, Delta and Kappa

Opioid Receptor.

M. Nozaki, M. Niwa and H. Fujimura . . . . . . . . . . . . 149

Enhancement of Perforant Path Transmission by Enkephalin

Analogues: Alteration of Granule Cell Inhibition/Potentiation.

J.B. Wiesner and S.J. Henriksen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

PHENCYCLIDINE AND ITS RECEPTOR

Modulation of Brain NMDA Receptors: Common Mechanism of

Delta/PCP Receptors and Their Exogenous and Endogenous Ligands.

R. Sircar, H. Samaan, R. Nichtenhauser, L.D. Snell,

K.M. Johnson, J. Rivier, W. Vale, R.S. Zukin

and S.R. Zukin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

An Endogenous Ligand for Phencyclidine Receptors: Isolated

from Human Brains.

W.X. Lu, Y.-F. Liu, Y.-X. Zhu, G.-L. Xi and

A.-Z. Zhang . . . . . . . . . . . . . . . . . . . . . . . . ......... 161

An Endogenous Ligand from Human CSF for the Phencyclidine Receptor.

G. Zhou, D. Gong, C. Jiang, J. Yao, H. Gao, Y. Zhu,

B. Pan and A. Zhang ................................. 165

Dextrorphan: An Antagonist for Phencyclidine Receptor.

F.-Y. Sun, H. Zhu, L.-Mei Zhang, X.-R. Xu, and

A.-Z. Zhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

High and Low Affinity Psychotomimetic Opioid Binding

Sites: Characterization by a Novel 3H-PCP-analog.

Y. ltzhak . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

NOVEL OPIOID LIGANDS

New Delta-Receptor Antagonists.

C.W. Thornber, J.S. Shaw, L. Miller, C.F. Hayward,

J.S. Morley, D. Timms and A. Wilkinson . . . . . . . . . . . 177

Dimeric Opioid Antagonists.

C.W. Thornber, J.S. Shaw, L. Miller, C.F. Hayward . . . . . . . . . . . . . 181

Melphalan-containing N,N-Dialkylenkephalin Analogs as Potential

Irreversible Antagonists of the Delta Opioid Receptor.

J.A. Lovett and P.S. Portoghese . . . . . . . . . . . . . . . . . . . . . . . 185

xii
Dihydromorphine-peptide Hybrids have Mu Receptor Antagonistic

and Delta Receptor Agonistic Activity on the Mouse Vas Deferens

and Bind with High Affinity to Opioid Receptors in Rat Brain.

C.B. Smith, F. Medzihradsky and J.H. Woods. . . . . . . . . . . . . 189

Characterization of D-Ala2, Leu5, Cys6-enkephalin: A Novel

Synthetic Opioid Peptide with Slowed Dissociation from Delta

Receptors.

W.D. Bowen, M. Kelemen, R. Huey and D. Stewart . . . . . . .193

3

H]Dstbulet, a New Linear Hexapeptide with Both an Improved

Selectivity and a High Affinity for -opioid Receptors.

P. Delay-Goyet, C. Seguin, V. Dauge, G. Calenco,

J.-L. Morgat, G. Gacel and B.P. Roquer . . . . . . . . . . . . . . .197

Anomalous Binding of DPDPE as a Result of Batch Variability.

L. Taylor, A. Mansour, S.J. Watson and H. Akil . . . . . . . . 201

Comparsion of the Opioid Antagonist Properties of a Cyclic

Somatostatin Analog In Vitro and In Vivo.

J.E. Shook, J.T. Peltont, W. Kazmierski, P.K. Lemcke,

W.S. Wire, V.J. Hruby and T.F. Burks. . . . . . . . . . . 205

H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2: A Potent and

Selective Antagonist for Mu Opioid Receptors.

K. Gulya, G.K. Lui, J.T. Pelton, W. Kazmierski,

V.J. Hruby and H.I.Yamamura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Opioid Agonist and Antagonist Effects of Configurational

Stereoisomers of 3-(Dimethylamino)-2,2-Dimethyl-7-Hydroxy1-Tetralol.

D.A. Lippman, F.F. Matos and D.Y. Shirachi . . . . . . . . . . . . . . . .213

Opioid Receptor Effects of Two 3-Amino-2,2-Dimethyl-Tetralin

Analogs in Guinea Pig Ileum Longitudinal Muscle.

F.F. Matos, D.A. Lippman and M.H. Malone. . . . . . . . 217

Preparation of /

3/-oxymorphazone and Its Binding to Rat

Brain Membranes.

A. Borsodi, E. Varga, G. Toth and S. Hosztafi . . . . . . . . . . . . . . .220

Comparsion of the Opioid Activity of Cam1 and Kappa Agonists.

S. Furst, T. Friedmann, A. Kovacs and T. Wagner . . . . . . . . . . . . . . 224

Comparative Effects of Dynorphin A(1-9) Pyrrolidine Analogs

on Isolated Tissues.

A. Rezvani, S.R. Kadambi, D.L. Rhoads, J.K. Chang

and E. Leong Way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

125I-OPDYN, Monoiodo [D-Pro10]-dynorphin (1-11), Is an

Effective and Useful Tool for the Study of Kappa Opioid Receptors.

J.E. Gairin, C. Jomary, J. Cros and J.-C. Meunier . . . . . . . . . . . .232

xiii
Influence of Y-20003, an Analgesic Agent, on the Endogenous

Opioid Peptide System in Rats.

T. Kudo, E.-Q. Wei, R. Inoki, M. Terasawa

and T. Nakao . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

OPIOID PEPTIDE SYNTHESIS AND PROCESSING

Prohormone Processing Reactions: Discrete Stages in the

Formation of -endorphin Related Peptides.

N.J. Darby, K. Maruthaninar and D.G. Smyth . . . . . . . . . . . . . . . .240

Bridge Peptide is a Cleavage Product of Pro-dynorphin Processing

in the Rat Anterior Pituitary.

R. Day and H. Akil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

A Processing Enzyme for Prodynorphin Derived Peptides.

K.M. Metters, J. Rossier, E.E. Oliveira, O. Toffoletto

and A.C.M. Camargo 247

Assay and Biochemical Characterization of a Dynorphin

Converting Enzyme in Human Cerebrospinal Fluid.

F. Nyberg, S. Lyrenas and L. Terenius . . . . . . . . . . . ..251

Presence of Enkephalin Precursor in Molluscan Neural

Tissue Extract.

B. Mixon, S. Kuruvilla, C.B. Stefano and M.K. Leung . . . . . . . . . . . 255

Inactiviation of [Leu5]-enkephalin in Three Isolated

Preparations: Relative Importance of Aminopeptidase,

Endopeptidase-24.11 and Peptidyl Dipeptidase A.

T. Oka, K. Aoki, M. Kajiwara, K. Ishii, Y. Kuno,

T. Hiranuma and T. Matsumiya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

Characterization of Enkephalin Degradation in Rat Plasma.

S.B. Weinberger, C.E. Zafuto, R.C. Walker and

J.L. Martinez, Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Characterization of the Enkephalin-degrading Aminopeptidase

of Neuroblastoma (N1E E-115) Cell Membranes.

H. Lentzen, R. Simon and I. Reinsch . . . . . . . . . . . . . . . . . . . . . . .267

The Endogenous Tripeptide Tyr-Gly-Gly as an Extracellular

Metabolite of Enkephalins in Rat Brain: Origin and Metabolism.

B. Giros, C. Llorens-Cortes, C. Gros

and J.-C. Schwartz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Evaluation of Striatal Enkephalin Release In Vivo

from Steady State Levels and Turnover Rates of the

Tripeptide Tyr-Gly-Gly.

C. Llorens-Cortes, C. Gros and J.-C. Schwartz. . . . . . . . . . . . . . 275

Isolation and Characterization of ACTH-related Peptides

from Porcine Pituitaries.

K. Voight and W. Stegmaier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

xiv
A Membrane Bound Substance P Degrading Endopeptidase

from Rat Brain.

A. Bergmann and K. Bauer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

CELLULAR LOCALIZATION

In Situ Hybridization Versus Northern Analysis:

Working Towards the Correlation of Two Quantitative

Techniques for Opioid and Vasopressin mRNAs in the Rat

Hypothalamus and Pituitary.

T.C. Sherman and S.J. Watson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

lmmunohistochemical Evidence for Subpopulations of

Dynorphinergic Neurons.

B. Quinn and E. Weber . . . . . . . . . . . . . . . . . ................ 291

Co-existence of Prodynorphin – Opioid Peptides and

Substance P in Primary Sensory Afferents of Guinea-pigs.

E. Weihe, A. Leibold, D. Nohr, T. Fink

and B. Gauweiler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .295

Distribution of Enkephalin-like Immunoreactivity in

the Feline Heart; Its Interrelation with Substance Pand Vasoactive Intestinal Polypeptide-like lmmunoreactive

Nerve Fibers.

S. Maeda, J. Nakamae, H. Ichikawa, S. Wakisaka,

M. Akai and R. Inoki .................................. 299

lmmunohistochemical and Subcellular Studies of Aminopeptidase

M Localization in Rat Brain: Microvessels and Synaptic Membranes.

C. Gros, B. Solhonne, B. Giros, H. Pollard, N. Sales,

J.-C. Schwartz, P. Ronco and P. Verroust . . . . . . . . . . . . . . . . . . . . . 303

Gamma-3-MSH and B-endorphin in Monkey Pituitary.

J. Meador-Woodruff, M. Knobloch, H. Vaudry,

N.G. Seidah, S.J. Watson and H. Akil . . . . . . . . . . . . . . . . . . .307

Multiple Opioid Receptor Subtypes in the Pituitaryadrenal Axis: A Cross-Species Study.

A. Mansour, M.E. Lewis, H. Khachaturian,

H. Akil and S.J. Watson . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Autoradiographic Localization of Opioid Receptor Types in

the Rat Small Intestine.

M.R. Dashwood, R.M. Sykes and C.S. Thompson . . . . . . . . . . . . . . . 315

Mu Opioid Receptor Binding Sites in Human Brain.

C. Pilapil, S. Welner, J. Magnan, N. Zamir

and R. Quirion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

xv
OPIOID/NON-OPIOID INTERACTIONS

Behavorial Interaction Between Endogenous Opioids, CCK-8

and Dopaminergic Systems in the Nucleus Accumbens of Rats.

J.M. van Ree, J. Igarashi and I. Kiraly. . . . . . . . . . . . . 323

Suppression of Sympathetic Nerve Activity by Methionine

Enkephalin: Additive Interaction with Clonidine in Intact

Anesthetized Rabbits.

H.M. Rhee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Beta-endorphin Modulates the Inhibitory Action of Corticotropinreleasing Factor on Luteinizing Hormone Secretion.

F. Petraglia, W. Vale and C. Rivier . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Leumorphin is a Potent Inhibitor of Vasopressin Secretion.

T. Yamada, K. Nakao, H. Itoh, N. Morii, S. Shiono,

M. Sakamoto, A.Sugawara, Y. Saito, G. Katsuura, M Eigyo,

A. Matsushitha and H. Imura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

The Effect of 17-alpha-estradiol, a Possible Endogenous

Opiate Antagonist, on D-Ala2-Met5-enkephalinamide-induced

Blood Pressure Responses in Conscious, Unrestrained Rats.

A. Chandra and W. Dixon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

Stress-induced-antinociception and Morphine Sensitivity in Mice

Following Chronic Treatment with Anabolic/Androgenic Steroids.

D.A. Cowan, S. Friend, A. Gibson, M. Ginsburg

and S.L. Hart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

Opioids and receptors Interaction: Further Studies in

Cultured Cells.

A. Reggiani, A. Carenzi and D.D. Bella . . . . . . . . . . . . . . . . . . . . . . . 347

Mu/Delta Opioid Site Selectivity of Some Antidepressants.

E.E. Codd and R.F. Walker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

Molecular Mechanisms of Ligand Binding to Opioid Receptors:

Selective Inhibition by Ethanol?

R.S. Rapaka, H.N. Bhargava, V. Renugopalakrishnan

and B.J. Collins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Opioids and Intake of Alcoholic Beverages.

L.D. Reid, S.A. Czirr, W.C. Milano, C.L. Hubbell

and N.A. Manha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

(+) – Naloxone: Antagonism of Pentobarbital-induced Narcosis.

D. Frost, A.R. Abadir, J. Cintautas and

B.J. Kraynack ................................ 363

Rat Striatal Dopamine Release Mechanisms of Cocaine.

P.A. Broderick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

xvi
Central Glucagon Antagonizes Morphine- and Stress-Induced

Antinociception in the Mouse.

D.S. Malcolm, S. Amir and J.W. Holaday . . . . . . . . . . . . . . . . . . . .371

Endorphins Contribute to the Loss of Glucose Homeostasis

in Anaphylactic Shock.

S. Amir ..................................... 375

Effects of Opioid Analgesics on Local Cerebral Glucose Utilization.

E. London, R. Fanelli, M. Szikszay and D. Jasinski. . . . . . .379

Effects of Morphine and Calcium Antagonists on Plasma Glucose

in Male Rats.

M. Szikszay, F.R. Snyder and E.D. London . . . . . . . . . . . . 382

REGULATION OF OPIOID SYSTEM5

Dynamic Changes in the Levels of Striatal Proenkephalin mRNA

and Large Molecular Weight Enkephalin Containing Peptides

Following Electroacupuncture.

K. Tsou, M. Zheng and X.-H. Ding . . . . . . . . . . . . . . . . . . . . . 385

Dopaminergic Regulation of Proenkephalin – a Gene Expression

in the Basal Ganglia.

S.P. Sivam, G.R. Breese, T.C. Napier, R.A. Mueller

and J.-S. Hong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

Increased Enkephalin Gene Expression in the Hippocampus

Following Seizures.

J.D. White and C.M. Gail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

Regulation of Proopiomelanocortin (POMC) mRNA Levels in

Primary Pituitary Cultures.

J.Ph. Loeffler, N.Kley, C.W. Pittius and V. Hollt . . . . . . . . . . . . . . 397

Regulation of Hypothalamic R-endorphin and Dynorphin

Release by Corticot ropin-releasing Factor (CRF).

O.F.X. Almeida, K.E. Nikolarakis and A. Herz. . . . . . . . . . . . . . . . .401

Inhibition of LH Release by CRF May Be Partially Mediated

Through Hypothalamic endorphin Release.

K.E. Nikolarakis, O.F.X. Almeida and A. Herz . . . . . . . . . . . . . . . . . 403

Increased Spinal Cord Dynorphin mRNA During Peripheral Inflammation.

M.J. ladarola, J.Douglass, O. Civelli and

J.R. Naranjo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406

Differential Effects of Selective Brain Lesions on Hypothalamic

and Pituitary IR-dynorphin.

S. Spampinato, S. Candeletti, E. Cavicchini, P. Romualdi,

E. Speroni and S. Ferri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

xvii
Modulation of ß-endorphin Secretion from Mouse Pituitary Tumor

Cells by Calmodulin Inhibitor W7.

B.C. Beaubien and E. Herbert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

Release of I-R Metenkephalin from Rat Amygdala Slices In Vitro.

A. Livingston and B. Morris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

Release of Opioid Peptides from the Spinal Cord of Rats

Subjected to Chronic Pain.

R. Przewlocki, W. Lason, J. Silberring, A. Herz and

B. Przewlocka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422

Relations Between Gabaergic and Enkephalinergic Systems:

Effects of Sodium Valproate on Rat Brain Enkephalins.

J. Vion-Dury, A. Cupo and T. Jarry . . . . . . . . . . . . . . . . . . . .426

[Met] Enkephalin-Arg6-Phe7 in Human Case CSF – Increased

Levels in Late Pregnancy.

S. Lyrenas, F. Nyberg, R. Folkesson, B. Lindberg

and L. Terenius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430

Expression and Regulation of Kappa Opiate Receptors in Rat

Spinal Cord-Dorsal Root Ganglion Coclultures.

B. Attali, D. Saya and Z. Vogel ....................... 434

ANTINOCICEPTION/ANALCESIA

Sensitivity of Antinociceptive Tests to Opioid Agontists and

Partial Agonists.

K.M. Delaney, J.D. Rourke and J.S. Shaw . . . . . . . . . . . . . . . . . . .438

Evidence for Delta Receptor Mediation of [D-Pen2,D-Pen5]-

enkephalin (DPDPE) Analgesia in Mice.

J.S. Heyman, H.I. Mosberg and F. Porreca. . . . . . . . . . . . 442

Intraventricular Administration of BAM-18: Antinociceptive

and Locomotor Activity in the Rat.

K.E. Stevens, F.M. Leslie, C.J. Evans, J.D. Barchas,

J.D. Belluzzi and L. Stein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

A Single ICV Injection of KCL Induces a Selective and Longlasting Inhibition of Opioid Analgesia in Mice.

J. Garzon and P. Sanchez-Blazquez . . . . . . . . . . . . . . . . . . . . . . . . . . 450

Increase in Met-enkephalin Level and Antinociceptive Effects

Induced by Kelatorphan in the Rat Spinal Cord.

M.-C. Fournie-Zaluski, 5. Bourgoin, F. Cesselin,

M. Hamon, D. Le Bars, A.H. Dickenson and

B.P. Roques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

Modulators of Pain in the Spinal Cord.

M.F. Jen, C.H. Lu, L. Huangand J.S. Han . . . . . . . . . . . . . 457

xviii
Similar Actions of Kappa and Mu Agonists on Spinal Nociceptive

Reflexes in Rats and Their Reversibility by Naloxone.

C.G. Parsons, D.C. West and P.M. Headley.. . . . . . . . . . . . . . . . . . .461

Opioid Analgesia in the Mouse: Evidence for Multiple Receptors

Using -FNA.

P. Sanchez-Blazquez and J. Garzon . . . . . . . . . . . . . . . . . . . . . . . . . . 465

Hyperalgesia Produced by Intrathecal Opioid Antagonists

Depends on Receptor Selectivity and Noxious Stimulus.

C.W.T. Pilcher and J.L. Browne . . . . . . . . . . . . . . . . . . . . . . . . 469

REHAVIOR

In Vivo Studies on Delta Opioid Receptors

A. Cowan, M.J. Rance and T.P. Blackburn . . . . . . . . . . . . . . . . . . . . 473

Effects of Naloxone and Neurotensin on Excessive Grooming

Behavior of Rats Induced by Bombesin, -endorphin and ACTH.

Tj.B. van Wimersma Creidanus . . . . . . . . . . . . . . . . . . . . . . . . . . . .477

Enkephalin Contained in Dentate Granule Cells is Important for

Kainic Acid-Induced Wet Dog Shakes.

L. Grimes, J. Hong, J. McGinty, C. Mitchell, J. Obie

and H. Tilson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481

Opiate Receptor Binding and Behavorial Effects of Morphine

in RHA/VERH and RLA/VERH Rats.

D. Lazega, P. Driscoll, H.-R. Frischknecht,

B. Siegfried and P.G. Waser . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

Action of Naltrexone on the Sexual Impairment of Obese

Cafeteria Rats.

A. Mandenoff, M.C. Bertiere, D. Betoulle and

M.Apfelbaum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Opiates Stimulate Food Consumption in the Land Snail Helix aspersa.

C. Ndubuka, D. Brown, S. Pratt, E. Braham,

M.K. Leung and C.B. Stefano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

Effect of Electrolytic and Chemical Ventromedial Hypothalamic

Lesions on Food Intake, Body Weight, Analgesia and the CNS

Opioid Peptides in Rats and Mice.

C.A. Tejwani and C.W. Richard III . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

Both Adrenal Opioids and Clucocorticoids Involved in the

Incorporation of Information into Memory Post-Stress.

D. Jeffreys and J.W. Funder . . . . . . . . . . . . . . . . . . . . . . . . . 501

Effect of Chronic Exercise on -endorphin Receptor Levels in Rats.

R.A. Houghten, S.M. Pratt, E.A. Young, H. Brown

and D.R. Spann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505

xix
AUTONOMIC AND NEUROLOGIC EFFECTS

Intrathecal Morphine and Blood Pressure in Preeclampsia.

C.E. Costas, N.W. Doss, S.G. Humayun, J. Cintautas,

A.R. Abadir and B.J. Kraynack . . . . . . . . . . . . . . . . . . . . . . . . 509

Centrally Administered Leumorphin Possesses Potent Depressor

Activity in Conscious Rats.

H. Itoh, K. Nakao, T. Yamada, N. Morii, S. Shiono,

M. Sakamoto, A.Sugawara, Y. Saito, G. Katsuura, T. Shiomi,

M. Eigyo, A. Matsushita and H. Imura . . . . . . . . . . . . . 512

Relationship Between Dihydroetorphine and Cholinergic System

in the Inhibition of Respiration and Heart Rate.

C.F. Bian, S.H. Xing and W. Qin . . . . . . . . . . . . . . . . . . . . . . . . 516

The Role of Mu and Delta Opioid Receptors on the

Intestinal Propulsion in Rats.

M. Sbacchi, A. La Regina, P. Petrillo and A. Tavani . . . . . . . . . . .520

Intrathecal Dynorphin A (1-13) and (3-13) Reduce Spinal

Cord Blood Flow by Non-opioid Mechanisms.

J.B. Long, R.C. Kinney, D.S. Malcolm, G.M. Craeber

and J.W. Holaday . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .524

Endogenous Opioids, Opiate Receptors and Traumatic Brain Injury.

T.K. McIntosh, S. Fernyak and A.I. Faden . . . . . . . . . . . . . . . . . . . . . 527

Treatment of Experimental Stroke with the Opiate Antagonist

WIN 44,441-3 Effects on Neurologic Function, Infarct Size,

and Survival.

D.S. Baskin and Y. Hosobuchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

Convulsant Action of Naloxone in the Rat Amygdala.

C. Ikonomikou-Turski, W.A. Turski, Z.A. Bortolotto,

L. Turski and E.A. Cavalheiro . . . . . . . . . . . . . . . . . . . . . . . . . . . . .535

Dynorphin A (1-13): In Vivo Opioid Antagonist Actions and

Non-Opioid Anticonvulsant Effects in the Rat Flurothyl Test.

F.C. Tortella and J.W. Holaday . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539

Mu- and Delta-Opioid Modulation of Electrically-Induced

Epileptic Seizures in Mice.

C. Pinsky, R. Bose and R.C.A. Frederickson. . . . . . . . . . . 543

The Threshold for Limbic Seizures in Rats is Decreased by

Intranigral Morphine.

W.A. Turski, Z.A. Bortolotto, C. Ikonomidou-Turski,

L.E.A. Moraes Mello, E.A. Cavalheiro and L. Turski. . . . . . . . .547

Opioids with Different Affinity for Subreceptors Induce

Different Effects on Early and Late Sensory Evoked Potentials

(SEP) in Man.

E. Freye, R. Buhl and F. Ciaramelli . . . . . . . . . . . . . . . . . . . . . . . . 551

xx
Central and Peripheral beta-endorphin Response to Transcutaneous

Electrical Nerve Stimulation.

F. Facchinetti, G. Sforza, M. Amidei, C. Cozza,

F. Petraglia, C. Montanari and A.R. Cenazzani . . . . . . . . . . . . . . .555

Limbic Regions of the Brain of Alzheimer’s Disease Patients

Show Selective Changes in Mu, Delta and Kappa Opioid Receptor

Binding.

J.M. Hiller, Y. Itzhak and E.J. Simon . . . . . . . . . . . . . . 559

ADDICTION, TOLERANCE AND DEPENDENCE

Differential Effects of Mu and Kappa Opioid Systems on

Motivational Processes.

T.S. Shippenberg and A. Herz . . . . . . . . . . . . . . . . . . . . . . . . . . . . .563

Aversive Properties of Opiate Withdrawal Studied in Rats.

R.F. Mucha, M.D. Critti and C. Kim . . . . . . . . . . . . . . . . . . . . . . .567

Morphine Withdrawal in Guinea-pig Ileum is Confined to

One Electrophysiologically Defined Class of Myenteric Neurone.

S.M. Johnson, M. Costa and J.B. Furness . . . . . . . . . . 571

Enkephalinase Inhibitors Attenuate Naloxone-Precipitated

Withdrawal Syndrome.

M.R. Dzoljic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .575

Presynaptic Alpha Adrenoreceptor Function in Dependent

Rats Before and After Morphine Withdrawal.

H.C. Moises, C.B. Smith, R.N. Spengler and

P.J. Hollingsworth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

The Effect of Chronic Met-Enk on Various Dopamine Systems

in the Rat.

K.A. Steece, F.A. DeLeon-Jones, J.M. Lee and

R.F. Ritzmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583

Effects of Prior Exposure to Morphine on the Opioid

Inhibition of the Stimulated Release of [

3H]norepinephrine

from Guinea Pig Cortex Slices.

L.L. Werling, S.R. Brown and Brian M. Cox. . . . . . . . . . . . . . . . .587

Tolerance to Opioids in Single Locus Coeruleus Neurons of the Rat.

M.J. Christie, J.T. Williams and R.A. North . . . . . . . . . 591

Effect of Maternally Administered Opiates on the Development

of the -endorphin System in the Offspring.

C. Gianoulakis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

Suppression of Immunological Functions in Morphine Addicted Mice.

W.K.K. Ho, K.W. Cheung, K.N. Leung and H.L. Wen . . . . . . . . . . . . 599

xxi
Morphine Metabolism in Mouse Brain.

A.W. Wahlstrom, L. Hammar, L.-G. Lundin and A. Rane . . . . . . . . 603

ABSTRACTS

Abstracts not submitted as papers – arranged alphabetically...........607

List of NIDA Research Monographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721

xxii
PURIFICATION OF KAPPA-OPIOID RECEPTOR SUBTYPE TO

APPARENT HOMOGENEITY FROM FROG BRAIN

A. Borsodi, A. Khan, J. Simon, S. Benyhe, J. Hepp*,

M. Wollemann, K. Medzihradszky*

Institute of Biochemistry, Biological Research Center of

the Hungarian Academy of Sciences, H-6701 Szeged,

P.O.Box 521, Hungary, and Central Research Institute

for Chemistry of the Hungarian Academy of Sciences,

II-1025, Budapest, P.O.:Box 17, Hungary.

ABSTRACT

Kappa-opioid receptor subtype was purified from digitonin

solubilized preparation of frog brain membranes by

hydrophobic and Sepharose 6B chromatography. 4800-fold

purification over the membrane-bound receptors was achieved.

The kappa receptor activity resides in a homo-oligomer of a

single polypeptide of Mr 65,000.

INTRODUCTION

The opioid receptor system displays heterogeneity and

consists of at least four major subtypes; i.e. mu, delta,

kappa and sigma (Zukin & Zukin 1934). Recently, several

laboratories have reported a partial purification of active

opioid mu and delta binding sites (Cho et al. 1983,

Gioannini et al. 1934 & 1985, Maneckjee et al. 1985, Fujioka

Fujioka et al. 1935, Simonds et al. 1985, Demoliou-Mason &

Barnard 1986). We have described earlier a succesful

solubilization of opioid receptors from frog brain by the

use of digitonin (Simon et al. 1984). This preparation was

found to be a rich source of the kappa-subtype (72%). In

solution this subclass was completely separated from the muand delta-sites by gel filtration on Sepharose-6B column

(Simon et al. 1985) and it was purified on an affinity

column (Simon et al. 1986). In the present work We purified

the receptor by the use of hydrophobic chromatography.

MATERIALS AND METHODS

3

H-Ethylketocyclazocine (

3

H-EKC: 0.74 TBq/mmol, 19.9

Ci/nmol) was purchased fron New England Nuclear. 3H-Dansyl

chloride (0.53 TBq/mmol, 14.2 Ci/mmol) was from Amersham.

1
Digitonin, bacitracin, phenylmethylsulfonyl fluoride

(PMSF), dansyl chloride and polyethyleneimine (PEI) were

obtained from Sigma Chemicals Co. Sepharose-6B beads, HMW

calibration kit for gel filtration and LMW kit for gel

electrophoresis were purchased by Pharmacia Fine

Chemicals. All other chemicals were of analytical grade.

Particulate membrane fraction of frog (Rana esculenta)

brain was prepared in Tris buffer and solubilized with 1%

digitonin as described earlier ($imon et al. 1984).

Specific binding was measured with 3

H-EKC in the presence

of blocking agents for mu and delta sites (Simon et al.

1934). Protein concentration was determined as described

by Bradford (1976), or with 3H-Dansyl chloride method

(Schultz & Wassarman 1977). SDS polyacrylamide gel

electrophoresis was carried out by the method of Laemmli

(1970) with slight modifications.

RESULTS AND DISCUSSION

Frog (Rana Esculenta) brain membranes were solubilized in

1% (ligitonin (Simon et al. 1984) and applied on PhenylSepharose CL-B column with high salt concentration (0.5 M

(NH4)2SO4), after the removal the excess of the detergent.

Proteins were eluted with decreasing salt concentration. 3

HKC binding of the fractions was measured. The highest

activity was found at low salt (0.06M-H2O) concentration

(fig. 1) indicating a hydrophobic character.

FIGURE 1. Phenyl-Sepharose CL-4B chromatography

Solubilized opioid receptors were applied on PhenylSepharose CL-4B column (32x0.8 cm) in 0.5M

(NH4)2S04. Elution was carried out by decreasing salt

concentration.EKC binding was measured.

2
The preparation exhibited high affinity for kappa-ligands as

it is shown in table 1.

TABLE 1.

Relative potencies of opioid ligands

in displacing 3H-EKC binding

Unlabelled ligands IC50(nM)

Ethylketocyclazocine

Dynorphin(1-13)

U-50, 488 H

30

10

11

The pooled and concentrated fractions were applied on

Sepharose 6B column, where a complete separation of the

kappa subtype can be obtained (Simon et al. 1985). Fractions

with high 3H-EKC specific binding were collected and their

protein contents were measured. Because of the very low

mount, the ultrasensitive (5-10 ng per assay) 3

H-Dansyl

chloride method was used .

FIGURE 2. SDS polyacrylanide gelelectrophoresis

Lane 1.: molecular weight markers, lane 2.: digitonin

extract, lane 3.: purified kappa fraction.

3
About 3360 pmol of 3

H-EKC per mg protein specific activity

was obtained compared to 0.7 pmol/mg in the original

membrane fraction. Approximately 4800-fold enrichment Of

kappa-subtype over the menbrane-bound receptors was

achieved. The kappa receptor activity resides in a homooligomer of a

shown on fig. 2.

single polypeptide of Mr 65,000, as it is

Results from other laboratories and our recent data suggest

a general pattern of opioid receptors in which each subtype

is due to a sinqle, specific type of subunit.

REFERENCES

Bradford, M.M. Anal Biochem, 72:248-254, 1976.

Cho, T.M., Ge, B.L., Yamato, G., Smith, A.P. and Loh, H.H.

Proc Matl Acad Sci USA, 80:5176-5189, 1953 .

Demoliou-Mason C. and Barnard, E.A. British Opioid

Colloquim, Notthingam, Abstr. 04, 1985.

Fujioka, T., Inoue, F. and Kuriyama, M. Biochem Biophys Res

Commun, 131:640-646, 1985.

Gioannini, T.L., Howard, A.D., Hiller, J.M. and Simon, E.J.

Biochem Biophys Res Commun, 119:624-629, 1984.

Gioannini, T., Howard, A.D., Hiller, J.M. and Simon, E.J.

J Biol Chem, 260:15117-15121, 1935.

Laemmli, U.K. Nature, 227:680-685, 1970.

Maneckjee, R., Zukin, R.S., Archer, S., Michael, J. and OseiGymah, P. Proc Natl Acad Sci USA, 82:594-598, 1985.

Schultz, R.M. and Wassarman, P.M. Anal Biochem, 77:25-32,

1977.

Simonds, W.F., Burke, T.R., Rice, K.C., Jacobson, A.E. and

Klee , W. A. Proc Natl Acad Sci USA, 32:4974-4973, 1985.

Simon, J., Szucs, M., Benyhe, S., Borsodi, A., Zeman, P. and

Wollemann, M. J.Neurochem, 43:957-963, 1984.

Simon J. Benyhe, S., Borsodi, A., Szucs, M. and Wollemann,

M. FEBS Lett, 183:335-337, 1995.

Simon, J. Benyhe, S., Hepp, J. Khan, A., Borsodi, A., Szucs,

M., Medzihradszky, K. and Wollemann, M. submitted to FEBS

Lett, 1986.

Zukin, R.S. and Zukin, S.R. Trends Neurosci, 7:160-164,

1984.

ACKNOWLEDGEMENTS

EKC donated by Sterling Winthrop Institute and DAGO kindly

provided by Dr. D. Roemer, Sandoz Ltd. are greatly

acknowledged.

4
CHARACTERIZATION, SOLUBILIZATION AND PURIFICATION OF AN OPIOID

RECEPTOR FROM THE BRAIN OF THE FROG RANA RIDIBUNDA.

A. Pascaud, C. Mollereau, A. Puget, G. Baillat, H. Mazarguil and

J.C. Meunier.

Laboratoire de Pharmacologie et de Toxicologic Fondamentales CNRS,

205, route de Narbonne, 31400 Toulouse, France.

ABSTRACT

The brain of the frog R. ridibunda contains a major opioid binding

site which in vitro pharmacological profile is different from those of

mammalian μ, - and -opioid sites. In digitonin extracts, this major

opioid site exists as two molecular forms -10S and 12S- which are

clearly resolved by sedimentation in sucrose gradients and which are

thought to represent the opioid receptor alone (10S) or associated

(12S) with a guanine nucleotide regulatory protein. Purification of

the digitonin extracts by affinity chromatography on immobilized

dynorphin, results in a single major protein component of apparent

M r  ̃ 64,000.

INTRODUCTION

Amphibian brain membranes display high levels of opioid binding (Ruegg

et al. 1981, Puget et al. 1983, Simon et al. 1984). In addition, .

amphibian brain opioid receptors are readily solubilized under active

form in reasonably high yields with digitonin alone (Ruegg et al.

1981, Simon et al. 1984). Therefore, the amphibian brain designates

itself as a suitable source wherefrom to purify opioid receptors by

affinity chromatography.

In this report, we have characterized the types of opioid sites

present in frog brain membranes and we have analyzed the sedimentation

behavior of the digitonin solubilized opioid receptors. Our results

suggest that in the preparation, there is a major opioid receptor type

different from mammalian μ, and types and which appears to exist

independently of as well as in association with a guanine nucleotide

regulatory protein.

MATERIALS AND METHOOS

Preparation and solubilization of the crude membrane fraction (O°C).

Freshly dissected whole brains from R. ridibunda frogs were

homogeneized in 50 mM tris-HCl pH 7.4 ("buffer") and the homogenate

was centrifuged in a Beckman rotor type 30 for 35 min at 28,000 rpm.

The pellet was washed and resuspended in enough buffer to obtain a

crude membrane fraction containing about 5 mg of protein per ml. The

suspension was made 1 % (w/v) with digitonin (Fluka AG) and gently

stirred for 30 min after which it was centrifuged in a Beckman rotor

type 30 for 30 min at 35,000 rpm. The supernatant (2 mg of protein/ml)

was taken as the soluble extract.

Equilibrium binding studies (25°C).

Equilibrium binding studies were carried out on 50 μl either of the

crude membrane fraction (diluted 1 : 5) or on 30 μl aliquots of the

soluble extract in a final volume of 0.5 ml using 3H-etorphine (30-60

Ci/mmol, Amersham) or 3

H-diprenorphine (25-50 Ci/mmol, Amersham). All

assays were in triplicate and incubation was for 1 hr at 25°C.

5
Nonspecific binding was determined in the presence of 10 μM diprenorphine. Free and bound radioligand were separated by rapid filtration

on glass fiber disks (Whatman GF/B) which, in the case of the soluble

extract, had been presoaked in PEI according to Bruns et al. (1983).

Sedimentation in sucrose gradients

0.4 ml aliquots of digitonin extract were layered on top of 10.6 ml

linear 5 to 20 % (w:v) sucrose gradients in 50 mM tris-HCl pH 7.4

containing 0.01 % (w:v) digitonin. Centrifugation was at 40.000 rpm

for 16 hrs in a Beckman rotor type SW 41 Ti after which fractions of

0.16 ml were collected. Each fraction was assayed for 3H-etorphine

(2 nM) or 3H-diprenorphine (2 nM) in a final volume of about 0.2 ml by

the rapid filtration method as modified by Bruns et al. (1983).

RESULTS AND DISCUSSION

The frog brain contains a major opioid receptor type that is neither

μ, nor or

Equilibrium saturation binding of the agonist 3H-etorphine (

3

H-ETO)

and of the antagonist 3H-diprenorphine (

3

H-DIP) in frog brain

membranes was clearly biphasic (figure 1). Sodium ions (120 mM NaCl)

selectively reduced binding of 3H-ET0 at the high affinity site but

not at the low affinity site suggesting the opioid nature of the

former but not of the latter.

Examination of the potency of various unlabelled opioid ligands to

compete with binding of 0.1 nM 3

H-ET0 at the high affinity site

revealed that the latter actually consisted of a mixture of several

types including a major one (70 %) "pharmacological profile" in vitro

was clearly different from those of mammalian μ-, and -opioid

sites. In particular, the major opioid site of the frog brain

displayed moderate affinity toward DAGO (Ki = 60 nM) and DTLET (Ki =

120 nM) and low affinity toward U-50488 (Ki = 1,200 nM), i.e. toward

selective ligands that normally bind μ, and sites respectively

with nanomolar affinities.

Frog brain opioid receptors exist independently of and in association

with a guanine nucleotide regulatory protein.

Because of the limited space allowed to the present report, only data

from assays with 3H-DIP in the presence cf 120 mM NaCl will be

presented. Under these conditions, digitonin extracts from frog brain

were found to contain in the range 1.2-1.5 pmol (/mg of protein) of a

homogenous population of sites that bind 3H-DIP with high affinity (Kd

= 0.2 nM). Interestingly, the binding characteristics of 3H-DIP were

not modified in the least had the soluble extract been derived from

NaCl (120 mM) or GppNHp (50 μM) pretreated membranes.

In contrast, the sedimentation profiles of opioid binding activity in

the soluble extract were clearly heterogenous (figure 2). Opioid

binding activity was found to be associated with a minor component at

position 12S and with a major component at position 10S.

In extracts from membranes that had been incubated with 120 mM NaCl or

with 50 μM GppNHp prior to solubilization, no activity was recovered

at position 12S while, under these conditions, higher levels of 3H-DIP

binding were observed in association with the 10S component.

A simple way to explain these results is that the frog brain opioid

receptor exists independently of (10S) as well as in association with

(12S) a guanine nucleotide regulatory protein (N). The antagonist

W-DIP does not discriminate the two forms in question hence linear

6
Figure 1: Scatchard transforms of the equilibrium adsorption

isotherms of 3H-etorphine (a) and of 3H-diprenorphine in the

crude membrane fraction from the brain of R. ridibunda. (a) in 50

mM tris-HCl pH 7.4. The insert is the Scatchard transform of an

isotopic dilution experiment using 0.1 nM 3H-etorphine. The

preparation contained 0.7-0.8 pmol/mg protein of a high affinity

site (Kd = 0.1 nM) and 3.2-3.3 pmol/mg protein of a low affinity

site (Kd = 10 nM). (b) in 50 mM tris-HCl pH 7.4 + 120 mM NaCl.

The histogram at the right indicates the proportions of the

various types of binding sites that were labelled in the presence

of 0.1 nM 3H-etorphine. Op represents the major opioid site and

LAS the low affinity site.

Figure 2: Sedimentation profiles of the opioid binding activity

in digitonin extracts from the crude membrane fraction of the

brain of R. ridibunda. Opioid binding activity was assayed for in

each fraction of the gradients with 3H-DIP (2 nM) in the presence

of 120 mM NaCl. Solid line: the detergent extract was from

untreated membranes. Dashed line: the detergent extract was from

NaCl (120 mM) or from GppNHp (50 μM) preincubated membranes. Note

that under these conditions no binding activity is recovered in

association with the component that is sedimented slightly faster

than catalase (C) used as velocity marker.

7
Scatchard transforms from equilibrium binding studies. 12S receptors

appear to be converted into 10S receptors in the presence not only of

sodium ions but also and most significantly of GppNHp, an effector of

N proteins : this conversion manifests itself, in sedimentation

studies, by a decreased binding of 3H-DIP to the 12S component and by

a concomittant increased binding of 3H-DIP to the 10S component.

Purification

Opioid receptors in digitonin extracts from several thousands of frog

brains are now being purified by affinity chromatography on

immobilized dynorphin 1-13 and following essentially the protocol of

Gioannini et al. (1985) for the purification of an active

opioid-binding protein from bovine striatum. Our results are still

very preliminary yet a purified preparation is routinely obtained that

contains a major protein component of apparent Mr = 64.000. However,

it has not been unambiguously shown yet that the purified protein

actually carries the opioid binding site.

CONCLUSION

In addition to the frog brain opioid receptor, the μ-opioid receptor

from rabbit cerebellum (Jauzac et al. 1986) and the -opioid receptor

from guinea-pig cerebellum (Frances et al., unpublished) display the

10S and 12S forms which are thought to represent the receptor alone

(10S) and the receptor associated (12S) with a N protein. It is

therefore possible that all opioid receptor types share the property

of interacting with a N-protein and it is tempting to speculate that

opioid agonists in general modulate the permeability of the neuronal

membrane (North 1986) through, primarily, the receptor-mediated

activation of a N-protein. In this respect, opioid agonists in general

would act, at the molecular level, in a way that is not any different

from that of classical neuromodulators, serotonin, acetylcholine or

noradrenaline (Siegelbaum and Tsien 1983).

REFERENCES

Bruns, R.F., Lawson-Wending, K. and Pugsley, T.A. Anal Biochem, 132 :

74-81, 1983.

Gioannini, T.L., Howard, A.D., Hiller, J.M. and Simon, E.J. J Biol

Chem, 260 : 15117-15121, 1985.

Jauzac, Ph., Frances, B., Puget, A., Moisand, Ch. and Meunier, J.C. J

Receptor Res, 6 : 1-25, 1986.

North, A. Trends in Neurosci, 9 : 114-117, 1986.

Puget, A., Jauzac, Ph. and Meunier, J.C. Life Sci, 33 : 199-202, 1983.

Ruegg, U.T., Cuenod, S., Hiller; J.M., Gioannini, T.L., Howells, R.D.

and Simon, E.J. Proc Natl Acad Sci USA, 78 : 4635-4638, 1981.

Siegelbaum, S.A. and Tsien, R.W. Trends in Neurosci, 6 : 307-313,

1983.

Simon, E.J., Szücs, M., Benyhe, S., Borsodi, A., Zeman, P. and

Wolleman, M.J. Neurochem, 43 : 957-963, 1984.

8
THE DIRECT DEMONSTRATION OF BINDING OF MU, DELTA AND KAPPA

AGONISTS TO DIGITONIN-SOLUBILIZED OPIOID RECEPTORS

FROM BOVINE STRIATUM

G. Crema, T.L. Gioannini, J.M. Hiller and E.J. Simon

Depts. of Psychiatry and Pharmacology

New York Univ. Med. Ctr., New York, New York, 10016,

U.S.A.

ABSTRACT

Active opioid binding sites, that retain the ability to

bind tritiated agonists have been obtained in good yield

in digitonin /NaCl/Mg2+ extracts of morphine protected

bovine striatal membranes. Ligand protection of binding

sites and the presence of Mg ions were found to be

absolute requirements for agonist binding in this

solubilized opioid receptor preparation. Soluble

preparations contained a ratio of mu: delta:kappa similar

to that in the membranes.

INTRODUCTION

Solubilization, attempted purification and separation of

the various opioid receptor types are important steps

toward the elucidation of the molecular basis of receptor

heterogeneity. Earlier studies from this laboratory

demonstrated successful solubilization of active opioid

binding sites from mammalian brain, using digitonin and

high concentrations of NaCl (Howells et al. 1982).

Binding was measured using antagonists because under these

conditions little or no tritiated agonist binding could be

detected. Recently Itzhak et al. (1984) were able to

obtain opioid agonist binding after fractionation of the

soluble binding sites on a sucrose density gradient.

Opioid agonist binding activity has also been reported to

Mg2+/digitonin solubilized receptors from rat brain

membranes (Demoliou-Mason and Barnard 1986) and to CHAPSsolubilized receptors (Simonds et al. 1980) In addition,

some progress has been made in the identification of the

ligand binding subunits of mu and delta receptors, using

the technique of affinity labeling (Newman and Barnard

1984; Howard et al. 1985, 1986). In the present study, we

describe a modification of our method for extracting from

bovine striatal membranes active opioid binding sites that

retain the ability to bind tritiated agonists as well as

antagonists, with high affinity and in good yield.

MATERIALS AND METHODS

Particulate membrane fractions from fresh bovine striatum

were

The

prepared as previously described by this laboratory.

membrane preparations were stored at -70° in 0.32 M

sucrose (1:6 w/v) until needed. For solubilization, crude

9
membrane preparations were thawed, diluted 1:l with 50 mM

Tris (buffer

A) and

HCl containing 1 mM K2 EDTA, and 10 mM MgSO4

incubated with 1 uM morphine sulfate, for 30 min at

room temperature. The mixture was diluted with an equal

volume of buffer A containing 1% digitonin/l M NaCl and

stirred on ice for 30 min followed by centrifugation at

100,000 x g at 4°C. The supernatant was removed and mixed

1:1 with 40% polyethylene glycol 6000 (PEG) in presence of

0.1% IgG. The suspension was then spun at 12,000 x g for

15 min at 4°C. The resulting pellet was rinsed twice with

7.5% PEG and resuspended in buffer A, 0.1 mM leupeptin

NaCl. Duplicate 1 ml samples were incubated with

tritiated ligands 1 uM unlabeled naloxone, for 60 min at

25°C. After incubation, samples were precipitated with 13%

PEG in the presence of 0.15% IgG prior to filtration.

Specific binding was assayed using membranes and

solubilized preparations derived from equal amounts of

tissue, at the same concentrations of radioligand and

under identical conditions. Protein was determined by

the method of Bradford (1972). 3H-bremazocine (30 Ci/mmoll

and 3

H[Tyr-D-Ser-Gly-Phe-Leu-Thr] enkephalin

Ci/mmol) were purchased from NEN and

(DSTLE, 30 3

H-[D-Ala2-Me-Phe4-

Gly-011 enkephalin (DAGO, 60 Ci/mmol) from Amersham. The

following unlabeled drugs were used: Naloxone (Endo

Laboratories), morphine sulfate (Merck). Other chemicals

used were: digitonin (US Biochemical Corp.), polyethylene

glycol 6000 (PEG) and bovine gamma-globulin (IgG) from

Sigma, guanosine-5'triphosphate (GTP) from Boehringer.

RESULTS AND DISCUSSION

In an attempt to protect the opioid agonist binding from

the detrimental effect of digitonin and NaCl present

during the extraction procedure (Method I), bovine

striatal membranes were incubated with morphine and MgSO4

prior to solubilization (Method II).

no specific binding of selective

As shown in table 1,

3

H-labeled

peptides occurred under conditions of method I,

opioid

while, as

expected for 3

H-bremazocine and H-naltrexone the specific

binding recovered represented 30-35%. In contrast,

solubilization of membranes under conditions of method II,

produced a good yield of soluble receptor activity for

opioid peptides.

binding

This represented an 11-30% yield of

activity. Saturation curves of 3H-bremazocine

binding yielded linear Scatchard plots for both soluble

preparations with KD = 1.9 nM, Bmax = 349 fmol/mg protein

and KD =1.5 nM, Bmax = 292 fmol/mg protein for methods I

and II respectively (table II). The binding of selective

opioid ligands to soluble binding sites (from method II)

was saturable and of high affinity as indicated by the

apparent KD

demonstrates

and Bmax values summarized in table II. This

that distinct subtype specificities,

characteristic of membrane-bound receptors, were still

retained in the soluble preparation. In addition, the

ratio of mu:delta:kappa was similar in the soluble

10
fraction as in the membranes.

Table I

Yields* of agonist and antagonist binding in soluble

receptors extracted from bovine striatal membranes

using methods I and II

Extract PEG Protein Recovery of specific binding (%)

pptation % Naltr. Bremaz. DAGO DSTLE

(3nM) (3nM) (6nM) (6nM)

Method I - ** 50 30 35 0 0

+ 30 18 21 0 0

Method I I + 25 21 19 20 11

*Percent yields were calculated as follows:

(Specific binding to soluble fraction/specific

binding to membrane fraction x 100)

**Binding in extract not precipitated by PEG was

carried out after 1:5 dilution to reduce digitonin

concentration to 0.1%.

Table II

Comparison of affinity constants and maximal binding

values or solubilized bovine striatal membranes,

processed by methods I and II

Method I Method II

Ligand Kd(nM) Bmax Kd(nM) Bmax

(fmol/mg (fmol/mg

protein) protein)

3

H-Bremazocine 1.9 0.2 349 32 1.5 0.13 292 21

3

H-Bremazocine

(+100 mMDAG0 N.D.* N.D. 1.5 135

+ 100mM DPDPE)

3

H-DAGO N.D. N.D. 2.5 0.2 65 12

3

H-DSTLE N.D. N.D. 6.2 45

*N.D. - not detectable

Values represent mean of at least 3 determinations

S.E.

When no S.E. is given only 2 determinations were done.

As shown in table III, specific mu opioid binding to the

solubilized receptors was still sensitive to the

inhibitory effect of GTP. In fact at 100 uM GTP it

seemed to be more sensitive than to membrane-bound

receptors. In the membrane Mg2+ was able to reverse the

GTP-induced reduction in 3

H-DAGO binding. A similar

11

172-683 O - 87 - 2 : QL 3
reversal of GTP-induced decrease in 3

H-dihydromorphine

binding by manganese was reported by Childers and Snyder

(1980).

Effect of GTP and Mg2+ on mu type opioid binding to

Table III

membranes* and solubilized fraction of

bovine striatum

Specific binding of 3

H-DAGO (fmol/mg protein)

Conc

NaCl Additions

(mM) None GTP(l00uM) Mg 2+(10mM) Mg2++GTP

0 64 45 60 74

10 50 30 56 85

Membranes 25 33 20 58 75

100 13 7.5 42 47

Soluble 0 0 0 43 0

(Method II) 100 0 0 42 0

Results are the average of duplicate experiments.

*Specific binding was determined following incubation

of 1 ml striatal membrane (1 mg/protein/ml) with 3HDAGO (1 nM) in the absence and presence of 1 uM

unlabeled naloxone for 60 min, 25°C.

**Specific binding was determined following

incubation of 1 ml solubilized stria al membranes

"Method II" (0.6 mg/protein/ml) with

the absence

3

H-DAGO (6 nM)

in and presence of 1 uM unlabeled

naloxone for 60 min, 25°C.

REFERENCES

Bradford, M. Anal Biochem. 72:248-254, 1976.

Childers, S., and Snyder, S. J Neurochem. 34:583-593, 1980.

Cuatrecasas, P. Proc Natl Acad Sci USA. 69:318-322, 1972.

Demoliou-Mason, C., and Barnard, E. J Neurochem. 46:1129-

1136, 1986.

Howard, A., DeLa Baume, S., Gioannini, T., Hiller, J., and

Simon, E.J. J Biol Chem 260:10833-10839, 1985.

Howard, A., Sarne, Y., Gioannini, T., Hiller, J., and

Simon, E.J. Biochemistry. 25:357-360, 1986.

Howells, R., Gioannini, T., Hiller, J., and Simon, E.J. J

Pharmacol Exp Ther, 222:629-634, 1982.

Itzhak, Y., Hiller, J., and Simon, E.J. Proc Natl Acad

Sci USA. 81:4217-4221, 1984.

Newman, E. and Barnard, E. Biochemistry, 23:5385-5389, 1984.

Simonds, W.F., Koski, G., Streaty, R.A., Hjelmeland, L.M., and

Klee, W.A. Proc Natl Acad Sci USA, 77:4623-4627, 1980.

12
IDENTIFICATION OF RAT BRAIN OPIOID (ENKEPHALIN) RECEPTOR BY

PHOTOAFFINITY LABELING

Clement W.T. Yeung

Playfair Neuroscience Unit and Department of Biochemistry, University

of Toronto, Toronto, Ontario M5T 2S8 CANADA

ABSTRACT

A photoreactive, radioactive enkephalin derivative was prepared and

purified by high performance liquid chromatography. Rat brain and

spinal cord plasma membranes were incubated with this radioiodinated

photoprobe and were subsequently photolysed. Autoradiography of the

sodium dodecyl sulfate gel electrophoresis of the solubilized and

reduced mernbranes showed that a protein having an apparent molecular

weight of 46,000 daltons was specifically labeled, suggesting that

this protein may be the opioid (enkephalin) receptor.

INTRODUCTION

Extensive studies have been made on the endogenous opioid peptides

and their interactions (Smith and Loh 1981; Barnard and DemolionMason 1983; Zukin 1984).

lin) receptor in situ,

In order to identify the opioid (enkephathe technique of photoaffinity labeling was

employed. The usse of photogenerated reagents for the labeling of

biological receptor sites has been reviewed (Bayley and Knowles 1977;

Eberle and de Graan 1985). A number of photoreactive enkephalin

derivatives designed to investigate the opioid receptor have been

reoorted (Hazum et al. 1979: Lee et al. 1979; Smolarskv and Koshland

1980; Garbay-Jaurequiberry et al. 1984; Zioudron et al.’ 1983; Fujioka

et al. 1984; Yeung 1985). In this communication, we report the

preparation and purification of a radioiodinated, para-azido-Lphenylalanyl derivative of enkephalin. This photosensitive and

radioactive enkephalin tracer was used to identify the opioid

receptor in the plasma membranes from rat neural tissues. A plasma

membrane protein with an apparent molecular weight of 46,000 daltons

was specifically labeled. It is likely that this protein is a

protein of the opioid (enkephalin) receptor.

MATERIALS AND METHODS

Preparation and Purification of 125I(D-Ala2, p-N3-Phe4-Met5)EnkephaPurified (D-Ala2

, p-N3-Phe4-Met5)enkephalin (AP-Enk) (Yeung 1985) was

radioiodinated with carrier free 125I (Amersham Radiochemicals) and

chloramine-T as described by Hunter and Greenwood (1962). Separation

of the iodinated mixture on a Sep-Pak C18 reverse phase cartridge was

carried out as described by Yeung (1984). The radioactive photoprobe

was further purified by high performance liquid chromatography (HPLC)

on a Lichrosorb RP-8 (5 micron) column (0.4 x 25 cm) using a linear

gradient of 0.05% trifluoroacetic acid in water and acetonitrile, pH

3.0. Fractions collected from the column were analyzed by thin-layer

chromatography (tlc) on a cellulose Chromagram sheet (Eastman type

13255), developed in I-butanol:ethanol:2 N NH4OH (v/v, 5:1:2),

followed by autoradiography on Kodak AR5-X-OMat film. The extent of

radioiodination of AP-Enk was determined by alkaline hydrolysis,

(

125I)diiodotyrosine on tlc.

followed by analyzing the hydrolysate for (

125I) monoiodotyrosine and,

13
Binding and Photoaffinity Labeling

Plasma membranes from the rat brain (minus cerebellum), spinal cord

and cerebellum were prepared as the microsomal fractions from 250 g

male Wister rats according to the method of Posner et al. (1974).

These membrane preparations (3-4 mg/ml) were incubated in the dark in

a polypropylene microfuge tube containing 275 μ1 of 0.05 M Tris-HCl,

pH 7.5, 0.05 mM each of benzamidine hydrochloride and phenylmethylsulfonyl fluoride and approximately 0.5 μCi/ml of the purified radioactive photoreactive enkephalin at 24°C for 30 minutes. (D-Ala2

Met5)Enkephalin (4.3 nmol) was added where appropriate to determine

the non-specific binding. The reaction mixture was photolysed for 30

seconds using a 100-W high pressure mercury lamp, and was analyzed by

sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis on a

gradient slab of 10-15% (Yip et al. 1982). The membrane was reduced

bv boiling for 15 minutes in U.l M dithiothreitol and 3% SDS.

solubilized material was run on the gel. After staining, destaining

and drying, the gel was exposed to Kodak AR5-X-OMat film, using an

intensifying screen (Swanstrom and Shank 1978).

RESULTS AND DISCUSSION

The crude radioactive photoreactive enkephalin analogue recovered

from Sep-Pak minicolumn (SP-2) was further ourified bv HPLC. Four

radioactive fractions were eluted from the Lichrosorb column (Figure

1a). The purity of the fractions were assessed by tlc. Figure lb

shows the relative mobilities of the fractions. The Rf values of

HPLC-1 and HPLC-2 were similar to that of radioactive iodine standard. HPLC-3 and HPLC-4 (Rf values 0.72 and 0.76 respectively)

showed single major component with a trace amount of free iodine.

Alkaline hydrolysis of HPLC-3 and HPLC-4 followed by tlc showed that

both of these fractions were monoiodinated. Although HPLC-1 was a

major radioactive component, both HPLC-1 and HPLC-2 did not show any

specific binding by rat brain plasma membranes. In contrast, the

specific binding for HPLC-3 and HPLC-4 were estimated to be 90% and

FIGURE 1. (a) Llution profile of 125I-AP-Enk recovered from

Sep-Pak minicolumn (SP-2). Radioactive fractions 1-4 were

pooled for further characterization.

thin-layer chromatography of pooled fractions.

(b) Autoradiogram of

carrier free Na125I standard in water; 0 and F indicate

origin and solvent front respectively.

14
72% respectively of the total binding. Since HPLC-3 was found to be

more active in radioreceptor binding assay, while HPLC-4 retained

some binding activity, HPLC-3 was used for the photoaffinity labeling

experiment.

Binding of HPLC-3 to rat brain (minus cerebellum) plasma membrane,

followed by photolysis, led to covalent labeling of membrane protein

as shown by autoradiography (figure 2). A radioactive band was

specifically labeled (lane 1) since the labeling of the band was

abolished in the presence of an excess (2.5 μg) of (D-Ala2, Met5)-

enkephalin (lane 20). Such labeling was most likely the result of net

cross-linking of 125I(AP-Enk) HPLC-3 to the protein through the

para-azido-L-phenylalanine. The apparent molecular weight of the

radioactive band was estimated to be 46 kDa (ranqe 44 to 48 kDa).

Considering the anomalous behavior of glycoprotein in SDS-gel electrophoresis, the molecular weight of the labeled band may be less

than 46 kDa. The labeling of this membrane protein was totally

dependent on photolysis. since a control not exoosed to liaht showed

no such labeling (lane 3). Prephotolysing the radioactive tracer for

5 minutes before use resulted in no specific labeling of the 46-kDa

protein (lanes 6-8). Exposure of the membrane to the light source

for 1 minute before use in incubation and subsequent experiments did

not seem to affect the ability of the photoprobe to label the specific 46-kDa protein. A similar 46 kDa radioactive band was also found

to be covalently cross-linked and identified in the rat spinal cord

plasma membranes. However, no such protein or any other protein band

were specifically labeled when rat cerebellum plasma membrane was

used. These observations strongly suggest that the 46-kDa protein

may be the opioid receptor. It is apparent that a wide range of

FIGURE 2.

brain plasma membranes labeled with

Autoradiogram of an SDS gradient gel of rat: 125I-Ap-Enk HPLC-3,

enatured and reduced. Excess (D-Ala2, Met5)enkephalin was

used in lanes 2, 5 & 7; while lines 3 and 8 served as dark

control. The band (Mr 46 kDa) specifically photolabeled is

indicated by an arrow. The molecular weight standards

(Biorad) used were myosin (200,000), galactosidase

116,250), phosphorylase B (92, 500), bovine serum albumin

(66,200), ovalbumin (45, 000), carbonic anhydrase (31,000),

soybean trypsin inhibitor (21,500) and lysozyme (14,400).

molecular weights for the opioid receptor complex and its subunit has

been obtained from a number of direct and indirect approaches. In

15
this study using the direct photoaffinity labeling approach, we

estimate the molecular weight of the opioid (enkephalin) receptor

protein to be 46 kDa. This molecular weight estimate is within the

range of the reported values of 25-65 kDa for the subunit.

REFERENCES

Barnard, E.A., and Demoliou-Mason, C. British Medical Bull, 39:37-45,

1983.

Bayley, H., and Knowles, J.R. Methods Enzymol, 46:69-114, 1977.

Eberle, A.N., and de Graan, P.N.E. Methods Enzymol, 109: 129-156,

1985.

Fujioka, T., Matsunaga, T., Nakayama, H., Kanaoka, Y., Hayashi, Y.,

Kangawa, K., and Matsuo, H. J Med Chem, 27:836-840, 1984.

Garbay-Jaureguiberry, C., Robichon, A., Dauge, V., Rossignol, P., and

Roques, B.P. Proc Natl Acad Sci USA, 81:7718-7722, 1984.

Hazum, E., Chang, K.J., Shechter, Y., Wilkinson, S., and Cuatrecasas,

P. Biochem Biophys Res Comm, 88: 841-846, 1979.

Hunter, W.M., and Greenwood, F.C. Nature (London), 194: 495-496,

1962.

Lee, T.T., Williams, R.E., and Fox, C.F. J Biol Chem, 254:11787-

11790, 1979.

Posner, B.I., Kelly, P.A., Shou, R.P.C., and Friesen, H.G. Endocrinology, 95:521-531, 1974.

Smith, A.P., and Loh, H.H. In: Li, C.H. ed. Hormonal Proteins and

Peptides. Vol. X. New York, Academic Press, 1981, pp. 89-170.

Smolarsky, M., and Koshland, D.E., Jr., J Biol Chem, 255:7244-7249,

1980.

Swanstrom, R., and Shank, P.R. Anal Biochem, 86:184-192, 1978.

Yeung, C.W.T. Int J Peptide Protein Res, 24:636-644, 1984.

Yeung, C.W.T. Int J Peptide Protein Res, 26:63-69, 1985.

Yip, C.C., Moule, M.L., and Yeung, C.W.T. Biochemistry, 21:2940-

2945, 1982.

Zioudron, C., Varoucha, D., Loukas, S., Nicolaou, N., Streaty, R.A.,

and Klee, W.A. J Biol Chem, 258:10934-10937, 1983.

Zukin, R.S. In: Marangos, P.M., Campbell, I.C., and Cohen, R.M. ed.

Brain Receptor Methodologies Part B. New York, Academic Press,

1984, pp. 77-100.

ACKNOWLEDGEMENTS

This study was supported by grants from the Medical Research Council

of Canada.

16
PHOTOAFFINITY LABELING OF OPIATE RECEPTORS WITH 3

H-ETORPHINE:

POSSIBLE SPECIES DIFFERENCES IN GLYCOSYLATION

Wayne D. Bowen and Grace Kooper

Section of Biochemistry

Division of Biology and Medicine

Brown University

Providence, R.I. 02912

ABSTRACT

Opiate receptors from whole rat brain (minus cerebellum) and cow

striatum were labeled irreversibly using the intrinsic photolability

of 3H-etorphine. After incubation with 2 nM 3H-etorphine and

centrifugal washing, membranes were irradiated with light of 254 nm.

Non-specific binding was determined by carrying out incubations in

presence and absence of 10 uM levallorphan. Specific binding in

photolabeled membranes was 75-80%, with a photo-incorporation yield

of approximately 50%. Photolabeled membranes were extracted with

CHAPS/Lubrol and unbound 3H-etorphine was removed by dialysis and

passage over Sephadex G-25. Solubilized proteins were then

subjected to chromatography on wheat germ agglutinin, and retained

proteins were eluted with N-acetyl D-glucosamine (NAG). Protein

profiles from rat brain and cow striatum were identical, with 89% of

the total protein flowing through unretained and 11% eluted by NAG.

However, the profile of radioactivity was markedly different in the

two species. With rat, the specific activity (cpm/A280) was the

same for flow-through and NAG-eluate. With cow, the specific

activity of the NAG-eluate was 17 times greater than the flowthrough. These results indicate that cow striatum and rat whole

brain contain populations of opiate receptors which are glycosylated

differently.

INTRODUCTION

Gioannini et al. (1982) have shown that opiate receptors from

several species are retained by wheat germ agglutinin and can be

eluted with N-acetyl D-glucosamine. This indicates that opiate

receptors are glycoproteins. These workers also found that varying

proportions of the total binding activity appeared in flow-through

and NAG-eluate in different species, indicating a possible

heterogeneity in receptor glycosylation across species. Howevever,

results are difficult to interpret in this regard since the 3Hligand could dissociate from receptor and appear in the flowthrough. We have developed a method to irreversibly label receptors

by utilizing the intrinsic photolability of various 3H-opiates

(Bowen et al. 1985, Kooper et al. 1985, submitted). Using this

method, opiate receptors can be irreversibly labeled with high

specificity and yield. Here we report that rat whole brain and cow

striatal receptors photolabeled with 3H-etorphine exhibit

differences in their ability to be retained on wheat germ

agglutinin. Therefore, opiate receptors may be glycosylated

differently across species or across brain regions.

17
METHODS

Lysed P2 membrane fraction was prepared from the brains (minus

cerebellum) of male Sprague-Dawley rats (150-200 gm) and freshly'

obtained cow striatum. Membranes (1 mg protein/ml) were incubated

with 2 nM 3H-etorphine in 10 mM TRIS-HCl pH 7.4 at 25°C for 90 min.

Non-specific binding was determined by incubating in presence of 10

uM levallorphan. Labeled membranes were washed twice by repeated

centrifugation and resuspension in ice-cold incubation buffer.

Resuspended membranes were either irradiated on ice for 10 min with

ultraviolet light of 254 nm (Mineralight R52G, Ultra-Violet

Products, San Gabriel, CA.) at a distance of 2 cm or allowed to

stand in room light (DARK-treated) for 10 min. Denaturation was

carried out by heating for 10 min at 90°C. To determine results of

these treatments, 500 ul aliquots were filtered over glass fiber

filters and washed with ice-cold TRIS-HCl pH 7.4. Filters were

counted after overnight extraction in scintillation cocktail.

Photolabeled membranes were solubilized by homogenization in 13 mM

CHAPS/1% Lubrol. The homogenate was incubated for 15-30 min at 25°.

The 105,000 x g supernatant was then dialyzed overnight at 4°C

against 10 mM TRIS-HCl pH 7.4 or gel filtered on Sephadex G-25

(eluted with buffer containing 10 mM CHAPS) to remove free 3Hetorphine.

Chromatography on wheat germ Lectin-Sepharose 6MB was carried out as

follows. Extract was applied to the column in 1 ml aliquots and

allowed to incubate in the bed for 15 min at 25°C. The column was

washed with 10 mM TRIS-HCl pH 7.4 containing 10 mM CHAPS and 1 mM

EDTA. Retained proteins were eluted with the same buffer containing

.5 M N-acetyl D-glucosamine. Protein was estimated by absorbance at

280 nm and a 50 ul aliquot of each 1 ml fraction was counted in a

scintillation spectrometer to detect tritium. Specific activities

of labeled proteins in the flow-through and NAG-eluate were

estimated by determining the areas under the cpm and A280 peaks and

taking their ratios.

RESULTS AND DISCUSSION

Table 1 shows results of photolabeling of rat whole brain and cow

striatal receptors with 3H-etorphine. After incubation with 3

Hetorphine and washing to remove unbound ligand, membranes were

irradiated at 254 nm or allowed to stand in room light.

Irreversible binding was assessed by heat denaturation.

Denaturation after dark treatment caused a loss of 97% and 95% of

specific binding in rat and cow membranes, respectively. However,

irradiation before denaturation markedly reduced the loss of

specific binding, For rat, 60% of specific binding in irradiated

membranes survived denaturation. For cow, 39% remained.

Importantly, the percent specific binding in irradiated/denatured

samples was 75% and 79% for rat and cow, respectively. Thus this

method produced covalent labeling of opiate receptors in rat and

cow. The receptors were labeled in high yield and with high signalto-noise ratio.

18
TABLE 1. Photolabeling of Rat and Cow Membranes with 3

H-Etorphine

RAT COW

B K TOT S B %SB B K TOT S B %SB

Dark 405 2363 1958 83 279 4007 3728 93

Dark/Denat. 376 433 57 13 192 365 173 47

UV 312 1455 1143 79 268 2465 2197 89

UV/Denat. 231 971 686 75 225 1076 851 79

Yield 60% Yield 39%

Membranes photolabeled with 3H-etorphine were solubilized with 13 mM

CHAPS/1% Lubrol and the soluble proteins were subjected to

chromatography on wheat germ Lectin-Sepharose 6MB. The elution

profile is shown in Figure 1. For both rat and cow, approximately

89% of the total protein flowed through the column unretained, while

11% was retained and eluted with NAG. However, the profile of

radioactivity was markedly different for the two preparations.

Table 2 shows an analysis of the specific activity in the flowthrough and NAG eluate from the two preparations, estimated by

determining the areas under the cpm and A280 peaks. For rat, the

specific activity of the flow-through and NAG eluate were the same.

However, for cow striatal receptors, the specific activity of the

NAG eluate was 17 times higher than that in the flow-through.

FIGURE 1. Wheat

germ agglutinin

chromatography of

CHAPS/Lubrol

extracts from

membranes

photolabeled with

3

H-etorphine.

19
TABLE 2. Comparison of Specific Activities of Peaks from WGAChromatography

RAT cow

area (cm2) area (cm2

)

cpm A 280 cpm/A280 cpm A 280 cpm/A280

FLOW-THRU 26.73 47.38 0.56 7.26 14.85 0.49

NAG-ELUATE 3.99 5.95 0.67 14.57 1.75 8.33

SP. ACT. RATIO

NAG-ELUATE : FLOW-THRU 1.2 : 1 17.0 : 1

These results are consistent with th se of Gioannini et al. (1982)

who observed that of total soluble 3

H-diprenorphine binding in rat,

23% was recovered in the flow-through and 31% was recovered in NAGeluate. For cow striatum, these values were 11% in flow-through and

47% in NAG-eluate. In the study described here, we utilized

covalently bound H-etorphine, so that dissociation of ligand is not

a factor. The results reported here have several interpretations.

Opiate receptors in different species may be glycosylated

differently. In this case, rat brain would contain a larger

population of receptors which lack affinity for wheat germ

agglutinin. Alternatively, different brain regions may contain

receptors which are glycosylated differently. The cow preparation

is from striatum while the rat preparation is from whole brain

(minus cerebellum). Striatal receptors from the two species would

have to be compared to resolve this issue. Finally, 3H-etorphine

will label mu, delta, and kappa receptors. The differences in the

species and/or regions may reflect a difference in glycosylation

among receptor types. Further purification of receptors will be

needed in order to determine glycosylation state of receptor types.

The method of photolabeling utilized here may facilitate achieving

this goal.

REFERENCES

Bowen, W.D., Kooper, G., Levinson, N., and Copeland, C. . __ INRC

Abstracts: 35, 1985.

Gioannini, T., Foucaud, B., Hiller, J.M., Hatten, M.E., and Simon,

E.J. Biochem Biophys Res Comm, 105:1128-1134, 1982.

Kooper, G., Levinson, N., Copeland, C., and Bowen, W.D. Fed Proc

Abstr, 44:494, 1985.

ACKNOWLEDGEMENT

This work was supported by NIDA Grant No. DA 03776 (W.D.B.).

20
BINDING TO MU AND DELTA OPIOID SITES BUT NOT KAPPA

SITES IS INHIBITED BY FAB FRAGMENTS FROM A MONOCLONAL

ANTIBODY DIRECTED AGAINST THE OPIOID RECEPTOR

Jean M. Bidlack

Center for Brain Research, University of Rochester,

School of Medicine and Dentistry, Rochester, NY 14642, U.S.A.

ABSTRACT

noncompetitively inhibited the binding of the selective peptide

[

3

H][D-Ala2,MePhe4,Gly-ol6] enkephalin (DAGO) and the selective 

peptide,[3H][D-Pen2,D-Pen5] enkephalin (DPDPE), to neural membranes.

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against

the opioid receptor selectively inhibited opioid binding to rat and guinea

pig neural membranes. In a titrable manner, the Fab fragments

The binding of [

125I-Tyr27] endorphin was also blocked by the Fab

fragments. In contrast, opioid binding, as measured by the binding of

[

3

H]bremazocine to rat neural membranes and guinea pig cerebellum in

the presence of μ and blockers was not significantly altered by the Fab

fragments. When μ sites were blocked with DAGO, the Fab fragments

suppressed the binding of [3H]DPDPE to the same degree as when the μ

binding sites was not blocked. The Fab fragments also inhibited binding

to the μ site regardless of whether or not the site was blocked. This

monoclonal antibody is directed against a 35,000 dalton protein. Since

the antibody is able to inhibit μ and binding but not opioid binding, it

appears that the 35,000 dalton protein is an integral component of μ and

opioid receptors but not receptors.

INTRODUCTION

As a tool to probe the molecular basis of the multiple opioid receptors, a

monoclonal antibody, OR-689.2.4, capable of partially inhibiting opioid

binding to rat neural membranes has been produced (Bidlack and Denton

1984). This IgM was able to block and displace opioid ligands from rat

neural membranes. The specificity of this immunoglobulin has been

demonstrated by its inability to inhibit the binding of 5 different

nonopioid ligands to neural membranes and the ineffectiveness of other

mouse immunoglobulins to block the binding opioids to neural membranes

(Bidlack and Denton 1985). The antibody, directed against a 35,000 dalton

protien, immunoprecipitated opioid binding sites from a solubilized

preparation (Bidlack and Denton 1985). Because it is an IgM with a

molecular weight of 980,000, the ability of the antibody to penetrate

tissue is hampered. As a consequence, a procedure was developed for

obtaining Fab fragments, with a molecular weight of 48,000, from this

IgM (Bidlack and Mabie in press). This study describes the selective

inhibition of opioid ligands by OR-689.2.4 Fab fragments.

21
MATERIALS AND METHODS

Generating the OR-689.2.4 monoclonal IgM and Fab fragments

The IgM, OR-689.2.4, was produced by immunizing a BALB/c mouse with

a partially purified opioid receptor complex consisting of proteins with

molecular weights of 43,000, 35,000 and 23,000 (Bidlack et al. 1981;

Harwell et al. 1984). The monoclonal IgM was detected by

radioimmunoassay using the 125I-labeled antigen and by its ability to

inhibit opioid binding to rat neural membranes (Bidlack and Denton 1984).

The Fab fragments from the IgM were generated by digesting the IgM

with trypsin in the presence of 10 mM cysteine and were purified by gel

filtration (Bidlack and Mabie in press).

Opioid binding to rat and guinea pig neural membranes in the presence of

OR-689.2.4 Fab fragments

Neural membranes from male Sprague-Dawley and Hartley guinea pigs

were prepared, excluding cerebellar tissue, as previously described by

Pasternak et al. (1975). Guinea pig cerebellar membranes were prepared

in a similar manner, In a final volume of 1 ml, 0.25 mg of neural

membranes

min at

protein was incubated wth OR-689.2.4 Fab fragments for 60

25 C. Radiolabeled ligand was

continued for 60 min. Binding of [

125I-Tyr27] h-endorphin was measured

then added and the incubation

in the presence of 0.2% bovine serum albumin and 100 μg/ml bacitracin.

The glass fiber filters were soaked in 0.2% polyethylenimine prior to use.

Bound radioactivity was determined by filtering samples through Whatman

GF/B glass fiber filters. All results are reported as specific binding- the

difference between binding in the presence and absence of 10 μM

unlabeled ligand. Binding of 0.1 nM [

3

H]bremazocine to rat neural

membranes and guinea pig cerebellum in the presence of 100 nM DAGO

and either 100 nM DADLE or 1 μM CPDPE was regarded as binding to

sites.

Measuring the ability of the OR-689.2.4 Fab fragments to inhibit

binding to membranes when binding sites are blocked

To determine if the Fab fragments could inhibit the binding to μ sites

when sites were blocked, the following protocol was followed.

Membranes were incubated with 100 nM DPDPE for 30 min to block

sites.

added.

OR-689.2.4 Fab fragments at a concentration of 250 nM were

Following a 60 min incubation, 0.5 nM [

3

H]DAGO was added.

After an additional 60 min incubation, the samples were filtered.

RESULTS AND DISCUSSION

As shown in Fig. 1, OR-689.34 Fab fragment% inhibited binding to the

binding of [

125I]endorphin, [

3

H]DAGO, and [

3

H]DPDPE to rat neural

membranes in a titrable manner. The IC50 value for the inhibition of

[

125I] endorphin binding was 400 nM, while the IC50 value for the

inhibition of binding of the μ and ligands was about 3-fold greater. As

previously shown, the Fab fragments are noncompetitive inhibitors of μ

and binding to rat neural membranes (Bidlack and O’Malley in press).

22
FIGURE 1. Inhibition of opioid binding to rat neural membranes

by OR-689.2.4 Fab fragments. Binding was measured as

described in “Materials and Methods.”

Kappa binding to rat neural membranes and guinea pig cerebellum was

determined by the binding of 0.1 nM [

3

H]bremazocine in the presence of

μ and blockers. In the absence of blockers, 500 nM OR-689.2.4 Fab

fragments inhibited the binding of 0.1 nM [3H]bremazocine to rat neural

membranes by 27% (Bidlack and O’Malley in press). However, in the

presence of 100 nM DAGO and 100 nM DADLE, the Fab fragments did

not have a significant effect on the binding of 0.1 nM [3H]bremazocine.

The Fab fragments did not alter the binding of 0.1 nM [3H]bremazocine

to guinea pig cerebellum regardless of whether μ and blockers were

present (Bidlack and O’Malley in press). The antibody inhibited the

binding of [3H]DAGO and [3H]DPDPE to guinea pig neural membranes.

Since the antibody is directed against a 3-dimensional epitope on the

35,000 dalton protein (Bidlack and Denton 1985), it may be possible to

use the antibody to probe conformational changes of the receptor. In an

attempt to address the question of interconversion between μ and

binding sites, the ability of the OR-689.2.4 Fab fragments to inhibit the

binding of 0.5 nM [3H]DAGO to neural membranes was determined after

suppression of sites by 100 nM DPDPE. As detailed in Bidlack and

O'Malley (in press), the Fab fragments inhibited the binding of 0.5 nM

[

3

H]DAGO to the same degree regardless of whether sites were or were

not blocked by DPDPE. The same results were obtained when μ sites

were block with DAGO and the ability of the Fab fragments to inhibit

the binding of [

3

H]DPDPE was measured. Under the experimental

conditions used, there is not an interconversion of μ and sites that

results in the inability of the antibody to inhibit binding to the other site

23
when one site is blocked.

The monoclonal antibody is directed against a 35,000 dalton protein

(Bidlack and Denton 1985). Evidence supporting the role of the 35,000

dalton protein as a component of the opioid receptor includes crosslinking

studies using enkephalin-like peptides (Zukin and Kream 1979; Zukin et

al. 1980), human [125I-Tyr27] endorphin (Howard et al. 1985; Howard

et al. 1986) and [

125I-Tyr27,Leu5

], endorphin (Helmeste et al. in .

press). Affinity labeling studies using [

125I]14-bromoacetamidomorphine

also resulted in the specific labeling of the 35,000 dalton protein (Bidlack

et al. 1982). Purification studies using an opioid agonist affinity column

(Bidlack et al. 1981; Maneckjee et al. 1985) and an opioid antagonist

affinity column (Bidlack et al. 1982) have resulted in the specific elution

of a 35,000 dalton protein. The monoclonal antibody directed against the

35,000 dalton protein immunoprecipitated opioid binding sites from a

solubilized preparation of rat neural membranes (Bidlack and Denton

1985). These studies strongly suggest that the 35,000 dalton protein is a

component of the opioid receptor, probably of the μ and receptors but

not the receptor.

REFERENCES

Bidlack, J.M., Abood, L.G., Munemitsu, S.M., Archer, S., Gala, D., and

Kreilick, R.W., Adv in Psychopharmacol, 33:301-309, 1982.

Bidlack, J.M., Abood, L.G., Osei-Gyimah, P., and Archer, S. Proc Natl

Acad Sci USA, 78:636-639, 1981.

Bidlack, J.M., and Denton, R.R. Neuropeptides, 5:225-228, 1984.

Bidlack, J.M., and Denton, R.R. J Biol Chem, 260:15655-15661, 1985.

Bidlack, J.M., and Mabie, P.C. J Immunol Methods, in press.

Bidlack, J.M., and O’Malley, W.E. J Biol Chem, in press.

Harwell, L.W., Bolognino, M., Bidlack, J.M., Knapp, R.J., and Lord, E.M.

J Immunol Methods, 66:59-67, 1984.

Helmeste, D.M., Hammonds, R.GJ., Jr., and Li, C.H. Proc Natl Acad Sci

USA, in press.

Howard, A.D., de la Baume, S., Gioannini, T.L., Hiller, J.M., and Simon,

E.J. J Biol Chem, 260:10833-10839, 1985.

Howard, A.D., Sarne, Y., Gioannini, T.L., Hiller, J.M., and Simon, E.J.

Biochem, 25:357-360, 1986.

Maneckjeee, R., Zukin, R.S., Archer, S., Michael, J., and Osei-Gyimah, P.

Proc Natl Acad Sci USA, 82:594-598, 1985.

Pasternak, G.W., Wilson, H.A., and Snyder, S.H. Mol Pharmacol,

11:340-351, 1975.

Zukin, R.S., Federoff, G.D., and Kream, R.M. In: Way, E.D., ed.

Endogenous and Exogenous Opiate Agonists and Antagonists. New

York: Pergamon Press, 1980. pp. 221-224.

Zukin, R.S., and Kream, R.M. Proc Natl Sci USA, 76:1593-1597,

1979.

ACKNOWLEDGEMENT

This work was supported by grant DA 03742 from the National Institute

on Drug Abuse.

24
MONOCLONAL ANTIIDIOTYPIC ANTIBODIES WHICH RECOGNIZED THE BINDING

SITE OF DELTA RECEPTOR :

FINE SPECIFICITY OF THE ANTIIDIOTYPIC ANTIBODIES

Anny Cupo and Pierre Kaldy

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy

Case 936, 13288 Marseille Cedex 9, France.

ABSTRACT

Monoclonal antiidiotypic antibodies (Mab2), specific for the binding

unit of the opiate receptor , were generated in rats immunized with

rabbit anti-DADLE antibodies (ab1). The fine specificity of the seven

clones screened in the fusion was studied using a solid phase test where

the abl antibodies were coated to the plate. In this respect, 4

different set of monoclonal antibodies can be distinguished. The

antibody affinities were comparable (Kd 1.10-8 M), but the specificity

patterns were completely different.

INTRODUCTION

A powerful approach to the production of antireceptor antibodies is now

fully exploited for a number of different cell receptors. This approach

employs the concept that antibodies directed against pharmacologically

active ligands have a three dimensional binding site which is somewhat

analogous to the natural receptor. Consequently, when antiidiotypic

antibodies (ab2) are produced against these anti-ligand antibodies

(ab1), some of the antiidiotypic antibodies, which mimic the original

ligand, are able to bind to cell surface receptors. Anti-idiotypic

antibodies, which have specificity of a variety of hormone and

neuro-transmetter receptors have been produced following immunization

against specific anti-ligand antibodies (Cleveland et al. 1983, Farid

and Lo 1985, David and Isom 1985). Using the antiidiotypic approach, we

developed monoclonal anti-idiotypic antibodies, specific for the binding

site of the receptor. These (ab2) antibodies were generated in rats

immunized with rabbit anti-DADLE antibodies. These anti-idiotypic

antibodies bind the cell surface of the NG108-15 cells and precipitated

a surface exposed 53Kd protein on the same cells (Cupo et al. data to be

published). The present study reports a detailed analysis of the

specificity of the seven monoclonal antiidiotypic antibodies screened in

the fusion and allows us to distinguish four different antiidiotypic

antibodies sets according to their structural requirements.

MATERIALS AND METHODS

Purification of monoclonal antibodies from ascitic fluids

3.106 hybridoma cells of each different clone were intraperitoneally

injected in nude mouse: (Swiss strain) in order to produce ascitic

fluids. After collection, 5 ml of each ascitic fluid were precipitated

by 50% ammonium sulfate and purified by DEAE-A52 chromatography

according to the Bazin's procedure for the IgG2a isotype (Bazin et al.

1974).

25
Anti-idiotypic test

DADLE was cross-linked by carbodiimide with bovine serum albumin (as

irrelevant carrier). Flexible PVC microtiter plates (Limbro plate SMRC

96) were coated with 50 μl of conjugate solution (1 nmole of DADLE) for

4 h at room temperature. The solution was then removed and the wells

were filled for 4 hours with PBS-0.5% BSA to saturate the remaining

binding site of the wells. The plates were washed 3 times with PBS, then

dried and stored at 4°C for weeks until used. A cascade of monoclonal

antibody dilutions and a solution of rabbit anti-DADLE immunserum

(diluted l/2000 in PBS-0.1% BSA) were preincubated at 37°C for 2 h 30 in

plastic tube. Then 50 μl of this mixture was transfered into

conjugate-coated wells at 4°C for 2 h. The wells were washed 3 times

with PBS, then incubated with 50 μl of iodinated protein A (1.105 cpm)

for 30 min at room temperature then washed again and counted. Polyclonal

rat immunserum (anti-anti-DADLE, diluted l/100) and irrelevant rat

monoclonal antibodies (anti-AMPc) served as controls.

Affinity and cross-reactivity measurements

The rabbit anti-DADLE antibodies (ab1) were purified onto a protein

A-Sepharose column. The total IgG solution (1.10 M) was coated on PVC

plates (50 μl/well) following the same procedure than in the

antiidiotypic test. The seven monoclonal antiidiotypic antibodies were

125I-iodinated, diluted in PBS-0.1% BSA and stored at 4°C. The

inhibition of binding of iodinated antiidiotypic antibodies Mab2, by ab2

was assessed by simultaneous incubation in abl (anti-DADLE antibodies)

coated wells of 25 μl of iodinated Mab2 (100,000 cpm) and 25 μl of ab2

at concentrations ranging from 10-5 M to 10-11 M. The incubation was

performed for 24 h at 4°C, the wells were then washed 3 times in PBS and

counted.

RESULTS AND DISCUSSION

Antiidiotypic activity

The antiidiotypic activity of each purified monoclonal antibody (Mab2)

was characterized by its ability to inhibit the interaction ab1-DADLE.

The 11 and 141 clones were able to inhibit 100% of the interaction

ab1-DADLE, The plateau was reached with an Mab2 concentration around

1.5 x 10-7M. For the 5 and 106 clones, the ab2 concentration necessary

to reach a plateau was identical, but these antibodies were able to

inhibit 85-90% of the abl-DADLE interaction. The 51 and 70 clones and 16

clone inhibited only 50% and 30% of the interaction ab1-DADLE,

respectively. To obtain this inhibition, the Mab2 concentration was

around 1.5.10-6 M. We can conclude, that the 11 and 141 antibodies

recognized all the abl population in a same manner or more probably the

same epitope exhibited by all the polyclonal anti-ligand antibodies

while the other clones 5-106, 51-70 and 16 recognized 85-90%, 50% and

30% of the abl population, respectively.

26
The results were shown in Figure 1.

FIGURE 1. Competition curve for the purified monoclonal antibodies.

Fine specificity of the anti-idiotypic antibodies (Mab2)

The affinity and the specificit of each purified Mab2 was realized by y

competition studies using 125I-labelled purified Mab2. The table

exhibited the results. The affinity (IC50) was expressed in term of

concentration (M) able to inhibit 50% of the 125I-MabZ-ab1 interaction.

The specificity pattern allows us to distinguish 4 different Mab2 sets.

Appeared to be identical 5 and 106 clones, 51 and 70 clones and 11 and

141 clones. Their affinities are similar and close to 1.10-8M but their

specificities are different. The 5, 11 and 16 Mab2 did not recognized

the epitope recognized by the 51 Mab2; moreover the fixation of these

Mab2 on their respective epitope did not interfere with the specific

binding of the 5 Mab2. For sake of clarity , we will named the epitope

recognized by the monoclonal antibody according to the number of the

antibody. The 11 and 51 Mab2 did not recognized the 16 epitope but the 5

Mab2 might be, either, exhibited a weak cross-reaction with the 16

epitope (f around 930) or its fixation on its specific epitope weakly

disturbed the specific binding of the 16 Mab2. We can propose the same

explanation for the specificity study of the 11 Mab2 ; the 16 and 51

Mab2 did not recognize the 11 epitope and the 5 Mab2 cross-reacted

weakly with the 11 epitope directly or not. The specificity pattern of

the 5 cloned showed that 16 and 51 clones did not recognize the 5

epitope but the 11 Mab2 strongly inhibited the 5 specific binding.

27
TABLE. FINE SPECIFICITY OF THE ANTI-IDIOTYPIC ANTIBODIES (versus ab1)

Iodinated M,ab

5 106 51 70 11 141 16

5 9.10-9M 7.10-9M nI nI 1.25 1.12 nI

106 7.10-9M 7.1.10-9M nI nI 2.5 2.5 nI

51 nI nI 1.2.10-8M 1.2.10-8M nI nI nI

70 nI nI 1.2.10-8M 1.2.10-8M nI nI nI

11 240 240 nI nI 1.10-8M 1.10-8M nI

141 150 75 nI nI 1.10-8M 9.7.10-8M nI

16 930 320 nI nI nI nI 3,10-9M

nI= no inhibition was observed using high concentration of Mab2 (C >

10-6M). The number expressed the cross-reactive factor f=IC5O analogue

versus IC5O homologue.

We can imagine either that the 11 and 5 epitopes are structurally

related epitopes or that the binding of the 11 Mab2 induced dramatically

structural change of the 5 epitope, but clearly the two epitopes were

carried by the same abl antibodies.

In conclusion, using the antiidiotypic approach, we have obtained 7

monoclonal antibodies (Mab2) which recognized the binding unit of the

receptor. These 7 Mab2 exhibit similar affinity but the antiidiotypic

activity and the specificity pattern allows us to distinguish four

different monoclonal antibody sets.

REFERENCES

Bazin, H., Beckers, A., and Querinjean, P. Eur J Immunol, 4:44-48, 1974.

Cleveland, W.L., Wassermann, N.H., Sarangarajan, R., Penn, A.S., and

Erlanger, B.F. Nature, 305:56-57, 1983.

David, S.S.N.G., and Isom, G.E. Biochemical Pharmacol, 16:2853-2858,

1985.

Farid, N.R., and Lo, T.C.Y. Endocrine Reviews, 6:1-23, 1985.

28
ANTI-IDIOTYPIC ANTIBODIES RAISED AGAINST

ANTI-METENKEPHALIN ANTIBODIES IN RABBITS

Jen Min-Feng, Liu Yan-Xin, and Chin Yin-Chang

Department of Pharmacology,

Institute of Basic Medical Sciences,

Chinese Academy of Medical Sciences, and

Faculty of Basic Medicine, Peking Union Medical College,

Beijing, China.

ABSTRACT

Anti-idiotypic antibodies were raised in rabbits immunized against immunoglobulins

purified from anti-metenkephalin antibody. The antibodies competed dose-dependently

with metenkephalin in binding to anti-metenkephalin antibody on a solid phase

metenkephalin enzyme-linked immunosorbant assay ( ELISA ). The titers of the antiidiotypic activity appeared characteristically in transient peaks and troughs in alternation with anti-metenkephalin activities and were retained after purification on various

affinity columns.

INTRODUCTION

Anti-idiotypes (anti-idiotypic antibody)

and antibodies have been proved to be useful tools in the study of receptor structure

and function (Venter et al. 1984). The

mechanism of interaction between a ligand

and its antibody might be quite different

from its binding to a receptor. However,

when an idiotypic antibody, raised from an

anti-ligand antibody, binds to the receptor,

it is always directed at the active binding

site of the receptor. This implies that,

under circumstances when it is difficult to

prepare anti-receptor antibodies from the

purified receptor directly, it might be feasible to use the anti-idiotypic approach. In

this way one obtains an anti-receptor antibody without purifying the receptor (fig. 1).

FIGURE 1. Anti-idiotypic approach of producing

anti-receptor antibodies. L: ligand, R: receptor,

Anti-R: anti-receptor antibody.

While working on opiate receptors, we have been trying to produce polyclonal and monoclonal anti-idiotypic antibodies as an immunological reagent and pharmacological tool to

study the structure and function of opiate receptors. We have succeeded in raising antimetenkephalin antibody (Ab1) in rabbits and use the purified anti-metenk immunoglobulin

as immunogen to produce polyclonal anti-idiotypic antibody (Ab2) against it.

METHODS

1. Immunization of rabbits:

Rabbits were immunized at multiple spots intradermally over the back with metenkephalin (metenk) conjugated to thyroglobulin at a dosage of 200 μg of free metenk emulsified

in complete Freund’s adjuvant for primary immunization, and 100 μg for monthly boostings thereafter. The animals were bled to death when a reasonable high titer of antimetenk antibody was detected in the serum. The antiserum was then purified down to its

IgG (IgG,) with a Protein A-Sepharose 4B column. This purified Ab1 was used as immunogen at 2 mg in complete Freund’s adjuvant for primary immunization and 1 mg

thereafter to raise Ab2 in rabbits. The best protocol we followed finally was to immunize

29
the rabbits once a week within the first month and biweekly thereafter or whenever

necessary.

2. ELISA for the characterization of anti-metenk antibody and Ab2 against it:

Multicell polystyrene plate coated with metenk-BSA conjugate 0.1 μg/well was incubated

with the rabbit anti-metenk antibody to be characterized, followed by goat-antirabbit

IgG horseradish peroxidase conjugate and then OPD added with H2O2 as its substrate, to

develop a color reaction readable at 492 nm with a spectrometer. For anti-idiotypic antibody against anti-metenk antibody, the anti-serum to be characterized was first incubated with a standard Ab1, before they were added to the well and proceed as mentioned

above.

3. Purification of the anti-idiotypic antiserum:

Normal rabbit IgG or metenk was coupled to CNBr-activated Sepharose 4B to form normal IgG-Spherose 4B and metenk-Sepharose 4B affinity columns. These are used to

separate anti-metenk and non-specific anti-IgG activities included in the anti-metenk

anti-idiotypic antiserum.

RESULTS

1. The specificity and sensitivity of the ELISA (Liu et a1. 1985), developed for the characterization of anti-metenk antibody and anti-idiotypic antibody against it:

The readings of O.D. at 492 nm on ELISA were linear with serial dilutions of anti-metenk

antibody or its IgG; it was even more sensitive than radio-immunoassay in monitoring the

titer of anti-metenk antibody (fig. 2). The binding of Ab1 or its IgG to the solid phase

metenk was displaceable by free metenk in the solution and is sensitive up to less than 0.5

μg or 1 nmol of free metenk (fig. 3). So it could also be utilized for the characterization

of the anti-idiotypic activities developed against Ab2.

FIGURE 2. Rabbit anti-metenk

antiserum dilution curve.

FIGURE 3. Inhibitory effect of free

metenk on the binding of antiserum or its

IgG to metenk-BSA conjugate coated on

the polystyrene plate. 50 μl of either

antiserum or its IgG were preincubated

with 50 μ1 of free metenk in twofold

serial dilutions at 37°, 1 hr, before

ELISA.

30
2. The development of anti-iditiotypic antibody against Ab, in rabbits:

Rabbits immunized against anti-metenk IgG, were bled at different intervals after

immunization and monitored for their titer of anti-idiotypic activities in their serum. As

shown in fig. 4A and 4B, in three out of four rabbits immunized with IgG1, their antiidiotypic activities went up and down in peaks and troughs, and did not follow closely the

protocol of immunization.

FIGURE 4. (A) The inhibitory effect of anti-metenk anti-idiotypic antiserum on the binding of

anti-metenk antiserum and solid phase metenk. (B) The binding of anti-metenk anti-idiotypic

antiserum on solid phase metenk. The anti-metenk anti-idiotype antiserum was drawn after

immunization with anti-metenk IgG at diIferent time intervals as indicated on the abscissa.

Number in parenthesis indicates the number of rabbits.

At the same time, there were corresponding fluctuations of anti-metenk activities in the

serum, interlocking with anti-idiotypic activities, as demonstrated clearly in fig. 5 for

individual rabbits.

FIGURE 5. The generation of anti-idiotypic antibodies following immunization with antimetenk IgG in Rabbit #2 (left) and #4 (right). (A) The inhibitory effect of anti-metenk antiidiotypic antiserum on the binding of anti-metenk antiserum and solid phase metenk. (B) The

binding of the antiserum to solid phase metenk.

31
3. The effect of purification of anti-idiotypic activities on normal rabbit IgG- and

metenk- Sepharose 4B columns:

The anti-idiotypic activities were retained in effluents when the anti-idiotypic antiserum

was added in batches to normal IgG- or metenk- Sepharose 4B affinity gels. At the

same time the eluates or the filtrates, obtained after the gels were treated with 0.1 M

acetic acid (pH 2.4) and neutralized immediately by saturated Tris buffer, showed no

significant anti-idiotypic activities (fig. 6).

FIGURE 6. The inhibition of purified anti-metenk anti-idiotypic antibody on the binding of

anti-metenk with solid phase metenk. serum, IgG, effluent

from normal IgG-Sepharose 4B, effluent from metenk-Sepharose 4B.

DISCUSSION

Our results indicate that anti-idiotypic antibody could be generated against antimetenk antibody in rabbits immunized against anti-metenk IgG. Their appearance is

in line with the “immune network theory” proposed by Jerne (1974). The characteristic peaks and troughs, in alternation with anti-metenk activities, were probably the

result of a self-regulatory mechanism within the immune system of the body. Analogous findings have been reported in the literature with rabbits immunized against

anti-alprenolol IgG by Strosberg’s group (Couraud et al. 1983). Our results also indicate a transient nature of the anti-idiotypic activity. No definite relation could be

found between its appearance in the serum and the protocol of immunization so that

optimal timing of its presence in the serum was only arbitrary. The amount of blood

that could be drawn from the animal was also limited. Further characterization of the

anti-idiotypic antiserum we obtained are now still underway, especially with regard to

its direct binding to opiate receptors. At the same time efforts are being made to

repeat the same procedure in BALB/c mice in an attempt to obtain an almost unlimited supply of monoclonal anti-idiotypic antibody.

REFERENCES

Couraud, P.O., Lu, B.Z., and Strosberg, A.D. J Exp Med, 157:1369-1378, 1983.

Jerne, N.K. Ann Immunol Inst Pasteur, 125:373-389, 1974.

Liu, Y.X., Yang, H.F., and Jen, M.F. submitted.

Venter, J.C., Fraser, C.M., and Lindstrom, J. Monoclonal and Anti-_

idiotypic Antibodies. Probes for Receptor Structure and Function, New York,

Alan R. Liss, 1984.

32
DEVELOPMENT OF A BIOTIN-AVIDIN PROBE FOR DETECTING

OPIOID RECEPTORS

Günther Hochhaus, Bradford W. Gibson and Wolfgang Sadée

Department of Pharmacy and Pharmaceutical Chemistry

University of California, San Francisco, CA 94143, U.S.A.

ABSTRACT

Biotinylated derivatives of -endorphin -EP) with C6 spacer arm,

inserted between biotin and -EP, were synthesized and isolated by

HPLC. Liquid secondary ion mass spectrometry (LSIMS) indicated the

presence of 1 to 4 biotin substituents per -EP molecule, and in

combination with the analysis of tryptic peptide fragments, specified

the location of the biotinylated lysine residue. Affinities to μ

receptors decreased with increasing biotinylation number.

Association of the biotinylated ligands with avidin retained or even

enhanced IC50 values at the μ site, thus, matching the relative

binding affinity of underivatized -EP with the monobiotinylated

derivatives. Hence, monobiotinylated -EP represents a versatile

opioid receptor probe.

INTRODUCTION

The avidin-biotin sandwich system represents a versatile tool in

biochemistry and molecular biology (Korpela 1984). Bifunctional

biotinylated receptor ligands have been employed for purification

(Finn et al. 1984) or histochemical detection of hormone and

neurotransmitter receptors with high resolution (Childs et al. 1983,

Atlas et al. 1978). Previously reported biotinylated leucineenkephalin derivatives failed to crosslink the opioid receptor and

avidin with sufficiently high affinity (Koman and Terenius 1980).

Here we report the synthesis of several biotinylated -EP analogs

with a C6 spacer arm and varying positon and number of

biotinylation. Among these, avidin-bound monobiotinylated

derivatives, modulated near the COOH terminal end were found to bind

to the μ receptor with a similar affinity to that of underivatized

-EP and hence, represent a promising probe for the opioid receptor.

MATERIALS AND METHODS

-EP was kindly provided by Dr. Chao Hao Li (University of

California, San Francisco). The following compounds were obtained

from the indicated sources: dimethylsulfoxide (DMSO), bacitracin,

trifluoroacetic acid (TFA), bovine serum albumin (BSA), 2-(4'-

hydroxyazobenzene)-benzoic acid (HABA) from Sigma, egg white avidin

(binding capacity: 12.8 μg biotin/mg), biotinyl- -aminocaproic acidN-hydroxysuccinimide ester (biotin-XNHS), D(+)-biotin from

Calbiochem, [3H]DAGO (50 Ci/mmol) from Amersham and diprenorphine-HCl

from NIDA.

Synthesis and identification

-EP (100 μg in 100 μl of 0.1 M NaHC03, pH:8.3) was mixed with

300 μl of biotin-XNHS (20 μg in DMSO). After 30 min, the reaction

was stopped by addition of 100 μl 1M acetic acid. Separation of

mono- and bis-biotinylated products was performed by reversed phase

HPLC (μ Bondpack Cl8 column, Waters) using a 40 min linear gradient

33
(initial solvent:acetonitrile/0.1% TFA in H2O (28/72, v:v); final

solvent: acetonitrile/0.1% TFA in H2O (34/66, v:v> with a flow rate

of 1 ml/min. UV detection was performed at 210 nM. Peaks were

collected and concentrated under a stream of nitrogen. Reaction

products were identified by LSIMS, as described by Gibson et al.

(1984), with or without prior tryptic digestion and subsequent HPLC

separation.

Binding studies

Ability of biotinylated analogs to bind to avidin was tested

spectrometrically, by displacing HABA from avidin (Green, 1970).

Competitive receptor binding studies were performed in washed rat

brain membranes (brain without cerbellum, Sprague-Dawley, male, 120-

140 g) with [

3

H]DAGO tracer (1 nM) at 20°C, as described by Yu et al.

(1986) with the exception of the incubation buffer (50mM Tris-HCl,

0.1% BSA, 0.01% bacitracin, pH:7.4). Ligands were used after

rechromatography of isolated peaks, shown in figure 1. Binding in

the presence of avidin was tested with preformed ligand-avidin

complexes at a fixed avidin concentration of 0.5 μM. Bound and

unbound tracers were separated by filtration through Whatman GF/B

filters. Relative receptor affinities were calculated from IC50

values according to Yu et al. (1986).

FIGURE 1. HPLC separation of monobiotinylated analogs.

For separation conditions see materials and methods (B1

B1 B1 monobiotinylated isomers; B2: mixture of bisbiotinylated products).

34
RESULTS AND DISCUSSION

We used -EP as parent compound for developing bifunctional ligands,

since it binds to the opioid receptor with high affinity (Ho et al.

1983) and slow dissociation kinetics (Howard et al. 1985). Further,

-EP

al.

can be covalently crosslinked to the receptor sites (Howard et

1985), which could serve to establish avidin-affinity

chromatography for receptor purification. -EP can be readily

biotinylated via free amino groups of lysine that are mainly located

in the a helical part, near the COOH terminal end, where modulations

may have little affect on receptor binding (Taylor et al. 1983).

Biotin was introduced via a C6 spacer arm, since systemic studies

showed the necessity of a spacer of at least 8 length to fully

retain avidin binding to macromolecule biotin-conjugates (Green et

al. 1971). Reaction of -EP with biotin-XNHS resulted in a mixture

of mono- to tetra-biotinylated analogs, that were separated by

reversed phase HPLC according to their biotinylation number (data not

shown). Mono-biotinylated derivatives were further resolved by twostep chromatography into several isomers (figure 1). Identification

was performed by LSIMS, with or without prior tryptic digestion.

Biotinylation of a lysine eliminates a potential tryptic cleavage

site; thus, in place of two peptides generated from trypsin cleavage

of native -EP only one new peptide containing biotin will be

generated. LSIMS of peptide fragments, after HPLC separation readily

specified number and position of the biotin residue (table 1).

TABLE 1. Relative IC50 values for μ receptor binding of

biotinylated -EP derivatives. Relative IC50 values are

calculated as described in materials and methods. IC50

values of -EP for μ sites were 1.6 0.7 (n=7) and were

identical in the presence of absence of avidin. B1 and B2

represent derivatives, separated according to the biotinylation number (mixture of isomers).

Ligand Rel IC50 Position of

biotinylation

without with

avidin avidin

-EP 1 1

B1 4 monobiotinylated

B1 4.3(4.1-4.5) 1.1(0.7-1.4) Lys 28, 29 or 24

B1 5.5(3.5-7.8) 3.0(2.0-4.0) Lys 9

B1 8.5(6.5-10.5) 2.9(1.7-4.2) Lys 19

B2 18 (16-19) 10 bisbiotinylated

Mono- and bis-biotinylated compounds displaced HABA from avidin in a

stoichiometric manner directly related to the biotinylation number,

which suggested that each biotin residue was accessible to avidin.

Affinities to μ sites were determined in rat brain homogenate, using

underivatized -EP (figure 1) as internal standard (IC50: 1.6 0.7

nM, n=7). There was a decrease of binding affinity as a function of

biotinylation number (table 1). Monobiotinylated derivatives

exhibited binding affinities approximately 5 times less than that of

the parent compound, with no significant effect of the biotin

position on the binding potency. The hydrophilic region between

35
residues 6-12 is thought to be responsible for the potency of

-EP binding to the μ sites (Taylor et al. 1982), however,

biotinylation at Lys 9 did not affect the binding potency relative to

biotinylation near the COOH terminal.

In order to test the ability of the bifunctional ligands to crosslink

receptor sites with avidin, binding studies were performed in the

presence of excess of avidin (0.5 μM). This insured the presence of

biotinylated -EP-avidin complexes at a 1:1 ratio over the whole

spectrum of ligand concentrations. Neither tracer binding nor the

IC50 value of -EP were affected by this concentration of avidin.

IC50 values of mono- and bis-biotinylated analogs shifted to higher

affinities by a factor of 2-4. A decrease of IC50 values in the

presence of avidin has not been previously reported for biotinylated

receptor ligands. However from these data alone, a higher receptor

affinity of the complex cannot be readily deduced. High nonspecific

binding to glass and plastic presents problems in receptor assays

with -EP. Assuming that nonspecific binding is reduced for avidin

associated ligand, a shift of the competition curve to lower IC50

values will be the result, without any change in intrinsic receptor

affinity of the complex over that of the free ligand. Because of the

high affinity of the monobiotinylated -endorphin Bla (with the

substituent near the COOH terminal), which is retained or even

enhanced after tight association with avidin, this derivative

represents the most promising probe of the opioid receptor.

REFERENCES

Atlas, D., Yaffe, D., and Skutelsky, E. FEBS Lett., 95:173-176,

1978.

Childs, G.V., Naor, Z., Hazum, E., Tibolt, R., Westlund, K.N., and

Hancock, M.B. J Histochem Cytochem, 31:1422-1425, 1983.

Finn, F.M., Titus, G., Horstman, D., and Hofmann, K. Proc Natl Acad

Sci USA 81:7328-7332, 1984.

Gibson, B.W., and Biemann, K. Proc Natl Acad Sci USA, 81:1956-1960,

1984.

Green, N.M. In: McCornick, D.B., and Wright, L.D., eds. Methods in

enzymology. Vol 18A. New York: Academic Press, 1970. pp. 418-424.

Green, N.M., Konieczny, L., Toms, E.J., and Valentine, R.C. Proc

Natl Acad Sci USA 74:2697-2700, 1971.

Ho, C.L., Hammonds, G., Jr., and Li, C.H. Biochem Biophys Res

Commun, 111:1096-1100.

Howard, D.A., de la Baume, S., Gionnanini, T.L., Hiller, J.M., and

Simon, E.J. J Biol Chem, 260:10833-10839, 1985.

Koman, A., and Terenius, L. FEBS Lett, 118:293-295, 1980.

Korpela, J. Med Biol, 62:5-26, 1984.

Taylor, J.W., Miller, R.J., and Kaiser, E.T. Mol Pharmacol, 22:651-

660, 1982.

Taylor, J.W., Miller, R.J., and Kaiser, E.T. J Biol Chem, 258:4464-

4471, 1983.

Yu, V., Richards, M.L., and Sadée, W. J Biol Chem, 261:1065-1070,

1986.

ACKNOWLEDGEMENTS

We would like to thank Dr. C.H. Li for the donation of EP. This

study was supported by Public Service Research Grant RO-1 DA 01095

from the National Insitute on Drug Abuse.

36
CONSIDERATIONS ON THE NATURE OF and RECEPTORS

Alexander Gero

Department of Pharmacology, Hahnemann University School of

Medicine, Philadelphia, Pennsylvania 19102

ABSTRACT

It is assumed that opioid receptors are flexible, distorted by

agonists but not by antagonists. Combined with Martin’s model of

the receptors, this assumption permits reasonable interpretation of experimental facts.

This is a progress report on an attempt to reinterpret Martin’s

picture of opioid receptors (1983) in light of the hypothesis

that receptors are flexible, inactive in their native conformation but activated when drugs distort them (Gero 1973). Drugs attaching without distortion have no effect and thus function as

competitive antagonists and may furnish information on the native

receptor. Agonists tell about the nature of receptor distortion.

Space limitation precludes reporting all results so far obtained.

Only some of the more important findings and the principal items

of evidence will be presented.

Martin distinguishes two “nuclear” sites in opioid receptors, an

aromatic ring binding site A and an anionic site B, and six major

(plus several minor) “satellite” sites which either reinforce

drug binding to the nuclear sites or (keeping the drug away from

the nuclear sites) hinder it. The location of the binding sites

around the morphine molecule is shown in the following sketch:

This writer also postulated two nuclear sites (Gero 1978), assuming further that the two sites are so located that they can

bind a drug molecule strainlessly if its cationic group is about 61⁄2 from the center of the benzene ring. The cation-binding

nuclear site in μ receptors is C, not B, and it binds not the N

37
atom but the methyl goup attached to it which, for instance in morphine, the quintessential μ agonist, carries the bulk of the cationic

charge (Kaufman et al. 1974). In morphine and its derivatives, including morphinans and benzomorphans, the N-methyl is only 41⁄2 from

the center of the benzene ring, and the rigidity of the polycyclic

ring system permits no alteration of this distance. Binding can

therefore occur only by forcing the receptor to narrow the A-C distance, hence such drugs are generally μ agonists. If, however,

allyl replaces the N-methyl, resonance shifts the cationic charge to

the terminal C atom of the allyl group. This C atom is 61⁄2 from the

center of the benzene ring, hence nalorphine, levallorphan, etc. do

not distort the receptor and are μ antagonists. A similar charge

shift occurs in N-cyclopropylmethyl and N-cyclobutylmethyl.

The first step in the attachment of the drug to the receptor is probably the attraction of the cationic head of the drug to the anionic

receptor site. Then the other sites come into play and add their

binding energy to the formation of the drug-receptor complex. It is

possible that not all other sites come close enough to the drug

molecule to contribute to binding. The final conformational adjustment of the drug-receptor interaction will be that which provides

the greatest free-energy decrease and therefore the most stable -

i.e. strongest - attachment. In this the benzene ring of the drug

plays a special role: if in the aggregate more free energy is lost

by binding it to site A even at the cost of receptor distortion,

the drug will be an agonist, otherwise an antagonist. The significance of the nuclear sites IS that their distance from each other

in the drug-receptor complex determines whether or not a drug is an

agonist. It is also apparent that there must be a great variety of

possibilities for antagonistic binding: a drug needs to occupy only

part of the binding sites in the receptor - of which site A may or

may not be one - in order to function as an antagonist.

Among the satellite sites, site F seems to be particularly important. We think that it is an electrophilic site with particular

affinity for a carbonyl oxygen at about 8 from the N-methyl (or

7 1⁄2 from the N), therefore it favors the attachment of hydromorphone, naloxone, etc. In some cases sites C and E may hold a drug

molecule quite strongly, and if no steric factors keep the drug

molecule from pivoting around the axis formed by these two sites

and away from site A, we have an antagonist. On the other hand, in

a drug like meperidine sites C and F can also bind the N-methyl

and the carbonyl 0, but then site A-binds the benzene ring with its

center 41⁄2 from the N-methyl. Hence meperidine is an agonist. In

fentanyl this distance is 71⁄2 , so here sites A and C are forced

apart rather than together. That is still a distortion, and fentanyl is also an agonist.

Site G appears to be a pocket that can accomodate both the polar

14-OH of oxymorphone, naloxone, etc. and the nonpolar hydrocarbon group of etorphine and other oripavines at the same place.

The receptor follows Martin’s model: the nuclear sites are A

and B, and C is a satellite site that can bind large alkyl -

38
groups. The electrophilic site F is important also: the antagonists

naloxone, naltrexone and WIN 44:441-3 all have a carbonyl group 71⁄2

from the N atom. The case of WIN 44,441-3 is particularly instructive: it is a benzomorphan with the center of the benzene ring 41⁄2

from the N-methyl (and 4 from the N) and should therefore be an

agonist, but since it has only a methyl group attached to the N,

binding to site C would be weak. On the other hand, just below the

N-methyl (when drawn like morphine in the illustration above) there

is a cyclopentyl ring which can bind strongly to site C. The molecule can therefore attach its N to site B, then turn clockwise so

that the cyclopentyl group faces site C and the carbonyl 0 site F,

but then the benzene ring is too far from site A to attract it and

to deform the receptor.

Where the receptor fits in remains to be seen.

As to receptors, a number of benzomorphans, morphinans and morphines have been listed as weak to moderate agonists or antagonists, but the really potent agonists are phencyclidine and ketamine, which have very little resemblance to opioid agonists, and

really potent antagonists are butyrophenones such as haloperidol and spiperone, which have just as little obvious resemblance

to opioid antagonists. However, if we write the structure of butyrophenones as N-H O=C chelates (which is certainly proper) we

see a benzene ring and an alkylated N in similar mutual position

as in μ antagonists. The receptor therefore may contain sites

A, B and C, also site E attracted to the fluorine atom, the strongly negative end of a dipole, and perhaps site F as welI (see

next paragraph).

Among morphine derivatives nalorphine is an agonist, naloxone an

antagonist, morphine a weak antagonist. Thus here, too, the allyl

is just a large alkyl group attaching to site C after site B has

bound the N. In nalorphine this places the benzene ring close

enough to site A to bind it, necessarily with distortion. If nalorphine distorts the receptor but naloxone does not, we must conclude that, as with receptors, the 6-carbonyl binds strongly to

site F and allows the molecule to pivot away from site A. Morphine-has no 6-carbonyl and only methyl on the N which does not

bind site C strongly enough to bring the benzene ring within

binding distance of site A. Hence morphine is attached only to

site B and functions simply as a weak antagonist.

Phencyclidine and ketamine can attach strongly to sites A, B

and C, but only at the cost of very great distortion: here

the N is closer to the benzene ring and sites A and B are

forced together to fit an N-to-phenyl distance-of only 3

in contrast to 4 in μ and agonists.

It is an intriguing question why μ, , and receptors have

so much in common that some of the same drugs can act on them.

This writer would answer that the basic features of these receptors - that is, sites A, B and C - must occur rather commonly in proteins, and when they are-present where appropriate

39
drugs can cause protein distortion with physiological consequences,

we are dealing with a receptor. The individual characteristics that

differentiate the several receptors will most probably lie in the

satellite sites. It will be an interesting challenge to molecular

pharmacology to unravel both the similarities and the differences

between the classes of opioid receptors and to find what features

they correlate with.

REFERENCES

Gero, A. Arch Int Pharmacodyn, 206:41-46, 1973.

Gero, A. In: van Ree, J.M. and Terenius, L. eds. Characteristics

and function of opioids. Elsevier/North Holland, 1978. pp.473-474.

Kaufman, J.J., Kerman, E. and Koski, W.S. Int L Quant Chem Quant

Biol Symp, 1:289-313, 1974.

Martin, W.R. Pharmacol Rev, 35:283-323, 1983.

40
A NOVEL COMPREHENSIVE OPIATE-RECEPTOR MODEL

Vera M. Kolb and James P. Snyder*

Department of Chemistry, University of Wisconsin-Parkside,

Kenosha, Wisconsin, 53141 U.S.A.

*Drug Design Section, Medicinal Chemistry

Searle Research and Development

Skokie, Illinois 60077, U.S.A.

ABSTRACT

A new, comprehensive opiate-receptor model based on calculations

of possible events at the molecular level is proposed. Agonist

versus antagonist action, ultra long-lasting activity and

N-dealkylation accompanying receptor binding are included.

MATERIALS AND METHODS

The syntheses and pharmacological characterization of the

ultra-long lasting compounds in this study are described by Kolb

et al. (1985) and Koman et al. (1986). Theoretical calculations

were carried out as described by Snyder et al. (1985, 1986).

RESULTS AND DISCUSSION

Recently we proposed a mu-opiate receptor model (Snyder et al.

1985) predicated on the concept that analgesia is the end-result

of proton transfer from nitrogen of piperidine ring-containing

opiates to the receptor (Dimaio et al. 1979). In this

communication we extend the outlines of the model to include the

phenomenon of long-lasting antagonist activity and

N-demethylation attending receptor action.

According to the present model the agonists bear equatorial N-CH3

and axial N-H functions, while the receptor features two negative

centers anchoring the positively charged ammonium head group by

means of intermolecular electrostatic bridges. Coulombic

attraction is dominated by the role of polar C-H bonds on the

rigid opiate (Sites 1 and 2, Fig. 1).

FIGURE 1. Antagonist and agonist cations bind to the

receptor through polar bonds. The antagonist N-R group hinders proton transfer; the

agonist N-H is readily deprotonated.

4 1
Biological action is initiated by transfer of the N-H proton from

the drug to the receptor. In response to the proton shift, the

receptor protein undergoes a conformational interconversion

inducing the amplification required for the ultimate effect.

Simultaneously, the agonist salt is converted to a neutral free

base. Positive charge at the N-head group is eliminated,

electrostatic binding is seriously weakened and the agonist is

released from the receptor binding pocket.

The positive, protonated head group of an antagonist, typically

carrying a somewhat bulky N-allyl or N-cyclopropylmethyl

substituent, binds in a similar fashion. However, by virtue of

the binding at Site 1 (fig. 1), the equatorial N-alkyl assumes a

rotational state which hinders proton transfer and displaces site

2 (Snyder et al. 1985). Head group charge is retained, receptor

conformation is conserved and the antagonists remain bound longer

than the agonists. The latter is supported by the experimentally

observed slower off-rates for antagonists (Kurowski et al.

1982). The former is substantiated by pharmacological evidence

that the mu-opiate receptor exists in two distinct molecular

forms assigned as agonist and antagonist (Snyder 1975).

Recently, we have discovered a series of novel ultra-long lasting

opiate antagonists (Kolb et al. 1985; Koman et al. 1986).

These compounds combine two chemically discrete and spatially

demanding molecular fragments. The one is an opiate antagonist,

e.g. naloxone. The second is a bulky moiety such as a

fluorescein or a steroid bound to the C-6 position of the rigid

antagonist via a thiosemicarbazone or an azine spacer,

respectively. The azino-compounds are prolonged blockers in the

brain opiate-receptor preparation, but not in the guinea pig

ileum. By contrast, the thiosemicarbazones are long lasting in

both preparations. Displacement studies with [3

H] DHM indicate

that all compounds in question access the same overall receptor

binding site.

Selected conformations of examples in the mono-axine,

double-azine and thiosemicarbazone classes were geometrically

relaxed by molecular mechanics. The naloxone moieties of each

were subsequently superimposed by a least squares fit.

Examination of the extended non-opiate fragments by computer

graphics portrays a qualitative structural differentiation among

the classes. In particular, the preliminary analysis suggests

distinct subsidiary binding sites for the various bulky groups.

Superposition of the double azines, e.g. the hybrid azine between

naloxone and androstene dione (NAL-AD-NAL), and the

naloxone-estrone mono-azine (NAL-E) intimates the presence of two

common binding sites. One is a hydrophobic patch which can

accept the nonpolar steroidal moieties. The other is a

hydrogen-bond-accepting site which can access the -OH group of

the phenol ring in either the estrone unit of NAL-E or the second

naloxone component of NAL-AD-NAL (fig. 2). In this scheme the

two classes -- mono and double azines -- are able to stimulate

similar receptor responses.

42
FIGURE 2. NAL-E (dark) and NAL-AD-NAL superimposed

at the naloxone fragment. The circled

OH’s may access a common receptor site.

The thiosemicarbazones, e.g. 6-fluoresceinyl- and 6-rhodaminyl

naloxone thiosemicarbazones (NAL-F and NAL-R respectively), are

characterized by a bulky 2,5-dibensopyran end group. The latter

is calculated to be nearly perpendicular to the rest of the

planar C-6 attachment (fig. 3).

FIGURE 3. NAL-E and NAL-F superimposed at the naloxone

fragment. The dibenxopyran group (light) is

perpendicular to the average molecular

plane.

No meaningful superposition was possible between the end groups

of the hybrid opiate-steroid mono-axines and the opiatefluorescein thiosemicarbazones because of profound structural

differences. The thiosemicarbaxone spacer is highly polar and

thus would appear incompatible with the above described

hydrophobic patch,

(fig. 4). Furthermore, the planar dibenzopyran moieties are

although it occupies the same molecular space

complemented by highly polar substituents that conceivably

associate with a polar peptide region of the receptor or possibly

the lipid head-groups of an adjacent membrane. In conclusion,

the thiosemicarbazones are proposed to achieve their long-lasting

effects by binding to either of the latter polar regions. This

suggests in addition that both the brain preparation and the gpi

contain similar environments for accommodating the fluorescein

43

172-683 O - 87 - 3 : QL 3
derivatives in the antagonist receptor conformation. The action

of long-lasting agonists (Koman et al., 1986) can likewise be

incorporated in this scheme as will be discussed in a forthcoming

paper.

FIGURE 4. NAL-F (dark) and NAL-E superimposed at the

naloxone fragment. The polar thiosemicarbazone of the former spatially matches the

nonpolar steroid of the latter.

Finally, transformation of the receptor protein to the agonist

conformation may activate N-dealkylases which convert a fraction

of the freed agonists to their nor-congeners. Evidence that this

reaction is initiated by oxidative electron transfer from

nitrogen has been summarized by Kolb (Kolb 1984). The process

was previously thought to be the genesis of the analgesic

response, though calculations on the energetics of N radical

cation formation render this speculation unlikely (Snyder et al.

1986). Presently, we consider oxidative N-dealkylation to be a

secondary event possibly associated with side effects such as

euphoria and addiction.

REFERENCES

Dimaio, J., Ahmed, F.R., Shiller, P. and Belleau, B. In:

Gualtieri, F., Giannella, M., and Melchiorre, C., eds. Recent

Advances in Receptor Chemistry. Amsterdam: Elsevier, 1979.

pp. 221-234.

Kolb, V.M. J Pharm Sci, 73: 715-719, 1984.

Kolb, V.M., Koman, A., and Neil, A. Pharmaceutical Res, 6:

266-271, 1985.

Koman, A., Kolb, V.M., and Terenius, L. Pharmaceutical Res, 3:

56-60, 1986.

Kurowski, M., Rosenbaum, J.S., Perry, D.C., and Sadee, W.

Brain Res, 249: 345-352, 1982.

Snyder, S.H. In: Matthysse, S. and Snyder, S.H. eds. Opiate

Receptor Mechanisms. Cambridge, Mass: MIT Press, 1975.

pp. 137-141.

Snyder, J.P., Mabilia, M., and Kolb, V.M. In: Dahlbom, R., and

Nilsson, J.L.G., eds. Proceedings from the VIIIth

International Symposium on Medicinal Chemistry. Stockholm:

Swedish Pharmaceutical Press, 1985. pp. 171-173.

Snyder, J.P., Halgren, T.A., and Kolb, V.M. submitted, 1986.

44
STEREOCHEMISTRY OF NOVEL DELTA SELECTIVE OPIATE

ANTAGONISTS

Vera M. Kolb, Duy H. Hua* and William L. Duax**

Department of Chemistry

University of Wisconsin-Parkside

Kenosha, Wisconsin 53141, U.S.A.

*Department of Chemistry

Kansas State University

Manhattan, Kansas 66506, U.S.A.

**Research Laboratories

Medical Foundation of Buffalo

73 High Street

Buffalo, New York 14203-1196, U.S.A.

ABSTRACT

We have discovered novel delta selective opiate

antagonists of a non-peptide nature. Our lead compound

at the present time is the mixed azine between estrone

and naloxone, the compound named EH-NAL. In this paper

we describe the stereochemistry of EH-NAL and an EH-NAL

analog, the mixed azine between pregnenolone and

naltrexone, the compound named PH-NX. The stereochemical

assignments are based on the high-resolution 13C-NMR

studies and on the X-ray structural determinations.

INTRODUCTION

Koman (1985) and Koman et al. (1986) studied the

interaction of the mixed azines between steroids and

naloxone with various opioid receptor types in vitro. We

summarize here their findings for the EH-NAL, the mixed

azine between estrone and naloxone. The potency of EHNAL as an antagonist of opioid effects on the

electrically evoked contractions of the mouse vas

deferens (M.v.d.) and guinea-pig ileum myenteric plexuslongitudinal muscle (G.p.i) preparations was compared to

that of naloxone. EH-NAL was 9-fold more potent than

naloxone in antagonizing the effects of D-Ala2-Leu5-

enkephalin in the M.v.d., and 22-fold less potent in

antagonizing normorphine in the G.p.i. The two compounds

were also compared for their capacity to displace the

binding of [

3

H]-dihydromorphine, [

3

H]-D-Ala2-Leu5-

enkephalin and [

3

H]-ethylketocyclazocine to rat brain

membranes. The relative affinities for delta-, mu- and

kappa-sites were 0.70, 0.16, 0.14 for EH-NAL and 0.05,

0.87, 0.08 for naloxone, respectively. Thus, in contrast

to naloxone which is mu selective, EH-NAL is a deltaselective antagonist.

In order to explain the unexpected delta selectivity of

EH-NAL, we undertook a detailed stereochemical analysis

of this molecule. Also, we designed another probe for

delta receptor, the mixed azine between pregnenolone and

naltrexone, the compound named PH-NX. In PH-NX the

steroidal unit is separated from the opiate unit by an

45
extra carbon as compared to EH-NAL. PH-NX has an OH

group at the same steroidal carbon as EH-NAL (C-3 of the

ring A), but lacks the aromatic pi cloud of the phenol

ring of EH-NAL (Fig. 1). A comparison of the delta

selectivities of EH-NAL and PH-NX thus may help elucidate

the biological roles of the phenolic pi cloud and the

absolute length of the steroidal unit in imparting the

delta selectivity.

MATERIALS AND METHODS

The 13C-NMR spectra were taken on a 400 MHz IBM-Bruker

WM-400 instrument. The X-ray measurements were performed

on a Nonius CAD4- and a Syntex P3 diffractometers.

The mixed azines between estrone and naloxone (EH-NAL)

and pregnenolone and naltrexone (PH-NX) were prepared by

reacting the free base of the opioid ketone naloxone or

naltrexone with the steroidal hydrazone estrone hydrazone

(EH) or pregnenolone hydrazone (PH), respectively. The

experimental procedure of Kolb and Hua (1984) was

followed. EH was prepared as described by Dandliker et

al. (1978). PH was synthesized by the same method.

Stereochemical determinations by 13C-NMR described below

were done in a manner analogous to that described by Kolb

and Gober (1983) and Kolb and Hua (1984).

RESULTS AND DISCUSSION

Stereochemistry of EH, a precursor of EH-NAL: Based on

the 13C-NMR, EH was a single hydrazone; only one C-17

(hydrazone carbon) was observed (at 157.76 ppm), and only

one azine with acetone-d, was found (C-17 of the azine at

173.56 ppm) (solvent CHCl3: acetone-d,). C-l 124.98; C2 111.66; C-3 153.73; C-4 114.01; C-5 136.32; C-6

29.08: C-7 25.79: C-8 37.25: C-9 42.98: C-10 129.84;

C-11 26.01; C-12 33.03; C-13 43.31; C-14 51.20; C-15

25.14; C-16 21.72; C-17 157.76 (the hydrazone carbon);

C-18 15.41. The stereochemistry of the hydrazone was

assigned to be anti since the syn isomer is sterically

very crowded. Namely, the distance between the syn NH,

and 12 H is only 1.2 (based on Dreiding models).

Stereochemistry of PH, a precursor of PH-NX: In the 13CNMR only one peak per carbon was observed, indicating the

presence of only one isomer. The inspection of the

Dreiding model of PH suggests the anti isomer as a more

favorable one, since the syn isomer is sterically crowded

due to a clashing between the NH, group of the hydrazone

and the angular C-18 methyl group and also the 16 H and

12 H. The X-ray structural determination confirms the

anti orientation in PH. Some characteristic 13C-NMR

shifts (dmso-d6): C-1 38.43; C-3 70.06; C-4 42.22; C5 141.31; C-6 120.42; C-9 49.83: C-10 36.16: C-11

22.73; C-13 43.13; C-14 55.75; C-16 36.99; C-17

58.35; C-18 13.04; C-19 19.20; C-20 147.28 (the

hydrazone carbon): C-21 15.81.

Stereochemistry of EH-NAL: Based on the 13C-NMR, EH was

a single azine; only one peak per carbon was observed.

46
The stereochemistry of the azine bond was assigned to be

anti on the steroidal C-17, because of the anti

orientation of EH, and anti on the opiate C-6 due to the

preference for the formation of the anti azine of

naloxone (Kolb and Hua 1984). Some characteristic 13CNMR shifts for the opiate and steroidal units of EH-NAL

are given (in CDCl3): The opiate unit: C-l 119.76; C-2

118.01: C-3 139.59; C-4 143.70; C-5 87.80; C-6 162.30

(the azine carbon); C-9 63.38: C-11 124.01; C-12

129.61; C-13 49.96: C-14 70.31; C-16 44.75: C-17

57.64: C-18 137.70; C-19 118.34. The steroidal unit:

C-l 126.29; C-2 113.83; C-3 153.52; C-4 115.90; C-5

135.20; C-8 37.95: C-9 43.68: C-10 131.51; C-13

44.25: C-14 52.12; C-17 175.79 (the azine carbon): C18 16.60.

Stereochemistry of PH-NX: The crude PH-NX was subjected

to a 13C-NMR analysis, which revealed the presence of two

azine isomers: steroid C-20 anti-opiate C-6 anti (major)

and steroid C-20 anti-opiate C-6 syn (minor). The

recrystallization of the crude PH-NX from EtOH-CHCl,

provided a single azine isomer (by 13C-NMR). The

structure of the latter isomer was determined by X-ray

and found to be anti (steroid)-anti (opiate) gauche azine

(Fig. 2). The C=N-N=C torsion angle was -123°,

indicating gauche geometry of the azine bond. Some

characteristic 1

3

C-NMR shifts (in dmso-d6) of the latter

anti-anti azine: The opiate unit: C-1 118.57; C-2

117.00; C-3 139.12; C-4 143.68; C-5 87.56: C-6 159.12

(the azine carbon): C-9 61.47; C-11 123.85; C-12

130.63; C-13 47.89; C-14 69.51; C-16 43.36; C-18

9.17; C-19 3.70; C-20 3.47 (the latter two shifts could

be interchanged). The steroidal unit: C-1 38.23: C-3

69.96: C-4 42.16; C-5 141.25; C-6 120.27: C-9 49.63;

C-14 55.85: C-18 13.16; C-19 19.09; C-20 157.24 (the

azine carbon): C-21 18.51. Some characteristic 13C-NMR

shifts for the opiate unit of the minor isomer: C-6

160.17 (the azine carbon): C-1 118.93; C-2 117.18; C-5

89.34: C-14 69.67: C-9 61.39. The steroidal azine

carbon: C-20 156.92.

Both EH-NAL and PH-NX are configurationally much more

rigid than typical delta substrates which are peptides.

Namely, both EH-NAL and PH-NX have as a major point of

flexibility in the molecule just one bond -- the N-N bond

of the azine linkage. Thus, these azines may be useful

as semi-rigid probes for mapping the delta receptor. EHNAL contains an aromatic ring (steroidal A ring) which

can be overlapped with Ph4-residue of enkephalins (Koman

1985; Koman et al. 1986; Pilipauskas et al. 1986). PHNX was found to be an active opiate antagonist (more

active than EH-NAL) on G.p.i. (Koman private

communication). The biological tests for the delta

selectivity of PH-NX are underway.

47
FIGURE 1. Structures of EH-NAL and PH-NX.

FIGURE 2. The X-ray structure of PH-NX.

REFERENCES

Dandliker, W. B., Braun, R. J., Hsu, M.-L., Brown, P.N.,

Levin, J., Meyers, C. Y., and Kolb, V. M. Cancer

Research, 38: 4212-4224, 1978.

Kolb, V. M., and Gober, J. R. Life Sci, 33, Suppl. 1:

419-422, 1983.

Kolb, V. M. and Hua, D.H. J Org Chem, 49: 3824-3828,

1984.

Koman, A. Ph.D. Dissertation, Uppsala University,

Sweden, 1985, ISBN 91-554-1753-1.

Koman, A., Kolb, V. M., and Terenius, L. submitted, 1986.

Pilipauskas, D., Snyder, J. P., and Kolb, V.M., 20th

Great Lakes Regional Meeting of Amer. Chem. Soc.,

Milwaukee, Wisconsin, June 2-4, 1986, Abstract MEDI268.

48
STRUCTURE ACTIVITY STUDIES OF A SERIES OF

4-(m-OH PHENYL) PIPERIDINES

G. Loew, J. Lawson, L. Toll, E. Uyeno, G. Frenking and W. Polgar

Life Sciences Division

SRI International, Menlo Park, California 94025, USA

ABSTRACT

A series of 4-(m-OH phenyl)-piperidine analogs with R4=methyl

or t-butyl and NR=methyl, allyl and phenethyl have been synthesized; and their receptor affinities, in vivo analgetic agonism and

antagonism, and energy-conformational profiles determined. These

analogs bind selectively and with moderate to high affinity to

opioid μ-receptors. Binding in their preferred phenyl axial

conformation appears to lead to meperidine-like agonism. In addition, for some R4=methyl but not t-butyl compounds, binding of an

energy-accessible phenyl equatorial conformation produces

antagonism.

INTRODUCTION

The 4 piperidine class of opioids, I, are thought to bind

and act at the same type of μ-receptors as morphine. Unlike morphine in which the phenyl ring is fused in a fixed axial position

with respect to the piperidine ring, these fragments are more flexible and can assume both phenyl axial and phenyl equatorial conformations. Consequently, in the 4 piperidines, antagonism was

found to be modulated primarily by the R4 substituent and the presence of a j-methyl and a m-OH phenolic group, rather than by Nsubstituents as in morphine. In particular, potent pure antagonists have been prepared in this family (Zimmerman et al. 1978),

for a number of R4 substituents with a 3-methyl group trans t o

them.

Previous theoretical studies (Burt and Loew 1980; Loew et al.

1981; Froimowitz and Kollman 1984), strongly indicate that the

observed antagonism is initiated by binding of an energy-preferred,

phenyl-equatorial conformer of these

analogs in an orientation relative to

morphine with overlapping m-OH phenyl

groups, as shown in figure la.

However, both phenyl equatorial and

phenyl axial conformers have been

implicated in agonist activity.

In order to further investigate

the role of relative stabilities of

phenyl axial versus phenyl equatorial

conformers in modulating relative

agonist/antagonist activity, a number

of 4-(m-OH ) piperidine derivatives

in which the R4 group alone could

affect conformational preferences

were selected for synthesis and

Fig. 1a: Proposed antagonist conformer

and receptorsite orientation relative

to morphine for 4 piperidines.

49
analyses. In particular, the effects of replacing a compact methyl

group by a bulky tertiary butyl group at the R4 position and of N-R

variations (analogs l-2a-c) on conformation, receptor affinity, and

in vivo analgetic agonism and antagonism were investigated.

METHODS AND PROCEDURES

A.

B.

C.

D.

CHEMICAL SYNTHESIS: All analogs, 1-2a-c were produced by

modification of a known procedure (McElvain and Clemens 1958).

OPIOID RECEPTOR BINDING: Competitive inhibition by analogs 1-

2a-c in rat brain homogenate of four tritium labled ligandc

(naloxone, D-Ala, D-Leu enkephalin, ethylketocyclazocine and

dihydromorphine) was determined by procedures and data analysis described previously (Cheng et al. 1986). Data obtained

were analysed by a modified version of the program LIGAND

(Munson and Rodbard 1980) which predicts a set of self consistent receptor binding affinities and capacities assuming different receptor site models.

IN VIVO PHARMACOLOGY: Analgesic agonist activity and antagonism to morphine analgesia of compounds 1-2a-c and of

morphine, meperidine and nalorphine as standards were evaluated in the mouse tail flick test by procedures which we have

described previously. (Cheng et al. 1986).

THEORETICAL STUDIES: All

calculations were performed

using a molecular mechanics

method called MOLMEC (Oie et

al. 1981). The initial geometries were optimized with

respect to all variables. To

obtain energy profiles for

phenyl ring rotation, constrained optimizations were

performed keeping the dihedral

angle C8C7C4Cm fig. 1b) fixed

Fig. 1b: Overlap of the optimized phenyl

axial conformer of 4 butyl-4-(m-OH)-

at a set of values with inter- phenyl piperidine with the benromorphan

vals of 30 degrees.

ring of morphine.

RESULTS AND DISCUSSION

The results of a 5-receptor site model obtained when all the

data was analyzed simulaneously are shown in table 1. The five

TABLE 1. Receptor Affinities(Kd n=) site model identifies sites we

Maximum Binding Capacities(pmol/gm) have called μ1, μ2, delta and

kappa. A fifth site, not

shown, with very high capacity

and low affinities for all

ligands is most likely unrelated to opioid activity. The

six 4-phenyl piperidines

studied (1-2a-c) are all μselective having high to moderate affinities at μ1 an μ2,

with values at delta and kappa

being one to two orders of

magnitude lower. The results

of in vivo studies of analgetic

agonist and antagonist potencies are shown in table 2.

50
The 4-t-butyl series are pure agonists with potencies about 1/10th

that of morphine, similar to meperidine, while two of the 4-methyl

analogs, 1b, 1c, show measurable antagonist activity, about 1/20

that of nalorphine. TABLE 2. Analgesic Agonist and Antagonist

The results of energy Potencies (μmol/kg.s.c.) in House Tallconformation studies of of flick Procedure

four 4-(m-OH-phenyl) piperidines with R4=t-butyl

(2a) , methyl (1a) and the 

latter with an a-or -3-

methyl substituent ( )

are summarized in table

3. As seen in this table,

a piperidine chair conformer is much more stable than

a twist boat conformer.

The analogs are ordered in

decreasing stability of the

optimized phenyl

axial piperidine

TABLE 3. Optimized Conformational Energiesa for 

4 -(m -OH phenyl) Piperdines

chair conformer.

The phenyl ring

torsion angle

( ), for the

lowest energy form

is given in

parenthesis. The

energy ( min/

morph) required to rotate the optimum axial phenyl ring conformer

to a morphine-like value ( =32’) is also shown.

Previous theoretical studies (Loew et al. 1980, Burt and Loew

1981) have led to the hypothesis that in meperidine- and prodinelike compounds, both agonist and antagonist activity appear to be

elicited by lowest energy piperidine-chair, phenyl equatorial conformers binding to the μ-receptor in a different orientations. For

the 4-alkyl-4-(m-OH phenyl)-piperidines, however, a different relationship between relative agonist/antagonist potency and phenyl

equatorial and axial conformations is emerging. As summarized in

table 3, our results reinforce the previous association of phenyl

equatorial conformers with antagonist activity but demonstrate for

the first time a clear association of phenyl axial conformations

with the initiation of agonist activity. The 4-t-butyl compounds

are the first simple 4-alkyl-4-arylpiperidines predicted to have a

definitive phenyl axial conformation. These compounds are pure

agonists, providing strong evidence that a phenyl axial conformation leads to agonist activity. As shown in table 3, the 4-methyl

compound, la, also has a preferred phenyl axial conformation but

with a much more accessible phenyl equatorial conformation. While

the N-methyl analog is in fact a pure agonist, the N-phenethyl

analog lc has significant antagonist activity.

axial and phenyl equatorial

Because both phenyl

conformers have energies within 1

Kcal/male, the N-substituent appears to affect the relative extent

to which each conformer binds to the receptor. Thus lc can bind in

both phenyl axial and phenyl equatorial conformations and exhibits

agonist and antagonist activity.

Further evidence for association of phenyl equatorial conformers with antagonist activity and phenyl axial conformers with ago51
nist activity comes from our energy conformation study of the

effect of adding a 3-methyl group cis and trans to the 4-

methyl group. As shown in table 3, the pure antagonism of 2

appears to be due to the preferential stabilization of the phenyl

equatorial conformer by 3.5 kcal/mole and verifies the role of

phenyl equatorial conformers in initiating antagonist activity. By

contrast, in the cis isomer, , the phenyl axial and phenyl

equatorial conformed are within 1kcal/mole, consistent with its

observed mixed agonist/antagonist activity.

Our studies not only provide further evidence for phenyl

equatorial conformers initiating antagonist activity and phenyl

axial conformers agonist activity, they also provide insight into

why N-substituent variations do not modulate agonist/antagonist

activity as they do in fused ring opioids. Previously, we have

postulated that important overlaps of both the amine nitrogen and

phenolic OH must be preserved in opioids for N-substituent modulation to correspond to that in fused ring analogs. Thus, since the

phenyl ring is axial in these rigid opioids, it is possible that,

at least in the 4-t butyl series in which the axial conformer is

the most definitive low energy form, N-substituent modification

could lead to an agonist/antagonist profile similar to morphine.

However, as shown in table 3 and in figure lb, for both 1a and 2a

in the lowest energy phenyl axial conformer, the phenylring is

nearly perpendicular to the piperidine ring, a very different relative orientation than in morphine. Thus, in neither series does

the optimum torsion angle of the phenyl group resemble that of

morphine and considerable energy is required (table 3) to rotate it

to that angle. As a results, no N-substituent variation effects

similar to those in fused ring opioids are observed in these 4-

alkyl piperidines.

REFERENCES

Burt, S.K. and Loew, G.H. In: Way, E. L., ed. Endogenous And

Exogenous Opiate Agonists And Antagonists. Pergamon Press, 1980

pp.25-28.

Cheng, A., Uyeno, E.T., Polgar, W., Toll, L., Lawson, J.A., DeGraw,

J.I. Loew, G.H., Camerman, A. and Camerman, N. J Med Chem,

29:531-537, 1986.

Froimowitz, M. and Kollman, P.J. Comput. Chem. 5:507-516, 1984.

Loew, G.H. and Jester J.R. J Med Chem, 18: 1051-1056, 1975.

Loew, G.H., Burt, S.K. and Hashimoto, G.M. In: Harris, L.S., ed.

Proceedings of the 42 Annual Scientific Meeting, The Committee

on Problems of Drug Dependence, NIDA Research Monograph 34,

1981. pp. 399-405.

McElvain, S.M., Clemens, D.H., J Am Chem Soc, 80:3915-3923, 1958.

Munson, P.J. and Rodbard, D. Anal Biochem, 107: 220-239, 1980.

Oie, T., Maggiora, G.M., Christoffersen, R.E., and Duchamp, C. Int

J Quantum Chem Quant Biol Symp, 8: 1-25, 1981.

Zimmerman, D.M., Smits, S. and Nickander, R. In: Harris, L.S.,

ed. Proceedings of the 39 Annual Scientific Meeting, The Committee

on Problems of Drug Dependence, 1978, p.237.

ACKNOWLEDGEMENTS

Support for this work from NIDA grant 02622 is gratefully

acknowledged.

52
THEORETICAL STRUCTURE-ACTIVITY STUDIES OF METHYL-SUBSTITUTED

4-(m-OH PHENYL) PIPERIDINES

Gernot Frenking, Gilda H. Loew and John Lawson

Life Sciences Division

SRI International, Menlo Park, California 94025, USA

ABSTRACT

The theoretically determined molecular structures of

N-protonated 1,3,4,6 methyl-substituted 4-(m-OH phenyl) piperidines

are correlated to their experimentally derived analgesic activities.

It is concluded that the orientation of the 3-methyl group plays a

crucial role in determining agonism and antagonism.

INTRODUCTION

The 4 piperidines are known since 1939 to be capable -of

exhibiting strong analgesic agonism. In 1977 the first example of

this class of compounds was found (Zimmerman and Nickander 1977)

which is a pure narcotic antagonist. Unlike the fused-ring

molecules, antagonism was not obtained by varying the N-methyl

substituent, but rather by introducing two methyl substituents in

the 3,4-trans position of the piperidine ring. Subsequently,

several analogs with methyl groups mainly in the 1,3,4,6 positions

have been synthesized and tested (Zimmerman et al. 1980, Huegi et

al. 1984), and it has been suggested (Zimmerman et al. 1980) that

antagonism is introduced by a phenyl equatorial conformer.

Molecular mechanics calculations of the various isomers were

performed in order to better understand conformational features

which modulate their agonist and antagonist activity.

METHODS AND PROCEDURES

All calculations were carried out

using the molecular mechanics program

MOLMEC (Oie et al. 1981). Unless

otherwise noted, all geometries and

energies are the result of a complete

optimization with respect to bond

lengths, bond angles, and torsion

angles. In all studies the piperidine

ring was calculated with a chair

conformation and protonated nitrogen

atom. In cases where several rotamers

exist with different values of the

torsion angle t (t = C8C7C4X, fig. 1)

we report only the energetically FIGURE 1

lowest-lying conformer.

RESULTS AND DISCUSSION

The theoretically obtained structures of the various isomers

are shown in figures 1 and 2. For each isomer only the global

minimum is shown, which represents one of the diastereomers

corresponding to the (-) 3-methyl and (+) 6-methyl group,

respectively. The value of E indicates the relative energy of the

higher energy conformer due to piperidine-ring inversion

(equatorial-axial) with the N-methyl group kept in an equatorial

53
position. The figures also exhibit the value for t calculated for

each global minimum. In some cases, the energy E to rotate the

phenyl group to a constrained value for t is given in parenthesis.

The experimentally derived analgesic activities were taken from Loew

et al. (this volume) for 1, from Zimmerman et al. (1980) for

compounds 2 - 7, and from Huegi et al. (1984) for compound 8. We

assume that the published results relate to racemates, since the

separation of the optical isomers was not reported.

Structure 1 has a methyl substituent only in the 4-position.

The phenyl-axial conformer is favored by 1.0 kcal/mol relative to

the phenyl-equatorial form, and this compound is reported as a pure,

moderate agonist (fig. 1). If an additional methyl group is added

in the 3-position trans to the 4-methyl (2, fig. 2), the

phenyl-equatorial conformer is now favored by 2.3 kcal. 2 is a

pure, potent antagonist. If still another methyl group is added to

2 in the equatorial 6-position, compound 3 results. The shapes of

2 and 3 are nearly identical except for- the additional methyl

group in 3. Like 2, 3 is a pure,

conclusion is that the methyl group

potent antagonist. The

in the 6-position of 3 is

unimportant for analgesic antagonism.

If the 3,4 methyl groups in 3 are not trans, but cis to each

other, compound 4 is formed. AS in 3, the phenyl equatorial

conformer of 4 is clearly favored over the phenyl axial form.

However, 4 is a pure, potent agonist! Two structural differences

may be related to this reversal of activity: The 3-methyl group is

equatorial in 4, but axial in 3, and the torsion angle t is very

different for both compounds. The energy to rotate the phenyl group

of 3 and 4 from their respective equilibrium values to the

constraint value of the other isomer is quite high (fig. 2). Thus,

at this point it can not be decided which structural difference

between 3 and 4 is responsible for the change in activity.

Compound 5 has an all-equatorial arrangement of the

3,4,6-trimethyl, groups. The phenyl-axial isomer is favored by 2.4

kcal/mol (fig. 2), and 5 is a pure, potent agonist. Comparing 5

with 4 shows that the-4-methyl and 4-phenyl groups are reversed,

while the rest of the structure is the same. The interesting

conclusion is that both phenyl axial and phenyl equatorial

conformers can lead to agonist activity.

In compound 6 the two methyl groups in the 3,4 position are

cis to each other. The phenyl equatorial form is favored for 6,

which is a mixed, moderate analgesic agonist/antagonist (fig. 2).

The structure of 6 is nearly identical to 4, except that the

methyl group in the 6 position is missing. What introduces

antagonism in 6? For 3, the 6-methyl group was found to be

unimportant for antagonism. Unless the 6-methyl group has a

different effect on antagonism in 4 compared to 6, the only

remaining explanation for the occurence of antagonism in 6 is the

reduced energy difference to the less stable conformer, which is

only 1.8 kcal/mol in 6, but 4.2 kcal/mol in 4. Thus, antagonism

would be introduced by the presence of the energetically higher,

phenyl axial form, 6a. This prediction can be tested by additional

studies of compound 7 (fig. 2). In 7, the additional methyl

group in the 6 position stabilizes the structure of the phenyl axial

minor isomer 6a sufficiently to become clearly the most stable form.

Unfortunately, 7 was only tested for analgesic agonism, not for

54
antagonism. If 7 were an antagonist, it would clearly implicate

phenyl axial conformers for the first time in such activities.

Analog 8, which has two methyl groups in the 3 position (fig.

2) does not bind to opiate receptor sites, and shows no analgesic

agonism or antagonism. The phenyl equatorial form is favored, and a

comparison with 2, a pure antagonist, and with 6 in which the

3-methyl equatorial form is most likely the agonist -(compare 4),

shows that the value of t in 8 is intermediate between 2 and 6.

However, the different t value of 8 should not cause the lack of

activity since rotation from the-equilibrium value of t = 36° to

the value for 2 (t = 68°) and 6 (t = 0°) is within the feasible

energy range (fig. 2). The only remaining explanation for the

inactivity of 8 as an opiate is the presence of two methyl groups

in the 3 position, each of which requires different orientations of

the compound at the receptor binding site to be accomodated.

The combination of all conclusions reached so far lead to a

picture which is different, but compatible with previous

suggestions. We conclude that for 1,3,4,6 methyl substituted

4 piperidines, a dominant factor in the structure activity

correlation of opiate activity is the orientation of the methyl

group in the 3 position of the piperidine ring. If this methyl

group is axial, the compound will be an antagonist, if it is

equatorial, it should be an agonist. If both orientations are

occupied by dimethyl groups, the compound is inactive. This points

to a mechanism where the 3-methyl group occupies a crucial steric

position in the molecule. It may be compared with a key which fits

in the "agonist key-hole" when the 3-methyl group is equatorial, and

it fits in the "antagonist key-hole" when the 3-methyl group is

axial. Both keys cannot fit into these key holes simultaneously if

methyl groups are found in both orientations in the same molecule.

This conclusion may now be used to predict activities of the

unknown compounds 9 and 10. In 9, the phenyl equatorial conformer

with the methyl group equatoria1 is clearly favored (fig. 2) and

thus, is expected to exhibit analgesic agonism. For 10, with the

methyl group in the axial position, the phenyl equatorial conformer

is the more stable form, but only by 1.5 kcal/mol. Consequently,

10 is expected to be an antagonist with the possibility to show

mixed activity since the less stable isomer with the methyl group in

the equatorial position might also be active.

REFERENCES

Huegi, B., Maurer, R., Römer, D., and Petcher, T.J. Eur J Med Chem

Chim Ther, 19:487-494, 1984.

Loew, G., Lawson, J., Toll, L., Uyeno, E., Frenking, G., and Polgar,

W., this volume.

Oie, T., Maggiora, G.M., Christoffersen, R.E., and Duchamp, C. Int

J Quantum Chem Quant Biol Symp, 8:1-25, 1981.

Zimmerman, D.M. and Nickander, R., Proc 39th Ann Mtg Committee on

Problems in Drug Dependence, 1977.

Zimmerman, D.M., Smits, S.E., Hynes, M.D., Cantrell, B.E., Reamer,

M., and Nickander, R. 43d Ann Mtg Committee on Problems in Drug

Dependence, 1981.

ACKNOWLEDGEMENT: Support for this work by NIDA Grant 02662 is

gratefully acknowledged.

55
FIGURE 2
CONFORMATIONAL STUDIES AND RECEPTOR BINDING OF DELTA

SELECTIVE OPIOID PEPTIDES

Christopher Keys, Peter Amsterdam, Philip Payne,

Lawrence Toll and Gilda Loew.

Life Sciences Division

SRI International, Menlo Park, California 94025, U.S.A.

ABSTRACT

Detailed energy-conformation studies of a linear (DTLET) and

four cyclic (DPDPE, DPLPE, DPLCE, DCLPE), selective opioid

peptides were combined with computer assisted detailed receptor

binding studies. The results of these studies have allowed the

identification of a low energy conformer common to all of these

analogs which could be responsible for their high affinity

receptor binding. This conformer contains multiple intramolecular

H-bonds and is very different from the beta-II type structure

previously postulated to lead to high affinity μ-receptor

binding. This μ-binding conformer was found either to have higher

energies or be greatly distorted in these selective analogs.

INTRODUCTION

selective

analogs has

been accomplished, the conformational requirements for high affinity binding

to receptors and how these are different from those favorable for

μ-receptor binding are not yet understood. Nor have commonalities

between linear and cyclic selective analogs been explored. In

previous studies of highly μ-selective morphiceptin analogs (Loew

et al. 1986) and enkephalins (Loew et al. 1982), we have identified

and characterized common candidate BII

1

-Type peptide conformers

leading to high affinity binding to μ-receptors. In a

complementary fashion, the energy conformational studies reported

here of one linear (DTLET) and the four cyclic selective peptides

binding has

been done

for the six

selective

peptides

shown in

table 1.

While

the successful design

of

Receptor binding studies (Chang and Cuatrecasas 1979) of

enkephalin analogs in rat brain homogenates have established that

these peptides bind to at least two distinct classes of opioid

receptor sites called μ and Further studies of the biochemistry

and pharmacology of receptors will be aided by the recent

discovery of two linear, elective, hexapeptides, Tyr-D-Ser(DThr)-Gly-Phe-Leu-Thr(DSLET, DTLET) (Zajac et al. 1983) as well as a

series of selective cyclic enkephalin pentapeptides (Mosberg et

al. 1983): (D-Pen2, D-Pen5 enkephalin, (DPDPE), its isomer

(DPLPE), and two analogs with a cysteine replacing one penicillaminone residue (DCLPE, DPLCE). To more clearly delineate

affinities to μ- and receptors, detailed computer assisted

TABLE 1. Receptor Affinitiesa and Calculated Relative Energiesb and 

Overlapc of Candidate High-Delta Affinity Conformers for Linear

and Cyclic Opioid Peptides.

57
shown in table 1, were designed to identify and characterize peptide conformations leading to high affinity binding to receptors.

METHODS AND PROCEDURES

OPIOID RECEPTOR BINDING: Competitive inhibition of all peptide analogs in rat brain homogenate of five labeled ligands [3

H]

Naloxone, DADL, DSLET, EKC AND DHM were determined by procedures

and data analysis described previously (Cheng et al. 1986). Data

obtained were analyzed by a modified version of the program LIGAND

(Munson and Rodbard 1980) which predicts a set of self consistent

receptor binding affinities and capacities assuming different

receptor site models.

Conformational energies were calculated using a modified version of the potential energy terms in the Empirical Conformational

Energy Program, ECEPP, (Momany et al. 1975), and optimized using a

new energy minimization program (PEP) developed in our

laboratory. Initial conformations to be optimized were constructed

using effficient interactive structure generating programs coupled

to graphics displays contained in the programs called MOLECULE

(Egan et al. 1982) Coordinates and atomic parameters of the unusual L-and D-penicillamines were developed systematically from a

related known X-Ray structure (Rosenfield and Parthasarathy

1975). The extent of overlap between any two conformers was determined by using a program called MOBLS, which determines the minimum

root mean square (rms) distance between user-selected, matched

atoms in two rigid molecules.

Different search strategies were used to span the conformational space for the linear and cyclic peptides studies. For the

linear haxapeptide, DTLET, an “aufbau” procedure was used, starting

with optimized conformers for each single amino acids, and linking

these to form initial hexapeptide conformers. This procedure led

to 30 different optimized structures with a relative energy range

of 7.5 kcal/mol. For the cyclic peptide DPLPE, the main

search strategy used involved a systematic exploration to determine

sets of 14 ring torsion angle values that allowed cyclic structures

to be formed, using a program developed in our laboratory based on

an existing ring closing algorithm (Go and Scheraga 1970). A

total of 248,000 conformations were subjected to cyclic ring

closure searches. These conformations were screened using

distance, energy and similarity criteria. Screening of all of the

successfully cyclized structures, resulted in 31 unique conformers

with optimized energies within 7.5 Kcal/mol of the lowest energy

structures. These 31 unique DPLPE conformers were used to construct initial conformations for the three remaining cyclic peptides DPDPE, DPLCE and DCLPE and additional conformers for the

linear peptide DTLET. Conversely, the 30 lowest energy DTLET

conformers were used as templates to construct additional initial

conformer for each of the four cyclic peptides. In this way a set

of 62 energy optimized conformers were obtained for DTLET and the

four cyclic peptides DPDPE, DPLPE, DPDCE and DCDPE.

RESULTS AND DISCUSSION

The receptor binding data was analyzed using a five-receptor

site model which gave the best fit to all of the experimental data.

called μ1, μ2, and and a fifth site with very high capacity and

low affinities for all ligands, most likely unrelated to opioid

activity. Table 1 shows the affinities of the six analogs studies

58
for sites labeled μ1,μ2 and verifying their high affinity at

and indicating that DPDPE and DPLPE have the most pronounced

selectivity with respect to both sites 31 cyclic structures. After

optimization of the three other cyclic analogs, using low energy

DPLPE conformers as templates, a single common low energy conformer

for all four cyclic analogs resulted. Moreover, when these 31 low

energy conformers were used as templates to construct initial conformers for the linear peptide DTLET, all resulting optimized conformers had energies more than 7.5 kcal/mole higher than the lowest

energy conformer found independently for DTLET by the aufbau procedure.

The search for

common low energy

conformers was much

more fruitful when the

reverse strategy was

tried, using the low

energy DTLET structures found from the

aufbau procedure as

templates for the

cyclic analogs. After

optimization, 16 conformers of DPLPE had

energies less than 7.5

kcal/mol above their

lowest energy conformers. These DPLPE

conformers were then

used as templates for

the remaining cyclic

peptides, and the

optimized conformers

were all examined for

similarities to their corresponding DTLET conformer using the rms

procedure. One low energy DTLET structure, with three strong

intramolecular H-Bonds (fig. 1) and a relative energy of 1.6

kcal/mol, was the template for the lowest energy conformer of DPLPE

found by any of the search strategies used. Furthermore, this

DPLPE conformer yielded low energy optimized structures for the

three other cyclic analogs with great similarity to DTLET. No

other conformer was found which was more similar, or had lower

energies for all 5 analogs. This conformer is then an excellent

candidate for high affinity binding to the receptor. Figure 1

shows the superposition of this candidate conformer for DTLET and

DPLPE and illustrates their similarity and internal H-bonding.

Given in table 1, is the correlation of receptor affinities,

with calculated relative energies and overlap of the candidate

conformer of each cyclic analog with that of DTLET. Consistent

with its lower affinity, only DPDPE seems appreciably different

from the others in its extent of similarity with DTLET. These

results indicate that a conformational effect, and perhaps the

position of the terminal COOH group and its involvment in two

internal H bonds, one to the TyrOH and the other to the DPEN-2

carbonyl, rather than an energy difference, could account for its

lower delta affinity. The candidate low energy conformers chosen

59
for DPDPE and DPLPE are also consistent with preliminary conformational results for these analogs using NMR methods (Mosberg 1986)

and should aid in their further interpretation.

The candidate peptide conformer leading to high affinity

receptor binding is very different from the form previously

proposed for favorable μ-receptor binding of D-Ala2-met-enkephalin

amide. Preliminary studies indicate that such conformers favorable

to μ-receptor binding are either higher energy forms or are greatly

distorted in these selective analogs.

The conformer identified in this study as responsible for high

affinity selective binding, clearly illustrates the key role for

the two threonine residues in DTLET. They are involved in two very

strong intramolecular H bonds: Thr2 with the Tyr-OH and Thr6 with

the Leu C=O. This latter moiety is also involved in H-Bonding with

the Tyr-OH. The multiple intramolecular H-bonds formed by these

residues apparently ensures a compact conformation very suitable to

selective binding. Cyclization appears to accomplish this same

purpose, cyclic analogs favoring a conformation very similar to the

multiply H-bonded linear peptide DTLET. This conformation retains

one strong H-bond between the TyrOH and the terminal carbonyl

group. The unique H-bonded conformations of these analogs must be

well suited for interaction with a receptor site. High affinity

could be the result of an excellent steric fit at this site,

assured by the internal H-bonds or the cyclization. Additionally,

once this fit is assured, the intramolecular H-bonds can be

replaced by more favorable interactions with appropriate receptor

residues to which the opioid peptide has been guided by its unique

conformat ion.

REFERENCES

Cheng, A., Uyeno, E.T., Polgar, W., Toll, L., Lawson, J.A., Degraw,

J.I. Loew, G.H., Camerman, A. and Camerman, N.J. J Med Chem, 29:

531-537, 1986.

Chang, K.J. and Cuatrecasas, P.J. J Biol Chem, 254:2610-1618, 1979.

Egan, J.T., Hart, J., Burt, S.K. and MacElroy, R.D. Comput

Graphics, 6:177-199, 1982.

Loew, G.H., Hashimoto, G., Williamson, L. Burt, S.K. and Anderson,

W. Mol Pharm, 22:667-677, 1982

Loew, G.H., Keys, C., Luke, B., Polgar, W., and Toll, L. Mol Pharm,

29 (#6): 546-553, 1986.

Momany, F.A., McGuire, R.F., Burgess, A.W. and Scheraga, H.A.

J Phys Chem, 79(22): 2361-2381, 1975.

Mosberg, H.J., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I.,

Galligan, J.J. and Burks, T.F. Life Sci, 33 Sup 1: 447-450,

1983.

Mosberg, H.J. private commm, 1986.

Munson, P.J. and Rodbard, D. Anal Biochem, 107:220-239, 1980.

Rosenfeld, R.E. and Parthasarathy, R. Aut, B31: 462-466,

1975.

Zajac, J.M., Gacel, G., Petit, F., Dodey, P.I., Rossignol, P. and

Roques, B.P. Biochem Biophys Res Commun, 111: 390-397, 1983.

ACKNOWLEDGEMENTS

Dr. H. Mosberg kindly supplied samples of the 4 cyclic analogs used

in binding studies. Support for this work from NIDA grant 02662 is

gratefully acknowledged.

60
SITE-DEPENDENT EFFECTS OF IONS ON μ-, AND OPIOID BINDING

IN SUSPENSIONS OF GUINEA-PIG BRAIN MEMBRANES

Linda E. Robson, Stewart J. Paterson and Hans W. Kosterlitz

Unit for Research on Addictive Drugs,

University of Aberdeen, Marischal College, Aberdeen, U.K.

ABSTRACT

NaCl, LiCl, NH4Cl and KC1 inhibit the equilibrium binding of peptide

agonists at each of the μ-, and sites; the orders of potencies

and the slopes of the dose-response curves are site-dependent. In

contrast, the monovalent salt choline chloride inhibits μ- and

binding but has little effect on binding. The profiles of activity of MnCl2, CaC12 and MgCl2 are also site-dependent but differ

markedly from those of the monovalent salts.

INTRODUCTION

In many of the early investigations of the effects of monovalent and

divalent salts on opioid binding, the opioids used were interacting

with more than one of the μ-, or sites. In more recent papers,

in which more selective labelling techniques have been used, sitedependent differences in the effects of salts on opioid binding in

rat brain have been indicated by several groups. The aim of this

investigation was to compare systematically the effects of the

chloride salts of monovalent and divalent ions on equilibrium binding at μ-, and K-binding sites of guinea-pig brain, using selective labelling conditions. A detailed report of the results will be

published elsewhere (Paterson et al. 1986).

MATERIALS AND METHODS

The effects of monovalent and divalent salts on equilibrium binding

at μ-, and opioid sites were investigated in membrane suspensions (10 mg tissue/ml 50 mM Tris-HCl, pH 7.4 at 25°C) of guinea-pig

brain. In the brain minus cerebellum the μ-sites were labelled with

[

3

HI-[D-Ala2

,MePhe4,Gly-o15]enkephalin (1 nM; 1221-2442 GBq/mmol,

Amersham International) and the sites with [

3

H]-[D-Pen2,D-Pen5]

enkephalin (1.5 nM; 1665 GBq/mmol, Amersham International). For

investigations at the site, binding of [

3

H]-dynorphin A (1-9) (0.1

nM; 1258 GBq/mmol, Amersham International) was determined in the

cerebellum, in which 84% of the opioid binding sites are of the

type; bestatin (30 μM) and captopril (300 μM) were added to reduce

the degradation of the labelled peptide. Specific binding was the

difference between the binding in the absence and presence of diprenorphine (1.2 μM; Reckitt and Colman). For a detailed account of

the experimental design see Paterson et al. (1986).

RESULTS

NaCl, LiCl, NH4Cl and KCl caused a dose-dependent inhibition of the

binding of the peptide agonists at each of the μ-, and sites.

On the other hand, choline chloride, which produced dose-dependent

61
inhibition of μ- and binding had, at concentrations up to 100 mM,

no inhibitory effects on binding. The orders of potencies of the

salts were different at each site, as seen from the values for threshold of inhibition (fig. 1). Furthermore, the slopes of the doseFIGURE 1. Thresholds of inhibition for effects of the

chloride salts of monovalent ions on binding at , μand opioid sites. Values are the means s.e.m. of 3-10

observations and were obtained by extrapolation of the linear

regression of log mM salt against change (%) in binding.

Ch: choline.

response curves also differed at each site. They were highest at

the site (mean = 51% change in binding/log mM salt), intermediate

at the μ-site (mean = 38% change in binding/log mM salt) and lowest

at the site (mean = 28% change in binding/log mM salt).

The effects of divalent salts differed from those of monovalent salts

and were also site-dependent. At the site, MnCl2, MgCl2 and CaCl2

markedly potentiated binding by up to 50%; no inhibition of binding

was observed at the highest concentration used (16 mM). In contrast, at the μ-site low concentrations of MnCl2 and MgCl2, but not

CaCl2, slightly potentiated binding and at higher concentrations all

three salts inhibited binding. In further contrast, binding was

inhibited by MnCl2, MgCl2 and CaCl2 but no potentiation was observed.

DISCUSSION

Since throughout this investigation chloride salts have been used the

62
differences in their effects can be attributed to the cations.

Since the orders of potencies and the slopes of the dose-response

curves are site-dependent, the mechanisms by which the monovalent

cations inhibit binding appear to differ at each site. Furthermore, the actions of divalent salts differ from those of monovalent

salts. For inhibition of μ- and binding by monovalent salts the

slopes of the dose-response curves are shallower than would be expected for competitive interactions, a finding in contrast to observations for NaCl in rat brain (Kouakou et al. 1982; Zajac and Roques

1985). These discrepancies may be due to differences in the species

or in the assay conditions.

REFERENCES

Pater

Kouakou, Y., Zajac, J.M., Moisand, C., and Meunier, J.C. Mol

Pharmacol, 21:564-569, 1982.

son, S.J., Robson, L.E., and Kosterlitz, H.W. Proc Natl Acad

Sci USA, 83:6216-6220, 1986.

Zajac, J.-M., and Roques, B.P. J Neurochem, 44:1605-1614, 1985.

ACKNOWLEDGEMENTS

Supported by grants from the U.K. Medical Research Council and the

U.S. National Institute on Drug Abuse (DA 00662).

63
BINDING CHARACTFRISTICS OF [D-Ala2

-MePhe4-Glyol5] ENKEPHALIN

AND [D-Pen2,D-Pen5] ENKEPHALIN IN RAT BRAIN: INHIBITION STUDIES

WITH FENTANYL-LIKE DRUGS

M. Yeadon and I. Kitchen

Department of Biochemistry, Division of Pharmacology & Toxicology,

University of Surrey, Guildford, Surrey GU2 5XH, U.K.

ABSTRACT

We have investigated the binding characteristics of two tritiated enkephalin

analogues [D-Ala2

-MePhe4-Glyol5]enkephalin (DAGO) and [D-Pen2, DPen5

]enkephalin (DPDPE) which are highly selective for μ and opioid sites

respectively, in homogenates of whole rat brain and of the pons/medulla region.

In addition, we have explored the binding profile of four 4-anilinopiperidine drugs

(fentanyls) by means of inhibition studies. Our studies show that the

involvement in pons/medulla of rat is small compared with that in whole brain

and with μ binding in both whole brain and pons/medulla. In addition the rank

order of affinities of the fentanyl series is the same at the μ and site, but the

affinities are between 1 and 600-fold greater at the former site.

INTRODUCTION

Foremost of the adverse effects observed with the use of opioid drugs as

analgesics or anaesthetic/analgesics is respiratory depression. Although this has

been known for a considerable time, little progress has been made in

determining the mechanism of action of opioids in this regard. Investigations at

the receptor level have until recently been impeded by the lack of highly

specific, high affinity ligands for μ- and -opioid sites. The advent at DAGO

and DPDPE has overcome this problem and these enkephalin analogues are now

available at usefully high specific activities.

We have begun in vitro experiments into the binding characteristics of these

ligands and of an homologous series of potent synthetic opioids, the fentanyls, in

rat brain homogenates, in particular from areas known to be involved- in

respiratory control.

METHODS AND MATERIALS

Adult male Wistar albino rats, University of Surrey strain (25Og) were killed by

decapitation, the brains rapidly removed and quickly dissected according to the

procedure of Glowinski and Iversen (1966). The procedure for preparation of

membranes for use in binding studies was identical to that of Gillan and

Kosterlitz (1982)

Saturation assay mixtures comprised a 2ml final volume of the membrane

preparation, containing 19mg original wet weight of tissue. Non-specific and

total binding of the 3H-DAGO and 3H-DPDPE was determined in the presence

and absence, respectively, of 100-fold excess of unlabelled diprenorphine or

naloxone. The assay tubes were incubated in a shaking water bath for 40 mins

(

3

H-DAGO) or 50 mins (3H-DPDPE) at 25°C, and incubation was terminated by

rapid filtration over glass fibre filters (Whatman GF/B), then immediately

washed three times with 5ml ice-cold buffer. The filters were transferred to

scintillation vials and extracted overnight in 0.5ml Triton X100: Toluene (20:80)

before addition of 3.5ml Unisolve 1 scintillant followed by liquid scintillation

counting. A

3

H-Hexadecane internal standard was included for quench

correction, and all assays were performed in triplicate.

64
For inhibition studies, the protocol above was followed, but the concentration of

3

H ligand was held constant whilst that of the cold displacing drug was

increased. Non-specific binding was assessed both in the presence and absence

of the displacing drug.

Data from saturation experiments were analysed by Scatchard plots and those

from inhibition assays by Hill plots. Slopes and intercepts were calculated from

the values of at least four independent experiments, derived by linear regression

of the data.

3

H-DAGO (57.5 Ci/mmole) and 3H-DPDPE (28.0 Ci/mmole) were obtained from

Amersham International, U.K. Alfentanil, carfentanil, fentanyl and sufentanil

were gifts fron Janssen Pharmaceuticals, Belgium. Naloxone was a gift from

Endo Laboratories, U.S.A., and Diprenorphine, a gift from Reckitt and Colman,

U.K. The labelled peptides were highly purified by HPLC.

RESULTS AND DISCUSSION

Binding of tritiated enkephalin analogues:

The specific binding of both 3H-DAG0 and 3H-DPDPE to whole brain and

pons/meduIla membranes demonstrated a rectangular hyperbolic relationship to

free ligand concentration, and Scatchard plots were always linear. However, it

was not possible to obtain acceptable correlation coefficients for Scatchard plots

from single experiments with 3

H-DPDPE in pons/meduIla homogenates.

Accordingly, binding constants were determined by simultaneous analysis of the

data from five separate experiments, with r = 0.95. There was no significant

difference between the Bmax or the K values for the μ specific ligand in the

two tissues (table 1). Whilst this was also true for the KD value for 3H-DPDPE

in pons/medulla homogenate, the maximum number of binding sites for 3HDPDPE in pons/medulla homogenates was less than one third that of the whole

brain. Moreover, the specific binding of 3H-DAGO in whole brain at 1nM

represented 91% of the total binding, but the equivalent value for H-DPDPE at

D in pons/medulla was only 38%. There was a positive correlation between the

non-specific binding of both ligands in both tissues and the time taken at 25°C

for half-maximal specific binding of the ligand to be reached (r = 0.98), and also

between the non-specific binding and the time taken for a constant ratio of

specific/non-specific binding to be reached (r = 0.99).

TABLE 1. Comparative binding characteristics of 3

H-DAG0 and 3H-DPDPE

in whole brain and pons/medulla homogenates of rat.

3

H-DAG0 3H-DPDPE

Whole brain pans/medulla Whole brain *pons/medulla

Bmax (fmol mg-1

protein) 167 11 145 6 175 23 54.9

D (nM) 1.1 0.2

Non-specific

binding (% of 8.7

total binding)

Time to halfmaximal specific 8.8

binding (mins)

Time to constant

ratio of specific to 15

non-specific binding (mins)

1.5 0.1 9.4 1.0 10.2

13.6 30.6 61.7

9.4 13.1 27.5

25 50 <120

Each value is the mean s.e.m. of at least four independent determinations.

*n = 5, but determined by simultaneous analysis of all the data.

65
Several studies have concluded that the depression of respiration produced by

opioids is mediated via μ or but not receptors (Santiago and Edelman

1985). However, the low selectivity of some of the ligands used has presented

difficulties in interpretation. Our quantitative studies show that the

involvement in rat pons/meduila is small compared with that in whole brain, and

with μ binding in both regions. This militates against mediation of respiratory

depression, but does not exclude it.

Inhibition studies:

The majority of inhibition studies produced Hill plots with near-integral slopes

and correlation coefficients, indicating interaction of the tracer Iigands with one

receptor population (table 2). We have thus far found no evidence for the

existence of μ-isoreceptors (Pasternak 3986) in rat brain.

The rank order of affinities of the fentanyl series is the same at the μ and

site, (and also the same as the rank order of potency in production of respiratory

depression by these drugs in rats (Janssen 1982), but the affinities are between 1

and 600-fold greater at the former site. This appears to implicate a μ receptor

involvement in the depression of respiration caused by the fentanyls. However,

the considerable differences in the relative affinities of the drug series at the μ

and sites provides a potential tool for testing this hypothesis.

TABLE 2. Binding profile of a series of 4-anilinopiperidine drugs at the μ and

opioid receptors of rat brain

I at

site (nM)

Carfentanil

0.44 0.02

Hill plot slope 1.00 0.03

I at

μ site (nM) 0.42 0.04

Hill plot slope 0.96 0.01

1.1 0.05

Relative μ

affinity

Relative

affinity

(fentanyl = 1.000)

7.9

47.8

Sufentanil Fentanyl

21.1 0.8 259 4.3

1.1 0.06

0.77 0.10

0.97 0.01

0.91 0.13

3.3 0.45

28 1.0

4.3

0.98 0.01

78 13

1.000

12.3 1.000

Alfentanil

22100 3700

0.83 0.07

38.9 3.7

0.97 0.01

570 90

0.085

0.012

Each value is the mean s.e.m. of at least four independent determinations.

*The marker ligand was 3H DPDPE at 0.5 KD.

**The marker ligand was 3H DAGO at KD.

REFERENCES

Gillan, M.G.C. and Kosterlitz, H.W. Br J Pharmacol, 77: 461-469, 1982.

Glowinski, J. and Iversen, L.L. J Neurochem, 13: 655-669, 1966.

Janssen, P.A.J. Acta Anaesth Stand, 26: 262-268, 1982.

Pasternak, G.W. Biochemical Pharmacol, 35: 361-364, 1986.

Santiago, T.V. and Edelman, M.H. J Appl Physiol, 59: 1675-1685, 1985.

66
DEVELOPMENT OF -OPIOID RECEPTORS IN RAT BRAIN

CHARACTERISED BY [

3

H]-[DPen2,D-Pen3]

ENKEPHALIN BINDING

Ian Kitchen and Julia McDowell

Department of Biochemistry, Division of Pharmacology & Toxicology,

University of Surrey, Guildford, Surrey, GU2 5XH, U.K.

ABSTRACT

The development of rat brain -opioid receptors has been studied in the

posnatal period using the highly selective -site ligand [

3

H]-[D-Pen2,DPen3

] enkephalin (DPDPE) in binding studies. Between day 10 and day 25 there

was a three-fold increase in the number of binding sites (expressed per mg

protein) and the D was constant throughout

binding could not be detected earlier than day 10.

the postnatal period. Specific

INTRODUCTION

There is good evidence that there is differential development of μ, - and

opioid binding sites (Spain et al. 1985). The understanding of this heterogeneity has

been hampered, however, because of the lack of selective ligands for the

receptor subtypes.

With the recent availabilty of 3H-DPDPE which has been shown to be a highly

selective binding ligand for the -opioid receptor (Cotton et al. 1985) an

accurate determination of -site ontogeny has become possible. We report here

3

H-DPDPE binding in the postnatal period and reveal inconsistencies with

previous ontogenetic studies using less selective ligands.

METHODS

Wistar albino rats of mixed sexes were used in all experiments and receptor

binding assays (in Tris HCl, pH 7.4 at 25°C for 40 mins) were carried out as

described by Cotton et al. (1985) in whole brain homogenates. 3

H-DPDPE (25-

27Ci/mmol, Amersham) was purified by HPLC prior to use. Scatchard analysis

of saturation curves was used to determine D and Bmax.

RESULTS AND DISCUSSION

Table 1 shows the maximal binding capacities and affinities for 3

H-DPDPE

during postnatal development. Specific binding was absent in animals less than

10 days old and this contrasts with data for [

3

H]-[D-Ala2,D-Leu5] enkephalin

(DADL) where binding was reported during the first postnatal week (Spain et al.

1985). In addition the three-fold increase in number of binding sites between the

second and fourth postnatal week is of a greater magnitude than reported for

3

H-DADL. In common with all ontogenetic binding studies using opioid ligands

the affinity of 3H-DPDPE remains constant during postnatal development.

In conclusion this study is the first report of A-receptor ontogeny using a ligand

highly selective for this receptor and confirm that the major development of

receptors occurs between the second and fourth postnatal weeks. It should be

pointed out however that difficulties were encountered in obtaining acceptable

Scatchard plots in young animals due to the low specific binding of 3H-DPDPE

and this poses problems in the use of this ligand for ontogenetic studies.

67
TABLE 1. Binding characteristics of [

3

H] - [D-Pen2, D-Pen5

I

enkephalin in the developing rat brain

Age Dissociation constant

(days) D, nM)

Binding capacity

(Bmax, fmol/mg protein)

10

15

21

25

30

35

40

45

50

10.5 1.9

10.2 1.7

9.7 1.1

12.7 2.7

9.7 0.9

9.7 1.4

8.5 0.5

11.1 1.8

7.2 0.4

34.5 6.2

49.3 4.6

78.6 7.5

102.8 13.6

107.6 6.1

111.8 15.2

103.3 11.5

119.4 11.2

99.9 12.0

Values are the mean s.e.m. of 4 Scatchard plots.

REFERENCES

Cotton, R., Kosterlitz, H.W., Paterson, S.J., Rance, M.J. and Traynor, J.R. Brit

J Pharmacol, 84: 927-932, 1985.

Spain, J.W., Roth, B.L. and Coscia, C.J., J Neurosci, 5: 584-588, 1985.

ACKOWLEDGEMENTS

This work was supported by a project grant from The Wellcome Trust.

68
COMPARISON OF THE EFFECTS OF CALCIUM CONCENTRATION ON MU

AND KAPPA AGONIST ACTIONS IN THE GUINEA PIG ILEUM

M.A.Johnson, R.G.Hill & J.Hughes.

Parke-Davis Research Unit,

Addenbrookes Hospital site,

Hills Rd,

Cambridge, CB2 2QB,

UK.

ABSTRACT

The effect of calcium on responses to selective mu

(fentanyl and DAGO) and selective kappa agonists was

investigated on guinea pig ileum. The dose-response

curves were shifted to the right in a non-parallel

fashion with increasing calcium concentration (0.9-3.6

mM). However U50488 was shifted more than either

fentanyl or DAGO. Both fentanyl and U50488 could not

produce 100% inhibition of twitch in 3.6mM calcium. It

is concluded that the efficacy of the agonists changes

with calcium concentration and that the kappa agonist

U50488 is affected more than the mu agonists because

kappa receptor activation directly inhibits calcium

entry whereas mu receptors inhibit calcium by increasing

potassium conductance.

INTRODUCTION

It has been shown that kappa receptor activation

probably inhibits calcium entry in neurons of the

myenteric plexus (Cherubini & North 1985). This is in

contrast to mu agonists which appear to indirectly

reduce calcium-entry by increasing potassium

conductance. Although there have been previous reports

of the effect of varying calcium concentration on

morphine actions in the guinea pig ileum (Opmeer & van

Ree 1979) there is little information available on the

effect of such manipulation on kappa agonists. We have

compared the effect of a range of calcium concentrations

on the actions of the kappa selective agonist U50488 and

the mu selective compounds fentanyl and

[D-Ala2

,MePhe4,Gly(ol)5]-enkephalin (DAGO) on the guinea

pig isolated ileum.

MATERIALS AND METHODS

Male Dunkin-Hartley guinea pigs (250-400g) were killed

by cervical dislocation and the ileum removed and placed

in Tyrode's solution of the following composition: (mM)

NaCl 136.9, NaH2PO4 0.42, NaHCO3 11.9, KCl 2.7, MgCl2

1.05, D-glucose 5.55 and CaCl2 1.8. The pieces of ileum

were then mounted on coaxial electrodes and electrically

69
stimulated with 0.5ms pulses at 0.1Hz at supramaximal

voltage. The 10ml organ baths were maintained at 37°C

and the solutions bubbled with 95%O2/5%CO2. Washing was

by overflow. The ability of the agonist to inhibit the

electrically evoked twitch was measured in Tyrode's with

0.9, 1.8 and 3.6mM calcium. The calcium concentration of

the solutions was altered by adding calcium chloride to

the reservoir. The osmolarity of the solution varied

between 282 and 287 milliosmoles.

RESULTS

FIGURE 1 Dose-response curves for the kappa

selective compound U50488. The curves were

shifted to the right in a non-parallel

fashion with increasing calcium

concentration.( =0.9mM, =1.8mM and

=3.6mM calcium). In 3.6mM calcium 100%

inhibition of the twitch was not seen.

The mu and kappa ligands tested in this study reduced

the electrically evoked twitch of guinea pig isolated

ileum. This effect was found to be dependent upon

external calcium concentration.

As is shown in table 1, the dose ratio produced by the

change from 1.8 to 3.6mM calcium for U50488 is

significantly greater than that for either fentanyl or

DAGO. The dose-response curves were shifted to the right

in a non-parallel fashion with increasing concentration

of calcium. At the highest calcium concentration (3.6mM)

it was not possible to obtain 100% suppression of twitch

with either fentanyl or U50488 (figures 1,2).

70
FIGURE 2 Dose-response curves for the mu

selective ligands fentanyl and DAGO in

increasing calcium concentration. The curves

were shifted to the right. Allthough the DAGO

curves appeared to be parallel the fentanyl

curves were not and did not reach maximum

inhibition.( =0.9mM, =1.8mM and

=3.6mM calcium)

DISCUSSION

The data presented suggest that mu and kappa agonists

may inhibit calcium entry by differing mechanisms. This

supports the finding that kappa agonists reduce

71
TABLE 1 The effect of calcium concentration

on the effectiveness of U5C488, fentanyl and

DAGO, in reducing the electrically evoked

twitch of guinea pig ileum. The potencies of

all three agonists are reduced by increasing

the calcium concentration. However the

dose-ratio for U50488 in 1.8 and 3.6 mM

calcium is significantly larger (*p<0.1,

Student's unpaired t-test) than for either

fentanyl or DAGO.

transmitter release directly by inhibiting calcium entry

(Cherubini & North 1985) whereas mu agonists act

indirectly by increasing potassium conductance to

hyperpolarise the nerve terminal and subsequently reduce

calcium entry. Hayes and Sheehan (1986) have shown that

compounds which appear to be partial agonists and

antagonists on the rat vas deferens, a tissue with a low

receptor reserve, increase their efficacy in lowered

calcium concentrations suggesting an increase in the

receptor reserve. Therefore it is possible that in

higher calcium concentrations the receptor reserve for

both mu and kappa receptors is reduced in guinea pig

ileum and so the efficacy of what appear to be full

agonists is reduced to that of partial agonists. This

could explain the reduced maximum responses seen with

fentanyl and U50488. The ability of DAGO to produce a

full inhibition of the twitch in the presence of 3.6mM

calcium suggests a high efficacy, in comparison with

fentanyl. The possibility that the guinea pig ileum

opioid receptor reserve is calcium dependent could only

be adequately investigated, however, by using an

irreversible receptor antagonist such as -CNA (Takemori

et al. 1986).

REFERENCES

Cherubini, E. and North, R.A. PNAS, 82:1860-1863, 1985.

Hayes, A.G., and Sheehan, M. Br J Parmacol, 88:276P, 1986.

Opmeer, F.A. and van Ree, J.M. Eur J Pharmacol, 53:395-397,

1979.

Takemori, A.E., Ikeda, M. and Portoghese, P.S. Eur J

Pharmacol, 123:357-361, 1986.

72
KAPPA OPIOID BINDING SITES IN RAT SPINAL CORD

John R. Traynor, M. Stuart Wood, Jacqueline A. Carroll*

and Michael J. Rance*

Department of Chemistry, University of Technology, Loughborough,

Leics. LE11 3TU, U.K.

*Bioscience Department 11, I.C.I. Pharmaceuticals Division,

Alderley Park, Macclesfield, Cheshire. Sk10 4TG, U.K.

ABSTRACT

The spinal cord of the rat is shown to contain only a low level of

kappa-binding sites, comparable to rat brain. [

3

H]Bremazocine

appears to label an additional class of sites which do not appear to

be of mu, delta, or kappa specificity.

INTRODUCTION

Intrathecally administered opioids are believed to act directly at

the spinal level to prevent the passage of nociceptive information

to higher centres (Yaksh 1981). Pharmacological studies of opioids

after intrathecal administration show that ligands with mu, delta,

and kappa selectivity are active in various test systems used'to

evaluate analgesia (Tung and Yaksh 1982, Schmauss and Yaksh 1984).

Characterisation of opioid receptors in the rat cord by ligand

binding assays have demonstrated the presence of a large number of

putative kappa sites (Gouarderes et al. 1981, Traynor and Rance

1984). Such sites have properties inconsistent with a single site

(Traynor and Rance 1985) and the existence of sub-types has been

suggested (Attali et al. 1982). In this paper we present a partial

characterization of these sites.

MATERIALS AND METHODS

The followinq labelled ligands were used: [

3

H]Bremazocine

(30 Ci.mmol-1) (New England Nuclear) and [3H]Dynorphin(1-9)

(26 Ci.mmol-1) (Amersham International). Unlabelled ligands used

were Tyr-D-Ala-Gly-MePhe-NHCH2-CH2OH (GLYOL), D-Ala2-D-Leu5-

Enkephalin (DADLE) (Sigma U.K.) and Trans-3,4-dichloro-N-methyl-N-

[2-(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide (U-50488H)

(Upjohn). Peptidase inhibitors used were Bestatin (Cambridge

Research Biochemicals) and Captopril (Merck).

Binding to rat lumbo-sacral spinal cord membranes was performed in

Tris buffer, pH 7.4, at 37°C or 25°C (for [3H]Dynorphin(1-9)) as

previously described (Traynor and Rance 1985). In the case of

[

3

H]Dynorphin(1-9) the peptidase inhibitors bestatin (30μM) and

captopril (300μM) were included to prevent degradation of the

labelled ligand. Binding to homogenates of guinea-pig cerebellum

(Robson et al 1984) was performed in 20mM Hepes buffer or

Hepes-buffered Krebs solution containing 10μM GppNHp for 40 min at

25°C. Non-specific binding was defined using 1μM Diprenorphine.

73
RESULTS AND DISCUSSION

[

3

H]Bremazocine exhibited high affinity binding to rat lumbo-sacral

cord membranes. However, after suppression of binding to mu and

delta sites, with 1OOnM each of GLYOL and DADLE per 0.5nM

[

3

H]ligand, displacement of the remaining bound [

3

H]Bremazocine was

not consistent with a single kappa site. Thus ligands for mu

(GLYOL), delta (DADLE) and kappa (U-50488H) sites displaced the

remaining specifically bound [3H]Bremazocine with low Hill

coefficients (table 1). Indeed for DADLE it was possible to discern

two definite components with affinities of 321nM and 25890nM respectively . Displacement by naloxone suggested all of the specifically

bound [3H]Bremazocine was opioid in nature.

TABLE 1. Inhibitory effects of opioids on the binding

of [3H]Bremazocine to rat lumbo-sacral membranes in the

presence of GLYOL (100nM) and DADLE (100nM).

Competing Ligand Ki(nM) Hill Coefficient

GLYOL 1338 309 0.83 0.04 (3)

DADLE 8945 825 0.61 0.07 (3)

U-50488H 57.3 7.8

Naloxone* 11.0

0.52 0.01(4)

* Naloxone displaced a mean of 99.1% of the specifically bound

[3H]Bremazocine in the two experiments.

In order to gain further insight into the heterogeneous site we have

studied the binding of [3H]Bremazocine in the presence of a high

concentration of DADLE (5-10μM) to suppress the higher affinity of

the two kappa components. The remaining binding is characteristic

of a classical kappa site (table 2).

TABLE 2. Inhibitory effects of opioids on the binding

of [3H]Bremazocine to rat lumbo-sacral membranes in the

presence of DADLE (5μM).

Competing Ligand Ki(nM) Hill Coefficient

GLYOL 6306 1333 1.12 0.13 (3)

DADLE 2573 638 0.84 0.10 (3)

U-50488H 15.63 2.84 0.77 0.08 (3)

Furthermore. in the presence of this high DAOLE concentration the

number of [3H]Bremazocine binding sites was markedly reduced bringing it into line with the number of sites labelled by the

kappa-selective peptide [3H]Dynorphin(1-9) (table 3). These

findings suggest that the true kappa site accounts for only 13% of

the total [3H]Bremazocine binding in the cord, rather than the much

higher percentage previously supposed (Gouarderes et al. 1981,

Traynor and Rance 1984). This low level of kappa binding agrees

with the findings of Lahti et al. (1985) who were unable to detect

binding of the kappa selective [

3

H]U69593 to rat cord membranes.

74
TABLE 3. Binding of [3

H]ligands to rat lumbo-sacral

spinal cord membranes. Bmax

[

3

H]ligand

KD

(nM)

(fmols,mg-l

protein)

Bremazocine 0.47 0.05 89.6 5.3 (3)

Bremazocine with 10μM DADLE* 0.20 0.02 11.9 0.4 (3)

Dynorphin(1-9) with 0.28 0.03 10.1 0.8 (4)

mu and delta suppression**

GLYOL+

DADLE+

1.95 0.2 24.2 1.3 (3)

1.92 0.3 11.1 0.6 (3)

* 10μM DADLE to 1 x KD of the [3H]ligand and ** 100 μM

each of GLYOL and DADLE to 1 x KD of the [3H]ligand.

+ Taken from Traynor and Rance, 984. 1

It is well known that apparently heterogeneous binding may be a

consequence of using non-physiological conditions (Creese 1985).

To determine if this could explain our findings we have used the

largely kappa containing guinea-pig cerebellum (Robson et al. 1984).

In this tissue, in Hepes buffer, [3H]Bremazocine (0.2nM) is

displaced by U-50488H with an IC5 0 of 10.3nM but a Hill coefficient

of only 0.59 which was similarly shallow even in the presence of

3μM DADLE. Using Hepes buffered Krebs containing the stable GTP

analogue GppNHp the dose-response curve for U-50488H is shifted

twenty-fold to the right but the slope remains at 0.59, suggestive

of true heterogeneity. Further studies are in progress to confirm

whether the nature of this heterogeneity is the same as that seen

in the rat cord.

The results demonstrate that the rat spinal cord contains a small

population of classical kappa sites, similar to that reported in rat

brain (Gillan and Kosterlitz 1982), and that [3H]bremazocine, but

not the peptide [3H]Dynorphin(1-9), labels a site which is

apparently not of mu, delta or kappa specificity. It is unlikely

that this should be considered as a sub-type of the kappa site in

view of its inability to reccgnise Dynorphin(1-9). It will be of

interest to determine what role, if any, this site plays in

spinally-mediated analgesia.

REFERENCES

Attali, C., Gouarderes, C., Mazarguil, H., and Cros. J.

Neuropeptides, 3:53-64, 1982.

Creese, 1. In: Yamamura, H.I., Enna, S.J., and Kuhar M.J., eds.

Neurotransmitter Receptor Binding. 2nd edn. New York, Raven

Press, 1985, pp.189-233.

Gillan, M.C.G., and Kosterlitz, H.W., Brit J Pharmac, 77:461-468,

1982.

Gouarderes, C., Attali, B., Audigier, Y. and Cros, J.

In: Takagi, H. and Simon, E.J., eds. Advances in Endoqenous and

Exogenous Dpioids. Tokyo: Kodansha Press, 1981, pp.18-20.

75

172-683 O - 87 - 4 : QL 3
Lahti, R.A., Mickelson, M.M., McCall, J.M., and Von Voightlander,

P.F. Eur J Pharmac, 109:281-284, 1985.

Robson, L.E., Foote, R.W., Maurer, R., and Kosterlitz, H.W.

Neuroscience, 12:621-627, 1984.

Schmauss, C., and Yaksh, T.L. J Pharmac Expt Ther, 228:1-12, 1984.

Traynor, J.R., and Rance, M.J. Neuropeptides, 5:81-84, 1984.

Traynor, J.R., and Rance, M.J. Br J Pharmac, 84:52P, 1985.

Yaksh, T.L., Pain, 11:293-346, 1981.

76
OPIOID RECEPTORS OF BOVINE POSTERIOR PITUITARY

NEUROSECRETOSOMES ARE EXCLUSIVELY KAPPA

Guido O. Pesce*, Michael A. Lang**, Ricardo A. Cruciani*,

James T. Russell**, Harold Gainer**, and David Rodbard*

*Laboratory of Theoretical and Physical Biology and

**Laboratory of Neurochemistry and Neuroimmunology,

National Institute of Child Health and Human Development,

National Institutes of Health, Bethesda, Maryland 20892,

U.S.A.

ABSTRACT

Intact neurosecretosomes (NSS) from bovine posterior pituitary were

prepared and characterized. Ligand binding studies were performed,

using 3H-[D-Ala2-D-Leu5]enkephalin (DADL), 3H-etorphine and

3

H-ethylketocyclazocine (EKC). The absence of specific binding of

3

H-DADL and the inability of DADL to displace 3H-etorphine,

implies the absence of mu, delta, or DADL-suppressible benzomorphan

("kappa-2") sites. Self- and cross displacement studies for

etorphine and EKC suggested receptor heterogeneity. EKC fails to

displace - 20% of specific binding of etorphine. Mathematical

modelling indicates the presence of three classes of sites. The

present findings imply that bovine posterior pituitary opioid

receptors are exclusively of the kappa type.

INTRODUCTION

Opioid peptides are present in the neural lobe of the pituitary.

Modulation of the secretion of oxytocin and vasopressin by opioid

peptides directly at the neural lobe level has been widely studied.

Although the coexistance of vasopressin and oxytocin with opioid

peptides in neurosecretory vesicles of nerve terminals themselves is

now accepted, the underlying mechanisms of the modulation of the

secretion and the type or subtype of opioid receptors involved

remains unclear. Several reports suggested that opioid receptors

are present primarily in the pituicytes (astrocytoglia), rather than

on the nerve terminals (Bunn et al. 1985; Lightman et al. 1983).

The present study was undertaken to determine whether opioid binding

sites are also present on neurosecretosomes (i.e., nerve terminals)

and to characterize the types of these receptors. We used DADL as a

probe of mu and delta receptors. Etorphine and EKC were selected,

following protocols similar to that used by another laboratory

(Castanas et al. 1985) and ourselves to characterize 3 subtypes of

kappa receptors in bovine adrenal medulla.

MATERIALS AND METHODS

Neurosecretosome preparation: Fifteen to twenty bovine posterior

pituitaries were suspended in 0.25 M sucrose, 10 mM HEPES-Tris

buffer (pH 7.3) and homogenized with a teflon-glass homogenizer.

The homogenate was centrifuged at 750 x g for 2 min and the

77
supernatant was centrifuged at 5.000 x g for 20 min. The pellet was

resuspended in sucroserpercoll (30%) medium and centrifuged at

50,000 x g for 45 min. The particulate band at the 1.045-1-055 g/ml

density region of the gradient was collected as purified

neurosecretosomes (NSS). The purity of the preparation was assayed

both by enzyme analysis and electron microscopy (Russell, in

preparation).

Opiate binding assay: Binding studies were performed in isotonic

sucrose solution with the following characteristics: sucrose 0.25 M;

HEPES-Tris, 10 mM (pH 7.3); PMSF, 3 pg/ml; aprotonin, 1000 U/ml;

bacitracin, 0.02% (w/v). The samples were incubated at 37°C for 30

min and then filtered through Whatman GF/B filters.

Mathematical and statistical analysis: Data were analyzed with the

computer program ALLFIT to estimate IC50 values and pseudo-Hill

coefficients, as described elsewhere (De Lean et al. 1978). Binding

parameters were obtained using program LIGAND, employing a weighted

non-linear regression analysis (Munson and Rodbard 1980; Cruciani

et al. in press).

Materials: 3

H-etorphine (40 Ci/mmol) was obtained from Amersham,

Arlington Heights, IL. 3

H-EKC (24 Ci/mmol) and 3H-DADLE (45

Ci/mmol) were obtained from New England Nuclear Corp., Boston, MA.

RESULTS AND DISCUSSION

Competition studies for 3H-etorphine by DADL were performed (fig. 1).

Fig. 1. Displacement of 3

H-etorphine from neurosecretosomes of bovine posterior pituitary by unlabeled etorphine

and DADL.

78
DADL failed to displace specific binding of etorphine even when

10-4M unlabeled ligand was used as displacer, implying the absence

of mu, delta, and benzomorphan binding sites. This observation was

further confirmed by the absence of specific binding for 3H-DADL.

Self-displacement curves for etorphine and EKC, using a fixed

concentration of labeled ligand showed a complex isotherm that

suggested hetereogeneity of binding sites. When each of these

curves were analyzed individually or by pooling results from several

experiments, one could reject a model involving a single class of

sites (p < 0.01). In each case a model involving two classes of

sites was sufficient, and further complexity was not supported by

analysis of data from homologous displacement curves considered

alone. Etorphine showed apparent Kd's of 1 and 100 nM; EKC showed

Kd's of 5 nM and > 100 μM.

Complete self- and cross-displacement study: We next sought to

study whether the two sites for etorphine and EKC were independent

or whether the two ligands shared the high affinity site.

Accordingly, a complete self- and cross-displacement study for

labeled etorphine and EKC was performed (table 1). Mathematical

modelling of the data with program LIGAND indicated that a model

involving three classes of sites was significantly better than a

model involving two classes of sites (p < 0.01). Parameters of the

"3-site" model are shown in Table 1. The data did not support a

more complex model with 4 classes of sites. R1 showed high affinity

for both etorphine and EKC, with approximately two-fold higher

affinity for etorphine. R2 has a 750-fold higher binding capacity

than R1, with low affinity for etorphine (320 nM) and negligible

affinity for EKC. The third site, R3 showed an even larger binding

capacity but with an extremely low affinity for both ligands (Kd's

in the μM range).

TABLE 1. Binding parameters of opioid binding to NSS.

Kd and binding capacities for a model involving three

classes of sites (R1-R3. A complete set of four selfand cross-displacement curves for labeled and unlabeled

etorphine and EKC was analyzed simultaneously using the

computer program LIGAND.

Class Receptor Etorphine EKC

of concentration K D K D

site (pmol/gr tissue) (nM) (nM)

R1 4 0.9 2

R2 320 150 > 10000

R3 1700 7300 2300

79
The present studies have shown that the neurosecretosomes from the

bovine posterior pituitary have kappa opioid receptors characterized

by high affinity for etorphine and EKC and complete lack of affinity

for the relatively delta/mu selective ligand DADL. It is possible

that R1 of the NSS (Table 1) corresponds to the "kappa-l" or

"kappa-3" subtypes of bovine adrenal medulla which have high

affinity for both etorphine and EKC, but are not suppressible by

DADL (Castanas et al. 1985). In contrast, in bovine adrenal

medulla, 80% of kappa receptors are present as "benzomorphan sites,"

also designated as the "kappa-2 subtype." This receptor was defined

operationally as sites with Kd - 1 nM for both etorphine and EKC,

but which could be suppressed by 1-100 μM DADL. The failure of

10-4M DADL to displace labeled etorphine from NSS implies that the

kappa receptor of bovine posterior pituitary NSS differs from the

major form in membranes from bovine adrenal medulla.

The present findings are in agreement with recent reports of

autoradiographic data (Herkenham et al. 1985, Gerstgerger and

Bardin 1986) and with other radioreceptor binding studies (Martin

and Falke 1985) that indicate predominance of kappa binding sites

in the neural lobe.

REFERENCES

Bunn, S., Hanley, M., and Wilkin, G. Neurosci Letts 55: 317,

1985.

Castanas, E., Bourhim, N., Giraud, P., et al. J Neurochem 45:

688, 1985.

Cruciani, R., Lutz, R., Munson, P., and Rodbard, D. J Pharmacol

Exp Ther , in press.

Gerstberger, R., and Barden, N. Neuroendocrinology 42: 376, 1986.

Herkenham, M., Rice, K., Jacobson, A., Bykov, V., and Rothman, R.

15th Ann Meet Soc Neurosci II: 582, 1985.

Lightman, S., Ninkovic, M., Hunt, S., and Iversen, L. Nature 305:

235, 1983.

Martin; R., and Falke, N. Washington D.C. Symposium on Peptides and

Receptors 6: 182, 1985.

Munson, P., and Rodbazrd, D. Anal Biochem 107: 220, 1980.

80
THE PHARMACOLOGICAL PROFILE OF BAM 18

D.E. Hurlbut, C.J. Evans*, J.D. Barchas* and F.M. Leslie

Department of Pharmacology

University of California, Irvine 92715, USA

*Department of Psychiatry and Behavioral Sciences

Stanford University, Stanford 94305, USA

ABSTRACT

The opioid receptor selectivity of BAM 18 was determined by radioligand

binding and peripheral tissue bioassay. Using selective radioligand binding

conditions, BAM 18 bound to the μ opioid receptor with an affinity twice that

of the K receptor and over 10 times that of the opioid receptor (Ki = 0.29,

0.84 and 3.9 nM, respectively). Ke values for naloxone antagonism of BAM 18

activity in the electrically stimulated guinea pig ileum and the mouse vas

deferens were 4.3 and 9.9 nM respectively. The pharmacological profile of

BAM 18 was similar to that of metorphamide.

INTRODUCTION

BAM 18 is an 18 amino acid peptide derived from proenkephalin A. Although

it is present in rat and guinea pig CNS in relatively high concentrations (Evans

et al. 1985), the pharmacological properties and physiological function of this

peptide are presently unknown. In the present study we have compared the

pharmacological properties of BAM 18 to that of another proenkephalin

A-derived peptide, metorphamide. Our data indicate that the pharmacological

profile of BAM 18 is similar to that of metorphamide, in that both peptides

display high affinity for the μ opioid receptor.

MATERIALS AND METHODS

Radioligand binding.

Male guinea pigs (350-400 g) were obtained from Simonsen Labs., Gilroy, CA.

Brain homogenates were prepared as described previously (Leslie et al. 1982).

Brain membranes (10 mg original tissue wet weight) were incubated for 90 min

at 22°C in 1 ml 50 mM Tris-HCl buffer (pH 7.4) containing radioligand,

appropriate competing ligand and enzyme inhibitors (bestatin, 10 μM, and

thiorphan, 0.1 μM). Selective binding conditions for each receptor subtype

were utilized. The μ binding site was labelled with [

3H] DAGO, the binding

site with [

3H] DADLE in the presence of 300 nM D-Pro 4

-morphiceptin

(PLO17) (to saturate all μ sites), and the K binding site with [

3H] EKC in the

presence of 100 nM DSLET and 300 nM PLO17 (to saturate all and μ binding

sites, respectively). Specific binding was defined as the difference in bound

radioactivity in the absence and presence of 1 μM levallorphan. Following

incubation, membranes were filtered through GF/C paper and rinsed with

16 ml of ice cold buffer. Saturation curves were constructed using increasing

concentrations of radioligand. The equilibrium dissociation constant (Kd) and

binding capacity (Bmax) of each radioligand were derived by non-linear, least

squares regression analysis using the computer program LIGAND (Munson and

81
Rodbard 1980). Both BAM 18 and metorphamide were used to competitively

displace radioligand binding. The dissociation constant of each inhibiting

ligand (Ki) was determined using the Cheng-Prusoff equation (Cheng and

Prusoff 1973).

Bioassay

Male guinea pigs and Balb C mice were obtained from Simonsen Labs., Gilroy,

CA. Myenteric plexus-longitudinal muscle strips (GPI) and mouse vas deferens

(MVD) were prepared as described previously (Leslie et al. 1980). Enzyme

inhibitors (bestatin, 1 μM, and thiorphan, 0.1 μM) and drugs were added to the

tissue baths at 20 min intervals for the GPI, and at 8 min intervals for

the MVD. Dose response curves were constructed by adding increasing

concentrations of the test drug to the organ bath and IC50 values determined.

Ke values for naloxone antagonism of agonist activity were determined by the

'single dose' method of Kosterlitz and Watt (1970).

RESULTS

Affinity constants for inhibition of radioligand binding are shown in table 1.

BAM 18 displaced the binding of all three radioligands. Although it exhibited

hi hest affinity for the μ binding site, it also potently displaced

[

3H] EKC binding from the K site. In contrast, BAM 18 had a 10-fold lower

affinity for the binding site. Metorphamide displayed a similar receptor

selectivity profile. It was a potent displacer of both [

3H] DAGO and

[

3H] EKC binding, with a lower affinity for the binding site. IC50 values

TABLE 1. Potencies of opioid peptides for displacement of

radioligand binding to guinea pig brain homogenates.

DRUG Ki

 (nM)

[

3H] DAGO [3H] EKC [3H] DADLE

BAM 18 0.29 0.063 0.75 0.26 3.17 0.60

Metorphamide 0.06 0.005 0.23 0.08 1.76 0.07

Values represent the mean SEM of 3-8 observations.

determined in bioassay preparations are shown in table 2. Both BAM 18 and

metorphamide were more potent inhibitors of twitch contraction in GPI than

MVD. In both tissues, Ke values for naloxone antagonism were intermediate

between those previously reported for antagonism of μ and K agonists (table 3;

Lord et al. 1977).

82
TABLE 2. Potencies of opioid peptides for inhibition of stimulated

twitch contraction in GPI and MVD.

DRUG IC50 (nM)

GPI MVD

BAM 18 1.37 0.30 26.2 6.34

Metorphamide 0.51 0.08 6.53 2.50

Values represent the mean SEM of 3-6 observations.

TABLE 3. Naloxone antagonism of BAM 18 and Metorphamide

agonist activity in GPI and MVD.

DRUG Naloxone Ke

 (nM)

GPI MVD

BAM 18 4.34 1.31 9.86 2.17

Metorphamide 4.88 1.43 10.6 0.97

Values represent the mean SEM of 3-6 observations.

DISCUSSION

Data from both receptor binding and bioassay indicate that BAM 18 has high

affinity for μ and K opioid receptors, with somewhat lower affinity for the

opioid receptor. This receptor selectivity profile is similar to that of

metorphamide (present study; Weber et al. 1983). In contrast, met-enkephalin

has a different pattern of pharmacological activity, exhibiting the highest

affinity for the receptor, with lower affinities for μ and K receptors.

BAM 18 is present in higher concentrations in the CNS of rat than is

metorphamide (Evans et al. 1985; Weber et al. 1983). Its regional distribution

throughout brain is similar to that of enkephalin (Evans et al. 1985; Sanders et

al. 1984; Hong et al. 1977; Zamir et al. 1985). It has previously been noted

that the anatomical distribution of proenkephalin-derived peptides does not

correspond precisely to that of receptors (Moss et al. 1986). In several brain

regions, however, there is a significant overlap between the pattern of

enkephalin immunoreactivity and that of μ receptors (Laughlin et al. 1985).

Based on our present findings that BAM 18 has highest affinity for the μ

binding site, we propose that this peptide may mediate its physiological

actions in certain brain regions via the μ opioid receptor. The functional

significance of BAM 18 remains to be determined.

63
REFERENCES

Cheng, Y. and Prusoff, W.H. Biochem Pharmacol, 22:3099-3108, 1973.

Evans, C.J., Erdelyi, E., Makk, G. and Barchas, J.D. Fed Proc, 44:422, 1985.

Hong, J.S., Yang, Y.T., Fratta, W. and Costa, E. Br Res, 134:383-386, 1977.

Kosterlitz, H.W. and Watt, A.J. Br J Pharmac, 39:398-413, 1970.

Leslie, F.M., Chavkin, C., and Cox, B.M. J Pharm Exp Ther, 214:395-402,

1980.

Leslie, F.M., Tso, S. and Hurlbut, D.E. Life Sci, 31:1393-1396, 1982.

Lord, J.A., Waterfield, A.A., Hughes, J. and Kosterlitz, H.W. Nature,

267:495-499, 1977.

Loughlin, S.E., Massamiri, T.R., Kornblum, H.I. and Leslie, F.M.

Neuropeptides, 5:469-573, 1985.

Moss, I.R, Denavit-Saubie, M., Eldridge, F.L., Gillis, R.A., Herkenham, M.

and Lahiri, S. Fed Proc, 45:2133-2147, 1986.

Munson, P.J. and Rodbard, D. Analyt Biochem, 107:220-239, 1980.

Sonders, M., Barchas, J.D. and Weber, E. Biochem Biophys Res Comm,

122:892-898, 1984.

Weber, E., Esch, F.S., Bohlen, P., Paterson, S., Corbett, A.D., McKnight, A.T.,

Kosterlitz, H.W., Barchas, J.D., and Evans, C.J. Proc Natl Acad Sci USA,

80:7362-7366, 1983.

Zamir, N., Plakovits, M. and Brownstein, M. Br Res, 326:1-8, 1985.

ACKNOWLEDGEMENTS

Supported by NIH Grant NS 18843.

84
CROSS-PROTECTION OF μ AND OPIOID RECEPTORS

IN THE MOUSE VAS DEFERENS.

M.J. Sheehan, A.G. Hayes and M.B. Tyers

Department of Neuropharmacology,

Glaxo Group Research Ltd., Ware, Herts., SG12 ODJ, U.K.

ABSTRACT

Opioid receptors in the C57/BL mouse vas deferens can be

irreversibly blocked by pre-treatment with -chlomaltrexamine.

Pre-incubation with the highly selective receptor antagonist

ICI 174864 concentration-dependently protected -receptors from

inactivation, but the same concentrations similarly protected μ

receptors. Pre-incubation with selective μ-receptor agonist,

[D-Ala2, MePhe4, Gly(ol)5

]enkephalin, also protected both μ and

receptors to the same degree. Neither ligand protected opioid

receptors. The results suggest that there may be a structural

interaction between μ and receptors in the mouse vas deferens.

INTRODUCTION

Much effort has been expended in demonstrating the existence of

multiple types of opioid receptor (Martin et al. 1976, Lord et

al. 1977). There is, nevertheless, a growing body of evidence

for interaction between the types, particularly μ and receptors

in vitro (Rothman and Westfall 1981) and in vivo (Holaday and

D’Amato 1983). It has even been suggested that the two receptors

may be interconvertible forms of a single macromolecule (Bowen et

al. 1981). In this study we have taken advantage of the presence

in the mouse vas deferens of all three established types of

opioid receptor (μ, and : Lord et al. 1977; Miller and Shaw

1984) to-attempt to selectively protect one opioid receptor type

from inactivation while irreversibly blocking the remainder. The

irreversible antagonist used was -chlornaltrexamine CNA), an

alkylating agent selective for opioid receptors but which does

not discriminate between the three types (Ward et al. 1982). The

protecting ligands used were the highly selective receptor

antagonist ICI 174864 (Cotton et al. 1984) and the selective

μ-agonist, [D-Ala2, MePhe4, Gly(ol)5

] enkephalin (DAGO; Corbett

et al. 1984). The results are interpreted as supporting an

interaction between μ and receptors in this tissue.

MATERIALS AND METHODS

Vasa deferentia from mature Glaxo-bred C57/BL mice were mounted

and field-stimulated as described in Hayes et al. (1985).

Cumulative concentration-response curves were constructed in

random order for the μ-selective agonist DAGO, the k-selective

agonist U50488 (trans- -3, 4-dichloro-N-methyl-[2-(1-pyroll85
idinyl) cyclohexyl] benzene acetamide methanesulphonate), and a

selective a onist, [D-Pen2, L-Pen5] enkephalin (DPLPE) or

[D-Pen2, D-Pen5] enkephalin (DPDPE). Tissues being treated with

irreversible antagonist were incubated with -CNA for 15 min,

after which any non-covalently bound antagonist was removed by

extensive washing at 5 min intervals for 90 min. For protection

experiments, the protecting ligand was incubated with the tissue

for 10 min (DAGO) or 30 min (ICI 174864) before and during the

CNA treatment. Control experiments showed that the 90 min

washout period was also enough to remove any remaining ICI

174864. Concentration-response curves for each agonist were

re-determined in the treated tissues and, where a parallel shift

was obtained, a dose-ratio was calculated.

DPLPE and DPDPE were supplied by Peninsula and DAGO by Cambridge

Research Biochemicals. U50488 was a gift from Dr. P.F. von

Voigtlander of the Upjohn Company, ICI 174864 was a gift from Dr.

R. Cotton of ICI. -CNA was synthesised by Dr. A. McElroy of the

Chemical Research Dept., Glaxo Group Research Ltd.

RESULTS

In the absence of any protecting ligand, -CNA (10-8M - 10-7M, 15

min) caused a concentration-dependent antagonism of the responses

to DAGO, DPLPE and U50488 which was not significantly reversed by

3hr of extensive washing of the tissues. The receptor reserve

for each agonist can be estimated by the extent to which an

irreversible antagonist can produce a parallel rightward shift of

the concentration-response curve without diminution of the

maximum available response (Furchgott 1966); these shifts were

about 30-fold for DAGO and 10-fold for DPLPE, while a parallel

shift for U50488 was rarely obtained.

Pre-incubation with the selective antagonist ICI 174864

(10-7-5x10-5M) protected the response to DPLPE in a

concentration-dependent manner, but also protected the response

to DAGO to a similar extent (table 1). There was no significant

protection of the response to U50488 at any but the highest

concentration of ICI 174864 (data not shown).

Similar experiments were performed using DAGO (10-6-3x10-5M) to

protect μ-opioid receptors. As before, there was little

protection of the response to U50488 (data not shown) but their

responses to DAGO and DPDPE were protected to a similar extent

(table 2).

In order to investigate whether there was an interaction between

the agonist effects mediated by μ and receptors,

concentration-response curves were constructed to one agonist in

the presence of low concentrations of the other.

The concentrations used were either just sub-threshold, causing

no inhibition of twitch by itself, or a concentration causing

about 10% inhibition of twitch. When tested thus, DAGO had no

significant effect on the concentration-response curve to DPDPE,

and was itself unaffected by low concentrations of DPDPE.

86
TABLE 1 Protection against --CNA by ICI 174864 in the mouse

vas deferens

The values given are the dose ratios (mean S.E.M., n = 4-8)

for the parallel rightward shifts in the

concentration-response curves for the μ-selective agonist,

DAGO, and the selective agonist, DPLPE. Below it is given

the ratio of the μ-shift/ -shift. The larger this figure,

the greater the selective protection of d-receptors. NT, not

tested.

TABLE 2 Protection against -CNA by DAGO in the mouse vas

deferens

Details are as in the legend to Table 1. In this context the

ratio of the μ-shift/ -shift is smaller for greater degrees

of μ-receptor protection.

DISCUSSION

The -receptor antagonist ICI 174864 was able to protect the

response to DPLPE from blockade by -CNA in the C57/BL mouse vas

deferens, but it also protected the response to DAGO to a similar

87
extent, even at concentrations which occupy only a small

proportion of μ-receptors (Ki at -receptors = 30nM and at

μ-receptors > 5000nM : Cotton et al. 1984). It thus appears

that occupation of -receptors by ICI 174864 can make μ-receptors

inaccessible to B-CNA. Similarly, DAGO was able to protect its

own agonist effect from -CNA but often produced an even greater

degree of protection of the response to DPDPE. The Ki value for

DAGO in displacing [

3

H]-naloxone from μ-receptors in a

physiological medium is 1300 nM (calculated from Carroll et al.

1984); the comparable value at -receptors has not been reported

but the binding selectivity of DAGO for μ-over -receptors in

Tris buffer is 185-fold (Corbett et al. 1984). Ward et al.

(1982) selectively protected -receptors from B-CNA in the mouse

vas deferens using an agonist, [D-Ser2

, Leu5] enkephalinyl-Thr,

as protecting ligand. Our own studies using this ligand did not

improve upon the low degree of selective -receptor protection

seen with some of the treatments shown in table 1 (unpublished

observations).

Our results suggest that there may be a structural interaction

between μ- and -opioid receptors in the C57/BL mouse vas

deferens, but not between k-opioid receptors and either of the

other types. However, since there was no synergism or antagonism

between DAGO and DPDPE in this tissue, this putative interaction

does not appear to alter the transduction of the agonist effects

mediated by μ and receptors.

REFERENCES

Bowen, W.D., Gentleman, S., Herkenham, M. and Pert, C.B.

Proc Natl Acad Sci USA, 78: 4818-4822, 1981.

Carroll, J.A., Miller, L., Shaw, J.S. and Downes, C.P.

Neuropeptides, 5: 89-92, 1984.

Corbett, A.D., Gillian, M.G.C., Kosterlitz, H.W., McKnight, A.T.,

Paterson, S.J. and Robson, L.E. Br J Pharmac, 83: 271-279,

1984.

Cotton, R., Giles, M.B., Miller. L., Shaw, J.S. and Timms, D.

Eur J Pharmac, 97: 331-332, 1984.

Furchgott, R.F. Adv Drug Res, 3: 21-55, 1966.

Hayes, A.G., Sheehan, M.J. and Tyers, M.B. Brit J Pharmac, 86:

899-904, 1986.

Holaday, J.W. and D’Amato, R.J. Life Sci, 33: 703-706, 1983.

Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.W.

Nature, 267: 495-499, 1977.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E. and

Gilbert, P.E. J Pharmac Exp Ther, 197: 517-532, 1976.

Miller, L. and Shaw, J.S. Neuropeptides, 5:93-96, 1984.

Rothman, R.B. and Westfall, T.C. Mol Pharmac, 21: 538-547, 1982.

Ward, S.J., Portoghese, P.S. and Takemori, A.E. Eur J Pharmac,

85: 163-170, 1982.

88
AFFINITY CROSSLINKING OF 125I-HUMAN BETA-ENDORPHIN

TO CELL LINES POSSESSING EITHER MU OR DELTA TYPE

OPIOID BINDING SITES

O. Keren, T.L. Gioannini, J.H. Hiller and E.J. Simon

Depts. of Psychiatry and Pharmacology

New York Univ. Med. Ctr, New York, New York 10016, U.S.A.

ABSTRACT

Affinity crosslinking of human 125I- -Endorphin to cell

lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these

crosslinked cell lines revealed that these two sites

contain major peptide subunits that differ in molecular

size. This confirms our earlier finding in mammalian

brain which demonstrated separate and distinct subunits

for mu and delta opioid receptors.

INTRODUCTION

To contribute to our understanding of the molecular basis

of opioid receptor heterogeneity our laboratory has used

affinity crosslinking of iodinated human beta-endorphin

(

125 I - -EndH) to membrane preparations. In previous

studies (Howard et al. 1985) two major peptide bands were

.

identified by autoradiography of SDS-PAGE gels namely a 65

kDa peptide which displayed mu characteristics and a 53

kDa peptide which displayed delta characteristics. The

assignment of mu or delta characteristics was based on

differential displacement by the highly mu-selective

ligand Tyr-D-DAla-Gly-MePhe-Gly-ol(DAGO) or by the highly

delta- selective ligand [D-Pen2-D-Pen5]-enkephalin (DPDPE)

and on the distribution and levels of the 65 kDa and 53

kDa peptides in membranes having various mu:delta ratios

(Howard et al. 1985, 1986).

In order to further support these findings, we have

studied the specific labeling pattern of opioid receptorrelated peptides in two cell lines, NG-108-15 which

contains exclusively delta binding sites (Klee and

Nirenberg 1974) and SK-N-SH which contains mainly mu

binding sites (Yu et al. 1986).

MATERIALS AND METHODS

The SK-N-SH cell line was a gift of Dr. W. Sadee

(University of California, San Francisco). The NG-108-15

cell line was a gift of Dr. A.J. Blume (Hoffman-La Roche).

125 I - -EndH (2,000 Ci/mmol) was purchased from Amersham

and 3H-bremazocine (30 Ci/mmol) was obtained from NEN.

Bis [2-(succinimidooxycarbonyloxy)ethyl]sulfone (BOSCOES)

was from Pierce Chemical Corp, electrophoresis chemicals

were from Bio-Rad.

89
Cell cultures - SK-N-SH cells were grown in RPMI 1640

medium supplemented with 10% fetal calf serum, 40 U/ml

penicillin and 40 U/ml streptomycin. NG-108-15 hybrid

cells were grown in Dulbecco' s modified Eagle's medium

(DMEM) containing HAT (0.1 mM hypoxanthine, 1 uM

aminopterin, 16 uM thymidine), supplemented with 10% fetal

calf serum, 1 mM sodium pyruvate, 40 U/ml penicillin and

40 U/ml streptomycin.

Membrane preoaration At confluency the SK-N-SH cells were

washed and harvested by mechanical agitation in a Ca++

Mg++-free phosphate buffered saline containing 0.04% EDTA.

After 10 min centrifugation at 1,000 x g, the cells were

homogenized in 50 mM Tris-HCl buffer pH 7.4 containing 1

mM EDTA. The homogenate was spun at 20,000 x g for 20 min

at 4°C and the resulting pellet was resuspended in 0.32 M

sucrose and stored at -70°C. At confluency, the NG-108-15

cells were washed and then harvested by mechanical

agitation in DMEM medium. After centrifugation at 1,000 x

g for 10 min, the cell pellet was washed with 25 mM TrisHCl buffer containing 290 mM sucrose, 1 mg/ml BSA and 0.1

mg/ml bacitracin and stored at -70°C. Membranes were

prepared, as described above, freshly before each

experiment,

0.2 mM PMSF,

in K2HP04 buffer pH 7.4 containing 1 mM EDTA,

10 ug/ml leu-peptin and 50 ug/ml bacitracin.

Affinity crosslinkinq - Membranes prepared from both cell

lines were diluted 4 fold (approx. 0.5 mg/ml) in K2HP04

buffer pH 7.4 containing protease inhibitors (see above).

125I- - EndH (1 nM) was bound and crosslinked to the membranes by the crosslinking reagent BOSCOES (1 mM) as dessolubilized membranes were subjected to SDS-polyacrylamide

by Howard et al. (1985). The crosslinked

gel electrophoresis (PAGE) and autoradiography of the gels

was performed [Howard et al. 1985).

RESULTS

Yu et al. (1986) have shorn by saturation binding experiments that the human neuroblastoma cell line SK-N-SH contains mainly mu opioid binding sites (mu:delta ratio 5:l).

Similar results were obtained by us in competition experiments with the mu-selective ligand DAGO and the deltaselective ligand, DPDPE against 3

H-bremazocine. When

crosslinking of 125I- -EndH to membranes prepared from SKN-SH cells was performed, 3 major bands were visible after

gel-electrophoresis of the solubilized membranes and

autoradiography of the gel, a large peptide band which

corresponds to molecular weight 65 kDa and two narrow

bands of 92 kDa and 25 kDa (Fig. 1).

All three peptide bands are opioid receptor related since

they are eliminated when binding of 125I- -EndH to the

membranes is conducted in the presence of 3 uM naloxone.

90
Crosslinking of 125I- -EndH to membranes prepared from NG108-15 cells, which contain exclusively delta opioid

binding sites, revealed a different labeling pattern of

peptides. Two major peptide-bands were observed,

corresponding to molecular weights of 53 and 25 kDa

(fig.1). Both peptides are opioid

their labeling was reduced when binding of 125I- -EndH

receptor-related, as

was conducted in the presence of 3 uM naloxone.

FIGURE 1. SDS-PAGE of NG-108-15 or

membranes crosslinked with 125I- -EndH. A. Binding

SK-N-SH cell

of 125I- -EndH was carried out in the absence or

presence of DAGO (20 nM, 50 nM). B. Binding of 125I-

-EndH was carried out in the absence or presence of

20 nM DPDPE.

In order to label selectively peptides containing mu or

delta opioid binding-sites, membranes prepared from both

cell lines were bound with 125I- -EndH in the presence of

20 nM and 50 nM of the mu-selective ligand DPDPE. Fig. 1A

presents the labeling patterns observed when binding of

125 I - -EndH was carried out in the presence of DAGO.

While DAGO, up to a concentration of 50 nM, had no effect

on the labeling of the peptides derived from the NG-108-15

cells, labeling of the 65 kDa peptide, and to a lesser

extent, the 92 kDa peptide, derived from SK-N-SH cells was

reduced.

when binding of 125I- -EndH was performed in membrane

Fig. 1B presents the labeling patterns observed

preparations from both cell lines in the presence of

DPDPE. As shown, DPDPE displaced the labeling of both 53

kDa and 25 kDa peptides, derived from NG-108-15 cells,

while it had no effect on the labeling of peptides derived

from SK-N-SH cells.

9 1
DISCUSSION

The labeling patterns observed after crosslinking of these

two cell lines with 125I- -EndH were different. In NG108-15 cells, two major peptides were labeled,

corresponding to molecular weights of 53 kDa and 25 kDa.

Both peptides displayed delta characteristics, i.e., their

labeling was reduced by DPDPE but not by DAGO. In SK-N-SH

cells, three major peptides were labeled by 125I- -EndH

corresponding to molecular weights of 92, 65 and 25 kDa:

The labeling of the 65 kDa peptide, and to a lesser

extent, the 92 kDa peptide, was reduced by DAGO but not by

DPDPE.

When 125I- -EndH was crosslinked to membranes prepared

from different brain regions of various species by Howard

et al. (1985,1986), 2 major peptides were labeled, a 65

kDa and a 53 kDa peptide, which displayed mu and delta

characteristics respectively. Our present results confirm

these findings. The major peptide labeled in SK-N-SH

cells was a 65 kDa peptide, while a 53 kDa peptide was

labeled in the NG-108-15 cells. Two other peptides were

also labeled in SK-N-SH cells. The labeling of both

peptides was eliminated by an excess of naloxone.

However, only the labeling of the 92 kDa peptide was

affected by DAGO. The identity of these peptides is not

clear. The 92 kDa peptide may represent a lower-affinity

binding site for mu-selective ligands. In agreement with

previous results (Howard et al. 1985), in NG-108-15 cells

a 25 kDa peptide was labeled in addition to the 53 kDa

peptide. Preliminary results suggest that this peptide

may be a breakdown product of the 53 kDa peptide, but

more experiments are needed to confirm this finding.

REFERENCES

Howard, A.D., de la Baume, S., Gioannini, T.L., Hiller,

J.M. and Simon, E.J. J Biol Chem. 260:10833-10839, 1985.

Howard, A.D., Sarne, Y., Gioannini, T.L., Hiller, J.M. and

Simon, E.J. Biochem, 25:357-360, 1986.

Klee, W.A. and Nirenberg, M. Proc Natl Acad Sci USA.

71:3474-3477, 1974.

Yu, V.C. Richards, M.L. and Sadee, W. J Biol

Chem, 261:1065-1070, 1986.

92
UPREGULATION OF THE MU-NONCOMPETITIVE DELTA BINDING SITE BY CHRONIC

MORPHINE ADMINISTRATION: EFFECT OF PREINCUBATING MEMBRANES IN

400 nM SODIUM CHLORIDE

Janine A. Danks*, ****, Frank C. Tortella**, Victor Bykov*, Arthur E.

Jacobson***, Kenner C. Rice***, John W. Holaday**

and Richard B. Rothman*

*Laboratory of Preclinical Pharmacology, St. Elizabeth's Hospital,

NIMH, Washington DC 20032; **Department of Neuropharmacology, Walter

Reed Army Institute of Research, Washington DC 20307; ***Laboratory

of Chemistry, NIADDK, Bethesda MD 20892; and ****Department of

Pharmacology, Monash University, Clayton, Australia '3168.

ABSTRACT

Rats treated chronically with morphine were found to have an

increased number of mu-noncompetitive delta binding sites. The

increase in the Bmax was 39% and the enhancement was blocked hy

pre-incubation in 50 mM Tris. pH 7.4 with 400 mM NaCl for one hour at

25oC. These findings are' .consistent with the removal of newly

manifested binding sites being a specific property of sodium, by

analogy with previous studies where sodium has been shown to extract

extrinsic proteins from red cell ghosts.

INTRODUCTION

We have previously shown that mu-noncompetitive delta binding sites

were upregulated after chronic morphine treatment (Rothman et al.

1986). These results were obtained in membranes that were not

pre-incubated in high concentrations of sodium, a technique reported

by Pasternak et al. (1975) to remove any endogenous ligand bound to

the opiate receptor.

The present studies were performed to investigate the possible effect

of pre-incubation,

hour at 25°C,

in 50 mM Tris, pH 7.4 with 400 mM NaCl for one

on the binding parameters of the two delta binding

sites in rats treated chronically with morphine. In these studies

pretreatment with the site-directed acylating agents, BIT

(1-[2-(diethylamino)-ethyl]-2-(4-ethoxybenzyl)-5-isothiocyanato--

benzi-midazole) and FIT (N-[1-[2(4-isothiocyanatophenyl]-

4-piperi-dinyl]-N-phenyl-propanamide) were used to enrich membranes

with the mu-competitive or mu-noncompetitive delta binding sites

respectively (Rothman et al. 1984).

MATERIAL AND METHODS

Male Sprague-Dawley rats (200-250 g; Zivic-Miller Labs., Allison

Park, PA) were acclimatized for six days. They were then implanted

with 2 morphine pellets (75 mg) on day 1 and 4 morphine pellets on

day 2 (Holaday et al. 1982). The rats were rapidly decapitated on

day 5, exactly 96 hours after the first implantation. A control

group of rats were implanted with placebo pellets with precisely the

same protocol.

A lysed mitochondrial P2 membrane preparation was made, by

differential centrifugation, using the whole rat brain (minus

cerebellum) following a modified method of Gray and Whittaker (1962).

93
The mitochondrial pellet was split into two aliquots; one aliquot was

incubated in 50 mM Tris, pH 7.4 with 400 mM NaCl for one hour at

25°C, while the other was kept on ice. The pre-incubated pellet was

centrifuged at 4825 xg for 15 minutes and the pellet resuspended in

ice-cold Tris, pH 7.97. This washing step (resuspension and

centrifugation) was repeated three times, in total, to ensure that

all sodium ions had been removed. After this point both aliquots

were treated identically.

The pellets were then treated with

(1 μM) for one hour at 25°C in 0.01

the acylating agents, BIT and FIT

M MOPS (3-(N-Morpholino)propanesulphonic acid) and 3 mM MnCl2. The pellets were centrifuged and

resuspended, as they were after the sodium pre-incubation. The final

pellet was resuspended in 0.75 ml/g original weight of Tris, pH 7.97.

Tritiated D-ala2-D-leu5-enkephalin was used as the ligand in the

binding assays. The incubation medium contained 10 mM Tris, pH 7.4,

100 mM choline chloride and 3 mM MnC12. [

3

H]DADL was added to the

assay in a protease inhibitor cocktail, which consisted of bacitracin

(0.1 mg/ml), Leupeptin (0.004 mg/ml), chymostatin (0.002 mg/ml),

bestatin (0.01 mg/ml), Captopril (1 μg/el), 2-mercaptoethanol (1 mM)

and phosphoramidone (1 pM).

'Binding surfaces' were used (Rothman et al. 1985), to describe the

binding sites. For both washed and control membranes, complete

saturation curves (0.25 nM to 95.6 nM) were constructed using

isotopic dilution; the Kd and Bmax were determined using computer

curve fitting methods.

RESULTS

The results set out in table 1 are from studies carried out in

membranes that had been pre-incubated in 50 mM Tris, pH 7.4 with 400

mM NaCl at 25°C for one hour. It should be noted that there is no

increase in the Bmax in either of the delta binding sites when rats

receive chronic morphine treated (n=3), although there were

significant increases in the dissociation constants of both the

mu-noncompetitive delta binding sites.

TABLE 1: The effect of Morphine tolerance on the two [

3

H]DADL.

binding sites in membranes that have been pre-incubated with 50

mM Tris, pH 7.4 and 400 mM NaCl for 1 hr at 25°C.

K d SD B max SD

(Nm) (fmol/mg prot)

MU-COMPETITIVE

(BIT-treated) Placebo 0.74 0.08 72 4

Tolerant 1.02 0.09 72 4

% of Control 158* 100

MU-NONCOMPETITIVE

(FIT-treated) Placebo 4.3 0.2 86 7

Tolerant 6.8 0.4 97 1 4

% of Control 158* 113

*P <0.05, n=3

94
The results of the studies with membranes that were not greincubated

in 50 mM Tris, pH 7.4 with 400 mM NaCl for one hour at 25°C are shown

in table 2. The Bmax of the mu-noncompetitive delta binding sites in

morphine pre-treated rats was significantly elevated (39%) above the

controls. In contrast, the Bmax of the mu-competitive delta binding

site was not significantly altered. The dissociation constants of

both the mu-competitive and the mu-noncompetitive delta binding sites

were significantly increased (52% and 111%, respectively).

TABLE 2: The effect of chronic Morphine treatment on the two

[

3

H]DADL binding sites in membranes that have not been

pre-incubated with 50 mM Tris, pH 7.4 and 400 mM NaCl for 1 hr at

25 C.

K d S D

(Nm)

B max S D

(fmol/mg prot)

MU-COMPETITIVE

(BIT-treated) Placebo 0.92 0.08 64 4

Tolerant 1.4 0.1 55 5

% of Control 152* 86

MU-NONCOMPETITIVE

(FIT-treated) Placebo 3.6 0.2 76 5

Tolerant 7.6 0.4 106 ll

% of Control 211* 139”

*P <0.05, n=3

DISCUSSION

The findings of this study demonstrate that our regime of chronic

morphine administration results in a significant increase in the

number of mu-noncompetitive delta binding sites, an increase which is

not seen when brain membranes are pre-incubated in 50 mM Tris, pH 7.4

with 400 mM NaCl for one hour at 25°C. In contrast, the elevation in

the dissociation constant for both delta binding sites was present,

regardless of whether or not the membranes were pre-incubated.

Our previous work (Rothman et al. 1986) indicated there was an

increase in the number of mu-noncompetitive binding sites afterchronic morphine treatment compared with rats not so treated. While

earlier studies (Klee and Streaty 1974) failed to find any

significant alteration in opioid binding parameters following chronic

morphine treatment, these studies used mu-selective radioligands, and

therefore the findings primarily concern the mu binding sites. There

are also reports in which [

3

H]DADL, the specific delta ligand, was

used in studies on membranes from chronically morphinized animals

(Holaday et al. 1982). These authors found that there was an

increase in the number of delta binding sites.

The experimental finding that pre-incubation in high sodium for one

hour was not followed by an increase in the Bmax of the

mu-noncompetitive delta binding site appears worthy of discussion.

Since the report by Pasternak et al. (1975) that pre-incubation of

membranes in sodium removed endogenous ligand bound to the opiate

95
receptor, this step has become an integral part of the preparation of

brain membranes for radioligand assay, despite the fact that the

range of radioligands have increased. The decrease in the number of

binding sites with pre-incubation may be explained by the finding

that the sodium ion reportedly extracts extrinsic membrane proteins

as has been shown for red cell ghosts (Capaldi 1972). This effect of

pre-incubation in high sodium upon delta opiate binding sites will

thus be investigated further, using appropriate biochemical

techniques. It is possible that these sodium-labile binding sites

may be different biochemically from those unaffected by the sodium

preincubation.

In summary, our findings are that chronic morphine treatment

upregulates the mu-noncompetitive delta binding site, and that this

upregulation can be blocked by pre-incubation in a high sodium

medium.

ACKNOWLEDGEMENT

We would like to thank Lydia Robles for her expert technical

assistance.

REFERENCES

Capaldi, R.A. Eur J Biochem, 24:74-79, 1972.

Gray, E.G., and Whittaker, V.P. J Anat, 92:79-82, 1962.

Holaday, J.W., Hitzemann, R.J., Curell, J., Tortella, F.C., and

Belenky, G.L. Life Sci, 31:2359-2362, 1982.

Klee, W.A., and Streaty, R.A. Nature, 248:61-63, 1974.

Pasternak, G.W., Wilson, H.A., and Snyder, S.H. Mol Pharmacol,

11:340-351, 1975.

Rothman, K.C., Tortella, F.C., and Holaday, J.W. Eur J Pharmacol (in

press), 1986.

Rothman, R.B., Bowen, W.D., Herkenham, M., Jacobson, A.E., Rice,

and Pert,

Rothman, R.B Danks, J.A., Pert, C.B., Jacobson, A.E., Burke, T.R.,

C.B. Mol Pharmacol, 27:399-408, 1985.

and Rice, K.C. Neuropeptides, 4:261-268.

96
INTERACTION BETWEEN HIGHLY SELECTIVE MU AND DELTA

OPIOIDS IN VIVO AT THE RAT SPINAL CORD

R.D. Russell*, J.B. Leslie*, Y.-F. Su*, W.D. Watkins*

and K.-J. Chang* **

*Department of Anesthesiology

Duke University Medical Center

Durham, N.C. 27710, U.S.A.

**Department of Molecular Biology

The Wellcome Research Labs

Research Triangle Park, N.C. 27709, U.S.A.

ABSTRACT

Possible interactions between μ- and receptors in the rat spinal

cord were studied using the radiant-heat-induced tail flick response

and the highly selective μ- or ligands: [NMePhe3,D-Pro4]

morphiceptin(PL-17) and cyclic[D-Pen2,D-Pen5]enkephalin(DPDPE).

Intrathecal infusions of PL-17(0.5μg/hr) for 5 days caused 61- and

6-fold shifts to the right of the dose-response curves for PL-17 and

DPDPE, respectively. Since PL-17 and DPDPE are highly selective

agonists for μ- and receptors, the partial cross-tolerance to the

agonist induced by PL-17 is probably not resultant from a

cross-reactivity between these ligands. These data suggest that

there may be an interaction between μ- and receptors in the rat

spinal cord.

INTRODUCTION

Several opioid receptor types and subtypes as well as their

endogenous opioid peptide ligands comprise complex pharmacologic

systems (Chang 1986). Despite the heterogeneity of these opioid

systems and a wide spectrum of biologic activities ascribed to them,

clinical interactions with opioid receptors are predominantly for

analgesia. The speculated roles of the endorphins in a variety of

physiologic and disease states are becoming more easily testable

with the development of highly receptor-specific ligands.

Such ligands are also powerful tools for examining the biochemical

nature of opioid receptor systems. For example, interactions

between opioid receptor types have recently been suggested by data

from D'Amato and Holaday (1984) and Rothman et al. (1985). Such

interactions, if present, may complicate the pharmacology of opioid

agonists' cross-reactivities. In order to relate the issues of

tolerance and cross-tolerance to the concept of μ- receptor

interactions we used two highly specific μ- or agonists: PL-17

(Chang et al. 1983) and DPDPE (Mosberg et al. 1983).

This study examines the cross-reactivity between these two highly

receptor-selective opioids by employing an assay of spinal analgesic

activity and a paradigm for inducing chronic opioid tolerance at the

rat spinal cord level. The μ- versus selectivity of our ligands

has allowed us to speculate regarding the mechanisms of ligand

cross-reactivities.

97
MATERIALS AND METHODS

Materials

The following ligands were used: [N-Methyl-Phe3

,D-Pro4]morphiceptin

(PL-17), a highly specific μ-agonist (Chang et al. 1983); cyclic

[D-Penicillamine2, D-Penicillamine5]enkephalin(DPDPE), a highly

specific agonist (Mosberg et al. 1983); funaltrexamine(R-FNA),

an irreversible μ-antagonist (Takemori et al. 1981) and naloxone

HCl, a competitive, relatively selective μ-antagonist. Ligands were

prepared in normal saline and diluted such that bolus doses equalled

twenty microliters.

Methods

400 gram male Sprague-Dawley rats had two polyethylene lo-gauge

catheters passed to the lumbar enlargement via the atlanto-occipital

membrane, following the technique of Yaksh and Rudy (1976). The

nociceptive assay was the tail-flick response to a radiant-heat

source beneath the tail (D'Armour and Smith 1941). The mean baseline latency response was 4 seconds: the cut-off time was 10 seconds. Each rat was tested 4-6 times over 6 minutes--beginning 30

minutes after intrathecal test doses. Tail-flick data were converted to maximum percent effects (MPE): (latency-response - baseline/cut-off - baseline) x 100. Responses of each rat were averaged

and a mean MPE was calculated based upon N=6-8 rats at each dose.

Spinal opioid tolerances were induced by infusing PL-17 (0.5 μg/μl)

intrathecally at 1 μl/hr for 5 days using subcutaneously implanted

osmotic pumps. Tolerance assessments were performed using the

second catheter while pumps were still attached. The dose-response

curve for PL-17 following a 5-day infusion of normal saline was not

shifted from the baseline curve for PL-17 (Figure 1). Scarcity of

materials precluded a similar vehicle-infusion control for DPDPE.

ED50's were calculated from the linear regression equations.

Dose-response curve shifts are reported as the ratios of

after-to-before treatment ED50 values. ED50's whose 95% C.I.'s do

not overlap are significantly different at the P<.05 level. Tests

of the null hypothesis, Ho=homogeneity of slopes, (conducted at

P<.05) for before- and after-treatment dose-response curves

indicated that all dose-response curve shifts were parallel.

RESULTS

Both PL-17 and DPDPE given as acute lumbar-intrathecal bolus doses

elicited antinociceptive effects. However, DPDPE was much less

potent and exhibited a flatter dose-response curve than PL-17

(Figure 1). The ED,, values were 0.06 μg (95% C.I., 0.05-0.07) and

3.56 μg (2.64-4.90) for PL-17 and DPDPE, respectively. Their μ- and

receptor selectivities were confirmed by the relative potencies of

naloxone or FNA in antagonizing their analgesic effects. The

doses of i.p. naloxone required to reduce the effects of an ED,,

dose of PL-17 or DPDPE to 50 percent effectiveness were 0.09

(0.08-0.11) and 2.71 (1.81-4.34) mg/kg, respectively, This difference is about 30-fold.

98
Sixty hours following a 2.3 μg intrathecal FNA pretreatment

(Figure 1) the dose-response curve for PL-17 was shifted parallel

and to the right 25-fold. PL-17-ED50 values before and after FNA

were 0.06 μg (0.05-0.07) and 1.50 μg (1.09-l-98), respectively.

FNA pretreatment had no significant effect upon the DPDPE

dose-response curve. DPDPE-ED50 values before and after FNA were

3.56 μg (2.64-4.90) and 5.65 μg (4.04-8.64), respectively. These

data suggest that PL-17 and DPDPE elicit antinociceptive effects

through μ- and receptors, respectively.

A chronic PL-17 infusion of 0.50 μg/hr for 5 days (Figure 2)

effected a parallel and right shift of the PL-17 dose-response

curve: ED50 after infusion, 3.65 μg (2.71-5.19)/ED50 before

infusion, 0.06 μg (0.05-0.07)--a 60.8-fold parallel shift.

Cross-tolerance to the highly selective agonist DPDPE was smaller

but significant at the P<.05 level: ED,, after infusion, 21.00 μg

(17.88-26.57)/ED50 before infusion, 3.56 μg (2.64-4.90)--a 5.9-fold

parallel shift.

DISCUSSION

The existence of some cross-tolerance between opioid agonists with

differing receptor-selectivities may be explained by crossreactivities of the compounds examined. However, the affinity of

PL-17 to receptors is 2000 times less than to μ-receptors (Chang

et al. 1983). The hourly infusion dose of PL-17 in this experiment

was only eight times a baseline PL-17 bolus-ED,, dose. It appears

unlikely that the partial cross-tolerance to DPDPE induced by PL-17

resulted from cross-reactivity of PL-17 to receptors. Furthermore, PL-17 has been shown to exert its activity via the μ-receptor

even in a receptor dominated tissue, the isolated mouse vas

deferens (Chang et al. 1983). Since the affinity of DPDPE to

receptors is at least 300 times greater than to μ-receptors

(Mosberg et al. 1983) and there is no FNA treatment effect upon

DPDPE-induced analgesia and the potency of naloxone against DPDPE is

30-times lower than that against PL-17, it is unlikely that DPDPE

interacts with μ-receptors at doses employed in this study.

These data suggest that the PL-17 infusion caused an interaction

between μ- and receptors resulting in a partial cross-tolerance to

DPDPE. This finding supports similar μ- receptor interactions

described in rat brain membrane receptor-binding studies by Rothman

et al. (1985) and postulated for an in vivo system from endotoxic

shock experiments in rats by D’Amato and Holaday (1984). The nature

of this μ- receptor interaction remains to be explored. The

suitability of spinal tissues for use in biochemical studies and the

in vivo nature of this model offer significant opportunities for

correlating the cellular biology of opioid receptor systems with

physiologic parameters.

99
FIGURE 1. Analgesic dose-response curves for PL-17 and

DPDPE Depicted is the curve for PL-17 and the data

points for DPDPE 60 hours following μ-receptor antagonism

with a 2.3μg i.t. bolus dose of FNA.

FIGURE 2. Analgesic dose-response curves: baseline curves for

PL-17 and DPDPE Depicted are the curves for PL-17

and DPDPE following 5 day i.t. infusions of PL-17, 0.5 μg/hr

and the data points for PL-17 following a vehicle-control

infusion

REFERENCES

Chang, K.-J. In: Conn, P.M. ed. The Receptors. Vol. I. Orlando:

Academic Press, 1986. pp.1-81.

Chang, K.-J. Wei, E.T., Killian, A. and Chang, J.-K. J Pharmacol Exp Ther, 227:403-408, 1983.

D'Amato, R. and Holaday, J.W. Proc Natl Acad Sci,

81:2898-2901, 1984.

D'Armour, F.E. and Smith, D.L. J Pharmacol Exp Ther,

72:74-79,1941.

Mosberg, H.E., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I.,

Galligan, J.J. and Burks, T.F. Proc Natl Acad Sci U S A,

80:5871-5874, 1983.

Rothman, R.B.. Bowen, W.D., Herkenham. M., Jacobson, A.E., Rice,

K.C. and Pert, C.B. Mol Pharmacol, 27:399-408, 1985.

Takemori, A.E., Larson. D.L. and Portoghese, P.S. Eur J Pharmacol, 70:445-451, 1981.

Yaksh, T.L. and Rudy, T.A. Physiol Behav, 17:1031-1036, 1976.

100
OPIOID RECEPTOR ACTIVITY IN THE DENTATE REGION OF THE RAT

HIPPOCAMPUS

John F. Neumaier and Charles Chavkin

Department of Pharmacology, University of Washington, Seattle, WA

98195

ABSTRACT

The electrophysiological actions of normorphine and dynorphin-A

were compared in the dentate gyrus and CA1 regions of the rat

hippocampus. In both regions, these opioids increased cell

excitability and induced afterpotentials following electrical

stimulation of synaptic afferents. Based upon apparent naloxone

dissociation constants, we conclude that normorphine activated μ

receptors in both regions. However, dynorphin-A appears to act via

μ receptors in CA1, but receptors in the dentate gyrus.

INTRODUCTION

High concentrations of opioid peptides have been detected in the

dentate region of the hippocampus, suggesting that this brain

region is a likely site of endogenous opioid action (McGinty et al.

1983, Chavkin et al. 1985b). μ, and binding sites have been

detected throughout the hippocampus (Zamir et al. 1985), and opioid

receptor mediated actions in the different hippocampal regions are

beginning to be described. In CA1, opioids have been found to

increase the excitability of pyramidal cells, probably by reducing

the inhibitory effects of GABAergic interneurons (Zieglgansberger

et al. 1979). The role of opioids in the dentate region was

previously studied (Tielen et al. 1981; Linseman and Corrigall

1982). To further characterize the potential sites of endogenous

opioid action, we have compared the receptor types mediating the

effects of opioids in the CA1 and dentate regions of the rat

hippocampus.

MATERIALS AND METHODS

Hippocampal slices (500 μm) were prepared from Sprague-Dawley rats

(150-250g) using methods previously described (Siggins and Schubert

1981). Extracellularly recorded responses were evoked by

stimulation of the dorsal blade of the dentate gyrus with a

concentric bipolar electrode placed in the center of the outer

molecular layer at least 400 μm away from the recording electrode

and recorded in the stratum granulosum using glass microelectrodes

(l-2 μm tip, 4-10 Healthy responses in the dentate granule

cell layer were typically 3-5 mV without afterpotentials even at

supramaximal stimuli (figure 1A). CA1 pyramidal cell responses

were similarly evoked and recorded as described (Chavkin et al.

1985a). Responses recorded in the cell body layers are likely to

be due to the orthodromic activation of presynaptic inputs as the

evoked responses were blocked by the addition of 10 mM MgC12 to the

superfusion buffer (data not shown).

101
RESULTS AND DISCUSSION

Dynorphin-A(1-17) (DYN) and normorphine (NM) were applied to the

tissue either by bath superfusion (0.01-10 μ) or by a pressure

micropipette (100 μM in 10 mM HEPES buffered saline, pH 7.4) placed

near the recording site. The effect of DYN delivered by pressure

pulse in the dentate region is shown in figure 1B. DYN and NM had

identical actions.

Figure 1. DYN increased cell excitability in the dentate

granule cell layer. Each response in A, B, and C was

evoked by a near maximal stimulus; arrows mark the

primary spike and asterisks mark the afterpotential. A)

control. B) 100 μM DYN was delivered by pressure pipette

(80 msec, 275 KPa) 10 sec prior to electrical

stimulation. C) DYN was delivered (as in B) in the

presence of 500 nM NAL. D) stimulus-response curve of

the primary population response before and during DYN

exposure (1 sec, 275 KPa).

The response to opioids was similar but not identical in the CA1

and dentate regions. As has been previously reported, opioids

induced both afterpotentials (APs) and a profound reduction in the

threshold for electrically stimulated primary spikes in the CA1

region. In the dentate region opioids did not effect the threshold

for primary spikes, but consistently induced APs (figure 1A-C). In

CA1 only sigmoidally shaped stimulus-response (S/R) curves were

observed; whereas, dentate S/R curves were either sigmoidal or

biphasic, depending on the precise placement of the stimulating

electrode. An example of a biphasic S/R curve is shown in figure

1D. When the dentate responses demonstrated biphaaic S/R curves,

opioids induced a change from a biphasic to a sigmoidal shape

(figure 1D). We presume that the biphasic response is due to the

activation of inhibitory pathways at higher stimulation

intensities. Differences in the nature of the opioid responses

between the CA1 and dentate region are likely to reflect

differences in synaptic circuitry.

102
Figure 2. The amplitude of the afterpotential induced by

DYN was measured in the dentate region. Log

dose-response curve for DYN (100 μM, 275 KPa) and

DYN in the presence of 100 nM NAL In this example

the NAL Ki value was 55 nM.

NAL (1 μM) completely blocked DYN and NM effects in both regions.

Apparent equilibrium dissociation constants (Ki) for NAL were

determined by Schild analysis at several NAL concentrations

(Arunlakshana and Schild 1959). We used bath application of NAL

(10-100 nM) while applying DYN or NM by pressure pipette (100 μM,

140-275 KPa) and measured the change in AP amplitudes. NAL caused

a parallel shift in the DYN sensitivity (figure 2); DYN sensitivity

returned to control levels following washout of NAL for 30-60 min.

The NAL Ki value for NM in CA1 was 2.7 0.3 nM (n=3) and 2.2 1.6

nM (n=4) in the dentate region (figure 3). These values are not

significantly different (p>0.5, Wilcoxon rank sum test), and

suggest the presence of μ receptors in both regions (Lord et al.

1977). The NAL Ki value for DYN in CA1 was 5.2 1.8 nM (n=3) and

32 11 (n=4) in the dentate gyrus. The NAL Ki value for DYN in

CA1 is not statistically different from the value for NM,

suggesting that DYN may also act at μ receptors in CAl. This

result is in agreement with our previous finding that

funaltrexamine, a μ-selective irreversible antagonist, inhibits

NM and DYN but not DADLE action in CA1 (Chavkin et al. 1985a). In

contrast, the NAL Ki value for DYN is significantly larger than the

value for NM in the dentate region (p<0.05), suggesting that DYN

acts at different receptors than NM in this region, presumably via

receptors. Therefore, although DYN is active in both the CA1

and dentate regions, it appears to act via different receptors in

these regions.

[D-Ser2,Leu5,Thr6]enkephalin and [D-Pen2,L-Pen5]enkephalin show

Preliminary studies using the selective peptides

that receptors exist in both the CA1 and dentate region and cause

very similar responses to those produced by NM and DYN.

The demonstration of opioid receptor mediated effects and the

detection of endogenous opioids in the dentate region support the

103
Figure 3. NAL Ki values for NM and DYN in the CA1 and

dentate regions.

hypothesis that endogenous opioids may be neurotransmitters at this

site. In addition, the presence of receptors suggests that

dynorphin may be released locally in the dentate gyrus. The

excitatory actions of opioids in the dentate gyrus imply that

opioids act via presynaptic disinhibition as has been shown in the

CA1 region, although the synaptic circuitry is likely to differ.

REFERENCES

Arunlakshana, O., and Schild, H. O. British J Pharmacology, 14:

48-58, 1959.

Chavkin, C., Henriksen, S. J., Siggins, G. R., and Bloom, F. E.

Brain Research, 331: 366-370, 1985a.

Chavkin, C., Shoemaker, W. J., McGinty, J. F., Bayon, A., and

Bloom, F. E. J Neuroscience, 5: 808-816, 1985b.

Linseman, M. A. and Corrigall, W. A. Neuropharmacology 21:

361-366, 1982.

Lord, J. A., Waterfield, A. A., Hughes, J., and Kosterlitz, H. W.

Nature, 267: 495-499, 1977.

McGinty, J. F., Henriksen, S. J., Goldstein, A., Terenius, L., and

Bloom, F. E. Proc Nat Acad Sci (USA), 80: 589-593, 1983.

Siggins, G. R., and Schubert, P. Neuroscience Letters, 23: 55-60,

1981.

Zamir, N., Quirion, R., and Segal, M. Neuroscience, 15: 1025-1034,

1985.

Zieglgansberger, W., French, E. D., Siggins, G. R., and Bloom, F.

E. Science, 205: 415-417, 1979.

ACKNOWLEDGEMENTS

This work was supported by a PMA research starter grant, by the

Alcoholism and Drug Abuse Institute of Washington, and by GM-07266.

104
DELTA AND KAPPA OPIOID RECEPTORS IN THE RABBIT EAR ARTERY

Ilona P. Berzetei, Alex Fong, and Sue Piper Duckles

Department of Pharmacology, College of Medicine

University of California, Irvine 92717, USA

ABSTRACT

Delta and kappa opioid receptors have been characterized in the central ear

artery of the rabbit. The selective D-Pen2,D-Pen5-enkephalin and the highly

selective antagonist ICI 174864 have been used for the opioid receptors. Ke

values were determined and compared to that of the electrically stimulated

mouse vas deferens preparation. In case of opioid receptors, Dynorphin (1-13)

and Ethylketazocine were used as agonists and Mr 2266 as antagonist. Because

of the relatively low selectivity of the antagonist (it is only twice as active at

the than at the μ receptor sites) the Ke values obtained in the rabbit ear

artery were compared to both the normal and beta-funaltrexamine treated vasa

deferentia. In the rabbit ear artery, the K2 values of Mr 2266 were also

determined in the presence of the selective antagonist ICI 174864. The Ke

values for the receptors were close in the two assay systems, suggesting that

the receptors have similar properties in rabbit ear artery and mouse vas

deferens. As for the receptors, the Ke values in the rabbit ear artery were not

significantly different in the presence or absence of the antagonist compound,

nor were they different from the Ke values obtained on the normal mouse vas

deferens. However, they were significantly different from that of the

funaltrexamine treated mouse vas deferens. Further experiments need to be

done to interpret the meaning of these results.

INTRODUCTION

Since the introduction of the isolated central ear artery of the rabbit (De la

Lande and Harvey 1965) it has been widely used as a pharmacological tool to

study the perivascular sympathetic neurotransmission. After the discovery that

some opioid peptides inhibit the electrically induced vasoconstriction of the

rabbit ear artery (REA) (Knoll 1976) much work has been done to investigate the

mode of action and characterize the opioid receptors present in this tissue (Illes

et al. 1983, Illes et al. 1985, Illes and Bettermann 19861. It has been established

that the postganglionic sympathetic neurons innervating the rabbit ear artery

possess inhibitory presynaptic receptors of the and but not of the μ subtypes.

In our present study we characterized the and opioid receptors with the help

of selective agonists and antagonist and the results obtained were compared to

that of the mouse vas deferens (MVD).

METHODS

Rabbit ear artery (REA). Male rabbits of 2.5-3 Kg weight were decapitated, and

the proximal 3 to 4 cm of the central ear artery was dissected. The arteries

were cleaned with the help of a dissecting microscope and cannulated at both

ends. To prevent longitudinal displacement, both cannulae were tightly fixed.

The vessels were perfused with Krebs solution at a steady rate of 2.4-2.6 ml/min

by means of a multichannel peristaltic pump. The perfusion pressure was

determined with a Statham P23 Db transducer and potentiometric recorder. The

preparation was equilibrated for 60 min before the beginning of electrical

stimulation. The platinum electrodes were placed at the top and the

105
bottom of the tissue-bath. A Grass S-88 electrostimulator was used for field

stimulation. The parameters of stimulation were as follows: short trains (5 HZ,

5 shocks) of supramaximal rectangular pulses of 1 msec duration were delivered

every minute. The composition of Krebs solution used was as follows (in mM):

NaCl, 118; KCl, 4.8; CaC12, 2.5; KH2PO4, 1.2; NaHCO3, 25; MgS04, 1.2 and

glucose 11.5. It was saturated with 95% O2 5% CO2 and kept at 37°C. The

agonists were added extraluminally into the tissue-bath, while the antagonists

were present both extra and intraluminally. The tissue was incubated for at

least 60 min with the antagonist.

Mouse vas deferens (MVD). Swiss Webster mice of 30-35 g weight were used.

The vasa deferentia were prepared according to Hughes et al. (1975) and bathed

at 31°C in Mg2+-free Krebs solution, bubbled with a mixture of 02 and CO2

(95:5). An initial tension of 100-150 mg was used. The experiments were carried

out in an organ-bath of 10 ml capacity. The parameters of field stimulation

were slightly modified (Ronai et al. 1977). Paired shocks with 100 msec delay

between the supramaximal rectangular pulses of 1 msec duration were used,

delivered at a rate of 0.1 Hz. Contractions were recorded using an isometric

transducer (Statham UC2) and potentiometric recorder. In order to eliminate

the μ receptors present in this preparation, the highly selective non-equilibrium

μ antagonist, -funaltrexamine (Takemori et al. 1981, Ward et al. 1.982) was

added into the tissue-bath for 30 min in the concentration of 2 x 10-7 M.

In kinetic studies complete dose-response curves were determined in the absence

and presence of antagonist. The antagonist activities were expressed in terms of

Ke (dissociation equilibrium constant) values (Kosterlitz and Watt 1968). The

antagonism was designated as competitive if it met the criteria proposed by

Arunlakshana and Schild (1959).

RESULTS AND DISCUSSION

Ke values are listed in table 1 for different agonists with the selective opioid

receptor antagonist ICI 174864 in rabbit ear artery and mouse vas deferens

preparations. The results show that Leu-enkephalin and D-Pen2,

D-Pen5-enkephalin (DPDPE) act on relatively homogenous receptor population in

both systems and that the receptors are similar in these two tissues.

TABLE 1. Comparison in MVD and REA of Ke values for the selective

antagonist, ICI 174864, using three different agonists

AGONISTS MOUSE VAS DEFERENS RABBIT EAR ARTERY

Ke (nM) Ke

 (nM)

MET-ENKEPHALIN 43.3 7.1 40.0 12.0

(16) (11)

LEU-ENKEPHALIN 17.5 3.1 12.3 2.5

(9) (9)

DPDPE 17.0 1.8 12.9 3.5

(10) (10)

106
To characterize the opioid receptors, Ke

 values were determined in "normal"

and -funaltrexamine ( -FNA) treated vasa deferentia for Dynorphin (1-13)

and Ethylketazocine with the non-selective receptor antagonist Mr 2266. The

significantly different Ke values (table 2) in the “normal” and “μ -less” MVD are

in good agreement with results of binding studies (Kosterlitz et al. 1981,

Paterson et al. 19831, which proved that Dynorphin (1-13) and Ethylketazocine as

well as Mr 2286 partially act on μ receptor sites.

TABLE 2. Comparison of Ke values of opioid agonists with

non-selective antagonist compound (Mr 2266) on “normal” and

-funaltrexamine treated mouse vas deferens preparations.

AGONISTS Ke

 (nM) and Schild slope

on "normal" MVD on -FNA MVD

DYNORPHIN (1-13) 3.86 0.4 -0.875 1.94 0.2 -0.979

(12) (8)

ETHYLKETAZOCINE 2.28 0.1 -0.925 0.63 0.1 -0.766

(6) (6)

According to the results obtained in the -FNA treated vasa deferentia, we

expected similar data on REA, which is known to contain only and receptor

subtypes. Surprisingly enough, the Ke values were (table 3) similar to the ones

obtained in the “normal” MVD. To eliminate the possible interaction of the

compounds with the receptors, the experiments were repeated in the presence

of 2 x 10-7 M ICI 174864.

TABLE 3. Characterization of the opioid receptor in the rabbit ear

artery with Mr 2266 in the presence and absence of ICI 174864.

AGONISTS Ke(nM) Schild slope Ke

 (nM) Schild slope

(in the presence of 2 x 10-7M

ICI 1748641

DYNORPHIN (1-13) 3.88 0.5 -0.98 3.49 1.1 -1.08

(8) (6)

ETHYLKETAZOCINE 2.91 0.2 -0.97 2.62 0.2 -1.02

(3) (5)

Further experiments need to be done to understand and correctly interpret the

data obtained for the receptors.

107

172-683 O - 87 - 5 : QL 3
ACKNOWLEDGEMENTS

We are grateful to Professor H.I. Yamamura for supplying us with compounds,

commercially not available. This work was supported in part by NIH grants

HL 36042 and AM 36289. SPD is an Established Investigator of the American

Heart Association.

REFERENCES

Arunlakshana, O. and Schild, H.O. Br J Pharmac, 14:48-58, 1959.

De la Lande, I.S. and Harvey, J.A. J Pharm Pharmac, 17:589-593, 1965.

Hughes, J., Kosterlitz, H.W. and Leslie, F.M. Br J Pharmac, 53:371-381, 1975.

IIIes, P., Pfeiffer, N., Limberger, N. and Starke, K. Life Sci, 33, Suppl.

I:30’-310, 1983.

IIles, P., Pfeiffer, N., Kugelgen, I.V. and Starke, K. J Pharmacol Exp Ther,

232:526-533, 1985.

Illes, P. and Bettermann, R. European J Pharmacol, 122:153-156, 1986.

Knoll, J. European J Pharmacol, 39:403-407, 1976.

Kosterlitz, H.W. and Watt, A.J. Br J Pharmac, 33:266-276, 1968.

Kosterlitz, H.W., Paterson, S.J. and Robson, L.E. Br J Pharmac, 73:939-949,

1981.

Paterson, S.J., Robson, L.E. and Kosterlitz, H.W. Br Med Bull, 39:31-36, 1983.

Ronai, A.Z., Graf, L., Szekely, J.I., Dunai-Kovacs, Zs. and Bajusz, S. FEBS Lett,

74:182-184, 1977.

Takemori, A.E., Larson, D.L. and Portoghese, P.S. European J Pharmacol,

70:445-451. 1981.

Ward, S.J., Portoghese, P.S. and Takemori, A.E. European J Pharmacol,

80:377-384, 1982.

108
A COMPARISON OF MICROSOMAL AND SYNAPTIC SITE BINDING KINETICS

James W. Spain, Dulcy E. Petta and Carmine J. Coscia

E.A. Doisy Department of Biochemistry

St. Louis University School of Medicine

1402 S. Grand Blvd., St. Louis, MO 63104, U.S.A.

ABSTRACT

High-affinity binding of the agonist, [

3

H]D-ala2-D-leu5-enkephalin

(DADLE), to sites on bovine hippocampal synaptic plasma membranes

(SPM) entails a multi-step association process. Microsomal binding

sites, which are thought to originate from internal membranes (golgi,

ser, etc.), display kinetic patterns that differ from SPMs. This is

evidenced by the absence of an association time dependent rate of

dissociation from microsomal binding sites. Although high affinity

steady state binding of agonists to microsomes occurs, kinetic

analysis indicates little or no formation of the high affinity slowly

dissociating complex. This slowly dissociating complex of SPMs is

most sensitive to guanine nucleotides. Consequently, the effect of

Gpp(NH)p on dissociation is significantly less for microsomes.

INTRODUCTION

We had previously demonstrated that agonist binding to μ and opioid

receptors displays a multi-step association while antagonists appear

to undergo simple bimolecular association (Schiebe et al. 1984). It

was determined that the dissociation rate for agonist binding is

association time dependent, i.e., a high affinity slowly dissociating

state is formed over time. The occurrence of high and low affinity

states for agonist binding was later corroborated by others using

different techniques (Law et al. 1985; Costa et al. 1985). We

postulated that the high affinity state may be linked to some aspect

of the signal-transduction functionality of the receptor such as

receptor-effector coupling. The hypothesis might be tested if a

preparation could be obtained which is composed of uncoupled

receptors.

A body of evidence has accumulated to suggest that opioid receptors

exist primarily in two subcellular pools; SPMs and microsomes (Pert

et al. 1974; Simantov et al. 1976; Smith and Loh 1976; Glasel et al.

1980; Roth et al. 1981; Klein et al. 1986). Despite their early

recognition, the functional significance of intracellular sites is

still not completely clear. We had shown that the binding

characteristics of opioid receptors on purified SPMs and microsomes

differ (Roth et al. 1981; Roth and Coscia 1984) with microsomes being

less sensitive to the effects of guanine nucleotides. These results

suggest that microsomal sites may be only partially coupled to a

guanine nucleotide binding protein. We now report an impaired

formation of a high affinity agonist state by microsomal receptors.

It is this slowly dissociating state which is most sensitive to

guanine nucleotides in SPMs and consequently there is a decreased

effect on microsomal dissociation rate.

109
METHODS

Subcellular fractions were prepared by sucrose density gradient as

previously described (Roth et al. 1981). Rapid filtration was used

to-quantify bound ligand while site selectivity for 3H-DADLE was

ensured by a 40 min pre-incubation of membranes with 20 nM d-Ala2-

mePhe4-Glyo15-enkephalin prior to addition of radioligand (Spain et

al. 1985). Dissociation was initiated by 1 μM DADLE.

RESULTS

As reported previously, the binding kinetics of 3H-DADLE to the

site in bovine hippocampal SPMs display characteristics which

suggest a multi-step association (Schiebe et al. 1984). During the

association process, there is an association time dependent formation

of a high affinity slowly dissociating state. GTP and its analogs

have been shown to increase the rate of agonist dissociation (Blume

et al. 1978; Roth et al. 1981). The effect of guanine nucleotides

appears to be limited to a transformation of the slowly dissociating

state to a lower affinity state (fig. 1A). This is demonstrated by

the profound effect of 50 μM Gpp(NH)p on the off-rate when added

following a 60 min incubation (fig. 1A) while there is little effect

on rate following a 7 min association (data not shown).

In contrast, the rate of 3

H-DADLE dissociation from receptors

present on highly purified microsomes is not dependent upon

association time (fig. 1B) suggesting a less complex association

process. Although high affinity agonist binding to microsomes is

still detected (KD=0.86 0.09 nM), the formation of the high affinity

slowly dissociating state occurs to a much lesser extent, resulting

in a rate of dissociation which is similar to that seen from SPMs

after a 7 min. association period. The addition of 50 μM Gpp(NH)p at

the onset of dissociation from microsomes resulted in a modest

acceleration of the off-rate (fig. 1B).

DISCUSSION

Past work had demonstrated that differences exist between the

receptors of SPM and microsomal origin (Roth et al. 1981; Roth and

Coscia 1984). In this study, we have found that the kinetics of

agonist binding to microsomal receptors also differ from SPMs. An

association time dependency for the rate of dissociation that is

readily demonstrated for SPM receptors was not detected with

microsomes. This suggests that the association process to the site

of microsomes does not procede in the same multi-step manner which

has been shown to occur to SPMs. In specific, the time dependent

formation of the high affinity slowly dissociation state does not

occur with microsomal receptors. Interestingly, in a previous study

(Moudy et al. 1985) we suggested that crude membranes (polytron, P20)

contain vesicles which are a mixture of internal and synaptic sites.

A single receptor type found in crude membranes can, therefore, be a

heterologous population of receptors and result in confused analysis.

Several studies have reported a decreased formation of the high

affinity state of opioid receptors in preparations which have become

expermentally uncoupled from guanine nucleotide binding protein, Ni.

110
FIGURE 1. Dissociation of DADLE from SPMs and microsomes.

(A) SPMs were incubated with 1 nM 3H-DADLE for 7 min or

60 min prior to dissociation with 1 μM DADLE. Binding

at 7 min was 46.0 2.6 % of the 60 min value (942 86 specific

dpm/tube). (B) Microsomes were incubated with radioligand

for 6 min or 60 min prior to dissociation with 1 μM

DADLE. Binding at 6 min was 68.9 10.2 % of the steady state

value (60 min, 652 100 specific dpm/tube). 50 μM Gpp(NH)p

was added to each preparation following a 60 min

incubation. Each line is the best fit for a bi-exponential

decay determine by non-linear regression.

111
Pertussis toxin (IAP) acts through ADP-ribosylation (Katada and Ui

1982) of Ni to uncouple receptors from the inhibition of adenylate

cyclase. The uncoupling procedure can result in a receptor

population which is much less able to form agonist high affinity

binding (Hsia et al. 1984) as determined by displacement of

antagonist. Similarly, desensitization by chronic agonist treatment

can produce a uncoupled receptor population (Sharma et al. 1975;

Blasig et al. 1979) and has been shown to decrease formation of the

agonist high affinity state (Law et al. 1983).

The decreased effect of guanine nucleotides on binding to microsomes

suggests that an interaction with a GTP binding protein is less

effective in this preparation and is analogous to membranes treated

with pertusis toxin or desensitized receptors. These results argue

for the existence of at least a three step association for agonists

to SPM opioid receptors whereas microsomes display one less step,

i.e., there is no formation of the slowly dissociating high affinity

complex which is most sensitive to guanine nucleotides. Studies are

now in progress to examine this hypothesis further.

REFERENCES

Blasig, J., Meyer, G., Hollt, V., Hengstenberg, J., Dum, J. and

Herz, A. Neuropharmacol, 18:473-481, 1979.

Blume, A.J. Proc Natl Acad Sci, 75:1713-1717, 1978.

Costa, T., Wuster, M., Gramsch, C. and Herz, A. Mol Pharmacol,

28:146-154, 1985.

Glasel, J.A., Venn, R.F. and Barnard, E.A. Biochem Biophys Res Comm,

95: 263-268, 1980.

Hsia, J.A., Moss, J., Hewlett, E.L. and Vaughan, M. J Biol Chem,

259:1086-1090, 1984.

Katada, T and Ui, M. J Biol Chem, 257:7210-7216, 1982.

Klein, C, Levy, R. and Simantov, R. J Neurochem, 46:1137-1144, 1986.

Law, P.Y., Horn, D.S. and Loh, H.H. Mol Pharmacol, 24:413-424, 1983.

Law, P.Y., Horn, D.S. and Loh, H.H. J Biol Chem, 260:3561-3569, 1985.

Moudy, A.M., Spain, J.W. and Coscia, C.J. Biochem Biophys Res Comm,

132:735-741, 1985.

Pert, C.B., Snowman, A.M. and Snyder, S.H. Brain Res, 70:184-188,

1974.

Roth, B.L., Laskowski, M.B. and Coscia, C.J. J Biol Chem,

256:10117-10123, 1981.

Roth, B.L. and Coscia, C.J. J Neurosci, 42:1677-1684, 1984.

Scheibe, S.D., Bennett, D.B., Spain, J.W., Roth, B.L., Coscia, C.J.

J Biol Chem, 259:13298-13303, 1984.

Sharma, S.K., Klee, W.A. and Nirenberg, M. Proc Natl Acad Sci,

72:3092-3096, 1975.

Simantov R., Snowman, A.M. and Snyder, S.H. Brain Res, 107:650-657,

1976.

Smith, A.P. and Loh, H.H. Res Comm Chem Path Pharmacol, 15:205-212,

1976.

Spain, J.W., Roth, B.L. and Coscia, C.J. J Neurosci, 5:584-588, 1985.

ACKNOWLEDGEMENTS

Supported by the National Science Foundation (NSF BNS8508933).

112
MULTIPLE SITES OF DIVALENT CATION MODULATION

OF OPIOID RECEPTOR BINDING

Maria Szücs, Gregory M. Oetting and Carmine J. Coscia

Department of Biochemistry, St. Louis University, Medical School

1402 S. Grand Blvd., St. Louis, MO 63104

ABSTRACT

Having established differential ontogeny for rat brain μ, and

opioid receptors (Spain et al. 1985) we have examined the effect of

divalent cations on receptors during postnatal development. The

pronounced inhibition of the binding of 1 nM [

3

H]D-Ala2-D-Leu5-

enkephalin (DADLE) by 100 mM Na was reversed by Mn to the levels

seen in the absence of ions both in adults and in lo-day-old

neonates. Mg++ and Ca++ on the other hand were much less potent in

adults, and ineffectjve in neonates.

the presence of Na+, regulation of

These results suggest that, in

emerges subsequent to Mn++ modulation in postnatal development.

binding with Mg++ or Ca++

Dose-dependency studies in adults performed with each ion in the

presence of fixed concentrations

that while Mg++ or Ca++ antagonized stimulation of

of either of the other ions revealed

and vice versa, Mg++ and Ca++ displayed no antagonism toward each

binding by Mn++

other. Our data suggest the existence of at least two divalent cation regulatory sites for opioid receptors, one of which appears to

be specific for Mn++ and can readily be detected in the presence of

Na+.

INTRODUCTION

Previous studies from our laboratory (Wohltmann et al. 1982; Spain et

al. 1985) and from others (Coyle and Pert 1976; Leslie et al. 1982;

Tsang et al. 1982) revealed that while high affinity μ and opioid

receptors are present in rat brain at birth, receptors appear

later. Using the bis-penicillamine enkephalin derivative, [3H]DPDPE

(thought to be highly specific, Mosberg et al. 1983) authentic

binding sites in low levels were detected in the 5-day-old neonates

(Szücs et al. 1986). In this report, divalent cation modulation of

neonatal opioid receptors in the presence and absence of Na+ is

described and compared with that of adult rat brain. In adults, Na+

inhibits opioid agonist binding and divalent cations (Me++) reverse

this effect (Pasternak et al. 1975; Simon et al. 1975; Simantov et

al. 1976). Divalent cations are also known to play an important role

in the physiological action of morphine (for a review see Chapmann

and Way 1980). The results presented in this paper suggest the

development of two divalent cation regulatory sites for the

receptors.

METHODS

Rat forebrain membranes (20,000 g x 25 min) were prepared as previously described (Spain et al. 1985). Binding assays were erformed

with 1 nM [

3

H]DADLE (in the presence of 10 nM D-Ala2-MePhe4Glyo15-

enkephalin) in a final volume of 1 ml for 1 hr at 25°C. Nonspecific

binding was determined with 10 nM unlabeled DADLE and subtracted.

113
RESULTS AND DISCUSSION

Delta receptors present in 10-day-old neonates were slightly, but

significantly, more inhibited (62 6% of control binding) by 100 mM

Na+ than those of adults (46 2%). figure 1. Manganese ion reversed

FIGURE 1: Divalent cation reversal of Na+

inhibition of 6

binding in adult (A) and 10-day-old neonate (B). Membranes

were incubated with 1 nM [3H]DADLE, 100 mM NaCl alone

or in the presence of Mn++ ; Mg++ ; or Ca++

Specific control binding (binding in the absence of ions)

was 2214 157 cpm x mg protein -1 in adult, and 763 283 cpm

x mg protein -1 in neonate. Each point is the mean S.E. of

3-7 experiments.

114
FIGURE 2: Effect of Mn++ in the presence of Mg++ on 1 nM

[

3

H]DADLE binding. Membranes were incubated with varying

concentrations of Mn++alone or in the presence of

2 mM or 20 mM Mg++

(p<0.05, T-test) inhibition of the Mn effect is shown

Statistically significant

by *. Each point is the mean of 3 experiments.

FIGURE 3: Effect of Ca++ in the presence of Mg++ on 1 nM

[

3

H]DADLE binding. Membranes were incubated with Ca++

alone or in the presence of 2 mM or 20 mM Mg++

Each point is the mean of 3 experiments.

115
the Na+

-induced decrease of binding to the level seen in the absence

of ions in adults (fig.

and Ca++ were much less potent than Mn++ in reversing inhibition of

1A); as well as in neonates (fig. 1B). Mg++

binding by Na+ in adults. These results are in a good agreement with

those of Pasternak et al. (1975) obtained for a μ selective ligand,

[

3

H]-dihydromorphine. In contrast, our data show that Mg++ or Ca++

are not able to reverse Na+ inhibition of [

3

H]DADLE binding in

10-day-old neonate (fig. 1B).

regulation of binding with Mg++ or Ca++ emerges later than Mn

Thus, these results suggest that

modulation during postnatal development.

To elaborate on these results, dose-dependency studies in adult rat

brain membranes for each ion were performed in the presence of fixed

concentrations of either of the other Me++. The fixed ion concentrations were chosen so that one represented the maximal, while the

other a submaximal effect.

summarized in Figs. 2-3. Ca++ or Mg++ antagonized the effect of Mn++

The results of these experiments are

and vice versa, especially at higher concentrations (>5 mM). Mg++

and Ca++ alternatively displayed no antagonism toward each other.

Thus, data presented in this report suggest the existence of two

divalent cation regulatory sites for opiod receptors: one site

binds Mg++ and Ca++, a distinct one binds Mn++ with an allosteric

interaction between the two.

REFERENCES

Chapman, D.B., and Way, E.L. Annu Rev Pharmacol Toxicol, 20:553-579,

1980.

Coyle, J.T., and Pert, C.B. Neuropharmacology, 15:555-560, 1976.

Leslie, F.M., Tso, S., and Hurlbut, D.E. Life Sci, 31:1393-1396,

1982.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I.,

Galligan, J.J., and Burks, T.F. Proc Natl Acad Sci USA, 80:5871-

5874, 1983.

Pasternak, G.W., Snowman, A.M., and Snyder, S.H. Mol Pharmacol,

11:735-744, 1975.

Simon, E.J., Hiller, J.M., Groth, J., and Edelman, I. J Pharmacol

Exp Ther, 192:531-537.

Simantov, R., Snowman, A.M., and Snyder, S.H. Mol Pharmacol,

12:977-986, 1976.

Spain, J.W., Roth, B.L., and Coscia, C.J. J Neurosci, 5:584-588,

1985.

Szücs, M., Oetting, G.M., and Coscia, C.J. Biochem Soc Transaction,

in press.

Tsang, D., Ng, S.C., and Ho, K.P. Dev Brain Res, 3:637-644, 1982.

Wohltmann, M., Roth, B.L., and Coscia, C.J. Dev Brain Res, 3:679-684,

1982.

ACKNOWLEDGEMENTS

This work was supported by NIH Grant MH40005.

116
EFFECTS OF MORPHINE AND B-ENDORPHIN ON

Ca2+-ATPASE ACTIVITY OF SYNAPTIC PLASMA MEMBRANES

Shih-chia C. Lin and E.Leong Way

Department of Pharmacology

School of Medicine, University of California,

San Francisco, CA 94143, USA

ABSTRACT

Morphine and B-endorphin were found to inhibit the high calcium

-affinity Ca2+-ATPase of synaptic plasma membranes which is

believed to be involved in the regulation of intracellular Ca2+ that

triggers the release of neurotransmittera. The inhibition was

blocked by naloxone and reversed by calmodulin. Kinetic analyses

revealed that opioida decreased the affinity of Ca2+-ATPase for

Ca2+ and calcium bound to the enzyme decreased the affinity of the

enzyme for opioida.

INTRODUCTION

It is well established that calcium antagonizes morphine analgesia,

but the mechanism of calcium-morphine interaction is still unknown.

However, morphine action has been hypothesized to be caused by

morphine's interference with neurotransmission through the

disturbance of intracellular calcium disposition.

The high Ca2+-affinity Ca2 +-ATPase of the synaptic plasma

membrane (SPM) is believed to be involved in the regulation of

intracellular Ca2+ (Lin and Way 1982a) which triggers the release

of neurotransmittera from synaptic vesicles (SV) (Miledi 1973).

This process involves the fusion of the SV with the SPM (Palade

1975). Nevertheless, the protein components for the attachment of

the two membranes have not been identified. The high

Ca2+-affinity Mg2+-independent Ca2+-ATPases of SPU and SV

are of particular interest because their Km values for Ca2+

assayed either in the absence or presence of Mg2+ are close to

the concentration of the intracellular Ca2+ (Lin and Way 1982)

and because Ca2+ binding is required for membrane attachment.

It should be noted that although the high Ca2+-affinity Ca2+

activity can be measured both in the absence and th presence of

Mg2+, Mg2+ is not required for the activation by Ca2+(Lin

1991; Lin and Way 1984). This differentiates it from the

Ca2+/Mg2+-ATPases reported by others (Schatzmann and Rossi 1971;

Duncan 1976) which require the simultaneus presence of Mg2+ for

stimulating ATP hydrolysis by Ca2+. The Ca2+-ATPase of SPM is

found to be localized on the cytoaolic aide (Lin and Way 1982) and

that on SV, dispersed in the cytosol near SPM. The localization of

the Ca2+-ATPases in the two organelles would appear to provide the

proper condition for membrane attachment. The present study was

designed to examine the effects of morphine and B-endorphin on SPM

Ca2+-ATPase activity.

117
MATERIAL AND METHODS

Morphine hydrochloride was purchased from Merck Chemical Division,

Rahway, New Jersey. All chemicals were of the highest grade

available and used without further purification. Calmodulin were

gifts from Dra. W.Y. Cheung and Jim Potter, B-endorphin, from Dr.

C.H. Li and naloxone, from Du Pont, Welmington, Delaware.

Synaptosomes from brain of male ICR mice were prepared by

differential and Ficoll gradient centrifugations as described

previously (Lin and Way 1984). SPM were fractionated by sucrose

gradient centrifugation of lysed synaptosomes and collected between

0.6M-0.8M sucrose-HEPES (SPM,) and 0.8M-1.1M sucrose-HEPES (SPM2)

solutions.

imidazole buffer, pH 7.5, at 37°C in 15 min (Lin and Way 1984). In

the inorganic phosphate (Pi) liberated from ATP by Ca in

experiments with morphine (or B-endorphin), naloxone, calmodulin,

alone or in combination, the enzyme preparation in HEPES buffer was

first mixed with the compound(a) and allowed to stand in ice-bath for

30 min before adding other constituents.

Ca2+-ATPase activity was determined by measuring colorimetrically

The double reciprocal plots were drawn by programmed least squares

linear regression. Ki and K'i

dissociation constants for morphine (or B-endorphin) complexes of

represent respectively the

enzyme and of enzyme-Ca2+ complex and were calculated according to

a model for a simplest mixed-type inhibition system (see Segel 1975).

It was considered that the enzyme-substrate-inhibitor complex was

non-productive.

RESULTS

Ca2 +-ATPase activity in SPM

10-6 morphine as shown in fig. 1A. The double reciprocol plots

was decreased in the presence of

intersect at a point at the left aide of vertical-axis above the

1/Ca2+-axis. Morphine increased Km and decreased Vmax values.

This suggests that the binding of morphine to the enzyme reduced the

affinity of the enzyme for Ca2+ and the enzyme-morphine-Ca2+

complex is less or non-productive. The morphine inhibition was

reversed by naloxone and calmodulin. The addition of 10-7M

naloxone restored the Km and Vmax to near control values.

Calmodulin, on the other hand, lowered Km and increased Vmax to

exceed their control values.

Similarly in SPM2, morphine was also shown to inhibit Ca2+-ATPase

activity. Again, the inhibition was reversed by naloxone and

calmodulin (fig. 1B) .

Fig. 1C shows the inhibition of Ca2+-ATPase activity of SPM2 by

B-endorphin (10-6M). The addition of 10-7M naloxone reversed the

Km and Vmax values of the enzyme in the presence of B-endorphin

to near control

Vmax further beyond the controls.

values and 6X10-7M calmodulin reversed the Km and

118
FIGURE 1. Effect of morphine and B-endorphin on

Ca2+-ATPase activity in synaptic plasma membranes.

(A),SPM1,(B) and (C), SPM2. The incubation medium

contained membrane protein of about 100 ug, 1mM ATP, 1mM

mixtures without or with 10-6M morphine (or

MgC12, various CaCl2 (0 to 950 uM) -EGTA (1.0mM)

B-endorphin) 10-6M morphine (or B-endorphin)+

1 0 - 7 M naloxone or 10-6M morphine (or B-endorphin) +

6X10-7M or 1.2X10-6M calmodulin as indicated

Incubation was at 37°C for 15 min. P represents the

difference between Pi liberated by (Ca12+ + Mg2+) and

by Mg2+ alone.

DISCUSSION

SPM Ca2+-ATPase is believed to be of special importance in synaptic

transmission because the enzyme has binding sites for both Ca2+ and

ATP (Lin and Way 1982a). Moreover, Ca2+-binding is required for

membrane fusion and ATP is required for secretion (Woodin and

Wienieke 1964; Douglas 1968). It has been shown that SPM Ca2+-ATPase

activity is regulated by calmodulin both in the absence (Lin,

unpublished data) and the presence (see Roufogalis 1979) of Mg2+ and

that calmodulin decreases Km and increased Vmax values as

compared to control values.

considered to interact with the catalytic site. The dissociation

constants Ki and K'i calculated (see Methods) from the slopes and

intercepts in fig.1 are, respectively, 2.84 and 5.05 (M-SPM1),

2.56 and 9.09 (M-SPM2) and 2.88 and 5.68 (B-endorphin -SPM2). Thus,

morphine and B-endorphin are less firmly bound to the enzyme-Ca2+

complex than to the free enzyme. However, the precise site for

morphine (or B-endorphin) binding remains unresolved at present.

The mode of morphine (or B-endorphin) inhibition of Ca2+-ATPase

suggests

Ca2+ at distinctly different sites, hence morphine cannot be

that morphine can bind to the enzyme simultaneously with

119
There are several possibilities that may explain the morphine and

B-endorphin effect. The opioid may interfere with the Ca2+

binding of calmodulin to the enzyme subunit and result in

a decrease of the amount of active form of the enzyme, or the opioid

may distort the enzyme conformation and render the

enxyme-substrate-opioid complex non-productive (or less productive).

In conclusion, the present results demonstrate that opioids decrease

the affinity of SPM Ca2+-ATPase for Ca2+, and Ca2+ decreases

the binding affinity of the enzyme for opioids. Since SPM

Ca2+-ATPase is believed to be regulated by intracellular Ca2+

which triggers the release of neurotransmitter through

Ca2+-dependent membrane fusion. The present findings may provide

the underlying basis for opioid action as well as the antagonistic

effect between calcium and opioids.

REFERENCES

Douglas, W.W. Biochem Soc Symp, 39:1-28, 1968.

Duncan, C.J. J Neurochem, 27:1277-1279, 1976.

Lin, S.C. Abtracts, 8th Int. Cong. Pharmacol. Tokyo, Japan,

1981, P.305.

Lin, S.C. and Way, E.L. J Neurochem, 39:1641-1651, 1982.

Lin, S.C. and Way, E.L. Brain Research, 235:387-392, 1982a.

Lin, S.C. and Way, E.L. J Neurochem, 42:1697-1706, 1984.

Miledi, R. Proc Roy Soc B, 183:421-425, 1973.

Palade, G. Science, 189:347-358, 1975.

Roufogalis, B.D. Canad J Physiol and Pharmacol,

57:1331-1349, 1979.

Schatzmann, H.J. and Rossi, G.L. Biophys Acta,

241:379-392, 1971.

Segel, I.H. Enzyme kinetics, John Wiley and Sons,

New York, 1975. pp.161-226.

Sobue, K., Ichica, S., Yoshida, H., Yamazaki, R. and Kakiuchi,

H. FEBS LETT 99:199-202, 1979.

Woodin, A.M. and Wienieke, A.A. Biochem J, 90:498-509, 1964.

ACKNOWLEDGEMENT

This work was supported in part by NIDA Grant DA 00037.

120
OPIOIDS AND RAT ERYTHROCYTE DEFORMABILITY

Donald L. Rhoads, Luxue Wei, Emil T. Lin, Ahmad Rezvani,

and E. L. Way

Department of Pharmacology, School of Medicine and Drug

Studies Unit, School of Pharmacy, University of California,

San Francisco, CA 94143, USA

ABSTRACT

In previous studies from this laboratory, it was noted that

opioids in vitro reduced human red blood cell deformability. The

effect was found to be dose-dependent, naloxone reversible and

preferentially selective kappa ligands exhibited the highest

potency. To extend these findings studies were carried out using

rat erythrocytes. The time required for erythrocytes to pass

through a 5.0 um pore membrane was determined and used as

an index of deformability. Opioids added in vitro produced

inhibition of deformability in a dose-dependent, naloxone

reversible manner. Injecting naive animals with morphine or

nalbuphine also produced dose related reductions in red cell

deformability. The degree of inhibition produced by nalbuphine

correlated well with its plasma concentrations as measured by

high performance liquid chromatography (HPLC). Chronic morphine

treatment by pellet implantation resulted in-the development of

tolerance as evidenced by a loss in the ability of morphine in

vitro to inhibit red cell deformability. Addition of naloxone

resulted in a decrease in filtration time. Thus, the data confirm

and extend previous findings on human red blood cells. In as

much as previous data from this laboratory demonstrated that

opioids inhibit calcium flux from erythrocytes by inhibiting

calcium-ATPase and calcium efflux is necessary for normal

deformability, it is concluded that opioids act to reduce red

cell deformability by inhibition of the calcium pump.

INTRODUCTION

Opioids cause a reduction in the ability of human red blood

cells to deform in a dose dependent, naloxone reversible,

stereoselective manner. Relatively selective kappa ligands

are more potent in eliciting this effect and extended opioid

incubation with erythrocytes produces tolerance and a rebound

phenomenon upon exposure to naloxone (Rhoads et al.1985).

To extend these findings studies were carried out using rat

erythrocytes.

MATERIALS AND METHODS

Heparinized blood was obtained from Sprague Dawley rats

(200-300g) under CO2 anesthesia by cardiac puncture. The

time necessary for 1 ml of blood to pass through a 5 um pore

Nucleopore membrane under 5 ml water pressure was measured and

used as an index of the ability of the erythrocytes to deform

121
(Reid et al 1976).

The effects of chronic opioid treatment on deformability were

studied by making rats morphine tolerant and dependent. One 75

mg morphine-base pellet was implanted on day 0 and 2 pellets on

days 1 and 2. Pellets were removed on day 3 and with some rats

naloxone was either injected intraperitoneally (5 mg/kg) or added

in vitro (0.2 uM) immediately afterwards.

In order to study the correlation between drug concentration

and deformability, rats were injected with nalbuphine HCl

intraperitoneally and the plasma concentration of nalbuphine was

determined according to the HPLC method described by Lo et al.

(1984). Nalbuphine was extracted from plasma and separated using

a C8, 5 um (4.6 x 250 mm) column, with a mobile phase of 27%

CH3CN, 0.035% H3PO4 at a flow rate of 1.4 ml/min using an

amperometric detector.

RESULTS

All opioids used in the study inhibited rat erythrocyte

deformability dose-dependently. Dynorphin A(1-13) and

1-pentazocine showed the highest potency, the IC50 being 0.3

nM and 0.5 nM respectively (figure 1). The lowest potency

was observed for D-ala-D-leucine enkephalin (800 nM) and

d-pentazocine (925 nM).

FIGURE 1. Dose response curves of opioids increasing

erythrocyte filtration time. Erythrocytes were incubated

with opioids in vitro at 37°C for 15 minutes prior to the

measurement of filtration time.

122
In erythrocytes obtained from morphine tolerant/dependent rats,

morphine added in vitro exhibited reduced ability to inhibit

deformability when compared to those from naive animals (figure

2). An increasing shift of the dose response curve to the right

occured with increasing pellet implantation time and when the

pellets were removed after the third day of implantation the

curves shifted back toward the naive state (figure 2). Addition

of naloxone resulted in a rebound effect with an increased

deformability of the red cells, as evidenced by a reduction in

the filtration time to one half (data not shown).

FIGURE 2. Chronology of development and loss of tolerance

to morphine inhibition of erythrocyte deformability. Rats

were implanted with one pellet of morphine base on day 0,

two pellets on days 1 and 2. Blood was withdrawn and the

filtration time of the erythrocytes in the presence of

varying concentrations of morphine was measured on day 0, 1,

2, 3 and on day 3, six and 12 hours after pellet removal.

HPLC measurement of the plasma concentrations of nalbuphine

yielded values that correlated directly with the degree of

inhibition of rat erythrocyte deformability (figure 3).

Dose (mg/kg)

FIGURE 3. Nalbuphine plasma concentrations (left y-axis,

open symbols and dotted lines) and changes in red cell

deformability (right y-axis, closed symbols and solid

lines) 40 and 80 minutes after 10, 30, and 100 mg/kg

intraperitoneally injected nalbuphine HCl.

123
DISCUSSION

Our results demonstrate that opioids inhibit rat erythrocyte

deformability in a dose dependent, stereoselective manner

with relatively selective kappa ligands exhibiting the highest

potency. Chronic treatment of rats with morphine resulted in

tolerance and a rebound effect occured upon addition of naloxone.

These results extend and confirm similar results with human

erythrocytes (Rhoads et al. 1985). The HPLC measurements revealed

that the deformability changes correlated well with the amount

of opioid available in plasma; higher nalbuphine plasma

concentrations produced a higher inhibition of deformability.

The change of deformability in erythrocytes is often postulated

as a symptom for an existing pathological condition and many

agents can increase or decrease the ability of erythrocytes to

deform. Intracellular calcium is believed to play a piv tol role

in the deformability of red cells and because Yamasaki and Way

(1983) found that the calcium-ATPase of rat red cells can be

inhibited by opioids, we suggest that such an action inhibits

calcium efflux and decreases erythrocyte deformability.

REFERENCES

Bernton, E.W., Long, J.B., Holaday, J.W. Fed Proc, 44:290-299,

1985. (Review)

Lo, M., Juergens, G.P., Whitney, C.C. Res Comm Chem Path

Pharmacol, 43:159-168, 1984.

Reid, H.L., Barnes, A.J., Jock, P.J., Dormandy, J.A. J Clin

Path, 29:855-858, 1976.

Rhoads, D.L., Yamasaki, Y., Way, E.L. Alcohol and Drug

Res. 6:229, 1985.

Yamasaki, Y., and Way, E.L. Life Sci, 33:723-726, 1983.

ACKNOWLEDGEMENTS

Endogenous peptides with opioid activity have been reported to

be released during stress and naloxone to be efficacious in

reversing some of debilitating effects of stress (Bernton et

al.1985). Since reduced red cell deformability is known to be

deleterious to systems by decreasing blood flow, we are presently

examining what role a reduced red cell deformability might play

under these circumstances.

This study was supported by NIDA Grant DA00037 and DAMD Grant

17-83C-3004.

124
Calmodulin Content in Rabbit Reticulocyte and the Influence of Opioid

Peptides on Cslmodulin Activity in its Membrane.

Liu Jingsheng, Liu Ye, Jiao Haiyan, and Chin Yinchang.

Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences,

Faculty of Basic Medicine, Peking Union Medical College. Beijing, China.

ABSTRACT

Calmodulin (CaM) content in rabbit reticulocyte and the influence of opioid peptides on

CaM activity in its membrane were studied by a highly sensitive assay of CaM activity

based on the stimulation of Calcium-dependent phosphodiesterase activity. The CaM

contents in reticulocytes were higher than those in normal erythrocytes, both in the cytosol fraction and in the membrane fraction. Among the opioid peptides, endorphin

EP) and dynorphin-A-(1-13) (dyn) had a significant inhibitory effect on CaM activity in

reticulocyte membrane. The effect was not antagonized by naloxone or Mr. 2286, nor

influenced by increase of Ca2+ concentration, but was reversed by the addition of exogenous CaM. This implies that the action of EP and dyn on reticulocyte membranes

probably involves an non-opioid mechanism, in which CaM may be an important key of

linkage.

INTRODUCTION

Calmodulin (CaM) has a wide distribution in the body. While calcium is important in the

stimulation-activation coupling of the central nervous system or hormonal effects, CaM

plays an important role in these calcium-dependent processes. It has been reported that

EGTA, the Ca2+

 chelating agent, inhibited phosphodiesterase (PDE) activity as a result

of CaM inactivation (Lin et al. 1975, Teo and Wang 1973). Neuropeptides or opioid peptides like ACTH, dynorphin, and beta-endorphin were also found to have an inhibitory

effect on CaM activity (Simantov et al. 1982, Barnette and Weiss 1983, 1984). Among

them, EP was the most potent, and its IC50 was 3 μM (Clouet et al. 1983).

Rat reticulocytes have been used as a convenient model for the study of the interaction of

receptor and second messenger systems. In the course of maturation, they showed a

marked loss of adenylate cyclase responsiveness to catecholamines and guanine nucleotides without significant loss of basal enzyme activity (Limbird et al. 1984). So we tried

rabbit reticulocyte membrane to study the molecular basis of the functional activity of

opioid peptides.

MATERIALS AND METHODS

1. Chemicals:

3

H-cGMP (23 ci/mmole) was obtained from Institute of Atomic Energy, Chinese

Academy of Science; endorphin, dynorphin-A-(1-13), met-enkephalin, leu-enkephalin,

were from Sigma; QAE-Sephadex A-25 was from Pharmacia; CaM-deficient PDE was

extracted from bovine heart (Liu et al 1985) at a concentration of 0.5 unit/μl; and

CaM was prepared from rat testes.

2. Solutions for CaM assay:

Assay buffer consisted of Tris-HCl 45 mmol/L, Imidazole 45 mmol/L, Mg-acetate 5.5

mmol/L, pH 7.5. All the following solutions were prepared with assay buffer: CaCl,

4.5 mmol/L, EGTA 4.5 mmol/L, cobra venom 10 u/ml, and 3H-cGMP 100,000 cpm /

50 μl (~250 pmole / 50 μl).

3. Generation of rabbit reticulocytes and the preparation of membranes (Tucker and

Young 1982):

125
Healthy male New Zealand white rabbits, about 2 kg in body weight, were used. Subcutaneous injection of vitamin B12 1 mg / 1 ml and folic acid 10 mg/l ml were given on the

first day, followed by subcutaneous injection of phenylhydrazine HCl in saline (10 mg/kg)

once daily for 5 days. On the 6th day, examination of blood sample taken from the ear

vein and stained on slide with brilliant cresyl blue showed that more than 98% of the red

blood cells were reticulocytes. The rabbit was bled from the carotid artery on the 7th

day, and blood collected and heparinized (1 mg heparin / 100 ml blood), centrifuged

under 1,000 x g for 15 minutes. After washing thrice with normal saline, the blood cells

were suspended in normal saline, counted and made up the EGTA-buffer to concentration

of 109 cells / ml. The cells were then homogenized under supersonic treatment (15 sec x

4) at 4° C, and centrifuged under 40,000 x g for GO minutes to yield sediments of cell

membranes, which were then kept, in aliquots and frozen under -65°C before use.

4. Determination of CaM activity in rabbit reticulocyte membrane:

CaM activity was determined by a highly

sensitive assay based on the stimulation of

calcium-dependent phosphodiesterase

activity (Liu et al. 1985). Rabbit reticulocyte membrane preparations (109

 cells /

ml) were serially diluted into five different

dilutions of 1:5, 1:10, 1:20, 1:40, and 1:80.

The CaM content in 20 μl of each were

measured according to the methods of

Thompson et al. (1971). The activity of

CaM in the rabbit reticulocyte membrane

which activated 20 μl of PDE from bovine

heart to 50% of its full activity was designated as 1 unit (Thompson and Appleman

1971). It can be seen clearly from fig. 1

that in our experimental conditions, the

CaM activity in 20 μl of 1:15 dilution of

membrane. preparation was equivalent to

FIGURE 1. CaM activity in rabbit reticulocyte

1 unit.

membrane.

RESULTS

1. CaM content in the cytosol and membrane fractions of normal rabbit erythrocyte and

reticulocyte:

The separated whole cells, suspended in EGTA-buffer and homogenized by supersonic

treatment, were kept in 100°C water bath for 5 minutes and centrifuged under 40,000 x g

for GO minutes. Assay of the supernatants (cytosols) as compared to the membrane fractions showed that most of the CaM content was found in the cytosol and the contents of

CaM in reticulocytes were much higher than those in normal erythrocytes both in the

cytosol fraction or within the cell membrane (table 1).

2. The effect of dyn on CaM activity in rabbit reticulocyte membranes:

Different amounts of dyn were added into reticulocyte cell membranes (1:15 dilution) to

make up a serial final concentrations of 0.5, 1.0, 2.0, 4.0, and 16.0 μM in the presence of

Ca2+ 2 mM, with 10 units (20 μl) of PDE extract from bovine heart and 3H-cGMP

(100,000 cpm / 50 μl) as substrate. Dose-dependent inhibition of PDE activation in reticulocyte membranes was observed (fig. 2). The inhibitory effect of dyn was not reversed

by 50 μl of naloxone, and Mr. 2266 (Vonvoigtlander et al. 1984), the antagonist, up to

a maximal concentration of 100 μM.

126
TABLE 1. CaM content of normal rabbit erythrocyte and and reticulocyte

(ng/109 cells)

Sample CaM( SD)

Erythrocyte Cytosol 6.05 2.37 (N=5)

Membrane 0.26 0.09 (N=2)

Retieulocyte Cytosol 37.40 8.69 (N=6)

Membrane 1.71 0.56 (N=6)

FIGURE 2. Effect of dyn on the activity of CaM in FIGURE 3. Activation of PDE by rabbit reticulocyte

rabbit reticulocyte membrane. membrane in the presence and the absence of dyn.

T)yn at 4 μM also exerts a significant inhibitory effect on the stimulation of PDE activity

by CaM present in reticulocyte membrane preparation across various amounts of PDE

from 10 to 60 μl, corresponding to 5 to 20 units. The curve of PDE activity shifts parallelly to the right in the presence of dyn (fig. 3). Similar results were obtained as the

experiment was repeated three times. The inhibitory effect of dyn on CaM-stimulated

PDE activity was not influenced by the addition of Ca2+ to 4 mM, but reversed by CaM

in a dose-dependent manner (fig. 4).

FIGURE 4. Effect of dyn on CaM activity in rabbit

reticulocyte membrane. 1: Control. 2: 4 μl dyn.

3,4,5: CaM 0.2,0.8,3.2 ng. 6: Ca2+added to 4 mM.

FIGURE 5. Activation of PDE by rabbit reticulocyte

membrane in the presence and the absence of EP.

3. The effect of EP on CaM activity in rabbit reticulocyte membranes:

EP had a similar and even stronger antagonistic effect on the activity of CaM in rabbit

reticulocyte membranes than dyn. EP, like dyn, does not affect PDE activity directly,

but inhibited the CaM PDE activating effect in 20 μl reticulocyte membranes preparation

(1:15 dilution). The effect of 1 μM of EP corresponded to 4 μM of dyn and caused a

127
parallel shift of the curve of CaM-activated PDE activity to the right (fig. 5). The effect

of EP was not antagonized by 50 nM of naloxone, not affected by an increase of Ca2+

concentration to 4 mM, but was reversed by the addition of CaM. The results implied

that EP antagonized the activating effect of CaM probably by competition with PDE

for CaM.

Besides, opiate alkaloids and endogenous opioid peptides like met-enkephalin, leuenkephalin, morphine, and etorphine were all not effective up to 10 μM. Naloxone had

also no effect on CaM activated PDE activity up to 50 μM.

DISCUSSION

CaM was assayed according to its ability to activate PDE and hydrolyze cGMP. Our

experimental results indicate that the CaM content in the cytosol and membranes of reticulocyte were much higher than those in normal erythrocytes. This is probably related to

the maturation process of red cells and coincides with the findings of Limbird who

reported a marked loss of adenylate cyclase responsiveness to catecholamines and guanine

nucleotides in the course of maturation (Limbird et al. 1984). High CaM content in reticulocyte facilitates its applications in the study of the mechanism of drug action upon

second messenger systems. Reticulocyte could be easily obtained in practically pure form

and changes involved during its maturation could be followed in cell culture.

Dyn at a concentration of 4 μM, which did not affect PDE basal activity, already showed

a significant inhibitory effect (around 50%) on the CaM activity of rabbit reticulocyte.

The effect was dose related (between 0.5 - 16.0 μM) and was not blocked by an increase of

Ca2+ concentration, but could be reversed by adding CaM. Naloxone and Mr 2266 did

not block the inhibitory effect of dyn on rabbit reticulocyte CaM. These implied that dyn

probably acts directly upon CaM and inhibits the activity of CaM in activating PDE.

EP, similar to but stronger than dyn, also inhibits CaM activity at a lower concentration of 1 μM. Although as opioid ligands, EP and dyn, which belonged to different

classes, had similar effect on CaM in cell membranes; morphine, met-enkephalin and leuenkephalin showed no significant effect. These are additional proofs to the fact the E P

and dynorphin act directly through CaM irrelevant to opiate receptors.

Known antipsychotics, e.g. fluphenazine, show a strong specific binding to CaM and competes with dyn and EP in binding with CaM (Prozialeck and Weiss 1982, Weiss et al.

1982). It is interesting to explore further the mechanism of action of EP and dyn on

CaM and its relation to anti-psychotic therapy.

REFERENCES

Barnette, MS. and Weiss, B. B. Psychopharmacol Bull, 19:387-397, 1983.

Barnette, M.S. and Weiss, B. Adv Cyclic Nucleotide Res, 16:261-276,

1984.

Clouet, D.H., Williams, N., and Yonehara, N. Life Sci, 33S:727-730, 1983.

Limbird, L.E., Gill, D.M., Stadel, J.M., Hickey, A.R., and Lefkowitz, R.J. J Biol Chem,

255:1854-1861, 1984.

Lin, J.S., Lin, Y., and Chin, Y.C. Acta Academiae Sinicae, 7:453-458, 1985.

Lin, Y.M., Liu, Y.P., and Cheung, W.Y. FEBS Lett, 49:356-360, 1975.

Prozialeck, W.C. and Weiss, B. J Pharmacol Exp Ther, 222:509-516, 1982.

Simantov, R., Nadler, H., and Levy, R. Eur J Biochem, 128:401-466, 1982.

Teo, T.S. and Wang, J.H. J Biol Chem, 248:5950-5955, 1973.

Thompson, W.J. and Appleman, M.M. J Biol Chem, 246:3145-3150, 1971.

Tucker, E.M. and Young, J.D. In: Ellory, J.C. and Young, J.D., eds.

Red cell membrane—a methodological approach. London:Academic Press. 1982. pp.

31-41.

Vonvoigtlander, P.F., Lahti, R.A., and Ludens, J.H. J Pharmacol Exp Ther, 224:7-12,

1984.

Weiss, B., Prozialeck, W.C., and Wallace, T.L. Biochem Pharmacol, 31:2217-2226, 1982.

128
ADRENAL MEDULLARY OPIOID RECEPTORS ARE LINKED TO

GTP-BINDING PROTEINS, PERTUSSIS TOXIN SUBSTRATES

Keita Kamikubo, Hiroshi Murase, Masayuki Niwa*, Kiyoshi Miura,

Masakatu Nozaki* and Kaito Tsurumi*

Departments of Internal Medicine (III) and *Pharmacology

Gifu University School of Medicine. Gifu 500. Japan

ABSTRACT

Effects of Gpp(NH)p and pretreatment of membranes with pertussis

toxin (PT) were studied on opioid binding to bovine adrenal medullary

opioid receptors. Gpp(NH)p inhibited [3

H]DADLE binding by increasing

the dissociation constant. PT treatment reduced [3H]DADLE binding and

almost abolished the Gpp(NH)p inhibition of [

3

H]DADLE binding.

Levorphanol did not show any significant effect on bovine adrenal

medullary adenylate cyclase activity. These results suggest that

bovine adrenal medullary opioid receptors are linked to PT-sensitive

GTP-binding proteins which are not directly coupled to adenylate

cyclase.

INTRODUCTION

At the present time, molecular mechanisms underlying opioid

inhibition of hormone or neurotransmitter release is still obscure.

Recently, GTP-related processes of signal transduction have been

suggested for the possible molecular mechanism of opioid action.

These suggestions are based on findings that GTP or its stable

analogs inhibits binding of opioid agonists to receptors. It has been

also shown that opioid receptors in neuroblastoma-glioma hybrid cells

(NG 108-15 cells) (Kurose et al. 1983) and-rat caudate nucleus

(Kamikubo et al. 1981, Law et al. 1981. Abood et al. 1985) are

coupled to inhibition of adenylate cyclase through a pertussis toxin

(PT)-sensitive GTP-binding protein.

In also adrenal medulla, it has been shown that opioids inhibit

hormone (catecholamine) secretion from chromaffin cells (Kumakura et

al. 1980, Barron and Hexum 1986). Because of the relative homogeneity

of composing cells, adrenal medulla may be a suitable model system to

study post-receptor mechanisms of opioid action. In the present

study, we have investigated the possible coupling of bovine adrenal

medullary opioid receptors to PT-sensitive GTP-binding proteins and

adenylate cyclase. The results suggest that adrenal medullary opioid

receptors are coupled to PT-sensitive GTP-binding proteins but not to

inhibition of adenylate cyclase.

MATERIALS AND METHODS

[

3

H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) (49.3 Ci/mmol) was from

Amersham. Guanylyl imidodiphosphate (Gpp(NH)p) was from Sigma. PT was

a generous Dr. Ui (Hokkaido University) and Kaken

Pharmaceuticals. [

125I]cAMP and anti-CAMP antibody were obtained from

Yamasa.

129
Pretreatment of membranes with PT

Bovine adrenal medullary crude plasma membranes, prepared as

described elsewhere (Kamikubo et al. 1986b), were incubated in 2 mM

NAD, 8 mM DTT, 1 mM ATP, 2 mM MgCl2, 10 mM thymidine and 50 mM TrisHCl (pH 7.4) with or without PT (20 pg/ml) at 37°C for 30 min and

washed twice with ice-cold 50 mM Tris-HCl (pH 7.4).

Opioid binding assay

Binding of [3H]DADLE to membranes was assayed in 50 mM Tris-HCl (pH

7.4) at 25°C for 30 min. Bound and free ligands were separated by

rapid vacuum filtration through Whatman GF/C filters. Non-specific

binding was determined in the presence of 1 pM DADLE. Saturation

binding data were analyzed by the computerized non-linear least

squares curve fitting procedure (Kamikubo et al. 1986a).

Adenylate cyclase assay

Adenylate cyclase activity was measured at 37°C for 10 min by the

method of Katada and Ui (1981). Incubation mixture contained 0.5 mM

ATP, 5 mM MgCl2, 0.1 mM EGTA, 0.5 mM isobutyl methylxantine, 7.2 mM

phosphocreatine, 40 U/ml creatine phosphokinase, 0.1% BSA, 10 μM GTP,

0.1 M NaCl and 25 mM Tris-HCl (pH 7.4). Cyclic AMP produced was

measured by sensitive and specific radioimmunoassay.

RESULTS AND DISCUSSION

Gpp(NH)p inhibited [

3

H]DADLE binding to bovine adrenal medullary

membranes in a concentration-dependent manner. The primary action of

Gpp(NH)p on the [

3

H]DADLE binding was an increase in the dissociation

constant (from 2.9 to 3.9 nM).

Pretreatment of the membranes with PT resulted in a 29% reduction of

[

3

H]DADLE binding (Table 1). In addition, the pretreatment abolished

almost completely the Gpp(NH)p inhibition of [3H]DADLE binding.

TABLE 1 Effects of pertussis toxin treatment on [3H]DADLE

binding to bovine adrenal medullary membranes

[

3

H]DADLE binding (%)

Addition PT (-) PT (+)

None 100 71 5

GPP(NH)p (10 uM) 36 3 65 3

Specific binding of 5 nM [

3

H]DADLE to bovine adrenal

medullary membranes treated with (PT (+)) or without

(PT (-))PT was shown. The control binding (100%) was 21

1 fmole/mg protein.

These results suggest that adrenal medullary opioid (DADLE) receptors

are regulated by Gpp(NH)p and that the Gpp(NH)p regulation may be

mediated by PT-sensitive GTP-binding proteins. The existence of PTsensitive GTP-binding proteins in bovine adrenal medulla was

130
demonstrated by PT-catalyzed [

32P]ADP-ribosylation of the proteins

(Kamikubo et al. manuscript in preparation). In several receptor

systems, it has been known that PT inhibits agonist binding to

hormone receptors and reduces GTP inhibition of agonist binding by

uncoupling PT-sensitive GTP-binding proteins from receptors (Kurose

et al. 1983, Hsia et al. 1984, Cote et al. 1984). From the present

observations, therefore, it seems likely that the PT-sensitive and

ADP-ribosylatable GTP-binding proteins mediate the Gpp(NH)p

regulation of adrenal medullary opioid receptors.

In NG 108-15 cells and rat caudate nucleus, it is likely that opioid

receptors are coupled to inhibition of adenylate cylase through the

PT-sensitive inhibitory GTP-binding protein (Ni) (Kurose et al. 1983,

Abood et al. 1985). In membranes from rat brain caudate nucleus,

levorphanol inhibited adenylate cyclase activity as previously shown

by us (Kamikubo et al. 1981) and by others (Law et al. 198l)(Table

2). However, in bovine adrenal medullary membranes, levorphanol

showed no clear inhibitory action on adenylate cyclase activity. PT

TABLE 2 Effects of levorphanol on adenylate cyclase

activity in rat caudate nucleus and bovine adrenal medulla

Adenylate cyclase activity

(% of control)

Addition Rat caudate nucleus Bovineadrenal medulla

None 100 100

Levorphanol 1 nM 92 4 96 5

Levorphanol 10 nM 83 3 98 4

Levorphanol 100 nM 76 5 97 3

Levorphanol 1 uM 74 5 93 6

Data are the means SD of triplicates. The control

activity was 148 9 pmole/mg protein/min for rat caudate

nucleus and 83 6 pmole/mg protein/min for bovine adrenal

medulla.

treatment showed only modest. if any, enhancing effect on GTP

activation of adrenal medullary adenylate cyclase.

The reason for the discrepancy between the actions of levorphanol in

rat caudate nucleus and in bovine adrenal medulla is not known.

However, it may be possible that adrenal medullary opioid (DADLE)

receptors are coupled mainly to other PT-sensitive GTP-binding

proteins than Ni. In addition to Ni, there are several functionally

or molecularly distinct classes of PT-sensitive GTP-binding proteins,

such as the stimulatory GTP-binding protein of phospholipase C in

certain cells (Nakamura and Ui 1985) and the GTP-binding protein with

39,000 dalton A subunit (No) (coupled to potassium channel?)

(Sternweis and Robinshaw 1984). PT treatment of adrenal medullary

membranes did not significantly modified the GTP activation of

adenylate cyclase. This finding may suggests that activity or content

of Ni in adrenal medulla is not high to significantly inhibit

adenylate cylase. Functional linkage of opioid (DADLE) receptors to

Ni may be scarce in adrenal medulla.

131
The present observations provide indirect evidence for the functional

linkage of adrenal medullary opioid (DADLE) receptors to PT-sensitive

GTP-binding proteins which are not directly coupled to adenylate

cyclase. Post-receptor mechanisms of signal transduction other than

adenylate cyclase regulation, such as K channel regulation etc., may

be involved in opioid (DADLE) action in bovine adrenal medulla

(Figure 1).

FIGURE 1 Linkage of bovine adrenal medullary opioid

(DADLE) receptors (OR)to pertussis toxin-sensitive GTPbinding protein (No?) not directly coupled to adenylate

cyclase (AC).

REFERENCES

Abood, M.E., Law, P.Y., and Loh, H.H. Biochem Biophys Res Commun,

127:477-483, 1985.

Barron, B.A., and Hexum, T.D. Life Sci, 38:935-945, 1986.

Cote, T.E., Frey, E.A., and Sekura, R.D. J Biol Chem, 259:8693-8698,

1984.

Hsia, J.A., Moss, J., Hewlett, E.L., and Vaughan, M. J Biol Chem,

259:1086-1090, 1984.

Kamikubo, K., Nozaki, M., and Fujimura, H. Japan J Pharmacol, 31:175-

184, 1981.

Kamikubo, K., Murase, H., Murayama, M., and Miura, K. Japan J

Pharmacol, 40:342-346, 1986a.

Kamikubo, K., Murase, H., Murayama, M., Miura, K., Nozaki, M., and

Tsurumi, K. Regul Peptides (in press), 1986b.

Katada, T. and Ui, M. J Biol Chem. 256:8310-8317, 1981.

Kumakura, K., Karoum, F., Guidotti, A., and Costa, E. Nature.

283:489-492, 1980.

Kurose, H., Katada, T., Amano, T., and Ui, M. J Biol Chem, 258:4870-

4875, 1983.

Law, P.Y., Wu, J., Koehler, J.E., and Loh. H.H. J Neurochem, 36:1834-

1846, 1981.

Nakamura, T.. and Ui, M. J Biol Chem 260:3584-3593, 1985.

Sternweis, P.C., and Robinshaw, J.D. J Biol Chem, 259:13806-13813,

1984.

ACKNOWLEDGMENTS

This work was supported in part by a grant from Japanese Ministry of

Health and Welfare. We thank Dr. Ui and Kaken Pharmaceuticals for the

generous gift of pertussis toxin. Dr. Masanori Murayama for

suggestions, Ms. Keiko Hirata and Ms. Yumi Kosaka for the expert

technical assistance are gratefully acknowleged.

132
VARYING EXPRESSION OF OPIOID RECEPTORS

AND GTP-BINDING PROTEINS IN HUMAN PHEOCHROMOCYTOMAS

Keita Kamikubo*#, Hiroshi Murase#, Masayuki Niwa##.

Kiyoshi Miura#, Yukio Miura+. Tatsuo Satoh++, Akio Tomita**,

Shigenori Nakamura*, Takehiko Miyashita*, Tatsuyuki Imai*.

Yasushi Kondo*, Masakatsu Nozaki## and Kaito Tsurumi##

*Dept. of Med., Gero Hot Spring Hospital. Gero, Gifu 509-22, Japan.

Dept. of #Internal Med. (III) and ##Pharmacol.,

Gifu University School of Medicine. Gifu 500, Japan.

+Dept. of Internal Med., Tohoku University School of Medicine,

++Dept. of Internal Med. Kumamoto University Medical School,

**Dept. of Internal Med. Nagoya University School of Medicine.

ABSTRACT

The expression of opioid receptors and GTP-binding proteins was

studied in 14 pheochromocytomas. The amount of [

3

H]diprenorphine

bound to membranes varied from 13 to 62 fmole/mg protein, but

significantly higher in adrenaline-secreting tumors than in noradrenaline-secreting tumors. None of [

3

H]DADLE, [

125I] endorphin or

[

3

H]ethylketocyclazocine binding was correlated with

[

3

H]diprenorphine binding. Gpp(NH)p inhibition of [

3

H]DADLE binding

was evident in all four normal human adrenal medullae but in only 8

out of 14 pheochromocytomas. The extent of Gpp(NH)p inhibition was

not correlated with the amount of pertussis toxin (PT)-sensitive GTPbinding proteins as measured by PT-catalyzed [

32P]ADP-ribosylation.

The present findings suggest that opioid receptors and PT-sensitive

GTP-binding proteins are variously expressed in transformed

chromaffin cells, pheochromocytoma.

INTRODUCTION

It has been shown that various amounts of opioid peptides derived

from proopiomelanocortin, proenkephalin A or proenkephalin B are

produced in transformed chromaffin cells, pheochromocytoma (Imura et

al. 1984). In addition to the production of opioid peptides,

expression of opioid receptors and GTP-binding proteins and their

functional coupling may be modified in pheochromocytomas. The present

study was undertaken to demonstrate such modification of the opioid

receptor system in pheochromocytomas. In addition, possible

correlation between the modification of opioid receptor system and

the clinical feature of pheochromocytoma has been studied.

MATERIALS AND METHODS

[

3

H]Diprenorphine (41 Ci/mmol). [

3

H]D-Ala2-D-Leu5-enkephalin

([3H]DADLE) (49.3 Ci/mmol), and [

125I] human endorphin

(approximately 2000 Ci/mmol) were from Amersham. [

3

H] Ethylketocyclazocine ([3

H]EKC) (23 Ci/mmol) was from New England Nuclear.

Pertussis toxin (PT) was a generous gift from Dr. Ui (Hokkaido

University) and Kaken Pharmaceuticals.

133
Subjects

In 7 out of 14 patients with pheochromocytoma, urinary excretion of

only noradrenaline was increased (Noradrenaline type). In other 7

patients, urinary excretions of both noradrenaline and adrenaline

were increased (Adrenaline type). Pheochromocytomas were obtained

surgically and kept -80°C until use. Four normal human adrenal

medullae were obtained at autopsy and kept -80°C until use.

Preparation of membranes

Pheochromocytomas were homogenized in 0.3 M sucrose and 10 mM TrisHCl (pH 7.4) and centrifuged at 800 g for 10 min. The supernatant was

centrifuged at 30,000 g for 20 min. The pelleted membranes were

washed with 50 mM Tris-HCl (pH 7.4) and resuspended in 50 mM Tris-HCl

(pH 7.4).

Opioid binding

The binding of [3H]diprenorphine. [

3

H]DADLE and [

3

H]EKC was assayed

in 50 mM Tris-HCl (pH 7.4) at 25°C for 30 min. The binding of

[

1251] endorphin was assayed in 0.1% BSA, 0.01% bacitracin and 50 mM

Tris-HCl (pH 7.4) at 25°C for 30 min (Kamikubo et al 1986). Bound and

free ligands were separated by rapid vacuum filtration through

Whatman GF/C filters. Non-specific binding was determined in the

presence of 1 μM of each unlabelled ligand.

Quantitation of PT-sensitive GTP-binding proteins

The amount of PT-sensitive GTP-binding proteins in pheochromocytoma

membranes was measured by the method of Pobiner et al. (1985) by

using the ability of PT to catalyze [

32P]ADP-ribosylation of the

proteins. GTP-binding proteins were extracted from membranes by

incubating membranes in 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 1% (w/v)

sodium cholate and 20 mM Tris-HCl (pH 8.0) at 4°C for 60 min. The

extract was centrifuged at 12,000 g for 15 min, and the solubilized

proteins were used as the source of GTP-binding proteins. PTcatalyzed [

32P]ADP-ribosylation was performed in 100 μl of solution

containing PT (25 μg/ml), 10 μM [32P]NAD (5-10 μCi), 10 mM DTT, 1 mM

ATP, 2 mM MgCl2, 10 mM thymidine and 50 mM Tris-HCl (pH 7.4) at 35°C

for 30 min. The reaction was terminated by adding 1 ml of 10%

trichloric acid. Proteins were precipitated by centrifugation at

12,000 g for 5 min and solubilized in Laemli's sample buffer. SDSpolyacrylamide gel electrophoresis was run on 10% polyacrylamide gel.

Autoradiography was performed at -80°C using Kodak XO-AR film. Gel

was cut, and radioactivity incorporated into proteins was counted in

a liquid scintillation counter.

RESULTS AND DISCUSSION

In all normal human adrenal medullae and pheochromocytomas examined,

specific binding sites for [

3

H]diprenorphine, [

3

H]DADLE, [

125I]

endorphin and [3H]DADLE were demonstrated in the membrane fraction. The

amount of [

3

H]diprenorphine bound to normal adrenal medullary

membranes was 40.5 5.5 fmole/mg protein (mean SD). The amount of

[

3

H]diprenorphine bound to pheochromocytoma membranes varied from 13

134
to 62 fmole/mg protein. As shown in figure 1, the adrenaline type

pheochromocytomas showed higher [

3

H]diprenorphine binding (40.6

11.7 fmole/mg protein) than the noradrenaline type (25.0 10.7

fmole/mg protein). These findings suggest that adrenaline-secreting

FIGURE 1 [

3

H]Diprenorphine binding to pheochromocytoma

membranes.

pheochromocytoma sells bear more [

3

H]diprenorphine-binding sites than

noradrenaline-secreting cells. Considering the relative nonselectivity of diprenorphine to opioid receptor subtypes, it seems

likely that the total content of opioid receptors is also higher in

adrenaline-secreting pheochromocytoma cells than in noradrenalinesecreting cells.

The amount of [

3

H]DADLE, [

3

H]EKC or [

125I] endorphin bound to pheochromocytoma membranes was not correlated with that of [

3

H]diprenorphine bound. These findings suggest that the subtype composition

of opioid receptors varies considerablely in pheochromocytomas.

Effects of Gpp(NH)p on [3H]DADLE binding were examined to study the

possible coupling of opioid receptors to GTP-binding proteins in

human adrenal medulla. Gpp(NH)p inhibited [

3

H]DADLE binding to normal

adrenal medulla, suggesting the coupling of opioid (DADLE) receptors

to GTP-binding proteins. The extent of Gpp(NH)p inhibition of

[

3

H]DADLE binding was 37 8% in normal adrenal medulla. In

pheochromocytomas, Gpp(NH)p inhibited [3H]DADLE binding in only 8 out

of 14 tumors. Apparent Gpp(NH)p inhibition of [

3

H]DADLE binding

varied from 19 to 76% inhibition in these 8 tissues.

PT-sensitive GTP binding proteins were demonstrated in normal human

adrenal medulla by using the ability of PT to [32P]ADP-ribosylate the

proteins (1.05 0.24 pmol/mg protein). In pheochromocytomas, the

amount of PT-sensitive GTP-binding proteins was not correlated with

the extent of Gpp(NH)p inhibition of [

3

H]DADLE binding.

In the present study, the presence of opioid receptors and PT135
sensitive GTP-binding proteins were demonstrated in normal and

transformed human adrenal medullae. Our results suggest that the

subtype composition of opioid receptors varies considerablely in

pheochromocytoma. The amount of [

3

H]diprenorphine bound, as a measure

of total opioid receptor content, was higher in adrenaline type than

in noradrenaline type pheochromocytomas. These findings may be in

line with the observations that naloxone increases plasma adrenaline

but not noradrenaline in the normal human (Mannelli et al. 1984) and

increases plasma catecholamines in adrenaline type but not in

noradrenaline type pheochromocytomas (Maggi et al. 1985). Interestingly, enkephalins are more rich in adrenaline cells than in

noradrenaline cells of bovine (Livett et al. 1982) and hamster

adrenal medulla (Pelto-Huikko et al. 1982). In addition, tissue

content of opioid peptides is higher in adrenaline type than in

noradrenaline type pheochromocytomas (Imura et al. 1984). Taken together, these findings may suggest that opioid peptides and opioid

receptor systems are located mainly in adrenaline-secreting

chromaffin cells in adrenal medulla and that this preferential localization is maintained in neoplasmic adrenal medulla.

The Gpp(NH)p inhibition of opioid binding was modifed variously in

pheochromocytomas. These findings suggest that functional aspects of

opioid receptor system is also important as the quantitative one to

consider modulatory action of opioid peptides in pheochromocytoma. In

addition. the observation that the extent of the Gpp(NH)p inhibition

was not correlated with the amount of PT-sensitive GTP-binding

proteins may suggest the uncoupling of opioid receptors from PTsensitive GTP-binding proteins. In conclusion, it was shown that

adrenal medullary opioid receptor-GTP-binding protein system is

variously modified by neoplasmic changes of chromaffin cells.

REFERENCES

Imura, H., Nakao, K., Yoshimasa, T., Suda, M., Sakamoto, M., and

Morii, N. Opioid Modulation of Endocrine Function New York, Raven

Press, 1984, 255 pp.

Kamikubo, K., Murase, H., Murayama, M., Miura, K., Nozaki, M., and

Tsurumi, K. Regul Peptides, (in press), 1986.

Livett, B.G., Day. R., Elde, R.P., and Howe, P.R. Neuroscience,

7:1323-1332, 1982.

Manneli, M., Maggi, M., De Feo, M.L., Cuomo, S., Delitala, G.,

Giusti, G., and Serio, M. Acta Endocrinol, 106:357-361, 1984.

Maggi, M., De Feo, M.L.. and Manneli, M. INRC '85 Abstract, 1985, 25

Pelto-Huikko, M., Salminen, T., and Hernoven, A. Histochemistry,

73:493-497, 1982.

Pobiner, B.F., Hewlett, E.L., and Garrison, J.C. J Biol Chem,

260:16200-16209, 1985.

ACKNOWLEDGEMENTS

This work was supported in part by a grant from Japanese Ministry of

Health and Welfare. We thank Dr. Ui and Kaken pharmaceuticals for

the generous gift of PT. Dr. Masanori Murayama for suggestions, Ms.

Keiko Hirata and Ms. Yumi Kosaka for the expert technical assistance

are gratefully acknowleged.

136
PERTUSSIS TOXIN TREATMENT RESULTS IN TOLERANCE

TO THE DEPRESSANT EFFECTS OF OPIOID, MONOAMINERGIC,

AND MUSCARINIC AGONISTS ON DORSAL-HORN NETWORK

RESPONSES IN MOUSE SPINAL CORD-GANGLION CULTURES

Stanley M. Crain*, Bea Crain*, and Maynard H. Makman**

Depts. of Neuroscience*, Biochemistry**, and Molecular Pharmacology**,

and the Rose F. Kennedy Center for Research in

Mental Retardation and Human Development,

Albert Einstein College of Medicine,

Yeshiva University, Bronx, New York 10461

ABSTRACT

After treatment of mouse spinal cord-ganglion explants with pertussis toxin (PTX), the acute depressant effects of opioids on sensoryevoked dorsal-horn network responses are markedly attenuated, and

characteristic cord discharges can then occur even in the presence

of >100-fold higher opioid concentrations, as observed after chronic

exposure to opioids. The usual acute depressant effects of

serotonin, norepinephrine, and oxotremorine on dorsal-horn discharges are similarly attenuated in PTX-treated cultures. These results

together with our previous physiologic and biochemical analyses of

adenylate cyclase (AC) and cyclic AMP (cAMP) activities in cordganglion cultures suggest that the neuromodulatory effects of

opioid, monoaminergic and muscarinic agonists on primary afferent

networks in the spinal cord may be mediated by binding to neuronal

receptor subtypes that are negatively coupled via Gi to a common

pool of AC.

INTRODUCTION

Exposure of cord-DRG explants to opiates or opioid peptides results

in stereospecific, naloxone-reversible, dose-dependent depression of

sensory-evoked dorsal-horn synaptic-network responses within a few

minutes (Crain et al.1978). When cord-DRG explants are treated

briefly with CAMP analogs or forskolin (a selective activator of AC;

Seamon & Daly 1981), the usual depressant effects of opioids, as

well as serotonin and norepinephrine, on sensory-evoked dorsal-horn

responses are markedly attenuated (Crain et al.1986), resembling

some aspects of the tolerance that develops in cord-DRG explants

after chronic exposure to opioids (Crain 1984; Crain et al.1979;

1982). PTX is known to interfere with the guanine nucleotide

protein Gi. that is required for opioid, -adrenergic, and muscarinic receptor-mediated inhibition of AC in various cells (Katada

& Ui 1982; Kurose et al. 1983; Ui 1984). The present study shows

that PTX selectively blocks the depressant effects of opioid and

other Gi mediated agonists on dorsal-horn responses in cord-DRG

explants, consonant with PTX effects on guinea pig ileum (Lujan et

al.1984; Tucker 1984) and isolated DRG neurons (Holz et al.1986).

MATERIALS AND METHODS

Spinal cord cross-sections with attached DRGs from 13-day fetal mice

were explanted onto collagen-coated coverglasses and incubated at

35°C in Maximow slide culture chambers. Extracellular recordings

137
were made with Ag-AgCl electrodes via micropipettes (3-5 um tips)

filled with isotonic saline. Electric stimuli were applied through

pairs of similar pipettes with 10 um tips. Recordings were made in

0.5 ml of BSS (pH 7.2; temp., 33°C; see technical details in Crain

1976; Crain et al.1978,1982). Pertussis toxin was obtained from

List Co., DADLE from Peninsula Co., oxotremorine from Research

Biochemicals Co., and other chemicals from Sigma Co.

RESULTS AND DISCUSSION

Focal DRG stimuli elicit negative slow-wave potentials restricted to

dorsal regions of spinal cord explants, arising abruptly after

latencies of 2-3 msec, and often lasting more than 500 msec. These

extracellularly recorded field-potentials resemble sensory-evoked

synaptic-network responses in dorsal spinal cord in situ (Crain

1976). Introduction of morphine or DADLE into these cultures

regularly led to sustained, dose-dependent, naloxone-reversible

depression of DRG-evoked dorsal cord responses within 3-10 min

(table 1). Measurements of the opioid sensitivity of dorsal cord

responses in these explants were made systematically in the present

study (n>40), (see also Crain et al.1978,1979,1982,1986). Similar

dose-dependent depressant effects on these cord responses were

produced by brief exposures (<10 min) to serotonin, norepinephrine,

carbamylcholine and the muscarinic agonist, oxotremorine (table 1).

The depressant effects of both cholinergic agonists were reversed or

prevented by atropine (1-10 uM), but not naloxone, whereas the

opioid-depressant effects were not antagonized by atropine.

In contrast, after pretreatment of cord-DRG explants with PTX (10

ug/ml for >1-2 days or 1 ug/ml for >4-5 days), the depressant

effects of all of these opioid, monoaminergic and cholinergic agents

failed to occur, and DRG-evoked cord responses were stably maintained during 10-30 min tests, often even with concentrations

>100-fold -higher than levels which strongly depressed control

explants (table 1). On the other hand, agents which produce depressant effects by more direct actions on ionic channels were still as

effective on PTX-treated explants as in controls, e.g. Mg++ (5 mM,

GABA (1 mM), xylocaine (ca. 100 uM), and ethanol (ca. 1-2%).

The blockade by PTX of the depressant effects of opioid, monoaminergic and muscarinic agonists on dorsal-horn responses in cord-DRG

explants is consonant with our demonstration that elevation of

intracellular CAMP levels produces similar, though less effective,

interference with these depressant actions (Crain et al. 1986). The

electrophysiologic data, together with correlative assays of AC

activity (see below), suggest that opioid, serotonergic, noradrenergic and muscarinic agonists may all act on receptor subtypes on DRG

and cord neurons that are negatively coupled via Gi to a common pool

of AC. The resulting decrease in AC/cAMP levels may, in turn,

138
TABLE 1

PTX-Blockade of Acute Depressant Effects of Opioid,

Monoaminergic, and Muscarinic Agonists on Dorsal-Horn Responses

Effective Depressant Concentration (uM)a

Agonist PTX-Treated

b *

Control n

morphine 0.1 - 1 >10 - 100 10

DADLE 0.03 - 0.3 >1 - 10 4

serotonin 0.01 - 0.1 >10 6

norepinephrine 0.1 - 1 >30 - 50 4

carbachol 0.3 - 3 >50 2

oxotremorine 0.1 - 1 >10 - 25 5

a

 Concentration required to depress sensory-evoked dorsal-horn

b

response in DRG-cord explant by >50% (Crain et al. 1978).

DRG-cord explants (ca. 2 wks in vitro) were pretreated with

*

10 ug/ml PTX for 1-2 days (see text).

Number of PTX-treated explants tested; 108 for each agent

in tests on control cultures.

regulate phosphorylation-dependent gates in K+

(e.g. Siegelbaum et

al.1982) and/or Ca++ channels (Reuter 1983) of DRG neurons, so that

Ca++ influx in presynaptic terminals as well as in the perikarya is

attenuated (Crain et al.1986). Our data do not preclude possible

PTX interference with Gi. coupling to other second messengers in

these neurons (Holz et al. 1986; Miller 1985).

The >100-fold tolerance to the depressant actions of opioids that

develops in cord-DRG explants after chronic exposure to PTX, as well

as to opioids (Crain et al.1979; Crain 1984), correlates well with

the stable increase in AC activity observed after similar treatments

of these explants (Crain & Makman 1986; Dvorkin et al.1985). In

addition, PTX-treatment attenuated the inhibition of AC produced in

control cord-DRG explants by levorphanol, and in preliminary assays,

by serotonin and norepinephrine (Crain & Makman 1986). Our studies

are consonant with the hypothesis that neurons may develop tolerance

and/or dependence during chronic opioid exposure by a compensatory

enhancement of their AC/cAMP system following initial opioid depression of AC activity (e.g. Collier 1980; Crain et al.1986; Klee et

al.1984; Sharma et al.1977). Further studies with this in vitro

model may help to determine the degree to which alterations in

opiate receptor linkages to AC/cAMP and other second messenger

systems are involved in the expression of physiological tolerance in

the CNS.

139
REFERENCES

Collier, H.O.J. Nature (London), 283: 625-629, 1980.

Crain, S.M. Neurophysiologic Studies in Tissue Culture.

New York, Raven Press, 1976. 280 pp.

Crain, S.M. In Sharp, C., ed. Mechanisms of Tolerance and

Dependence. National Institute on Drug Abuse Research Monograph.

DHEW Pub. No. (ADM) 84-1330. Washington, D.C.: Supt. of Docs.

U.S. Govt. Print. Off. 1984. pp. 260-292.

Crain, S.M. and Makman, M.H. In Ehrlich, Y.H., ed. Molecular

Mechanisms of Neuronal Responsiveness. New York, Plenum Press,

1986 (in press).

Crain, S.M., Crain, B., Finnigan, T. and Simon, E.J. Life Sci, 25:

1797-1802, 1979.

Crain, S.M., Crain, B., Peterson, E.R. and Simon, E.J. Brain Res,

157: 191-201, 1978.

Crain, S.M., Crain, B. and Peterson, E.R. Life Sci, 31: 241-247,

1982.

Crain, S.M., Crain, B., and Peterson, E.R. Brain Res, 370: 61-72,

1986.

Dvorkin, B., Crain, S.M. and Makman, M.H. Soc Neurosci Abstr, 11:

1198, 1985.

Holz, G.G., Rane, S.G., and Dunlap, K. Nature, 319: 670-672, 1986.

Katada, T. and Ui, M. Proc Nat Acad Sci U S A, 79: 3129-3133,

1982.

Klee, W.A., Milligan, G., Simonds, W.F. and Tocque, B. In Sharp, C.,

ed. Mechanisms of Tolerance and Dependence. National Institute on

Drug Abuse Monograph.

- -

DHEW Pub. No. (ADM) 84-1330.

Washington, D.C.: Supt. of Docs., U.S. Govt. Print. Off. 1984.

PP. 109-118.

Kurose, H., Katada, T., Amano, T. and Ui, M. J Biol Chem,

258: 4870-4875, 1983.

Lujan, M., Lopez; E., Ramirez, R., Aguilar, H., Martinez-Olmedo, M.A.

and Garcia-Sainz, J.A., Eur J Pharmacol, 100: 377-380, 1984.

Miller, R.J. Trends in Neurosci, 8: 462-465, 1985.

Reuter, H. Nature (London), 301: 569-574, 1983.

Seamon, K.B. and Daly, J.W. J Cyclic Nucleot Res, 7: 201-224,

1981.

Sharma, S.K., Klee, W.A. and Nirenberg, M. Proc Natl Acad Sci USA,

74: 3365-3369, 1977.

Siegelbaum, S., Camardo, J.S. and Kandel, E.R. Nature (London),

299: 413-417, 1982.

Tucker, J.F. Br J Pharmacol, 83: 326-328, 1984.

Ui, M. Trends in Pharmacol Sci, 5: 277-279, 1984.

ACKNOWLEDGEMENTS

This work was supported by research grants DA-02031 to S.M.C. and

NS-09649 and AG-00374 to M.H.M.

140
INHIBITION OF ADENYLATE CYCLASE AND INDUCTION OF HETEROLOGOUS

DESENSITIZATION BY KAPPA AGONISTS IN RAT SPINAL CORD.

Bernard Attali and Zvi Vogel

Neurobiology Department,

Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT

Using crude P2 membranes of adult rat spinal cord we were able to

show that the opiate agonist U50488 significantly and dose-dependently inhibited the basal cyclase activity, while μ (DAGO) and

(DADL) agonists were ineffective. The regulatory action was stereospecific and required the presence of GTP plus Na+

 as well as

Ca2+ ions. This inhibitory effect of K agonists was also observed when

the cyclase activity was stimulated by forskolin. Similar inhibition

was observed in spinal cord-dorsal root ganglion cocultures.

Following chronic exposure of cultured cells to etorphine or U50488,

the agonists lost their ability to inhibit the cyclase.

Furthermore, the desensitization process appeared to be. heterologous,

since the 2 adrenergic agonist, norepinephrine and the muscarinic

agonist, carbachol exhibited significant lawer potency for inhibiting

cyclase activity when compared to control cultures. These data suggest that in spinal cord, opiate receptors of the K type are negatively coupled to adenylate cyclase and the induction of tolerance

produced by K agonists is related to alterations of post-receptor

regulatory components.

INTRODUCTION

Many lines of evidence suggest that the spinal cord is an important

site for the mediation of the antinociceptive action of opiates and

endogenous opioids (Yaksh 1983). Pharmacological studies showed

antagonism of opiate analgesia by injections of cAMP (Ho et al. 1973)

or inhibitors of cAMP phosphodiesterase (Juma 1984). Furthermore,

biochemical studies demonstrated that opiate agonist binding in rat

spinal cord membranes is regulated by GTP and Na+ (Kelly et al. 1982)

suggesting a possible coupling to the adenylate cyclase system. The

rat spinal cord contains high concentrations of K receptor sites

(Gouarderes et al. 1985). However, contrary to the receptors very

little is known about the modulation of adenylate cyclase activity by

opiate ligands. The present communication shows that K opiate

receptors are negatively coupled to adenylate cyclase in rat spinal

cord and that K agonists induce heteroloqous desensitization upon

chronic treatment.

METHODS

Rat (Sprague Dawley, male adult) spinal cords were homogenized in 10%

sucrose (wt/wt) containing 1mM DTT and 1M Tris-HCL (pH 7.4). Nuclei

and cell debris were discarded by centrifugation (800xg, 10min) end

the supematant was centrifuged at 27,500xg for 3min. The crude P2

membranes were washed twice and assayed for cyclase activity

141
according to the procedure of Salomon et al. (1974). Rat dorsal root

ganglion-spinal cord primary cocultures were prepared according to

routine methods (Ransom et al. 1977). After 3 weeks in culture the

cells were washed twice with PBS and homogenized in 1mM DTT, 5mM

MgCl2 and 10mM Tris-HCL (pH=7.4).

RESULTS

We found that the K selective opiate agonist U50488 significantly and

dose-dependently inhibited the basal cyclase activity of membranes

from adult rat spinal cord (table 1), while μ (DAGO 10 μM) and

(DADL10μM) agonists were ineffective. Etorphine (1μM) and levorphanol (1μM), two non-selective opiate agonists significantly affected

the enzyme activity as well. The regulatory action was stereospecific

since dextrorphan was unable to inhibit the basal cyclase activity.

In all cases GTP (10μM) and NaCl(100mM) were required for the expression of agonist inhibitory action (data not shown). Naloxone, a non

selective opiate antagonist fully antagonized etorphine inhibition

whereas it partially reversed the U50488 response.

TABLE 1. Effect of opiates on basal adenylate cyclase activity in

adult rat spinal cord membranes.

Untreated membranes EGTA-treated membranes

(% of control) (% of control)

etorphine 1μM

etorphine 1μM

+naloxone 10μM

U50488 10μM

U50488 10μM

+naloxone 10μM

naloxone 10 μM

Levorphanol 1μM

dextrorphan 10μM

80 3** 99 4

97 2 109 9

69 5 * 102 3

87 4

102 3 101 3

79 2** 91 5

103 5 95 5

The experiments were performed in the presence of 10μM GTP

and 100mM NaCl. When EGTA(was included, it was present at

1mM concentration throughout the membrane preparation and

the assay. The adenylate cyclase activity of EGTA-treated

membranes and untreated membranes was 0.81 0.03 and

3.89 0.01 nmoles cAMP/15 min/mg protein, respectively. The

results are expressed as percent of control. The data represent the mean +SEM of 2-6 separate experiments performed

in triplicate. The significance of the data was calculated

by unpaired student t tests (*p<0.01; **p<0.001).

When the membranes were treated with 1mM EGTA, the basal cyclase

activity decreased by 4-fold and the opiate inhibition was no

longer observed. To study opioid regulation of cyclase following

chronic agonist treatment, we used homogenates of rat spinal corddorsal root ganglia primary cocultures (3 weeks in vitro). In control

cultures, we found that the K agonist inhibitory action exhibited

similar properties to those displayed in adult spinal cord preparations.

142
TABLE 2. Effect of chronic treatment of rat spinal cord-dorsal root

ganglion primary cultures with etorphine on adenylate cyclase inhibition by opiates, carbachol and norepinephrine.

untreated cells

(% activity)

Etorphine treated cells

(% activity)

basal activity

+U50488 10μM

+U50488 10μM

+naloxone 100μM

+naloxone 100μM

Forskolin stimulated

activity (25μM)

+U50488 10μM

+U50488 10μM

+naloxone 100μM

+naloxone 100μM

+carbachol 1mM

+norepinephrine 1mM

100 (4.97 0.14) 100 (4.87 0.33)

86 2**

98 1

99 2

102 7

+

100 1

102 1

100 (13.76 0.84)

78+10*

100 (13.66 0.43)

101 4

++

94 7

98 3

79 3 

57+5

97 4

97 6

97 7

++

761;2

+++

Cells were grown for 3 weeks in vitro, the last 4 days in

the presence of etorphine (1μM). Assays were performed in

the presence of 10μM GTP, 100mM NaCl and included the opiates, forskolin, carbachol and norepinephrine in the concentration stated. Cells remained in the. presence of

etorphine. until the assay. Then numbers in parentheses correspond to the adenylate cyclase activity in moles/15 min/

mg protein. Results are expressed as percent of activity.

The data correspond to the mean SEM of 2-6 separate experiments performed in triplicate. For each experiment, at

least 2 different cultures were used. The significant of

thedatawas calculated by unpaired student t test (significance versus basal or forskolin stimulated activities in

control cells*, *p<0.01; **p<0.001; significance versus

untreated cells in etorphine treated cells +p<0.05,

++p<0.02, +++p<O.O01).

In addition, as with the spinal cord tissue the forskolin stimulated

cyclase was inhibited by norepinephrine and carbachol (43 and 21%

respectively). Following chronic exposure of cells to U50488 (10μM)

or etorphine (1μM) for 4 days, the K agonist U50488 lost its ability

to inhibit the cyclase, while the basal as well as the forskolinstimulated enzyme activities remained identical to those of paired

control cultures (table 2). Furthermore, the addition of opiate

antagonists such as naloxone (100μM) did not produce any overshoot of

adenylate cyclase activity. Interestingly, the desensitization of

the agonist action on cyclase activity was found to be heteroloqous

since norepinephrine and carbachol exhibited significant lower

potency for inhibiting spinal adenylate cyclase following chronic

treatment by the opioid ligands as compared to control untreated cultures.

143
DISCUSSION

Our biochemical data demonsirate that in the spinal cord, opiate

receptors are negatively coupled to adenylate cyclase. The ineffectiveness of μ and ligands as well as the ability of K agonists to

modulate the enzyme activity suggest that the receptors involved are

likely of the K type. The lack of effect of K agonists on basal

enzyme level upon EGTA treatment of membranes suggests the participation of Ca2+-calmodulin in K opiate-dependent inhibitory circuits.

such involvement of C a2 + -Calmodulin has been suggested for adenosine

and enkephalin inhibition of rat hippocampal cyclase (Girardot et al.

1983). Interestingly, the desensitization process was shown to be

heteroloqous, since the 2 adrenergic agonist norepinephrine and the

muscarinic agonist carbachol exhibited lower potency on cyclase

activity after chronic exposure of cultured cells to opiate agonists.

This is in good agreement with recent electrophysiologic studies

showing that cAMP or forskolin not only attenuate the depressant

effects of opioids on sensory-evoked dorsal horn responses in mouse

spinal cord-ganglion explants but antagonized as well the depressant

action of norepinephrine and serotonin (Crain et al. 1986). Such a

pattern of regulation completely differs from that observed in NG108

15 cells, where chronic agonist treatment leads to homologous desensitization with a concomittant increase in adenylate cyclase activity

and subsequent receptor down-regulation (Law et al. 1983). In contrast, K receptors in spinal cord cultures only poorly down-regulate

(Attali et al. this volume). Furthermore, the lack of compensatory

increase in adenylate cyclase activity as well as the heterologous

pattern of desensitization rather suggest that the induction of tolerance produced by K opiates in the spinal cord is related to alterations of post-receptor regulatory mechanisns such as changes in the

balance of Gi/Gs or in the functional state of Gi.

REFERENCES

Crain,S.M., Crain,B., and Peterson, E.R. Brain Res, 370:61-72, 1986.

Girardot,J.M., Kempf, J.,and Cooper, D.M.F. J Neurochem, 41:848-859,

1983.

Gouarderes, C., Cros,J., and Quirion, R. Neuropeptides, 6:331-342,

1985.

Ho, I.K., Loh, H.H., and Way, E.L. J Pharmacol Exp Ther, 185:336-345,

1973.

Juma, I. Naunyn-Schmiedeberg's Arch Pharmacol, 327:23-30, 1984.

Kelly, P.D., Rance, M.J., and Traynor, J.R. Neuropeptides,

2:319-324,1982.

Law, P.Y., Hom, D.S., and Loh, H.H. Mol Pharmacol, 24:413-424, 1983.

Ransom B.R., Neale, E., Henkart, M., Bullock, P.N., and Nelson P.G.

J Neurophysiol, 40:1132-1150, 1977.

Salomon,Y., Londos, C., and Rodbell, M. Anal Biochem, 58:541-548,

1974.

Yaksh, T.L. J Pharmacol Exp Ther, 226:303-316, 1983.

144
SPARE LIGAND BINDING SITES IN THE COUPLING OF OPIOID

RECEPTOR TO BRAIN GTPase

Mary J. Clark and Fedor Medzihradsky

Departments of Biological Chemistry and Pharmacology

The University of Michigan Medical School

Ann Arbor, Michigan 48109-0010

ABSTRACT

Receptor alkylation with 10 μM superfit (SF), an irreversible deltaspecific opioid ligand, in rat brain membranes abolished 73% of

available binding sites for 3H-DSLET without affecting the stimulation of high affinity GTPase by DSLET. Alkylation with 25 μM SF

further decreased the number of low affinity sites and revealed a

slight inhibition of GTPase stimulation. Membrane treatment with 75

μM SF abolished high affinity 3

H-DSLET binding and reduced GTPase

stimulation to below 50% of control accompanied by a 10-fold decrease

in the potency of DSLET. Protective alkylation with 25 μM CNA in

the presence of DSLET yielded similar results. The findings describe

spare delta receptors in their coupling to the GTPase effector

component in brain, and reveal possible functional significance of

the low affinity ligand binding sites.

INTRODUCTION

In rat brain membranes opioid agonists stimulate high affinity GTPase

(Barchfeld et al. 1984), recently identified as the inhibitory

guanine nucleotide binding protein in brain (Milligan and Klee 1985).

The stereospecific effect is blocked by opioid antagonists, and the

stimulation by mu, kappa, or delta ligands can be prevented by alkylating the respective opioid receptors (Clark and Medzihradsky 1985,

Clark et al. 1986). In the present study, we have investigated the

correlation between delta receptor occupancy and GTPase stimulation

by gradual alkylation of the receptor with superfit, a

delta-selective (Smith et al. 1985) irreversible ligand.

MATERIALS AND METHODS

Membrane preparation (Fischel and Medzihradsky 1981). Membranes from

rat cerebrum were isolated bv differential centrifuaation as described. Aliquots of the membrane suspension were kept frozen until

use.

Receptor alkylation (Clark et al. 1986). Membranes were incubated at

25" with different concentrations of SF (methylfentanrlisothiocyanate) and subsequently washed by centrifugation and resuspension.

Alternatively, the membranes were incubated with CNA in the

presence of protecting concentrations of DSLET.

Binding assay (Fischel and Medzihradsky 1981). After incubation at

25° to reach binding equilibrium, the suspension of brain membranes

145
in the assay medium containing 120 mM Na+

 was quickly filtered and

the bound radioactivity determined. Opioid receptor binding of

3

H-DSLET was determined as the difference between binding in the

absence and presence of 50 μM unlabeled DSLET. Kd and Bmax were

estimated from Scatchard plots after resolving the high and low

affinity binding components according to Neal (1972). Final values

for the binding parameters were derived through the use of computer

analysis and statistical evaluation as described (Fischel and

Medzihradsky 1981 1986). Presented are the means of results

obtained in 3-5 experiments, with each sample carried out in duplicate.

GTPase assay (Clark et al. 1986). The assay is based on the release

of inorganic phosphate from (gamma- 32P)GTP in the presence of different concentrations of opiates (and 120 mM NaCl). The released

32P-phosphate was separated from the nucleotides by adsorption on

charcoal, and the radioactivity determined by liquid scintillation

counting. The results are expressed as concentration of a compound

to produce half-maximal stimulation of GTPase (KS) and as maximal

stimulation of basal rate (Smax). Shown are the means of data from

3-5 experiments, each sample run in triplicate

RESULTS AND DISCUSSION

Pretreatment with 10 μM SF markedly reduced 3H-DSLET binding to

opioid receptor (fig. 1). Scatchard analysis showed the presence of

both high and low affinity sites with little effect on the respective

Kd's (table 1). In these membranes the ratio of Kd/Ks for the high

affinity binding component was essentially unchanged from that in

control membranes (table 1), suggesting the importance of these

binding sites for coupling to the effector component (Fantozzi et al.

1981). However, following alkylation with 75 μM SF, high affinity

3

H-DSLET binding was undetectable but GTPase stimulation, although

impaired relative to control, still occurred. Work is in progress to

elucidate the role of the high and low affinity opioid receptor sites

in the interaction with the guanyl nucleotide binding protein and

adenylate cyclase.

The possibility was considered that at the higher concentration of

SF, partial alkylation of the mu receptor could interfere with the

interpretation of our results. However, membrane treatment with 25

μM of the nonspecific alkylator CNA in the presence of 400 μM DSLET

(Clark et al. 1986) reduced 3H-DSLET binding by 72% without affecting

GTPase stimulation by the delta opiate (KS 6.5 μM, Smax 33%).

In the receptor alkylated membranes GTPase stimulation by DSLET was

blocked by ICI 174,864, a delta specific antagonist. The treatment

of membranes did not increase the basal rate of GTPase activity, and

similar correlations between occupancy of delta receptor and GTPase

stimulation were observed if 3H-DSLET binding was determined in the

GTPase assay medium. It should also be emphasized that the treatments with superfit did not significantly alter the Kd values of

either the high or low affinity receptor binding sites for 3H-DSLET

(table 1), thus indicating the lack of allosteric binding of the

alkylator.

146
FIGURE 1. Opioid receptor binding of 3

H-DSLET in rat

brain membranes. Cerebral membranes were incubated

with and without )1O μM superfit as described

under MATERIALS AND METHODS. Subsequently, the

membranes were incubated with different concentrations

of 3H-DSLET to reach binding equilibrium at 65 min.

Plotted is specifically bound radiolabeled ligand

against the concentration of free DSLET in the medium.

TABLE 1

Parameters of opioid receptor binding and GTPase

stimulation in brain membranes

Receptor Binding GTPase stimu-

(

3

H-DSLET) lation (DSLET)

Membrane Available

treatment Bmax1 Bmax2 receptor

Kd1 Kd2 (fmol/ (fmol/ sites KS Smax

(μM SF) (nm) (nm) mg) mg) (% control) (μM) (%)

0 1.43 19.3 1 5 122 100 5.6 27

10

10

1.37 19.2 5 34 27 7.5 2 5

1.20 13.5 6 23 21 11.0 19

75 18.3 *

100 100 - *

18 13 57.0 1 3

* * - *

* undetectable

147
To what extent maximal GTPase stimulation reflects inhibition of

adenylate cyclase, the putative effector, is uncertain at this time.

It is of interest to note the similar magnitude of Smax (table 1) and

of maximal inhibition of adenylate cyclase in rat brain membranes

(Law et al. 1981). Spare receptors have been implicated in the

coupling of opioid receptor to adenylate cyclase in NG108-15 hybrid

cells (Fantozzi et al. 1981, Law et al. 1983), and as determinants of

dynorphin potency in smooth muscle preparation (Cox and Chavkin

1983).

ACKNOWLEDGEMENTS

This work was supported in part by USPHS grant DA 00254.

REFERENCES

Barchfeld, C.C., and Medzihradsky, F. Biochem Biophys Res Comm,

121:641-648, 1984.

Clark, M. J., Levenson, S.D., and Medzihradsky, F. Life Sci,

19: 1721-1727, 1986.

Clark, M.J., and Medzihradsky, F. Abstracts, INRC, North Falmouth,

1985.

Cox, B.M., and Chavkin, C. Mol Pharmacol, 23:36-43, 1983.

Fantozzi, R., Mullikin-Kilpatrick, D., and Blume, A.J. Mol Pharmacol,

20:8-15, 1981.

Fischel, S.V., and Medzihradsky, F. Mol Pharmacol, 20:269-279, 1981.

Fischel, S.V., and Medzihradsky, F. Neuropharmacol, 25:351-359,

1986.

Law, P.Y., Horn, D.S., and Loh, H.H. Mol Pharmacol, 23:26-35, 1983.

Law, P.Y., Wu, J., Koehler, J.E., and Loh, H.H. J Neurochem,

36:1834-1836. 1981.

Milligan, G., and Klee, W.A. J Biol Chem, 260:2057-2063, 1985.

Neal, J.L. J Theor Biol, 35:113-118, 1972.

Smith, C.B., Jacobson, T.E., Burke, T.R., Rice, K.C., and Woods, J.H.

Fed Proc, 44:610, 1985.

148
COMPARATIVE PROPERTIES OF SH-GROUPS

AT MU. DELTA AND KAPPA OPIOID RECEPTOR

Masakatsu Nozaki, Masayuki Niwa and Hajime Fujimura*

Dept. of Pharmacology, Gifu Univ. Sch. of Med.

Gifu 500, Japan

*Kyoto Pharm. Univ., Kyoto 607, Japan

ABSTRACT

Crude synaptosomal membrane from rat brain was treated with three

kinds of DTNB analogs. The receptor bindings of opioids were inhibited on a concentration dependent manner by 5-nitro-2-PDS and DTNHEB,

regardless of the receptor types. DTNB showed complete inhibition of

the bindings of mu and delta agonists. But, the effect of DTNB on the

binding of kappa agonist was incomplete, resulting in approximately

50% inhibition at the maximal effective concentration. Treating the

membrane with DTNB, but not with DTNHEB and 5-nitro-2-PDS. completely

eliminated inhibitory effects of guanine nucleotides on agonist binding. These results suggest that one SH-group, being sensitive to

DTNHEB, exists at an active center of the receptor binding site(R) and

another SH-group, blocked by DTNB, forms a binding site for GTP in

GTP-binding protein(Ni), and that a coupling mechanism between R and

Ni at kappa receptor differs from those at mu and delta receptor.

INTRODUCTION

It is well known that sulfhydryl covalent linking agents such as

NEM and DTNB discriminate between agonist and antagonist bindings to

membrane bound and partially purified opioid receptor, particularly

when DTNB is used (Pasternak et al. 1975, Nozaki and Cho 1985). DTNB

specifically reacts with all the surface SH-groups of protein which

is not influenced by negative charge of DPNB. A SH-group in the opioid

receptor binding site should interact with positively charged N-atom

of opiate. Therefore, without too great difficulty, DTNB can react

with the SH-group in the binding site. We compared the effects of

differently charged DTNB analogs on the opioid receptor binding.

MATERIALS AND METHOD

DTNB and 5-nitro-2-PDS, positively charged analog of DTNB, were

obtained from Dojindo Laboratories (Kumamoto, Japan). DTNHEB was newly

synthesized. Tritiated DHM(80Ci/mmol), DADLE(45Ci/mmol), EKC(23.3Ci/

mmol) and phencyclidine(43.5Ci/mmol) were purchased from New England

Nuclear. Tritiated naloxone(45Ci/mmol). diprenorphine (25Ci/mmol) and

dynorphin-A(1-9)(25.3Ci/mmol) were obtained from Amersham.

Abbreviations used: NEM, N-ethylmaleimide; DTNB, 5,5'-dithiobis-

(2-nitrobenzoic acid); DTNHEB, 5,5'-dithiobis-(2-nitro-n-2'-hydroxyl

ethylbenzamide); 5-nitro-2-PDS, 2.2'-dithiobis-(5-nitropyridine);

DHM, dihydromorphine; EKC, ethylketocyclazocine.

149
Crude synaptosomal membrane from rat brain without cerebellum was

preincubated at 20°C for 20 minutes with DTNB analog. After washing

the membrane by centrifugation, the membrane was incubated at 20°C for

30 minutes with 3H-opioid ligand and filtered over glass filter

(GF/B). In the case of dynorphin-A(1-9). the binding assay was carried

out with incubation at 4°C for 90 minutes. All assay tubes were

coated with silicon to prevent adsorption of dynorphin. The filter was

pre-soaked with pH 7.4, 50 mM Tris-buffer containing 0.4% BSA and 0.1%

polylisine. The filtrated residue was washed with the ice-cold buffer

containing 0.1% BSA and 100 mM cholin chloride.

RESULTS AND DISCUSSION

The P2-membrane was pretreated with varying concentrations of DTNB,

DTNHEB or 5-nitro-2-PDS. The inhibition profiles of the receptor bindings of a variety type of opioid by DTNB analogs are shown in fig.1.

Positively charged analog of DTNB, 5-nitro-2-PDS, and neutral

analog, DTNHEB, inhibited the receptor bindings of all types of opioid

ligands except phencyclidine on a concentration dependent manner. The

effect of 5-nitro-2-PDS was more potent than that of DTNHEB. DTNB

inhibited agonist bindings of mu and delta ligands. and showed no

effect on the binding of naloxone. In the case of EKC and dynorphinA(1-9) bindings, the inhibitory effects of DTNB were incomplete,

resulting in only 50% at the maximum inhibition. When DTNHEB was

applied to the DTNB-treated membrane which showed the maximum inhibition on the binding of EKC, DTNHEB exerted an additional inhibitory

effects on EKC and dynorphin bindings (Niwa et al. 1983).

These suggest that a state of SH-group at kappa receptor, being

sensitive to DTNB, might be different from that of mu or delta

receptor. No influences of DTNB and its analogs were observed at the

binding of phencyclidine.

After preincubating the membrane with 50 nM DADLE for 20 minutes,

500 μM DTNB or 10 μM DTNHEB was added. At appropriate time, the

membrane was washed and DADLE binding was evaluated. The inhibitory

effect of DTNB on DADLE binding to the membrane was not modified by

the preincubation with DADLE. However, the effect of DTNHEB decreased

at the DADLE-pretreated membrane.

DTNB and its analogs potentiated sodium effects on opioid agonist

binding. There is no difference in the potentiation effects between

DTNB- and DTNHEB-treatment.

Guanine nucleotide inhibits opioid agonist binding to the receptor.

When the membrane was pretreated with DTNB, the inhibitory effect of

guanine nucleotide completely disappeared, but DTNHEB showed no effect

(fig. 2). Preteating the membrane with guanine nucleotide diminished

the effect of DTNB, but not DTNHEB. A SH-group, blocked by DTNB, may

be different on a role from a SH-group, blocked by DTNHEB.

These results suggest that DTNB-sensitive SH-group exists in a

binding site of guanine nucleotide in GTP binding protein and DTNHEBsensitive SH-group is a functional group in the center of the receptor

binding site.

150
FIGURE 1

Inhibitory effects of DTNB analogs on the receptor binding

of a variety type of opioid; A, dihydromorphine: B, DADLE;

C, naloxone; D, ethylketocyclazocine; E, dynorphin-A(1-9)

and F, phencyclidine.

DTNB, ; DTNHEB, and 5-nitro-2-PDS,

151
FIGURE 2

Effects of guanine nucleotides on 3H-DADLE specific bindings

to (A) 500 μM DTNB- and (B) 10 μM DTNHEB-treated membranes.

Open symboles represent results at non-treated membrane

(control) and closed symbols represent results at DTNB- and

DTNHEB-treated membranes: GTP, and GPP(NH)p,

Reactivity of a SH-group in protein depends on its environmental

factor, such as steric interactions and electrostatic effects. There

are little differences in molecular size and general reactivity,

except the effect of attracting force of charged group, among DTNB.

DTNHEB and 5-nitro-2-PDS.

One SH-group in the opioid receptor may be located in an active

center of an anionic binding site for the ligand binding. This SHgroup is blocked by DTNHEB and 5-nitro-2-PDS. Another SH-group

which is located in GTP-binding protein, is in a positively charged

environment and specifically blocked by DTNB. The fact that opioid

agonist binding is selectively inhibited by DTNB supports this

conclusion.

Finally, since the maximum inhibitory effect of DTNB on kappa

agonist binding is approximately 50%, a coupling mechanism at kappa

receptor between the ligand binding site and GTP-binding protein may

be different from those of mu and delta receptors.

REFERENCES

Niwa, M. et al. Life Sci, 33: 211-214. 1983.

Nozaki. M. et al. Life Sci. 31: 1339-1342, 1982.

Nozaki, M. and Cho, T. M. J Neurochem, 45: 461-464, 1985.

Pasternak, G. W. et al. Mol Pharmacol, 11: 340-351, 1975.

152
ENHANCEMENT OF PERFORANT PATH TRANSMISSION BY ENKEPHALIN

ANALOGUES; ALTERATION OF GRANULE CELL INHIBITION/POTENTIATION

James B. Wiesner and Steven J. Henriksen

Preclinical Neuroscience and Endocrinology

Research Institute at Scripps Clinic

La Jolla, California 92037, U.S.A.

ABSTRACT

Enkephalin analogues were electrophoretically applied into the dentate granule cell layer of the hippocampus in vivo to determine

opioid effects on granule cell responsiveness to afferent input. The

results indicate that opioids, probably acting at μ receptors,

enhance dentate responsiveness to perforant path transmission. This

enhancement includes decreased inhibition and increased potentiation.

INTRODUCTION

Immunocytochemical studies have shown that the dentate gyrus of the

hippocampus of the rat contains endogenous opioid peptides (Gall

et al. 1981, McGinty et al. 1984). To begin to understand the roles

of these opioids, we have applied opioid peptides locally to the

dentate granule cells, in vivo, to determine the effects of opioids

on responsiveness of these cells to afferent input. In addition, we

examined the relative potencies of selective opioid agonists in

exerting these effects.

MATERIALS AND METHODS

These studies involved stimulation of the perforant path, which

evokes an excitatory response by the granule cells. The excitatory

response was recorded as a 'population spike' (PS) within the evoked

field potential. Using halothane-anesthetized rats (250-350), a

concentric bipolar stimulating electrode was stereotactically positioned within the perforant path (AP -8.1, L 4.4, V dura-3.5), and a

glass 5-barrel recording pipet (8-12 m) was positioned within the

ipsilateral granule cell layer (AP -4.0, L 2.5, V dura-2.7 to -3.5).

The center barrel of the pipet contained Pontamine Sky Blue (2% in

0.5 M sodium acetate), used for marking the recording site. The

outside barrels contained opioid peptides (5mM, pH 7-8 in saline) for

electrophoresis; one barrel contained 3 M NaCl for current balancing.

Input/output (I/O) curves were constructed by plotting PS amplitude

as a function of stimulus intensity. Paired-pulse data were obtained

by presenting pairs of identical stimulus pulses (a Conditioning

pulse followed by a Test pulse) at varying interpulse intervals; the

paired-pulse curves represent the amplitude of the second evoked PS

(Test PS) expressed as a percentage of the first evoked PS

(Conditioning PS) for each interpulse interval.

RESULTS AND DISCUSSION

To obtain an initial assessment of opioid effects on dentate

responsiveness to single or paired-pulse stimulation, analogs of

153
methionine-enkephalin or leucine-enkephalin were electrophoresed

within the granule cell layer. The analogs, [2-D-Ala,5-

Met]enkephalinamide (DAMEA) and [2-D-Ala,5-D-Leu]enkephalin exerted

similar effects. Figure 1 illustrates the effect of DAMEA (25 and 75

nA) on responsiveness to single-pulse stimulation. DAMEA shifted the

I/O curve to the left and increased the maximum PS amplitude,

indicating an increased responsiveness to perforant path stimulation.

This effect was reversed by intravenous naloxone administration

(figure 1) as well as by local electrophoretic naloxone

administration (not shown).

FIGURE 1. Effect of DAMEA (25 and 75 nA) on I/O curves, and

blockade by naloxone (5 mg/kg i.v.); N=5.

* p<.05 vs. Control; + p<.05 vs. 75 nA DAMEA [ANOVA].

In order to investigate potential opioid modulation of local

hippocampal circuitry characteristic of the dentate gyrus, we have

employed a standard paired-pulse paradigm of stimulation. When the

stimulus current employed is adjusted to evoke a half-maximal

conditioning PS, two local circuitry effects characteristic of the

dentate gyrus can be observed (figure 2, Control). One such effect,

that of recurrent (or feed-forward) inhibition, is associated with

interpulse intervals of less than 45 msec and is marked by an

inhibited test response. A second, potentiating effect is observed

at interpulse intervals ranging from 50-150 msec.

DAMEA increased the Test PS relative to the Conditioning PS at

interpulse intervals ranging from 30 to 120 msec (figure 2). This

effect could be antagonized by both intravenous and local naloxone

administration. Since the effect of DAMEA is observed over a wide

range of interpulse intervals, the increase could be due to a change

in the sequential inhibition or in the sequential potentiation. We

have found, however, that these two phenomena can be partially

dissociated by altering the experimental parameters. When the

electrophoretic application of the opioid is restricted to a short

time period (eg. less than 11 min.), the relative enhancement of the

154
test response is restricted to the portion of the curve dominated by

the sequential inhibitory effect (Wiesner and Henriksen 1986). This

result suggests that DAMEA is able to exert a disinhibitory action in

the dentate similar to that seen in the CA subfields

(Zieglegansberger et al. 1979). Alternatively, when the stimulus

current is adjusted to evoke only a threshold conditioning PS, the

effect of sequential inhibition is minimized, essentially isolating

the sequential potentiation effect. Under these experimental

conditions, DAMEA greatly increased the relative amplitude of the

test response over a broad range (not shown). It therefore appears

that DAMEA increases sequential potentiation as well as decreasing

sequential inhibition.

FIGURE 2. Effect of DAMEA (25 and 75 nA) on paired-pulse

curves (see MATERIALS AND METHODS); N=5. * p<.05

To obtain an initial indication of the specific opioid receptor

type(s) which may be involved in these opioid effects on perforant

path transmission, we applied various selective opioid agonists to

the granule cell layer. Figure 3 shows a comparison of effects on

the I/O curve of a few prototypic agonists, along with DAMEA (25 nA

for 4-6 min). The most effective of the selective agonists was the μ

agonist PL017 morphiceptin. Neither the 6 agonist D-pen,L-penenkephalin (DPLPE), nor the agonist U-50488H were effective at this

dose level. Similar differential effects were observed using paired

pulse paradigm. These results are suggestive that the opioid effects

may be mediated at μ receptors.

We conclude from these results that opioid peptides,by acting at

opioid (possibly μ) receptors, can enhance dentate responsiveness to

perforant path input. Components of this enhanced responsiveness are:

(1) increased primary responsiveness to single stimuli; (2) decreased

sequential inhibition; and (3) increased sequential potentiation. In

excitatory effects of opioids in the CA subfields (Zieglegansberger

et al. 1979), it seems likely that the disinhibitory effect indicated

in the present studies may in turn be responsible for the increased

155
FIGURE 3. Effect on the I/O curve of DAMEA, PL017 (μ

agonist), DPLPE ( agonist), and U-50488H( agonist),

(25 nA, 4-6 min, N=3-6). Stimulus levels were threshold

(T), half-maximal (1/2 MAX), or maximal (MAX) for response

levels of each animal; responses were normalized as a

percentage of the Control maximum for each test.

primary responsiveness. Finally, our studies of single unit activity

have shown that, while exerting these facilitatory effects on

perforant path transmission, opioids, in fact, suppress the

spontaneous activity of individual granule cells (Wiesner and

Henriksen 1986). The facilitating effect reported here, therefore,

may be specific to perforant path transmission.

ACKNOWLEDGEMENTS

Sources of peptides were: Dr. N. Ling (DAMEA), Endo Laboratories

(naloxone), Peninsula (PL017, DPLPE), Upjohn, Inc. (U-50488H). The

work was supported by NIDA grant DA03365.

REFERENCES

Gall, C., Brecha, N., Karten, H.J., and Chang, K.-J. J Comp Neurol

198:335-350, 1981.

McGinty, J.F., van der Kooy, D., and Bloom, F.E. J Neurosci,

4:1104-1117, 1984.

Wiesner, J.B., and Henriksen, S.J. Neurosci Lett, 1986, in press.

Zieglgansberger, W., French, E.D., Siggins, G.R., and Bloom, F.E.

Science, 205:415-417 (1979).

156
MODULATION OF BRAIN NMDA RECEPTORS: COMMON MECHANISM OF/PCP

RECEPTORS AND THEIR EXOGENOUS AND ENDOGENOUS LIGANDS

Ratna Sircar*, Hany Samaan*, Roxanne Nichtenhauser*,

Laurence D. Snell***, Kenneth M. Johnson***, Jean Rivier ,

Wylie Vale** **, R. Suzanne Zukin** and Stephen R. Zukin*,**,*****

Departments of *Psychiatry and **Neuroscience,

Albert Einstein College of Medicine, and *****Bronx Psychiatric

Center, Bronx, NY 10461

***Department of Pharmacology and Toxicology

University of Texas Medical Center Branch Galveston, TX 77550

****Clayton Foundation Laboratories for Peptide Biology

The Salk Institute, San Diego, CA 92138

ABSTRACT

Quantitative autoradiographic distribution patterns of /PCP and

NMDA receptors show striking similarities but not in all brain

areas. We have identified an endogenous ligand of brain PCP receptors which noncompetitively antagonizes NMDA-induced neurotransmitter release from brain slices. These data support the concept that

the ( /PCP receptor ligands may exert their unique behavioral effects

by indirect modulation of NMDA-mediated transmission.

INTRODUCTION

Binding of SKF10,047-like benzomorphans, phencyclidine (PCP) derivatives, dioxolanes, and benz-f-isoquinolines to /PCP receptors selectively modulates the excitatory effects of the prototypic excitatory amino acid agonist n-methyl-d-aspartate (NMDA). Such drugs

potently and stereoselectively antagonize the excitatory effects of

NMDA on spinal neurones noncompetitively (Berry et al. 1984). In

addition PCP-like drugs potently inhibit NMDA-stimulated acetylcholine and dopamine release from rat striatal slices (Snell and Johnson 1985). These effects are selective in that these drugs fail to

modulate the excitatory responses to quisqualate (q) or kainate (k)

type excitatory amino acid receptors. In this study we investigate

the neuroanatomical and functional relationships between /PCP and

NMDA receptors by autoradiographic and biochemical techniques using

exogenous and endogenous ligands of the ( /PCP receptor.

MATERIALS AND METHODS

1. Comparative Quantitative Autoradiographic Localizations:

Adult male Sprague-Dawley rats were decapitated and brains were

rapidly removed and frozen on powdered dry ice, 6 μ sections were

cut at various levels of the brain. Alternate sections were radiolabeled for PCP and NMDA receptors.

a) [3H]TCP Binding to PCP Receptors:

Brain sections were preincubated in 5 mM Tris-HCl at 37°C for 30

min, then incubated with 5nM [3H]TCP (52.9 Ci/mmol, NEN, MA) in 5 mM

Tris-HCl ph 7.4 at 4°C for one hr (Sircar and Zukin 1985). Adjacent

sections were incubated under the same conditions but in the presence of 10 μm non-radioactive PCP. At the end of incubation,

157
slides were washed sequentially through 6 rinses (6 sec each) of 5

mM Tris-HCl, pH 7.4 At 4°C. Slides were rapidly dried under a

stream of cold air. They were tightly juxtaposed against tritium

sensitive film (Ultrofilm, LKB Instruments, Gaithersburg, MD) at

room temperature for 4 weeks. 10 μ thick sections were cut from a

tritium standard (

3

H-microscale block, Amersham, IL). These sections were dry-fixed to slides and at least one such 3

H-microscale

containing slide was placed against each film.

b) [3H]L-Glutamate Binding to NMDA Receptors:

Slides containing brain sections were preincubated at 30°C for 10

min to remove endogenous

30 min at 4°C in 100 nM [3H]L-glutamate

lutamate. The sections were incubated for

(47.7 Ci/mmol, NEN, MA) in

50 mM Tris-acetate ph 7.2 at 4°C (Monaghan and Cotman 1985). Alternate sections were incubated under the same conditions but in the

presence of 100 um NMDA. Following incubation the sections were

washed four times in ice-cold 50 mM Tris-acetate buffer for a total

time of 30 sec. Sections were dried and tightly juxtaposed against

tritium-sensitive film for 1-3 weeks at 4°C. Each film also carried

3

H-microscale-containing slides.

c) Development and Quantification of Films:

Following exposure, films were developed in Kodak D19 for 5 min at

20°C, rinsed in water for 20 sec and fixed for 10 min. Autoradiograms were analyzed by an image processor (Quantimet 920, Cambridge Instruments, UK) which produced an electronic image of the

autoradiographic section with point-to-point digitization to give

optimal resolution and gray-scale separation. The system was then

calibrated to convert gray levels into optical density values by

using standards of known optical density. Color-coded images of the

brain section for receptor density (fmol/mg tissue) were generated.

FIGURE 1: Effect of Hippocampal HPLC Fractions 23-38 (1) and 56-

66 (2) on NMDA-stimulated [3H]DA and [14C] ACh Release

2. Extraction of Endogenous PCP-like Ligand:

Six hundred bovine hippocampi were freshly dissected from fresh

158
whole brains, immediately frozen on dry ice and lyophilized. Dried

fragments (1 fragment = 1 hippocampus) were acetone-defatted and

hot-acid extracted using the method of Vale et al. (1983). Extracts

were further purified with 300 g Vydac reverse phase C18 sorbent.

Prior to assay, aliquots of starting material were again purified by

adsorption to hydrophobic derivatized gel using prepacked 6-ml bond

elute columns containing 500 mg C18 sorbent. The crude starting

material showed dose-dependent inhibition of [

3

H]TCP binding at

doses up to 100 millifragments (mf) per assay tube. This material

was further purified by HPLC on a Vydac C18 preparative cartridge (5

X 30 cm) using a TFA/CH3CN solvent system consisting of buffer A:

0.1% TFA and buffer B: 40% buffer A and 60% CH3CN at a flow rate of

75 ml/min (Zukin et al. submitted for publication).

3. Effect of Endogenous Ligand on NMDA-Induced ACh and NE Release:

Male Sprague Dawley rats were decapitated and the corpora striata

were removed, sliced and preincubated in a modified Tyrode's buffer

adjusted to pH 7.4 for 10 min at 4°C. [

3

H]choline hydrochloride or

[

3

H]dopamine was added to the buffer at final concentrations of 50

and 100 mM, respectively. Slices were then su erfused with the

buffer solution containing 10 μM hemicholinium ([3H]choline release)

or 10 μM pargyline ([3H]DA release). The superfusate was then

collected and radioactivity was determined. NMDA-stimulated tritium

release was calculated by the method of Snell and Johnson (1985).

Assays were carried out in the absence of magnesium ions, which

inhibit NMDA-induced neurotransmitter release.

RESULTS AND DISCUSSION

TABLE 1: Regional Distributions:A Similarities

REGION RECEPTOR DENSITY RECEPTOR DENSITY

[

3

H]TCP* [

3

H]L-glutamate**

CA1

98.4% 100.0%

CA2

100.0% 97.0%

Dentate gyrus 87.7% 93.3%

Medial amygdala 71.9% 55.9%

CA3 54.3% 62.0%

VMH 53.3% 34.8%

Post. cingulate cortex 38.2% 37.6%

Entorhinal cortex 36.4% 48.0%

Median eminence 34.2% 44.9%

Arcuate nucleus 33.9% 36.3%

Subiculum 28.7% 24.8%

Substantia nigra 25.4% 13.1%

Superior colliculus 17.9% 29.4%

Cerebellum 8.1% 13.7%

Ventral tegmental area 6.6% 20.9%

Pontine ret. Formation 5.1% 13.1%

Globus pallidus 1.3% 10.6%

* data expressed relative to CA2 (receptor density = 100%)

** data expressed relative to CA1 (receptor density = 100%)

We have demonstrated neuroanatomical and functional relationships

between /PCP and NMDA receptors by autoradiographic, biochemical

and bioassay techniques. Regional distribution of NMDA and PCP

159
receptors show striking similarities in certain brain areas (table

1) in agreement with Maragos et al. (1986). Hippocampus had the

highest level of both types of receptors with hippocampal fields

CA1, CA2, and dentate gyrus having higher levels than CA3. Other

regions that showed similar distributions included medial amygdala,

VMH, various cortical areas, substantia nigra, cerebellum, ventral

tegmental area, arcuate nucleus, median eminence, subiculum, pontine

reticular formation and globus pallidus. NMDA and PCP receptor

distributions were dissimilar in certain regions, most notably central gray, anterior cingulate cortex, caudate-putamen, nucleus accumbens, dorsal thalamus, septal area and preoptic nucleus (table

2). The endogenous ligand of /PCP receptors which we have identified mimics the effects of PCP-like drugs on the NMDA system. Partially purified bovine hippocampal extract was found to have PCPlike inhibitory effects on both NMDA-stimulated DA and ACh release

(figure 1).

TABLE 2: Regional Distributions: Dissimilarities

REGION RECEPTOR DENSITY RECEPTOR DENSITY

[

3

H]TCP* [

3

H]L-glutamate**

Dorsomedial hypothalamus 68.9% 44.5%

Central gray 30.5% 11.0%

Ant. cingulate cortex 26.9% 88.1%

Auditory cortex 22.0% 53.1%

Interpeduncular nucleus 21.9% 4.2%

Caudate-putamen 12.4% 60.7%

Preoptic 11.0% 33.5%

Laterodorsal thalamus 6.4% 81.5%

Mediodorsal thalamus 6.3% 36.4%

Nucleus accumbens 5.3% 83.8%

Lateral septum 3.9% 87.4%

Medial septum 1.7% 22.9%

* data expressed relative to CA2 (receptor density = 100%)

** data expressed relative to CA1 (receptor density = 100%)

REFERENCES

Berry, S.C., Anis, N.A., and Lodge, D. Brain Res, 307: 85-90,

1984.

Maragos, W.F., Chu, D.C.M., Greenamyre, J.T., Penney, J.B., Young,

A.B. Europ J Pharmacol, 123: 173-174, 1986.

Monaghan, D.T., and Cotman, C.W. J Neurosci, 5: 2909-2919, 1985.

Sircar, R., and Zukin, S.R. Brain Res, 344:142-145, 1985.

Snell, L.D., and Johnson, K.M. J Pharmac Exp Ther, 235: 50-57,

1985.

Vale, W., Vaughan, J., Yamamoto, G., Bruhn, T., Douglas, C., Dalton,

D., Rivier, C., and Rivier, J Methods in Enzymology, 103: 565-577,

1983.

ACKNOWLEDGMENTS

Supported in part by PHS (NIDA) grants DA03383 to SRZ, DA01843 and

DA00069 to RSZ and DA02073 to KMJ and by the Department of Psychiatry, Albert Einstein College of Medicine/Montefiore Medical Center, Herman M. van Praag, M.D., Ph.D., Chairman.

160
AN ENDOGENOUS LIGAND FOR PHENCYCLIDINE

RECEPTORS: ISOLATED FROM HUMAN BRAINS

Wen-Xiao Lu, Ya-Fang Liu, *Yun-Xiang Zhu,

*Guo-Liang Xi and An-Zhong Zhang

Shanghai Medical University, *Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai, 200032, China

ABSTRACT

Using preparative HPLC, Sephadex G-25 and analytic HPLC, we

obtained an active peak inhibiting (

3

H) PCP receptor binding, with

a molecular weight of 3,000 daltons. Another isolation process

included acetic acid extraction, Toyopearl HW gel filtration,

Sephadex G-10 chromatography and analytic HPLC. An active peak was

also observed, which was consistent with that from the first

isolation process.

INTRODUCTION

Phencyclidine (1-(1-phenylcyclohexyl) piperidine, PCP) is a psychotomimetic drug. The existence of specific, high-affinity, stereoselective PCP binding site in mammalian brains has been reported by

various groups since 1979 (Zukin and Zukin 1979, Vincent et al. 1979).

Elucidation of endogenous ligand for PCP receptor may bring important

imformation to the studies on mental activities and related diseases.

Quirion et al. (1984) reported a fraction of PCP-like peptide isolated

from the porcine brains. But the sequence has not been published yet.

In our laboratory, an active peak inhibiting (

3

H) PCP receptor

binding was observed from human brains (Lu et al. 1986). I- this

paper, we report further isolation and purification of this fraction.

METHODS

First isolation process: Human brains (willed contribution) were

processed into acetone powder, extracted by acids and chromatographed

on a preparative HPLC (Lu et al. 1986). The fractions containing

activity were lyophilized and chromatographed by Sephadex G-25 (total

volume 55 ml, superfine), and eluted with 0.5 M acetic acid. Then the

active fraction from Sephadex G-25 was further purified by analytic

HPLC with μ Bondapak Cl8 column, and eluted with a gradient of

acetonitrite.

Second isolation process: Sample was extracted by 0.5 M acetic acid

from acetone powder, and chromatographed on Toyopearl HW column

(total volume 400 ml) at a flow rate of 40 ml/hr. The active fraction

was processed through Sephadex G-10 for desalination and analytic HPLC

for further purification.

(

3

H) PCP receptor binding assay: Same as which described in our

privious paper (Lu et al. 1986).

RESULTS

The 10th min fraction eluted by 30% acetonitrile in preparative HPLC

161
Fig. 1. Active peak (B) eluted

from Sephadex G-25 column and

peaks monitored by U.V. 254 nm

(A); a further purification for

active fraction obtained from

preparative HPLC. The column

was eluted by 0.5 M acetic acid,

and collected by O.D. peaks and

screened with (3H) PCP binding

assay.

Fig. 2. Molecular weight of endogenous PCP-like substance. MW of

fraction 3 from Sephadex G-25

(in fig.1) was determined on

Sephadex G-25 column, checked

with standards: insulin (MW

5,200), insulin B-chain (MW

3,200) and LH-RH (MW 1,000).

was chromatographed on a Sephadex G-25 column. An active peak

(fraction 3) was overlapped with the main peptide peak monitored

under U.V. 254 nm (fig.1). The molecular weight of the active

substance is around 3,000 daltons (fig.2). The fraction 3 underwent through analytic HPLC; the active peak was located at around

30% acetonitrile elutions and the recovery was about 1 %, so

another isolation process was used. As shown in fig. 3, desalted

fraction G from Toyopearl HW gel filtration potently inhibited (

3H)

PCP receptor binding, to non-specific binding level. The fraction

G was not degraded by pronase (Sigma) (fig. 4). After the fraction

G was chromatographed with analytic HPLC, the activity was

decreased about 99 %.

162
Fig. 3. Chromatogram (A) and peaksdetecting (3

H) PCP receptor

activity (B), sodium (C) and potassium (D) content of fractions

from Toyopearl HW gel filtration. Sample was extracted by 0.5

M acetic acid from acetone powder, and eluted by the same acid.

Fractions were collected according to peak of O.D. under 254 nm

ultraviolet spectrum. The fraction G and H were processed

through Sephadex G-10 for desalination before (3H) PCP receptor

binding assay.

DISCUSSION

Many authors reported that known neurotransmitters and neuropeptides

could not inhibit (

3

H) PCP receptor binding (Quirion et al. 1984).

In our experiments, the endogenous PCP-like substance was obtained

with both isolatIon processes. Other factors, pH and salts, which

interfere with (

3

H) PCP binding, were ruled out in our experiments.

So we consider that the active peak obtained in present studies

represents the endogenous ligand for PCP receptors.

Quirion et al. (1984) reported that the PCP-like substance was a

peptide. Analytic HPLC with reverse phase C18 column was usually

used for purification of peptides, but in our experiment, the

recovery of both preparative HPLC and analytic HPLC were as low as

1 %. Now we are trying new methods for purification of PCP-like

substances.

163
Fig. 4. Effect of pronase on fraction G from Toyopearl HW gel

filtration and Sephadex G-10. In control (below), sample of

fraction G was incubated with unactive pronase, which pretreated

in boiling water for 10 min, in Tris-HCl buffer (pH 7.5) at

37°C for 30 min, then isolated by Sephadex G-25. Only one peak

was observed, with receptor binding activity and a MW of around

3,000 daltons. But in incubation with enzyme (upper), the 3,000

daltons peak with (

3

H) PCP binding activity was remained intact.

Beside, there was a smaller peak (without binding activity,

MW around 1,000) behind the active peak.

REFERENCES

7615372-5376, 1979.

Lu, W.X., Lai, Q.H., Lu, R.L., Wen, Y.X., Li, Y.N., Rao, Y., Zhou, G.Z.

and Zhang, A.Z. Chinese J Physiol Sci, 2:98-100, 1986.

Quirion, R., Dimaggio, French, E.D., Contreras, P.C., Shiloach, J.,

Pert, C.B., Everist, H., Pert, A. and O'Donohue, T.L. Peptides,

5:967-973,1984.

Vincent, J.P., Kartalovski, B., Geneste, P., Kamenka, J.M. and

Lazdunski, M. Proc Natl Acad Sci USA, 76:4678-4682, 1979.

Zukin, S.R. and Zukin, R.S. Proc Natl Acad Sci USA,

ACKNOWLEDGEMENTS

The authors thank Dr. DK Huang, Mr. WM Xu and SD Lu for brains

collection, and Ms. GZ Du for her excellent technician work.

This work is sponsored by the Chinese Academy of Sciences and

W.H.O.

164
AN ENDOGENOUS LIGAND FROM HUMAN CSF FOR

THE PHENCYCLIDINE RECEPTOR

Guangzhao Zhou, Dawei Gong, Chengchuan Jiang*, Jinli Yao*, Hui Gao

Yunxiang Zhu**, Baisen Pan*** and Anzhong Zhang

Depts. of Neurobiology, Neurology* and Clinic

Biochemistry***, Shanghai Medical Univ., Shanghai

**Shanghai Inst. of Materia Medica Academia Sinica

ABSTRACT

anceran just before the salts in Sephadex G-25 column and was

luted with low concentrations of acetonitrile in reverse phase HPLC

predicating that it may be a small hydrophilic compound with a MW.

of less than 1,000 daltons.

A fraction which potently and specifically competed-with 3H-PCP

binding to rat membranes was isolatedfrom human CSF. This substINTRODUCTION

Phencyclidine(PCP) has received increasing attention recently not

only because of its abuse hazards, but also its unique CNS effects.

PCP-induced psychosis resembles schizophrenia. Various laboratories

described the specific binding sites of PCP in rat and human brains

with characteristic regional distribution (Vincent et al.1979;Zukin

and Zukin 1979;Sircar and Zukin 1983). Several papers suggested that

PCP receptors were well related to its psychotomimetic effects and

that endogenous ligands for the receptors might be present in CNS

(Quirion et al. 1984). Therefore, a search for this kind of ligand

will cast some light on the pathogenesis of schizophrenia. Quirion

et al.(1984) reported a peptide ligand in porcine brain. In our

laboratory, with preparative HPLC, a fraction which inhibited 3HPC? binding to rat membrane was found in human brain (Lu et al.

1986). Here, we report a fraction isolated from human CSF with

potent 3-PCP binding activity.

MATERIALS AND METHODS

(+)-3H-PCP (48 Ci/mmol; 1 Ci=37 G Bq, New England Nuclear). PCP

was synthesized by Dept. of Pharmaceutical Chemistry of this Univ.

CSF was collected via lumbar puncture at 8-9 A.M. from patients suffering from peripheral neuritis or myelopathy without any psychotropic medication. It was frozen immediately after the collection,

then lyophilized (5 ml in each tube) for use.

The lyophilizsd samples were resuspended in 0.5 M acetic acid and

chromatographed on column of Sephadex G-25 fine (1.2x60 cm, 9 ml/

hr) eiuted with 0.5 M acetic acid using LKB automatic collector,

Aliquots were collected, lyophilized for H-PCP receptor binding

assay. The fractions containing 3H-PCP binding activity were pooled

and chromatographed in 0.1% heptafluorobutyric acid, They were chromatographed on 3.9x30 cm u Bondapak C18 column at a flow rate of

0.6 ml/min. eluted with gradient 0-40% acetonitrile and were moni165
tored under 230 nm ultraviolet wave spectrum. The samples from analytical HPLC were further screened by 3

H-PCP binding assay.

Rat brain membranes were prepared according to Quirion et al.(1984)

with a few modifications. For 3H-PCP receptor binding assay, duplicate assay tubes contained 8 nM 3

H-PCP, membrane preparation of 10 mg

tissue and chromatographic fractions (their pH was adjusted to 7.6)

or standards(PCP) all in 5 mM Tris-Hcl/5O mM sucrose pH 7.6 at 4°C

in a final volume of 0.5 ml. Incubation took 45 min.at 4°C, then the

samples were filtered under reduced pressure through GF/C fiters(Whatman) which presoaked for at least two hrs. at 4°C in 0.05% polyethlenimine, then rapidly washed with 2x5 ml aliquots of ice cold 5.0 mM

Tris-Hcl buffer. Each filter was dried by infrared lamp, then placed

in vials containing 5 ml of liquid (TP 0.3%, POPOP

and counted by liquid scintillation spectrometry.

0.03% in toluene)

3

H-PCP specific

binding to synaptic membranes was defined as the difference in radioactivity bound in the-absence and presence of 20 uM PCP. In a typical

experiment, total binding was 2,400 cpm and non-specific binding was

800 cpm.

RESULTS

1. Human CSF went through Sephadex G-25 fine column

Human CSF was applied to a Sephadex G-25 fine column at a flow rate

of 9 ml/hr and monitored under UV 254 nm. Aliquots of the 3 ml eluate in each tube were collected and lyophilized. Each fraction was

assayed with 3H-PCP receptor binding and salt concentrations measured

by Beckmenn system E-4A electrolites analyze.(See fig.1)

Fig.1 Human CSF went through Sephadex

G-25 fine column

2. Active peak and salt peaks

As fig.2 shows, human CSF contains an active fraction No.17-19 and

salts peaks No.20-21. The pH of all fractions for binding assay

were 7.5. Some were adjusted by 5 mM Tris solution(pH 8.5 at room

temperature). The experiment was repeated three times. Since the

salt will interfere the 3H-PCP binding, the salt containing fractions

(20,21) were discarded. The fractions No.17-19 were combined and

lyophilized for analytical HPLC.

166
3. Analytical HPLC elution pattern of fraction No.17-19

Fig.2. Active peaks and salt peaks screened by

3

H-PCP binding assay and salt analyzer respectively

active peaks

sodium peak

potassium peak

Fig.3 Analytical HPLC elution pattern of fraction

No.17-19 from Sephadex G-25 chromatography

Fig.3 showed the peaks within 30 min. elution after injection of

fraction No.l7-19(see Fig.2). Elutes were collected by eaks monitored with UV 230 nm and lyophilized, then screened by 3

H-PCP binding

assay. The bared peaks possessed activity for inhibiting 3H-PCP

binding.

DISCUSSION

According to work of many laboratories, including our past work, 3HPCP binding assay was specific, and none of the known neurotransmitters

and neuropeptides could displace its binding in reasonable concentrations(Quirion et al.1984;Lu et al.1986). In this experiment, we excluded the factors which might interfere with binding, such as pH

167
and salts. Therefore, we consider the endogenous ligand in human CSF

is conceivable. It may be released directly from brain or as active

metabolite of some authentic forms of PCP-like substance. Unlike the

neuropeptide reported by Quirion et al., our fraction may be small

hydrophilic compound, since it ran just before the salts in Sephadex

G-25 column and was rapidly eluted with low concentrations of acetonitrile in reverse phase HPLC. Its molecular weight may be less than

1,000 daltons. The biochemical properties of the ligand are to be

determined.

REFERENCE

Lu, W.X., Lai, Q.H., Wen, Y.X., Li, Y.N., Rao, Y., Zhou, G.Z. and

Zhang, A.Z. Chinese J Physiol Sci, 1(1):98-100, 1986.

Quirion, R., DiMaggio, D.A., French, E.D., Contreras, P.C., Shiloach,

J., Pert, A., and O'Donohue, T.L. Peptides, 5:967-973, 1984.

Sircar, R., and Zukin S.R. Life Sci, 33(Supp.l):259-262, 1983.

Vincent, J.P., Karialovski, B., Genesete, J.M., Kamenka, J.K., and

Lazdunski, M. Proc Natl Acad Sci USA, 76:4672-4682, 1979.

Zukin, S.R., and Zukin, R.S. Proc Natl Acad Sci USA, 67:5372-5376,

1979.

ACKNOWLEDGEMENTS

The grant was from the Chinese Academy of Science

168
DEXTRORPHAN: AN ANTAGQNIST FOR PHENCYCLIDINE RECEPTOR

Feng-Yan Sun, Han Zhu, Ling-Mei Zhang, Xiu-Rong Xu*

and An-Zhong Zhang

Dept. of neurobiology, Shanghai Medical University.

*Shanghai Institute of Meteria Medica, Shanghai,

200032 China

ABSTRACT

Radio-binding assay, bioassay and HPLC detection were used to observe

the antagonistic effects of dextrorphan on PCP's actions. Dextrorphan

displayed high affinity to PCP receptors and it had weak PCP-like

bioactivity, but could antagonize PCP's action dose-dependetly in

vitro and shift the dose-response curve to the right. PCP increased

the contents of norepinephrine in bath medium, which was reversed by

dextrorphan. Thus, the results suggest that dextrorphan is a partial

antagonist for PCP receptors.

INTRODUCTION

Using radio-binding assay, we have demonstrated phencycliding(PCP)

receptors in the blood vessel and in vitro bioassay showed that PCP

markedly enhanced the electrical field stimulated construction of

rabbit central ear artery(ESC)(Zhu et al.1986). Dextrorphan, a d-isomer of levorphanol, could displace PCP binding to the membrane preparation of blood vessels and it could decrease the excitatory effects

of PCP on ESC. In this paper, we used three different ways to demonstrate dextrorphan being a partial antagonist for PCP receptors.

METHODS AND MATERIALS

Animal: New Zealand rabbit weighting 2-2.5 Kg.

Materials: 3H-PCP(38Ci/mmol) and PCP were made by Drs. Gong JL and

Sun OF in the school of pharmacy of this university. Dextrorphan and

levorphanol were generous gifts of Dr. Avram Goldstein, Addition Research Foundation, U.S.A. Phentolamine was made by the pharmaceutic

factory of this university, and norepinephrine bitartratis, by Shanghai Tenth Pharmaceutic Factory.

Radio-binding assay: Rabbit mesenteric blood vessels were homogenized

in ice-cold 5mM Tris-HCl, pH7.4. The homogenate was centrifuged at

500xg for 10 min and at 16,500xg for 30 min(4°C). The pellet (2.4mg

ptotein) and 3H-PCP(5.2nM) were incubated with various ligands with a

final volume of 0.5ml at 4°C for 60 min and terminated by rapid filtration(GF/C or GF/B filters preincubated in 0.05& polyethylenimine).

Non-specific binding and specific binding

sence or absence of unlabelled PCP (20μM). Filters were counted by

were abserved in the preliquid scintillation spectrometry.

Bioassay: The central ear artery of rabbit was prepared in a length

of 4-5mm. The preparation was suspended under 0.5g of tension and

incubated in 4ml bath organ containing Krebs solution at 37°C through

a mixture of 95% O2 and 5% CO2 and equilibrated for 4-5hrs prior to

169
receiving electrical field stimulation(25v, 5-6Hz, trains of 5 pulses,

lms per pulse, 2.5 min of interval between trains). Dextrorphan and

levorphanol were given 10 min before administration of PCP. Doseresponse curves of PCP were constructed by increasing bath medium

concentration of PCP cumulatively.

High performance liquid chromatography(HPLC): The amine was separated

on a GYT-C18 column (20x0.5cm I.D., 1OμM partical size) and 0.15M

chloracetic acide-sodium hydroxide buffer with 0.83 nM EDTA, 9 nM DCampher sulfonic acide, pH 4.2 and methanol (92:8) at a flow rate of

1.5ml/min and detected by a LC-4B/17 electro-chemical detector.

RESULTS

Radio-binding assay showed that 3H-PCP could bind with high specificity to rabbit blood vessel membranes with Kd=31.9±3.0nM, Bmax=4.96±

0.29pmol/mg protein. Dextrorphan and levorphanol could displace 3HPCP binding to rabbit blood vessel with IC50=3.01±0.19 and 18.2±1.2

μM, respectively (fig.1).

In bioassay, the ESC was enhanced by PCP in a dose-dependent manner.

Dextrorphan reduced, even abolished the effect of PCP on ESC and

shifted its dose-response curve to the right (fig.2 and fig.3). Dextrorphanand levorphanol decreased the maximum effect of PCP, the pD2'

for them were 6.8463 and 5.5862, respectively.

The contents of norepinephrine in bath medium were 16.53±0.37ng/ml

after 20 min electrical field stimulation only. The contents were increased to 20.36±1.13 nq/ml after administration of 10-5M of PCP, and

decreased to 17.04±0.698 ng/ml after using 10-6M of dextrorphan (fig.

4).

displacing 50% of specific

bound. The experiment has

been replicated three times.

DISCUSSION

Using the binding assay, bioassay and HPLC detection, we have demonstrated that dextrorphan is a specific antagonist for PCP receptors.

Fig.1 Displacement curves

of ligands inhibiting 3HPCP binding to the homogenate of mensenteric blood

vessel.

The ordinate represents

persentage inhibition of

specific binding of 3H-PCP

(5.2nM) to the homogenate(

2.4mg protein per assay

tube). IC50s represent cone

centrations of ligands that

In radio-binding assay, we have found that specific, saturable and

stereoselective PCP binding sites in the blood vessels(Zhu et al.

1986). The binding sites could be displaced by PCS analogs and some

benzomorphan derivatives. Here, we observed that H-PCP binding to

170
the blood vessel could be displaced by dextrorphan dose-dependently

and sereospecifically, thus demonstrating that dextrorphanhad high

affinity to PCP receptor.

Fig.2 The effects of dextrorphan

on ESC and the reversal effects

of it on PCP's action.

The vasoconsitriction was induced

by electric-field stimulation. PCP

enhanced such vasoconstriction

and dextrorphan reversed the effect of PCP(left). Dextrorphan alone

has a slight excitatory effect on

the vasoconstriction(right).

Fig.3 Right-shited dose-response curves of PCP by dextrorphan

(left) and levorphanol(right).

The ordinates showed vasoconstrictive responses of ligands

compared with the maximum effect of PCP. Dextrorphan or levorphanol was added in the medium 10 min before administration

of PCP. Dextrorphan(1O-8M) significantly shifted the dose-response curves to the right with a pD2'=6.8463, while levorphanol

showed an effect about 10 times less potent(pD2'=5.5864).

Bioassay showed that PCP could markedly and dose-dependently enhance

the ESC. The concentrations for half maximum effect of PCP(pD2) were

6.5928 to 6.6665. Dextrorphan showed very weak PCP-like bioactivity

(fig.2), but it could antagonize the enhancing ESC by PCP and shifte

the dose-response curves of PCP to the right. Levorphanol, a l-enantimer, also had such antagonizing effect, but with lower pontency(fig.

171
33). The bioactivity of d- and l-isomers in antagonizing PCP's action

on ESC was consistent with the affinity of those ligands to PCP receptor, which also was the same as that obtained from radio-binding

assay in the rat brain(Tam 1983). In both binding assay and bioassay,

dextrorphan was about 10 times more potent than that of levorphanol,

which is another evidence for dextrorphan being an antagonist for PCP

receptors.

Fig.4 The norepinephrine contents

in the medium after administration

of PCP or PCP and dextrorphan.

The bath medium was collected after

20 min of electric-field stimulated

without any drugs or with PCP(10-5M

or/and dextrorphan(10 M). The nore

pinephrine contents in the bath medium were measured by PHLC.

The effects of PCP on ESC could be abolished by phentolamine and the

norcpinephrine contents in bath medium detected by HPLC was markedly

increased after administration of PCP, suggesting that the vasoconstriction induced by PCP may be produced by increasing the release of

norepinephrine from the nerve terminals. On the contrary, dynorphin

could inhibit the ESC and its action may produced by inhibiting the

release of norepinephrine(Sun et al 1983 and Illes et al 1983). In

this paper, we showed that dextrorphan could antagonize the increment

of norepinephrine content in bath medium by PCP. This result have

further supplied the evidence that dextrorphan is a PCP antagonist.

Since dextrorphan per se showed weak PCP-like effect and it decreased

the maximum effect of PCP, we considered it a partial antagonist for

PCP receptor.

REFERENCE

Illes, P., Pfeiffer, N., and Limberger, N., Life Sci, 33(Sup I):307-

310, 1983.

Sun, F. Y., Yu, G. H., and Zhang, A, Z. Acta Pharmacologica Sinica,

4:100-102, 1983.

Tam, S. W. Proc Natl Acad Sci USA, 80:6703-6707, 1983.

Zhu, H., Zhang, A. Z., Zhang, L. M., Xu, X. R., and Te, W. L. Acad

Physilogica Sinica, 1986.

ACKNOWLEGEMENT

This work is sponsored by the Chinese Academy of Sciences.

172
HIGH AND LOW AFFINITY PSYCHOTOMIMETIC OPIOID BINDING SITES:

CHARACTERIZATION BY A NOVEL 3H-PCP-ANALOG

Yossef Itzhak

Dept. of Pharmacology, Hadassah School of Medicine, The Hebrew

University, Jerusalem, Israel

ABSTRACT

1-[1-(3-Hydroxyphenyl)cyclohexyl]piperidine (PCP-3-OH) is one of the

most potent analogs of phencyclidine (PCP). In the present study we

describe the binding properties of 3H-PCP-3-OH to guinea pig brain

membranes. Scatchard analysis of saturation binding studies revealed

the existence of high (0.44nM) and low (17nM) affinity binding

components. High affinity binding sites were completely blocked in

the presence of (+)SKF 10047 (50nM). In competition studies PCP

analogs compete for 3

H-PCP-3-OH specific binding in a monophasic

manner whereas psychotomimetic opioid ligands compete for this

binding in a biphasic manner. Results from both saturation and

competition experiments suggest the existence of a common high

affinity binding site for psychotomimetic opioid ligands and PCP

analogs and a low affinity binding component primarily for

phencyclidines.

INTRODUCTION

On the basis of behavioral experiments (Shanon 1981; Brady et al.

1982) and binding studies (Zukin and Zukin 1979; Vincent et al. 1979;

Quirion et al. 1981; Zukin and Zukin 1981; Itzhak et al. 1981a) it

has been suggested that PCP and psychotomimetic opioids, such as

SKF-10047, may share a common binding site in CNS. Sigma opioid

receptors, originally proposed for SKF 10047 and related benzomorphans (Martin et al. 1976), are now termed "sigma opioid/PCP"

receptors. The biochemical characterization of these binding sites is

somewhat hampered, compared to mu, delta and kappa opioid receptors,

primarily by the lack of highly potent and/or selective "sigma"

ligands. The high dissociation constant of 3H-PCP (ca. 200nM) (Zukin

and Zukin 1979; Vincent et al. 1979) may give rise to difficulties in

receptor binding studies involving such a ligand. However, recently

by utilizing 3H-(+)SKF 10047 we reported the existence of common high

affinity (Kd=20nM) dextrorotatory-opioid binding sites for psychotomimetic opioids and phencyclidines in rat CNS (Itzhak et al. 1985).

The aim of this study was to determine whether "sigma opioid/PCP"

binding sites represent a single or multiple binding components.

Utilizing PCP-3-OH, which produces strong psychotomimetic PCP-like

effects (Kalir et al. 1978) in its radiolabeled form (

3

H-PCP-3-OH),

we present evidence for the existence of multiple binding components

for psychotomimetic opioids and phencyclidines in guinea pig brain.

MATERIALS AND METHODS

3

H-PCP-3-OH (9.8 Ci/mmole) was purchased from Israel Nuclear Research

Center-Negev, Israel. The purity of the drug as determined by thin

layer chromatography and U.V. spectrophotometer was > 99%. PCP and

its derivatives: PCP-3-OH, 1-[1-(3-aminophenyl)cyclohexyl]piperidine

173
(PCP-3-NH2), 1-phenylcyclohexylamine (PCNH2), N-ethyl-phenylcyclohexylamine (PCNHEt), and 1-(1-phenylcyclohexyl)-4-piperidinol (PCP4'-OH), were synthesized as previously described (Kalir et al. 1978;

Itzhak et al. 1981b). The two stereoisomers of SKF 10047 were

generously supplied by Dr. R. Hawks National Institute on Drug Abuse.

Other drugs were purchased from commercial sources. Crude guinea pig

brain (minus cerebellum) membranes were prepared as previously

described (Simon et al. 1975). Binding assays were carried out in

HEPES-KOH buffer 1OmM (pH 7.7; 25°C) as previously described (Itzhak

et al. 1985). Specific binding of 3H-PCP-3-OH was assessed in the

absence and presence of 1μM unlabeled PCP-3-OH or PCP, which resulted

in similar levels of non specific binding.

RESULTS AND DISCUSSION

In typical experiments specific binding of 3H-PCP-3-OH (3nM) to

guinea pig brain membranes represented 80±3% of total binding. Radioactivity absorbed to GF/B filter blanks was usually less than 5% of

the total binding. Specific binding increased linearly within protein

concentration of 0.2-1.7mg/ml and was reduced in the presence of

trypsin (20μg/ml) or N-ethylmaleimide (0.5mM) by 66 and 54%, respectively, from control binding. Preincubation of membranes at 60°C/20

min resulted in 90±5% decrease in specific binding. These findings

are consistent with the requirements for drug-receptor interaction

and indicate the protein nature of the binding sites. Saturation

binding experiments of 3H-PCP-3-OH were carried out at concentrations

of 0.2-40nM and revealed two saturable binding components. Scatchard

analysis of the specific binding resulted in a biphasic plot (Fig.

1). Apparent Kd values, calculated as described by Munson and Rodbard

(1980), are 0.44nM and 17nM for high and low affinity binding

components, respectively.

The ability of various PCP-analogs and psychotomimetic opioid ligands

to compete for 3H-PCP-3-OH (3nM) specific binding was also studied.

The rank order of potency among PCP analogs was as follows: PCP-3-OH>

PCP=PCNHEt>PCP-3-NH2>PCNH2>PCP-4'-OH. The IC50 determined for PCP-3-

OH was 7nM, whereas the I? 50 for the least potent analog, PCP-4'-OH,

was 860nM. This rank order of potency is consistent with that found

in behavioral studies (Kalir et al. 19 8; Shanon 1981; Solomon et al.

1982) and competition studies for 3H-(+)SKF 10047 binding sites

(Itzhak et al. 1985). All PCP analogs competed for 3H-PCP-3-OH

specific binding in a monophasic manner yielding Hill coefficient

values near 1.

Psychotomimetic opioids, such as (+)SKF 10047, ketocyclazocine,

pentazocine, nalorphine and dextrallorphan competed for 3H-PCP-3-OH

specific binding in a biphasic manner (Fig. 2). About 30% of the

binding was readily inhibited by low concentrations (1-25nM) of these

opioids, whereas the residual binding of 3

H-PCP-3-OH was far less

sensitive (Fig. 2). The levorotatory isomer of SKF 10047 (Fig. 2),

levorphanol and levallorphan inhibited 3H-PCP-3-OH specific binding

only at high concentrations and in a monophasic manner, yielding IC

values of 350-800nM. However, morphine (u-agonist) naloxone, DADL

( -prefering ligand) and tifluadom (K-agonist) as well as non-opioid

ligands, such as haloperidol and atropine (each at 1nM-10μM) did not

affect the binding of 3H-PCP-3-OH.

174
FIGURE 1. Scatchard plot of 3

H-PCP-3-OH specific binding to

guinea pig brain membranes. Binding of tritiated ligand

(0.2-40nM) was carried out at 25°C for 40 min. Specific binding

was assessed in the absence and presence of PCP-3-OH (1μM).

Points represent a typical experiment which was repeated three

times with similar results.

FIGURE 2. Inhibition of 3

H-PCP-3-OH (3nM) specific binding to

guinea pig brain membranes by unlabeled PCP-3-OH and selected

opioids. Assays were carried out at 25°C for 40 min. Control

specific binding was 1630±80 cpm. Results represent the mean of

three separate determinations (SEM < 10%).

NANM = N-allylnormetazocine (SKF 10047)

175
The ability of (+)SKF 10047 and the racemic mixture of other psychotomimetic opioids, at rather low concentrations, to inhibit a portion

of 3H-PCP-3-OH specific binding,

levorphanol

and the inability of (-)SKF 10047,

and levallorphan to inhibit this binding, at low

3

H-PCP-3-OH displays dextrorotatory stereospecificity. Moreover,

concentrations, suggests that the high affinity binding component of

saturation binding studies of 3

H-PCP-3-OH in the presence of (+)SKF

10047 (50nM) yielded a linear Scatchard plot (Kd=21nM). These results

indicate that high affinity binding sites for 3H-PCP-3-OH were

completely blocked in the presence of (+)SKF 10047, whereas the low

affinity binding component was practically unaffected.

We have previously demonstrated that 3H-(+)SKF 10047 binds to a

single high affinity binding component and both psychotomimetic

opioids and PCP analogs competed for 3H-(+)SKF 10047 binding sites in

a monophasic manner (Itzhak et al. 1985). These findings together

with the present results strongly support the view that the high

affinity binding component, labeled by 3H-PCP-3-DH, corresponds to a

common high affinity binding site for both phencyclidines and psychotomimetic opioids. The low affinity binding component may correspond

to subpopulation of the sigma binding sites, which interact primarily

with PCP analogs. Thus, it is proposed that "sigma-opioid/PCP

receptor" may represent two distinct subtypes of binding sites.

REFERENCES

Brady, K.T., Balster, R.L., and May, E.L. Science, 215: 178-180,

1982.

Itzhak, Y., Kalir, A., and Sarne, Y. Eu J Pharmacol, 73: 229-233,

1981a.

Itzhak, Y., Kalir, A., Weissman, B.A., and Cohen, S. J Med Chem, 24:

496-499, 1981b.

Itzhak, Y., Hiller, J.M., and Simon, E.J. Mol Pharmacol, 27: 46-52,

1985.

Kalir, A., Maayani, S., Rehavi, M., Elkavetz, R., Pri-Bar, I.,

Buchman, O., and Sokolovsky, M. Eur J Med Chem, 13: 17-24, 1978.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., and

Gilbert, P.E. J Pharmacol Exp Ther, 197: 517-532, 1976.

Munson, P.J. and Rodbard, D. Anal Biochem, 107: 220-239, 1980.

Quirion, R., Hammer, R., Herkenham, M., and Pert, C.B. Proc Natl Acad

Sci USA, 78: 5881-5885, 1981.

Shanon, H.E. J Pharmacol Exp Ther, 216: 543-551, 1981.

Simon, E.J., Hiller, J.M., Groth, J., and Edelman, I. J Pharmacol Exp

Ther, 192: 531-537, 1975.

Solomon, R.E., Herling, S., and Woods, J.H. Eur J Pharmacol 82:

233-237, 1982.

Vincent, J.P., Kartalovski, B., Genest, P., Kamenka, J.M. and

Lazdunski, M. Proc Natl Acad Sci USA, 76: 4678-4682, 1979.

Zukin, R.S., and Zukin, S.R. Proc Natl Acad Sci USA, 76: 5372-5376,

1979.

Zukin, R.S. and Zukin, S.R. Mol Pharmacol 20: 246-254, 1981.

ACKNOWLEDGMENT

Dr. J.M. Hiller (NYU, Medical Center) assisted in the Computer

analysis of the binding data.

176
NEW RECEPTOR ANTAGONISTS

Craig w. Thornber , John S. Shaw+

, Lynne Miller+,

Christopher P. Hayward , John S. Morley , David Timms and

Anthony Wilkinson

Departments of Chemistry and Bioscience+,

ICI Pharmaceuticals Division,

Mereside, Alderley Park, Cheshire, UK

ABSTRACT

A new selective opiate antagonist has been synthesised in which the

two glycine residues of diallyl leucine enkephalin have been replaced

by 4-aminobenzoic acid. The compound has a different conformation to

that of ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu).

INTRODUCTION

Diallyl enkephalin analogues have been reported by Shaw et al. (1982)

and by Cotton et al. (1984) with selective antagonist activity at the

-opioid receptor in which the amidic link between Gly3 and Phe4 has

been replaced by a methylenethio link (ICI 154.129, structure 1 in

the results table) or in which the two glycine residues have been

replaced by two aminoisobutyric acid residues (ICI 174,864, structure

2 in the results table). We now report the synthesis of the series

of diallyl enkephalin analogues in which the two glycine residues

have been replaced by a single 4-aminobenzoic acid residue or one of

its close relatives. The objectives of the study were to examine the

structure activity relationships of the compounds as opiate

antagonists and to compare the conformation of the most potent and

selective agents with that of the selective -receptor antagonist,

ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu).

MATERIALS AND METHODS

Synthetic methods

The novel compounds indicated in the results table were prepared by

standard chemical methods. The route to the 4-aminobenzoic acid

analogue, 4, is indicated in figure 1. The 4-methylaminobenzoic acid

analogue, 6, was prepared by N-methylation (NaH/DMF/MeI) of the amide

formed by the mixed anhydride coupling of 4-aminobenzoic acid ethyl

ester with N,N-diallyl-Tyr(But)-OH. The 3-aminobenzoic acid

derivative, 7, the 4-aminophenylacetic acid derivative, 8, and the 4-

carboxybenzylamine derivative, 9. were made by initial chain

extension at the carboxy-terminus (H-Phe-Leu-OBut, mixed anhydride)

of the starting benzyloxycarbonyl protected amino-acid and subsequent

N-acylation with N,N-diallyl-Tyr(But)-OH. For compound 10, the

coupling of 4-benzyloxycarbonylaminobenzoic acid with N—

methylphenylalanylleucine methyl ester was accomplished with

dicyclohexylcarbodiimide and hydroxybenztriazole. The 4-amino-2-

methylbenzoic acid analogue was prepared starting from 2-methyl-4-

nitrobenzoic acid which was used to acylate the dipeptide ester,

H-Phe-Leu-OBut, by mixed anhydride coupling. Catalylic hydrogenation

(Pd/H2) then reduced the nitro-group to an amino-group for subsequent

177
acylation with N,N-diallyl-Tyr(But

)-OH. Mixed anhydride (M/A)

couplings were carried out using ethyl chloroformate and

triethylamine and t-butyl ethers and esters were cleaved by 90%

trifluoroacetic acid.

FIGURE 1. Synthetic route

Biological methods

The compounds were tested as opioid antagonists on the field

stimulated mouse vas deferens against the selective -agonist, [Leu5]

enkephalin and the μ-agonist, normorphine, as previously described

by Miller et al. (1986)

RESULTS

The results in the table indicate that the 4-aminobenzoic acid

derivative, 4, is the most potent -receptor antagonist with a

selectivity ratio of about 100 between the - and μ-receptors. The

presence of methyl substituents on the aromatic ring (11) on the

amino-group (6) or on the nitrogen of the adjacent phenylalanine (10)

all led to loss of potency at the -receptor. Similarly the

inclusion of an extra methylene group as in compounds 8 and 2 caused

loss of activity. These observations were of value in studying the

conformation of the most potent -receptor antagonist, the 4-

aminobenzoic acid derivative, 4, (ICI,167,853).

178
TABLE 1. Assay for opiate antagonist activity on field stimulated

mouse vas deferens

Conformational Studies

A major source of flexibility within the diallyl-[Leu5]-enkephalin

molecule is the central Gly2-Gly3 dipeptide unit which can adopt more

than 100 conformations. Energy calculations using ECEPP (Momany et

al. 1975) identified 18 distinct, accessible conformers. Following

earlier studies with L- and D-alanine at the 2 and 3 positions, where

it was shown that only the D-Ala3

analogue was as potent as the

parent compound, two conformers were selected for further study where

the conformation of residue 2 was the same (C7 ax: phi = 80, psi = -

80). The other residue of conformer 1 was relatively extended (phi

= 65, psi = -175), whereas in conformer 2 the right-handed alpha

helical conformation (AR: phi = -54, psi = -60) was postulated.

Substitution of a-aminoisobutyric acid (Aib) for glycine at the 2

and 3 positions led to a 6 fold increase in -antagonist potency.

Further energy calculations on N,N-diallyl-Tyr-Aib-Aib-Phe-Leu

suggested that the conformation adopted by the central dipeptide was

helical but could be either right- or left-handed. Indeed the righthanded form could be related to conformer 2, in that the tyramine and

phenylalanine entities could be superposed with an RMS deviation of

about 1.5 , however the relative orientations of the leucine side

chains were different.

179
In the current work, aminobenzoic acids and homologues were

incorporated as replacements for the two glycine residues. These

"dipeptide mimics" are conformationally restricted ie. the

conformation of the p-aminobenzoic acid (PABA) analogue can be

defined in terms of two torsions corresponding to rotation about the

N-C bond (phi2) and the C-CO bond (psi3). The energetically

accessible values for these torsions are:

phi2 +45,-45,+135,-135

psi3 +50,-50,+130,-130

There are 16 possible combinations of these angles, however, because

of the symmetry and planarity of the phenyl ring, there are only 4

distinct conformations. In addition to the considerable reduction in

flexibility when compared with the Gly2-Gly3 unit (>100

conformations), the PABA moiety cannot mimic the spatial

characteristics of a helical dipeptide such as that suggested for the

Aib2-Aib3 analogue. It was thus intriguing to discover that the

PABA derivative was equipotent with diallyl-[Leu5]-enkephalin, whilst

the meta counterpart (MABA) was an order of magnitude poorer.

CONCLUSIONS

Although energy calculations have suggested a low energy conformation

of the PABA analogue which can be related to the originally

identified conformer 2. the spatial relationships between the PABA

and Aib2-Aib3 analogues are, at best, unclear.

It would appear that the two types of conformational restriction have

both generated potent delta-receptor antagonists, but the restricted

conformations adopted differ in the two cases. This conclusion is

unusual if not unique and was certainly unexpected.

REFERENCES

Cotton, R., Giles, M.B., Miller, L., Shaw, J.S. and Timms. D. Eur

J Pharmacol, 97: 331-332, 1984.

Miller, L., Shaw, J.S., and Whiting, E.M. Br J Pharmac, 87: 595-601,

1986.

Momany, P.A., McGuire, R.P., Burgess, A.N., and Sheraga, H.A. J Phys

Chem, 79: 2361-2381, 1975.

Shaw, J.S.. Miller, L., Turnbull, M.J., Gormley, J.J., and Morley,

J.S. Life Sciences, 31: 1259-1262, 1982.

180
DIMERIC OPIOID ANTAGONISTS

Craig W. Thornber , John S. Shaw+, Lynne Miller+ and

Christopher F. Hayward

Departments of Chemistry and Bioscience+,

ICI Pharmaceuticals Division,

Mereside, Alderley Park, Macclesfield, Cheshire, UK

ABSTRACT

Symmetrical and unsymmetrical dimeric pentapeptide opioid antagonists

have been prepared and studied on the mouse vas deferens preparation.

The findings do not support the hypothesis that such agents bind to

dimeric -opioid receptors.

INTRODUCTION

Dimeric opioid peptides have been reported which have enhanced

selectivity for the opioid b-receptor compared to their monomeric

analogues. Those dimers, prepared by linking two tetrapeptides as

amides of -alkylenediamines, have receptor binding affinities at

the -receptor similar to those of pentapeptide monomers but some

achieve high &selectivity through loss of affinity for the μreceptor. However, where two molecules of the pentapeptide Tyr-DAla-Gly-Phe-Leu. were linked as amides of ethylenediamine, a compound

was obtained with a ten-fold enhanced affinity for -receptors and

similar affinity for p-receptors as Tyr-D-AlaXly-Phe-Leu-NH2, as

measured by binding studies. (Shimohigashi et al.1982).

The hypothesis has been advanced that the -selectivity observed is

as a consequence of the dimeric ligands interacting with two, closely

associated, receptors. The objective of this study was to explore

the hypothesis that dimeric peptides have enhanced b-selectivity

because of interaction with a dimeric -receptor. Antagonists

rather than agonists were chosen for investigation so that Ke values

obtained from isolated tissue assays would be a reflection of

affinity rather than affinity and intrinsic activity. The key

experiment was the comparison of symmetrical and unsymmetrical

dimeric pentapeptides as antagonists of [Leu5] enkephalin and

normorphine in the electrically stimulate mouse vas deferens

preparation.

Theoretical Considerations

For the binding of a ligand to a receptor, the free energy of

interaction, G, is related to the affinity constant by the

expression :-

where AH is the change in enthalpy, AS the change in entropy and T

the absolute temperature. Thus, if it were possible to dimerise a

ligand and to allow it to associate with a dimeric receptor such that

the value of G were to be doubled, then the doubling of log K would

be reflected in a squaring of the value of K. Consequently, one

181
might see a change of affinity from say 10-7M to 10-14M.

Such enhancements in potency have not been observed for opioid

peptide dimers. On the dimeric receptor theory this could be

explained by entropic factors involved in the association of two

receptors into a dimer, by entropic factors associated with the

flexible linkage in the dimer between the two pharmacophores, or by

unfavourable interactions between the receptor and the polymethylene

chain linking the two monomeric pentapeptides.

MATERIALS AND METHOD

Synthetic methods

Peptide dimers were prepared in which the carboxyl termini of the two

peptide chains were held together as amides of ethylenediamine. The

synthesis used an orthoganol protection strategy in which one arm of

the dimer was elaborated using t-butyloxycarbonyl nitrogen protection

(deprotection with acid) and the other arm was elaborated using

benzyloxycarbonyl nitrogen protection (deprotection with catalytic

hydrogenation). Standard peptide methodology was used throughout the

synthesis.

Thus t-butyloxycarbonyl ethylenediamine, 1a, was elaborated first to

the unsymmetrically protected leucine dimer 1b, and thence to the

phenylalanylleucine dimer, 1c. Peptide chain extension was

accomplished either by the use of protected amino-acid active esters

(4-nitrophenol or 3, 4, 5 - trichlorophenol esters) or by the mixed

anhydride method employing ethyl chloroformate and triethylamine.

The intermediate 1c, was extended to give 2 by a fragment coupling

employing the t-butyl ether of N,N-diallyltyrosinylglycylglycine and

mixed anhydride activation. Similar extension on the second

phenylalanine gave the symmetrical dimer. 3, the unsymmetrical dimer,

4, and the truncated acetyl derivative, 5. The fragment couplings to

convert 2 to 3 and 4 were accomplished using dicyclohexylcarbodiimide

and hydroxybenztriazole activation.

Biological methods

The compounds were tested as opioid antagonists on the field

stimulated mouse vas deferens against the selective -agonist, [Leu5

]

enkephalin and the p-agonist. normorphine, as previously described by

Miller et al. (1986).

182
RESULTS AND DISCUSSION

FIGURE 1. Assay for opioid antagonist activity on the field

stimulated mouse vas deferens

The symmetrical dimer (3) is a more potent -antagonist than the

monomeric ethylamide but is only comparable in potency to and less

selective as a -antagonist than the monomeric pentapeptide ICI

174,864 (diallyl-Tyr-Aib-Aib-Phe-Leu). The unsymmetrical dimer (4)

is 80 times less potent than 3 but retains some -selectivity. The

most surprising finding is that the truncated dimer (5) is almost as

potent as the symmetrical dimer (3) and has a high -selectivity.

The increase in potency at the -receptor caused by dimerisation is

about nine-foid in this series and is comparable to that obtained in

other compounds with a single tyrosine moiety such as ICI 194,864.

The 80-fold loss of potency at the -receptor caused by the removal

of one hydroxyl moiety from 3 to give 4 is of interest. If the two

peptide chains do not interact with each other, then such a compound

would be expected to be two-fold less potent for a monomeric receptor

system. However, the 80-fold loss of potency going from compound 3

to 4 and the nine-fold difference between the monomeric and dimeric

amide antagonists are still small compared to the orders of magnitude

which might be expected. The high potency and selectivity of the

truncated dimer (5) suggests that the -receptor, but not the μreceptor, can tolerate lipophilic residues beyond the normal binding

site of the pentapeptide carboxy-terminus.

183
Thus part, if not all, of the potency and selectivity of dimeric

enkephalins can be explained without invoking a dimeric -receptor

system. The differences in potency between dimers and monomers and

between symmetrical and unsymmetrical dimers could be a consequence

of conformational factors whereby a dimer folds as a single large

molecule rather than as a linked pair of monomers each with the

tertiary structure it has in the monomeric state.

REFERENCES

Cotton, R., Giles, M.B., Miller, L., Shaw, J.S., and Timms, D.

Eur J Pharmacol, 97: 331-332, 1984.

Miller, L., Shaw, J.S., and Whiting, E.M. Br J Pharmac, 87: 595-

601, 1986.

Shimohigashi, S., Costa, T., Chen, H.-C., and Rodbard, D. Nature

297: 333, 1982 and Molec Pharmacol, 21, 558-563, 1982.

184
MELPHALAN-CONTAINING N,N-DIALKYLENREPHALIN

ANALOGS AS POTENTIAL IRREVERSIBLE ANTAGONISTS

OF THE OPIOID RECEPTOR

Jane A. Lovett1 and Philip S. Portoghese

Department of Medicinal Chemistry, College of Pharmacy

University of Minnesota, Minneapolis, MN 55455, U.S.A.

ABSTRACT

N,N-Dialkylated leucine enkephalin analogs containing melphalan in

place of Phe4 were synthesized as potentially irreversible antagonists of

the opioid receptor. These compounds, along with the corresponding

Phe4 pe tides, were tested for both agonist and antagonist activity in the

GPI and MVD smooth muscle preparations. All but one of the compounds showed antagonist activity at 10-6M against [D-Ala2

,D-Leu5]-

enkephalin in the MVD when tested under reversible conditions; in all

cases the Mel4 peptide had lower activity against DADLE than did the

corresponding Phe4 peptide. At high concentrations (10-5M) the active

Mel4

 analogs, (benzyl)2Tyr-Gly-Gly-Mel-Leu and (allyl)2Tyr-Aib-AibMel-Leu, both showed weak irreversible antagonism at the receptor.

INTRODUCTION

Selective irreversible opioid receptor antagonists can be valuable

pharmacological tools, as evidenced by the use in a variety of studies of

funaltrexamine -FNA) (Portoghese et al. 1980, Takemori and

Portoghese 1985), an irreversible antagonist of the μ receptor.

N,N-Diallyl leucine enkephalin analogs are selective reversible

antagonists of the opioid receptor (Shaw et al. 1982, Cotton et al. 1984)

which have been useful in studying the role of the receptor in various

physiological processes. In a combination of these two approaches,

several enkephalin analogs with the eneral structure

R2Tyr-Y-Y-X-Leu, where R = allyl or benzyl, X = Gly or Aib

aminoisobutync acid) and X = Mel (melphalan), the nitrogen

mustard-containing analog of phenylalanine, were synthesized and

tested as potential irreversible antagonists of the opioid receptor. Here

we present the results of the biological evaluations for this series on the

guinea pig ileum and the mouse vas deferens preparations.

MATERIALS AND METHODS

Synthesis

Melphalan-containing peptides were synthesized in solution by mixed

anhydride couplings of R2Tyr(OtBu)-X-X with Mel-LeuOtBu, followed

by deprotection with trifluoroacetic acid. Except for compound 2b (ICI

174,864), which was a gift from ICI Pharmaceuticals Division,

Macclesfield, Cheshire, England, the corresponding phenylalanine1

Current address: Department of Chemistry, California State

University Sacramento, Sacramento, CA 95819, U.S.A.

185
containing peptides were synthesized in a similar manner, or were

prepared by dialkylation of a protected pentapeptide followed by

deprotection. Details of these syntheses will be published elsewhere.

Biological Evaluation

Compounds were evaluated in the electrically stimulated longitudinal

muscle of the guinea pig ileum (GPI) (Rang 1965) and in the mouse vas

deferens (Henderson et al. 1972). The melphalan-containing

compounds were added to the tissues as a solution in methanol; in the

concentrations used methanol had no effect on the tissues. Agonist

activity is expressed as the percent inhibition of the twitch after a 10

minute incubation of the tissue with the test compound. The reversible

antagonist potency was determined against morphine, ethylketazocine

(EK), or [D-Ala2,D-Leu5]enkephalin (DADLE), and is expressed as the

ratio of the IC50 of the agonist in the presence of the test compound to the

IC50 in the absence of the test compound in the same preparation.

Irreversible antagonism was evaluated by incubating the MVD with the

antagonist for 10 minutes, washing the tissue 30 times, and then

determining the IC50 of DADLE; the activity is expressed as the ratio of

the IC of DADLE after exposure to the antagonist to that of the same

tissue before exposure.

RESULTS AND DISCUSSION

All but one of the compounds were reversible antagonists in the MVD

when tested against DADLE, a receptor agonist (table 1). Of the Phe4

peptides, all of which were active, 2b (ICI 174,864) was significantly

more potent than the corresponding dibenzyl analog 3b. The N,N-dibenzyl modification was chosen for further examination because for the

Gly-containing peptides the reverse was true; lb was 2.5-fold more

potent than N,N-diallyl leucine enkephalin (IC50 ratio = 2.7±0.6, n = 3).

Possible contributing factors to this reversal are conformational changes

in the peptide chain imposed by the Aib residues which alter the position

of the alkyl groups attached to the amine terminus and the greater

lipophilicity of peptides containing both the benzyl groups and the Aib

residues. The two dibenzyl peptides lb and 3b showed similar antagonist

activities against DADLE.

Replacement of phenylalanine by melphalan, the para-substituted

nitrogen mustard analog of phenylalanine, resulted in decreased affinity

for the receptor. The decrease in activity was only 2.2-fold in the case

of the dibenzyl leucine enkephalin analog la, but was larger for the Aibcontaining peptides. The 4.7-fold decrease in activity for 3a resulted in

an inactive compound at 10-6M. The decrease for 2a was approximately

3.1-fold, but the resulting compound was still more potent than la.

Most of the compounds showed little or no activity in the remaining

tests. Only one compound, 2a, had an IC50 ratio against morphine in the

MVD which was greater than one. The compounds did show some

agonism in the MVD at 10-6M, but the activity was weak (7.4% to 25.7%

inhibition of the twitch, see table 1). One compound, 3b, showed

significant agonist activity in the GPI (68.7±6.7% inhibition of the twitch

at 10-6M (n = 3), IC50 = 2.7 x 10-7±1.4 x 10-7M (n = 4)); the remaining

compounds at 10-6M showed some agonism in the GPI, but the activity

186
was much weaker (less than 10% inhibition of the twitch for compounds

la, 1b, and 3a, 11.4±3.3% inhibition (n = 2) for 2a, and 27.0±10.8%

inhibition (n = 2) for 2b). These compounds were inactive as antagonists

at 10-6M in the GPI against both morphine and EK (data not shown).

TABLE 1. Reversible Agonism and Antagonism in the MVD

of N,N-Dialkyl Leucine Enkephalin Analogs,

R2Tyr-Y-Y-X-Leu.

Agonism: Antagonism:

Percent IC5 0 Ratio1

Cmpd. R X Y Inhibition1 DADLE Morphine

1a benzyl Gly Mel 16.2 1.7%2

3.0 0.521.0 0.4

1 b " " Phe 11.4 5.9% 6.6 1.42

0.6 0.2

2a allyl Aib Mel 20.6 6.5% 5.0 0.9 2.2 0.72

2 b " " Phe 7.4 7.4% 15.6 2.4 0.8 0.1

3 a benzyl " Mel 22.5 5.2% 1.2 0.2 1.0 0.3

3 b " " Phe 25.7 4.9% 5.6 1.7 1.0 0.1

1

Compounds were tested at 10-6M. Results are ± standard

error of the mean (S.E.M); n = 3 unless otherwise indicated.

2n = 4.

The two melphalan-containing peptides which showed reversible

antagonist activity were tested for irreversibility against DADLE in the

MVD. At 10-6M no irreversible antagonism was observed (table 2), but

at 10-5M both compounds did show weak irreversible antagonist activity.

The irreversible antagonism was similar for the two compounds in spite

of the 2.5-fold higher reversible antagonism of 2a. Although extremely

high concentrations were required to demonstrate any irreversibility,

these results do demonstrate the potential feasibility of developing

irreversible antagonists based on the enkephalins. Other less bulky and

TABLE 2. Reversible and Irreversible antagonism of 1a and

2a against DADLE in the MVD.

1 0- 6 M

DADLE IC50 Ratio1

1 0- 5 M

Cmpd Reversible Irreversible Reversible2

Irreversible

1a 3.0 0.5(4) 1.2 0.2 (4)3 7.7 3.3(4) 2.4 0.8 (5)

2 a 5.0 0.9 (3) 1.1 0.2 (3) 19.4 4.7 (4) 2.9 0.9 (5)

1

S.E.M. (n).

2

At 10-5M both 1a and 2a showed significant reversible

agonism (47.8±5.7% and 42.9±7.0% inhibition of the twitch,

respectively).

3

After 30 minutes incubation.

187
more reactive functional groups at the para position of the phenylalanine ring may result in increased affinity of the peptide for the

receptor and greater irreversible antagonism at lower concentrations.

REFERENCES

Cotton, Giles, M.G., Mil

 Henderson, G., Hughes, J., and Kosterlitz, H.W., Br J Pharmacol,

ler, L., Shaw, J.S., and Timms, D. Eur J

Pharmacol, 97:331-332, 1984.

46:764-766, 1972.

Portoghese, P.S., Larson, D.L., Sayre, L.M., Fries, D.S., and Takemori,

A.E. J Med Chem, 23:233-234, 1980.

Rang, H.P. Br J Pharmacol, 22:356-365, 1965.

Shaw, J.S., Miller, L., Turnbull, M.J., Gormley, J.J., and Morley, J.S. Life

Sci, 31:1259-1262, 1982.

Takemori, A.E., Portoghese, P.S. Annu Rev Pharmacol Toxicol,

25:193-223, 1985.

ACKNOWLEDGEMENTS

This research was supported by individual service award grant DA-05263

(J.A.L.) and research grant DA-01533 from the National Institute on Drug

Abuse. We thank Michael Powers and Barbara Taylor for their capable

assistance in the biological testing and Prof. A.E. Takemori for his advice.

188
DIHYDROMORPHINE-PEPTIDE HYBRIDS HAVE MU RECEPTOR ANTAGONISTIC AND

DELTA RECEPTOR AGONISTIC ACTIVITY ON THE MOUSE VAS DEFERENS AND

BIND WITH HIGH AFFINITY TO OPIOID RECEPTORS IN RAT BRAIN

C.B. Smith, F. Medzihradsky and J.H. Woods

Departments of Pharmacology and Biological Chemistry

The University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT

The actions of three morphine derivatives with short peptide side

chains were evaluated upon the contraction of the isolated, electrically stimulated mouse vas deferens preparation and upon displacement of specifically bound 3

H-etorphine in rat brain membranes. NIH-9834 (N-[6, 14-endoetheno-7, 8-dihydromorphine-7-alpha-carbonyl]-L-phenylalanyl-L-leucinol) and its ethyl ester, NIH9833, were potent agonists upon the vas deferens. ICI-174864, 100

nM, markedly antagonized the actions of both NIH-9833 and NIH-9834

which indicates that these are delta receptor agonists. NIH-9835

(N-[6, 14-endoetheno-7, 8-dihydromorphine-7-alpha-carbonyl]-L-glycyl-L-phenylalanyl-L-leucine ethyl ester HCl) differs from NIH-9833

and NIH-9834 by the presence of a single amino acid residue. Although this drug had no agonistic activity on the vas deferens, it

was a potent antagonist of mu agonists. All three hybrids were potent displacers of 3

H-etorphine in rat cerebral membranes. The observation that addition of a single glycyl residue changes dihydromorphine-peptide analogs from potent delta receptor agonists to

equally potent mu receptor antagonists suggests that the two receptor sites might be structurally quite similar.

INTRODUCTION

Morphine is a mu receptor agonist on the mouse vas deferens. In

contrast, the enkephalins are most potent as delta receptor agonists in that preparation (Lord et al. 1977). The purpose of the

present study was to evaluate the effects of several dihydromorphine-peptide analogs upon the mouse vas deferens preparation and

examine their binding to opioid receptor in rat brain membranes.

These analogs incorporate peptide fragments of the enkephalins into

the dihydrcmorphine molecule.

METHODS

In the binding assay, the competition of the investigated compounds

with 3H-etorphine for opioid receptor sites in a membrane

preparation from rat brain cerebrum was determined (Medzihradsky et

al. 1984). After incubation at 25°C to reach binding equilibrium,

the suspension of brain membranes in the assay medium was quickly

filtered and the bound radioactivity determined. The binding

affinity of the tested compounds is expressed as their EC 50 (from

log-probit plots) in displacing specifically bound 3H-etorphine.

For the vas deferens experiments, male, albino ICR mice, weighing

between 25 and 30 g, were used. The mice were decapitated, the

vasa deferentia removed, and 1.5 cm segments were suspended in or189
gan baths which contained 30 ml of a modified Kreb's physiological

buffer. The buffer contained the following (mM): NaCl, 118; KCl,

4.75; CaCl2 2.54; MgSO4, 1.19; KH2PO4, 1.19; glucose, 11; NaHCO3,

25; pargyline HCl, 0.3, tyrosine, 0.2; ascorbic acid, 0.1; and

Na2EDTA, 0.03.

kept at 37 C.

The buffer was saturated with 95% 02 - 5% CO2 and

The segments were attached to strain gauge transducers and suspended between two platinum electrodes. After a 30-min

equilibration period, the segments were stimulated once every 10

sec with pairs of pulses of 2 msec duration, 1 msec apart and at

supramaximal voltage. Naltrexone, ICI-174864 or NIH-9835 were

added to the organ baths 15 minutes before the determination of cumulative concentration-effect relationships for the various agonists. beta-FNA was added to the organ baths after the initial

equilibration period. Thirty min later the beta-FNA was removed

from the organ baths by repeated washings with fresh buffer. The

tissues were washed three times every 5 min for 15 min. Cumulative

concentration-effect relationships for the various agonists were

then determined 20 min after the last wash (i.e. 30 min after the

beta-FNA was removed from the organ baths). The EC 50's were calculated by probit analysis. All values are the means of at least 6

determinations ± the standard error of the mean. The following

drugs were studied: ethylketazocine, etorphine, DSLET [Tyr-D-SerGly-Phe-Leu-Thr], NIH-9824, NIH-9833, the ethyl ester of NIH-9834,

NIH-9835, naltrexone HCl, ICI-174864 [N,N-diallyl-Tyr-Aib-Aib-PheLeu-OH], morphine sulfate, U50, 488H [trans-3, 4-dichloro-N-methyl-N

(2- (1-pyrrolidinyl) cyclohexyl) benzeneacetamide methanesulfonate

hydrate] and beta-funaltrexamine (beta-FNA).

RESULTS AND DISCUSSION

NIH-9833 was an extremely potent agonist upon the mouse vas deferens preparation with an EC 50 of 0.85 + 0.53 nM (n=8, FIGURE 1).

Naltrexone, 100 nM, beta-FNA, 1 μM, and ICI-174864, 100 nM, antagonized the actions of NIH-9833. EC 50's were 1.7 0.3 nM in the

presence of naltrexone and 2.4 0.5 nM in the presence of betaFNA. Naltrexone competitively blocks mu, kappa and delta receptors, and beta-FNA blocks mu and delta, but not kappa receptors in

the mouse vas deferens preparation (Smith in press). In the presence of ICI-174864 the EC 50 for NIH-9833 was 1.9 nM 0.5. ICI174864 is a highly selective delta receptor antagonist (Cotton et

al. 1984, Smith et al. 1984), and blockade by ICI-174864 suggests

that NIH-9833 acts primarily upon delta opioid receptors. Similar

results were obtained with NIH-9834.

NIH-9835 was a potent mu receptor antagonist upon the mouse vas

deferens preparation. It caused a parallel shift (12.3-fold shift)

to the right in the morphine concentration-effect curve. In control experiments the EC 50 for morphine was 350 nM 117 (n=3), and

in the presence of NIH-9835, 100 nM, the EC 50 for morphine was 4.3

μ M 1.48. NIH-9835 caused a slight shift (3-fold) to the right

in the DSLET concentration-effect curve. In control experiments,

the EC 50 for DSLET was 0.49 nM 0.08 (n=4), and in the presence

of NIH-9835, 100 nM, was 1.6 nM 0.2. NIH-9835 did not significantly shift the U50,488H concentration-effect curve. In control

experiments, the EC 50 for U50,488H was 473 nM 145, and in the

presence of NIH-9835, 100 nM, the EC 50 was 473 nM 125. Thus,

190
FIGURE 1. Antagonism of the effects of NIH-9833 upon the

electrically stimulated mouse vas deferens. Abscissa:

concentration of NIH-9833; Ordinate: percent inhibition

of the twitch. Each point represents the mean of 6-9

preparations in the absence of antagonist (circles) or in

the presence of 100 nM naltrexone (triangles), 100 nM

ICI-174864 (squares), or after pretreatment with 1 μM

beta-FNA (diamonds). Vertical bars, S.E.M.

FIGURE 2. Antagonism by NIH-9835 of the effects of morphine upon the mouse vas deferens preparation. Abscissa:

concentration of morphine; ordinate: percent inhibition

of the twitch. Circles, controls; triangles, observations in the presence of 100 nM NIH-9835. Each value is

the mean of 6-9 determination. Vertical bars, S.E.M.

191
NIH-9835 is a potent opioid antagonist which appears to act primarily upon mu receptors, although it is slightly active upon delta

receptors. Its activity is different than that of naltrexone which

blocks mu, kappa and delta receptors and which is more potent as an

antagonist at kappa receptors than at delta receptors.

All three hybrids displaced bound 3H-etorphine in rat cerebral membranes. The antagonist, NIH-9835, was less potent than naltrexone

as a displacer (TABLE 1). The two agonists, NIH-9833 and NIH-9834

were extremely potent displacers of 3H-etorphine with EC 50's in

the range of etorphine.

TABLE 1. EC 50's of NIH-9833, NIH-9834 and NIH-9835 compared to those of representative opiate agonists and antagonists in displacing 0.5 nM 3

H-etorpine in a membrane

preparation from rat cerebrum (in the presence of Na+).

Compound EC 50 (nM)

Morphine 23.60

Ethylketazocine 6.60

NIH-9835 1.70

Naltrexone 0.87

NIH-9833 0.58

Etorphine 0.37

NIH-9834 0.34

The observation that the addition of a single glycyl residue

changes a dihydromorphine-peptide analog from a potent delta receptor agonists to an equally potent and quite specific mu receptor

antagonists suggests that the two receptor sites on noradrenergic

nerve terminals in the mouse vas deferens might be structurally

quite similar.

ACKNOWLEDGEMENTS

Supported by USPHS grants DA-00254 and MH-36336.

REFERENCES

Cotton, R., Giles, M.G., Miller, L., Shaw, J.S., and Timms, D. Eur

J Pharmacol, 97:331-332, 1984.

Lord, J.A., Waterfield, A.A., Hughes, J., and Kosterlitz, H.W.

Nature (Lond), 267:495-499, 1977.

Medzihradsky, F., Dahlstrom, P.J., Woods, J.H., Fischel, S.V. and

Mitsos, S.E. Life Sci, 34:2129-2138, 1984.

Smith, C.B., Bennett-Kelly, L., and Woods, J.H. Neuropeptides,

5:161-164, 1984.

Smith, C.B. In Harris, L.S., ed. Problems of Drug Dependence 1986:

Proceedings of the 48th Annual Scientific Meeting, The

Committee on Problems of Drug Dependence, Inc. National

Institute on Drug Abuse Research Monograph. Washington, D.C.:

Supt. of Docs., U.S. Govt. Print. Off. (in press)

192
CHARACTERIZATION OF D-ALA2

,LEU5,CYS6-ENKEPHALIN:

A NOVEL SYNTHETIC OPIOID PEPTIDE WITH SLOWED DISSOCIATION FROM DELTA

RECEPTORS

Wayne D. Bowen, Mark Kelemen,

Roger Huey, and D. Stewart

Section of Biochemistry

Division of Biology and Medicine

Brown University

Providence, Rhode Island 029 12

ABSTRACT

D-Ala2,Leu5,Cys6-enkephalin (DALCE) is a synthetic enkephalin analog

which contains a reduced sulfhydryl group. It exhibited moderate

delta selectivity (mu/delta IC50 ratio 13), but was not as selective

as the disulfide-containing peptide, D-Pen2,5-enkephalin (DPDPE)

(mu/delta ratio 1121). However, unlike other delta-selective

peptides, DALCE exhibited a markedly slowed dissociation from

receptors after pretreatment of membranes with micromolar

concentrations. Pretreatment of membranes with 10 uM DALCE,

followed by extensive

binding sites. D-Ala2,D-Leu5-enkephalin (DADLE), D-Ser2,Leu5,Thr6-

washing produced an 85-90% loss of 3H-DPDPE

enkephalin (DSTLE) and DPDPE produced losses of 59%, 70%, and 19%,

respectively. The effect of DALCE was not reversed by a 60 min

post-incubation in buffer containing 250 mM NaCl + 100 uM GMPPNP, a

condition which produced nearly complete reversal of loss of sites

by DADLE and DSTLE. DPDPE could be dissociated merely by postincubation in TRIS-buffer alone for 15 min. The order for ease of

dissociation after preincubation was DPDPE > DADLE > DSTLE >>>

DALCE. The effect of DALCE was selective for delta sites, although

higher concentrations of DALCE produced loss of mu sites. DALCE

pretreatment had no effect on recovery of kappa sites. These

results indicate that DALCE binds essentially irreversibly to delta

receptors.

INTRODUCTION

In the search for more selective and stable delta receptor ligands,

several analogs of enkephalins have been synthesized. Examples are

DADLE and DSTLE (Kosterlitz et al. 1980, Gacel et al. 1980) which

make use of the incorporation of D-amino acids for proteolytic

stability. Recently, conformationally restricted enkephalin analogs

have been produced by disulfide bridge cyclization of the peptide

chain. This is accomplished by introduction of cysteine or

penicillamine at positions 2 and 5, followed by oxidation to form

Pen2,5-enkephalin is the most delta selective enkephalin analog to

the disulfide bond (Schiller et al. 1981, Mosberg et al. 1983). Ddate (Mosberg et al. 1983). Although these disulfide-containing

peptides have been thoroughly characterized in binding assays and

bioassays, the effect of introducing a reduced thiol group has not

yet been studied. We report here the synthesis and characterization

of D-Ala2,Leu5,Cys6-enkephalin (DALCE). This peptide makes use of

the D-Ala2 residue for stability, but is unique in that it contains

a sulfhydryl group at the C-terminus. Although this analog is not

193
as delta-selective as the disulfide-containing peptides, the free

sulfhydryl group endows this peptide with a property lacking in

other enkephalin analogs. DALCE has a markedly slowed rate of

dissociation from delta and mu receptors. This is quite interesting

in view of the fact that delta and mu opiate receptors contain an

essential sulfhydryl group (Smith and Simon 1980). DALCE may bind

covalently by formation of a disulfide bond to receptor.

METHODS

Tyr-D-Ala-Gly-Phe-Leu-Cys was custom synthesized by Peninsula Labs

using solid phase techniques. Purity was established by TLC, paper

electrophoresis, and reversed phase HPLC. The peptide tended to

dimerize during storage due to sulfhydryl oxidation. This was

prevented by storage in presence of 20 mM beta-mercaptoethanol in

acidic solution at -20°C.

Lysed P2 membrane preparations were prepared from brains (minus

cerebellum) of male Sprague-Dawley rats (150-200 g). Membranes (650

ug protein/ml) were incubated with peptides for 60 min at 25° in 50

mM TRIS-HCl pH 7.4 containing 100 mM NaCl, 3 mM Mn acetate, 2 uM

guanosine 5'-triphosphate (GTP), and the following protease

inhibitors to protect susceptible peptides: bacitracin (100 ug/ml),

leupeptin (4 ug/ml) chymostatin (2 ug/ml), bestatin (10 ug/ml).

Following pelleting and resuspension in an equal volume of the

buffers specified in table 1 and figure 1, membranes were incubated

for the specified times to effect dissociation of the bound peptide.

After this dissociation period (post-incubation), the membranes were

washed and incubated with 3H-ligand to assess recovery of binding

sites compared to controls which were incubated with no peptide.

Except where indicated otherwise, incubations with 3H-ligand were

oarried out for 90 min at 25°C in a total volume of 500 ul

containing 10 mM TRIS-HCl pH 7.4 325 ug of membrane protein, and

the components specified below. 3H-DPDPE. 100 mM NaCl, 3 mM Mn

acetate, 2 uM GTP; 3H-DAGO (D-Ala2,NMe-Phe4,Gly-ol5-enkephalin): 3

mM Mn acetate; H-BREM (bremazocine):

DAGO to suppress mu and delta binding. 3H-Ligand concentration was

100 nM unlabeled DSTLE and

2 nM. Non-specific binding was determined in presence of 10 uM

unlabeled levallorphan. The incubation was terminated by filtration

of inoubation mixture through glass fiber filters under reduced

pressure and washing with ice-cold buffer.

RESULTS AND DISCUSSION

The receptor selectivity of DALCE was compared to other enkephalin

analogs by competition binding assays using 3H-DPDPE, 3H-DAGO, and

3

H-BREM to label delta, mu, and kappa receptors, respectively (10 mM

TRIS-HCl pH 7.4, 90 min, 25°C). Delta, mu, and kappa IC50 values

for the following unlabeled peptides were: DALCE - 4.1, 55; >10,000

nM; DADLE - 3.4, 151, >10,000 nM; DSTLE - 4.0, 250, kappa not

determined; DPDPE - 5.5, 6167, >10,000 nM. Therefore, DALCE

exhibited moderate delta selectivity with roughly a 10-fold

selectivity for delta sites over mu sites. Like the other

enkephalin derivatives, DALCE had very low affinity for kappa

receptors. As reported by others (Mosberg et al. 1983), the

disulfide-containing peptide DPDPE exhibited the highest delta

194
selectivity. All the analogs had similar IC50 values at the delta

site.

Table 1 shows the effect of various dissociation conditions on

occupation of delta sites after preincubation with various deltaselective peptides at a concentration of 10 uM. After a 15 min

dissociation period in 50 mM TRIS-HCl, all peptides produced a

residual blockade of sites. However, DALCE produced the greatest

loss of sites while DPDPE produced the least. Increasing the

dissociation time in this buffer to 60 min caused moderate increases

in recovery of sites after preincubation with DADLE and DSTLE.

Recovery of sites with DALCE was unaffected and remained low.

Sodium ion and guanine nucleotides have been shown to lower the

affinity of opiate receptors for ligand, therefore inhibiting

binding or promoting dissociation (Simon et al. 1975, Childers and

Snyder 1980, Zukin and Gintzler 1980). Dissociation in buffer

containing 250 mM NaCl an

nearly complete recovery of 3H-DPDPE binding sites with DADLE and

d 100 uM GMPPNP resulted in complete and

DSTLE, respectively. However, the marked loss of sites produced by

DALCE was unaffected by post-incubation in this buffer. Therefore,

DADLE, DSTLE, and DPDPE can readily dissociate from delta receptors,

while DALCE remains tightly bound. This effect required both opiate

receptor recognition as well as a thiol group since preincubation

with 25 uM beta-mercaptoethanol or dithiothreitol had no effect on

recovery of sites (not shown).

TABLE 1. Pretreatment of membranes with peptides and effect of

various dissociation conditions on recovery of delta binding sites

Dissociation Condition and

% Recovery of 3H-DPDPE Binding

Peptide

Pretreatment (10 uM) 15 min

60 min,

60 min NaCl/GMPPNP

DADLE 41 50 100

DSTLE 30 50 80

DPDPE 81 -- --

DALCE 11 17 14

Figure 1 shows the receptor selectivity of the DALCE effect.

Membranes were preincubated with various concentrations of DALCE,

post-incubated in buffer containing 250 mM NaCl and 100 uM GMPPNP

for 60 min, and recovery of mu, delta, and kappa sites was assessed

by binding of 3H-DAGO, 3H-DPDPE, and 3H-BREM, respectively. Delta

sites were most sensitive to DALCE. Half maximal loss of sites

occurred at 2-3 uM DALCE. Mu sites were not affected at 2 uM and

only reached 50% loss at 20 uM DALCE. Kappa sites were not affected

by DALCE pretreatment at concentrations up to 20 uM. These results

are consistent with the receptor selectivity of DALCE (delta > mu >>

kappa).

195
FIGURE 1. Receptor

selectivity of the

DALCE effect.

In conclusion, DALCE is a sulfhydryl-containing enkephalin analog

with preference for delta receptors. It exhibits a markedly slowed

rate of dissociation from delta and mu receptors compared to other

delta receptor peptides. Taken together with the presence of a

critical sulfhydryl group at the ligand binding site of mu and delta

receptors (Smith and Simon 1980), it is possible that DALCE binds

covalently by forming a disulfide bond with receptor. This is

supported by preliminary results suggesting that the effects of

DALCE are attenuated by post-incubation with a reducing agent. It

would be interesting to ascertain whether incorporation of free

sulfhydryl groups into other opioid peptides results in slowed

dissociation from receptors.

REFERENCES

Childers, S.R., and Snyder, S.H. J Neurochem, 34:583-593, 1980.

Gacel, G., Fournie-Zaluski, M.C., and Roques, B.P. FEBS Lett,

118:245-247, 1980.

Kosterlitz, H.W., Lord, J.A.H., Paterson, S.J., and Waterfield, A.A.

Br J Pharmacol, 68:333-342, 1980.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.F.,

Galligan, J.J., and Burks, T.F. Proc Natl Acad Sci USA 80:5871-

5874, 1983.

Schiller, P.W., Eggimann, B., DiMaio, C.L., and Nguyen, T.M-D.

Biochem Biophys Res Commun., 101:337-343, 1981.

Simon, E.J., Hiller, J.M., Groth, J., and Edelman, I. J Pharmacol

Exp Ther, 192:531-537, 1975.

Smith, J.R., and Simon, E.J. Proc Natl Acad Sci USA, 77:281-284,

1980.

Zukin, R.S., and Gintzler, A.R. Brain Res, 186:486-491, 1980.

ACKNOWLEDGEMENT

This work was supported by NIDA Grant No. DA03776 (W.D.B.)

196
[

3

H]DSTBULET, A NEW LINEAR HEXAPEPTIDE WITH BOTH AN IMPROVED

SELECTIVITY AND A HIGH AFFINITY FOR -OPIOID RECEPTORS.

Philippe Delay-Goyet*, Christine Seguin*. Valérie Daugé*,

Geneviève Calenco, Jean-Louis Morgat,

Gilles Gacel* and Bernard P. Roques*.

* Département de Chimie Organique, U 266 INSERH, UA 498 CNRS,

Faculté de Pharmacie, 4 av. de l'Observatoire, 75006 Paris.

** Biochimie, CEA Saclay, 91190 Gif Sur Yvette.

ABSTRACT

The binding properties of the new agonist Tyr-D-Ser(OtBu)-GlyPhe-Leu-Thr, DSTBULET, in rat brain tissue shows that insertion of a

bulky t.butyl group into the sequence of DSLET leads to a

conformationally-induced large increase in selectivity for opioid

receptors (KI( )/KI(μ) = 0.012). In addition to its similar

selectivity to that of the cyclic enkephalins DPDPE and DPLPE. the

affinity of [

3

H]DSTBULET for sites (KD = 2.9 nM) is significantly

better than that of [3H]DPDPE (KD ̃10.5 nM) or DPLPE (KI( )  ̃ 19 nM).

DSTBULET is therefore the most appropriate probe for both binding

studies and pharmacological investigations of -receptors as

illustrated by a comparison of the analgesic properties of DAGO,

DSTBULET and DPLPE.

INTRODUCTION

A clear distinction between the pharmacological responses induced by

stimulation of opioid μ or -receptors requires highly potent and

selective ligands. In the case of receptors, linear hexapeptides

(DSLET and DTLET) were the first really -selective ligands

(Fournié-Zaluski et al. 1981; Gacel et al. 1980; Zajac et al.

1983). Recently, two cyclic enkephalins DPDPE and DPLPE have been

shown to exhibit even better -selectivity than the linear peptides

(Mosberg et al. 1983). Unfortunatly this is associated with a large

decrease in affinity, preventing the use of [

3

H]DPDPE for

competition studies or for acute quantitative determinations of

-sites in brain after biochemical lesions or pharmacological

manipulations (Delay-Goyet et al. 1985). We report here the binding

properties in rat brain tissue, of a new -ligand [

3

H]DSTBULET which

shows both a high selectivity and a high affinity.

197
MATERIALS AND METHODS

The linear hexapeptide Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr, DSTBULET, was

prepared by the liquid phase method as described for DSLET and DTLET

(Gacel et al. 1981). [3

H]DSTBULET (70 Ci/mmol) was obtained from the

2,5-dibromotyrosyl precursor. [

3

H]DPDPE was from Amersham and

[

3

H]DTLET and [

3

H]DAG0 from CEA (France). Binding assays (in

triplicate) were carried out at 37°C using a crude rat brain membrane

preparation as described (Zajac and Roques 1985). KD and KI values

were obtained from computerized linear regression analysis.

Inhibition of electrically evoked contractions of GPI or MVD was

performed as described. Analgesic potency of the various compounds

was evaluated using the hot-plate test in mice.

RESULTS AND DISCUSSION

Pharmacological profile of DSTBULET v DPDPE. The selectivity of

DSTBULET was compared to that of DSLET, DTLET and DPDPE using : i)

bioassays on peripheral organs (GPI enriched in μ sites and MVD in

sites), ii) inhibition of the binding to rat brain tissue of [3H]DAGO

(μ selective ligand) and [

3

H]DTLET (nearly -selective ligand). AS

shown in table 1, the introduction of the bulky O.tBu. group on DSLET

leads to a large improved -selectivity, as occurs with the cyclic

enkephalins. At the level of the brain receptors, DSTBULET was at

least as selective as DPDPE but its apparent affinity was

significantly better. Besides, DSTBULET is unable (IC50>10,000 nM) to

interact with receptors labelled with [

3

H]EKC.

Binding characteristics of [

3

H]DSTBULET on rat brain. Binding

equilibrium of [

3

H]DSTBULET (5 nM) was reached in 30 min at 35°C.

Saturation experiments were performed under these conditions and

binding parameters (KD and Bmax) calculated from linear regression

analysis of the saturation isotherms. As shown in fig.1, [3H]DSTBULET

interacts with a single class of site (0.5-15 nM) (equilibrium

dissociation constant KD = 2.90 0.33 nM and Bmax = 98.8 9.4

femtomoles/mg. In addition to its better -affinity than [

3

H)DPDPE

(KD = 10.5 nM) it is very important to observe that [

3

H]DSTBULET

displayed 55% specific binding at 63% saturation against 26% at 67%

saturation for [

3

H]DPDPE. Moreover, as expected from its high

selectivity, the Bmax of [3H]DSTBULET was not affected by blocking of

u-sites with high concentration of DAGO (300 nM) and as a consequence

of its higher affinity, the binding parameters can be determined with

greater accuracy.

198
TABLE 1. Pharmacological profile of linear and cyclic

enkephalins on peripheral organs and rat brain tissue.

FIGURE 1. Eadie-Hofstee and Scatchard analyses of the

specific binding of [3H]DSTBULET to rat brain membranes.

199
Correlation between supraspinal analgesia and affinity for opioid μ

sites. As expected, the pharmacological properties of DSTBULET are

in agreement with its binding characteristics to brain tissue. This

is clearly demonstrated by its 130 fold lower potency than DAGO, a

typical μ agonist, to inhibit nociceptive stimuli induced in mice

through the hot plate test after i.c.v. administration. The low

analgesic potency of DSTBULET and DPLPE, contrasting with their

affinity for -sites, reinforces the suggested preferential

involvement of μ-receptors in the supraspinal control of pain

(Chaillet et al. 1984).

CONCLUSION

The new linear hexapeptide DSTBULET possesses the main requirements

for both accurate biochemical and pharmacological investigations on

delta opioid receptors : - a high affinity with a low non specific

binding ; - a good selectivity for the delta sites ; - a

satisfactory resistance to peptidases ; - a favourable

hydrophobic-hydrophilic balance. Finally DSTBULET belongs to a large

series of linear analogues with /μ selectivities ranging between 1

and 120. Taken together [

3

H]DSTBULET and its analogues behave as

useful compounds for a complete study of the physiological relevance

of delta opioid receptors.

REFERENCES

Fournie-Zaluski, M.C., Gacel, G., Maigret, B., Premilat, S. and

Roques, B.P. Mol Pharmacol, 20:484-491, 1981.

Gacel, G., Fournie-Zaluski, M.C. and Roques, B.P. FEBS Lett,

118:245-247, 1980.

Zajac, J.M., Gacel, G., Dodey, P., Morgat, J.L., Chaillet, P.,

Costentin, J. and Roques, B.P. Biochem Biophys Res Commun,

111:390-397, 1983.

Mosberg, H.I., Hurst, R., Hruby, V.J., Kelvin, G., Yamamura, H.J.,

Galligan, J.J. and Burks, T.F. Proc Natl Acad Sci USA,

80~5871-5874, 1983.

Delay-Goyet, P., Zajac, J.M., Rigaudy, P., Foucaud, B. and

Roques, B.P., FEBS Lett, 183:439-443, 1985.

Gacel, G., Fournié-Zaluski, M.C., Fellion, E. and Roques, B.F. J

Med Chem, 24:1119-1124, 1981.

Zajac, J.M. and Roques, B.P. J Neurochem, 44:1605-1614, 1985.

Chaillet, P., Coulaud, A., Zajac, J.M., Fournie-Zaluski, M.C.,

Costentin, J. and Roques, B.P. Eur J Fharmacol, 101:83-90, 1984.

200
ANOMALOUS BINDING OF DPDPE AS A RESULT OF BATCH VARIABILITY

Larry Taylor, Alfred Mansour, Stanley J. Watson, and Huda Akil

Mental Health Research Institute and Department of Psychiatry

University of Michigan Medical Center

205 Washtenaw Place, Ann Arbor, MI, USA 48109-0720

ABSTRACT

The binding of the delta selective agonist [3H]DPDPE to diencephalic

rat brain tissue was examined. Although most material examined gave

rise to a single receptor model, one commercial preparation

(Amersham, batch 8) suggested a two receptor model. Purification by

HPLC removed a minor component and restored a single receptor model

to the major component.

INTRODUCTION

The endogenous opioid peptides are known to interact with several

classes of opioid receptors. The synthetic peptide

(2-D-penicillamine, 5-D-penicillamine) enkephalin (DPDPE) is a

conformationally restricted enkephalin analogue which has been shown

to be a selective delta agonist (Mosberg et al. 1980) and is useful

in autoradiographic differentiation of delta receptors in rat brain

(Mansour et al. 1986). During the course of these studies several

different batches of [

3

H]DPDPE were utilized. One of these, Amersham

batch #8, exhibited binding characteristics different from previous

and subsequent batches of synthetic [3H]DPDPE.

METHODS AND MATERIALS

Slide-mounted brain sections (25 um) from the diencephalon were

incubated with 200 ul of [3H]DPDPE (Amersham, 41.5 Ci/mmol, batch 8)

in a 50 mM Tris buffer (pH 7.5 at 25°C). The concentrations of

[

3

H]DPDPE ranged from 0.1 to 14 nM. After a 60 minute incubation,

the slides were drained and washed in four consecutive 250 ml 50 mM

Tris washes (pH 7.6, 4°C) for 30 seconds. The slides were then dried

with a portable hair dryer and the binding was quantified using

liquid scintillation counting (Packard, 2000 CA). Non-specific

binding was evaluated by treating a parallel set of slides with the

same [

3

H]DPDPE concentrations with a 1 uM final concentration of

unlabelled DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr). The data were graphed

as Scatchard plots and the KD and Bmax values determined with the

LIGAND program (Munson and Rodbard 1980). Similar studies were done

on other batches of [

3

H]DPDPE (Amersham, batch 9) and purified

[

3

H]DPDPE from batch 8.

The liquid chromatography was done on an HPLC system comprised of two

Altex 110 pumps coupled to an Altex model 420 controller, a Rheodyne

7125 injection valve, a Waters 3.9 mm X 30 cm uC18 reverse phase

column and a Pharmacia model 100 fraction collector. The flow rate

was 1.5 ml/min; the elution solvent was 10-60% (over 80 minutes)

acetonitrile/0.1% TFA. Samples were collected every minute and a 50

ul aliquot was examined for [

3

H] activity by liquid scintillation

counting.

201
FIGURE 1. Scatchard plots of selected DPDPE batches

202
Figure 2. HPLC Chromatograms for DPDPE Batches

203
RESULTS

The Scatchard plot from the "LIGAND" analysis for DPDPE binding to

rat brain diencephalic tissue for Amersham batch 8 is shown in figure

1(A). The best "LIGAND" fit for this data was a two receptor model

with a KD1 of 2.4 nM and a KD2 of 13.8 nM. The corresponding Bmax

values were 44 and 189 pM respectively.

The Scatchard plot from the "LIGAND" analysis for binding of the

major component of batch 8 (which had the same elution volume as

authentic DPDPE, figure 2(A) and (C)) to diencephalic tissue is shown

in figure l(B). The best "LIGAND" fit was for a single receptor site

having a KD of 7.6 nm with a Bmax of 146 pM.

The Scatchard plot from the "LIGAND" analysis of the binding of most

batches of DPDPE was typified by batch 9 as shown in figure 1(C).

The HPLC chromatogram of this material is shown in figure 2(B). This

gives rise to a single receptor model with a KD = 17 nM and a Bmax =

157 pM.

CONCLUSIONS

Amersham DPDPE, batch 8, contained an easily removable impurity which

altered the binding characteristics of this material.

The binding of purified DPDPE to diencephalic sections of rat brain

is consistent with a single receptor model having a KD = 7.6 nM and a

Bmax of 146 pM.

KEFERENCES

Mansour, A. et al. Brain Res, in press.

Mosberg, H. et al. Proc Natl Acad Sci USA, 80:5871-5874, 1980.

Munson, P.J. and Rodbard, D. Anal Biochem, 107:220-229, 1980.

ACKNOWLEDGMENTS

Financial assistance from NIDA Grants #DA02265 and DA00254 is

gratefully acknowledged.

204
COMPARISON OF THE OPIOID ANTAGONIST PROPERTIES OF A CYCLIC

SOMATOSTATIN ANALOG IN VITRO AND IN VIVO

Jennifer E. Shook, John T. Peltont, Wieslaw Kazmierski*,

Paul K. Lemcke, William S. Wire, Victor J. Hruby* and

Thomas F. Burks

Departments of Pharmacology and Chemistry*,

University of Arizona, Tucson, Arizona 85724

Merrell Dow Research Institutet

Merrell Dow Pharmaceuticals

Indianapolis, Indiana 46268-0470

INTRODUCTION

We have recently developed a conformationally constrained analog of

somatostatin-14 called CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2)

which shows a high degree of affinity and selectivity for the mu

opioid receptor in binding assays using homogenized rat brains

(Pelton et al. 1985; 1986; Gulya et al. 1986). The purpose of

these investigations was to determine the opioid pharmacology of CTP

in the guinea pig isolated ileum, mouse isolated vas deferens, and in

analgesic and gastrointestinal transit time tests in mice.

MATERIALS AND METHODS

In Vitro Bioassays: The guinea pig ileum longitudinal musclemyenteric plexus (GPI) and mouse vas deferens (MVD) were used to

determine the mu/kappa and mu/delta selectivity of CTP respectively.

The GPI (Kosterlitz et al. 1970) and MVD (Hughes et al. 1975) were

run according to standard procedures.

In Vivo Bioassays: CTP was tested for antagonism of mu, delta and

kappa selective agonists in two models of analgesia in male ICR mice

(20-25 g), the hotplate and abdominal stretch tests. The hotplate

(55°C) test was run according to the methods of Porreca et al. (1984).

For the abdominal stretch test, mice received intrathecal (5 ul)

distilled water or drug, followed immediately by an intraperitoneal

(i.p.) injection of 1.2% acetic acid. Mice were then observed for

stretching behaviors for 5 min.

The rate of gastrointestinal transit in mice was determined by the

geometric center method as described by Porreca et al. (1984).

Peptides: The following peptides were used as mu, delta and kappa

selective agonists respectively: PLO17 (Chang et al, 1983), DPDPE

(Mosberg et al. 1983) and U50,488H (VonVoigtlander 1983). PLO17 and

DPDPE were purchased from Peninsula Labs., Belmont CA., and U50,488H

was purchased from Upjohn, Kalamazoo, MI. CTP was synthesized as

previously described (Pelton et al. 1986). All peptides were

dissolved in distilled water.

205
FIGURE 1. The effect of pretreatment with 1.0 uM CTP on the inhibitory

actions of PLO17 (0.1 uM) and U50,488H (0.1uM) in the guinea pig ileum

and of PLO17 (1.0 uM) and DPDPE (0.01 uM) in the mouse vas deferens.

Significant differences between control (open bars) and CTP treated

(hatched bars) are indicated by **, p<0.01, n=4.

FIGURE 2. The effect of icv distilled water (open bars) or icv 1.0 ug

CTP (hatched bars) pretreatment on the analgesic actions of PLO17

(0.3 ug, icv). DPDPE (3.0 ug, icv) or U50,488H (0.1 ug, icv) in the hotplate

test and PLO17 (0.3 ug, it). DPDPE (1.0 ug, it) or U50,488H (0.1 ug, it) in

the abdominal stretch test. Significant differences are indicated by **,

p< 0.01, n = 6/dose.

206
RESULTS

In Vitro Bioassays: In the GPI, pretreatment with CTP antagonized the

actions of PLO17 but not U50,488H (figure 1). The actions of PLO17

were also blocked by CTP in the MVD. In contrast, CTP did not

antagonize the effects of DPDPE in this tissue (figure 1).

In Vivo Bioassays: PL017, DPDPE and U50,488H given intracerebroventricularly (icv) all produced increases in the latency to display

escape behaviors in the hotplate test (figure 2). The increased

latency caused by U50,488H appeared to be related to the production

of immobility rather than true analgesia. Pretreatment with CTP

antagonized the analgesic actions of PLO17 and DPDPE in the hotplate

test, but did not block the actions of U50,488H (figure 2). Schild

analysis of the interaction of supraspinal CTP with PLO17 or DPDPE

showed that CTP antagonized PLO17 in a competitive fashion (pA2 11.9

1.3, slope -0.85 0.5) but antagonized DPDPE in some complex,

noncompetitive fashion (slope -0.55 ± 0.3). Likewise, in the

abdominal stretch test, CTP again blocked the analgesic actions of

PLO17 and DPDPE, but did not block that produced by U50,488H (figure

2). The actions of all 3 agonists were reversed by naloxone in both

analgesic tests (data not shown).

Both PLO17 and DPDPE also slowed the rate of gastrointestinal transit

after spinal administration to mice (table 1). While pretreatment

with CTP antagonized the antitransit effects of PL017, it did not

block the effects of DPDPE (table 1). Naloxone (1.0 ug, it)

antagonized the antitransit effects of both agonists (data not shown)

Table 1. The effect of intrathecal CTP (1.0 ug)

pretreatment on the rate of gastrointestinal transit.

CTP 26 10* 34 9

*p<O.O5; n=5/dose.

% Inhibition of Gastrointestinal Transit

Pretreatment PLO17 (3 ug, it) DPDPE (3 ug, it)

vehicle 60 16 40 8

DISCUSSION

Results from the GPI and MVD studies indicate that CTP possesses mu

selective antagonist properties with no delta or kappa antagonist

actions. These findings support the conclusions of Pelton et al.

(1985, 1984; Gulya et al. (1984, who have shown that CTP displays a

high degree of selectivity for the mu receptor in binding assays

using homogenized rat brains.

The selective antagonism of mu but not delta related inhibition of

gastrointestinal transit shows that CTP retains its mu selectivity in

vivo. And, as expected from the results of the GPI studies, CTP had

no kappa antagonist properties when tested in vivo.

207
Despite the consistent mu selectivity of CTP (no delta or kappa antagonism) seen in the binding assays, GPI, MVD and gastrointestinal

transit time tests, CTP antagonized the analgesia evoked by both mu

and delta agonists. The antagonism of mu related analgesia was shown

to be of a competitive nature while that of the delta agonist was not

competitive. These findings may indicate that certain mu and delta

receptors which mediate analgesia are functionally coupled, and that

through its interaction with the mu receptor, CTP can also block

delta related analgesia.

In conclusion, CTP appears to produce mu selective antagonism both

in vitro and in vivo. CTP may prove useful in studying the possible

link between analgesic mu and delta receptors, and may also help in

determining if mu and delta receptors which mediate other functions

are also coupled.

ACKNOWLEGEMENTS

This work was supported by DA 02163, NS 19972, AM 36289 and AM 33547.

REFERENCES

Chang, K.J., Wei, E.T., Killian, A., and Chang, J.K. J Pharmacol

Exp Ther, 227:403-408, 1983.

Gulya, K., Pelton, J.T., Hruby, V.J., and Yamamura, H.I. Life

Sciences, 38:2221-2229, 1986.

Hughes, J., Kosterlitz, H.W., and Leslie, F.M. Br J Pharmacol,

53:371-381, 1975.

Kosterlitz, H.W., Lydon, R.S., and Watt, A.J. Br_ J Pharmacol,

39:398-413, 1970.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I.,

Galligan, JJ., and Burks, T.F. Proc Natl Acad Sci USA,

80:5871-5874, 1983.

Pelton, J.T., Gulya, K., Hruby, V.J., Duckles, S.P., and Yamamura,

H.I. Proc Natl Acad Sci USA, 82:236-239, 1985.

Pelton, J.T., Gulya, K., Yamamura, H.I., and Hruby, V.J. J Med

Chem, In press, 1986.

Porreca, F., Mosberg, H.I., Hrust, Hruby, V.J., and Burks, T.F. J

Pharmacol Exp Ther, 230:341-348, 1984.

208
H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2: A POTENT AND

SELECTIVE ANTAGONIST FOR MU OPIOID RECEPTORS

K. Gulya1, G.K. Lui1, J.T. Pelton2, W. Kasmierski2,

V.J. Hruby2 and H.I. Yamamura1

Department of 1Pharmacology and 2Chemistry,

University of Arizona,

Tucson, AZ 85724, U.S.A.

ABSTRACT

H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2(CTOP) exhibited high

affinity (IC50 = 2.80 nM) in displacing [

3

H]naloxone binding

(nH = 0.89 0.1) and showed an exceptional selectivity for mu

opioid receptors with an IC50(DPDPE)/IC50(naloxone) ratio of

4,840, while it displayed very low affinity for somatostatin

receptors (IC50 = 22,700 nM) in rat brain binding assays.

[

3

H]CTOP was recently custom synthesized (spec. act.: 84 Ci/mmol)

and evaluated for its in vitro binding properties towards the mu

opioid receptors in rat brain membrane preparations. Association

and dissociation of [

3

H]CTOP binding to mu opioid receptors were

rapid at 25°C with a kinetic Kd value of 0.67 nM. Saturation

experiments gave apparent Kd value of 1.11 nM and Bmax value of

136 13 fmol/mg prot at 25°C. Specific [

3

H]CTOP binding was

inhibited by a number of different opioid and opiate ligands. Among

them, putative mu opioid receptor-specific ligands, such as

naloxone, naltrexone and CTOP inhibited the binding with high

affinity, while delta opioid receptor-specific compounds or

non-opioid drugs inhibited specific [

3

H]CTOP binding with low

affinity or they were ineffective.

INTRODUCTION

In an attempt to study the selectivity, potency and conformational

requirements of somatostatin-related peptides for the mu opioid

receptor, we prepared a series of cyclic conformationally restricted

penicillamine ( -dimethylcysteine) containing somatostatin

octapeptide analogues by standard solid phase synthetic methods and

tested them for their ability to inhibit specific [

125I]CGP 23,996

(des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3,14-somatostatin),

[

3

H]naloxone or [

3

H]DPDPE ([D-Pen2,D-Pen5]enkephalin) binding

in rat brain membrane preparations: Our most potent and mu receptorselective octapeptide CTOP was recently custom labeled and its

binding characteristics were evaluated toward the mu opioid receptors.

MATERIALS AND METHODS

[

3

H]CTOP (84 Ci/mmol), [

3

H]naloxone (57 Ci/mmol) and [

3

H]DPDPE

(33.6 Ci/mmol) were from New England Nuclear. [

125I]CGP 23,996, a

relatively nonhydrolysable analogue of somatostatin, was prepared

according to Czernik and Petrack (1983). Inhibition studies

employing [

125I]CGP 23,996, [3

H]naloxone or [

3

H]DPDPE were

carried out as described elsewhere (Pelton et al. 1985a, b; Gulya et

al. 1986). [

3

H]CTOP binding was carried out in 50 mM Na/K-PO4

209
Table 1. IC50 value and selectivity of CTOP for

somatostatin, mu- and delta opioid receptors in

rat membrane preparation

IC50 values represent the geometric mean with the range

of values from at least three separate experiments done in

duplicates shown in parentheses. The average Hill values

represent the Hill coefficients from the unweighted bestfits of the computer-generated regression curves. N.D. =

not determined.

1

IC5O(somatostatin)/IC5O(mu); 2IC50(delta)/IC50(mu).

buffer (pH 7.4 at 25°C) containing 5 mM MgCl2, 1 mg/ml bovine

serum albumin (BSA) and 100 uM phenylmethylsulfonylfluoride (PMSF).

For kinetic studies or

[

3

H]CTOP binding,

experiments involving the inhibition of

a total concentration of 0.5 nM [

3

H]CTOP was

incubated with 400-450 ug protein. For saturation experiments,

twelve concentrations of [

3

H]CTOP ranging from 0.1 nM to 16 nM

were used. 1 uM naltrexone was used to determine nonspecific

binding. The reaction was terminated by rapid vacuum filtration

through 0.1% polyethylenimine-pretreated Whatman GF/B glass

filters. The filters were then rinsed three times with 3 ml saline

solution and dried. Scintillation fluid (6 ml) was added and the

samples were counted by liquid scintillation spectrophotometry (44%

efficiency).

RESULTS AND DISCUSSION

The potency and selectivity of CTOP on inhibiting somatostatin, muand delta opioid receptors are shown in table 1.

[

125I]CGP 23,996 and [3

H]DPDPE bindings were inhibited by CTOP

Specific

with low affinity, while [

3

H]naloxone binding was displaced with

high affinity from an apparently single population of binding sites

(nH = 0.89 ± 0.1). The exceptional selectivity of CTOP toward mu

opioid receptors (IC5O(delta)/IC5O(mu) = 4,840) is the result of

its high affinity for mu opioid receptors combined with very low

affinity for the delta sites. No substantial interaction to

somatostatin receptors was observed at low concentrations of CTOP.

Because of its high affinity and selectivity for the mu opioid

receptor binding sites, it seemed to be the ligand of choice for

further characterization of this class of opioid receptors using

direct radioligand binding studies.

210
Table 2. Determination of the nonspecific binding of

[

3

H]CTOP using selected mu opioid compounds in rat brain

membranes at 25°C

Displacer Nonspecific binding

drug (% of total)

naloxone 37

naltrexone 39

naloxonazine 40

DAG0 34

PL017 38

CTOP 24

[

3

H]CTOP (0.5 nM) was incubated with 400-450 ug protein

in 50 mM Na/K-PO4 buffer (pH 7.4 at 25°C) containing 5

mM MgCl2, 1 mg/ml BSA and 100 uM PMSF for 120 min at

25°C. Nonspecific binding was determined in the presence

of different putative mu opioid receptor-selective drugs at

a final concentration of 1 uM.

Table 3. Inhibition of specific [

3

H]CTOP binding by

selected opioid and non-opioid compounds in rat brain

membrane preparations

Drug IC50 (nM) Ki (nM) nH

opioids

naltrexone

naloxone

CTOP

DAGO

morphine

PLO17

DADLE

DSLET

DPDPE

0.37 (0.33-0.42) 0.26

0.97 (0.59-1.64) 0.67

4.62 (3.52-4.19) 2.19

1.33 (0.78-2.05) 0.92

7.32 (6.06-8.14) 5.08

44.1 (34.1-70.1) 30.4

14.3 (10.5-21.0) 9.9

46.5 (31.1-63.4) 32.1

845 (645-1,246) 583

1.112 0.06

1.084 0.08

1.344 0.25

0.826 0.12

0.713 0.04

0.707 0.03

0.817 0.13

0.883 0.20

0.711 0.11

non-opioids (at 10 uM) % of control

somatostatin 94

substance P 70

phenoxybenzamine 69

picrotoxin 68

atropine 86

The inhibition of [

3

H]CTOP binding by 1 uM naltrexone was used to

define nonspecific binding (table 2) to the mu opioid receptor since

CTOP inhibited [

3

H]CTOP binding an additional 15% from a putative

non-opioid site.

Results of kinetic experiments performed to determine association

(k+1) and dissociation (k-1) rate constants at 25°C show that

specific [

3

H]CTOP binding reached steady-state after 45 min of

211
incubation (k+1 = 2.04 M

-1 min-1), while dissociation of

specific [

3

H]CTOP binding was

value of 1.62x10-1 min-1.

rapid (t1/2=4.40 min) with a k-l

The kinetic Kd was found to be 0.67

nM at this temperature.

Computer-assisted nonlinear least squares regression analyses of

saturation isotherms of specific [

3

H]CTOP binding gave an apparent

dissociation constant (Kd) of 1.11 nM and a Bmax value (maximal

number of binding sites) of 136 13 fmoles/mg protein in rat brain

membranes.

Inhibition curves were obtained for several opioid and opiate

compounds as well as non-opioid drugs in order to determine the

specificity of [

3

H]CTOP binding to mu opioid receptors (table 3).

[

3

H]CTOP binding was displaced with high affinity (Ki values from

0.26 nM to 2.19 nM) by opioid antagonists such as naloxone,

naltrexone and CTOP from a single population of binding sites, while

putative mu opioid agonists such as

ol), morphine and PLO17 ([NMePhe3, D-Pro5]morphiceptin) had nH

DAGO (Tyr-D-Ala-Gly-NMePhe-Glyvalues significantly less than one, indicating the presence of

multiple affinity states of mu opioid receptors. Delta opioid

receptor-specific compounds like DADLE ([D-Ala2,

D-Leu5]enkephalin), DSLET ([D-Ser2

]Leu-enkephalin-Thr) or DPDPE

Inhibited [

3

H]CTOP binding with much lower affinity (Ki values

from 9.90 to 583 nM) and Hill coefficients less than one. Non-opioid

compounds were very weak in displacing [

3

H]CTOP binding, or they

were ineffective.

In summary, we designed and synthesized a somatostatin-related

conformationally restricted octapeptide analogue highly specific for

the mu opioid receptor site. This peptide antagonist has high

affinity and exceptional selectivity for this class of opioid

receptors virtually without any interaction to other neurotransmitter

systems at lower concentrations. This octapeptide might be the

ligand of choice for further characterization of mu opioid receptors.

REFERENCES

Czernik, A. and Petrack, B. J Biol Chem, 258:2325-2330, 1983.

Gulya, K., Pelton, J.T., Hruby, V.J. and Yamamura, H.I. Life Sci,

38:221-229, 1986.

Pelton, J.T., Gulya, K., Hruby, V.J. and Yamamura, H.I. Peptides,

6(Suppl. 1):159-163, 1985a.

Pelton, J.T., Gulya, K., Hruby, V.J., Duckles, S.P. and Yamamura, H.I.

Proc Natl Acad Sci USA 82:236-239, 1985b.

212
OPIOID AGONIST AND ANTAGONIST EFFECTS OF CONFIGURATIONAL

STEREOISOMERS OF 3-(DIMETHYLAMINO)-2,2-DIMETHYL-7-HYDROXY-1-TETRALOL

David A. Lippman, F. Fatima Matos* and Donald Y. Shirachi*

Synthetic Research Labs

1407 West Oak Street, Stockton, CA 95203, U.S.A.

*Department of Physiology and Pharmacology

University of the Pacific, Stockton, CA 95211, U.S.A.

ABSTRACT

The opioid agonist and antagonist effects of two configurational

aminotetralin stereoisomers, namely, the cis-(MRSAL) and trans-

(RMG) diastereo isomers of 3-(dimethylamino)-2,2-dimethyl-7-hydroxy1-tetralol were studied using the guinea pig ileum longitudinal

muscle preparation. MRSAL demonstrated opioid antagonist activity

but failed to show agonist effects. In contrast, RMG showed agonist

activity which was partially reversed by NX and MRSAL. RMG also exhibited weak antagonist activity towards DHM, NM and DADLE. These

results showed that MRSAL was an opioid antagonist while RMG possessed weak mixed opioid agonist-antagonist activities.

INTRODUCTION

Aminotetralins are considered A, B ring structural analogs of morphine. Martin and Kandeel (1973) showed that these compounds had

opioid agonist activity. Subsequently, Fries and Bertelli (1982)

synthesized aminotetralin analogs which were capable of producing

mixed opioid agonist-antagonist activities. More recently, we have

reported on several structural modifications of aminotetralins

(Lippman et al. 1985) , the most potent being 3-(dimethylamino)-2,2-

dimethyl-7-hydroxy-1-tetralone (J). In vitro and in vivo testing of

J showed only mu agonist activity. Reduction of the C-1 carbonyl on

J to a hydroxyl group resulted in an opioid antagonist, MRSAL

(Lippman et al. 1985/1986).

Recently we purified the diastereo isomers of 3-(dimethylamino)-2,2-

dimethyl-7-hydroxy-1-tetralol. We now report on the pharmacological

properties of these two isomers on the guinea pig ileum longitudinal

muscle (GPI).

MATERIALS AND METHODS

Male guinea pigs (400-500 g) were decapitated and the ileum longitudinal muscle was removed and prepared as described by Kosterlitz et

al. (1981). The longitudinal muscle with its myenteric plexus was

mounted in a 5 ml tissue bath (37°C) between two platinum electrodes

containing oxygenated (95% 02 :5% CO2 ) Krebs-bicarbonate buffer solution with the following composition (mM): NaCL, 118.40; KCl, 4.75;

CaCl2, 2.54; MgSO4, 1.20; KH2PO4, 1.19; NaHC03, 25; glucose, 11.10;

choline chloride, 20 uM; and pyrilamine maleate, 0.125 uM. The

muscle strip was connected to a Grass transducer (FT.03) and isometric contractions were induced by a Grass Model S44 stimulator at 0.1

Hz, 0.25 msec, 80-90v and recorded on a Grass Model 7B polygraph.

The tension was set at 1.0 g. After equilibrium was reached the

213
compounds were added to the bath and the effects recorded for 5 min.

In agonist experiments the maximum effect (5 min) of the compound

was recorded, then naloxone (NX) was added to the bath. In antagonist experiments, the compounds were incubated for 5 min before the

agonists prototypes dihydromorphine (DHM), normorphine (NM) and

DADLE were added to the bath.

The diastereo isomers were assigned relative configurations by FTNMR and mass spectroscopy.

RESULTS

The effects of MRSAL and RMG on the electrically stimulated contraction of the GPI are shown in table 1. RMG caused a maximal decrease

TABLE 1. Effects of MRSAL and RMG in the electricallystimulated guinea pig ileum.

Concentration % Change in Effect (mean S.E.)

(uM)

N MRSAL N RMG

42.5 19 3.21 0.5 -- NT

85.0 55 9.94 1.1 18 21.6 1.7

170.0 7 11.34 1.2 5 17.6 2.0

NT= not tested

in contraction of 21% at 85 uM, while MRSAL caused increases in contractions of 3-11% at concentrations of 42.5-170 uM. The maximal

agonist effect of RMG was partially antagonized by NX (53%) and

MRSAL (63%) (table 2). suggesting that RMG had some opioid agonist

Table 2. Antagonism of RMG agonist activity by naloxone

and MRSAL.

RMG N % Antagonist Concn %

(uM) Decrease Blockade

85.0 7 21.6 1.7 NX 176 nM 53.9 2.9

170.0 3 17.6 2.0 NX 704 nM 44.3 3.0

85.0 4 20.7 1.9 MRSAL 85 uM 63.3 3.1

% is expressed as the mean S.E.

activity. Both compounds were able to antagonize selected opioid

agonists (table 3). However, MRSAL was more potent than RMG. MRSAL

was able to antagonize nearly equally NM and DHM by 89% and DADLE by

214
81%, while RMG antagonized NM by 65%, DHM by 47% and DADLE by 37%.

The above data demonstrated that MRSAL was an opioid antagonist devoid of agonist activity, while RMG had mixed opioid agonist-antagonist activities at equiconcentrations of the two isomers.

Table 3. Effect of Opioid Antagonist Activity of MRSAL and

RMG Against Selected Opioid Agonists in guinea pig ileum.

Opioid N Opioid %

Antagonist Agonist Blockade

MRSAL 14 NM 89.9 1.9

MRSAL 4 DHM 89.0 1.8

MRSAL 7 DADLE 81.1 1.8

RMG 4 NM 65.5 7.7

RMG 6 DHM 47.8 3.8

RMG 3 DADLE 37.5 1.9

Concentration used for MRSAL and RMG was 85 uM. Results

are expressed as the mean S.E.

DISCUSSION

We had previously demonstrated that 3-(dimethylamino)-2,2-dimethyl7-hydroxy-1-tetralone (J) to be a mu opioid agonist. When the C-l

carbonyl on J was reduced to a hydroxyl, at least two diastereo

isomers were formed as shown in figure 1. When these isomers were

FIGURE 1. Cis-(MRSAL) and trans-(RMG) isomers of 3-(dimethylamino)-2,2-dimethyl-7-hydroxy-1-tetralol.

tested in the GPI bioassay in this study, it was clear that the agonist activity present in J was significantly decreased by this very

slight alteration in the molecular structure. The trans-isomer

(RMG) inhibited the muscle contraction only 21% at 85 uM of which

53% was reversed by NX as compared to 70% inhibition by J at 30 uM

and was completely reversed by NX (Matos et al. this volume). The

cis-isomer (MRSAL) was devoid of agonist activity and instead increased slightly the muscle contraction of the ileum. This latter

effect is similar to that of NX as has been shown by many investigators. When these two isomers were tested against prototype opioid

215
agonists, both isomers showed antagonist activity.

The orientation of the hydroxyl group about the C-l position appeared to be important for both agonist and antagonist activities. If

the hydroxyl was trans to the nitrogen, then agonist and antagonist

activities were conferred. Whereas if the hydroxyl was cis, only

antagonist activity was observed. In addition the cis-isomer reversed the opioid agonist activities of DHM, NM and DADLE more effectively than did the trans-isomer. Thus, it appeared that the preferred configuration for antagonism of opioid agonists was the cisisomer.

Since the optical isomers of these diastereo isomers were also pressent, final conclusions about antagonism of opioid agonists by these

compounds must await final determination of the absolute configuration of the active isomer. Nevertheless, since the cis-isomer effects in the GPI were very similar to that of NX, it is possible

that these two antagonists are acting via similar mechanisms at the

level of the mu-opioid receptor. If this is true, then these small

molecules might be good pharmacological tools to be used in increasing our knowledge about the interaction of opioid agonists and antagonists at the molecular level of the opioid receptor(s).

REFERENCES

Fries, D.S. and Bertelli, D.J. J Med Chem 258:216-220, 1982.

Lippman, D.A., Matos, F.F., Fries, D. and Shirachi, D.Y. Alcohol

and Drug Research, 6:208 (2-3), 1985/86.

Lippman, D.A., Matos, F.F., Fries, D., and Taubert, K. Proc

West Pharm Soc, 28:147-149, 1985.

Martin, A.R. and Kandeel E.M. J Med Chem 16:947-948, 1973.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. Jim Shoolery and Varian Scientific

for their assistance in obtaining and interpreting the FT-NMR data.

216
OPIOID RECEPTOR EFFECTS OF TWO 3-AMINO-2,2-DIMETHYLTETRALIN ANALOGS IN GUINEA PIG ILEUM LONGITUDINAL MUSCLE

F. Fatima Matos, David A. Lippman and Marvin H. Malone

Department of Physiology and Pharmacology

School of Pharmacy, University of the Pacific

Stockton CA 95211, U.S.A.

ABSTRACT

Two substituted analogs of 3-amino-2,2-dimethyltetralin,

namely 3-dimethylamino-2,2-dimethyl-7-hydroxy-1-tetralone HBr (J) and 3-dimethylamino-2,2-dimethyl-7-hydroxy1-tetralol (MRSAL), were evaluated for opioid agonist

and antagonist activity using the electrically driven

guinea pig ileum longitudinal muscle preparation (GPI).

Compound J appeared to be an opioid agonist with a

preference for mu receptors while MRSAL was an opioid

antagonist with-little selectivity for mu or kappa receptors,

INTRODUCTION

Aminotetralins with opioid analgesic activity were first

studied by Martin et al. (1969). Subsequently, Kandeel

and Martin (1973) reported a number of aminotetralins

with potent opioid agonist activity. Such compounds

also have the potential for opioid antagonist as well as

agonist activity. In the present report, two new aminotetralin analogs, J and MRSAL (figure 1), were studied

for both opioid agonist and antagonist activities using

FIGURE 1. Aminotetralin chemical structures.

the electrically stimulated GPI. Opioid receptor selectivity for the agonist was studied by using naloxone

(NX) as a prototype antagonist. The potency of MRSAL

antagonism was evaluated by using agonist prototypes

such as N-normorphine HCl (NM), dihydromorphine (DHM),

ethylketocyclazocine methanesulfonate (EKC) and eptides

such as dynorphin 1-13 (DYN 1-13) and (D-alanine2, Dleucine5

) enkephalin (DADLE).

MATERIALS AND METHODS

Male guinea pigs (400-550 g) were decapitated and ileums

217
removed. Each longitudinal muscle with myenteric plexus

attached was prepared as described by Kosterlitz et al.

(1970) from a 6-cm segment 10-15 cm proximal to the ileocaecal valve using the method of Su et al, (1981). The

muscle was set up between two platinum electrodes in a 5-

ml bath containing oxygenated (95% O2:5% CO2) Krebs bicarbonate buffer solution with the following composition

(mM): NaCl 118.4, KCl 4.75, CaCl2 2.54, MgSO4 1.2, KH2PO4

1.19, NaHCO3 25.0, glucose 11.1 plus choline chloride 20

μM and pyrilamine maleate 0,125 μM. The strip was connected to a transducer, allowed to equilibrate with constant electrical stimulation (0.1 Hz, 0.25 msec, 80-90 V)

and isometric contractions polygraphically recorded. The

resting tension was 1 g. After equilibrium, the agonists

were added to the bath and the effects recorded until the

maximum effect had been reached. The antagonists were

either preincubated for 3 min (NX) and 5 min (MRSAL) or

added after the agonist.

RESULTS AND DISCUSSION

The results of this study demonstrate that J behaved as

an opioid agonist producing a concentration-response related decrease in contractions with the effects antagonized by NX. On the other hand, MRSAL demonstrated significant opioid antagonist activity in a manner similar

to that of NX. Figure 2 shows the concentration-effect

agonist curves for NM (mu-receptor prototype) and NM

FIGURE 2. Agonist capacity in GPI. Slope (b)

values are: NM 38.6 (r-0.62, N = 300), MRSAL +

NM 42.9 (r = 0.86, N = 50), J 34.1 (r = 0.74,

N = 174), MRSAL + J 33.5 (r = 0.93, N = 49), NX

+ J 38.8 (r = 0.90, N = 57).

218
TABLE 1: Calculated pA2 values for NX and MRSAL

antagonism of selected opioid agonists

Antagonist Agonist N pA2 S.E.M.

NX NM 15 8.40 0.06

NX EKC 14 7.62 0.08

NX J 30 8.17 0.071

MRSAL NM 28 5.41 0.02

MRSAL DHM 21 5.53 0.05

MRSAL EKC 25 4.50 0.07

MRSAL J 20 5.09 0.04

MRSAL DYN 1-13 24 5.10 0.05

MRSAL DADLE 20 5.22 0.03

1

Significantly different from corresponding

values for NX + NM (P <0.05) and NX + EKC

(P <0.001).

antagonism by MRSAL. In addition, the concentrationeffect curve for J is shown and the antagonism of J by NX

and MRSAL. The concentrations (nM) of the agonists causing 50% inhibition of maximum response (IC50) as calculated from the respective regression-line equations are:

NM 110.2, DHM 45.1, EKC 1.3, DYN 1-13 1.8, DADLE 19.9

and J 9625.0. Compound J was completely and reversibly

antagonized by low concentrations of NX (88 nM) suggesting a specificity for mu receptors (pA2 = 8.17, table 1).

The pA2 values for MRSAL antagonism of selected opioid

agonists are also shown in table 1. MRSAL demonstrated

low affinity but behaved as a full antagonist of opioid

receptors. Its highest affinity was seen toward mu rather than kappa opioid receptors. Prototypes selective for

mu receptors (NM, DHM) and J were antagonized with concentrations of 21.2 μM while kappa agonists (EKC, DYN

1-13) required higher concentrations such as 85 μM.

ACKNOWLEDGEMENT

Supported in part, by Conselho National de Pesquisa of

Brazil (CNPq).

REFERENCES

Kandeel, E.M., and Martin, A.R. J Med Chem, 16:947-948,

1973.

Kosterlitz, H.W., Lydon, R.J., and Watt, A.J. Br J

Pharmacol, 39:398-413, 1970.

Martin, A.R., Parulkar, A.P., Gusseck, D.J., Anderson,

L.J., Grunewald, G.L., and White, A.I. J Pharm Sci,

58:340-347, 1969.

Su, T.-P., Clements, T.H., and Gorodetzky, C.W., Life Sci,

28:2519-2528, 1981.

219
PREPARATION OF /3

H/-OXYMORPHAZONE AND ITS BINDING TO RAT BRAIN

MEMBRANES

A. Borsodi, E. Varga, G. Tothx, S.Hosztafixx

Institute of Biochemistry and xIsotope Laboratory, Biological

Research Center of the Hungarian Academy of Sciences, 6701

Szeged, P.O.B. 521. Hungary

xxAlkaloida Chemical Works, 4440 Tiszavasvari, Hungary

ABSTRACT

Oxymorphazone is a 14-hydroxydihydromorphinone derivative which

contains C-6 hydrazone group and hence could serve as an irreversible

lahel for opioid receptors.

3

H-oxynorphazone was synthesized by the reaction of 3H-oxymorphone

with excess hytirazine, with 640 GBq/mmol specific radioactivity. Both

the unlabelled compound and the tritiated ligand show high affinity to

mu and kappa opiate receptor subtypes in rat brain membranes. TWO

binding sites were detected by equilibrium binding studies. About 60%

of the 3H-oxymorphazone specific binding is irreversible at 10nM

ligand concentration. Preincubation of the membranes with unlabelled

oxymorphazone resulted in an irreversible blockade of the high

affinity 3H-naloxone binding sites.

INTRODUCTION

Using ligands of high specific radioactivity two classes of binding

sites - low and high affinity - is detectable for mu and delta

opioid receptor subtypes. 14-Hydroxydihydromorphinone hydrazones and

azines, first synthesized by Pasternak et al.(1980) have been proved

to be a useful tools in elucidation of their pharmacological role and

involvement in opiate analgesia.

For the detailed investigation of the mechanism and kinetics of the

irreversible binding tritiated oxymorphazone was needed.

We have synthesized 3H-oxymorphazone and studied its binding

characteristics on rat brain membranes.

MATERIALS AND METHODS

Synthesis of 8-3

H-oxymorphazone:

a/ Synthesis of 8-3H-oxymorphone: The precursor molecule (14-hydroxycodeinone) was prepared by the method of Iiyima et al.(1978), and Odemethylated by BBr3 to give 14-hydroxy-morphinone, which was

tritiated in the psesence of PdO catalyst.

b/ Synthesis of 8-3H-oxymorphazone:

The product was reacted with tenfold excess of hydrazine at room

temperature. Specific radioactivity of 640 GBq/mmol was obtained.

220
Chemicals:

3

H-EKC was purchased from New England Nuclear (specific activity: 0.74

TBq/mmol), 3H-DAGO was from Amersham (specific activity: 1.67

TBq/mmol), 3H-DALE (1.37 TBq/mmol) and 3H-naloxone (3.09 TBq/mmol) was

synthesized by Toth (1982,1985). DADLE was purchased from Sigma,

morphine was a commercial product. Unlabelled oxymorphazone was

synthesized by our laboratory, all other chemicals were of analytical

grade.

Membrane preparation:

Crude membrane fraction from rat (PVG/C) brain was prepared as

described earlier (Simon et al. 1986). Protein content was estimated

by the method of Bradford (1976).

Binding Assay:

Membrane suspension in 50 mM Tris-HCl buffer, (pH=7.4) was incubated

with 3H-oxymorphazone in the presence or absence of unlabelled ligands

in a final volume of 1 ml. Specific binding was defined as the

difference between the bound radioactivity in the absence and presence

of unlabelled ligand. Incubation was started by addition of the

membrane protein and stopped by rapid filtration of the mixtures

through Whatman GF/C filters, which were presoaked in 10 μM unlabelled

oxymorphazone solution for 1 hour.

Radioactivity was determined in toluene based scintillation cocktail

by LKB Minibeta Liquid scintillation spectrometer.

Irreversibility studies:

500 ul of membrane suspension was incubated with 200 μl 3Hoxymorphazone solution in the presence or absence of excess

levorphanol (10 μM)in a final volume of 2 ml. After 90 min

incubation at 0°C samples were diluted with ice cold Tris-HCl buffer

and centrifuged. The pellets were resuspended in 30 ml buffer,

incubated for 10 min and recentrifuged. This washing step was

repeated 4 times. 1 ml aliquots of the homogenized final pellets were

filtered through presoaked GF/C filters. The washing experiments with

unlabelled ligands were performed as previously described (Szucs et

al. 1983).

RESULTS AND DISCUSSION

The binding reached equilibrium at about 90 min at 0°C. All further

experiments were carried out at 0°C in order to minimize the

possibility of azine formation.

Dissociation of the radiolabelled ligand was extremely slow as

measured by dilution or after addition of excess of reversible

(levorphanol) or irreversible (unlabelled oxymorphazone) compounds

(data not shown).

Scatchard analysis of 3-H-oxymorphazone saturation curve is shown on

fig.1. On the basis of computerized fit of data two classes of

binding sites were identified with dissociation constants of 0.62 and

28 nM, and Bmax: 18 and 134 fmol/mg respectively.

221
FIGURE 1.

of 3H-oxymorphazone

Scatchard plot

binding.

Rat brain membranes were

incubated at 0°C for 90

min with 3H-oxymorphazone

in the presence or absence

of 10 μM unlabelled

oxymorphazone. B: bound

ligand (fmol/mg protein)

F: free ligand (nM).

Competition experiments were carried out with 3H-oxynorphazone and

with sub-type specific tritiated ligands (Kosterlitz et al. 1951).

From these data it became evident that oxymorphazone exhibits high

extremely low affinity for delta (IC50:2000 nM) sites.

affinity for mu IC50:0,4 nM) and kappa (IC50:2 nM) sites and

FIGURE 2.

of 3H-oxymorphazone

Irreversibility

binding.

Irreversible labelling of

rat brain membranes was

carried out as described

in Materials and Methods.

Control values repres nt

specific binding of 3Hoxymorphazone on membrane

preparations incubated

with buffer and washed in

the same way.

As shown in fig. 2. the binding of 3H-oxymorphazone is irreversible

in about 20% at 1nM isotope concentration despite of extensive

washings, and this irreversible binding greatly increased at higher

concentrations. The irreversible binding blocks mainly the high

affinity (mu,) opioid binding site as shown in fig. 3.

222
FIGURE 3. Scatchard

analysis of 3H-naloxone

binding.

Rat brain membranes

preincubated with 10 μM

oxymorphazone were

labelled with naloxone

after extensive washing

Control was treated

in the same way without

addition of oxymorphazone

In this case the membrane was incubated with unlabelled

oxymorphazone (10 μM) and the remaining 3H-naloxone binding was

measured. These results are in agreement of earlier investigations

(Pasternak et al. 1980).

REFERENCES

Benyhe, S., Toth, G., Kevei, J., Szucs, M., Borsodi, A., DiGleria,

K., Szecsi, J., Suli-Vargha, Ii., Medzihradszky, K., Neurochem

Res, 10:627-635, 1935.

Bradford, M. Anal Biochem, 72:248-251, 1976.

Iijima, I., Minamikowa, J., Jacobson, A.A., Brossi, A., Rice K.C.

J Med Chem, 21:398-400, 1978.

Kosterlitz, H., Paterson, S., Robson L. Br J Pharmacol, 73:939-949,

1981.

Pasternak, G.W., Hahn E.F. J Med Chem, 23:674-676, 1980.

Pasternak, G.W., Childers, S.R., Snyder S.H. J Pharm Exp Ther,

214:455-462, 1980.

Toth, G.. Kramer, M., Sirokman, F., Borsodi, A., Ronai A. J Label

Comp Radiopharm, 14:1021-1030, 1982.

Simon, J., Benyhe, S., Abutidze, K., Borsodi, A., Szucs, M., Toth,

G. , Wolleman M. J Neurochem, 46:695-701, 1986.

Szucs, M., DiGleria, K., Medzihradszky K. Life Sci, 33:435-438,

1983.

ACKNOWLEDGEMENT

Levorphanol donated by Hofmann La Roche, EKC provided hy Sterling

Winthorp Institute and naloxone supplied by DuPont de Nemours are

greatly acknowledged. We thank Dr. Cs. Bagyinka for help with

computer programs.

223
COMPARISON OF THE OPIOID ACTIVITY OF CAM1

 AND KAPPA AGONISTS

Susanna Furst, Thomas Friedmann, Aniko Kovacs and Tibor Wagner

Department of Pharmacology, Semmelweis University of Medicine

1089 Budapest, Nagyvarad ter 4. Hungary

ABSTRACT

CAM has been shown to produce opiate agonist and antagonist activities. Based on its potent agonist activity in rabbit vas deferens

and the low potency of naloxone to antagonize its agonist effect in

the guinea pig ileum and mouse vas deferens, CAM can be considered

as kappa agonist. It is further supported by its diuretic effect and

the estimated "apparent pA2" values with naloxone and Mr-2266BS. However, in the rabbit ear artery and cat nictitating membrane, ethylketazocine (EK) produced agonist activity, while CAM produced antagonist activity, Also, EK differs from CAM in mouse vas deferens in

the presence of 4-aminopyridine. It is concluded, that CAM might be

an agonist on kappa type (subtype) of opiate receptors, although its

spectrum of activity was found to be different from EK or hremazocine.

INTRODUCTION

The most widely accepted classification of opioid receptors (OR) is

mu, kappa and delta (Iwamoto and Martin 1981, Lord et al. 1977). Previously we reported the peculiar spectrum of activity of CAM, producing strong agonist and antagonist effects in several in vivo and in

vitro tests /Knoll 1977, Furst and Knoll 1982, 1985). In an attempt

to define OR subtype(s) involved in the effects of CAM, various isolated organ preparations, containing different OR-s were used, together with some in vivo tests. The sensitivity to naloxone (NX) and

Mr-2266BS was also studied in various assays.

MATERIALS AND METHODS

The effects of the test substances on OR-s in isolated tissue preparations were compared using the electrically stimulated myenteric

plexus-longitudinal muscle of the guinea pig ileum (GPI), electrically

stimulated mouse, rat and rabbit vasa deferentia (MVD, RVD and LVD,

resp.), electrically stimulated isolated cat nictitating membrane

(CNM) and rabbit ear artery (REA) with the following parameters;

0.1 HZ, 1 ms (GPI,RVD); 0.01 HZ, 1 ms (CNM); 0.05 HZ, 1.0 ms (LVD) 3

pulses of 10 Hz, 1.0 ms per 10 s (MVD); 5 pulses of 5 Hz, 1.0 ms per

60 s (REA). Supramaximal voltage of field stimulation was used in all

experiments. The antinociceptive activity of the test substances was

assessed using the acetic acid writhing test, in the mouse. Urine

output measures were carried out according to Leander (1983a). Intrathecal injections (i.t.) were made to rats as described by Yaksh and

Rudy (1976).

Drugs: Morphine HCl, normorphine, N-cyclopropylmethylnorazidodihydroisomorphine (Alkaloida, Hungary), naloxone HCl (Endo), ethylketazocine

EK (Sterling Winthrop), Mr-2266BS (Boehringer, Ingelheim) hremazocine

1

N-cyclopropylmethylnorazidodihydroisomorphine

224
HCl, BR (Sandoz) 4-aminopyridine 4-AP (Sigma), ThiobutabarbitalNatrium (INACTIN, Byk-Gulden, Hamburg), [D-Ala2

,Leu5] enkephalin

(DALE), [D-Ala2, D-Leu5] enkephalin (DADL), [D-Ala2, MePhe4, Gly-ol5]

enkephalin (DAGO). (Central Res. Inst. for Chemistry, Budapest,

Hungary)

RESULTS AND DISCUSSION

The rabbit vas deferens has been shown to be especially useful for

identifying kappa agonists. Such compounds are potent inhibitors of

electrically induced contraction in this tissue, whereas mu and

delta agonists are virtually ineffective (Oka 1981). CAM vas found to

be agonist, similarly to EK and other kappa agonists (table 1). For

the antagonism of the agonist action of CAM and EK in GPI, 10-11

and in MVD 6-7 times more NX was required than was needed to

TABLE 1. The agonist activities of CAM and EK in isolated organs (IC50;nM S.E.M.)

GPI MVD

4-AP1

LVD CNM

control

CAM 0,3 0.07 1.7 0.2 * 18.3 1.6 **

E K 1.2 0.22 3.9 0.4 19.8 5.3 5.7 0.3 3.8 21.0

*the maximum inhibition <40% 1with 4-AP 2x10-5M

**agonist activity undetectable n:5-12

antagonize the effect of normorphine. In RVD, CAM and EK had no

agonist activities but antagonized the effect of DALE. However, in

REA and CNM, EK produced opiate agonist action while CAM showed only

antagonist activity. In CNM the agonist actions of morphine, DALE

and EK were antagonized by CAM. In REA the mu agonists normorphine

and DAGO did not show agonist effect. The delta agonist DADL was

FIGURE 1. The effects of various doses of CAM S.C.

i.t. nalorphine S.C. bremazocine, S.C. on

cumulative urine output at 2 hr (left) and 5 hr (right)

in the Sprague Dawley rats weighing between 160-220 g.

The points and bars are the mean S.E. for 8-14 rats.

Controls above 0 

225
potent agonist indicating a lack of mu receptors in this preparation

supporting the existence of enkephalinergic receptors as it was

first suggested by Knoll (1976). The difference observed between the

effects of CAM and EK was further supported by the facts that in MVD

in the presence of 4-AP (2x10-5M) which increased the electrically

evoked noradrenaline release (Friedmann et al. 1994) CAM proved to

be only partial agonist. When the maximal agonist effect of CAM was

already reached (<40%) DADL and EK further reduced the contractions.

In the presence of 4-AP, CAM (2.5x10-7M) diminished the marked

agonist actions of DADL and EK. Recently, it has been shown (Slizgi

and Ludens 1982, Leander 1983a) that kappa opioid agonists -such as

EK, BR, etc. - have a marked diuretic effect in rat, and it was

antagonized by opioid antagonists in a potency order which indicated

that the effects were due to an action at kappa OR-s. Figure 1 shows

the effect of CAM on urine output in normally hydrated rats, compared

to other drugs. CAM produced dose related (0.01-2.5 mg kg-1) diuresis

similarly to BR, by S.C. administration, This effect was more pronounced when CAM was given i.t. This diuretic effect was antagonized

by kappa antagonist dose of NX(10 mg kg-1, 0.5-2.5 mg kg-1 dose of

Mr-2266BS, and blocked by 20 mg kg-1 morphine administration

(table 2). Mr-2266BS was more potent than NX in antagonizing the

TABLE 2. Antagonism of CAM (0.5 mg kg-1 s.c.) induced

urination. Data are the mean S.E. in mls for 8-15 rats.

dose mg kg-1 0.5 mg kg-1CAM + antagonists

of antagonist 2 hr 5 hr

+ 0 (control) 6.6 0.3 7.7 0.6

+ 1 naloxone 5.6 0.8 6.8 0.6

+10 naloxone 3.4 0.5 4.7 0.4

+2.5 Mr-2266BS 1.19 0.2 2.3 0.25

+20 morphine 1.73 0.2 5.5 3.6

diuretic effect of CAM, as it was found against BR and U-50488H,

reported by Leander (1983b). It is interesting, however, that while

the diuretic effects of two other kappa agonists: proxorphan or BR

are not affected significantly by morphine, supposingly because of

TABLE 3. Sedative effects of morphine, CAM and bremazocine and their antagonism by naloxone and Mr-2266BS

(n=10 rat/dose)

increase of control sleeping time. The AD50 is defined

as the dose of the antagonist needed to reduce by 50% the

narcosis potentiating effect of equi-active doses (ED90)

of the agonist.

A D 5 0

(mg kg-1 naloxone 1.2 3.8 3.5

s.c.) MR-2266BS 0.1 0.1 0.3

The ED50 is defined as the dose that induced a fivefold

morphine CAM bremazocine

ED50 (mg kg-1i.v.) 0.7 0.28 0.036

226
effects by naloxone and Mr-2266BS is also presented in table 3. It is

remarkable, that Mr-2266RS was 10 times more potent than NX in antagonizing morphine and BR induced prolongation of sleeping time. Our

data demonstrate that the opioid agonist effects of CAM are mediated

by similar type of receptor (kappa or subtype of kappa receptors) as

the effects of classical kappa agonists: EK or BR in GPI, LVD and MVD

preparations and in the sedation, diuresis and writhing test. However,

CAM failed to show agonist activity in CNM and REA and we could not

elicit full effect in MVD in the presence of 4-AP in tests where the

classical kappa agonist drugs e.g. EK or BR proved to be agonists.

These results indicate the existence of OR population of kappa receptor subtype/s/ or others/?/ which are stimulated by EK or BR but not

by CAM. In the RVD, BR, CAM and EK in some analgesic-tests CAM (Furst

and Knoll 1985) and BR (unpublished observation) proved to be antagonists while in some assays, where CAM showed antagonistic activity

(CNM, REA and MVD in the presence of 4-AP) EK did not (mu, delta,

kappa subtype). In the latter two tests BR also exhibited antagonist activity (unpublished data).

their mu opioid receptor antagonist activity (Leander 1983b). The

effect of CAM was found to be suppressed by morphine in spite of its

strong mu antagonist activity demonstrated by several tests, as it

was published elsewhere (Furst and Knoll 1982, 1985) and in this

paper. The analgesic effect of CAM in the writhing test was found to

be 37 times stronger than that of morphine (ED50 s.c.: 0.015 and

0.43 mg kg-1 resp.) The pA2 for naloxone antagonism of morphine(6.9)

was much higher than that for CAM (5,6). Conversely, Mr-2266BS a

reported kappa antagonist (Romer et al. 1980), has a higher pA2 for

CAM (6.2) than for morphine (5.8) in the acetic acid induced writhing

test. This observation also supports the concept, that CAM and

morphine exert their analgesic effect through different receptors.

As another possible measurement of kappa activity, we also evaluated

the sedative effects of this class of drugs, as the prolongation of

barbiturate narcosis (table 3.) BR was observed as the most potent

drug, while morphine the less active one. The reversal of these

REFERENCES

Friedmann, T., Kerecsen, L. and Knoll, J. IUPHAR 9

th Int Cong Pharm

Abstr. 1409P, 1984.

Furst, S., and Knoll, J. Pol J Pharmacol Pharm, 34:115-125, 1982.

Furst, S., and Knoll, J. Int J Tiss Reac, VII/1/:69-77, 1985.

Iwamoto, E.T., and Martin, W.R. Med Res Rev, 1:411-440, 1981.

Knoll, J. Eur J Pharmacol, 39:403-401, 1976.

Knoll, J. Pol J Pharmacol Pharm, 29:165-176, 1977.

Leander, J.D. J Pharmacol Exp Ther, 224:89-94, 1983a.

Leander, J.D. J Pharmacol Exp Ther, 227:35-41, 1983b.

Lord. J.A.H.. Waterfield, A.A.. Hughes. J. and Kosterlitz, H.W.

Nature (London), 267:495-499, 1977.

Oka, T., Negishi, K., Suda, M., Matsumiya, T., Inazu, T. and Ueki, M. Eur

J Pharmacol, 73:235-236, 1981.

Romer, D., Buscher, H., Hill, R.C., Maurer,R.,Petcher,T.J.,Welle,H.B.A.,

Bakel, H C.C.K. and Akkerman, A.M. Life Sci, 27:971-978, 1980.

Slizgi, G.R., and Ludens,J.H. J Pharmacol Ther, 220:585-591, 1982.

Yaksh, T.L., and Rudy, T.A. Physiol Behav, 17:1031-1036, 1976.

ACKNOWLEDGEMENTS

The authors thanks Dr.H.Merz, Boehringer Ingelheim am-Rhein, GFR,

for the gift of Mr-2266BS.

227
COMPARATIVE EFFECTS OF DYNORPHIN A(1-9) PYRROLIDINE

ANALOGS ON ISOLATED TISSUES

Ahmad Rezvani, Srinivasa R. Kadambi*,

Donald L. Rhoads, J. K. Chang* and E. Leong Way

Department of Pharmacology

School of Medicine, Univesity of California,

San Francisco, CA 94143, USA

*Peninsula Laboratories, Inc., Belmont, CA 94002, USA

ABSTRACT

Three dynorphin A(1-9) pyrrolidine analogs were

synthesized by substituting isoleucine at position 8

with alanine (Ala), D-alanine (D-ala), and D-leucine

(D-leu), and assayed for their effect on electrically

induced twitches of the isolated guinea pig ileum and

mouse vas deferens. All three dynorphin A(1-9)

pyrrolidine analogs caused inhibition of the evoked

twitches of the two preparations dose-dependently that

was reversed by naloxone. However, the substitution by

alanine or D-alanine resulted in a decrease in potency

on guinea pig ileum when compared to dynorphin A(1-9)

pyrrolidine and substitution by D-leucine caused a

considerable loss of potency in both mouse vas deferens

and guinea pig ileum. Although these substitutions

reduced overall potency, a change of potency ratio

towards more delta selectivity resulted.

INTRODUCTION

It has been shown that incorporating amino-acids at

different positions of dynorphin A(1-13) and its

fragments greatly modifies potency and affinity of the

resulting peptides to multiple opiate receptors.

For example, Chavkin and Goldstein (1981) reported

that substitutions at arg7 and lys11 reduce

selective binding of dynorphin in the guinea pig ileum.

Also, Rezvani et al. (1984) evaluated seven dynorphin

A(1-9) analogs on the guinea pig ileum and mouse vas

deferens and concluded that protection of the carboxy

terminal with a pyrrolidine group results in

prolongation of dynorphin A(1-9) action. Furthermore,

Turcotte et al. (1984) replaced the amino acids in

positions 1-12 of dynorphin A(1-13) by ala and concluded

that only replacement at position 8 results in a higher

potency of the peptide in the mouse vas deferens. In

order to investigate whether other substitutions at

position 8 will change the potency of dynorphin (1-9) we

have synthesized three dynorphin A(1-9) pyrrolidine

analogs by substituting iso-leucine at position 8 with

228
alanine, D-alanine or D-leucine and evaluated their

effects on the guinea pig ileum and mouse vas deferens.

The results are presented in this communication.

MATERIALS AND METHODS

Ala8 dynorphin A (1-91, D-ala8 dynorphin A(1-9) and

D-leu8 dynorphin A(1-9) were synthesized by the solid

phase method and the pyrrolidine group was attached by

treatment of the peptide-resins with pyrrolidine-DMF

(1:1 mixture). The peptides were purified by

reverse-phase column chromatography using C-18 resin.

The purity was checked by HPLC, TLC and electrophoresis.

The isolated longitudinal muscle with the attached

myenteric plexus from the guinea pig ileum was mounted

in a chamber containing Ringers' solution and the

excised mouse vas deferens in Krebs' solution. The

tissues were stimulated electrically and the induced

twitches recorded. Agonists were added to the media in

increasing concentrations and a dose response curve was

plotted. The concentration which inhibits maximally

induced contractions by one-half (IC5O) was then

determined from the dose response curve.

RESULTS

High performance liquid chromatography showed the three

peptides to be pure as evidenced by a single peak for

each (figure 1).

FIGURE 1: High performance liquid

chromatography of three dynorphin A(1-9)

pyrrolidine analogs

229
All three dynorphin A(1-9) pyrrolidine analogs

inhibited electrically induced twitches of the guinea

pig ileum and the mouse vas deferens dose-dependently.

Ala8 dynorphin A(1-9) pyrrolidine showed the highest

potency in the guinea pig ileum, while D-ala8

dynorphin A(1-9) pyrrolidine and Ala8 dynorphin A

(1-9) pyrrolidine showed the highest potency in the

mouse vas deferens. However, based on their relative

activities in both tissues the D-ala8 analog exhibited

more selective delta activity. D-leucine8 dynorphin A

(1-9) pyrrolidine showed the lowest potency both in the

guinea pig ileum and mouse vas deferens. The IC50 and

relative potencies of three peptides in guinea pig ileum

and mouse vas deferens are shown in Table 1. The effect

of all three compounds could be blocked by naloxone.

TABLE 1. Potency of dynorphin A(1-9)

pyrrolidine analogs on the guinea pig ileum

and mouse vas deferens

Peptide IC50 nM

GPI MVD Ratio

G/M

2.6

10

0.2

0.04

0.2

ALA8dynorphin A(1-9) pyrrolidine 200 75

D-ALA8dynorphin A(1-9) pyrrolidine 750 75

D-LEU8 dynorphin A(1-9) pyrrolidine 250 1250

Dynorphin A(1-9) pyrrolidine 7.5 160

Dynorphin A(1-13) 0.8 2.8

DISCUSSION

Our results demonstrate that all three modified

dynorphin A(1-9) pyrrolidine analogs inhibit

electrically induced twitches of the guinea pig ileum

and mouse vas deferens in a dose-dependent manner.

However, the substitution of iso-leucine by alanine or

D-alanine decreases potency in the guinea pig ileum but

increases potency in the mouse vas deferens. These

results are consistent with those obtained by Turcotte

230
et al. (1984) who observed an increase in potency for

dynorphin A(1-13) in mouse vas deferens by replacing

iso-leucine with alanine at position 8. Other

substitutions on dynorphin A(1-13) resulted in decreased

potency in both the guinea pig ileum and mouse vas

deferens. For example, replacement of residues at

positions 1,2, and 4 inside the leu-enkephalin core

causes a decrease in biological activity (Turcotte et

al. 1984), and positions 6 and 7 are found to be the

most important site outside the enkephalin moiety

(Chavkin et al.1982) for biological activity.

With respect to substitution at position 8,

replacement of iso-leucine by D-leucine causes a

decrease of potency of the resulting peptides both in

the guinea pig ileum and mouse vas deferens. On the

other hand, substitution of iso-leucine by alanine or

D-alanine, seems to favor delta-like activity, since the

alteration decreases potency in the guinea pig ileum

much greater than in the mouse vas deferens.

REFERENCES

Chavkin, C. and Goldstein, A. Proc Natl Acad Sci

USA, 78:6543-6547, 1981.

Chavkin, C., James, I.F., Goldstein, A. Science,

215:413-14 1982.

Rezvani, A., Miller, J., Kadambi, S.R., Chang, J.K.,

Lee, N.M., Way, E.L. Neuropeptides, 5:153-156, 1984.

Turcotte, A., Lalonde, J.M., St. Pierre, S., Lemaire,

S. Int J Pevtide Protein Res, 23:361-369, 1984.

ACKNOWLEDGEMENTS

This study was supported by Grant DA03777 from the

National Institute of Drug Abuse.

231
125I-DPDYN, MONOIODO [D-PRO10]- DYNORPHIN (1-11), IS AN EFFECTIVE AND

USEFUL TOOL FOR THE STUDY OF KAPPA OPIOID RECEPTORS.

Jean Edouard Gairin, Catherine Jomary

Jean Cros and Jean-Claude Meunier

Laboratoire de Pharmacologic et Toxicologic Fondamentales, CNRS

205, route de Narbonne - 31077 Toulouse, France.

ABSTRACT

Iodination of the -selectfve peptide DPDYN, [D-Pro10]-dynorphin

(1-11), has been performed. The non radioactive monoiodo derivative of

DPDYN retains -selectivity ( /μ = 48 and = 140), despite a

general but moderate decrease in affinity. Radioiodination of DPDYN

leads to the monoiodinated peptide (S,A 700-800 Ci/mmol) which

interacts specifically and reversibly with the -sites in guinea-pig

cerebellum membranes with high affinity (KD = 0.12-0.18 nM). In

guinea-pig brain ( ) and rabbit cerebellum ( <<μ), 125I-DPDYN

discriminates between - and other (μ, ) binding sites. We have used

this new labelled probe for the direct, precise and rapid (exposure

time less than 100 hours) visualization of -sites in guinea-pig and

rabbit cerebellar slices using autoradiography.

INTRODUCTION

Biochemical and pharmacological studies on a particular opioid

receptor type (μ, or ) are greatly

ligands. DPDYN ([D-Pro10]-dynorphin (1-11)),

facilitated by use of selective

derived from dynorphin A,

displays high affinity and selectivity for the opioid receptor type

(Gairin et al. 1984). So, it was of interest to obtain a labelled

K-probe derived from this selective ligand. Here we would like to show

that the radiolabelled monoiodo derivative 125I-DPDYN appears to be an

effective and useful tool for the biochemical studies on kappa opioid

receptors.

MATERIALS AND METHODS

Preparation of iodinated derivatives of DPDYN.

Radioiodination of DPDYN was performed as described elsewhere (Gairin

et al. 1986). Non radioactive iodination was performed according to

the same method however on milligram quantities.

Binding studies

Crude membrane fractions were prepared according to Meunier et al.

(1983). Affinities of DPDYN and of its non radioactive mono and diiodo

derivatives at μ, and sites were determined by measuring their

inhibitory effects respectively on the binding of [

3

H]-DAGO (1.5 nM)

and [3

H]-DSLET (2.5 nM) in rat brain membranes and of [3H]-bremazocine

(0.5 nM) in guinea-pig cerebellum membranes at 25°C in tris-HCl

pH 7.4. Saturation and kinetic binding studies of 125I-DPDYN to

-sites were carried out on crude guinea-pig cerebellum membranes at

25°C as previously described (Gairin et al. 1986).

Autoradiographic studies

After preincubation at room temperature in 50 mM tris-HCl buffer

pH 7.4, slices (20 μm section) of guinea-pig or rabbit cerebellum were

incubated with 125I-DPDYN for 40 min. at 25°C in the presence (non

specific binding) or absence (total binding) of 1μM unlabelled

232
peptide. Sections were then washed (6 x 40 min.) in cold tris-HCl

buffer -2 % BSA and quickly dried. Autoradiograms were exposed for 3-5

days at 4°C.

RESULTS AND DISCUSSION

Binding properties of mono an diiodo derivatives of DPDYN

As shown in table 1, incorporation of one iodine atom on the tyrosyl

phenolic ring of DPDYN results in a decrease of affinity for binding

site (KI = 0.38 nM). Interestingly, weaker affinities are also

observed for μ- and binding sites, so that selectivity /μ = 48

and = 140) is preserved. Incorporation of an additional bulky

iodine atom (diiodo derivative) leads to a further decrease in

affinity and selectivity of the peptide for binding sites.

TABLE 1. Binding selectivity profilesa of DPDYN and of its non

radioactive mono- and diiodo- derivatives.

peptide Binding site

μ

DPDYN 62 233 [10.49]

monoiodo-DPDYN 48 139 [ 9.42]

diiodo-DPDYN 25 102 [ 8.57]

a

Number in brackets is -log Ki(nM) at the preferred site.

Other numbers are binding selectivities defined as the ratio of

Ki at each non-preferred site over Ki at the preferred site.

Saturation equilibrium and kinetic binding studies

Equilibrium binding experiments have been performed at 25°C in

guinea-pig cerebellum membranes, containing essentially (> 80 %)

binding sites. The linear Scatchard transformation of the data

indicates that 125I-DPDYN interacts specifically with one (kappa)

single class of site with high affinity (KD = 0.12 nM).The maximum

binding capacity value (Bmax = 120 fmol/mg protein is quite

comparable to the ones observed by Robson et al. (1984) (110 fmol/mg

protein) or Frances et al. (1985) (180 fmol/mg protein) in the same

tissue preparation. The low proportion of other opioid sites (μ or

was not detected.

TABLE 2. Parameters of the binding of 125I-DPDYN in guinea-pig

cerebellum membranes at 25°C.

parameter value

thermodynamic

kinetic

Bmax 118 11 fmol/mg protein

KD 0.12 0.01 nM

k a

1.9 0.2 x 107

 M-1s

-1

k d 3.5 0.2 x 10-3 s-l

KD=kd/ka 0.18 nM

Equilibrium of the binding of 125I-DPDYN (0.2 nM) to guinea-pig brain

membranes is reached after 10-15 minutes at 25°C. In the presence of

233
an excess of unlabelled peptide, 125I-DPDYN dissociates from its

complex with the receptor,

The linear

as a result of a reversible binding

process. semi-logarithmic representation of the

dissociation kinetic data indicates a first-order reaction. The

calculated association and dissociation rate constants (ka,kd) are

reported in table 2. It is interesting to note that KD values from

kinetics or equilibrium experiments are very close to ea

and 0.12 nM) and in good agreement with the KI

ch other (0.18

value (0.38 nM) of the

non radioactive monoiodo derivative at -site s .

Specific binding of 125I-DPDYN (0.15 nM) to guinea-pig brain and

rabbit cerebellum membranes

In guinea-pig brain, containing equal proportions of μ-, and

SiteS, 125 I-DPDYN (0.15 nM) binds selectively to sites. As shown

in table 3, the binding capacities of the labelled peptide are

rigorously comparable without (B (μ + + ) = fmol/mg protein)

or with suppression of μ- and binding 7 1 9 fmol/mg

protein). No specific binding at μ- or sites can be detected after

suppression of binding. A comparable binding pattern is obtained in

the rabbit cerebellum containing essentially (> 75 %) u-binding sites

and low content (< 20 %) of sites.

TABLE 3. Binding capacity (fmol/mg protein) of 125I-DPDYN

(0.15nM) at 25°C pH 7.4 to crude membrane fractions of

guinea-pig brain and rabbit cerebellum in presence (single

binding site) or absence (multiple binding sites) of selective

(μ, o r ) unlabelled liganda.

multiple binding sites single binding site

μ + + μ

guinea-pig brain 73 9 n.d. n.d. 71 9

rabbit cerebellum 63 10 n.d. n.d. 47 4

a

 μ-, or K-binding were respectively suppressed by 200nM

DAGO, 100 nM DSLET or 3 nM DPDYN. n.d.: not detectable.

These results clearly show that 125I-DPDYN at 0.15 nM selectively

discriminates between and other (μ, ) sites in nerve tissues

containing various proportions of opioid binding sites.

Autoradiographic studies

Figure 1 shows that binding sites labelled with 125I-DPDYN (0.3 nM)

are localized mainly in the molecular layer of the guinea-pig

cerebellum (A,B). This result is in complete agreement with that found

by Robson et al. (1984) in the same tissue. In the rabbit cerebellum,

very similar autoradiograms are obtained with (C,D) or without (E,F)

suppression of μ- and binding. Interestingly, in the rabbit

cerebellum, the 125DPDYN binding sites are restricted to the Purkinje

cell layer while in the guinea-pig cerebellum they are present

throughout the molecular layer. All these results clearly confirm the

selectivity of the labelled probe.

In summary, 125I-DPDYN can be considered as an useful tool for the

study of opioid receptor, particularly for their direct (no

suppression of μ- and binding is needed), precise and rapid

234
(exposure time less than 100 hours) visualization in nerve tissues by

autoradiography.

FIGURE 1: Autoradiographic localization of 125I-DPDYN (0.3nM)

binding sites to sagittal sections of guinea-pig (A,B) and

rabbit (C,D,E,F) cerebellar slices. Left side of the figure

(A,C,E) represents total binding and right side (B,D,F) non

specific binding. Autoradiograms of rabbit cerebellum slices

were obtained in presence (C,D) or absence (E,F) of 1OOnM DAGO +

200nM DSLET (suppression (or not) of μ- and binding).

REFERENCES

Frances, B., Moisand, C., and Meunier, J.C. Eur J Pharmacol,

117:223-232, 1985.

Gairin, J.E., Gouardères, C., Mazarguil, H., Alvinerie, P., and Cros, J.

Eur J Pharmacol, 106:457-458, 1984.

Gairin J,E, Jomary, C., Pradayrol, L., Cros, J., and Meunier, J.C.

Biochem Biophys Res Commun, 134:1142-1150, 1986.

Meunier J.C. Kouakou, Y., Puget, A., and Moisand, C. Mol Pharmacol,

24:23-29, 1983.

Robson, L.E., Foote, R.W., Maurer, R.;

12:621-627, 1984.

and Kosterlitz, H.W. Neuroscience,

235

172-683 O - 87 - 9 : QL 3
INFLUENCE OF Y-20003, AN ANALGESIC AGENT, ON THE ENDOGENOUS OPIOID

PEPTIDE SYSTEM IN RATS

T. Kudo, E.-Q. Wei, R. Inoki, *M. Terasawa and *T. Nakao

Department of Pharmacology, Osaka University Faculty of Dentistry

1-8, Yamadaoka, Suita, Osaka 565, Japan

*Research Laboratories, Yoshitomi Pharmaceutical Industries, Ltd.

Yoshitomi-cho, Chikujo-gun, Fukuoka 871, Japan

ABSTRACT

Changes of endogenous opioid peptide system by pain stimuli were examined using adjuvant-induced arthritic rats. Following the 5th day

after adjuvant inoculation, met-enkephalin(ME)-like peptide content

in plasma decreased, while after the 12th day, the peptide content

increased in adrenal medulla. A significant correlationship was

found between pain threshold and plasma ME-like peptide content.

Y-20003, a new nonsteroidal agent without inhibition of prostaglandin biosynthesis, showed an analgesic effect which was significantly

antagonized by naloxone or Win 44,441-3, suggesting that endogenous

opioid peptide system may be involved in the analgesic mechanism of

Y-20003.

INTRODUCTION

It has been reported that the contents of opioid peptides in plasma

or cerebrospinal fluids decreased in patients with chronic pain (

Panerai et al. 1982, Simonnet et al. 1986), although the physiological significance was unclear. On the other hand, it has been also

reported that endorphin(B-ED) content in human plasma was increased by diclofenac, a nonsteroidal antiinflammatory agent, (Martini et

al. 1984). ED content in rat pituitary gland was reduced and the

content in rat hypothalamus was increased by this agent (Sacerdote

et al. 1985), suggesting that ED might be involved in the analgesic mechanism of diclofenac, in addition to inhibition of prostaglandin synthesis. Furthermore, it has been known that in vitro

experiments, indomethacin enhanced release of ED from neurointermediate lobe of the rat pituitary gland (Knepel et al. 1985). In the

present study, it was examined what changes of the endogenous

opioid peptide contents could be shown in adjuvant-induced arthritis

of the rat, a model of chronic pain diseases, and then whether the

analgesic action of Y-20003 [10-amino-6-methyl-1,2,3,4,7,8,9,10-

tetrahydrobenzo(f)quinolin-3-one] in the rat could be mediated

through the endogenous opioid peptide system or not.

MATERIALS AND METHODS

Male rats (Lewis strain) were used. Animals were intradermally injected at the base of the tail with 0.1 ml of adjuvant. The adjuvant

was a 0.5 % suspension of dead tubercle bacilli (R35H5) in liquid

paraffin. The foot was pressed to measure pain threshold and the

foot volume was determined by volumetry before and 5, 12 and 20 days

after inoculation. In addition, the adrenal medulla, incisor pulps

and blood were collected. Plasma from the blood, including heparin

and actinonin(20 μM), were deproteinized with HCl-ethanol. Adrenal

236
TABLE 1. % Changes of endogenous opioid peptide contents after adjuvant inoculation

Opioids Tissues Days after adjuvant inoculation

0 5 12 20

ME-like adrenal medulla 100.0 13.6(7) 110.0 9.5(7) 191.9 17.9(7)** 186.8 9.7(7)**

peptides

tooth pulp 100.0 5.1(6) 90.8 5.5(6) 96.5 5.8(7) 98.2 7.1(7)

plasma 100.0 6.1(5) 63.2 4.3(7)** 59.8 4.1(7)** 47.5 4.9(7)**

**p<0.01, significantly different from the corresponding 0 day value, respectively. Each value represents the

mean SEM. Numbers in parentheses are the numbers of animals used.

TABLE 2. Influences of Y-20003 on pain threshold and foot volume

Treatments

Days after adjuvant inoculation

15 16 18 20

pain threshold control -63.1 0.6(10) -64.8 15.7(10) -57.7 13.7(10) -48.3 16.9(10)

( g ) Y-20003 -61.1 13.0(10) -10.2 16.7(10)* -10.1 15.6(10)* -9.4 12.7(10)*

foot volume control 1.84 0.06(10) 1.98 0.06(10) 1.87 0.07(10) 1.95 0.08(10)

( ml ) Y-20003 1.87 0.05(10) 1.93 0.05(10) 1.94 0.06(10) 2.01 0.07(10)

*p<0.05 significantly different from the corresponding control value, respectively. Each value represents

the mean SEM. Numbers in parentheses are the numbers of animals used. Y-20003 was daily administered in

a dose of 50 mg/kg/day, p.o. from 15th day after adjuvant inoculation.
medulla and tooth pulps were homogenized with 0.1 N HCl. These

materials were centrifuged and the supernatants were lyophilized.

The lyophilized materials were radioimmunoassayed to measure the

ME-like peptide contents, as described previously (Kudo et al.

1983).

FIGURE 1. Correlationship between pain threshold and plasma

ME-like peptide content. The data from control, 12th and

20th days after adjuvant inoculation were plotted.

TABLE 3. Antagonism of naloxone or Win 44,441-3 against

the analgesic effect of Y-20003

Treatments Doses(mg/kg) n g of pain threshold

control 0 10 -75.1 21.3

Y-20003 50 10 - 7.2 16.9*

Y-20003 50

+ } 10 -55.0 14.0#

naloxone 2

Y-20003 50

+ } 10 -78.6 15.6##

Win 44,441-3 1

*p<0.05, significantly different from the control. #p<0.05

and ##p<0.01, significantly different from the Y-20003 group.

Each value ( g ) represents the mean SEM. Y-20003 was

orally administered. Naloxone and Win 44,441-3 were injected

subcutaneously 10 min before Y-20003. The data are from

those of 18th day after adjuvant inoculation.

238
RESULTS AND DISCUSSION

In the present study, it was examined what changes of endogenous opioid peptide contents could be shown in adjuvant-induced arthritis of

the rats. It was found that pain threshold decreased and foot volume

increased significantly following the 12th day after adjuvant inoculation. These indices did not show any changes at the 5th day, suggesting that the day was on the latent stage of the arthritis (data

not shown). However, the content of plasma ME-like peptides were

significantly reduced following 5th day, while the content of adrenal

medullary ME-like peptides was elevated after the 12th day(table 1).

These results may suggest that a long term of lowering of plasma

opioid peptide levels is necessary to lower the pain threshold. Furthermore, it is conceivable that at the initial stage of adjuvant

inoculation(5th day), the lowering of the peptide level is due to

activation of some degrading enzymes in plasma, followed by compensatory promotion of opioid peptide processing in adrenal medulla

since 12th day. In fact, as shown in fig. 1, a significant correlationship was found between pain threshold and plasma ME-like peptide

content. On the other hand, in the present study, we investigated

whether analgesic action of Y-20003 which did not inhibit prostaglandin biosynthesis could be mediated through the endogenous opioid

peptide system in the adjuvant-induced arthritis or not. The pain

threshold, but not the foot volume, was recovered near the normal

level after Y-20003(table 2). The analgesic effect of Y-20003 was

antagonized by naloxone or Win 44,441-3, a antagonist(table 3). In

addition to these results, we have previously certified that adrenal

medullary ME-like peptide content was markedly reduced and in contrast, plasma ME-like peptide content showed a tendency to be increased

by Y-20003 in normal rats(data not shown). From these findings, it

may be concluded that 1) plasma opioid peptide levels are closely

related to pain threshold in the arthritic rats and 2) the analgesic

action of Y-20003 on the adjuvant-induced arthritis is mediated

through endogenous opioid peptide.

REFERENCES

Knepel, W., Nutto, D., Vlaskovska, M. and Kittel, Ch. J Endocr, 106:

189-195, 1985.

Kudo, T., Chang, H.-L., Maeda, S., Uchida, Y., Nakamae, J. and Inoki, R.

LifUci, 33, Sup.I:677-680, 1983.

Martini, A., Bondiolotti, G.P., Sacerdote, P., Pierro, L., Picotti, G.B.

and Panerai, A.E. J Int Med Res, 12:92-95, 1984.

Panerai, A.E., Martini, A., De Rosa, A., Salerno, F., Di Giulio, A.M. and

Mantegazza, P. in: Costa, E. and Trabucchi, M., eds. Regulatory

Peptides: From Molecular Biology to Function. New York, Raven

Press, 1982, pp.139-149.

Sacerdote, P., Monza, G., Mantegazza, P. and Panerai, A.E. Pharmacol

Res Commun, 17:679-684, 1985.

Simonnet, G., Taquet, H., Floras, P., Caille, J.M., Legrand, J.C.,

Vincent, J.D. and Cesselin, F. Neuropeptides, 7:229-240, 1986.

ACKNOWLEDGEMENTS

We thank Dr. T. Aoyagi for his gift of actinonin and Dr. A.E. Soria,

Sterling-Winthrop Research Institute, for his gift of Win 44,441-3.

239
PROHORMONE PROCESSING REACTIONS: DISCRETE STAGES

IN THE FORMATION OF ENDORPHIN RELATED PEPTIDES

N.J. Darby, K. Maruthainar and D.G. Smyth

National Institute for Medical Research

The Ridgeway, Mill Hill, London NW7 1AA, U.K.

ABSTRACT

Analysis of lipotropin from the pars intermedia of

bovine pituitary showed that it contained the full COOHterminal sequence of endorphin. In contrast the

endorphin related peptides exhibited a high degree of

C-terminal proteolysis. The implications of these

findings are discussed with reference to the cellular

compartments where the processing reactions take place.

INTRODUCTION

It is well appreciated that peptide hormones and transmitters are formed by proteolytic cleavage of prohormone

Precursors and their formation may involve the proteolysis of more than one peptide bond. It is not known,

however, whether these cleavages take place simultaneously and competitively or whether they take place

independently and in a predetermined order. The

formation of endorphin 1-27 offers an example of a

prohormone fragment that requires two processing

reactions for its release, one cleavage to free the NH2-

terminus of the peptide and the second to release the

COOH-terminus. We present evidence that the two

reactions take place consecutively. They may occur in

different cellular compartments.

METHODS

Peptides were extracted from 4 bovine anterior pituitaries

or pars intermedia by homogenisation in acid acetone

(Smyth et al. 1978). The extracts were dried and

fractionated by gel exclusion chromatography on Sephadex

G75 (100 x 2cm) in 20% acetic acid. Lipotropin and

endorphin related peptides were located by RIA with a

endorphin antiserum (Zakarian and Smyth 1979) and the

lipotropin containing fraction was re-chromatographed

under the same conditions to ensure complete removal of

the endorphin related peptides.

To determine whether the lipotropin molecules contained

the full 31 residue sequence of endorphin or were

shortened at the COOH-terminus, the lipotropin containing

fractions were subjected to citraconylation and trypsin

digestion. This procedure allowed limited proteolytic

cleavage to take place at arginine residues, thus

releasing the C-terminal sequence. After removal of

240
the citraconyl groups, the corresponding forms of

endorphin were identified chromatographically. The

lipotropin obtained from 2 pituitaries was reacted with

40μ1 of citraconic anhydride (in 160μl dioxan) in 2ml of

400mM sodium phosphate at pH 9. The reaction mixture

was vortexed until no further change in pH was observed,

the pH being maintained by automatic addition of 1M-NaOH.

The reaction mixture was then treated with TPCK trypsin

(2μg) and digestion carried out at 37°C for 16 h. The

reaction was terminated by addition of acetic acid and

gel exclusion chromatography was employed to resolve

endorphin related peptides from residual lipotropin.

The peptides were located by RIA with a endorphin

antiserum, the citraconyl groups being removed by the

exposure to acetic acid during chromatography. The

endorphin containing fraction was resolved by ion

exchange chromatography on SP-Sephadex C25 (sodium form)

in 50% acetic acid with a sodium chloride gradient (0 to

0.5M, mixer volume 1OOml). The peptides related to

endorphin again were located in the eluted fractions

by RIA.

The lipotropin fractions from the two regions of

pituitary, obtained by gel exclusion chromatography,

were further examined by HPLC (Smyth 1984) using a C18

column in 10mM HCl with an acetonitrile gradient (0 to

25% in 2 min, and 25 to 50% in 25 min). Aliquots of the

eluted fractions were dried in vacuo and the lipotropin

located by RIA.

RESULTS AND DISCUSSION

The lipotropin fractions obtained from both the anterior

pituitary and pars intermedia, after citraconylation and

trypsinisation, gave rise only to endorphin 1-31; none

of the shortened forms of endorphin endorphin 1-26

and endorphin 1-27) were formed (figure 1).

Similarly no evidence for the presence of shortened forms

of lipotropin could be detected by HPLC; only a single

immunoreactivity component was observed. It can be

concluded that lipotropin is generated in both regions of

pituitary in a form that contains the full 31 residue

sequence of endorphin. The results show that the

proteolytic processing reaction that releases the Nterminus of lipotropin goes to completion and the lipotropin is converted to endorphin before any detectable

C-terminal proteolysis takes place.

It is well known that the endorphin related peptides

that are present in the pars inter-media exhibit a high

degree of C-terminal proteolysis (Smyth and Zakarian

1980). The major peptides are the 26 and 27 residue

forms together with the corresponding N-acetyl derivatives, whereas the 31 residue form of endorphin in the

pars intermedia is only a minor component. The present

241
FIGURE 1. Ion exchange chromatography of

endorphin forms released by tryptic

digestion of citraconylated lipotropin from

(a) bovine pars intermedia and (b) anterior

pituitary. The peptides were chromatographed as described in the text.

results show, in marked contrast, that lipotropin in

the pars intermedia and in the anterior pituitary

exhibits no C-terminal shortening of the peptide chain.

It is clear that the processing reactions that release

lipotropin (Reaction 1), and endorphin from lipotropin

(Reaction 2), take place and go to completion before

proteolysis at the C-terminus commences. It could be

that the combined rates of Reactions 1 and 2 are

several orders of magnitude greater than the rate of

Reaction 3. Alternatively the final processing step

may take place in a different cellular compartment from

that where lipotropin is formed, or it may take place

in the same compartment but under different conditions.

It seems likely, furthermore, that the enzyme that

generates lipotropin and endorphin is different from

the enzyme that is involved in C-terminal shortening

of the peptide chain.

The molecular events involved in the conversion of

lipotropin to endorphin 1-27 are seen to comprise a

series of three cleavage reactions which take place

consecutively. Thus shorter forms of lipotropin do not

POMC LPH 1-91 end 1-31 end 1-27

seem to occur to a measurable extent in the pituitary

and the conversion of lipotropin to endorphin 1-31

appears to be a mandatory step which must take place

before C-terminal proteolysis of the peptide chain can

242
occur.

The results suggest that processing reactions take place

in ordered sequence, each step going to completion before

the succeeding step commences. The ordered processing

could, of course, be related to conformational restriction

of certain cleavage sites which would determine the nature

of the fragments produced; alternatively the cleavage

reactions may be catalysed by different enzymes. In

either case the possibility should be considered that

proteolytic processing reactions may be regulated

independently.

REFERENCES

Smyth, D.G. Analyt Biochem, 136:127-135, 1984.

Smyth, D.G., Snell, C.R. and Massey, D.E. Biochem J,

175:261-270, 1978.

Smyth, D.G. and Zakarian, S. Nature, 288:613-615, 1980.

Zakarian, S. and Smyth, D.G. Proc Natl Acad Sci, 76:

5972-5976, 1979.

243
BRIDGE PEPTIDE IS A CLEAVAGE PRODUCT OF PRO-DYNORPHIN PROCESSING IN

THE RAT ANTERIOR PITUITARY

Robert Day and Huda Akil

Mental Health Research Institute

University of Michigan

205 Washtenaw Place, Ann Arbor, MI, USA 48109-0720

ABSTRACT

Bridge peptide, the sequence of amino acids which joins

alpha-neo-endorphin and dynorphin A 1-17, was determined to exist as

a free molecule in the rat anterior pituitary. Gel filtration

chromatography revealed the presence of the free peptide as well as a

high molecular weight intermediate of 8-9 kilodaltons.

INTRODUCTION

Pro-dynorphin (PRO-DYN) is the common precursor to a series of

leucine-enkephalin extended opioid peptides, including dynorphin

(DYN) A 1-17, DYN A 1-8, DYN B 1-13 and alpha-neo-endorphin (for

review see Dores et al. 1984). These products are generated when

PRO-DYN undergoes a series of proteolytic cleavages at basic amino

acid pairs. The order in which these events occur is not well

understood. However, nucleotide sequence analysis of cloned

complementary DNA disclosing the primary sequence of PRO-DYN

(Kakidani et al. 1982) combined with knowledge of Lys-Arg as a

cleavage site permits certain predictions. One of these predictions

would be the liberation of the amino acid sequence which lies in

between alpha-neo-endorphin and DYN A 1-17, and is referred to in

this text as "bridge peptide."

The bridge peptide sequence is highly heterogeneous across species

(Civelli et al. 1985). In rat, it is composed of 23 amino acids,

while porcine and human bridge peptide are 21 and 19 amino acids,

respectively. However, the 6 amino acids which compose ethe

C-terminal of bridge peptide are conserved across species.

PRO-DYN end product immunoreactivity is known to be present in the

anterior lobe of the pituitary (cf. Khachaturian et al. 1986) and has

been characterized as existing mainly in high molecular weight

intermediates (Seizinger et al. 1984). Two intermediates have been

described, an 8 kdal molecule with alpha-neo-endorphin in its

C-terminal and a 6 kdal molecule with DYN A 1-17 in its N-terminal.

This would suggest the presence of free bridge peptide in the

anterior pituitary. This study was undertaken to verify if bridge

peptide occurred as an intact entity.

METHODS

A radioimmunoassay (RIA) to bridge peptide was developed. Porcine

bridge peptide synthesized by Dr. Richard Houghten (Scripps Clinic,

La Jolla, CA) was conjugated to thyroglobulin and injected s.c. to

rabbits. The labelled antigen for this RIA was iodinated (I 125) rat

244
bridge peptide (rat bridge peptide was also synthesized by Dr.

Houghten). The iodination was carried out in the presence of

chloramine-T and the unreacted cold, peptide was separated from

labelled bridge peptide by HPLC. The RIA was carried out in a 300 ul

volume, consisting of 2 parts - 150 mM PO buffer (pH 7.2) with 0.1%

BSA, 0.9% NaCl, 0.06% triton and 0.01% sodium azide; 1 part -

methanol: 0.1 N HCl (1:1, v/v). The assay was incubated 24 hrs at

4°C and bound labelled antigen was separated from free labelled

antigen by the addition of 100 ul of 0.5% bovine gamma globulins and

500 ul of 20% polyethylene glycol. Under these conditions the

antibody was used at a final dilution of 1/18,000. The limit of

sensitivity, defined here as the amount of unlabelled antigen

required to displace 10% of labelled antigen, was 30 fmoles. The

antibody was determined to be C-terminal directed.

Fifty anterior pituitaries from adult male Sprague-Dawley rats were

homogenized in 10 ml of methanol: 0.1 N HCl (1:1, v/v). The

extracts were chromatographed on Sephadex G-50, superfine column (1.5

X 90 cm) in 1% formic acid and 0.01% BSA. Two ml fractions were

collected, lyophilized and resuspended in methanol: 0.1 N HCl (1:1,

v/v). Aliquots of these fractions were analyzed for bridge peptide

immunoreactivity.

RESULTS AND DISCUSSION

Gel filtration chromatography of rat anterior pituitaries combined

with the bridge peptide RIA revealed the presence of two peaks; Peak

I and Peak II, (Table I).

TABLE I. Distribution of bridge peptide immunoreactivity (IR) in the

rat anterior pituitary after gel filtration

chromatography.

Apparent MW Bridge Peptide-IR1% of Total

(kilodaltons) (fmoles) Bridge Peptide-IR

Peak I 8-9 864 7.0

Peak II22-3 11,475 93.0

1

Sum of immunoreactivities measured in fractions of Peaks I and II.

2

Co-migrates with rat bridge peptide.

Ninety-three percent of the bridge peptide immunoreactivity was

contained in Peak II, a Peak which co-eluted with synthetic rat

bridge peptide standard. Seven percent of the immunoreactivity was

contained in Peak I, a higher molecular weight species of 8-9 kdal.

When accounting for column recovery ( 80%) and the number of

pituitaries chromatographed, one rat anterior lobe would contain

approximately 280 fmoles of free bridge peptide. However, the total

content of bridge peptide is most likely higher, since we observed a

245
high molecular weight species with bridge peptide immunoreactivity

(Peak I). It is possible that this molecule contains bridge peptide

with a blocked C-terminal, i.e. extended with DYN A 1-17. If this

were the case, the amount of immunoreactivity detected for Peak I,

would be largely underestimated since our antibody was determined to

be C-terminal directed and a C-terminal extended bridge peptide is

poorly crossreactive.

The biological effects of bridge peptide are not known and will have

to be explored. This peptide does not contain the Tyr-Gly-Gly-Phe

amino acid sequence associated with opioid function. Modulation of

opioid function, however, may still be possible. It is not known

what role bridge peptide fulfills in regards to existing in the same

precursor molecule as the leucine-enkephalin extended opiate peptides

known as dynorphins.

REFERENCES

Civelli, O., Douglass, J., Goldstein, A., Herbert, E. Proc Natl Acad

Sci USA, 82:4291-4295, 1985.

Dores, R.M., Akil, H., Watson, S.J. Peptides, 5:9-17, 1984.

Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y.,

Hirose, T., Asai, M., Inayama, S., Nakanishi, S., Numa, S. Nature,

298:245-249, 1982.

Khachaturian, H., Sherman, T.G., Lloyd R.V., Civelli, O., Douglass,

J., Herbert, E., Watson, S.J., Akil, H. Endocrinology, in press,

1986.

Seizinger, B.R., Hollt, V., Herz, A. Endocrinology, 115:662-671,

1984.

ACKNOWLEDGMENTS

This work was supported by DA02265 to H.A. R.D. is a fellow of the

Fonds de la Recherche en sante du Quebec (FRSQ).

246
A PROCESSING ENZYME FOR PRODYNORPHIN DERIVED PEPTIDES

Kathleen M. Metters* , Jean Rossier*,

Eduardo E. Oliveira+ Odaly Toffoletto and Antonio C.M. Camargo**

* Laboratoire de Physiologie Nerveuse, C.N.R.S.

91190 - Gif-sur-Yvette, France

** Departamento de Farmacologia and + Departamento

de Imunologia, Instituto de Ciencias Biomédicas,

Universidade de Sao Paulo, Cidade Universitaria

and the ++ Instituto Butanta, 05508 Sao-Paulo, SP, Brazil

ABSTRACT

Endo-oligopeptidase A known to hydrolyse the Phe5-Ser6 bond of

bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to

produce, by a single cleavage, leucine5-enkephalin from small

prodynorphin derived enkephalin-containing peptides. The specificity

constants (kcat /km) obtained for the hydrolysis of bradykinin,

neurotensin arnd dynoprphin B are of the same order, suggesting that the

substrate amino acid sequence is not the only factor determining the

cleavage site of this enzyme.

INTRODUCTION

Biologically active peptides are generated from large inactive

precursor molecules by proteolytic cleavages. It has been proposed

that biologically active sequences bracketed by pairs of basic amino

acid residues are liberated by the action of a trypsin-like

endopeptidase followed by a carboxypeptidase B type exopeptidase

(Steiner et al. 1980). In this study, we provide evidence that this

pattern of sequential cleavage may not be the only mechanism for

liberating enkephalin from low molecular weight enkephalin-containing

peptides. Previous reports have described the purification and

characterization of endo-oligopeptidase A, a 75000 Dalton thiol

peptidase (Camarqo et al. 1973; Carvalho and Camargo 1081) which

hyrolyses the Phe5-Ser6 bond of bradykinin (Camargo et al. 1973), the

Arg8-Arg9 bond of neurotensin (Carmargo et al. 1983) and liberates

methionine5-enkephalin (ME) from BAM 12P by cleavage at the Met5-Arg6

position (Camargo et al. 1985). Following this latter observation, we

report here that bovine brain endo-oligopeptidase A can produce, by a

single cleavage, leucine5-enkephalin (LE) from small precursor

molecules.

MATERIALS AND METHODS

Endo-olipopeptidase A assay

Endo-oligopeptidase A used for enzyme assays was highly purified by

immunoaffinity chromatography (data not shown). Enzyme assays. were

performed in a final volume of 100 μl of 17.5 mM Tris-HCl pH 7.6

containing 7 μg of endo-oligopeptidase A (specific activity 1370

μmol/min/mg towards bradykinin), 0.35 mM dithiothreitol and 10-20 nmol

of the required peptide. Samples were incubated at 37°C and the

247
reaction terminated by addition of 5 ul of concentrated H3PO4.

Aliquots from each incubation medium were analysed by reverse phase

HPLC over a

Corporation).

C18 μBondapak column (4.6 x 250 mm; Millipore

Peptides were separated by an initial 3 min isocratic

elution in 0.1% H3PO4 pH 2.7 containing 10% (v/v) acetonitrile

followed by a 15 min linear gradient of 10-50% (v/v) acetonitrile in

the 0.1% H3PO4 pH 2.7, at a flow rate of 2 ml/min. Reaction products

were monitored by optical density at 214 nm.

RESULTS

The ability of endo-oligopeptidase A to hydrolyse several

enkephalin-containing prodynorphin derived peptides was studied. With

dynorphin(DYN)-B as substrate the product formed was LE, demonstrating

that this peptide is hydrolysed at the Leu5-Arg6 bond (fig.1A; table

1). Furthermore, the amount of LE released accounted for all the LE

contained within DYN-B fig.1B. LE formation was inhibited in a

concentration dependent manner by bradykinin, indicating that the

purified endo-oligopeptidase A contained only one enzyme activity.

fig. 1. Hydrolysis of peptide substrates by purified

endo-oligopeptidase A was analysed by reverse phase HPLC

with (A) a typical chromatogram showing the production of LE

from DYN-B and (B) the formation of LE from DYN-A1-8,

-NEO , DYN-B and -NEC

Standards : Tyr-Gly-Gly-Phe (1), LE-Arg6-Arg7 (2) LE-Arg6

(3) and LE (4).

Similar results were obtained using the prodynorphin derived peptides

DYN-A1-8, -neoendorphin(NEO) and -NEO. LE formation again accounted

for around 100 % of the total peptide LE content, except in the case

of -NE0 where this could not be measured due to the slower rate of

hydrolysis over the time course of the experiment (fig. 1B; table 1).

The complementary basic peptides produced by endo-oligopeptidase A

hydrolysis of these species eluted as single peaks on another HPLC

system described elsewhere (Camargo et al. 1985), substantiating the

248
observation that cleavage occurs only at one position. In contrast,

the related peptides DYN-A1-17 and DYN-A1-13 were not detectably

cleaved by endo-oligopeptidase A. Since smaller peptides DYN-A1-6

(LE-Arg6) and LE itself were also found to be resistant to cleavage by

this enzyme, whereas the heptapeptide DYN-A1-7 was hydrolysed to

liberate Tyr-Gly-Gly-Phe, but not LE, these results indicate that in

general, the optimum for the release of LE is between 8-13 amino acid

residues and that substrate size may be important in determining both

the cleavage position and the susceptibility tc hydrolysis.

TABLE 1

Prodynorphin peptides hydrolysed by endo-oligopeptidase A

DYN A1-7 Tyr-Gly-Gly-Phe-Leu-Arg-Arg

DYN A1-8 Tyr-Gly-Gly-Phe-Leu Arg-Arg-Ile

DYNE B Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr

-NEO Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys

-NEO Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro

Arrows indicate the observed cleavage points. The sequences

of LE, LE-Arg6, DYN-A1-13 and DYN-Al-17, which were

resistant to hydrolysis, are not shown.

Specificity of endo-oligopeptidase A

These results indicate an optimum substrate size for catalysis by

endo-oligopeptidase A. However, the requirement for amino acids around

the subceptible bond is unclear since the enzyme can cleave the

Phe5-Ser6 bond in bradykinin (Camargo et al. 1973, the Arg8-Arg9 bond

in neurotensin (Camargo et al. 1983) and the Leu5

-Arg6 bond in DYN-B,

where the amino acid residues found surrounding the cleavage sites

bear little similarity. Therefore, the hydrolysis of these peptides

was further investigated using a >20000 fold purified enzyme. As shown

in table 2 all three peptides prove good substrates for this enzyme,

with km values in the lower μM range. In addition the high specificity

constants (kcat/km) are not strikingly different between substrates,

suggesting that the primary amino acid sequence of the peptides alone

is not responsible for determining the cleavage site.

TABLE 2

Bradykinin

Neurotensin

DYN-B

Specificity of endo-oligopeptidase A

Km(μM) kcat(s-1) kcat/Km(M-1

s

-1)

8.78 9.8 1.12 x 106

12.49 1.55 0.28 x 106

5.23 21.52 4.11 x 106

249
Endo-oligopeptidase A catalysis of bradykinin, neurotensin

and DYN-B was stopped when less than 15 % of the substrates

had been hydrolysed and the products analysed over HPLC.

Assays were conducted in triplicate at 0.5, 1.0, 1.5, 2.0,

3.0 and 4.0 μM substrate concentrations. km and kcat values

were obtained using Lineweaver-Burk plots with initial rates

expressed as molar rates.

DISCUSSION

Recently it has been reported that the processing of dynorphin

precursors into dynorphins may occur via a single-step reaction

involving cleavage at a single arginine (Wallace et al. 1984; Devi and

Goldstein 1985) which differs from the general mechanism of

trypsin-like followed by carboxypeptidase-B like hydrolysis (Steiner

et al. 1980). Since several biologically active peptides are now known

to be released by proteolysis at cleavage sites other than dibasic

residues (Devi and Goldstein 1985; Niall et al. 1982; Kreil et al.

1980), it may be that processing does not necessarily follow the

trypsin-carboxypeptidase B-like enzyme hypothesis. In this study it

has been shown that endo-oligopeptidase A, an enzyme which displays

selectivity towards small intermediates, could be involved in this

type of processing scheme and, more particularly, in the processing of

the prodynorphin precursor.

REFERENCES

Camargo A.C.M., Caldo H. and Emson P.C. Biochem Biophys Res Comm,

116:1151-1159 (1983).

Camargo A.C.M., Carvalho M.J.V. and Schwartz W. Biochem Biophys Res

Comm, 130:932-938 (1985).

Camargo A.C.M., Shapanka R. and Greene L.J. Biochemistry,

12:1838-1844 (1973).

Carvalho K.M. and Camargo A.C.M. Biochemistry, 20:7082-7088 (1981).

Devi L. and Goldstein A. Proc Natl Acad Sci USA, 81:1892-1986 (1985).

Kreil G., Mollay C., Kaschnitz R., Haiml L. and Vilas V. Ann NY Acad

ci, 343:338-346 (1980).

Niall H., Hudson P., Haley J., Cronk M. and Shine J. Ann NY Acad Sci,

380:13-21 (1980).

Steiner D.F., Quinn D.S., Chan S.J., Marsh J. and Tager H.S. Ann NY

Acad Sci, 343:1-16 (1980).

Wallace E.F., Weber E., Barchas J.D. and Evans C.J. Biochem Biophys

Res Comm, 119:415-422 (1984).

250
ASSAY AND BIOCHEMICAL CHARACTERIZATION OF A DYNORPHIN

CONVERTING ENZYME IN HUMAN CEREBROSPINAL FLUID

Fred Nyberg1, Sven Lyrenäs2

and Lars Terenius1

1

Department of Pharmacology, University of Uppsala, P.O.

2

Box 591, S-751 24 UPPSALA, Sweden

Department of Obstetrics and Gynecology, University of

Uppsala, Akademiska Sjukhuset, S-751 05 UPPSALA, Sweden

ABSTRACT

An endopeptidase hydrolyzing dynorphins A and B

alpha-neo-endorphin at the Arg6-Arg7 or Arg6-Lys7 bonds,

and

was partially purified from human cerebrospinal fluid and

further characterized by various biochemical techniques

including HPLC gel permeation (UltroPac TSK G3000SW) and

ion exchange (TSK DEAE-3SW) chromatography. A procedure

for quantitative analysis of the enzyme in individual CSF

samples is also described. The activity in lumbar CSF of

women in late pregnancy was significantly lower than that

in control samples.

INTRODUCTION

The endogenous opioid peptides alpha-neo-endorphin,

dynorphin A (Dyn A) and dynorphin B (Dyn B) derive from a

common precursor polypeptide, proenkephalin B. As with

other neurotransmitters or neuromodulators their actions

are likely to be under enzymatic control. Several peptidases capable of converting dynorphins have been reported

(e.g. Devi and Goldstein 1984; Mizuno and Matsuo 1984).

We recently reported an endopeptidase in human cerebrospinal fluid (CSF) capable of releasing the N-terminal

hexapeptide Leu-enkephalin-Arg6 from dynorphin A (Nyberg

et al. 1985). The enzyme, identified as a serine protease, cleaves at pairs of consecutive basic amino acid

residues and showed close similarity with an endopeptidase previously purified from yeast (Mizuno and Matsuo

1984). Here we describe further biochemical characterization of this activity and a procedure for its quantitation in individual CSF samples.

MATERIALS AND METHODS

Materials. Reference peptides were from sources previously noted (Nyberg et al. 1985). Labelling of dynorphin B

with 125I was performed as described (ChristenssonNylander et al. 1985). The chromatographic material

(SP-Sephadex) was purchased from Pharmacia Fine Chemicals

(Uppsala, Sweden). The peptidase inhibitors bestatin and

phosphoramidon were from Sigma and captopril from Squibb

(Princeton, NJ, U.S.A.). All other chemicals and solvents

were of analytical reagent grade from commercial sources.

Enzyme preparation. The dynorphin converting endopeptidase was purified from pooled human CSF by ion exchange

251
TABLE 1. Conversion of proenkephalin B derived peptides

by the purified CSF endopeptidase1.

Peptide Structure Release of Leuenkephalin-Arg6

(pmol/min)

Dyn A Tyr-Gly-Gly-Phe-Leu-Arg-Arg- 12.3

-Ile-Arg-Pro-Lys-Leu-Lys-Trp-

-Asp-Asn-Gln

Dyn A 4.1

(1-8)

Dyn B Tyr-Gly-Gly-Phe-Leu-Arg-Arg- 10.1

-Gln-Phe-Lys-Val-Val-Thr

Alpha- Tyr-Gly-Gly-Phe-Leu-Arg-Lys- 7.4

neo-end. -Tyr-Pro-Lys

1

Peptides (10 uM) were incubated in triplicates with the

CSF enzyme (1 mg/ml) and aliquots were removed at

different times and analyzed by RIA for the hexapeptide

peptide product. For other details see text.

chromatography, hydrophobic interaction chromatography,

and molecular sieving (Nyberg et al. 1985).

High performance liquid chromatography (HPLC). The HPLC

system (LKB Produkter AB, Bromma Sweden) was used for gel

permeation (TSK G3000SW, 7.5 x 600 mm), ion exchange

(TSK DEAE-3SW, 7.5 x 150 mm) and reversed phase (Spherisorb C-18, 5u, 4.6 x 250 mm) chromatography. Eluted fractions were collected and analyzed for enzyme activity.

All columns were calibrated with peptide and protein

standards.

Enzyme assay. All incubations were performed in Eppendorf

tubes at 37°C. Enzyme fractions or pure CSF (20 ul) were

incubated with a mixture of 125I-labelled (about 5000

cpm) and unlabelled (40 pmol) dynorphin B in a final

volume of 40 ul Tris-HCl (20 mM, pH 7.8) in the presence

of 100 uM bestatin, 10 uM captopril, and 20 uM phosphoramidon. The reaction was terminated by dilution with 1.5

ml cold pyridine(0.018 M)-formate(0.1 M) buffer (pH 3.2)

before product separation on SP-Sephadex minicolumns. The

generated enkephalyl hexapeptide was eluted with 0.35 M

pyridine-formate buffer (pH 4.4) and collected for counting. In assays of individual CSF samples incubations (in

triplicates) were allowed to proceed for 60 min. Product

formation was also monitored by radioimmunoassay (RIA)

(Nyberg et al. 1985).

RESULTS

Fig. 1A illustrates the the UV-profile and the pattern of

252
FIGURE 1. (A) HPLC gel permeation chromatography of

a crude CSF sample (200 ul). The column was eluted

with 40 mM ammonium acetate (pH 7.0) and fractions

of 0.5 ml were collected at a flow rate of 30 ml/h.

(B) HPLC ion exchange chromatography of peak 2

material. Gradient, 0.05-0.25 M NaCl in 20 mM TrisHCl (pH 7.2); fraction size, 1 ml; flow rate, 60

ml/h. (C) RP-HPLC reaction mixtures of the CSF

enzyme and Dyn A, Dyn B or alpha-neo-endorphin. The

column was eluted at a flow rate of 1 ml/min with a

40 min linear gradient of acetonitrile (15-45%)

containing trifluoroacetic acid (0.04%).

dynorphin converting activity obtained after HPLC gel

filtration of a crude CSF sample. As earlier shown

(Nyberq et al. 1985)) the enzyme activity was resolved

in two major peaks, one eluting ahead of albumin (> 68

kdaltons) and the other at a position corresponding to

40 kdaltons (peak 2). In this study, material corresponding to peak 2 was subjected to further characterization.

On HPLC ion exchange chromatography (Fig. 1B), peak 2

separated into two distinct fractions. The major fraction

eluted as a comparatively basic protein, thus showing

similar properties as those of the yeast enzyme (Mizuno

253
and Matsuo 1984). This enzyme fraction was capable of

releasing the N-terminal fragment, Leu-enkephalin-Arg6,

from Dyn A, Dyn B and alpha-neo-endorphin, as shown in

Fig. 1C, as well as from Dyn A (1-8). The enkephalyl

hexapeptide appeared as a major product in all experiments. The formation of this fragment was confirmed by

amino acid analysis. Studies performed with RIA amounts

of substrate indicated a different rate of conversion of

the various proenkephalin B products (Table 1). Dyn A and

Dyn B were converted at a higher rate than alpha-neoendorphin and Dyn A (1-8). A KM-value of 0.45 mM for conversion of Dyn B was estimated for the purified enzyme.

The enzyme could be analyzed in less than 100 ul CSF

samples. Values obtained by product quantitation with RIA

parallelled those obtained by the labelled substrate

procedure, which was used here. The enzyme activity was

analyzed in lumbar CSF of normal women of fertile age and

of women in late pregnancy. Mean levels in the pregnant

group (6.7 units, n = 12) were significantly lower (p <

0.01) than those in the nonpregnant subjects (11.7 units,

n = 10, one unit was defined as the release of 1 pmol

Leu-enkephalin-Arg6 per ml CSF and min).

DISCUSSION

The enzyme described here acts on the proenkephalin B

family of opioid peptides with specificity towards consecutive pairs of basic amino acid residues. The enzyme

more efficiently converted Dyn A than e.g. its (1-8)

fragment suggesting that the sequences beyond the local

cleavage site are important. We have therefore named it a

dynorphin converting enzyme. The lower levels found in

CSF of pregnant women is of interest since they correlate negatively to the increased levels of opioid peptides observed in this group (Lyrenäs et al. to be published). It should be noted that the product, Leu-enkephalin-Arg , maintains opioid activity but with different receptor preference. Therefore, this enzyme may not

only terminate the action of dynorphin peptides but also

generate a fragment with different activity profile.

REFERENCES

Christensson-Nylander, I., Nyberg, F., Raqnarsson, U. and

Terenius, L. Req Peptides, 11:65-76, 1985.

Devi, L. and Goldstein, A. Proc Natl Acad Sci USA, 81:

1892-1896, 1986.

Minzuno, K. and Matsuo, H. Nature, 309:558-560, 1984.

Nyberq, F., Nordström, K. and Terenius, L. Biochem BioRes Commun, 131:1069-1074.

This study was supported by the Swedish Medical Research

Council (Grant 04X-3766) and Bank of Sweden Tercentenary

Foundation.

254
PRESENCE OF ENKEPHALIN PRECURSOR IN MOLLUSCAN NEURAL TISSUE EXTRACT

Barry Mixon, Sunita Kuruvilla, George B. Stefano and

Michael K. Leung

Departments of Chemistry and Biological Sciences

State University of New York/Old Westbury

Old Westbury, NY 11568, U.S.A.

ABSTRACT

Mytilus pedal ganglia extract was treated sequentially with TPCKtrypsin and carboxypeptidase B. The treated sample and an untreated

sample were purified separately with Sep-Pak C18 and subjected to

HPLC. Fractions with Rt's corresponding to the Met-, Leu-enkephalin

and Met-enkephalin-Arg6-Phe7 were assayed for enkephalin activities

by displacement studies using pedal ganglia membrane and 3H-DAMA.

The data showed the activities in the regions of Met-, Leuenkephalin and Met-enkephalin-Arg6

-Phe7 of the treated sample

increased over that of the untreated sample by 2.3, 2.5 and 1.6

folds respectively. These results provided strong evidence for the

presence of enkephalin precursor material in Mytilus.

INTRODUCTION

Recently, we reported the isolatio and identification of Met-, Leuenkephalin and Met-enkephalin-Arg6

-Phe7 in Mytilus pedal ganglia

(Leung and Stefano 1984; Stefano and Leung 1984). These results

demonstrated directly the presence of an enkephalinergic system in

the invertebrates. In addition, this system must be similar to the

mammalian one. Direct biochemical evidence, obtained by HPLC-RIA

technique, for the presence of enkephalin in shore crab has also

been reported by Jaros et al. (1986). Thus, the opioid system must

be developed at a very early stage of evloution.

Based upon current knowledge, all known opioid peptides are

processed from three precursors--proenkephalin, prodynorphin and

pro-opiomelanocortin. The nucleotide sequences as well as the amino

acid sequences for all three precursors had been deduced by cDNA

technique (Noda et al. 1982; Nakanishietal. 1979; Kakidani et al.

1982). Comparison of the gene and protein sequences reviewed

tremendous homology among the precursors (Horikawa et al. 1983). It

is, therefore, likely that all three precursors share a common

evolutionary origin.

Structurally, all opioid peptides in the precursors are bracketed by

a pair of basic amino acids on both the N- and C-side. This

indicated a similar processing scheme for all three opioid

precursors. Opioid peptides can be processed from their precursors

by sequential treatment with trypsin and carboxypeptidase B (Lewis

et al. 1980. The isolation and identification of Met-enkephalinArg6

-Phe7 in Mytilus is especially strong evidence for the presence

of proenkephalin-like precursor in this organism. This is true

since the heptapeptide represents the last seven amino acid sequence

255
in the mammal proenkephalin. In this present report data will be

presented which strongly suggested the existence of opioid precursor

material in Mytilus pedal ganglia.

MATERIALS AND METHODS

Subtidal Mytilus were harvested from Long Island Sound at Northport,

N.Y. Pedal ganglia from 425 fresh animals were excissed and stored

at -160° C before use. The ganglia were homogenized with a Brinkman

Polytron Homogenizer with four 3-sec bursts in 7.5 ml of 50 mM TrisHCl buffer, pH 8.0; containing 0.1 M NaCl and 10 mM CaCl2. The

homogenate was centrifuged at 10,000xg for 10 min at 4° C and the

supernatant was filtered with a 0.22 um membrane. The protein

content of the supernatant was determined by Bio-Rad (Richmond, CA.)

dye assay. TPCK-Trypsin was added to an aliquot of the filtrate in

an amount equal to 1% of the protein content. This mixture was

incubated overnight at 37° C. The enzyme activity was inactivated

by heating at 80° C for 15 min. After clarification by

centrifugation, carboxypeptidase B was added in an amount equal to

2% of the protein content. It was incubated at 37° C for 4 hr and

then inactivated as described earlier.

The incubation mixture was purified by Sep-Pak C18 cartridge

(Waters). The cartridge was activated first with methanol and

water. After the sample was injected into the column, it was rinsed

with 1% acetic acid and then eluted with methanol. The eluant was

lyophilized, dissolved in water and filtered through a 0.45 um

membrane. An aliquot of the extract without enzymatic treatment was

processed in similar manner for use as the control,

Purified samples of the treated and untreated extracts were

fractionated by HPLC on a Brownlee RP-300 reverse-phase column. The

HPLC system used was a Waters model 840. The solvent system used

was consisted of 0.1% trifluoroacetic acid (TFA) as A and 80%

acetonitrile in 0.1% TFA as B. The column was initially eluted

isocratically for 5 min with 20% B and followed by linear gradient

of 20-80% B in 20 min. One-ml fractions of both samples were

collected. Fractions with Rt's coresponding to Met-, Leu-enkephalin

and Met-enkephalin-Arg6-Phe7

were subjected to displacement assay

using Mytilus pedal ganglia membrane and 3H-DAMA. The displacement

assay procedure was the same as described previously (Leung and

Stefano 1984).

RESULTS AND DISCUSSION

The chromatograms, monitored at 280 um, of the untreated and treated

samples are shown in figure 1. The untreated sample contained

numerous small peaks and several major peaks (fig. 1A). After

enzyme treatment significant changes in the peak pattern was

observed (fig 1B). As a result of the enzymatic actions some of the

major peaks were significantly reducedin size. Results from the

displacement assays are also represented in figure 1. The

displacement activities in fractions from the untreated sample are

in general significantly lower than those in the treated sample.

256 .
The combined displacement activities found in the Met-, Leuenkephalin and Met-enkephalin-Arg6

-Phe7 regions as compared to the

3

H-DAMA standard were 35, 23 and 30% respectively. The combined

activities in these same regions in the treated sample were 81, 57

and 48%. They represented an .pa increased of 2.3, 2.5 and 1.6

folds of activities in Met-enkephalin, Leu-enkephalin and Metenkephalin-Arg6

-Phe7, respectively, in the treated sample over that

of the untreated. These data strongly suggested the presence of

opioid precursor material in the Mytilus pedal ganglia extract.

FIGURE 1. HPLC and displacement assays of untreated (A)

and treated (B) samples of Mytilus pedal ganglia extracts.

Procedure were as described in the text. One hundred

percent displacement is 3H-DAMA bound in the presence of

10 uM levorphanol minus 3H-DAMA bound in the presence of

10 UM dextrorphan.

Evidence has been gathered which demonstrate the presence of opioid

system in the invertebrates. Great-structural and physiological

similarities also exist between the vertebrate and invertebrate

opioid peptides (review, Leung and Stefano 1986). This preliminary

report provides additional support in this area by showing the

existence of enkephalin precursor in Mytilus. Even though only

limited information is available concerning the precursor material

in Mytilus, one significant similarity with the mammalian precursors

is observed. Since the enkephalin peptides in the Mytilus precursor

can be released by trypsin and carboxypeptidase B they must be

257
bracketed by basic amino acids as in the mammalian precursor.

However, it is not known if the amino acids at both ends are paired

and if more than one precursor is present in the tissue.

In conclusion, our current and earlier findings clearly demonstrated

the existence of an opioid mechanism in Mytilus. The enkephalin

peptides in Mytilus areidenticaltothose of mammals. As in the

mammals, the enkephalin peptides in Mytilus appear to exist in a

precursor form when first synthesized. The enkephalin peptides in

the Mytilus precursor are also bracketed by basic amino acids and

can be released by trypsin and carboxypeptidase B treatment. These

results suggest that the entire opioid mechanism, including the

structure of the precursor and the processing scheme, is a product

of early evolutionary development. Its importance as a signal

system is emphasized by its conservation through evolution.

REFERENCES

Horikawa, S., Takai, T., Toyosato, M., Takahashi, H., Noda, M.,

Kakidani, M., Kubo, T., Hirose, T., Inayama, S., Hayashida, M.,

Miyata, T., and Numa, S. Nature (London), 306:611-614, 1983.

Jaros, P.P., Dircksen, H., and Keller, R. Cell Tiss Res,

241:111-117, 1985.

Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y.,

Hirose, T., Asai, M., Inayama, S., Nakanishi, S., and Numa, S.

Nature (London), 298:245-249, 1982.

Leung, M.K. and Stefano, G.B. Proc Natl Acad Sci USA, 81:955-958,

1984.

Leung, M.K., and Stefano, G.B. Progr in Neurobiol, in press, 1986.

Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S., and

Udenfriend, S. Science, 208:1459-1461, 1980.

Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y.,

Cohen, S.N., and Numa, S. Nature (London), 278~423-427, 1979.

Noda, M., Furutami, Y., Takahashi, H., Toyosato, M., Hirose, T.,

Inayama, S., Nakanishi, S.,and Numa, S. Nature (London),

295:202-206, 1982.

Stefano, G.B. and Leung, M.K. Brain Res, 298:362-365, 1984.

ACKNOWLEDGEMENTS

This work was supported by NIH-MBRS Grant RR08180 and ADAMHA-MARC

Grant 17138. Mr. B. Mixon is a MBRS participant and Ms. S.

Kuruvilla is a ADAMHA-MARC fellow.

258
INACTIVATION OF [LEU5

]-ENKEPHALIN IN THREE ISOLATED PREPARATIONS:

RELATIVE IMPORTANCE OF AMINOPEPTIDASE, ENDOPEPTIDASE-24.11 AND

PEPTIDYL DIPEPTIDASE A

Tetsuo Oka, Kazuko Aoki, Midori Kajiwara, Kaori Ishii,

Yoshiki Kuno, Toyokazu Hiranuma and Teruhiko Matsumiya

Department of Pharmacology, School of Medicine, Tokai University,

Isehara 259-11, Japan

ABSTRACT

Enkephalin had been shown to be almost exclusively hydrolyzed by

three peptidases in the previous studies. In the present investigation, the relative importance of three enzymes in the inactivation of

[Leu5]-enkephalin was examined in three isolated preparations. Results showed that amastatin-sensitive aminopeptidase played the

greatest role in both guinea-pig ileum and rat vas deferens while it

played the similar role to either phosphoramidon-sensitive

endopeptidase-24.11 or captopril-sensitive peptidyl dipeptidase A in

mouse vas defer-ens.

INTRODUCTION

Three distinct enzymes, aminopeptidase, endopeptidase-24.11 ("

enkephalinase"), and peptidyl dipeptidase A (angiotensin converting

enzyme), had been shown to inactivate enkephalin in three isolated

preparations, guinea-pig ileum (Aoki et al. 1984), mouse vas deferens

(Aoki et al. 1986), and rat vas deferens (Cui et al. 1986). Additionally, the enkephalin-hydrolyzing aminopeptidase, endopeptidase-24.11,

and peptidyl dipeptidase A had been reported to be inhibited almost

completely with either 100 μM of bestatin or 1 μM of amastatin, either 1 μM of thiorphan or 1 μM of phosphoramidon, and 1 μM of

captopril, respectively (Aoki et al. 1984; Aoki et al. 1986; Cui et

al. 1986). Moreover, approximately 98% of enkephalin had been shown

to remain intact when enkephalin was incubated with an ileal membrane

fraction for 60 min at 37°C in the presence of three peptidase inhibitors, amastatin, captopril, and thiorphan (Hiranuma and Oka 1986).

Thus, enkephalin had been demonstrated to be almost exclusively hydrolyzed by three distinct enzymes. In the present investigation, the

relative importance of three enzymes in the inactivation of [Leu5]-

enkephalin was examined in three in vitro isolated preparations,

guinea-pig ileum, mouse vas deferens, and rat vas deferens.

MATERIALS AND METHODS

Chemicals

A gift, gratefully received, was captopril from Sankyo Company

(Tokyo, Japan). Amastatin, phosphoramidon and [Leu5

]-enkephalin were

purchased from Peptide Institute, Inc. (Minoh, Japan).

In vitro isolated preparations

The vasa deferentia from mice or rats, and the myenteric plexus-lon259
gitudinal muscle strip of guinea-pig ileum were prepared and set up

for electrical stimulation as described previously (Oka et al. 1982).

The % inhibition of the stimulated muscle twitch produced by

enkephalin was plotted against the log concentration of the opioid to

estimate the IC50 (concentration of the opioid peptide to produce 50%

inhibition of the twitch). When the effect of the peptidase inhibitor

on the potency of enkephalin was studied, the inhibitor was given at

least ten minutes before the enkephalin administration. The % difference shown in the tables was calculated from the formula % difference

= [(IC50 before additional treatment - IC50 after additional treatment) / IC50 before additional treatment] x 100. The significance of

% differences between IC50 values of two adjacent groups was determined by the paired Student's t-test.

RESULTS AND DISCUSSION

[Leu5]-Enkephalin significantly inhibited the electrically-evoked

contractions of guinea-pig ileum. Since its IC50 value in one preparation was sometimes significantly different from that in another,

the IC50 values in the absence and the presence of the peptidase inhibitor were estimated in the same preparation.

TABLE 1. The greater enhanced effects of amastatin than

either phosphoramidon or captopril on the inhibitory potency of [Leu5

]-enkephalin in guinea-pig ileum

Inhibitors IC50(nM) Ratio of % Difference

potency

None 220 73 1

Amastatin 55.0 13 3.82 0.31 73.3 2.0***

None 275 81 1

Phosphoramidon 227 68 1.20 0.084 16.9 1.9***

None 194 59 1

Captopril 165 43 1.18 0.093 16.4 1.7***

Each peptidase inhibitor at the final concentration of 1 μM

was given 10 min before the enkephalin administration. Each

value represents the mean S.E. of 4 experiments.

***P < 0.01.

The inhibitory potency of [Leu5]-enkephalin was significantly enhanced by the pretreatment of the guinea-pig ileum with either

amastatin, phosphoramidon or captopril (table 1). The magnitude of

the enhancement by amastatin was significantly higher than that by

either phosphoramidon or captopril (table 1). This shows that

amastatin-sensitive aminopeptidase plays the major role in the inactivation of exogenously given enkephalin in guinea-pig ileum, being

consistent with the previous report (Geary et al. 1982).

In the mouse vas deferens, the inhibitory potency of [Leu5

]-

enkephalin was also significantly enhanced by each peptidase inhibitor (table 2). The magnitude of the enhancement by amastatin, however, was similar to that by either phosphoramidon or captopril in

mouse vas deferens (table 2). This shows that three enkephalin-hydrolyzing peptidases play the similar role in the inactivation of

260
[Leu5

]-enkephalin in mouse vas deferens.

TABLE 2. The similar magnitude of the enhanced effects of

three peptidase inhibitors, amastatin, phosphoramidon and

captopril, on the inhibitory potency of [Leu5

]-enkephalin

in mouse vas deferens

Inhibitors IC50 (nM) Ratio of

potency

% Difference

None

Amastatin

9.54 1.5 1

4.76 0.56 2.01 0.19 47.5 4.6***

None 11.7 1.7 1

Phosphoramidon 7.19 0.98 1.63 0.061 37.7 2.5***

None 10.7 1.7 1

Captopril 5.39 0.61 1.93 0.16 45.3 4.0***

Each peptidase inhibitor at the final concentration of 1 μM

was given 10 min before the enkephalin administration. Each

value represents the mean S.E. of 8 experiments.

***P < 0.01.

TABLE 3. The highest, the intermediate, and the lowest

magnitude of the enhanced effects of amastatin (A),

phosphoramidon (P), and captopril (C), respectively, on the

inhibitory potency of [Leu5]-enkephalin in rat vas deferens

pretreated with the residual two peptidase inhibitors

Inhibitors IC5o (μM) Ratio of % Difference

potency

P+C 59.2 13 1

P+C+A 2.16 0.19 37.3 7.9 96.5 1.2***

C+A 9.45 2.1 1

C+A+P 5.43 1.6 1.99 0.15 46.6 3.9***

A+P 1.95 0.44 1

A+P+C 1.43 0.26 1.32 0.093 22.8 6.1*

The mixture of peptidase inhibitors was given 10 min before

the enkephalin administration at the final concentration of

1 μM each. Each value represents the mean S.E. of 4

experiments. *P < 0.05, ***P < 0.01.

In the rat vas deferens, the presence of the enzyme with low

enkephalin-hydrolyzing activity was sometimes difficult to detect when the residual enzyme with high enkephalin-hydrolyzing activity was not inhibited in the preliminary experiments. When the

enkephalin-hydrolyzing ability of a particular enzyme among three enzymes was examined, therefore, the residual two enzymes were inhibited in advance. The IC50 values of [Leu5

]-enkephalin in the rat vas

deferens pretreated with three peptidase inhibitors were significantly lower than those pretreated with any combination of two peptidase

inhibitors (table 3). The data in table 3 show that amastatin-sensitive aminopeptidase, phosphoramidon-sensitive endopeptidase-24.11 and

captopril-sensitive peptidyl dipeptidase A play, respectively, the

261
greatest, the intermediate and the least role in the inactivation of

[Leu5]-enkephalin in rat vas deferens.

Previous studies (Aoki et al. 1984; Aoki et al. 1986; Cui et al.

1986) have shown that, when three peptidase inhibitors are given individually to the bath after the enkephalin administration, the

enkephalin-induced inhibition of contractions of the isolated preparation is enhanced every time by the administration of each peptidase

inhibitor although each inhibitor by itself does not inhibit the contractions at all when an opioid peptide is absent in the bath. Additionally, the observation that the inhibition produced by peptidase

inhibitors is completely reversed by naloxone, an opioid antagonist,

indicates that the inhibition must be caused by the increased amount

of the enkephalin in the vicinity of opioid receptors by the administration of each peptidase inhibitor. Therefore, previous results

(Aoki et al. 1984; Aoki et al. 1986; Cui et al. 1986) have indicated

that three enkephalin-inactivating peptidases are all located very

close to opioid receptors and produce a significant concentration

difference of enkephalin between the surrounding organ bath and the

vicinity of opioid receptors. Additionally, these enzymes are suggested not only to inactivate the exogenously given enkephalin in the

vicinity of opioid receptors but also to play important roles in the

termination of the action on opioid receptors of the enkephalin released from presynaptic nerve terminals. Interestingly, the results

in the present investigation have shown that the relative importance

of the [Leu5]-enkephalin-inactivating enzymes is dependent on the

tissue examined. The significance of the difference in the relative

importance of the inactivating enzymes from one tissue to another remains to be elucidated in the present investigation.

ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan.

A generous gift of a compound mentioned in MATERIALS AND METHODS is

gratefully acknowledged.

REFERENCES

Aoki, K., Kajiwara, M., and Oka, T. Japan J Pharmacol, 36:59-65,

1984.

Aoki, K., Kajiwara, M., and Oka, T. Japan J Pharmacol, 40:297-302,

1986.

Cui, S., Kajiwara, M., Ishii, K., Aoki, K., Sakamoto, J., Matsumiya,

T., and Oka, T. Japan J Pharmacol, in press.

Geary, L.E., Wiley, K.S., Scott, W.L., and Cohen, M.L. J Pharmacol

Exp Ther, 221:104-111, 1982.

Hiranuma, T., and Oka, T. Japan J Pharmacol, 41:437-446, 1986.

Oka, T., Negishi, K., Suda, M., Sawa, A., Fujino, M., and Wakimasu,

M. Eur J Pharmacol, 77:137-141, 1982.

262
CHARACTERIZATION OF ENKEPHALIN DEGRADATION IN RAT PLASMA

Susan B. Weinberger, Cara E. Zafuto, Robert C. Walker

and Joe L. Martinez, Jr.

Department of Psychology, University of California,

Berkeley, CA 94720

ABSTRACT

Approximately 80% of the hydrolysis of [leu]enkephalin in rat plasma

can be attributed to bestatin-sensitive aminopeptidase activity, and

an additional 5% is due to angiotensin converting enzyme.

Thiorphan-sensitive enkephalinase hydrolysis of [leu]enkephalin could

not be detected in plasma. On the other hand,

2-d-ala-1-[leu]enkephalin is metabolized approximately 35% by an

unidentified bestatin-sensitive enzyme and approximately 15% by

thiorphan-sensitive enkephalinase in rat plasma, while

captopril-sensitive angiotensin converting enzyme is without

measurable activity against this substrate.

INTRODUCTION

We reported previously (Martinez et al. 1985) that a very high

correlation exists in rats between the rate of [leu]enkephalin (LE)

hydrolysis in plasma and the latency to escape on the first trial in

an active avoidance task. The activity of the plasma enzyme system is

altered by shock experience, suggesting that the enzyme system may

influence behavior by regulating the concentration of

[leu]enkephalin, or its substrate, in blood and hence at a receptor.

The specific enzymes involved in this relationship are as yet

unidentified.

Data from brain tissue suggest a major role for aminopeptidases and a

dipeptidyl carboxypeptidase ("enkephalinase") in the cerebral

metabolism of endogenous and exogenously-administered enkephalin

(Schwartz et al. 1983; Hambrook et al. 1976; Hazato et al. 1983).

Studies by Hambrook et al. (1976) implicate the same enzymes in rat

plasma as well. In vitro studies with the guinea pig ileum attribute

enkephalin hydrolysis in this tissue to the combined action of

aminopeptidases, enkephalinase, and angiotensin converting enzyme

(ACE) (Aoki et al. 1984). In the isolated perfused rat lung,

aminopeptidases and ACE, but not enkephalinase, are responsible for

enkephalin metabolism (Gillespie et al. 1985). In the present study

we characterize in more detail the enzymes associated with the

hydrolysis of LE in rat plasma.

MATERIALS AND METHODS

Male Sprague-Dawley rats (300-400 gm) were anesthetized with

pentobarbital and implanted with femoral artery cannulae. In vitro

enkephalin metabolism was measured in blood samples collected two

days later through the indwelling cannulae. For this assay, plasma

was rapidly separated, incubated 5 min at 37 deg C to permit

263
metabolism of endogenous enkephalin, and then combined in 115 or 230

μl aliquots with one or more of the following inhibitors: bestatin

(prepared in saline to achieve final concentrations of 5 - 1500 μM),

thiorphan (prepared in 5% N,N-dimethyl formamid in saline to achieve

final concentrations of 0.1 - 10 μM), and captopril (prepared in

saline to achieve final concentrations of 0.1 - 10 μM).

Following a 15 min incubation period, either 3H-LE (3 or 6 μM,

specific activity 43.6 Ci/mmol) or 3

H-2-d-ala-1-LE (3 or 6 μ1,

specific activity 58 Ci/mmol) was added to the reaction mixture.

Aliquots of plasma were removed at several time points after label

addition; the reaction was terminated in these aliquots with two

volumes of 0.1% trifluoroacetic acid in methanol. Unmetabolized LE or

2-d-ala-1-LE was separated from its combined metabolites by thin

layer chromatography, according to the method of Ziring et al.

(1983), and quantified by liquid scintillation counting.

RESULTS

The half-life of LE in rat plasma was determined to be 2.4 min, which

is in close'agreement with the 2.5 min half-life determined by

Hambrook et al. (1976) in a vas deferens bioassay. The plasma

half-life of 2-d-ala-1-LE was between 3.5 and 7.5 hr.

The aminopeptidase inhibitor bestatin, in concentrations between 5

and 1500 μM, produced concentration-dependent decreases in the rate

of LE metabolism, to a maximum of 80% inhibition. The dipeptidyl

carboxypeptidase ("enkephalinase") inhibitor thiorphan, in

concentrations up to 10 μM, was without detectable effect on the rate

of LE hydrolysis. The combination of bestatin and thiorphan produced

no further inhibition beyond that attributable to bestatin alone. On

the other hand captopril (0.1 - 10 μM), an ACE-selective inhibitor,

when combined with bestatin (500 μM), produced an additional

concentration-dependent decrease in LE hydrolysis. The maximal

inhibition produced by this combination was approximately 25% beyond

that produced by bestatin alone. See Table 1 for summary of these

data.

A different pattern of enzyme activities was revealed when

2-d-ala-1-LE replaced LE as substrate for the reaction. Although this

substrate is reportedly insensitive to aminopeptidase degradation in

brain (Llorens et al. 1982), a concentration-dependent decrease in

2-d-ala-1-LE hydrolysis was seen in the presence of bestatin (50 -

1500 μM), to a maximum of approximately 30 - 40% inhibition (although

in one animal more than 90% inhibition was observed). Thiorphan (0.1

- 10μM) produced small concentration-dependent decreases in

hydrolysis, to a maximum of 10 - 20% inhibition. The combination of

bestatin (500 μM) and thiorphan (0.1 - 10 μM) produced no further

inhibition beyond that attributable to bestatin alone. ACE hydrolysis

of 2-d-ala-1-LE was not detectable, since captopril (0.1 - 10 μM)

produced no measurable inhibition. These data are summarized in Table

1.

264
TABLE 1: Effects of Inhibitors on Enkephalin Metabolism

Substrate Inhibitor Concentration Maximum %

(Highest con- Dependent Inhibition

centration;μM) Inhibition? (Estimate)

3

H-LE bestatin (1500)

thiorphan (10)

best (500) + thio (10)

best (500) + cap (10)

3H-2-d-ala-1-LE bestatin (1500)

thiorphan (10)

captopril (10)

best (500) + thio (10)

yes 80

no 0

no 80

yes 85

yes

yes

no

no

35

15

0

35

Combined inhibitor studies = bestatin (best) at a single

concentration, with thiorphan (thio) or captopril (cap) in

increasing concentrations

DISCUSSION

These data indicate that bestatin-sensitive aminopeptidase activity

accounts for approximately 80% of the [leu]enkephalin hydrolysis in

rat plasma. This is in agreement with the report by Venturelli et al.

(1985), based on the concentrations of resulting metabolites, that

aminopeptidases hydrolyze approximately 75% of the enkephalin in rat

blood. Approximately 5% of the LE in blood is metabolized by

captopril-sensitive ACE, while thiorphan-sensitive enkephalinase

activity could not be detected. This pattern of enzyme activities is

similar to that in rat lung (Gillespie et al. 1985), but different

from that in brain and guinea pig ileum (Schwartz et al. 1983;

Hambrook et al. 1976; Hazato et al. 1983; Aoki et al. 1984). The

enzyme(s) responsible for the remaining 15% of LE metabolism in blood

are as yet unidentified.

Our data also suggest an unusual pattern of enzyme activities against

the 2-d-ala-1-LE substrate. This substrate reportedly is selective

for enkephalinase and insensitive to aminopeptidase activity in brain

tissue (Llorens et al. 1982), and its hydrolysis in brain is potently

inhibited by thiorphan (Roques et al. 1983). In addition, brain

enkephalinase is not inhibited by bestatin (van Amsterdam et al.

1983). However, in our studies, only 10 - 20% of the hydrolysis of

265
2-d-ala-1-LE could be attributed to a thiorphan-sensitive

enkephalinase; 30 - 40% of the hydrolysis was due to an unidentified

bestatin-sensitive enzyme, captopril-sensitive ACE activity could not

be detected, and the source of the remaining 40 - 60% of the

degradation is unknown.

Taken together, these data indicate that rat plasma has its own

unique pattern of enkephalin hydrolysis. The enzymes active in plasma

may be isoenzymes to the enzymes found in other tissues, differing

from these latter enzymes in their sensitivities to inhibitors.

Alternatively, the plasma enzymes may only appear to be different

because of differences in conformation or in the availability of a

substance that competes with, or alters the binding of,

[leu]enkephalin or the inhibitors to the enzymes.

REFERENCES

Aoki, K., Kajiwara, M., and Oka, T. Japan J Pharmacol, 36:59-65,

1984.

Gillespie, M.N., Krechniak, J.W., Crooks, P.A., Altiere, R.J., and

Olson, J.W. J Pharmacol Exp Therap, 232:675-681, 1985.

Hambrook, J.M., Morgan, B.A., Rance, M.J., and Smith, C.F.C.

Nature, 262:782-783, 1976.

Hazato, T., Shimamura, M., Katayama, T., Kasama, A., Nishioka, S.,

and Kaya, K. Life Sciences, 33:443-448, 1983.

Llorens, C., Malfroy, B., Schwartz, J.C., Gacel, G., Roques, B.P.,

Roy, J., Morgat, J.L., Javoy-Agid, F., and Agid, Y. J Neurochem,

39:1081-1089, 1982.

Martinez, J.L., Weinberger, S.B., Morrow, S., and Dana, R.C.

Neurosci Abstr, 11:982, 1985.

Roques, B.P., Lucas-Soroca, E., Chaillet, P., Costentin, J., and

Fournie-Zaluski, M.C. Proc Nat Acad Sci USA, 80:3178-3182, 1983.

Schwartz, J.C., De La Baume, S., Yi, C.C., Malfroy, B., Chaillet, P.,

Marcais-Collado, H., and Costentin, J. In:Ehrenpreis, S., and

Sicuteri, F., eds. Degradation of Endogenous Opioids: Its

Relevance in Human Pathology and Therapy. New York: Raven Press,

1983. pp 51-66.

van Amsterdam, J.G.C., van Buuren, K.J.H., de Jong, A.M., and

Soudijn, W. Life Sciences, 33(Sup.I):109-112, 1983.

Venturelli, F., Roscetti, G., Possenti, R., Vita, F., and Roda, L.G.

Neurochem Res, 10:333-342, 1985.

Ziring, B., Sheppard, S., and Kreek, M.J. Int J Pep Prot Res, 22:

32-38, 1983.

ACKNOWLEDGMENTS

This research was supported by ONR #N00014-83-K-0408. Captopril was

donated by The Squibb Institute for Medical Research.

266
CHARACTERIZATION OF THE ENKEPHALIN-DEGRADING

AMINOPEPTIDASE OF NEUROBLASTOMA (N1E-115) CELL MEMBRANES

Hans Lentzen, Reimund Simon and Ingeborg Reinsch

Institut für Pharmakologie, Freie Universität Berlin,

Thielallee 69/73

D-1000 Berlin 33, F. R. G.

ABSTRACT

A membrane-bound enkephalin (ENK) -hydrolyzing aminopeptidase (AP) was partially purified from neuroblastoma

(clone N1E-115) cell membranes; enzyme activity was

assayed by determination of the leu-enkephalin (LENK)

degradation product, tyrosine (Tyr), with HPLC. The

enzyme was extracted with Triton X-100, resolved by

anion-exchange chromatography and further purified by

gel filtration. The overall purification was about

100-fold with a yield of 43%. The apparent Mr value of

the AP by gel filtration in the presence of 0.3% Triton

X-100 was approx. 400 kDa. In the absence of detergent

the apparent Mr value was about 305 kDa. In the elution

buffers, where Triton X-100 was omitted, the peptidase

activity was lost. The enzyme had a Km of 0.13 mM and a

Vmax of 450 nmoles per mg protein per min at 25°C for

LENK and exhibited little sensitivity to bestatin (IC50:

200 μM) and puromycin (IC50: 500 μM), but it was

strongly inhibited by amastatin (IC50: 8 μM). The enzyme

is an amphiphilic membrane protein; the native primary

structure is preserved only in the 'detergent form'. It

seems to be AP N (EC 3.4.11.2) with an optimum of pH 7.2

to 7.4. We assume that this AP plays the important role

in inactivating ENKs on the neuronal level.

The ENK-degrading AP, partially purified from primary

rat brain astrocyte cell membranes, exhibited a smaller

apparent Mr value (130 kDa) and a higher sensitivity to

amastatin (IC50: 0.4 μM), bestatin (IC50: 90 nM) and

puromycin (IC50: 5 μM) than did the N1E-115 enzyme. From

these data we conclude that there are at least two

membrane-bound APs responsible for the ENK degradation

in the brain.

INTRODUCTION

In this paper we report the existence of an AP N like

enzyme in membranes of neuroblastoma cells (N1E-115) and

of a bestatin-sensitive AP in cell membranes of primary

astrocytes of 2-day old rats. In case of the neuronal

enzyme the entire AP molecule in the cell membranes of

the NlE-115 cells is extracted by the neutral detergent

Triton X-100 leading to an amphiphatic form called the

detergent form. The catalytically active site of the

enzyme can also be solubilized by proteolytic cleavage

at the hydrophilic-hydrophobic junction of the protein

chain (protease form). Both forms of the AP have been

267

172-683 O - 87 - 10 : QL 3
purified and some of its properties have been

investigated and compared. The comparison includes the

partially purified glial AP, in which a detergent and

protease form was not distinguishable.

MATERIALS AND METHODS

(Tyr-3,5-3H)-LENK (36.5 Ci/mmol) was purchased from

DuPont (NEN, Dreieich, Germany). Mono Q monobeads and

superose 6 were from Pharmacia Fine Chemicals AB

(Uppsala, Sweden). LENK, bestatin and puromycin were

from Sigma Chemical Co. (Munich, Germany). Mouse

neuroblastoma cells, clone N1E-115, were grown as

monolayers. The media were changed three times a week

and the experiments performed at the confluent stage,

usually 7 days after plating. Primary astrocytes of

2-day old rats were cultured as previously described

(Lentzen and Palenker 1983). After washing the cells

were scraped off in 10 mM dipotassium phosphate buffer,

pH 7.4, containing 1 mM EDTA with 2.8 μg aprotinin/ml

and homogenized in a Potter-Elvehjem homogenizer

(glass/Teflon). The homogenate was centrifuged at 4°C at

1000 x g for 15 min, the 1000 g supernatant at 8000 x g

for 20 min, the 8000 g supernatant at 40000 x g for 30

min. The 40000 g pellet was washed once with 10 mM

dipotassium phosphate buffer, pH 7.4, containing 1 mM

EDTA with 2.8 μg aprotinin/ml and once more in

dipotassium phosphate buffer without EDTA. The pellet

was resuspended in 10 mM dipotassium phosphate buffer

(40 mg/ml) containing 1 mM DT, 1 mM PMSF, 100 nM

pepstatin A, 0,3% Triton X-100, 20% ethylene glycol, 2.8

μg aprotinin /ml, pH 7,7, solubilized at 4°C for 1 h and

subsequently centrifuged at 4°C at 100000 x g for 1 h.

The 100000 x g supernatant was loaded on an

anion-exchange column (Mono Q, Pharmacia, Uppsala,

Sweden) and eluted with 10 mM dipotassium phosphate, 20%

ethylene glycol, 1 mM DT, 1 mM PMSF and 0.3% Triton

X-100 and an NaCl gradient from 0 to 1 M with a flow

rate of 1 ml/min. The pooled enzyme fractions of the

Mono Q chromatography were concentrated by

ultrafiltration and applied to a Superose 6 column

(Pharmacia; 1.0 x 30 cm). Protein was eluted with 10 mM

dipotassium phosphate buffer containing 0.1 M NaCl, 0.3%

Triton X-100, 20% ethylene glycol, pH 7.4, at a flow

rate of 0.5 ml/min. 40 μl of enzyme fractions were

preincubated with 40 μl dipotassium phosphate buffer, pH

7.4, for 15 min at 25° C and further incubated with

3H-LENK (20 μl) for 15 min. The incubation was stopped

with 10 μl of 1 N HCl and by deep-freezing of the

samples in liquid nitrogen. The 3H-labeled Tyr was

detected as described (Lentzen and Palenker 1983).

RESULTS AND DISCUSSION

The enzyme was purified within 10 h after homogenization

of the cells. The optimal time was 60 min for

268
solubilizing the AP from the neuroblastoma cell

membranes incubated with 0.3 % Triton X-100 at 4°C.

Under these conditions 95% of the whole AP activity was

solubilized. In the supernatant after solubilization

only one activity peak was detected using anion-exchange

chromatography. At a concentration of 0.25-0.35 M NaCl

about 77% of the whole activity loaded on the column was

eluted. In the absence of detergent in the elution

buffer, the peptidase activity was lost. The relatively

naked protein molecules tend to bury their hydrophobic

regions by clustering together, forming large aggregates

under the loss of enzyme activity. The fractions from

anion-exchange chromatography were loaded after

concentrating by ultrafiltration on a gel filtration

column. The Mr value of the enzyme in the presence of

Triton X-100 was approx. 400 kDa. The following proteins

were used for calibration: thyroglobulin 669 kDa,

ferritin 440 kDa, catalase 232 kDa, aldolase 158 kDa.

After solubilization, using Triton X-100 as detergent,

the hydrophobic domain binds a micelle of detergent,

thereby increasing the molecular weight by about 95 kDa,

corresponding to approx. 150 molecules of detergent.

Therefore, the Mr value of the native enzyme after gel

filtration seems to be 305 kDa. The Michaelis constants,

Km and Vmax, for LENK as substrate was calculated by

Lineweaver-Burk analysis using substrate concentrations

from 1.8 to 800 μM. The Km and Vmax for LENK were 125 μM

and 450 nmoles per mg protein per min at 25°C. The AP

inhibitors, amastatin (IC50: 8 μM), bestatin (IC50: 200

μM) and puromycin (IC50: 500 μM) had inhibitory effects.

The enzyme is active between pH 5.9 and 8.2 with maximal

activity for LENK hydrolysis between pH 7.2 and 7.4.

These properties of the enzyme are not in accordance

with the studies about ENK-degrading APs in synaptosomal

preparations (Hui et al. 1983; Hersh 1985 ). However,

they are in agreement with the studies of Maroux et al.

(1973), Gray and Santiago (1977), Kenny and Maroux

(1982) and others on the AP N from microvilli from

intestinum and kidney of pigs and rats. Experiments of

Gros et al. (1985) suggest, that AP N is present in the

brain. If solubilization and purification of the enzyme

was made without suitable protease inhibitors or if

trypsin was added to the detergent extract, some

catalytically active fractions were isolated in the

anion-exchange chromatography, which showed activity

both in the presence and in the absence of the detergent

in the elution buffer. We selected a low-molecular

weight activity peak (66 kDa) for determination of the

kinetic parameters for the substrate LENK and for

estimation of the IC50 values for the inhibitors

bestatin and puromycin. This fraction showed a six-fold

higher affinity (Km: 25 μM) and more sensitivity against

bestatin (IC50: 0.6 μM).

Our studies with the so-called detergent- and protease

form of the enzyme demonstrate that there obviously

exists an amphiphatic integral membrane protein

269
comparable to the AP N. Correspondingly this peptidase

has a lipohilic, catalytically inactive anchor in the

membrane and a hydrophilic catalytically active head

projecting into the extracellular space. The hydrophilic

head can be cut off by proteases like trypsin and can be

further fragmented dependent on the incubation time. It

was surprising that the hydrophilic part of the AP shows

a lower Km value and lower inhibitor constants than did

the detergent form of the enzyme. We suggest that this

enzyme is the membrane-bound, ENK hydrolyzing, neuronal

AP, which must be distinguished from a glial AP.

We isolated an AP from cell membranes of primary

astrocytes. Solubilization and purification of the glial

enzyme were performed as described above for the

neuronal enzyme. As in the case of the neuronal enzyme

the glial AP is an membrane protein as several

solubilization trials with different detergent

concentrations revealed. Using anion-exchange

chromatography, only one AP activity peak was eluted at

a sodium chloride concentration of 0.3 M. In contrast to

the neuronal AP the glial enzyme was catalytically

active independent on the presence or absence of Triton

X-100 in the elution buffer. The pooled Mono Q fractions

containing the AP activity were concentrated by

ultrafiltration and then further purified by gel

filtration. After calibrating the column with standard

proteins, this peptidase corresponded to a molecular

weight of approx. 130 kDa.

The enzyme had an optimum of about pH 6.7. Its affinity

to the substrate LENK with a Km of 14 μM is relatively

high. It was strongly inhibited by amastatin (IC50: 0.4

μM), bestatin (IC50: 90 nM) and puromycin (IC50: 5 μM).

Similar results for a synaptosomal bestatin-sensitive AP

were reported by Hui et al. (1983), Hersh (1985) and

Giros et al. (1986). In contrast to the neuronal enzyme,

a detergent- and protease form of the glial AP is not

distinguishable. (This work was supported by the DFG).

REFERENCES

Giros, B., Gros, C., Solhonne, B., and Schwartz, J.-C.

Mol Pharm, 29:281-287, 1986.

Gray, G.M., and Santiago, N.A. J Biol Chem,

252:4922-4928, 1977.

Gros, C., Giros, B., and Schwartz, J.-C. Biochemistry,

24:2179-2185, 1985.

Hersh, L.B. J Neurochem, 44:1427-1435, 1985.

Hui, K.-S., Wang, Y.-J., and Lajtha, A. Biochemistry,

22:1062-1067, 1983.

Kenny, A.J., and Maroux, S. Physiol Rev,

62:91-128, 1982.

Lentzen, H., and Palenker, J. FEBS Lett, 153:93-97,

1983.

Maroux, S., Louvard, D., and Baratti, J. Biochim Biophys

Acta, 321:282-295, 1973.

270
THE ENDOGENOUS TRIPEPTIDE TYR-GLY-GLY AS AN EXTRACELLULAR

METABOLITE OF ENKEPHALINS IN RAT BRAIN : ORIGIN AND METABOLISM

Bruno Giros, Catherine Llorens-Cortes, Claude Gros

and Jean-Charles Schwartz

Unite 109 de Neurobiologie et Pharmacologie

Centre Paul Broca de l'INSERM 2 ter rue d'Alésia 75014 PARIS (FRANCE)

ABSTRACT

The tripeptide Tyr-Gly-Gly (YGG) was established as an endogenous

constituent in rat brain. Its origin from enkephalin neurons is

suggested by its regional distribution paralleling that of (Met5)-

enkephalin (YGGFM), its decrease following kainate-induced ablation

of the striato-pallidal neurons and its enhanced formation following

depolarization of pallidal slices. Enkephalinase (EC 3.4.24.11) is

selectively responsible for endogenous YGG formation in vitro and in

vivo.

INTRODUCTION

Using a sensitive RIA (Llorens-Cortes et al. 1985b) YGG, a potential

metabolite of opioid peptides (OP), was characterized as an extraneuronal constituent of mouse brain (Llorens-Cortes et al. 1985a).

In this present work we have characterized YGG in rat brain regions

and explored the hypothesis that it may originate from OP by assessing the effects of lesions of a known enkephalin pathway and studying its formation in depolarized slices.

MATERIALS AND METHODS

In most cases male Wistar rats were killed by decapitation, brain

regions dissected out in the cold in less than 3 min and homogenized

in 0.4 N HClO4. Experiments with slices of globus pallidus were

performed as described (Giros et al. 1986).

RIAs for YGGFM and YGG were performed as described (Gros et al. 1978;

Llorens-Cortes et al. 1985a)

RESULTS AND DISCUSSION

YGG immunoreactivity was detected in all regions of rat brain (table

1) and appears to correspond to authentic YGG as indicated by its

chromatographic patterns in various systems (namely HPLC on C18

μBondapak columns). Its levels in striatum did not differ when rats

were killed by decapitation or microwave irradiation, indicating that

it was not formed artefactually by hydrolysis of larger peptides

brought into contact with peptidases at the homogenization step.

In most brain areas YGG levels corresponded to about 10 % of YGGFM

levels (table 1). This parallel regional distribution is consistent

with the idea tnat the tripeptide constitutes a metabolic product of

enkephalins or related OP.

Ablation of proenkephalin-A-containing neurons in the striatopallidal pathway elicited by intrastriatal kainate was accompanied

by significant decreases in YGG in both the injected and the

projection areas (table 2).

271
TABLE 1: Regional distribution of YGG and YGGFM immunoreactivities

in rat brain

REGIONS YGG YGGFM YGG

(pmol/mg protein) YGGFM

Globus pallidus 3.50 0.40 38.2 0.38 0.09 0.01

Substantia nigra 1.71 0.25 10.0 0.80 0.17 0.03

Caudate putamen 1.01 O.11 14.8 0.16 0.07 0.01

Hypothalamus 0.47 0.03 12.7 0.70 0.04 0.01

Cerebral cortex 0.45 0.05 3.62 0.30 0.12 0.01

Hippocampus 0.29 0.02 2.40 0.28 0.12 0.02

Brainstem 0.05 0.01 0.46 0.03 0.11 0.04

Cerebellum 0.02 0.02 0.10 0.01 0.20 0.02

Means S.E.M. of data from 5 to 18 experiments.

TABLE 2: Effect of kainic acid injections on YGG-ir and YGGFM-ir

levels in caudate-putamen and globus pallidus

ANALYSED REGION YGG LEVELS YGGFM LEVELS

(pmol/mg protein)

Caudate putamen

Control side

Injected side

Variation

Globus pallidus

Control side

1.06 O.11 19.7 0.8

0.77 0.09* 15.4 1.3=

- 27 % - 23 %

4.12 0.36 28.2 1.9

Injected side 2.09 0.21** 19.7 1.3

Variation - 49 % - 30 %

* P < 0.05 ; ** P < 0.01 ; *** P < 0.001 compared to control

Kainic acid (1.5 μg/1 μl), dissolved in distilled water adjusted

to pH 7.4 was microinjected into the right anterior caudateputamen at a rate of 0.6 μl/min. Animals were sacrificed by

decapitation 14 days later.

Means S.E.M. of data from 18 separate experiments.

272
When pallidal slices were incubated in the absence (not shown) or

the presence (table 3) of bestatin, an aminopeptidase inhibitor

preventing the hydrolysis of YGG (De la Baume et al. 1983), the

tripeptide was predominantly found in the incubation medium. This

extracellular localization of YGG is consistent with data from subcellular

1985a). K

+

-induced depolarization of the slices elicited a strong

fractionation studies in mouse brain (Llorens-Cortes et al.

elevation of YGG in the medium (in which it was identified by HPLC)

as well as in slices (in which it might be locates in the

extracellular space). The amount of YGGFM released by the K+ stimulus

(corresponding to the difference of YGGFM tissue levels at 5 mM K+

and 50 mM K

+

, respectively) was notably inferior to the increase of

the sum (YGGFM + YGG) recovered in the medium (table 3). This indicates that released peptides other than YGGFM, i.e. presumably other OP

derived from proenkephalin A also contribute in YGG formation.

TABLE 3: Effects of a depolarizing stimulus on tissue and incubation

medium levels of YGG-ir and YGGFM-ir of pallidal slices

CONDITIONS

LEVELS IN SLICES

(pmol/mg protein)

LEVELS IN MEDIUM

(pmol/mg protein)

YGGFM YGG YGGFM YGG

5 mM K+

23.8 0.9 1.2 0.2 0.8 0.1 2.6 0.5

50 mM K+17.9 0.2 2.1 0.2 2.6 0.2 10.2 0.7

Difference - 5.9 1.0 + 0.9 0.3 + 1.8 0.2 + 7.6 0.9

*P < 0.05 ; ** P < 0.01 ; *** P < 0.001 compared to 5 mM K+

Slices were preincubated for 10 min in the presence of 100 μM

bestatin), and incubated for a further 5 min with 50 mM K+, when

stated. At the end of incubations, slices were separated from

medium by a short centrifugation and peptides extracted into

HClO4 were radioimmunoassayed in both fractions.

Means SEM from 6 experiments

In the presence of Thiorphan in increasing concentrations, the K

+

-

induced rise in YGG was progressively abolished while the recovery of

YGGFM in the incubation medium rose to reach the amount of YGGFM

released from the slices (figure 1). The IC50 value of Thiorphan (9

nM) was consistent with its effect being se;lectively mediared by

inhibition of enkephalinase. In addition acetorphan (10 mg/kg), a

parenterally-active derivative of Thiorphan, strongly reduced YGG

levels in various brain regions (by 55-70 % after 1h), establishing

enkephalinase as the YGG forming enzyme in vivo as well as in vitro.

This peptidase selectively cleaves amide bonds comprising the amino

group of en aromatic (or hydrophobic) amino acid residue which is

consistent with the idea that OP (which all have in common the YGGF

sequence) are precursors of YGG. Also the fact that enkephalinase is

an ectoenzyme in brain (De La Baume et al. 1983) is consistent with

273
the idea that YGG is formed after release of OP, derived from

proenkephalin A precursor since other OPs are poor substrates.

the

FIGURE 1: Effects of Thiorphan, en "enkephalinase" inhibitor,

on levels of YGG and YGGFM imnunoreactivities in the incubation

medium of K

+

-depolarized slices of rat globus pallidus.

Pallidal slices were preincubated for 10 min in the presence of

100 μM bestatin and increasing concentrations of Thiorphan and

then exposed for 5 min to 50 mM K

+

. After centrifugation, YGG

and YGGFM were radioimmunoasssyed in media.

In conclusion, all data were consistent with the idea that YGG represents an extracellular metabolite of endogenous OPs derived from

proenkephalin-A for which its measurement may constitute an index of

release.

REFERENCES

De la Baume, S., Yi, C.C., Schwartz, J.-C., Chaillet, P., MarcaisCollado, H., and Costentin, J Neuroscience, 8: 143-151, 1983.

Giros, B., Gros, C., Solhonne, B., and Schwartz, J.-C. Mol Pharmacol. 29 :281-288, 1986.

Gros, C., Pradelles, P., Rouget, C., Bepoldin, O., Dray, F., FournieZalusKi, M.-C., Roques, B.P., Pollard, H., Llorens, C., and

Schwartz, J.-C. J Neurochem, 31:29-39, 1978.

Llorens-Cortes, C., Schwartz, J.-C., and Gros, C. FEBS Lett, 189:

325-328, 1985b.

Llorens-Cortes, C., Gros, C., and Schwartz, J.-C. Eur J Pharmacol,

119:183-191, 1985a.

Llorens-Cortes C., Gros, C., and Schwartz, J.-C. Proc Natl Acad Sci

(USA), 83:6226-6230, 1986.

274
EVALUATION OF STRIATAL ENKEPHALIN RELEASE IN VIVO FROM STEADY

STATE LEVELS AND TURNOVER RATES OF THE TRIPEPTIDE TYR-GLY-GLY

C. Llorens-Cortes, C. Gros and J-C. Schwartz

Unite 109 de Neurobiologie et Pharmacologie,

Centre Paul Broca de l'INSERM,2ter rue d'Alésia- 75014 PARIS (FRANCE)

ABSTRACT

The tripeptide Tyr-Gly-Gly (YGG), an extraneuronal metabolite of

opioid peptides (OP) derived from proenkephalin A is in a highly

dynamic state in mouse striatum. Inhibition of its synthesis by

Thiorphan reduced its levels with a t 1/2 of 12 min. Inhibition of

its degradation by bestatin elicited its rapid accumulation consistent with a t 1/2 of enkephalins in the one-hour range. Pentobarbital

anesthesia markedly reduced its steady state level end turnover

rates.

INTRODUCTION

Using a sensitive RIA, we recently characterized YGG as an endogenous

constituent of mouse (Llorens-Cortes et al. 1985a,b) and rat brain

tissues (Giros et al. this volume). Endogenous YGG appears to be

formed extraneuronally by enkephalinase degradation of the OPs derived from the proenkephalin A precursor

distribution parallels that of YGGFM, ii)

because i) its regional

its levels decrease after

ablation of these neurons iii) its extracellular localization has

been shown by subcellular fractionation and studies with brain

slices; iv) its level rises considerably as a result of depolarization of proenkephalin A neuron terminals, which are abundant in

slices from globus pallidus, and v) inhibitors of the ectoenzyme

enkephalinase inhibit YGG formation selectively.

We have presently attempted to evaluate YGG turnover in mouse striatum by measuring its rates of decline and rise following inhibition

of enkephalinase and aminopeptidase respectively. To validate these

measurements under conditions of altered neuronal activity, we

assessed the effects of pentobarbital anesthesia which is known to

modify the activity of a variety of neuronal systems.

MATERIALS AND METHODS

Male Swiss mice were killed after i.c.v. administration of saline or

peptidase inhibitors. The striata were dissected out in less than 2

min, homogenized in 0.4 N HClO4 and YGG measured in extracts by a RIA

(Llorens-Cortes et al. 1985a,b).

RESULTS AND DISCUSSION

Thiorphan elicited a monoexponential decline of striatal YGG with a

fractional rate constant of 3.5 0.5 hr corresponding to a t 1/2

of 12 2 min end a turnover-rate of 18 3 pmol/mg protein/hr

(figure 1). A similar rate of decline was found with 1 mg/kg acetorphan a parenterally-active enkephalinase irhibitor (Lecomte et al.

1986) (not shown).

275
FIGURE 1: Decline of YGG levels in mouse striatum after

administration of the enkephalinase inhibitor Thiorphan.

Mice were given saline or 100 ug of Thiorphan i.c.v. and were

killed at different times after injection. Values are means

S.E.M. of five to eight experiments.

* P < 0.05 ; ** P < 0.01 versus the respective controls.

After bestatin administration, striatal YGG increased linearly for

15-20 min and then reached a plateau at about twice its steady-state

level (figure 2).

pmol/mg protein/hr.

The initial rate of YGG accumulation was 18 3

FIGURE 2: YGG accumulation in mouse striatum after administration of the aminopeptidase inhibitor bestatin.

Amimals received saline or 150 ug of bestatin i.c.v. and were

killed at different times after injection. Values are means

S.E.M. of 5-11 experiments.

*P < 0.05 ; ** P < 0.01 versus the respective controls.

276
From these two consistent estimations it may be inferred that YGGFM

release and its degradation into YGG occur at a rather high rate,

implying that the turnover of striatal enkephalins is comparable to

that of "classical" neurotransmitters. This conclusion is somewhat

unexpected since the t 1/2 of neuropeptides, although never reported

before, is often assumed to be longer due to processes of ribosomal

synthesis, maturation and axonal transport which precede their exocytosis.

However the above values should be considered with caution since they

are based upon a series of assumptions that may not be verified.

Peptidase inhibition may not be complete immediately after treatments and may trigger feedback control mechanisms regulating OP

release. Bestatin blocks the degradation of not only YGG but also of

OPs. Hence the fairly good agreement between the two estimations of

YGG turnover rate may be to a certain extent fortuitous. Nevertheless

the observation (Llorens-Cortes et al. 1986) that a similar value is

found for the rate of YGGFM accumulation in an extrasynaptosomal

fraction of mouse striatum following complete blockade of its metabolism (by Thiorphan plus bestatin) strenghthen our conviction that

these represent reasonable estimates of the rate of enkephalin

release in vivo.

In mice treated with pentobarbital the striatal YGG levels were

found to decrease progressively, a plateau being reached after 30

min (figure 3) without significant modification of YGGFM levels (not

shown).

FIGURE 3: Steady-state levels end bestatin-induced accumulation of YGG in mice anesthetized with pentobarbital.

Mice were given pentobarbital i.p. at 60 mg/kg either alone

(solid line) or with 150 ug of bestatin, administered 30 min

later i.c.v. (broken line) and were killed at various intervals

thereafter. Values are means S.E.M. of 5-15 experiments.

N.S. non significant

* P < 0.01 versus saline ; ** P < 0.01 versus pentobartital

alone.

277
When this new steady-state level was reached, administration of

bestatin elicited, for the next 15 min, linear accumulation of YGG at

a rate (pmol/mg protein/hr) of 7 1 versus 18 2 in controls.

A similar effect was observed when mice were anesthetized with chloral. This decrease in steady-state YGG level during anesthesia seems

attributable to impairment of enkephalin release and of YGG formation

shown with peptidase inhibitors. Barbiturates reduce the activity of

several classes of aminergic neurons. As far as we know, this is the

first time that their effects on peptidergic neuron activity have

been reported. The drastic reduction in the activity of enkephalin

neurons, shown by the present data, might be relevant to the hyperalgesic and anticonvulsive effects of barbiturates since enkephalins

induce opposite effects.

Hence the steady-state level of YGG appetis to constitute a useful

index of changes in enkephelin neuron activity in the brain, just as

the levels of certain extraneuronal metabolites reflect changes in

monoamine neuron activity. Measurement of this simple index offers

the great advantage of avoiding the administration of any metabolic

inhibitor. A similar experimental approach could conceivably be

applied to other classes of cerebral neuropeptides as soon as their

metabolic pathways are identified.

REFERENCES

Llorens-Cortes, C., Schwartz, J-C., and Gros, C. FEBS Lett,

189: 325-328, 1985a.

Llorens-Cortes, C., Gros, C., and Schwartz, J-C. Eur J Pharmacol,

119:183-191, 1985b.

Llorens-Cortes, C., Gros, C.,

(U.S.A.), 83:6226-6230, 1986.

and Schwartz, J-C. Proc Natl Acad Sci,

Lecomte, J-M., Costentin, J., Vlaiculescu, A., Chaillet, P., MarcaisCollado, H., Llorens-Cortes, C., Leboyer, M., and Schwartz, J-C. J

Pharmacol Exp Ther, 237:937-944, 1986.

278
ISOLATION AND CHARACTERIZATION OF ACTH-RELATED PEPTIDES

FROM PORCINE PITUITARIES

Karlheinz Voigt and Wolf Stegmaier

Institut für Klinische Pharmakologie, Freie Universität,

D-1000 Berlin 45, F.R.G.

ABSTRACT

We report on the isolation of new variants of porcine

ACTH with corticotropic activity. Two of these variants

differ in their amino acid sequence from porcine ACTH at

one position (Arg8). This implies that they cannot be

derived from the known precursor proopiomelanocortin

(POMC).

INTRODUCTION

The adrenocorticotropic hormone (ACTH) is synthesized as

part of its precursor POMC. In the pituitary intermediate

lobe ACTH 1-39 can be processed to -MSH (acetylACTH 1-13-amide) and CLIP (ACTH 18-39). None of these

peptides possesses corticotropic activity.

However, recently corticotropic active fragments of ACTH

were isolated from porcine anterior pituitaries and

identified as ACTH 1-38, ACTH 1-37, ACTH 7-39 and

ACTH 7-38 (Ekman et al. 1984).

We supposed that there could be more and yet shorter

ACTH-variants showing significant biological activities.

We will report on the isolation and sequence determination of five new ACTH-variants from porcine pituitaries, two of them differing in their primary structure

from the corresponding ACTH-sequence.

MATERIALS AND METHODS

Isolation of ACTH-like peptides

Fresh porcine pituitaries were obtained from a

slaughterhouse. They were extracted and prepurified as

described by Schleyer et al. (1976) and by additional gel

filtration on Fractogel TSK HW-40 (S) (Merck, F.R.G.).

The resulting fraction P-LF II D3 was further

chromatographed in a number of successive reversed phase

HPLC-systems.

The semipreparative system using an Ultrasphere ODS

column (250x10 mm, particle size 10μ, Beckman, U.S.A.)

yielded three main peaks eluting in a gradient of 0.2 %

TFA and acetonitrile. These main fractions were further

chromatographed in one or two analytical HPLC-systems

using either a 250x4.6 mm column packed with Ultrasphere

ODS 10μ and a gradient of 0.2 % TFA and acetonitrile

and/or a 250x4 mm column packed with Hypersil WP 300

butyl 5μ (Shandon, U.K.) and a gradient of 50 mM

279
potassium phosphate pH 2.5 and acetonitrile. This

resulted in 6 pure fractions F1 to F6. All peptides were

detected by their UV-absorption at 210 and 280 nm.

Peptide mapping

Fractions F1 to F6 were digested by TPCK-treated trypsin

(Sigma, F.R.G.), staphylococcus aureus protease (Miles,

U.S.A.), proline specific endopeptidase (Miles, U.S.A.)

and mouse submaxilliary protease (Sigma, F.R.G.)

following slightly modified standard procedures.

The digests were subjected to reversed phase HPLC on a

250x4 mm column packed either with HD-Gel (Gynkotek,

F.R.G.) or Hypersil WP 300 butyl 5μ applying a gradient

of 0.2 % TFA and acetonitrile.

Amino acid analysis

The amino acid compositions of the desalted and

hydrolyzed peptides were determined by precolumn

derivatization with ortho-phthalaldehyde (OPA) and

mercaptoethanol. The procedure of Umagat et al. (1982)

was modified and optimized for the routine analysis of

50 pmol samples.

Sequence analysis

The pure peptides were manually sequenced following the

4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC)

method of Wittmann-Liebold and Kimura (1984).

The C-termini were enzymatically sequenced using carboxypeptidase Y (Sigma, F.R.G.) as descibed by Allen (1981).

Biological activity and RIA

Corticotropic activity was tested by the dose dependent

secretion of corticosteron from isolated rat adrenal

cells (Sayers et al. 1971)

Radioimmunoassays using 125I-labelled tracers were performed for systems specific for -MSH, CLIP and the

middle portion of the ACTH-molecule.

RESULTS AND CONCLUSIONS

The prepurified extract of porcine pituitaries was

subjected to reversed phase HPLC in a number of

successive HPLC-systems with increasing separation power.

The HPLC-systems were monitored by region specific ACTHradioimmunoassays and by the corticotropic activity

tested with isolated rat adrenal cells. Finally we

obtained fractions F1 to F6, all showing a strong

crossreactivity to ACTH-antisera directed against the

middle portion of ACTH.

F1 to F6 were proven to be pure by OPA amino acid

analysis . They were digested by trypsin, staphylococcus

280
aureus protease, proline specific endopeptidase and mouse

submaxillary protease. The resulting peptide fragments

were separated by means of HPLC and subjected to amino

acid analysis and manual microsequencing.

FIGURE 1. Schematic representation of the amino

acid sequences of pACTH 1-39 and its variants

F1 to F6.

The results are shown in figure 1. The amino acid

sequence of F1 relates to ACTH 7-31 with the uncertainty

of Asx in ACTH position 29. F3, F5 and F6 proved to be

identical to ACTH 1-31, ACTH 7-35 and ACTH 7-38,

respectively. The latter variant has been already

described by Ekman et al. (1984).

F2 and F4 are ACTH-fragments with close sequence

homologies but differ from the above mentioned variants

by an exchange of Thr for Arg8 , thus resembling

(Thr8-)ACTH 7-31 and (Thr8-)ACTH 1-31.

Our peptides F1 to F6 exhibited a corticotropic activity

of about 2 to 25 % compared to porcine ACTH 1-39.

These by now unknown ACTH-variants could be generated

from ACTH by truncating it C-terminally in an unspecific

manner. In contrast the N-terminal cleavage site between

His6 and Phe7 seems to be very specific since no other

N-terminally truncated ACTH-variants have been isolated

(Ekman et al. 1984). To our knowledge such a specifity

has not been attributed to one of the known proteases.

The occurrence of the two (Thr8-)ACTH-variants points to

the existence of a gene differing from the known POMCgene.

Both genes could occur together in one animal or in a

subpopulation of animals lacking the ordinary POMC-gene

and having instead the gene for the ACTH-variants.

Oates and Herbert (1984) and Gossard et al. (1986) found

only one porcine POMC-gene. Boileau et al. (1983)

isolated two POMC mRNA species which coded for two

different N-terminal POMC-sequences but they did not show

differences in the ACTH-coding region.

The mutation at position 8 of the ACTH molecule might be

of functional relevance because the partial sequence

about it is crucial for the biological activity of a

number of POMC derived peptides (ACTH, -MSH, -MSH,

-MSH).

281
REFERENCES

Allen, G. Methods in Molecular Biology. Amsterdam,

Elsevier, 1981. 41 pp.

Boileau, G., Gossard, F., Seidah, N.G., and Chretien, M.

Can J Cell Biol, 61:333-339, 1983.

Ekman, R., Noren, H., Hakanson, R., and Jörnvall, H.

Regulatory Peptides, 8:305-314, 1984.

Gossard, F.J., Chang, A.C.Y., and Cohen, S.N.

Biochim Biophys Acta, 866:68-74, 1986.

Oates, E., and Herbert, E.

J Biol Chem, 259:7421-7425, 1984.

Sayers, G., Swallow, R.L., and Giordano, N.D.

Endocrinology, 88:1063-1068, 1971

Schleyer, M., Voigt, K.H., and Pfeiffer, E.F.

Horm Metab Res, 8:175-181, 1976.

Umagat, H., Kucera, P., and Wen, L.F.

J Chromatogr, 239:463-474, 1982.

Wittmann-Liebold, B., and Kimura, L.-F.

In: Walker, J.M., ed. Methods in Molecular Biology,

Vol. I. Clifton N.J.: Humana Press, 1984. pp. 221-242.

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 87/B2; Vo 253/6-1).

282
A MEMBRANE BOUND SUBSTANCE P DEGRADING ENDOPEPTIDASE

FROM RAT BRAIN

Andreas Bergmann and Karl Bauer

Institut für Biochemie und Molekularbiologie D-1000

Berlin 10, Franklinstra e 29, F.R.G.

ABSTRACT

From rat brain, a membrane bound substance P-degrading

endopeptidase (SPE) was purified 1580 fold to near

homogeneity. After extraction with 1OmM CHAPS, the enzyme

preparation was subjected to ion exchange chromatography

on DEAE-cellulose, adsorption chromatography on hydroxyapatite, gelfiltration through Ultrogel AcA 44 and FPLC

on Mono Q. This enzyme of 70000 molecular weight is

optimally active at pH 7.5. Metal chelators( EDTA and EGTA)

and sulfhydryl modifying reagents (N-ethylmaleimide and

p-chloromercuriphenylsulfonic acid) are strongly inhibitory while the serine-protease inhibitor diisopropylfluorophosphate does not effect the enzyme activity.

The enzyme is strongly inhibited by bacitracin but not

by phosphoramidon and captopril. Degradation of substance P

is strongly inhibited by neurotensin, somatostatin, ACTH

1-39, and less effectively by LHRH but not by Leucineenkephalin. Substance P is preferentially hydrolyzed at the

Gln6-Phe7 peptide bond but fragmentation at the Pro4-Gln5,

Gln5-Gln6,Phe7-Phe8 and Glyg-Leu1O bonds was also observed.

INTRODUCTION

The mechanism involved in the inactivation of the

putative neurotransmitter substance P are unknown. A

number of peptidases capable of degrading substance P have

been identified (Akopyan et al. 1979, Chrètien et al. 1980,

Leeet al. 1983, Matsas etal. 1983, Skidgelet al. 1984,

Yokosawaetal. 1983), but their functions for the

inactivation of synaptically released substance P

remain unclear. We previously reported that substance P

is hydrolyzed by a bacitracin sensitive enzyme localized

on neuronal cells in primary culture (Horsthemke etal.

19841 and now report on the purification of a membrane

bound and bacitracin sensitive substance P degrading

endopeptidase from rat brain.

MATERIALS AND METHODS

Enzyme assay: The enzyme solution was diluted with buffer

A (25mM K-diethylmalonate, pH 7.5, containing 0.04% NaN3)

and supplemented with 1OOpM diisopropylfluorophosphate

(DFP) and 1OOμM bestatin (final volume=19μl). After preincubation at 35°C for 15 minutes the reaction was started

by addition of O.1μCi (

3

H)-Phe8-substance P (1μl;24.5Ci/

mmol). At given time intervals 10μl of the reaction

mixture was spotted on silica gel plates (Merck, F.R.G.).

The plates were developed in ethyl acetate:pyridine:acetic

acid:H20 (125:100:20:60). Theamount of degraded substance P

was determined as described (Horsthemke etal. 1984).

283
Enzyme purification: All steps were performed at 4°C.

Frozen rat brains (1OOg) were thawed and homogenized in

380ml buffer B (5mM NaP04, pH7.5 containing 1mM DTE and

O.O4%NaN3). The homogenate was centrifuged for 1h at

1OOOOOg. The resulting pellet was rehomogenized in buffer

B (380ml) and washed again for additional 5 times. The

last homogenate was adjusted to 1OmM CHAPS (Sigma) and

stirred for lh. The membrane extract obtained by centrifugation at 1OOOOOg for 1h was then fractionated by

ion exchange chromatography on DE-52 cellulose (equilibrated in buffer B) using a linear NaCl gradient

(0-250mM). The substance P degrading activity eluting

at 4.5 to 8.5 mSi conductivity was pooled and applied

to a hydroxyapatite column, equilibrated in buffer B.

After washing with 50mM NaP04, two substance P degrading

activities were desorbed in a linear gradient from 50

to 180mM NaP04. The enzyme eluting at higher NaP04-

concentrations (6-9mSi conductivity) was pooled, concentrated by ultrafiltration (Amicon PM 10 membrane) and

subjected to gel filtration through Ultrogel AcA 44.

The substance P degrading endopeptidase eluting with a

molecular weight of approx. 70000 was clearly separated

from another substance P degrading activity of low

molecular weight and was further purified over FPLC on

Mono Q (Pharmacia) . The enzyme eluted as a single peak

at approx. 120mM NaCl. After rechromatography on Mono Q

this enzyme preparation was used for all further

investigations.

RESULTS AND DISCUSSION

Although the enzyme assay contained 100pM DFP to

inhibit the degradation of substance P by dipeptidylaminopeptidase IV and other serine-proteases, several

substance P degrading activities could be separated by

the purification procedure described. By this procedure

the substance P degrading endopetidase was purified

1580 fold to yield an enzymatically homogenous-enzyme

preparation.

The SPE was found to be strongly inhibited by metalchelating compounds such as EDTA and EGTA and also by

the -SH reactive reagents N-ethylmaleimide (NEM) or

p-chloromercuriphenylsulfonic acid (pCMBS) but not by

DFP, an inhibitor of serine-proteases (tab.1). SPE is

strongly inhibited by bacitracin, a general inhibitor

of various peptidases which also inhibits the substance

P degrading activity associated with neuronal cells in

primary culture (Horsthemke et al. 1984). These results

suggest that SPE is different from the substance P

degrading activity described by Leeet al. (1981) which

is not inhibited by 1mM Bacitracin. SPE is obviously

also different from other peptidases known to hydrolyze

substance P and other peptides (e.g. Leu-Enkephalin),

namely angiotensin converting enzyme and endopeptidase

24.11, since it is not inhibited by captopril and also not

by phosphoramidon, the known inhibitors of these enzymes.

284
Test substance concentration %inhibition

EDTA

EGTA

NEM

pCMBS

DFP

Captopril

Phosphoramidon

Bacitracin

I

1mM

1mM

100μM

100μM

100μM

1OμM

1OμM

100μM

93

70

96

100

0

0

0

72

Substance P

Neurotensin

Somatostatin

ACTH 1-39

LHRH

Leu-enkephalin

TABLE 1.

1OOμM 83

100μM 92

100μM 85

100μM 81

1OOμM 24

1OOμM 0

Test substances (2μl, 10-fold concentrated in

buffer A) were mixed with 17μl of buffer A

containing 1μl enzyme. Except when peptides

were tested, the mixture was preincubated for

15 min at 35°C. The reaction was started by

adding O.1μCi (1μl) (

3

H)-substance P and stopped

after incubation for 15 min at 35°C by spotting

1μl aliquots on silica gel plates. The amount

of substance P degraded was determined as

described under "materials and methods".

In agreement with these results, the degradation of

(

3

Hl-substance P was not effected by addition of unlabeled Leu-enkephalin. In contrast, the degradation of

the radiolabeled tracer was strongly inhibited not only

by unlabeled substance P but also by neurotensin,

somatostatin and ACTH 1-39 while LHRH was considerably

less effective. These results strongly indicate that

SPE is not a substance P-specific enzyme but a more

general peptidase which also hydrolyzes other peptides

by recognizing only certain structural elements of

their substrates. This notion is supported by the

observation that SPE catalyzes the hydrolysis of

substance P at various peptide bonds. Identification

of the enzymatically formed fragments by HPLC on a

μBondapak C18-column and amino acid analysis clearly

demonstrated that substance P is preferentially hydrolyzed by SPE at the Gln6

-Phe7 bond additionally also at

Pro4-Gln5,Gln5-Gln6, Phe7-Phe8 and Gly9-Met10 peptide

bonds.

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2

285
The present studies clearly demonstrated that SPE is

distinctly different from the substance P degrading

activities described so far and furthermore suggest

that this bacitracin-sensitive endopeptidase is a good

candidate likely to be important in the synaptic

inactivation of substance P and/or other neuropeptides.

However, since the physiological function of neuropeptide degrading enzymes appears to be primarily

determined by their specific localisation, only

immunocytochemical investigations at the electronmicroscope level will finally answer the question as

to the physiological function of this enzyme.

REFERENCES

Akopyan T.A., Arutunyan A.A., Organisyan A.I., Lajtha A.,

Galoyan A.A. J Neurochem, 32:629-631, 1979.

Chrètien M., Lee C.M., Sandberg B.E.B., Iversen L.L.,

Boucher R., Seidah N.G., Genest J. FEBS Lett, 113:

173-176, 198O.

Horsthemke B., Schulz M., and Bauer K. Biochem Biophys

Res Commun, 125:728-733, 1984.

Lee C.M. Sandberg B.E.B., Hanley M.R., Iversen L.L.

Eur J Biochem, 114:315-327, 1981.

Matsas R. Fulcher I.S., Kenney A.J., Turner A.J. Proc

Natl Acad Sci USA, 80:3111-3115, 1983.

Skidgel R.A., Engelbrecht S., Johnson A.R., Erdös E.G.

Peptides, 5:769-776, 1984.

Yokosawa H., Endo S., Ogura Y., Ishii S. Biochem Biophys

Res Commun, 116:735-742, 1983.

ACKNOWLEDGEMENTS

We gratefully acknowledge the valuable gift of (

3

H)-

substance P generously provided to us by Dr.R.Wade and

Dr.P.E.Brundish, Ciba Geigy, Horsham, U.K. We thank R.Hirt,

W.Stegmaier and Dr. K.H. Voigt, FU Berlin for performing

the amino acid analysis. This work was supported by the

Stipendienfonds des Verbandes der Chemischen Industrie

and by the Deutsche Forschungsgemeinschaft.

286
IN SITU HYBRIDIZATION VERSUS NORTHERN ANALYSIS:

WORKING TOWARDS THE CORRELATION OF TWO QUANTITATIVE TECHNIQUES FOR

OPIOID AND VASOPRESSIN mRNAs IN THE RAT HYPOTHALAMUS AND PITUITARY

Thomas G. Sherman and Stanley J. Watson

Mental Health Research Institute

University of Michigan

205 Washtenaw Place

Ann Arbor, MI 48109-0720

ABSTRACT

Several physical and technical aspects of in situ hybridization are

examined to help ascertain to what extent the total specific mRNA

population of interest in a tissue section is actually detected using

complementary RNA probes. Two experimental paradigms, Rot curve

hybridization analysis and varying the tissue section thickness,

indicate that complementary RNA (cRNA) probe hybridization to a

specific mRNA in a tissue section can be made saturable, but remains

so only for those mRNAs within a limited distance from the surface of

the section. Direct comparison with Northern analysis, however,

shows that in situ hybridization still gives comparable relative

quantitative results for mRNA levels changing due to physiological

stimulation.

INTRODUCTION

The application of in situ hybridization for the autoradiographic

identification of cellular mRNAs has gained general acceptance as a

useful tool in studies of the anatomical localization of specific

gene products. If this qualitative information could be extended to

either the absolute or relative quantitation of specific mRNA

transcripts, then in situ hybridization could be an even more useful

tool for the regulation of gene expression in tissues possessing

extreme cellular heterogeneity, such as the brain. Towards this end,

several technical aspects of in situ hybridization, including probe

hybridization kinetics, tissue section thickness, probe length and

photographic emulsion preparation are being further characterized in

hopes that such an understanding will aid in the application of in

situ hybridization as a quantitative method. In this brief

manuscript, we will discuss the application of [32P]-labeled cRNA

probes, generated with the pSP6 vector system (Promega Biotec), for

in situ hybridization studies of pro-opiomelanocortin (POMC) mRNA in

the intermediate lobe of the rat pituitary, or a [32P]-labeled

synthetic oligonucleotide complementary to rat vasopressin mRNA in

the rat magnocellular hypothalamus.

MATERIALS AND METHODS

Many of the in situ hybridization methods presented in this

manuscript have been extensively described elsewhere (Brahic and

Haase 1978; Cox et al. 1984; Lewis et al. 1985; Sherman et al. in

287
press; Watson et al. in press; Watson et al. 1985). Northern gel

analysis was conducted as previously described by Sherman et al.

(1986).

RESULTS AND DISCUSSION

The kinetics of cRNA hybridization to mRNA immobilized in a tissue

section, as is the case with in situ hybridization, more closely

approximates the situation existing with Northern or dot-blot

analyses than with classical solution-phase hybridization. The laws

of nucleic acid hybridization kinetics tell us that the rate of

duplex formation of excess cRNA probe to its complementary mRNA in an

immobile phase should follow pseudo first-order kinetics as described

by equation 1 (Young and Anderson 1985):

H = Do (1 - e

-kRoT) Equation 1

where H is the amount of duplex RNA formed, [Do] is the initial

amount of mRNA in the immobile phase, [Ro] is the initial amount of

cRNA probe in the mobile phase, T is time and k is a constant

possessing the units of 1/(sec-moles/liter). Practical application

of this equation takes the form of the so-called Rot curve, where

percent or amount of hybridization is plotted against the product of

probe concentration and time of hybridization (Ro x t = Rot). That

this can be shown to hold approximately true for in situ

hybridization is shown in figure 1, in which varying concentrations

of a mouse POMC cRNA probe are hybridized for varying lengths of time

against rat POMC mRNA in the intermediate lobe of the pituitary.

Figure 1. Rot curve for POMC cRNA hybridization to rat

pituitary intermediate lobe. To each 10 micron section was added

250,000 to 5,000,000 dpms of [32P]-labeled cRNA probe (800

nucleotides). Hybridizations were conducted for 2 to 7 days.

288
In its simplest terms, figure 1 demonstrates that the extent of

[32P]-cRNA:mRNA duplex formed increases with time or [32P]-cRNA

concentration, and that with sufficient amount of either or both of

these, the hybridization is saturable.

While it is good to know that in situ hybridization follows orderly

kinetics, and is, to a limited extent, predictable, the situation is

more complicated than it appears. It is more accurate to conclude

from figure 1 that the POMC mRNA accessible by the cRNA probe is

saturable; for recent preliminary experiments suggest that only the

POMC mRNA localized in the top 2-4 microns of the tissue section is

available for hybridization. Probe hybridization to pituitary

sections ranging in thickness from 4.5 to 20 microns resulted in

specific signals all lying within the same (relatively large)

standard error. Thicker sections clearly did not give appreciably

greater signal than much thinner sections. That this results from an

effective limit of probe penetrance is a likely explanation.

Although in situ hybridization, as of this stage, remains nonquantitative in absolute terms, it still may be useful for relative

quantitation studies where many uncharacterized sampling problems

essentially cancel each other out of the results. Even Northern gel

analysis remains at this level, since neither the efficiencies of RNA

transfer from gel to membrane nor the hybridization to these

immobilized RNAs are quantitative.

Figure 2. Correlation between Northern and in situ hybridiza

tion quantitation of salt-loading induced increases in vasopressin

mRNA in the supraoptic and paraventricular nuclei. The Northern gel

data is published in Sherman et al. (1986). The in situ data rep

resents average values from 2-4 sections for each nuclei. *p 0.005.

289
The analysis is effective, nonetheless, because each sample suffers

the same disadvantages experienced by all samples. This situation

may, in some situations, be true for in situ hybridization analysis

also. Figure 2 depicts the results of an experiment comparing the

relative quantitation of vasopressin mRNA in hypothalamic

magnocellular nuclei by either Northern gel or in situ hybridization

using the same synthetic oligonucleotide probe (Sherman et al. 1986).

The induction of cytoplasmic vasopressin mRNA occurring with chronic

salt-loading, as detected by Northern gel analysis, is similarly

detected by in situ hybridization. Both Northern hybridization bands

and in situ positive nuclei were quantitated with a digital image

analysis system using [32P] autoradiographic standards. At a first

approximation, therefore, it appears the two results give comparable

results. A similar comparison was made in the quantitation of POMC

mRNA in the intermediate lobe of the pituitary during chronic

haloperidol treatment (Kelsey et al. 1986). Clearly, however, there

remains the question of tissue section sampling as a source of great

error with in situ hybridization, which is not a problem with

Northern analysis.

In conclusion, we have shown that in situ hybridization follows

orderly hybridization kinetics, and can be a useful tool for the

relative quantitation of cellular mRNAs in some circumstances. We

feel, that with additional characterization and optimization

(including the development of good autoradiographic standards), the

capability of accurate, absolute, quantitation exists.

REFERENCES

Brahic, M, and Haase, A.T. Proc Natl Acad Sci USA, 75: 6125-6129,

1978.

Cox, K., Deleon, D., Angerer, L., and Angerer, R. Developmental

Biology, 101: 485-502, 1984.

Kelsey, J.E., Watson, S.J., Burke, S., Akil, H., and Roberts, J.L. J

Neurosci, 6(1): 38-42, 1986.

Lewis, M.E., Sherman, T.G., and Watson, S.J. Peptides, 6(2): 75-87,

1985.

Sherman, T.G., Kelsey, J.E., Khachaturian, H., Burke, S., Akil, H.,

and Watson, S.J. In: Uhl, G., ed. In Situ Hybridization in Brain.

New York: Plenum Press, in press.

Sherman, T.G., McKelvy, J.F., and Watson, S.J. J Neurosci, 6(6):

1685-1694, 1986.

Watson, S.J., Burke, S., Sherman, T.G., Lewis, M.E., and Akil, H.

In: Modern Neuroanatomical Methods, Society for Neuroscience Short

Course. Syllabus I, 1985. pp. 73-93.

Watson, S.J., Sherman, T.G., Kelsey, J.E., Burke, S., and Akil, H.

In: Proceedings of the meeting on In Situ Hybridization Methods.

MacArthur Foundation and the Nancy Pritzker Laboratory of the

Stanford Department of Psychiatry, in press.

Young, B.D., and Anderson, M.L.M. In: Hames, B.D., and Higgins,

S.J., eds. Nucleic Acid Hybridization, a practical approach.

Oxford: IRL Press, 1985. pp. 47-71.

290
IMMUNOHISTOCHEMICAL EVIDENCE FOR SUBPOPULATIONS OF

DYNORPHINERGIC NEURONS

Bruce Quinn and Eckard Weber

Institute for Advanced Biomedical Research

Oregon Health Sciences University

Portland, OR 97201

ABSTRACT

Biochemical studies have shown that the dynorphin precursor can generate at least seven opioid peptides. We

report here a study of dynorphin products analyzed on

adjacent semithin sections of rat brain, showing evidence

for differential peptide localization among magnocellular

neurons. In the oxytocinergic nucleus of the anterior

commissure, staining was strong for dynorphin B but very

weak for other prodynorphin markers; in the supraoptic

nucleus, a discrete subset of dynorphin neurons were positive for leu-enkephalin.

INTRODUCTION

Several recent studies have suggested regional heterogeneity in the immunohistochemical localization of products from the POMC (Bloch et al. 1979; McGinty and Bloom

1983) and proenkephalin precursors (Williams and Dockray

1983.) Heterogeneity of hypothalamic magnocellular

neurons has been reported for oxytocin/CRF expression

(Sawchenko et al. 1984) and for vasopressin in the supraoptic nucleus (Richards et al. 1985.) When studied by

RIA, the processing of the dynorphin precursor varies

markedly among brain regions (Weber et al. 1982b); however, immunohistochemical heterogeneity has not been

reported. Therefore, we have examined hypothalamic magnocellular dynorphin neurons for differential immunohistochemical staining of prodynorphin products.

MATERIALS AND METHODS

Male Sprague-Dawley rats were colchicine-treated (50 ug

icv) 48 hrs before perfusion with 4% paraformaldehyde,

and brains were immersed 24 hrs in 20% sucrose/3% PEG MW

400 before freezing in Cl2F2 Me (chilled in a liquid

nitrogen bath). Serial sections were cut at 1-2 u in a

-25° C cryostat. Sections were stained as previously

described at 1:400-1:800 (Weber and Barchas 1983) and

visualized with fluoroscein-labeled second antibodies.

Antisera were raised in our laboratory and characterized

previously: dynorphin A (Dyn A R2-4), Dyn A(1-8) (R2-2),

Dyn B (R2-4), alpha-neo-endorphin (a-NEp R2-4), leuenkephalin (L-Enk R1-3) and met-enkephalin-arg-gly-leu

(MERGL R2-2). In each case staining was blocked by preincubation with 10-20 uM of the homologous peptide, but

not by the other peptides.

291
Dyn B Dyn A Dyn A(1-8) a - NEp

Figure 1. Serial sections, nucleus of the anterior

commissure.

RESULTS AND DISCUSSION

We have previously colocalized oxytocin and Dyn B in the

nucleus of the anterior commissure (ACN; Quinn and Weber

1985.) As shown in figure 1, there was only weak staining in these cells for Dyn A, Dyn A(1-8), and a-NEp.

Blocking controls suggest relative C-terminal specificity

for these antisera. McGinty (1985) has reported cortical

neurons staining with Dyn B(R2-4); after pretreatment

with cholinesterase, other Dyn antisera produced staining

in her preparations (J. McGinty pers. comm.) Dyn B

(R2-4) may thus recognize its epitope in perikarya where

other Dyn products are uncleaved.

In the paraventricular nucleus (PVN), the four Dyn antisera produced staining differing somewhat in intensity

but consistent across PVN magnocellular neurons for each

antiserum. In contrast, in the supraoptic nucleus (SON),

isolated groups of neurons had sharply reduced staining

for a-NEp, despite consistent staining with the other

antisera (figure 2.) The same weak/strong aNEp cells

could be followed on several adjacent sections, and this

finding was similar in all animals studied. This result

could reflect either differential processing of a-NEp

among SON neurons, or possibly, different absolute precursor expression, to which this antisera may have been more

sensitive. In support of the former hypothesis, dilution

of Dyn A(1-8) antiserum to extinction (1:6400) never

produced a pattern similar to a-NEp (data not shown.)

a - NEp Dyn A(1-8) a - NEp

Figure 2. Serial sections, supraoptic nucleus

292
Dyn A (1-8) Leu - Enk

Figure 3. Adjacent sections, supraoptic nucleus.

In the SON, isolated Dyn-positive neurons stained

brightly for Leu-Enk, against a background of other Dyn

neurons with no detectable Leu-Enk staining (figure 3);

the majority of SON Dyn occurs in vasopressin neurons

(Watson et al. 1982a; our obs.) Consistent with earlier

work (Weber et al. 1982a; Watson et al. 1982b; Williams

and Dockray 1983) we saw no MERGL staining in this study

in the SON. Production of Leu-Enk from Dyn has been

shown by RIA/lesion studies by Zamir et al. (1984.) We

saw only scattered Leu-Enk-positive neurons in this

nucleus, and in only about half of these colchicinetreated animals. However, in noncolchicine-treated rats,

Martin and Voigt (1982) have reported a subset of Leu-Enk

positive terminals in the vasopressin fibers of the

neurohypophysis, while all vasopressin terminals were

Leu-Enk positive after trypsin pretreatment. Our results

further support a process for Leu-Enk production in a

specific subset of the vasopressin/dynorphin pathway.

Heterogeneity of neuropeptide expression in the magnocellular system has been documented several times. Oxytocin neurons express CRF in the ACN, sometimes in the

PVN, and occasionally in the SON (Sawchenko et al. 1984.)

All ACN, but only rare SON oxytocin neurons contain Dyn

(Quinn and Weber 1985.) Interestingly, in the mapping of

several antisera to regions of the brainspecific precursor 1B236 Bloom et al. (1985) reported similar staining

in almost all brain regions except the magnocellular system, where only the C-terminal immunogen (P7) was reactive; this finding resembles our observation for Dyn in

the ACN. McCabe et al. (1986) found evidence of SON subgroups with differential vasopressin mRNA response, evaluated in situ under dehydration stress. Opioid peptides

are thought of as co-transmitters with potential modulatory effects on terminals at sites of release (Pittman

et. al. 1983.) Possibly, the subgroups reported here

allow release of oxytocin/vasopressin with alternate complements of opioids, if these subgroups have distinct

afferent pathways such as for norepinephrine (Sladek and

Zimmerman 1982.) Alternatively, these opioid subgroups

could reflect functional groups of oxytocin/vasopressin

neurons selectively colocalized with yet-undescribed

neuropeptides.

293
REFERENCES

Bloch, B., Bugnon, C., Fellman, D., Lenys, D., and

Gouget, JG. Cell Tissue Res, 204:1-15, 1979.

Bloom, F.E., Batterberg, E.L.F., Milner, R.J., and

Sutcliffe, J.G. J Neurosci, 5:1781-1802, 1985.

Martin, R., and Voigt, K.H. Life Sci, 31: 1729-1732,

1982.

McCabe, J.T., Morrell, J.I., and Pfaff, D.F. In: Fink,

G., Harmer, A.J., and McKerns, K.W., eds. Neuroendocrine Molecular Biology. New York: Plenum Press, 1986

(in press).

McGinty, J.F. Neuropeotides, 5:465-468, 1985.

McGinty, J.F., and Bloom, F.E. Brain Res, 278:145-153,

1983.

Pittman, Q.J., Lawrence, D., and Lederis, K. Prog Brain

Res, 60:319-332, 1983.

Quinn, B., and Weber, E. Soc Neurosci Abst, 15:336.3,

1985.

Richards, S.-J., Morris, R.J., and Raisman, G. Neuroscience, 16:617-623, 1985.

Sawchenko, P.E., Swanson, L.W., and Vale, W.W. J

Neurosci 4:1118-1129, 1984.

Sladek, J.R., and Zimmerman, E.A. Brain Res Bull, 9:431-

440, 1982.

Watson, S.J., Akil, H., Fischli W., Goldstein, A., Zimmerman. E.. Nilaver, G., and van Wimersma Greidanus.

T.B. Science, 216:385-87, 1982a.

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Goldstein, A. Science, 228:1134-1136, 1982b.

Weber, E., Roth, K.A., Evans, C.J., Chang, J.-K., and

Barchas, J.D. Life Sci, 31:1761-1764, 1982a.

Weber, E., Evans, C.J., and Barchas, J.D. Nature, 299:

77-79, 1982b.

Weber, E., and Barchas, J.D. Proc Natl Acad Sci, 80:

1125-1129, 1983.

Williams, R.G., and Dockray, G.J. Neuroscience, 9:563-

586, 1983.

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Brownstein, M.J. Nature, 307:643-645, 1984.

ACKNOWLEDGEMENTS

Supported by a predoctoral fellowship from the MacArthur

Foundation to BQ and by a grant from the National

Institute of Mental Health (MH 40303) to EW.

294
CO-EXISTENCE OF PRODYNORPHIN - OPIOID PEPTIDES AND SUBSTANCE P IN

PRIMARY SENSORY AFFERENTS OF GUINEA-PIGS

Eberhard Wefhe, Antje Lefbold, Donatus Nohr, Thorsten Fink

and Bernd Gauweller

Dept. of Anatomy

Johannes Gutenberg-Universität

D-6500 Mainz, F.R.6.

ABSTRACT

Light microscopic immunoenzymatic and immunofluorescence

histochemistry revealed co-existence of opiofd peptfdes with

substance P in primary sensory neurons of all segmental dorsal root

and trigainal ganglia of guinea-pig. Sensory opioid peptides

appeared to be exclusively processed from prodynorphin and include

[leu]enkephalin, neoendorphlns, and dynorphin A. We suggest the

presence of presynaptfc opfoid autoreceptors in prlmary sensory

afferences.

INTRODUCTION

It is a widespread concept that enkephalins in terminals of the

dorsal horn presynaptically inhibit the release of substance P (SP)

from spinal endings of primary sensory afferences although the

neuroanatomical evidence for such a synaptic circuitry is missing

(Basbaum and Fields 1984; Jessel 1983; Kosterlitz and NcKnight 1981).

It was concluded that there is no primary sensory origin of spinal

opfoid peptfdes (Botticelli et al. 1981; Vincent et al. 1984).

Contrary to this view are immunohistochemical investigations with

cultured mouse dorsal root ganglia demonstrating a few dynorphinimmunoreactive (ir) ganglfonic cells (Sweetnam et al. 1982). Also in

neonatal rats very few enkephalfn ir small ganglfonic cells were

found (Senba et al. 1982). First morphological evidence for a general

opfofdergic primary sensory pathway in adult animals was obtained in

an immunohistochemical study with guinea-pigs (Weihe et al. 1985a).

Measurements by radioimmunoassay indicated the presence of low

amounts of ir dynorphin and ir -neoendorphin in human spinal ganglia

(Przewlocki 1983). The main focus of this study was to investigate

the posslble co-existence of opioid peptides, apparently exclusively

derived from prodynorphin (Weihe et al. 1985a), with substance P in

primary sensory afferences.

MATERIALS AND METHODS

Six male guinea-pigs of 200-250 g were deeply anaesthetized with

Nembutal (i.p.) and fixed by perfusing 500 ml Bouin's solution

retrogradcly through the distal abdominal aorta. Both trigeminal

ganglia, the entire spinal cord and the medulla, the segmental

cervical, thoracfc and lumbosacral ganglia, peripheral somatic

nerves, somatic (e.g. skin) and visceral (e.g. heart) tissues were

dehydrated in propan-2-ol, and embedded in paraplast. 4-6 μ thick

serial sections were Incubated with the primary antisera at room

temperature. Several rabbit polyclonal primary antisera directed

against a variety of proenkephrlin-opiofd peptides ([Met]enkephalin,

295
heptapeptide, octapeptide, BAM 12P, metorphamide), against various

prodynorphin-opioid peptides i.e. dynorphln A (1-8), dynorphin A

(1-17), dynorphln B, /ß-neoendorphin, and against [Leu]enkephalin

were used as previously described (Weihe et al. 1985a). Additional

polyclonal antisera were against dynorphln A (1-179, -neoendorphin

(Millan et al. 19869, amidorphin (Seizinger et al. 19859. A

monoclonal antlbody against enkephalln which was found to stain the

pentapeptides non-differentially (Cuello et al. 19849 but not

dynorphln and neoendorphins or larger molecular forms of

proenkephalin- or prodynorphin-opiolid peptides was also used. SP was

immunoreacted with commercial monoclonal antlbody NCl/34HL.

Specifities of antisera were tested by preincubating them at 37°C for

2 hours or more with 100 nmol homologous or heterologous peptides per

ml working dilutions. Biotlnylated species-specific secondary

antisera and Streptavidin- peroxldase-complexes were used in 1:50

respectively 1:200 dilutions. The diaminobentidine reaction was as

described (Weihe et al. 1985a). Co-existence and co-distribution of

ir material was preferentially determined using paired consecutive

sections alternately stained immunoenzymatically with the antibody

against SP vs. the various antisera against different opioid

peptides. In order to assess co-ex istence on Identical sections the

monoclonal SP antlbody and the different polyclonal and monoclonal

antisera against opioid peptldes were applied slmultaneously and

visualized by consecutive immunofluorescence double-staining using

either Fluorescein-Streptavidin or Texas-Red-Streptavidin.

RESULTS AND DISCUSSION

Out of all opioid antisera Investigated that against [Leu]enkephalin

produced the strongest immunoreactions in numerous small and some

medium-sized dorsal root and trigeminal ganglionic cells and their

processes even In dilutions up to 1:80000. Our novel preabsorption

studies Indicated that It was less specific In immunohistochemistry

than it could be expected from previous radioimmunoassay data (Weber

et al. 19839. It crossreacted completely with C-terminal extenstons

of [Leu]enkephalin including the neoendorphfns and with dynorphln A

(1-13) and (1-17). There was less crossreaction with [Met]enkephalin,

the hepta- and the octapeptide. BAM 12 P had a lower blocking potency

than the shorter fragments. Contrary to the immunohistochemically

relatively unspecific [leu]enkephalin antiserum those against

dynorphln A (1-17) and -neoendorphln were highly specific and

neither crossreacted with proenkephalin peptides nor with

[Leu]enkephalin, dynorphln A (1-6), (1-7), (1-8), (1-13) but were

fully preabsorbed with low concentrations (1-10 nmol) of homologous

peptides. Equivocal results were obtained with higher molecular

weight forms containing the dynorphin A (1-179 or neoendorphin

sequences. In spite of the partial crossreactivity of the

[Leu]enkephalin antiserum with [Met]enkephalin containing

proenkephalin-sequencer we argue that the ir material In sensory

ganglia visualized with this antiserum does not appear to be

proenkephalln-derived because none of the numerous antisera against

proenkaphalin-peptides revealed ir material In sensory ganglia. Since

the monoclonal antibody against enkephalln was found to stain sensory

gangllonlc cells and fibers we conclude that the pentapeptlde

[Leu]enkephalin itself is processed which is in progress to be

296
substantiated by other methods as used for the identification of

putative sensory opioid peptides in skin, heart and peripheral nerves

(Weihe et al. 1983; 1985a,b).

Out of the antisera against prodynorphin opioid peptides we selected

the most effective (against [leu]enkephalin, dynorphin A 1-17 and

-neoendorphin) for the evaluation of co-existence. Both, the

immunoenzymatic and the immunofluorescence detection demonstrated

co-existence of ir opioid peptides with ir SP in all segmental dorsal

root and trigeminal ganglia. It appeared that a major population of

small ganglionic cells which contained ir opioid peptides exhibited

also SP immunoreactivity. Opioid-antisera did not crossreact with SP

and the SP antibody was not preabsorbed with any of the opioid

peptides. There was a substantial population of non-opioid but SP ir

ganglionic cells. Preliminary morphometric data indicate that the

cell diameters of the non-opioid SP ir cells are large. Assessment of

the variations in the number of ir cells depending on the segmental

level of ganglia or even on a non-random distribution pattern within

individual ganglia (Price 1985) are needed for precise quantification

of opioidergic SP-ergic primary sensory neurons. The co-existence of

opioid- and SP-ir material in primary sensory ganglionic cells and

their juxtaperikaryal processes was paralleled by co-distribution in

small diameter fibers (C, A ) of dorsal horn, dorsal roots, Litsauer

tract, peripheral somatic and visceral nerves, and of vascular and

non-vascular nerves of many somatic (e.g. skin) and visceral (e.g.

heart) tissues. Preliminary data indicate that the ir opioid material

not unexpectedly was also co-existing with calcitonin gene related

peptide (CGRP), a recently identified co-transmitter candidate of SP

in primary sensory neurons (Skofitsch and Jacobowitz 19859. Further

co-existence patterns of opioid and non-opioid peptides can be

expected based on the current knowledge of the neurochemistry of

primary sensory neurons (Jessel 1983).

We suggest that the well known presynaptic opioid receptors on spinal

endings of primary sensory afferences are, at least in part,

autoreceptors and not heteroreceptors as generally believed.

Interestingly opioid receptors seem to be present also on perikarya

of sensory ganglionic cells (Ninkovic and Hunt 1985; Werz and

Macdonald 1984) which may be the target of opioidergic fibers

occasionally observed in this study surrounding non-opioidergic

sensory ganglionic cells. Primary sensory opioid peptides, to our

opinion, are exclusively processed from prodynorphin and include

[leu]enkephalin. All may inhibit the release of co-transmitters like

SP or other peptides (e.g. CGRP) or non-peptide substances not only

centrally but also peripherally (Dale's principle) by acting on

mainly kappa, but also some delta, presynaptic autoreceptors, and

perhaps, also on postsynaptic heteroreceptors. Experimental evidence

for opioid receptors being functionally important in mechanisms of

reflex vasodilation and extravasation has been obtained recently

(Smith and Buchan 1984). The main function of primary sensory

prodynorphin opioid peptides (Weihe 1985; Weihe et al. 1985a) may be

antinociceptive as suggested for spinal segmental or

ascending/descending opioid systems (Cruz and Basbaua 1985; Han and

Xie 1984; Herman and Goldstein 1984; Jessel 1983; Kosterlitz and

McKnight 1981; Millan et al. 1986). It will be interesting to see

whether there are also trophic functions of sensory opioid peptides.

297
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Basbaum, A.I., and Fields, H.L. Ann Rev NeurosciI 7:309-38, 1984.

Botticelli, L.J., Cox, B.M., and Goldstein, A. Proc Natl Acad Sci,

78(12):7783-7786, 1981.

Cruz, L., and Basbaum, A.I., J Co Neural 240:331-348, 1985.

Cuello, A.C., Milstein, C., Couture, R., Wright, B., Priestley, J.V.,

and Jarvis J. J Histochem Cytochem, 32(9):947-957, 1984.

Han, J., and Xie, G., Pain, 18:367-376, 1984.

Herman, B.H., and Goldstein, A. J Pharmacol Exp Therapeutics,

232(1):27-32, 1984.

Jessell, T.M. In: Krüger, D.T., Brownstein, M.J., and Martin, J.B.

eds. Brain Pe tides. J. Wiley & Sons, NY., Chichester, Brisbane,

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Kosterlitz, H.W., and McKnight, A.T. In: Autrum, H., Ottoson, D.,

Perl, E.R., and Schmidt, R.F., ed. Progress in Sensory Physiology,

Springer Berlin-Heidelberg, 1981. pp. 31-95

Millan, M.J., Millan, M.H., Czlonkowski, A., Höllt, V., Pilcher,

C.W.T., Herz, A. and Colpaert, F.C. J Neurosci, 6(4):899-906, 1986.

Ninkovic, M., Hunt, S.P. Neurosci Lett, 53:133-237, 1985.

Price, J. J Neurosci, 5(8):2051-2059, 1985.

Przewlocki, R., Gramsch, C., Pasi, A., and Herz, A. Brain Res,

280:95-103, 1983.

Seiringer, B.R., Liebisch, D.C., Gramsch, C., Herz, A., Weber, E.,

Evans, C.J., Esch, F.S., Böhlen P. Nature, 313(5997):57-59, 1985.

Senba, E., Shfosaka, S., Hara, Y., Inagaki, S., Sakanaka, M.,

Takatsuki, K., Kawai, Y., and Tohyatna, M. J Comp Neurol, 208~54-66,

1982.

Skofitsch, G., and Jacobowitz, D.M. Peptides, 6:747-754, 1985.

Smith, T.W., and Buchan, P. Neuropeptides, 5:217-220

Sweetnam, P.M., Neale, J.H., Barker, J.L., and Goldstein A. Proc Natl

Acad Sci USA, 79:6742-6746, 1982

Vincent, S.R., Dalsgaard, C.-J., Schultzberg, M., Hökfelt, T.,

Christensson, I., Terenius L. Neurosci, 11(4):973-987, 1984.

Weber, E., Evans, C.J., Barchas, J.D. TINS August 1983, 333-336.

Weihe, E. Neurosci Lett, Suppl 22: p5, 1985.

Weihe, E., McKnight, A.T., Corbett, A.D., Hartschuh, W., Reinecke, M.,

and Kosterlitz, H.W. Life Sci 33(1):711-714, 1983

Weihe, E., Hartshuh,W., and Weber, E. Neurosci Lett, 58:347-352,

1985a.

Weihe, E., McKnight, A.T., Corbett, A.D., and Kosterlitz, H.W.

Neuropeptides, 5:453-456, 1985b.

Werz, M.A., and Macdonald, R.L. Neuropeptides, 5:253-256, 1984.

ACKNOWLEDGEMENTS

For the donation of antisera we would like to thank E. Weber, Oregon

(antisera against [leu]enkephalin, -neoendorphin, heptapeptide,

octapeptide, BAM 12P, metorphamide); A. Goldstein, Stanford (antisera

against dynorphin A and B); A. Herz and co-workers C. Gramsch, B.

Seiringer and D.C. Liebisch, Munich (antisera against /&neoendorphin, dynorphin A 1-17, heptapeptide). Immunochemical reagents

were from Amersham. Peptides from Peninsula, CRB and Bachem,

commercial antisera from Serotec, Seralab, Immunonuclear, CRB. The

study was supported by "Stifterverband der Deutschen Wissenschaft",

"Freunde der Universität Mainz", and Schering AG, Berlin.

298
DISTRIBUTION OF ENKEPHALIN-LIKE IMMUNOREACTIVITY IN THE FELINE HEART;

ITS INTERRELATION WITH SUBSTANCE P- AND

VASOACTIVE INTESTINAL POLYPEPTIDE-LIKE

IMMUNOREACTIVE NERVE FIBERS

Sadaaki Maeda, Junji Nakamae, Hiroyuki Ichikawa*,

Satoshi Wakisaka*, Michio Akai* and Reizo Inoki

Department of Pharmacology and Oral Anatomy I*,

Osaka University Faculty of Dentistry,

1-8, Yamadaoka, Suita, Osaka, 565 JAPAN.

ABSTRACT

Distribution of enkephalin (Enk)-like immunoreactivity in the feline

heart was investigated by indirect immunofluorescence method. Enk-like

immunoreactive (Enk-IR) nerve fibers and cells predominated in the

atria but were rare in the ventricles. Enk-IR cells were mainly

distributed near sinoatrial (SA) node and between aorta and pulmonary

artery. These cells were usually grouped in clusters near blood

vessels or cardiac ganglia. Some Enk-IR cells appeared to extend their

processes to other Enk-IR cells, cardiac ganglia, blood vessels and

rarely cardiac muscles in the atria. Furthermore, substance P (SP)-

and vasoactive intestinal polypeptide (VIP)-IR nerve fibers were

closely associated with some Enk-IR cells. Moreover, the distribution

and morphological profiles of Enk-IR cells were very similar to those

of catecholamine (CA)-containing cells.

INTRODUCTION

Recent radioimmunological and immunohistochemical studies have

revealed the wide, but uneven distribution of several neuropeptides in

the central and peripheral nervous system. In the heart, the

occurrences of neuropeptides were reported by several investigators

using radioimmunoassay or immunohistochemical method. Biochemical

studies of Lang et al. (1983) detected the existence of methionine

(Met)- and leucine (Leu)-Enk-like immunoreactivity in guinea pig

heart. Weihe et al. (1983) characterized opioid peptides in guinea pig

heart. Lundberg et al. (1983) reported that Enk-IR nerve fibers were

localized in vagal nerve. Little is known, however, on the detailed

distribution of Enk-like immunoreactivity in the heart. In this study,

we elucidated the distribution of Enk-like immunoreactivity in the

feline heart and examined the interrelation between Enk-IR cells and

SP- or VIP-IR nerve fibers. Furthermore, we presented the possible

coexistence of Enk and CA in the heart.

MATERIALS AND METHODS

Immunohistochemistry: Young cats, weighing 2.5-3.0 kg, were used in

this study. Animals were anesthetized and perfused through the left

299

172-683 O - 87 - 11 : QL 3
cardiac ventricle with ice-cold Zamboni fixative. Whole heart was

carefully removed, separated cardiac atria under dissecting microscope

and stored in the same fixative at 4°C overnight. Specimens were then

frozen in isopentane cooled by the liquid nitrogen and serial frozen

sections were cut at the thickness of 20-30 um with a cryostat.

Indirect immunofluorescence method was applied to the sections.

Sections were first incubated either with rabbit anti Met-Enk serum

Ucb, diluted 1:1600), rabbit anti Leu-Enk serum (Ucb, 1:1600),

monoclonal rat anti SP serum (Sera Lab, 1;1000) or rabbit anti VIP

serum (INC, 1:1000) at room temperature overnight in a humid

atomosphere. After rinsing several times, sections were then incubated

in FITC-conjugated anti rabbit IgG (Miles, 1:1000), or FITC-conjugated

anti rat IgG (Cappel, 1:1000) for 60 min at 37°C. For double

immunostaining of Enk and SP, sections were first incubated with the

mixture of rabbit anti Enk serum (final dilution 1:1600) and rat

monoclonal anti SP serum (final dilution 1:1000) at room temperature

overnight, rinsed several times and then incubated with the mixture of

RITC-conjugated anti rabbit IgG (Cappel, final dilution 1:1000) and

FITC-conjugated anti rat IgG (final dilution 1:1000). Sections were

viewed with fluorescence microscope using appropriate exciting filter.

After photography, sections were stained with hematoxylen-eosin to

determine the structure of the tissue.

Catecholamine histochemistry: For demonstration of CA, glyoxylic acidinduced fluorescence method of Imai et al. (1982) was applied. Cats

were killed by bleeding and hearts were removed immediately. Cardiac

atria were cut into small pieces and fixed in FA-GLU-PA-GA-S solution

(4% paraformaldehyde, 0.5% glutaraldehyde, 0.2% picric acid, 2%

glyoxylic acid and 15% sucrose in 0.1M phosphate buffer, pH 7.3) for 2

hours at 4°C. The specimens were then frozen and cut at the thickness

of 20- 30 um with a cryostat. Sections were immersed in FA-GA-S

solution (4% paraformaldehyde, 2% glyoxylic acid and 15% sucrose in

0.1M phosphate buffer, pH 7.3), air-dried for 60 min at 45°C, sealed

with xylene and viewed with fluorescence microscope.

RESULTS

As the distribution of Met-Enk-IR and Leu-Enk-IR structures was very

similar, we simply described as "Enk-IR." Enk-IR structures

predominated in the atria but were rare in the ventricles. In the

atria, many Enk-IR cells were localized near SA node and between aorta

and pulmonary artery. Enk-IR cells were also distributed between

coronary sinus and pulmonary vein and near inferior vena cava. Several

thin Enk-IR nerve fibers with varicosities were observed in the atria1

muscles (fig. 1), along the blood vessels and near the ganglionic

cells. Enk-IR cells were distributed in clusters or, in some cases, in

single cells. Many Enk-IR cells were localized at the walls of small

blood vessels (fig. 2) and near the ganglia (fig. 3). Some Enk-IR

cells were observed to extend their processes toward other Enk-IR

cells, blood vessels, ganglionic cells and rarely cardiac muscles.

Double immunostaining sections revealed that many SP-IR nerve fibers

300
FIGURES. 1-3: Immunofluorescence micrographs of Enk-like

immunoreactivity.

FIGURE 1: Thin varicose Met-Enk-IR nerve fibers are

observed in the atria1 muscles (M) near SA node.

FIGURE 2: Several Leu-Enk-IR cells are localized at the

walls of small blood vessels (BV) between aorta and

pulmonary artery. FIGURE 3: Met-Enk-IR cells are present

over the ganglion near SA node.

FIGURE 4: Histofluorescence micrograph between aorta and

pulmonary artery. Several CA-containing cells are observed

near the blood vessels. Some CA-containing cells extend

their processes.

301
with varicosities were observed around the Enk-IR cells. Adjacent

sections incubated with anti VIP serum showed that many varicose VIPIR nerve fibers formed dense network over the Enk-IR cells. The

distribution and morphological profiles of CA-containing cells were

very similar to those of Enk-IR cells (fig. 4). Moreover, CAcontaining nerve fibers were observed in close association with some

CA-containing cells.

DISCUSSION

Our study demonstrated the detailed distribution of Enk-IR structures

in the feline heart. Enk-IR nerve fibers with varicosities were

present in cardiac muscles, at the walls of blood vessels and near the

ganglionic cells. Some Enk-IR cells were observed to extend their

processes to the ganglionic cells, blood vessels and rarely cardiac

muscles. These findings suggest that Enk may directly control the

cardiac functions. Recently Laurent et al; (1985) reported that Enk

had a direct positive inotropic effect on cultured cardiac myocytes.

Our present findings support their results. Moreover, our immunohistochemical evidences that SP- and VIP-IR nerve fibers were closely

associated with Enk-IR cells suggest that SP and VIP may regulate the

effects of Enk in the heart. It was reported that Enk coexisted with

CA in the chromaffin cells of adrenal gland and Enk might regulate the

release of CA from the chromaffin cells. Although the coexistence of

Enk and CA was not investigated in this study, it was shown that the

distribution and morphological profiles of CA-containing cells were

very similar to those of Enk-IR cells. Thus, our observation suggests

that in the heart Enk-IR cells may also contain CA and regulate the

release of CA.

REFERENCES

Imai, H., Kimura, H. and Maeda, T. Acta Histochem Cytochem, 15: 798-

806, 1982.

Lang, R.E., Hermann, K., Dietz, R., Gaida, W., Ganten, D., Kraft, K.

and Unger, Th. Life Sci, 32: 399-406, 1983.

Laurent, S., Marsh, J.D. and Smith, T.W. Proc Natl Acad Sci USA, 82:

5930-5934, 1985.

Lundberg, J.M., Hökfelt, T., Kewenter, J., Petterson, G., Ahlman, H.,

Edin, R., Dahlstrom, A., Nilsson, G., Terenius, L., VvnasWallenstein, K. and Said, S. Gastroenterology, 77: 468-471.

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and Kosterlitz, H.W. Life Sci, 33 (suppl): 711-714, 1983.

302
IMMUNOHISTOCHEMICAL AND SUBCELLULAR STUDIES OF AMINOPEPTIDASE M

LOCALIZATION IN RAT BRAIN : MICROVESSELS AND SYNAPTIC MEMBRANES

C. Gros, B. Solhonne, B. Giros, H. Pollard, N. Salès, J-C. Schwartz,

P. Ronco* and P. Verroust*

Unite 109 de Neurobiologie et Pharmacologic,

Centre Paul Broca de l'INSERM,ilter rue d'Alésia - 75014 PARIS (FRANCE)

*INSERM U 64, Hôpital Tenon - 75970 PARIS (PRANCE)

ABSTRACT

A predominantly microvascular localization of aminopeptidase M (APM)

was established both by immunohistochemistry and biochemical studies

of isolated microvessels. In addition a high relative specific

activity of APM was evidenced in fractions enriched in synaptic

membranes.

INTRODUCTION

APM (EC 3.4.11.2) which is sensitive to bestatin but relatively

insensitive to puromycin has recently been identified in rat cerebral membranes (Schwartz et al. 1984 ; Gros et al. 1985). Its major

role in the inactivation of endogenous enkephalins released from

brain slices was demonstrated by the protecting effects of several

inhibitors including antibodies directed against the active site of

APM (Giros et al. 1986). Since the KM values of enkephalins for APM

and another cerebral aminopeptidase more sensitive to puromycin do

not greatly differ, the preferential role of APM is likely to reflect topological rather than catalytic differences. We have therefore studied the localisation of APM in rat brain by immunohistochemical and subcellular fractionation methods.

MATERIAIS AND METHODS

Polyclonal antibodies (Ab) directed against an homogeneous preparation of rat kidney APM were obtained in rabbits (Gros et al. 1985).

Monoclonal Ab against "enkephalinase" (EC 3.4.24.11) were obtained

after injecting to mice a preparation of rat kidney brush borders.

The recognition of "enkephalinase" was assessed by immunoprecipitation of the enzyme activity (in preparation).

Immunolocalizations were established by incubating unfixed frontal

sections of rat brain (or isolated microvessels) with either polyclonal anti APM Ab followed by antirabbit immunoglobulins (labelled

with [125I] or peroxidase) or [125I] monoclonal anti-enkephelinase Ab.

Subcellular fractions were prepared and named according to Gray and

Whittaker (1962).

Aminopeptidase activity was measured using 40 nM [3

H] (Met5)enkephalin as substrate. Incubations were for 10 min at 37°C in the presence

of 1 μM Thiorphan and 20 μM puromycin, [

3

H]Tyr being isolated by

polystyrene bead column chromatography. APM activity was evaluated as

the difference between values obtained in the presence and absence of

APM antibodies.

303
RESULTS AND DISCUSSION

Immunostaining of rat brain sections (figure 1) indicates a rather

uniform localization of APM within the brain parenchyma which contrasts with the highly heterogeneous localization of enkephalinase,

the other major enkephalin-degrading peptidase.

In agreement, assay of APM activity in homogenates from several brain

regions revealed a rather uniform distribution of the enzyme activity

which represented approximately 1 % of the total aminopeptidase

activity (not shown).

FIGURE 1: Autoradiography of APM (upper picture) and enkephalinase (lower pictures) immunoreactivities in frontaI sections

of rat brain.

The [

125I] Labelled enkephalinase Ab was obtained as described

(H. POllard, P. Ronco, P. Verroust and J-C. Schwartz

-

submitted).

am : amygdala ; Cg : cingulate cortex ; Ch : choroid plexus ;

cp : caudate putamen ; GP : globus pallidus ; hip :

hippocampus ; SN : subtantia nigra

304
However a strong APM immunoreactivity was detected in the walls of

vessels in all studied cerebral areas as well as within choroid

plexuses such as those of the lateral ventricles (figures 1 and 2).

FIGURE 2: Immunoperoxidase staining of APM in a frontal

section of rat brain (left) and in isolated microvessels

(right).

Ch : choroid plexus ; v : vessel

The microvascular localisation of APM was confirmed after isolation

of cortical microvessels by a sieving procedure. On this relatively

pure fraction a dense immunostaining of all vascular elements was

observed (figure 2). After solubilization of the isolated microvessels with Triton X-ICC, APM activity was clearly identified by

establishing the potencies of various inhibitors.

APM antibodies inhibited about 80 % of the eminopeptidase activity of

this fraction at a dilution (1/50,000) similar to that required to

inhibit purified APM. Also the IC50 values of bestatin (2 μM), amastatin (5 μM) and puromycin (78 μM) were similar on the two

preparations.

This major microvascular localization of APM suggests that the

enzyme may participate in the inactivation of neuropeptides regulating the permeability and tone of cerebral vessels. Together with its

presence in choroid plexuses, it also suggests that it participates

in the enzymatic "blood-brain barrier" which prevents the access of

circulating regulatory peptides into the CSF and brain extracellular

fluids.

305
FIGURE 3: Subcellular distribution of APM into fractions from

rat cerebral cortex.

Fractions were obtained after two different density gradient

centrifugatiohs end designated according to Gray and Whittaker

(1962). The relative specific activity (RSA) is the ratio

percent of enzyme activity over percent of recovered proteins.

In primary subcellular fractions from cortical homogenates, a major

portion (78 %) of APM was found to sediment with the heavy P1 fraction containing microvessels whereas 7 % and 10 % sedimented with

the P2 and P3 fractions respectively and no significant APM activity

would be detected in soluble form. However upon density gradient

analysis of the shocked synaptosomal fraction a clear enrichment (by

about 5-fold as compared to the homogenate) was found in those

subfractions containing synaptic membranes (figure 3). However it

is difficult to exclude a contamination of these fractions by elements of the predominant vascular pool in brain.

In conclusion this study indicates a dual localization of APM in

rat brain. The synaptic localization would be more consistent than

the microvascular one with the major participation of APM in neuronal

enkephalin inactivation.

REFERENCES

Giros, B., Gros, C., Solhonne, B., and Schwartz, J-C. Mol Pharmacol,

29:281-287, 1986.

Gray, E.G., and Whittaker, V.P. J Anat, 96:79-88, 1962.

Gros, C., Giros, B., end Schwartz, J-C. Biochemistry, 24:2179-2185,

1985.

Joo, F., and Karnushina, I. Cytobios, 8:41-48, 1973.

Schwartz, J-C., Gros, C., Giros, B., Llorens, C., Malfroy, B., Rose,

C., and Zuzel, K. In: Vizi E.S and Magyar, K., eds. Regulation

of Transmitter function. Elsevier Amsterdam, 1984. pp. 217-224.

306
GAMMA-3-MSH AND B-ENDORPHIN IN MONKEY PITUITARY

J. Meador-Woodruff, M. Knobloch, H. Vaudry*, N.G. Seidah**,

S.J. Watson, and H. Akil

Mental Health Research Institute and Department of Psychiatry

University of Michigan Medical Center

205 Washtenaw Place, Ann Arbor, MI, USA 48109-0720

*Laboratorie d' Endocrinologie, Faculte des Sciences

et Techniques, 76130 Mon-Saint-Aignan, France

**Protein and Pituitary Hormone Laboratory,

Clinical Research Institute of Montreal, Canada

ABSTRACT

The content and extent of processing of gamma-3-MSH and B-endorphin

were determined in the anterior (AL) and intermediate (IL) lobes of

three monkey pituitaries. The peptides existed in equimolar amounts

in each lobe. The processing of gamma-3-MSH paralleled that of

B-endorphin in each lobe; the primary products in AL were

B-lipotropin and a 9K form of gamma MSH, whereas in IL the

predominant products were N-acetyl-B-endorphin and gamma-3-MSH.

INRODUCTION

Gamma-3-melanocyte stimulating hormone (MSH) is a 27-residue peptide

found in the N-terminal region of proopiomelanocortin (POMC),

containing the MSH core sequence His-Phe-Arg-Trp (Nakanishi et al.

1979). There are two major cleavage sites in the N-terminal domain

of POMC, which potentially generate the extreme N-terminal peptide,

gamma-3-MSH, and joining peptide, which is the segment found between

gamma MSH and ACTH. Gamma-3-MSH thus can be found associated with

joining peptide (molecular weight 8-10K, depending on the extent of

glycosylation), the N-terminal fragment of POMC (molecular weight

10-12K), or alone (molecular weight 3-4K). Bovine and human (but not

rat) gamma-3 MSH have an additional cleavage site that potentially

generates the C-amidated II-residue peptide, gamma-1-MSH (Ling et al.

1979).

Gamma-3-MSH has been shown to exist in several forms in bovine

(Tanaka et al. 1980; Shibasaki et al. 1980a, 1980b) and rat (Pedersen

et al. 1982) AL and IL, as well as being present in whole human

pituitary extract (Tanaka et al. 1980). To date, however, the nature

of gamma-3-MSH in monkey pituitary has not been studied. In the

present investigation we examined the content and degree of

processing of gamma-3-MSH in monkey AL and IL, and compared these to

the corresponding parameters for B-endorphin.

307
MATERIALS AND METHODS

Pituitaries

Three post-menopausal Rhesus monkeys (Macaca mulatta) were

anesthetized and sacrificed. The pituitaries were immediately

removed, placed on wet ice, and dissected into the AL and the IL.

The IL was trimmed as closely as possible, but due to the anatomy of

monkey pituitary, a clear separation between anterior/posterior and

intermediate pituitary was not feasible, hence the IL had

contaminating fragments of both anterior and posterior lobes. After

dissection, the pituitaries were frozen on dry ice and stored at

-70°C.

Extraction

The peptides were extracted from the pituitaries into acetone/0.1N

HCl (3:1). The extract was dried by lyophilization, and the residue

resuspended in 1.0% formic acid. For column chromatography, the

formic acid extract was used. For radioimmunoassay, aliquots of

formic acid extracts were dried and resuspended in the appropriate

assay buffer.

Radioimmunoassays

human B-MSH, human B-endorphin, and ACTH, <10-6.

B-endorphin and N-acetylated B-endorphin were assayed by previously

established radioimmunoassays (Cahill et al. 1983a, Akil et al.

1981a). Gamma-3-MSH was measured using an assay recently developed.

Samples were diluted in a 50 mM sodium phosphate buffer, pH 7.6

containing 0.12M NaCl, 0.004M EDTA, 0.016% NaN3, 1.6% normal rabbit

serum, and 0.1% polylysine. The assay was run at disequilibrium

conditions for 72 hours at 4°C; the antiserum, raised in rabbits

using synthetic bovine gamma-3-MSH conjugated to bovine serum albumin

with bis-diazatized benzidine as an antigen, was used at a final

dilution of 1/25000. The radioactive trace was human gamma-3-MSH,

iodinated by the chloramine T method (Hunter and Greenwood, 1962).

Separation of free from bound material was by the second antibody

method, using sheep-anti-rabbit gamma-globulin. This assay has an

intrassay CV of 9.2%, and an interassay CV of 5.0%. It has a useable

range of 6-300 fmoles/tube, with an IC of 40 fmoles/tube. On a

molar basis, its crossreactivity profile is: gamma-3-MSH = 1;

Lys-gamma-1-MSH, <10-3; gamma-1-MSH and gamma-2-MSH, <10-4; a-MSH,

Column Chromatography

Samples were applied to a sephadex G-50 column (1.5 x 90 cm) that had

been precalibrated with molecular weight standards. Aliquots of each

fraction were dried, resuspended in the appropriate buffer, and

assayed for B-endorphin and gamma-3-MSH immunoreactivity. The

fractions with immunoreactivity in the void volume of this column

were pooled, and rechromotagraphed on a sephadex G-100 column (1.5 x

90 cm) to separate larger-sized fragments. The percentage of the

total immunoreactivity found in each peak was calculated for each

peptide.

308
RESULTS AND DISCUSSION

As shown in table 1, the immunoreactive gamma-3-MSH and B-endorphin

concentrations were not significantly different fcr each pituitary

lobe. In addition, much of the B-endorphin immunoreactivity in

intermediate lobe was N-acetylated, whereas very little was in

anterior lobe, similar to previous findings in rat pituitary (Akil et

al. 1981b).

TABLE 1. Gamma-3-MSH and B-endorphin Content of Monkey

Pituitary Lobes

Lobe Peptide Content (pmoles/mg tissue)

gamma-3-MSH B-endorphin N-acetyl-B-endorphin

anterior 120 33 91 2 6 5 3

intermediate 69 20 98 46 6 2 52

The size distribution for B-endorphin and gamma-3-MSH

immunoreactivity in each lobe are shown in table 2.

TABLE 2. Percent of Total B-endorphin and gamma-3-MSH Found In

Each Peak

PEAK

(3-5K)

II III

(9-11K) (15-20K) (Void)

Anterior lobe

gamma-3-MSH 22 10% 6 0 7% 18 3 % 1 1 %

B-endorphin 37 15% 54 17% 8 3 % 1 1%

Intermediate lobe

gamma-3-MSH 48 12% 3 5 6 % 1 6 11% l l %

B-endorphin 65 15% 29 14% 5 3 % 2 1 %

Peak I corresponds to 3-5 kdaltons, representing B-endorphin or

gamma-3-MSH. Peak II is 9-11 kdaltons, and contains B-lipotropin or

(tentatively) glycosylated gamma-3-MSH plus joining peptide. Peak

III is 15-20 kdaltons, and in the case of gamma-3-MSH

immunoreactivity probably represents the entire 16K N-terminal POMC

fragment; the remaining 15K C-terminal piece may be the peak measured

by the B-endorphin assay. Peak IV is the void volume of the G-100

column, presumably representing intact POMC. The processing for each

peptide is quite similar, with a tendency toward the presence of

smaller fragments in both lobes. The AL manifests predominately

B-lipotropin-sized material when B-endorphin-immunoreactivity is

measured, although a significant portion of the immunoreactivity is

B-Endorphin-sized, as has been previously reported (Cahill et al.

1983b). Gamma-3-MSH-immunoreactivity in AL is found in a similar

sized fraction which is tentatively gamma-3-MSH extended with joining

peptide. The IL shows a pattern of smaller, more processed material

309
(B-endorphin or gamma-3-MSH-sized), and the B-endorphin is

N-acetylated.

These findings are in agreement with previous studies on lower

animals. Pedersen et al. (1982) demonstrated that gamma-3-MSH exists

in 6K and 11K forms in rat AL and IL; the smaller form predominates

in IL, whereas the larger form is more predominant in AL. Similar

findings were reported by Shibasaki et al. (1980a) from their studies

on bovine AL and IL. Fractions of 15K, 8K, and 4.5K daltons have

been shown to have gamma-3-MSH immunoreactivity in bovine whole

pituitary, but in humans the predominant form of gamma-3-MSH

immunoreactivity is 9K daltons (Tanaka et al. 1980). The presence of

gamma-3-MSH immunoreactivity in 16K, 9K, and 4K fractions observed in

monkey pituitary is consistent with these previous reports in other

species.

In summary, we have shown that gamma-3-MSH immunoreactivity is

detectable in monkey AL and IL, is present in equimolar amounts with

B-endorphin, and is processed into primarily 4K and 9K fragments,

paralleling the processing of B-endorphin. The smaller fragment

predominates in IL, while the 9K fraction is the primary product in

AL, similar to the way B-endorphin is handled by the pituitary lobes.

We are currently developing an assay for gamma-1-MSH to determine if

gamma-MSH in monkey pituitary is processed to this smaller form.

REFERENCES

Akil, H., Ueda, Y., Lin, H.S., Watson, S.J. Neuropeptides, 1:429-426,

1981a.

Akil, H., Young, E., Watson, S.J., Coy, D. Peptides, 2:289-292,

1981b.

Cahill, C.A., Matthews, J.D., Akil, H. J Clin Endocrinol Metab,

56:992-997, 1983a.

Cahill, C.A., Watson, S.J., Knobloch, M., Akil, H. Life Sci,

33:53-55, 1983b.

Hunter, W.M. and Greenwood, F.C. Nature, 194:495-496, 1962.

Ling, N., Ying, S., Minick, S., Guillemin, R. Life Sci, 25:1773-1780,

1979.

Nakanishi, S., Inove, A., Kita, T., Nakamura, M., Chang, A.C.Y.,

Cohen, S.N., and Numa, S. Nature, 278:423-427, 1979.

Pedersen, R.C., Ling, N., Brownie, A.C. Endocrinology, 110:825-834,

1982.

Tanaka, I., Nakai, Y., Jingami, H., Fukata, J., Nakao, K., Oki, S.,

Nakanishi, S., Numa, S., and Imuna, H. Biochem Biophys Res Commun,

94:211-217, 1980.

Shibaskai, T., Ling, N., Guillemin, R. Nature, 285:416-417, 1980a.

Shibasaki, T., Ling, N., Guillemin, R. Life Sci, 26:1781-1785,

1980b.

ACKNOWLEDGEMENTS

Supported in part by NIDA Grant #DA00254 (to H.A. and S.J.W.), and

the Department'of Psychiatry, University of Michigan.

310
MULTIPLE OPIOID RECEPTOR SUBTYPES IN THE PITUITARY-ADRENAL AXIS: A

CROSS-SPECIES STUDY.

Alfred Mansour, Michael E. Lewis*, Henry Khachaturian, Huda Akil and

Stanley J. Watson

University of Michigan

Mental Health Research Institute

205 Washtenaw Place, Ann Arbor, MI, 48109

*E.I. Du Pont de Nemours & Co., Inc.

Central Research and Development Department

Wilmington, Delaware 19898

ABSTRACT

This study examines the multiple opioid receptor subtypes present in

the pituitary-adrenal axis of several species using in vitro

autoradiography. The monkey neural lobe was found to contain all

three subtypes with a preponderance of kappa sites, while the rat and

bovine neurohypophyses contained only kappa sites. In the adrenal,

opioid receptor sites were found exclusively in the medulla and

varied with species. Monkey adrenal contained predominantly delta

sites with a relatively small number of kappa sites present, while

bovine adrenal medulla had predominantly kappa sites and only a

relatively small proportion of delta receptor sites. No specific

opioid receptor binding could be detected in the rat adrenal. These

results emphasize the importance of species-differences in

understanding the role of opioids in neuroendocrine regulation.

INTRODUCTION

Several lines of evidence suggest that the endogenous opioid systems

may modulate the release of pituitary hormones. Acute administration

of mu opiates, for instance, has been reported to increase the

release of ACTH, growth hormone, and prolactin while decreasing the

release of luetinizing hormone and thyroid stimulating hormone

(Martin et al. 1975, Bruni et al. 1977, Cicero et al. 1977). Kappa

agonists, on the other hand, have been reported to antagonize the

antidiuretic action of vasopressin (Leander, 1983) and modulate the

release of oxytocin (Bicknell et al. 1985, Samson et al. 1985).

While the neuroendocrine effects of the endogenous opioids are well

established, the opioid receptor subtypes mediating these actions and

their anatomical localization is not clear. To clarify this issue

futher, this study has examined the opioid receptor subtypes found in

the pituitary-adrenal axis across several species using in vitro

receptor autoradiography.

MATERIALS AND METHODS

Pituitaries and adrenals of rats, monkeys, and cows were frozen,

sectioned on a Bright cryostat (25 um) and thaw-mounted on precleaned

and subbed microscope slides. The tissue sections were then dried

311
overnight at 4°C under reduced pressure in a glass desiccator and

stored in a -80°C freezer. Immediately prior to using the tissue,

the slide-mounted sections were gradually brought up to room

temperature and incubated with various opioid ligands in 50 mM Tris

buffer (pH 7.5 at 25°C). The tritiated opioids used were DAGO

(Tyr-D-Ala-Gly-MePhe-Gly-ol, 57.5 Ci/mmol 20.0 - 0.6 nM) to label mu

sites, DPDPE (D-Pen2, D-Pen5-enkephalin, 29 Ci/mmol, 30.0-0.9 nM) to

label delta sites, and (-)bremazocine (41.4 Ci/mmol, 10.0 - 0.3 nM)

to label kappa sites. As bremazocine does not selectively bind to

kappa receptors, all [

3

H]bremazocine binding was conducted in the

presence of IGO nM DAGO and DPDPE in order to block the mu and delta

sites. The slides were incubated for 60 minutes, drained, and washed

in four consecutive 250 ml, 50 mM Tris washes (pH 7.6, 4°C). Slides

incubated with [

3

H]DAG0 and [

3

H]DPDPE were given four 30 second

washes, while those incubated with [3

H]bremazocine were given four 4

minute washes. Following the Tris washes, all slides were rinsed (2

seconds) in a 250 ml wash of distilled water (4°C) and quickly dried

with a portable hair dryer set to cool. Nonspecific binding was

evaluated by treating a parallel set of slides with the same

concentration of [

3

H]ligand with a 1 uM final concentration of

unlabelled competitor: levorphanol to displace [

3

H]DAG0, DSLET

(Tyr-D-Ser-Gly-Phe-Leu-Thr) to displace [

3

H]DPDPE, and UM 1071 (MR

2034) to displace [

3

H]bremazocine. Slides were then arranged in

X-ray cassettes and apposed to tritium-sensitive LKB Ultrofilm for

179 days.

RESULTS AND DISCUSSION

Marked species differences were observed in the distribution of the

opioid receptor subtypes in the pituitary and adrenal. In the

pituitary, opioid receptor binding was found only in the neural lobe,

but the subtypes present varied with species (table 1).

TABLE 1. MULTIPLE OPIOID RECEPTOR SUBTYPES IN PITUITARY AND

ADRENAL

I. Pituitary (Neural Lobe)

Rat Monkey Bovine

Kappa ++ +++ +++

M u --- + ---

Delta --- ++ ---

II. Adrenal

Kappa --- + +++

M u --- --- +

Delta --- +++ +

(+++ = dense, ++ = moderate, and + = light labelling)

Rat Monkey Bovine

312
More specifically, the monkey neurohypophysis contained all three

opioid receptor subtypes with a preponderance of kappa sites, while

the rat and bovine pituitaries contained only kappa receptor sites.

The lack of opioid receptors in the anterior lobe would suggest that

the endogenous opioids do not act directly on the pituitary to

moderate hormonal release, but most likely have their effects on the

hypothalamus itself. While debate in the literature has centered

around whether the opioid receptors present in the neural lobe are on

pituicytes (Lightman et al. 1983, Bunn et al. 1985) or nerve

terminals (Falke and Martin 1985), their localization within the

neurohypophysis provides an anatomical basis for the modulation of

neural lobe hormones, such as, vasopressin and oxytocin.

In the adrenal gland, both kappa and delta receptors were observed in

the monkey and bovine medulla (table 1). The animals differed,

however, in the relative proportions of these sites in the medulla.

In the bovine adrenal, kappa sites were prominent with a relatively

small proportion of mu and delta sites present, while in the monkey,

delta sites were predominant, with a small proportion of kappa sites

present. In contrast, no opioid receptor binding was observed in the

rat adrenal owing, most likely, to the relative paucity of opioid

receptor sites in this tissue. Illustrative examples of opioid

receptor autoradiography in the monkey pituitary and adrenal are

present below in figure 1.

FIGURE 1. Darkfield autoradiograms of opioid receptors in the

monkey pituitary (A) and adrenal (B). Note the labelling of

kappa sites in the neural lobe of the pituitary and the delta

sites in the adrenal medulla. Abbreviations include AL =

anterior lobe, Ctx = cortex, Med = medulla and NL = neural

lobe.

313
These results emphasize the importance of considering

species-differences in understanding the role of opioid receptors in

neuroendocrine regulation. In the adrenal, for instance, where the

predominant receptor subtype varies with species, different opioid

receptor subtypes may serve the same function in different animals.

If catecholamine (CA) release is the functional endpoint, kappa

receptors may be important in modulating CA release in bovine adrenal

medulla (Dumont and Lemaire 1985), while delta receptors may mediate

these actions in the monkey adrenal.

REFERENCES

305:235-237, 1983.

Cicero, T.J., Bell, R.D., Meyer, E.R. and Schweitzer, J. J Pharm

Expt Ther, 201:76-83, 1977.

Dumont, M. and Lemaire, S. Neuropeptides, 6:321-329, 1985.

Falke, N. and Martin R. Neurosci Lett, 61:37-41, 1985.

Leander, J.D. J Pharm Expt Ther, 227:35-41, 1983

Lightman, S.L., Ninkovic, M., Hunt, S.P. and Iversen, L.L. Nature,

Bruni, J.F., Vugt, D.V., Marshall, S. and Meites, J. Life Sci,

21:461-466, 1977.

Bunn, S.J., Hanley, M.R. and Wilkin, G.P. Neurosci Lett, 55:317-323,

1985.

Bicknell, R.J., Chapman, C. and Leng G. Neuroendocrinol, 41:142-148,

1985.

Martin, J.B., Audet, J. and Saunders, A. Endocrinology, 96:839-847,

1975.

Samson, W.K., McDonald, J.K. and Lumpkin, M.D. Neuroendocrinol,

40:68-71, 1985.

ACKNOWLEDGMENTS

This work was supported by the NIH Training Grant #MH15794 (AM),

Scottish Rite Grant (SJW), NIDA Grant #DA02265 (HA), NIMH Grant

#MH39717 (HA and SJW), Theophile Raphael Research Fund (HA), and the

Michigan Gastrointestinal Hormone Research Core Center Grant. We are

also grateful to Henry Mosberg and James Woods of the University of

Michigan for their generous gifts of DPDPE and UM 1071,

respectively.

314
AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTOR TYPES

IN THE RAT SMALL INTESTINE

Michael R. Dashwood, Rachel M. Sykes and Cecil S. Thompson

Department of Physiology,

Royal Free Hospital School of Medicine,

Rowland Hill Street, London NW3 2PF, England

ABSTRACT

The selective μ and ligands [3H]DAGO and [3H]DPDPE have been used

to investigate the distribution of specific opioid subtypes in the

rat small intestine by in vitro autoradiography. There was a

greater density of [3H]DPDPE binding at regions of the villi and

crypts than [

3

H]DAGO binding. These results suggest that the opioid

receptors located in these regions are predominantly of the

subtype.

INTRODUCTION

It has been suggested that opioid peptides are important in

regulating the transport of water and electrolytes across the rat

small intestine (Warhurst et al. 1983). Recently, using in vitro

autoradiography, we have demonstrated dense [3H]naloxone and [3H]

dihydromorphine binding in proximal and distal regions of the rat

small intestine (Dashwood et al. 1985). In the present autoradiographic study we have used [3

H]DAG0 and [3H]DPDPE in order to

determine which opioid receptor subtype is most prominent in the

villi and crypt regions of the small intestine of the rat.

MATERIALS AND METHODS

Radioligand binding studies

Sprague Dawley rats (male, 150-200 g) were obtained from Charles

River. All radiolabelled ligands were obtained from Amersham.

DAGO, (D-Ala2-Mephe4-Glyol5)-enkephalin and DPDPE, (D-Peu2,D-Peu5)-

enkephalin were obtained from Cambridge Research Biochemicals (UK).

Rats were stunned and decapitated and the whole brain, minus

cerebellum, was removed. Tissue was homogenised (Polytron Setting

5, 20 sec) in 50 vols Tris-HCl buffer (50 mM pH 7.7 at 25°C) and

centrifuged for 20 min at 40,000 g. The resultant pellet was

resuspended in 100 vols Tris buffer, incubated at 37°C for 45 min

and centrifuged. The final pellet was suspended in 100 vols Tris

buffer and used immediately. The membrane suspension was incubated

for 45 min at 25°C in the presence of [3

H]ligand and either drug or

buffer. The reaction was terminated by filtration under reduced

pressure through Whatman GF/B filters and washed with buffer.

Specific binding was that component displaced by 10 μM diprenorphine. Displacement data were analysed using the ALLFIT computer

programme (DeLean et al. 1978). Saturation data were analysed using

the Ligand programme (Munson and Rodbard, 1980).

315
In vitro autoradiography

Serial 20 μM longitudinal frozen sections of the rat proximal small

intestine were taken and thaw mounted onto gelatinised microscope

slides. These sections were then incubated in 4 nM [3H]naloxone,

[

3

H]DPDPE and [

3

H]DAGO (Amersham). Alternate sections were

incubated in the presence of 1 μM unlabelled naloxone in order to

determine the degree of binding to non-specific sites. The

incubation conditions used were those described by Gillan and

Kosterlitz (1982). After washing in buffer and drying, the sections

were placed in 24 x 30 cm X-ray cassettes and apposed to LKB [3H]

Ultrofilm for 4-6 weeks at 4°C. Tritium standard scales (American

Radiolabelled Chemicals Inc.) were included in each cassette. At

the end of the exposure period the film was processed and areas of

high [3H]ligand binding were evident as accumulation of silver

grains on the film. Photographs of autoradiographs were taken

where appropriate and preliminary densitometric measurements were

carried out on a Cambridge Quantimet 970 image analysis system.

RESULTS

Radioligand binding studies

Saturation analysis of [3H]opioid binding revealed that both DAGO

and DPDPE bound to one class of site with KDS of 1.7 0.5 (n=4) and

1.6 0.6 (n=0.4) nM respectively. Unlabelled DPDPE was a potent

and selective inhibitor of [3H]DPDPE. Whereas, as expected, DAGO

potently and selectively interacted with the [3H]DAGO binding site.

Naloxone interacted with both sites but with some selectivity for

the [3H]DAGO binding site. Data on the selective antagonist ICI

174,864 is shown for comparison (Table 1). Thus, it can be

concluded that, under the conditions used in the autoradiographic

studies, DPDPE will label exclusively sites whereas DAGO will

label exclusively μ sites. Naloxone, at the concentration used,

although labelling a mixture of μ and sites will interact

primarily with the μ population.

μ site = [

3

H]DAGO

KI (nM)

site = [

3

H]DPDPE

DPDPE 780 40 (n=4) 1.3 0.4 (n=5)

DAGO 1.7 0.3 (n=4) 570 80 (n=4)

Naloxone 1.8 0.4 (n=3) 14.3 0.5 (n=3)

ICI 174,864 > 10,000 (n=4) 62 14 (n=4)

TABLE 1: Inhibition of [3H]opioid binding

Autoradiography

On development of the autoradiographs there was a striking degree of

[

3

H]naloxone binding to the villi and crypts as reported previously

(Dashwood et al. 1985). From the raw autoradiographic data the

degree of [3H]DPDPE binding to the intestine was far greater than

[

3

H]DAGO binding indicating that a higher population of opioid

316
receptors are present than μ receptors. In all cases [3

H]ligand

was displaced when incubated with 1 μM naloxone (see Figure 1).

Preliminary densitometric analysis was carried out whereby

measurements of the grey levels of the autoradiographs were compared

to densities of the tritium standard scales included with each

cassette. These measurements showed that the rank order of opiate

ligand binding to villi and crypt regions of the rat small intestine

was [3H]DPDPE > [3H]naloxone > [3H]DAGO where:

DPDPE = 225 fmol/mg protein

naloxone = 65 fmol/mg protein

DAGO = <34 fmol/mg protein

FIGURE 1. Binding of [3

H]naloxone, [3H]DPDPE and [3H]DAGO to

longitudinal sections of rat small intestine (left). All

[

3

H]ligands were displaced when incubated in the presence of

1 μM unlabelled naloxone (right). Scale bar = 500 μm.

317
DISCUSSION

Opiates are known to exert profound effects on fluid and electrolyte

transport across the epithelium of the small intestine (Dobbins

et al. 1980; Hughes et al. 1982; Turnberg 1983). The possible

location of the opioid receptors involved in the regulation of fluid

transport has emerged from the demonstration of dense [3H]naloxone

and [3H]dihydromorphine binding to the villi and crypt regions of

the rat intestinal mucosa using an in vitro autoradiographic

technique (Dashwood et al. 1985).

In the present study we have used [3H]DAGO and [3H]DPDPE, specific

ligands for the μ and opioid receptors respectively in order to

determine which subtypes are located in the villi and crypt regions

of the rat small intestine. The results indicate a greater binding

of [3H]DPDPE (at least eight times greater) to the villi and crypts

compared to [3H]DAGO binding. These data are consistent with a

previous study which suggested that the effects of opiates on

intestinal fluid movement are mediated by the specific opiate

receptor (Kachur et al. 1980). Recently we have demonstrated a

marked increase in opioid receptor density in the villi and crypts

of rat small intestine with streptozotocin-induced diabetes mellitus

when compared to normal rats (Dashwood and Thompson 1986).

Although the functional significance of the identification of opioid

receptors in the small intestine awaits clarification it is

suggested tentatively that the intestinal opioid receptors are

capable of adaptation in certain pathological conditions.

REFERENCES

Dashwood, M.R., Debnam, E.S., Bagnall, J. and Thompson, C.S.

J Pharmacol, 107: 267-269, 1985.

Dashwood, M.R. and Thompson, C.S. J Physiol, in press, 1986.

DeLean, A., Munson, P.J. and Rodbard, D. Am J Physiol, 235:

E98-E102, 1978.

Dobbins, J., Racusen, L. and Binder, H.J. J Clin Invest, 66:

19-28, 1980.

Gillan, M.G.C. and Kosterlitz, H.W. Brit J Pharmacol, 77: 461-469,

1982.

Hughes, S., Higgs, N.B. and Turnberg, L.A. Gut, 25: 931-935, 1984.

Kachur, J.F., Miller, R.J. and Field, M. Proc Nat Acad Sci USA, 77:

2575-2756, 1980.

Munson, P.J. and Rodbard, D. Analyt Biochem, 107: 220-239, 1980.

Turnberg, L.A. ScAnd J Gastroent, 18 (Suppl 84): 79-83, 1983.

Turnberg, L.A., McKay, J. and Higgs, N.B. Electrolyte and water

transport across gastrointestinal epithelial cells. New York,

Raven Press, 1982. pp 287.

Warhurst, G., Smith, G., Tonge, A. and Turnberg, L.A. European J

Pharmacol, 86: 77-82.

Acknowledgement

This work was supported by a grant from the British Heart

Foundation.

318
MU OPIOID RECEPTOR BINDING SITES IN HUMAN BRAIN

Carmencita Pilapil, Sharon Welner, Jacques Magnan*, Nadav Zamir** and

Rémi Quirion

Douglas Hospital Research Centre and Dept. of Psychiatry, McGill

University, 6875 Blvd LaSalle, Verdun, Quebec Canada H4H 1R3

*Dept. of Pharmacology, Faculty of Medicine, University of Montreal,

Quebec Canada

**NHLBI, NIH, Bethesda, MD, U.S.A.

ABSTRACT

Our experiments focused on the examination of the distribution of mu

opioid receptor binding sites in normal human brain using the highly

selective ligand [3H]DAG0, in both membrane binding assay and in

vitro receptor autoradiography. Mu opioid binding sites are very

discretely distributed in human brain with high densities of sites

found in the posterior amygdala, caudate, putamen, hypothalamus and

certain cortical areas. Moreover the autoradiographic distribution

of [3H]DAG0 binding sites clearly reveals the discrete lamination

(layers I and III-IV) of mu sites in cortical areas.

INTRODUCTION

Various investigators have reported on the presence of opioid receptor binding sites in human brain. For example, the gross regional

distribution of mu and delta receptor subtypes and the predominance

of kappa receptors have been shown (Kuhar et al.1973; Bonnet et al.

1981; Pfeiffer et al.1982). However, the characterization of these

various opioid receptor types has not been studied in detail. As a

first series of experiments, we investigated the distribution of mu

opioid receptors in normal human brain using a highly selective

ligand [

3

H]D-Ala2, (Me)Phe4, Gly-ol5 enkephalin (DAGO; Peterson et al.

1983) in both membrane binding assays and in vitro receptor autoradiography.

MATERIALS AND METHODS

Membrane binding assays were performed using normal human brains

obtained at autopsy, frozen on dry ice and then kept at -80°C until

used (Quirion 1985). Subsequently, punches (500-750 mg) were taken

from various areas, homogenized in 50 mM Tris.HCl buffer, pH 7.4 at

4°C using a Brickman polytron (setting 5, 20 s). The homogenate was

then centrifuged for 15 min at 49,000 x g. The supernatant was

discarded and the pellet resuspended in buffer and homogenized as

above. This preparation was then incubated at 37°C for 40 min and

centrifuged for 15 min at 49,000 x g. The final pellet was washed

twice with buffer and resuspended in buffer to a final concentration

of 0.8 - 1 mg protein/ml (Lowry et al.1951). For binding assays, 0.1

ml of membrane preparation was added to 0.85 ml of 50 mM Tris.HCl

buffer, pH 7.4 at 25°C, with various concentrations of [3H]DAGQ (44.7

Ci/mmol, New England Nuclear) ranging between 0.05 - 1 nM in 0.05 ml,

to a total incubation volume of 1,0 ml. After 60 min, incubations

were terminated by rapid filtration through Whatman GF/B filter

strips followed by 3 x 5 ml rinses using cold incubation buffer.

Filter strips were pre-soaked in 0.05% polyethyleinine to reduce

binding to filters. Specific binding was calculated as the dif319
ference in radioactivity bound in presence and absence of 5 μM

cyclazocine. Binding was quantitated by counting filters in 6.0 ml

Scinti-Verse II (Fisher Scientific Ltd., Canada) scintillation

cocktail.

For receptor autoradiography, human brain slide-mounted sections were

prepared as described before (Quirion 1985). Sections were then incubated for 60 min at room temperature in 50 mM Tris.HCl, pH 7.4 containing 1 nM [

3

H]DAG0. Specific binding was determined as above. At

the end of incubation, slides were rinsed for 5 min in cold incubation buffer, dipped in cold distilled water to remove salts, dried

and then juxtaposed tightly against tritium-sensitive film (Ultrofilm, LKB Instruments). After 12 weeks, films were developed as

previously described (Quirion et al.1981).

RESULTS AND DISCUSSION

As shown in table 1, mu opioid binding sites are discretely distributed in human brain. More specifically, high densities of binding

sites are found in the posterior amygdala, caudate, putamen, various

thalamic and hypothalamic nuclei (table 1). Much lower densities are

present in various brainstem nuclei including the pontine, hypoglossal and trigeminal nuclei (table 1). In the cortex, Brodmann's areas

#30, 24, 23, 22, 21, 20, 17, 9, 6 and 4 (in descending order) are

especially enriched in mu opioid binding sites (table 1).

Preliminary autoradiographic data support results obtained in membrane binding assay. As shown in figure 1, high densities of

[

3

H]DAG0 binding sites are seen in the caudate, putamen, ventral

pallidum-substantia innominata area and cortical layers I and IV.

Much lower densities are found in the globus pallidus, internal

capsule and claustrum (figure 1).

The distribution of mu sites in human brain reported here using a

highly selective ligand correlates fairly well with previous results

obtained using less selective probes (Kuhar et al.1973; Pfeiffer et

al.1982; Bonnet et al.1981). In all these studies, it was found that

the amygdala, hypothalamus, thalamus, caudate and putamen were enriched in mu sites. Additionally, we provided preliminary data on

the distribution of these sites in multiple cortical areas and brainstem.

The autoradiographic distribution of mu sites in human brain is also

most interesting. As observed in rat brain (Goodman et al.1981;

Quirion et al.19831, high densities of sites are found in caudate,

putamen and certain cortical layers. However, it appears that the

"patchy" distribution of mu sites in the striatum is less apparent in

human brain. The possible significance of this observation remains

to be established. We are currently studying the comparative

distribution of other opioid binding sites (delta and kappa) in

normal and pathological human brain.

320
Table 1. Distribution of [3

H]DAG0 binding sites in different

regions of the human brain using membrane assay.

Region Kd (nM)

Caudate 1.1

Posterior amygdala

Hippocampus

Hypothalamus

Putamen

Thalamus

Substantia nigra

Internal capsule

Cortex - Brodmann area

9

44

24

8

6

22

20

24

4

23

31

21

17

30

39

19

18

Cerebellum

Pontine nucleus

Bmax (fmol/mg protein)

32.3

1.0 53.8

1.1 19.1

4.2 49.3

2.2 25.2

1.1 30.5

2.2 11.1

0.4 7.8

0.4 35.2

0.6 26.2

0.9 36.1

0.2 24.7

0.5 37.5

0.4 32.3

0.5 46.7

0.2 25.3

1.3 39.9

0.9 78.9

0.3 27.1

0.5 42.6

1.0 25.6

0.6 30.0

0.6 28.9

0.3 12.6

0.4 11.4

8.1 46.8

1.0 23.6

Data represent mean of 2-3 experiments using 6 different

concentrations of ligand. All data were analyzed by

computerized linear regression analysis (Bio-Soft, Elsevier,

Cambridge, England).

ACKNOWLEDGEMENTS

Supported by the Douglas Hospital Research Centre. C. Pilapil, holds

a studentship of the Fonds pour la Formation de Chercheurs et 1'Aide

a la Recherche, S. Welner is a fellow of the Medical Research Council

of Canada and R. Quirion is a "Chercheur-Boursier" of the Fonds de la

Recherche en Santé du Québec.

321
Figure 1. Photomicrograph of the autoradiographic distribution

of [

3

H]DAG0 opioid binding sites in human brain. Abbreviations

used: C, caudate; CL, claustrum; GP, globus pallidus; P,

putamen; SI, substantia inominata, I and IV, layers I and IV of

the cortex.

REFERENCES

Bonnet, K.A., Groth, J., Gioannini, T., Cortes, M., and Simon, E.J.

Brain Res, 221:437-440, 1981.

Goodman, R.R., Snyder, S.H., Kuhar, M.J., and Young, W.S. Proc Natl

Acad Sci USA, 77:6239-6243, 1980.

Kuhar, M.J., Weiss, A.S., Pert, C.B., and Snyder, S.H. Nature,

245:447-450, 1973.

Lowry, O.H., Rosebrough, N.J., Farr, A.L.. and Randall, R.J. J Biol

Chem, 193:265-275, 1951.

Paterson, S.J., Robson, L.E., and Kosterlitz, H.W. Brit Med Bull,

39:31-36, 1983.

Pfeiffer, A,, Pasi, A., Mehraein, P., and Herz, A. Brain Res, 248:87-

96, 1982.

Quirion, R., Hammer, R.P., Jr., Herkenham, M., and Pert, C.B. Proc

Natl Acad Sci USA, 78:5881-5885, 1981.

Quirion, R., Zajac, J.M., Morgat, J.L., and Roques, B.P. Life Sci,

33(1):227-230, 1983.

Quirion, R. Eur J Pharmacol, 117:139-142, 1985.

322
BEHAVIORAL INTERACTION BETWEEN ENDOGENOUS OPIOIDS, CCK-8 AND

DOPAMINERGIC SYSTEMS IN THE NUCLEUS ACCUMBENS OF RATS

Jan M van Ree, Joshio Igarashi* and Ildiko Király**

Rudolf Magnus Institute for Pharmacology,

Medical Faculty, University of Utrecht,

Vondellaan 6, 3521 GD Utrecht, The Netherlands

*On leave of absence from Saitama Medical School Saitama, Japan

**On leave of absence from Inst. of Drug Research, Budapest, Hungary

ABSTRACT

Injection of apomorphine into the nucleus accumbens of rats elicits

hypermotility, presumably by activating postsynaptically located dopamine receptor systems. This behavioral response is inhibited by local

treatment with CCK-8 or endorphin. The action of both peptides is

antagonized by intra-accumbal treatment with naloxone. These findings

suggest that endogenous opioids are implicated in certain neurolepticlike effects of CCK-8 and that in the nucleus accumbens a functional

interaction exists between CCK-8, endogenous opioids and dopaminergic

systems.

INTRODUCTION

The influence of peptides related to endorphin and cholecystokinin

(CCK) on behaviors of rodents suggest that these peptides induce effects that can also be elicited by neuroleptic drugs (De Wied et al.

1978; Van Ree et al. 1980; Zetler 1983; Van Ree et al. 1983).

Clinical studies have revealed that treatment with these peptides

results in an antipsychotic effect in a number of schizophrenic

patients (Van Ree et al. 1986; Nair et al. 1986). Since brain dopamine has been implicated in the antipsychotic action of neuroleptics,

extensive studies have been performed to investigate the interaction

between these peptides and dopaminergic systems in the brain. One

approach to study this interaction is to determine the behavioral

effect of dopaminergic drugs micro-injected into restricted brain

areas, and to investigate whether known or purported antipsychotic

substances interfere with this behavioral effect. Following this approach and focussing on the nucleus accumbens area, that contains

terminals of the dopaminergic mesolimbic pathway, we have found that

relatively small doses of apomorphine induce hypomotility in rats.

This behavioral response that may be mediated by presynaptically located dopamine receptor systems, can be blocked by local pretreatment

with neuroleptic drugs (e.g. haloperidol and sulpiride) and peptides

related to endorphin and cholecystokinin (Van Ree et al. 1982,

1983, 1984). The active peptides in this respect are the opioid

endorphin and the non-opioid -acetyl endorphin, des-Tyr1endorphin and desenkephalin endorphin and both sulphated and desulphated

CCK-8. When higher doses of apomorphine are injected into the nucleus

accumbens, the rats show hypermotility, a behavioral response probably mediated by postsynaptically located dopamine receptor systems.

This response is antagonized by pretreatment with endorphin and

sulphated CCK-8 (CCK-8-S) but not by the non-opioid type endorphins

and desulphated CCK-8 (CCK-8-DS) (Van Ree et al. 1983, 1984; Van Ree

and Gaffori 1983). Since the effect of endorphin can be blocked by

323
naloxone and is not mimicked by the non-opioid related peptides

acetyl endorphin and des-Tyr1endorphin), the effect is likely

mediated by opioid receptor systems. Interestingly this action of

endorphin is unique in that other opioid peptides, like enkephalins

and and endorphin, are not active in this respect (Van Ree and

Gaffori 1983). Evidence has been presented that CCK-8-S counteracts

opioid-induced analgesia (Faris et al. 1983), suggesting an interaction between CCK-8-S and endogenous opioids. The present experiments

were designated to investigate whether opioid receptor systems are

involved in the antagonizing action of CCK-8-S on the apomorphineinduced hypermotility following injection into the nucleus accumbens.

MATERIALS AND METHODS

Male Wistar rats weighing 130 - 140 g were equipped with a stainless

steel cannula at each side of the brain. The cannulae were aimed at the

nucleus accumbens (for details see Van Ree et al. 1982, 1983). At

least 7 days after the operation, the rats were three times injected

bilaterally with 1 μl. Groups of rats received placebo, CCK-8-S (10

ng) or endorphin (10 ng), after 30 min placebo or naloxone (2 μg)

and 10 min later placebo or apomorphine (10 μg). Twenty minutes after

the last injection the rats were placed in a small open field, a perspex circular test cage, the bottom of which was divided into 4 equal

sections. Motor activity was measured for 3 min by counting the number

of sections explored. After experimentation the sites of injection

were evaluated histologically. Data of rats outside the nucleus accumbens were discarded from the analysis. Group means and standard errors

were calculated and the statistical significance was determined using

ANOVA and subsequently Newman-Keuls analysis. The peptides were

donated by Organon International B.V., Oss, The Netherlands and naloxone by Endo, New York, U.S.A. Drugs and peptides were dissolved in

saline just prior to experimentation.

RESULTS AND DISCUSSION

Injection of apomorphine (10 μg) into the nucleus accumbens resulted

in hypermotility (table I). This behavioral effect was completely

antagonized by local pretreatment with either CCK-8-S or endorphin.

As already mentioned desulphated CCK-8 and non-opioid peptides related

to endorphin e.g. acetyl endorphin are inactive in this respect

(Van Ree et al. 1983, 1984). Thus, small changes (i.e. desulphation

and acetylation) in the peptide structure lead to inactivation with

respect to this action of the peptides. Interestingly, both sulphated

and desulphated CCK-8 and both the opioid and non-opioid type endorphins interfere with another dopaminergic system in the nucleus accumbens, which can be activated by small doses of apomorphine (Van Ree

et al. 1983, 1984). Desulphation and acetylation thus result in more

selectively acting peptides in this case. Whether these enzymatic

processes occur before or after the release of the peptides, thus

intra- or extraneuronally, is unknown, although the present knowledge

favors intraneuronal processing.

The opioid antagonist naloxone antagonized the inhibitory effect of

both CCK-8-S and endorphin on apomorphine-induced hypermotility

(table I). Naloxone did not interfere with the apomorphine response

per sé (Van Ree et al. 1984). The apomorphine-induced hypermotility

324
TABLE I

Effect of naloxone on the antagonistic action of CCK-8-S and

endorphin on apomorphine-induced hypermotility following

injection into the nucleus accumbens

Treatment Motor activity

scores

(mean SEM)

- 60' - 30' - 20'

placebo placebo placebo 15.2 0.4 (28)

placebo placebo apomorphine 10 μg 23.1 0.6* (27)

CCK-8-S 10 ng placebo apomorphine 10 μg 15.5 0.7+(23)

CCK-8-S 10 ng naloxone 2 μg apomorphine 10 μg 22.6 1.7° (10)

YE 10 ng placebo apomorphine 10 μg 14.8 0.7+(14)

Y E 10 ng naloxone 2 μg apomorphine 10 μg 23.9 1.2° ( 7)

( ) number of observations

ANOVA: F (5,103) = 32.48 (p < O.OO1), Newman-Keuls:

* different from placebo, placebo, placebo (p < 0.05),

+ different from placebo, placebo, apomorphine (p < 0.05),

° different from peptide, placebo, apomorphine (p < 0.05).

is thought to be mediated by activation of postsynaptically located

dopamine receptor systems. Thus, CCK-8-S and endorphin may act in

this brain area as a dopamine antagonist. Indeed the dopamine antagonist haloperidol blocked the hypermotility induced by apomorphine

following injections into the nucleus accumbens, but the effect of

haloperidol (100 pg) was not affected by similar treatment with naloxone. It may therefore be postulated that activation of opioid receptor systems or release of endogenous opioids is involved in the effect

of CCK-8-S on the apomorphine-induced hypermotility. Candidates in

this respect are endorphin receptors or endorphin respectively,

since endorphin in contrast to other opioid peptides mimics this

effect of CCK-8-S. Endogenous opioids may be involved in some, but

not all interactions between CCK-8-S and dopaminergic systems in the

nucleus accumbens. In fact, the CCK-8-S-induced inhibition of the

hypomotility elicited by small doses of apomorphine is not affected

by local treatment with naloxone. CCK-8-S may thus have multiple actions on the function of dopaminergic neurons in the nucleus accumbens, and in one or some of these actions endogenous opioids are concerned. The functional interaction between CCK-8, endogenous opioids

and dopaminergic systems may contribute to further elucidate the antipsychotic action of certain endogenous peptides.

325
REFERENCES

De Wied, D., Kovács, G.L., Bohus, B., Van Ree, J.M. and Greven, H.M.

Eur J Pharmacol, 49: 427-436, 1978.

Faris, P.L., Komisaruk, B.R., Watkins, L.R., Mayer, D.J. Science,

219: 310-312, 1983.

Nair, N.P.V., Lal, S., and Bloom, D.M. Progr Brain Res, 65: 237-258,

1986.

Van Ree, J.M., and Gaffori, O. Life Sci, 33: suppl. I: 619-622, 1983.

Van Ree, J.M., Bohus, B., and De Wied, D. In: Leong Way, E., ed.

Endogenous and Exogenous Opiate Agonists and Antagonists. New York:

Pergamon Press, 1980. pp. 459-462.

Van Ree, J.M.; Gaffé, A.M., and Wolterink, G. Neuropharmacology, 21:

1111-1117, 1982.

Van Ree, J.M., Gaffori,O., and De Wied, D. Eur J Pharmacol, 93: 63-

78, 1983.

Van Ree, J.M., Gaffori,O., and Király, I. Life Sci, 34: 1317-1324,

1984.

Van Ree, J.M., Verhoeven, W.M.A., Claas, F.H.J., and De Wied, D.

Progr Brain Res, 65: 221-235, 1986.

Zetler, G. Eur J Pharmacol, 94: 261-270, 1983.

326
SUPPRESSION OF SYMPATHETIC NERVE ACTIVITY BY METHIONINE ENKEPHALIN:

ADDITIVE INTERACTION WITH CLONIDINE IN INTACT

ANESTHETIZED RABBITS

Hee M. Rhee

Department of Pharmacology

Oral Roberts University, School of Medicine

Tulsa, OK 74137 U.S.A.

ABSTRACT

In order to study the role of methionine enkephalin (met-enkephalin)

in cardiovascular system, interaction between clonidine and metenkephalin on the rate of sympathetic nerve discharge was investigated. In anesthetized rabbits multiunit renal nerve activity (RNA)

was monitored after the treatment of met-enkephalin alone or metenkephalin plus clonidine pretreatment. Met-enkephalin suppressed

RNA, and reduced heart rate and blood pressure. Treatment of metenkephalin after clonidine (2 to 20 μg/kg) increased the duration of

RNA suppression. The data suggests that the cardiodepressive action

of met-enkephalin is mediated by the stimulation of central presynaptic adrenergic receptor as proposed for clonidine.

INTRODUCTION

Met-enkephalin has profound effects on the cardiovascular system in

addition to diverse other physiological actions (Evans et al. 1986;

Simon and Hiller 1978). Met-enkephalin decreased blood pressure and

heart rate in parallel with the suppression of sympathetic nerve

activity in anesthetized intact rabbits (Rhee et al. 1985), although

the response to the peptide in conscious animal may be different

(Yukimura et al. 1981). This cardiodepressive effects of the peptide was blocked by naloxone, but not by naloxone methobromide,

which is a quaternary analog of naloxone. The cardiovascular action

of the peptide was also partially blocked by phentolamine, a nonspecific alpha-adrenergic receptor blocker (Eulie and Rhee 1984).

It is understood that met-enkephalin effect is mediated by opioid

receptors and alpha adrenergic system is involved in the hypotensive

action of met-enkephalin, but the exact nature of interaction

between the peptide and clonidine is not clear. Therefore, the main

object of this research was to establish potential synergistic or

additive interaction of the two drugs.

MATERIALS AND METHODS

In pentobarbital (30 mg/kg i.v.) anesthetized rabbits, the right

femoral artery and vein were cannulated for the measurement of

arterial pressure and subsequent delivery of drugs. The rabbits

were allowed to respire spontaneously through a tracheostomy.

Arterial blood pressure was monitored by a Statham P23 pressure

transducer coupled to a pressure processor amplifier (Gould Instruments, Cleveland, OH, model 13-4615-52) which computes the systolic,

327
mean, diastolic, and pulse pressures. Heart rate (HR) was monitored

by a Gould Biotec ECG amplifier (model 13-4615-65).

After the basic surgery, the animals were placed on their right side

and the kidney was exposed retroperitoneally. A branch of the left

renal nerve was carefully dissected from the surrounding connective

tissue, cleaned and suspended on a bipolar electrode as reported

Rhee et al. (1985). Multiunit renal nerve activity (RNA) was monitored, and integrated for a given time before analysis. All of the

above parameters were recorded on a Gould 16-channel electrostatic

recorder (model ES 1,000). Renal blood flow was determined electromagnetically.

Animals were given met-enkephalin (Sigma Chemical Co., St. Louis,

MO) in bolus doses of 3, 10, and 30 μg/kg in ascending order with a

5-min interval between doses. After establishing the control doseresponse curve of met-enkephalin, the same dose-response test was

repeated 10 min after pretreatment with 1, 2, 5, 10, and 20 μg/kg of

clonidine. The mean values for arterial pressure, HR and RNA were

calculated and time for half-maximal recovery (T50) of systolic

blood pressure was also determined. Each datum point was analyzed

by unpaired student t test and significant difference was considered

as P is lesser than .05.

RESULTS AND DISCUSSION

In anesthetized rabbits an intravenous injection of met-enkephalin

decreased systolic, mean and diastolic blood pressure in the peptide

dose dependently, which confirms our previous reports (Rhee et al.

1985; Eulie and Rhee 1984). However, pulse pressure was slightly

increased in many experiments as shown in fig. 1. Heart rate and

renal nerve activity (RNA) were reduced by the peptide so that

integrated RNA per min was also significantly reduced. Renal blood

flow was reduced by the treatment of met-enkephalin from the control

26 ml/min for a kidney to 21 ml/min. It is not clear whether this

effect on renal blood flow is secondary to its primary effect on

blood pressure or its potential direct effect on renal blood vessel. Clonidine initially increased blood pressure which was followed by a gradual but a profound reduction in blood pressure and

heart rate in the drug dose-dependent manner. Clonidine also suppressed RNA in a manner similar to met-enkephalin, which usually

manifested at the time of initial increase in blood pressure by

clonidine. Simultaneous administration of clonidine and metenkephalin produced an additive action in duration of RNA suppression. Clonidine also increased the time that is required 50% recovery from the maximal reduction of blood pressure (T50). But it was

not always possible to see clonidine additively interact with metenkephalin in the reduction of blood pressure or heart rate due to

presumable interference with baroreflex and other physiological

compensating mechanisms (fig. 2).

Antihypertensive effect of clonidine is known to be mediated by the

stimulation of central adrenergic receptors which will decrease

peripheral sympathetic outflow. The similarity between the action

328
of the two drugs, particularly at low doses of clonidine (e.g.,

under 5 μg/kg) suggests the mechanism of met-enkephalin on cardiovascular action indeed involves central stimulation of receptors. It is also supported by the fact that adrenergic blocker such

as phentolamine antagonized cardiovascular effects of met-enkephalin

instead of potentiating the effects (EuLie and Rhee 1984).

Figure 1. Effects of met-enkephalin (ME) on cardiac

parameters in intact anesthetized rabbit. Animal was

prepared as in "methods" and ME (300 μg/kg) was loaded

at the first bar in a volume of .25 ml of saline and

flushed with saline as shown at the second bar. RNA,

SP, MP, DP, PP and BPfa indicate renal nerve activity,

systolic, mean, diastolic and pulse pressures, and

femoral arterial blood pressure, respectively. Note

that there is no unit in integration of RNA.

329
Figure 2. Interaction between clonidine and metenkephalin on mean blood pressure in anesthetized

rabbits. Animals were prepared as in fig. 1 and metenkephalin was given 10 min after the treatment of

clonidine. Vertical bars indicate S.E. and numbers

indicate the number of animal used.

REFERENCES

Eulie, P., and Rhee, H.M. Br J Pharmacology, 83:783-790, 1984.

Evans, C.J., Erdelyi, E., and Barchas, D. In: Plotnikoff et al.,

eds. Enkephalins and Endorphins. N.Y. Plenum Press, 1986.

pp.3-15.

Rhee, H.M., Eulie, P.J., and Peterson, D.F. J Pharmacol Exp Ther,

234:534-537, 1985.

Simon, E.J., and Hiller, J.M. Ann Rev Pharmacol and Toxicol,

18:371-394, 1978.

Yukimura, T., Stock, G., Stump, H., Unger, T., and Ganten D.

Hypertension, 3:528-533, 1981.

ACKNOWLEDGEMENTS

This work was supported in part by an intramural research grant from

Oral Roberts University School of Medicine.

330
BETA-ENDORPHIN MODULATES THE INHIBITORY ACTION OF CORTICOTROPINRELEASING FACTOR ON LUTEINIZINC HORMONE SECRETION

Felice Petraglia, Wylie Vale and Catherine Rivier

The Clayton Foundation Laboratories

for Peptide Biology

The Salk Institute for Biological Studies

La Jolla, CA 92037

ABSTRACT

(2 nmole, icv),

nmole, icv), a mu1

effect of CRF on LH.

opiate receptor antagonist, also reversed the

ip),

Either kappa (Mr1456 and Mr2266) (10 mg/kg,

or delta (ICI 154,129) (10nmole, icv) opiate receptor

antagonists did not significantly modify the’inhibitory effect of CRF

on circulating LH levels. These results suggest that central END

participates in the inhibitory action of CRF on LH secretion.

The present study investigates the possible role of endogenous opioid

peptides in mediating the inhibitory effect of corticotropinreleasing factor (CRF) on circulating Iuteinizing hormone (LH) . The

centraI injection of an antiserum against endorphin END)

reversed CRF-induced LH decrease in castrated male rats, while

antisera raised against DYN-A or M-Enk were inactive. END-(6-31)

a END antagonist, and beta-funaltrexamine (4.8

INTRODUCTION

Endogenous opioid peptides (EOP) exert a tonic inhibitory effect on

luteinizing hormone (LH) secretion. They modulate the hypothalamic

secretion of gonadotropin-releasing hormone (GnRH), and participate

in the negative feedback action of gonadal steroids on hypothalamicpituitary function (Cicero et al. 1979; Van Vugt et al. 1982).

Recently, the central injection of corticotropin-releasing factor

(CRF) has been shown to decrease plasma LH levels in castrated rats

Rivier and Vale 1984; Ono et al. 1984). The action of CRF on LH

secretion is independent from the stimulation of the pituitaryadrenal axis, and appears to be due to an effect of CRF on the

mechanisms that control GnRH secretion (Rivier and Vale 1985).

Interestingly, the coexistence of CRF and EOP has been described in

the arcuate-ventromedial hypothalamus (Watson et al. 1982; Millan et

al. 1986), thus suggesting a possible functional interaction in

between these pathways into these brain areas, particularly rich of

GnRH neurons. The aim of the present study was to evaluate whether

the inhibitory action of CRF on LH secretion involves EOP activation.

Passive immunoneutralization of the three most important brain EOP

(beta-endorphin, END; dynorphin-A, DYN-A; methionine-enkephalin, MEnk) and specific antagonists for the main opiate receptor subtypes

(mu/epsilon, kappa, delta) were used to antagonize the action of CRF

on circulating LH.

331

172-683 O - 87 - 12 : QL 3
MATERIALS AND METHODS

Male Sprague-Dawley rats (220-240 g) were used. One week before the

bioassay, rats were castrated and a permanent cannula was implanted

into lateral ventricle of the brain. The implantation of a jugular

catheter was done 24 hours before the experiments. Ovine CRF (2

nmole) (Rivier et al. 1983) was dissolved in water and injected icv

(5μl) In groups of 6-8 rats antiserum raised against END or DYNA or M-Enk was injected icv (5 μI) two hours before the CRF

administration. In another set of experiments the following opiate

receptor antagonists were administered centrally: END(6-31), a

END antagonist (2 nmole) (gift of Dr. C.H. Li, San Francisco, CA);

beta-funaltrexamine ( -FNA) (4.8 nmole) (Res. Biochem. Inc., Wayland,

MA), a long acting irreversible mu1 receptor antagonist; ICI 154,129

(10 nmole) (gift of Dr. P. Sacerdote, Bethesda, MD), a delta antagonist. Two different kappa opiate receptors antagonists were

injected intraperitoneally: Mr1456 MS and Mr2266 BS (10 mg/kg BW)

(gift of Drs. Plauth and Elich, Boerhinger Ingelheim, Ingelheim an

Rhein, FRG). Blood samples were collected before and every 30

minutes following CRF injection. Plasma LH levels were measured by

radioimmunoassay, using materials provided by NIADDK (Bethesda, MD).

The assay sensitivity was 10 pg; the inter- and intrassay

coefficients of variation were 6% and 3.5% respectively.

Statistical analysis of the results was performed using the one-side

multiple comparison test of Dunnett and the multiple range test of

Duncan.

RESULTS AND DISCUSSION

The central injection of CRF significantly decreased plasma LH levels

in castrated male rats from 6.8 + 0.7 ng/ml to 3.4 + 0.3 ng/ml (M +

SEM) (P < 0.01). The inhibitory effect of CRF on LH secretion was

measurable over a 60 minute period following the administration of

the peptide (4.6 + 0.7 ng/ml, P < 0.019, reaching the nadir values

after 180 minutes. The central injection of an antiserum raised

against END completely reversed the inhibitory effect of CRF (fig.

1). In END immunoneutralized rats, plasma LH levels were in the

same range as control rats. By contrast, in rats injected with DYN-A

or M-Enk antiserum the inhibitory effect of CRF on LH was not

significantly modified (fig. 1). These results showed that CRF

interact centrally with END in modulating LH decrease. In support

of this hypothesis, we have observed that the central injection of

END(6-31) or FNA also reversed the decrease of LH levels induced by

icv injection of CRF (fig. 1). However, in rats pretreated with

FNA a slight CRF-induced LH decrease was still evident (fig. 1), thus

suggesting a secondary role of mu1 receptor subtype regulating LH

secretion. Kappa (Mr1452 or M-2266) and delta (ICI 154,129) opiate

receptor antagonists did not significantly influence the LH decrease

following CRF injection (fig. 1). These results suggest that CRF

decreases plasma LH levels by stimulating the inhibitory action of

END on GnRH neurons. Moreover, these results agree with the

observation that END is the main EOP involved in the control of LH

secretion. The use of a specific antiserum to END (Schultz et al.

1981; Forman et al. 1983) or of mu/epsilon opiate receptor

antagonists (Petraglia et al. 1984; Panerai et al. 1985) have

332
indicated a major role of END pathway in tonically inhibiting LH

secretion. These results may also explain recent reports showing

that either a CRF antagonist (Rivier et al. 1986) or naloxone (Hulse

et al. 1982; Brisky et al. 1984) completely reversed stress-induced

LH decrease in rats. It is possible that stress activates

hypothalamic CRF/ MND pathways, leading to a decrease in GnRH as

well as LH secretion. It is therefore possible that the

antireproductive actions of stress activation of pituitary-adrenal

axis may be explained not only by a pituitary (Suter and Schwartz

1985) or gonadal (Sapolsky 1985) action, but also by the central

activation of CRF/ END pathways. In conclusion these results show

that CRF and END interact to modulate LH secretion.

Figure 1: Effect of various antisera (AS) raised against END,

DYN or M-Enk or of antagonists for the different opiate receptor

subtypes on CRF-mediated LH inhibition. *P < 0,01 vs. control rats

(NRS, normaI rabbit serum).

333
ACKNOWLEDGMENTS

This research was partially supported by the HD13527 grant from the

National Institutes of Health. F.P. was partially supported by

Consiglio Nazionale delle Ricerche, No. 350.542. Permanent address:

Depr. Obstet. Gynecol., University of Modena, School of Medicine, Via

del Pozzo 71, 41100 Modena, Italy. Research conducted in part by the

Clayton Foundation for Research, California Division. W.V. is a

Clayton Foundation investigator.

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Sapolsky, R.M.

Schultz, R., Wilhelm, A., Pirke, K.M., Gamsch, G., and Herz, A.

Nature, 294:757-759, 1981.

Suter N.E., and Schwartz, N.B Endocrinology, 117:849-853, 1985.

Van Vugt, D.A., Sylvester, P.W., Aylsworth, C.F., and Meites, J.

Neuroendocrinology 34:274-278, 1982.

Watson, S.J Fischi, W.,

216:85-87; 1982.

Zimmerman, E., and Nilaver, G. Science,

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Med, 173:14-17, 1983.

Proc Soc Exp Biol and

Hulse, G., Coleman, G., Nicholas, J., and Greenwood, K. J Reprod

Fert, 66:451-453, 1982.

Millan, M.A., Jacobowitz, D.M., Hauger, R.L., Catt, K.J., and

Aguilera, G. Proc Natl Acad Sci USA, 83:1921-1925, 1986.

Ono, N., Lumpkin, M.D., Samson, W.L., McDonald, J.K., and McCann,

S.M. Life Sci, 35:117-122, 1984.

Brizky, K.P., Quigley, and K., Meites, J. Life Sci, 34:2485-2492,

Cicero, T.J., Schainker, B.A., and Mayer, E.R. Endocrinology,

104:1286-1290, 1979.

Panerai, A.E., Petraglia, F., Sacerdote, P., and Genazzani, A.R.

Endocrinology, 117:1096-1099, 1985.

Petraglia, F., Locatelli, V., Penalva, A., Cocchi, D., Genazzani,

A.R., and Muller, E.E. J Endocrinol, 101:33-39, 1984.

Rivier, C., and Vale, W. Endocrinology, 114:914-918, 1984.

Rivier, C., and Vale, W. J Clin Invest, 75;689-694, 1985.

Rivier, C., Rivier, J., and Vale, W. Science, 131:607-609, 1986.

Rivier, J., Spiess J., and Vale, W. Proc Natl Acad Sci USA,

Endocrinology, 116:2273-2276, 1985.

334
LEUMORPHIN IS A POTENT INHIBITOR OF VASOPRESSIN SECRETION

Takayuki Yamada1, Kazuwa Nakao1,2, Hiroshi Itoh1, Narito Morii1,

Shozo Shiono Makoto Sakamoto1, Akira Sugawara1, Yoshihiko Saito1,

Goro Katsuura3, Masami Eiguo3, Akira Matsushita3 and Hiroo Imura1

1

2nd Division, Department of Medicine,

Kyoto University School of Medicine,

2

Radioisotope Research Center, Kyoto University, Kyoto 606,

3

Shionogi Research Laboratories,

Shionogi & Co., Ltd., Osaka 553, Japan.

ABSTRACT

The effects of intracerebroventricular (i.c.v.) injection of leumorphin on the vasopressin secretion induced by angiotensin II or

dehydration were studied in conscious unrestrained rats. The vasopressin secretion induced by angiotensin II (100 pmol) was significantly inhibited by the simultaneous injection of leumorphin (6 pmol600 pmol) in a dose-dependent manner. In 72-hour water-deprived

rats, the i.c.v. injection of leumorphin (60 pmol or 600 pmol)

significantly suppressed the vasopressin secretion dose-dependently.

These results suggest that leumorphin is involved in the regulation

of the vasopressin secretion.

INTRODUCTION

Leumorphin is a proenkephalin B-derived 29-amino acid polypeptide

containing leucine-enkephalin sequence at its N-terminus (Kakidani et

al. 1982, Suda et al. 1983). Using a specific radioimmunoassay (RIA)

for leumorphin coupled with high performance liquid chromatography,

we demonstrated the presence of leumorphin in the pituitary and brain

(Nakao et al. 1983, Suda et al. 1984) and parallel distribution of

leumorphin and dynorphin in the brain (Suda et al. 1985). We also

reported that leumorphin acts as an agonist at the type opioid

receptor (Suda et al. 1983) and that intracerebroventricular (i.c.v.)

injection of leumorphin inhibits water drinking induced by water

deprivation or angiotensin II (AII) in rats, suggesting its involvement in water and electrolyte balance (Imura et al. 1985, Imura et

al. 1986). In addition, dynorphin derived from the same precursor as

that of leumorphin, is known to co-exist with vasopressin (AVP) in

magnocellular neurons in the supraoptic and paraventricular nuclei

(Watson et al. 1982). In this study, to further elucidate the

involvement of leumorphin in fluid balance, we have examined the

effect of i.c.v. injection of leumorphin on the AVP secretion in

conscious unrestrained rats.

MATERIALS AND METHODS

Peptides

Synthetic angiotensin II (AII) was purchased from Protein Research

Foundation, Osaka, Japan. Human leumorphin was synthesized by a

conventional solid phase method (Yamamoto et al. 1983). These

peptides were dissolved in physiological saline solution for intracerebroventricular (i.c.v.) injection at a volume of 5 μl.

335
Experimental animals

Male Wistar rats, weighing 200-230 g, were housed in a temperaturecontroled room (25 1°C) with a 12-hour light-dark cycle (light on

7:00 A.M.). The rats were fed standard rat biscuits and given free

access to food and water. They were anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories, Chicag, 50 mg/kg, intraperitoneal) and a stainless steel cannula was implanted stereotaxically into the left lateral ventricle as reported elsewhere

(Ikeda and Matsushita 1980). Four days later, a jugular catheter was

positioned into the superior vena cava under the pentobarbital

anesthesia. The experiments were performed after a 3-day recovery

period. At the end of experiments, 5 μl of 1 % Evans blue solution

was injected through the cannula to confirm that the placement of the

cannula was appropriate.

Effect of leumorphin on AII-stimulated AVP secretion

Rats were simultaneously injected i.c.v. with AI1 (100 pmol) and

leumorphin (0, 6 pmol, 60 pmol or 600 pmol). Blood (250 μ1) samples

were taken from the jugular catheter four times, just before the

injection and 1.5, 5 and 10 minutes after the injection.

Effect of leumorphin on dehydration-induced AVP secretion

After 72-hour water-deprivation, the effect of i.c.v. injection of

leumorphin (0, 60 pmol or 600 pmol) on the AVP secretion was examined. Blood (250 μl) samples were drawn from the jugular catheter at

0, 1.5, 5 and 10 min. An equal volume of physiological saline

(250 μ1) was injected intravenously just after the blood sampling.

Radioimmunoassay (RIA)

Blood samples were collected into polypropylene tubes containing

Na2EDTA (1 mg/ml). Plasma was separated immediately and stored at

TABLE 1. Effect of i.c.v. injection of leumorphin on AVP

secretion induced by AII

No. of

Treatment Dose

Time after i.c.v. injection

rats before 1.5 min 5 min 10 min

AII 100 pmol 7 3.1 16.6††† 8.9 ††† 6.1 †

±0.4 ±1.2 ±0.7 ±0.5

Leumorphin§ 6 pmol 4 4.7 13.3† † 6.0 5.1

±0.7 ±1.6* ±1.2* ±0.7

60 pmol 6 3.3 6.8 3.5 3.4

±0.4 ±1.2** ±0.4** ±0.4**

600 pmol 7 3.4 4.0 3.4 3.4

±0.4 ±0.6** ±0.4** ±0.4**

Values are means ±SE. § AII as given simultaneouly.

(pg/ml)

* P(0.05, ** P(0.01 vs AII (Duncan's multiple range test).

†P<0.05, ††P<0.01, †††P<0.001 vs before injection

(paired t test).

336
-20°C. The plasma vasopressin concentration was measured by RIA

(Yamada et al. 1986).

Statistical analysis

The data were analyzed by paired t test or Duncan's multiple range

test following one way analysis of variance.

RESULTS AND DISCUSSION

I.c.v. injection of AII (100 pmol) stimulated the AVP secretion from

the posterior pituitary in conscious unrestrained rats. This AVP

secretion was significantly inhibited by the simultaneous i.c.v.

injection of leumorphin (6 pmol-600 pmol) in a dose dependent manner

(table 1). The plasma AVP concentration was markedly elevated in 72-

hour water-deprived rats. As shown in table 2, the i.c.v. injection

of leumorphin (60 pmol or 600 pmol) also suppressed the dehydrationinduced AVP secretion dose-dependently.

The present study has demonstrated that leumorphin possesses a potent

inhibitory action on the AVP secretion in conscious unrestrained

rats. This observation is consistent with the inhibitory effects of

i.c.v. administration of endogenous opioid peptides such as endorphin and enkephalins on the AVP secretion (Van Wimersma Greidanus et

al. 1979, Summy-Long et al. 1981). In the present study, a relatively small dose of leumorphin (6 pmol) significantly inhibited the

AVP secretion, when compared with those of endorphin and enkephalins (Van Wimersma Greidanus et al.1979, Summy-Long J.Y. et al.

1981). This potent inhibitory effect of leumorphin on the AVP secretion corresponds to our previous findings that leumorphin inhibits

water drinking induced by i.c.v. injection of AII or water deprivation much more effectively than neo-endorphin or leucine-enkephalin

(Imura et al. 1985, Imura et al. 1986).

The potent inhibitory effect of leumorphin on the AVP secretion

together with its potent antidipsogenic action indicates that leumorphin plays a regulatory role in fluid homeostasis in the central

nervous system.

TABLE 2. Effect of i.c.v. injection of leumorphin on AVP

secretion induced by 72-hour water deprivation

No. of

Treatment Dose

Time after i.c.v. injection

rats before 1.5 min 5 min 10 min

Leumorphin 0 5 13.8 - 14.2 -

1.5 2

60 pmol 5 15.4 14.8 9.5 7.9

2.0 3.4 1.9* 2.2

600 pmol 5 14.0 6.7 4.8 5.7

1.5 1.2 0.5** 1.3

(pg/ml)

Values are means SE.

* P<0.05, ** P(0.01 vs Leumorphin 0

(Duncan's multiple range test).

P<0.05, P<0.01 vs before injection (paired t test).

337
ACKNOWLEDGEMENTS

This work was supported in part by research grants from the Japanese

Ministry of Education, Science and Culture, the Japanese Ministry of

Health and Welfare "Disorders of Adrenal Hormone" Research Committee,

Japan, 1986, Japan Tabacco Inc. and Yamanouchi Foundation for

Research on Metabolic Disorders, by a research grant for cardiovascular diseases (60A-3) from the Japanese Ministry of Health and

Welfare and by Japan Heart Foundation Research Grant for 1985.

REFERENCES

Ikeda, M., and Matsushita, A. J Neurosci Methods, 2:9-17, 1980.

Imura, H., Kato, Y., Nakai, Y., Nakao, K., Jingami, H., Tanaka, I.,

Koh, T., Tsukada, T., Yoshimasa, T., Suda, M., Sakamoto, M., Morii,

N., Takahashi, H., Tojo, K., and Sugawara, A. J Endocr, 107:147-

157, 1985.

Imura, H., Nakao, K., Yanaihara, N., Katsuura, G., and Nakamura, M.

Neuroendocrinology, in press, 1986.

Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y.,

Hirose, T., Asai, M., Inayama, S., Nakanishi, S., and Numa, S.

Nature, 298:245-249, 1982.

Nakao, K., Suda, M., Sakamoto, M., Yoshimasa, T., Morii, N., Ikeda,

Y., Yanaihara, C., Yanaihara, N., Numa, S., and Imura, H. Biochem

Biophys Res Commun, 117:695-701, 1983.

Suda, M., Nakao, K., Yoshimasa, T., Ikeda, Y., Sakamoto, M.,

Yanaihara, N., Numa, S., and Imura, H. Life Sci, 32:2769-2775,

1983.

Suda, M., Nakao, K., Sakamoto, M., Yoshimasa, T., Morii, N.,

Yanaihara, N., Numa, S., and Imura, H. Biochem Biophys Res Commun,

123:148-153, 1984.

Suda, M., Nakao, K., Sakamoto, M., Yoshimasa, T., Morii, N.,

Yanaihara, N., Numa, S., and Imura, H. Neuropeptides, 5:461-464,

1985.

Summy-Long, J.Y., Keil, L.C., Deen, K., Rosella, L., and Severs, W.B.

J Pharmacol Exp Ther, 217:619-629, 1981.

Van Wimersma Greidanus, T.B., Thody, T.J., Verspaget, H., de Rotte,

A., Goedemans, H.J.H., Croiset, G.C., and Van Ree, J.M. Life Sci,

24:579-586, 1979.

Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E.A.,

Nilaver, G., and van Wimersma Griedanus, T.B. Science, 216:85-87,

1982.

Yamada, T., Nakao, K., Morii, N., Itoh, H., Shiono, S., Sakamoto, M.,

Sugawara, A., Saito, Y., Ohno, H., Kanai, A., Katsuura, G., Eigyo,

M., Matsushita, A., and Imura, H. European J Pharmacol, in press,

1986.

Yamamoto, Y., Yanaihara, C., Katsumaru, Y., Mochizuki, T., Tobe, A.,

Egawa, M., Imura, H., Numa, S., and Yanaihara, N. Regulatory

Peptides, 6:163-168, 1983.

338
THE EFFECT OF 17-ALPHA-ESTRADIOL, A POSSIBLE ENDOGENOUS OPIATE

ANTAGONIST, ON D-ALA2-MET5-ENKEPHALINAMIDE-INDUCED

BLOOD PRESSURE RESPONSES IN CONSCIOUS, UNRESTRAINED RATS

A. Chandra and W. R. Dixon

Department of Pharmacology and Toxicology

School of Pharmacy, University of Kansas, Lawrence, KS 66045

ABSTRACT

D-ala2-met5-enkephalinamide (DAME) produced a dose-related increase

in the mean arterial blood pressure of conscious, unrestrained

rats. Intravenous injection of DAME (0.5, 1, 2, and 4 mg/kg)

resulted in mean systemic arterial blood pressures of 138±2, 146±5,

141±4, 156±5 mmHg, respectively. 17-alpha-estradiol and its

derivatives are known to be inactive in target tissues responsive

to estrogenic hormones such as 17-beta-estradiol. However, LaBella

et al. (1978) found after testing a large number of steroid

hormones and their metabolites that only 17-alpha-estradiol

significantly inhibited binding of 3H-naloxone, an opiate

antagonist, in rat-brain homogenates. The present study was

designed to determine whether 17-alpha-estradiol could antagonize

the cardiovascular responses elicited by intravenous injections of

DAME. Intravenous infusion of 17-alpha-estradiol (1.5 mg/kg) every

2 hours for 24 hours (total infusion time was 2 minutes for each

infusion) did not change the mean systemic arterial blood pressure

(94±5 mmHg) compared to the blood pressure prior to infusion of 17-

alpha-estradiol (99±7 mmHg). Intravenous infusion of 17-alphaestradiol (1.5 mg/kg) 10 minutes prior to DAME (1 mg/kg, i.v.)

resulted in a blood pressure of 106±9 mmHg, which is significantly

less than the blood pressure of 146±5 mmHg seen with DAME (1 mg/kg,

i.v.) alone. Intravenous injection of DAME (1 mg/kg) 8 hours after

the last infusion of 17-alpha-estradiol produced an increase in

mean systemic arterial blood pressure of 136±8 mmHg. These results

indicate that 17-alpha-estradiol may function as an opiate

antagonist.

INTRODUCTION

Pinsky et al. (1975) studied the role of endogenous sex steroids in

morphine antinociception. They showed that morphine was ineffective in relieving the stress of mild pain in gonadectomized rats.

However, adrenalectomy alone enhanced morphine antinociception.

The authors suggested that mild pain may mobilize an endogenous

steroid of gonadal origin which acts to reduce the nociception

response and that the adrenals may release a substance that

antagonizes the effects of both the antinociceptive steroid and

morphine. LaBella et al. (1978) showed that a large number of

steroids of the estrane, androstane, pregnane, and etianic-acid

series, including their sulfate and glucuronide conjugates, were

inactive in an opiate radioreceptor assay (displacement of 3Hnaloxone). In their experiments, only 17-alpha-OH estrogens showed

significant affinity for displacement of 3H-naloxone in rat-brain

homogenates.

339
The present study investigated the possibility that the 17-alphaestradiol (a potent competitor for binding of 3

H-naloxone and other

opiate drugs--such as morphine, methionine-enkephalin, and leucineenkephalin--to brain membranes or brain slices [see LaBella 1985])

may function as an opiate antagonist and may antagonize the cardiovascular responses elicited by d-ala2

-met5-enkephalinamide in

conscious, unrestrained rats.

MATERIALS AND METHODS

Male Sprague-Dawley rats weighing 300-350 gm were used in the

study. Under ether anesthesia, the femoral artery and vein were

catheterized with PE-50 tubing in order that blood pressure might

be measured and drugs injected. The PE-50 tubing was guided

through subcutaneous fat on the back and was exited from the

neck. A heparin solution (285 units/ml) was injected into the

tubing to prevent blood clotting. The PE-50 tubing was then

connected to a 21-gauge needle connected to a three-way stopcock

fastened to a transducer, which in turn was connected to a Model 7

Grass Polygraph used to record blood pressure. 17-alpha-estradiol

was dissolved in vehicle (propylene glycol/absolute ethanol/glacial

acetic acid/ascorbic acid; 95/5/0.6/0.01; v/v/v/w). D-ala2-met5-

enkephalinamide (DAME) was dissolved in 0.9% normal saline. Three

groups of animals were used in these experiments.

(1) Rats were given intravenous injections of various doses

of DAME (0.5, 1, 2, and 4 mg/kg).

(2) An intravenous infusion of 17-alpha-estradiol

(1.5 mg/kg) was given every 2 hours for 24 hours (total

infusion time, 2 minutes for each infusion). One hour

after the last infu-sion, a bolus injection of 17-

alpha-estradiol (1.5 mg/kg, i.v.) was given 10 minutes

prior to DAME (1 mg/kg, i.v.). Eight hours later, the

injection of DAME (1 mg/kg, i.v.) was repeated.

(3) An intravenous infusion of vehicle was given at the

rate of 0.1 ml every 2 hours for 24 hours (total

infusion time, 2 minutes per infusion). One hour after

the last infusion, a bolus injection of vehicle

(0.1 ml, i.v.) was given 10 minutes prior to DAME

(1 mg/kg, i.v.). Eight hours later, the injection of

DAME (1 mg/kg, i.v.) was repeated.

Statistical evaluation of the data was performed by ANOVA and by

the Student Newman-Keul test for multiple comparisons of means

(Zar 1974).

RESULTS AND DISCUSSION

Intravenous injection of d-ala2-met5-enkephalinamide (DAME,

primarily a delta-receptor agonist) at 0.5, 1, 2, and 4 mg/kg

caused a dose-related increase in mean arterial blood pressure

(fig. 1) with a duration of 0.98±0.14, 2.25±0.42, 1.90±0.72, and

9.17±0.79 minutes, respectively. However, the heart exhibited

bradycardia (165±15, 150±16, 146±14, 135±15). 17-alpha-estradiol

340
has been found not to have biological activity in target tissues

responsive to estrogenic hormones, such as 17-beta-estradiol

(Ruediger et al. 1983; Foy and Teyler 1983; Pert et al. 1973).

It was also shown that in a family of steroids, only 17-alphaestradiol was effective in displacing 3

H-naloxone in rat-brain

homogenates (LaBella et al. 1978, 1985). This observation is

consistent with the idea that 17-alpha-estradiol might have opioid

antagonist properties. Intravenous infusion of 17-alpha-estradiol,

1.5 mg/kg (infused every 2 hours for 24 hours; total infusion time

of 2 minutes for each infusion), did not significantly alter blood

pressure and heart rate in the conscious rat (fig. 2). One hour

after the last infusion of 17-alpha-estradiol (1.5 mg/kg, i.v.), a

bolus injection of 17-alpha-estradiol (1.5 mg/kg, i.v.) was given

10 minutes prior to DAME (1 mg/kg, i.v.). The hypertension

produced by DAME was significantly inhibited. Eight hours later,

the injection of DAME (1 mg/kg, i.v.) produced a mean arterial

blood pressure of 136±9 mmHg, which was significantly higher than

the blood pressure response to DAME (106±9 mmHg) given 10 minutes

after 17-alpha-estradiol (fig. 3). An interesting observation was

that a bolus injection of 17-alpha-estradiol alone could significantly decrease the mean arterial blood pressure response

(78±5 mmHg) with a duration of 6.00±0.41 minutes. This is in

contrast to the lack of effect of the same dose of 17-alphaestradiol infused over two minutes. Moreover, 17-alpha-estradiol

produced sedation after the infusion of the steroid or after a

bolus injection. It has been reported that the gonadal steroid

progesterone and the mineralocorticoid deoxycorticosterone, as well

as several of their metabolites, caused sedation and anesthesia in

the rat (Atkinson et al. 1965). Recently, Majewska et al. (1986)

found that reduced metabolites of progesterone and deoxycorticosterone are potent modulators of the GABA-receptor complex in crude

synapto-somal membranes from rat brain and that these metabolites

interact with the receptor complex in a manner similar to that of

the barbiturates. In our results, 17-alpha-estradiol antagonized

the hypertension resulting from intravenous injections of DAME,

suggesting that 17-alpha-estradiol may function as an endogenous

opioid antagonist.

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Prentice-Hall, Inc., 1974.

341
FIGURE 1. The effect of intravenous injection of d-ala2-met5-enkephalinamide (DAME)

on the blood pressure of conscious, unrestrained rats.

**P < 0.01 compared to the saline injection. n-=-number of animals.

FIGURE 2. The effect of intravenous infusion of 17-alpha-estradiol. 1.5mg/kg

(infused every 2 hours for 24 hours; total infusion time, 2 min. for each in usion) f

on the blood pressure and heart rate response of conscious. unrestrained rats. There

was no significant difference in the blood pressure and heart rate during the 24-hours.

FIGURE 3. The effect of intravenous infusion of 17-alpha-estradiol, 1.5mg/kg (infused

every 2 hours for 24 hours; total infusion time. 2 min. for each Infusion) on the mean

blood pressure response to DAME (1mg/kg. i.v.) of conscious, unrestrained rats.

**P < 0.01 compared to control. n = number of animals.

342
STRESS-INDUCED-ANTINOCICEPTION AND MORPHINE SENSITIVITY

IN MICE FOLLOWING CHRONIC TREATMENT WITH ANABOLIC/

ANDROGENIC STEROIDS

David A. Cowan, Susan Friend, Alan Gibson,

Michael Ginsburg, and Stephen L. Hart

Drug Control Centre, and Department of Pharmacology,

Kings College London (KQC), Manresa Road, Londn SW3 6LX UK.

ABSTRACT

In male mice treated chronically with anabolic/androgenic teroids,

morphine produced changes in exploratory behaviour similar to those

normally associated with anxiolytic drugs. In addition, steroid

treatment attenuated opioid-mediated stress-induced antinociception.

INTRODUCTION

It has been recognised for some time that opioid drugs disturb normal endocrine function (Meites 1980). In the case of male gonadal

hormones, opioid agonists reduce circulating levels of testosterone

by inhibiting the release of hypothalamic LHRH (Cicero 1980). Conversely, administration of opioid antagonists elevates plasma LH and

testosterone. These findings, together with the detection of opioidcontaining neurones in the hypothalamus, have led to the proposal

that endogenous opioids are involved in the negative feedback control of LHRH release exerted by circulating testosterone (Cicero et al.

1982). It follows that administration of male sex hormones might

influence endogenous opioid function in brain. In this paper, we

report that chronic treatment of mice with anabolic/androgenic steroids alters some behavioural responses to opioids.

MATERIALS AND METHODS

Male mice (LACA strain ; 20 - 25 g) were used throughout. Steroids

were given by weekly i.m. injections as slow-release preparations

in ethyl oleate. Controls received appropriate volumes of ethyl

oleate alone.

Antinociception

Testosterone, methyl-testosterone, or methenolone (each 0.4 mg/Kg)

were given for 4 weeks. 3 - 4 days after each steroid injection

morphine-induced antinociception (20 mg/Kg, i.p.) was assessed on

a hot-plate (54°C).

Motor activity

Methyl-testosterone or methenolone (5, 10, and 20 mg/Kg) were given

for 6 weeks. On the seventh week, motor activity was assessed 24 hr

before and 30 min after 20 mg/Kg morphine i.p. Animals were injected

with saline or morphine 30 min prior to being placed in an activity

cage. After a 10 min settling in period X - Y motor activity was

counted for 10 min.

Exploratory behaviour

Methyl-testosterone or methenolone (5 mg/Kg) were given for 6 weeks.

On the seventh week, exploratory behaviour was assessed 24 hr before

and 30 min after 20 mg/Kg morphine i.p. Animals were given saline

343
or morphine 30 min before being placed in a box maze (Davies and

Wallace 1976). After a 1 min settling in period exploratory behaviour was counted for 3 min. Parameters measured were, dipping (pushing head down hole in middle of box section), rearing (placing both

front paws on dividing walls), and crossing (moving from one box

section to the next).

Stress-induced antinociception (SIA)

Methyl-testosterone or methenolone (1 and 5 mg/Kg) were given for 6

weeks. On the seventh week, SIA was assessed by recording reaction

time on a hot-plate (54°C) before and 2 min after a 3 min swim at

20°C (Hart et al. 1983).

RESULTS

Antinociception

In naive mice, morphine increased hot-plate reaction time by 35 sec.

In control mice, treated with ethyl oleate, there was a gradual decrease in morphine-induced antinociception such that, by the fourth

week, there was no significant increase in reaction time; this was

presumably due to tolerance induced by the weekly exposure to morphine for the antinociceptive test. A similar pattern was shown by mice

treated with the steroids, there being no significant difference

among the groups.

Motor activity

In control mice, morphine increased motor activity from 192 ± 10 to

252 ± 15 counts/10 min (mean ± s.e.). Methyl-testosterone treatment

per se did not affect motor activity, but it abolished the stimulatory effect of morphine. A similar, but less pronounced, inhibition of

morphine-induced motor activity was observed in methenolone treated

mice.

Exploratory behaviour

The results from these experiments are shown in figure 1. In control

mice, morphine reduced the number of dips, but did not affect rearing

or crossing. Morphine also reduced the number of dips in mice treated with methyl-testosterone or methenolone. Further, in methyltestosterone treated mice, morphine reduced the number of rears,

while in methenolone treated mice morphine increased the number of

crossings.

SIA

These results are given in table 1. In ethyl oleate treated mice

the initial median reaction time on the hot-plate (T1) was 12 sec;

after a 3 min swim the reaction time (T2) was increased by 9 sec.

Treatment with methyl-testosterone or methenolone did not alter

the initial reaction time; however, in methyl-testosterone treated mice (5 mg/Kg) the increased reaction time following a swim

was attenuated. Methenolone (5 mg/Kg) also reduced the prolongation of reaction time after a swim, but in this case the reduction

was just outside the levels of significance (0.01>P>0.05).

DISCUSSION

The results clearly indicate that chronic administration of

344
FIGURE 1. Histogram showing (mean ± s.e.) exploratory

behaviour of mice on a box maze for 3 min. Open columns -

mice given 6 weekly injections as indicated (steroids at

5 mg/Kg) ; hatched columns - same mice 30 min after 20 mg/Kg

morphine. * P<0.05

anabolic/androgenic steroids alters the behavioural responses of mice

to opioids. Two results are of particular interest. First, on the

box maze morphine increased the number of crossings in mice treated

with methenolone. Few drugs have been shown to have this effect, and

the common property of those that do seems to be anxiolytic activity

(J. K. Davies personal communication). The difference in morphine

activity in methyl-testosterone and methenolone treated mice on the

box maze may be a reflection of the different anabolic/androgenic

ratios of the two steroids. Secondly, methyl-testosterone and to a

lesser extent methenolone decreased SIA in a model which is known

345
Median Reaction Times

Saline

Ethyl Oleate

Methyl Testosterone 1mg/Kg

5mg/Kg

Methenolone 1mg/Kg

5mg/Kg

T 2 - T 1

(12) T 1 +10

(12) 12 + 9

(6) 12 + 9

(12) 11 +2.5*

(12) 9.5 +12

(12) 12.5 +6.5

TABLE 1. Median reaction times (sec) of mice on a hotplate. T1 was measured after 6 weekly injections of the

treatment indicated. T2 was measured 2 min after a 3 min

swim. The increased reaction time (T2 - T1) represents SIA.

Numbers in parentheses give number of mice.

* P<0.05

to involve the release of endogenous opioids (Hart et al. 1983).

Thus, steroid administration not only influences responses to exogenous opioids but also behavioural activity resulting from endogenous

opioid function. Both these observations may have relevance to one

group of individuals known to take regular doses of anabolic steroids; that is athletes involved in strength-related events (Goldman

1984). In such athletes, the interaction between male sex hormones

and opioids may be of importance during competition since both stress and exercise are potent stimuli for endogenous opioid release

(Gibson et al. 1979 ; Gambert et al. 1981).

REFERENCES

Cicero, T.J. Fed Proc, 39:2551-2554, 1980.

Cicero, T.J., Meyer, E.R., and Schmoeker, P.F. J Pharmac exp

Ther, 223:784-789, 1982.

Davies, J.K., and Wallace, P. Br J Pharmac, 57:474P, 1976.

Gambert, S.R., Garthwaite, T.L., Pontzer C.H., Cook, E.E.,

Tristam, F.E., Duthie, E.H., Martinson, D.R., Hagan, T.C., and

McCarty, D.J. Proc Soc exp Biol Med, 168:1-4, 1981.

Gibson, A., Ginsburg, M., Hall, M., and Hart, S.L. Br J Pharmac,

65:139-146, 1979.

Goldman, R. Death in the Locker Room London, Century Publishing,

1984.

Hart, S.L., Slusarczyk, H., and Smith, T.W. Eur J Pharmac, 95:

283-285, 1983.

Meites, J. Fed Proc, 39:2531-2560, 1980.

ACKNOWLEDGEMENTS

This work was supported by The Sports Council

346
OPIOIDS AND E-RECEPTORS INTERACTION: FURTHER STUDIES IN

CULTURED CELLS

Angelo Reggiani, Angelo Carenzi and Davide Della Bella

Zambon Farmaceutici Research Laboratories

Via Lillo Del Duca 10, 20091 Bresso-Milan, Italy

ABSTRACT

In the present paper a further evidence of a functional link between

opioids and ß-receptor system in cultured rat C6 cells is presented.

We showed that C6 cell exposure to Desmethylimipramine (DMI) causes

expression of opioid receptors and loss of ß-receptors. We now

show that C6 cells exposure to DMI causes an increase of intracellular levels of opioid peptides and an increase of aminopeptidase

activity. In addition, the presence of the aminopeptidase inhibitor

bestatin during cell exposure inhibits DMI-induced R-receptor loss.

These results indicate that cell exposure to DMI causes the full

expression of the opioid peptide system. This effect could be

related to changes of ß-receptor system.

INTRODUCTION

It has been recently found that C6 cell exposure to DMI causes

expression of cell membrane opioid receptors (Tocque et al. 1984)

and we have recently found that this effect was associated to the

loss of R-receptors (Reggiani et al. in preparation). We also found

that the simultaneous presence of opioids during DMI-exposure

prevents ß-receptors down regulation and a similar modulation

can be demonstrated on isoproterenol (ISO) induced desensitization

in DMI-exposed cells. All these results suggest that, besides the

actual mechanism of inducing ß-receptors loss opioids could operate

to preserve ß-receptor function. In order to investigate whether

DMI-induced opioid receptors appearance was associated to a wider

influence on opioid peptide system experiments were carried out

to study the effect of cell exposure to DMI on the intracellular

synthesis of opioids and on their metabolic degradation. Furthermore, the effect of peptidase inhibitors on DMI induced loss of

ß-receptors was tested in the attempt to verify whether peptidase

inhibitors could mimic the exogenous opioid agonist action.

MATERIALS AND METHODS

Confluent monolayers of rat C6 glioma cells between passage 47th

to 75th were used. Cells were grown in a humidified atmosphere

5% CO2 - 95% air, using EMEM supplemented with 10% FBS and antibiotics as growth medium. 3

H-DHA and 3H-DHM bindings were estimated

as previously described by Fishman et al. (1981) and by Albouz

et al. (1982) respectively. CAMP levels were measured on acid

347
extracts from cell suspension by using the RIA method of binding

protein as recommended by Amersham. Met-Enkephalin (ME) content

was measured on acid extracts from cell suspension by using rabbit

antibody from Amersham (code RPM-1561) and 10 nM 3H-ME as radioactive tracer. The hydrolysis of 3

H-Leu-Enkephalin (

3

H-Le) by

cultured C6 cells was estimated as follows: at the end of the

proper treatment cells were challenged for their ability to hydrolyze peprides by exposing cultures for 30 min to 20 nM LE containing

10.000 cpm 3H-LE. At the end, the medium was collected, boiled

for 3 min and aliquots were applied for TLC separation by comparison

with the proper standard mixtures.

RESULTS AND DISCUSSION

The influence of cell exposure to DMI on the intracellular content

of opioid peptides was investigated by measuring ME content as

probe of the opioid peptide concentration. As shown in table 1

naive cells have an intracellular level of ME comparable to the

lowest estimate in whole brain and in other cultured cell lines

(Gilbert et al. 1982, Glaser et al. 1982, Miller et al. 1978,

Yang et al. 1977, Hughes et al. 1977). 24 hrs of cell exposure

to different concentrations of DMI increased peptide content up

to 5-6 times as much, suggesting that DMI exposure would trigger

the expression of opioid peptides synthesis.

TABLE 1. Effect of C6 cell-exposure to different concentrations of DMI on the intracellular content of ME.

TREATMENT

(24 hrsl [M]

ME-CONTENT

(fmol/mg protein)

Vehicle 277 31

DMI 5 . 10-5 1784 110 *

DMI 5 . 10-6 1055 97 *

DMI 5 . 10-7 630 78 *

* P < 0.05 vs vehicle treated cells

Kesults are the mean ± SE of 3 determinations in duplicate.

C6 exposure to DMI also influenced cell associated neuropeptidase

activity as indicated by the results reported in table 2. According

to these experiments, both in vehicle and in DMI treated cells

most, if all, the neuropeptidase activity was due to an aminopeptidase since the major fragment formed was 3

H-Tyrosine (3H-T).

However, after cell exposure to DMI, the aminopeptidase activity

was increased as indicated by the time dependent decrease in the

348
amount of intact 3

H-LE recovered after cell challenge associated

to the time dependent increase of 3H-T formed.

TABLE 2. Influence of cell exposure to DMI (5 . 10-5M) on cell

associated neuropeptidase activity versus exogenous 3H-LE.

TREATMENT TIME OF EXPOSURE RECOVERY AFTER CHALLENGE

(hrs) 3

H-LE (cpm) 3

H-T(cpm)

Vehicle 8 6549 2663

16 6263 2606

24 6423 2680

DMI 8 5345 3090

16 4969 4270

24 3770 5015

At the end of the treatment cells were challen ed for 30 min

with 20 nM LE containing 10.000 cpm of intact 3H-LE.

Among the neuropeptidase profile of the various peptidase inhibitors

tested, only bestatin was found effective in interfering on DMI

induced R-receptors loss of density and function. Table 3 summarizes

the effects of bestatin on DMI induced decline of either O-receptors

density or ISO stimulated CAMP accumulation in intact cells. In

both cases the aminopeptidase inhibitor protected against DMI

induced effects suggesting that the newly synthesized peptides

mimic the action of exogenous peptides on ß-receptors system.

TABLE 3. Effect of bestatin on DMI induced decrease of R-receptors density and ISO-stimulated CAMP accumulation in intact C6 cells.

TREATMENT CAMP a)

(24 hrs) (pmol/mg protein)

3

H-DHA BOUND Bmax

(fmol/mg protein)

Vehicle 2157 169 255 41

DMI 957 181 * 123 23 *

DMI+bestatin 1691 171 ns 221 35 ns

Bestatin 2258 183 ns 241 47 ns

* P < 0.05 vs vehicle ns: not significant vs vehicle

a) CAMP after 10 min of challenge with ISO 10-6M

DMI concentration was 50 μM, bestatin was 1O μM

Results are the mean ± SE of 3 determinations in duplicate.

349
At the present time it is not known which is the actual biochemical

mechanism involved in the reported effects of cell exposure to

DMI. However, all these results could suggest that a common

mechanism would activate opioid receptors' appearance, opioid

peptide synthesis and aminopeptidase activity. Since the inhibition

of endogenous opioid peptide degradation by bestatin prevents DMI

induced B-receptor loss it can be speculated that the opioid system

activation could be related to specific changes in B-receptor

function.

ACKNOWLEDGEMENTS

This work was partially supported by Consiglio Nazionale delle

Ricerche, Progetto Finalizzato Chimica Fine e Secondaria,

REFERENCES

Albouz, S., Tocque, B., Hauw, J.J., Boutry, J.M., LeSaux, F.,

Bourdon, R., and Baumann, N. Life Sci, 31:2549-2554, 1982.

Fishmann, P.H., Mallorga, P., and Talman, J.P. Mol Pharmacol,

20:310-318, 1981.

Glaser, T., Hübner, K., and Hamprecht, B. J Neurochem, 39:59-69,

1982.

Gilbert, J.A., Knodel, E.L., Stenstrom, S.D., and Richelson, E.

J Biol Chem, 257:1274-1281, 1982.

Hughes, J., Kosterlitz, H.W., and Smith, T.W. Br J Pharmacol,

61:639-647, 1977.

Miller, R.J., Chang, K.-J., Cooper, B., and Cuatrecasas, P. J Biol

Chem, 253:531-538, 1978.

Tocque, B., Albouz, S., Boutry, J.M., LeSaux, F., Hauw, J. J.,

Bourdon, R., Baumann, N., and Zalc, B. J Neurochem, 42:1101-1106,

1984.

Yang, H.-Y., Hong, J.S., and Costa, E. Neuropharmacology, 16:303-

307, 1977.

350
MU/DELTA OPIOID SITE SELECTIVITY OF SOME ANTIDEPRESSANTS

Ellen E. Codd and Richard F. Walker

Department of Reproductive and Developmental Toxicology

Smith Kline and French Laboratories

1500 Spring Garden Street

Philadelphia, PA 19101, USA

ABSTRACT

Since pain and depression are influenced by opioids and

antidepressants, it was of Interest to explore their interaction

with common CNS receptors. A method for binding site selectivity

was used to evaluate antidepressant specificities in inhibiting mu

versus delta opioid binding. With the very specific opioid probes

used, DAGO and DPDPE, a wide range of antidepressant selectivities

was found. These may represent different modes of interaction

between mu and delta opioid sites and antidepressant compounds.

INTRODUCTION

Chronic pain and depression are frequently associated, but the

mechanisms underlying the efficacy of antidepressants in the

management of pain syndromes are not clearly understood. The

effectiveness of these drugs in pain relief may be secondary to

their antidepressant activity, or may result from their own

intrinsic analgesic activity. Since certain antidepressants inhibit

opioid binding in vitro (Biegon and Samuel 1980; Somoza et al. 1981;

Baraldi et al. 1983; Isenberg and Cicero 1984), it was of interest

to examine the mu/delta opioid specificity of the interaction. The

site selectivity method (Bárány 1973, Terenius and Wahlström 1976)

was used to circumvent the potentially confounding effects of

al losteric interactions between μ and opioid binding sites

(Vaught et al. 1981, 1982; Rothman and Westfall 1982a and b).

MATERIALS AND METHODS

Male Sprague-Dawley rats (2-3 months of age) were decapitated and

their brains removed. The forebrains were homogenized in 50 mM

Tris-HCl (pH 7.4 at 25°C) in a teflon-glass homogenizer. The

homogenate was centrifuged at 27,000 x g for 15 min and the pellet

resuspended in Tris buffer (100 ml buffer/g brain tissue). One ml

of this crude membrane fraction was used per assay tube; the final

assay volume was 1.1 ml. Incubations (25°C, 1 hr) were terminated

by filtration over Whatman GF/B filters with a Brandel cell

harvester. Tubes and filters were rinsed 3 X 4 ml buffer.

Radioactivity was counted in Protosol/Econofluor (New England

Nuclear).

The experimental design was based on the site selectively method

(Barany 1973; Terenius and Wahlström 1976). DAGO and DPDPE were

used as indicator ligands for μ and opioid binding sites

respectively. In each of two test solutions, both ligands were

present at about their Kd values (previously determined to be:

351
DAGO, 0.4 nM; DPDPE 1.8 nM). In one solution DAGO was in the

tritiated form, while DPDPE was radiolabeled in the other. A range

of concentrations of the antidepressants examined was incubated in a

series of tubes under each test condition. Controls for

nonsaturable binding contained nonradlolabeled indicator ligand at a

concentration of 0.1 μM. Binding of the indicator ligand in the

presence of antidepressant was calculated as a percent of that

observed when no test drug was present. The results were graphed

according to Bárány (1973) and Terenius and Wahlström (1976).

Tritiated DAGO and DPDPE were from New England Nuclear, while the

nonradiolabeled peptides were from Peninsula Labs. Imipramine and

desipramine were from Sigma. Other antidepressants were kindly

donated: fluoxetine by Eli Lilly, mianserin by Organon,

amitriptyline by Merck, chlorimipramine by CIBA-GEIGY and

nomifensine by Hoechst.

RESULTS

Previous work has demonstrated the excellent specificity of DAGO and

DPDPE for μ and opioid binding sites, respectively (Mosberg et

al. 1983; Paterson et al. 1984). In the present study, nonradiolabeled DAGO and DPDPE were also highly selective for the sites

labeled by their tritiated analogs (fig. 1). DAGO inhibited only 5%

of 3H-DPDPE binding at a concentration which blocked 85% of

3

H-DAGO binding, while DPDPE prevented 98% of 3

H-DPDPE binding

at a concentration which blocked only 5% of 3H-DAGO binding.

FIGURE 1. Relative inhibition of 3

H-DAGO and 3H-DPDPE

binding by nonradioableled DAGO and DPDPE (1 nM - 0.1 mM).

Each point shown represents the mean of triplicate

determinations of specific binding.

352
The mu/delta site selectivity of several antidepressants was

evaluated in this test system and a range of selectivities was

observed (fig. 2). Amitriptyline and mianserin competed equally well

for DAGO and DPDPE binding sites; the graph of their inhibition data

fell on the diagonal, indicating no preference for mu versus delta

opioid binding sites. In contrast, fluoxetine had a 10-fold

selectivity for sites labeled by the delta site ligand, DPDPE since

its data points fell well above the diagonal. The other

antidepressants tested Including imipramine, nomifensine,

chlorimipramine and desipramine, had greater selectivities for mu

binding sites since their competition data points were below the

diagonal line (fig. 2). Thus, although all the antidepressants

tested inhibited opioid binding in the range of concentrations

tested, the drugs varied greatly in their preference for mu versus

delta binding sites.

FIGURE 2. Relative inhibition of 3

H-DAGO and 3H-DPDPE

binding by some antidepressants. - fluoxetine. 1- 300 PM,

- amitrptyline, 1 - 1000 μM; - mianserin 6 - 100 PM;

- nomifensine, 10 - 1000 μM; - chlorimipramine, 30 -

1000 μM; - imipramine, 0.1 - 1000 PM; - desipramine.

0.1 - 1000 μM. The values shown are the means of triplicate

determinations of specific binding.

353
DISCUSSION

Modulatory interactions between mu and delta ligands have been

demonstrated in analgesic responses (Vaught and Takemori 1979; Lee

et al. 1980; Vaught et al. 1982) and receptor binding (Vaught et al.

1981; Rothman and Westfall 1982a and b). Because of the proposed

allosteric interactions between mu and delta opioid binding sites

(Rothman and Westfall 1982a,b; Vaught et al. 1982). the site selectivity method was chosen to investigate antidepressant preference

for opioid receptor subtypes. Because the molecular constituents of

the test solutions used in this method are identical, their effects

on the receptors will be identical (Terenius and Wahlström 1976).

Under such conditions, DAGO and DPDPE exhibited a high specificity

for their respective binding sites (fig. 1). An almost 100-fold

concentration range exists for each ligand within which there is

virtually no overlap in ligand binding between sites. Since the

Kd of each ligand is well within that concentration range, the

site selectively method is a powerful analytical tool when DAGO and

DPDPE are used as the indicator ligands at their Kd concentrations.

A range of selectivities was found among the antidepressants

tested. Patterns of selectivity included delta preferring

(fluoxetine), mu preferring (desipramine, imipramine, chlorimipramine, nomifensine) and unselective (amitriptyline,

mianserin). A similar range of preferences was observed by Isenberg

and Cicero (1984) using dihydromorphine and DADL as mu and delta

ligands. respectively. Presumably the use of more selective

indicator ligands in the present study yielded greater

discrimination of the selectivities of compounds tested.

REFERENCES

Baraldi, M., Poggioli, R., Santi, M., Vergoni, A.V., and Bertolini, A.,

Pharmacol Res Communic, 15:843-857, 1983.

Bbrdny, E.H., Acta Physiol Scand, 88:412-429, 1973.

Biegon, A., and Samuel, D., Biochem Pharmacol, 29:460-462, 1980.

Isenberg, K.E., and Cicero, T.J. Eur J Pharmacol, 103:57-63, 1984.

Lee, N.M., Leybin, L., Chang, J.-K.,and Loh, H.H., Eur J Pharmacol,

68:181-185, 1980.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee.K., Yamamura, H.I., Galligan, J.J.,

and Burks, T.F., Proc Natl Acad Sci USA, 80:5871-5874, 1983.

Paterson, S.J., Corbett, A.D., Gillan, M.G.C., Kosterlitz, H.W., McKnight,

A.T., and Robson, L.E., J Receptor Res, 4:143-154, 1984.

Rothman, R.B., and Westfall, T.C., Molec Pharmacol, 21:538-547, 1982a.

Rothman, R.B., and Westfall, T.C., Molec Pharmacol, 21:548-557, 1982b.

Somoza, E., Galindo,A., Bazan, E., Guillamon, A., Valencia, A., and Fuentes,

J.-A., Neuropsychobiol, 7:297-301, 1981.

Terenius, L., and Wahlström, A., Eur J Pharmacol, 40:241-248, 1976.

Vaught, J.L., and Takemori, A.E., J Pharmacol Exptl Ther, 211:280-283,

1979.

Vaught, J.L., Kitano,T., and Takemori, A.E., Molec Pharmacol, 19:236-

241, 1981.

Vaught, J.L., Rothman, R.B., and Westfall, T.C., Life Sci, 30:1443-1455,

1982.

354
MOLECULAR MECHANISMS OF LIGAND BINDING TO OPIOID RECEPTORS:

SELECTIVE INHIBITION BY ETHANOL ?

Rao S. Rapaka, Hemendra. N. Bhargava*, Venkatesan

Renugopalakrishnan**, and Bradley J. Collins***

Division of Preclinical Research , National Institute on Drug

Abuse, Room 10A-13, Rockville, MD 20857

*College of Pharmacy, university of Illinois at Chicago, Health

Sciences Center, Chicago, IL 60612

**Laboratory for the Study of Skeletal Disorders and

Rehabilitation, Harvard Medical School and Children's

Hospital, Boston, MA 02115

***Radian Corporation, Morrisville, NC 27560

ABSTRACT

Effects of varying concentrations of ethanol on the conformations

of DAGO, a D-specific opioid ligand were monitored by FT-IR

spectroscopy. The binding of 3

H-labeled DAGO (u-agonist) and

DSLET ( agonist) to opioid receptors in the presence of varying

concentrations of alcohol was also determined. The results

indicate that the conformational features of the ligands are

altered and conformational factors may play a role in

ligand-receptor interaction.

INTRODUCTION

The concept of multiple receptors for the opioid ligands was

originally proposed by Portoghese (1965). However, this concept

was more widely accepted only after Martin et al. (1976) proposed

that there are at least 3 types of opioid receptors, i.e., μ,

and from their pharmacological experiments. Later Lord et al.

(1977) put forth evidence for a fourth type of receptor,

Presently, there are several methods that differentiate the various

receptor types (Paterson et al. 1984). Hiller et al. 1981 proposed

that alcohol selectively inhibits binding to receptors, as

&receptors are more strongly influenced by the alcohol-induced

membrane fluidity changes than are the μ-receptors. They further

stated that it is likely that alcohol affects the receptor rather

than the peptides (ligands). As drug-receptor interaction involves

rigid stereochemical and structural requirements, and in view of

the importance of the conformational features for drug-receptor

interaction, we have previously investigated the effects of ethanol

on the secondary structural features of Met5-enkephalinamide

(Rapaka et al. 1986). We proposed that conformational features of

the ligand may also play a role in drug-receptor interaction. We

have presently extended these studies to both μ and specific

opioid peptide ligands.

MATERIALS AND METHODS

DAGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) and DSLET (Tyr-D-Ser-Gly-PheLeu-Thr-OH) were purchased from Bachem Inc., Switzerland.

355
3

H DAGO was purchased from Amersham, Boston, MA, and 3H DSLET

was purchased from New England Nuclear Corporation. FT-IR

Spectroscopy was performed as reported by Rapaka et al. (1986).

Rat brain membrane preparation was prepared as reported by Bhargava

and Das (1986).

RESULTS AND DISCUSSION

FT-IR spectra of DAGO were obtained at room temperature with 0%.

2%, and 5% (v/v) ethanol in water. The background subtracted

spectra are presented in figure 1. In water, DAGO manifests a

doublet amide I band with an intense band at 1651 cm-l and a less

intense band at 1636 cm-1 with a shoulder around 1670-1680

cm

-1

. From the conformational dependence of IR amide bands

(Renugopalakrishnan et al. 1986; Byler et al. 1986), the intense

band at 1651 cm-1 is assigned to either -helical or "unordered"

structure, the 1636 cm-1 band to ß-sheet structure, and the broad

shoulder at 1670-1680 cm-1 to ß-turn structure. The occurrence

of helical structure seems less likely, due to the short peptide

backbone of DAGO. Hence, DAGO is expected, based on IR studies, to

exist in a conformational equilibrium involving ß-turn, ß-sheet,

and "unordered structures. In 2% aqueous ethanol, the pattern of

IR bands remains unchanged but the intensities of the bands are

slightly diminished. At higher concentrations of ethanol, i. e.,

5% (v/v), the resolution-enhanced amide I region is simplified by

the occurrence of two bands, an intense band at 1635 cm-1 and a

less intense band at 1660 cm-l. Although the spectrum is

simplified, further studies are required to clarify the details of

assignment. In order to correlate the ethanol-induced changes in

conformation with receptor binding parameters, binding of the

tritiated ligands to receptors in the rat brain membrane

preparation was studied. The effect of alcohol on binding of the

tritiated ligands to the rat brain membrane preparation is

presented in table 1.

TABLE 1

Effects of Varying Concentrations of Alcohol on the Binding

of 3H-Labeled Opioid Peptides to Opioid Receptors.

Alcohol Specific Binding

Concentration 3

H DAGO

% v/v % Control

Specific Binding

of 3

H DSLET

% Control

Control 100 100

1 63.1 89.6

2 59.0 83.0

3 54.6 70.8

5 43.1 48.5

In our earlier studies (Rapaka et al. 1986), we investigated the

conformational changes of Met5-enkephalinamide upon the addition

of different concentrations of ethyl alcohol to an aqueous solution

356
of Met5

-enkephalinamide. Met5-enkephalinamide is an ideal

molecule for these studies, as it exists in both ß-turn and ß-sheet

conformations. FT-IR is ideally suited to follow the

conformational transitions as changes in both ß-turn and ß-sheet

conformations can be monitored simultaneously and the technique is

simple and fast. Hence, in our continuing studies FT-IR was the

method of choice.

Schiller and colleagues (Schiller and DiMaio 1980; DiMaio and

Schiller 1980; DiMaio et al. 1982), from their studies on cyclic

enkephalin analogs and the corresponding linear analogs, proposed

that the opioid receptor subtypes differ in their conformational

requirements of the ligand. Later several groups proposed that

indeed the conformational features such as the ß-turns, ß-sheets,

or other extended structures may play a role in receptor subtype

recognition (for a review see Rapaka et al. 1985). Ethanol is

Figure. 1. Resolution-enhanced FT-IR spectra in the

amide I region of DAGO in water (3 mM) at room

temperature and in the presence of ethanol. (A) without

ethanol; (B) with 5% ethanol.

357
known to induce conformational changes in polypeptides and proteins

(Mishra and Ahluwalia 1983; Conio et al. 1970). Although Hiller et

al. (1981) believe that alcohol may not affect the peptide, but

rather the receptor, it appears likely that the effect may be

primarily on the ligand. The ligands are usually small molecules

and, especially, the enkephalins and their analogs are only

pentapeptides and are known to exist in a conformational

equilibrium. The FT-IR results indicate that both DAGO and

Met5-enkephalinamide exist in a conformational equilibrium

involving ß-turns and ß-sheets. Addition of alcohol destabilizes

these structures. Binding studies indicate that binding to both μand ligands is inhibited in the presence of alcohol. We believe,

the inhibition of binding by alcohol is not specific to ligands

only. Finally, it is also possible that alcohol may induce

conformational changes in both the ligand and the receptor. Hence,

the inhibition of binding may be due to loss of conformational

features of the ligand or the receptor or both. Further studies

are in progress.

REFERENCES

Bhargava, H.N., and Das, S. Life Sci, 1986 In Press.

Byler, D.M., Brouillette, J.N. and Susi, H. Spectroscopy, 1:29-32,

1986.

Conio, G., Patrone, E., and Brighetti, S. J Biol Chem, 245:

335-3360, 1970.

DiMaio, J., and Schiller, P.W. Proc Natl Acad Sci USA,

77:7162-7166, 1980.

DiMaio, J., Nguyen, T.M.-D., Lemieux, C., and Schiller, P.W.

J Med Chem, 25: 1432-1438, 1982.

Hiller, J.M., Angel, L.M., and Simon, E.J. Science, 214:468-469,

1981.

Lord, J.A. H., Waterfield, A.A., Hughes, J., and Kosterlitz, H.W.

Nature, 267:495-499, 1977.

Martin, W.R., Eades. C.G., Thomson, J.A., Huppler, R.E., and

Gilbert, P.E. J Pharmacol Exp Ther, 197:517-532, 1976.

Mishra, A.K., and Ahluwalia, J.C. Int J Peptide Protein Res, 21:

322-330, 1983.

Paterson, S.J., Robson, L.E., and Kosterlitz, H.W. In: Udenfriend,

S., and Meienhofer, J., eds. The Peptides: Analysis, Synthesis,

Biology, Vol. 6, Opioid Peptides: Biology, Chemistry, and

Genetics. New York: Academic Press, 1984, pp. 147-189.

Portoghese, P.S. J Med Chem, 8:609-616, 1965.

Rapaka, R.S., Renugopalakrishnan, V., and Bhatnagar, R.S. Am

Biotech Lab, 3:8-21, 1985.

Rapaka, R.S., Renugopalakrishnan, V., Goehl, T.J., and Collins,

B.J. Life Sci, 1986, In Press.

Renugopalakrishnan, V., Strawich, E.S., and Glimcher, M.S.

Biochemistry, 1986, In Press.

Schiller, P.W., and DiMaio, J. Nature, 297:74-76, 1982.

Schiller, P.W. Conformational analysis of opioid peptides and the

use of conformational restriction in the design of selective

Analogs, In: Rao S. Rapaka et al. eds. Opioid Peptides: Medicinal

Chemistry. National Institute on Drug Abuse Research Monograph

69. Washington. D.C.: Supt. of DoCS., U.S. Govt. Print. Off.,

1986, In Press.

358
OPIOIDS AND INTAKE OF ALCOHOLIC BEVERAGES

Larry D. Reid, Stephanie A. Czirr, William C. Milano,

Christopher L. Hubbell and Nicholas A. Manha

R.P.I., Troy, New York 12180, U.S.A.

ABSTRACT

Rats were given a daily opportunity to consume a

sweetened ethanol solution (ES) or water until they took

about 2.0 g/kg of pure ethanol (E) during 1.5 hr/day.

Then, rats were given one of a number of opioids before

an opportunity to drink. Some opioids, e.g., morphine

(MOR), methadone, and fentanyl, at small doses, increase

ES-intake; some decrease intakes, e.g., naloxone (NX) and

MR 2266. Diprenorphine, a mixed agonist-antagonist

having no analgesic potential and blocking other opioids'

analgesia, increases ES-intake across a wide range of

doses beginning with 0.003 mg/kg.

INTRODUCTION

From older studies of a time when people could legally

buy opium as well as alcohol (Seigel 1985), to studies of

E's metabolism (Davis and Walsh 1970, Cohen and Collins

1970) and modern studies of

Hiller et al. 1981, Tabakoff 1981), a relationship

receptor-dynamics (e.g.,

between processes of opioids and E have been proposed.

Not all E's effects are mediated by way of opioid

receptors, since naloxone (NX) has limited or no effects

on many E-induced events (Severs 1970). E has, however,

many effects and endogenous opioids are multifaceted. It

is unknown, however, whether or not all effects of E are

unrelated to opioid-mechanisms. Since, there are no

known E-receptors, E may achieve its ability to sustain

its own use by way of opioid-mechanisms. If this is the

case, at least some opioids should increase and NX should

decrease E-intake. Large doses of morphine (MOR),

however, decreased E-intake (Sinclair 1974) and NX

reduces intake of many ingesta including ESs (Reid 1985).

So, these early studies of opioids' effects on E-intake

did not support the idea that opioids had specific

effects on taking ES.

A problem with testing drugs' effects on E-intake is that

rats do not readily take meaningful amounts of ESs across

the brief time that relevant doses of drugs might be

effective. To overcome this, a sweetened ES was used and

it was found that rats eventually take, over days,

substantial amounts of E during brief daily opportunities

to do so. After rats were taking considerable ES, small

doses of MOR (e.g., 2 mg/kg) were tested and they

produced marked increments in E-intake. Studies of the

small-dose-MOR-effect indicate that (a) the increment in

intake is not related to flavoring of ES, (b) it does not

359
tolerate, and (c) it is not dependent on variables such

as housing conditions, age, or sex of the subjects

(Hubbell et al. 1985, Reid et al. 1986). ES-intake was

also shown to be more sensitive to NX than water-intake.

So, the basic theoretical requirement of an agonist

increasing and an antagonist decreasing E-intake was met.

Also, benzodiazepines and a barbiturate did not produce

similar effects and a dopamine receptor antagonist did

not modify the intake of an ES (Mudar et al. 1986).

These initial results led us to test other opioids and

some of those tests are reported here.

MATERIALS AND METHOD

Male, Sprague-Dawley rats were placed on a daily regimen

for more than 21 days before testing. The daily regimen

involved water-deprivation until the time of presentation

of ES and water for 1.5 hr/day (food was always

available). ES was 5 g sucrose, 12 g pure E and water

added to yield 100 g of solution. Across days, rats

gained weight as those given unlimited water. So, these

healthy rats can take or not take ES. At first, rats

take very little, but, across days they take more and

more until they take about 2.0 g/kg of E during the 1.5

hr-period (an amount often producing visible

drunkenness).

After stable intakes are achieved, rats were given

placebo, and then the next day, an opioid. Subsequent to

a test of one dose, a number of days intervened and then

another placebo and dose were tested. Rats were given a

limited number of doses before being retired to avoid

carryover-effects; although in direct tests for

carryover, we have yet to observe any as long as 2 days

intervened between tests. Each dose was tested with 10

or more rats. Injections were given subcutaneously, 15

min before a test. Doses range from very small to those

large enough to interfere with drinking of any kind.

Consequently, the largest doses of all opioids are

expected to reduce intakes.

A number of indices of rats' intake of E are possible,

e.g., (a) preference ratio, i.e., the amount of ES

taken/total fluid-intake, or (b) g of pure E taken/kg of

body weight (g/kg). Since these two measures were highly

correlated, only g/kg are reported in detail here, but

preference ratios are mentioned when they give a

different perspective. The mean difference between

intake under placebo and a dose is determined and

dependent t-tests calculated.

RESULTS AND DISCUSSION

Small doses of MOR as well as small doses of methadone

and fentanyl increase intake of ES. With large doses of

MOR, preference ratios are often increased since large

360
doses reduce intake of water more than ES. These agents

can be analgesics and are readily self-administered. Not

all opioid analgesics, however, increase E-intake, e.g.,

ethylketocyclazocine (EKC) did not, except in a narrow

range of doses. Also, levorphanol and dextrorphan both

increase E-intake at small doses. NX reduces E-intake.

When MOR and NX are given concurrently, the resultant Eintake is as if NX alone was given. MR2266, in doses

similar to NX. decreases intake.

Diprenorphine is not an analgesic and blocks other

opioids' analgesia. Diprenorphine, however, increments

pressing for rewarding brain stimulation and establishes

a conditioned place preference, both of which are indices

of an opioids' ability to induce positive affect (Beaman

et al. 1984, Pollerberg et al. 1983). Diprenorphine

across a wide range of doses increases E-intake and, if

anything, reduces water-intake. When diprenorphine and

NX are given concurrently, the effect is that of NX.

FIGURE 1. Mean difference scores are depicted (drug-placebo),

so a positive score indicates the dose produced that increment

in g/kg of E-intake. Only the value for 0.2 mg/kg for levorphanol tartrate (LEV) and 0.2 and 2.0 mg/kg dextrorphan tartrate (DEX) represent reliable (p < 0.05) differences. Only

the value for 0.1 for (-)Ethylketocyclazocine (EKC) is reliably

different than placebo-scores of all the values plotted in the

top-right panel. Doses greater than 0.3 mg/kg of NX HCl represents

reliable decreases in E-intake. All values representing diprenorphine's effects, except the smallest (0.001 mg/kg) and the

largest dose (10 mg/kg), are statistically significant. Doses

are represented as the salt, except for diprenorphine and EKC.

361
When diprenorphine and MOR are given concurrently, the

increase in E-intake is greater than when either is given

alone. Diprenorphine increases intakes of unflavored ES

(Reid et al. 1986). It is clear, small doses of some

opioids produce marked increments in E-intake. The

pattern of different opioids' effects indicate that the

ability to increment E-intake is not related to opioids'

analgesic potential: some analgesics produce decreases

in intake and others little or no effect. Diprenorphine,

an antagonist with respect to analgesia, produces

increases. A pattern that seems to relate to opioids'

enhancement of drinking ES is the pattern of opioids'

effects on pressing for rewarding brain stimulation. In

general, the doses of opioids that increase pressing also

increase E-intake.

These data lead to the conclusion that an opioidmechanism could be involved in E's ability to sustain its

own intake and involved in promoting "excessive" Eintake. Perhaps, it is the same mechanism that is

involved with increasing pressing for rewarding brain

stimulation. The receptor for which diprenorphine is an

agonist, which is also sensitive to NX's antagonism,

seems particularly germane.

REFERENCES

Beaman, C.M., Hunter, G.A., & Reid, L.D. Bull Psychon

Soc, 22: 354-355, 1984.

Cohen, G., & Collins, M. Science, 167: 1749-1751, 1970.

Davis, V.E., & Walsh, M.J. Science, 167: 1005-1007,

1970.

Hiller, J.M., Angel, L.M. & Simon, E.J. Science, 214:

468-469, 1981.

Hubbell, C.L., Czirr, S.A., Hunter, G.A., Beaman, C.M.,

LeCann, N.C., & Reid, L.D. Alcohol, 3: 39-54, 1986.

Mudar, P.J., LeCann, N.C., Czirr; S.A., Hubbell, C.L., &

Reid, L.D. Alcohol, in press, 1986.

Pollerberg, G.E., Costa, T., Shearman, G.T., Herz, A., &

Reid, L.D. Life Sciences, 33: 1549-1559, 1983.

Reid, L.D. Am J Clin Nut, 42: 1099-1132, 1985.

Reid, L.D., Czirr, S.A., Bensinger, C.C., Manha, N.A., &

Volanth, A.J., paper presented at the Annual Meeting of

the EPA, April 18, 1986.

Reid, L.D., Hubbell, C.L., Dunn, L.L., & Costa, T.

Neuropeptides, 5: 331-334, 1985.

Reid, L.D., & Hunter, G.A. Alcohol, 1: 33-37, 1984.

Seigel, S. In: Cappell, H., Israel, Y., Kalant, H.,

Schmidt, W., Sellers, E., & Smart, R., eds. Research

Advances in Alcohol & Drug Problems. Vol. 9. New

York: Plenum, 1985.

Severs, M.H. Science, 170: 113-115, 1970.

Sinclair, J.D. Pharm Bio Behav, 2: 409-412, 1974.

Tabakoff, B. Pharm Bio Behav, 18(1): 335-339, 1981.

362
(+)-NALOXONE: ANTAGONISM OF PENTOBARBITAL-INDUCED NARCOSIS

David Frost, Adel R. Abadir, Jonas Gintautas and

Barry J. Kraynack*

Anesthesiology Research Laboratories,

Brookdale Hospital Medical Center,

Brooklyn, N.Y. 11212, U.S.A.

*Department of Anesthesiology,

University of South Carolina School of

Medicine, Columbia, S.C. 29203, U.S.A.

ABSTRACT

Intracerebroventricular injection of (+)-naloxone

shortened pentobarbital-induced sleeping times in rats.

It does not appear that opiate receptors regulate the

duration of narcosis.

INTRODUCTION

The role of opiate receptors and the endogenous opioid

peptides in the production of general anesthesia is

unclear. Intracerebroventricular administration of

racemic (+)-naloxone dose-relatedly shortened the duration

of sleeping time induced by pentobarbital, halothane and

ketamine in rats. The fact that equivalent molar doses of

naltrexone, a more potent opiate antagonist, had no

antianesthetic effect suggested that naloxone's activity

was unrelated to an opiate receptor mechanism (Kraynack

and Gintautas 1982). Naloxone exhibits biopharmacological

effects unrelated to opiate receptor activity. Parallel

experiments with the inactive enantiomer, (+)-naloxone,

may provide additional evidence concerning the opioid

agonism theory of anesthesia.

This study was designed to investigate the modification of

narcosis in laboratory animals by the inactive enantiomer

of naloxone, (+)-naloxone. The effects of centrally

administered (+)-naloxone on the duration of sleeping time

(ST) has been determined in rats anesthetized with

pentobarbital.

METHODS AND MATERIALS

Male Sprague-Dawley rats (Harlan Sprague-Dawley,

Indianapolis, Indiana) weighing 125-150 g, were given an

intraperitoneal injection of sodium pentobarbital (50

mg/kg). Immediately after the loss of righting reflex

(LRR), groups of rats received an intracerebroventricular (ICV) injection of 120 or 240 ug of

(+)-naloxone according to a method described previously

(Kraynack and Gintautas 1982). Control ST were determined

in rats receiving 0.9 percent saline ICV. A total volume

363

172-683 O - 87 - 13 :QL 3
of 12 or 24 microliters of the test drug or 0.9 percent

saline was injected into the right lateral ventricle of

the brain. The (+)-naloxone was dissolved in 0.9 percent

saline for injection. There were 10 rats per treatment

group. Awakening was defined as regaining the righting

reflex (RRR). Sleeping time (ST), defined as the period

between LRR and RRR, was recorded for all experimental

groups.

Data were expressed as the mean ± SEM. Bonferroni (DUNN)

t- test was used to evaluate pairwise differences of

naloxone induced changes. Critical differences was set at

alpha = 0.05. Significant differences between values was

assumed when p<0.05.

RESULTS

There was no statistically significant difference between

mean sleeping times of saline treated rats receiving 12 or

24 ul injections. The data were pooled

for comparisons. The mean sleeping time induced with

pentobarbital (ICV saline treatment) in this study

demonstrated no statistically significant differences from

that previously reported by us (pairwise differences

p<0.05; t-test).

Intracerebroventricular administration of (+)-naloxone

shortened the ST duration of rats anesthetized with

pentobarbital. A dose-related shortening of ST was not

observed. Sleeping time was shortened by 17 and 21

percent in rats receiving 120 or 240 ug ICV of

(+)-naloxone (table 1).

Treatment group

Saline

(+)-naloxone

120 ug

240 ug

Mean Sleeping Time (ST)

(min ± SEM)

140.9 ± 3.7

116.3 ± 6.9*

111.0 ± 12*

*Significantly different from control at 0.05

level

Thirty and forty percent of the rats in the groups treated with 120 or 240 ug, respectively, of (+)-naloxone

died. These animals exhibited gasping respiratory movements accompanied by cyanosis, defecation and micturition.

364
DISCUSSION

The present study clearly demonstrates that centrally

administered (+)-naloxone shortens ST induced by

pentobarbital. Enantiomeric specificity was not observed.

As shown by Ijima et al. (1978), (+)-naloxone has no more

than 1/1.000 - 1/10,000th the activity of (-)-naloxone as

an opiate receptor antagonist in severe1 in vitro systems.

Our studies revealed that siqnificantly higher doses of

(+)-naloxone, and (+)-naloxone are needed to shorten ST,

than would be expected if the drugs acted strictly as

opiate receptor antagonists. In addition, naltrexone, a

more potent narcotic antagonist, has no antianesthetic

effect in rats anesthetized with pentobarbital, halothane

or ketamine. In accordance with our data, is the fact

that barbiturates do not modify the stereo-specific opioid

receptor binding of 3H-naloxone (Pert and Snyder 1973).

The mechanism of naloxone's antianesthetic action remains

obscure, but we may conclude that narcosis (as measured by

the righting reflex), the predominant effect of general

anesthetics, is not related to pharmacologic competition

for opiate receptors. Clearly, pentobarbital does not

mediate its effect on the righting reflex (i.e., narcosis)

through pharmacologic antagonism of a receptor sharing

comparable affinities for naloxone, naltrexone and

morphine. Furthermore, Way et al. (1984) found no

evidence that anesthetics act by increasing endogenous

opioid peptide activity and concluded that analgesia

associated with general anesthesia is not mediated by

beta-endorphin; nor, as suggested by Shingu et al. (1981)

do general anesthetics alter sensitivity or facilitate

opioid peptide actions on nociceptive neurons. These

findings concur with those of Deady et al. (1981) who

concluded that the righting reflex is depressed through a

mechanism separate from that responsible for analgesia.

Consequently, these in vivo findings enhance our understanding of the many inconsistant and often contradictory

results reported concerning naloxone antagonism when

different indices (narcosis, analgesia, cardiorespiratory

effect) are used. The inference that these effects of

general anesthesia share a common mechanism mediated by

opiate receptors appears unwarranted.

REFERENCES

Deady, J.E., Kublin, D.D., Eger, E.I., Heavner, J.E.,

and D'Aoust, B. Anesth Analg, 60:380-384, 1981.

Ijima, I., Minami Kowa, J., Jacobson, A.E., Brussi, A.,

and Rice, K.S. J Med Chem 21:398-400, 1978.

Kraynack, B.J. and Gintautas, J. Anesthesioloqy,

56:251-253, 1982.

Pert, C.B. and Snyder, S.H. Science, 179:1011-1014,

1973.

365
Shingu, K., Owawa, M., Omatsu, Y., Komatsu, T.,

Urabe, N., and Mori, K. Acta Anaesthesiol Scand,

25:526-532, 1981.

Way, W.L., Hosobuchi, Y., and Johnson, B.H.

Anesthesioloqy, 60:43-45, 1984.

366
RAT STRIATAL DOPAMINE RELEASE MECHANISMS OF COCAINE

Patricia A. Broderick

Department of Neurology

Cornell Univ. Med. Coll., Burke Rehab. Ctr.

White Plains, New York 10605, U.S.A.

ABSTRACT

Centrally problematic in the study of drugs of abuse is

the addictive process itself. Addictive drugs affect dopaminergic nigrostriatal and mesolimbic neuronal circuitry during brain reward. These findings show that cocaine produces a decrease in dopamine release from rat

striatum and prevents the surge of dopamine release which

occurs post-mortem. The results suggest that dopaminergic control over striatal neuronal membrane may be important in neurochemical mechanisms underlying brain reward.

INTRODUCTION

Cocaine's commonality with opiates is "abuse potential."

A neuronal reward circuitry in brain exists (Olds and

Milner 1954) and activation of this circuitry is necessary for reinforcement (Wise 1982). Psychomotor stimulants and opiates both appear to have their effect in

the nigrostriatal and mesolimbic areas of the descending

reward fibers of the medial forebrain bundle (Corbett

and Wise 1980;Wise 1981). Whereas opiates affect the

level of cell bodies, stimulants affect terminals of the

dopaminergic nerve fibers (Wise 1984).

The critical response for the rewarding property of cocaine is an action at the dopaminergic synapse. Cocaine

actually prolongs the activity of dopamine by presynaptic reuptake inhibition, an action shared by amphetamine

(Fuxe et al. 1967). Although amphetamine has been shown

to augment dopamine release (Broderick et al. 1983),

there have been no previous in vivo studies on the dopaminergic release mechanisms of cocaine. The present

study serves to further elucidate the relationships between cocaine, striatal dopaminergic presynaptic mechanisms and brain reward.

METHODS

The effect of cocaine (20 mg/kg SC) on dopamine release

from striata of male, Sprague-Dawley rats was studied by

semiderivative electroanalyses, an in vivo electrochemi- - -

cal methodology. Chloral hydrate, anesthetized rats

(body temperature maintained at 37° C) underwent stereotaxic surgery for positioning of a teflon-coated working

graphite electrode (150-175μ) (stearate modification)

(Blaha and Lane 1983) in anterior striatum. A Ag/AgCl

reference electrode and stainless steel auxiliary electrode was placed in contact with the rat cortex. Semiderivative voltammograms were recorded every ten minutes.

367
Potentials were applied between -200 and +500 mv, at a

scan rate of 10 mv sec-1. In calibration experiments,

the electrochemical response increased linearly with elevated concentrations of dopamine without interference

from serotonin, other amines, associated metabolites and

ascorbic acid.

RESULTS AND DISCUSSION

The results show that cocaine, at a dose sufficient to

produce euphoria, caused a decreased dopamine release

from rat striatum. The dopamine signal returned to normal within the hour period studied. These results are

consistent with the known half-life of cocaine (Nayak et

al. 1976).

FIGURE 1. A line graph showing the time course

characteristics of the effect of cocaine (20

mg/kg SC) on dopamine release from rat striaturn. The x axis represents time (in minutes)

before and after the administration of cocaine.

The y axis represents dopamine release as percent of control. The percent of controls were

calculated by averaging the first four scans

and dividing all values by that average. This

was done for each animal. The results were

considered statistically significant (p<0.05)

if the data points were outside the 95% confidence limits of the control.

Other addicting drugs, such as morphine and enkephalinamides, also decrease dopamine release from rat striatum

(Broderick et al. 1983; Broderick et al. 1984;Broderick

1985). Post-mortem studies of rat striatal dopamine re368
lease after cocaine treatment show that cocaine caused an

inhibition of the significantly increased surge of dopamine release which occurs at death (Phebus et al. 1986;

Broderick 1986).

FIGURE 2. Semiderivative voltammograms showing

post-mortem differences between dopamine release in cocaine treated (left voltammogram)

and untreated animals (right voltammogram).

The x axes represent increasing oxidation potentials in volts;

rent in nA sec-1.

the y axes represent curThe electrochemical signal

for dopamine (1) represents dopamine release

pre-mortem. The electrochemical signals for

dopamine (2,3,4,5) represent dopamine release

post-mortem, (ten minute intervals), in a cocaine treated animal. In contrast, the right

voltammogram represents dopamine release ten

minutes post-mortem in an untreated animal.

The signal is increased approximately sixfold over that dopamine release indicated by

voltammogram (1) pre-mortem.

369
Taken together with previous studies which show that cocaine inhibits reuptake (Fuxe et al. 1967), these findings lend explanation to a report showing unchanged striatal dopamine concentrations after cocaine (Lasley et al.

1985). In aqreement with others (Rice et al. 1985),

these studies show that in vivo electrochemistry primarily estimates release. Finally, the results suqqest

that control of dopaminergic function, perhaps across

membrane (Sershen et al. 1984), may be as important in

brain reward mechanisms than is augmentation of dopaminergic function.

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Blaha, C.D., and Lane, R.F. Brain Res Bull, 10:861-864,

1983.

Broderick, P.A. Life Sci, 36:2269-2275, 1985.

Broderick, P.A. The Hastings Center Report, in preparation, 1986.

Broderick, P.A., Blaha, C.D., and Lane, R.F. Brain Res,

269:378-381, 1983.

Broderick, P.A., Gardner, E.L.. and Van Praaq, H.M.

Biol Psychiat, 19(1):45-54, 1984.

Corbett, D., and Wise, R.A. Brain Res, 185:1-15, 1980.

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Lasley, S.M., Emmett-Oglesby, M.W., and Lane, J.D. Soc

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Nayak, P.K., Misra, A.L., and Mule, S.J. J Pharmacol Exp

Therap, 196(3):556-569, 1976.

Olds, J., and Milner, P. J Comp Physiol Psychol, 47:419-

427, 1954.

Phebus, L.A., Perry, K.W., Clemens, J.A., and Fuller, R.W.

Life Sci, 38:2247-2453, 1986.

Rice, M.E., Oke, A.F., Bradberry, C.W.,

Brain Res, 340:151-155, 1985.

Sershen, H., Reith, M.E.A., Hashim, A.,

and Adams, R.N.

and Lajtha, A.

Eur J Pharmacol, 102:175-178, 1984.

Wise, R.A. Behav Brain Sci, 5:39-53, 1982.

Wise, R.A. Brain Res, 213:190-194, 1981.

Wise, R.A. Neuralmechanisms of the reinforcing action

of cocaine. In Grabowski, J., ed. Cocaine: 1984. National Institute on Drug Abuse Research Monograph 50. DHHS

Pub. No. (ADM) 84-1326. Washington, D.C.: Supt. of

Docs. U.S. Govt. Print. Off., 1984. pp. 15-33.

ACKNOWLEDGEMENTS

Supported in part by a grant from the Upjohn CO. Please

address reprint requests to the author: Dr. P.A. Broderick Dept. of

Pharmacology, City University of NY Medical School, Covent Ave. at

138th Street, New York, New York 10031

370
CENTRAL CLUCACON ANTAGONIZES MORPHINE- AND STRESSINDUCED ANTINOCICEPTION IN THE MOUSE

Diana S. Malcolm, S. Amir* and John W. Holaday

Neuropharmacology Branch

Dept. of Medical Neurosciences, Div. of Neuropsychiatry

Walter Reed Army inst. of Res., Washington, D.C. 20307-5100, U.S.A.

*Dept. of Isotope Research, Weizmann Inst. of Science,

Rehovot, Israel

ABSTRACT

Previously, we have demonstrated that glucagon antagonizes morphineinduced antinociception in the rat (Malcolm et al. 1985). In the present

studies we have shown that central injection of glucagon antagonizes

morphine- and stress-induced antinociception in the mouse. Since stressinduced antinociception is mediated by the activation of endogenous opioid

systems, these findings provide the first evidence for glucagon’s ability to

antagonize endogenous opioid actions.

INTRODUCTION

Clucagon and its receptor have been identified within the mammalian central

nervous system (CNS)(Tager et al. 1980, Hoosein and Curd 1984). However,

with the exception of reports indicating that intracerebroventricular (icv)

injections of glucagon elevate circulating levels of somatostatin (Abe et al.

1978) and glucose (Marubashi et al. 1985), very little is known about the

physiological and pharmacological actions of glucagon in the CNS.

We have previously reported that central injection of glucagon antagonizes

morphine-induced bradycardia and antinociception in the rat (Malcolm et al.

1986) suggesting that glucagon may functionally interact with endogenous

opioid systems. This interpretation is supported by the observations that (1)

glucagon and opioid receptors share many similarities in neuroanatomical

distribution (Akil and Watson 1983, Hoosein and Curd 1984) and (2) other

brain-gut peptides (e.g. ACTH, somatostatin, cholecystokinin) have been

shown to possess some anti-opioid properties (Terenius 1975, 1976, Faris et al.

1983, ltoh et al. 1982). In the present studies we sought to determine whether

central injection of glucagon antagonizes opioid actions in another species,

the mouse, and furthermore to determine whether central glucagon can

modulate the action of endogenous opioid systems. Since endogenous opioid

systems are thought to mediate immobilization stress-induced

antinociception, we chose to test glucagon’s ability to antagonize stressinduced antinociception as a means to assess glucagon’s effects on endogenous

opioid actions.

MATERIALS AND METHODS

All studies were carried out using male mice (ICR, CD-l strain) weighing

approximately 30 g and having free access to food and water. Central (icv)

injections of drugs were performed in conscious mice according to the method

of Haley and McCormick (1957). Immobilization stress was induced by

restraining animals for 30 minutes in a restraining apparatus for mice (Amir

371
et al. 1981). Animals were injected icv with either saline (control) or

glucagon (1 or 10 μg, n=5-10; Eli Lilly and Co., Indianapolis, IN) just prior to

subcutaneous (SC) injection of morphine sulfate (5 mg/kg, n=8; Merck and Co.,

Inc., West Point, PA) or immobilization. Thirty minutes after morphine or 30

min after the onset of immobilization stress, animals were placed on a hot

plate (52°C) and jump/escape latencies were recorded. Maxiumum time

allowed for an animal to remain on the hot plate was 90 seconds.

Data were analyzed using analysis of variance followed by Duncan’s Multiple

Range test for comparison. The level of statistical significance was chosen at

p < 0.05.

RESULTS AND DISCUSSION

Compared to control nociceptive response latencies (29.3±11 sec), morphine

administration elevated jump/escape latencies 2-fold (65.1±11 sec) by 5

minutes post-injection (fig. 1). Pretreatment with 10 but not 1 μg glucagon

icv significantly attenuated morphine-induced antinociception at 15 and 30

minutes by 66% and 68%, respectively. Mice not treated with morphine (fig.

1, saline/saline group) exhibited a progressive decrease in nociceptive

response latencies when tested over time, and this decrement most likely

reflects the normal learning curve for these animals. These findings in the

mouse confirm our previous observations in the rat and support the hypothesis

that central glucagon antagonizes morphine actions and furthermore indicate

that glucagon’s anti-opioid effects occur across species.

SALINE + SALINE

SALINE + MORPHINE

GLUCAGON ( 1 μg , ICV ) + MORPHINE

GLUCAGON ( 10 μg , ICV) + MORPHINE

Figure 1. Effects of icv injection of glucagon on morphineinduced antinociception in the mouse. Clucagon at 10 μg but

not 1 μg icv, significantly (p<0.05) attenuated morphine-induced

antinociception at 15 and 30 min as compared to the saline plus

morphine-treated group (n=5-10).

372
Thirty minutes of immobilization elevated nociceptive response latencies 2.3-

fold from 32.6±6 to 76.6±8 sec. (fig. 2). Pretreatment with 1 but not 10 μg

glucagon icv completely prevented the stress-induced antinociception (fig.

2). In both studies, glucagon treatment (1 and 10 μg, icv) alone did not alter

baseline nociceptive response latencies at the times tested. This suggests

that glucagon’s antagonism of these opioid effects is not due to a direct

lowering of the threshold to pain but rather may represent a functional

antagonism between glucagon and the opioids. Since glucagon receptors have

been localized in areas of the brainstem (Hoosein and Curd 1984) known to be

involved in the regulation of pain transmission, it is possible that glucagon’s

antagonism of opioid-induced antinociception may be mediated through

glucagon receptors at these brainstem sites.

Figure 2. Effects of icv injection of glucagon on stress-induced

antinociception in the mouse. Clucagon at 1 μg but not 10 μg,

icv, completely prevented the stress-induced antinociception at

30 minutes post-injection. Clucagon treatment alone (1 and 10

μg, icv) did not alter baseline nociceptive response latencies at

the times tested.

In summary, these data confirm our previous findings in another species and

more importantly demonstrate that central glucagon blocks stress-induced

ant inociception. Since stress-induced antinociception is mediated by the

activation of endogenous opioid systems, these findings provide the first

evidence for glucagon’s ability to antagonize endogenous opioid actions.

373
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Abe, H., Kato, Y., Chiba, T., Taminato, T., and Fujita, T. Life Sci, 23:1647-

1654, 1978.

Akil, H., and Watson, S. In: Handbook of Psychopharmacology. New York:

Plenum Press, 1983. pp. 209-251.

Amir, S., Amit, Z., and Brown, Z.W. Physiol Behav, 26:335-336, 1981.

Faris, P., Komisaruk, B.R., Watkins, L.R., and Mayer, D.J. Science, 219:310-

312, 1983.

Haley, T.J., and McCormick, W.C. Brit J Pharmacol, 12:12-15, 1957.

Hoosein, N.M., and Curd, R.S. Proc Natl Acad Sci USA, 81:4368-4372, 1984.

Itoh, S., Katsuura, C, and Maeda, Y. Eur J Pharmacol, 80:421-425, 1982.

Malcolm, D.S., Zaloga, C.P., Teich, S., Chernow, B., and Holaday, J.W. INRC,

Alcohol and Drug Research, 6:246, 1985.

Malcolm, D.S., Zaloga, C.P., Chernow, B., and Holaday, J.W. Life Sci, (in

press, 1986).

Marubashi, S., Tominaga, M., Katagiri, T., Yamatani, K., Yawata, Y., Hara,

M., and Sasaki, H. Acta Endocrinol, 108:6-10, 1985.

Tager, H., Hohenboken, M., Markese, J., and Dinerstein, R.J. Proc Natl Acad

Sci USA, 77:6229-6233, 1980.

Terenius, L. J Pharm Pharmacol, 27:450-452, 1975.

Terenius, L. Eur J Pharmacol, 38:211-213, 1976.

374
ENDORPHINS CONTRIBUTE TO THE LOSS OF GLUCOSE HOMESTASIS IN

ANAPHYLACTIC SHOCK

Shimon Amir

Neuroscience Center

The Weizmann Institute of Science

Rehovot, Israel

ABSTRACT

Central injection of naloxone attenuated the development of hyperglycemia following induction of non-fatal anaphylaxis in mice, but it

failed to reverse the hyperglycemia in this model. In contrast, central TRH both blocked as well as reversed the hyperglycemic response.

These results suggest a possible role for central endorphin mechanisms in the hyperglycemic response to anaphylactic shock in mice.

Further, they demonstrate a novel action of TRH to block shock-induced hyperglycemia. This antihyperglycemic action of TRH is independent of opiate mechanisms.

INTRODUCTION

Endorphins have been implicated in the cardiovascular depression of

circulatory shock because treatment with the opiate antagonist naloxone reversed the hypotension and improved survival in several experimental shock states, including endotoxic, hemorrhagic, spinal, and

anaphylactic shock (Holaday 1983). More recent studies have shown

that endorphins may also contribute to the glucose dishomeostasis

which characterizes shock since naloxone was found to block the hyperglycemic response to endotoxin administration (Amir and Harel

1983) or hemorrhage (Bereiter et al. 1983). The involvement of endorphins in the hyperglycemic response to shock was further investigated in the present study by evaluating the effect of central or

systemic naloxone on the plasma glucose responses to non-fatal anaphylaxis in mice. In addition, the effect of the physiologic opiate

antagonist thyrotropin-releasing hormone (TRH) on the plasma glucose

responses to anaphylaxis was investigated. TRH was found in earlier

studies to block fatal anaphylaxis in mice (Amir et al. 1984) and to

antagonize opiate-induced hyperglycemia in this species (Amir 1985).

MATERIALS AND METHODS

Subjects were normally fed male ICR mice, 28-30 g. Anaphylaxis was

induced by subcutaneous (SC) injection of the histamine releaser compound 48/80 (C 48/80 [Sigma] 4 mg/kg in 0.2 ml saline). Naloxone

(Endo) and TRH (Sigma) were dissolved in sdline and injected intracerebroventricularly (ICV) or intravenously (IV) one min before or 15

min after C 48/80. The ICV injections were made according to the

method of Haley and McCormick (1957) using a Hamilton microsyringe

bearnig a 27 gauge needle 2.5 mm long. Animals were sacrificed by

decapitation at different times after C 48/80 administration. The

trunk blood was collected and centrifuged to obtain plasma. Plasma

375
glucose concentrations were detenmined by the glucose oxidase method

using a Bekckman glucose analyzer.

RESULTS AND DISCUSSION

Induction of anaphylaxis by SC challenge with 4 mg/kg C 48/80 resulted in a rapid and sustained increase in the levels of circulating

glucose (fig. 1). This hyperglycemia was attenuated by 10 ug naloxone given ICV one min before C 48/80. In contrast, naloxone increased the plasma glucose of shocked mice when given IV (100 ug) one

min before C 48/80 (fig. 2).

FIGURE 1. Effect of ICV naloxone or TRH pretreatment on

plasma glucose of C 48/80-treated mice. The symbols and

vertical lines represent means ± SEM of plasma glucose concentrations. Asterisks indicate significant difference

from C 48/80 alone (p<0.05 t tests).

As with naloxone, ICV pretreatment with 10 ug TRH strongly blocked C

48/80-induced hyparglycemia (fig. 1). However, TRH also blocked the

hyperglycemic response when given IV (100 ug, fig. 2).

Finally TRH (1 ug ICV), but not naloxone (1 ug ICV), reversed the hyperglycemic response to C 48/80 when given 5 min after induction of

shock (fig. 3).

376
FIGURE 2. Effect of IV naloxone or TRH prtreatment on

plasna glucose of C 48/80-treated mice. See legend to fig.

1 for details.

The results with naloxone suggest that activation of edorphin mechanisnms in the CNS may contribute to the loss of glucose homeostasis

and development of hyperglycemia following induction of anaphylactic

shock in mice. Morever, failure of systemic naloxone to mimic the

central action of the drug on hyperglycemia further suggests that endorphins may affect central and peripheral glucoregulatory mechanisms

differentially. In contrast to naloxone TRH not only blocked C

48/80-induced hyperglycemia but also rapidly reversed it. This suggests that the antihyperglycemic action of TRH is independent of endorphin mechanisms. Thus, while naloxone probably affects the development of hyperglycemia following C 48/80 by antagonizing a central

opiate receptor-mediated action of endorphins to stimulate glucose

production (Van Loon and Appel 1981), TRH may act in the CNS to physiologically oppose the hyperglycemia by stimulating insulin secretion

and enhancing glucose uptake. This possibility is consistent with

recent finding that TRH lowers the plasma glucose of normoglycemic

mice by a parasympathetic-mediated insulin-dependent mechanism (Amir

et al. 1985).

377
FIGURE 3. Action of ICV naloxone or TRH to reverse C

48/80-induced hyperglycemia. See legend to fig. 1 for details.

REFERENCES

Amir, S. INRC Abst, pp. 10, 1985.

Amir, S., and Harel, M. Neurophamacology, 22:1117-1120, 1983.

Amir, S., Harel, M., and Schachar, A. Brain Res, 298:219-224, 1984.

Amir, S., Rivkind, A.I., and Harel, M. Brain Res, 339:382-385, 1985.

Bereiter, D.A., Plotsky, P.M. and Gann, D.S. Endocrinoloqy,

113:1439-1446, 1983.

Haley, T.J. and McCormick, W.G. Br J Pharmacol, 12:12-15, 1957.

Holaday, J.W. Annu Rev Pharm Toxicol, 23:541-594, 1983.

Van Loon, G.R., and Appel, N.M. Brain Res, 204:236-241, 1981.

378
EFFECTS OF OPIOID ANALGESICS ON LOCAL CEREBRAL GLUCOSE UTILIZATION

Edythe London, Richard Fanelli, Margit Szikszay and Donald Jasinski

Addiction Research Center, National Institute on Drug Abuse

Baltimore, MD 21224, U.S.A

ABSTRACT

The autoradiographic 2-deoxy-D-[1-[4C]glucose ([14-C]DG) method

was used to map the effects of morphine, oxymorphone and nalbuphine

on local cerebral glucose utilization (LCGU), an index of local

brain function. The mu agonists (morphine, oxymorphone) decreased

LCGU in thalamic nuclei, including some which have been implicated

in somatosensory processing. Nalbuphine did not produce these

effects, but stimulated LCGU in nuclei of the spinal trigeminal

tract. The findings suggest that different supraspinal mechanisms

mediate the actions of mu vs. kappa opioids, and that the [14-C]DG

procedure is helpful in elucidating the anatomical areas involved.

INTRODUCTION

Opioid analgesics vary in their spectrum of side effects and abuse

potential. The differences may reflect varying activities at opioid

receptor subtypes. Oxymorphone is a relatively pure mu agonist,

morphine has mu and kappa agonist properties, and nalbuphine is a

partial agonist at ka a receptors with minimal sigma agonist and mu

antagonist properties (Jaffe and Martin 1985). The purpose of this

study was to compare the distributions of the in vivo effects of

these drugs on rates of LCGU, an index of local cerebral function.

Studies of LCGU have been used to elucidate the in vivo metabolic

responses to drugs of various classes, including dopaminergic agents

(McCulloch 19821, cholinomimetics (Dam et al. 1982), and GABAmimetics (Palacios et al. 1982).

MATERIALS AND METHODS

Male Fischer-344 rats, 3 mo of age (Charles River Breeding Laboratories) were acclimated to a vivarium (25°C, 12h:12h light:dark

cycle) at the Addiction Research Center for about 4 weeks before the

drug studies. [14-C]DG, specific activity 59 mCi/mmol, Amersham

Corp.) was rechromatographed to ascertain purity. Oxymorphone HCl

and nalbuphine HCl were supplied by DuPont Pharmaceuticals; morphine

sulfate was obtained from Mallincbrodt. Inc.

The rats were fasted overnight before being prepared with

indwelling, femoral venous and arterial catheters under light

halothane anesthesia. After surgery, they were placed in a soundinsulated box where they were allowed to recover from surgery for

about 3 h before drug treatments.

Opioids were administered S.C. 15 min before the radiotracer.

Control animals received S.C. injections of 0.9 % NaCl. Effects on

arterial blood gases and pH were assessed at several times.

379
LCGU was determined after injection of [14-C]DG (125 μCi/kg body

weight, i.v.) as previously described (Sokoloff et al. 1977).

arterial blood samples were collected for assesment of plasma

glucose and [14-C]DG. Animals were killed by a pentobarbital

overdose 45 min after the [14-C]DG injection, and brains were

rapidly removed and frozen in 2-methylbutane at -5O°C.

Frozen 20 μM cryostatic sections were cut from the brains at -18 to

-20°C thaw-mounted onto glass coverslips, and exposed to x-ray film

(Kodak SB5) for one week in a cassette containing [14-C]methylmethacrylate standards. Brain radioactivity was determined by

quantitative autoradiography using a Leitz microscope and the

MPV-DADS scanning microdensitometry system (E. Leitz, Inc.). Brain

structures were identified using standard rat brain atlases. LCGU

was calculated from brain and plasma radioactivities and plasma

glucose concentrations using an operational equation (Sokoloff et

al. 1977).

RESULTS

Morphine and oxymorphone, but not nalbuphine decreased the pH of

arterial blood (mean pH ± SEM: control, 7.403 ± .006; morphine,

7.336 ± .013; oxymorphone, 7.336 ± .009; nalbuphine, 7.391 ± .008).

Oxymorphone significantly increased arterial blood PaCO2 and

greased PaCO2 51 min after the drug administration (PaCO2,

PaO2: control, 32.8 ± 0.9, 94.0 ± 2.0; oxymorphone 38.0 ± 1.4,

80. 0 ± 4.0). At this time, morphine produced similar changes in

blood-gases (35.9 ± 2.8, 82.0 ± 3.0), but to a smaller degree which

did not reach statictical significance.

Drug effects on LCGU were examined in 62 brain regions, and

significant differences from control were obtained in twelve of the

regions (Table I). Morphine and oxymorphone decreased LCGU in

several thalamic nuclei and in the dorsal tegmental nucleus and the

median eminence. Nalbuphine stimulated LCGU in the spinal nuclei of

the trigeminal nerve, and in the globus pallidus.

DISCUSSION

The results support the view that opioid systems are involved in

several distinct analgesic mechanisms that are mediated by different

subpopulations of opioid receptors. The LCGU decrements in thalamic

nuclei induced by the mu agonists are consistent with mu receptor

localizations and with the hypothesis that these opioids can produce

analgesia not only by activating descending inhibitory pathways but

also by inhibiting ascending somatosensory pathways at a supraspinal

level. There are especially high concentrations of mu receptors in

midline and ventral thalamic nuclei (Goodman et al. 1980). In

contrast, the kappa system has been localized to other brain areas,

including the striatum, hypothalamus, and trigeminal sensory nuclei

(Goodman and Snyder 1982; Watson et al. 1982). Nalbuphine's effect

on LCGU in sensory nuclei of the spinal trigeminal tract is

consistent with an active inhibition of pain pathways originating at

a spinal level (Dubner and Bennett 1983).

380
The findings indicate that the [14-C]DG method can be used to

delineate CNS areas mediating the effects of opioid analgesics,

distinguishing between effects of ligands for various opioid

receptor subtypes.

79 ± 2.3* 107 ± 6.3

62 ± 1.3* 83 ± 5.3

63 ± 1.3* 90 ± 5.7

58 ± 1.8* 81 ± 3.9

79 ± 2.8* 114 ± 7.7

37 ± 1.7* 51 ± 3.2

70 ± 3.2* 84 ± 7.8

52 ± 3.3 63 ± 4.6*

43 ± 2.3 61 ± 6.6*

46 ± 3.4 64 ± 4.6*

TABLE 1. EFFECTS OF OPIOID AGONISTS ON GLUCOSE UTILIZATION

Saline Morphine Nalbuphine

Control 8 mg/kg 16 mg/kg

Paratenial Nucleus 95 ± 8.9 69 ± 3.4* 94 ± 6.6

Central Medial Nucleus

Ventrolateral Nucleus

Ventroposterior Nucleus

Medial Part

Lateral Part

Interanteromedial Nucleus

Median Eminence

Dorsal Tegmental Nucleus

Spinal Trigeminal Nerve

Oral Part

Interpositus Part

Caudal Part

Globus Pallidus

86 ± 7.4

74 ± 6.1

100 ± 8.8

46 ± 3.1

90 ± 5.6

51 ± 2.9

49 ± 3.3

52 ± 2.7

41 ± 3.9

104 ± 7.5

79 ± 8.3

43 ± 1.9 53 ± 3.2*

min).

Each value represents the mean ± SEM for 6 rats (μmol/100 g/

Values obtained in rats treated with 0.4 mg/kg oxymorphone

were similar to those from morphine-treated rats.

*Significant from control by one-way analysis of variance and

Duncan's multiple range test, p <0.05.

REFERENCES

Dam, M., Wamsley, J.K., Rapoport, S. I., and London, E.D. J

Neurosci, 2:1072-1078, 1982.

Dubner, and Bennett, G.J. Ann Rev Neurosci, 6: 381-419, 1983.

Goodman, R.R., and Snyder, Proc Natl Acad Sci, 79:5703-5707,

1982.

Goodman, R.R., Snyder, S.H., Kuhar, M.J., and Young, W.S. III. Proc

Natl Acad Sci, 77: 6239-6243, 1980.

Jaffe, J.H., and Martin, W.R. In: Gilman, A.G., Goodman, L.S., Rall,

T.W., and Murad, F., eds. The Pharmacological Basis of Therapeutics. 7th edn. New York: Macmillan Press, 1985. pp.491-531.

McCulloch, J. In: Iversen, L.L., Iversen, S.D., and Snyder, S.H.,

eds. Handbook of Psychopharmacology. Vol. 15. New York: Plenum

Press, 1982. pp. 321-410.

Palacios, J.M., Kuhar, M.J., Rapoport. S. I., and London, E.D. J Neurosci, 2:853-860, 1982.

Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.

S., Pettigrew, K.D., Sakurada, O., and Shinohara, M. J Neurochem,

28:897-916, 1977.

Watson, S.J., Khachaturian, H., Akil, H., Coy, D.H., and Goldstein,

A., Science 218:1134-1136, 1982.

ACKNOWLEDGEMENT

Supported in part by a grant from E. I. DuPont de Nemours and Co.

381
EFFECTS OF MORPHINE AND CALCIUM ANTAGONISTS

ON PLASMA GLUCOSE IN MALE RATS

Margit Szikszay*, Frederick R. Snyder and Edythe D. London

Neuropharmacology Laboratory, Addiction Research Center,

National Institute on Drug Abuse, Baltimore, Maryland 21224, U.S.A.

*Department of Physiology, University Medical School

Szeged, Hungary

ABSTRACT

Subcutaneous administration of morphine sulfate (4-16 mg/kg) did

not significantly alter the plasma glucose concentration in fasted

adult male Fischer-344 rats. All rats treated with calcium channel

antagonists, verapamil or diltiazem (10 mg/kg, with or without

morphine), showed marked and significant hyperglycemia, persisting

at high levels throughout the 2 h experiment.

INTRODUCTION

Calcium channel antagonists reduce calcium influx through voltagedependent calcium channels (for reviews see Chaffman and Brogden

1985; Schramm and Towart 1985). Opioids also inhibit Ca2+ influx

in a naloxone-reversible manner (for reviews see Ross and Cardenas

1979; Chapmand and Way 1980), suggesting that opioids might interact

with calcium channel antagonists in affecting various physiological

parameters. In this regard, calcium antagonists potentiate morphine

induced antinociception and hypothermia in rats (Benedek and

Szikszay 1984), but attenuate the respiratory depressant effect of

morphine (Szikszay et al. 1986). The major objective of the present

work was to determine if calcium antagonists and morphine interact

to alter plasma glucose levels in rats.

MATERIALS AND METHODS

Seventy seven male 3-4 month old Fischer-344 rats were fasted for

approximately 15 h prior to surgery, and then were prepared with a

femoral arterial catheter under halothane anesthesia. They were

partially immobilized as described previously (London et al. 1981),

so that results would be pertinent to future studies of cerebral

glucose utilization. Three hours later, arterial blood samples were

taken from awake rats 5 min before and at 0.25, 0.5, 1 and 2 h after

drug treatments to assess plasma glucose concentrations by a Beckman

Glucose Analyzer 2.

Morphine sulfate, verapamil HCl and diltiazem HCl were dissolved in

0.9% NaCl. Morphine sulfate was administered at 4, 6, 8 and 16

mg/kg, and calcium antagonists were given at 10 mg/kg, expressed as

the salts. All doses of morphine were tested with verapamil, but

only 4 mg/kg was tested with diltiazem. Drugs were injected S.C.

either alone or in combination (5-6 rats/group). Effects of the

treatments were assessed by a two-way (diltiazem) or three-way

(verapamil) analysis of variance with repeated measures over time.

Post-hoc comparisons were performed using Tukey's w-procedure.

382
RESULTS AND DISCUSSION

At all doses tested, morphine did not significantly alter the plasma

glucose concentration as compared to values in saline-treated rats

[main effect of morphine (4,43); F = 0.61. Although a hyperglycemic

effect of morphine has been recognized for many years, most of the

previous experiments were performed in cats and dogs (Borison et

al. 1962; Feldberg and Shaligram 1972). In rodents, some studies

have shown altered glucoregulation in response to morphine,

depending upon the strain, gender and dose. For example, Simon et

al. (1981) did not show significant effects of 4 or 8 mg/kg morphine

on blood glucose in female mice. In immature (120-140 g) female

Sprague-Dawley rats, 30 mg/kg morphine increased the glucose level

by about 40% (Muller et al. 19671, and in male non-fasted,

anesthetized Wistar rats, 20 mg/kg morphine increased plasma glucose

by about 50% (Nakaki 1981). The dosages of morphine used in our

experiments were much smaller and apparently were not sufficient to

cause hyperglycemia in awake male Fischer-344 rats.

FIGURE 1. Effects of different morphine doses and verapamil

on plasma glucose levels. Measurements were taken before (0)

and at four times after the S.C. injection of 0.9% NaCl

10 mg/kg verapamil morphine alone and in combination

with verapamil . Abscissae: time in hours. Ordinate:

mean deviation from the baseline values (114.3 ± 4.1 mg/dl).

*Significant p<0.05 (verapamil vs. morphine + verapamill.

In contrast, rats treated with verapamil, alone or in combination

with morphine, showed marked and significant elevations of arterial

plasma glucose levels [main effect of verapamil (1,43); F = 2141.

Moreover, there were significant interactions of verapamil not only

with time but also with dose of morphine (Fig 1). The highest

plasma glucose levels were observed in verapamil-treated rats after

the lower doses of morphine; whereas rats treated with 16 mg/kg of

383
morphine + verapamil had approximately the same plasma glucose level

as rats treated with verapamil

interaction: (4,43); F = 5.81.

alone [morphine by verapamil

Plasma glucose levels rose more

sharply during the first hour and tended to level off or increase

at a lower rate during the second hour after treatment [verapamil by

time interaction: (3,129); F = 20.21. The significant three-way

interaction of morphine by verapamil by time (12,129; F =3.5)

reflected the difference between verapamil alone as compared to

verapamil in combination with the three lower doses of morphine at

2 h only. Diltiazem alone or in combination with 4 mg/kg morphine

produced a level of hyperglycemia similar to that obtained with

verapamil (52-60 mg/dl over baseline, data not shown).

Although verapamil inhibits both calcium entry and uptake in the

pancreatic beta cells and glucose-induced insulin release in vitro

(Malaisse et. 19771, effects of calcium channel antagonists on

blood glucose concentrations in vivo have been inconsistent. For

example, nifedipine produced hyperglycemia and increased blood

glucagon levels in human subjects (Charles et al. 1981), but

verapamil (0.5 mg/kg, i.p.) did not alter blood glucose in rats

(Luyckx and Lefebvre 1976). Our results indicate that a much higher

dose of verapamil or diltiazem can produce hyperglycemia in rats,

suggesting an overall hyperglycemic effect of calcium antagonists.

The authors thank Dr. Ronald K. Browne of Marion Laboratories, Inc.

for supplying diltiazem.

REFERENCES

Benedek, G. and Szikszay, M. Pharmacol Res Commun, 16: 1009-1018,

1984.

Borison, H.L., Fishburn, B.R., Bhide, N.K. and McCarthy, L.E.

J Pharmacol Exp Ther, 138: 229-235, 1962.

Chaffman, M. and Brogden, R.N. Drugs, 29: 387-454, 1985.

Chapman, D.B. and Way, E.L. Annu Rev Pharmacol Toxicol, 20:

553-579, 1980.

Charles, S., Ketelslegers, J.M., Buysschaert, M. and Lambert, A.E.

Br Med J, 283: 19-20, 1981.

Feldberg, W and Shaligram, S.V. Br J Pharmacol, 46: 602-618, 1972

Luyckx, A.S: and Lefebvre, P.J. Biochem Pharmacol, 25: 2703-2708,

1976.

London, E.D., Nespor, S.M., Ohata, M. and Rapoport, S.I.

J Neurochem, 37: 217-221, 1981.

Malaisse, W.J., Herchuelz, A., Levy, J. and Sener, A. Biochem

Pharmacol, 26: 735-740, 1977.

Muller, E.E., Saito, T., Arimura, A. and Schally, A.V.

Endocrinology, 80: 109-117, 1967.

Nakaki, T., Muraki, T., Tokunaga, Y. and Kato, R. Biochem Pharmacol,

30: 2217-2220, 1981.

Ross, D.H. and Cardenas, H.L. Adv Biochem Psychopharmacol, 20:

301-336, 1979.

Schramm, M. and Towart, R. Life Sci, 37: 1843-1860, 1985.

Simon, G.S., Borzelleca, J. and Dewey, W.L. J Pharmacol Exp Ther,

218: 324-329. 1981.

Szikszay, M., Snyder, F.R. and London, E.D. J Pharmacol Exp Ther,

in press.

384
DYNAMIC CHANGES IN THE LEVELS OF STRIATAL PROENKEPHALIN mRNA

AND LARGE MOLECULAR WEIGHT ENKEPHALIN CONTAINING PEPTIDES

FOLLOWING ELECTROACUPUNCTURE

Xang Tsou, Min Zheng and Xiao-Hong Ding

Shanghai Institute of Materia Medica, Academia Sinica

Shanghai, 200031, China

ABSTRACT

Electroacupuncture has been shown to increase met- and leu-enkephalin

contents in rat striatum and hypothalamus. Its mechanism was further

studied by measuring the levels of the large molecular weight enkephalin containing peptides (LECP), processed intermediates, free

leu-enkephalin as well as proenkephalin mRNA in the striatum of rats

decapitated at different times after the termination of 30 min

electroacupuncture. A prominent increase of leu-enkephalin was seen

in rats decapitated immediately after electroacupuncture. This

increase returned to control level at 0.5 h. The levels of LECP and

intermediates began to elevate at 1 h after electroacupuncture,

reached a plateau at 8 h and lasted for 96 h. A second phase of

smaller but longer lasting increase of leu-enkephalin appeared at

8-96 h. The proenkephalin mRNA amount measured by dot-blot procedure

increased 3-5 folds starting at 1 h and lasting for at least 48 h.

INTRODUCTION

It has been shown that acupuncture increases the contents of metand leu-enkephalin immunoreactivities in the striatum and the hypothalamus (Tsou et al. 1980). This effect was attenuated by icv

administration of the protein synthesis inhibitor cycloheximide

suggesting that acupuncture accelerates enkephalin synthesis (Wu et

al. 1980). In order to gain some insight into this mechanism, we

followed the levels of the enkephalin precursors, the processed

intermediates and leu-enkephalin in the rat striatum simultaneously

after electroacupuncture for 96 h. To answer if it also involves

gene transcription, the proenkephalin mRNA was quantitated by dotblot procedure at various times after electroacupuncture.

MATERIALS AND METHODS

Electroacupuncture

Rats weighing 200-250 g were restrained in lucite containers. One

stainless steel electrode was implanted in the sciatic nerve region

of each hind limb. The electrodes were connected to a therapeutic

acupuncture stimulator, which produces intermittent high and low

frequency biphasic waves. The rats were stimulated for 30 min with

pulses just above the threshold of slight vocalization.

Chromatographic fractionation and enzymatic digestion

The striata of 10 rats from the same group were pooled and homogeni385
zed in a 10 times volume of 1 M acetic acid.

centrifuged for 20 min at 10,000 rpm, 4°C.

The homogenate was

The supernatant was

fractionated on a Sephadex G 75 column. From each collecting tube

1 ml of eluates was lyophilized. The lyophilized material was

resuspended and incubated first with TPCK treated trypsin solution

and then with carboxypeptidase B. The enzymes were subsequently

inactivated in a 90°C water bath for 20 min. Radioimmunoassay was

carried out as described (Tsou et al. 1930).

Proenkephalin mRNA quantitation

RNA was extracted and purified according to Feramisco et al (1982).

A 918 base pair cDNA complementary to the proenkephalin mRNA sequence

of human pheochromocytoma was a gift of Professor E. Herbert. This

cDNA was cloned into the Pst 1 site of pBR 322 (Comb et al. 1982 and

propagated in E. coli RRI. The cDNA probe was labelled with 32P)

MT? by nick-translation to a final specific activity of 1-5x108

cpm/μg. Equivalent amount (20 μg) of RNA from each sample was used

for dot-blot assay on a nitrocellulose filter paper, which was then

washed and exposed on to a X-ray film at -7O°C. The autoradiograms

were scanned with a Shimadzu CG-930 scanning densitometer.

RESULTS AND DISCUSSION

When the rat striatal eluates from the Sephadex G 75 column were

monitored at 254 μM, two peaks of absorption were observed. Radioimmunoassay of leu-enkephalin content in each collecting tube

revealed immunoreactivities only in those tubes containing small

molecular size peptides. This peak of immunoreactivites coincided

with the second peak of UV absorption. In the tubes containing

higher molecular weight peptides and proteins, leu-enkephalin immunoreactivities only appeared after trypsin and carboxypeptidase B

digestion. Therefore the eluates from tube No. 3-24 (corresponding

to the first peak of UV absorption) were pooled as the total amount

of the enkephalin precursors, from tube No. 25-48 as the intermediates and from tube No. 49-68 (corresponding to the second peak of UV

absorption) as the free leu-enkephalin. A composite figure (figure

1) could be drawn with changes of precursors, intermediates and free

leu-enkephalin levels in the rat striatum after the termination of

30 min electroacupuncture. A prominent increase of leu-enkephalin

content appeared immediately (0 h) after electroacupuncture. This

rapid increase declined quickly to the control level at 0.5 h.

During this initial period no change in the levels of precursors and

intermediates was found. At 1 h after the termination of electroacupuncture, there were elevations of both the precursor and

intermediates levels, which rose steadily until reaching a plateau

at about 8 h and lasted for at least 96 h. A second phase of smaller

hut longer lasting increase of leu-enkephalin appeared at 8 h and

lasted for more than 96 h. These results suggest that a huge amount

of enkephalin is released during acupuncture. The high level of

leu-enkephalin represents the released and the newly formed but still

unreleasable forms. The quick decline of leu-enkephalin was due to

rapid degradation of the released form. Although during this initial

period no change was found with the precursor and the intermediate ,

the rate of biosynthesis and processing might have already increased

386
FIGURE 1. Changes in the levels of enkephalin

precursors, processed intermediates and leuenkephalin after electroacupuncture. Each column

represents the content in the extract of pooled

striata of 10 rats.

so that their levels could remain unchanged. After the cessation

of electroacupuncture for about 1 h, the rate of enkephalin

utilization might slow down leading to an increase in the levels of

enkephalin precursors and processed intermediates. Their elevation

would eventually lead to a second phase of smaller but longer lasting

increase of leu-enkephalin.

Proenkephalin mRNA was quantitatad by hybridization with a human

proenkephalin cDNA probe using dot-blot procedure. A 3-5 fold

increase of proenkephalin mRNA level in the striatum could be detectcd as early as 1 h after the termination of 30 min electroacupuncture.

This proenkephalin mRNA induction continued for at least 48 h (fig

2).

387
FIGURE 2. Temporal effect of electroacupuncture

on rat striatal proenkephalin mRNA level.

C: conrol; 0.5, 1,2, 16, 48 and 96 are the

time intervals (h) between the termination of

electroacupuncture and the sacrifice of the

animals.

Since no change in the proenkephalin mRNA level was observed in the

first hour after electroacupuncture, the initial rapid enkephalin

increase in the striatun is most likely due to accelerated translation of the mRNA and post-translational processing of the precursor.

It is also postulated that the large release of enkephalin during

electroacupuncture may lead to a depletion of the releasable form.

'This depletion in turn brings about an enhanced transcription of the

proenkephalin gene. Such a mechanism may play an important part to

compensate the depletion by electroacupuncture.

REFERENCES

Comb, M., Seeburg, P., Adelman, J. and Herbert, E. Nature, 295:663-

666, 1982.

Feramisco, J. R., Helfman, D. M. and Smart, J. E. J Dial Chem, 257:

11024-11031, 1982.

Tsou, K., Yi, Q.-C., Wu, S.-X., Lu, Y.-X., Wang, F.-S., Yu, Y.-G.,

Ji, X.-Q., Zhang, Z.-X. and Zhao, D.-D. Scientia Sinica, 23:

1197-1207.

ACKNOWLEDGES

We wish to thank Professor E. Herbert for providing the proenkephalin

cDNA probe.

388
DOPAMINERGIC REGULATION OF PROENKEPHALIN-A GENE EXPRESSION

IN THE BASAL GANGLIA

Subbiah P. Sivaml, George R. Breese2, T. Celeste Napier2,

Robert A. Mueller2 and Jau-Shyong Hong1

1

 Neuropharmacology Section

Laboratory of Behavioral and Neurological Toxicology

National Institute of Environmental Health Sciences

P.O. Box 12233

Research Triangle Park, North Carolina 27709, USA

2

 Biological Sciences Research Center

of the Child Development Institute

Departments of Psychiatry, Pharmacology and Anesthesiology

University of North Carolina

School of Medicine

Chapel Hill, North Carolina 27514, USA

ABSTRACT

Dopaminergic denervation by 6-hydroxydopamine (6-OHDA) increased

proenkephalin-A gene expression in the striatum as evidenced by

increases in the levels of Met5-enkephalin (ME), precursor and

preproenkephalin mRNA (PE-mRNA). This and other lines of evidence

support the hypothesis that nigrostriatal dopamine input is inhibitory to striatal enkephalin neurons and that the removal of this

inhibitory influence leads to an acceleration of ME biosynthesis.

These findings suggest that the enkephalin system may be associated

with the progression of adaptive changes subsequent to dopaminergic

dysfunction in basal ganglia.

INTRODUCTION

Enkephalins are found in high concentrations in the same forebrain

structures which contain high levels of dopamine (Atweh and Kuhar,

1977; Hong et al. 1977; Johnson et al. 1980). Treatments that

alter dopaminergic transmission also alter the enkephalin system, for

example, chronic dopamine receptor blockade with haloperidol leads to

an increase in ME biosynthesis (Hong et al. 1978; Tang et al. 1983;

Sabol et al. 1983; Hong et al. 1985; Mocchetti et al. 1985; Sivam

et al. 1986). In addition, unilateral substantia nigra lesions also

increase the ME level in the ipsilateral striatum (Thal et al.

1983). Parkinson's disease and Lesch-Nyhan syndrome are two clinical

disorders in which dopamine deficiency has been found to be the major

component. Therefore, elucidation of the interrelationship between

enkephalin and dopamine in basal ganglia function is important

and the present work addresses this issue.

389
METHODS

Adult Sprague-Dawley rats received 6-OHDA (200 μg, free base) intracisternally in a volume of 25 μl. Behavioral evaluation to assess

dopaminergic supersensitivity following 6-OHDA lesions was made by

measuring locomotor activity. Animals were decapitated, brains were

removed, the striata and hippocampi were dissected out and used for

biochemical determinations.

Dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid

(HVA) were determined utilizing the reverse phase high-performance

liquid chromatography (HPLC) procedure. The content of ME (native

ME) was determined by radioimmunoassay (RIA) as described earlier

(Hong et al. 1978). Precursor content as reflected by the cryptic

ME level was determined following sequential digestion of the

tissue proteins with trypsin and carboxypeptidase-B (Sivam et al.

1986). Dynorphin-A (1-8)-like immunoreactivity was determined by

RIA.

Total RNA was isolated from striatal tissues following the method

described by Chirgwin et al. (1979). Northern blot and dot-blot analyses were carried out using a nick-translated 32P cDNA probe from

rat brain (Yoshikawa et al. 1984). The blots were subjected to

autoradiography followed by densitometry. The data were evaluated

for significance by analysis of variance. Post hoc comparisons between group means were made with Fisher’s Least Significant Test.

RESULTS AND DISCUSSION

Rats lesioned with 6-OHDA exhibited an enhanced locomotor response

(table 1) to LY-171555, a D2-dopamine receptor agonist. These data

Table 1. Locomotor responses to dopamine agonist in rats

lesioned with 6-OHDA.

Drug Activity Counts

Treatment (counts/150 min)

Unlesioned control Saline 884 ± 92

Unlesioned control LY-171555 2954 ± 757*

6-OHDA lesion Saline 876 ± 176

6-DHDA lesion LY-171555 31403 ± 2000*

LY-171555 (0.3 mg/kg), a D2 agonist. There are at least 8 rats in

each group. p<0.05 compared to the respective saline group.

document a functional supersensitivity of dopamine receptors and confirm previous observations (Breese et al. 1985a,b). The 6-OHDAinduced lesion reduced dopamine (>90%) in striatum; the dopamine

metabolite DOPAC and HVA were also markedly reduced. These data

clearly indicate the effectiveness of 6-OHDA treatment to destroy

dopaminergic fibers.

390
Table 2. Effect of 6-OHDA-induced lesions on various biochemical

parameters in the striatum.

Proenkephalin-A systemb

Native ME 1.34 ± 0.07 (12) 1.99 ± 0.17* (13)

Cryptic ME 0.19 ± 0.02 (12) 0.31 ± 0.02* (12)

PE-mRNA 100 ± 0 (4)

Parameters

Dopamine systema

Dopamine

DDPAC

HVA

Treatment

control 6-OHDA lesion

41.8 ± 1.8 (8) 3.2 ± 0.6 (8)

5.8 ± 0.4 (8) 0.7 ± 0.1* (8)

2.2 ± 0.2 (8) 0.5 ± 0.1* (8)

157 ± 5* (4)

Dynorphin systemC

Dynorphin-A (1-8) 20.6 ± 1.43 (12) 21.3 ± 1.53 (12)

* p<0.01 compared to respective control group. Values are mean ± SE

of the indicated number of samples given in parentheses. a) Dopamine

and its metabolites were estimated by HPLC; b) ME was estimated by

radioimmunoassay and PE-mRNA was quantitated by RNA-cDNA hybridization; c) dynorphin-A (1-8) was measured by radioimmunoassay.

The 6-OHDA lesioned group had elevated levels of ME in the striatum

as compared to levels observed in unlesioned controls (table 2).

Dynorphin-A (1-8) content was also evaluated, but no significant

change was observed between the groups. The hippocampal content of

ME or dynorphin-A (1-8) was not altered by the 6-DHDA treatments.

These results indicate regional specificity and selectivity with

regard to the increase in ME as a result of 6-DHDA-induced lesions.

In order to investigate the mechanism of this increase, the

proenkephalin content as reflected by the cryptic ME content, as well

as the PE-mRNA abundance, was assessed in the striatum. It was

found that both precursor content and PE-mRNA abundance were

increased after 6-DHDA-induced lesions (table 2).

These data provide evidence that dopaminergic denervation by 6-OHDA

administration increases major biosynthetic indices of ME in the

basal ganglia, suggesting increased transcription and/or translation

processes for this peptide. These results are consistent with other

lines of evidence (see Introduction) that dopamine can modulate the

enkephalinergic neurons in the striatum. These studies support the

hypothesis that dopaminergic neurons are inhibitory to ME-containing

neurons in the striatum and that the removal of this control results

in disinhibition of enkephalinergic neurons (Tang et al. 1983; Thal

et al. 1985; Hong et al. 1985) which, in turn, leads to enhanced

synthesis of ME. The adaptive changes in the enkephalin system

resulting from the dopamine deficiency may have relevance to clinical

syndromes with dopamine deficiency.

391
REFERENCES

Atweh, S.F. and M.J. Kuhar. Brain Res, 134: 393-405, 1977.

Breese, G.R., Baumeister, A., NapiK T.C., Frye, G.D. and Mueller,

R.A. J Pharmacol Exp Ther, 235: 287-295, 1985a.

Breese, G.R., Napier, T.C. and Mueller, R.A.. J Pharmacol Exp

Ther, 234: 447-455, 1985b.

Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rulter, W.J.

Biochemistry, 18: 5294-5299, 1979.

Hong, J.S., Yang, H.Y.T.,

383-386, 1977.

Fratta, W. and Costa, E. Brain Res, 134:

Hong, J.S., Yang, H.Y.T., Fratta, W. and Costa, E. J Pharmacol

Exp Ther, 205: 141-147, 1978.

Hong, J.S., Yoshikawa, K., Kanamatsu, T. and Sabol, S.L. Fed Proc,

44: 2535-2539, 1985.

Johnson, R.P., Sar, M. and Stumpt, W.E. Brain Res, 194: 566-571,

1980.

Mocchetti, I., Schwartz, J.P. and Costa, E. Mol Pharmacol, 28:

86-91, 1985.

Sivam, S.P., Strunk, C., Smith, D.R. and Hong, J.S. Mol Pharmacol,

In press, 1986.

Tang, F., Costa, E. and Schwartz, J.P. Proc Natl Acad Sci (USA),

80: 3841-3844, 1983.

Thal, L.J., Sharpless, N.S., Hirschhorn, I.D., Horowitz, S.G. and

Makman, M.H. Biochem Pharmacol, 32: 3297-3301, 1983.

Sabol, S.L., Yoshikawa, and Hong. J.S. Biochem Biophys Res

Comm, 113: 391-399, 1983.

Yoshikawa, K., Williams, C. and Sabol, S.L. J Biol Chem, 259:

14301-14308, 1984.

392
INCREASED ENKEPHALIN GENE EXPRESSION IN THE HIPPOCAMPUS

FOLLOWING SEIZURES

Jeffrey D. White and Christine M. Gall*

Division of Endocrinology, Health Sciences Center

SUNY, Stony Brook, NY 11794

*Department of Anatomy and Neurobiology

University of California, Irvine, CA 92717

ABSTRACT

Bilateral, recurrent seizures were induced in adult male rats by a

unilateral, electrolytic lesion of the dentate gyrus hilus. This

treatment led to a bilateral depletion of enkephalin-like

immunoreactivity in the hippocampal mossy fibers by 12 hours (h)

post-lesion, a rebound above normal levels by 24 h, a further rise by

4 days then a fall to control values by 10 days post-lesion. The

mRNA for preproenkephalin was elevated in the granule cells by 3h

post-lesion, reached a maximal 24-fold rise by 30 h then fell again

to control values by 4-10 days post-lesion.

INTRODUCTION

The mossy fiber axons of the rat hippocampus have been shown to

contain the opioid neuropeptides enkephalin and dynorphin (Gall et

al. 1981, McGinty et al. 1983). This topographic and exclusively

ipsilateral projection from dentate gyrus granule cells to CA3

pyramidal cells represents a principal relay in the circuit of

excitatory connections within the hippocampal formation. In contrast

to enkephalin's typically observed inhibitory actions, in hippocampus

enkephalin has been demonstrated to lead to a net excitation of

pyramidal cells either through inhibition of inhibitory neurons

(Zieglgansberger et al. 1979) or by changing somatic dendritic

coupling (Lynch et al. 1981). Several previous studies have

demonsytrated that enkephalin-like immunoreactivity or

preproenkephalin mRNA change in the hippocampus in response to a

variety of seizure-producing stimuli (Gall et al. 1986; Hong et al.

1980; Hong et al. 1985 a,b). Similarly, we have demonstrated that

enkephalin peptide biosynthesis is stimulated 14-fold following a

unilateral electrolytic lesion of the dentate gyrus hilus and that

this rise in peptide biosynthesis is preceeded by a rise in the

content of preproenkephalin mRNA (White et al. 1986). In the present

study we wished to monitor the time course over which enkephalin-like

immunoreactivity and preproenkephalin mRNA change in the granule

cells following the seizure-producing hilus lesion.

MATERIALS AND METHODS

Materials

Male Sprague-Dawley rats (200-250 gm) were used for these studies.

Reagents for nucleotic acid techniques were generally from Sigma and

Fisher. [ ] 32P-CTP (300 - 600 Ci/mmol) was from Amersham or ICN.

GeneScreen Plus membrane was from New England Nuclear.

393
Surgical procedures

Experimental animals were anesthetized and a lesion was placed in the

right dentate gyrus hilus using an insulated steel wire and an anodal

current of 0.8mA for 8 sec. as described (White et al. 1986).

Control (sham lesion) animals received identical surgical procedures

without current. Behavioral seizures were rated using the scale of

Racine (1972) and only those animals displaying scale 4-5 seizures

were used for the study.

Immunohistochemistry

Coronal sections through the hippocampus were processed for

enkephalin-like immunoreactivity by the peroxidase antiperoxidase

technique as described elsewhere (Gall et al. 1981).

Tissue Harvest and Nucleic acid techniques

At designated intervals following the lesion, the rats were

sacrificed by decapitation, the hippocampus contralateral to the

lesion was dissected free and divided longitudinally into region CA3

and CAl/dentate gyrus. The latter samples were used for RNA

measurement.

Total nucleic acid was prepared by extraction in urea/SDS followed by

phenol/chloroform extraction and ethanol precipitation. Total RNA

was obtained by treating these samples with RNase-free DNase,

phenol/chloroform extraction, and ethanol precipitation. Identical

concentrations of RNA was electrophoresed on denaturng agarose gels,

transferred to nylon membrane and hybridized with 32P-labeled cRNA

probe to rat preproenkephalin mRNA. Following washing, the blot was

apposed to X-ray film. The resulting autoradiographs were scanned

densitometrically for quantitation. Details are given in White et

al. (1986).

RESULTS AND DISCUSSION

In previous studies, we have demonstrated that a unilateral lesion of

the hilus leads to the generation of bilateral,

electrophysiologically identified episodes of epileptiform activity

which are initiated approximately 1-2 hours post-lesion and that

recur for 12 hours thereafter (Gall et al. 1986). Additionally, we

have demonstrated that this seizure-producing paradigm leads to a

dramatic increase in enkephalin biosynthesis in the granule cells.

Thus, 24 hours post-lesion, there is a 14-fold increase in enkephalin

peptide biosynthesis, as measured by the technique of in vivo

radiolabeling, and a similar rise in preproenkephalin mRNA (White et

al. 1986). Preliminary studies suggested that enkephalin levels

gradually returned to normal levels and the present studies were

initiated to verify and extend these initial observations.

Figure 1 illustrates the changes in the intensity of

immunohistochemical staining for enkephalin-like immunoreactivity in

intact, contralateral mossy fibers at various times post-lesion. As

394
the figure indicates, enkephalin levels are low in control animals

(a) but fall rapidly to non-detectable levels by 12-hours post-lesion

(b). By 24 hours post-lesion the staining has attained supranormal

levels and enkephalin-like immunoreactivity increases further by 2-4

days post-lesion (c). However, by 10 days post-lesion the staining

has returned to control values.

FIGURE 1: Enkephalin-like immunoreactivity in the mossy fibers of

region CA3 of a control animal (a) and, rats sacrificed at

12-hours (b), 2 days (c) and 1 month (d) post-lesion. Side

contralateral to lesion is shown.

Preproenkephalin mRNA levels

The above described changes in enkephalin-like immunoreactivity were

accompanied by a similar change in the content of preproenkephalin

mRNA in the dentate gyrus granule cells following the hilus lesion.

As shown in figure 2, preproenkephalin mRNA was elevated in the

granule cells as early as 3 hours post-lesion (2-fold above control),

reaching a peak by approximately 30 hours post-lesion (24-fold) then

declining to near baseline levels by 10 days post-lesion.

FIGURE 2: Preproenkephalin mRNA in the granule cells: The

autoradiogram of the Northern blot analysis of preproenkephalin

mRNA content in animals sacrificed at the indicated post-lesion

intervals is shown above. C=control, h=hours, d=days.

395

172-683 O - 87-14 :QL 3
The present experiments have demonstrated that enkephalin gene

expression is rapidly influenced by the consequences of the hilus

lesion. These changes appear to be relatively transient in that

enkephalin peptide and preproenkephalin mRNA levels return to control

values by 10 days following the cessation of seizures. It is worth

noting that the hilus lesion-induced changes in enkephalin gene

expression are not accompanied by changes in the relative amounts of

the several Met-enkephalin containing peptides present in the mossy

fibers. That is, despite the large increase in enkephalin peptide

biosynthesis induced by the seizures, the ratio of Met-enkephalin,

Met-enkephalin-ArgGlyLeu, Met-enkephalin-ArgPhe, and BAM 18P does not

change, suggesting a tight physiological control of the

post-translational processing of proenkephalin (White et al. 1986).

The rapid and dramatic increase in enkephalin biosynthesis within

identified hippocampal neurons in response to seizure activity makes

the hilus lesion a promising paradigm for the elucidation of

mechanisms which regulate enkephalin gene expression in brain. Given

the well-known anatomy, physiology and putative neurotransmitter

makeup of hippocampal circuitry, it will now be possible to evaluate

the separate roles played by peptide release and depletion of

cellular storage pools, neuronal excitation per se and the activation

of specific post-synaptic receptors in the regulation of enkephalin

gene expression.

REFERENCES

Gall, C., Brecha, N., Karten, H.J. and Chang, K-J. J Comp Neurol,

198:335-350, 1981.

Gall, C., Pico, R.M., and Lauterborn, J.C. Peptides, in press, 1986.

Hong, J.S., Kanamatsu, T., McGinty, J.F., Obie, J., Dyre, R.S. and

Mitchell, C.L. Fed Proc 44:425, 1985a.

Hong, J.S., Wood, P.L., Gillin, J.C., Yang, H.Y.T. and Costa, E.

Nature, 285:231-232, 1980.

Hong, J.S., Yoshikawa, K., Kanamatsu, T., McGinty, J.F., Mitchell,

C.L. and Sabol, S.L. Neuropeptides, 5:557-560, 1985b.

Lynch, G., Jensen, R.A., McGaugh, J.L., Davila, K., and Oliver, M.

Exp Neurol, 71:527-540, 1981.

McGinty, J.F., Henriksen, S.J., Goldstein, A., Terenius, L. and

Bloom, F.E. Proc Natl Acad Sci, USA, 80:589-593, 1983.

Racine, R. Electroencephalogr Clin and Neurophysiol, 32:281-294,

1972.

White, J.D., Gall, C.M. and McKelvy, J.F. J Neurosci, submitted,

1986.

Zieglgansberger, W., French, E., Siggins, G. and Bloom, F. Science,

205:415-417, 1979.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. J.F. McKelvy for assistance in

completion of this work. This work was supported by NSF BNS 8417098

and RCDA NS00915 to CMG.

396
REGULATION OF PROOPIOMELANOCORTIN (POMC) mRNA LEVELS

IN PRIMARY PITUITARY CULTURES

J.Ph. Loeffler, N. Kley, C.W. Pittius and

V. Höllt*

Dept. of Neuropharmacology, Max-Planck-Institut

*Physiologisches Institut, University of Munich,

fiir Psychiatric, D-8033 Martinsried, F.R.G.

Pettenkoferstr. 12, D-8000 München 2, F.R.G.

ABSTRACT

The effect of a variety of neurotransmitters and of corticotropin releasing factor (CRF) upon levels of mRNA

coding for proopiomelanocortin (POMC) was studied in

primary cultures of rat pituitary. Total nucleic acid was

extracted from cells, fixed on nitrocellulose and hybridized to a single-stranded (

32P-labeled DNA complementary

to 150 bases of mouse POMC mRNA. After two days incubation with CRF (10 M), the levels of POMC mRNA in both

the anterior (AL) and the intermediate lobe (IL) cells

increased by about 100%. A similar increase in the IL

cells was seen after incubation with the ß-adrenergic

agonist isoprenalin (10-6M). In contrast, dopamine (10-6

M) and GABA (10-6 M) decreased POMC mRNA levels in the IL

cells to about 50% of control levels without affecting

POMC mRNA levels in the AL cells.

lobes, POMC mRNA was decreased by the Ca2+-channel

In both pituitary

blocker D600 (10-5M) and increased by forskolin (10-6M).

This indicates that both CAMP and Ca2+ ions play an important role as intracellular messengers for the control

of POMC gene expression in normal melanotrope and corticotrope cells.

INTRODUCTION

The proopiomelanocortin (POMC) gene is expressed in both

the melanotrope cells of the intermediate (IL) and the

corticotrope cells of the anterior (AL) lobe of the pituitary. Processing of the peptide precursor, however, is

different in the two structures. In addition, there exist

major differences in the regulation of the release of

POMC-derived peptides from the two pituitary lobes. Thus,

the secretion of POMC peptides from corticotrope cells is

stimulated by CRF and inhibited by glucocorticoids. The

secretion from the melanotrope cells of IL is under inhibitory control of dopamine (DA) and aminobutyric acid

(GABA) (Demeneix et al. 1986). In contrast to CRF, which

stimulates both melanotrope and corticotrope cells (Vale

et al. 1983, Proulx-Ferland et al. 1982), DA and GABA

only interact with IL cells. There is accumulating evidence that these factors not only modulate peptide secretion but also induce a readjustment of peptide biosynthesis. Recently, we and others have shown that POMC gene

expression at the mRNA level can be modulated by pro397
longed treatment with glucacorticoids, CRF and dopaminergic drugs in vivo (Birnberg et al. 1983, Bruhn et al.

1984, Höllt and Haarmann 1984, Höllt et al. 1982, Chen et

al. 1983) (for a review see Civelli et al. 1984). However, from these in vivo results it is difficult to interpret whether these drugs act directly at the level of

pituitary cells to change POMC mRNA levels or indirectly

by altering factors released by the hypothalamus. Therefore, we measured the effect of various drugs on primary

cultures of IL and AL of rat pituitary.

MATERIALS AND METHODS

Cells were cultured as previously described (Loeffler et

al. 1985). Briefly, intermediate (IL) and anterior pituitary lobes (AL) from 30 male Sprague-Dawley rats (200 g)

were dispersed and washed with phosphate-buffered saline

(PBS) supplemented with kanamycin (50 μg/ml), 0.1% collagenase, 0.2% dispase II, 0.1% hyaluronidase, 0.05% DNase

I and 1% bovine serum albumin (BSA). Dispersion was completed after shaking for 1 h at 37°C. IL and AL cells

were plated on 24 and 48 dishes (Costar), respectively,

in BM 86 WISSLER medium (Boehringer, Mannheim, F.R.G.)

supplemented with 10% foetal calf serum. After two days

of culture, cells were kept in serum-free medium to which

drugs were added during two additional days. The cultured

cells were lysed in 300 μl of 20 mM HEPES, 1 mM EDTA and

1% SDS and the total nucleic acids (TNAs) were extracted

with phenol-chloroform. The TNAs were bound to

nitrocellulose filters according to the dot-blot

technique of White and Bancroft (1982). For

hybridization, a single-stranded cDNA complementary to

150 nucleotides of mouse POMC mRNA was used. The mouse

POMC DNA originally cloned in pBR 322 (a generous gift

from Dr. J. Roberts, New York) (Roberts et al. 1979) was

subcloned in M13 mp8 vector. A single-stranded M13 recombinant containing the message-sense insert was used as a

template for the synthesis of a cDNA with high specific

activity (109

cmp/μg DNA). Relative POMC mRNA levels are

expressed as percentage of control.

RESULTS

Treatment of cells for two days with dopamine (10-6 M)

and GABA (10-6 M) resulted in a marked decrease in POMC

mRNA levels in the IL, but not in the AL cells of the pituitary (Fig. 1). Isoprenaline (10-6 M), a B-receptor

agonist, increased the POMC mRNA levels in the IL cells

by about 100% (its effect on the AL cells has not been

measured). CRF (10-8 M) doubled POMC mRNA levels in both

IL and AL (Fig. 1).

The Ca++ channel blocker D600 (10-5 M) reduced the POMC

messenger in both pituitary lobe cells (Fig. 2) by about

50%, whereas forskolin (10-5 M), an agent which stimulates the adenylate cyclase, increased the POMC mRNA

levels in AL and IL by more than 100% (Fig. 2).

398
Fig. 1: After two initial days of culture, IL and

AL cells were treated for 48 h in serum-free medium with various drugs in the presence of 1‰

ascorbic acid. Dopamine (10-6 M) (DA), GABA

(10-6 M), CRF (10-8 M) and isoprenalin (10-6 M)

(Isop.) were used. Data are given as percentage of

control. Significance of differences are assessed

with Student's t test (*when p < 0.05).

Fig. 1 Fig. 2

Fig. 2: IL and AL cells were treated for 48 h in

serum-free medium with Ca+ + channel antagonist D600 (10-5 M) or with the adenylate cyclase

activation forskolin (10-6 M) (FK). Data are given

as percentage of control. Significance of differences are assessed with Student's t test (*when

p < 0.05).

DISCUSSION

The results of this study showed that in primary cultures of rat pituitary, various neurotransmitters directly

modulate the gene expression of POMC at the mRNA level.

This regulation can be different in the distinct pituitary lobes; thus, dopamine and GABA decrease POMC mRNA in

the IL but not in AL cells. However, for CRF a similar

increase in the POMC mRNA levels in both AL and IL has

been found (Fig. 1). In earlier experiments, we found a

marked decrease in the levels of POMC-mRNA in the IL of

rats which had been chronically infused with low doses of

CRF (Höllt and Haarmann 1984). Thus, it appears that CRF

induces changes in the activity of unknown transmitters

in vivo, which counteract its own up-regulating effect on

POMC mRNA in the IL. In both lobes, CAMP appears to play

a major role as second messenger for the changes in POMC

gene expression at the mRNA level. Indeed, forskolin,

399
which elevates intracellular CAMP by activating the adenylate cyclase, increases POMC mRNA levels. Moreover,

isoprenalin, a ß-receptor agonist, increases CAMP levels

and enhances POMC mRNA in the IL. Similarly, CRF, which

increases CAMP levels in both lobes, increases POMC mRNA

levels. The direct involvement of CAMP in the POMC mRNA

enhancing effect of CRF was recently demonstrated in

AtT20 cells (Reisine et al. 1985). Besides CAMP, Ca2+

appears to have an important function in the control of

POMC gene expression in both lobes, since the Ca2+-antagonist D600 inhibited POMC mRNA levels. It may be that

GABA exerts its effect on IL cells by decreasing their

electrical activity and thus causing a decrease in Ca

entry (Demeneix et al. 1986, Taraskewich and Douglas

1982).

 In conclusion, we have shown that a variety of substances

(dopamine, GABA, CRF, ß-adrenergic agonist) directly interact with melanotrope or corticotrope cells to modulate

POMC gene expression. These effects appear to be mediated

by CAMP and/or Ca2+ ions.

REFERENCES

Birnberg, N.C., Lissitsky, J.C., Hinmann, M., and Herbert, E. Proc Natl Acad Sci USA, 80:6982-6986,

1983.

Bruhn, T.O., Sutton, R.E., Rivier, C.L., and Vale, V.

Neuroendocrinology, 39:170-175, 1984.

Chen, C.L.C., Dionne, F.T., and Roberts, J.L. Proc Natl

Acad Sci USA, 80:2211-2215, 1983.

Civelli, O., Douglas, J., and Herbert, E. In: Gross, E.,

and Meienhofer, J., eds. The Peptides. Vol. 6. New

York: Academic, 1984. pp. 69-94.

Demeneix, B.A., Taleb, O., Loeffler, J.P., and Feltz, P.

Neuroscience, 17(4):1275-1285, 1986.

Höllt, V., Haarmann, I., Seizinger, B.R., and Herz, A.

Endocrinology, 110:1985-1991, 1982.

Höllt, V., and Haarmann, I. Biochem Biophys Res Comm,

124:407-415, 1984.

Loeffler, J.P., Kley, N., Pittius, C.W., and Höllt, V.

Neuroscience Lett, 62:383-387, 1985.

Pittius, C.W., Kley, N., Loeffler, J.P., and Höllt, V.

EMBO J, 4:1275-1260, 1985.

Prouxl-Ferland, L., Labrie, F., Dumont, D., Cote, J.,

Coy, D.H., and Sveiraf, J. Science, 217:62-63, 1982.

Reisine, T., Rougon, G., Barbet, J., and Affolter, H.U.

Proc Natl Acad Sci USA, 82:8261-8266, 1985.

Roberts, J.L., Seeburg, P.H., Schine, J., Herbert, E.,

Baxter, J.D., and Goodman, H.M. Proc Natl Acad Sci

USA, 76:2153-2157, 1979.

Taraskewich, P.S., and Douglas, W.W. Nature, 299:733-734,

1982.

Vale, W., Vaughan, J., Smith, M., Yamamoto, G., Rivier,

J and Rivier, C. Science, 312:1394-1397, 1981.

White, B.A., and Bancroft, F.C. J Biol Chem , 257: 8569-

8572, 1982.

400
REGULATION OF HYPOTHALAMIC ß-ENDORPHIN AND DYNORPHIN RELEASE

BY CORTICOTROPIN-RELEASING FACTOR (CRF)

Osborne F.X. Almeida, Kostas E. Nikolarakis and Albert Herz

Department of Neuropharmacology

Max-Planck-Institut für Psychiatric

D-8033 Martinsired. F.R.G.

ABSTRACT

Synthetic rat-human corticotropin releasing-factor (CRF), in

doses between 10-12M and 10-8M, significantly stimulates the

release of immunoreactive ß-endorphin and dynorphin-A from

rat hypothalamic slices superfused in vitro: With prolonged

exposure to CRF, there is a reduction in the release of both

opioids which can be reversed by the addition of naloxone.

The latter result indicates the involvement of autoregulatory mechanisms in the control of opioid neuronal function.

INTRODUCTION

The opioid peptide ß-endorphin is found in both the pituitary and hypothalamus (Rossier et al. 1977). While several

studies have shown that corticotropin releasing factor (CRF)

is one important modulator of pituitary ß-endorphin (Bruhn

et al. 1984; Höllt and Haarmann 1984; Vale et al, 1980), the

role of CRF in regulating hypothalamic ß-endorphin has been

only speculated upon (Nikolarakis et al. 1986; Sirinathisinghji 1985). The studies described here were done to test

the effects of CRF upon hypothalamic ß-endorphin release.

In addition, CRF effects upon the release of dynorphin-A

(dynorphin) were also investigated. Dynorphin is found in

pituitary and brain (Höllt et al. 1980), but there is no information available as to whether its secretion is modulated

by CRF.

MATERIALS AND METHODS

Hypothalami were obtained from adult male Wistar rats,

sliced (250 μM) and superfused, in pairs, with Medium 199

(100 ul.min-1) in chambers maintained at 37°C. Superfusates

were collected every 10 min., snap-frozen and later assayed

for ß-endorphin and dynorphin immunoreactivities. CRF was

added to the superfusion medium at concentrations ranging

from 10-18 M to 10-8M . In separate experiments, naloxone

(10-6M) was added to the slices together with CRF (10-8M).

RESULTS AND DISCUSSION

CRF was found to significantly stimulate the release of ßendorphin and dynorphin from rat hypothalamic slices invitro, when presented at doses of 10-12M to 10-8M. The data

therefore support earlier speculations about the stimulatory

effects of CRF upon hypothalamic ß-endorphin (Nikolarakis et

al. 1986; Sirinathisinghiji 1985) and provide evidence that

CRF also stimulates the release of dynorphin, an opioid

derived from a precursor different to the one from which

ß-endorphin is derived.

401
Fig. 1. Time course of ß-endorphin release after superfusion

with CRF 10-8M. Basal release

rates were in the range of 3-5

fmol•100μl-l.10 min-1.

Figure 1 shows the ß-endorphin response to CRF; the pattern

of response for dynorphin was similar, except that stimulation of release occured some 30-40 min. later. Of particular interest in fig. 1 is the decline in the amount of opioid released upon continued exposure to CRF. This feature

suggested to us that CRF stimulates the release of opioids

which may then act presynaptically to turn off their own

release (reminiscent of the way neurotransmitters limit

their own release). Indeed, when the opiate receptor antagonist naloxone was superfused together with CRF the attenuation of opioid peptide release was not observed. Furthermore,

there was a tendency for opioid release to increase when

naloxone was presented on its own: and these effects persist

in the presence of tetrodotoxin (TTX, 10-6M), suggesting

them to be presynaptic events. Current studies are focussed

on finding the type of ligand(s) involved in this inhibition

of opioid peptide release.

We conclude that: (1) CRF can stimulate the release of ßendorhin and dynorphin from the rat hypothalamus; (2) prolonged exposure to CRF leads to an attenuation of opioid

peptide release; and (3) naloxone prevents the loss of

response to CRF, demonstrating the involvement of autoregulatory mechanisms in the control of opioid peptide neurones.

REFERENCES

Bruhn, T.O., Sutton, R.E., Rivier, C.L., and Vale, W.W.

Neuroendocrinology, 39:170-175, 1984.

Loeffler, J-P., Kley, N., Pittius, C.W., and Höllt, V.

Neurosci Lett, 62:383-387, 1986.

Nikolarakis, K.E., Almeida, O.F.X., and Herz, A. Brain Res,

in press. 1986.

Rossier, J., Vargo, T.M., Scott, M., Ling, N., Bloom, F.E.

and Guillemin, R. Proc Natl Acad Sci USA, 74:5162-5165,

1985.

Sirinathsinghji, D.J.S. Brain Res, 336:45-55, 1985.

Vale, W.W., Spiess, J., Rivier, C., and Rivier, J. Science,

213:7394-1396, 1980.

ACKNOWLEDGEMENTS

This work was supported by the DFG (SFB220), Bonn.

402
INHIBITION OF LH RELEASE BY CRF MAY BE PARTIALLY MEDIATED

THROUGH HYPOTHALAMIC ß-ENDORPHIN RELEASE

Kostas E. Nikolarakis, Osborne F.X. Almeida and Albert Herz

Department of Neuropharmacology

Max-Planck-Institut für Psychiatric

D-8033 Martinsried, F.R.G.

ABSTRACT

Corticotropin releasing factor (CRF) given i.c.v. at doses

of 0.02-2 nmol, significantly inhibited luteinising hormone

(LH) secretion in castrate male rats. The luteinising hormone releasing hormone (LHRH) release from hypothalamic

slices Superfused in vitro with CRF (10-12M - 10-8Ml was

markedly reduced. In rats that had been infused with naloxone 2 days prior to testing with 0.2 nmol CRF, the effect

of the CRF was significantly reduced. Together, these findings indicate that CRF inhibits LH secretion by inhibiting

LHRH release from the hypothalamus, and that the latter

action involves endogenous opioid peptides.

INTRODUCTION

Injections of corticotropin-releasing factor (CRF) into the

mesencephalic central gray area of the brain inhibits sexual receptivity in the female rat (Sirinathsinghji 1985),

and its injection into the third ventricle reduces serum

luteinising hormone (LH) concentrations (Rivier and Vale

1984). Since opioids inhibit LH secretion (see Millan and

Herz 1985) and CRF modulates ß-endorphin levels in the

pituitary (Bruhn et al. 1984; Höllt and Haarmann 1984) and

hypothalamus (Almeida et al. this volume), an involvement

of opioids in the CRF inhibition of LH has been postulated

(Sirinathsinghji 1985; Nikolarakis et al. 1986). In view of

our recent data, showing CRF stimulating the release of ßendorphin and dynorphin from the hypothalamus (Almeida et

al. this volume), the possibility that hypothalamic, rather

than pituitary, opioids might mediate the CRF action arises.

MATERIAL AND METHODS

1. In vivo experiment:

Adult male Wistar rats were implanted with i.c.v.

stainless steel guide cannulae. 5 days later they were

bilaterally castrated and had a cannula placed into a

jugular vein. Some rats were also implanted with osmotic minipumps containing naloxone (released at a rate

of 9.6 mg/kg/day), starting 48h prior to, and during,

treatment with CRF. CRF was applied i.c.v. 5 days

after castration in doses ranging from 0.02 to 2 nmols.

Blood was withdrawn at various time intervals via the

jugular cannula and the serum was assayed for LH concentrations.

2. In vitro experiment:

Hypothalami were dissected from normal adult male

Wistar rats following decapitation, and superfused as

described elsewere (Nikolarakis et al. 1986). CRF was

403
added in the superfusion medium at concentrations ranging from 10-12 to 10-8M. Ten minute pools of superfusate were collected and assayed for LHRH.

RESULTS AND DISCUSSION

CRF at all doses tested (0.02, 0.2 and 2 nmoles) produced

significant reductions in serum LH levels within 30 min of

i.c.v. administration. LH levels remained significantly

lower in rats receiving 0.2 or 2 nmoles for up to 6 hours,

whereas LH secretion was not significantly different 2 hours

after treatment with the lowest dose. In the presence of the

opioid antauonist naloxone the CRF (0.2 nmol) inhibition of

LH was significantly reduced (fig. 1).

Fig. 1. Serum LH

levels after CRF in

castrates pretreated

with either water or

naloxone via osmotic

mini-pumps. LH levels

in untreated castrates

shown for comparison.

Furthermore, rats treated with naloxone showed a faster

recovery of LH levels than did the animals that received

CRF alone. In our in vitro experiments basal LHRH release

was suppressed in a dose dependent way after CRF application (fig. 2).

Our findings are in accordance with a previous report

(Rivier and Vale 1984) showing that centrally administered

CRF is a potent inhibitor of LH secretion in recently castrated male rats. Although naloxone could block the CRF inhibition of lordosis behaviour in the rat (Sirinathsinghji

1985) , suggesting an opioid involvement, the application of

a different antagonist, naltrexone, did not affect the CRF

inhibition of LH (Rivier and Vale 1984), leading the latter

authors to question an involvement of opioids. Our results,

showing that chronic opiate blockade leads to a reduced

inhibition at LH by CRF, indicates an opioid involvement in

this CRF action. CRF probably inhibits LH by reducing the

release of hypothalamic LHRH. The inhibitory actions of CRF

within the hypothalamic neuronal pathways affecting LHRH

404
Fig. 2. Dose-response inhibition of LHRH release

from hypothalamic slices in vitro.

release could include the release of opioids. In fact, we

have found that CRF increases both ß-endorphin and dynorphin

release from hypothalamic slices (Almeida et al. this volume.

In conclusion, we suggest that CRF inhibits LHRH and

secretion by stimulating the release of opioids such

ß-endorphin and dynorphin from hypothalamic sites.

REFERENCES

LH

as

Bruhn, T.D., Sutton, R.E., Rivier, C.L., and Vale, W.W.

Neuroendocrinology, 39:170-175, 1984.

Höllt, V., and Haarmann, I. Biochem Biophys Res Commun,

124:407-415, 1984.

Millan, M.J., and Herz, A. Neurobiology, 26:1-84, 1985.

Nikolarakis, K.E., Almeida, O.F.X., and Herz, A. Brain Res

in press, 1986.

Rivier, C., and Vale, W. Endocrinology, 114:914-921, 1984.

Sirinathsinghji, D.J.S. Brain Res, 336:45-55, 1985.

405
INCREASED SPINAL CORD DYNORPHIN mRNA

DURING PERIPHERAL INFLAMMATION

Michael J. Iadarola, J. Douglass,* O. Civelli* and J. R. Naranjo**

Neurobiology and Anesthesiology Branch,

National Institute of Dental Research,

NIH, Bethesda, MD 20892

*Institute for Advanced Biomedical Research,

Oregon Health Sciences University

Portland, OR 97201

**FIDIA-Georgetown Institute for the Neurosciences,

Georgetown University Medical and Dental Schools,

Washington, D.C. 20007

ABSTRACT

Injection of a complete Freunds adjuvant-saline emulsion into the

hindpaw of the rat induces a peripheral inflammatory process

accompanied by pronounced increase (3 fold) in spinal cord dynorphin

content. A marked increase (-9 fold) in the mRNA coding for the

preprodynorphin precursor accompanies the increase in peptide. The

parallel elevations of the mRNA and peptide product suggest that an

increased activity of dynorphin neurons occurs in spinal cord in

response to altered afferent input due to the peripheral inflammatory

process.

INTRODUCTION

Chronic inflammatory states, as exemplified by the adjuvant-induced

arthritis model, have been associated with an increase in spinal cord

met5-enkephalin content (Cesselin et al. 1980) and more recently

dynorphin (Millan et al. 1985, 1986). Using a peripheral, more acute

inflammation model we have found that spinal dynorphin is subject

to a rapid elevation in its content (Iadarola et al. 1985). The

increase is specific to the spinal segments innervating the inflamed

limb and peaks between 5-8 days. A basic problem in interpreting

these data revolves around whether an elevated peptide content reflects an increase or decrease in peptide utilization. We have

measured the content of preprodynorphin mRNA in spinal cord tissue

during the peripheral inflammation. Our results indicate that the

dynorphin peptide increase is accompanied by a pronounced increase in

preprodynorphin mRNA accumulation.

MATERIALS AND METHODS

Inflammation of one hind paw was induced by intraplantar injection

(100 ug) of a saline-Freunds adjuvant (Calbiochem) emulsion in 350 g

male Sprague Dawley rats. Treated rats were killed by decapitation

after 5 days of inflammation; a group of non-treated rats was killed

at the same time. The whole spinal cord was removed from the spinal

canal by injection of saline at the sacral vertebral level (De Sousa

& Horrocks 1979). The dorsal lumbar spinal cord was dissected and

frozen on dry ice until use. RNA was prepared as described by

Chirgwin et al. (1979) using homogenzation in guanidine thiocyanate

406
and centrifugation of RNA through cesium chloride. Dynorphin peptides

were assayed in the guanidine thiocyanate supernatants from the above

RNA preparation as described by Naranjo et al. (1986); the specificity of the antibody is described in Iadarola et al. (1986).

Northern blots were prepared with poly A

+

 enriched RNA and were

hybridized with plasmids pBgBa and pBaBa labeled with 32P-dCTP and

-dATP to a specific activity of 1-3 x 105 cpm/ng. The two plasmids

each contain 800-900 base pairs of genomic DNA from the major exon

and represent, respectively, the protein coding and non-coding regions of rat preprodynorphin mRNA (Civelli et al. 1985). After

autoradiography the blots were washed (Byrd et al. 1986; Milner and

Sutcliffe 1983) and probed again with a cDNA to the rat ß-actin

message (Nudel et al. 1983) in order to standardize each lane for

slight variation in the amount of poly A

+

 RNA applied. Both

autoradiograms were scanned with a laser densitometer and the data

are expressed as a ratio of the dynorphin densitometric signal to the

ß-actin signal.

RESULTS

A single hybridization band was observed after agarose gel electrophoresis conducted under denaturing conditions (Tang et al. 1983).

This indicates that no heterogeneity in message size (Civelli et al.

1985) occurs in dorsal spinal cord preprodynorphin mRNA in the

inflamed condition compared to control (Fiagure 1). At 5 davs of

inflammation a marked elevation of preprodynorphin mRNA is evident.

The RNA blot was hybridized

with labeled dynorphin DNA

probes. Dorsal spinal cord

RNA was extracted at day 5

of inflammation (treated) and

processed in parallel with

untreated rats (control). The

increased density of the band

from the inflamed condition

is apparent. There appears to

be some nonspecific binding

or tailing into the nearby

band of 18 S rRNA; this was

not included in the densitometric analysis.

Figure 1

Increase in preprodynorphin mRNA

after inflammation

This same blot was rehybridized with a rat ß-actin cDNA (not shown).

Normalization of densitometric scan from the dynorphin blot with that

407
from the ß-actin blot showed that about a 9 fold increase in dynorphin mRNA had occurred in the inflamed condition (Figure 2).

Measurement of dynorphin A 1-8 content from the guanidine thiocyanate

supernatent from this experiment and several other similar RNA preparations showed that the mRNA increase was accompanied by approximately a 3 fold increase in this dynorphin peptide (Figure 2).

The peptide content

of several guanidine

thiocyanate RNA

preparations was

analyzed (Naranjo et

al. 1986) for

dynorphin A 1-8

content (left side).

The histograms at

the right show the

ratio of dynorphin

mRNA to ß-actin

mRNA based on the

areas under the respective peaks after

densitometric scanning. The ß-actin

cDNA probe was used

to standardize the

lanes for the amount

of poly A

+

 RNA applied.

FIGURE 2

Comparison of increase in dynorphin

A 1-8 peptide and dynorphin mRNA

after inflammation

DISCUSSION

The data presented here show that a peripheral inflammation induces

an increase in dynorphin mRNA content and dynorphin A 1-8 immunoreactivity. These observations are consistent with the idea that

the increase in peptide occurs as a result of an activation of

dynorphin gene transcription in neurons located in dorsal lumbar

spinal cord. Since mRNA transcription and translation generally

occur in the neuronal cell body, our data also minimize the possibility that the dynorphin peptide increase arises from alternative

neuronal sources such as the dorsal root ganglia (Botticelli et al.

1981, Basbaum et al. 1986) or a supraspinal nucleus.

The data suggest that spinal cord dynorphin-containing neurons are in

an altered state of activity produced by the afferent input arising

from the inflamed limb. The concurrent elevation of both mRNA and

peptide product support the idea of an increase in peptide synthesis

and utilization. However, the precise mechanism whereby the increase

408
in mRNA occurs (for review see Schwartz and Costa 1986) and whether

peptide release is increased requires further examination.

In conclusion, the data presented suggest an enhanced activity of

spinal cord dynorphin neurons during a peripheral inflammation and

indicates the participation of these neurons in the sensory processing or modulation of pain at the spinal level.

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Millan M.J.,

Brain Res, 340:156-159, 1985.

Millan, M.H., Czlonkowski, A., Hollt, V., Pilcher,

C.W.T., Herz, A., and Colpaert, F.C. J Neurosci, 6:899-906, 1986.

Milner, R.J., and Sutcliffe, J.G. Nucleic Acid Res, 11:5497-5520,

1983.

Naranjo, J.R., Iadarola, M.J., and Costa, E. J Neurosci Res, in

press.

Nudel, U., Zakut, R., Shani, M., Neuman, S., Levy Z., and Yaffe, D.

Nucleic Acid Res, 11:1759-1771, 1983.

Schwartz, J.P., and Costa E. Ann Rev Neurosci 9:277-304, 1986.

Tang, F., Costa, E., and Schwartz, J.P. Proc Natl Acad Sci USA, 80:

3841-3844 1983.

409
DIFFERENTIAL EFFECTS OF SELECTIVE BRAIN LESIONS ON HYPOTHALAMIC

AND PITUITARY IR-DYNORPHIN

Santi Spampinato, Sanzio Candeletti, Emanuela Cavicchini,

Patrizia Romualdi, Ester Speroni and Sergio Ferri

Institute of Pharmacology, University of Bologna, Irnerio 48

40126 Bologna, Italy.

ABSTRACT

Selective radiofrequency and neurotoxic lesions of the serotonergic

pathways caused in the rat a significant reduction of ir-dynorphin A

and Bin the hypothalamus but not at pituitary level. These data confirm that dynorphin-related peptides are regulated independently in

these areas.

INTRODUCTION

Several studies support the view that differential neural and endocrine factors participate in the regulation of immunoreactive dynorphin (ir-DYN) at hypothalamic and pituitary level (Höllt et al.1981,

Spampinato et al. 1986a. Spampinato et al. 1986b). Thus, it seems

reasonable to hypothesize that hypothalamic and pituitary ir-DYN pools

may be influenced by aminergic neurons, since biogenic amines act as

neurotransmitters in these areas. Millan et al. (1984) reported that

noradrenergic neurons regulate ir-DYN in the neurointermediate lobe

of the pituitary. However, little is known about possible interactions between the dynorphinergic and serotonergic systems. To ascertain this possibility we examined the changes of hypothalamic and pituitary ir-DYN A and B following neurotoxic and radiofrequency lesions

of serotonergic neurons.

MATERIALS AND METHODS

Neurotoxic lesions

Male Sprague-Dawley rats (220-250 g) bearing chronic intracerebroventricular (icv) cannulae received 5,7 dihydroxytryptamine creatinine

sulphate (5,7 DHT, 200 μg/rat, icv) combined with desipramine (25

mg/kg, i.p., 60 min prior to 5,7 DHT).

Lesions of the medial and dorsal raphe nuclei

Male Sprague-Dawley rats (220-250 g) were anaesthetized and placed in

a stereotaxic apparatus. Radiofrequency lesions were performed by

nickel-chrome electrodes, according to the Pellegrino et al. (1979)

stereotaxic atlas and pilot studies. The coordinates adopted for the

medial raphe nucleus were: antero-posterior (AP) -6.3 mm, lateral (L)

1.3 mm, depth (D) -8.6 mm at an angle of 8°; for the dorsal raphe nucleus were: AP -6.2 mm, L 0.8 mm, D -6.3 mm, at an angle of 7°.

410
Sham lesions were performed in an identical manner with unactivated

electrodes. Histological examinations were made in all lesioned animals to ascertain the correct position and size of lesions.

Preparation and processing of tissue extracts

Two weeks after lesions rats were killed by decapitation. Hypothalamic

and pituitary tissues were processed and ir-DYN A and B measured by

radioimmunoassay as extensively reported (Spampinato et al.l986a, Cone

and Goldstein 1982).

RESULTS AND DISCUSSION

As shown in table 1, ir-DYN A and B were significantly reduced in the

hypothalamus after the 5,7 DHT treatment, while there was no change

in the pituitary gland. This neurotoxin causes a prolonged reduction

of serotonin in whole brain, including the hypothalamic area, as a

consequence of the destruction of serotonergic neural terminals

(Baumgarten et al. 1977). It is likely therefore, that the reduction

of serotonin levels in the hypothalamus might affect in some way the

dynorphinergic system.

Table 1

Effect of 5,7 DHT treatment on hypothalamic and pituitary

content of ir-DYN A and ir-DYN B.

Area examined

ir-DYN A ir-DYN B

Treatment (fmol/μg protein) (fmol/μg protein)

Hypothalamus Control 0.44±0.09 1.81±O.11

Treated 0.27±0.04** 1.25±0.13**

Anterior pituitary Control 1.70±0.11 7.51±0.76

lobe Treated 2.44±0.29 9.05±1.42

Neurointermediate Control 9.30±0.96 47.04±4.09

pituitary lobe Treated 11.56±1.81 51.14±5.76

Each group consisted of 8-10 rats.

** p < 0.05 vs control rats of the same group (Student's two-tailed

t-test).

After destruction of dorsal and medial raphe nuclei, from which originates the major serotonergic input to the hypothalamus (Van de Kar and

Lorens 1979). a decrease of ir-DYN A and ir-DYN B occurred in this

area (table 2). On the contrary, the levels of both peptides remained

unmodified in the pituitary gland (table 2). Thus, these results are

411
in agreement with those reported above and, taken together, give evidence of an influence of the serotonergic system on hypothalamic dynorphins.

This hypothesis is further supported by the well known interactions

between other opioid peptides and the serotonergic neurons at hypothalamic level (Spampinato et al. 1979).

Table 2

Effect of medial and dorsal raphe nuclei lesions on hypothalamic and pituitary content of ir-DYN A and ir-DYN B.

Area examined ir-DYN A ir-DYN B

Treatment (fmol/μg protein) (fmol/μg protein)

Hypothalamus Control 0.40±0.08 1.98±0.17

Lesioned 0.26±0.05** 1.47±0.27**

Anterior pituitary Control 1.34±0.05 8.79±0.39

lobe Lesioned 1.28±O.08 9.53±0.84

Neurointermediate Control 10.01±1.37 64.08±1.60

pituitary lobe Lesioned 8.64±1.54 63.22±3.25

Each group consisted of 8-10 rats.

** p < 0.05 vs control rats of the same group (Student's two-tailed

t-test).

The existence of a differential regulation of dynorphin-related peptides by the serotonergic system is consistent with a recent paper of

Majeed et al. (1985), who found a decrease of duodenal, but not hypothalamic, ir-DYN after an acute treatment with drugs known to activate

the serotonergic system. However, the discrepancy with our findings

as regards hypothalamic ir-DYN, may be related to the fact that we

achieved a chronic and marked depletion of brain serotonin.

REFERENCES

Baumgarten, H.G., Lachenmayer, L., and Björklund, A. In: Myers, R.D., ed. Methods in Psychobiology. Vol.I. New York: Academic Press, 1977. pp.49-98.

Cone, R.J., and Goldstein, A. Neuropeptides, 3:97-106, 1982.

Höllt,V.,Haarman,J.,Seizinger,B.R.,and Herz, A. Neuroendocrinol, 33:333-

-339,1981.

Majeed, N.H., Lason, W., Przewlocka, B.,and Przewlocki, R. Life Sci, 37:339-

-343,1985.

Millan, M.J., Millan, M.H., Czlonkowski, A.,and Herz, A. Brain Res, 298:243-

-252,1984.

412
Pellegrino, L.J., Pellegrino, A.S., and Cushman, A.J. A Stereotaxic Atlas of

the Rat Brain. New York, Plenum Press, 1979.

Spampinato, S., Locatelli, V., Cocchi, D., Vicentini, L., Bajusz,S., Ferri, S.,

and Müller, E.E. Endocrinol, 105:163-171, 1979.

Spampinato, S., Candeletti, S., Russo, A., Leanza, G., Stanzani, S., and Ferri, S.,

Life Sci, 38:403-408, 1986a.

Spampinato, S., Stanzani, S.,and Ferri, S. Neurosci Lett, 65:109-113, 1986b.

Van de Kar, L.D., and Lorens, S.A. Brain Res, 162:45-54, 1979.

413
MODULATION OF ß-ENDORPHIN SECRETION FROM MOUSE PITUITARY TUMOR

CELLS BY CALMODULIN INHIBITOR W7

Beatrice C. Beaubien and Edward Herbert

Institute for Advanced Biomedical Research

The Oregon Health Sciences University

3181 SW Sam Jackson Park Road

Portland, Oregon U.S.A.

ABSTRACT

The involvement of calmodulin in the secretion of ß-endorphin from

the mouse anterior pituitary tumor cell line, AtT-20, was

investigated. The calmodulin inhibitor W7 potentiated secretion

produced by 8-BrcAMP, and induced a secretory response to arginine

vasopressin, which did not elevate ß-endorphin levels when added

alone. Release of hormone in response to CRF was not affected.

Calmodulin phosphodiesterase inhibitor 8-MeOMeMIX produced a dosedependent increase in 8-BrcAMP stimulation, suggesting that

inhibition of cAMP degradation is the mechanism of enhancement of

8-BrcAMP-induced secretion in the presence of W7.

INTRODUCTION

The mouse anterior pituitary tumor cell line AtT-20 secretes ACTH

and R-endorphin in response to a variety of receptor-mediated

events and to cAMP analogues (Reisine et al. 1982; Axelrod and

Reisine 1984). The participation of the adenylate cyclase/protein

kinase A pathway AtT-20 cells share with corticotrophs and other

endocrine cells in the anterior pituitary (Bilezikjian and Vale

1983; Brazeau et al. 1982; Labrie et al. 1982; Aguilera et al.

1983). It has recently been demonstrated that Ca2+-channel

antagonists are capable of elevating cAMP levels and CAMP-dependent

responses in the presence of growth hormone releasing factor and

forskolin in somatotrophs and GH4 cells respectively (Barinaga et

al. 1985; Waterman et al. 1985). This Ca2+-dependent modulation of

cAMP processes suggests a Ca2+ -sensitive component in either the

biosynthesis or degradation of the cyclic nucleotide. As there is

evidence that the calmodulin (CaM)-dependent phosphodiesterase can

contribute to decreasing cAMP levels (Miot et al. 1984; Erneux et

al. 1985), and Murakami et al. (1985) have demonstrated the

involvement of CaM in ACTH secretion in anterior pituitary

(Murakami et al. 1985), experiments were conducted with a CaM

inhibitor and an inhibitor of the CaM-dependent phosphodiesterase

to investigate any contribution this system might have in AtT-20

cell secretion.

MATERIALS AND METHODS

Release Experiments

AtT-20 cells were plated in 24 well Primaria plates (Falcon) at an

initial density of 1 x 105

cells/ml/well in Dulbecco's Modified

Eagle Medium (DMEM, Gibco) with 10% fetal calf serum (HiClone), and

grown at 37°C in 5% CO2. Incubations with regulators were

conducted three or four days after plating in 0.1% bovine serum

albumin (BSA, Sigma) in DMEM. 414
CaM inhibitor W7 (6-aminohexyl-5-chloronapthalene-1-sulfonamide,

Sigma) and CaM-dependent phosphodiesterase inhibitor 8-MeOMeMIX (1-

methyl-3-isobutyl-8-methoxy methylxanthine (a gift from Dr. Jack

Wells, Vanderbilt University, Nashville) were dissolved in ethanol

and DMSO respectively, diluted in DMEM and added 15 minutes before

the secretagogues. Corticotropin releasing factor (CRF,

Penninsula) arginine vasopressin (AVP, Sigma) and 8-BrcAMP (8 bromo

adenosine 3':5'-cyclic monophosphate, Sigma) were introduced to the

wells and the incubation continued for a further three hours at

37°C. The medium was then removed and stored at -20°C or assayed

immediately.

RIA

The ß-endorphin immunoreactivity (B-End I.R.) was assayed using

Sugar antisera (a gift of Dr. Richard Allen). The antibody

recognizes the mid portion of ß-endorphin and has a titre of

1:2,000.

Aliquots of medium were incubated with antibody and 125I-labelled

ß-endorphin overnight in 200 ul PBS, 0.1% BSA, 0.1% Triton X-LOO at

4°C and antibody-bound label was precipitated the next day with 500

ul 2% goat antirabbit gamma globulin, 0.2% normal rabbit serum

(both from Breit Laboratories) in 1.2 M ammonium sulphate in 10 mM

sodium phosphate buffer, pH 7.2, at 4°C. Tubes were centrifuged in

a Beckman J6B centrifuge and pellets were counted on a Micromedic

Apex 10/600 gamma counter.

RESULTS

AVP.

CaM inhibitor W7 at 10 uM did not affect basal secretion, and no

effect on CRF-stimulated secretion was observed (fig. la), however,

the response to 8-BrcAMP was considerably enhanced, and the

addition of W7 appeared to increase R-End I.R. in the presence of

FIGURE 1a. Incubation of AtT-20 cells with regulators

of secretion in the presence (closed bars) and absence

(open bars) of 10 uM W7 and conducted as described in

materials and methods. Concentrations of compounds

were as follows: CRF, 30 nM; 8-BrcAMP, 500 uM; AVP,

100 nM. Bars show the mean and standard deviation of

at least two experiments conducted in triplicate.

4l5
FIGURE 1b. Dose-response curve of W7 in the presence

(closed bars) and absence (open bars) of 500 uM 8-Br

CAMP. Each point represents the mean and standard

deviatio of a representative experiment conducted in

triplicate.

Figure lb shows the dose-related increase in ß-end I.R. in response

to W7 in the presence of 500 uM 8-BrcAMP. The increase over

control is particularly marked at 30 uM W7, which represents the

ID50 for this inhibitor when tested in intact cells (Hidaka and

Tanaka 1985).

The capacity of 8-MeOMeMIX to potentiate 8-BrcAMP secretion is

shown in figure 2. No increase is apparent at 2 uM, the reported

ID50 for this compound (Miot et al. 1984); only at 30 uM and 100 uM

8-MeOMeMIX is an increase over 8-BrcAMP alone apparent.

FIGURE 2. Dose-response curve of 8-MeOMeMIX in the

presence (closed bars) and absence (open bars) of 500

uM 8-BrcAMP. The experiments were conducted twice

with similar results. The values shown are the means

and standard deviations from a representative

experiment conducted in triplicate.

DISCUSSION

The data presented here suggests the involvement of an

inhibitory calmodulin function, perhaps at the level of cAMP

degradation, in AtT-20 cells.

Murakami et al. (1985) found that in pituitary halves, W7

inhibited CRF-stimulated secretion at 100 uM, a concentration

which potentiated 8-BrcAMP-stimulated secretion in AtT-20 cells

in the experiments described in figure 2. This difference could

be due to an altered signal transduction mechanism in AtT-20's

or to the difference in secretagogues used. In the above paper

a small increase in both ACTH release and cAMP levels in

response to CRF was observed at 10 uM W7.

In the absence of direct measurement of CAMP levels, the role of

a calmodulin dependent phosphodiesterase can only be inferred by

the data presented here. In thyroid and astrocytoma cells this

type of phosphodiesterase activity is associated with capacity

of muscarinic agonists to attenuate increases in cAMP (Miot et

al. 1983; Miot et al. 1984; Meeker and Harden 1982), and occurs

416
concomitantly with phosphatidylinositol turnover (Masters et al.

1984). Although the contribution of this system in AtT-20 cells

has not been described, the basal activity of the CaM

phosphodiesterase may indicate that this activity could be

increased in response to a receptor mediated event.

It is clear from the data presented here and elsewhere that

further work is needed to clarify the effects observed when cAMP

and CAMP-mediated functions are elevated by calcium-channel

blockers, calmodulin inhibitors and calmodulin-sensitive

phosphodiesterase inhibitors.

REFERENCES

Aguilera, G., Harwood, J.P., Wilson, J.X., Morell, J., Brown,

J.H., and Catt, K.J. J Biol Chem, 258:8039-8045, 1983.

Axelrod, J. and Reisine, T. Science, 224:452-459, 1984.

Barinaga, M., Bilezikjian, L.M., Vale, W.W., Rosenfeld, M.G.,

and Evans, R.M. Nature, 314:279-280, 1985.

Bilezikjian, L.M, and Vale, W. Endocrinology, 113:1726-1731,

1983.

Brazeau, P., Ling, N., Esch, F., Bohlen, P., Mougin, C., and

Guillemin, R. Biochem Biophys Res Comm, 109:588-594, 1982.

Erneaux, C., Van Sande, J., Miot, F., Cochaux, P., Decoster, C.,

and Dumont, J.E. Mol Cell Endocrinol, 43:123-134, 1985.

Hidaka, H. and Tanaka, T. In: Hikada, H. and Hartshorne, J.D.,

eds. Calmodulin Antagonists and Cellular Physiology. New York,

Academic Press, 1985.

Labrie, F., Veilleux, R., Lefevre, G., Coy, D.H., Sueiras-Diaz,

J., and Schally, A.V. Science, 216:1007-1008, 1982.

Masters, S.B., Harden, T.K., and Brown, J.H. Mol Pharmacol,

26:149-155, 1984.

Meeker, R.B. and Harden, T.K. Mol Pharmacol, 22:310-319, 1982.

Miot, F., Dumont, J.E., and Erneux, C. FEBS Lett, 151:273-276,

1983.

Miot, F., Erneux, C., Wells, J.N., and Dumont, J.E. Mol

Pharmacol, 25:261-266, 1984.

Murikami, K., Hashimoto, K., and Ota, Z. Neuroendocrinology,

41:7-12, 1985.

Reisine, T., Heisler, S., Hook, V.Y.H., and Axelrod, J. Biochem

Biophys Res Comm, 108:1251-1257, 1982.

Waterman, M., Murdoch, G.H., Evans, R.M., and Rosenfeld, M.G.

Science, 229:267-269, 1985.

ACKNOWLEDGEMENTS

The kind gift of 8-MeOMeMIX from Dr. Jack Wells, Vanderbilt

University, Nashville, Tennessee, is gratefully acknowledged.

Thanks are due to Dr.

125I-ß-endorphin,

Richard Allen for gifts of antiserum,

and ß-endorphin standard, and to Olli

Vuolteenhao for sharing his RIA methodology. B.C.B. is a

recipient of a Medical Research Council of Canada Research

Fellowship. E.H. work was supported by NIH grant #AM37231.

417
RELEASE OF I-R METENKEPHALIN FROM

RAT AMYGDALA SLICES IN VITRO

A. Livingston & B. Morris *

Dept. of Pharmacology,

Medical School, Bristol, BS8 ITD, U.K.

*Present address Max Planck Institut für Psychiatric,

Am Klopferspitz 18A, D8033 Planegg-Martinsried, Germany.

ABSTRACT

Release of immuno-reactive metenkephalin has been demonstrated from

slices of rat amygdala in vitro. This release was calcium dependent

and the amount released was related to the concentration of

potassium ions in the releasing stimulus. The effect of drugs such

as clonidine, an alpha 2 adrenoceptor agonist, and idazoxan, an

antagonist, on this release was measured. Neither drug had a

significant effect on the amount of ir-metenkephalin released. Thus

although release of noradrenaline can be affected by opioids it seems

that release of endogenous opioids is not affected by drugs which

interact with noradrenaline.

INTRODUCTION

Several groups of workers have studied the stimulated release of irmetenkephalin (ir-ME) from slices of rat brain taken from various

regions e.g. Henderson et al. (1978) corpus striatum, Iversen et al.

(1978) qlobus pallidus, Bayon et al (1978) qlobus pallidus, Richter

et al. (1980) corpus striatum, Lindberg and Dahl (1981) corpus

striatum and Del Rio et al, (1983) corpus striatum and periaqueductal

grey. The majority of the groups have confined their studies to

areas which are relatively rich in their content of enkephalins. In

this study we have investigated an area of the brain which has a

lower content of enkephalins (Hong et al.1977) but which shows a

high level of alpha-2 adrenoceptor agonist binding (Unnerstall et al,

(1984), and we have examined the effects of drugs active at alpha-2

adrenoceptor sites on the potassium stimulated release of irmetenkephalin in order to examine if these drugs can have any effects

on enkephalin release. There have been reports of interaction

between alpha-2 agonist druqs and opioid induced changes in neuronal

function (Aghajanian 1978). and also in behavioural studies (Tsenq

et al. 1975), but the exact nature of the sites of interaction of

these two groups of drugs is still not clear.

MATERIALS AND METHODS

Adult male Wistar rats (200-300 g body wt.) were decapitated and the

brains removed onto ice. The amygdalae were dissected out, and

tissue slices (1.0x0.4x0.4mm) were prepared on a McIlwain tissue

chopper. The slices were placed in superfusion chambers and superfused with an oxygenated physiological saline solution (PSS) pH7.2

containing (mM) NaCl 118, KCl 5, KH2P04 1.2, MgSO4 1.0, CaCl2 0.75,

NaHCO3 25, glucose 11, ascorbic acid 1.0. In addition 1g/l bovine

serum albumen and 50mg/l bacitracin were included to reduce peptide

breakdown. The slices were superfused for ten minutes to

418
equilibrate at a rate of 0.5ml/min. Four eight minute fractions

were then collected with the first four minutes of the third

fraction beinq exposed to PSS containing an elevated potassium ion

concentration (balanced bv a reduction in sodium ion concentration).

Normally this elevated K+-concentration was 35mM. At the end of

each experiment the tissue was removed from the chambers and

processed for radioimmunoassay as described by Morris & Livingston

(1984). The fractions were extracted on Sep-Pak disposable ODS

columns and also processed for radioimmunoassay. The radioimmunoassay used an antibody to the sulphoxide form of metenkephalin which

was generously provided by the group working at St. Bartholomew's

Hospital, London. This antibody was highly specific and showed

negligible cross-reactivity, and a sensitivity down to 1 femtomole

per assay tube (Clement-Jones et al.1980). The amounts of ir-ME

released were calculated as a fractional release of the total

present in the tissue at the start of the release period for each

fraction.

RESULTS

Preliminary experiments showed that the ir-metenkephalin released

produced parallel displacement curves in the radioimmunoassay with

authentic metenkephalin and that the PSS and raised K+

had no

intrinsic effects on the radioimmunoassay. The recovery of ir-ME,

calculated by adding a known amount of ME to the PSS, was 35%.

Basal release was 5-20 pq/min representing about 0.1-0.2% of tissue

stores. The amount of ir-ME released was dependent on the K+

concentration used, 25mM K+ produced only a slight rise in release,

35mK+ raised release to about 0.8%, 45mM K+ raised release to about

1.2% and 55mM K+ raised it to about 2%. The release of ir-ME was

also dependent on the presence of Ca++ions and omission of Ca++ ions

completely abolished 35mM K+stimulated release (Fig. 1). 35mM K+ was

chosen as the release stimulus for the subsequent experiments. In

the presence of 1 μm clonidine, the alpha-2 adrenoceptor agonist, from

the start of perfusion there was no significant change in 35mM K+

stimulated release of ir-ME. similarly in the presence of 1 μm

idazoxan (RX781094), the alpha-2 adrenoceptor antagonist, there was

no significant change (Fig. 2).

DISCUSSION

The recovery rates of ir-ME found in these experiments are similar to

those obtained by Iversen et al.(1978) and Bayon et al.(1978),

however some workers (Richter et al.1979) have found higher

recoveries using faster perfusion rates. It would appear that

recovery rates do vary with the experimental conditions used. The

values for basal release of 0.1-0.2% agree well with most of the

other reports (Iversen et al.1978, Sawynok et al.1980, Lindberg and

Dahl 1981). Similarly,

response to K+

the degree of increase in release in

stimulation was comparable with the other reports

although comparison is difficult because of the variety of values for

K

+

 utilised in the different studies. The concentrations of

clonidine and idazoxan used were sufficient to produce significant

effects in studies using stimulated [3H] noradrenaline release from

rat amygdala under similar experimental conditions, but at these

levels had no effect on ir-ME release. These findings are in accord

419
FIGURE 1.

Effect of K+

concentration and _presence of Ca++ ions on

release of ir-ME from rat amygdala. Each example represents

a single experiment.

with our earlier report (Morris & Livingston 1986) that administration of clonidine to rats did not affect ir-ME levels in this

region. Thus we would conclude that although opioids may affect

noradrenaline release and there are reports of alpha-2 adrenerqic

modulation of the effects of chronic opioid administration, there is

no evidence that alpha-2 adrenergic active drugs can effecf the

release of endogenous metenkephalin from the rat amygdala in vitro.

REFERENCES

Aghajanian, C.K. Nature, 276:186-188. 1978.

Bayon, A., Rossier, J., Mauss, A., Bloom, F.C., Iversen, L.L.,

Ling, N. and Guillemin, R. P N A S, 75:3503-3506, 1978.

Clement-Jones, V., Lowry, P.J., Rees, L. and Besser. G.M.

J Endocr, 86:321-243, 1980.

Del Rio, J., Naranjo, J.R., Yang, H.Y.T. and Costa, E. Brain Res,

279:121-126, 1983.

Henderson, G., Hughes, J. and Kosterlitz, H.W., Nature, 271:677-679,

1978.

Kong, J.S., Yang, H.Y.T., Fratta, W. and Costs, E. Brain Res,

134:383-386, 1977.

Iversen, L.L., Iversen, S.D., Bloom, F.E., Vargo, T. and

Guillemin, R. Nature, 272:679-681, 1978.

Lindberg, I. and Dahl, J. J Neurochem, 36:506-512, 1981.

Morris, B. and Livingston, A. Neurosci Lett, 45:11-14, 1984.

Morris, B. and Livingston, A. Neuropeptides, 7:27-30, 1986.

Osborne, H. and Herz, A. Naun Schmied. Arch Pharmacol, 310:

203-209, 1980.

420
FIGURE 2.

Effect of clonidine and idazoxan on the release of ir-ME

from rat amygdala slices in vitro. Mean ± SEM from 6-8

determinations of the fractional rate of release

stimulated by 35mM K+.

Richter, J.A., Wesche, D.L. and Frederickson, R.C.A. Eur. J.

Pharm. 56:105-113, 1979.

Sawynok, J., LaBella, F.S. and Pinsky, C. Brain Res, 189:489-493,

1980.

Tseng, L.F., Loh, H.H. and Wei, E.T. Eur. J. Pharm. 30:93-99,

1975.

Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J. Brain Res. Rev.

7:69-101, 1984.

421
RELEASE OF OPIOID PEPTIDES FROM THE SPINAL CORD OF RATS SUBJECTED TO

CHRONIC PAIN

Ryszard Przewlocki, Wladyslaw Lason. Jerzy Silberring, Albert Herz

and Barbara Przewlocka

Polish Academy of Sciences, Inst. of Pharmacol., 31-343 Kraków,

Poland, *Dept. of Neuropharm., Max Planck Inst. für Psychiatric,

D-8033 Martinsried, F.R.G.

ABSTRACT

Chronic localized pain increased the level of the opioid peptides,

dynorphin (DYN), neoendorphin (ANEO), Met-enkephalin (MET) and

Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), in the lumbar enlargement of

the rat spinal cord. It was accompanied with a reduction of the

spontaneuos and K

+

-stimulated release of ANEO and MEAGL from spinal

cord slices in vitro and a decreased release of ANEO from the spinal

cord in vivo. The results indicate that the reduction in the activity

of endogenous opioid peptide systems might occur in the spinal cord

of rats subjected to chronic pain.

INTRODUCTION

Chronic localized pain, as well as chronic polyarthritis increase the

level of opioid peptides derived from prodynorphin (Przewlocki et al.

1984, Millan et al. 1985) and proenkephalin (Cesselin et al. 1980,

Faccini et al. 1984, Przewlocki et al. 1984) in rats. these changes

occur mostly in the lumbar enlargement of the spinal cord. They are

accompanied with an increase in the analgesic potency of various

opioid agonists and opioid peptides when applied directly into the

spinal cord (Przewlocki et al. 1984). The obtained results indicate

that supersensitivity of opioid receptors develops in rats subjected

to chronic pain. Furthermore, the above observation posed a question

whether chronic pain was associated with the enhancement or decrease

of the activity of spinal opioid peptide systems. Therefore we

studied an in vitro and in vivo release of opioid peptides from the

spinal cord of rats subjected to prolonged localized pain. In

addition, we evaluated the activity of enkephalin convertase, an

enzyme thought to be responsible for the enkephalin synthesis in the

nervous system (Strittmatter et al. 1984).

MATERIALS AND METHODS

Male Wistar rats (250-300 g) were intradermally injected with 0.1 ml

of Freund's adjuvant into the hind limb sole in order to induce a

chronic local inflammation. Three weeks after inoculation the rats

were killed by decapitation and their spinal cord was immediately

dissected and divided, and the tissue was processed to estimate

immunoreactive DYN, ANEO, MET and MEAGL by specific RIAs as described

previously (Przewlocki et al. 1983). For the in vitro release the

tissue from dorsal halves of the lumbar enlargement was sliced (300

μm) with MC Ilwain tissue chopper. Slices weighing about 100 mg were

transferred to 1.5 ml superfusion chambers filled with a Ca2+-free

medium. After 20 min of washing the slices were continously super422
fused (flow rate 0.25 ml/min, temp 30°C with Krebs-bicarbonate

solution modified according to Chavkin et al. (1983). Fractions (1 ml)

were collected and ANEO and MEAGL were measured by RIAs. The in vivo

release was performed according to the techniques described by Yaksh

and Tyce (1980). To determine the stimulated release 30 and 57 mM K+

and 20 μM capsaicin were used in respective solutions. Aliquots

(usually 250 μl) of the collected frations were used directly for

RIAs. The enkephalin convertase activity, as well as the 3

Hguanidinoethylmercaptosuccinic acid (GEMSA - an inhibitor of enkephalin convertase) binding to the enzyme were assessed according to

Strittmatter et al. (1984).

RESULTS

As is shown in tab. 1, a chronic localized pain induced an increase

in the level of DYN, ANEO, MET and MEAGL locally in the lumbar (but

not cervical or thoracic) part of the spinal cord.

TABLE 1. The influence of chronic localized pain (CP) on

the levels (pmol/g) of opioid peptides in lumbar spinal

cord of rats

DYN ANEO MEAGL NET

C P 55.8±5.5* 26.5±1.8* 139.0±13.0* 551.5±62.6

p* <0.05

control 26.1±2.3 12.8±1.3 93.6±3.6 401.2±34.0

FIGURE 1. In vitro release of ANEO (A) and MEAGL (B) from the

spinal cord of rats subjected to chronic pain. Control

chronic pain

No changes in the peptide level were observed when tested in

different brain structures (hypothalamus, thalamus, cortex). An

increase in the -endorphin content was noticeable in the anterior,

423
but not intermediate part of the pituitary (not shown). Increased

levels of the peptides were accompanied with a slight increase in the

basal in vitro release of ANEO and MEAGL from the dorsal part of the

lumbar spinal cord. The stimulated release (57 mM K

+

) was not

significantly different when both groups of rats were compared (fig.

1). When the release was calculated as per cent of the tissue

content, it showed that the basal and K

+

-stimulated release was

significantly lower in rats subjected to chronic pain (0.16 ± 0.01 vs

0.1 ± 0.01, and 0.34 ± 0.04 vs 0.15 ± 0.02 in controls and rats with

chronic pain, respectively). The release of ANEO was further examined

using an in vivo superfusion technique. The results are shown in fig.

2. As can be seen, in viva basal and stimulated release (30 mM K+ or

20 μM capsaicin) of ANEO was significantly lower in rats subjected to

chronic pain.

The activity of enkephalin convertase, an enkephalin synthetizing

enzyme was not significantly changed though a slight decrease in the

binding affinity of the enzyme inhibitor 3H-GEMSA occurred in the

lumbar spinal cord of rats subjected to chronic pain (KD = 4.46 ± 0.1

vs KD = 6.17 ± 0.2, respectively).

FIGURE 2. In vivo release of ANEO from the spinal cord of rats

subjected to chronic pain. Control chronic pain

DISCUSSION

The present results demonstrate that chronic localized pain induces

changes in the level and release of opioid peptides from the spinal

cord. An elevated level of the peptides in the lumbar part of the

spinal cord was accompanied with a decrease in the release of

peptides derived from prodynorphin and proenkephalin in both in vitro

and in vivo studies. Similar results were currently obtained by

Cesselin et al. (1984), who examined release of Met-enkephalin in

polyarthritic rats. It appears that prolonged painful stimuli most

probably induced a decrease in the opioid peptide release, which

424
leads to their accumulation in neuronal terminals and, in consequence, decreases the availability of the peptides at a synaptic cleft.

This might further induce supersensitivity of opioid receptors;

actually, this phenomenon was demonstrated in our laboratory in rats

subjected to chronic localized pain (Przewlocki et al. 1984), as well

as in polyarthritic rats (Kayser and Guilbad 1983). Similar phenomena

might also occur in man, since some authors reported reduction in the

opioid peptide content in the cerebra-spinal fluid of patients

suffering from chronic pain (Almay et al. 1978).

In conclusion, our results indicate that both prodynorphin and

proenkephalin systems in the rat spinal cord adapt themselves to

chronic painful stimuli by reducing the system activity, which in

consequence, may result in their insufficiency in physiological

mechanisms of pain control.

REFERENCES

Almay, B.G.L., Johansson, F., Von Knorring, L., Terenius, L., and

Walhstrom, A. Pain, 5:153-162, 1978.

Cesselin, F., Bourgoin, S., Artaud, F., Gozlan, H., Le Bars, D., Clot,

A.M., Besson, J.-M., and Hamon, M. In: Besson, J.-M., Lazorthes,

Y., eds. Spinal Opioids and the Relief of Pain. Paris, INSERM, 127:

241-264, 1984.

Cesselin, F., Montastruc, J.L., Gros, C., Bourgoin, S., and Hamon, M.

Brain Res, 191:289-293, 1980.

Chavkin, C., Bakhit, C., Weber, E., and Bloom, F.E. Proc Natl Acad

Sci USA, 80:7669-7673, 1983.

Faccini, E., Uzumaki, H., Govoni, S., Missale, C., Spano, P.F.,

Covelli, V., and Trabuchi, M. Pain, 18:25-31, 1984.

Kayser, V., and Guilbaud, G. Brain Res, 267:131-138, 1983.

Millan, M.J., Millan, H.H., Pilcher, C.W.T., Czlonkowski, A., Herz,

A., and Colpaert, F.C.

Przewlocki, R.,

Brain Res, 340:156-159, 1985.

Gramsch, C., Pasi, A., and Herz A. Brain Res, 280:95-

103, 1983.

Przewlocki, R., Przewlocka, B., Lason, W., Stala, L., and Herz A. In:

Besson, J.-M., Lazorthes, Y., eds. Spinal Opioids and the Relief

of Pain, Paris, INSERM, 127:159-170, 1984.

Strittmatter, S.M., Lynch, D.R., and Snyder, S.H. J Biol Chem, 259:

11812-11817, 1984.

Yaksh, T.L., and Tyce, G.M. Brain Res, 192:133-146, 1980.

425
RELATIONS BETWEEN GABAERGIC AND ENKEPHALINERGIC SYSTEMS:

EFFECTS OF SODIUM VALPROATE ON RAT BRAINENKEPHALINS.

Jean Vion-Dury *, Annie Cupo ** and Therese Jarry **

* Laboratoire de Pharmacologie, Faculté de Médecine,

Bd. J Moulin, 13385 Marseille FRANCE.

** Centre d'Imnunologie INSERM/CNRS , Luminy,

Case 906,13288 Marseille FRANCE.

ABSTRACT

The acute effects of a Gaba-mimetic drug, sodium Valproate, are

studied on brain enkephalin contents in rat brain. Valproate

administered at both 300 and 600 mg/kg doses induces an increase of

enkephalin levels in striatum, hypothalamus, brain stem and cortex.

No effect is seen in the hypophysis.

INTRODUCTION

When relations between monoaminergic and enkephalinergic systems are

extensively studied, the interactions between opioidergic and

Gabaergic systems present an interesting development of opiate

physiology. Both anatomical evidences (Aronin et coll 1984) and

pharmacological arguments (De Feudis 1984) underly the concept of a

Gaba/enkephalin link. Some studies were realized about the effects

of Gaba-mimetic drugs on opiates. Roughly, administration of a Gaba

A agonist is generally followed by a reduction of opioid 'levels

(Duka and coll 1980) or release (Bourgoin and coll 1982). In

opposition, the Gaba-B mimetic drugs as Baclofen enhance enkephalin

release from brain rat slices (Sawynok and Labella 1981).

Sodium Valproate (VPA) is an extensively used anti-epileptic drug,

enhancing Gaba levels by blocking the degradation enzymes of this

neuro-transmitter (Johnston and Slater 1982). We studied the acute

effect of this drug on Mat-Enkephalin and Leu-Enkephalin levels in

five areas of rat brain. VPA was administered at doses eliciting an

anti-epileptic effect on kindled seizures in rat (Tulloch and coll

1982)

MATERIEL AND METHODS

Three groups of 7 albinos male rats (300g +/- 20) from Iffa Credo

were used for the experiments. Animals of each group were

administered I.P. respectively by two doses of 300 and 600 q/kg of

VPA (Depakine, Labaz laboratories, France), and 0.5 ml of

distillated water (drug vehicle). The rats were killed by

decapitation, without anaesthesia, between 10:00 and 12:00 am, to

avoid a possible effect of circadian ryhtms.

Both tissue samples preparation and RIA procedure was carried out as

previously described (Cupo and Jarry 1985). ME antibodies exhibit

following properties: IC50 = 9.1 pM and cross-reactive factor (f)

with analog peptides always greater than 175. LE antibodies were

obtained by an original method (Cupo and coll to be published) and

present the following characteristics: IC50 = 23 pM and f > 80 in

426
all cases.

RESULTS

Injection of VRA at anti-epileptic doses is followed by an increase

of both LE (table 1) or ME (table 2). This effect is significant at

a 600 mg/kg dose for LE levels and significant for the 2 doses for

ME levels. Note the lack of effect in the hypophysis.

TABLE 1. Acute effect of sodium valproate on Leucine-enkephalin

levels in five areas of rat brain. Two doses were injected

intra-peritonealy: 300 and 600 mg/kg. Animals were sacrified 30

minutes after injection. Results are given in picomole of wet

tissue (+ or - sem). St= striatum, Ht=Hypothalamus,

Hph=Hypophysis, Br.St.=Brain stem, and Cx=cortex.

Groups St. Ht. Hph. Br St. Cx

Controls 0.033 0.033 0.26 0.013 0.0023

(0.004) (0.003) (0.012) (0.001) (0.001)

VPA 300 0.038 0.039 0.25 0.012 0.005

mg/kg (0.004) (0.006) (0.016) (0.012) (0.002)

VPA 600 0.065 0.068 0.23 0.029 0.005

mg/kg (0.010) (0.021) (0.016) (0.005) (0.004)

*** *** *** **

Statistical analysis: variance analysis. ***, p<0.001;

**,p<0.01; *, p<0.05.

When administration of two doses of VPA induces an increase of

enkephalinergic system activity in four brain areas, the lack of

this effect in hypophysis suggests that, in 30 minutes, VPA presents

store of a neuro-neuronal action than a neuro-endocrine one. In this

point of view, if the effect of VPA per se on enkephalinergic

neurons must not be discarded, there are many evidences that there

is a close correlation between plasmatic concentrations of VPA and

synaptic Gaba levels (Iadarola and Gale 1981). The nature of the

effect of VPA on ehkephalinergic systems seems to mimic the effects

of Baclofen and suggests a preferential implication of Gaba B

receptors (Sawynok and Labela 1981).

The small delay between the injection of VPA and the sacrifice of

the animals is a good argument for an increase of enkephalin levels

in relation to an increase in pro-enkephalin consumption more than

to an increase of pro-enkephalin processing. Preliminary results on

octapeptide (an other peptide derived from pro-enkephalih) show that

the same doses of VRA induce a reduction of octapeptide levels

suggesting a precursor consumption and/or a differential maturation

of the pro-ehkephalin related peptides (Vion-Dury and coll to be

published)

427

172-683 O - 87 15 : QL 3
This work raises an important question: are the therapeutic or

adverse effects of VPA partially related to enkephalinergic pathway

activation ? When action of opioid peptides or agonists on epilepsy

is very complex and very discussed (Frenk 1983), it is probable that

the anti-epileptic action of VPA is mainly related to an increase of

Gaba-ergic transmission (Johnston and Slaters 1982).

TABLE 2. Acute effect of sodium valproate on

Methionine-Enkephalin levels in the same areas of rat brain.

For legend, see table 1.

Groups St. Ht. Hph. Br.St. Cx

Controls 0.58 0.52 2.49 0.19 0.033

(0.06) (0.016) (0.42) (0.016) (0.003)

VPA 300 0.82 0.77 2.51 0.24 0.041

mg (0.11) (0.08) (0.34) (0.03) (0.003)

** ** *

VPA 600 0.80 0.76 2.37 0.33 0.051

mg (0.13) (0.11) (0.48) (0.04) (0.004)

*** **

Yet VPA (as others Gaba-mimetic drugs) exhibits also important

analgesic properties at lower doses than those used for the

anti-epileptic treatment (Mesdjian and coll 1983) Some preliminary

dosages show that VPA a 100mg/kg (analgesic doses), induces an

increase of opioid peptides in striatum(Vion-Dury and coll to be

published). In this point of view, it must be noted that Mesdjian

and coll (1983) suggested an non implication of opioidergic

transmission because the effect of VPA was not reversed by naloxone.

They conclude that the effects of VPA was related to an increase of

Gabaergic transmission. But, if it is assumed that 1) weak doses of

VPA induce an increase of enkephalinergic transmission, and 2) the

synaptic organization of Gaba/enkephalin link is underlyed by a

parallel circuitry (Andree and coll 1983), the lack of the effect of

naloxone can be easily explained: the naloxone antagonism of

enkephalin effect would be masked by Gaba-mimetic action of VPA on

pain pathways.

Increase of enkephalinergic transmission after VPA treatment might

also explain the apparition of a tolerance and dependence to

analgesic action of both morphine and Gaba agonists in mice

receiving injections of THIP(Andree and coll 1983). If the action of

VPA on enkephalinergic pathways is underlyed by a preferential

activation of Gaba-B receptors, our results might also explain the

syndrome of a Baclofen overdose, which mimics the syndrome of opiate

overdose (De Feudis 1984).

So, from these data, it is possible to try to complete the schema

proposed by Andrée and coll (fig 1) and to suggest the hypothesis of

a parallel organization of Gaba-ergic and enképhalinergic pathways

with a fine modulation of enkephalinergic activity by Gaba-ergic

428
system involving several

Bourgoin and coll (1981).

Gaba-ergic receptors as suggested by

FIGURE 1: Possible organization of the relations between Gabaergic

and enkephalinergic systems (from Andree and coll 1983): Commentary

in the text

REFERENCES

Aronin, N, Difiglia, M, Graval, G.A., Schwartz, W.J., and Wu, J.Y.

Brain Res, 300(2) :376-380, 1986.

Bourgoin, S., Cesselin, F., Artaud, F., Glowinski, J., and Hamon, M.

Brain Res, 248:321-330, 1982.

Cupo, A ., and Jarry, T. J Neuroimmunol, 8:57-67, 1985.

De Feudis F.V. Drug Alcohol Depend, 14:101-111, 1984.

Duka, T., Wuster M., and Herz A. Life Sci, 26:771-776, 1980.

Frenk, H. Brain Res Rev, 6:197-210, 1983.

Iadarola, M.J., and Gale, K Mol Cel Biochem, 39:305-330, 1981.

Johnston D., and Slater G.E., In:Woodburry , P.M., Penry, J.K.,

Andree, T., Kendall, D.A., and Enna, S.J. Life Sci,

32:2265-2272, 1983.

and Pippenger, C.E., eds. Antiepileptic drugs.

New York:Raven Press , 1982. pp. 611-616.

Mesdjian, E., De Feudis, F.V., Valli, M., Jadot, G., and Mandel, P.

Gen Pharmac, 44:697-699, 1983.

Sawynok, J., and Labella, F.S. Eur J Pharmacol, 70:103-110, 1981.

Tulloch, I.F., Walter, D.S., Howe G.M., and Howe , S.J.

Neuropharmacology, 21:555-562, 1982.

429
[MET]ENKEPHALIN-ARG6

-PHE7 IN HUMAN CSF - INCREASED LEVELS IN

LATE PREGNANCY

Sven Lyrenäs1

, Fred Nyberg2, Ronnie Folkesson2, Bo Lindberg1 and

Lars Terenius2

1

Dept. of Obstetrics and Gynecology, Uppsala University, Akademiska

Sjukhuset, S-751 85 Uppsala, Sweden.

2

Dept. of Pharmacology, Uppsala University, S-751 85 Uppsala,

Sweden.

ABSTRACT

The opioid heptapeptide, [Met]enkephalin-Arg6-Phe7 was measured in

CSF of women at term pregnancy and during the luteal phase by RIA.

Levels turned out to be significantly increased in pregnant women.

These results are in agreement with an earlier paper in which we reported increased levels of receptorassayed opioid peptides deriving

from proenkephaln A in CSF of women in term pregnancy compared to

levels in nonpregnant, nonpuerperal women.

INTRODUCTION

C-terminal extensions of the pentapeptides [Met]enkephalin and [Leu]-

enkephalin (so called ECPs) were in fact isolated from brain and

peripheral tissues a few years prior to the sequencing of the opioid

peptide precursors proenkephalin A and proenkephalin B. One of these

C-terminally extended peptides, [Met]enkephalin-Arg6-Phe7, was described by Stern et al. 1980. On a molar basis, Rossier et al. (1980)

found this heptapeptide eight times more analgesic than [Met]enkephalin in the tail flick test. In a previous study we reported elevated

levels of so-called FII endorphins both in term pregnancy and in

early puerperium compared to levels in nonpregnant, nonpuerperal women. We suggested these elevated FII endorphins to derive from the

[Met]enkephalin precursor (Lyrenäs et al. 1986). In the present study

we measured CSF levels of [Met]enkephalin-Arg6-Phe7 by RIA in individual pregnant women and in nonpregnant women during the luteal phase

of the menstrual cycle.

MATERIAL AND METHODS

The study included CSF from two groups of women:

1. This group comprised of 12 pregnant women who underwent elective

cesarean section at term due to pelvic disproportion. The operations

were performed during spinal anesthesia and five ml samples of CSF

were removed prior to administration of the anesthetic drug. CSF

levels of [Met]enkephalin-Arg6

-Phe7 were measured by RIA.

2. A control group composed of eight nonpregnant nonpuerperal healthy

female volunteers; CSF samples were collected during the luteal phase

of the menstrual cycle. Lumbar punctures were performed at the level

of L:3-L:4 with the same technique as used in group 1. CSF levels of

[Met]enkephalin-Arg6-Phe7 were estimated by RIA.

CSF samples were taken in ice cold polyethylene tubes prepared with

EDTA, immediately frozen and kept at -70°C until analyzed.

Materials: Peptide standards used in this study were obtained from

430
sources previously noted (Nyberg et al. 1983). Reversed phase silica

gel cartridges (SepPak C-18) were purchased from Waters Associates

(Milford, Mass., U.S.A.). All other chemicals and solvents were of

analytical reagent grade from commercial sources.

Sample work up: Prior to RIA, the thawed CSF sample (two ml) was subjected to SepPak separation. Before sample application, the cartridge was washed consecutively with 10 ml methanol containing 0.04 %

trifluoroacetic acid (TFA), 10 ml methanol and 10 ml water. The CSF

sample was applied in a volume of two ml and the cartridge washed

with 2.5 ml of water. Elution was carried out with aqueous mixtures

of TFA (0.04 %) and increasing concentrations of methanol. The heptapeptide-containing fraction was eluted with 60 % methanol and evaporated. The dried fractions were redissolved in 100 μl 1M acetic acid

and incubated for 30 min. at 37°C with 1 % H2O2. All samples were

evaporated a second time before RIA analvsis.

The radioimmunoassay for [Met]enkephalin-Arg6-Phe7 was based on the

charcoal adsorption technique and conducted in conformity with those

described for other peptides (Christenson-Nylander et al: 1985). The

antibodies were raised against the heptapeptide sulfoxide

([Met]-O-enkephalin-Arg6-Phe7) and the iodinated oxidized peptide was

used as tracer. The detection limit of the RIA was around

20 fmol/tube. Crossreaction with [Met]enkephalin and other

proenkephalin A or B derived peptides was less than 0.1 %. Runs with

the synthetic heptapeptide added to artificial CSF at a concentration

of 50 fmol/ml indicated a recovery of approximately 70 % of the extraction procedure.

High performance liquid chromatography (HPLC): A HPLC system (LKB,

Bromma, Sweden) was used for identification of the measured peptide.

Pooled aliquots of the assayed fractions were analyzed in a single

run. The column (Spherisorb C-18, 5 μ, 4.5 x 250 mm) was eluted

with a 40 min. linear gradient of acetonitrile (10-45 %) containing TFA (0.04 %). Fractions of 0.5 ml were collected at a flow rate

of 0.5 ml/min. and evaporated prior to RIA analysis.

RESULTS

fmol/ml. The difference is statistically significant (p < 0.05;

Student's t-test). HPLC identification showed that the major part

of RIA activity eluted like the standard (figure 2). Minor activity

eluting with higher retention time was also observed.

Figure 1 shows the levels of radioimmunoassayable CSF [Met]-O-enkephalin-Arg6

-Phe7 in groups 1 and 2. The mean level (SD) in term pregnancy was 32.8 (12.0) fmol/ml and in the luteal phase 22.4 (7.1)

DISCUSSION

The complexity of the proenkephalin systems is considerable, and a

whole family of enkephalin peptides can be identified in human CSF

(Nyberg et al. 1986). We have earlier used a receptorassay to

identify so-called Fraction II opioid peptides, which mainly derive

from proenkephalin A. In CSF from women at term pregnancy and in the

early puerperium, fraction II levels were increased (Lyrenäs et al.

1986). One major component in Fraction II is [Met]enkephalin-Arg6-Phe7

(Nyberg et al. 1983). The present study using a specific RIA for this

peptide, confirmed the elevation of enkephalin peptides at term pregnancy. Assuming this elevation to reflect increased activity,

431
FIGURE 1. CSF levels of [Met]enkephalin-Arg6

-Phe7 in

term pregnancy and during the luteal phase measured

by RIA. Antibodies used in the RIA were raised using

the heptapeptide sulfoxide.

FIGURE 2. High performance liquid chromatography of

[Met]enkephalin-Arg6-Phe7 sulfoxide immunoreactivity.

Calibration runs were performed with synthetic standards as indicated by arrows (ENK = enkephalin).

Further details are given in the text.

432
enkephalin pathways appear to be under higher tonic activity in late

pregnancy and the early puerperium than in non-pregnant, non-puerperal women. Similar information is available with regard to plasma

levels, i.e. the hormonal secretion of -endorphin and [Met]enkephalin in late pregnancy and early puerperium (Pancheri et al. 1985).

The general elevation of endorphin activity in late pregnancy and

early puerperium may be considered a primary reaction subserving

a protective role or be a secondary reaction to the stress involved.

Further studies will be needed to distinguish between these possibilities and to elucidate the basic mechanisms.

REFERENCES

Christenson-Nylander, I., Nyberg, F., Ragnarsson, U. and Terenius, L.

Reg Peptides, 11:65-76, 1985.

Lyrenäs, S., Nyberg, F., Willdeck-Lund, G., Lindström, L., Lindberg, B.,

and Terenius, L. Uppsala J Med Sci, accepted for publ. 1986.

Nyberg, F., Wahlström, A., Sjölund, B., and Terenius, L. Brain Res, 259:

267-274, 1983.

Nyberg, F., Nylander, I., and Terenius, L. Brain Res, 371:278-286,

1986.

Pancheri, P., Zichella, L., Fraioli, F., Carilli, G., Perrone, G., Biondi,

M., Fabbi, A., and Moretti, C. Psychoendocrinol, 10:289-301, 1985.

Rossier, J. Audigier, Y., Ling, N., Cros, J., and Udenfriend, S. Nature,

288: 88-96, 1980.

Stern, A.S., Lewis, R.V., Kimura, S., Rossier, J., Stein, S., and Udenfriend, S. Arch Biochem Biophys, 205:606-613, 1980.

ACKNOWLEDGEMENTS

This study was supported by Syskonen Svenssons Fund and by the Swedish Society of Medicine.

433
EXPRESSION AND REGULATION OF KAPPA OPIATE RECEPTORS

IN RAT SPINAL CORD-DORSAL ROOT GANGLION COCULTURES

Bernard Attali, Danielle Saya and Zvi Vogel

Department of Neurobiology,

Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT

We have been using rat spinal cord-dorsal root ganglion primary

cocultures (SC-DG) as a model system for exploring K receptor regulation. During the first 10 days in culture, the total number of opiate receptors increased markedly, reaching a Bmax of 180 fmoles/mg

protein for K sites and a Bmax of 50 fmoles/mg protein for μ sites.

Following this period of development, the K and μ receptor number did

not change significantly. No detectable sites were observed at any

time of culture. The binding of [

3

H]diprenorphine to K sites was

found to be saturable, of high affinity and stereospecific. After

chronic agonist treatment of cultured cells, K receptors did not

down-regulate, whereas more than 50% of the μ receptor sites did.

Following chronic antagonist treatment, μ receptors were markedly

up-regulated (260% of control), while K sites exhibited a weaker upregulation response (160% of control). These data demonstrate that K

opiate receptors are expressed at high concentrations in SC-DC cultures andthatcontraryto μ receptors in spinal cord and receptors

in NG10815 cells, K binding sites are less susceptible to modulation

following chronic agonist or antagonist treatment. This suggests

that K receptors may be regulated by different control mechanisms.

INTRODUCTION

Exposure of cells to agonists or antagonists often results in a

change in the concentration of their surface receptors.

Up-regulation of μ and opiate receptors in rat brain has been

induced by chronic administration of antagonists (Tempel et al.

1985). Ample documentation of μ and receptor down-regulation has

been provided by agonist treatment of aggregated fetal brain cells

(Lenoir et al. 1984) and of cultured. NG10815 cells (Law et al. 1983).

On the other hand, very little is known about the regulation of K

opiate receptors in nervous tissue. We have been using SC-DG cultures as a model system for exploring K receptor regulation since

spinal cord has been proved to be a rich source of K sites (Attali et

al. 1982).

METHODS

SC-DG cultures were prepared according to Ransom et al. (1977). After

2 weeks in culture, the cells were treated for the time indicated.

Cells were washed 3 times with PBS and incubated 15 min at O°C in 100

mM NaCl and 50mH Tris-HCl (pH=7.4). Cells were homogenized and centrifuged for 30 min at 27,500xg, pellets resuspended in 100mM NaCl,

50mM Tris-HCl (pH=7.4) and incubated for 15 min at 37°C to allow

bound drug to dissociate from the receptor. The membranes were further washed twice in 50mM Tris-HCl (pH=7.4) and resuspended in the

434
same buffer for binding assay. In control untreated cells, equal

concentrations of the same agonists or antagonists were added to culture dishes immediately before cell harvesting. K binding sites were

laballed by [3H] diprenorphine (30 min, 37°C) in the presence of DAGO

(0.1μM) and DADL (0.1μM) to suppress the contribution of μ and

sites. [3H] DAGO and [3H]DSTLE were used for labeling (30 min, 37°C)

μ and receptor sites, respectively. Non-specific binding was

determined in the presence of 10 μM diprenorphine.

RESULTS

sites were found to be saturable and of high affinity

(Kd=O.85±0.15nM, n=11 and Kd=2.4±0.5nM, n=3, respectively).

No detectable μ, or K sites were observed on day of plating.

During the first 10 days in vitro, the total number of opiate receptors increased markedly, reaching a Bmax of 180±44 fmoles/mg protein

(n=11) for K sites and a Bmax of 50±6 fmoles/mg protein (n=3) for μ

sites. Following this period of development, the K and μ receptor

number did not change significantly. Interestingly, no detectable

sites were observed at any time of culture (up to 4 weeks in vitro).

The binding of [

3

H] diprenorphine to K sites and that of [3H]DAGO to μ

FIGURE 1. Pharmacology of K opiate binding sites in SC-DG

culturess. The pharmacological profile of various ligands

was assessed by competition experiments. K receptors in

membranes of 2 weeks old SC-DG cultures were labeled by

[

3

H]diprenorphine (1.5nM) in the presence of DAGO (0.1μM)

and DADL (0.1μM). The data correspond to a typical experiment replicated two to three times with similar values.

435
Treatment

TABLE 1. Regulation of opiate receptors upon chronic agonist or

antagonist treatment

Etorphine 1 μM, 1 day 47±2  96±7 0±0** **

Etorphine 1 μM, 3 days N D 80±1 *

U50488 10 μM, 1 day N D 95±11

Naloxone 10 μM, 5 days 261±17*** 164±3 ****

30±11* *

123±3 ***

Regulation of μ and receptors was assayed in 2 week old

SC-DG cultures and that of sites in NG108-15 cells.

Results are expressed as percent of control binding to

untreated paired cultures. Values were deduced from saturation experiments; the control binding values for μ, and

receptors are 48±3, 187±36 and 435±21 fmoles/mg protein,

respectively (n=3). Significance of data was calculated by

the unpaired student t test (*p<0.05, **p<0.02, ***p<0.01,

****p<0.001). ND (not determined).

Displacement experiments revealed that the binding to K sites was

stereospecific since MR 2034 and levorphanol were potent competitors,

while their respective inactive enantianers, MR2035 and dextrorphan

were ineffective (figure 1A). K selective ligands such as MR2034,

ethylketocyclazocine and tifluadom were able to compete with

[

3

H]diprenorphine binding at low concentrations, whereas μ or

ligands were shown to be very weak displacers (figure 1B). The

affinity of the K agonist U50488 decreased significantly in the presence of 100μM GTP and 100mM NaCl (data not shown). Following chronic

exposure to the non-selective opiate agonist etorphine (1μM, 24h), K

receptors did not down-regulate, whereas under the same experimental

conditions, 53% of μ sites down-regulated (table 1). The same

results were obtained by using the K selective agonist U50488 (10 μM,

24h). In contrast, we found that in NG10815 cells which contain predominantly sites, one day treatment with 1 μM etorphine or 10 μM

U50488, a concentration with also occupies receptors induced

receptor down-regulation by more than 90% and 70%, respectively.

Prolonging the time of agonist exposure(etmphine 1 μM, 3 days), we

observed a weak but significant down-regulation of receptors which

amounted now to 20% (table 1). Followinq chronic antagonist treatment (naloxone 10 μM, 5 days), μ receptors strongly up-regulated

(261% of control), while sites exhibited a weaker up-regulation

(164% of control). Similar experiments performed in NG108-15 cells

showed that receptors slightly up-regulated (125% of control).

SC-DG CELLS NG108-15 CELLS

(% of control) (% of control)

DISCUSSION

In agreement with previous studies showing that the adult spinal cord

contains high amounts of receptors (Attali et al. 1982; Czlonkowski

et al. 1983), our data now demonstrate that after neuronal differentiation in vitro, opiate receptors are expressed at high concentrations (more than 70% of total receptors) in SC-DC cultures. The

opioid nature of these binding sites is demonstrated by the saturability, high affinity and stereospecificity of ligand binding. The

properties of the binding sites is revealed by the pharmacological

436
profile of various compounds in competition experiments. selective

ligands were good displacers, while μ and ligands were relatively

ineffective. The regulation of K agonist binding (U50488) in cultured cells by GTP and Na+

correlates well with previous studies

showing a similar regulation in adult rat spinal cord (Kelly et al.

1982), which suggests a possible couplinq of K receptors to adenylate

cyclase (see also Attali and Vogel this volume). Our data concerning

K receptor regulation reveal striking differences with μ and receptors. Contrary to μ receptors in SC-DG cells or receptors in

NG10815 cells, K receptors were not down-regulated upon chronic exposure of SC-DG cultures to etorphine or U50488. Only after 3 days of

agonist exposure, a slight down-regulation of K sites is observed

(20%). This lack of apparent down-regulation for K receptors suggests that the regulation upon chronic activation might occur at a

different level such as post-receptor binding regulatory compnents

(Attali and Vogel this volume). Upon chronic antagonist treatment, K

receptor regulation, exhibited again a different feature when cmpared to μ receptors. However, although K sites displayed a weaker

up-regulation, the significant increase in K opiate binding might be

the result of several mechanisms. These may include either increased

receptor synthesis, lowered degradation, unmasking of active receptor

molecules, or even conversion of newly synthesized non-binding receptors to active receptors. It is interesting to note that antagonistinduced μ receptor up-regulation in fetal mouse spinal cord-ganglion

explants does not require the synthesis of new receptor molecules

(Tempel et al. 1986). Taken together, these data reveal that contrary to μ or receptirs, K binding sites are less susceptible to

modulation upon chronic agonist or antagonist treatment, suggesting

that they are regulated by different control mechanisms.

REFERENCES

Attali, B., Gouarderes, C., Mazarguil, H., Audigier, Y. and Cros, J.

Neuropeptides, 3:53-64, 1982.

Czlonkowski, A., Costa, T., Przewlocki, R.,

Brain Res, 267:392-396, 1983.

Pasi, A. and Herz, A.

Kelly, P.D., Rance, M.J. and Traynor, J.R. Neuropeptides,

2:319-324, 1982.

Law, P.Y., Hom, D.S. and Loh, H.H. Mol Pharmacol, 24:413-424, 1983.

Lenoir, D., Barg, J.and Simantov, R. Brain Res, 304:285-290, 1984.

Ransom, B.R., Neale, E., Henkart, M., Bullock, P.M. and Nelson, P.G.

J Neurophysiol, 40:1132-1150, 1977.

Tempel, A., Gardner, E.L. and Zukin, R.S. J Pharmacol Exp Ther,

232:439-444, 1985.

Tempel, A., Grain, S.M., Peterson, E.R., Simon, E.J. and Zukin, R.S.

Dev Brain Res, 25:287-291, 1986.

437
SENSITIVITY OF ANTINOCICEPTIVE TESTS TO OPIOID AGONISTS

AND PARTIAL AGONISTS

Karen M. Delaney, Janet D. Rourke and John S. Shaw

Bioscience Department

ICI Pharmaceuticals Division

Alderley Park, Macclesfield, Cheshire SK10 4TG. U.K.

ABSTRACT

There is considerable variation in the potency of opioids across

different animal models of antinociceptive activity. In the less

sensitive tests the partial agonist analgesics behave as antagonists.

The activity of opioids in antinociceptive tests appears to be

dependent on both the intensity of the noxious stimulus and the

intrinsic activity of the drug.

INTRODUCTION

It has long been recognised that the potencies of analgesic agents

exhibit considerable variation between different animal models. For

example, the ED50 for morphine in the hot plate test has been

reported to be 30 times greater than in the abdominal constriction

model (Ward and Takemori 1983). In addition, several drugs (eg.

nalorphine) which display potent antfnociceptive activity in the

abdominal constriction test are inactive in tests employing heat as

the noxious stimulus (Tyers 1980). It has been suggested that the

neural pathways involved in the transmission of heat and non-heat

stimuli may possess different types of opioid receptor, and that

drugs which are ineffective against heat stimuli act predominantly at

k-sites.

However, this hypothesis does not explain why the potencies of drugs

such as morphine vary between tests. Neither does it explain the

observation made by several groups (Zimet et al. 1986. Sewell and

Spencer 1976, Luttinger 1985) that heat tests can be made to detect

the agonist-antagonist analgesics by lowering the temperature of the

stimulus.

The present study was designed in an attempt to clarify the relative

contribution of receptor selectivity, intrinsic activity and test

methodology to the analgesic efficacy of opioids.

METHODS

Dose responses to each of the test drugs were performed on 4

analgesic tests employing different noxious stimuli : 55° hot plate

(mouse), acetic acid induced abdominal constriction (mouse), paw

pressure (rat) and tail flick (rat). The animals used were either

female mice of the Alderley Park strain weighing 18-20g or newly

weaned female Alderley Park strain rats weighing 40-60g. In all

studies the test drug was administered subcutaneously 30 minutes

prior to test.

438
Mouse hot plate

Reaction times of mice placed on a hot plate maintained at 55°C were

determined. Any drugs devoid of activity were tested as antagonists

of the k-agonist tifluadom. In this situation dose responses to

tifluadom were performed in the presence and absence of a constant

dose of antagonist. Groups of at least 6 mice were used in all hot

plate studies.

Acetic acid induced abdominal constriction

Acetic acid (0.4 ml) was administered intraperitoneally 30 minutes

after the test drug. Animals were left for 2 minutes before counting

the cumulative number of responses over the next 15 minute period. A

minimum group size of 10 was employed. In the first part of the

study the acetic acid concentration was 0.4%, but in later

experiments 0.25% and 0.6% concentrations were also used.

Paw pressure

Thresholds for each hind paw were measured using a modified

"Analgesimeter" (Ugo Basile, Milan) with the endpoint being taken as

the first struggle or vocalisation by the rat.

Tail flick

The method used was essentially the same as that described by D'Amour

and Smith (1941). The rat's tail was placed over a focussed light

beam and the latency to flick recorded. A cut-off time of 20 seconds

was imposed. In both rat studies at least 10 animals were used per

group.

RESULTS AND DISCUSSION

The activity of each of the drugs varied according to the test

situation (table 1) with only the full agonists and high intrinsic

activity partial agonists displaying full dose response curves across

the complete range of tests. U50488H produced a flattened dose

response curve on the hot plate test but complete dose responses on

the rat paw pressure and tail flick tests and the mouse abdominal

constriction test. The lower intrinsic activity partial agonists

xorphanol, nalbuphine, bremazocine, levallorphan and nalorphlne were

effective only on the abdominal constriction test.

The potencies of the drugs varied across the range of tests making it

possible to rank the assays in order of decreasing sensitivity as

follows: abdominal constriction > tail flick = paw pressure > hot

plate. These findings prompt the suggestion that each model provides

a different noxious stimulus intensity with hot plate providing the

highest and abdominal constriction test the lowest. If, by

increasing the stimulus intensity, the apparent number of "spare"

receptors is reduced, then it is possible to explain the current

observations. In the hot plate test, which provides the highest

stimulus intensity as reflected by the low potencies of the test

drugs. only the full agonists fentanyl and tifluadom and the high

439
intrinsic activity partial agonist morphine have sufficient efficacy

to generate a full dose response. U50488H is less efficacious and

this is reflected by a flattened dose response on the hot plate test

although full dose responses were produced against the other lower

intensity stimuli. The low intrinsic activity partial agonists have

sufficient efficacy to be effective against only the lowest

stimulus.

TABLE 1 Antinociceptive potencies of a range of opiolds

If this explanation is correct, the lower efficacy drugs should

antagonise the effects of agonists acting at receptors for which the

partial agonists have affinity. This hypothesis was tested by

determining the effects of nalorphine, levallorphan and bremazocine

on the dose response to tifluadom in the hot plate test. The

tifluadom dose response was shifted to the right in a parallel manner

in the presence of each of these partial agonists (results not shown)

thus providing support for the hypothesis.

These findings do not support the suggestion of Tyers (1980) that

μ-agonists are equipotent against heat and non-heat stimuli whereas

k-agonists are less effective against heat. In the light of the

current findings it is obvious that μ and k-drugs with sufficient

efficacy are equally effective against heat and non-heat stimuli as

demonstrated by their similar potencies in the paw pressure and tail

flick assays, employing mechanical and thermal stimuli respectively.

The most likely explanation for this discrepancy is that many drugs

regarded as k-agonists are, in fact, partial agonists (Miller et al.

1985). The current data suggest that it is the efficacy of the drug

rather than its receptor selectivity which determines its profile

against different noxious stimuli.

440
TABLE 2 Effect of acetic acid concentration on potency

Further support for the hypothesis is provided by the second part of

the current study which examined the effects of changing the stimulus

intensity within one assay - the mouse abdominal constriction test

(table 2). Responses to the μ- and k-agonists, morphine and

tifluadom were not significantly influenced by the acetic acid

concentration. In contrast the k-partial agonists xorphanol,

nalorphine and levallorphan, and the p-partial agonist nalbuphine

exhibited lower potency and flattened dose response curves when

tested against the higher acetic acid concentrations.

In conculsion, antinociceptive tests exhibit a wide range of

sensitivity to opioids. Some models such as the hot plate assay are

only sensitive to high doses of full agonists, whilst partial

agonists display antagonist properties. The more sensitive assays

such as the abdominal constriction test appear to have a high

receptor reserve in the abdominal constriction model. These findings

highlight the need for caution when comparing data from different

tests, or even from the same test performed under slightly different

conditions.

REFERENCES

D'Amour, P.R. amd Smith, D.L. J Pharmacol Exp Ther, 72: 74-79, 1941,

Luttinger, D. J Pharmacol Meth, 13: 351-357, 1985.

Miller, L., Shaw. J.S. and Whiting, M. Br J Pharmacol, 87: 595-601.

1985.

Sewell, R.D.E. and Spencer, P.S.J. Neuropharmacol. 15: 683-688, 1976.

Tyers, M.B. Br J Pharmacol, 69: 503-512, 1980.

Ward, S.J. and Takemori, A.E. J Pharmacol Exp Ther, 224:525-530,

1983.

Zimet, P.O., Wynn, R.L., Ford, R.D. and Rudo, F.G. Drug Dev Res,

7:277-280. 1986.

441
EVIDENCE FOR DELTA RECEPTOR MEDIATION OF [D-PEN2

,D-PEN5]-

ENKEPHALIN (DPDPE) ANALGESIA IN MICE

Julius S. Heyman, Henry I. Hosberg* and Frank Porreca

Department of Pharmacology, University of Arizona Health Sciences

Center, Tucson, AZ 85724, U.S.A.

*College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, U.S.A.

ABSTRACT

Possible involvement of cerebral delta opioid receptors in

antinociceptive processes was studied in a test utilizing heat as the

noxious thermal stimulus. The investigation focused on selective

agonists and antagonists for mu and delta opioid receptors. Morphine

and [D-Ala2,NMPhe4,Gly-ol]enkephalin (DAGO) were used as agonists for

the mu receptorwhile [D-Pen2,D-Pen5]enkephalin (DPDPE) was the agonist

for the delta receptor. Two approaches were employed: first, the

intracerebroventricular (i.c.v.) analgesic activity of the agonists was

determined in the absence, and in the presence of graded i.c.v. doses of

the selective delta antagonist, ICI 174,864 (N, N diallyl-Tyr-Aib-AibPhe-Leu-OH)(where Aib is alpha-aminoisobutyric acid); second, acute

tolerance to morphine was produced and the possible presence of acute

cross-tolerance between subcutaneous (s.c.) morphine and the delta

agonist investigated. ICI 174,864 antagonized the analgesia produced

by DPDPE, but not that resulting from morphine or DAGO. biorphine

pretreatment resulted in the development of acute tolerance to i.c.v.

morphine, and acute cross-tolerance to i.c.v. DAGO, but not to i.c.v.

DPDPE. These results provide evidence that both cerebral delta and mu

opioid receptors are responsible for the mediation of analgesia in tests

utilizing heat as the nociceptive stimulus.

INTRODUCTION

Traditional views regarding the involvement of supraspinal opioid

receptorsintests ofanalgesiawhere heat is employed as the nociceptive

stimulus have focused on the exclusive involvement of mu, rather than

delta and kappa, receptors (Audigier et al. 1980; Chaillet et al. 1984;

Changetal. 1982). While the analgesic effects of i.c.v. kappa agonists

are generally difficult to detect, both mu and delta agonists produce

effective analgesia by this route (Porreca et al. 1984). Thus, it is of

interest to determine whether even highly delta selective agonists such

as DPDPE (Mosberg et al. 1983) produce supraspinal analgesia in thermal

tests by activation of mu receptors, or whether cerebral delta receptors

are also involved in this effect. The present investigation attempts to

determine the possible involvement of cerebral delta opioid receptors in

thermal analgesic tests using direct antagonism with the highly

selective delta antagonist, ICI 174,864 (Cotton et al. 1984) as well as

the determination of possible cross-tolerance between morphine and the

selective delta agonist, DPDPE.

MATERIALS AND METHODS

Analgesic assay: Male, ICR mice (20-25 g) were tested forthelatencytoa

442
rapid flick of the tail following immersion into warm (55°C) water. The

mice then received graded i.c.v. doses of agonist and distilled water, or

agonist and antagonist. The i.c.v. injection was made following the

induction oflightether anesthesia by an incision in the scalp, location

of bregma and injection directly into the lateral ventricle using a

microliter syringe in a volume of 5 μl. Twenty min after the i.c.v.

injection, a time determined to be close to the peak analgesic effect of

the agonists, the mice were retested for the latency to tail-flick. A

cut-off time of 15 sec was employed in order to minimize damage to the

tail. Post-drug latency was comparedtotheindividual pre-drug latency

and percent analgesia determined.

Acute tolerance: The procedure used was that described by Vaught and

Takemori (1979). Mice received a single S.C. injection of morphine

sulfate (100 mg/kg) or water 5 hr prior to testing. The morphine

pretreatmentnolongerhadanyagonisteffectatthis time. Graded doses

of morphine, DAGO and DPDPE were then given i.c.v. andtestingtook place

after 10 min.

RESULTS

Co-administration of ICI 174,864 (at 1 or 3 μg, i.c.v.) with i.c.v. DPDPE

resulted in a dose-related antagonism of the analgesic effect (fig. 1).

In contrast, ICI 174,864 (3 μg) failedtoantagonizethemorphine (fig.1)

or DAGO (not shown) dose-response lines.

Figure 1. Analgesic dose-response lines for i.c.v. morphine

(circles) or DPDPE (squares) in the absence (closed symbols) or presence

of ICI 174,864 (open symbols) at 1 or 3 μg (DPDPE) or 3 μg (morphine).

Pretreatment with morphine (100 mg/kg, S.C. at -5 hr) did not result in

any analgesic effect following the i.c.v. administration of water. The

i.c.v. morphine dose-response line was displaced to the right by a factor

of 6.6, indicating the developmentofacutemorphine tolerance (table 1).

443
In contrast, the i.c.v. DPDPE dose-response line was not affected (table

1), indicating a lack of i.c.v. acute cross-tolerance between morphine

and the selective delta agonist.

TABLE 1. Analgesic A50 (and 95% C.L.'s)(μg/mouse) for 3 agonists

given intracerebroventricularly to control or morphine pretreated mice

(100 mg/kg, S.C. at -5 hr) in the warm-water tail-withdrawal test.

Agonist

Potency

Control Morphine-pretreated

Morphine 0.25 (0.107 - 0.598) 1.65 (0.79 - 3.47) 6.6

DPDPE 5.71 (3.13 - 10.43) 7.84 (4.53 - 13.56) 1.37

DPLPE 6.77 (0.01 -2.8-16.4) 7.68 (5.17 - 11.41) 1.13

DISCUSSION

The present study has investigated the possible involvement of delta

opioid receptors in the inhibition of responses to a noxious thermal

stimulus. Both mu (morphine, DAGO) and delta (DPDPE) agonists produced

full dose-response effects after i.c.v. administration, suggesting that

both mu and delta receptorsmightbe responsible forthemediation of this

effect. Previous studies, however, have suggested that all supraspinal

analgesia determined in tests utilizing heat as the noxious stimulus

relied on the sole activation of mu opioid receptors (Audigier et al.

1980; Chailletetal 1484; Chang etal. 1982). Ward and Takemori (1983)

found that the naloxone pA2 value against [D-Ala2, D-Leu5]enkephalin

(DADLE) was slightly lower than that against morphine. Additionally,

those investigators found that the i.c.v. analgesic effects of DADLE were

antagonized by beta-funaltrexamine (beta-FNA)(Takemori et al. 1981),

leading to the suggestion of involvement of both i.c.v. mu and delta

opioid receptors in the tail-flick test. Our results using DPDPE, a

highly selective delta agonist (Mosberg et al. 1983) together with ICI

174,864, a highly selective delta antagonist (Cotton et al. 1984),

support this conclusion.

The co-administration of ICI 174,864 with DPDPE produced a dose-related

antagonism of i.c.v. DPDPE, but not morphine or DAGO, analgesia.

Administration of ICI 174,864 alone, at the doses studied, didnotaffect

the tail-flick response. Differential antagonism has historically been

taken as the strongest evidence fortheinvolvementofmultiple receptors

inapharmacological effect. Thus, the present results strongly suggest

that both cerebral mu (morphine, DAGO) and delta (DPDPE) receptors

mediate analgesia in this test.

Determination of the possible presence or absence of acute crosstolerance between morphine and DPDPE was also employed as a second

approach in an attempttoconfirmthe results oftheexperiments using the

444
delta antagonist. While the presence of cross-tolerance suggests

either activity at a common receptor or some common post-receptor

pathway, the absence of cross-tolerance is reasonable evidence of

activity of separate receptor types. Acute pretreatment with a bolus

injection of morphine resulted in the development of acute tolerance as

shown by the rightward displacementofthe i.c.v. morphine dose-response

line. Similarly, morphine pretreatment displaced the i.c.v. DAGO doseresponse line to the right demonstrating acute cross-tolerance. In

contrast, however, the i.c.v. DPDPE dose-responselinewas not affected.

The lack of i.c.v. cross-tolerance between DPDPE and morphine, while not

establishing activity at separate receptors, supports the concept of

involvement of two receptors in this effect.

The finding of a lack of i.c.v. acute cross-tolerance between DPDPE and

morphine, when taken together with the antagonism of DPDPE, but not

morphine or DAGO analgesia by ICI 174,864, provides strong and direct

evidence of involvement of both cerebral delta, and mu opioid receptors

in this analgesic test. This concept has been previously suggested on

the basis of a lack of i.c.v. inhibition of gastrointestinal transit by

DPDPE, butnotby mu agonists (Porreca et al. 1984). The involvement of

kappa receptors in this thermally-initiated endpoint seems unlikely as

kappa agonists do not produce reliable effects after i.c.v.

administration. Thus, it appears thatbothmu and delta receptors in the

brain participate in this analgesic effect; the identification of the

spinal opioid receptors in this effect remains to be investigated.

REFERENCES

Audigier, Y., Marzarguil, H., Gout, R. and Cros, J. Eur J Pharmacol,

63: 35-46. 1980.

Chaillet, P., Coulaud, A., Zajac, J.M., Fournie-Zaluski, M.C.,

Costentin, J. and Roques, B.P. Eur J Pharmacol, 101:83-90, 1984.

Chang, K.J., Cuatrecasas, P., Wei, E.T. and Chang, J.K. Life Sciences,

30:1547-1551, 1982.

Cotton, R., Giles, M.G., Miller, L., Shaw, J.S. and Timms, D. Eur J

Pharmacol, 97:331-332, 1984.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I., Galligan,

J.J. and Burks, T.F. Proc Natl Acad Sci (U S A ), 80:5871-5874,

1983.

Porreca, F., Mosberg, H.I., Hurst, R., Hruby, V.J. and Burks, T.F. J

Pharmacol Exp Ther, 230:538-547, 1984.

Takemori, A.E., Larson, D.L. and Portoghese, P.S. Eur J Pharmacol,

70:445-451, 1981.

Vaught, J.L. and Takemori, A.E. J Pharmacol Exp Ther, 208:86-90, 1979.

Ward, S.J. and Takemori, A.E. J Pharmacol Exp Ther, 224:525-530, 1983.

ACKNOWLEDGEMENTS

This work was supported by USPHS Grants NS 21193 (F.P.) and DA 03910

(H.I.M.).

445
INTRAVENTRICULAR ADMINISTRATION OF BAM-18:

ANTINOCICEPTIVE AND LOCOMOTOR ACTIVITY IN THE RAT

K. E. Stevens, F. M. Leslie, C. J. Evans*, J. D. Barchas*,

J. D. Belluzzi and L. Stein

Dept. of Pharmacology, University of California,

Irvine, CA 92715

*Depts. of Psychiatry and Behavioral Science,

Stanford University, Stanford CA 94305

ABSTRACT

BAM-18, a new endogenous opioid containing 18 amino acid residues,

was tested in 3 behavioral paradigms. Tail-flick analgesia, a

spinally mediated response, hot-plate analgesia, a centrally mediated

response, and open-field locomotor activity. Rats were

stereotaxically implanted with a unilateral cannula aimed at the

lateral ventricle. Following recovery, each animal was tested in one

of the paradigms after receiving an intraventricular injection of

BAM-18, morphine or the Ringer's vehicle. BAM-18 produced

significant tail-flick analgesia only at doses (50 ug) 50 times

higher than those needed with morphine (1 ug). BAM-18 produced an

extended hyperalgesia at lower doses (5 ug) that was also seen

transiently at the high dose. The analgesia but not the hyperalgesia

was reversed by naloxone (10 mg/kg, s.c.). BAM-18 produced

significant naloxone-reversible hot-plate analgesia, but again it was

less potent than morphine (50 ug for BAM-18 vs. 5 ug for morphine).

There was no evidence of hyperalgesia in this paradigm. Locomotor

activity, following 50 ug of BAM-18, resembled control injections for

the first 18 minutes, then became reduced in a manner similar to

morphine (5 ug). This reduction in activity was completely reversed

by naloxone. These data suggest that BAM-18 is indeed an opioid

molecule but is at least 10 times less potent at altering behavior

than morphine.

INTRODUCTION

BAM-18 is a new endogenous opioid molecule of 18 amino acid residues

which has recently been isolated from bovine adrenal medulla (Evans

et al. 1985). BAM-18 is a derivative of the pro-enkephalin A

molecule (Pittius et al. 1984), and is found in high concentrations

in many areas of the central nervous system including striatum,

hypothalamus, pons/medulla and spinal cord (Evans et al. 1985). We

have found that BAM-18 binds with high affinity to both u and k

receptors (ki-0.29 nM and 0.75 nM, respectively) and might therefore

be expected to exhibit potent analgesic activity. After central

administration of BAM-18 or morphine, we tested rats in 2 analgesia

paradigms, tail-flick which is a spinally mediated response and

hot-plate which is centrally mediated (Jensen and Yaksh 1985), and

also assessed drug effect on spontaneous locomotor activity. All

observed behavioral changes after BAM-18 administration were tested

for naloxone reversibility.

446
MATERIALS AND METHODS

Seventy male Sprague-Dawley rats (Charles Rivers, Portage, Michigan)

were unilaterally implanted with a 24 gauge stainless steel injection

cannula aimed at the lateral ventricle (-1.0 mm from bregma, +1.7 mm

from midline and -3.0 mm from dura). Following recovery, the animals

were placed in one of the following experimental paradigms.

Experiment 1.

Animals were placed in the tail-flick restrainer and allowed to

acclimate for 30 minutes. The tip of the tail was blackened to

reduce variability of heat absorbtion. The intensity of the heat

lamp was adjusted for each animal to yield a mean baseline tail-flick

latency of 7-9 seconds. Following 3 baseline readings, BAM-18 or

morphine, dissolved in 20 ul of modified Ringer's solution, or

Ringer's alone, was administered through the cannula and tail-flick

latencies were performed every 2 minutes for a total of 30 minutes.

Naloxone reversibility was determined by administration of naloxone

(10 mg/kg, s.c.) 15 minutes before icv BAM-18 or Ringer's injections.

Experiment 2.

The hot-plate testing apparatus consisted of a Corning hot-plate

covered with a 0.25 inch aluminum plate for even heat distribution,

and a plexiglass open-ended box on the plate. The temperature was

maintained at 45° C and monitored continually with a YSI thermistor

thermometer. Animals were allowed to habituate to the apparatus on a

cold plate. Testing was performed by lowering the animal, by its

tail, onto the hot-plate. When the hind paws touched the plate,

timing began and was terminated when the animal licked a hind paw.

Three readings were taken and averaged to yield the pre-drug

baseline. BAM-18 or morphine, dissolved in 20 ul of Ringer's

solution, or Ringer's alone was administered through the cannula, and

latencies were performed every 5 minutes for a total of 30 minutes.

Naloxone reversibility was determined by administration of naloxone

(10 mg/kg, s.c.) 15 minutes before icv BAM-18 or Ringer's injections.

Experiment 3.

Spontaneous locomotor activity was recorded in an Opto-Verimex-Minor

activity monitor (Columbus Instruments). Animals were injected with

BAM-18 or morphine dissolved in 20 ul of Ringer's solution or

Ringer's alone, as in previous experiments, and placed in the animal

activity monitor. Locomotor activity was recorded every 6 minutes

for a total of 60 minutes. Naloxone reversibility was assessed by

administration of naloxone (10 mg/kg, s.c.) 15 minutes prior to icv

BAM-18 or Ringer's solution injection.

RESULTS

BAM-18 produced a significant analgesic response in the tail-flick

paradigm (figure la) similar to that seen with morphine. However,

the dose of BAM-18 required was 50 times that needed with morphine

(50 ug and 1 ug respectively). At lower doses of BAM-18 (5 ug), and

447
transiently at higher doses, a hyperalgesic response was

observed. The analgesia but not the hyperalgesia was reversed by

naloxone (10 mg/kg, s.c.)(figure 1b), unmasking the hyperalgesia at

the 50 ug dose. A significant analgesia was also observed with

BAM-18 in the hot-plate paradigm (figure 2a). The dose required was

vs. controls)

FIGURE 1. a. Mean tail-flick latency (sec) after

intraventricular administration of 2 doses of BAM-18.

b. Mean tail-flick latency (sec) after pretreatment

with naloxone (10 mg/kg, s.c.) and icv administration

of either BAM-18 or Ringer's solution. Scores

represent change from baseline (drug score - baseline

score). Bars are S.E.M.'s. (* p < 0.05, F-test,

administration of BAM-18. b. Mean hot-plate latency

(sec) after pretreatment with naloxone (10 mg/kg,

s.c.) and icv administration of either BAM-18 or

Ringer's solution. Scores represent change from

baseline (drug score - baseline score). Bars are

S.E.M.'s. (* p < 0.05, F-test, vs. control)

10 times greater than an analgesic dose of morphine (50 ug BAM-18, 5

ug morphine). The BAM-18-induced analgesia was completely reversed

by naloxone (10 mg/kg, s.c.)(figure 2b). BAM-18 was 50 times less

potent than morphine in producing analgesia in the tail-flick test (a

spinally mediated response) and 10 times less potent in the hot-plate

test (a centrally mediated response). These data suggest that the

degradation of BAM-18 plays a significant roll in determining its

potency in the production of analgesia. BAM-18 (50 ug) did not

affect locomotor activity during the first 18 minutes following

injection (figure 3a). During the later time frames, BAM-18 caused a

reduction in spontaneous activity similar to that seen with morphine,

albeit at a larger dose than morphine (5 ug). The reduction in

spontaneous activity caused by BAM-18 was completely reversed by

naloxone (10 mg/kg s.c.)(figure 3b). Occasionally, seizure-like

activity was seen with the 50 ug dose of BAM-18. The seizures were

448
of three types, wet-dog shakes, head and fore-paw shakes, and

barrel-rolling. The barrel-rolling behavior was observed in one

animal even after naloxone. Non-naloxone reversible barrel-rolling

activity is routinely seen with 100 ug intraventricular injections of

Dynorphin (1-13) (Herman et al. 1980) which has some sequence

homology with BAM-18.

icv administration of BAM-18. b. Mean open-field

locomotor activity after pretreatment with naloxone (10

mg/kg, s.c.) and icv administration of either BAM-18 or

FIGURE 3: a. Mean open-field locomotor activity after

Ringer's solution. Bars are S.E.M.'s. (* p < 0.05,

F-test, vs. control)

In conclusion, BAM-18, when administered centrally, is capable of

producing analgesia in 2 different behavioral tests. However, it is

consistantly less potent than morphine. Reliable analgesia has been

achieved with as little as 0.5 ug ivc in the tail-flick paradigm

(McGregor et al. 1978) whereas in the present work a dose of 50 ug

BAM-18 was required. Similarily, BAM-18 reduces spontaneous activity

but, again, at a larger dose than is required for morphine. A

hyperalgesia in the tail-flick paradigm was observed at low doses of

BAM-18 and was unmasked by naloxone at higher doses. All observed

effects of BAM-18 (except the hyperalgesia) were reversed by

naloxone.

REFERENCES

Evans, C.J., Erdelyi, E., Makk, G., and Barchas, J. D. Fed Proc,

44:422, 1985.

Herman, B. H., Leslie, F., and Goldstein, A. Life Sci, 27:883-892,

1980.

Jensen, T. S., and Yaksh, T. L. Neurosci Abst, 11:910, 1985.

McGregor, W. H., Stein, L., and Belluzzi, J. D. Life Sci,

23:1371-1378, 1978.

Pittius, C. W., Seizinger, B. R., Pasi, A., Mehraein, P., and Herz,

A. Br Res, 304:127-136, 1984.

ACKNOWLEDGEMENTS

The authors wish to thank Columbus Instruments International

Corporation for their generous loan of the Opto-Verimex-Minor

activity monitoring system. The naloxone was kindly supplied by Endo

Laboratories Inc., Garden City, New York. Research was supported by

AFOSR Grant 84-0325 and NIH Fellowship DA-05288-01.

449
A SINGLE icv INJECTION OF KCl INDUCES A SELECTIVE AND

LONG-LASTING INHIBITION OF OPIOID ANALGESIA IN MICE

Javier Garzón and Pilar Sánchez-Blázquez

Department of Neuropharmacology, Cajal Institute

28006 Madrid, Spain.

ABSTRACT

The icv administration of potassium chloride a few min

before the icv injection of opioids, antagonized the

supraspinal analgesia induced by these substances in the

mouse. The antagonistic effect of a single injection of

KCl lasted for 3 to 4 days and was accompanied by

opposite changes in 3H-DADLE binding to mesencephal ic

(decrease) and spinal cord (increase) membranes.

INTRODUCTION

Potassium ion has usually been considered as an inert

agent on properties of the opioid receptor in vitro (Pert

and Snyder 1974). However, electrophysiological studies

have recently shown that op io ids cause hyperpolarization

of neurons by opening certain potassium channels in the

neural membrane. This action of opioids was reduced by

increasing the extraneuronal concentration of ion

(Willians et al. 1982). On the basis of these findings,

the influence of icv administration of KCl on the

analgesia induced by opioids at supraspinal level in the

mouse was investigated.

MATERIALS AND METHODS

Pept ides were obtained from Bachem (Bubendorf,

Switzerland). Tritiated dihydromorphine (DMH) and

D-Ala2,D-Leu5-enkephal in (DADLE) were purchased from

Amersham Corporation and New England Nuclear

respectively. Albino male mouse CFLP. 22-27 g were used

throughout. Analgesia was evaluated by the tail-flick

method in a water bath at 52° C. and expressed as a

percent of the maximum possible effect after setting a

cut off time of 10s. Binding experiments were carried out

on crude P2 fractions at 25°C for 30 min in 2 ml of 50mM

TRIS buffer pH 7.7 in the presence of several

concentrations of labeled 1 igands. Samples were filtered

through Whatman GF/B filters and counted in a

scintillation counter Beckman LS-5801.

450
RESULTS

KCl or NaC (0.1-1 μmole/mouse,icv) given 30 min before

the icv administration of opioids, did not modify the

analgesia induced by morphine and related alkaloids

(normorphine, levorphanol...), which agrees with previous

reports (Chapman and Way 1980). Similarly, the ions did

not alter the analgesic properties of DTLET and

morphiceptin. However, potassium but not sodium (1

μmole/mouse), clearly diminished in a dose-dependent and

non-competitive manner the analgesia induced by several

opioid peptides of different receptor selectivities

(table 1): μ, FK 33-824; , DADLE, DPLPE. In mice

pretreated with KCl icv 0.3, 0.5 and 1 μmole/mouse, DADLE

only reached 85, 60 and 20% of the maximum analgesic

effect. KCl treatment also reduced the analgesia elicited

by endogenous opioid peptides derived from the

Proenkephalin precursor. In contrast, the analgesic

action of ß-endorphin remained practically unchanged.

TABLE 1. Effect of KCl and NaCl on the analgesia induced

by opioids in the mouse

Opioid

test time NaCl KCl

min ED50 ED50 slope

Morphine 30 3.00 0.26

DADLE 15 0.10 0.02

DPLPE 15 20.9 3.10

DTLET 15 1.14 0.17

FK 33-824 30 0.0027 0.0003

Morphiceptin 5 6.91 0.75

Met-ENK-Arg-Phe 5 37.1 4.20

Adrenorphin 15 28.1 2.53

Peptide E 30 1.51 0.72

ß-endorphin 30 0.042 0.004

3.30 0.22 0.90

> 30* 0.24

>200* 0.54

1.44 1.15 1.15

0.15 0.09* 0.51

7.22 0.60 1.06

>300* nd

> 100* nd

> 10* nd

0.070 0.006 0.80

Salts (1μmole/mouse) were given icv 30min prior

to the opioids. Groups of 8 to 12 animals were

used. Slopes and ED50 were computed from the

Hill transformation of experimental data. ED5Os

are in nmoles/mouse. * Statistically different

from the group treated with NaCl, Student's t

test;p<0.05. Abbreviations,nd:non determinated;

DPLPE: (D-Pen2, L-Pen5)-enkephalin; DTLET:

(D-Thr2, Leu5)-enkephalin-Thr6; FK 33-824:

(D-Ala2, N-Me-Phe4, Met-(o)5-ol)-enkephalin.

The antagonistic effect of a single icv injection of KCl

(1 nmole/mouse) was found to be of a remarkable long

duration. The analgesia was still diminished 3 days later

and after a slow recovery phase the animals showed a

normal response to opioids on the 5th day (table 2).

451
TABLE 2. Time-course of KCl antagonism on opioid-induced

supraspinal analgesia

Ratio Na/K

Opioid Dose Day: 1 2 3 4 5

Morphine 3.0 0.86 0.89 1.12 1.15 0.88

DADLE 0.1 5.80* 3.20* 2.26* 1.67* 1.20

FK 33-824 0.003 2.80* 2.34* 2.14* 1.75* 1.37

Salts <1μmole/mouse,icv) were given once on day

1, 30 min prior to the opioids. Opioids were

administered icv at ED50 (nmole/mouse) and the

analgesic effect studied daily for 5

consecutive days. Ratios between the percent of

the maximum analgesic effect induced by opioids

in groups of mice (n=10) pretreated either with

NaCl or KCl are shown. * Statistically

different from the group treated with NaCl,

Student's t test; p<0.05.

TABLE 3. Time-course of KCl effect on 3H-DADLE binding

Ratio Na/K

Structure Day: 1 2 3 4 5

Mesencephalon Kd 1.00 1.11 1.02 1.03 1.10

Bmax 0.95 1.53* 1.35* 1.12 1.11

Spinal cord Kd 1.05 1.00 0.95 0.95 0.98

Bmax 1.03 0.75* 0.80* 0.96 1.10

Salts (1μmole/mouse.icv) were given once a day

1. Groups of mice were killed 30 min after the

salt injection on day 1 and at the intervals

above indicated. Ratios between parameters

obtained from NaCl and KCl treated mice are

shown. The experiment was repeated four times. Kd

and Bmax for 3H-DADLE specific binding to

mesencephalic and spinal cord membranes are

respectively: 3.2 0.2 nM, 115 5 fmoIe/mg

protein and 1.9 0.1 nM, 56 3 fmole/mg protein.

* Statistically different from 1, Student's t

test; p<0.05.

The specific binding of 3H-DHM and 3H-DADLE to mouse

brain and spinal cord membranes was also monitored during

the 5 days after the single icv injection of the salts

(1 μmole/mouse). No change was shown when the whole brain

was studied. However, in KCl treated mice, 3H-DADLE

specific binding decreased in mesencephalon and increased

in spinal cord. These changes were evident 24 h after the

KCl injection and slowly returned to the initial values

on day 4 (table 3). In contrast, 3H-DHM binding to these

structures remained unchanged.

452
DISCUSSION

The apparent selectivity of the antagonism induced by KCl

on the opioid analgesia and the changes found in the

number of opioid receptors labeled by DADLE, agree with

the proposed involvement of, multiple receptors in the

supraspinal analgesia induced by opioids (Höllt et al.

1985). Assuming that hypothesis, the "potassiumsensitive" opioids would induce at least a part of the

analgesic effect after binding to a receptor(s) whose

function is susceptible of being reduced by that ion. The

'potassium-sensitive' receptor appears not to be the one

preferred by either the opiate alkaloids, morphiceptin or

DTLET to promote analgesia. Interestingly, DTLET and its

analog DSLET seem to induce analgesia through the

morphine receptor (Chaillet et al. 1984). On the other

hand, it has been shown that morphine- and ß-endorphininduced analgesia can be modulated without altering the

antinociceptive properties of the ligand DADLE or the μ

ligand FK 33-824 (Tulunay et al. 1981). In the present

study, it was evidenced the antagonism by KCl of DADLEand FK 33-824-evoked analgesia but not of morphine, thus

confirming the possibility of modulating the analgesic

effects of FK 33-824 and morphine independently. Of

interest is a recent report describing the existence of

an additional type of binding site to which μ- and

d-selective enkephalin analogs display their highest

affinities (Lutz et al. 1985). We find particularly

interesting the antagonism by KCl of the analgesia

elicited by the Proenkephalin-derived peptides. This

result might indicate that a common mechanism underlies

the effect of that family of endogenous opioids. Although

further work is required to identify the opioid receptor

whose function is decreased by potassium as well as the

processes responsible for that antagonism, these results

suggest a regulatory role for that ion on the function of

the opioid receptor in vivo.

REFERENCES

Chaillet, P., Coulaud, A., Zajac, J.M., Fournie-Zaluski,

M.C., Coustentin, J. and Roques, B.P. Eur J Pharmacol,

101:83-90, 1984.

Chapman, D.B. and Way, E.L. In:George, R., Okun, R. and Cho,

A.K. ed. Annual Review of Pharmacology and Toxicology.

Vol. XX. Palo Alto, Annual Reviews Inc., 1980. pp 553-579.

Höllt,V., Sánchez-Blázquez, P. and Garzón, J. Phil Trans

R Soc Lond, B308:299-310, 1985.

Lutz, R.A., Cruciani, R.A., Munson, P.J. and Rodbar, D. Life

Sci, 36:2233-2238, 1985.

Pert, C.B. and Snyder, S.H. Mol Pharmacol, 10:868-879, 1974.

Tulunay, F.C., Jen, M.F., Chang, J.K., Loh, H.H. and Lee, N.M.

J Pharmacol expt Ther, 219:296-299, 1981.

Willians, J.T., Egan, T.M. and North, R.A. Nature, 299:74-77,

1972.

453
INCREASE IN MET-ERKEPHALIN LEVEL AND ANTINOCICEPTIVE EFFECTS

INDUCED BY KELATORPHAN IN THE RAT SPINAL CORD.

Marie-Claude Fournié-Zaluski*, Sylvie Bourgoin**

François Cesselin**, Michel Hamon**, Daniel Le Bafs*** ,

Antony H. Dickenson and Bernard P. Roques .

* Chimie Organique, U 266 INSERM, UA 498 CNRS, Faculté

de Pharmacie, 4 Av. de l'Observatoire, 75006 Paris, France.

** U 288 INSERM, CHU Pitié-Salpérière, 75013 Paris,

*** U 161 INSERM, Centre Paul Broca, 75014 Paris,

**** University College, London.

ABSTRACT

Kelatorphan, a mixed inhibitor of “enkephalinases” is used for

demonstrating the implication of endogenous enkephalins in

nociceptive transmission at the spinal level.

INTRODUCTION

The high levels of enkephalins in the superficial dorsal horn of

spinal cord, together with dense opiate receptor binding sites, may

be related to a role in the modulation of nociceptive transmission

at the spinal level. However, demonstration of this putative role is

prevented by the rapid degradation of enkephalins by three

peptidases : enkephalinase, aminopeptidase M and a dipeptidylaminopeptidase whose specific role is not clearly established. Mixed

inhibitors of these three enzymes were developed and kelatorphan is

the first of a new series of compounds exhibiting high analgesic

properties by both i.c.v. and i.v. routes (Fournié-Zaluski et al.

1985). Consequently, kelatorphan was also studied for its ability to

protect endogenous enkephalins (Bourgoin et al. 1986) and to depress

the responses of nociceptive neurons in the spinal cord (Dickenson

et al. 1986).

RESULTS AND DISCUSSION

Effect of kelatorphan on the in vitro outflow of endogenous

met-enkephalin from rat spinal cord slices. Superfusion of dorsal

horn slices with 2 and 20 μM kelatorphan, produces an increase of

82% and 175% in the spontaneous and 130% and 230% in the K

+

evoked

outflow of enkephalin. The association of bestatin (20 μM) and

thiorphan (1 μM) is inactive on the spontaneous release.

454
Effect of kelatorphan on the in vivo release of met-enkephalin like

material (MELM) from the rat spinal cord. The subarachnoidal space

of anesthetized rats was perfused with artificial CSF, with or

without, the various inhibitors. As shown in table 1, kelatorphan

increases both the spontaneous outflow of MELM and the outflow

evoked by K+

 depolarization or by a noxious treatment.

TABLE 1. Comparison of the effect of kelatorphan and the

association bestatin + thiorphan on the in vivo release

of MELM from the rat spinal cord.

Treatment Control Th(1μM)+B(20 μM) Kelatorphan(20μM)

None 4.33 ± 0.4 9.47 ± 0.66 10.82 ± 1.38

K

+ (40mM) 10.97 ± 1.59* 23.65 ± 3.29*

Muzzle pinching10.10 ± 1.26* 19.82 ± 2.42* 22.28 ± 4.60”

* P < 0.05.

Inhibition of nociceptive neurones in rat spinal cord. The

inhibitors were applied at the indicated concentrations onto the

surface of the dorsal horn of anaesthetized rats and the neurons

were recorded and characterized by their responses to electrical

stimulation of A and C fibres. As observed in figure 1, the effect

of kelatorphan was significant with a dose as low as 2.5 μg and the

inhibition of C fibres was clearly dose dependent. The maximal

effects were seen with 25 and 50 μg.

FIGURE 1. Inhibition of the A evoked activity and

C-fibre responses by bestatin, thiorphan and kelatorphan.

455
The A fibre responses were only significantly altered by the highest

doses. The effects of kelatorphan were greater than those produced

by the same doses of bestatin and thiorphan and were reversed by

naloxone.

Analgesic effects of kelatorphan on hot plate and tail flick tests.

The properties of kelatorphan were also studied on the tail flick

test, a spinal model of analgesia and on the hot plate test, which

is considered as a supraspinal model. As shown in table 2,

kelatorphan induces high analgesic responses on the jump latency

time but also on both the licking, and the tail flick tests for the

highest doses. All these effects are reversed by naloxone.

TABLE 2. Effects of kelatorphan on analgesic tests in

mice.

Hot plate test Tail flick test(s) c)

Licking time(s) a) Jump latency time(s) b)

control 5 ±0.3

10 μg 4.3±O.3 (NS)

40 μg 6.1±0.7 (NS)

80 μg 8.4±0.8 *

114 μg 8.6±_0.9 **

200 μg 9.2±1.3 ***

50±5 7.3±0.3

82±8 ** NT

123±16 *** 7.6±0.5 (NS)

188±1O *** 7.9±0.6 (NS)

200±16 *** 11.9±0.6 ***

206±15 *** 12.2±0.6 ***

a) cut off time, 30 s ; b) cut off time, 240 s ; c) cut

off time, 24 s.

CONCLUSION

The experiments performed in both CNS and spinal cord clearly

indicated that the use of mixed inhibitors of "enkephalinase" are

the most efficient method for the in vivo study of physiological

properties of enkephalin.

REFERENCES

Fournié-Zaluski, M.C., Coulaud, A., Bouboutou, R., Chaillet, P.,

Devin, J., Waksman, G., Costentin, J. and Roques, B.P. J Med Chem,

28:1158, 1985.

Bourgoin, S., Le Bars, D., Artaud, F., Clot, A.M., Bouboutou, R.,

Fournié-Zaluski, M.C., Roques, B.P., Hamon, M. and Cesselin, F. J

Pharmacol Exp Ther, 238:360, 1986.

Dickenson, A.H., Sullivan, A.F., Fournié-Zaluski, M.C. and Roques,

B.P., Brain Res, in press, 1986.

456
MODULATORS OF PAIN IN THE SPINAL CORD

Jen, M.F.*, Lu, C.H., Huang, L. and Han, J.S.

Department of Physiology, Beijing Medical University

*Department of Pharmacology, Institute of Basic Medical Sciences,

Chinese Academy of Medical Sciences, Beijing, China.

ABSTRACT

Intrathecal injection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1-13)

(1.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick

latency in the rat. The analgesic effect of the composite of dynorphin/morphine was

dose-dependent in serial dilutions and not accompanied by any signs of motor dysfunction. Synergism between dynorphin and morphine can also be demonstrated without

motor dysfunction, when dynorphin is injected together intrathecally with a serial

increasing dosages of morphine. Metenkephalin showed an analogous synergistic effect

with dynorphin and also morphine in the spinal cord, although dynorphin (and also

metenkephalin) antagonized morphine analgesia by the intraventricular route. The

underlying mechanism of the interactions among different classes of opioid ligands at

different levels of the central nervous system deserves further study.

INTRODUCTION

The analgesic effect of dynorphin upon intrathecal injection has been well documented

(Han and Xie 1982, Piercey et al. 1982, Przewlocki et al. 1983); but large dose of intrathecal dynorphin often causes motor paralysis of hind limbs, which might interfere with the

tail flick analgesic test. Intraventricular injection of dynorphin and also metenkephalin

have been proved to have an inhibitory effect on morphine or beta-endorphin analgesia

(Lee et al. 1980, Friedman et al. 1981). So it would be interesting to compare the interaction of dynorphin, metenkephalin, and morphine in the spinal cord and the brain. This

may provide important clues about the pain modulatory effects of different types of opioid

ligands and their interactions.

METHODS

Rats, 200-250 gm in body weight were used. Intrathecal injections were given via PE-10

catheter within 24-48 hours after its implantation into the spinal subarachnoid space

(Yaksh and Rudy 1976). Intraventricular injections (ICV) were given to the lateral ventricles through a stainless steel cannula inserted along an appropriate guide anchored to

the skull. Tail flick latencies (TFL) were assessed (Jen and Han 1979) prior to drug

administration and thereafter; the percent change of pain threshold was determined by

taking the pre-drug level as 100 %. Statistical significances between groups were assessed

by Student’s t-test.

RESULTS

I. Synergistic effect between dynorphin and morphine given intrathecally:

Different groups of rats, 8 to 11 in each group, were given 1.25 nmol of dynorphin-A-(1-13)

or 7.5 nmol of morphine separately or in combination at a total volume of 10 μl. The

combined effect was much stronger than the mathematical additive effect of the two components, which were both not significantly different from the saline control (fig. 1).

The combination of morphine and dynorphin gave rise to a marked increase in TFL over

100 % of the basal level 10 min after injection, which was maintained at a height

between 104±15 % and 119±7 % for the whole observation period of 80 minutes (fig. 2).

The effect was also dose-related upon serial dilution. None of the rats showed any signs

of motor dysfunction when they were allowed to move freely out of the holders.

457
FIGURE 1. Analgesic effect of dynorphin-A-(1-13)

1.25 nmol, morphine 7.5 nmol and both drugs in

combination. *** Indicates p <0.001 compared to

either dynorphin or morphine alone.

FIGURE 2. Dose-response effect of seriall dilutions of

intrathecal dynorphine/morphine composite.

Numerals in parentheses indicate number of

animals.

Similar synergistic effect between dynorphin and morphine could also be observed when

dynorphin was given on top of serial increasing dosages of morphine of 2, 4, 8, 16, and 32

nmol, given in succession every 10 min (fig. 3). Again, none of these animals showed any

signs of motor dysfunction.

FIGURE 3. Synergistic effect between

dynorphin and serial increasing dosages

of morphine given intrathecally.

II. Synergistic effect between metenkephalin and morphine or dynorphin given intrathetally:

Analogous synergistic effect was observed between metenkephalin and serial increasing

dosages of morphine or dynorphin given intrathecally (fig. 4 and 5). Subanalgesic dose of

metenkephalin (100 nmol) was effective in potentiating morphine analgesia across various

dose ranges of morphine or dynorphin.

FIGURE 4. Synergistic effect between metenkephalin FIGURE 5. Synergistic effect between metenkephalin

and serial increasing dosages of morphine given and serial increasing dosages of dynorphin given

intrathecally. intrathecally.

458
III. Antagonism of morphine analgesia by dynorphin given intraventricularly:

Different groups of rats, 6-11 in each group, were given ICV injection of normal saline 8

μl, dynorphin-A-(1-13) 20 nmol or morphine 30, 60, and 120 nmol respectively. Saline and

dynorphin did not cause any significant change in basal pain threshold. However, morphine, at different dose levels, produced a dose-dependent increase of 19±12, 75±18, and

139±7 % of basal pain threshold.

When dynorphin 10 and 20 nmol was given ICV together with 120 nmol of morphine in

another 2 groups of rats, the analgesic effect of morphine was attenuated. A reduction of

88 % was noticed with 20 nmol of dynorphin and was statistically very significant (p <

0.01) from the group given morphine alone (fig. 6).

FIGURE 6. The effect of ICV injection of

various doses of dynorphin-A-(1-13) and

morphine in combination.

When dynorphin 20 nmol was given 30 min after 120 nmol of morphine was injected ICV,

a drastic suppression of the analgesic effect of morphine was noticed within 20 min (68 %

reversal, p < 0.001, as shown in fig. 7). Similar suppressibe effect was noticed when dynorphin 20 nmol was injected ICV together with 30, 60, and 120 nmol of morphine given in

serial succession to rats (fig. 8). The differences in analgesic effect between the group

given morphine and dynorphin in combination and the group given saline plus morphine

were statistically significant or very significant across all dosages of morphine.

FIGURE 7. The effect of ICV injection of

dynorphin-A-(1-13) on morphine analgesia. M, morphine 120 nmol; D,

dynorphin-A-(1-13) 20 nmol; NS, normal

saline.

FIGURE 8. Antagonistic effect of dynorphin on increasing dosages of morphine

given intraventricularly.

459

172-683 O - 87 - 16 : QL3
DISCUSSION

Our data clearly showed that a subanalgesic dose of dynorphin-A-(1-13) produced a

marked potentiation on morphine analgesia whereas none of the animals had any sign of

motor dysfunction. Thus different effects of dynorphin-A on sensory and motor function

may occur depending on the dose: a small amount. of exogenously applied or endogenously

released (Han and Xie 1984) dynorphin-A primarily affects nociception. Motor activities

are impaired only when a relatively large dose is administered or huge amount of dynorphin is released in case or cord injury (Faden et al. 1983). The supposition that

dynorphin-A in spinal cord may be more involved in sensory rather than in motor function seems to be in line with the findings that the dynorphin-A content in the dorsal spinal cord is much higher than in the ventral cord (Botticelli et al. 1981).

Synergism between different classes of opioid ligands dynorphin, metenkephalin, and morphine at the spinal level implies that they may act’ synergistically in the spinal cord and

are important modulators of pain at that level. These findings strongly suggest that

dynorphin-A-(1-13), morphine and metenkephalin are acting on different receptor populations or different binding sites of the receptor, resulting in a synergistic effect. Another

interesting finding in our study is the sharp contrast between brain and spinal cord for

dynorphin/morphine interaction. While dynorphin-A-(1-13) potentiated morphine analgesia in the spinal cord, it antagonizied morphine analgesia in the brain. This

phenomenon was first reported in mice (Friedman et al. 1981, Tulunay et al. 1981) and is

here confirmed in the rat. The same was true for the interaction between metenkephalin

and morphine. The underlying mechanism of the intriguing interactions among different

classes of opioid ligands at different levels of the central nervous system deserves further

study.

REFERENCES

Botticelli, L.I., Cox, B.M., and Goldstein, A. Proc Natl A.cad Sri, USA, 78:7783-7786,

1981.

Faden, A.I., Jacobs, T.P., Smith, G.P., Gree, B. and Zivin, J.A. Peptises, 4:631634, 1983.

Friedman, H.J., Jen, M.F., Chang, J.K., and Lee, N.M. Eur J Pharmacol, 69:357-380,

1981.

Han, J.S. and Xie, C.W. Life Sci, 31:31781-1783, 1982.

Han, J.S. and Xie, C.W. Pain, 367-376, 1984.

Jen, M.F. and Han, J.S. Chin Me d J, 92:576-582, 1979.

Lee, N.M., Leybin, L., Chang, J.K., and Loh, H.H. Eur J Pharmacol, 68:181-185, 1980.

Piercey, M.F., Varner, K., and Schroeder, L.A. Eur J Pharmacol, 80:283-284, 1982.

Przewlocki, R., Shearman, G.T., and Herz, A. Neuropeptides, 3:233-240, 1983.

Tulunay, F.C., Jen, M.F., Chang, J.K., Loh, H.H., and Lee, N.M. J Pharmacol Exp Ther,

219:296-298, 1981.

Yaksh, T.L. and Rudy, T.A. Physiol Behav, 17:1031-1036, 1976.

460
SIMILAR ACTIONS OF KAPPA AND MU AGONISTS ON SPINAL NOCICEPTIVE

REFLEXES IN RATS AND THEIR REVERSIBILITY BY NALOXONE

Chris G. Parsons, David C. West* and P. Max Headley

Dept. of Physiology, The Medical School, Bristol BS8 1TD, UK

* Dept. of Physiology, University College, Cardiff CF1 1XL, UK

ABSTRACT

The mu agonist fentanyl and the kappa agonists U-50,488 and tifluadom

were tested intravenously in electrophysiological experiments on

spinal nociceptive reflexes in anaesthetised, spinalised rats.

Responses of single motoneurones to thermal and mechanical stimuli

were reduced to a similar degree by both mu and kappa agonists and

these effects were reversed by low doses of naloxone.

INTRODUCTION

Most studies addressing the role of opiate receptor subtypes in the

modulation of spinal nociceptive responses have used behavioural

tests. Interestingly, systemic kappa agonists have been reported to

be relatively ineffective against reflexes to thermal noxious stimuli

in tail flick and hotplate tests, whilst having clear effects against

reflexes to mechanical and visceral noxious stimuli (Tyers 1980,

Upton et al. 1982, Ward and Takemori 1983). Several reports from

Yaksh and his coworkers have indicated that this is also the case

when kappa agonists are administered intrathecally, whereas mu

agonists are effective against reflexes to thermal as well as to

mechanical and other noxious stimuli (see Yaksh and Noueihed 1985).

Other workers have reported that kappa opiates show less difference

in effectiveness between thermal and non-thermal nociceptive tests

using either systemic (VonVoigtlander et al. 1983) or intrathecal

administration (Przewlocki et al. 1983).

We have been using an electrophysiological correlate of these

behavioural studies so as to be able to record the responsiveness of

single motoneurones to intravenously administered mu and kappa

ligands. We have selected agonists on the basis that they show

receptor selectivity, that they are suitable for systemic

administration and that they are rapidly metabolised. We have used

fentanyl as a mu agonist (see Magnan et al. 1982) and U-50,488

(VonVoigtlander et al. 1983) and tifluadom (Römer et al. 1982) for

kappa receptors. We have used the opiate antagonist naloxone to test

the opioid receptor specificity of the observed agonist effects.

MATERIALS AND METHODS

Experiments were performed on alpha-chloralose anaesthetised,

spinalised rats. Using silver wire electrodes, recordings were made

from the axons of single motoneurones in fine ventral root filaments.

The motoneurones were excited by natural peripheral stimuli which

were electronically controlled for duration, repetition rate and

constancy of intensity. The opioids were tested for selectivity

against responses to alternating thermal and mechanical noxious

stimuli. Particular care was taken to adjust the stimulus intensities

461
so as to elicit similar action potential discharge rates in response

to the different stimuli. Analysis was of the total number of spikes

evoked by the stimulus.

All drugs were given intravenously in these spinalised preparations,

both so as to expose the whole polysynaptic pathway to the

motoneurone to even concentrations of the drug and so as to allow

direct comparison with doses used in behavioural tests. Doses were

augmented logarithmically; normally until the cumulative dose was

sufficient to reduce the response to less than 25% of the control

mean count of spikes per response. Tests were only considered

acceptable if the response recovered by more than 50% of the

reduction. More than one drug was tested on nearly all cells.

RESULTS

As predicted, fentanyl reduced thermal and mechanical nociceptive

responses to a similar degree. In fact at 2 ug/kg on 23 motoneurones

in 19 rats the thermal and mechanical nociceptive responses were

reduced to means of 44% (±7 SEM) and 51% (±8) of control

respectively. The kappa agonists U-50,488 and tifluadom showed a

similar lack of selectivity when tested against alternating thermal

and mechanical responses in 14 and 10 rats respectively. For example,

at 2 mg/kg, U-50,488 reduced noxious heat responses of 23 cells to a

mean of 45% (±7) and noxious pinch responses to 54% (±7). Tifluadom

was more potent than U-50,488, 0.2 mg/kg being sufficient to reduce

noxious heat responses of 10 cells to a mean of 47% (±12) and noxious

pinch responses to 30% (±10) of control values. Such non-selectivity

by the kappa agonists was however dependent on the neuronal firing

rates during the two stimuli; this is illustrated in figure 1. The

left side shows an apparently 'selective' reduction of the mechanical

response by U-50,488. The right side shows that when firing rates

of alternating responses of the same cell were matched by reducing

the heat stimulus, this selectivity was no longer apparent.

FIGURE 1. Responses of a motoneurone to noxious heat and

pinch alternated in a regular 3 minute cycle. Alphachloralose anaesthetised, spinalised rat.

462
When tested in opiate naive rats, the opiate antagonist naloxone (0.1

to 10 mg/kg) had no effect on the responses of motoneurones to

noxious heat or pinch stimuli. We were unable to distinguish between

the effects of mu and kappa agonists with naloxone at 0.1 mg/kg i.v.

since this dose effectively reversed the actions of fentanyl,

U-50,488 and tifluadom. Thus, in 7 cells, naloxone at 0.1 mg/kg

reversed the depression of heat responses by U-50,488 (1-16 mg/kg)

from a mean of 16% (±7 SEM) to 81%(±12) of control. An example of

such a result is illustrated in figure 2.

FIGURE 2. Responses of a motoneurone to noxious pinch

stimuli applied every 3 minutes. The squares indicate

responses to pinch during a test with U-50,488 and show the

subsequent time course of recovery. The diamonds illustrate

superimposed data from a subsequent, similar test on the

same cell. In this case reduction of the pinch response by

U-50,488 was rapidly reversed by a low dose of intravenous

naloxone. Alpha-chloralose aneasthetised, spinalised rat.

DISCUSSION

Our electrophysiological experiments have shown that the kappa

agonists U-50,488 and tifluadom reduce thermally-evoked reflexes to

just as great a degree as mechanically-evoked reflexes. This nonselectivity contrasts with the results of most of the behavioural

tests which have utilised spinal administration of opioids. This

contrast between electrophysiological and behavioural tests can best

be explained by two factors. Firstly, in our experiments great care

was taken to adjust stimulus intensities so as to match the responses

in terms of motoneuronal firing rate. The relative strength of

reflexes in behavioural tests cannot be assessed in terms of neuronal

firing rate and therefore cannot be matched in the same way. Figure 1

illustrates that the apparently selective depression by U-50,488 of a

motoneuronal response to noxious pinch was entirely dependent on the

463
relative stimulus intensities. It is thus clear that stimulus

intensity is a crucial factor in the assessment of the

antinociceptive effects of opioids.

The second factor is the different receptor access by a drug between

systemic and intrathecal or epidural administration. For most opioid

drugs, the diffusion from intrathecal or epidural sites into the cord

has not been assessed. For fentanyl it is apparently very poor, for

the effective dose intrathecally can exceed the effective systemic

dose (Durant and Yaksh 1986, Yaksh et al. 1986). In the absence of

specific data for the kappa agonists it is thus possible that the

relative ineffectiveness of topically administered kappa agonists in

thermally-evoked reflexes is due largely to inadequate penetration of

the cord to the appropriate opioid receptor sites. Systemically

administered kappa agonists certainly can reduce such reflexes by an

action restricted, via spinal transection, to the spinal cord.

The reversal of kappa opioid mediated effects by low doses of

naloxone illustrates the problem of using this antagonist in attempts

to distinguish between mu and kappa mediated effects in vivo.

ACKNOWLEDGEMENTS

This project was supported by the British MRC and the Wellcome trust.

We wish to thank Dr. P.F. VonVoigtlander for a gift of U-50,488 and

Dr D. Römer for a gift of tifluadom.

REFERENCES

Durant, P.A.C., and Yaksh, T.L. Anesthesioloev, 64: 43-53, 1986.

Magnan, J., Paterson, S.J., Tavani, A., and Kosterlitz, H.W.

Naunyn-Schmiedebern's Arch Pharmacol, 319: 197-205, 1982.

Przewlocki. R., Stala, L., Greczek, M., Shearman. G.T..

Przewlocka, B., and Herz, A. Life Sci, 33:(Suppl 1): 649-652, 1983.

Römer, D., Büscher, H.H., Hill, R.C., Maurer, R., Petcher, T.J.,

Zeugner, H., Benson, W., Finner, E., Milkowski, W., and Thies, P.W.

Nature, 298: 759-760, 1982.

Tyers, M.B. Brit J Pharm, 69: 503-512, 1980.

Upton, N., Sewell, R.D.E., and Spencer, P.S.J. Eur J Pharm, 78:

421-429, 1982.

VonVoigtlander, P.F., Lahti, R.A., and Ludens, J.H. J Pharm exp Ther,

224: 7-12, 1983.

Ward, S.J., and Takemori, A.E. J Pharm exp Ther, 224: 525-530, 1983.

Yaksh, T.L., and Noueihed, R. Ann Rev Pharmacol Toxicol, 25: 433-462,

1985.

Yaksh, T.L. Noueihed, R., and Durant, P.A.C. Anesthesioloïy, 64:

54-66, 1986.

464
OPIOID ANALGESIA IN THE MOUSE: EVIDENCE FOR MULTIPLE

RECEPTORS USING ß-FNA

Pilar Sánchez-Blázquez and Javier Garzón

Department of Neuropharmacology, Cajal Institute

28006 Madrid, Spain

ABSTRACT

The icv injection of morphine or DADLE ED50 a few min

before the alkylating agent ß-FNA resulted in complete

protection of their respective analgesic effects when

evaluated 24h later, although a 1 ittle cross-protection

could be observed. The analgesia evoked by DADLE was

partially protected using higher doses of morphine before

R-FNA. However, higher doses of DADLE did not protect the

analgesia induced by morphine. On the other hand, the

antagonistic action of KCl on opioid analgesia was found

to be dependent on the opioid utilized to protect the

opioid receptor against the effect of R-FNA. These

results are discussed in terms of multiple receptors

mediating opioid analgesia at supraspinal level in the

mouse.

INTRODUCTION

Frequently it has been assumed that opioids induce their

antinociceptive effect at the supraspinal level after

binding to the μ class of opioid receptor. However,

several laboratories have evoked the mediation of

multiple receptors in that phenomenon (see Höllt et al.

1985). With respect to that concept, we have recently

reported (Garzón and Sánchez-Blázquez, this volume) that

a single icv injection of KCl antagonized the supraspinal

analgesia elicited by certain opioids. In the present

study, we have investigated further the issue by

analyzing the analgesic effect of different opioids on

mice previously exposed to the effect of ß-FNA in the

presence of various protecting opioids. Moreover, the

antagonistic action of KCl on those animals treated with

ß-FNA was studied.

MATERIALS AND METHODS

Albino male mouse CFLP, 22-27 g were used throughout.

Opioids were obtained from commercial sources.

ß-funaltrexamine from N.I.D.A.. Analgesia was evaluated

by the tail flick test using a water bath at 52° C as

noxious stimulus. Latencies were measured both before and

after any given treatment and analgesia expressed as a

percent of the maximum possible effect after setting a

cut off time of 10s.

465
RESULTS AND DISCUSSION

The ß-fumarate methylester of nal tre xone (ß-FNA) given

icv 0.2 nmole/mouse, antagonized the analgesia induced 24

h later by icv injection of morphine or DADLE ED50s (fig

1). Morphine or DADLE ED50s given a few min prior to

ß-FNA produced a good protect ion of their respective

analgesic effect, but little cross-protection could be

observed. This result agrees with the proposed

multiplicity of opioid receptor mediating analgesia at

supraspinal level (see Höllt et al. 1985). Therefore,

these two opioids at ED50 seem to mainly act on each

selective receptor in order to induce analgesia. Although

higher doses of morphine induced a good protection of

DADLE ED50 analgesic effect, in contrast, DADLE at ED90

did not improve the analgesic action of morphine ED50 24h

after the ß-FNA challenge. Thus indicating a low

cross-binding of that opioid peptide to the morphine

receptor. The μ ligand FK 33-824 and the ligand DTLET

protected to a similar extent the analgesia elicited by

morphine and DADLE ED50s (fig 1). This result suggests

that both enkephal in derivatives might have access to

morphine and DADLE analgesic receptors.

As previously described (Garzón and Sánchez-Blázquez,

this volume) a single icv injection of KCl, but not NaCl,

(1 μmole/mouse) reduced the analgesic activity of DADLE

and FK 33-824 in naive mice (table 1). That treatment did

not alter significantly the analgesia evoked by either

morphine or DTLET, confirming that this pept ide induces

analgesia through the same receptor used by the alkaloid

(Chaillet et al. 1984). In mice treated with morphine

ED90, 30 min before ß-FNA, it could be observed that the

analgesia induced by these four opioids was much less

sensitive to KCl. The 13-FNA mainly blocked the

"potassium-sensitive" opioid receptors. Conversely, with

DADLE ED90 given 15 min be fore ß-FNA, the analgesic

effect of those opioids 24h later was now reduced by KCl

administration. Thus, it is 1 ikely that opioids

displaying a better affinity to the morphine receptor

will induce analgesia insensitive to KCl antagonism. On

the other hand, a simultaneous binding to both receptors

will result in a slight decrease of analgesic potency

which will be difficult to detect experimentally. That

might be the case for DTLET. Therefore, the possibility

of the "potassium-sensitive" receptor being an additional

type of binding site to which μ- and d-selective

enkephalin derivatives display a high affinity appears to

be very attractive (Lutz et al. 1985).

466
FIGURE 1. Protection against the antagonistic

effect of 13-FNA on the supraspinal analgesia

elicited by opioids in the mouse.

Protecting opioids at doses inducing various

degrees of analgesia were administered 30 min

(morphine and FK 33-824) or 15 min (DADLE and

DTLET) be fore R-FNA respectively. Analgesia

induced by morphine and DADLE ED50s was

evaluated 24h later. All substances were

injected icv in a final volume of 4 μl. Values

are the MEAN SEM of percent of maximum

analgesic effect from various groups each of 10

mice. Student’s t test was applied for

statistical analysis. a and b refer to saline

and ß-FNA treated groups respectively.

467
TABLE 1. Effect of ß-FNA and KCl administration on the

analgesia elicited by opioids in the mouse

ß-FNA

Control Morphine ED90 DADLE ED90

test

Opioid dose time - + + +KCl

Morphine 27.0 30 90 1 4 8 7 4 76 6 74 5 50 4 25 3*

DADLE 1.7 15 89 5 33 2* 55 4 45 3 80 5 33 3*

FK 33-824 0.03 30 90 3 43 2* 71 6 65 6 90 3 39 3*

DTLET 1.5 15 61 3 5 0 Z 45 4 46 3 58 3 29 2*

Morphine and DADLE ED9Os (27 and 1.7

nmole/mouse) were given 30 or 15 min before

ß-FNA (0.2 nmole/mouse) respectively. Opioid

doses are in nmole/mouse. Analgesia was

evaluated 24h after ß-FNA. KCl (1 umole/mouse)

was administered 24h after ß-FNA and 30min

before the opioids. All substances were

injected icv in a final volume of 4 μl. Values

are the MEAN SEM of percent of maximum

analgesic effect from various groups each of 10

mice. * Statistically different from the group

not receiving KCl. Student’s t test; p<0.05.

Abbreviations:

DADLE : (D-Ala2, D-Leu5)-enkephalin.

DTLET : (D-Thr2, Leu5)-enkephalin-Thr6.

FK 33-824: (D-Ala2, N-Me-Phe4, Met-(o)5-ol)-

enkephalin.

REFERENCES

Chaillet, P., Coulaud, A., Zajac, J.M., Fournie-Zaluski,

M.C., Coustent in, J. and Roques, B.P. Eur J Pharmacol,

101:83-90, 1984.

Höllt, V., Sánchez-Blázquez, P. and Garzón, J. Phil Trans

R Soc Lond, B308:299-310, 1985.

Lutz, R.A.. Cruciani, R.A., Munson, P.J. and Rodbar, D. Life

Sci, 36:2233-2238, 1985.

ACKNOWLEDGEMENTS

This work was supported by the CAICYT and the FISS

(Spain). Pilar Sánchez-Blázquez is a recipient of the

Fellowship from Plan de Formacidn del Personal

Investigador (MEC).

468
HYPERALGESIA PRODUCED BY INTRATHECAL OPIOID ANTAGONISTS DEPENDS ON

RECEPTOR SELECTIVITY AND NOXIOUS STIMULUS

C.W.T. Pilcher and Julia L. Browne

Department of Pharmacoloqy

Faculty of Medicine

P.O. Box 24923 - Safat

KUWAIT 13110

ABSTRACT

Opioid antagonists selective for , k- and μ-receptor subtypes were

administered intrathecally in rats prior to determination of response

thresholds to noxious heat, pressure and chemical visceral stimulation. All antagonists induced hyperalqesia differentially with two or

more stimuli but and μ-blockade failed to alter writhing activity.

Thus, the extent of involvement of an opioid receptor subtype in

antinociception depends on the type of noxious stimulation.

INTRODUCTION

The multiplicity of endogenous opioid systems is reflected in the

fact that there are at least three families of endogenous opioid

peptides (EOPs) and at least three different opioid receptors are

widely recoqnised (Höllt et al. 1983). Among the questions raised by

this complexity are: does any one EOP modulate pain processes and are

they mediated by a specific receptor? If EOPs have physiological

roles in antinociception and these are expressed through activation

of specific receptors, antagonists selective for those receptors

should produce altered nociceptive responding. Previously we studied

opiate antagonists in rats never before exposed to exogenous opioids

and showed that systemic administration of k-selective agents induced

hyperalqesia to pressure without affecting nociceptive heat thresholds, whereas naloxone was equipotent in producing hyperalqesia with

both stimuli (Pilcher and Browne 1983). In the CNS the recognised

receptor subtypes are differentially distributed; k-agonists are

ineffective analgesics when given intracerebrally (Wood et al. 1981)

but they are active on intrathecal injection, as are and μ-opioids

(Przewlocki et al. 1983). Since it appears that spinal populations of

, k- and μ- receptors participate in modulation of pain we have now

extended our studies to examine the effects of their antagonists

applied directly to the spinal cord. For selective antagonism at

k- and μ-receptors ICI 174,864 (Traynor et al. 1985), Mr 1452 (Merz

personal commun.) and naloxone (Vonvoiqtlander et al. 1983) were used

respectively.

MATERIALS AND METHODS

Male hooded rats were used. They were housed at about 22°C in a

light-dark cycle with lights on 06.00 - 18.00 h. Food and water were

freely available. On attaining a body weight of 300-350 g they were

implanted with a polyethylene cannula in the subarachnoid lumbar sac

according to Schmauss and Yaksh (1984). Injections of drugs and

469
analgesic tests were carried out 48 h after surgery. Drugs were

injected in a volume of 10 μl followed by a flush with 10 μl isotonic

saline over a period of 45 sec. Naloxone hydrochloride and ICI

174,864 (Allyl2-Tyr-Aib-Aib-Phe-Leu-OH) were dissolved in isotonic

saline. Mr 1452 ((-)-N(3-Furylmethyl)- -normetazocine methanesulphonate) was first dissolved in distilled water and diluted 1:10 with

isotonic saline. Doses are expressed as the weights of the salts.

Analgesic Testing

Analgesic testing was carried out between 09.00 and 12.00 h. In the

paw pinch and tail immersion tests baseline (control) thresholds were

obtained 15 min before drug injections.

Paw pinch Initial nociceptive pressure thresholds of non-inflamed

hind paws were determined using an 'Analqesy-Meter' (Uqo Basile,

Milan).

Tail immersion Tail imnersion tests were carried out at 50 (±0.2)°C.

Latencies to the nociceptive response, a flicking or withdrawing of

the tail from the water, were determined.

Writhing test The number of writhing responses made during the 5 min

immediately following an intraperitoneal injection of 4% sodium

chloride was determined.

In all tests results were expressed as a percentage of the control

value. Statistical analyses were performed using analysis of variance

and post hoc t-tests.

RESULTS

All three antagonists produced their maximal effects in the tail

inmersion and paw pressure tests within 1-4 min after cessation of

injections. ICI 174,864 produced a significant reduction in paw pressure thresholds (p<0.01), which was maximal at 25% with 0.1 μg after

only 1 min. The dose-response curve was almost flat over the range

examined (0.01-1.0 μg) and doses above 2.0 μg caused hind limb flaccidity followed by severe rigidity. In the tail immersion test ICI

174,864 produced a significantly greater reduction in threshold (37%)

than with pressure (p<0.01). In contrast to these hyperalgesic

effects with heat and pressure, at 0.1 μg this antagonist had no

effect on writhing scores.

The hyperalgesia induced by Mr 1452 was maximal at 1.0 μq and that

obtained with pressure (49%) was significantly greater (p<0.01) than

with heat (33%). This antagonist increased writhing responses by 18%

but the effect was not significant. The hyperalqesic effect of Mr

1452 was greater than that of either of the other two antagonists

with pressure and visoeral stimulation. Naloxone also gave peak

effects at 1.0 μg and was most effective in the tail immersion test,

when latencies were reduced by 70%. Naloxone produced significantly

greater (p<0.01) reductions in tail flick latencies than the other

antagonists. Marked hyperalqesia was also seen with the pressure

stimulus (42%) and a weak, insignificant effect occurred in the

470
writhing test. Thus the order of hyperalgesic effectiveness of receptor blockade in the three tests was: tail imüersion, p > > k; paw

pressure, k > μ > ; writhing, k > μ > = 0.

FIGURE 1. Hypóralqesic effects of opioid antagonists selective for , k- and μ-receptors with different stimuli. P=

paw pressure: H=heat; W=writhing. (n=5-8)

DISCUSSION

The results reported herein demonstrate that opioid antagonists

administered to the spinal cord of opioid-naive rats produce hyperalgesia to different kinds of noxious stimuli. This heightened sensitivity is interpreted as resulting from prevention of antinociceptive

modulation by endogenous ligands. It is also clear that the magnitude

of the hyperalqesia depended not only on the antagonist's selectivity

for a receptor subtype, but also on the nature of the noxious

stimulus. Moreover, the time-course of an antagonist's action in the

same animal was not identical for all stimuli. These differences in

time-courses and effectiveness support two related notions: i) that

neural systems concerned with nociception are distinguishable on the

basis of their receptor subtype, ii) that the extent to which spinal

, k- and μ- receptors participate in modulation of pain processing

depends on the qualitative nature of the stimulus. The present

findings support our earlier work in which, using systemic administration of naloxone and the k-antagonists Mr 1452 and Mr 2266, it was

shown that the latter are 10 to 15-fold more potent in producing

hyperalgesia with pressure than heat.

In general, the above results are compatible with those of studies

using selective agonists. Martin et al. (1976) first showed that

the prototypic k-agonist, ethylketocyclazocine, was twice as potent

as morphine, the p-prototype, in suppressing the mild pressure-evoked

flexor reflex in spinal dogs. It was later reported that k-agonists

were analgesic against noxious visceral stimulation and pressure but

not heat, whereas μ-agonists were effective against all three

471
stimuli (Tyers 1980). When administered intrathecally, μ- and kagonists inhibited responses in the writhing test but agonists were

ineffective (Schmauss and Yaksh 1984). In the hot plate and tail

flick tests μ- and but not k-agonists were analgesic. If these

differential analgesic actions of exogenous agonists reflect reliably

the physiological activation of receptors by their respective endogenous liqands, then the corollary, that selective antagonists will

produce hyperalqesia differentially, should hold true. The results

presented here support this proposition.

REFERENCES

Höllt, V., Seizinger, B.R., Garzón, J., and Loh, H.H. In: Koch, G.,

and Richter, D., ed. Biochemical and clinical aspects of neuropeptides synthesis, processing and qene structure. New York: Academic

Press, 1983. pp.59-72.

Martin, W.R., Fades, C.G., Thompson, J.A., Huppler, R.E. and Gilbert,

P.E. J Pharmac exp Ther, 197:517-532, 1976.

Pilcher, C.W.T., and Browne, J.L. Life Sci, 33(Suppl.1):697-700,

1983.

Przew ocki, R., Stala, L., Greczek, M., Shearman, G.T., Przew ocka,

A., and Herz, A. Life Sci, 33(Suppl.1):649-652, 1983.

Schmauss, c., and Yaksh, T.L. J Pharmac exp Ther, 228(1):1-12, 1984.

Traynor, J.R., Simon, E.J., Appelmans, R., Carroll, J.A., and Rance,

M.J. Abs INRC, p3, 1985.

Tyers, M. Br J Pharmac, 69:503-512, 1980.

Vonvoigtlander, P.F., Lahti, R.A., and Ludens, J.H. J Pharmac exp

Ther, 224(1):7-12, 1984.

Wood, P.L., Rackham, A., and Richard, J. Life Sci, 28:2119-2125,

1981.

ACKNOWLEDGEMENTS

We thank Dr H. Merz of Boehringer Ingelheim for generously donating

Mr1452 and Dr M.J. Rance of Imperial Chemical Industries (UK) for the

kind gift of ICI 174,864. This work was supported by Grant No. MR 015

from Kuwait University Research Council.

472
IN VIVO STUDIES ON DELTA OPIOID RECEPTORS

Alan Cowan, Michael J. Rance* and Tom P. Blackburn*

Department of Pharmacology, Temple University School of

Medicine, Philadelphia, PA 19140, U.S.A. and *Bioscience

Department II, ICI Pharmaceuticals Division, Alderley

Park, Macclesfield, Cheshire SK10 4TG, England

ABSTRACT

[D-Pen2, D-Pen5]enkephalin (DPDPE), a selective agonist at

delta opioid receptors, causes excessive vertical rearing

when given icv to rats or s.c. to mice. Tolerance develops to this behaviour. Rats do not rear excessively when

injected icv with the following prototype agonists at

opioid receptors: OAGO, dynorphin A, U-50488H or SK&F

10047. The incidence of OPDPE-induced rearing is reduced

when rats are pretreated S.C. with ICI 174864 (a selective

antagonist at delta opioid receptors) (A50=0.09 mg/kg) but

not by ICI 178173 (an inactive analogue of ICI 174864);

this finding suggests that delta binding sites mediate the

behaviour. Pretreatment with naloxone attenuates rearing

but the antagonism is unimpressive over the dose range

tested (0.05-1 mg/kg, s.c.). Low doses of haloperidol

(A50=0.05 mg/kg, s.c.) antagonize the rearing. Dopamine

may therefore mediate the behaviour through delta receptor

modulation of dopamine release. The practical gain from

this study is as follows: a simple, discriminating test is

now available for evaluating novel delta agonists and

antagonists in vivo.

INTRODUCTION

[D-Pen2, D-Pen5]enkephalin (DPDPE) (Mosberg et al. 1983) is

a selective agonist and ICI 174864 (Cotton et al. 1984) is

a selective antagonist at delta opioid receptors. Initial

experiments in vivo (Cowan et al. 1985) have shown that

the icv administration of DPDPE to rats causes a marked

increase in vertical rearing. This behaviour was studied

in the present work as a possible in vivo index of delta

agonist activity.

We used the following approaches to clarify the pharmacological basis of DPDPE-induced rearing: (a) interaction

studies between DPDPE and ICI 174864, ICI 178173, naloxone

and haloperidol, respectively! (b) tolerance experiments

involving multiple icv injections of DPDPE, and (c) comparative behavioural studies with agonists selective for

mu (DAGO), kappa (dynorphin A, U-50488H) and sigma (SK&F

10047) opioid binding sites, respectively.

MATERIALS AND METHODS

Male albino Sprague Dawley rats (180-200 g; n=4-8) were

each stereotaxically implanted with an icv cannula. Five

days later, the animals were placed singly in perspex

boxes (25 cm long; 20 cm wide; 30 cm high) for an hour

then challenged with test agent (5 μl icv). Animals were

473
observed and scored for behavioural changes for a period

of 50 min after agonist administration. Potential antagonists were given s.c. 5 min before a standard dose of

DPDPE (20 μg).

Female mice (20-25 g; ICI strain; n=6) were injected S.C.

with DPDPE (3-30 mg/kg) and observed regularly over the

following hour. The number of rears displayed by each

mouse was counted at +1O min for 1 min.

In a tolerance study! DPDPE (20 μg) [Group 1] or saline

[Group 2] was given icv to rats twice daily (at 9 a.m.

and 5 p.m.). Animals in Group 1 were tested for 50 min

in observation boxes after the 1st, 7th and 13th (final)

injection. Animals in Group 2 were similarly challenged

(with 20 μg of DPDPE) after the 7th and 13th injection.

The following compounds were used: [D-Ala2, MePhe5, Gly-ol5]

enkephalin (DAGO), DPDPE and dynorphin A (all from Peninsula); ICI 174864 (Diallyl-Tyr-Aib-Aib-Phe-Leu-OH, Aib is

aminoisobutyric acid; ICI); ICI 178173 (Diallyl-Tyr-Aib

-D-Ala-Phe-Leu-OMe; ICI); naloxone hydrochloride (Endo);

N-allylnormetazocine (SK&F 10047; Sterling Winthrop) and

trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)

cyclohexyll-benzeneacetamide methanesulphonate (U-50488H;

Upjohn).

RESULTS

In rats, the DPDPE dose-response relation for rearing was

curvilinear with the 20 μg dose being most effective (fig.

1). Behavioural depression was associated with 50 μg of

DPDPE; lower doses caused increased yawning, licking of

penis, sniffing and biting/licking (forepaws, wood chips

and faecal pellets). Behavioural activation was essentially over by +50 min. After s.c. injection to mice,

DPDPE provoked increased rearing (table 1), grooming and

restlessness for about 30 min.

FIGURE 1. Curvilinear dose-response curve for

DPDPE-induced rearing in rats.

474
TABLE 1. DPDPE-induced rearing in mice.

Dose Rears during 10th min

(mg/kg, s.c.) (mean ± s.e.)

Saline 7.33 ± 1.17

3 10.83 ± 0.83*

10 14.33 ± 1.17**

30 13.17 ± 2.24*

*P<0.05; **P<0.01 (Dunnett's test).

TABLE 2. Antagonism of DPDPE-induced rearing in rats.

Test agent *A50 (mg/kg, s.c.)

Haloperidol 0,053 (0.022-0.13)

ICI 174864 0.087 (0.031-0.24)

ICI 178173 Inactive at 1 mg/kg

Naloxone Flat dose-response curve

*Tallarida and Murray (1981).

ICI 174864 caused an unusual behaviour in rats - hindleg

stretching. When the animals were given ICI 174864 (0.01,

0.10 and 1 mg/kg, s.c.) 5 min before the standard dose of

DPDPE (20 μg), rearing was antagonized in a dose-dependent

manner (table 2). ICI 178173 (an inactive analogue of ICI

174864) (1 mg/kg, s.c.) did not antagonize DPDPE-induced

rearing. Pretreatment with naloxone (0.05-1 mg/kg, s.c.)

attenuated rearing but the antagonism (35% at 1 mg/kg)

was unimpressive.

Tolerance developed to DPDPE-induced rearing by the 7th

injection (49±8 rears from 91±12 rears) and had fully

developed by the 13th injection (25±16 rears). Control

(saline-injected) rats reared excessively when challenged

with DPDPE (20 μg) on the 7th and 13th injections.

When rats were injected icv with DAGO (0.125 and 0.50 μg),

dynorphin A (0.50 and 10 μg), U-50488H (7.5 and 100 μg)

and SK&F 10047 (5 and 50 μg) increased rearing was not

observed.

DISCUSSION

Early behavioural experiments with [D-Ala2, Met5]enkephalin

(amide) [DALA] revealed that this opioid peptide, when

injected into the nucleus accumbens (Pert and Sivit 1977)

or ventral tegmental area (Broekkamp et al. 1979) of rats,

increases locomotor activity and vertical rearing. These

brain areas, part of the mesolimbic dopamine system, play

an important role in regulating exploratory and locomotor

behaviours. Kalivas et al. (1983) have recently summarized evidence that favours enkephalinergic modulation of

the mesolimbic dopamine system.

DPDPE, the selective delta agonist (Mosberg et al. 1983),

475
is behaviourally active when given icv to rats, and evokes

a rich array of signs that are reminiscent of those described for amphetamine and apomorphine. Perhaps the most

notable behaviour is repetitive vertical rearing, which

can also be observed in mice after s.c. DPDPE. This behaviour has been the focus of the present communication.

Our results with ICI 174864, ICI 178173, naloxone and

haloperidol lead to the conclusion that DPDPE-induced

rearing is controlled by delta receptor modulation of

dopamine release. Future studies with newly available

dopamine receptor antagonists will no doubt sharpen our

views on the dopaminergic basis of DPDPE-induced rearing.

Follow-up experiments with ICI 174864 should be started

so as to unravel the in vivo pharmacology of this interesting agent. For example, a high dose of naloxone (10

mg/kg) (Haber et al. 1978) attenuates amphetamine-induced

rearing in rats (Gupta and Gregory 1967; Bryan and Ellison

1975). What can we expect with low doses of ICI 174864 in

this paradigm?

Two practical gains result from our study. First,

DPDPE-like compounds can be discriminated from agents with less selectivity for delta binding

sites, e.g, DALA (Kalivas et al. 1983), as well as prototype agonists at mu, kappa and sigma recognition sites.

Second, DPDPE-induced rearing provides a simple in vivo

endpoint against which the next generation of potential

delta antagonists may be tested and compared.

REFERENCES

Broekkamp, C.L.E., Phillips, A.G., and Cools, A.T.

Nature, 278: 560-562, 1979.

Bryan, K.S., and Ellison, G. Psychopharmacologia, 43:

169-173, 1975.

Cotton, R., Giles, M.G., Miller, L., Shaw, J.S., and

Timms, D. Eur J Pharmac, 97: 331-332, 1984.

Cowan, A., Zhu, X.Z., and Porreca, F. Neuropeptides, 5:

311-314, 1985.

Gupta, B.D., and Gregory, K. Psychopharmacologia, 11:

365-371, 1967.

Haber, S., Matsukami, T., Berger, P.A., Barchas, J.D.,

and Akil, H. Prog Neuropsychopharmac, 2: 425-430, 1978.

Kalivas, P.W., Widerlöv, E., Stanley, D., Breese, G., and

Prange, A.J. J Pharmac exp Ther, 227: 229-237, 1983.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura,

H.I., Galligan, J.J., and Burks, T.F. Proc Nat Acad

Sci, 80: 5871-5874, 1983.

Pert, A., and Sivit, C. Nature, 265: 645-647, 1977.

Tallarida, R.J., and Murray, R.B. Manual of Pharmacologic

Calculations. N.Y., Springer-Verlag, 1981, procedure 8.

ACKNOWLEDGEMENTS

Thanks are extended to ICI for providing facilities and

animals for this work which began while A.C. was on study

leave at Alderley Park.

476
EFFECTS OF NALOXONE AND NEUROTENSIN ON EXCESSIVE GROOMING BEHAVIOR

OF RATS INDUCED BY BOMBESIN, -ENDORPHIN AND ACTH

Tj.B. van Wimersma Greidanus

Rudolf Magnus Institute for Pharmacology,

Medical Faculty, University of Utrecht,

Vondellaan 6, 3521 GD Utrecht, The Netherlands

ABSTRACT

Bombesin, endorphin and ACTH-(1-24) induce excessive grooming behavior in rats. Whereas ACTH increases the frequency of all components of grooming behavior (head washing, bodily grooming and paw

licking), the most pronounced element of bombesin- and endorphininduced excessive grooming is scratching. Naloxone counteracts

peptide-induced grooming and in particular excessive scratching is

reduced by this opiate antagonist. Also neurotensin suppresses

peptide-induced grooming behavior and in particular scratching. It is

concluded that the element scratching is the component of grooming

behavior which is mainly displayed by activation of opiate receptors

and that neurotensin is able to interfere with opiate receptor mediated behavior.

INTRODUCTION

ACTH and MSH are the first peptides reported to induce excessive

grooming (Ferrari 1958; Ferrari et al. 1963). Later also other peptides appeared to be active in this respect. Neurohypophyseal hormones

(Delanoy et al. 1978; Rees et al. 1976; Drago et al. 1986), prolactin (Drago and Bohus 1981; Drago et al. 1981), substance P (Katz

1979), bombesin (Gmerek and Cowan 1983; Katz 1980; Kulkosky et al.

1982; Van Wimersma Greidanus et al. 1985a), dynorphin and endorphin

(Aloyo et al. 1983) have recently been shown to induce excessive

grooming behavior. Interestingly, the various grooming-inducing peptides have been reported to induce structurally different grooming

bouts. ACTH and related peptides produce excessive grooming, which is

as far as the composition of the grooming behavior is concerned, comparable to normally occurring grooming behavior (Gispen and Isaacson

1981; Van Wimersma Greidanus et al. 1985a, 1986), whereas grooming

induced by endorphin and bombesin is characterized by the predominant display of scratching (Gispen and Isaacson 1981; Gmerek and

Cowan 1983; Van Wimersma Greidanus et al. 1985a). Naloxone, haloperidol and neurotensin have been shown to suppress the excessive

grooming induced by ACTH (Van Wimersma Greidanus and Rinkel 1983;

Van Wimersma Greidanus et al. 1986) and by bombesin (Van Wimersma

Greidanus et al. 1985b), and it has been suggested that opioid and

dopaminergic systems are involved in peptide-induced excessive

grooming (Wiegant et al. 1977a, 1977b; Spruijt et al. 1986; Cools

et al. 1978; Dunn et al. 1981; Gispen and Wiegant 1976). The present study deals with the nature of the excessive grooming induced by

ACTH, endorphin and bombesin and with the effects of neurotensin

and naloxone on thus induced excessive grooming. In particular the

suppressive effects of neurotensin and naloxone on the various components of the excessive grooming are discussed.

477
MATERIAL AND METHODS

Male rats (150 g) of an inbred Wistar strain, equipped with a canula

in the right lateral ventricle, were used. Bombesin was intracerebroventricularly (i.c.v.) injected in doses grading from 10 ng, 30 ng to

100 ng, endorphin in doses of 10 ng, 30 ng, 100 ng, 300 ng, 1 μg

and 3 μg, whereas ACTH was administered in a dose of 1 μg. Pretreatment with naloxone was performed at 20 min prior to the i.c.v. injection of peptides, using a dose of 1 mg /kg. Pretreatment with

neurotensin was performed immediately before peptide administration

in a dose of 1 μg i.c.v. For observation of grooming behavior rats

were placed into perspex boxes (26 x 20 x 13 cm). Using a 15 sec

sampling time, the display of grooming elements (head washing: H;

bodily grooming: B; sexual (= anogenital) grooming: A; paw licking:

P; scratching: S) was recorded for a period of 50 min, starting 10

min after peptide administration and placing of the animals into the

observation boxes.

RESULTS AND DISCUSSION

Bombesin induces a dose dependent increase of grooming behavior, with

scratching as the most pronounced element. Even administration of the

relatively low dose of 10 ng results in excessive grooming and in

particular excessive scratching. Naloxone suppresses significantly

the excessive grooming induced by bombesin. This suppression of bombesin-induced excessive grooming by naloxone is mainly due to a reduction in scratching and, to a lesser extent, bodily grooming. As a

result of this differential suppression of the various elements of

bombesin-induced excessive grooming by naloxone, a strong shift in

the relative distribution of grooming elements occurs in bombesintreated rats following pretreatment with this opiate antagonist. From

these results it is concluded that excessive scratching as induced by

bombesin is the component of grooming behavior which is mainly displayed by activation of opiate receptor systems, Neurotensin also

suppresses bombesin-induced excessive grooming, mainly by reducing

the element scratching. Consequently neurotensin induces a change in

the relative distribution of grooming elements displayed by bombesintreated rats, which is more or less similar as the one induced by

naloxone. Thus, it is tempting to assume that neurotensin interferes

with opiate receptor systems, at least with one of the behavioral

results of such a system, i.e. scratching.

Administration of endorphin induces excessive grooming behavior as

well. However, the endorphin induced excessive grooming is less

pronounced and the frequency of grooming is quite lower than observed

following similar doses of bombesin. Doses of endorphin exceeding

200 ng are even less powerful than doses of 30 ng and 100 ng and a

dose of 3 μg does not exert excessive grooming at all during the observation period of 50 min. Time course studies reveal that this bellshaped dose-response curve for the grooming inducing effect of

endorphin is due to the endorphin induced immobility of the animals

during the first half of the observation period. Doses of 30 ng and

100 ng endorphin induce excessive grooming with scratching as one

of the predominant elements. This confirms the idea that excessive

scratching is mainly due to activation of opiate receptor systems.

Both naloxone and neurotensin suppress the endorphin induced ex478
cessive grooming, mainly by reduction of the element scratching. This

differential suppression of the various components of endorphininduced excessive grooming by naloxone and neurotensin again results

in a shift in the relative distribution of the various grooming elements.

Administration of ACTH results in excessive grooming behavior in which

all elements are more or less equally involved. In this respect ACTHinduced excessive grooming differs markedly from the excessive grooming

behavior displayed by rats treated with bombesin or endorphin. Interestingly both naloxone and neurotensin suppress ACTH-induced excessive

grooming without affecting the relative distribution of the various

grooming elements.

From these data it is concluded that different neuroanatomical and/

or neurophysiological systems in the brain are involved in excessive

grooming induced by bombesin, endorphin or ACTH. In addition it

appears that scratching is the behavioral element which is mainly

displayed by activation of opiate receptor systems. Moreover, it seems

likely that neurotensin interferes with opiate receptors.

REFERENCES

Aloyo, V.J., Spruijt, B.M., Zwiers, H. and Gispen, W.H. Peptides, 4:

833-836, 1983.

Cools, A.R., Wiegant, V.M. and Gispen, W.H. Eur J Pharmacol, 50:

265-268, 1978.

Delanoy, R.J., Dunn, A.J. and Tintner, R. Horm Behav, 11: 348-362,

1978.

Drago, F. and Bohus, B. Behav Neural Biol, 33: 117-122, 1981.

Drago, F., Bohus, B., Canonico, P.L. and Scapagnini, U. Pharmacol

Biochem Behav, 15: 61-63, 1981.

Drago, F., Pedersen, C.A., Caldwell, J.D. and Prange Jr., A.J. Brain

Res, 368: 287-295, 1986.

Dunn, A.J., Childers, S.R., Kramarcy, N.R. and Villiger, J.W. Behav

Neural Biol, 31: 105-109, 1981.

Ferrari, W. Nature, 181: 925-926, 1958.

Ferrari, W. Gessa, G.L. and Vargiu, L. Ann NY Acad Sci, 104: 330-345,

1963.

Gispen, W.H. and Isaacson, R.L. Pharmacol Ther, 12: 209-246, 1981.

Gispen, W.H. and Wiegant, V.M. Neurosci Lett, 2: 159-164, 1976.

Gmerek, D.E. and Cowan, A. Peptides, 4: 907-913, 1983.

Katz, R.J. Neurosci Lett, 12: 393-401, 1979.

Katz, R.J. Neuropharmacology, 19: 143-146, 1980.

Kulkosky, P.J., Gibbs, J. and Smith, G.P. Physiol Behav, 28: 505-512,

1982.

Rees, H.D., Dunn, A.J. and Iuvone, P.M. Life Sci, 18: 1333-1340, 1976.

Spruijt, B.M., Cools, A.R., Ellenbroek, B.A. Gispen, W.H. Eur J

Pharmacol, 120: 249-257.

Van Wimersma Greidanus, Tj.B. and Rinkel, G.J.E. Eur J Pharmacol, 88:

117-120, 1983.

Van Wimersma Greidanus, Tj.B., Donker, D.K., Van Zinnicq Bergmann,

F.F.M., Bekenkamp, R., Maigret, C. and Spruijt, B. Peptides, 6:

369-372, 1985a.

479
Van Wimersma Greidanus, Tj.B., Donker, D.K., Walhof, R., Van Grafhorst,

J.C.A., De Vries, N., Van Schaik, S.J., Maigret, C., Spruijt, B.M.

and Colbern, D.L. Peptides, 6: 1179-1183, 1985b.

Van Wimersma Greidanus, Tj.B., Maigret, C., Ten Haaf, J.A., Spruijt,

B.M. and Colbern, D.L. Behav Neural Biol, 1986, in press.

Wiegant, V.M., Gispen, W.H., Terenius, L. and De Wied, D. Psychoneuroendocrinology, 2: 63-69, 1977a.

Wiegant, V.M., Cools, A.R. and Gispen, W.H. Eur J Pharmacol, 41: 343-

345, 1977b.

480
FOR KAINIC ACID-INDUCED WET DOG SHAKES.

L. Grimes*, J. Hong, J. McGinty**, C. Mitchell,

J. Obie, and H. Tilson

*Curriculum in Toxicology, UNC-CH, Chapel Hill, NC 27514;

Laboratory of *Behavioral and Neurological Toxicology,

NIEHS, RTP, NC 27789; and **Department of Anatomy,

School of Medicine, ECU, Greenville, NC 27834

ABSTRACT

Kainic acid (KA) caused an initial decrease and a subsequent rebound in hippocampal enkephalin (ENK) in rats

exhibiting wet dog shakes (WDS) without behavioral convulsions. Antibody to methionine-enkephalin but not

dynorphin A(1-8) injected into lateral ventricles attenuated KA-induced WDS, as did naloxone. Granule-cell

destroying injections of colchicine into ventral but not

dorsal hippocampus caused a 60% reduction of hippocampal

ENK and a complete elimination of KA-induced WDS. These

studies suggest that release of granule cell ENK, which

is 3 times more concentrated in ventral than dorsal hippocampus, plays an important role in KA-induced WDS.

INTRODUCTION

In the hippocampal formation, opioid peptides of the ENK

family are contained in: a) projections from lateral

entorhinal cortex to dentate gyrus and Amman's horn, b)

dentate granule cells, and c) scattered neurons

throughout hippocampus (Gall et al. 1981).

Concentrations of ENK are several times higher in ventral

than in dorsal hippocampus (Hong and Schmid 1981). The

dynorphin (DYN) family of opioid peptides is contained in

dentate granule cells (McGinty et al. 1983).

Concentrations of DYN A(1-8) in ventral hippocampus are

twice those in dorsal hippocampus (Kanamatsu et al., in

press). KA causes an initial seizure-associated depletion of hippocampal ENK and DYN and a subsequent rebound

at 48 hr that corresponds to changes in ENK and DYN immunostaining of the mossy fiber axons of the dentate

granule cells (Kanamatsu et al. in press). The purpose

of the present studies was to determine whether ENK or

DYN contained in dentate granule cells is important for

KA-induced WDS or behavioral convulsions. Studies were

designed to determine : a) whether KA-induced changes in

hippocampal ENK or DYN are associated with WDS as well as

with seizures, and b) whether the effects of icv injections of antibody to methionine enkephalin (ME) or DYN,

sc injections of naloxone, or intrahippocampal injections

of colchicine that destroy dentate granule cells

(Goldschmidt and Steward 1981) have an effect on

KA-induced WDS or seizure behavior.

481
METHODS

Male Fischer-344 rats between 12 and 15 weeks of age were

used in these studies. KA was administered SC (8 mg/kg),

and rats were observed for WDS and limbic seizures (rearing, clonus, hypersalivation, and falling) for 2.5 hr.

At this time, rats were anesthetized with pentobarbital

(50 mg/kg) to prevent seizure-related mortality. Data

were analyzed with analysis of variance significant

main effects were followed with post hoc Fisher's LSD

tests.

Study I. Rats (n=4) were injected with KA and were

with a radioimmunoassay described previously (Hong et al.

1978) in hippocampal tissue that had been frozen at -70

Stydy II. Rats (n=40, 10/group) were injected with 0,

2.5, 5.0, and 10.0 mg/kg of naloxone (NAL) SC 5 min prior

to injection of KA.

Study III.

body to ME, DYN A(1-8),

Rats (n-38) were injected with 20 ul of antior normal rabbit serum into the

lateral ventricles 6 min prior to injection of KA. The

ME antiserum crossreactivity was: ME, 100%; leucine

enkephalin (LE), 0.47%; DYN A (1-8) < 0.06; ME

-Arg-Phe (MEAP), < 4.0% and B-endorphin (BE), < 0.2%.

The DYN A (1-8) antiserum crossreactivity was: DYN A

(1-81, 100%; DYN A (1-13), 0.02%; DYN A (1-17), 0.01%;

ME, 0.005%; and LE, 0.005%. For the ME antiserum, the

IC-50 was 644 fmoles, and for antiserum to DYN A (1-8),

it was 25.5 fmoles.

Study IV. Rats (n=86) were bilaterally injected with

colchicine (COL) (2.5 μg/site) into dorsal and ventral

hippocampus, dorsal hippocampus alone, or ventral hippocampus alone. After a 2 week recovery period, rats were

injected with KA and killed 48 hr later. Rats (n-40)

injected with COL into the dorsal or ventral hippocampus

alone were killed by perfusion with 4% formalin, and

brains were processed for cresyl violet and Luxol fast

blue staining. Rats injected with COL into dorsal and

ventral hippocampus were perfused with 1.2% sodium sulfide followed by 4% paraformaldehyde (n-19) for Timms

staining of zinc and immunocytochemical staining of ME

and DYN or were killed by decapitation for radioimmunoassay of ME and DYN (n-27):

RESULTS

Study I. Rats included in data analysis were those that

exhibited WDS but not behavioral convulsions following

systemic injection of KA. Those killed 1-2 hr post

injection had an averaae of 45 WDS and those killed 48

post injection had an average of 55 WDS across the 2.5 hr

observation period. Hippocampal ME was decreased by 23%

482
at the early time point and was increased by 40% at the

later time point. DYN was initially decreased by 50% and

returned to baseline at 48 hr.

Study II. NAL caused a dose and time-dependent decrease

in KA-induced WDS. The maximal effect (59% reduction)

was achieved with a dose of 5.0 mg/kg. This effect was

observed from the onset of WDS 30 min post KA injection

to approximately 90 min post injection.

Study III. Pretreatment with antibody to ME but not

A (1-8) caused a

DYN

30% reduction in KA-induced WDS when

compared to rats receiving only control serum.

Study IV. COL injections into ventral hippocampus alone

or combined injections into dorsal and ventral hippocampus caused a virtually complete elimination of

KA-induced WDS. COL injections into dorsal hippocampus

did not attenuate KA-induced WDS. None of the injections

had an effect on the onset or topography of behavioral

convulsions. COL caused a 60% depletion of ME and a 100%

depletion of DYN in dorsal and ventral hippocampus. ME

in ventral hippocampus is approximately 3.7 times that in

dorsal hippocampus; therefore the absolute reduction of

ME in ventral hippocampus was greater than that in dorsal

hippocampus. COL blocked the KA-induced increase in hippocampal ME observed in saline-injected controls. Timms

and Nissl staining combined with immunostaining for ME

and DYN revealed severe (>75%) destruction of granule

cells and their mossy fiber axons.

DISCUSSION

Several lines of evidence suggest that release of ENK

within the hippocampal formation plays an important role

in KA-induced WDS. WDS follow KA-induced epileptiform

bursting of hippocampal neurons (Lothman and Collins

1981); electrical stimulation of dentate granule cells

sufficient to cause a population spike also leads to WDS

(Damiano and Connor 1984). Lesions of the CA3 region of

hippocampus attenuate electrically-induced (Damiano and

Connor, 1984) and KA-induced WDS (Isaacson and Lanthorn

1981). These studies suggest that release of a

transmitter from granule cells onto CA3 pyramidal neurons

plays an important role in KA- or electrically-induced

WDS. Injections of ENK into the lateral ventricles cause

naloxone-reversible WDS (Drust and Connor 1983), suggesting that this behavior involves actions at an opiate

receptor. The granule cells of the dentate gyrus contain

two families of opioid peptides, ENK and DYN (See Introduction) either of which could play an important role in

the initiation of KA-induced WDS.

In these studies, we found that SC injections of KA that

lead to WDS but not behavioral seizures cause initial

depletions of ME and DYN and 48 hr later a rebound in ME.

We also found that pretreatment with NAL attenuated

483
KA-induced WDS. These results suggest that release of an

opioid peptide, possibly ME or DYN could play a role in

KA-induced WDS. Therefore, we injected antibody to ME or

DYN A(1-8) into the lateral ventricles prior to SC injection of KA and found that antibody to ME but not DYN

attenuated KA-induced WDS. Selective COL lesions of the

dentate granule cells of the hippocampal formation were

performed to determine whether ME contained in these neurons was important for KA-induced WDS. We found that COL

lesions of ventral but not dorsal hippocampus were sufficient to eliminate KA-induced WDS. Because systemic KA

causes greater increases in metabolic activity in ventral

than in dorsal hippocampus (Lothman and Collins 1981), we

propose that release of ENK from dentate granule cells in

ventral hippocampus onto neurons of the CA3 region plays

an important role in the generation of KA-induced WDS but

does not appear to be essential for subsequent behavioral

convulsions. [5-T32-ES-07126]

REFERENCES

Damiano, B.P. and Connor, J. D. Brain Research, 308:

383-386, 1984.

Drust, E. G. and Connor, J. D.

458-459, 1983.

J Pharmacol Exp Ther, 224:

Gall, C., Brecha, N., Karten, H. J., and Chang, K.-J.

J Comp Neurol, 198:335-350, 1981.

Isaacson, R. L. and Lanthorn, T. H. Federation Proc, 40:

1508-1512, 1981.

Goldschmidt, R. B. and Steward, O. Proc Natl Acad Sci

USA, 77(5):3047-3051, 1981.

Hong, J. S., Yang, H.-Y.T., Fratta, W., and Costa, E. J.

Pharmacol Ther, 205:141-147. 1978.

Hong, J. S., an Schmid, R. Brain Research, 205:415-418,

1981.

Kanamatsu, T., Obie, J., Grimes, L., McGinty, J. F.,

Yoshikawa, K., Sabol, S., and Hong, J. S. J Neuroscience, (in press).

Lanthorn, T., Isaacson, R.L. Life Sci, 27:171-178, 1978.

Lothman E.W. and Collins, R.C. Brain Research, 218:

299-318, 1981.

McGinty, J. F., Henriksen. S.J., Goldstein, A.,

Terenius, L.; and Bloom, F. E. Proc Natl Acad Sci

USA, 80:589-593, 1983.

484
OPIATE RECEPTOR BINDING AND BEHAVIORAL EFFECTS OF

MORPHINE IN RHA/VERH AND RLA/VERH RATS

D. Lazega, P. Driscoll*, H.-R. Frischknecht,

B. Siegfreid and P.G. Waser

Inst. of Pharmacology, Univ. of Zurich

Gloriastrasse 32, 8006 Zurich, Switzerland

*Inst. of Anatomy, Univ. of Lausanne

1005 Lausanne, Switzerland

ABSTRACT

Large baseline differences were found in open field activity and rearing

behavior between 2 psychogenetically selected rat lines, as well as differences

in the effects of peripherally administered morphine (2,4 and 10 mg/kg) on

those behaviors and on body temperature. The 2 lines also showed different

capacities of tritiated DACO binding to preparations of striatal membranes.

INTRODUCTION

Roman high-avoidance (RHA/Verh) rats are selectec and bred for the rapid

acquisition of two-way active avoidance, whereas Roman low-avoidance

(RLA/Verh) rats are selected and bred for the non-acquisition of that

response. Considerable literature has accumulated documenting

neurochemical correlates of behavior in these rats, and profound differences

exist between them in baseline activity and in stress situations, in which

RHA/Verh rats become hyperactive and RLA/Verh rats tend to ‘freeze’

(Driscoll and Battig 1982, Driscoll 1986). Genetic differences in opioid

peptide systems, in relation to known differences in behavior, have been

extensively studied in several species (e.g. Neil et al. 1986, Frischknecht et al.

1986), and it is likely that genetically related behavioral modification, through

selective breeding, can also lead secondarily to opiate changes. We have

therefore compared the effects of morphine on open field behavior, nocieption

and body temperature in RHA/Verh and RLA/Verh rats, and also compared

both lines in regard to opioid receptor binding to preparations of crude

membranes of striatum and hippocampus.

MATERIALS AND METHODS

28 male RHA/Verh (mean body weight 340 g) and 28 male RLA/Verh rats

(332 g) were divided into groups of 7 and injected i.p. with either saline or

morphine (2,4 or 10 mg/kg). 30 min later, the numbers of squares crossed and

rearing behavior were monitored, individually, during 5 min in an open field

(60 x 60x 30 cm, divided into 9 equal squares). 40 min after injection, the

nocieptive response was measured with a tail-flick apparatus. The intensity

of the heat beam was adjusted to obtain baseline response latencies of about 2

sec. A cut-off time of 15 sec was used to prevent tail damage. Rectal body

temperature was measured 90 min after injection with a telethermometer

probe. Statistical analyses were performed by 2-way ANOVAs followed by

Duncan’s multiple range test.

485
The brains of other groups of RHA/Verh and RLA/Verh male rats of the

same age were removed, following decapitation, and striata and hippocampi

were extracted and weighed. 4 strata were pooled for each replication. The

tissue was homogenized in Tris buffer (pH 7.4 at 2°C), centrifuged at

39,000g for 15 min, the pellet suspended in Tris buffer and incubated at 37°C

for 45 min and centrifuged again.

buffer at 25°C.

The resulting pellet was suspended in Tris

For the assay, .8 ml final homogenate was added to .1 ml of

the labeled ligand solution and .1 ml of Tris buffer of cold displacer ligand

solution. The final tissue concentration was 5.5 mg fresh tissue/ml,

corresponding to .43 mg protein/ml. The mixture was incubated for 2.5 hr at

25°C, filtered through a Whatman GF/B filter disc and washed 3 times with

5 ml ice-cold Tris buffer. The filters were dried and, after the addition of

10 ml scintillation cocktail (toulene: Triton X-100 at 2:1, and Butyl-PBD

5g/l3), were cou

(D-Ala2, MePhe4, Gly-ol5) enkephalin (

ted at an efficiency of 42-45%. The ligands used were Htritiated DAGO) for mu-binding

determination, and 3H-(D-Ala2, D-Leu5) enkephalin (tritiated DADLE),

added with cold DACO (7nM/nM DADLE) for delta binding. Cold DAGO was

added to suppress the mu-fraction of tritiated DADLE binding. Non-specific

binding was defined as the bound radioactivity in the presence of cold

etorphine at a constant ratio of 1000x the concentration of labeled ligand.

Scatchard analyses (striatum only) were performed by the use of LIGAND

program (Dr. Munson, NIH, Bethesda, MD, USA).

RESULTS

In regard to both open field activity and rearing, it was found that RHA/Verh

rats were more active than RLA/Verh rats (p< .001). Perhaps as a result of

the large baseline (NaCI injection) differences, morphine administration did

not significantly decrease behavior in RLA/Verh rats, while most dosages

significantly diminished both horizontal and vertical activities in RHA/Verh

rats. The analysis of tail-flick response latencies showed no significant

genetic differences, only a pronounced drug effect (p< .0001). A significant

TABLE 1

Effects of morphine on open field behavior, nociception and

body temperature in RHA/Verh and RLA/Verh rats (Results are

expressed as the mean 2 s.e.m.; n=7 per treatment group)

Number of Number of Tail-flick Body

Squares Rearings Latency Temperature

crossed (sec) (°C)

RHA NaCl 63.3±6.2 43.4±5.4 1.88±0.40 37.85±0.14

2mg/kg 46.3±7.8 28.4±4.4 4.28±1.88 38.35±0.13

4mg/kg 40.0±12.1 21.6±4.6 10.10±2.15 38.72±0.21

10mg/kg 33.8±8.2 9.0±2.2 15.00±0.00 39.14±0.14

RLA NaCl 19.7±5.2 11.8±3.2 2.48±0.67 37.74±0.16

2mg/kg 14.0±4.9 11.6±1.9 7.91±1.92 37.98±0.16

4mg/kg 17.7±4.0 8.3±2.2 9.09±1.82 38.02±0.17

10mg/kg 6.6±3.1 23.1±1.8 13.46±1.54 37.52±0.29

486
antinociception was found in RHA/Verh rats with 4mg/kg, but already at

2mg/kg in RLA/Verh rats. In addition, a significant increase in body

temperature after morphine, as compared to the NaCl controls, was found

only in RHA/Verh rats (4mg/kg: p< .005, 10mg/kg: p< .001).

The 2 lines also differed significantly in tritiated DAGO binding to

preparations of striatal membranes. As visualized in figure 1 A, the binding

capacities differed significantly, Bmax being 1.72x greater in RHA/Verh

than in RLA/Verh rats. The affinities did not differ (Student-t-test). No

difference was found in tritiated DADLE binding to the same preparation

(figure 1B). Curve-fitting by LIGAND program revealed a single binding site

saturated by tritiated DAGO and 2 binding sites saturated by tritiated

DADLE. In addition to table 2, the second binding site of tritiated DADLE

was characterized by a Kd of 7.1±2.5 and 4.9±2.1 and a Bmax of 11.9±1.0 and

11.6±0.9 for RHA/Verh and RLA/Verh rats, respectively. The meaning of

this

FIGURE 1

Scatchard plot of specific binding of tritiated DAGO (A)

and tritiated DADLE (6) to preparations of striatal membranes of RHA/Verh (o) and RLA/Verh ( ) rats. Each point

represents the mean of a triplicate incubation. Concentration ranges of labeled ligands were 0.3-12nM for tritiated DAGO and 0.3-17nM for tritiated DADLE. The thin

lines in figure IA represent the 95%-condidence limits (±2

sigma) of the means.

TABLE 2

Binding results of tritiated DAGO and tritiated DADLE to

preparations of striatal membranes of RHA/Verh and RLA/Verh

rats, expressed as mean value (± s.e.m.) of 3 separate

experiments. 4 striata were pooled for each experiment. Kd

is expressed as nM and Bmax as fmol/mg of tissue.

3

H-DAG0 3

H-DADLE

Kd Bmax Kd Bmax mu/delta

RHA 0.39±0.40 9.6±0.44 0.06±0.14 4.19±0.4.3 2.3

RLA 0.36±0.08 5.56±0.35 1.02±0.24 4.62±0.77 1.2

487
second binding site is not yet known. The first fraction of the Scatchard was

therefore considered to define delta binding. Single-point binding of tritiated

DAGO (5nM) and tritiated DADLE (1.5 nM) to preparations of hippocampal

membranes from RHA/Verh and RLA/Verh rats did not differ significantly.

DISCUSSlON

The genetic differences seen here in regard to the behavioral and

thermoregulatory effects of morphine, as well as in the striatal mu-receptor

binding capacities, provide valuable information which not only adds to the

steadily increasing state of knowledge regarding this type psychogenetic

selection, but also which indicates some potentially interesting directions for

future research. First of all, opioids are believed to exert a neuromodulatory

role, particularly in regard to the striatum and locomotor behavior, where

they have long been known to participate in the regulation of dopaminergic

and cholinergic transmission (Cheney et al. 1974, Frischknecht et al. 1986).

Morphine also affects the production and metabolism of brain serotonin

(Perez-Cruet et al. 1975). All 3 of these neurotransmitter systems (as well as

locomotor activity) are known to differ in RHA/Verh and RLA/Verh rats in

that the former have a more active striatal dopamine metabolism, a more

active serotonergic system in several brain areas, and have lower levels of

hippocampal and, possibly, striatal choline acetyltransferase activity than do

the latter (Driscoll and Battig 1982, Driscoll 1986, Scatton et al. unpublished

results). Therefore, a more detailed analysis of endogenous opioid peptides, as

well as additional opioid receptors and regions, would definitely be of interest

in these lines of rats.

Secondly, the mu opiate receptor, particularly in the striatum, appears to be

involved in the initiation of acute opiate dependence (Eisenberg 1985, Neil et

al. 1986) and, as genetic background appears to be important with respect to

predisposition to dependence (Jacob and Ramabadran 1981). The finding in

the present study that the Bmax of mu binding differs by almost 2 to 1

between RHA/Verh and RLA/Verh rats would seem to make a comparsion of

these 2 rat lines, in regard to opiate dependence studies, an undertaking of the

utmost interest, with clinical implications.

REFERENCES

Cheney, D.L., Trabucchi, M., Racagni, G., Wang, C., and Costa, E. Life Sci,

15:1977-1990, 1974.

Driscoll, P. Behav Genet, 16:355-364, 1986.

Driscoll, P., and Battig, K. In: Lieblich, I., ed. Genetics of the Brain.

Amsterdam, Elsevier Biomed Press, 1982. pp. 95-123.

Eisenberg, R.M. J Pharmacol exp Ther, 233:863-869, 1985.

Frischknecht, H.-R., Siegfried, B., and Waser, P.G. To be published in:

Genetically Determined Animal Models in Biological Psychiatry. Basel,

Karger, 1986.

Jacob, J.J.C., and Ramabadran, K. Pharmacol Ther, 14:177-196, 1981.

Neil, A., Terenius, L., Ternes, J.W., Ehrman, R.N., and O’Brien, C.P. Eur J

Pharmacol, 122:143-147, 1986.

Perez-Cruet, J., Thoa, N.B., and Ng, L.K.Y. Life Sci, 17:349-362, 1975.

488
ACTION OF NALTREXONE ON THE SEXUAL IMPAIRMENT OF

OBESE CAFETERIA RATS

A. Mandenoff, M.C. Bertière, O. Betoulle,

M. Apfelbaum

Laboratoire de Nutrition Humaine INSERM U 286 -

Facultè X. Bichat - 16 rue H. Huchard, 75018 Paris,

France

ABSTRACT

Two experiments were performed to explore 1) the

sexual behavior of male obese cafeteria rats, 2) a

possible role of endogenous opiates in the regulation

of their sexual behavior. In obese cafeteria rats the

proportion of ejaculators and the number of

ejaculations per hour were significantly lower

compared to controls. Naltrexone provoked an increase

in the number of ejaculations/h both in control and

cafeteria rats due to an induction of copulatory

behavior in sexually inactive rats. Thus nutritional

obesity provokes an impairment of sexual behavior and

an injection of naltrexone (2.5 mg/Kg IP) lifts this

inhibition in a similar way as in inactive, control

rats.

INTRODUCTION

Two kinds of obesity in rodents, the genetic obesity

of Zucker fa/fa rats and the obesity nutritionally

induced by a choice of highly palatable foods, include

hyperphagias ; both these hyperphagias are suppressed

by opiate antagonists, naloxone or naltrexone

(Margules et al. 1978, Mandenoff et al. 1982). It is

known that opiate antagonists enhance sexual behavior

in non obese rats (Hetta 1977, Myers and Baum 1979) ;

agonists inhibit it and antagonists lift this

inhibition (Gessa et al. 1979, Hetta 1977, McIntosh et

al. 1979, Mumford and Kumar 1979, Myerson and Terenius

1977, Pellegrini et al. 1978). Therefore we made the

hypothesis that the decrease in sexual performances

could be linked to a hyperendorphinism in nutritional

obesity as well as in genetic obesity. In genetic

obesity a sexual impairment is documented (Edmonds and

Withyachumnarnkul 1980), and it has been recently

discovered that sexuality and fertility are restored

by an injection of naloxone (personal communication :

J. Walter, D. Margules). However no information is

available concerning the sexual behavior during

nutritional obesity. The present work has two aims :

to evaluate sexual behavior in cafeteria obese rats

and to determine the effect of an opiate antagonist,

naltrexone, on this behavior.

489
MATERIALS AND METHODS

Animals

56 male Wistar rats weighing between 200 and 250g were

housed in groups of 4. They were maintained at 24°C

with a 12 hour light/dark cycle.They were distributed

in two groups : a group "control" (n=28) received

ordinary laboratory chow (Extra Labo M25) ad lib; a

group "cafeteria" (n=28) was given 4 palatable foods

in addition to chow. The 4 supplementary items were

changed each day on a weekly rotation. The animals

were weighed regularly. At week 9 the mean body weight

of "cafeteria" rats was 514.25±6.09g vs 438.69±5.22g

for the controls (mean ± sem < 0.001). The first

sexual behavior test was then performed. One week

later control and cafeteria rats were divided in two

subgroups: one IP injected with NaCl (1 ml/Kg of body

weight), the other with naltrexone (NTX 2.5 mg/Kg),

The sexual behavior tests were conducted 30 mn after

the injections.

Sexual behavior tests

The tests were carried out during light period

(between 02.00 and 06.00 pm) over one hour. 3 males

were placed in a cage. Ten minutes later four 3 month

old female rats, made sexually receptive by an IN

injection of oestradiol benzoate (10μg/rat) 48 hours

and 24 hours prior, were introduced. During the sexual

test, some rats were sexually active while others were

inactive. The parameters observed were:-intromission

latency (latency of the occurence of the first

intromission after the introduction of the females in

the cage) ; -pre-ejaculatory latency (latency from the

first intromission to ejaculation), - refractory

period (post-ejaculatory period between an ejaculation

and the next intromission) - number of ejaculations

per hour (Nb ejac/h).

Statistical Analysis

The proportions of sexually active and inactive

control and cafeteria rats were compared by X2 test.

The parameters studied in control and cafeteria rats

were compared with the Student T test. Effects of

naltrexone on the different parameters were analyzed

with the T paired test.

RESULTS

Table 1 shows that at week nine the proportion of

active cafeteria rats is significantly smaller than

that of active control rats (*P< 0.01). The number

of ejaculations/h in the cafeteria group is smaller

than in the control group (** P< 0.025).

490
Table 1 Control Cafeteria

Active n

%

Inact n

%

Act + Inact n

Nb ejac/h

(mean + sem)

14 5

50% 17.8%*

14 23

50% 82.2%

28 28

1.96±0.42 0.67±.31*”

When the active cafeteria rats are considered alone,

the number of ejaculations/h and the parameters

characterizing the sexual behavior are not

significantly different from those of the active

control rats ("control" (n=14) vs “cafeteria” (n=5):

intromission latency 12.33±2.15 vs 10.22±3.02, pre

ejaculatory latency 19.71±4.37 vs 18.4±4.50,

refractory period 4.38±0.71 vs 5.0±0.71, expressed in

minutes + sem,

3.8±0.667.

intromission frequency 3.92±0.33 vs -

Tabie 2 shows that naltrexone increases the percentage

of active rats both in cafeteria and control qroups,

though this increase is not statistically siqnifi -

cant. Naltrexone significantly increases the global

number of ejaculations in both groups. But if the

sexually active rats are considered alone the number

of ejaculations is not significantly increased by

naltrexone (4.57±0.97 for controls and 5±1.41 for

cafeteria), nor are any of the other parameters

studied.

Table 2

Control Cafeteria

NaCl NTX NaCl NTX

Active 7

% 50% 78.6%

2 5

14.3% 35.7%

Inact 7 3 12

% 50% 21.4% 85.7% 6493%

Act+Inact n 14 14 14 14

Nb ejac/h 1.92±2.2 3.5±2.17**0.5±1.29 1.28±1.98*

 (mean+sem)

NTX vs NaCl *P< 0.05, ** P < 0.025

DISCUSSION

The first finding was quite expected: a nine week

cafeteria diet inhibits the sexual behavior since out

of 28 rats only 5 ejaculate during a one hour sexual

test. Thus an impairment of sexual behavior occurs in

various types of obesity, both genetic and

nutritional, suggesting an effect of obesity itself.

The results concerning the involvement of the opiate

system in such an impairment are less clearcut. In

control rats naltrexone does not modify sexual

behavior of active rats, but does induce sexual

activity in inactive rats. This result is quite

491

172-683 O - 87 - 17 : QL 3
consistent with those of the literature : it has been

reported that in sexually active rats, small or medium

doses of opiate antagonists do not modify the sexual

behavior (Gessa et al. 1979, McIntosh et al. 1979,

Myerson and Terenius 1977), while high doses

facilitate it (McIntosh et al. 1977, Myers and Baum

1979). In inactive rats high (McIntosh et al. 1979)

and even low doses (Gessa et al. 1979) induced

mounting and ejaculations. In inactive cafeteria rats,

naltrexone increases sexual performances, however the

amplitude of such an increase is not significantly

different from that observed in control rats. The dose

of 2.5 mg/Kg of naltrexone used to test an eventual

hypersensitivity to opiate antagonists in sexual

behavior of cafeteria rats could appear to be high

since we previously showed that a dose as low as 0.5

mq/Kg is efficient for suppressing hyperphaqia induced

by cafeteria diet without modifying food intake in

chow fed rats (Mandenoff et al. 1982). However 0.5

mq/Kq of naltrexone did not provoke any change in

sexual performances either in cafeteria or in control

rats (unpublished results). Thus there is no

experimental evidence that in terms of sexual behavior

cafeteria rats are more sensitive to naltrexone than

control rats. In conclusion, like genetic obesity,

nutritional obesity provokes an impairment in sexual

behavior and an opiate abtaqonist induces the

copulatory behavior in cafeteria inactive rats as well

as in controls. Although Naltrexone lifts the

inhibition of sexual behavior induced by the cafeteria

diet, it is not proved that the impairment of sexual

behavior in cafeteria obese rats is related to an

hyperendorphinism.

REFERENCES

EDMONDS, E.S., WITHYACHUMNARNKUL, B Physiol Behay,

24 : 1139-1141. 1980.

GESSA, G.L., PAGLIETTI, E., PELLEGRINI QUARANTOTTI, 8.

Science, 204 : 203-205, 1979.

HETTA, J. Acta Pharmacol Tax, 41 : suppl 4, 53, 1977.

McINTOSH, T.K., VALLONO, M.L., BARFIELD, R.J.

Pharmacol Biochem Behav, 13 : 435-441 , 1979.

MANDENOFF, A., FUMERON, F., APFELBAUM, M., MARGULES,

O.L. Science, 215 : 1536-1538, 1982.

MARGULES, D.L., MOISSET, B., LEWIS, M.J., SHIBUYA, H.,

PERT, C.B. Science, 202 : 988-991, 1978.

MUMFORO, L., KUMAR, R. Psychopharmacol, 65 : 179-185,

1979.

MYERS, B.M., BAUM, M.J. Pharmacol Biochem Behav, 10:

615-618, 1979.

MYERSON, B.J., TERENIUS, L. Eur J Pharmacol, 42: 191

192, 1977.

PELLEGRINI QUARANTOTTI, B., CORDA, M.G., PAGLIETTI,

E., BIGGIO, G., GESSA, G.L. Life Sci, 23 : 673-678,

1978.

492
OPIATES STIMULATE FOOD CONSUMPTON IN THE LAND SNAIL HELIX ASPERSA

Chris Ndubuka, Derrick Brown, Stacy Pratt, Ericka Braham,

Michael K.Leung and George B. Stefano

Departments of Biological Sciences and Chemistry, State

University of New York/College at Old Westbury, Old Westbury, New

York 11568 U.S.A.

ABSTRACT

Morphine stimulates food consumption in the land snail Helix

aspersa. This stimulation of food consumption can be blocked by

naloxone, the potent opiate antagonist. In addition, this opiateinduced food consumption exhibits tolerance by the sixth day of

treatment. This study further highlights opioid mechanisms in

relatively simple organisms and also suggests that these

mechanisms are "ancient" signal systems.

INTRODUCTION

Opioid substances have been shown to have diverse functions that

transcend their original proposed role in nociceptive/analgesic

systems. The importance of this family of compounds is

highlighted even more with their discovery in invertebrates.

Recently we have reported the isolation and identification of

Met-enkephalin, Leu-enkephalin and Met-enkephalin Arg6-Phe7 in

Mytilus pedal ganglia (Leung & Stefano 1984; Stefano and Leung

1984). The same neural tissue also has been shown to contain

opioid receptors that are similar to those reported to exist in

mammals (Kream et al. 1980; Stefano et al. 1980). As a result, it

is no longer surprising to find opiate functions in various

invertebrates. In mammals opiate mechanisms have been shown to

be involved in the regulation of normal ingestive activities

(Morley et al. 1983). Opiates have been shown to not only

stimulate food consumption but various ingestive behaviors as

well (Sanger 1981; Sanger and McCarthy 1981; Morley et al. 1983).

Naloxone, the potent opiate antagonist, can block these

morphine-induced activities (Brands et al. 1979) as well as

decrease feeding in food deprived mammals (Brown and Holtzman

1979). In an earlier study Kavaliers et al. (1984) demonstrated

that opiates could induce feeding in the slug Limaz maximus.

Thus, this study complements the earlier study in that morphine

also stimulates this activity in Helix aspersa.

MATERIALS AND METHODS

Helix aspersa were obtained from the Pacific Biomarine Company in

California. The animals were maintained in the laboratory at 21±

1°C under a 12-hr light and 12-hr dark cycle. They were housed

prior to experimentation in a large Styrofoam box containing

moistened paper towels, lettuce, carrots and rat chow. The

organisms were divided into four groups and placed in individual

1L beakers containing moistened paper toweling clinging to the

493
side and bottom. For each experiment there was a minimum of five

groups of organisms. A substrate of lettuce and carrots were

pureed in a blender and poured into a 5 cm petri dish. This was

then weighed before and after a 3-hr feeding period and corrected

for evaporation. Animals receiving drug treatments were injected

through the epidermis in a region superior to the anterior

ganglionic complex. The organisms received either drug, vehicle

or no injection.

Table 1. The effects of opiates on food consumption

in the land snail Helix aspersa

OPIATE DOSE(M) %CONSUMED OPIATE DOSE(M) %CONSUMED

vehicle ---- 100 naloxine 10-7 100

morphine 10-7 100 naloxone 10-6 90

morphine 10-6 124analoxone 10-5 50b

morphine 10-5 140bnaloxone 10-4 70b

morphine 10-4 135b

etorphine 10-4 138b

Morphine 10-5 and Naloxone 10-5 95% Consumed

Animals were maintained in groups of four for a

3-hour interval. Each group consumed .7g of food.

The variation between groups was no greater than 4%

Statistical analysis was by Student's t-test, a =

P<0.05, b = P< 0.0.1 Vehicle and control values

were identical (.7g/3 hr).

Table 2. Demonstration of tolerance to morphine in

reference to its induced stimulation of food ingestion.

DAYS MORPHINE DOSE (10-6M) %CONSUMED

1 AS NOTED 125

2 AS NOTED 125

3 AS NOTED 120

4 AS NOTED 113

5 AS NOTED 99

6 1 0 - 5 130

Experiments were as noted in table 1. These animals

were injected with morpine 4x/day for the indicated

number of days. Statistical analysis was by Student'st-test, a = P<0.05, b = P< 0.01.

RESULTS

Vehicle and control organisms ate 0.7g/3 hr of the prepared food.

Morphine treated organisms exhibited a significant dose-dependent

increase in food consumption (table 1). This increase in food

consumption occurred within the first 1.5 hr following morphine

injection. Interestingly, organisms observed for 24-hr (vehicle

494
and morphine treated) were found to consume the same amount of

food, indicating that the morphine groups' food consumption

decreased following the initial morphine-induced stimulation of

ingestion. Naloxone, an opiate antagonist, when administered

alone was found to decrease food ingestion in a dose dependent

manner (table 1). In addition, when co-injected with morphine it

blocked the opiate induced stimulation of food consumption.

Again, the maximal effects of naloxone occurred during the first

1.5 hr of treatment.

In another sec of experiments, organisms treated with morphine

(10 M) 4x/24 hrs) for 5 days gradually lost the morphine

induced stimulation of food consumption. However, increasing the

dose of morphine on the 6th day brought it back (table 2). This

may serve to indicate that this behavior was made tolerant to

morphine.

DISCUSSION

The present report suggests that an endogenous opioid system may

be present in Helix aspersa and that it may be involved with the

organisms feeding responses and food intake. The same type of

regulatory role of opioids in regard to food consumption has been

reported in Limax maximus by Kavaliers et al. (1984). In both

studies, naloxone was able to antagonize the opiate stimulation

of food ingestion, indicating the presence of specific opiate

receptors. In addition, in both organisms naloxone alone was able

to reduce food intake, strongly suggesting the presence of an

endogenous opioid system regulating feeding behaviors.

Interestingly, this system can be made tolerant to chronic

morphine treatments, a finding which clearly demonstrates the

cellular complexity of invertebrate opioid activities.

In general the growing body of evidence for the presence of

endogenous opioid regulatory systems in invertebrates is

increasing (for review, Leung & Stefano 1986; Stefano 1986). The

data to date suggests that opioid evolvement had an early

development and that this "old" status highlights the importance

of these particular signal molecules.

REFERENCES

Brands, B., Thornhill, J.A., Hirst, M., and Gowdy, C.W., Life

Sci, 24:1773-1778, 1979.

Brown, D.R., and Holtzman, S.G. Pharmacol Biochem Behav, 11:567-573,

1979.

Kavaliers, M., Hirst, M., and Teskey, G.C. Physiology and Behavior

33:765-767, 1984.

Kream, R.M., Zukin, R.S., and Stefano, G.B. J Biol Chem, 225(19):

9218-9224, 1980.

Leung, M.K., and Stefano, G.B. Proc Nat'l Acad Sci (USA), 81:955-

958, 1984.

Leung, M.K., and Stefano, G.B. Prog in Neurobiol, in press, 1986.

Morley, J.E., Levine, A.S., Yim, G.K. and Lowy, M.T. Neurosci

Biobehav Rev, 7:281-305, 1983.

495
Sanger, D.J. Appetite, 2:193-208, 1981.

Sanger, D.J., and McCarthy, P.S. Psychopharmacology, (Berlin)

74:217-220, 1981.

Stefano, G.B. Cell Mol Neurobiol, 2:167-178, 1982.

Stefano, G.B. Comparative Opioid and Related Neuropeptide Mechanisms.

Boca Raton, FL., CRC Press, 1986. 576 pp.

Stefano, G.B., Kream, R.M., and Zukin, R.S. Brain Res, 181:445-458,

1980.

Stefano, G.B., and Leung, M.K. Brain Res, 298:362-365, 1984.

ACKNOWLEDGEMENTS

This work was supported by NIH Grant RR 08180 and ADAMHA-MARC

Grant 17138. Mr. C. Ndubuka and Mr. D. Brown are ADAMHA-MARC

Fellows. Ms. E. Braham is a MBRS student assistant.

496
EFFECT OF ELECTROLYTIC AND CHEMICAL VENTROMEDIAL

HYPOTHALAMIC LESIONS ON FOOD INTAKE, BODY WEIGHT, ANALGESIA

AND THE CNS OPIOID PEPTIDES IN RATS AND MICE.

Gopi A. Tejwani and Charles W. Richard III

Department of Pharmacology

College of Medicine

The Ohio State University

Columbus, Ohio 43210

ABSTRACT

The hyperphagia and obesity induced by ventromedial hypothalamic (VMH) electrolytic lesions in female rats were asscciatedwitha

70-94% decrease in the level of ß-endorphin (ß-E) in the hypothalamus and other regions of brain, but not in the pituitary. Dynorphin

(Dyn) and methionine-enkephalin (ME) levels were also decreased.

Rats with VMH lesions were less sensitive to the inhibitory effect

of naloxone on their food-intake. Mice injected with gold

thioglucose (GIG) also showed a decrease inthehypothalamiccontent

of ß-E and Dyn and exhibited 30% less analgesia compared to control

mice after cold swim stress.

INTRODUCTION

Lesions in the VMH region of brain cause hyperphagia, obesity

and several other behavioral and physiologic abnormalities

(Hetherington and Ranson 1940; Powley 1977; Bray and York 1979). In

view of the role of opioid peptides in appetite control (Morley et

al. 1983), the objectives of this study were to examine the effects

of VMH lesions on the CNS opioid peptides and correlate these

changes with specific behavioral characteristics such as food intake

and analgesia.

MATERIALS AND METHODS

Female Sprague-Hawley rats (240-260 g) were obtained from lab

Supply (Indianapolis, IN) and were maintained as described earlier

(Vaswani et al.1983). The details of the stereotaxic VMH lesioning

technique are described by Richard III, 1984. Briefly, the rats

were anesthetized with sodium pentobarbital (40 mg/kg) and were

positioned in a Kopf 1404 research stereotaxic instrument. Prophylactic antibiotics (ampicillin 70/mg/kg i.m., and gentamicin, 5 mg/kg

i.m.), were administered immediately and 24 hr after surgery. The

electrode was positioned 0.2 mm posterior to bregma, ± 0.7 mm

lateral to the mid-sagittal suture and 10 mm below the surface of

the skull. Bilateral VMH lesions were produced by passing an anodal

current between the 0.5 mm uninsulated tip of an epoxy coated

stainless steel electrode (0.11 mm diameter) and a rectal cathode

for 20 sec (2 ma x 20 sec current = 40 millicoulombs). control rats

were subjected to sham operation. Sold thioglucose (GIG), 800

mg/kg, was given i.p. to female outbred CD-1 mice (Charles River),

21-25 g, to produce bilateral VMH lesions. For histological verification of the extent of VMH and GTG lesions, animals were perfused

497
with 10% formaldehyde solution in saline. The brains were removed,

blocked and sectioned in 20p sections, cut at -20°C on a cryostat

and picked up on gelatin coated slides. Slides were stained with

cresyl violet and an anatomical comparison and verification of

lesion placement was done. Upon gross inspection, the median

eminence was found intact in all VMH-lesioned rats. Opioid peptides

were measured by RIA as reported previously (Tejwani et al. 1985).

Analgesia in mice was measured by using a hot plate at 55°C and the

movement of the hind limb was used as the criterion of acute

exposure. Measurement of opiate receptor (μ-receptor) birding was

done by using [15,16-3H]-etorphine, according to the method of Pert

and Snyder (1973) as modified by Bardo et al (1982) and Young et al.

(1983) .

RESULTS AND DISCUSSION

Since ithasbeenreportedthatweightgain in animals with VMH

lesions is accentuated by a high fat diet (Sawchenko et al. 1981)

rats in our study were maintained on a high fat diet (Vaswani et al.

1983).

Daily consumption of a high fat diet increased by 136% in rats

with VMH lesions (n=22, P<0.001). Fifteen days later these rats

weighed 35% more than sham operated animals (P<0.001, data not

shown) . VMH lesions in rats (n=9) significantly reduced B-E levels

in the hypothalamus (-85%), striatum (-94%), midbrain/thalamus

(-88%), medulla/pons (-97%), hippocampus (-74%) and cortex (-70%)

but not in the pituitary (fig. 1A). Dynorphin level decreased

(fig. 1B) in the hypothalamus (-54%), pituitary (-66%) and

hippocampus

(-28%). Brain ß-E cell bodies are confined to the medial basal

hypothalamus and arcuate nucleus. Destruction of cell bodies or

fibers eminating from this area would conceivably reduce brain ß-E

content. Watson et al. (1978) observed that unilateral arcuate

lesions produced a 40% decrease in D-E in the central gray area,

while Kerdelhue et al. (1982) showed that mediobasal hypothalamus

(MBH) lesioned rats had an 89% decrease in ß-E in the central gray

area. Milan et al.(1980) have found that small discrete lesions of

the arcuate nucleus produced a marked reduction in the hypothalamic

ß-E and Dyn levels. In the present study the reduction in pituitary

Dyn most probably represents a destruction of neurointermediate lobe

Dyn fibers originatihg in hypothalamus. The reduction in hippocampal Dyn may represent a yet unidentified hypothalamic-hippocampal pathway. We observed (fig. 2A) a decrease in ME level in the

hypothalamus (-37%) and hippocampus (-38%). In spite of a decrease

in the level of opioid peptides, lesioned rats were still sensitive

to the effect of naloxone, although to a lesser extent. Naloxone

decreased the food intake in rats with VMB lesions by 33%, compared

to 67% in control rats (fig. 2B).

Eleven weeks after GTG administration, mice weighed 32% more

than control (fig. 3A). GTG lesions significantly reduced hypothalamic ß-E (-80%) and Dyn (-19%) but did not affect ME level.

Mice with GTG lesions had no statistically different sensitivity to

naloxone or levorphanol but showed 30% less analgesia compared to

control mice after 1.5 min of cold swim stress (fig. 3B). We

498
FIGURE 1 Effect of VMH lesions on ß-endorphin1-31 (A) and

dynorphin1-13 (B) levels in female rats 12 days after electrolytic

lesions (n=9) orshamlesions (n=7).

FIGURE 2 A. Effect of VMH lesions on methionine-enkephalin

level. B. Effect of naloxone on food-intake. Rats were tested 9

days after lesioning, after 12 hr of food-deprivation. Naloxone (1

mg/kg) was given i.p. 30 min before testing. Food-intake was

measured for 1hr.

FIGURE 3 A. Effect of GTG lesions on weight gain in female CD1 mice. B. Analgesic response of mice with GIG lesions to 1 mg/kg,

i.p. injection of naloxone or levorphanol

analgesia (1.5 min swim in H2O at 4°C).

or stress-induced

499
conclude that electrolytic or GTG lesions in VMH decrease CNS opioid

peptide levels which is reflected by an alteration in the feeding

behavior and analgesia in these animals.

Compensatory up-regulation and down-regulation of neurotransmitter receptors have been shown to occur with chronic neurotransmitter decreases and increases, respectively. Compensation of

initially perturbed behaviors has been postulated to occur in

parallel with changes in receptor affinity or number. In our study,

examination of 3

H-etorphine receptor binding in VMH and GTGlesioned animals revealed no change in the opioid receptor affinity

or number in any of the brain regions where a decrease in the opioid

level was observed (data not shown).

ACKNOWLEDGEMENTS

Supported in part by a grant from the Weight Watchers Foundation, Inc. CWR was a recipient of OSU Neuroscience Predoctoral

Research Fellowship Award.

REFERENCES

Bardo, M.T., Bhatnagar, R.K. and Gebhart, G.F. J Neurochem,

39:1751-1754, 1982.

Bray, G.A. and York, D.A. Physiol Rev, 59:719-809, 1979.

Hetherington, A.W. and Ranson, S.W. Anat Rec, 78:149-172, 1940.

Kerdelhue, B., Bethea, C.L., Ling, N., Chretien, M. and Weiner, R.I.

Brain Res, 231:85-91, 1982.

Millan, M.J., Gramsch, C., Przewlocki, R., Hollt, V. and Herz, A.

Life Sci, 27:1513-1523, 1980.

Morley, J.E., Levine, A.S., Yim, G.K. and Lowy, M.T. Neuroscience

Behav Rev, 7:281-305, 1983.

Pert, C.B. and Snyder, S.H. Proc Natl Acad Sci, 70:2243-2247, 1973.

Powley, T. Psycho1 Rev, 84:89-126, 1977.

Richard, C.W. III Ph.D. Thesis, The Ohio State University, 1984.

Sawchenko, P.E., Gold, R.M. and Alexander, J. J Physiol Behav,

26:293-300, 1981.

Tejwani, G.A., Vaswani, K.K., Barbacci, J.C., Richard, C.W. III and

Bianchine, J.R. Life Sci, 33(1):519-522, 1983.

Tejwani, G.A., Vaswani, K.K. and Barbacci, J.C. Peptides, 7:555-

561, 1985.

Vaswani, K.K., Tejwani, G.A. and Mousa, S. Life Sci, 32:1983-1996,

1983.

Watson, S.J., Akil, H., Richard III, C.W. and Barchas, J.D. Nature,

275:226-228, 1978.

Young, E., Olney, J. and Akil, H. J Neurochem, 40:1558-1564, 1983.

500
BOTH ADRENAL OPIOIDS AND GLUCOCORTICOIDS INVOLVED IN THE

INCORPORATION OF INFORMATION INTO MEMORY POST-STRESS

D. Jefferys and J.W. Funder

Medical Research Centre, Prince Henry's Hospital, Melbourne,

Australia 3004

Various behavioral paradigms have clearly shown that glucocorticoids

and opiates influence the acquisition and maintenance of several

behaviors in the rat. Recently, in an experimental paradigm

originally designed to screen for antidepressants (4), we have shown

that manipulation of steroid and opioid levels profoundly influence

the maintenance of an immobile floating response. This response

develops when rats are swum in a restricted space from which they

cannot escape. Rats are placed in a narrow plexiglass cylinder for

15 minutes; initially they swim vigorously, but progressively spend

more and more time relatively immobile. Normal, naive rats are

immobile for 70% of the last 5 minutes of the test period, and for a

similar percentage of a 5 minute retest period 24 hrs later.

FIGURE 1: Rat showing characteristic immobile posture.

We have shown that rats have no difficulty in acquiring the

immobility response after adrenalectomy, hypophysectomy or concurrent

501
hypophysectomy and adrenalectomy (adrx-hypox). As is shown in figure

2, hypophysectomized animals showed levels of immobility similar to

intact animals on retest. In contrast, adrenalectomized rats (or

adrx-hypox) showed a significant reduction in immobility, remaining

immobile for only 28%-30% of the time.

FIGURE 2: Percent immobility during the 5 minute retest by

intact, adrenalectomized, hypophysectomized and hypophysectomized-adrenalectomized rats. Columns represent mean ± SEM

values, the number of animals in each group is shown within each

column. Significant differences from control are shown. **P

<0.01.

This behavioral effect of adrenalectomy was reversed by the

glucocorticoids corticosterone or dexamethasone (DM), but not by the

mineralocorticoid deoxycorticosterone, administered within an hour- of

the initial test period. The catecholamines adrenaline and

noradrenaline were shown to be without effect. The non-specific

opiate agonist D[Ala2-Met5]enkephalinamide (DAMEA), at 5 or 50 μg,

reversed the behavioral effect, with animals remaining immobile for

68% of the retest period (3).

On the basis of these findings we suggested that maintenance of

immobility was dependent on glucocorticoids and/or adrenal opioid

peptides, and that in vivo these mechanisms may have a coordinate

effect.

To verify the specificity of the opiate effect we administered

naloxone (0.1-10 mg/kg) to adrx rats with the DAMEA, and as a control

tested the effect of equivalent doses of naloxone on the dexamethasone

reversal. With increasing doses of naloxone there was a progressive

fall in immobility on retest in both DAMEA and DM treated animals. At

502
10 mg/mg naloxone, DAMEA treated animals were immobile for 31% and DM

treated animals for 36% of the retest period. We therefore tested

naloxone at similar doses in the intact animal. Over the dose range

used, naloxone lowered immobility equivalently in intact and in adrx

rats treated with DAMEA or DM; with 10 mg/kg intact animals were

immobile for only 36% of the 5 minute retest.

This inhibitory action of naloxone was seen only when it was given

within 1 hour of the initial swimming exposure. That the opioidergic

mechanisms involved in the maintenance of the immobility were within

the CNS was strongly indicated by the inability of MRZ2593, a

quaternary naloxone analogue which does not cross the blood-brain

barrier, to affect the swimming response (2).

On the basis of these studies, we proposed a model of maintenance of

immobility involving at least two opioidergic pathways, and that the

integrity of the second of these pathways is an absolute requirement

for the response to occur. In this model there are sensors for

peripheral opioids putatively outside the CNS, and sensors for adrenal

corticosteroids either central or peripheral. Both of these relay

onto a common effector which is itself central and opioidergic.

Activation of either pathway is sufficient to stimulate the common

effector and facilitate maintenance of the immobile response.

DAMEA is a relatively non-specific opiate agonist, and naloxone

though with a higher affinity for mu receptors than kappa or delta

receptors - is by definition a non-specific opiate receptor

antagonist. Accordingly we have undertaken studies to delineate the

receptor specificity of the opioidergic pathways involved in the

reversal of the effect of adrenalectomy. Equimolar doses of morphine

sulphate (mu-selective), [D-Ala2

-D-Leu5]enkephalin (delta-selective)

and ketocyclazocine, dynorphin 1-8, dynorphin 1-17 and the

heptapeptide [Met5]enkephalin [Arg6-Phe7], all kappa-selective

agonists, were administered to adrenalectomized rats and the

behavioral effect observed. Reversal of the effect of adrenalectomy

occurred with ketocyclazocine,

[Arg6-Phe7],

dynorphin 1-17 and [Met5]enkephalin

but not with 10-fold higher doses of the mu- or

delta-selective ligands. The reversal of the effect of adrenalectomy

by ketocyclazocine was antagonized by MR2266, a kappa-selective

partial agonist/predominant antagonist (1).

That it is the peripheral opioidergic pathway which is kappa-selective

was shown by studies in which equivalent doses of MR2266 were without

effect in intact rats, or in adrx rats treated with DM.

Further evidence for glucocorticoids and opioids of adrenal medullary

origin being involved in the maintenance of the immobile behavior was

shown when intact animals were treated with the anti-glucocorticoid

RU38486 (3.75 mg/rat) and/or the nicotinic antagonist mecamylamine (10

mg/kg) - Administered alone, they were without effect; administered

together immobility was significantly reduced, with animals remaining

immobile for 40% of the 5 minute retest period. Under these

condititons both adrenal medullary secretion and glucocorticoid action

is blocked. RU38486 and MR2266, simultaneously administered to the

intact animal, similarly impaired maintenance of the immobile

response.

503
FIGURE 3: Hypothetical model showing pathways for maintenance of

immobility.

Additional evidence in support of our hypothesis was the finding that

hypophysectomized animals given mecamylamine showed similar levels of

immobility on retest as found in adrenalectomized animals; MR2266

administered to hypophysectomized rats equivalently inhibited the

maintenance of immobility. These findings strongly support the

hypothesis that when animals are swum under conditions of moderate

stress, the immobile response which is acquired is maintained by

either glucocorticoids or adrenal medullary kappa-selective opioids,

with both of these endocrine effects being mediated through a final,

common, non-kappa selective opioidergic pathway.

REFERENCES

Jefferys, D., Boublik, J., and Funder, J.W. Eur J Pharmacol,

107:331-335. 1985.

Jefferys, D.; Copolov, D., and Funder, J.W. Eur J Pharmacol,

103:205-210, 1984.

Jefferys, D., Copolov, D., Irby, D., and Funder, J.W. Eur J Pharmacol,

92:99-103, 1983.

Porsolt, R.D., Le Pichon, M., and Jalfre, M. Nature, 266:730-732,

1977.

504
EFFECT OF CHRONIC EXERCISE ON ENDORPHIN RECEPTOR LEVELS IN RATS

Richard A. Houghten, Suzanne M. Pratt, Elizabeth A. Young,

Helen Brown and Dennis R. Spann

Department of Molecular Biology

Research Institute of Scripps Clinic

La Jolla, California 92037

ABSTRACT

A radioreceptor assay using [3H]2- endorphin (B-EP) was used to

examine binding in the brains of rats that had been exercised on a

treadmill for 5 months. The binding of [3H]2 EP to brain

homogenates gave an average Kd of 0.830 ± 0.055 nM for the controls

and 0.826 ± 0.022 nM for the runners, and respective Bmax's of 166 ±

20 pM and 159 ± 27 pM.

INTRODUCTION

There has been a great deal of press on the effects of "endorphins"

on "runners high", "exercise withdrawal syndrome", and "depression"

connected with cessation of running. It has been well documented

that exercise increases plasma endorphin levels in man (Colt et al.

1981 and Gambert et al. 1981) and that an up-regulation of opiate

receptors is known to occur when the opiate antagonist naltrexone is

chronically implanted in rats (Zukin et al. 1982). These facts

prompted us to examine the effect of chronic running on opiate

receptor behavior in rats. The results of this study are presented.

MATERIALS AND METHODS

Thirty six male Fisher 344 (Simonsen) rats weighing from 150-170 gms

were run on a treadmill. To habituate the rats to the treadmill they

were run for 3 weeks (10-20 min). Thirty of the most consistent

runners were randomly divided into 3 groups of 10, two groups were

designated as runners and one as the control. Dry ice was placed at

the back of each runway to keep the animals running. Electric shock,

initially used, was more stressful and injurous to the animals than

dry ice. The experimental groups were run daily for 22 weeks,

gradually working up to 1.4 km/hr for 60-70 min. The control group

was placed on the other half of the treadmill but not run. At the

end of the training period the animals were weighed, decapitated and

the following tissues were removed: brain, pituitary, soleus muscle

and plasma. The levels of endorphin in plasma and pituitary was

determined by RIA as described earlier (Watson et al. 1982). The

soleus muscles were assayed for the amount of citrate synthase

activity (Srere 1982) as an indicator of each rats conditioning level

(Lewis et al. 1980).

Radioreceptor assays (RRA) with [3H]2 EP were performed on brain

homogenate prepared as described previously (Ferrara et al. 1979).

The final homogenate was resuspended at a concentration of 10 mg

wet-weight of brain per ml of cold Tris buffer. Binding experiments

505
were performed on 0.666 ml aliquots of membranes (0.5mg protein).

Membranes were incubated with Tris buffer or unlabeled EP (500 nM)

and then with varying amounts of [3H]2 EP (0.3 - 3.0 nM) for 60 min

at 22°C then 10 min at 0°C in an ice water bath. The incubation was

terminated by rapid filtration. Receptor-ligand interaction was

examined by Scatchard analysis (Scatchard 19491. Results were

analyzed for significance by Student's T-test.

RESULTS AND DISCUSSION

The biochemistry of the changes which occur due to chronic

physiological conditions, such as aging, stress, excessive food

intake, habitual exercise and the major chronic functional psychoses

are poorly understood. Although ill defined, there appears to be a

relationship between the central and peripheral opioid peptides

and/or their receptors in many age related disorders.

While increases in endorphin levels in plasma drop to normal in

30-60 minutes after cessation of exercise (Gambert et al. 1981), any

changes in the receptor number for endorphin would be expected to

return to normal over a period of five to seven days (Zukin et al.

1982). This decrease in receptor number, with a concomittant

periodic increase in opioid peptide level could possibly explain the

"addiction" felt by runners, and may also be one factor in a number

of compulsive behavior patterns or the general phenomenon of

addiction. Very little work has been carried out which addresses the

possibility of change in the receptor systems in chronic

physiological states.

Our research began with a working hypothesis that receptor dynamics

occur for a variety of physiological changes, and very likely occur

differently for the various opiate receptors and brain areas. These

changes may therefore effect physiological/psychological variables

such as mood, sleep, sexual behaviors, etc. to different degrees at

different times.

A convenient natural means for inducing receptor changes was needed,

and since there has been much press on the effects of the endogenous

opioid peptides (usually lumped together as "endorphins") on

"runner's high" or "addiction to running", it was decided to examine

the possible changes in the receptor behavior in chronically

exercised versus sedentary rats. The rat has long been used as a

model for physical fitness studies (Harpur 1980). The RRA for

endorphin was selected because it has been used extensively in this

laboratory (Ferrera et al. 1979). The exercise regimen used, is

known to give substantial exercise conditioning to heart and leg

muscle in the rat (Baldwin et al. 1972). When the receptor assay for

endorphin was carried out using 9 runners and 9 sedentary controls

and their Scatchard plots examined, it was found that an apparent

small decrease of receptor number for the runners (Bmax = 159 pM

versus Bmax = 166 pM) had occurred with no change in affinity of

binding (Fig. 1). The differences were consistent throughout the

nine points of the Scatchard analysis, although the individual points

reached 95% significance by Student's T test in only three instances.

While the difference in receptor number was small, the decrease for

506
the runners is in the direction one might expect if an agonist

down-regulation were operative (Zukin et al. 1982). A loss of body

weight of approximately 10% was found for the running rats, the

effect of which will be examined in subsequent experiments.

Figure 1

Scatchard plot of [3H]2- endorphin binding to rat brain

homogenates of the running group (o) and the control group

(o). Each point represents the mean of values obtained from

nine animals.

Our results also indicated a substantial increase in B-endorphin

levels in plasma for runners versus controls, but no apparent

difference in levels in the whole pituitary (Table I). Since it is

known that endorphfn levels increase in some brain areas in rats

(Wardlaw and Frantz 19801, while decreasing in others (Gambert et al.

1980), the small decrease in receptor number found for the runners

versus the controls indicates that regional brain areas need to be

examined for both differences in receptor numbers and endorphin

levels. The difference in endorphin levels in anterior and

posterior pituitary versus whole pituitary also need to be examined.

These studies are in progress.

507
Table I

Plasma, EP levels in Pituitary, Citrate syntase in Soleus

and Body weight.

Runners Controls

Plasma EP (fmoles/ml) 14.5 ± 3.56 2.95 ± 0.55

Pituitary EP (pmoles) 1020 ± 85 865 ± 63

Soleus-citrate syntase

(μM/g/min)

13.04 ± 0.32 5.16 ± 0.14

Body weight (g) 311.0 ± 2.95 355.3 ± 2.60

Results are presented as means ± SEM.

LITERATURE CITED

Baldwin, K.M., Klfnkerfuss, G.H., Feyung, R.L., Mole, P.A., and

Holloszy J.O. Amer J Physiol, 222:373-378, 1972.

Colt, E.W., Wardlaw, S.L., and Frantz, A.G.

28:1637-1640, 1981.

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508
INTRATHECAL MORPHINE AND BLOOD PRESSURE IN PREECLAMPSIA

Carl E. Costas, Nabil W. Doss, Shaesta G. Humayun,

Jonas Gintautas, Adel R. Abadir and Barry J. Kraynack*

Anesthesiology Research Laboratories,

Brookdale Hospital Medical Center,

Brooklyn, N.Y. 11212, U.S.A.,

*Department of Anesthesiology,

South Carolina School of Medicine,

Columbia, S.C. 29203, U.S.A.

ABSTRACT

Ten term parturients with moderate-severe preeclampsia

received intrathecal morphine for labor analgesia.

Significant reductions in blood pressure and heart rate,

which were unrelated to analgesia, were observed.

INTRODUCTION

Preeclampsia is a serious disease of pregnancy which is

associated with proteinuria, hypertension and edema.

Toxemia is responsible for up to 20% of reported fetal

and maternal deaths (Bonica 1965). For toxemic

patients, the ideal technique of analgesia for labor and

delivery does not exist. Each method has significant

disadvantages (James and Wheeler 1982). In the case of

normal parturients, it has been demonstrated that prolonged analgesia (8-11 hours) for labor can be achieved

with intrathecal morphine (ITM) without any adverse

effects to mother or newborn. No change in maternal

heart rate or blood pressure was observed (Abboud et al.

1982, Baraka et al. 1981).

Anesthetic management of preeclamptic patients during

the labor and delivery period remains controversial. It

was of interest to determine whether ITM afforded any

advantages as an analgesic method for parturients with

severe toxemia.

MATERIALS AND METHODS

Ten parturients (average age, 20 years), greater than 34

weeks gestation, in early labor and undergoing oxytocin

augmenation of labor were studied. Moderate to severe

preeclampsia was defined by the following criteria: 1)

systolic blood pressure >160 mm Hg or diastolic blood

pressure >100-110 mm Hg; 2) proteinuria; 3) oliguria.

Treatment consisted of intravenous hydration and continuous infusion magnesium therapy (prior to intrathecal morphine). Each patient received 1.0 - 1.2 mg of

morphine sulfate (preservative free) in 2.0 cc of 10%

dextrose solution via a 25 gauge spinal needle at the

L2-3 interspace while in the sitting position. After

509
one minute, the patient was placed supine with the head

elevated at 30 degrees and with a left lateral tilt by

wedge. Oxygen (5 L/minute by face mask) was administered continuously. The patient's blood pressure

(systolic, diastolic, mean) and heart rate were monitored continuously using an Ohio 2100 Adult Noninvasive Blood Pressure Monitor.

Progress of labor was estimated by continuous external

cardiotocography and by hourly vaginal determination of

cervical dilation. Oxytocin augmentation was used in

each case. Pain level was evaluated by the patient

using the visual linear analog scale (Revill et al.

1976). Also, an investigator independently assessed the

intensity of pain and its relief, and time of onset

(Writer et al. 1981). These assessments were made

before ITM injection, every 15 minutes for one hour,

every 1/2 hour for the next seven hours and then every

hour until delivery. Maternal venous blood gases were

obtained before ITM injection and every 4 hours afterwards up to 24 hours. Maternal arterial blood gases

were determined at delivery. Fetal scalp pH determinations were obtained in one patient during labor. The

condition of the infants was evaluated by Apgar scores

at 1, 5, and 15 minutes, by measuring time to sustained

respirations (TSR) and determining umbilical venous

blood gases from doubly clamped cord at delivery.

Maternal vital signs were monitored for a total of 24

hours post-intrathecal injection. Anesthesia for delivery was provided by pudendal block (1% lidocaine).

One patient underwent caesarian section due to failure

to progress. Maternal side effects attributed to morphine were rated absent, mild or severe every 60

minutes.

RESULTS AND DISCUSSION

We observed that 1.0 and 1.2 mg of morphine intrathecally administered provided prolonged and complete

analgesia (up to 8 hours) during oxytocin stimulated

labor in moderate and severely preeclamptic patients.

The average diastolic blood pressure and mean arterial

blood pressure were reduced 22% and 15% respectively.

Heart rate averaged a 14% decrease. These autonomic

changes and pain relief occurred within 60 minutes of

ITM injection. The decrease in blood pressure and heart

rate occurred prior to the onset of oxytocin-induced

labor. Although magnesium sulfate was used, other

antihypertensive therapy was not required. Fetal and

maternal acid-base status remained normal during labor.

Time to sustained respirations for the infants was less

than 30 seconds. Apgar scores were greater than 8 at 5

minutes in all infants. In the post-partum period (up

to 30 hours) no significant adverse effects in mother or

newborn were noted. Maternal and neonatal blood gases

510
were normal at delivery. Maternal blood gases were

unchanged up to 24 hours post-partum. No respiratory

distress was observed. Several patients had loss of pin

prick and cold sensation over the neck, chest, abdomen,

legs and feet. Following ITM some patients complained

of nausea, vomiting and itching.

Although Baraka et al. (1981) and Abboud et al. (1982)

have demonstrated the efficacy of intrathecal morphine

as an analgesic during labor, it is still considered an

experimental technique. Dosage, as well as other

factors, remain to be determined. In this small series

of preeclamptic patients, we observed satisfactory

analgesia for labor (up to 8 hours) and up to 30 hours

post-cesarian section. No significant maternal or

fetal/neonatoal side effects were seen. More important

was our observation of a temporally related decrease in

diastolic and mean blood pressures and heart rate in

preeclamptic women during labor. This decrease was

unrelated to pain relief since induction with oxytocin

had not begun.

These findings suggest that ITM may be of therapeutic

benefit to toxemic patients. Abouleish et al. (1981)

observed moderate blood pressure decrease in monkeys

receiving ITM (0.07 mg/kg). No such changes have been

observed in normal human parturients. The mechanisms by

which blood pressure and heart rate are lowered by ITM

in preeclamptic patients remains speculative. Laubie et

al. (1977) noted that morphine-like agents reduced

sympathetic tone (lowered blood pressure and heart rate)

by an action on the CNS. The clinical significance of

these preliminary findings remains unknown, but appears

promising.

REFERENCES

Aboud, T.K., Shniaer, S.M., Dailey, P.A., Raya, J.,

Khou, S.S., Sarkis, S., DeSausa, B., Baysinger, C.L.,

and Miller, F. Anesthesiology, 57:A384, 1982.

Abouleish, E., Barmada, M.A., Nemota, E.M., Tung, A.,

and Winter, P. Br J Anaesth, 53:1027-1032, 1981.

Baraka, A., Noveihid, R., and Hajj, S. Anesthesiology,

54:136-140, 1981.

Bonica, J.J. Clin Anesth, 3:5-37, 1965.

James, F.M. and Wheeler, A.S., eds. Obstetric

Anesthesia: The Complicated Patient. Philadelphia,

F.A. Davis Co., 1982. pp. 372-378.

Laubie, M., Schmitt, H., Vincent, M., and Remund, G.

Eur J Pharmacol, 46:67-71, 1977.

Revill, S.J., Robinson, J.O., and Rosen, M. Anesthesia,

31:1191-1198, 1976.

Writer, W.D.R., James, F.M., and Wheeler, A.S.

Anesthesiology, 54:215-219, 1981.

511
CENTRALLY ADMINISTERED LEUMORPHIN POSSESSES POTENT DEPRESSOR ACTIVITY

IN CONSCIOUS RATS

Hiroshi Itoh1, Kazuwa Nakao1,2, Takayuki Yamada1

, Narito Morii1,

Shozo Shiono1, Makoto Sakamoto1, Akira Sugawara1

, Yoshiko Saito1,

Goro Katsuura3, Teruo Shiomi3, Masami Eigyo3,

Akira Matsushita3 and Hiroo Imura1

1

Second Division, Department of Medicine

Kyoto University School of Medicine, Kyoto 606, Japan,

2

Radioisotope Research Center

Kyoto University, Kyoto 606, Japan,

3

Shionogi Research Laboratories

Shionogi & Co., Ltd., Osaka 553, Japan.

ABSTRACT

The effect of intracerebroventricular (i.c.v.) administration of

leumorphin on basal blood pressure and angiotensin II (AII)-

stimulated increase in blood pressure was examined in conscious

unrestrained rats. The i.c.v. injection of leumorphin (0.06 and

0.6 nmol) elicited significant depressor effect. The i.c.v. administration of leumorphin with AI1 (0.1 nmol) significantly attenuated

the AII-induced pressor response and, furthermore, lowered blood

pressure below the basal level. These results suggest that leumorphin is involved in the central control of blood pressure.

INTRODUCTION

The possibility that opioid peptides are involved in the central

modulation of blood pressure homeostasis stems from the cardiosuppressive effects of opiate alkaloids and is strengthened by the

neuroanatomical findings that opioid peptides are distributed in

brain regions especially in the areas involved in cardiovascular

regulation (Khachaturian et al. 1985). Several experiments of

central administration of opioid peptides suggest the integral role

of opioids in blood pressure control, but no unanimous conclusion on

their effects has been drawn, since their effects largely depend upon

the state of consciousness of the experimental animals (Yukimura et

al. 1981) or the sites of administration (Feuerstein et al. 1982).

The possible existence of leumorphin with 29 amino acids was

predicted by the nucleotide sequence of preproenkephalin B (Kakidani

et al. 1982) and we demonstrated its existence in the pituitary and

brain using radioimmunoassay coupled with gel filtration and high

performance liquid chromatography (Nakao et al. 1983, Suda et al.

1984, Suda et al. 1985). We also reported that leumorphin is a

ligand (Suda et al. 1983) and possesses potent actions on consummatory behaviors in rats (Imura et al. 1986). In the present study,

we examined the effects of intracerebroventricular (i.c.v.) administration of leumorphin on blood pressure using conscious unrestrained

rats.

MATERIALS AND METHODS

Animals Thirty-seven male Slc: Wistar rats (220-270 g) were used. A

stainless steel cannula was stereotaxically implanted into the left

512
lateral ventricle 1-2 weeks before the experiments. One day before

the experiment, a polyethylene catheter was placed in the abdominal

aorta through the left femoral artery. For i.c.v. injection, the

peptide dissolved in isotonic saline or saline as a vehicle was

injected (5 μl) for 10 sec. Blood pressure (mean arterial pressure)

was monitored while the animals were conscious and freely moving in

their cage as previously reported (Itoh et al. 1986).

Experiment 1. Effect on basal blood pressure Twenty-one rats were

divided into 4 groups, and rats in each group received one of the

following administrations: saline, 6 nmol of leucine-enkephalin (Leuenkephalin), 0.06 nmol or 0.6 nmol of leumorphin after a 30-min

stabilization period. Blood pressure following the administration

was monitored for 60 min and compared with the basal level.

Experiment 2. Effect on angiotensin II (AII)-induced pressor response

Sixteen rats were divided into 3 groups and rats in each group

received 2 i.c.v. injections with a minimal interval of 100 min:

first, the administration of 0.1 nmol of AI1 alone (control), and

second, the simultaneous administration of AI1 (0.1 nmol) and of

saline (Group I), 0.06 nmol of leumorphin (Group II) or 0.6 nmol of

leumorphin (Group III) (treated). Pressor responses to AI1 were

compared between the first and second administrations.

Peptides AII and Leu-enkephalin were purchased from Protein Research

Foundation (Osaka, Japan) and human leumorphin was synthesized by a

conventional solid phase method.

Statistical analysis Results are expressed as means ± S.E. Corresponding values on the same animal were assessed by Student's paired

t-test and values among groups were compared by Duncan's test

following a one-way analysis of variance for multiple comparisons.

RESULTS

Experiment 1 The i.c.v. injection of leumorphin at the doses of

0.06 nmol and 0.6 nmol caused significant decreases in blood

pressure, while 6 nmol of Leu-enkephalin or saline had no apparent

effect on basal blood pressure (table 1). Two doses of leumorphin

manifested the same magnitude of maximal depressor effects but the

temporal profiles were different. The duration of significant

suppression of blood pressure was 2 min in 0.06 nmol of leumorphin

and 10 min in 0.6 nmol of leumorphin. There were no apparent changes

in heart rate (2.8 ± 3.5 % for Leu-enkephalin, 4.4 ± 0.6 % for 0.06-

nmol leumorphin, and 9.3 ± 2.2 for 0.6-nmol leumorphin).

Experiment 2 Hypotensive effects of leumorphin were more notable

when blood pressure was elevated by centrally administered AII. As

shown in table 2, pressor response to central AII was reduced by the

two doses of leumorphin by 60-70 %. Both doses of leumorphin even

lowered blood pressure below the basal level (-8.1 ± 3.1 mmHg for

0.06 nmol and -19.0 ± 1.8 mmHg for 0.6 nmol) and this depressor

effect persisted more than 20 min.

DISCUSSION

The present study has demonstrated potent suppressive effects of

i.c.v. administration of leumorphin on both basal blood pressure and

pressor response to centrally administered AII in conscious rats. In

our preliminary study, the depressor effects of leumorphin were

partially blocked by the preadministration of naloxone, indicating

513
TABLE 1. Effects of i.c.v. injection

of Leu-enkephalin and leumorphin on basal blood pressure

in conscious unrestrained rats

Treatment Baseline

Group (mmHg)

Maximal Change

(mmHg)

Saline 127.8 ± 2.6 0.9 ± 2.9

Leu-enkephalin 6 nmol 117.5 ± 4.4 2.5 ± 1.9

Leumorphin 0.06 nmol 116.8 ± 4.2 -6.5 ± 2.7†

Leumorphin 0.6 nmol 119.1 ± 3.5 -7.2 ± 1.7†

Values are the means ± S.E.

Significantly different from saline-control:

† = P<0.01, Duncan's test.

TABLE 2. Effect of i.c.v. injection of leumorphin

on blood pressure increase induced by i.c.v. administration

of angiotensin II in conscious unrestrained rats

Treatment

group

Baseline Maximal % reduction Recovery

(mmHg) response of maximal time

(mmHg) response(%)§

 (min)

Group I control 121.7 22.9 68.4

(Saline) ±3.0 ±2.7 2.2 ±9.9

treated 122.6 21.4 ±6.0 53.1

±2.6 ±1.6 ±6.4

Group II control 129.8 18.1 51.0

(Leumorphin ±4.5 ±1.9

0.06 nmol) treated 131.5 5.6

65.3 ±15.4

214.4†2.1

±4.6 ±2.0* ±1.2*

Group III control 120.8 24.0 58.6

(Leumorphin ±4.0 ±2.8 ±69.2 ±9.6

0.6 nmol) treated 125.2 7.8 ±4.9†

2.5

±4.0 ±2.2*** ±1.3**

Values are the means±S.E.

§: 100 x ((Maximal response) control - (Maximal response)

treated) / (Maximal response) control.

Significantly different from control: *=P<0.05, **=P<0.05,

***=P<0.001, Student's paired t-test.

Significantly different from Group I: †=P<0.01,

Duncan's test.

that the observed effects were mediated by opioid receptors. Little

is known about the role of ligand on blood pressure regulation. In

anesthetized rats, the microinjection of agonist, U50488H into the

nucleus tractus solitarius was reported to increase blood pressure

and decrease heart rate (Carter and Lightman 1985), while

dynorphin(1-13) microinjected into the hypothalamic periventricular

nucleus was found to cause hypotension and bradycardia (Feuerstein

and Faden 1982). Our results using conscious rats further indicate

the involvement of leumorphin, agonist, on the maintenance of

blood pressure. Sites of action or mechanisms of the depressor

514
effect of leumorphin cannot be ascertained from our present experiment since leumorphin administered intracerebroventricularly may

affect multiple opioid sensitive circumventricular sites (Atweh and

Kuhar 1977). However, we have observed the inhibition of vasopressin

secretion induced by centrally administered AII by i.c.v. injection

of leumorphin (Yamada et al. 1986). This finding together with our

observation that leumorphin is present in the hypothalamus (Suda et

al. 1985) suggests that the depressor effect of leumorphin is in part

mediated by the inhibitory modulation of vasopressin secretion, as

observed with other opioid peptides (Summy-Long et al. 1981).

The potent inhibitory effect of leumorphin on blood pressure demonstrated in this study and our previous works of its marked suppressive actions on water intake (Imura et al. 1985) and vasopressin

secretion (Yamada et al. 1986) imply the involvement of leumorphin in

body fluid and blood pressure homeostasis.

REFERENCES

Atweh, S.F., and Kuhar, M.J. Brain Res, 134:393-405, 1977.

Carter, D.A., and Lightman, S.L. J Physiol, 367:363-375, 1985.

Feuerstein, G., and Faden, A.I. Life Sci, 31:2197-2200, 1982.

Imura, H., Kato, Y., Nakai, Y., Nakao, K., Tanaka, I., Jingami, H.,

Koh, T., Yoshimasa, T., Tsukada, T., Suda, M., Sakamoto, M., Morii,

N., Takahashi, H., Tojo, K., and Sugawara, A. J Endocr, 107:147-

157, 1985.

Imura, H., Nakao, K., Yanaihara, N., Katsuura, G., and Nakamura, M.

Neuroendocrinology, in press, 1986.

Itoh, H., Nakao, K., Morii, N., Yamada, T., Shiono, S., Sakamoto, M.,

Sugawara, A., Saito, Y., Katsuura, G., Shiomi, T., Eigyo, M.,

Matsushita, A., and Imura, H. Brain Res Bull, in press, 1986.

Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y.,

Hirose, T., Asai, M., Inayama, S., Nakanishi, S., and Numa, S.

Nature, 298:245-249, 1982.

Khachaturian, H., Lewis, M.E., Schafer, M.K.H., and Watson, S.J.

Trends Neurosci, 8:111-119, 1985.

Nakao, K., Suda, M., Sakamoto, M., Yoshimasa, T., Morii, N., Ikeda,

Y., Yanaihara, C., Yanaihara, N., Numa, S., and Imura, H. Biochem

Biophys Res Commun, 117:695-701, 1983.

Suda, M., Nakao, K., Sakamoto, M., Yoshimasa, T., Morii, N.,

Yanaihara, N., Numa, S., and Imura, H. Biochem Biophys Res Commun,

123:148-153, 1984.

Suda, M., Nakao, K., Sakamoto, M., Yoshimasa, T., Morii, N.,

Yanaihara, N., Numa, S., and Imura, H. Neuropeptides, 5:461-464,

1985.

Suda, M., Nakao, K., Yoshimasa, T., Ikeda, Y., Sakamoto, M.,

Yanaihara, N., Numa, S., and Imura, H. Life Sci, 32:2769-2775,

1983.

Summy-Long, J.K., Keil, L.C., Deen, K., Rosella, L., and Severs, W.B.

J Pharmacol Exp Ther, 217:619-629, 1981.

Yamada, T., Nakao, K., Itoh, H., Morii, N., Shiono, S., Sakamoto, M.,

Sugawara, A., Saito, Y., Katsuura, G., Eigyo, M., Matsushita, A.,

and Imura, H. The proceeding of the 17th Annual Meeting of INRC,

1986.

Yukimura, T., Unger, T., Rascher, W., Lang, R.E., and Ganten, D.

Clinical Science, 61:347S-350S, 1981.

515
RELATIONSHIP BETWEEN DIHYDROETORPHINE AND CHOLINERGIC

SYSTEM IN THE INHIBITION OF RESPIRATION AND HEART RATE

C.F. Bian, S.H. Xing and W. Qin

Department of Pharmacology

Xuzhou Medical College

Xuzhou, Jiangsu 221000, China

ABSTRACT

The respiratory inhibition caused by dihydroetorpine (DHE) was fully

antagonized by pilocarpine or physostigmine, but the bradycardia was

not. When atropine (icv) was combined with DHE, the respiratory

inhibition was antagonized but the bradycardia persisted. However,

intravenous atropine antagonized both the respiratory and cardiac

inhibition caused by DHE.

Key Words : dihydroetorpine, atropine, pilocarpine, physostigmine,

respiration, heart rate

INTRODUCTION

Dihydroetorpine (DHE) is a potent analgesic agent. When given

intravenously, it causes significant respiratory and cardiac

inhibition. B-7601, a new muscarinic blockade, antagonizes the

bradycardia after DHE (Bian et al. 1985). Bethanechol, a

cholinomimetic, acts on the respiratory center, causing initial

excitation and subsequent inhibition, of which the latter can be

antagonized by atropine, but not the former (Gonsalves and Borison

1980). Considering that opioids decrease the release of Ach and

thus depress respiration (Pepeu 1973), we studied the effects of

DHE, atropine and the cholinomimetics on respiration and heart rate

to explore the relationship between DHE's inhibitory effects on

respiration, heart and cholinergic system.

MATERIALS AND METHODS

Hybrid rabbits were used; body weight was 1.8-2.5 kg and six groups

with 5-7 in each group.

Pilocarpine hydrochloride and physostigmine salicylate were

purchased from the Medical Supply Station of Shanghai, and atropine

sulfate from the Chengdou First Pharmaceutical Factory. DHE was

given by the Academy of Military Medical Sciences of China.

Respiration and ECG were recorded with RM-86 polygraph. The tidal

volume was estimated according to the amplitude of respiration.

516
RESULTS AND DISCUSSION

Effects on respiration:

DHE (0.5 μg/kg, iw) inhibited rabbits’ respiration, the effect being

most apparent 5-10 min after injection. The frequency was decreased

by 70±12% or more (mean ± SD, p<0.01) with no significant change in

tidal volume and the change lasted over 40 min.

Pilocarpine (2.5 mg/kg, icv) accelerated respiration from 53±14 to

94±21 times/min (p<0.01) 10 min after its administration without

significant change in tidal volume. When DHE was given at this

point, respiration became slower but still more rapid than the

control. After physostigmine (0.3 mg/kg, iv), the respiration

increased markedly by 10 min (from 59±9 to 159±48 times/min,

p<0.01); the tidal volume was decreased from 56±29 to 33±27 ml, but

the ventilation volume remained higher than the control. When DHE

was then given, the frequency became lower (still higher than the

control) and the tidal volume matched that of the control (fig.

1). These results suggest both pilocarpine icv and physostigmine iv

stimulate the respiratory center and antagonize the respiratory

inhibition of DHE. This may be related to the possibility that DHE

could resemble morphine in that it agonizes opiate receptors and

retards the release of Ach (Pepeu 1973).

Atropine (0.1 mg/kg, icv) increased the frequency of respiration

from 51±11 to 75±37 times/min (p>0.05) 5 min after injection, with

no significant change in tidal volume. Five minutes after or before

DHE, atropine antagonized the inhibitory effect of DHE on

respiration, the changes being significant (p<0.01 and p<0.05

respectively). When a bigger dose of atropine (1 mg/kg) was given

intravenously, its antagonistic effect also appeared, but rather

slowly (30 min after injection). It is clear that atropine and DHE

are antagonistic in modifying respiration. Gonselves demonstrated

that bethanechol first stimulates and then inhibits respiration, and

proposed the possibility that there are 2 subtypes of muscarinic

cholinoreceptors in respiratory center, excitatory M1 and inhibitory

M2, of which atropine blocks the latter selectively (Gonsalves and

Borison 1980). We postulate that Ach is the excitatory transmitter

for the respiratory center, while DHE agonizes opiate receptor,

depressing the release of Ach in the CNS (Bian et al. 1985) and

blocks M2 receptor, it antagonizes the inhibitory effect of DHE on

respiration.

517
TABLE 1. Antagonist effect of atropine on respiratory

inhibition of DHE in rabbits (n-6 in each group)

Group Change of respiration (%, mean±SD)

minutes after DHE

A -70±12 -72± 11 -71±10 -65±10 -62±l2

B 51±77 46±55 36±54 40±40 37±44

C -55±ll -57±17 -49±5** -38±10 -29±8

D -54±12 -53±12 -4O±20* -30±34 -23±8**

E -60±16 -61±l5 -58±9 -50±11 -39±19**

compared with group A: *p<0.05; **p<0.01

A: DHE 0.5 μg/kg, iv. B: Atropine 0.1 mg/kg, icv;

C: Atropine 0.1 mg/kg, icv 5 min after DHE iv;

D: Atropine 0.1 mg/kg, icv 5 min before DHE iv;

E: Atropine 1 mg/kg, iv 10 min after DHE iv.

Effects on heart rate:

DHE (0.5 μg/kg, iv) as well as pilocarpine (2.5 mg/kg, icv)

decreased the heart rate of rabbits from 262±26 to 187±32 beats/min

and from 268±27 to 182±12 beats/min respectively (p<0.01).

Subsequent DHE following pilocarpine further decreased the heart

rate to 167±14 beats/min, showing no significant alteration (fig

2.). The bradycardia faded gradually and the heart rate remained

slower than the control at 40 min. Physostigmine alone did not

decrease heart rate significantly (p>0.05), nor did it affect the

bradycardia following DHE significantly. The bradycardia following

pilocarpine icv is secondary to the raised blood pressure, which is

induced when pilocarpine agonizes the central M receptor, and

therefore is reflex in nature (Henry and Rachel 1982).

Atropine (0.1 mg/kg, icv) made the heart rate decrease from 276±29

to 227±4 beats/min (p<0.05). Its addition after or before DHE

showed no remarkable interaction between them. However, intravenous

atropine (1 mg/kg) antagonized DHE’s bradycardia almost

completely. In view of the fact that the bradycardia of DHE was

abated by atropine iv, but not icv, it may be inferred that atropine

blocks the M receptors in the cardiac muscles and thus nullifies the

elevated vagal tone over the heart, which is elicited by DHE through

its stimulation of the μ or receptor in the cardiovascular center

(Feldberg and Wei 1978; Xia 1985).

518
Figure 1. Antagonist effect of

pilocarpine and physostigmine on

respiratory inhibition by DHE in

rabbits (n=6). DHE

0.5μg/kg,iv; pilocarpine

2.5 mg/kg,icv;

physostigmine 0.3 mg/kg, iv.

* pilocarpine or physostigmine

Figure 2. Effect of DHE

and pilocarpine (pill on

heart rate in rabbits

(each group=6)

REFERENCES

Bian, C.F., Xing, S.H., Xu, P.C. and Wang, Y. National Medical J of

China, 65(9):545, 1985.

Bian, C.F., Ye, M., Xing, S.H., Teng, Y.D. and Xu, P.C. Acta

Pharmacologica Sinica, 6(3):149-151 1985.

Feldberg, W. and Wei, E. J Physiol (London), 275:57p, 1978.

Gonsalves, S.F. andf Borison, H.L. J Pharmacol Exp Ther, 214:297-

305, 1980.

Henry, E.B. and Rachel, G. Ann Rev Pharmacol Toxicol, 22:341-381,

1982.

Pepeu, G. Progress in Neurobiology, 2(3):259-288, 1973.

Xia, Y. Progress in Physiological Sciences, 16(1):27-31, 1985.

519
THE ROLE OF μ- AND OPIOID RECEPTORS ON THE

INTESTINAL PROPULSION IN RATS

M. Sbacchi, A. La Regina, P. Petrillo, A. Tavani

Istituto di Ricerche Farmacologiche "Mario Negri"

Via Eritrea 62 - 20157 Milan, Italy

ABSTRACT

The agonist effects on intestinal transit of relatively selective

μ- and ligands, administered intraperitoneally, and their antagonism

by the preferentially μ- and antagonists naloxone and ICI 174,864

were studied in rats 5 min after a charcoal meal. The dose-response

curves of the preferential μ- ligands morphine and [D-Ala2, MePhe4,

Gly-ol5] enkephalin (DAGO) were shifted by naloxone at low doses but

not by ICI 174,864. The preferential peptide [D-Pen2, D-Pen5]

enkephalin (DPDPE) had no significant agonist activity. [D-Ala',

D-Leu5] enkephalin [DADLE) induced dose-related effects that were

weakly antagonized by ICI 174,864 and partly by low-dose naloxone.

Thus the inhibition of intestinal transit induced by opioids may

depend mainly on the interaction of the agonists at the u-receptors,

while the receptors may play only a secondary role. DADLE agonist

effects probably depend on interaction at μ-, and at non-opioid

receptors.

INTRODUCTION

Inhibition of intestinal transit in rats is a well-known opioid

effect currently attributed to local and CNS-mediated mechanisms

(Manara et al. in press). It has been reported that the CNS-mediated

antipropulsive activity of opioids depends on their interaction at

u-receptors or at μ- and receptors when respectively supraspinal

or only spinal mediations are involved (Porreca and Burks 1983;

Galligan et al. 1984; Porreca et al. 1984).

To clarify the contributions of opioid receptor types in the local

component of opioid-induced inhibition of intestinal transit, we

showed that k-receptors probably play no major role (Gambino et al.

1983; Petrillo et al. 1984; Tavani et al. 1984; Petrillo et al.

1985). In this study, in order to determine the contribution of μand receptors, we investigated the effects on intestinal propulsion

of relatively selective μ- and agonists and antagonists after intraperitoneal injection.

MATERIALS AND METHODS

Overnight fasted male CD-COBS rats (Charles River, Italy) 180-200 g,

were tested for intestinal transit (Manara et al. in press). Five min

after a charcoal meal (Tavani et al. 1980) rats were killed and the

520
distance travelled by the charcoal meal was recorded as a percentage

of the total length of the small intestine (% intestinal transit).

Drugs and peptides were dissolved in distilled water and injected

intraperitoneally (i.p.). The following drugs and peptides were used:

morphine HCl (Salars, Italy), naloxone HCl (gift from Endo Laboratories, USA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI l74,864)(gift

from Dr. J.S. Shaw, ICI, U.K.), [D-Pen2, D-Pen5] enkephalin (peninsula, USA), [D-Ala2

, MePhe4, Gly-ol5

] enkephalin (DAGO) and [D-Ala2,

D-Leu5] enkephalin (DADLE)(Bachem, Switzerland).

RESULTS AND DISCUSSION

The two relatively selective u-agonists morphine (Magnan et al. 1982)

and DAGO (Handa et al. 1981) injected i.p. into rats at the peak

time or a plateau of the effect (5 and 1 min before charcoal respectively, data not shown) induced dose-related inhibition of intestinal

transit, fitting a straight regression line (Fig. 1). The doses

reducing transit to 50% that of drug-free controls (ED50) were about

15.2 and 6.2 μg/kg for morphine and DAGO respectively. Pretreatment

with the preferential p-antagonist naloxone (Magnan et al. 1982)(50

μg/kg i.p. 6 min before charcoal) shifted the dose-response curves

to the right and the ED50 were 239 μg/kg for morphine and 153 μg/kg

for DAGO. The relatively selective antagonist ICI 174,864 (Cotton

et al. 1984; Corbett et al. 1984) at the dose of 1 mg/kg 6 min

before charcoal, did not inhibit intestinal transit (110% of controls)

and did not significantly shift the dose-response curves of the two

agonists. This leads us to conclude that both morphine and DAGO i.p.

inhibited intestinal transit in rats mainly through the u-type opioid

receptor.

The relatively selective agonist DPDPE (Mosberg et al. 1983,

Corbett et al. 1984) at doses from 60 to 2000 μg/kg i.p. 5 min

before charcoal did not reduce intestinal transit dose-dependently

(Fig. 1). A very slight agonist effect was seen at a few doses but

there was never any significant difference from controls. No stronger

agonist effects were observed when DPDPE was injected 1 or 10 min

before the meal (data not shown). Metabolic inactivation of the

peptide in our conditions cannot be excluded, although it is unlikely

as DPDPE is reported to be stable in the hamster vas deferens prepation (McKnight et al. 1985).

Conversely DADLE, which has been reported to act at sites with

cross reactivity at μ- sites (Magnan et al. 1982), injected i.p.

5 min before the meal, induced a dose-related inhibition of intestinal

transit with an ED50 of about 23 μg/kg (Fig. 1). When 50 μg/kg of

naloxone was injected 6 min before the meal, DADLE's effect was partly

antagonized, but with no dose relation. A dose of 5 mg/kg of naloxone

was necessary to antagonize the effect of 80 μg/kg of DADLE from 41%

to 69% of control value. In the presence of 1 mg/kg of ICI 174,864

i.p. 6 min before charcoal the dose-response curve of DADLE showed a

521
group. All curves except naloxone + DADLE and DPDPE significantly fitted a straight line. Comparison of regression lines by analysis of variance showed that morphine vs ICI + morphine and DAGO vs ICI + DAGO were the same,

whereas DADLE vs ICI + DADLE (p<0.05), morphine vs naloxone + morphine and DAGO vs naloxone + DAGO (p<0.01).

Fig. 1 -

Overnight fasted rats were injected i.p. with water naloxone

Dose-related inhibition of the intestinal transit induced by morphine, DAGO, DPDPE and DADLE.

min before a charcoal meal, with morphine, DPDPE or DADLE 5 min before or with DAGO 1 min before the meal. Con-

(50 μg/kg) or ICI 174,864 (1 mg/kg) 6

trols received water. % intestinal transit was measured 5 min after the meal. Mean and S.E. of 5-8 rats per
very slight but statistically significant shift to the right and the

ED50 was 38.5 μg/kg. Because of the low solubility the highest dose

of ICI 174,864 in our conditions was 4 mg/kg i.p. This dose itself had

no antipropulsive effect and partly prevented the antipropulsive effects of 40 μg/kg i.p. of DADLE, reducing the inhibiting effects of

the agonist from 41% to 64% of control value. The very weak antagonism

of ICI 174,864 on DADLE-induced constipation and the slight tendency

of some doses of DPDPE to inhibit intestinal transit suggest that the

d-receptor probably plays a secondary role in the antipropulsive action

of i.p. opioids. Thus DADLE's effect might also depend on its interaction at u-sites and probably at some other non-opioid sites not

easily displaceable by opioid-antagonists.

In conclusion, the inhibition of intestinal transit in rats induced by

i.p. opioids appears to depend mainly on the agonists' interactions

at the p-receptor, while the receptor seems to play only a secondary

role. However more selective compounds are needed to determine the

exact role of μ- and receptors in locally elicited intestinal transit

inhibition.

REFERENCES

Corbett, A.D., Gillan, M.G.C., Kosterlitz, H.W., McKnight, A.T.,

Paterson, S.J., and Robson, L.E. Br J Pharmacol, 83:271-279, 1984.

Cotton, R., Giles, M.G., Miller, L., Shaw, J.S., and Timms, D. Eur J

Pharmacol, 97:331-332, 1984.

Galligan, J.J., Mosberg, H. I., Hurst, R., Hruby, V.J., and Burks, T.F.

J Pharmacol Exp Ther, 229:641-646, 1984.

Gambino, M.C., Petrillo, P., and Tavani, A. Life Sci, 33 suppl.1:

461-464, 1983.

Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J., and

Smith, C.F.C. Eur J Pharmacol, 70:531-540, 1981.

Magnan, J., Paterson, S.J., Tavani, A., and Kosterlitz, H.W. Naunyn

Schmiedebergs Arch Pharmacol, 319:197-205, 1982.

Manara, L., Bianchi, G., Ferretti, P., and Tavani, A. J Pharmacol

Exp Ther, in press.

McKnight, A.T., Corbett, A.D., Marcoli, M., and Kosterlitz, H.W.

Neuropharmacology, 24: 1011-1017, 1985.

Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I.,

Galligan, J.J., and Burks, T.F. Proc Natl Acad Sci USA, 80:

5871-5874, 1983.

Petrillo, P., Amato, M., and Tavani, A. Neuropeptides, 5:403-406, 1985.

Petrillo, P., Gambino, M.C., and Tavani, A. Life Sci, 35:917-927, 1984.

Porreca, F., and Burks, T.F. J Pharmacol Exp Ther, 227:22-27, 1983.

Porreca, F., Mosberg, H. I., Hurst, R., Hruby, V.J., and Burks, T.F.

J Pharmacol Exp Ther, 230: 341-348, 1984.

Tavani, A., Bianchi, G., Ferretti, P., and Manara, L. Life Sci, 27:

2211-2217, 1980.

Tavani, A., Gambino, M.C., and Petrillo, P., J Pharm Pharmacol, 36:

343-344, 1984.

523

172-683 O - 87 - 18 : QL 3
INTRATHECAL DYNORPHIN A (1-13) AND (3-13) REDUCE SPINAL CORD

BLOOD FLOW BY NON-OPIOID MECHANISMS

Joseph B. Long, Richard C. Kinney*, Diana S. Malcolm,

Geoffrey M. Graeber* and John W. Holaday

Neuropharmacology Br., Dept. of Medical Neurosciences

Div. of Neuropsychiatry, *Div. of Surgery,

Walter Reed Army Inst. of Res., Washington, D.C. 20307-5100, U.S.A.

ABSTRACT

Dynorphin A (Dyn A)-related peptides have been implicated in the

pathophysiology of spinal cord injury in part because their intrathecal (i.t.)

injection causes hindlimb paralysis. The effects of paralytic doses of i.t. Dyn

A (1-13) and Dyn A (3-13) on spinal cord blood flow and cardiac output were

examined in rats using radiolabeled microspheres. Both Dyn A (1-13) and Dyn

A (3-13) significantly reduced blood flow in lumbosacral spinal cord without

altering cardiac output. Pretreatment with naloxone failed to block these

reductions in blood flow. Thus, the paralytic effects of Dyn A may result

from non-opioid actions of Dyn A to reduce spinal cord perfusion.

INTRODUCTION

Endogenous opioids have been implicated in the pathophysiology of spinal cord

injury based on evidence that the opioid antagonist naloxone improves spinal

cord blood flow (Faden et al. 1981a; Young et al. 1981) and neurological

function (Faden et al. 1981b) following experimental spinal trauma. Among

the opioids found in spinal cord, dynorphin A (Dyn A)-related peptides have

been specifically implicated as potential mediators in the pathogenesis of

spinal cord injury for several reasons: 1) trauma-induced loss of motor

function is reduced following treatment with the relatively selective opioid

receptor antagonist WIN44,441-3 (Faden et al. 1986) suggesting involvement

of an endogenous ligand such as Dyn A; 2) in contrast to other opioids, Dyn

A immunoreactivity progressively increases at the injury site following

trauma (Faden et al. 1985); and 3) Dyn A-related peptides are unique among

opioids in producing a flaccid hindlimb paralysis following intrathecal (i.t.)

injection in rats (Przewlocki et al. 1983; Faden and Jacobs 1984; Herman and

Goldstein 1985; Long et al. 1985). Dyn A may promote injury mechanisms by

altering spinal cord blood flow following trauma, and in turn, the therapeutic

actions of naloxone may result from protection against Dyn A contributions to

post-traumatic ischemia. Alternatively, the paralytic effects of i.t. Dyn A

have been shown not to involve actions at opioid receptors (Faden and Jacobs

1984; Herman and Goldstein 1985; Long et al. 1985). Consequently,

deleterious actions of Dyn A in injured spinal cord may result from the nonopioid actions of this peptide. Therefore, the present studies were conducted

to determine 1) if i.t. Dyn A reduces spinal cord blood flow, and 2) whether

Dyn A-induced blood flow alterations involve opioid or non-opioid

mechanisms. Using radiolabeled microspheres, we examined the effects of

Dyn A (1-13) and the non-opioid fragment Dyn A (3-13) on spinal cord blood

flow. Additionally, we evaluated the ability of naloxone to block these

actions of Dyn A (1-13).

524
MATERIALS AND METHODS

Male Sprague Dawley rats (300-350g) were anesthetized with ketamine

hydrochloride (100 mg/kg, i.m.). Saline-filled polyethylene catheters (PE IO)

were introduced into the spinal subarachnoid space through the atlantooccipital membrane and advanced to the level of the lumbar enlargement (8.5

cm). On the following day, rats were again anesthetized with ketamine (100

mg/kg, i.m.). A PE 50 catheter was advanced through the carotid artery into

the left ventricle of the heart for injection of microspheres, and a PE 50

catheter was inserted into the tail artery for mean arterial pressure

recordings and reference blood sampling.

Approximately one hour following catheterization, a 0.5 ml microsphere

suspension containing 150,000 to 250,000 microspheres labeled with either

141Ce or 86Sr was injected over a 15-20 second period, followed immediately

by a 0.5 ml heparinized saline flush delivered over 15-20 seconds. Reference

blood withdrawal was begun IO seconds prior to microsphere injection and was

continued for a total of 75 seconds (withdrawal rate of 1.0 ml/min). Two

microsphere injections (20 min preceding and 10 min following i.t. injections)

provided cardiac output (CO) and blood flow data. lntrathecal injections of

Dyn A (1-13) (25 nnmoles), Dyn A (3-13) (50 nmoles), or saline were made in a

total volume of 18 μl (including catheter flush). Following the second

microsphere injection, brains and cervical, thoracic, and lumbosacral portions

of the spinal cord were removed and weighed, and the activity of each isotope

was measured in a gamma counter. Cardiac output (ml min-1) was calculated

as total injected radioactivity times withdrawal

radioactivity in the reference blood sample.

rate divided by the

Tissue blood flow (ml min-1

100g-1) was calculated as tissue radioactivity times withdrawal rate divided

by reference blood radioactivity.

Differences between post- and pre-treatment measurements in each rat were

evaluated by analysis of variance. Significant differences among treatment

group means were determined by the least significant difference test.

RESULTS AND DISCUSSION

Dyn A (1-13) significantly reduced blood flow to the lumbosacral spinal cord

(49-66%) without altering cardiac output or blood flows to the brain or other

spinal cord regions. These reductions in spinal cord perfusion were limited to

those regions where direct exposure to the intrathecally injected Dyn A would

be expected on the basis of the measured distribution of 125I-BSA in the

spinal cord following its i.t. injection (results not shown). Naloxone

pretreatment 10 minutes prior to injection of Dyn A (1-13) failed to block the

reductions in lumbosacral blood flow. Additionally, lumbosacral blood flow

was significantly reduced by the non-opioid fragment Dyn A (3-13). Thus, the

Dyn A effects on spinal cord perfusion appear to involve non-opioid

mechanisms. Similarly, the dose-related hindlimb paralytic actions of Dyn A

(1-13) are not blocked by naloxone, and are shared by Dyn A (3-13)

(Przewlocki et al. 1983; Faden and Jacobs 1984; Herman and Goldstein 1985;

Long et al. 1985). Consequently, the paralytic actions of Dyn A may result

from non-opioid actions of Dyn A to reduce spinal cord perfusion. Assuming

that intrathecally injected Dyn A (1-13) mimics the actions of endogenous Dyn

A in injured spinal cord, it would appear likely that the therapeutic actions of

the opioid antagonist naloxone in spinal cord injury (Faden et al. 1981a; Faden

et al. 1981b; Young et al. 1981) do not result from protection against Dyn Ainduced ischemia, since these effects appear to involve non-opioid

mechanisms.

525
TABLE I. (A) Cardiac outputs’ (CO) and (B) blood flows2

 in brain,

cervical, thoracic, and lumbosacral spinal cord 20 min preceding (Pre) and

10 min following (Post) i.t injections of Dyn A (1-13) (25 nmoles), Dyn A

(3-13) (50 nmoles), or saline. Rats treated with naloxone (10 mg/kg, s.c.)

were injected 10 minutes prior to i.t. administration of either saline or

Dyn A (1-13) (25 nmoles). Data are presented Ias means ± S.E.M. for

groups of 8 rats. * p< 0.05, 1

ml min-1, 

2

ml min-1 100g-1

TREATMENT

SAL Dyn (1-13) Dyn(3-13) Nal/Sal Nal/Dyn

187±1.5 204±12 197±29 200±26

149±11 160±17 203±25 193±26

Brain

Pre 211±35 245±30 280±35 208±25 287±25

Post 194±23 207±18 217±26 203±28 212±19

Cervical

Pre 96±7 108±11 113± 11 107±15 118±11

Post 104±10 103±8 112±12 106±12 95±9

Thoracic

Pre 76±4 96±11 95±7 82±8 92±5

Post 80±10 83±5 79±8 83±7 57±7*

Lumbosacral

Pre 75±4 100±8 109±10 91±9 102±8

Post 82±10 51±12* 61±13* 100±14 34±11*

REFERENCES

A. CARDIAC OUTPUT

Pre 179±13

Post 176±30

B. BLOOD FLOW

Faden, A.I., Jacobs, T.P., Mougey, E.H., and Holaday, J.W. Ann Neurol,

10:326-332, 1981 a.

Faden, A.I., Jacobs, T.P., and Holaday, J.W. Science, 211:493-494, 1981b.

Faden, A.I., and Jacobs, T.P. Eur J Pharmacol, 91:321-324, 1984.

Faden, A.I., Molineaux, C.J., Rosenberger, J.C., and Cox, B.M. Ann Neurol,

17:386-390, 1985.

Faden, A.I., Knoblach, S., Mays, C., and Jacobs, T.P. Peptides, 6, Suppl 1:15-

17, 1985.

Herman, B.H., and Goldstein, A. J Pharmacol and Exp Ther, 232:27-32, 1985.

Long, J.B., Echevarria, E.E., Mobley, W.C., and Holaday, J.W. Alcohol and

Drug Res, 6:136, 1985/86.

Przewlocki, R., Shearman, C.T., and Herz, A. Neuropeptides, 3:233-240,

1983.

Young, W., Flamm, E.S., Demopoulos, H.B., Tomasula, J.J., and DeCrescito,

V. J Neurosurg, 55:209-219, 1981.

526
ENDOGENOUS OPIOIDS, OPIATE RECEPTORS AND TRAUMATIC BRAIN INJURY

Tracy K. McIntosh, Susan Fernyak and Alan I. Faden

Center for Neural Injury

Department of Neurology

University of California, San Francisco

Veterans Administration Medical Center

San Francisco, California 94121, U.S.A.

ABSTRACT

The present study examined the role of endogenous opioid peptides in

the pathophysiological sequelae of fluid percussion head injury in

the cat. Two hours following injury, tissue concentrations of

dynorphin-like immunoreactive material (ir-Dyn) were significantly

elevated in specific brain regions where injury, as evidenced by

histological examination, was most severe. Changes in ir-Dyn but not

beta-endorphin-like immunoreactive material (ir-End) were significantly correlated with a fall in regional cerebral blood flow (CBF)

that occurred 2 h following injury. Administration of the opiate

antagonist WIN44,441-3 (with enhanced activity at kappa-receptors)

stereospecifically increased cerebral blood flow to the injured

regions.

INTRODUCTION

Endogenous opioids have been implicated as secondary pathophysiological factors in shock and trauma (Faden 1984a,b). Traumatic injury

to the central nervous system (CNS) may cause functional deficits

through the release of endogenous factors, including endogenous

opioids. We have provided evidence that the kappa-receptor/dynorphin

opioid system may play a pathophysiological role in traumatic spinal

cord injury (Faden et al. 1983; 1985a,b). Although the non-selective

opiate antagonist naloxone has been found to improve several physialogical variables (mean arterial pressure, brain perfusion pressure)

following fluid percussion head injury in the cat (Hayes et al.

1984), there is no information available regarding the role of

specific opioid peptides in traumatic brain injury or of the effects

of opiate antagonists on CBF. In the present study, we utilized a

fluid percussion device to induce traumatic head injury in the

anesthetized cat and measured regional concentrations of ir-Dyn and

ir-End by radioimmunoassay at 2 h after injury. Beta-endorphin was

chosen as a comparison neuropeptide because: (1) it is derived from

a different prohormone system (Li 1981); (2) it has a different

distribution in the brain from that of dynorphin (Khachaturian

et al. 1985); and (3) it has been implicated in the pathophysiolagy

of shock and trauma (Faden 1984a,b). Changes in the regional

concentration of opioid peptides were compared with changes in

regional CBF at 2 h following injury in order to gain insight into

the possible involvement of endogenaus opioid systems in the pathophysiological sequelae of traumatic brain injury. We also examined

the effectiveness of the opiate-receptor antaganist WIN44,441-3 with

its dextrostereoisomer WIN44,441-2 (which is inactive at the opiate

receptor) on local CBF after traumatic brain injury.

527
MATERIALS AND METHODS

Male and female cats (2.75 - 3.25 kg) were anesthetized with sodium

pentobarbital (30 mg/kg). Animals were then intubated, paralyzed

with pancuronium (0.6 mg/kg) and artificially ventilated with

70% N2O/30% 02. A femoral cutdown was performed far placement of

venous (drug administratian) and arterial (blood gas) catheters.

A catheter was also inserted into the left atrium far administration

of radiolabelled microspheres. A hallow injury screw was rigidly

fixed to the skull aver an 8 mm craniectomy centered over the

sagittal suture midway between lambda and bregma. Head injury was

induced by infusing a rapid epidural saline bolus, produced by the

fluid percussion device, through the central injury screw. At 2 h

past-injury (3.2 - 3.6 atmospheres [atm]), one group of animals

(n=6/group) were sacrificed, brains removed, dissected and assayed

far regional concentrations of endogenous opioids using radioinmunoassays as previously described (Ghazarossian et al. 1980; McIntosh

et a?. 1985; Molineaux et al. 1982). Antisera for radioimnunoassay

was generously supplied by Drs. A. Goldstein (dynorphin) and

G. Mueller (beta-endorphin). A second group of animals (n=5/group)

were assigned to treatment with an intravenous bolus of either

WIN44,441-3 (-) (0.2 mg/kg) or its dextrostereoisomer WIN44:441-2 (+)

(0.2 mg/kg) administered 1.5 min following fluid percussion injury

(3.2 -3.6 atm). Sequential measurement of regional CBF using

intra-atria1 injections of radiolabelled microspheres as previously

described (McIntosh et al. 1986) was performed 10 min prior to drug

administration, 30 min, 1 h and 2 h following drug treatment. At 2 h

following injury, animals were sacrificed for CBF measurements.

RESULTS AND DISCUSSION

Tissue concentration of ir-Dyn were significantly elevated in the

caudate nucleus (p < 0.05), frontal cortex (p < 0.05) and medulla

TABLE 1

Endogenous opioid response 2 h after head injury.

Dynorphin Beta-endorphin

(fmol/mg protein) (fmol/mg protein)

Baseline Post-injury Baseline Past-injury

Anterior Pituitary 4917 ± 704 1043 ± 539** 6938 ± 947 3393 ± 1185

Frontal Cortex 10 ± 1 17 ± 2* 13 ± 6 24 ± 16

Caudate Nucleus 301 20 ± 521 ± 50* 61 ± 24 93 ± 23

Parietal Cortex 55 ± 12 68 ± 5 16 ± 6 30 ± 21

Midbrain/Thalamus 60 ± 26 64 ± 6 77 ± 4 33 ± 9*

Medulla 40 ± 10 69 ± 4* 9±3 18 ± 14

* = p < 0.05; ** = p < 0.01

528
(p < 0.05) where injury, evidenced by histological examination, was

most severe. Tissue concentrations of ir-End were not significantly

altered following injury (table 1). A significant fall in whole

brain and regional CBF to caudate nucleus, frontal cortex and pons

(p < 0.05) was observed in WIN (+) animals at 2 h post-injury

(table 2). The change in ir-Dyn but not ir-End was correlated with

the fall in regional CBF that was observed. In addition, WIN (-)

caused a significant increase in CBF to frontal cortex, caudate

nucleus, midbrain/thalamus and medulla.

TABLE 2

The effects of WIN(-) an cerebral blood flaw (ml/100 g/min

± S.E.M.).

10 min 30 min 1 h 2 h

Region Baseline Post-drug Post-drug Post-drug Post-drug

Frontal Cortex 60 ± 14 59 ± 15 82 ± 10 94 ± 18* 93 ± 12*

Caudate Nucleus 55 ± 15 54 ± 8 72 ± 15 77 ± 10 84 ± 10*

Parietal Cortex 34 ± 7 33 ± 1 41 ± 8 41 ± 6 46 ± 7

Midbrain/

Thalamus 41 ± 10 44 ± 11 52 ± 13 60 ± 13 65 ± 10*

Medulla 38 ± 8 47 ± 11 44 ± 10 46 ± 12 54 ± 8*

Whole Brain 46 ± 4 42 ± 4 49 ± 8 53 ± 9 56 ± 7

The effects of WIN(+) on cerebral blood flow (ml/100 g/min

± S.E.H.).

10 min 30 min 1 h 2 h

Region Baseline Post-drug Post-drug Post-drug Post-drug

Frontal Cortex 59 ± 8 55 ± 15 54 ± 15

Caudate Nucleus 69 ± 17 67 ± 18 64 ± 17

Parietal Cortex 43 ± 18 39 ± 11 39 ± 8

Midbrain/

Thalamus 57 ± 16 54 ± 18 47 ± 10

Medulla 46 ± 10 45 ± 14 40 ± 15

Whole Brain 54 ± 6 40 ± 13 44 ± 10

* = p < 0.05 when ompared to baseline values.

529

52 ± 12 43 ± 10*

61 ± 14 49 ± 16*

36 ± 7 32 ± 7

49 ± 4 34 ± 9*

34 ± 8 33 ± 10

42 ± 9 37 ± 5*
Our results suggest that tissue concentrations of dynorphin but not

beta-endorphin are elevated in brain regions where the fall in CBF is

most pronounced following injury. This fall in CBF observed at 2 h

post-injury could be reversed by administration of the opiate antagonist WIN (-), which has enhanced activity at kappa-receptors (Michne

et al. 1978), but not WIN (+) (table 2). The lack of effect shown by

the dextro-stereoisomer in this model implies that the efficacy of

WIN (-) is mediated through an action at opiate receptors. These

results are consistent with the hypothesis that dynorphin, acting

through specific opiate receptors, may contribute to the pathophysiology of secondary brain injury following head trauma. In addition,

it appears that opiate antagonists with increased activity at kappareceptor sites may be effective in the treatment of reduced cerebral

blood flow often associated with acute head injury.

REFERENCES

Faden, A., JAMA, 252:1177-1180, 1984a.

Faden, A., JAMA, 252:1452-1454, 1984b.

Faden, A., and Jacobs, T. Eur J Pharmacol, 91:321-324, 1983.

Faden, A., Molineaux, C., Rosenberger, J., Jacobs, T., and Cox, B.

Ann Neurol, 17:386-390, 1985a.

Faden, A.I., Knoblach, S., Mays, C., and Jacobs, T.P. Peptides,

6(Suppl 1):15-17, 1985b.

Ghazarossian, V., Chavkin, C., and Goldstein, A., Life Sci, 27:75-86,

1980.

Hayes, R., Galinet, B., Kulkarne, P., and Becker, D.J. Neurosurgery,

58:720-728, 1984.

Khachaturian, H., Lewis, M.E., Schafer, M., and Watson, S.J., Trends

Neurosci, 1:111-119, 1985.

Li, C.H., In: Li, C.H., ed., Hormonal Proteins and Peptides, Academic

Press: New York, 1981, pp. 2-30.

McIntosh, T., Bush, H., Palter, M., Hay, J., Aun, F., Yeston, N., and

Egdahl, R., J Surg Res, 38:210, 1985.

McIntosh, T., Agura, V., Hayes, R., and Faden, A., Am J Physiol,

(submitted) 1986.

Michne, W.R., Lewis, T.R., Michalec, S.J., Person, A.K., Gillan,

M.G.C., Person, S.J., Robson, L.E., and Kosterlitz, H.W., In: van

Ree, J.M. and Terenius, L., eds., Characteristics and Functions of

Opioids. Elsevier Press: North-Holland, 1978, pp. 197-206.

Molineaux, C. Jl., Feuerstein G., Faden, A., and Cox, B., Neurosci

Lett, 33:179-184, 1982.

ACKNOWLEDGMENTS

This work was supported by USAMRDC Contract #DAMD17-85-PP-5843 and

Merit Review Grant from the Veterans Administration.

530
TREATMENT OF EXPERIMENTAL STROKE WITH THE OPIATE ANTAGONIST WIN

44,441-3 EFFECTS ON NEUROLOGIC FUNCTION, INFARCT SIZE, AND SURVIVAL

David S. Baskin* and Yoshio Hosobuchi**

*Department of Neurosurgery, Baylor College of Medicine

6501 Fannin, Suite A404, Houston, Texas 77030

** Department of Neurosurgery, University of California, San Francisco

San Francisco, California 94143

ABSTRACT

Cats were treated using a blinded protocol with either the

dextrorotary or levorotary form of WIN 44,441-3 injected

subcutaneously six hours after middle cerebral artery occlusion,

followed by continuous subcutaneous infusion. The levorotary form

acutely improved neurologic function in 90% of animals, whereas the

dextrorotary form had no effect. Survival rate and infarct size were

the same in both groups. These results further implicate the kappa

receptor in the pathophysiology of cerebral ischemia.

INTRODUCTION

The results of our previous work indicate that endogenous opioid

peptides may contribute to the pathophysiology of neurological

deficits caused by cerebral ischemia. In humans, the opiate

antagonist, naloxone, reversed neurological deficits secondary to

cerebral ischemia, whereas morphine exacerbated them; in both

situations, there was no change in vital signs or level of

consciousness (Baskin and Hosobuchi 1981). The neurological deficits

produced by unilateral carotid ligation in gerbils were reversed by

the intraperitoneal administration of naloxone (Hosobuchi et al.

1982). In baboons, naloxone partially reversed and morphine

exacerbated ischemic neurological deficits resulting from focal

cerebral ischemia; the effect was not due to a change in systemic

parameters (Baskin et al.1982, Baskin et al. 1984, Zabramski et al.

1984). In cats with focal ischemia, naloxone and naltrexone improved

neurologic function and prolonged survival (Baskin et al. 1986);

dynorphin 1-13 also prolonged survival, suggesting that the kappa

receptor is involved in this phenomenon (Baskin et al. 1984). This

study was designed to further elucidate the role of the kappa

receptor in focal cerebral ischemia by using the relatively selective

kappa antagonist, WIN 44,441-3 to treat cats with experimentally

induced focal cerebral ischemia.

MATERIALS AND METHODS

20 adult male cats were randomly assigned in blinded fashion to one

of two groups, and were treated with the dextrorotary or levorotary

form of WIN 44,441-3. The cats were sedated with ketamine, and

anesthesia was induced by a mixture of halothane, nitrous oxide, and

oxygen, administered by mask. The trachea was then intubated, but

the cat was allowed to breathe spontaneously. One million units of

penicillin G was then administered intramuscularly. Transorbital

occlusion of the right middle cerebral artery (MCA) was performed

using the technique described in cats by O'Brien and Waltz (1973).

531
In all cases, the MCA was occluded proximal to the take-off of the

lenticulostriate arteries.

An incision was made in the mid-lumbar region and a subcutaneous

pocket was created for the later placement of an osmotic pump

designed to deliver drug solution at a constant volume. The cat was

allowed to awaken, and was examined 6 hours following MCA occlusion.

Neurological function was assessed independently by two individuals

who were unaware of the experimental protocol. Sensory and motor

function, level of consciousness, and pupillary reaction were graded

using an 18-point scale (Baskin et al. 1986).

Cats then received an intraperitoneal injection of 10 mg/kg of the

dextrorotary or levorotary form of WIN 44,441-3. A repeat

neurological assessment was performed 20 mintues later. The cat was

then sedated and an ALZET osmotic pump was implanted sterilely in the

subcutaneous pocket in the lumbar region to deliver either the

dextrorotary or levorotary isomer at 0.5 mg/kg/Hr. The investigators

were blinded as to which treatment was being administred, but the

pumps were coded in such a way that each cat received the same drug

in both the acute injection and the chronic infusion phases of the

study.

Neurological assessments were performed daily for as long as the cats

were alive, or until 7 days after occlusion, at which time all were

sacrificed. One million units of penicillin G was administered

intramuscularly daily along with subcutaneous injections of lactated

Ringer's solution calculated to provide adequate daily fluid

maintenance. Once a cat began to eat or drink, subcutaneous fluids

were discontinued. If a cat was found dead, a craniectomy was

performed, the brain removed, and a coronal section made at the level

of the optic chiasm. At the end of 7 days, all surviving cats were

sacrificed, the brains removed, and slices prepared. The slices were

incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride

(TTC) for 25 minutes according to the method of Han et al.(1983). A

TTC solution has been used extensively to demonstrate the presence

and extent of acute myocardial infarcts (Ramkisson 1966), and gives a

vivid indication of cerebral infarction in the acute stage. The

reaction product of TTC and viable tissue is a deep red fonnazan that

stains normal gray matter, whereas normal white matter stains with

less intensity. Infarcted tissue does not stain.

Color slides were then made of the stained brain slices. A

neuropathologist who was unaware of the experimental protocol made

tracings of the entire affected hemisphere and infarcted area from

projected images of the slide, A digitizing pad was used to

calculate the percentage of infarcted tissue relative to the entire

hemisphere for both sections in each cat; this defined the infarct

size.

The Wilcoxon signed rank analysis was performed to compare

neurological scores obtained before the acute drug injection with

those obtained 20 minutes after injection. A Kruskall-Wallis

analysis was performed to compare neurological scores before and

after injection between groups. A two-way analysis of variance was

532
used to compare infarct size in all groups of cats. Kaplan-Meier

survival curves were constructed, and Gehan's statistic was used to

compare survival rates among groups.

RESULTS

Following acute administration, the levorotary form of WIN 44,441-3

produced statistically significant improvement in motor function in

all animals tested, whereas the dextrorotary form had no effect

(Figure 1).

FIGURE 1

Median Motor Function in Cats Tested 6 Hrs. After

Middle Cerebral Artery Occlusion

(-) WIN 44,441-3 (+) WIN 44,441-3

BEFORE INJECTION 2 2

(25/75th Percentiles) 2/3 2/3

AFTER INJECTION 4 2

(25/75th Percentiles) 4/5 2/3

There was no effect of either drug on sensory function, level of

consciousness, or pupillary reaction to light. There were no

statistically significant differences in survival rates or size of

infarction between the detrorotary or levorotary groups.

DISCUSSION

Evidence for the involvement of the kappa receptor in central nervous

system dysfunction is increasing. Tang (1985) has shown that the

kappa agonist U50,488 can improve survival and hyperactivity

associated with global cerebral ischemia in the rat. Tortella et al.

(1986) have demonstrated that U50,488 can increase our seizure

threshold either secondary to electroshock or drugs with convulsant

activities. Faden and Jacobs (1985) have shown that WIN 44,441-3 can

improve motor function in unanesthestized rabbits with spinal cord

ischemia secondary to temporary occlusion of the abdominal aorta.

In our previous work, we have shown that a variety of opioid

compounds can influence either neurologic function and/or survival in

experimental focal cerebral ischemia. Indeed, it is possible to see

a dissociation of these effects: diprenorphine improved neurologic

function without altering survival (Baskin et al. 1986), whereas

dynorphin 1-13 improved survival without changing the neurologic

deficit (Baskin et al.1984). The fact that such dissociation exists

suggests that naloxone and other compounds that alter both survival

and function may do so at either different receptor subtypes or

perhaps in several different as yet unidentified ways.

We selected WIN 44,441-3 for these experiments because of our recent

experience with dynorphin 1-13 and the dramatic improvement in

survival associated with its use in focal cerebral ischemia (Baskin

533
et al. 1984). As there is much evidence that dynorphin 1-13 binds

mainly at the kappa receptor, and that WIN 44,441-3 has significant

antagonist properties at the kappa receptor (Michne et al. 1978), we

chose this compound for testing.

Our results clearly show biologic significance of the effect of WIN

44.441-3 on neurologic function in cerebral ischemia, as only the

levorotary form produced the change. The lack of an effect on

survival may be due to an inadequate dose for this to occur, or

because of a differentially selective effect on neurologic function

alone. While WIN 44,441-3 does bind at the mu, delta, and kappa

receptor subtypes, its relatively selective kappa antagonist activity

(Michne et al.1978) provides additional supportive evidence for the

role of the kappa receptor in cerebral ischemia. Further studv of

the compound in focal cerebral ischemia including dose-response

testing is in progress.

REFERENCES

Baskin, D.S.,

Baskin, D.S.,

1982.

Baskin, D.S.,

1984.

Baskin, D.S.,

1984.

Baskin, D.S.,

and Hosobuchi, Y. Lancet, 2:272-275, 1981.

Kieck, C.F., and Hosobuchi, Y. Life Sci, 31:2201-2204,

Hosobuchi, Y., Loh, H. H., et al. Nature, 312:551-552,

Kieck, C.F., and Hosobuchi, Y. Brain Res, 290:289-296,

Kuroda, H., Hosobuchi, Y., et al. Neuropeptides

5:307-310, 1985.

Baskin, D.S., Grevel, J., Hosobuchi, Y., J. Neurosurg, 64:99-103,

1986.

Faden, A.I., Jacobs, T.P., Neurology 35:1311-1315, 1985.

Han, D.H., Zervas, N.T., Geyer, R.P, et al. In: Reivich, M., and

Hurtig, H.I., eds. Thirteenth Research (Princeton) Conference.

New York: Raven Press, 1983. pp. 409-419.

Hosobuchi, Y., Baskin, D.S., and Woo, S.K. Science 215:69-71, 1982.

Michne, W.F., Lewis, T.R., Michalec, S.J. In: Van Ree, J.M. and

Terenius, L., eds. Characteristics and Functions of Opioids. North

Holland: Elsevier Press, 1978. pp. 197-206.

O'Brien, M., Waltz, A.G., Stroke 4:201-206, 1973.

Ramkisson, R.A., J Clin Pathol 19:479-407, 1966.

Tang, A.H., Life Sciences 37:1475-1482, 1985.

Tortella, F.C., Robles, L., Holaday, J.W. J Pharm Exp Ther, 237:49-53,

1986.

Ward, S.J., Pierson, A.K., Michne, W.F., Life Sciences, 33:303-306,

1983.

Zabramski, J.M., Spetzler, R.F., Selman, W.R., Stroke, 15:621-627,

1984.

ACKNOWLEDGEMENTS

We thank the Sterling-Winthrop Company for the gift of WIN 44,441-3.

This research was supported by Grants to Dr. Baskin from the Smith,

Kline, and French Fellowship of the American College of Surgeons, the

Baylor Research Support Grant, and the Regional Advisory Group and

Merit Review Board of the Veterans Administration, and to Dr.

Hosobuchi from the National Institute of Neurological and Communicative Disease and Stroke, Grant NS 20620.

534
CONVULSANT ACTION OF NALOXONE IN THE RAT AMYGDALA

Chrysanthy Ikonomidou-Turski, Waldemar A. Turski, Zuner A. Bortolotto,

Lechoslaw Turski and Esper A. Cavalheiro

Escola Paulista de Medicina, Department of Neurology and Neurosurgery,

Laboratory of Experimental Neurology,

R. Botucatu 862, CEP-04023 Sao Paulo, S.P., Brazil

ABSTRACT

Increasing doses of naloxone hydrochloride (100-1000 nmol) were microinjected unilaterally into the rat amygdala and the behavioral, neuropathological and electrographic responses were studied. Microinjections of low doses of naloxone (100-250 nmol) produced staring, gustatory automatisms and wet shakes whereas higher doses additionaly resulted in motor limbic seizures and status epilepticus. The electroencephalogram showed a sequence of alterations characterised by high

voltage fast activity, spiking, bursts of polyspiking, electrographic

seizures and postictal depression which first appeared in the amygdala and rapidly spread to hippocampal and cortical areas. The neuropathological analysis of frontal forebrain sections by means of light

microscopy revealed seizure-related brain damage in amygdala, olfactory cortex, thalamus, hippocampal formation, substantia nigra and neocortex. Diazepam, 10 mg/kg i.p., when given prior to the microinjection of naloxone into the amygdala, abolished the epileptogenic and

neurotoxic effects of the drug. The results suggest that naloxone,

when microinjected into rat amygdala elicits electrographic and motor

limbic seizures followed by seizure-related brain damage.

INTRODUCTION

There is a fairly large body of evidence to suggest that derangements

in the function of endogenous opioid mechanisms in the brain may play

a role in experimental, and human epilepsy. Opiates and opioid peptides can modulate the threshold for experimentally induced seizures in

a way which depends upon the art of convulsions, the route of drug application and the species used (Frenk 1983). Intracerebroventricular

application of high doses of morphine produce electrographic and

behavioral manifestation of limbic seizure activity (Frenk 1983).

Interestingly, the opiate antagonist naloxone, when given systemically in high doses causes generalised clonic convulsions which resemble

stage 5 kindled seizures and result in prominent increase of 2-deoxyglucose metabolism in several limbic forebrain structures (Chugani et

al. 1984). The amygdaloid complex has been shown to play a pivotal

role in activating epileptic limbic circuits (Watson et al. 1983).

Recently we reported that intraamygdaloid microinjection of morphine

results in motor limbic seizures and brain damage (Turski et al.

1983a). The present study provides an electroencephalographic, behavioral and morphological analysis of the effects produced by application of the opiate antagonist naloxone into the amygdaloid complex

of the rat.

535
MATERIALS AND METHODS

For intraamygdaloid injections adult male Wistar rats weighing 200-

220 g were used. Naloxone hydrochloride was supplied by Endo Labs

Inc (Garden City, N.Y., USA) was dissolved in saline and administered at doses of 100-1000 nmol into the amygdala. Diazepam (Polfa,

Kutno, Poland) was suspended in a 3% solution of Tween 81 (Loba,

Vienna, Austria) and given i.p. in the dose of 10 mg/kg 15 min prior

to intraamygdaloid injection of naloxone, 1000 nmol. The methods for

stereotactic implantation, the electroencephalographic registration

and the morphological examination of the brains are described elsewhere (Turski et al. 1985).

RESULTS

Microinjection of low doses of naloxone hydrochloride, 100 nmol (n=4),

into the amygdala presented no abnormal behavior. Higher doses, 250-

1000 nmol, produced hyperactivity, gustatory automatisms and occasionally scratching or myoclonic movements of fore- or hindlimbs and wet

shakes. This pattern of behavior persisted up to 30-40 min in animals

FIGURE 1. The evolution of electrographic activity elicited

by unilateral injection of naloxone hydrochloride (NAL), 500

nmol, into the rat amygdala. (a) Pre-drug control recordings.

(b-f) High voltage fast activity, spiking and bursts of polyspiking represent electrographic patterns registered in the

amygdala 3-20 min after the injection of NAL. (g-i) Electrographic seizures dominate the EEG patterns within 20-90 min

after NAL. (j-l) Progressive normalisation of electrographic

activity is observed 2-12 h after the injection of NAL. (m)

The EEG returns to the pre-drug pattern by 12-24 h post-injection. AMG, amygdala; CX, cortex.

536
receiving 250 nmol (n=6) of naloxone. With higher doses, 500 and 1000

nmol, the behavioral alterations increased and built up into seizures

and status epilepticus. The ED50 (effective dose) of naloxone for

generation of motor limbic seizures is 359.5 nmol (259.3-498.4)(n=24).

Motor limbic seizures commenced after a mean delay of 17.2 - 2.2 min †

following 1000 nmol (n=6) and 43.0 - 12.5 min (n=5) following 500 nmol †

of naloxone, recurred every 3-10 min and lasted up to 2-3 h following

the intraamygdaloid injection of the drug. After the intraamygdaloid

injection of 500-1000 nmol of naloxone the electrographic background

activity in the amygdala was replaced with high voltage fast activity

which progressed in high voltage spiking and bursts of polyspiking

(Fig. Ic-e), and developed into electrographic seizures. Ictal periods

lasted for 1-3 min, recurred every 3-5 min and were followed by periods of depression of the electrographic activity (Fig. 1g,i). Within

24-48 h the electroencephalographic activity slowly returned to the

pre-drug pattern (Fig. 1m). Examination of frontal forebrain sections

with light microscopy 2-15 days after microinjection of naloxone, 500

and 1000 nmol, revealed widespread damage in amygdala, hippocampus,

thalamus, olfactory cortex, substantia nigra and neocortex. The damage to the amygdala was most severe in the region immediately surrounding the injection cannula tip and entended into adjacent regions

i.e. pyriform cortex, entorhinal cortex and claustrum. With shortlasting survival times (48-72 h) the damage consisted of prominent

edema of the tissue as well as darkened and shrunken neurons. At longer survival time (10-15 days) the morphological structure of the

amygdala undervent severe breakdown with loss of most neurons. The

damage in the thalamus was limited to dorsomedial, reuniens, rhomboideus and paraventricular nuclei. Neuronal destruction in the hippocampus affected CA1 subfield of the intermediate and rostral hippocampus. The CA3 and CA4 subfields were affected considerably less.

The damage in the substantia nigra extended into most caudal portions

of the pars reticulata, whereas pars compacta was affected only very

rarely. The most injured parts of the neocortex included areas located dorsally to the rhinal sulcus and cingulate cortex. Pretreatment

with diazepam, 10 mg/kg, completely blocked the behavioral, electrographic and morphological alterations produced by intraamygdaloid injection of naloxone, 1000 nmol (n=5).

DISCUSSION

Our results demonstrate potent epileptogenic and brain damaging properties of naloxone when injected into the amygdala of rats. High doses of naloxone have been reported to cause generalized clonic convulsions when given systemically (Chugani et al. 1984, Dingledine et al.

1978) whereas lower doses block epileptogenic properties of opiates

(Frenk 1983). Two possible explanations for the convulsant action of

naloxone in the amygdala exist. The first one implies that naloxone

blocks an endogenous anticonvulsant system which is mediated by endogenous opioids (Frenk 1983). In fact, anticonvulsant action of morphine and enkephalins has been reported in animal models of reflex epilepsy and against pentylenetetrazol- and fluorothyl-induced seizures

(Frenk 1983; Frey 1986). The second proposal explaining convulsant

action of naloxone implies that the drug behaves as a aminobutyrate

(GABA)-antagonist. It is known that the opiate antagonist displaces

GABA from its binding sites in rat cerebral membranes and facilitates

bicuculline induced seizures (Dingledine et al. 1978, Sagratella and

537
Massoti 1982). Since antagonism at the GABA-receptor could also be

demonstrated for morphine, it seems tempting to speculate that both,

the opiate as well as the opiate antagonist might act in the amygdala

at the same site to initiate generalised limbic seizures, namely the

GABA receptor. Studies with intraamygdaloid injections of the GABA

antagonists bicuculline and picrotoxin demonstrate that both drugs

induce motor limbic seizures and brain damage closely resembling those

seen after morphine or naloxone (Turski et al. 1983a; 1985). Diazepam,

a compound which enhances GABA-mediated synaptic inhibition in the

brain (Haefely et al.1981) abolished the epileptogenic and brain damaging potential of naloxone. This finding however is not indicative

of a specific GABA antagonistic action of naloxone, since the benzodiazepine also protects against convulsions produced by systemic or intraamygdaloid injections of kainic acid and cholinomimetics (Ben-Ari

et al. 1980; Olney et al. 1983; Turski et al. 1983b).

REFERENCES

Ben-Ari, Y., Tremblay, E., Ottersen, O.P., and Meldrum, B.S. Brain

Res, 191: 79-97, 1980.

Chugani, H.T., Ackermann, R.F., Chugani, D.C., and Engel, J. In:

Fariello, R.G., Morselli, P.L., Lloyd, K.G., Quesney, L.F., and

Engel, J., eds. Neurotransmitters, Seizures and Epilepsy II.

New York, Raven Press, 1984. op. 315-325.

Dingledine, R., Iversen, L.L., and Breuker, E. Eur J Pharmacol, 47:

19-27, 1978.

Frey, H.-H. Pharmacology, 32: 335-339, 1986.

Frenk, H. Brain Res Rev, 6: 197-210, 1983.

Haefely, W., Pieri, L., Polc, P., and Schaffner, R. In: Hoffmeister,

F., and Stille, G., eds. Psychotropic Agents. Handbook of Experimental Pharmacology. Vol. 55/II, Berlin, Springer, 1981. pp. 13-262.

Olney, J.W., deGubareff, T., and Labruyere, J. Nature, 301: 520-522,

1983.

Sagratella, S., and Massoti, M. Neuropharmacology, 21: 991-1000, 1982.

Turski, W.A., Cavalheiro, E.A., Calderazzo-Filho, L.S., Kleinrok, Z.,

Czuczwar, S.J., and Turski, L. Neuroscience, 14: 37-53, 1985.

Turski, W.A., Czuczwar, S.J., Kleinrok, Z. Schwarz, M., and Turski, L.

Life Sci, 33 (Suppl 1): 615-618, 1983a.

Turski, W.A., Czuczwar, S.J., Kleinrok, Z., and Turski, L. Experientia,

39, 1408-1411, 1983b.

Watson, R.E., Troiano, R., Poulakos, J., Weiner, S., Block, C.H., and

Siegel, A. Brain Res Rev, 5: 1-44.

538
DYNORPHIN A (1-13): IN VIVO OPIOID ANTAGONIST ACTIONS AND NONOPIOID ANTICONVULSANT EFFECTS IN THE RAT FLUROTHYL TEST

F.C. Tortella and J.W. Holaday

Neuropharmacology Branch

Dept. of Medical Neurosciences, Div. of Neuropsychiatry

Walter Reed Army Inst. of Res., Washington, D.C. 20307-5100, U.S.A.

ABSTRACT

Dynorphin A (1-13) acutely elevated the seizure threshold (ST) to the

convulsant flurothyl, and this action was not blocked by naloxone. Increases

in ST were also observed following i.c.v. injections of the non-opioid fragment

dynorphin A (3-13). Pretreatment with dynorphin A (1-13), but not dynorphin

A (3-13), non-competitively blocked the anticonvulsant effect of the μ

selective opioid DAGO. Furthermore, pretreatment with dynorphin A (1-13)

antagonized the antagonist properties of naloxone or ICI 154,129 in this

seizure model. Thus, in addition to its non-opioid anticonvulsant effects,

dynorphin A (1-13) exhibits unique antagonist actions which appear to be

specific for the active opioid fragment.

INTRODUCTION

Dynorphin A (1-13). the endogenous ligand for opioid binding sites, exhibits

activity in receptor binding assays, in vitro bioassays, and in vivo analgesia

systems. It has also been shown that dynorphin A (1-13) demonstrates opioid

antagonist effects in opiate naive and tolerant rats (Lee and Smith 1984).

Hence, under the appropriate conditions, dynorphin A (1-13) appears to possess

mixed agonist/antagonist properties.

Recently, using the flurothyl seizure test as an in vivo model to differentiate

multiple opioid receptor systems (Cowan et al. 1979; Tortella et al. 1985), we

demonstrated that FNA, another agonist with μ antagonist properties

(Ward et al. 1982), attenuates the antagonist properties of naloxone or ICI

154,129. Therefore, considering the possible similarities between FNA and

dynorphin A as opioids possessing mixed agonist and antagonist properties, the

present studies were aimed at establishing the pharmacological profile of

dynorphin A (1-13) in this in vivo model. For these studies, we used the μ

selective agonist DAGO (Handa et al.

agonist DADL (D-Ala2

-D-Leu5-enkephalin) (Tortella et al. 1985), peptide

1981) and the mixed μ receptor

ligands possessing potent μ (DAGO; Robles et al. 1986) and (DADL; Tortella

et al. 1985) receptor-mediated anticonvusant profiles in the rat flurothyl test.

MATERIALS AND METHODS

Male Sprague-Dawley rats (225-300 g) obtained from Zivic Miller laboratories

(Allison Park, PA) were used. Three to five days prior to testing, rats were

anesthetized with ketamine HCL (100 mg/kg, i.p.) and stereotaxically

prepared with i.c.v. cannulae aimed at the right lateral ventricle. Three to

five days following surgery, the rats (n=8-10 per group) were randomly

assigned to treatment groups for testing. The effects of dynorphin A (1-13)

(3.9-15.6 nmol, i.c.v.) or dynorphin A (3-13) (15.6 nmol, i.c.v) on flurothyl ST

were determined at various times postinjection. Additional rats were

539
pretreated with naloxone (1 or 10 mg/kg, s.c.) 10 min prior to an i.c.v.

challenge of dynorphin A (1-13) (15.6 nmol) and exposed to flurothyl 15 min

later. In separate experiments, the μ antagonist actions of dynorphin A (1-13

or 3-13) (15.6 nmol; -2hr pretreatment) were determined in DAGO (0.275-4.4

nmol, i.c.v.) treated animals. Additionally, antagonist-interaction studies

were conducted in naive rats pretreated at -2 hr with dynorphin A (1-13) (15.6,

nmol), subsequently injected with ICI 154,129 (50 nmol, i.c.v.) or naloxone (290

nmol, i.c.v) 10 min prior to receiving an i.c.v. injection of DADL (35 nmol) and

tested 20 min later in the rat flurothyl test.

To determine flurothyl ST’s, rats were exposed to the volatile convulsant

flurothyl and tested as previously described (Tortella et al. 1985). All i.c.v

injections were given in a maximum volume of 7.5 μl (including a 2.5 μl

flush). All the control groups received i.c.v. injections of saline at the

appropriate treatment times.

RESULTS AND DISCUSSION

Mean ST’s for all the control groups ranged from 340 to 370 seconds. The

administration of dynorphin A (1-13) (fig. 1A) caused a significant dose- and

time-dependent increase in ST. At the highest dose tested (15.6 nmol), ST’s

were increased 140 + 11 % of control. Peak effect occurred 15-30 min

postinjection, with a duration of action of 60 min. The anticonvulsant action

of dynorphin A (1-13) was not antagonized by naloxone (fig. 16). Moreover,

dynorphin A (3-13) (15.6 nmol) was as effective as dynorphin A (1-13) as an

anticonvulsant in this seizure model (data not shown). Collectively, these

data clearly indicate that the actions of dynorphin A to increase the ST to

flurothyl do not require the active opioid fragment, suggesting that a nonopioid mechanism of action is responsible for dynorphin’s anticonvulsant

effect in rats.

FIGURE 1. A. Dose- and time-dependent anticonvulsant effect of

dynorphin A (1-13). B. Failure of naloxone to antagonize dynorphin A

(1-13). *p<0.05, compared to saline control (Student’s test).

The μ antagonist actions of dynorphin A (1-13) are seen in table 1. In rats

540
pretreated with dynorphin A (1-73)(15.6 nmol), but not (3-13)(15.6 nmol), the

ability of DAGO to increase the threshold to flurothyl seizures was completely

antagonized. Although not shown, dynorphin pretreatment shifted the DAGO

anticonvulsant dose-response curve to the right and decreased its maximal effect,

indicating a non-competitive antagonism. The results of these experiments

demonstrate a μ selective antagonist action of dynorphin A which, unlike the nonopioid actions of dynorphin A, is dependent upon the intact opioid fragment.

TABLE 1. Dynorphin A (1-13) antagonism of the anticonvulsant effects of DAGO.

Antagonist Dose Range of DAGO Maximal increase in ST

(nmol, i.c.v.) (% of Control)

Saline 0.275-2.2 164 ± 11

Dyn A (1-13) 0.275-4.4 115 ± 5

Dyn A (3-13) 0.275-2.2 158 ±12

FIGURE 2. Dynorphin A (1-13)

blockade of the 6 antagonist

actions of naloxone or ICI

154,129 on DADL-induced

increases in seizure threshold.

All rats were injected with

DADL in combination with

Qther substances as the

indicated dosed (substcripts).

*p<0.05, compared to control,

+

p<0.05, compared to the 35

nmol DADL group (Dunnett’s

test). #p<0.05, compared to

lCI50 + DADL and NLX290 +

DADL groups (Student’s test).

The effect of dynorphin A (1-13) to diminish the ability of either naloxone or ICI

154,129 to antagonize the anticonvulsant actions of DADL (Tortella et al. 1985) is

shown in figure 2. In the absence of dynorphin A (1-13), pretreatment with i.c.v.

naloxone (290 nmol) or ICI 154,129 (50 nmol) antagonized the anticonvulsant effect of DADL (157±13 %) to 106±6 % and 120±8 % of control, respectively. However, in the presence of dynorphin A (1-13), which alone had no effect on DADL

anticonvulsant activity (151±9 %), the DADL-induced increase in ST was only

541
partially antagonized by naloxone (132±6 %) and no longer antagonized by ICI

154,129 (166±9 %). Since the doses of naloxone and ICI 154,129 used here have

been previously shown to selectively antagonize opioid receptor activity in this

seizure model (Tortella et al. 1984; Tortella et al. 1985), we conclude that under

the appropriate conditions of pretreatment time and dose, dynorphin A (1-13) is

capable of antagonizing the selective antagonist actions of naloxone and ICI

154,129 in the rat flurothyl test.

In addition to the agonist properites of dynorphin A (1-13), the in vivo pattern of

results described in the present report are suggestive of three different actions

for this opioid ligand: 1) an anticonvulsant action which is clearly non-opioid, 2) μ

antagonist activity and 3) the ability to antagonize the actions of antagonists

(ICI 154,129 or naloxone), but not the agonist properties of DADL.

Interestingly, a remarkably similar pattern of results has been observed with the

agonist/irreversible μ antagonist FNA (D’Amato and Holaday 1984; Tortella et

al. 1985). Given the noncompetitive nature of the μ antagonist properties of

dynorphin A reported here, it seems possible that irreversible μ receptor blockade

may be a prerequisite for the types of antagonist interactions observed in the

present report. In this regard, dynorphin A and FNA may share a common

mechanism of action at an opioid receptor complex.

Obviously, several mechanisms can be proposed to explain the interactions

observed between dynorphin A and FNA and the antagonists. These include

act ions upon second messenger systems, altered metabolism, or simple

pharmacological interactions of the various ligands at individual μ and

receptors (Holaday et al. 1986). Alternatively, since several investigators have

performed in vivo, in vitro and binding studies indicating that μ and receptor

sites are isverconvertible (Bowen et al. 1981), and may interact through noncompetative molecular mechanisms (see Holaday et al. 1986), we and others have

postulated that a functional coupling (possibly via allosterism) of opioid binding

sites on a common receptor macromolecule may account for these interactions.

Regardless of the mechanisms involved, this consistent pattern of interactions

observed with dynorphin A in the present report, and elsewhere with FNA (see

above), may have important significance relevant to the concept of multiple

opioid receptors. At least in the case of dynorphin A, an endogenous neuropeptide

ligand, the results reported in this study strongly support the hypothesis proposed

by Lee and Smith (1984) suggesting that dynorphin may function in the CNS

primarily as a modulator of opioid receptor systems.

REFERENCES

Bowen, W.D., Gentleman, S., Herkenhan, M., and Pert, C. Proc Nat’l Acad Sci,

78:4818-4822, 1981.

Cowan, A., Geller, E.B., and Adler, M.W. Science, 206:465-467, 1979.

Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J. and Smith,

C.F.C. Br J Pharmacol, 70:531-540, 1981.

Holaday, J.W., Tortella, F.C., Maneckjee, R., and Long, J.B. NIDA Monograph: In

Press, 1986.

Lee, N.M. and Smith, A.P. TIPS, 5: 108, 1984.

Robles, L., Tortella, F.C., and Holaday, J.W. Pharmacologist, 28:424, 1986.

Tortella, F.C., Robles, L., and Holaday, J.W. Life Sci, 37:497-503, 1985.

Tortella, F.C., Robles, L., Holaday, J.W., and Cowan, A. Eur J Pharmacol,

97:141-144, 1984.

542
MU- AND DELTA-OPIOID MODULATION OF ELECTRICALLY-INDUCED

EPILEPTIC SEIZURES IN MICE

Carl Pinsky, Ranjan Bose and Robert

C.A. Frederickson*

Department of Pharmacology and Therapeutics

Faculty of Medicine, University of Manitoba

770 Bannatyne Avenue, Winnipeg, Manitoba

Canada R3E OW3

*Monsanto Company G3WE; CNS Research, Health

Care Division, 800 North Lindbergh Blvd,

St. Louis MO 63167 U.S.A.

ABSTRACT

Graded seizure responses to suprathreshold cerebral

electroshock in mice were modified by drugs acting at muand at delta-opioid receptors. Morphine exerts a

proconvulsant effect at a non-mu opioid receptor and may

exert a simultaneous anticonvulsant effect at a mu-opioid

receptor. Delta-opioid receptor blockade increases

seizure severity, suggesting a predominantly

anticonvulsant nature of the delta-opioid system in the

seizure model tested here.

INTRODUCTION

Dua et al. (1985) demonstrated that blockade of central

delta-opioid receptors can attenuate

electroencephalographic seizures initiated by mu-opioid

receptor stimulation in rats. In contrast, Tortella and

Long (1985) had earlier reported on a putative endogenous

delta-opioid agonist which can diminish flurothylprovoked seizures. We have therefore examined for this

study the effects of mu- and delta-opioid receptor

agonists and antagonists on the severity of electroshock

seizure (ECS) activity in mice, to determine whether

modulation of seizure activity might be a general

property of such opioid agents.

MATERIALS AND METHODS

Electrical stimulation and seizure response measurement.

ECS was produced in Swiss-Webster albino mice by passing

60-Hz sinusoidal constant current (generator output

impedance > 50 k ohms) for 200 msecs via electrode jellycoated pinnae through spring-loaded metal clips. We

determined from pilot experiments in untreated mice that

a stimulus strength of 5.6 mA root mean square could

produce full seizure activity while even slightly higher

currents often produced immediate death. With 5.6 mA

strength all untreated mice displayed a gradeable

continuum of motorobehavioral responses which permitted

543
us to guantitate seizure response severity in treated and

untreated animals on a scale of from 0 to 5, as follows:

0 = no observable seizure activity: 1 = mouse stunned,

immobile, may subsequently run, Straub tail; 2 = brief

(<2 sec) clonic seizure, stunned, runs and becomes

hyperactive; 3 = sustained (>2 sec) clonic seizure,

stunned: 4 = brief tonic seizure, stunned, running, all

often accompanied by brief clonic activity; 5 = sustained

tonic-clonic seizure, stunned, running, followed by

postictal depression and, occasionally, death. The

seizure severity grade for each mouse was multiplied by

total response duration (from initial application of

current until cessation of seizure activity) to obtain a

Seizure Index (SI) score. Animals which died during

seizure were assigned the mean of the two highest SI

scores for surviving animals in the same treatment group.

Effects of drugs were examined on mean SI scores for each

group.

Drugs and administration. Mice were pretreated with

morphine sulfate (MOR; BDH, at 0, 8, 32 or 128 mg/kg

intraperitoneally (ip) administered 20 min prior to

electroshock), naloxone hydrochloride (NIX; Endo, at 0,

0.16, 0.64, 1.0 or 2.56 mg/kg ip administered 10 min

prior to electroshock) and ICI 154,129 (I/l; at 0, 6 or

24 mg/kg ip administered 30 min prior to electroshock).

All drugs were dissolved in saline and injected in

volumes of 10.0 ml/kg ip.

Statistical procedures. Differences between treatment

groups were evaluated by analysis of variance (ANOVA)

followed by Duncan's test for multiple comparisons.

Linear regression was done by the method of least

squares.

RESULTS

Reproducibility and reliability of SI measurement. There

were no statistical differences between SI values

obtained by two different observers who evaluated SI in

different groups of saline-treated (control) mice in

separate experiments. Statistical inferences concerning

the effects of MOR 8 mg/kg on SI were identical between

the two experimenters and identical for tests done at

different times in separate experiments.

The effects of pretreatment with opioid drugs on SI.

These results are summarized in Table 1. NLX 1.0 mg/kg

ip by itself did not alter SI but the combination of NLX

with MOR, in a fixed molecular NLX:MOR ratio of

approximately 1:50, produced significantly greater SI

scores than did MOR alone at 32 and 128 mg/kg. The

NLX/MOR combination with MOR at 8 mg/kg produced SI

scores numerically greater than those with MOR alone at

544
mainly from in vitro opioid receptor kinetics data

(Kosterlitz and Watt 1968; Lord et al. 1977) and was

aimed at maintaining essentially complete blockade of muopioid receptors at all doses of MOR used in combination

with NLX. Differences between SI scores with MOR alone

and with the given combinations of MOR and NLX were

plotted against the dose of MOR over the range from 0 to

128 mg/kg. The data resulted in a straight line of best

fit (forced through origin) with slope = 2.5, r = 0.65

and p<0.001, to indicate the slope as being significantly

different from zero. Administration of I/l 6 mg/kg by

itself provoked Severity Index scores significantly

greater than those from untreated mice. SI in mice

treated with a combination of I/l and MOR was always

numerically greater, by at least 117 SI points, than with

MOR by itself at corresponding doses. Differences for

these comparisons were statistically significant with MOR

at 8 and at 128 mg/kg.

TABLE 1

Effects of Pretreatment with

Opioid Drugs on Seizure Index

8 mg/kg. The chosen NLX:MOR ratio of 1:50 was arrived at

PRETREATMENT

GRP MOR NLX I/1

A 0 0 0

B 0 1.0 0

C 0 0 6

D 8 0 0

E 8 0.16 0

F 8 0 6

G 32 0 0

H 32 0.64 0

J 32 0 6

K 128 0 0

L 128 2.56 0

M 128 0 24

SEIZURE

INDEX

mean ± S.E.

99 ± 26

129 ± 30

338 ± 8

219 ± 11

305 ± 57

405 ± 49

128 ± 16

508 ± 30

245 ± 49

85 ± 19

330 ± 55

312 ± 83

For all groups n=6, except A and D where

n=12. Mean SI for Grp A is significantly

different (p<0.05; ANOVA) from C, D, E,F, H,

J, L. M; B from C, E, F, H, L, M; C from A,

B, D, G, H; D from A, C, F, H, K; E from A,

B, H, K; F from A, B, D, K; G from C, E, F,

H, L, M; H from all except F; J from A, F, H,

K; K from C, D, E, F, H, J, L, M; L from A,

B, G, H, K; M from A, B, G, H, K.

545
DISCUSSION

The approximated linear relationship between dose of

morphine and seizure severity in the presence of a fixed

mu-opioid receptor-blocking NLX:MOR ratio supports the

possibility that morphine exerts a proconvulsant action

at a non-mu opioid receptor. The significant

proconvulsant effect of morphine, which with doses

greater than 8 mg/kg was unmasked only in the presence of

naloxone, suggests the existence of a mu-receptor related

anticonvulsant mechanism. This mechanism becomes evident

only after blockade of mu-opioid receptors and not with

morphine administration alone. Our results suggest also

the existence of an anticonvulsant delta-opioid receptor

which is tonically activated by endogenous delta-opioid

ligand(s), a concept supported here by the significantly

proconvulsant effect of I/l, a relatively specific deltaopioid antagonist (Gormley et al. 1982), by itself at 6

mg/kg. This is consistent with the work of Tortella et

al. (1984) and Tortella and Long (1985) who maintain that

delta-opioid agonism can be anticonvulsant. Thus,

depending upon the particular seizure model being tested

(Dua et al. 1985), the central delta-opioid receptor

system may support both anticonvulsant and proconvulsant

mechanisms.

REFERENCES

Dua, A.K., Pinsky, C. and LaBella, F.S. EEG Clin

Neurophysiol, 61:569-572, 1985.

Gormley, J.J., Morley, J.S., Priestley, T., Shaw, J.S.,

Turnbull, M.J. and Wheeler, H. Life Sci, 31:1263-1266,

1982.

Kosterlitz, H.W. and Watt, A.J.

33:266-276, 1968.

Br J Pharmac Chemother,

Lord, J.A.H., Waterfield, A.A., Hughes, J. and

Kosterlitz, H.W.

Tortella, F.C., Robles,

Nature, 267:495-499, 1977.

L.E., Holaday, J.W. and Cowan, A.

Europ J Pharmacol, 97:141-144, 1984.

Tortella, F.C. and Long, J.B. Science, 228:1106-1108.

ACKNOWLEDGEMENTS

This work was supported by the Medical Research

Council of Canada, Term Grant No. MT7223 and by the

Centre on Aging, University of Manitoba. We thank R.

Cotton and J.S. Shaw of ICI Pharmaceuticals for gifts of

ICI 154,129; Dupont de Nemours for naloxone

hydrochloride: Mr. Egon Brockhausen for his outstanding

work in these experiments, Grigory Karmy for assistance

with statistical analysis and Carol Joseph for excellent

technical support.

546
THE THRESHOLD FOR LIMBIC SEIZURES IN RATS IS DECREASED BY INTRANIGRAL

MORPHINE

Waldemar A. Turski, Zuner A. Bortolotto, Chrysanthy Ikonomidou-Turski,

Luiz E.A. Moraes Mello, Esper A. Cavalheiro and Lechoslaw Turski

Department of Pharmacology, Medical School, Jaczewskiego 8,

PL 20-090 Lublin, Poland

ABSTRACT

Microinjection of morphine hydrochloride into the substantia nigra

pars reticulata, bilaterally, converts non-convulsant dose of pilocarpine hydrochloride, 100 mg/kg, into a convulsant one. The ED50 of

morphine for the generation of seizures after pilocarpine, 100 mg/kg,

is 3.8 nmol (2.5-5.8). Electrographic and behavioral monitoring both

show a pattern of convulsant activity similar to those produced by pilocarpine in doses exceeding 350 mg/kg. Morphological analysis of

frontal forebrain sections reveals epilepsy-related damage to the hippocampus, thalamus, olfactory cortex, substantia nigra, neocortex and

amygdala. The proconvulsant action of morphine in the substantia nigra is reversed by co-administration of naloxone hydrochloride. The

results show that the threshold for limbic seizures may be modulated

by opiates in the substantia nigra.

INTRODUCTION

The basal ganglia are concerned with the propagation of motor seizures

and in the generation of seizure activity in the limbic system. Clinical observations in man and lesion studies in primates established

that substantia nigra governs the spread of seizures elicited from the

motor cortex (Hayashi 1952). The substantia nigra undergoes irreversible degeneration in rodents subjected to status epilepticus (Turski

et al. 1986). Aminobutyrate (GABA), substance P and dynorphin are

tentatively suggested to subserve neurotransmitter role in the striatonigral pathway (Fallon and Loughlin 1985). An excitatory amino acid,

L-glutamate and/or L-aspartate, are the most likely transmitter candidates of corticonigral pathway (Fallon and Loughlin 1985). Manipulating GABAergic functions in the substantia nigra (SN) modulates the

threshold for limbic seizures (Turski et al. 1986). Blockade of excitatory amino acid- or substance P-mediated excitation in the SN raises

the threshold for seizures (Gale 1985). It remains uncertain whether

opiates in the SN modify the threshold for limbic seizures. Morphine

(MF) potentiates the convulsant action of systemically administered

kainic acid and pilocarpine and facilitates the rate of development of

amygdala kindling (Fuller and Olney 1979; Turski et al. 1985; Stone et

al. 1982). The morphological substrate of this action is largely unknown, although preliminary observations indicate that the concentration of opioid peptides changes in the SN in the course of amygdala kindling (Iadarola et al. 1986). The SN is essential for the motor regulatory function of opiates. There is electromyographic evidence that

MF elicits increase in the muscle tone when microinjected into the SN

pars reticulata in rats (Turski et al. 1983). Application of opiate

receptor antagonist naloxone into the SN blocks the muscle rigidity

produced by systemic morphine (Turski et al. 1982). These experiments

furnish corroborative evidence for the role of nigral opiates in the

547
control of spinal motor functions. The purpose of this study is to investigate the function of opiates in the SN in the motor expression of

pilocarpine-induced seizures. Seizures produced by pilocarpine in rats

provide a useful animal model of temporal lobe epilepsy which permits

studying mechanisms of initiation of and pathways operative in the propagation of convulsive activity in the brain (Turski et al. 1986).

MATERIALS AND METHODS

Male Wistar rats, 230-250 g in weight, were subject to stereotaxic surgery for the implantation of stainless-steel guide cannulae directed towards the SN (AP 1.61; L ±

 1.9; V -2.6)(Turski et al. 1986). After

a 5-day recovery period microinjections into the SN were performed bilaterally in unanesthetised rats. The drugs were delivered in a volume

of 0.5 μ1 (0.1 μl/min). Morphine hydrochloride (MF)(Polfa) was administered into the SN in doses of 1, 2, 5 and 10 nmol, while naloxone hydrochloride (NAL)(Endo) was microinjected into the SN in the dose of 10

nmol, 15 min prior to i.p. injection of pilocarpine hydrochloride (Sigma), 100 mg/kg. Methylscopolamine nitrate (Sigma), 1 mg/kg, was administered S.C. 30 min prior to the injection of pilocarpine. MF, NAL, pilocarpine and methylscopolamine were dissolved in saline. In all experiments the animals were used once only. For depth recordings, bipolar

twisted electrodes were positioned in the dorsal hippocampus. Surface

recordings were led from screws positioned bilaterally over the occipital cortex. The brains were processed for morphological examination by

light microscopy 1-3 and 5-15 days after the administration of pilocarpine. The methods for histological processing of the tissue were similar

to those described elsewhere (Turski et al. 1986).

RESULTS

Microinjection of MF into the SN pars reticulata, bilaterally, increased the severity of behavioral and EEG alterations produced by pilocarpine, 100 mg/kg, in a dose-dependent manner. None of four rats injected with MF, 1 nmol, presented motor limbic seizures or status epilepticus after pilocarpine, 100 mg/kg. Two of seven rats injected

with 2 nmol of MF developed motor limbic seizures, while microinjection of 5 nmol resulted in seizures and status epilepticus in six out

of eight rats. Seven out of eight animals receiving MF, 10 nmol, disFIGURE 1. Schematic reconstruction of the injection sites

of morphine (MF) in the substantia nigra of the rat. MF, 10

nmol was microinjected bilaterally into the substantia nigra

15 min prior to the i.p. administration of pilocarpine, 100

mg/kg. Dark squares represent sites from which microinjections of MF convert non-convulsant dose of pilocarpine, 100 mg/

kg, into a convulsant one. Open squares represent sites from

which microinjections of MF do not affect the action of pilocarpine, 100 mg/kg.

548
FIGURE 2. Electrographic recordings demonstrating the effect of morphine hydrochloride (MF) on the convulsant action

of pilocarpine (PILO) in rats. MF, 10 nmol, was microinjected bilaterally into the SN pars reticulata 15 min prior to

systemic administration of PILO, 100 mg/kg. (a) Pre-drug

control recordings. (b-d) Unchanged records 7-15 min after

microinjection of MF into the SN. (e-o) Electrographic recordings to illustrate the alterations registered during 3 min -

18 h after the administration of PILO in rats given MF into

the SN. HPC, hippocampus; CX, cortex.

played severe status epilepticus (Fig. 1). The ED50 (effective

dose) for MF, as a factor triggering seizures in rats treated with pilocarpine, 100 mg/kg (n=27), was 3.8 nmol (2.5-5.8). The proconvulsant effect of MF, 10 nmol, was blocked by NAL, 10 nmol, when co-injected into the SN (n=4) prior to pilocarpine, 100 mg/kg. The range of

EEG changes produced by pilocarpine, 100 mg/kg, in rats pretreated

with microinjection of saline into the SN is described elsewhere (Turski et al. 1986). The nature of EEG changes induced by pilocarpine,

100 mg/kg, in MF, 10 nmol, pretreated rats is best illustrated in Fig.

2. The electrographic activity produced by pilocarpine, 100 mg/kg, in

rats pretreated intranigrally with MF, 10 nmol, resembles the build-up

and EEG components of seizures produced by pilocarpine, 380 mg/kg (Turski et al. 1986). The pattern and topography of the damage to the forebrain produced by pilocarpine, 100 mg/kg, in rats pretreated with MF,

10 nmol, in the SN resemble those found in the brains of animals treated with pilocarpine, 380 mg/kg (Turski et al. 1986). Extensive morphological alterations were found throughout the hippocampus (subfields CA

1 and CA 4), thalamus (mediadorsal lateral, reuniens, rhomboideus, paratenial and paraventricular nuclei), olfactory cortex, amygdala, neocortex and substantia nigra pars reticulata.

DISCUSSION

A deep controversy surrounds the function of endogenous opioids in the

pathogenesis of seizure disorders. The present report on proconvulsant action of MF in the SN pars reticulata in pilocarpine-model of

549
limbic seizures in rats provides further proof for differential actions of opiates in the complex neuronal networks regulating the seizure threshold of the forebrain. Evidence is presented that the action of MF in the SN is opiate specific since naloxone reverses anticonvulsant action of the drug. This finding is in apparent contrast

however with anticonvulsant action of intranigral morphine against maximal electroshock (MES)-induced seizures in rats. Garant and Gale

(1985) showed that very high doses of MF sulphate when injected into

the SN protect rats from tonic hindlimb extension in the MES-test.

This action of MF was blocked by systemic naloxone. Surprisingly, anticonvulsant action of MF in the SN in the MES fits well protective

effect of the opiate against MES when administered systemically (Czuczwar and Frey 1986). Similarly, proconvulsant effect of intranigral

MF in pilocarpine-induced seizures reflects perfectly the proconvulsant

action of systemic MF in this model of convulsions (Turski et al. 1985).

Several mechanisms have been proposed as explanation of the pro- and

anticonvulsant action of MF in different experimental situations (Frenk

1983). It seems likely that SN may be proposed as a site at which opiates are operative in the propagation and generation of seizures. The

apparent disparity between proconvulsant action of MF in the SN in an

animal model of limbic seizures and potent anticonvulsant action of the

drug in MES-induced convulsions probably reflects basic differences between pathways and neurotransmitters determining the spread of seizures

in these two models of epileptogenesis. Alternatively, selective interactions with precise neuronal networks differentially involved in regulation of the seizure threshold may well provide a basis for differential response to intranigral MF in various epilepsy models. The emerging picture of the function of opiates in the SN in the modulating of

the seizure threshold is further complicated by multiplicity of opiate

receptors in this brain region. Further studies are therefore urgently

required to dissect out the contribution these receptors make to the

epileptogenesis.

REFERENCES

Czuczwar, S.J., and Frey, H.-H. Neuropharmacology, 25: 465-469, 1986.

Fallon, J.H., and Loughlin, S.E. In: Paxinos, G., ed. The Rat Ner- and Loughlin, S.E. In: Paxinos, G., ed. The Rat Nervous System. Vol. 1. Sydney: Academic Press, 1985. pp. 353-374. vous System. Vol. 1. Sydney: Academic Press, 1985. pp. 353-374.

Frenk, H. Brain Res Rev, 6: 197-210, 1983. Frenk, H. Brain Res Rev, 6: 197-210, 1983.

Fuller T.A., and Olney, J.W. Life Sci, 24: 1793-1798, 1979.

Gale, K. Fed Proc, 44: 2414-2424, 1985.

Garant, D.S., and Gale, K. J Pharmacol Exp Ther, 234: 45-48, 1985.

Hayashi, T. Jap J Pharmacol, 3: 46-64, 1952.

Iadarola, M.J., Shin, C., McNamara, J.O., and Yang, H.-Y.T. Brain Res,

365: 185-191.

 L.,Ikonomidou, C., Cavalheiro, E.A., Kleinrok, Z., Czuczwar,

Brain Res, 370: 294-309, 1986.

Turski,

S.J., and Turski, W.A. Neuropeptides, 5: 315-318, 1985.

Turski, L., Havemann, U., and Kuschlnsky, K. Behav Brain Res, 5: 415-

422, 1982.

Turski, L., Havemann, U., and Kuschinsky, K. Neuropharmacology, 22:

1039-1048, 1983.

Turski, L., Cavalheiro, E.A., Schwarz, M., Turski, W.A., Moraes Mello. L.E.A., Bortolotto, Z.A., Klockgether, T., and Sontag, K.-H.

Stone, W.S., Eggleton, C.E., and Berman, R.F. Pharmacol Bioch Behav,

16: 751-756.

550
OPIOIDS WITH DIFFERENT AFFINITY FOR

SUBRECEPTORS INDUCE DIFFERENT EFFECTS ON

EARLY AND LATE SENSORY EVOKED POTENTIALS (SEP) IN MAN

Enno Freye, Rolf Buhl and Francesco Ciaramelli

Dept. of Central Diagnostics,

University Clinic of Psychiatry,

4300 Essen 1, Hufelandstr.55, F.R.G.

Dept. of Clinical Anesthesia

University Clinic of Duesseldorf,

4000 Duesseldorf 1, Moorenstr. 5, F.R.G.

ABSTRACT

Amplitude changes of event-related sensory potentials (SEP's) present

objective measurements for the evaluation of pain perception in

man.

Alfentanil, an opioid with dominant binding to the mu-receptor

was given in graded doses (5-, 10- μg/kg) to volunteers. Nalbuphine,

an opioid with reported kappa-agonist activity, was given in graded

doses (100-, 500-, 1000- μg/kg) to another group i.v. Both groups

were also exposed to progressively increased electrical stimulations

(mA) in order to determine tolerance threshold before and after

drug administration. In the alfentanil group (n=5) there was a

dose- related decrease of the amplitude of the early N20-peak.

The late N100-peak was not, however, affected. This correlated

closely (r=0.87) with a naloxone-reversible increase of tolerance

to electrical current. In the nalbuphine group (n=15), there was

a dose-related, naloxone non-reversible reduction of amplitude

of the late N1OO-peak which showed a good correlation (r=0.9) with

an increase in current threshold. As the early evoked potentials

are primarily generated in the pontine-thalamic region, it is suggested that the mode of action of alfentanil resembles that of

a blockade of sensory impulses. The latter takes place before impulses are transmitted to more rostrally located CNS structures which

are responsible for pain identification. The late evoked potential

affected by nalbuphine indicates an activity on thalamo-cortical

projection sites which are involved in subjects’ cognitive activities. Kappa-ligands, therefore, seem to exert a different mode

of action than mu-opioids. which results in a modulation of the

negative emotional response associated with pain, rather than an

attenuation of pain impulses.

INTRODUCTION

Monitoring of somatosenory-evoked potentials (SEP's) has gained

accep- tance as a suitable tool for the objective measurement of

efficacy of analgesics in man (Buchsbaum et al.1981; Chapman et

al.1982). As central analgesics differ in regard to their interaction with opioid receptor sites, the study was undertaken in order

to evaluate the possible mode of action of alfentanil, a predominant

mu-opioid,

551
and that of nalbuphine, an agonist-antagonist which mediates analgesia through kappa-receptor interaction (Schmidt et al.1985).

MATERIALS AND METHODS

After institutional approval and informed consent, twenty adult

male volunteers, aged 38-45 years, were studied. Unilateral right

meadian nerve SEP's were obtained from Ag/AgCl surface electrodes

fixed with collodion to the contralateral sensory cortex site (FpZC3', ground right arm strap). Stimulation was of square wave nature

with a duration of 0.2 ms, at a rate of 5/s, a stimuli repetition

of 1024, and a current twice above motor threshold (supramaximal).

At a sampling rate of 256 KHz, and a filter setting between 5-800

Hz, potentials were averaged over a period of 150 ms (Neurotrec,

Interspec Medical, Wisconsin, USA). The cortical wave forms were

assayed in regard of early (less than 50 ms) and late (more than

70 ms) major deflection changes in terms of base to peak amplitude

(μV), compared to control, and identified as either N(=negative)

or P(=positive) polarity. Alfentanil was administered as a slow

bolus of 5 μg/kg, followed by 10 μg/kg 20 min later. 5 min thereafter, naloxone was given (5 μg/kg) i.v. SEP's were derived at

peak drug activity 1 min after injection. Nalbuphine was given

in graded doses (100-, 500-, 1000- μg/kg) every 15 min. Prior to

the next dose, SEP's were derived. After the last injection, naloxone

(5 μg/kg) was given. In addition, subjective pain was evaluated

by determination of tolerance threshold to progressively increased

stimulations from an electrical current (mA), applied to the median

nerve before and after each dose.

RESULTS AND DISCUSSION

Compared to control, alfentanil induced a dose-related decrease

in the amplitude of the early N20-, however, not of the late N100-

wave. This effect correlated closely (r=0.87) with a concomitant

increase in tolerance to electrical current threshold (table 1). The

opioid antagonist naloxone reversed the effects of early amplitude

suppression and the concomitant increase of tolerance to current

threshold.

TABLE 1.

The effect of increasing doses of alfentanil on early

and late SEP changes and the corresponding increase

of tolerance to electrical stimulation (mean +/-SD).

dose μg/kg

(mA)

control

5

10

naloxone

N2 0 (μV) N100 (μV) tolerance

1.5 +/-0.6 1.9 +/-0.9 13.2

1.2 +/-0.6 1.9 +/-1.4 18.5

0.6 +/-0.5 1.9 +/-1.4 24.8

0.9 +/-0.8 2.4 +/-2.2 11.9

552
Nalbuphine induced a dose-related suppression of the amplitude

of the late N100-peak which showed a good correlation (r=0.90)

to an increase of threshold to painful stimuli. This effect could

not be reversed by naloxone (table 2).

TABLE 2.

The effect of increasing doses of nalbuphine on early

and late amplitude changes of median nerve SEP's and the

corresponding increase of tolerance to electrical current

(mean +/-SD).

dose(μg/kg) N2 0 (μV) N100 (μV) tolerance

control 1.5 +/-1.8 1.4 +/-1.1 13.8

100 1.6 +/-1.4 1.4 +/-0.9 18.2

500 1.3 +/-1.1 1.2 +/-0.8 21.7

1000 1.7 +/-2.3 1.1 +/-0.6 23.5

naloxone 1.3 +/-1.1 1.0 +/-0.8 23.5

The peaks of the evoked potentials are the result of stimuli, which,

along their way through the CNS, generate an excitatory response

in different anatomical structures (Buchsbaum et al.1981). Thus,

a possible funtional deficit as induced by an analgesic is reflected

in the generated SEP. By means of identifying the peaks which present drug-induced changes, the possible site(s) of action may be

localized (fig.1).

FIG.1

Representative example of increasing doses of nalbuphine

on late evoked potentials. Note, there is a dose-related

depression of Peak 4 (N100-peak).

553
In addition, by the use of an antagonist, an identification of

the type of receptor mediating the observed neurophysiological

changes is possible.

Early evoked potentials appearing less than 50 ms post stimuli

are generated in the pontine-thalamic region (Allison et al.1980).

This suggests that any change in the early peak (N20) is initiated

in these structures.Since the mu-specific opioid alentanil depresses early evoked potentials in a dose-related fashion, the main

mechanism seems to be that of a blockade of sensory impulses in

subcortical structures. As a result, less activity will reach the

more rostrally located CNS structures which are responsible for

the identification of incoming impulses. The net effect is that

of a loss of pain sensations. One possible subcortical structure

involved in the identification of pain is the pallidum, a part

of the limbic system and the alleged psychomotoric center for pain

sensations and associated conflict feelings (Hassler 1970). As

a result of mu-opioid activity, limbic structures are deprived

from incoming impulses and so the pain is not felt as an aggrevation. Mu-receptor sites seem to play an important part in mediating

this effect.

The suppression of amplitude of the late evoked potential (N100)

by nalbuphine, which was not naloxone-reversible, suggests a receptor site which is not of mu, but rather of kappa nature (Schmidt

et al.1985). Late-evoked potentials are generated in more rostrally

located thalamo-cortical projection sites, where the subjects'

cognitive capabilities are situated (Chapman et al.1982). The

agonist-antagonist, therefore, seems to exert its analgesic activity

mainly through a modulation of impulses coming from the extralemniscal sensory pathway. These neurophysiological findings are in line

with displacement and autoradiographic studies demonstrating a

predominance of kappa-binding sites in the deep layers of the cortical projection sites (Maurer et al.1963; Goodman and Snyder 1984).

Nalbuphine, contrary to the mu-opoid alfentanil, although inducing

a centrally mediated effect, is functionally different than the

pure agonist. This difference is reflected mainly in the supression

of the late and not the early SEP, an effect which is not reversible

by naloxone. Thus, an attenuation of the endless discomfort

ted with pain is induced. A blockade of pain impulses as

with alfentanil is not present.

REFERENCES

Allison, T., Goff, W.R., Williamson, P.D., Wan Gilder, J.C.

Clin Neurophysiol, Vo1.7. Basel, Karger, 1980. pp.51-68.

Eluchsbaum, M.S., Davis, G.C., Coppola. R., Naber, D. Pain, 10:

357-366, 1981.

Chapman, C.R., Colpitts, Y.M., Benedetti, C., Butler, S. Pain,

14:327-337, 1982.

Goodman, R., Snyder S. Life Sci, 31:1291-1294, 1984.

Hassler, R. In: Hassler, R., and Walker; A.E., eds. Trigeminal

Neuralgia. Stuttgart, Thieme, 1970. 123 pp.

Maurer, R., Cortes, R., Probst, A., Palacios, J.M. Life Sci, 33:

231-234, 1983.

Schmidt, W.K., Tam, S.W., Shotzberger, G.S., Smith, D.H.,

Clark, R., Vernier, V.G. Drug Alcohol Depend, 14:339-362, 1985.

associaobserved

Prog

554
CENTRAL AND PERIPHERAL B-ENDORPHIN RESPONSE TO TRANSCUTANEOUS

ELECTRICAL NERVE STIMULATION

F. Facchinetti, G. Sforza*, M. Amidei, C. Cozza*, F. Petraglia,

C. Montanari*, A.R. Genazzani

Department of Obstetrics and Gynecology, University of Modena

via del Pozzo 71, 411OO Modena

*Division of Anesthesia, USL16, S.Agostino Hospital,

Piazzale S.Agostino 228, Modena ITALY

ABSTRACT

The effects of transcutaneous electrical nerve stimulation on plasma

and cerebrospinal fluid (CSF) levels of B-Endorphin (B-EP) were

studied in 6 groups of pain-free patients. Different modes of TENS

were applied for 20 minutes. Regardless of mode and/or frequency of

the stimulation plasma and/or CSF B-EP levels were significantly

increased. Plasma and CSF responses were not correlated suggesting

that TENS activates different opiatergic pathways at central and

peripheral level.

INTRODUCTION

The efficacy of antalgic stimulations are known from thousands.

However, one of their mechanism of action has been elucidated only

after the discovery of the endogenous opioid systems in our species.

Chinese Acupuncture (Mayer et al. 1977; Facchinetti et al. 1981).

electrical acupuncture (Clement-Jones et al. 1980; Nappi et al.

1982), transcutaneous electrical nerve stimulation (TENS) (Facchinetti et al. 1984) all are believed to induce analgesia through an

activation of peptides related to Proopiomelanocortin (POMC) such as

B-endorphin (B-EP). Substantial differences seems to exist between

the various kind of stimulation.

A concomitant release of B-EP and ACTH in peripheral plasma is detectable after electroacupunture (Abbate et al. 1980; Nappi et al.1982)

and stress situations (Guillemin et al.1977), while only B-EP is

increased upon traditional Chinese acupuncture (Facchinetti et

al.1981). As far as TENS is concerned, both opioid and non-opioid

mechanisms have been hypothesized depending on frequency of stimulation (Sjolund and Eriksson 1979; Cheng and Pomeranz 1979). Anyway,

either low frequency (LoF) TENS or high frequency (HiF) TENS are

accompanied by a significant increase of pain threshold (Facchinetti

et al.1984). In this study we compared the effect of TENS application

on the changes of plasma and CSF B-EP in pain free patients.

555

172-683 O - 87 - 19 : QL 3
MATERIALS AND METHODS

Subjects

Six groups of eight patients entered the study.They were pain-free

patients (aged 46-83 ys ; 65.4 on average) undergoing transurethral

resection of benign prostatic hyperplasia,performed in local peridural anesthesia. No premeditation was given and any drug treatment

was stopped for at least two weeks. TENS electrodes were applied

paravertebrally, at Th 12-L 2 level ( 2.29 cm of surface). The

different kinds of stimulation (lasting 20 min) were reported in the

table. TENS units were EPIX model kindly supplied by EMPI Inc (Minneapolis/MN). Ten ml heparinized blood and 5 ml CSF samples were

collected either before or at the end of treatment.Plasma and CSF

samples were added of Trasylol and then stored at -20 C until

assays.

MODE FREQUENCY

(Hertz)

Multimod 80-100

Continous 2-10

Continous 80-100

Continous 2-10

Burst (2Hz) 85

Burst (2Hz) 2-4

INTENSITY WAVE CSF BEP

(mA) (% M ± SE)

50-99 Changing 40.7 ± 18.8

10-30 square,250μsec 71.6 ± 16.2

50-99 52.8 ± 22.5

50-99 54.0 ± 27.7

50-99 109.2 ± 29.2

10-30 13.4 ± 10

Table 1: Details on the different TENS used and effects on

CSF B-EP as % on basal value.

Hormone Assays

Silicic acid extracts of plasma samples and 2 ml freeze-dried CSF

samples were reconstituted to 0.5 ml with 0.1 M Acetic Acid, 0.01%

BSA and applied on Sephadex G-75 column (1.5 x 45 cm) eluted through

the same solution. According to the retention coefficient of cold BEP (0.81), a 18 ml fraction was collected, freeze-dried, redissolved

in 0.12 M phosphate buffer, 0.1% BSA, pH 7.4 and submitted to Radioimmunoassay (RIA) as previously described in detail (Facchinetti et

al.l984). Cortisol was measured in plasma samples through a commercially available RIA Kit (Radim-Italy).

RESULTS AND DISCUSSION

Both plasma and CSF B-EP levels were increased by TENS, with the

exception of burst LoF-LoI stimulation which should be considered

like a placebo. Regardless of mode of stimulation (Bursted, Continous,

Multimod) there is no difference in the B-EP changes in either CSF or

plasma (table 1). Similarly, regardless of the frequency of stimulation, plasma and CSF B-EP levels were increased by at least 40% in

556
respect to basal values (fig.1). Interestingly, similar data were

obtained in plasma by Hughes et al.(1984). Thus these data are opposite to the hypothesis that LoF-HiI TENS would induce analgesia

through an opiate mediation, while HiF-LoI analgesia is supported by a

Figure 1: CSF and plasma B-EP changes after different kinds

of TENS (M±SE).

non-opiate mechanism (Sjolund and Eriksson 1979; Cheng and Pomeranz

1979).

It should be pointed out that few studies reported the direct measurement of endogenous opioids and the hypotesis was build only on

indirect evidences, i.e. the antagonism of analgesia through naloxone. However, as reviewed by Sawynok et al. (1979), naloxone reversal is not a sufficient criterion since different subclasses of

opiate receptors exist and it is still unknown if just one of them

mediate analgesia (Panerai et al.1984). Moreover, many studies were

not performed in healthy controls, but in chronic pain patients (

Abraham et al.1981) where an impairment of endogenous opioid system

has been demonstrated (Genazzani et al.1984). Accordingly, we previously reported that patients affected by malignant diseases were

unable to release B-EP into CSF upon bursted HiF TENS (Facchinetti et

al. 1985). Like electroacupuncture (Nappi et al. 1982), the TENS

557
induced plasma B-EP increase is accompained by a concomitant release

of cortisol levels in many cases, indicating the anterior pituitary

cleavage of POMC in this condition.If this is depending on corticotrophin-releasing hormone release from the hypothalamus or to a direct

effect on the pituitary remain to be clarified. However, despite TENS

increases both plasma and CSF B-EP levels, there is no correlation

between the two phenomena.Indeed, there are cases with a poor response in CSF (less than 20%) and an important release in plasma(more

than 100%) and viceversa. In conclusion regardless of mode and/or

frequency of the stimulation plasma and/or CSF B-EP levels were

significantly increased. Plasma and CSF responses were not correlated

suggesting that TENS activates different opiatergic pathways at central and peripheral level.

REFERENCES

Abbate, D., Santamaria, A., Brambilla, A., Panerai, A.E., Di Giulio, A.M.

Lancet, ii:1309, 1980.

Abram, S.E., Reynolds, A.C., Cusick, J.F. Anesth Analg Curr Res,

60:81-84, 1981.

Cheng, R.S.S., Pomeranz, B. Life Sci, 25:1957-1962, 1979.

Clement-Jones, V., Tomlin, S., Rees, L.H., McLoughlin, L., Besser, G.M.,

Wen, H.L. The Lancet, ii: 946-948, 1980.

Facchinetti, F., Nappi, G., Savoldi, F., Genazzani, A.R.

Cephalalgia, 1: 196-201, 1981.

Facchinetti, F., Sandrini, G., Petraglia, F., Alfonsi, E., Nappi, G.,

Genazzani, A.R. Pain, 19: 259-303, 1984.

Facchinetti, F., Montanari, C., Cioni, A., Cozza, C., Barbieri, F.,

Sforza, G., Petraglia, F., Genazzani, A.R. In: Carraro, U. and

Angelini, C. Functional Electro Stimulation of Neurones

and Muscles. Cleup Editore, 1985. pp.133-138.

Gazzani, A.R., Nappi, G., Facchinetti, F., Micieli, G., Petraglia, F.,

Bono, G., Monittola, C., Savoldi, F. Pain, 18: 127-133, 1984.

Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier, C.,

Vale, W., Bloom, F. Science, 197:1367-1369, 1977.

Hughes, G.S., Lichstein, P.R., Whitlock, D., Harker, C. Physical Therapy,

64:1062-1065, 1984.

Mayer,D.J.,Price, D.D.,Rafii, A. Brain Res, 121: 368-372, 1986.

Nappi, G., Facchinetti, F., Legnante, G., Parrini, D., Petraglia, F.,

Savoldi, F., Genazzani, A.R. Acupuncture & Electro-Therapeutic Res,

Int. J. 7: 93-103, 1982.

Panerai, A.E., Martini, A., Sacerdote, P., Mantegazza, P. Brain Res,

304:153-156, 1984.

Sawynok, J., Pinsky, C., La Bella, F.S. Life Sci., 25:1621-1632, 1979.

Sjolund, B.H., Eriksson, M.B.E. Brain Res, 173:295-301, 1979.

558
LIMBIC REGIONS OF THE BRAIN OF ALZHEIMER'S DISEASE PATIENTS

SHOW SELECTIVE CHANGES IN MU, DELTA AND KAPPA OPIOID

RECEPTOR BINDING

Jacob M. Hiller, Yossef Itzhak* and Eric J. Simon

Departments of Psychiatry and Pharmacology,

New York University Medical Center, New York, NY 10016

*Department of Pharmacology, Hadassah Medical Center

Hebrew University, Jerusalem, Israel

SUMMARY

Total opioid binding and levels of the three major types of

opioid binding sites were measured in homogenates of various

limbic structures from post-mortem brains of Alzheimer's

Disease (AD) patients and age-matched control individuais.

AD brains showed an increase in kappa binding in ail six

areas of the limbic system examined, with the putamen and

caudate regions showing significant increases of 114% and

53%, respectively. In addition, the AD putamen showed a

significantly higher level of total binding (85% increase).

The amygdala of AD patients exhibited significantly lower

levels of mu and delta binding (41% and 55% decrease, respectively).

INTRODUCTION

Deterioration of cognitive function in AD has been imputed in

part to neurochemical deficiencies and pathological changes

in limbic regions of the brain, an area involved in the

complex processes of memory storage and recali. We have

previously reported (Hiller et al. 1973) that in the human

brain, most areas containing high leveis of opioid receptors

lie within or are closely associated with the limblc system.

The possible involvement of the endogenous opioid system in

memory storage and physiological amnesia has been indicated

in several studies. The similarity between the anatomical

distribution of opioid receptors and pathoiogical markers of

AD and the putative role of the opioid system in cognitive

functions prompted us to undertake a study comparing opioid

receptor populations in various limbic structures obtained at

autopsy from AD patients and age matched individuais unaffected by this disease.

MATERIALS AND METHODS

Brain tissue samples, obtained at autopsy less than 24 hours

after death, from control individuals and AD patients were

stored at -70°C until processed into crude membrane preparations (Lin and Simon 1978). The regions dissected for this

study, using standard anatomical landmarks, were : caudate,

frontal cortex, amygdala, temporal cortex, putamen and hippocampus. Brain samples were examined for the presence of

pathology by Drs. D.J. Selkoe (Harvard Medical School. Boston, MA) J. Pearson (New York University Medical Center, New

York, N.Y.) and J. Uirich (Basel University, Basel, Switzer559
land), from whom these post mortem brain samples were obtained.

Binding assays were carried out essentially as previously

described (Hiller et al. 1973) at 25° for one hour in the

presence and absence of a 1000-fold excess of naloxone. Mu

site binding was measured with 3H-D-Ala2

MePhe4-Gly-ol5-

enkephalin (

3

H-DAGO, 2 nM, 49 Ci/mmol, Amersham, Des Plaines,

IL). To assess delta site binding, 3

H-D-Ala2-D-Leu5 enkephalin (

3

H-DADLE, 2 nM, 43 Ci/mmol, New England Nuclear, Boston,

MA) was used. 3

H-bremazocine (2 nM 32 Ci/nmol, New England

Nuclear, Boston, MA) possessing almost equal affinity for mu,

delta and kappa binding sites, was used to assess total

binding levels. Selective binding to kappa sites was determined using 2 nM 3

H-bremazoclne in the presence of 100 nM

each of unlabeled DAGO (Reckitt and Coleman, Hull, England)

and DADLE (Peninsula Lab., Inc., San Carlos, CA). AnalySiS Of

data was carried out using the Mann-Whitney U test.

RESULTS AND DISCUSSION

Binding studies were carried out on samples from twelve

autopsied brains, seven from AD patients (ages 58-68 years)

and five from control individuals (ages 54-60 years). The

presence of the characteristic neuropathological changes,

consisting of neurofibrillary tangles and neurotic plaques

in AD samples were found as well as the absence of anomalies

in control samples. In those cases where the number of individual determinations of binding site levels differs from the

number Of autopsied cases. the amount of material recerved

was insufficient to carry out the assay. Results from equilibrium binding studies indicated that, in some regions of the

brain of AD patients, statzistically significant differences

from control samples were observable. A summary of results

from AD and control samples is presented in tabie 1.

Total opioid receptor binding, as determined by the binding

of 3

H-bremazocine, was increased in the AD putamen over

control samples by 85% (p<0.02). Assessment of mu opioid

binding sites with 3

-DAGO demonstrated a 41% decrease in AD

amygdaia (p<0.06). Delta opioid binding, as determined by 3HDADLE binding was decreased in the AD amygdaia by 55%

(P<0.03). In addition, delta opioid binding was reduced in

AD temporal cortex by 41%. This change from control level,

however, was not significant (p<0.09). Kappa opioid binding

was assessed by the binding of 3

H-bremazocine in the presence

of saturating concentrations of DAGO and DADLE. In all six

areas of the AD limbic system kappa binding was increased

(12% to 114%) over levels seen in control samples. Of these

six areas the caudate and the putamen showed statistically

significant increases of 53% (p<0.03) and 114%.

560
TABLE 1

Opioid receptor binding in limbic areas of control (C)

and Alzheimer's Disease (AD) brains

fMoles/as protein

3

H-DAGO 3H-DADLE 3H-BREM

BLOCKED

Caudate(C)

±S.E.

(N)

Caudate(AD)

±S.E.

(N)

P

15.45

1.17

(4)

14.40

1.95

(4)

N.S.

20.02

1.66

(5)

25.29

6.00

(7)

N.S.

Frontal Cortex(C) 17.50 24.66

±S.E. 2.10 3.74

(N) (5) (5)

Frontal Cortex(AD) 24.93 27.16

±S.E. 5.61 4.84

(N) (7) (7)

P N.S. N.S.

Amygdala(C) 25.74

±S.E. 3.45

(N) (5)

Amygdala(AD) 15.14

±S.E. 4.77

(N) (7)

P <0.06

30.48

6.90

(5)

13.69

4.31

(7)

<0.03

Temporal Cortex(C) 26.40

±S.E. 5.51

(N) (4)

Temporal Cortex(AD)20.41

±S.E. 4.21

(N) (7)

P N.S.

37.90

8.18

(4)

22.33

3.60

(7)

<0.09

Putamen 9.63

±S.E. 3.37

(N) (3)

Putamen(AD) 14.77

±S.E. 2.63

(N) (7)

P N.S.

13.60

2.69

(3)

16.04

3.04

(7)

N.S.

Hippocampus(C) 4.20

±S.E. 0.02

(N) (3)

Hippocampus(AD) 4.18

±S.E. 1.04

(N) (6)

P N.S.

6.83

0.54

(3)

8.72

2.31

(6)

N.S.

49.36

6.70

(5)

75.59

8.71

(7)

<0.03

60.94

11.08

(5)

94.64

15.08

(7)

N.S.

117.50

27.83

(5)

131.53

23.60

(7)

N.S.

92.47

26.74

(3)

113.45

27.29

(4)

N.S.

27.97

5.49

(3)

59.84

7.12

(7)

<0.05

39.53

2.97

(3)

64.18

11.52

(4)

N.S.

3

H-BREM

124.40

7.84

(5)

155.20

17.28

(7)

N.S.

152.80

17.59

(5)

208.37

21.17

(7)

N.S.

239.20

18.29

(5)

191.41

31.58

(7)

N.S.

188.65

36.02

(4)

171.44

23.41

(4)

N.S.

66.23

8.16

(3)

122.66

8.84

(7)

<0.02

77.83

7.26

(3)

93.52

26.89

(6)

N.S.

561
(p<0.02), respectrveiy. if total kappa binding in all control

and AD brain areas is compared, an increase in total kappa

binding of 35% is noted in the AD group with a highly significant p value of <0.01.

The simplest explanation for our observation of lowered levels of mu and delta binding is that the manifestation of

AD involves cellular patterns of pathology that affect those

cells bearing mu and delta type opioid receptors. However,

other possible explanations do exist. One that seems quite

plausible is "homospecific up- or down-reguiation" in which

certain ligands can modulate the number or affinity of their

own receptors. For example, chronic exposure of HG108-15

cells to opiate agonists leads to a down-regulation of opioid

binding (Chang et al. 1982). In contrast, chronic exposure

of rats to opiate antagonists caused a 140% increase in 3

Hetorphine binding in limbic regions (Zukin et al. 1982).

This last phenomenon may be explained by a mechanism which

up-regulates the receptors by chronic displacement of endogenous opioid ligands. It is possible that our observed increase in kappa receptor binding may be due to an up-regulation caused by a decrease of dynorphin content in the AD

limbic system. Support for this theory may be derived from

the report (Pongdhana et al. 1985) showing that intraperitoneal injection of ketamine increased the dynorphin content of

rat brain cortex while decreasing it in the septal area.

Simultaneously, the binding affinity of the kappa ligand,

ethylketocyciarocine, was decreased in the cortex but increased in the septal area. Dynorphin content of the human

caudate and putamen is among the highest in human brain

(Gramsh et al. 1982).

REFERENCES

Chang, K-J., Eckel, R.W., and Blanchard, S.G. Nature,

296:446-448, 1982.

Gramsch, C., Holtt, V., Pasi, A., Mehraein, P., and Herz, A.

Brain Research, 233:65-74, 1942.

Hilier, J.M., Pearson, J. and Simon, E.J. Res Commun Chem

Pathol Pharmacol, 6:1052-1061, 1973.

Lin, H-K. and Simon, E.J. Nature, 271:383-384, 1978.

Pongdhana, K., Ogawa, N., Hirose. Y., Ohara, S., Kosaka, F.

and Tachibana, S. Neurochem Res, 10:945-952, 1985.

Zukin, R.S., Sugarman, J.R., Fitz-Syage, M.L.. Gardner,

E.L. , Zukin, S.R. and Gintzier, A.R. Brain Research, 245:285-

292, 1982.

ACKNOWLEDGMENTS

This work was supported by grant 83-003 from the Alzheimer's

Disease and Related Disorders Association to Y.I. and grant

DA 00017 from the National Institute on Drug Abuse to E.J.S.

562
DIFFERENTIAL EFFECTS OF MU AND KAPPA OPIOID

SYSTEMS ON MOTIVATIONAL PROCESSES

T.S. Shippenberg and A. Herz

Department of Neuropharmacology,

Max-Planck-Institut für Psychiatrie,

D-8033 Martinsried, F.R.G.

ABSTRACT

The role of multiple opioid receptor types in

mediating the motivational properties of opioid

agonists and antagonists was investigated by means

of a place-preference conditioning procedure.

Mu-agonists produced positive reinforcing effects

in drug-naive animals whereas kappa agonists and

the opioid antagonist, naloxone, were aversive.

Chronic infusion of naloxone during conditioning,

at a dose sufficient to block mu- but not

kappa-receptors, antagonized the effects of both

morphine and naloxone. This treatment did not alter

the aversion produced by the kappa agonist, U69593.

These data demonstrate that opioid reinforcement

results from an activation of mu-receptors whereas

aversive effects are produced by either an antagonist action at this receptor or an activation of

kappa receptors. Such results indicate that mu and

kappa opioid systems differentially modulate

motivational processes.

INTRODUCTION

There is now substantial evidence to indicate the

existence of multiple opioid receptor types within

the mammalian CNS (Lord et al. 1977; Martin et al.

1976). However, despite extensive study, the role

of these various receptor types in the regulation

of motivational processes remains ill-defined. In

the present study, a place preference conditioning

procedure was utilized to characterize the

motivational properties of opioids and examine the

role of mu (μ)- and kappa ( )-receptors in mediating their reinforcing and/or aversive effects. Conditioning produced by the μ-agonist, morphine; the

K-agonist, U69593; and the opioid antagonist,

naloxone (NLX) were evaluated in drug-naive animals

and those chronically treated with NLX at a dose

which selectively blocks mu-receptors. Evidence is

presented to indicate that μ- and - opioid systems

exert opposite effects upon motivational processes.

MATERIAL AND METHODS

Subjects were male Sprague-Dawley rats weighing

120-160g. One day prior to the commencement of

563
experiments, rats were implanted, under light ether

anethesia, with osmotic minipumps (Alzet Model

2001) containing either the opioid antagonist, NLX,

at a dose (0.55 mg/kg/hr) sufficient to block μbut not K-receptors (Millan et al. submitted) or

distilled water. Using a place-preference conditioning procedure (Mucha et al. 1982), separate

groups of implanted and drug-naive (control) rats

were administered (s.c.) various doses of morphine,

U69593, or NLX. Each rat received alternate day

injections of drug or vehicle for 6 days. Following

drug injections, rats were immediately confined to

a visually and texturally distinct environment i.e.

black box with smooth black floor for either 60 min

(morphine and U69593) or 30 min (NLX). Following

injections of vehicle, rats were confined to a

different environment, i.e. white box with rough

white floor. The order of injections and drugpaired environments were counterbalanced within

groups. On day 7, preference for a particular place

was determined by allowing uninjected rats free

access to both environments and measuring the time

spent in each. Analyses of variance and the

Student- Newman Keuls test were used to determine

the statistical significance of results (Zarr

1974).

RESULTS AND DISCUSSION

Figure la demonstrates that, in control rats,

morphine was positively reinforcing as indicated by

a significant preference for the drug-paired place.

However, in rats chronically treated with NLX, at a

dose which selectively blocks B-receptors, the

reinforcinq effect of morphine (3.0 mg/kg) was

abolished. These results suggest that the

reinforcing properties of opioid agonists results

specifically from an activation of the μ-opioid

receptor.

564
FIGURE 1: Motivational properties of μ- and

receptor agonists in control and chronic NLXtreated rats. Each point represents the conditioning score (mean ± S.E.M.) of 8-10 rats.

**p < 0.001: significant difference between

control and NLX-treated rats.

In contrast to morphine, U69593 produced dose-related aversions for the drug-paired place (figure

lb). If the aversion resulted from an agonist action at k-receptors, selective p-receptor blockade

should be ineffective in altering its motivational

properties. As shown in figure lb, 0.16 mg/kg

U69593 was aversive in both control and NLX-treated

rats. Taken together, these data indicate that

μ-and opioid systems have opposing motivational

effects: activation of the former is reinforcing

while activation of the latter is aversive.

Previous studies using the place conditioning procedure (Bechera and Van der Kooy 1985, Mucha et al.

1982) have demonstrated that NLX is aversive in

drug-naive animals. As the reinforcing effect of

morphine is mediated by an activation of μ- receptors, we hypothesized that the aversive property of

NLX results specifically from an antagonism of this

receptor. Therefore, chronic p-receptor blockade

during training should mask the b-antagonist activity of acute NLX, resulting in an attenuation of

its aversive effects. The results shown in figure 2

confirm this prediction. Administration of NLX (5.0

mg/kg) was highly aversive in control animals.

Mu-receptor blockade abolished this effect. These

data indicate the aversive properties of NLX result

from antagonist effects at the μ-opioid receptor.

565
FIGURE 2: Effect of b-receptor blockade on the

aversive properties of NLX. Each point

represents the mean ± S.E.M. of 10 rats (**P <

0.001).

In conclusion, these studies demonstrate that opioids with agonist actions at the μ-receptor function

as positive reinforcers whereas agonists are

aversive. Such results suggest the existence of

opposing opioid systems which regulate motivation.

Moreover, these data reveal that contrasting mechanisms underlie the aversive effects of NLX as compared to agonists.

REFERENCES

Bechara, A., and van der Kooy, D. Nature,

314:533-534, 1985.

Lord, J.A., Waterfield, A.A., Hughes, J., and

Kosterlitz, H. Nature, 267:495-499, 1977.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler,

R.E., and Gilbert, P.E. J Pharmac Exp Thr,

197:517-532, 1976.

Millan, M.J., Cz onkowski, A., and Herz, A. submitted.

Mucha, R.F., van der Kooy, D., O'Shaughnessy, H.

and Bucenieks, P. Brain Res, 243:91-105, 1982.

Zarr, J.H. Biostatistical Analysis, Englewood

Cliffs, Prentice Hall Inc., 1974. 620 pp.

ACKNOWLEDGEMENTS

Morphine sulfate was purchased from Merck Inc.,

Darmstadt, F.R.G. Naloxone hydrochloride and U69593

were qenerously supplied bv DuPont Inc., Wilminqtion, DE. and The UpJohn Co., Kalamazoo, Mi., respectively.

These studies were supported by the Bundesgesundheitsamt, Berlin.

566
AVERSIVE PROPERTIES OF OPIATE WITHDRAWAL

STUDIED IN RATS

Ronald F. Mucha*, **Mary D. Gritti** and Chul Kim*

*Biobehavioral Research Department

Addiction Research Foundation, Toronto, Ontario

**Department of Pharmacology

Canada M5S 2S1

Univ. of Toronto, Toronto, Ontario, Canada M5S 2S8

ABSTRACT

Preference conditioning models were applied to the

study of the motivational properties of opiate withdrawal. Conditioned place and taste aversions were

produced by a single injection of an opiate antagonist

in rats implanted with a morphine but not a placebo

pellet. The hypothesis that withdrawal-produced jumping reflects the aversive properties of withdrawal was

not confirmed. The use of central and peripheral

administration of antagonists suggest that the aversive

effects reflect antagonist activity in the brain.

INTRODUCTION

Although threats of drug intake reduction or of withdrawal are powerful stimuli for activating drug seeking

behaviors in humans, the motivational properties of

withdrawal have received little attention in the

experimental literature on opiate physical dependence.

The best examined models are contractures in the

isolated opiate-treated guinea pig ileum (Schulz and

Herz 1976), and "wet dog shakes", body weight loss and

jumping or escape attempts in opiate-treated rodents

(Blasig et al. 1973, Wei et al. 1973). However, only

in the case of jumping has there been any attempt to

relate a withdrawal sign to the discomfort described by

withdrawing addicts; withdrawal-produced jumping is

said to be similar to behavior elicited by electric

shock and can be modified by variables believed to be

motivational (Siegel et al. 1975).

Previous work suggested that taste (Pilcher and

Stolerman 1976) and place preference conditioning procedures (Mucha et al. 1982) could be used to directly

assess motivational properties of opiate withdrawal,

but no attempt has been made to systematically extend

these findings. Accordingly, we describe here progress

on studies designed 1) to establish efficient models of

withdrawal aversiveness in rodents, 2) to test the

hypothesis that withdrawal jumping reflects withdrawal

aversiveness and 3) to determine the general location

of the receptors involved in the aversions.

567
MATERIALS AND METHODS

Male Sprague-Dawley rats obtained from St. Constant,

Quebec, were used. Conditions of housing, testing, and

general experimental design were outlined previously

(Mucha and Herz 1986, Mucha and Iversen 1984), except

that the composition of the flavors for the taste

conditioning here comprised a) 92.7 mM NaCl, and 9.36

mM MSG and b) 1.13 mM citric acid and 0.71 mM

saccharine. All subjects were implanted using halothane

with a morphine (containing 75 mg morphine base) or

placebo pellet. Where necessary, a cannula was

implanted stereotaxically in the lateral ventricle.

Drug administration was SC (approximately 1 ml/kg) or

ICV through the cannula (4 μl). Peripheral doses of

antagonists were expressed as free base.

Withdrawal aversiveness was assessed by measuring the

preference of the subject for a cue paired with a

single administration of an opiate antagonist as

opposed to a different cue paired with the

corresponding vehicle administration. With place

training, animals were injected first with antagonist

or vehicle and then placed for 1 h into one of the two

place-training boxes and 7-24 h later injected with

vehicle or antagonist and placed into the other box.

For the taste training, rats were given 3 mls of the

appropriate flavor to drink after water deprivation

overnight and then given the necessary injection.

Unless otherwise stated, training was carried out on

the 5th and 6th day after pellet implantation; testing

was always one day after training. Place testing

involved giving individual rats access for 15 min to a

rectangular box with the two sets of training boxes

(cues) separated by a neutral grid platform. Taste

testing involved one day ad lib access to the two

flavors. Other assessment of withdrawal signs was

carried out after the antagonist administration during

the place training sessions. For the first 0.5 h after

injection rats were observed for diarrhea, jumping,

writhing and head or body shakes.

The place conditioning score was the time on the side

of the test box where withdrawal was experienced

(administration of antagonist) minus time on the

control side. The taste score comprised the amount

consumed of antagonist-paired flavor minus the amount

consumed that paired with vehicle, expressed as a

percent of total intake. An effect was considered

significant with a p<.05.

RESULTS AND DISCUSSION

As assessed with taste conditioning using a low

training dose (0.015 mg/kg) in animals trained at

568
different times after implantation with morphine

pellets, naloxone was most potent when the training was

4-8 days after pellet implantation. Mean serum

morphine levels measured (Kim and Kats 1984) in tail

vein blood of 6 rats run in parallel were 126.0 ± 15.2

ng/kg on the 4th and 70.9 ± 19.0 ng/ml on the 9th day

after implantation but fell sharply thereafter (Day 11:

18.0 ± 10.1 ng/ml). On Day 14 naloxone produced almost

no effects (X = +17.3 ± 24%, n = 8) in contrast to

significant conditioning on Day 7 (X = -55.3 ± 16,4%,

n = 11).

Dose response curves were measured during the abovementioned period of peak naloxone sensitivity (fig. 1)

and revealed maximal aversions with 0.05 mg/kg in

pellet animals; however, even 1.5 mg/kg failed to

produce significant aversions in placebo rats. As seen

in fig. 2, potent aversive effects in pellet animals

were not limited to the use of naloxone nor the SC

route; corresponding significant aversive effects were

not seen in placebo controls (not shown). Given the

different conditions and the dose ranges where the

drugs were effective, it was concluded that the potent

aversive effects of opiate antagonists in morphine

pellet-implanted animals are probably due to their

antagonistic action and therefore reflect the aversive

properties of opiate withdrawal.

FIGURE 1 Taste conditioning scores in rats (ns =

8-10) implanted for 5 days with a morphine or

placebo pellet.

In addition, the jumping response and the aversive

state produced by withdrawal were contrasted over

different conditions of antagonist administration.

With SC naltrexone, the aversion paralleled jumping,

but in rats trained with ICV quaternary-naltrexone it

did not (fig. 2). Accordingly, we could not support

the hypothesis that presence of aversive effects was

reflected in jumping. Also, none of the other signs of

withdrawal showed a good correlation with the place

aversions (not shown).

569
FIGURE 2 Place conditioning scores and jumping

measured in the same animals after naltrexone

given SC (left) or quaternary naltrexone ICV

(right). Implantation: circles, pellet; squares,

placebo.

It should be noted that quaternary naltrexone does not

readily cross the blood brain barrier but produced

clear place aversions when given centrally (fig. 2).

Therefore, it is likely that the receptors responsible

for the withdrawal-related aversive effects are

somewhere in the brain.

REFERENCES

Bläsig, J., Herz, A., Reinhold, K., and

Zieglgansberger, S. Psychopharmacol, 33:19-38, 1973.

Kim, C., and Kats, T. J Anal Toxicol, 8:135-137, 1984.

Mucha, R.F., and Herz, A. Life Sci, 38:241-249, 1986.

Mucha, R.F., and Iversen, A. Psychopharmacol,

82:241-247, 1984.

Mucha, R.F., van der Kooy, D., O'Shaughnessy, M., and

Bucenieks, P. Brain Res, 243:91-105, 1982.

Pilcher, C.W.T., and Stolerman, I.P. P B &B,

4:159-163, 1976.

Russell, J., Bass, P., Goldberg, L.I., Schuster, C.R.,

and Merz, H. Eur J Pharmacol, 78:255-261, 1982.

Siegel, R.K., Gusewelle, B.E., and Jarvik, M.E.

Int Pharmacopsychiat, 10:17-23, 1975.

Wei, E. Psychopharmacol, 28:35-44, 1973.

Wei, E., Loh, H.H., and Way, E.L. J Pharmacol

Exptl Therap, 198:398-403, 1973.

ACKNOWLEDGEMENTS

Supported by MRC grant MA-9552. Gifts of drugs from du

Pont deNemours Ltd. and Dr. Merz are gratefully

acknowledged.

570
MORPHINE WITHDRAWAL IN GUINEA-PIG ILEUM IS CONFINED TO ONE

ELECTROPHYSIOLOGICALLY DEFINED CLASS OF MYENTERIC NEURONE.

Stephen M. Johnson, Marcello Costa and John B. Furness

Addiction Research Laboratory

Centre for Neuroscience and Depts. of Physiology

and Anatomy

Flinders University of South Australia

Bedford Park 5042 Australia

ABSTRACT

Intracellular microelectrodes were used to record changes in

myenteric neurones underlying morphine dependence in the guinea-pig

ileum. Of the two electrophysiologically defined classes of

neurones (S and AH), only a proportion of S-neurones were involved

in the naloxone-induced withdrawal responses. Withdrawal caused

direct depolarization and discharge of action potentials in Sneurones and also resulted in synaptic activation of neurones of

this class. Both fast and slow synaptically mediated

depolarizations were evoked by naloxone. The experiments suggest

that morphine withdrawal is expressed in quite specific neuronal

circuits in the myenteric plexus.

INTRODUCTION

The analysis of opiate dependence in the guinea-pig myenteric

plexus has been based largely on studies of the nerve-mediated

contraction of the longitudinal muscle evoked in morphinepretreated tissues by naloxone. The naloxone-induced contraction

is due predominantly to activation of cholinergic motor neurones

since it is depressed by tetrodotoxin and atropine (Collier et al.

1981). The residual non-cholinergic component of the contraction

may be due in part to the release of substance P (Gintzler 1980;

Tsou et al. 1985). The present study aimed to analyse the changes

underlying morphine dependence more directly by using intracellular

microelectrodes to record from myenteric neurones.

MATERIALS AND METHODS

Guinea-pigs were pretreated either by injections three times daily

with increasing doses of morphine (10 - 100 mg/kg s.c.) for 3-5

days, or by subcutaneous implantation of a pellet containing a

mixture of morphine base (120 mg) and morphine hydrochloride (30

mg) for 7 days. Longitudinal smooth muscle-myenteric plexus (LSMMP) preparations from the treated animals were set up in

physiological saline solution (PSS) containing 1 μM morphine in

order to maintain continuous exposure to morphine in vitro (Schulz

and Herz 1976). These preparations responded to naloxone (1 μM)

with a powerful nerve-mediated contraction of the longitudinal

muscle, the characteristic index of morphine dependence in the

guinea-pig ileum (Schulz and Herz 1976; Collier et al. 1981).

Control preparations, from untreated guinea-pigs, were unaffected

571
by naloxone.

Similar LSM-MP preparations were mounted in a tissue chamber for

intracellular recording from myenteric neurones. In addition to

morphine (1 μm), the PSS perfusing the preparations contained

hyoscine (1 μm) and nicardipine (3 μm) to prevent contractions of

the longitudinal muscle during intracellular recording from

myenteric neurones.

Intracellular recordings were made from both S and AH neurones

(Hirst et al. 1974). Neurones in which electrical stimulation of

fibre tracts evoked fast cholinergic excitatory postsynaptic

potentials (e.p.s.p.'s) were classified as S-neurones. AH neurones

received no fast synaptic input and were defined by the long

lasting (2 - 30 sec) after-hyperpolarization accompanying an

action potential evoked in the soma. After a stable impalement of

a neurone had been obtained, naloxone (1 m) was added to the

perfusate to displace morphine from its receptors so that

electrophysiological changes associated with withdrawal could be

recorded.

RESULTS AND DISCUSSION

1) S-neurones

Intracellular recordings were obtained from 53 S-neurones and 40

AH-neurones in LSM-MP preparations from 51 morphine-pretreated

guinea-pigs.

50% of S-neurones in morphine-dependent LSM-MP preparations showed

changes in their properties during naloxone-induced withdrawal.

Naloxone did not alter the electrophysiological properties of Sneurones in control tissues. The most frequent withdrawal response

was a membrane depolarization (12.1 ± 1.2 mV; mean ± S.E.M.)

observed in 40% of neurones. The change in membrane potential,

which was sustained throughout the 5-10 minute perfusion with

naloxone, was associated with a slight but not statistically

significant increase in input resistance (120 ± 30 M cf. 101 ±

22 M ; means ± S.E.M.). Twenty per cent of S-neurones responded

with a depolarization which was accompanied by discharge of action

potentials (0.5 - 10 Hz).

In 12% of S-neurones, naloxone evoked fast depolarizations which

were similar in amplitude (5 - 20 mV) and time course (20 - 50

msec) to the fast cholinergic e.p.s.p. These fast depolarizations

dissapeared after synaptic transmission was blocked by raising the

concentration of Mg++ in the PSS from 1.7 mM to 10 or 20 mM, or by

lowering the concentration of Ca++ from 2.5 mM to 0.5 mM, or by a

combination of reduced Ca++ and raised Mg++. This observation

suggested that the withdrawal response was initiated by excitation

of a cholinergic interneurone synapsing with the impaled Sneurone. The activation of interneurones during withdrawal was

further suggested by the observation that the naloxone-induced slow

depolarization was also reduced in some cells after synaptic

572
transmission blockade. Naloxone-induced withdrawal may therefore

involve activation of not only the final motor neurones (leading to

contraction of the longitudinal muscle) but also of cholinergic

interneurones and interneurones producing slow

depolarizations in S-neurones (Johnson et al. 1980).

synaptic

In a small proportion (6%) of neurones naloxone evoked action

potentials which were not associated with any change in the

underlying membrane potential or with fast e.p.s.p.'s. The

discharge of action potentials persisted after synaptic

transmission blockade. These neurones therefore represent those in

which withdrawal responses are initiated.

FIGURE 1. Possible arrangement of myenteric Sneurones involved in naloxone-induced withdrawal

responses. During withdrawal the impaled neurone

receives synaptic input from cholinergic interneurones

(Ach) and from interneurones which release transmitter(s) producing slow depolarizations. Excitation of

final motor neurones, either directly or by synaptic

activation, leads to contraction of the muscle by

release of Ach and substance P (SP) (not necessarily

from the same motor neurone). The axon of the impaled

neurone has been interrupted in the diagram to denote

that it is not identified as a motor neurone by the

intracellular recording technique.

573
2) AH-neurones

In contrast to its effects on S-neurones, naloxone did not alter

the membrane potential or evoke action potential discharge in AHneurones of morphine-dependent LSM-MP preparations. Furthermore,

naloxone did not alter the input resistance, soma action potential

configuration or the slow hyperpolarization following a soma spike.

The specificity of the withdrawal responses for S-neurones and

their restriction to only a proportion of those neurones suggests

that morphine withdrawal may occur in quite specific circuits in

the myenteric plexus. Fig. 1 shows the possible arrangement of

neurones participating in the withdrawal responses. The further

characterization of these neurones will provide a better

understanding of the cellular basis of morphine dependence.

REFERENCES

Collier, H.O.J., Cuthbert, N.J. and Francis, D.L. Brit J Pharmac,

73: 921-932, 1981.

Gintzler, A.R. Brain Res, 182: 224-228, 1980.

Hirst, G.D.S., Holman, M.E. and Spence, I. J Physiol, 236: 303-

326, 1974.

Johnson, S.M., Katayama, Y. and North, R.A. J Physiol, 301: 505-

516, 1980.

Schulz, R. and Herz, A. Life Sci, 19: 1117-1128, 1976.

TSOU, K., Shi-Xiang, W., Ti-An, H. and Way, E.L. Eur J Pharmac,

110, 155-156, 1985.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Health &

Medical Research Council of Australia and from the Flinders Medical

Centre Research Foundation.

574
ENKEPHALINASE INHIBITORS ATTENUATE NALOXONE-PRECIPITATED

WITHDRAWAL SYNDROME

M.R. Dzoljic

Department of Pharmacology, Faculty of Medicine,

Erasmus University Rotterdam, P.O.Box 1738,

3000 Rotterdam, The Netherlands

ABSTRACT

Enkephalinase inhibitors phosphoramidon, thiorphan or phelorphan

suppressed naloxone-precipitated withdrawal symptoms in acute and

chronic morphine dependent mice and rats. It is suggested that

decreased biotransformation of the endogenous opioid peptides might

compensate the relative shortage of exogenous opiates during

withdrawal in opiate dependent subjects and could be a new and

promising way to attenuate the severity of abstinential syndrome.

INTRODUCTION

Exogenous administration of opioid peptides

withdrawal in animals (Blasig and Herz

suppress the opiate

1976) and man (Wen and Ho

1982). Similarly, blockade of biodegradation of endogenous opioid

peptides by the unspecific peptidase inhibitors aprotonin and

bacitracin or by the relatively specific enkephalinase inhibitor

phosphoramidon, suppressed the severity of the opiate withdrawal syndrome in rats and nitrous oxide convulsant withdrawal in mice,

respectively (Pinsky et al. 1982, Dzoljic et al. 1984). These data

indicate that enhancing of endogenous opioid peptides, by decreasing

a biotransformation of these substances, might have an attenuating

effect on the narcotic withdrawal syndrome, similar to the exogenously administered opioid peptides or opiates.

In order to study the role of endogenous opioid peptides in morphine

addicted animals we examined the effect of relatively specific

enkephalinase inhibitors, phosphoramidon, thiorphan or phelorphan,

on the withdrawal syndrome in acute or chronic morphine dependent

in mice and rats.

METHODS AND MATERIALS

Rats. Male wistar rats (200-225 g) were used in experiments. A steel

cannula for intracerebroventricular (i.c.v.) administration of

artificial cerebrospinal fluid (CSF) or enkephalinase inhibitors

was implanted stereotaxically into the left lateral ventricle.

Chronic morphine dependence was induced by subcutaneously (s.c.)

implanted pellets (75 mg morphine), according to the method described

by Blasig et al. (1973).

Mice. Adult male mice of B10A strain were used. Administration of CSF

or enkephalinase inhibitors was carried out by means of a stainless

guide cannula stereotaxically implanted in the left lateral ventricle.

Acute morphine dependence was induced by a single s.c. injection of

morphine sulphate (32 mg/kg). In order to observe the withdrawal

syndrome, two hours later mice were injected intraperitoneally (i.p.)

with naloxone hydrochloride (16 mg/kg). In general, the experimental

575
model of Kosersky et al. (1974) for acute withdrawal jumping in mice

was followed.

Chronic morphine dependence was induced by two daily s.c. injections

of morphine sulphate with a starting dose of 50 mg/kg and increasing

daily by 25 mg/kg to reach a final dose of 200 mg/kg on the fifth day.

On the 8th day a last morphine injection of 225 mg/kg was given,

followed by naloxone 16 mg/kg i.p. (Takemori and Sprague 1978).

Drugs. Phosphoramidon (Cambridge Research Biochemical, New York).

thiorphan (CRB, Cambridge) and phelorphan (donated by the Division

of Pharmaceutical Chemistry, University of Amsterdam) were dissolved

in CSF before i.c.v. administration. Naloxone hydrochloride (Endo

Lab.) and morphine sulphate (Chemiefarma NV, Maarsen) were dissolved

in saline.

Statistical analysis. The significance of the differences in the

different group means are evaluated by the Mann-Whitney U test.

Significance was accepted at P<0.05.

RESULTS

Phosphoramidon (50-200 μg i.c.v.) suppressed, in a dose-dependent

manner, the naloxone precipitated withdrawal jumping but potentiated

forelimb shakes in both acute and chronic dependent mice. Both

thiorphan and equimolar doses of phelorphan decreased the severity

of the naloxone-precipitated withdrawal syndrome in rats (table 1).

However, some symptoms remained unchanged or even potentiated

(table 1).

DISCUSSION

The essential observation in this study is that enkephalinase

inhibitors, phosphoramidon, thiorphan or phelorphan attenuate the

severity of morphine withdrawal syndrome in mice and rat. However,

some of the withdrawal symptoms were either not altered or even

potentiated. For example, phosphoramidon suppressed withdrawal

jumping in the mice, while forelimb shakes were potentiated. The

failure of phosphoramidon to inhibit the forelimb tremor is not clear.

The withdrawal syndrome is a complex behavioural pattern, mediated by

a balance of many transmitter systems in various brain regions.

Unequal distribution of i.c.v. administered phosphoramidon and other

enkephalinase inhibitors might be a reason that various opiate withdrawal symptoms were unequally affected. Furthermore, a comparison of

the effects of equimolar concentrations of thiorphan and phelorphan

shows that phelorphan has a less prominent antiwithdrawal effect than

thiorphan. These differences might be due to the unequal penetration

into the various brain regions or to the unequal capacity to inhibit

the biodegradation of the enkephalins. It is of interest to note

that some sexual manifestation during naloxone-precipitated

withdrawal were potentiated by administration of thiorphan or

phelorphan (penile licking, erection, ejaculation). It supports the

idea that sexual response might be potentiated by any condition

activating the endogenous opioid system (Henry 1982), including the

enkephalinase inhibitors (this study). The decrease or abolition of

the abstinential diarrhea by i.c.v. administered enkephalinase

inhibitors is in accordance with suggestion that opioid peptides

might regulate the intestinal transit by a central mechanism

(Galligan and Burks 1982).

576
Incidence of the withdrawal signs (counted signs)

CSF THIORPHAN PHELORPHAN

2 ul 40 ug/2 ul i.c.v. 62 ug/2 ul i.c.v.

WITHDRAWAL SIGNS (n=9) (n=9) (n=8)

teeth chattering 5.6 ± 2.4

wet dog shakes 10.5 ± 1.7

grooming 4.0 ± O.7

scratching 0.2 ± O.1

penile licking 5.5 ± O.4

writhing 2.3 ± 0.5

rearing 9.5 ± 2.1

digging 31. ± 5.3

head hiding 27.1 ± 5.7

chewing 69.1 ± 11.0

paw tremor 1.8 ± O.5

head shakes 1.7 ± O.3

stretching 13.2 ± 3.1

1.9 ± 0.6*

10.0 ± 1.7

3.2 ± 0.9

0.4 ± 0.2

8.7 ± 1.0*

6.8 ± 1.4

4.0 ± 0.9”

1.2 ± 0.8”

32.9 ± 8.0*

2.2 ± 0.8

3.0 ± 1.8

8.9 ± 2.4

6.7 ± 1.8

12.5 ± 1.5

5.3 ± 1.2

0.9 ± 0.5*

9.2 ± 1.5*

0.6 ± 0.3*

8.7 ± 2.0

11.1 ± 4.0*

5.6 ± 2.4

33.8 ± 10.0*

5.0 ± 0.5*

3.9 ± 1.1*

3.0 ± 1.0*

Presence of the withdrawal signs (checked signs)

erection

ejaculation

diarrhea

Straub tail

3/9 3/9 8/8*

9/9 2/9*

7/8*

*

8/9 3/9* *

Table 1. The effects (mean ± SEM) of equimolar dose of

the enkephalinase inhibitors thiorphan or phelorphan on

naloxone-precipitated (4 mg/kg, i.p.) withdrawal symptoms in

chronic morphine dependent rats. The control group received

artificial cerebrospinal fluid (CSF) instead of an

enkephalinase inhibitor. Observation period: 30 min after

naloxone administration. *Means significant difference

compared to control (P<0.05). Note that enkephalinase inhibitors attenuated some of the withdrawal signs.

Concluding, the saturation of the opiate receptors by enkephalins,

following administration of enkephalinase inhibitors, alleviates the

several symptoms of morphine withdrawal syndrome in mice and rat.

Although not all withdrawal symptoms were alleviated by enkephalinase

inhibitors, it seems that the potentiation of the functional activity

of endogenous opioid peptides could be a promising tool in future for

treatment of opiate dependence.

REFERENCE

Blasig, J., Herz, A., Reinhold, K. and Zieglgansberger, S.

Psychopharmacologia, 33:19-38, 1973.

Blasig, J. and Herz, A. Naunyn-Schmied Arch Pharmacol, 249:297-300,

1976.

Dzoljic, M.R., Ukponmwan, O.E., Baas, A.A.G., v.d. Berg, D.J. and

Rupreht, J. In: Degen, R. and Niedermeyer, E. eds. Epylepsy,

Sleep and Sleep Deprivation. Elsevier, 1984. pp.305-310.

577
Galligan, J.J. and Burks, T.F. Eur J Pharmacol, 85:61-68, 1982.

Henry, J.L. Neurosci Biobehav Rev, 6:229-245, 1982.

Kosersky, D.S., Harris, R.A. and Harris, L.S. Eur J Pharmacol,

26:122-124, 1974.

Pinsky, C., Dua, A.K. and La Bella, F.S. Brain Res, 243:303-307,

1982.

Takemori, A.E. and Sprague, G.K. J Pharm Pharmacol, 30:585-590,

1978.

Wen, H.L. and Ho, W.K.K. Eur J Pharmacol, 82:183-186, 1982.

578
PRESYNAPTIC ALPHA2 ADRENORECEPTOR FUNCTION IN DEPENDENT RATS BEFORE

AND AFTER MORPHINE WITHDRAWAL

H.C. Moises, C.B. Smith, R.N. Spengler and P.J. Hollingsworth

Departments of Physiology and Pharmacology

University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT

Changes in the number of alpha2 adrenoreceptors in the hippocampus,

as measured by the specific binding of 3H-clonidine, were compared

to changes in presynaptic alpha2 adrenoreceptor function in electrically stimulated hippocampal slices. In dependent rats the specific binding of 3

H-clonidine was reduced significantly. At 32 h

after withdrawal binding began to return toward normal values, and

at 72 h after withdrawal was significantly greater than that seen

in saline-treated rats. In hippocampal slice experiments sensitivity to clonidine was significantly decreased in morphine-dependent

animals and returned toward normal values after withdrawal. At 72

h after withdrawal, the sensitivity to clonidine of the presynaptic

alpha2 adrenoreceptor remained depressed although receptor density

as measured in the binding studies was increased. This study suggests that the changes in the function of presynaptic alpha2

adrenoreceptors might be important in the development of dependence

upon morphine.

INTRODUCTION

Acute administration of morphine stimulates markedly the turnover

of catecholamines in the brains of rats (Garcia-Sevilla et al.

1978) and mice (Smith et al. 1972). Tolerance develops to this effect of morphine, and after withdrawal, norepinephrine (NE)

turnover is decreased in the brains of dependent mice (Rosenman and

Smith, 1972). Neuronal release of NE is regulated in part by the

presynaptic alpha2 adrenoreceptor. Several investigators have reported that chronic treatment with morphine decreases the number of

alpha2 adrenoreceptors in specific areas of the rat brain as measured by the binding of 3

H-clonidine, an agonist at alpha2

adrenoreceptors (Smith et al. 1983, Vincentini et al. 1983). The

purpose of the present study was to determine whether changes in

3

H-clonidine binding to hippocampal membranes that occur after

chronic morphine treatment and during withdrawal result in corresponding alterations in presynaptic alpha2 adrenoreceptor mediated

inhibition of stimulation-evoked NE release from hippocampal

slices.

METHODS

Male Sprague-Dawley rats (220-240 g) were injected i.p. every 8 h

with saline or with morphine sulfate as follows (day,

mg/kg/injection): 1-3, 10; 4-6, 20; 7-9, 40; 10-12, 70; and 13-14,

100. Hippocampi were isolated from 6 rats (220-240 g). Specific

binding of 3H-clonidine to hippocampal membranes was measured as

579
described previously (Smith et al. 1983). The non-linear regression analysis program, LIGAND ( Menson and Rodbard 1980) was used

to develop the final values for KD and Bmax For the electrically

stimulated brain slice experiments two sections, 0.5 mm thick, were

made perpendicular to the longitudinal axis of each hippocampus.

The slices were incubated with 3H-NE, 300 nM, for 15 min, washed

twice with fresh Krebs buffer and transferred to superfusion chambers. Each slice was superfused at a rate of 0.5 ml per minute for

a 30 min equilibration period prior to the onset of field stimulation. Field stimulation consisted of trains of square-wave pulses

(100 mA, 2 msec duration) at 4 Hz for 2 min periods. There were

nine stimulation periods at 16 min intervals in each experiment.

Two of each set of four slices were stimulated but did not receive

any drug. For the other 2 slices drugs were introduced into the

superfusion buffer immediately after the second (the baseline response period) and each subsequent period of stimulation. Drug

concentrations were increased by approximately 3-fold increments in

order to construct complete concentration-effect curves. Each

slice was equilibrated in the presence of each drug concentration

for a period of 13.5 min after which the slice was stimulated for

an additional 2 min. Aliquots of the superfusate (2 ml) were collected at 4 min intervals. Immediately after the last aliquot of

superfusate was collected, the tissue slice was removed from the

superfusion chamber, blotted and weighed. Release of 3H-NE was expressed as the amount of labelled amine in each aliquot given as a

percentage of the amount of labelled amine in the tissue immediately before collection of that aliquot. The stimulationevoked release was calculated as the difference between the total

release during the period of stimulation less the estimated basal

release. EC 50’s were calculated by probit analysis.

RESULTS

After treatment of rats with morphine for 14 days, the specific

binding of 3H-clonidine was reduced significantly. Eight h after

the last injection of morphine, the Bmax for 3H-clonidine was decreased by 26.3 % (TABLE 1) . At this time the rats were dependent

but did not show signs of withdrawal. Thirty-two h after the last

morphine injection the Bmax had returned only slightly toward control values. At this time the rats were at the peak of the withdrawal syndrome. Seventy-two h after the last morphine injection,

no signs of withdrawal were noted. At this time there was a marked

(45.8%) increase above normal control values in the specific binding of 3

H-clonidine.

Prolonged treatment of rats with morphine caused a decrease in the

sensitivity to clonidine of presynaptic alpha2 adrenoreceptors in

the electrically stimulated hippocampal slice preparation. Eight h

after the last injection of morphine the clonidine concentrationeffect curve was shifted to the right (FIGURE 1) and the EC50

for clonidine was increased from a control value of 3.4 ± 0.8 nM

to a value of 26.3 ± 9.8 nM in the dependent rats. Thirty-two h

after the last morphine injection the clonidine concentration580
TABLE 1. Specific binding of 3

H-clonidine to hippocampal

membranes isolated from rats treated with morphine or after withdrawal from morphine.

TreatmentaBmax

 b % Change

K D

C n

Duration in Bmax

Control 96.5 ± 2.6 0 3.71 ± 0.54 12

1 Day 83.5 ± 4.9d

-13.5 3.31 ± 0.35 4

14 Days

8 hr 71.1 3.2f ± -26.3 3.36 ± 0.69 7

32 hr 78.3 ± 4.5e

-18.9 2.64 ± 0.41 5

72 hr 140.7 ± 7.5g

+45.8 3.22 ± 0.25 6

a

 Chronically treated animals were sacrificed at 8, 32 or

72 h after the last injection of morphine. b fmoles/mg

protein. c nM. Values represent the mean of n

observations ± the

d

 P < .05, e P< .0005, f P < .0001, g P < .00001.

standard error of the mean.

FIGURE 1. Effects of clonidine upon electrically evoked

release of 3H-NE from hippocampal slices from dependent

rats before and after morphine withdrawal. Abscissa:

con entration of clonidine.

of 3H-NE release

Ordinate: percent inhibition

. Open circles: controls; solid squares,

8 h after the last injection; open squares, 32 h after

581
withdrawal; open triangles, 72 h after withdrawal. Each

point represents the mean of 5-9 values. Vertical bars,

standard error of the mean.

effect curve was shifted back toward the left. At that time the

EC50 for clonidine was 8.1 ± 2.2 nM. Seventy-two h after the last

morphine injection there was no further change in the clonidine

concentration-effect curve, and the EC50 for clonidine was 12.9 ±

3.4 nM.

DISCUSSION

Both the density and function of alpha2 adrenoreceptors in the

brain change when rats are made dependent upon morphine and after

withdrawal of dependent rats from morphine. Although Hamburg and

Tallman (1981) reported an increase in the density of alpha2

adrenoreceptors in cerebral cortex from rats implanted with morphine pellets for 4 days, the present study and previous studies

(Smith et al. 1983, Vincentini et al. 1983) indicate that the maximum number of specific binding sites for 3

H-clonidine is decreased

in morphine-dependent rats. This decrease in binding is well-correlated with a decrease in the sensitivity to clonidine of

presynaptic alpha2 adrenoreceptors in electrically stimulated

hippocampal slices. Seventy-two h after withdrawal from morphine

the sensitivity to clonidine of presynaptic alpha2 adrenoreceptors

in the hippocampus remains depressed although there is a marked

increase in the number of specific binding sites for 3H-clonidine.

This observation suggests that during withdrawal an increase in the

number of postsynaptic alpha2 adrenoreceptors occurs in the

hippocampls. The finding of changes in alpha2 adrenoreceptor

number and sensitivity in the hippocampus of morphine-dependent

rats suggests that alterations in central presynaptic alpha2

adrenoreceptor function might be have an important role in opioid

dependence.

ACKNOWLEDGEMENT

Supported by USPHS grants DA-03365 and MH-36336 and by an award to

HCM from The Chicago Community Trust/Searle Scholars Program.

REFERENCES

Hamburg, M. and Tallman, J.F. Nature, 240:153-155, 1981.

Munson, P.J. and Rodbard, D. Anal Biochem, 107:220-239, 1980.

Smith, C.B., Sheldon, J.I., Bednarczyk, J.H. and Villarreal, J. J

Pharmacol Exp Ther, 180:547-557, 1972.

Smith, C.B., Hollingsworth, P.J., Geer, J.J. and Moises, H.C. Life

Sci, 33: 369-372, 1983.

Vincentini, L.M., Miller, R.J. and Robertson, M.J. Eur J Pharmacol,

95:265-270, 1983.

582
THE EFFECT OF CHRONIC MET-ENK ON VARIOUS DOPAMINE

SYSTEMS IN THE RAT

Kathy A. Steece, Frank A. DeLeon-Jones, John M. Lee,

and Ronald F. Ritzmann.

Neurobiology Laboratories, West Side VA Hospital,

University of Illinois, Health Sciences Center,

Research & Development (151), P.O. Box 8195

Chicago, Illinois, USA

ABSTRACT

Morphine and enkephalins act preferentially via different

receptors, mu and delta, respectively. Chronic administration of

morphine or methionine enkephalin results in development of

physical dependence. Data from our laboratory suggests that

there are some differences in the effect of these two opioids

with regards to certain neurochemical systems.

INTRODUCTION

Several types of opioid receptors have been demonstrated. Among

these are the mu (μ) and delta (6) receptors which interact with

morphine and enkephalins, respectively. Distinctions between

these 2 classes of receptors have been clearly defined on the

basis of pharmacological differences, localization studies, and

peripheral bioassays (for review see Paterson et al. 1983).

There is a dense population of μ as well as 6 receptors in the

striatum and hypothalamus. Here neuroregulatory interactions

occur with various neurotransmitter systems. Both μ and

receptor agonists can elicit alterations in dopaminergic

transmission in these areas. The relationship between

catecholamines and responses induced by opioids have been

investigated extensively to determine whether amines play an

important role in the adaptive mechanism(s) of physical

dependence. Chronic administration of either morphine or

enkephalin peptides results in development of physical

dependence. Continuous exposure to morphine has been

demonstrated to result in an increased sensitivity of striatal

and hypothalamic dopamine (DA) systems as measured by apomorphine

(APO)-induced stereotypy and hypothermia, respectively (Ritzmann

et al. 1983). Changes in the high affinity component of D2-DA

receptor binding for agonists in these brain regions were found

to correlate with these APO-induced behavioral/physiological

parameters in morphine-dependent rats (Ritzmann et al. 1983). It

remains to be determined whether various opioid receptors are

regulated by or linked to different molecular processes.

Therefore, the many differences in characteristics of μ and

receptors leads one to question whether the mechamism(s) of

adaptation during chronic exposure to morphine and enkephalins

are similar. Thus, using a method which produces physical

dependence, the effect of chronic methionine enkephalin

administration on striatal and hypothalamic dopamine systems was

investigated.

583
MATERIALS AND METHODS

Male Wistar rats, (Fredricks Cancer Center) weighing between 200-

300 grams were housed in a controlled environment (temperature 23

± 2°C; light 0800 to 1800 hours). Animals were anesthetized with

Equithesin (2.7 cc/kg; Jen Sail) and a stainless steel cannula

was implanted into the lateral ventricle of the brain.

Methionine5-enkephalin (met-enk) was administered into the

ventricle at a rate of 10 μg/μl/hr for 5 days via an osmotic

mini-pump (Tm, Alzet; model 2001). Control animals received 0.9%

sterile saline in place of a met-enk solution and underwent the

same procedures as the met-enk treated animals. A mini-pump was

connected to a cannula via polyethylene tubing. Withdrawal was

initiated by disconnecting the tubing and sealing off the

cannula. Rectal measurements of body temperature were monitored

every 2 hours for the first 8 hours and at the 24 hour time

point. Twenty four hours after initiation of withdrawal, animals

were either tested for behavioral/physiological responses to the

dopamine agonist apomorphine (0.5 mg/kg, i.p.) or sacrificed for

binding studies. In animals receiving an APO injection, behavior

was monitored at 5 minute intervals for 30 minutes post-injection

and rated according to the following scale: a score of 0 -

normal behavior; 1 - increased locomotor behavior; 2 - rearing

and head bobbing (non-continuously); 3 - rearing and head bobbing

(continuously); 4 - stereotyped sniffing continously; 5 - licking

and gnawing behavior. An animal was considered stereotypic if a

score of at least 4 was obtained. Rectal temperatures were

monitored in these animals prior to and 30 minutes after APO

injection. Animals for DA binding assays were sacrificed by

decapitation at the 24 hour time point. Hypothalamus and striata

were dissected out for determination of specific D2-DA receptor

binding. Binding assays were performed as previously described

(Ritzmann et al. 1983). Parametric data was analyzed by t-tests.

Values are expressed as means ± SEM. Non-parametric data was

analyzed using Fisher's Exact Probability Test. Binding data was

analyzed by the computer program LIGAND (Munson and Rodbard

1980).

RESULTS AND DISCUSSION

Chronic 5 day exposure to met-enk resulted in a marked degree of

physical dependence as evidenced by a significant (p<0.01) degree

of withdrawal hypothermia up to 8 hours after initiation of

withdrawal. The maximum temperature change occured in the

peptide treated animals at the 4 hour time point (-0.69 ± 0.14°C

met-enk group; 0.09 ± 0.07°C control group). Temperatures

returned to control levels in the met-enk group 24 hours postwithdrawal. Animals dependent on met-enk were more sensitive

than controls to the hypothermic response to APO 24 hours after

initiation of withdrawal. This was evident by a temperature

change of -0.67 ± 0.22°C in the met-enk treated animals compared

to a change of -0.08 ± 0.18°C in control animals (Fig. 1). This

increased sensitivity to APO-induced hypothermia is similar to

that observed in animals physically dependent on morphine

(Ritzmann et al. 1983). In contrast, met-enk treatment groups

584
exhibited a response to APO-induced stereotypy that was not

statistically different from their controls (0% stereotypic, metenk group; 12% stereotypic, control group; Fig. 1). This is

unlike the effect produced by chronic morphine treatment which

results in an increased sensitivity to APO-induced stereotypy

(Ritzmann et al. 1983). Chronic morphine exposure produces an

increase in percentage of receptors and/or increase in affinity

of D2-DA receptors in the high affinity state in both

hypothalamus and striatum for DA (Ritzmann et al. 1983).

Similarly, 5 day met-enk treatment increased the affinity in the

hypothalamus of the high affinity component for DA compared to

controls <KD - 10.0nM control group; KD - 0.008nM, met-enk group;

Table 1). This increased affinity of hypothalamic DA receptors

correlates positively with the increased sensitivity to APOinduced hypothermia observed in these animals. In contrast to

morphine treatment (Ritzmann et al. 1983), the high affinity

striatal D2-DA receptor binding was not different in control

versus met-enk treated animals (KD - 53nM, control group; KD - 46

nM, met-enk; Table 1). Again this corresponds with the lack of

change in response to APO-induced stereotypy seen in these

animals compared to controls. There was no change in receptor

binding capacity (Bmax) in either striatal or hypothalamic D2-DA

agonist binding with chronic met-enk similar to that observed

previously with chronic morphine (data not shown). Although metenk preferentially interacts with receptors it also interacts

with μ receptors at higher concentrations. Recently Pasternak

and Wood (1986) have postulated the existence of 3 receptor types

that bind morphine and enkephalins selectively: μl, which has

very high affinity (<1 nM) for both morphine and enkephalins; μ2,

a site which preferentially binds morphine far more potently than

enkephalins; and which selectively binds enkephalins. The

hypothalamus has been demonstrated to contain a high

concentration of μ1 receptors and these have been linked to

effects on DA turnover in this region (Pasternak and Wood 1986).

On the other hand, the striatum contains high concentrations of

μ2 and receptors which have been demonstrated to effect DA

turnover in this area (Pasternak and Wood 1986). Therefore, metenk may be affecting the DA system in the hypothalamus via μl

receptors and via receptors initially in the striatum.

However, desensitization or down-regulation of but not μ

receptors can occur during chronic exposure to opioid peptides in

various cell cultures and brain slice preparations (for review

see Zukin and Temple 1986). Therefore the possibility exists

that the effect on DA turnover by 6 receptors during chronic

opioid exposure is different than that produced by either μ1 or

μ2 receptors. Recent data from our laboratory suggests that this

phenomena of opioid receptor down-regulation does indeed occur in

vivo in the striatum during 5 day met-enk exposure (Steece et al.

1986). Hence, such an adaptive mechanism of 6 receptors in the

striatum could conceivably account for the overall lack of effect

of chronic met-enk on the DA system in the striatum.

585
FIGURE 1. The effect of 5 day met-enk treatment on

APO-induced hypothermia and stereotypy 24 hours after

initiation of withdrawal. (n > 12; *p<0.05)

EFFECT OF MET-ENK ON THE AFFINITY (KD) OF D2-DA RECEPTORS

Treatment Striatum Hypothalamus

Control 46.0 10.0

Met-Enk 53.0 0.008

TABLE 1. The effect of 5 day met-enk treatment on the

high affinity component of D2-DA receptors in the striatum

and hypothalamus. Each group represents at least four

determinations. Data is KD expressed in nM.

REFERENCES

Munson, P., and Rodbard, D. Anal Biochem 107:220-239, 1980.

Pasternak, G.W., and Wood, P.J. Life Sci, 38:1889-1898, 1986.

Paterson, S.J., Robson, L.E., and Kosterlitz, H.W. British Med

Bull, 39(1):31-36, 1983.

Ritzmann, R.F., Lee, J.M., and Fields, J.Z. Psvchopharm Bull,

19(3):321-324, 1983.

Zukin, R.S., and Temple, A. Biochem Pharmacol, 35:1623-1627,

1986.

Steece, K.A., DeLeon-Jones, F.A., Meyerson, L.R., Lee, J.M.,

Fields, J.Z., and Ritzmann, R.F. Brain Res Bull, (in press).

586
EFFECTS OF PRIOR EXPOSURE TO MORPHINE ON THE OPIOID INHIBITION

OF THE STIMULATED RELEASE OF [3H]NOREPINEPHRINE

FROM GUINEA PIG CORTEX SLICES

Linda L. Werling, Stephen R. Brown and Brian M. Cox

Department of Pharmacology, Uniformed Services University

Bethesda, Maryland 20814, U.S.A.

ABSTRACT

The potassium stimulated release of [

3

H]norepinephrine ([3H]NE) from

terminal fields of locus coeruleus projections can be inhibited in a

dose-dependent manner by mu and kappa selective opioids. Chronic

exposure to morphine for six days decreases the maximum achievable

depression by the mu selective agonist Tyr-D-Ala2-Gly-Me(Phe)-Gly-ol

(DAGO) , but has no effect on the degree of inhibition produced by the

kappa selective opioid U50,488H.

INTRODUCTION

Opioids inhibit release of several neurotransmitters in the central

nervous system, presumably by interactions with receptors located

presynaptically on the nerve terminals. There are reports of the

opioid inhibition of the release of dopamine and acetylcholine

(Mulder et al. 1984), and of norepinephrine (Monte1 et al. 1974;

Hagan and Hughes, 1984). In the rodent, cell bodies of the locus

coeruleus project to several areas of the brain, including the cortex

(Ungerstedt 1971). Locus coeruleus neurons are hyperpolarized by

the application of opioids in electrophysiological studies using

slices of brain tissue from the rat (Williams and North 1984) and

the guinea pig (Pepper and Henderson 1980). The terminal fields of

these noradrenergic fibers contain opioid receptors, as shown

autoradiographically (Lewis et al. 1983) and in binding studies

(Werling et al. 1985). We therefore investigated the possible

opioid regulation of the release of [

3

H]NE by the mu selective

agonist DAGO and the kappa selective agonist U50,488H in naive guinea

pigs compared to those implanted with osmotic pumps which delivered

chronic morphine to the animals for six days.

METHODS

Male Hartley guinea pigs (300 - 600 g) were subcutaneously implanted

with Alzet osmotic pumps (Alza Corp., Palo Alto, CA) containing

either 0.9% saline or morphine-HCl at the appropriate concentration

to deliver 100 μg/hr. After 6 days, animals were sacrificed, their

brains removed to ice, and the cortices dissected. The cortices were

chopped in two planes into 250 μm slices on a Sorvall TC2 tissue

slicer (DuPont Instruments, Newtown, CT), then suspended in oxygenated modified Krebs Ringers (MKR) solution (127 mM NaCl, 5 mM KCl,

1.3 mM NaH2P04, 1.2 mM MgS04, 2.5 mM CaCl2, 15 mM HEPES, 10 mM

Glucose). Subsequently, all steps of the preparation and experiment

were performed in a warm room which was maintained at a constant

temperature of 37°C. The tissues were washed 3 times in MKR (20 ml/

half cortex), resuspended in MKR and incubated for 15 min with 15 nM

587

172-683 O - 87 - 20 : QL 3
[

3

H]NE (New England Nuclear, Boston, MA). The tissues were then

washed twice for 5 min each in MKR, and once for 5 min in MKR containing 1 μM each desiprimine and yohimbine. All subsequent steps

were carried out in buffer containing these drugs to prevent feedback

inhibition of release through presynaptic alpha receptors and reuptake of released [3

H]NE. The tissue slices were resuspended in MKR

and distributed onto a series of nylon mesh baskets attached to

Costar (Cambridge, MA) multi-well plastic culture plate lids. The

baskets rested in the wells which contain 2 ml each MKR. To change

treatment, the lids were lifted from one plate of wells to the next,

which contained buffer with or without drug as appropriate. In all

cases, MKR had been pregassed with 95 % O2/5% CO2 and pipetted into

the wells immediately before tranferring the baskets. In this way,

24 different conditions could be examined simultaneously in

quadruplicate by employing 4 plates of 24 baskets each. Preliminary

experiments established that a stable baseline release was achieved

by 10 min incubation time in wells. After this initial 10 min

incubation, baskets were transferred to another set of wells

containing MKR, this time containing drug as appropriate, for 10

min. The tissues were then stimulated to release [

3

H]NE by transferring the baskets to wells containing 20 mM potassium plus or

minus drug as appropriate for 10 min. Finally, tissue was

transferred to wells containing 0.2 N HCl for 45 min to extract the

remainder of the loaded [

3

H]NE. All releasates were sampled and

counted, and fractional release calculated according to the total

amount of radioactivity in the tissue samples during the release

interval.

RESULTS

Both mu and kappa selective opioids produced a dose-related depression of stimulated [

3

H]NE release. The maximum inhibition produced

by DAGO was 60% in animals which received saline containing pumps,

and that produced by U50,488H was about 85% (Fig 1). The inhibition

Of release produced by the mu agonist DAGO was more readily

reversible by naloxone than the inhibition produced by the kappa

agonist U50,488H, consistent with their action through mu and kappa

receptors, respectively. However, at doses of agonist greater than

500 nM, the inhibition produced by either agonist could not be

completely reversed. In the absence of any added agonist, naloxone

did not produce an increase in control resting or stimulated release

at doses between 1 and 1000 nM.

In the cortices of animals chronically treated with morphine, the

maximum achievable depression by DAGO was about 40%, but that by

U50,488H was unchanged at 85% (Fig 1). The shape of the DAGO dose

response curve was changed in the morphine tolerant tissues relative

to that in controls. Whereas in cortex from naive animals, DAGO

produced a broad displacement over the concentration range 0.05 - 500

nM in chronic morphine treated animals, lower concentrations showed

similar potency in inhibiting the release of [

3

H]NE while higher

doses produced relatively little additional depression. At

concentrations of 5.0 nM and above, means of values representing per

cent control release from tissues of saline treated (n = 6) animals

were significantly different from those from chronic morphine treated

(n = 3) animals as determined by a two-tailed T test (p < 0.05). The

T statistic employed a pooled variance estimate derived from a two

way analysis of variance. As in the control animals, in the absence

of added agonist, naloxone failed to increase resting or stimulated

588
Fig. 1 Inhibition of the stimulated release of [3

H]NE by

DAGO (A) and U50,488H (B) in chronic morphine treated

and saline treated guinea pigs. Data are expressed

as percent of release of [

3

H]NE elicited by 20 mM K

+

 in

the absence of any added agonist. Control release from

the cortices of morphine tolerant animals was approximately 70% that of naive animals. Data are means from

three morphine treated and three naive animals.

589
[

3

H]NE release in tissue from chronic morphine treated animals. Thus

the overall effect of the chronic treatment was a reduction in the

maximum effect of the mu agonist, while the inhibition of [

3

H]NE

release by the kappa agonist was not changed significantly.

DISCUSSION

The data presented suggest that both mu and kappa opioid receptors

may be involved in the regulation of release of norepinephrine in the

cortex of the guinea pig. These observations are consistent with

electrophysiological studies on neurons of the locus coeruleus and

receptor binding studies. Presumably opioid peptides within the

brain are available to interact with the receptors which modulate

this release. However, naloxone was not able to increase either

resting or stimulated release, suggesting that the release of

norepinephrine from the cortex is not under tonic suppression by

these opioids. In the morphine dependent state, the regulation of

release of this neurotransmitter by opioids which work through

interaction with mu type receptors is apparently altered. DAGO

produces a lower maximum inhibition of release in the morphine

tolerant animal. However, the ability of kappa opioids to inhibit

release remains relatively unaffected. This result is not unexpected

since morphine exerts its effects primarily through mu opioid

receptors. Further characterization of the effects of opioids on

release of catacholamines in the dependent state may help to

elucidate adaptive mechanisms of tolerance and dependence.

REFERENCES

Hagen, R.M. and Hughes, I.E. Neuropharmacol, 23(5):491-495 (1984).

Lewis, M.E., Pert, A., Pert, C.B. and Herkenham, M. J Comp Neurol,

216(12-13) 1347-1350 (1983).

Montel, H., Starke, K. and Weber, F. L-S. Arch Pharmacol, 283:372-377

(1974).

Mulder, A.H., Wardeh, G. Hogenboom, F. and Frankhuyzen, A.L. Nature,

308:278-280 (1984).

Pepper, C.M. and Henderson, G. Science, 209:394-396 (1980).

Ungerstedt, U. Acta Physiol Scand Suppl, #367 1-48 (1971).

Werling, L.L., Zarr, G.D., Brown, S.R. and Cox, B.M. J Pharmacol Exp

Ther, 233(3):722-728 (1985).

Williams, J.T. and North, R.A. Molec Pharmacol, 26:489-497 (1984).

ACKNOWLEDGEMENTS

This work was supported by a grant from the National Institute on

Drug Abuse. The opinions or assertions contained herein are the

private ones of the authors and are not to be construed as official

or reflecting the views of the Department of Defense or the Uniformed

Services University of the Health Sciences.

590
TOLERANCE TO OPIOIOS IN SINGLE LOCUS COERULES NEURONS OF THE RAT

Macdonald J. Christie, John T. Williams and R. Alan North

Neuropharmacology Laboratory

M.I.T.(56-245), Cambridge, MA 02139, U.S.A.

ABSTRACT

Chronic treatment of rats with morphine caused tolerance to the

membrane hyperpolarizing action of morphine in single locus coeruleus

neurons in vitro. Tolerance was less pronounced for

Try-D-Ala-Gly-MePhe-Gly-ol (DAGO) than for normorphine. No changes

were found in affinity of naloxone for μ-receptors on these neurons,

nor in the properties of the potassium conductance increase caused by

μ-receptor activation. These results suggest that the mechanisms

underlying tolerance involve either a reduction of the number of μreceptors on each cell, or a reduced coupling of μ-receptors to

potassium channels.

INTRODUCTION

Opioid inhibition of firing of locus coeruleus (LC) neurons is due to

membrane hyperpolarization (Williams et al. 1982). This

hyperpolarization results from increase in conductance to

potassium ions and is brought about by activation of p-receptors

(Williams et al. 1982; Williams and North 1984). -Adrenoceptor

agonists also hyperpolarize LC neurons by increasing the same

potassium conductance as that which is activated by μ-receptor

agonists (North and Williams 1985). The mechanisms underlying the

development of opioid tolerance could involve adaptations of μreceptor binding sites, the coupling of binding sites to potassium

channels, potassium channels themselves, or subsequent processes.

Some of these possibilities were investigated in the present study by

measuring the potassium conductance increased by μ-receptor and

-adrenoceptor agonists in LC neurons from rats chronically treated

with morphine.

METHODS

Rats were treated with morphine by subcutaneous implantation of

morphine-containing pellets (75 mg base) on alternate days for 7 days

(1,3,5 and then 7 pellets/day). Intracellular recordings were made

as previously described (North and Williams 1985) from the LC of

rats killed 2 to 4 days after the last implantation. Slices of pons

containing the LC were submerged in a continuously flowing

artificial cerebrospinal fluid at 37°C to which drugs could be added.

Membrane current was measured by single electrode voltage clamp

technique (Axoclamp II)(North and Williams 1985). All data are

presented as mean ± SEM. The unpaired Student t-test was used.

591
RESULTS

Membrane properties of LC neurons from animals chronically treated

with morphine were not distinguishable from controls. Both fired

action potentials spontaneously with amplitudes close to 80 mV, which

arose from a threshold potential of -55 mV and had durations of about

1.5 ms. Steady-state conductances were plotted directly on an X/Y

recorder by applying a ramp potential change (-130 to -45 mV, at 1

mV/s). Cells from both control and treated animals had similar

steady-state current/voltage (I/V) relationships. Between -90 and

-60 mV the I/V plot was linear (resting conductance denoted Grest,

table 1). At potentials negative to -90 mV the slope of the I/V

relation was increased (inward rectification).

Control Morphine-treated

Grest (nS) 8.34 ± 0.51 (38) 7.59 ± 0.54 (27)

Go p (nS) 3.35 ± 0.28 (38) 2.79 ± 0.24 (27)

Iop (pA) 286 ± 19.3 (38) 242 ± 20.0 (27)

Io p/I 1.16 ± 0.05 (14) 1.10 ± 0.05 (11)

TABLE 1. Membrane properties of LC neurons following chronic

treatment with morphine. Grest is resting conductance between

-60 and -90 mV. Gop is maximal additional opioid conductance

at same potential range. Iop is maximal opioid current at

-60 mV. I is maximal current induced by UK14304 or

noradrenaline at -60 mV. Number of cells in parentheses.

No significant differences were found.

The increase in conductance induced by maximal concentrations of DAGO

(3 μM) or Met-enkephalin (ME, 30 μM) was determined both by measuring

the outward current induced by superfusion of the agonist while

holding the membrane potential at -60 mV, and by constructing steadystate I/V plots in the absence and presence of DAGO (3 μM) or ME (30

μM) and then subtracting the resting conductance (table 1) from that

found in the presence of agonist over the same potential range (-60

to -90 mV). The increase in conductance and outward current induced

by DAGO (3 μM) or ME (30 μM) was not different from that induced by

maximal concentrations of agonists (noradrenaline in the presence

of cocaine (10 μM) or 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline

(UK14304, 1 μM)). I (table 1) was the same in cells from control

and morphine-treated rats.

The EC50 for DAGO was shifted to the right two-fold in the morphinetreated group although the maximal outward current induced by DAGO

was not significantly affected (figure 1). In control tissues, the

maximum outward current induced by normorphine (30 μM) was similar to

that produced by DAGO. In contrast, the maximum response evoked by

592
normorphine in cells from morphine-treated rats was only 55 ± 2%

(figure 1) of that produced by DAGO, and the EC50 was shifted to the

right two-fold (figure 1). The degree of tolerance to normorphine

ranged from about six-fold at the foot of the concentration-response

curve to infinite at about 55% of the maximum response (figure 1).

Such reductions in the maximal response to normorphine were observed

for up to 6 h after removal of tissue from the animal.

FIGURE 1. Maximum outward currents induced by superfusion of

DAGO and normorphine following chronic morphine treatment are

expressed as percentage of maximum possible current for the

same cell. EC50 for DAGO were: control 91 ± 17 nM (8);

treated 167 ± 26 nM (11)(p < 0.05). EC50's for normorphine

were: control 0.91 ± 0.23 μM (8); treated 1.9 ± 0.17 μM (7)

(p < 0.005).

Naloxone was without effect on the membrane current of neurons for

either control or morphine-treated animals. Concentration-respone

relations for DAGO or ME were constructed in the presence of

increasing concentrations of naloxone in both control and treated

tissues. Chronic morphine treatment had no effect on the naloxone

pA2 (control 8.8 ± 0.1 (10): treated 8.7 ± 0.1 (8); Schild analysis

or method of Kosterlitz and Watt 1968).

DISCUSSION

The present results demonstrate that chronic treatment of rats with

morphine induces opioid tolerance in single LC neurones when their

properties are later measured in vitro with an intracellular

electrode. These results are consistent with extracellular

recordings of LC neurons both in vivo and in vitro (Aghajanian 1978;

Andrade et al. 1983).

593
Because no significant effects of treatment were observed on the

properties of the potassium conductance, nor on the ability of

maximal concentrations of full μ-or -agonists to increase potassium

conductance, it can be concluded that the mechanisms underlying

tolerance do not directly involve the potassium channel which is

activated by μ- and -receptors. More likely, the mechanisms

underlying tolerance development occur between p-receptor and

potassium channel.

Two conclusions can be drawn from the effects of naloxone on LC

neurones following chronic morphine treatment. Firstly, consistent

with extracellular recordings from LC slice preparations (Andrade et

al. 1983), there was no change in membrane conductance or potential

following application of naloxone to tissue from treated animals.

With extracellular recording from LC neurones in vivo, a meagre

increase in firing rate was observed (Aghajanian 1978). This

discrepancy could result from the presence of circulating morphine,

or from increased activity of excitatory afferents to the LC during

in vivo experiments. Secondly, no change was observed in the

affinity of naloxone for μ-receptors.

The observation that tolerance to normorphine is more pronounced than

that to DAGO following chronic treatment is consistent with the

interpretation that the site of adaptation resides between k-receptor

occupancy and potassium channel activation. This interpretation is

based on two findings. First, full activation of potassium channels

in normal LC neurons occurs with low fractional occupancy of μreceptors by full agonists (Williams and North 1984), i.e. the tissue

posesses considerable receptor reserve. Second, normorphine must

occupy a greater fraction of μ-receptors than DAGO to elicit an

equivalent response; ie. it posesses lower intrinsic efficacy (Miller

et al, 1986). Thus, in control tissue both normorphine and DAGO are

capable of eliciting the same maximal response since there is

considerable receptor reserve. However, in treated tissue

normorphine is no longer capable of eliciting the same maximal

response as DAGO because fewer receptor sites are present, or because

coupling of receptor to channel has become less efficacious. In

either case the net result is a decrease in the μ-receptor reserve of

LC neurons following chronic morphine treatment.

REFERENCES

Aghajanian, G.K. Nature, 276: 186-188, 1978.

Andrade, R., Vandermaelen, C.P., and Aghajanian, G.K. Eur J

Pharmacol, 91: 161-169, 1983.

Kosterlitz, H.W., and Watt, A.J. Br J Pharmacol, 33: 266-276, 1968.

Miller, L., Shaw, J.S., and Whiting, E.M. Br J Pharmacol, 87:

595-601, 1986.

North, R.A., and Williams, J.T. J Physiol, 364: 265-280, 1985.

Williams, J.T., Egan, T.M., and North, R.A. Nature, 299: 74-76,

1982.

Williams, J.T., and North, R.A. Mol Pharmacol, 26: 489-497, 1984.

ACKNOWLEDGEMENTS This work was supported by NIDA grant DAO3161. MJC

was supported by a Fogarty International Fellowship.

594
EFFECT OF MATERNALLY ADMINISTERED OPIATES ON THE DEVELOPMENT OF

THE ENDORPHIN SYSTEM IN THE OFFSPRING

Christina Gianoulakis

Department of Psychiatry, McGill University, Douglas Hospital

Research Centre, 6875 LaSalle Blvd., Verdun, Quebec, Canada

ABSTRACT

Pregnant rats received subcutaneous implantation of either

morphine or placebo pellets at various stages of gestation.

Untreated animals served as basic controls. The effect of

morphine treatment on the growth of the offspring as well as on

the ontogeny of endorphin in the pituitary gland and the hypothalamus was investigated. Results obtained indicated that

pregnant rats treated with morphine from day 1 of pregnancy to day

21 presented complete resorption. Subcutaneous implantation of a

morphine pellet on days 11, 13 and 19 of gestation allowed

pregnancy to term, however the post-natal mortality rate was very

high. Implantation of a morphine pellet on days 9 and 13 of

gestation allowed pregnancy to term and after birth the withdrawal

reaction of the offspring was mild and the mortality rate was low.

At early stages of development there were differences in the

pituitary and hypothalamic endorphin content between the

offspring of the morphine treated animals and the offspring of the

control animals, but by day 22 no significant differences among

the various groups were noticed.

INTRODUCTION

Prenatal exposure to morphine has been shown to induce a number of

morphological and behavioural changes (Kirby and Holtzman 1982;

Lichtblau et al. 1984). Since prenatal exposure to opiates alters

the responsivity of mature rats to noxious stimuli e.g. hot plate,

inducing a decreased analgesic response (Lichtblau et al. 1984),

opiates may alter the development and function of the endogenous

opiate system (opiate peptides and opiate receptors) in the

pituitary gland and specific areas of the brain. Thus the

objective of the present studies was to investigate the effect of

exposure to morphine in utero on the ontogeny of the endorphin

related peptides in the pituitary gland and hypothalamus.

MATERIALS AND METHODS

Female Sprague Dawley rats at the stage of late proestrous were

placed individually into breeding cages with experienced male

breeders (Sprague Dawley). The following morning the presence of

sperms in the vagina was tested. If sperms were detected in the

vagina this was considered as day 1 of pregnancy. The pregnant

animals were divided into three major groups. Animals of group I

received subcutaneous (s.c.) implantation of morphine pellets (75

mg morphine base) at the following stages of gestation: (a) on

days 1, 4, 7, 10, 13, 16 and 19; (b) on days 11, 13 and 19; and

595
(c) on days 9 and 13 of gestation. Animals of group II received

s.c. implantation of placebo pellets while animals of group III

received no treatment and served as basic controls. All animals

were given water and food ad lib. Following birth the offspring

were counted and weighed. On day 2 of post-natal life the number

of offspring per litter were limited to eight. The offspring were

weighed and sacrificed on days 1, 4, 8, 14 and 22 post-natally.

The pituitary and hypothalamus were dissected free and extracted

by sonication in 0.2 N HCL with protease inhibitors (Gianoulakis

and Gupta 1986). The extracts were stored at -75°C for estimation

of the total content of immunoreactive endorphin. Some animals

receiving a morphine or a placebo pellet on days 9 and 13 as well

as some untreated control animals, were sacrificed on days 19 and

20 of gestation. The pituitary and hypothalamus were dissected

and extracted as described previously. For the estimation of the

total endorphin like inununoreactivity EPLIR) in the

peripheral circulation trunk blood was collected at the time of

sacrifice, centrifuged and the serum was stored at -75°C till the

time of radioimmunoassay. The content of EPLIR in the tissue

extracts and the unextracted serum was estimated by

radioimmunoassay using a specific antiserum to endorphin at

1:30,000 final dilution (Gianoulakis et al. 1981). The

specificity of the antiserum was directed to the C-terminal

portion of endorphin (Gianoulakis et al. 1981).

RESULTS

In general, prenatal exposure to morphine increased the rate of

mortality of the offspring. Animals receiving morphine treatment

from day 1 of pregnancy and throughout the whole gestation period

presented complete resorption. In fact, the process of resorption

took place at the early stages of pregnancy as was shown by the

changes in body weight. Subcutaneous implantation of a morphine

pellet on days 11, 13 and 19 allowed pregnancy to term, however,

the mortality rate was very high ( 66%). Implantation of a

morphine pellet on days 9 and 13 of pregnancy allowed pregnancy to

term and after birth the withdrawal reaction of the offspring was

mild, and the mortality rate was low ( 10%). Offspring of

morphine treated animals had lower body weight at the time of

birth, however at later stages of development no significant

difference was observed among the various groups. On days 19 and

20 of gestation as well as on day 4 post-natally, the content of

EPLIR in the pituitary gland was lower in the offspring exposed

to morphine in utero. No significant difference was noticed in

the other stages of development (figure 1). The pattern of

ontogeny of endorphin was similar in the pituitary of the three

treatment groups, showing a sharp increase between days 1 to 4, a

smaller increase between days 4 to 8 and a sharp increase between

days 14 to 22. On days 19 and 20 pre-natally and day 1 postnatally, a lower content of EPLIR was found in the hypothalami

of the animals exposed to morphine in utero, while on day 4 postnatally a higher content of EPLIR was noticed in the hypothalami

of the offspring of both the morphine and the placebo treated

animals (figure 1). No significant difference in the content of

596
EPLIR in the hypothalamus was noticed at the other stages of

development investigated. Likewise to the pituitary gland, a

similar pattern of the ontogeny of endorphin in the hypothalamus

was presented by the offspring of all treatment groups, showing a

small rate of increase between days 4 to 8 followed by a sharp

increase between days 8 to 14 and 14 to 22. On day 1 post-natally

the content of EPLIR in the serum of the offspring exposed to

morphine in utero was significantly higher than in the offspring

of the control groups. No significant difference was noticed in

the serum EPLIR content at the other stages of development among

the various groups.

FIGURE 1. Content of endorphin-like immunoreactivity

in the pituitary gland (A) and hypothalamus (B) at

various stages of pre-natal and post-natal development.

Ex = Expanded scale. *Significantly different from

controls p < 0.05

DISCUSSION

The present results clearly indicate that pre-natal exposure to

morphine may induce pre-natal death and increase the post-natal

597
mortality. The high incidence of post-natal mortality may be

attributed to the withdrawal reaction of the offspring following

birth, however, the cause of the pre-natal mortality is not clear.

Since offspring of animals receiving s.c. morphine pellets on days

9 and 13 showed mild withdrawal reaction and high survival rate,

they were used for estimation of tissue and serum EPLIR content.

Though a lower content of EPLIR was observed on pre-natal days

19 and 20 in the morphine exposed offspring at later stages of

post-natal development no significant differences were noticed

among the offspring of the three treatment groups. These

observations indicate that the effect of pre-natal exposure to

morphine on the EPLIR content in both the pituitary gland and

the hypothalamus is transient and complete recovery to control

levels occurs at early post-natal life (day 8). Similar changes

in the hypothalamic endorphin content were noticed in the

offspring of both the placebo and the morphine treated animals,

suggesting that the changes observed in the content of EPLIR may

be partially due to the stress of ether exposure and surgery.

Furthermore, both the pituitary and the hypothalamus showed a

slower rate of increase in the total content of EPLIR between

days 4 and 8, the period of development known as the stress non

responsive period (Childs et al. 1982). The higher content of

serum EPLIR on day 1 of development observed in the offspring

exposed to morphine in utero, may be attributed to the withdrawal

reaction following birth.

In summary, prenatal exposure to morphine alters the content of

EPLIR in the pituitary and hypothalamus pre-natally and at early

stages of post-natal life with complete recovery to control levels

by day 8 post-natally. However, though no significant difference

is present in the total pituitary and hypothalamic EPLIR

content, the ability of the endorphin system to respond to

stress may have been modified by prenatal exposure to morphine.

REFERENCES

Childs, V.G., Ellison, D.G. and Ramaley, J.A. Endocrinology,

110:1676-1692, 1982.

Gianoulakis, C. and Gupta, A. Can J Physiol and Pharmacol,

64:430-437, 1986.

Gianoulakis, C., Drouin, J.N., Seidah, N.G., Kalant, H. and

Chretien, M. Eur J Pharmacol, 72:313-318, 1981.

Kirby, M.L. and Holtzman, S.G. Pharmac Bioch and Behav, 16:261-

269, 1982.

Lichtblau, L., Messing, R.B. and Sparber, S.B. Life Sciences,

34:1725-1730, 1984.

ACKNOWLEDGEMENTS

Supported by the Medical Research Council of Canada, grant MA6923.

598
SUPPRESSION OF IMMUNOLOGICAL FUNCTIONS IN MORPHINE ADDICTED MICE

Walter K. K. Ho, K. W. Cheung, K. N. Leung and *H. L. Wen

Department of Biochemistry, Chineses University of Hong Kong and

*Neurosurgical Unit, Kwong Wah Hospital, Hong Kong

ABSTRACT

Lymphocytes isolated from morphine addictedmice show a number of

impaired immunological functions. They include Con A stimulated

mitogensis, NK cell activity, plaque forming cell activity and

delayed-type of hypersensitivity. In addition, the met-enkepalin and

endorphin level in the lymph nodes of addicted mice were also

depressed.

INTRODUCTION

The role of the opioid peptides in modulating immune response has

received much attention since Wybran et al. (1979) first

demonstrated the existence of opiate receptors in human blood

lymphocytes. Since then, the action of the opioid peptides,

particularly the enkephalins and endorphin, in stimulating a

variety of immune response has been documented (Fischer and Falke

1984). Chronic opiate addicts are known to have higher rates of

infection (Brown et al. 1974). In human as well as in animal

studies, it has been shown that lymphocytes isolated from addicted

subjects have depressedmitogenic activity (Brownetal. 1974, Ho

and Leung 1979). As chronic addiction can also depressed the level

of enkephalin and endorphin (Clement-Jones et al. 1979, Ho et al.

1980), it is notunreasonableto expect that there may be a causal

relationship between the lower level of circulating opioid peptides

and the depressed immune status of the addict. The present study

aims to 1) establish if other immunological parameters, besides

mitogensis, can be depressed by addiction and 2) to determine

whether a depressed immune response is correlated with a lower level

of opioid peptides in the lymphoid tissues.

MATERIALS AND METHODS

Addiction of animals: Inbred female Balb/c mice weighing between 25

to 30 gm were used in this study. The mice were randomly divided

into three groups; untreated, saline-treated and morphine addicted.

Addiction tomorphine was inducedby two daily injections of 1 ml

saline containing morphine-HCl for three weeks. The dose used ranged

from 10 to 50 mg/kg/day. As 30 mg/kg/day turned out to give the

Optimal result, this dose was used for most of the experiments. The

development of addiction was confirmed by behavioral criteria. The

body weight of the animals remained constant throughout the entire

experimental period and there was no sign of malnutrition.

Assay of immune functions: Con A stimulated mitogensis was performed

according to Ng et al. (1978). Endogenous and activated natural

killer (NK) cell activity wasmeasured by the release of Cr-51 0n

599
prelabeled YAC-1 cells. Plaque forming cell (PFC) activity was

determined by the formation of plaques on lyzed sheep red blood

cells after the addition of complement. Delayed-type of

hypersensitivity was measured by the swelling of the foot pad when

animals previously sensitized to sheep red blood cells were

challenged with a dose of the antigen. Unless specified otherwise,

lymphocytes isolated from the spleen were used in these studies.

Radioinmunoassay and enzyme assay: The level of met-enkephalin and

endorphin in the lymph nodes were determined by RIA according to

Clement-Jones et al. (1980) and Guillemin et al. (1977). Metenkephalin degrading activity of lymph node membranes was determined

by the rate of disappearance of met-enkephalin in the assay mixture.

HPLC was used to separate met-enkephalin from contaminants and byproducts.

RESULTS AND DISCUSSION

In this study we have used four imnune function tests to determine

the effect of addiction on immunity. The results are presented in

table 1 and 2 and fig. 1. In the Con A stimulation test, the

mitogenic activity of lymphocytes isolated from addicted mice was

significantly depressed. In order to prove that this effect was

mediated by morphine, the antagonist, naloxone, was coadministered.

As indicated in table 1, mitogenic activity was suppressed to a

significantly lower level when animals were treated in this manner.

TABLE 1 Effect of Addiction on Con A Stimulated Mitogensis

Treatment H-3 Thymidine Uptake

(x 10-3 cpm/105 cells)

% of Untreated

Control

Untreated 140 ± 2.8 100

Saline treated 140 ± 5.0 100

Morphine addicted 73 ± 7.0 52

Naloxone 131 ± 9.5 93

Morphine/nax 114 ± 4.5 81

The dose of morphine and naloxone used was 50 mg/kg/day. In the

naloxone experiment, it was given immediately before morphine.

Spleen cells were used in this study.

The suppressive effect of addiction on mitogensis was determined to

be dependent on the dose of morphine used (data not shown). At a

daily dose of 10 mg/kg, there was no significant suppression of

activity. Only when the dose was increased to 30 mg/kg/day did the

effect on mitogensis become noticeable. Other than dose, the organ

from which the lymphocytes were isolated may also influence the

results. Thus, cells isolated from the spleen were in general less

suppressed than those isolated from the lymph nodes. This may be due

to a difference in the response of the lymphoid tissue to

neuroendocrine control (to be discussed later).

As in the case of Con A stimulated mitogensis, addiction also

suppressed both NK and PFC activities (table 2). In the delayed-type

600
of hypersensitivity assay, addicted animals had a slower and less

active response in general. Maximum swelling of the footpads was

delayed by one day on the average and the degree of inflammation was

never as severe as the controls (fig. 1).

Table 2. Effect of Addiction on Natural Killer Cell and Plague

Forming Cell Activities

Treatment

NK Activity (% Cr-51 released)

Endogenous Activated

PFC per

10 Cells

Untreated 19 45 354

Saline-treated 20 (105) 41 (91) 364(102)

Addicted 14 (73) 25 (55) 77(22)

NK activity of animals was preactivated by injection of C. parvum

(350 ug/mouse) 4 days prior to measurement. Similar results were

obtained from at least 3 independent trails. Values in parethesis

are % of untreated control.

Fig. 1. Effect of Addiction

on Delayed-Type of Hypersensitivity.

The mechanism of how opiate addiction can suppress immune response

may be related to the inhibition of endogenous opioid peptide level.

In both animal and human studies, it is known that chronic addiction

can decrease the level of enkephalin and endorphin (Clement-Jones

et al. 1979, Ho et al. 1980). Since these peptides can activate

lymphocyte response (Wybran 1985), it is not unreasonable to

speculate that factors which can reduce the level of opioid peptides

would also have a negative influence on the immune system. To

assess this, we have measured the level of met-enkephalin and

endorphin in the lymph nodes of addicted and control animals. As

indicated in table 3, the concentration of both peptides were

significantly reduced in the addicted group.

Presumably, this would not be due to a higher level of degrading

enzyme activity since membranes isolated from lymph nodes of

addicted mice hydrolyzed met-enkephalin at the same rate as that of

the controls. Further work is in progress to evaluate the role of

601
the opioid peptides in modulating immune responses.

Table 3 Effect of Addiction on Opioid Peptide Level and MetEnkepalin Degrading Activity of the Lymph Nodes

Treatment E P Met-ENK Met-ENK Degrad.Act.

Untreated 120 ± 6 1.82 ± 0.43 8.92 ± 4.75

Saline-treated 105 ± 22 1.75 ± 0.03 8.82 ± 3.68

Addicted 29 ± 11* 1.33 ± 0.22* 9.48 ± 2.74

EP was measured as ng/mg protein, met-ENK as ng/mg wet weight

and met-ENK degrading activity as nmol/min/mg protein.

* A high level of significance of difference compared with

control (p<0.01).

REFERENCE

Brown, S.M., Stimmel, B., Taub, R.N., Kochwa, S. and Rosenfield,

RE. Arch Intern Med, 134:1001-1006, 1974.

Clement-Jones, V., McLaughlin, L., Lowry, P.J., Besser, G.M.,

Rees, L.H. and Wen, H.L. Lancet, 2:380-383, 1979.

Clement-Jones, V., Lowry, R.J., Rees, W.H. and Besser, G.M.

J Endocr, 86:231-243, 1980.

Fischer, E.G. and Falke, N.E. Psychother Psychosom, 42:195-204,

1984.

Guillemin, R., Ling N. and Vargo, T. Biochem Biophys Res Comm,

77:361-366, 1977.

Ho, W.K.K. and Leung, A.M. Pharmacol Res Commun, 11:413-419,

1979.

Ho, W.K.K., Wen, H.L. and Ling, N. Neuropharmacology, 19:117-

120. 1980.

Ng, M.H., Ng, W.S., Ho, W.K.K., Fung, K.P. and Lamelin, J.P.

Exptl Cell Res, 116:387-395, 1978.

Wybran, J., Appelboom, T., Famaey, J.P. and Govaerts, A. J

Imnunol, 123:1068-1070, 1979.

Wybran, J. Fed Proc, 44:92-94, 1985.

ACKNOWLEDGEMENTS

Financial support from the Lee Foundation of Singapore and Mr. Lee

Wing Tat is gratefully acknowledged. Naloxone was a gift from

DuPont Pharmaceutical.

602
MORPHINE METABOLISM IN MOUSE BRAIN

Agneta Wahlström, Lena Hammar*, Lars-Gustav Lundin* and

Anders Rane

Div. of Clinical Pharmacology, University Hospital, Uppsala, Sweden

* Dept. of Medical and Physiological Chemistry,

Biomedical Center, Uppsala, Sweden

ABSTRACT

Morphine UDP-glucuronyltransferase activity was demonstrated in the

brain of mice from recombinant inbred strains of the BXD series. The

formation rate of morphine-3-glucuronide was about 4 fold higher in

the progenitor DBA as compared to the C57BL strain.

INTRODUCTION

Morphine is still the mainstay in the treatment of chronic severe

pain. When used as an oral medication its effects vary between patients. To a large extent this is due to wide interindividual variation in the presystemic elimination of this drug (Säwe et al. 1985).

The possibility that also the formation of active morphine metabolites may contribute to the pharmacological effects and its variation

has previously been put forward (Joel et al. 1985, Osborne et al.

1986). A genetic difference in the quantitative metabolism as well

as a difference in metabolic pattern would be compatible with a variation in effect. Whereas the major metabolite morphine-3-glucuronide (M3G) is inactive, the morphine-6-glucuronide (M6G) (Shimomura

et al. 1971), and morphine-6-sulphate (M6S) (Mori et al. 1972) have

been demonstrated to possess analgesic activity in mice. However,

these metabolites represent minor metabolic pathways of morphine.

The present study was designed to investigate if there is a measurable morphine UDP-glucuronyltransferase activity (UDP-GT) in the

mouse brain and if the enzyme activity i affected by one or several

genes.

MATERIALS AND METHODS

Microsomes from the whole male mouse brain were isolated by standard

procedures. The glucuronidation assay was performed according to Pacifici et al. (1982). The morphine metabolites were analyzed by high

performance liquid chromatography according to Svensson et al. (1982).

The reproducibility of the whole assay was 11% determined as coefficient of variation from 5 repeated assays of the same sample. The

detection limit of M3G in the incubate was 2 ng. The genetical model

used was the BXD series of recombinant inbred (RI) strains and the

two progenitor strains C57BL/6J and DBA/2J.

RESULTS

The morphine UDP-GT activity was measurable in the brain microsomes

from the progenitor strains as well as in the brain of all the RIstrains. The reaction was linear with protein up to a concentration

of 3 mg per ml incubate (fig 1). The reaction proceeded linearly

with time from 1 to at least 4 hours (fig 1). The rate of glucuronidation was 4 times higher in the DBA than in the C57BL mice (p<0.001).

603
The variation between individuals was also larger in the DBA strain;

6.8 ± 4.01 pmol/mgxmin (n=10) versus 1.8 ± 0.681 pmol/mgxmin (n=9)

in the C57BL strain. The morphine UDP-GT activity in the different

RI-strains is shown in fig 2. Each bar represents one strain and the

mean of four animals. As is seen in the figure, the activity profile

from one experiment to another was not fully reproducible. The values

of the RI-strains were within the range of the two progenitor strain

values.

FIGURE 1.

Morphine UDP-GT activity in brain of DBA

and C57BL mouse

strains, at different

protein concentrations (upper panel)

and the time course

of morphine-3-glucuronide (M3G) formation (lower panel).

Standard incubations

(100 ul final volume) were carried out

at 37°C for 2 hours,

0,2 mg protein in

TRIS-buffer pH = 7.4

with 8.3mM Mg Cl2,

3mM morphine and

15mM uridine diphosphoglucuronic acid

(UDPGA).

1

Mean ± standard deviation

604
FIGURE 2. Morphine UDP-glucuronyltransferase activity in mouse

brain. The profile of 13 RI-strains is shown. The averages of

two experiments each including two animals are shown as black

cuts in the histogram. The activities of the progenitor strains

were determined in 10 (DBA) or 9 (C57BL) male individuals. Mean

values and standard error of the mean for these are indicated

to the right. (Details in fig 1.)

DISCUSSION

Morphine UDP-GT activity was measurable in brain microsomes from all

strains tested. The activity differed by a factor of 4 in the two

progenitor strains indicated a strong genetic influence. However,

the distribution of morphine UDP-GT activity among the RI-strains

does not allow us to easily interpret the mode of inheritance. One

explanation might be that there is a family of morphine UDP-GT enzyme forms (Mackenzie et al. 1985), of which more than one contributes to the variation in our results. Environmental factors may also influence the activity. We did not attempt to monitor the crowding conditions in the cages which may bear importance for the brain

metabolism. Such factors may effect certain pain mechanisms as suggested by Rodgers and Hendrie (1983). They showed that social conflict in male mice is a potent biologically relevant stimulus in the

activation of endogenous naloxone-sensitive pain mechanisms. These

authors also found that social status is an important determinant of

nociceptive response to such experience. Indeed the DBA strains that

had a higher and more variable activity "seemed" more aggressive

than the other progenitor strain.

The hypothesis that different metabolic routes of morphine could be

responsible for the analgesic effect and side effects has not been

605
fully investigated. The other pathways of morphine metabolism, as the

formation of morphine-ethereal sulphate and normorphine are currently

investigated in our laboratory. Although these metabolites are formed

in minor amounts they may contribute quantitatively to the side effects by virtue of their high potency. In the light of the recent evidence of biosynthesis of morphine in the mammalian body (Donnerer et

al. 1986) more information is neededd about the metabolism of this

drug and its relation to the pharmacological effects.

REFERENCES

Donnerer, J., Oka, K., Brossi, A., Rice, K.C. and Spector, S. Proc

Natl Acad Sci, 83:4566-4567, 1986.

Joel, S.P., Osborne, R.J., Nixon, N.S. and Slevin, M.L. The Lancet,

May 11, 1099-1100, 1985.

Mackenzie, P.I., Joffe, M.M., Munson, P.J. and Owens, I.S. Biochemical Pharmacology, 34:737-746, 1985.

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Ueki, S. Life Sci, 11:525-533, 1972.

Osborne, R.J., Joel, S.P. and Slevin, M.L. Br Med J, 292:1548-1549

1986.

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Rodgers, R.J. and Hendrie, C.A. Physiology & Behaviour, 30:775-780,

1983.

Shimomura, K., Kamata, O., Ueki, S., Ida S., Oguri, K., Yoshimura, H.

and Tsukamoto, H. Tohoku. J Exp Med, 105:45-52, 1971.

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ACKNOWLEDGEMENTS

Supported by the Swedish Cancer Society, the Swedish Medical Research

Council (14X-04496) and the Swedish Natural Science Research Council

(B-BU 2992-109).

606
OPIOID REGULATION OF PHOSPHOINOSITIDE TURNOVER. M.E. Abood, N.M.

Lee. H.H. Loh, Depts. of Pharmacology and Psychiatry, University

of California, San Francisco, CA 94143, USA.

Since a number of studies indicate the involvement of Ca++ in

the action of opioids, as well as a role for G proteins, the

regulation of phosphoinositide (PI) turnover may be a possible

second messenger system for opioids in the brain. This possibility was addressed by measuring PI turnover in rat brain cerebral

cortical slices. In the presence of 10 μM etorphine, a small (23

- 83%) but significant (p< .01, n = 9) increase in all three

products of PI hydrolysis was observed. It is worth noting that

with smaller sample numbers (n = 5), it was difficult to

demonstrate a significant increase in PI turnover in response to

etorphine. However, with smaple numbers of n = 9 or more the

increase was always statistically significant. Etorphine at 1 μM

produced a 10% increase in phosphoinositides. The etorphine

effect was a specific opioid effect, as it was reversible with 20

μM naloxone. This data indicate another second messenger system

in the rat brain through which opioids may act.

607
OPIOID REGULATION OF PHOSPHOINOSITIDE TURNOVER. M.E. Abood, N.M.

Lee, H.H. Loh, Dept of Pharmacology, UCSF, San Francisco, CA

94143, USA

Since a number of studies indicate the involvement of

calcium in the action of opioids, as well as a role for G

proteins, the regulation of phosphoinositide (PI) turnover may be

a possible second messenger system for opioids in the brain.

This possibility was addressed by measuring PI turnover in rat

brain cerebral cortical slices. In the presence of 10 μM

etorphine, no significant increase in the three products of PI

hydrolysis was observed (n = 15). It is worth nothing that with

smaller sample numbers (n = 5), it was difficult to demonstrate

reproducible effects on PI turnover in response to etorphine.

The effect of etorphine on PI turnover in the midbrain was also

examined.

608
SYNAPTOSOMAL MEMBRANES CONTAIN A MEMBRANCE-BOUND FORM OF

ENDOPEPTIDASE 24.15 THAT GENERATES LEU- AND METENKEPHALIN FROM LARGER PEPTIDES. G.R. Acker, C.J.

Molineaus and M. Orlowski. Dept. of Pharmacol., Mount

Sinai Sch. of Med. New York, NY 10029.

There is evidence that enkephalins are derived not

only from proenkephalin, but also from prodynorphin. A

soluble metalloendopeptidase (SMEP) (EC 3.4.24.15) from

rat brain was previously shown to cleave dynormphin

A-(1-8) (D8); and -neo-Enaorpnin and -nE) to

Leu enkephalin (LE) (Chu and orlowski, Endocrinol.,

116: 1418-1425, 1985). We have found that a membranebound form of this enzyme accounts for 20-25% of the

total activity. The specific activity of this

membrane-bound form was higher in synaptosomal

membranes than that of endopeptioase-24.11

("enkephalinase"). Purified synaptosomal membranes were

incubated with the prodynorphin-derived peptides D8, -

and -nE, and the proenkephalin-derived Met-enkephalinArg-Gly-Leu (ME-RGL), in the presence of bestatin,

captopril and N-01-(R,S)-carboxy-2-phenylethyl]-Phep-AB, specific inhibitors of aminopeptioase,

engiotensin-converting enzyme (EC 3.4.-15.1) and

memberane-bound MEP (EC 3.4.24.11), respectively. After

3 h, the rate of enkephalin generation was greater than

70% from ME-RGL, D8, -nE. A slower rate of conversion

was observed for -nE, consistent with the turnover

rate constants found for the purified SMEP (above

ref.). Addition of N-[1-(R,S)-carboxy-2-phenyletny1]-

Ala-Ala-Phe-p-AB, an active site-directed inhibitor of

endopeptidase-24.15 innibited LE and ME formation by

80% indicating the involvement of this enzyme in the

observed conversions. These results demonstrate that

synaptosomes contain a membrane-bound form of

enaopeptidase-24.15 that efficiently generates

enkephalin from proenkephailn and prodynorphin sources.

609
EFFECTS OF MONOSODIUM GLUTAMATE ON THE LEVELS OF BETA-ENDORPHIN

IMMUNOREACTIVE MATERIAL IN RAT DORSAL AND VENTRAL CAUDAL MEDULLA.

N. E. Alessi and H. Khachaturian, Mental Health Research Institute, University of

Michigan, Ann Arbor, Michigan 48109, U.S.A.

Neuronal perikarya containing beta-endorphin immunoreactivity (BE-IR) are found in

two regions of the brain, the hyopthalamic arcuate nucleus and nucleus tractus

solitarius (NTS) in the dorsal caudal medulla (Khachaturian et al., Trends Neurosci.

8:111, 1985). BE-IR is also found in fibers in both the dorsal caudal medulla (DCM) and

ventral caudal medulla (VCM). The exact origin of these fibers, i.e. arcuate versus NTS

is currently not known. Biochemical studies which have characterized BE-IR in medulla

have used either the entire caudal medulla or the DCM (Alessi and Quinlan, Peptides. 6

Suppl 2:137, 1985; Dores et al., Brain Res, In press). In order to clarify the

contributions of the arcuate versus NTS to total BE-IR in caudal medulla, we have used

monosodium glutamate (MSG) lesions which selectively damage the arcuate neurons.

Neonatal male Sprague-Sawley rats were injected with MSG (4 mg/gm body weight) on

postnatal days P2, P4, P6, and P8. Control animals were injected with 0.9% saline. At

100 days of age, the animals were decapitated and the brain stem rapidly frozen in

powdered dry ice. The caudal medulla was cut at the central canal into DCM and VCM.

Levels of BE-IR was determined with a BE RIA using antiserum Brenda (Drs. Watson

and Akil). In the saline injection group BE-IR in the DCM and VCM was 7.53 ± 0.99

(mean ± SEM) and 4.23 ± 0.44, respectively, with BE-IR being significantly higher in the

DCM (paired t-test. p < 0.025). MSG treatment resulted in a decrease in BE-IR in DCM

(3.51 ± 0.53). but not in VCM (3.38 ± 0.67) (n.s.). A comparison of the DCM in the

saline versus MSG groups demonstrates a significant drop in the BE-IR levels with MSG

(p<0.005). A similar comparison of the VCM demonstrates no significant change (p<

0.375). The extent of cell damage in the arcuate nucleus and NTS was also

determined immunohistochemically using a 16K antiserum. MSG and control rats were

treated with colchicine prior to sacrifice. The preliminary results indicate a significant cell

loss in the arcuate nucleus (approximately half of control), whereas no perikaryal loss

was apparent in the NTS. The results of this study suggest that a significant portion of

the BE-IR identified in the DCM oriainates from cells in the arcuate nucleus which might

be affected by MSG. Furthermore: since no significant drop in BE-IR was noted in the

VCM, BE fibers in this region of the medulla may originate either from NTS or portions of

the arcuate nucleus not affected by MSG.

610
HIGH EXTRACELLULAR K+

 ION AND THE DOPAMINE (DA) AGONIST

BROMOCRIPTINE INHIBIT ENDORPHINS SECRETION FROM PERIFUSED RAT

NEUROINTERMEDIATE (NIL) PITUITARY CELLS IN A VERY DIFFERENT

MANNER. Richard G. Allen and Julianne Stack. Departments of

Medicine and Physiology, Oregon Health Sciences University,

Portland, Oregon 97201, U.S.A.

The secretion of endorphins derived from the pro-hormone

pro-opiomelanocortin (POMC) is tonically inhibited by the

neurotransmitter dopamine in vivo. It has been demonstrated

that ion fluxes, voltage dependent ion channels and membrane

potentials contribute to the mechanism(s) of release of

neuropeptides from secretory vesicles. We measured the effects

of potassium, sodium and bromocriptine on endorphins release

from the NIL-cells to investigate the relationship between ion

fluxes, membrane potential and neurotransmitter actions. Male

rat NILs were enzymatically dispersed and perifused with a

continuous flow for 5 hrs. Twenty min pulses of control medium

were interrupted with 10 min pulses of experimental medium.

Control medium was a complete medium with 5.4 mM KCl and 155mM

NaCll. Experimental megiums were composed of control medium

plus additives: high K+=60.4mM KCl, high Na+=209mM NaCl,

bromocriptine(10-6M). Two min fractions were collected and

analyzed for endorphins immunoactivity. The high K

+

 caused

an immediate inhibition of basal release and in 10 min had

reduced secretion to 40% of the expected basal release. When

control medium was reintroduced, the secretion rate returned to

100% of the pre-inhibited rate. The high Na+ had no

significant effect on basal release, but when control medium

was reintroduced there was an immediate, transient (8 min)

increase of secretion, 407% above the predicted basal rate.

Bromocriptine had an immediate 20-fold inhibitory effect on

secretion of endorphins that was never reversed with control

medium. The inhibitory effects of high K+ were specific for

this ion and reversible. Inhibition of secretion by

bromocriptine was much greater than high K+ and not reversible,

implying that other intracellular mechanisms may be involved in

the inhibitory effects of bromocriptine. (Supported by NIH

grant NS 19397-02).

611
THE PURIFIED μ OPIOID RECEPTOR AND ITS RELATIONSHIP TO

OTHER OPIOID-RELATED BINDING SITES. E.A. Barnard, C.D.

Demoliou-Mason and Y. Wong, MRC Molecular Neurobiology

Unit, MRC Centre, Cambridge CB2 2QH, U.K.

Soluble highly active opioid receptors from rat

brain membranes have been purified by affinity

chromatography on 14- -(C-glycyl)-amido-(N-cyclopropylmethyl)-norcodeinone (GANC) immobilized on Affi-Gel-10,

or on D-ala2-leu5-enkephalin chloromethylketone

(DALECK) immobilized on thiol-Sepharose 4B. After

elution by opiates and a lectin affinity chromatography

step, the completely purified receptors bind opiate

agonists and antagonists with high affinity and also

opioid peptides, the latter with lower affinity. The

subtype specificity depends upon the conditions used in

the chromatography. The purified receptor from either

column shows a single protein band (apparent mol. wt.

65,000) after SDS-gel electrophoresis and silver

staining, when the affinity chromatography was in the

presence of Mg2+ and Na+. [

3

H]DALECK itself acts as a

u-specific affinity reagent and labels a single

polypeptide. Comparison will be made between the

receptor subunits as identified thus in the membrane

and after purification under various conditions.

Separation from other opioid subtypes in solution, and

from the initially-associated guanine-nucleotide

binding protein, can be demonstrated. Approaches

directed towards the cloning of the cDNAs encoding

these receptor subunits will also be discussed.

612
EXPRESSION OF OPIOID RECEPTORS IN CULTURED EMBRYONIC

NEURONS IS REGULATED BY COMPONENTS IN THE CULTURE MEDIUM. J.

Barq, R. Levy and R. Simantov, Department of Genetics, Weizmann Institute of

Science, Rehovot 76100, Israel.

Cultured aggregating rat brain neurons express mu, delta and kappa opioid

receptors (Lenoir, Barg and Simantov, Brain Res., 304, 285-290, 1984). A more

recent study shows that chemical depolarization alters the number and possibly

the type of receptors expressed (Simantov and Levy, Develop. Brain Res., 1986,

in press). It appears, therefore, that the profile of receptors expressed by a

given tissue, at least in culture, depends on its neurochemical activity. This

study was undertaken to further analyze the factors that regulate the

expression of these receptors. Hindbrain, forebrain or whole brain aggregating

cell cultures were prepared from dissociated E-15 rat brain ceils and grown in

serum containing or serum free media. 3H-Diprenorphine was used to

determine the total number of opioid receptors, and DAGO and DPDPE, the

number of mu and delta sites, respectively. Cultures grown for only 7 days

have different receptor density and subtype profile than cultures grown for 19

days. Serum alters the receptor number, and aggregates cultured in serum free

medium have different response to depolarizing concentration of potassium ions

than serum containing cultures. Moreover, opiate antagonists have a selective

effect on the number of different receptor subtypes. These and additional data

using cultures treated with conditioned media from several cell sources indicate

that the phenotypic expression of opioid receptors in culture may be regulated

by multiple factors.

613
POSSIBLE IMPLICATIONS OF PITUITARY THYROTROPIN

RELEASING HORMONE RECEPTORS IN MORPHINE

TOLERANCE IN THE RAT. Hemendra N. Bhargava, Sumantra

Das, M. Bansinath and R. Prasad, Departments of Pharmacodynamics

and Pathology, University of Illinois at Chicago, Health Sciences

Center, Chicago, Il 60612, U.S.A.

The effect of chronic treatment of rats with morphine and

subsequent withdrawal on brain and pituitary receptors for

thyrotropin releasing hormone (TRH) was investigated. Rate were

implanted with four morphine pellets (75 mg each) during a three

day period. Placebo pellet implanted rate served as controls. Both

tolerance to and dependence on morphine developed as a result

of this procedure. The binding of 3H-MeTRH to membranes

prepared from whole brain minus cerebellum was unaffected in

morphine tolerant-dependent rata as demonstrated from the

unaltered Kd and Bmax values when compared to placebo pellet

implanted animals The binding of 3H- MeTRH to five different

regions of the brain am well as pituitary were also investigated.

Binding of 3H-MeTRH to pituitary was increased during

development of morphine tolerance and naloxone preceipitated

withdrawal, whereas, binding to the different regions of brain was

unaffected under these conditions. There was a decrease in

circulating levels of thyroid hormones (T8

 and T4). However,

serum TSH level remained unaltered. In contrast, 24 hour

withdrawal of morphine renulted in an increase in 3H-MeTRH

binding in pons + medulla whereas binding in other regions of

the brain as well as in pituitary was unaffected. Serum T3, T4

and TSH levels were also unaffected during 24 hour

withdrawal of morphine. The results suggest that the development

of tolerance to morphine may be associated with changes in the

pituitary- thyroid axis (Supported by a grant DA-02698 from the

National Institute on Drug Abuse).

614
SELECTIVE INHIBITION OF OPIOID BINDING BY FAB FRAGMENTS

FROM A MONOCLONAL ANTIBODY DIRECTED AGAINST THE OPIOID

RECEPTOR. J.M. Bidlack and W.E. O'Malley, Center for Brain

Research, University of Rochester, School of Medicine and Dentistry,

Rochester, NY 14642, U.S.A.

Fab fragments from a monoclonal antibody, OR-689.2.4, directed

against the opioid receptor selectively inhibited opioid binding to rat

and guinea pig neural membranes and to the NG108-15 neuroblastoma x

glioma cell line. In a titrable manner, the Fab fragments

noncompetitively inhibited the binding of the μ selective peptide,

[

3H][D-Ala2

,MePhe4,Gly-ol5]enkephalin, and the selective peptide,

[

3H][D-Pen2

,-Pen5]enkephalin, to neural membranes. The Fab

fragments were competitive inhibitors of [

125I-Tyr27] -endorphin binding

to neural membranes and to membranes from the NG108-15 cell line.

In contrast, opioid binding, as measured by the binding of

[

3H]bremazocine to guinea pig cerebellum in the presence of μ and

blockers, was not significantly altered by the Fab fragments. The

OR-689.2.4 Fab fragments blocked the binding of [

3H]bremazocine to μ

and binding sites. In addition to blocking the binding of μ and

ligands, the Fab fragments displaced bound opioids from the

membranes. When μ sites were blocked with [D-Ala2,MePhe4,Gly-ol5]

enkephalin, the Fab fragments suppressed the binding of

[

3H][D-Pen2

,D-Pen5]enkephalln to the same degree as when the μ

binding site was not blocked. The Fab fragments also inhibited binding

[D-Pen2,D-Pen5]enkephalln This monoclonal antibody is directed

to the μ site regardless of whether or not the site was blocked with

.

against a 35,000 dalton protein. Since the antibody is able to inhibit μ

and binding but not opioid binding, it appears that this 35,999

dalton protein is an integral component of μ and opioid receptors but

not receptors.

615
INTERACTIONS OF A MORPHINE (μ) RECEPTOR SPECIFIC LIGAND, [

3H]TyrPro-NMePhe-D-Pro-NH2

, WITH RAT BRAIN MEMBRANES. S. G. Blanchard,

P. Lee, J. S. Hong and K.-J. Chang, Molecular Biology Department,

The Wellcome Research Laboratories and N.I.E.H.S., Research

Triangle Park, NC 27709, U.S.A.

The highly u-receptor selective ligand Tyr-Pro-NMePhe-D-ProNH, (PL017) was custom tritiated and its binding to rat brain

membranes was directly measured. The data were well fit by a

model assuming the existence of a single homogenous population of

binding sites. Scatchard analysis yielded values for Kd and

binding sites of 6 nM and .16 pmole/mg, respectively. As

expected for binding to an opiate receptor, addition of sodium,

magnesium and guanyl nucleotides to the assays resulted in a

modulation of ligand binding. In all cases tested, however, no

significant deviations of the Scatchard plots from linearity were

observed. Furthermore, displacement of 3H-PL017 by all opioid

ligands tested was monophasic, consistent with simple competitive

inhibition at a single binding site. The IC50 values thus

obtained showed good agreement with previously published values

determined using less selective radiolabeled ligands. The

regional distribution of 3H-PL017 binding to rat brain was

examined by in vitro autoradiography. The labeling pattern was

similar to that seen for other μ-type ligands. Because of its

high selectivity, the binding of 3H-PL017 to the μ-type receptor

can be measured directly without the need to suppress binding to

other sites. As such, 3H-PL017 should be a useful ligand for

dissecting the actions of multiple opiate receptors.

616
IRREVERSIBLE BINDING OF IODO AZIDO DTLET TO RAT BRAIN DELTA

OPIOID RECEPTOR.

P. Bochet*, C. Icard-Liepkalns*, F. Pasquini+, C. GarbayJaureguiberryo

, B.P. Roqueso, A. Beaudet+ and J. Rossier*.

*Physiologic Nerveuse, CNRS, Gif-sur-Yvette, France ; + Institut

Neurologique de Montreal, Canada ; o

 INSERM, CNRS, Faculté de

Pharmacie, Paris, France.

Azido DTLET (Tyr-DThr-Gly-pN3Phe-Leu-Thr) is a photoactivable

derivative of the delta specific ligand DTLET and is itself a

potent opioid agonist. The iodination of Azido DTLET by the

chloramine T method, followed by separation over reverse phase

HPLC, yields a pure monoiodinated species, as verified by mass

spectrometry. 125I Azido DTLET displays high affinity, levorphanol

displaceable binding to rat brain membranes. The behaviour of the

ligand in inhibition experiments is very similar to that of DTLET

(Table). Irradiation with u.v. light (254 nm), following

incubation with 125I Azido DTLET, results in irreversible binding

to several rat brain proteins. SDS polyacrylamide gel

electrophoresis shows that one of these proteins (apparent

molecular weight 43000) is not labeled when the incubation is

carried out in presence of a high concentration of levorphanol.

125IAzido DTLET also binds to rat brain slices (20 um thick) with

a distribution similar to the localisation of delta receptors.

125IAzido DTLET has also been used to label NG 108-15 cell

membranes. The labelled proteins from both rat brain and this

opioid receptor rich cell line do not display the same patern.

Inhibition of binding by DAGO, DTLET and 127IAzido DTLET (Ki nM)

Radioactive ligand

DAGO 3.9 700 668

127IAzidoDTLET 25 1.4 2.5

136 14.9 

competing 3HDAGO 3HDTLET 125I Azido DTLET

ligand

13.8

617
KAPPA OPIOID BINDING SITES ON PITUITARY ASTROCYTES.

S.J. Bunn, M.R. Hanley, and G.P. Wilkin, Dept. Biochem. Imperial College of

Sci. and Tech. London SW7, England.

Opioid binding sites in the rat pituitary are predominantly located

over the neural lobe. We have demonstrated that the majority of these

sites are of the kappa subtype, and are located on the pituitary

astrocytes, the pituicyte. This unexpected observation raises the

interesting possibility that astrocytes elsewhere in the CNS may also

possess kappa opioid binding sites. A limited study suggests however

that this is not the case. Kainic acid injection of the rat striatum

induces a marked gliosis, but a concurrent reduction in the density of

opioid binding sites. The rat striatum has only very low levels of

kappa opioid binding sites. In contrast, the guinea-pig cerebellum

ehibites a high density of these sites, located predominantly over

the molecular layer (Robson et al. 84). Such a distribution would be

consistent with a location on radial Bergman glia, a type of

cerebella astrocyte. Once again, although injections of kainic acid

produced a very considerable gliosis, there was an almost total

abolition of kappa opioid binding sites.

These and other observations suggest that the pitucicytes may be unique

among astrocytes in possessing binding sites for kappa opioid ligands.

Pituicytes also exhibit a remarkable morphological plasticity,

engaging in a dynamic interaction with the neurosecretory nerve

terminals (Hatton et al. 84). It is interesting to speculate as to the

role of this interaction in regulating neurohypophyseal secretion and

as to the possible influence of opioids upon this relationship. As a

first step in addressing these questions, and as part of a wider

interest in the actions of opioids on astrocytes, we have attempted to

establish pituicytes in tissue culture. A wide variety of culturing

procedures have been evaluated and the resulting cells identified

using immunohistochemical cell-specific markers. Monolayer cultures of

three day old rat pituitary gave rise to a number of cell types,

including a population of cells intensely expressing the astrocytic

marker glial fibrillary acidic protein. The striking stellate

morphology often displayed by these astrocytes made them easily

identifible in histologically stained preparations. Autoradiograms

produced from these cultures show that kappa directed 3H-EKC binding

was selectively localized over these cells.

It is our working hypothesis that these in vitro astrocytes are

derived from the in vivo pituicytes, an hypothesis supported by the

continuity of GFAP and kappa opioid binding site expression. We

suggest that the cultured pituicyte preparation may provide a useful

model to examine the action of kappa opioid agonists.

Robson et al 1984.

Hatton et al 1984.

Neurosci. 12, 621.

Peptides 5, 121.

618
DIRECT EVIDENCE FOR THE COMPLEXATION OF A GTP-BINDING PROTEIN

WITH DELTA-OPIOID RECEPTORS. K.-J. Chang. 2. Y. Li and

S. G. Blanchard, Molecular Biology Department, The Wellcome

Research Laboratories, Research Triangle Park, NC 27709, U.S.A.

Opioid-receptors in brain are regulated by GTP and cations.

The binding affinity of opioid-agonists to receptors is decreased

by GTP and sodium ions and increased by divalent cation, Mg2+.

In neuroblastoma cells, delta-receptors are similarly regulated

by GTP and cations. Enkephalins inhibit the activity of adenylate cyclase in a GTP- and Na+

-dependent manner. Pretreatment of

cells with pertussis toxin eliminates the opioid inhibitory

effect on adenylate cyclase and decreases the affinity of

agonists to delta-receptors. These effects of pertussis toxin

are correlated with the ADP-ribosylation of a GTP-binding

protein. These data suggest that delta-receptors are associated

with a GTP-binding protein. In this study, we have used an

opioid-affinity column to obtain direct evidence that suggests

the complexation of a GTP-binding protein with delta-receptors.

The solubilized receptors were assayed with 125I-labeled [D-Ala2, -

D-Leu5]enkephalin and the GTP-binding proteins were measured by

the binding activity of 35S-labeled GTP S . Delta-receptors were

solubilized from neuroblastoma cell membranes with 10 mM CHAPS

and subsequently applied to an opioid-coupled affinity column.

Nearly 90% of solubilized delta-receptors and significant amounts

of GTP-binding proteins are retained by the affinity column.

Significant activity of a GTP-binding activity was specifically

eluted by [D-Ala2,D-Leu5]enkephalin or naloxone. No significant

activity of either protein was retained and subsequently eluted

in a control column. These data suggest that a GTP-binding

protein can be adsorbed to an opioid-affinity column and subsequently be specifically eluted by opioid-ligands. This result

provides direct evidence that a GTP-binding protein is a part of

the delta-receptor system.

619

172-483 O - 87 - 21 : QL 3
RELATIONSHIP OF OPIATE RECEPTOR BINDING SITES AND OPIATE-INHIBITED

ADENYLATE CYCLASE IN BRAIN MEMBRANES. S.R. Childers, P. Nijssen

and J. Harris, Dept. of Pharmacology, Univ. of Florida Coll. of

Med., Gainesville, Fl 32610.

Opiate agonists inhibit adenylate cyclase in both brain and

neuroblastoma cell membranes. Adenylate cyclase appears to be

coupled to delta opiate receptors in NG108-15 cells, but the

pharmacological nature of the adenylate cyclase-linked opiate

receptor in mammalian brain has yet to be established. To

determine the relationship between high affinity multiple opiate

receptor binding sites and opiate-inhibited adenylate cyclase in

brain, we assayed adenylate cyclase in membrane preparations after

treatment with various agents which are known to block high

affinity opiate agonist binding. Incubation of membranes with

phospholipase A (500 ng/ml) inhibited 93-100% of binding of

[

3

H]-DAGO, [3H]-DSTLE and [3H]-EKC to mu, delta, and

kappa sites, respectively. However, adenylate cyclase in treated

membranes was still inhibited by D-Ala enk (with inhibition

blocked by naloxone); moreover, the D-Ala enk agonist dose

response curve was not affected by phospholipase treatment. To

obtain more specific receptor blockade, membranes were incubated

with irreversible opiates, then washed to remove traces of

reversibly-acting drugs; control membranes contained equal

concentrations of naloxone to control for washing. Incubation of

membranes with several irreversible opiates, including antagonists

like naloxone azine and beta-FNA, and agonists like

p-nitro-phenyl-oxymorphone, did not affect agonist dose response

curves in inhibiting adenylate cyclase, but blocked high affinity

receptor binding by 75-100%. However, incubation of membranes with

beta-CNA (10 μM), which blocked binding by 95-1OO%, shifted the

adenylate cyclase dose response curve for D-Ala enk by approx.

ten-fold. These results indicate an irreversible loss in adenylate

cyclase-linked receptors by beta-CNA, but not by other

irreversible opiates, and suggest that receptors linked to

adenylate cyclase in brain do not correspond to any of the sites

labeled with nM concentrations of [3H]-agonists.

Suppported by grant DA-02904 from the National Institute on

Drug Abuse.

620
DEVELOPMENT OF A SELECTIVE MU1

 OPIATE RECEPTOR BINDING ASSAY.

J.A. Clark and G.W. Pasternak, The Cotzias Laboratory of NeuroOncology, Memorial Sloan-Kettering Cancer Center, New York, NY

10021, U.S.A.

Previous studies have identified a binding site of very high

affinity which bound both opiates and enkephalins with very high

affinity. In most routine assays this binding site corresponded

to approximately 20-30% of total specific binding, precluding

detailed studies of mu1 binding. Therefore, we have developed a

simple binding assay in which greater than 75% of total specific

binding corresponds to mu1 sites. There are currently no

suitable radiolabeled mu1-selective ligands. An alternative

approach is to obtain tissue enriched in mu1 sites relative to

either mu2 or delta sites. Detailed computerized digital

subtraction autoradiograms reveal relatively high levels of mu1

and mu2 sites in the thalamus, but few delta sites. We took

advantage of the low density of delta sites in the thalamus by

using 3H[D-ala2,D-leu5]enkephalin which labels mu1 and delta

sites. The low density of delta sites relative to mu1 sites

increased the proportion of mu1 to delta binding compared to

whole brain homogenates. In this assay system, 3H[D-ala2

,Dleu5

]enkephalin labels the mu1 sites with an affinity constant

(KD) of 0.4 nM and a Bmax of approximately 3 fmoles/mg tissue.

Both morphine and [D-ala2,D-leu5]enkephalin compete 3H[D-ala2,Dleu5

]enkephalin binding with IC50 values under 1 nM and

saturation studies performed in the presence and absence of

morphine reveal competitive inhibition. Other opiates and opioid

peptides also compete binding effectively. Mu1

 binding in this

assay is very sensitive to sodium chloride and guanyl nucleotides

and is irreversibly inhibited by naloxonazine. N-Ethylmaleimide

at 25 μM lowers binding by greater than 50% and trypsin (1 μg/ml)

virtually abolishes binding. The development of this assay

system should provide a means of examining the mu1 binding sites

more extensively.

621
Na+

 and GTP have

on the kinetics of 3

similar but not identical effects

H-DPR binding: they both - and syTarget size analysis of the opioid receptor in NG

108-15 cells reveals that a molecular unit of 56 kDa

accounts for the binding activity determined with 3Hdiprenorphine (

3

H-DPR) in the presence of Na and GTP,

A UNIT WHICH IS NOT SUBSTRATE OF PERTUSSIS TOXIN MAY

MEDIATE SODIUM EFFECTS ON OPIOID RECEPTORS IN NG 108-15

CELLS. T. Costa, S. Ott and A. Herz, Department of Neuropharmacology, Max-Planck-Institut für Psychiatric,

D-8033 Martinsried, F.R.G.

whereas in the absence of these agents a structure of

98 kDa is measured. This suggests that an additional

42 kDa subunit is required for opioid binding in the

absence of ions and nucleotide.

nergistically - appear to increase the proportion of a

fast-dissociating form of the receptor, but only Na+

enhances the absolute rate of association and dissociation. The slowly dissociating form is likely to represent in part a N-protein receptor complex, since in

membranes exposed to low concentrations of N-ethylmaleimide (NEM) or obtained from cells pre- treated with

pertussis toxin (PT) the receptor exists predominantly

in a fast dissociating form and no or little GTP effect

can be detected; however, the effect of Na+ is still

present. In these membranes, as well as in those from

cells treated with cholera toxin (CT), the target size

is identical to that assessed in native membranes,

whether or not GTP is present in the binding reaction.

Conversely, in the presence of Na+

, a 40% reduction of

the apparent target size is observed in+ control, PT,

CT, and NEM treated membranes. Thus, Na is the sole

agent responsible for the change in apparent target

size of the opioid receptor. We conclude that a

 ̃

42

kDa membrane unit, which is not substrate of bacterial

toxins, is also necessary for high affinity binding of

the opioid receptor and may mediate the effect of Na+.

Supported by the DFG, Bonn.

622
DEMONSTRATION AND CHARACTERIZATION OF 3 SUBTYPES OF KAPPA

OPIOID RECEPTORS IN BOVINE ADRENAL MEDULLA MEMBRANES.

R. A. Cruciani, G. O. Pesce, P. J. Munson, and D. Rodbard,

LTPB, NICHD, NIH, Bethesda, MD 20892 USA.

Opiopeptides and their receptors are present in high concentration in adrenal medulla,and may play a role in the regulation of catecholamine release and chromaffin cell function.

Previous studies have shown predominance of (relative to μ and

) sites, but there has been controversy about whether these

sites represent 1, 2 or 3 classes of sites. We have used frozen bovine adrenal medulla membranes in Tris.Cl pH 7.4, 37°, 30

min with 3H-etorphine (Et), 3H-ethylketocyclazocine (EKC), and

[

3H]-[D-Ala2

,D-Leu5]enkephalin (DADL) as tracers, displaced by

all 3 unlabeled ligands. Studies were done "unblocked" (μ,

sites), and with 100 nM DADL to block μ and sites,

and with 1, 10 and 100μM DADL to progressively block the

(or benzomorphan) site. Mathematical modeling with the LIGAND

computer program was used to analyze data from more than 100

curves, to compare models with 1 to 5 classes of sites. Results

indicate 3 subtypes of receptors. is selective for EKC

relative to Etorphine and has binding capacity of 4 fmoles/mg

prot. is EKC-Etorphine

nonselective (Kd=1 nM), and

represents 80% of the sites

(84 fmoles/mg protein) and

can be blocked by 1 to 100

micromolar DADL. i s

Etorphine selective, with

a binding capacity of 32

fmoles/mg protein. DADL at

high concentration (100 μM)

does not block Etorphine or

MC binding to o r

Conclusion: Multiple subtypes of kappa opioid receptors are present in bovine adrenal medulla membranes.

623
UPREGULATION OF BRAIN KAPPA OPIATE RECEPTORS IN

SPONTANEOUSLY HYPERTENSIVE RATS. Sumantra

Das and Hemendra N. Bhargava, Department of Pharmacodynamics,

University of Illinois at Chicago, Health Sciences Center, IL 60612,

U.S.A.

The binding of tritiated ligands for various opiate receptor

subtypes to brain membranes prepared from spontaneously

hypertensive rats and normotensive Wistar-Kyoto rats was

determined. The density (Bmax) or the apparent

dissociation constant (Kd) for the binding of the mu-ligand

(naltrexone) and delta-ligand (Tyr-D-Ser-Gly-Phe-Leu-Thr)

to brain membranes of hypertensive and normotensive rats did not

differ. However, the Bmax for the binding of kappa-ligand

(ethylketocyclazocine) to brain membranes after the suppression of

mu- and delta-sites by 100 nM each of unlabeled D-Ala2-

MePhe4-Gly-ol5-enkephalin and D-Ala2-DLeu5

-enkaphalin, respectively, was significantly greater in hypertensive

rats compared to normotensive rats. The Kd

 values for the binding

of 3H-ethylketocyclazocine (EKC) in the two groups did not differ.

The binding of 3H-EKC in brain regions was in the order:

hypothalamus> midbrain> striatum> cortex> pons + medulla. The

increase in the binding of 3H- EKC in the brain of hypertensive rats

compared to normotensive rats was due to increased binding in the

hypothalamus and cortex. These results provide for the first time

evidence for selective proliferation of kappa-opiate receptors in

the brain of hypertensive rats, and suggest that brain

kappa-opiate receptors may play an important role in the

pathophysiology of hypertension. (Supported in parts by grants

from the National Institute on Drug Abuse, DA-02598 and Chicago

Heart Association A83-30).

624
PRODYNORPHIN DERIVED PEPTIDES AND mRNA IN RAT LEYDIG CELL LINES.

L. Devi, J. Douglass, R. Thompson, O. Civelli and E. Herbert,

Institute for Advanced Biomedical Research, The Oregon Health

Sciences University, 3181 SW Sam Jackson Park Road, Portland,

OR 97201, U.S.A.

The primary structure of the rat precursor for dynorphin

peptides has recently been deduced using cDNA hybridization

technology. The precursor protein, prodynorphin, contains

several opioid peptides that have been isolated as separate

products from mammalian neural tissues. In addition,

imnunoreactive dynorphins have been found in various reprductive

tissues of rat, guinea pig and rabbit. In the rat testis, the

immunoreactive dynorphin peptides have been localized to Leydig

cells. These observations prompted us to determine the presence

of prodynorphin mRNA and dynorphin peptides in two stably

transformed rat Leydig cell lines, R2C and LC540. Prcdynorphin

transcripts were observed in poly (A+) mRNA isolated from R2C

cells but not in LC540 cells. Neither cell lines expressed the

proenkephalin gene while both cell lines expressed the

proopiomelanocortin gene. Immunoreactive dynorphin A and (Leu)

enkephalin were measured in R2C cells by radioimmunoassay. The

estimated concentration of 33fmol immunoreactive dynorphin A/mg

protein in R2C cells corresponds to a tissue concentration of

14fmol/mg protein in rat testis. Trypsin and carboxypeptidase B

treatment of the cell extract did not substantially increase the

(Leu)enkephalin immunoreactivity suggesting the absence of any

large molecular weight precursor that might have cross reacted

poorly with the dynorphin antibody. The observations that the

R2C cells contain prodynorphin transcripts and translated

products suggest that this Leydig cell line could be used to

study the dynorphin gene at the level of transcription,

translation and the processing of the propeptide.

625
THE ROLE OF ENDOGENOUS OPIOID PEPTIDES IN SUDDEN INFANT DEATH

SYNDROME AND OTHER CLINICAL SITUATIONS. William L. Dewey, Edwin

C. Myer, Dale Morris and Michael Adams. Departments of

Pharmacology and Neurology, Medical College of Virginia, Virginia

Commonwealth University, Richmond, VA 23298 U.S.A.

Sudden infant death syndrome occurs in approximately two out

of every one thousand births. The cause of death is not known

but appears to be associated with respiratory malfunction. Since

opioid peptides have been shown to cause respiratory depression

and because at least one child with documented apneic spells has

been treated successfully with a narcotic antagonist, we have

investigated whether a correlation exists between endorphin

levels in CSF and/or plasma and risk for SIDS. The CSF level of

endorphin in the siblings of SIDS was 78.8 pg/ml, and children

with interrupted SIDS had levels averaging 128.9 pg/ml. The CSF

level of endorphin in children not at a high risk for SIDS

(controls) was 32.8 pg/ml. The highest control value was below

the lowest value in the high risk group. Thus no overlap existed

between the two groups. Two children with high CSF levels of

endorphin were treated with naltrexone, which resulted in

normalization of life-threatening respiratory patterns. These

results support the hypothesis that in humans endogenous opioids

in excess may result in respiratory events which, under certain

circumstances in infants, may result in SIDS. The level of

endorphin in plasma was not elevated in the high risk group.

CSF endorphins also were found to be elevated in five children

with Rett's Syndrome, and in one child with nonketotic

hyperglycemia with ongoing seizures. Two children admitted in

status epilepticus had high endorphin levels. CSF endorphins

would appear to be the result of CNS stress events and may have

many etiologic causes. (Supported in part by USPHS Grant No.

DA01647).

626
EXPRESSION OF OPIOID PEPTIDE GENES IN MALE AND FEMALE

REPRODUCTIVE TISSUES. J. Douglass, B. Quinn, B. Cox, O. Civelli

and E. Herbert, Institute for Advanced Biomedical Research, The

Oregon Health Sciences University, 3181 SW Sam Jackson Park Road,

Portland, OR 97201, U.S.A.

Recent studies suggest a functional role for opioid peptides

as modulators of the hypothalamus-pituitary-qonadal axis at a

variety of levels in both males and females. We report here the

expression of all three opioid peptide genes in rat male and

female reproductive tissues. Prodynorphin mRNA is present in the

rat ovary, uterus and testis. Proenkephalin mRNA can be detected

in the epididymis and vas deferens, in addition to the ovary,

uterus and testis. POMC mRNA can be detected in the ovary,

testis and epididymis. Immunohistochemical analysis reveals that

prodynorphin derived peptides are expressed in Leydig cells in

the testis. The distribution of immunoreactive dynorphins in

reproductive tissues was also determined by RIA. Dynorphin A and

B immnoreactivity was detected in the testis, epididymis and vas

deferens of the rat, guinea pig and rabbit. These observations

suggest that opioid peptides may exert paracrine and/or autocrine

effects in mammalian reproductive tissues.

627
ACTION AT THE MU RECEPTOR IS SUFFICIENT TO EXPLAIN

THE SUPRASPINAL ANALGESIC EFFECT OF OPIATES.

Fernette G. Fang. H.L. Fields, and Nancy M. Lee,

Departments of Neurology and Physiology and Langley

Porter Psychiatric Institute and Department of

Pharmacology, University of California, San

Francisco, CA 94143, U.S.A.

Mu, delta, and kappa opiate receptors have been

implicated in the production of analgesia. In order

to study the relative contributions of these

receptor types to supraspinally-mediated analgesia,

apparent pA2 values and rank order potencies were

determined f or intracerebroventricularly-injected

highly selective opioid agonists in the mouse using

a thermal nociceptive assay. Drugs used included

the prototypical mu agonist morphine, put tive mu

agonists DAGO [D-Ala2, N-methyl-Phe4, Gly5-ol]

enkephalin and BAM 2 P, putative delta agonists

DPDPE [D-Pen2, D-Pen5] enkephalin, DTLET [D-Thr2,

Thr6, Leu5] enkephalin, and DSLET [Tyr-D-Ser-GlyPhe-Leu-Thr] enkephalin, and the putative kappa

agonists U50488H [trans-3,4-dichloro-N-methyl-N[2-

(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide

methane sulfonate hydrate] and dynorphin A (1-13).

We were unable to demonstrate significant

analgesic potencies for intracerebroventricularlyinjected (i.c.v.) dynorphin A (1-13) or BAM 22P in

the absence of marked behavioral abnormalities. The

rank order potency of the remaining compounds

studied was found to be: DAGO > DTLET > DSLET >

Morphine > DPDPE > U50488H. Apparent pA2 values of

morphine, DAGO, and DPDPE in naloxone antagonism

trials did not differ significantly.

These results indicate that while both mu- and

delta-selective ligands produce potent analgesia, a

single receptor (mu) is sufficient to mediate the

supraspinal effects of opiate drugs.

628
ABSENCE OF HIGH AFFINITY DELTA AND KAPPA BINDING IN RAT

PERIAQUEDUCTAL GRAY ENRICHED P2 MEMBRANE. J. P. Fedynyshyn, G.

Kwiat. and N. M. Lee, Department of Pharmacology, University of

California, San Francisco, CA 94143, U.S.A.

The periaqueductal gray (PAG) region of the midbrain has been

implicated in both stimulation produced and opioid induced

analgesia. In the present study the opioid binding

characteristics of the PAG were examined with an in vitro

radioligand binding technique. 2nM 3H-Ethylketocylazocine (EKG)

was used to nonselectively label μ, and binding sites in PAG

enriched P2 membrane. The μ selective ligand [D-Ala2,N-Methyl

Phe4,Glyol5] enkephalin (DAGO) displaced 3

H-EKC from more than

one population of binding sites with both high and low affinity.

In contrast the selective ligand [D-Pen2

,D-Pen5]enkephalin

(DPDPE) and the selective ligand U50,488 displaced 3H-EKC from

a single population of binding sites with low affinity. DPDPE

and U50,488 also displaced 2 nM 3H-DAGO from a single population

of binding sites with similar low affinity. DAGO and not DPDPE

displaced 2 nM 3H-[D-Ala2,D-Leu5] enkephalin (DADLE) with high

affinity. This binding data is consistent with the presence of a

single population of μ selective high affinity binding sites in

PAG enriched P2 membrane to which ligands and ligands have

low affinity. This data is also consistent with opioid induced

supraspinal analgesia being a primarily μ opioid receptor

mediated event. This research was supported in part by National

Institute of Drug Abuse Grants DA 02643 and 1 F31 DA

05281-01(JPF).

629
GASTRIC ACID SECRETORY EFFECTS SEPARATE OPIOID RECEPTOR TYPES.

D.A. Fox, E.A. Ayres and T.F. Burks, Department of Pharmacology,

University of Arizona, Tucson, AZ 85724, U.S.A.

Morphine and the endogenous opioid peptides produce variable

effects on gastric acid secretion. Part of this variability may

be due to the activation of different opioid receptor types. The

aim of the present investigation was to evaluate the effects of

central and peripheral administration of the mu selective agonists DAGO and PL017, the delta selective agonist DPDPE, the

kappa selective agonist U50,488H and morphine on basal gastric

acid secretion in the rat. Gastric secretion was measured in 24

hour fasted, unanesthetized, pylorus-ligated rats. Drugs were

administered immediately after pylorus ligation either intracerebroventricularly (ICV) through an indwelling guide cannula or

intravenously (IV) through the tail vein. Eats were sacrificed 2

hours after drug administration and stomach volume and acid were

measured. Acidity was determined by titration with 0.01M NaOH to

a pH of 7.0. ICV administration of DAGO, PL017 and morphine

produced a dose-dependent (0.3-10ug) decrease in both gastric

secretion volume and acidity. In contrast, central administration of DPDPE (100ug) or U50,488H (600ug) did not alter gastric

acid secretion. IV administration of morphine and PL017

(0.3-3mg/kg) significantly decreased gastric acid secretion.

U50,488H administered IV caused a dose-dependent (1-10mg/kg)

increase in gastric acid secretion, whereas DPDPE and DAGO did

not alter gastric secretion when given IV. The central

inhibitory effect of PL017 and the peripheral stimulatory effect

of U50,488H on gastric acid secretion were antagonized by

naloxone 3mg/kg administered subcutaneously 15 minutes prior to

the agonists. These results suggest that stimulation of central

and possibly peripheral mu receptors decreases acid secretion,

stimulation of peripheral kappa receptors increases gastric

secretion and delta opioid receptors are not involved in the

regulation of gastric acid secretion. Because of the clear

separation of gastric secretory effects observed with these

selective agonists, measurement of gastric acid secretion may be

useful as an in vivo bioassay to determine relative opioid

receptor selectivity. (Supported by AM36289 and AM33547.)

630
MOLECUIAR BIOLOGY OF ENKEPHALIN CONVERTASE (CARBOXYPEPTIDASE E).

L. D. Fricker and E. Herbert, Institute for Advanced Biomedical

Research, Oregon Health Sciences University, Portland OR 97201 USA

Enkephalin convertase (carboxypeptidase E) is the carboxypeptidase B-like enzyme involved in the biosynthesis of the opiate

peptides, as well as other peptide hormones and neurotransmitters.

This enzyme has been purified to homogeneity from bovine

pituitary, and a partial amino acid sequence has been determined.

Oligonucleotide probes corresponding to the amino acid sequence

have been used to isolate a cDNA clone from a bovine pituitary

cDNA library. The predicted amino acid sequence of the cDNA clone

contains several pairs of basic amino acids, and displays some

homology with carboxypeptidases A (CPA) and B (CPB). Although the

overall homology is low, all of the amino acids thought to be

essential for the catalytic activity of CPA and CPB have been

conserved in enkephalin convertase. Major differences between

enkephalin convertase and the other carboxypeptidases include an

additional 120 amino acids at the C-terminal of enkephalin

convertase, and numerous changes in amino acids near the substrate

binding sites. These changes may reflect the different

specificities of the enzymes: enkephalin convertase is very

specific for the enkephalin precursors, and does not further

degrade the enkephalin peptides. The homology between the

carboxypeptidases suggests a common evolutionary origin, which

supports the theory that the peptide processing enzymes existed

before the first opiate peptides.

The cDNA encoding enkephalin convertase hybridizes primarily to

mRNA of approximately 3.3 kb in bovine brain, pituitary, and

adrenal medulla. Restriction fragment analysis of bovine genomic

DNA indicates a single gene encodes enkephalin convertase.

Results of studies on the regulation of this gene will be

presented.

631
DYNORPHIN AND ANTERIOR PITUITARY HORMONES: EFFECTS ON

PLASMA CONCENTRATIONS IN THE MALE RHESUS MONKEY.

P. M. Gilbeau, Y. Hosobuchi and N. M. Lee, Departments

of Neurological Surgery and Pharmacology, University of

California, San Francisco, CA 94143, U.S.A.

The role of dynorphin-(1-13) and dynorphin-(1-10)-

amide in the neuroendocrine control of primate anterior

pituitary hormones was studied in nonrestrained, adult

male rhesus monkeys. The effects of these opioids on

plasma concentrations of prolactin (PRL), luteinizing

hormone (LH), follicle stimulating hormone (FSH), thyrotropin (TSH) and growth hormone (GH) were assessed.

Dynorphin or its saline vehicle were administered and

blood was collected through an indwelling jugular catheter to avoid the stress associated with these procedures. Blood samples were drawn at 20 min intervals 1

hr prior to and 4 hr post administration, additional

blood samples were collected at 5 and 10 min post

administration. Opioid doses ranged from 0.1 to 120

ug/kg. Hormones were measured by RIA. Intravenous

administration of dynorphin-(1-13), 1 to 120 ug/kg,

significantly increased plasma PRL levels. Average

maximal increases of 100% to 225% occurred within 5 min

and levels remained significantly elevated for up to 2

hr. A plateau was reached following the 30 ug/kg dose.

No effect was seen with doses lower than 1 ug/kg.

Dynorphin-(1-13) had no observable effects on plasma

concentrations of LH, FSH, TSH or GH at any dose level

studied. Administration of dynorphin-(1-10)-amide

significantly increased plasma PRL concentrations.

Dose levels of 1 to 120 ug/kg produced average peak

increases from 100% to 225%, 5 to 10 min post

administration. Dynorphin-(1-10)-amide had no significant effect on plasma concentrations of LH, FSH, TSH or

GH. The increases in PRL by dynorphin were naloxone

reversible. These results indicate a selective effect

of dynorphin on the regulatory mechanisms of PRL secretion over that of other anterior pituitary hormones.

632
Abstract

DIRECT ANALYSIS OF THE RELEASE OF METHIONINE-ENKEPHALIN

FROM GUINEA PIG MYENTERIC PLEXUS. A.R. Gintzler, W.C. Chan and

J. Glass. Department of Biochemistry, Downstate Medical Center, Brooklyn,

NY 11203, U.S.A.

The in vitro release of methionine-enkephalin (met-enkephalin) from 2

longitudinal muscle myenteric plexus (LMMP) strips from guinea pig ileum has

been obtained during continuous superfusion and quantitated directly using a

radioimmunoassay specific for this opioid peptide. Electrical stimulation

(5-80 Hz) produced a significant increase in the rate of release of metenkephalin the magnitude of which was not dependent on the frequency of

stimulation. Analysis of the release of met-enkephalin per pulse as a

function of the frequency of stimulation indicated that the release of this

opioid peptide from the myenteric plexus is inversely proportional to the

frequency of stimulation. Electrically evoked release (40 Hz) of metenkephalin was reduced by greater than 80% by substituting CoCl2

 for CaCl2

or by pretreatment with tetrodotoxin (TTx; 1 ug/ml for 15 min. Evoked

release was also substantially reduced by pretreatment with morphine (1 uM,

1.5 min). Alternatively, pretreatment of naive LMMP strips with the opiate

antagonist (-) naloxone caused a significant increase in the rate of metenkephalin release in the absence of electrical stimulation. In contrast,

(+)-naloxone was devoid of any activity. These data, in combination with

indirect pharmacological experiments, strongly indicate that met-enkephalin

functions as a neurotransmitter in the enteric nervous system. Moreover,

the activity of neurons that transmit via met-enkephalin appear to be under

opioid regulation.

633
INTRATHECAL DYNORPHIN (1-13) ATTENUATES WITHDRAWAL IN

MORPHINE-DEPENDENT RATS. P.G. Green and N.M. Lee. Department of

Pharmacology, University of California, San Francisco, Ca 94143,

U.S.A.

Dynorphin is an endogenous opioid peptide and studies have

suggested that it may be involved in tolerance and dependence

mechanisms (Lee and Smith, TIPS 5 108-110, 1984). Systemically

administered dynorphin has been shown to substitute for morphine

in morphine-dependent monkeys (Aceto et al., Eur. J. Pharmacol. 83

139-142, 1982). However, it is not known whether dynorphin acts

on spinal or supraspinal systems; both these have been implicated

in the development of opioid dependence (Yaksh et

al., Eur. J. Pharmacol. 42 275-284, 1977).

Male Sprague-Dawley rats (220-240g) were implanted either with

chronic catheters to permit injection of dynorphin directly into

the spinal subarachnoid space (i.t.) or cannulae for

intracerebroventricular (i.c.v) administration. At least 5 days

following surgery, rats were made tolerant to morphine by the

implantation of morphine pellets. Six hours after removal of the

pellets, rats were given either dynorphin or vehicle i.t. or

i.c.v. and assessed for withdrawal over a period of time.

The relative importance of spinal and supraspinal sites in the

modulation of opioid withdrawal could be determined by the

effectiveness of dynorphin administered i.t. and i.c.v. The data

from these experiments suggest that dynorphin is able to

attenuate morphine withdrawal signs by acting primarily at spinal

sites.

634
BRAIN OPIOID RECEPTOR REGULATION IN THE DEVELOPING ANIMAL.

J. Habas and A. Tempel2, 

2

Dept. of Neuroscience Albert

Einstein College of Medicine, Bronx, NY 10461, U.S.A. 1Dept. of

Medicine SUNY/Downstate Medical Center, Brooklyn, NY 11203,

U.S.A.

Chronic treatment with opiate agonists and antagonists have

been shown to induce changes in brain opioid receptor density

in adult rats. The development of these receptors and their

regulation by exogenously administered opioids in perinatal

pups has been less well characterized. We examined opioid

receptor regulation in brains of neonatal rats after chronic

agonist and antagonist treatment. Two regimens of drug treatment were used: naltrexone was administered to 1) mothers prior

to conception or 2) pups of non-treated mothers starting on day

of birth. Pups (ages 1-7 days) from each treatment group were

sacrificed and brains assayed for specific opioid receptor

types. Aliquots of brain homogenate (1.0 ml, .45 mg/ml protein) were incubated with various concentrations of [

3

H]DAla2

,Gly-ol5-enkephalin (μ specific opioid) in the presence or

absence of a 1000-fold excess of levorphanol. Scatchard analysis revealed a 139% increase in brain opioidy receptor density

on the day of birth in offspring of naltrexone-treated mothers.

Naltrexone administration to offspring of non-treated mothers

induced a 60% increase in brain u receptor number at day 7.

In a separate experiment, young rats were treated with morphine

sulfate (5 mg/kg). Results of [

3

H] etorphine (a universal

ligand) binding to brains of pups treated postnatally with

morphine sulfate revealed a 30% decrease in receptor density

after 8 days of treatment; subsequently, the number of receptors increased to control levels. These data indicate that

neonatal rats exhibit the ability to regulate brain opioid

receptors and that these receptors are in a plastic state both

in utero and for the first two weeks postnatal. (This work is

supported by NIH grant No. No. NS21973).

635
LIPID REQUIREMENT FOR OPIOID RECEPTOR BINDING. J.

Hasegawa, N. M. Lee and H. H. Loh, Depts. of Pharmacology and

Psychiatry, University of California, San Francisco, San

Francisco, CA 94143, USA.

We have recently purified to homogeneity an opioid receptor

from rat brain that shows selectivity for mu-type opioid ligands

(Cho et al., Proc. Natl. Acad. Sci., 1986, in press). While

this receptor is a protein, it requires lipid for expression of

high opioid binding activity. In order to define this requirment precisely, we have tested the ability of various lipid

components to reconstitute binding to the partially purified

protein. Several fatty acids were able to restore binding, with

linoleic (18:2), arachidonic acid (20:4) and linoleic (18:3) the

most potent; stearic (18:0), and oleic (18:l) were inactive.

Phospholipids were active if, and only if, they contained a)

active fatty acids; and b) acidic polar head groups. Thus

phosphatidic acid (PA) and phosphatidyl inositol (PI) containing

linoleic acid (18:2) restored opioid binding, while dioleoyl PA

and dilineoyl phosphatidylcholine (PC) did not. Confirming our

earlier findings, no lipids tested exhibited significant opioid

binding activity in the absence of the protein component.

These results indicate that mu opioids bind to a lipid-protein complex, and that both the polar head group and the fatty

acid of the lipid component are critical to this binding.

Because of the greater degree of specificity possible in a

protein, and because several lipids can combine with the protein

in an opioid binding complex, we believe that the opioid binding

site itself is on the protein; the role of the lipids, then,

would presumably be to stabilize a particular conformation of

the protein. However, we cannot rule out the possibility that

the roles of protein and lipid are reversed, with lipids binding

the opioid and proteins ordering the lipids in a specific array.

To decide between these two possibilities, it will be necessary

to label the receptor covalently with an opioid ligand.

636
LUMBAR INTRATHECAL MORPHINE AFFECTS PUTATIVE PAIN

MODULATORY NEURONS IN THE RAT MEDULLA. M.M. Heinrincher, K.

Drasner, F. Fang and H.L. Fields, Departments of Physiology,

Anesthesiology and Neurology, University of California, San

Francisco, CA 94143, U.S.A.

On- and off-cells are putative pain modulating neurons in

the rostral ventromedial medulla (RVM) of the rat. On-cell

activity increases, while that of off-cells decreases,

immediately prior to the tail flick reflex (TF) elicited by

noxious heat. We have previously shown that systemic administration and PAG microinjection of morphine produce an

increase in activity of all off-cells and a decrease in

activity of all on-cells concomitant with inhibition of the

TF. The present experiments investigate whether spinal

opiates similarly affect the activity of on- and off-cells in

the RVM.

A lumbar intrathecal catheter was implanted under pentobarbital anesthesia. The animals were allowed to recover for

5 to 10 days, and were then used in the electrophysiological

experiment. Activity of off- and on-cells was recorded with

a microelectrode stereotactically placed in the RVM.

Measures of spontaneous and TF-related activity were obtained

before and after intrathecal administration of 10 or 15 μg of

morphine sulfate in 5 μl of a balanced salt solution.

Intrathecally-administered morphine that inhibited the TF

(10 s cut-off) differentially affected the spontaneous

activity of on- and off-cells. Activity of all off-cells was

increased and that of all on-cells ceased following morphine.

The slowing of the off-cells and acceleration of the on-cells

in response to heat were attenuated or abolished. These

changes were reversed by naloxone (0.25-0.5 mg/kg, i.v.).

Administration of the vehicle had no effect on the activity

of these cells.

These results raise the possibility that intrathecallyadministered opiates produce analgesia in part by activation

of the sane brainstem neurons that are activated by opiates

given systemically or in the PAG.

637
IMMUNOHISTOCHEMICAL LOCALIZATION OF AMINOPEPTIDASE M

IN RAT BRAIN: RELATIONSHIP OF ENZYME LOCALIZATION AND

ENKEPHALIN METABOLISM. Louis B. Hersh and Stanley Watson,

Department of Biochemistry, Universitv of Texas Health Science

Center at Dallas, Dallas, Texas 75235 and the Mental Health

Research Institue, University of Michigan School of Medicine, Ann

Arbor, Michigan, 48109, U.S.A.

It is currently believed that the physiological action of

synaptically released enkephalins is terminated by enzymatic

degradation. Two peptidase activities have been identified in

synaptic membrane preparations which could act as

“enkephalinases” in vivo; a neutral metalloendopeptidase

(“enkephalinase”) and an aminopeptidase referred to as the

“enkephalin degrading aminopeptidase”. Recently aminopeptidase

M activity has been found in brain synaptic membranes

preparations. This aminopeptidase has been proposed to represent

the true enkephalin degrading aminopeptidase. If aminopeptidase

M could be shown to exist at enkephalinergic synapses its

involvment in the degradation of synaptically released enkephalins

would be more firmly established. Using a monospecific polyclonal

antibody to aminopeptidase M the localization of the enzyme in

several brain regions has been determined immunohistochemically.

The results clearly demonstrate a localization restricted to blood

vessels. The evidence that this aminopeptidase functions as an

“enkephalinase” must thus be reconciled with its localization on

blood vessels. A model which is suggested is one in which

diffusion of enkephalins away from their receptors is a primary

event in terminating the action of enkephalins. Distal to

enkephalin receptors several peptidases, including aminopeptidase

M, hydrolytically inactivate enkephalins. This action can serve

two functions. First it maintains a high concentration gradient

for diffusion of enkephalins away from the receptor, and secondly

it prevents free enkephalins from acting distal to the site of their

release.

638
REGULATION OF GENE EXPRESSION OF OPIOID PEPTIDES. V. Höllt, N.

Kley, J.Ph. Loeffler and C.W. Pittius, Department of Neuropharmacology, Max-Planck-Institut für Psychiatrie, D-8033 Martinsried and

Department of Physiology, University of Munich, D-8000 München,

F.R.G.

The concentrations of messenger ribonucleic acids (mRNAs) coding

for the three opioid peptide precursors proopiomelauocortin (POMC),

proenkephalin A and prodynorphin were measured in a variety of

tissues by hybridization to cloned nucleic acid sequences complementary to the opioid precursor mRNAs. Regulation of opioid peptide

gene expression at the mRNA level was investigated in the following

sets of experiments:

1. The effects of chronic stress and/or pain on the levels of

POMC mRNA in the pituitary and on proenkephalin A and prodynorphin

mRNA in the spinal cord was studied in rats in vivo. An inductive

effect of chronic stress/pain on the POMC mRNA levels in the anterior pituitary was found which could be mimicked by infusion of

corticotropin-releasing factor (CRF) and by drugs which release CRF

from the hypothalamus (e.g. morphine).

2. The influence of CRF and other secretagogues on POMC mRNA

levels was analysed in vitro using cultured rat anterior and intermediate lobe cells. CRF induced an increase in POMC mRNA levels in

both pituitary lobe cells - an effect which was found to be mediated by cAMP. The expression of POMC mPNA levels in intermediate

lobe cells was negatively regulated by dopamine and GABA and positively by ß-receptor agonists. cAMP was found to be a critical

mediator for these effects.

3. Proenkephalin A gene expression was studied in cultured bovine adrenal medullary cells. An increase in the levels of proenkephalin A mRNA was seen after activation of nicotinic

after treatment with depolarizing agents

receptors,

(veratridine, K

+

) and

after treatment with agents that stimulate the adenylatecyclase

system. These effects were inhibited by Ca2+ channel blockers indicating that, besides cAMP, Ca2+ plays a major role in mediating

proenkephalin A gene expression.

639
FEEDBACK INHIBITION OF CARBOXYPEPTIDASE H. V.Y.H. Hook and E.F.

LaGamma*, Laboratory of Cell Biology, NIMH, Bethesda, MD. 20892

and *Division of Developmental Neurology, Cornell University

Medical College, New York 10021, USA.

Carboxypeptidase H (CPH) is one of several processing enzymes

required for the conversion of peptide hormone and neuropeptide

precursors into the smaller active peptides. CPH removes COOHterminal lysine and arginine residues to result in the formation

of peptides such as (Met5)enkephalin (ME), ACTH, vasopressin, and

others. It has been refered to as 'carboxypeptidase B-like' and

'enkephalin convertase' but has now been designated 'carboxypeptidase H' ("H" for Hormone, Enzyme Nomenclature Committee of IUB)

to distinguish it from other carboxypeptidases. We demonstrate

in this study that CPH activity can be regulated by its products

by a feedback inhibition mechanism. CPH activity in bovine adrenal medulla chromaffin granules and in rat adrenal medulla homogenates, which contain enkephalin peptides, was inhibited by ME

with an inhibition constant, Ki, of 12.0 ± 0.8 ( ± s.e.m.) mM.

The known intragranular concentration forenkephalin peptides has

been estimated to be in the millimolar range. Therefore, CPH may

be inhibited by endogenous enkephalins as their concentration

rises. In contrast to ME, the other proenkephalin-derived peptides (Leu5

)enkephalin (LE), ME-Arg6-Gly7-Leu8, and ME-Arg6-Phe7

inhibited CPH with lower Ki values of 6.3 ± 0.4, 7.4 ± 0.6, and

4.4 ± 0.9, respectively. One proenkephalin precursor-molecule

contains 4 copies of the sequence ME, but contains only one copy

of each of the sequences LE, ME-Arg6-Gly7-Leu8, and ME-Arg6-Phe7.

Thus, the single copy peptides are present within the granule at

lower concentrations than ME. The difference in Ki values for

the single copy compared to the multiple copy sequences demonstrates that CPH can be inhibited by lower concentrations of LE,

ME-Arg6-Gly7-Leu8, or ME-Arg6-Phe7 compared to ME. It appears

that the final products that result from complete processing of

proenkephalin can equivalently alter CPH activity by feedback

inhibition.

640
METHAQUALONE: SPECIFIC BINDING IN CENTRAL TISSUES.

R. A. Houghten and S. M. Pratt, Scripps Clinic and Research

foundation, 10666 N. Torrey Pines Rd., La Jolla, CA 92037, U.S.A.

Tritiated analogs of methaqualone having high specific

activity were prepared for use in binding studies. Specific

binding of [3H]3-MQ (70 Ci/mmolel was found in synaptosomal

preparations of mouse, rat, and bovine brain with an apparent

dissociation constant (Kd) of 0.18-0.39 μM and a Bmax of 5.6

pmoles/mg protein. This binding was satdrable, reversible,

temperature sensitive and exhibited subcellular and regional

variations. The specific binding was inhibited by drugs that

share the pharmacological actions of methaqualone but was not

inhibited by those in unrelated classes. The ability of various

classes of opioid peptides to displace [3H]3-MQ binding will be

discussed.

641
PREPROENKEPHALIN mRNA LEVELS IN TESTIS UNALTERED BY HYPOPHYSECTOMY. R.D. Howells, B.D. Shivers, P. Morris and S.

Udenfriend, Dept. Neuroscience, Roche Institute of Molecular

Biology, Nutley, NJ 07110, U.S.A. and The Rockefeller

University, New York, NY 10021, U.S.A.

We have reported recently that the proenkephalin gene is

expressed in rat testis (Kilpatrick et al., PNAS 82:7467-7469,

1985). Since the major physiological functions of the testis,

viz. production and release of androgens and spermatogenesis,

are dependent on luteinizing hormone and follicle-stimulating

hormone derived from the anterior pituitary gland, we have

compared the steady state levels of preproenkephalin mRNA in

hypophysectomized and sham-operated rats. Four week old rats

were hypophysectomized (n = 7) then sacrificed nine days later

with age-matched, sham-operated controls. Hypophysectomy was

verified by observing a lack of weight gain over the nine day

period and by visual inspection of the sella turcica following

sacrifice. ETestes were removed, frozen in liquid nitrogen and

total RNA was isolated following CsCl centrifugation. Steady

state levels of preproenkephalin mRNA were compared between

groups using Northern blot analysis with 32P-labeled rat

preproenkephalin cDNA as probe (Howells et al., PNAS

81:7651-7655, 1984). Hypophysectomy did not alter the levels or

the size of the mRNA in testis, which is longer by about 500

bases than preproenkephalin mRNA extracted from other tissues.

Thus, the expression of the proenkephalin gene in testis is not

influenced by gonadotropins or androgens. Currently, we are

determining the cellular localization of this mRNA in testis

using in situ hybridization and enriched cell populations

maintained in culture conditions. Knowing which cell type

expresses the proenkephalin gene should provide a better

understanding of the function of peptides derived from proenkephalin in the male reproductive tract.

642
AGONIST ACTION OF THE AGONIST/ANTAGONIST ANALGESIC

BUTORPBANOL ON MESOLIMBIC DOPAMINE METABOLISM IN THE

RAT. S. Iyengar, H.C. Kim and P.L. Wood, Neuroscience

Research, Research Dept., Pharmaceuticals Division,

Ciba-Geigy Corp., Summit, N.J. 07901, U.S.A.

Agonist/antagonist (Ag/Ant) analgesics have been

shown to exhibit bell-shaped dose-response curves

with regard to analgesia and nigrostriatal dopamine

metabolism (Wood et al., Life Sci. 33(1):759-762,

1983). Throughout this bell -shaped curve, these

agents act as mu and delta opioid antagonists and

demonstrate a lack of cross-tolerance between mu,

delta and ag/ant drugs. Studies with both local drug

injections as well as parenteral drug after

hemisection showed that this phenomenon was not the

result of auto-inhibition. To determine the action

of butorphanol on mesolimbic and mesocortical DA

neuronal pathways, various doses of the drug were

administered intraperitonially and dopamine

metabolism was studied by gas chromatography- mass

fragmentography. Areas studied included pyriform

cortex, prefrontal cortex, cingulate cortex,

entorhinal cortex, olfactory tubercle, nucleus

accumbens and striatum. The effect of 16 mg/kg

morphine was studied in these regions as well.

Butorphanol had no effect on the cortical regions as

well as the olfactory tubercle, even though morphine

caused potent increases in three of the cortical

regions and olfactory tubercle. Thus, this drug had no

mu opioid action in these regions. The previously

observed bell shaped dose-response curve was

reconfirmed in the striatum. However, in the nucleus

accumbens, this drug exhibited a dose-dependent

linear increase in dopamine metabolism. It thus has

an agonist effect on a mesolimbic DA pathway which

seems to be an action of this drug that is different

from that in the nigrostriatal dopamine pathway. The

receptor affinities of this action need to be

elucidated.

643
ANTINOCICEPTIVE ACTIVITIES OF MORPHINE (A MU AGONIST) AND OF U

50,488 H (A KAPPA AGONIST) IN VARIOUS STRAINS OF MICE. J.J.C

Jacob, G Michaud, M.A Nicola and N. Prudhomme, Unit of Pharmacology, Pasteur Institute, F75724 Paris Cédex 15, France.

Previous studies have shown, in mice, genetic variations in

the latencies of some nociceptive reactions and in the amplitude,

duration and naloxone sensitivity of stress induced analgesias.

Differences were also found in the amounts of tritiated naloxone

or EKC bound to their brain extracts. In this work, range of doses

of two agonists, Morphine (Mo) and U 50,488 H, were studied in 4

strains (CXBH, CXBK, C3H and C57B16

); doses-and times-effects

curves were drawn and ED50 calculated. With the hot plate test

(response: licking) Mo had much greater effects in two strains

(CXBH - C3H) then in the two others (CXBH - CXBK) U 50,488 H was

the most effective in a different pair of strains (C3H - C57B16)

Results were different with the tail flick test. CXBK mice were

still low responsive to Mo but C57B16 mice were than high responsive. The latter strain in contrast was hardly responsive to U

50,488 H (whereas it was much more in the hot plate test). The

tail flick test was also used in spinalectomized mice (C5-C8).

Surprisingly, effects of 10 mg s.c. of Mo and of U 50,488 H were

almost similar, the pair CXBH-C3H being more responsive than the

two other strains. Assuming that Mo triggered mu receptors and U

50,488 H kappa ones, it appears that in the hot plate test, opioid

mu receptor systems (including related efferent and modulatory

pathways) would be more efficient in CXBH-C3H mice than in

C57B16-CXBK whereas kappa opioid system(s) would be more in

C3H-C57B16 than in CXBH-CXBK. When considering the tail flick

test in intact animals different mu system(s) would be involved,

at least in C57B16, a strain which would then on the contrary lack

a kappa system. The lumbo-sacral opioid system appeared to be more

developed in CXBH and C3H but no mu or kappa functional selectity

was observed; it cannot be excluded that in this latter case, the

dose used was no more selective. These observations add a new

link to previous ones.

644
EFFECT OF ADRENERGIC BLOCKERS ON THE DEVELOPMENT OF

ANALGESIC TOLERANCE TO MORPHINE IN MICE. H. Kaneto

and T. Kihara. Department of Pharmacology, Faculty of

Pharmaceutical Sciences, Nagasaki University, Nagasaki

852, JAPAN.

A single dose of reserpine dose-dependently reduced

the brain content of catecholamines and antagonized

the analgesic effect of morphine. At the peak of the

effect, 24 hr after 2.5 mg/kg i.p., the brain content

of norepinephrine was lowered to 30 % of the control

Level, and the analgesic effect of morphine was also

reduced to 40 % of that in naive animals. The reduced

effect of morphine was maintained almost the same

level during 10 daily repetitions. Low dose, 0.1 mg/kg,

of reserpine neither affected the brain content of

norepinephrine nor modified the analgesic effect of

morphine. Daily treatment of animals with this low dose

of reserpine did not induce any appreciable changes in

brain norepinephrine level during 10 days, but effectively blocked the development of tolerance to morphine.

Similarly, and blockers, phentolamine and propranolol, at the dose of 0.5, 1 and 10 mg/kg, did not

affect morphine analgesia but concomitant administration of 1 or 10 mg/kg of each blocker with 10 mg/kg

morphine suppressed the development of tolerance to

morphine analgesia. These results clearly indicate

that the suppression of the development of tolerance

to morphine analgesia is not attributed to the reduction of brain narepinephrine content by reserpine.

Maintenace of analgesic effect of morphine in reserpinized animals without development of tolerance

during repeated administration may support our previous finding that morphine analgesia and tolerance is

separable from each other by underlying mechanisms.

Effects of other adrenergic blockers are now under

investigation.

645
MU-1 OPIATE RECEPTOR: DO DISPLACEMENT EXPERIMENTS INDICATE

ITS EXISTENCE IN THE BRAIN? A. Kenner & Y.Sarne Department

of Physiology and Pharmacology, Tel-Aviv University,

Tel-Aviv 69-978 Israel.

Displacement from brain membranes of labeled opiate

alkaloids by low concentrations of enkephalins and of

labeled enkephalins by low concentrations of alkaloids , has

been previously explained by the existance of a common high

affinity site termed mu-l (Wolozin & Pasternak, PNAS

78,6181, 1981). An alternative interpretation of the same

results is that the trough seen in the low concentration

zone of the displacement curves represents cross binding of

mu and delta opioid ligands to delta and mu receptors,

respectively. That such cross binding takes place is

suggested by our experiments in which the same low

concentrations of [3H]-morphine bind to NG-108 membranes

which contain a pure population of delta receptors. In two

sets of experiments with brain membranes , the size of the

trough (previously interpreted as mu-l) is shown to be

dependent on the labled ligand used ; the ratio between the

size of troughs seen with [3H]-D-ala-D-leu-enkephalin

(DADLE) and [3H]-morphine varies with experimental

conditions (storage of membranes at 4°C for 72h ) and with

the ratio of mu:delta receptors (e.g. in thalamus and cortex

which are enriched in mu and delta sites, respectively).

These results cannot be explained by a common high affinity

site , but rather by binding of [3H]-DADLE to mu and of

[3H]-morphine to delta opioid receptors.

646
COMPARISON OF THE INHIBITORY EFFECTS OF CORTICOTROPIN-RELESING

FACTOR (CRF) AND OPIOID PEPTIDES ON THERMAL INJURY IN RATS.

J.G. Kiang, J.Q.Z. Tian and E.T. Wei, University of California,

Berkeley, CA 94720, U.S.A.

Increased exudation of plasma proteins, as measured by Evans

Blue leakage into the innervated pawskin, can be produced by

antidromic stimulation of the saphenous nerve in the anesthetized

rat. This condition, termed neurogenic plasma extravasation

(NPE), is inhibited by opioid agonists, dynorphin (1-13), and

CRF (Europ. J. Pharmacol. 114:111, 1985). Inhibition of NPE has

been attributed to interference with the actions or release of a

putative neurotransmitter, substance P, from sensory nerve endings and it has been shown by Saria (Brit. J. Pharmacol. 82:217,

1984) that administration of a substance P antagonist or depletion of substance P by pretreatment of neonatal rats with

capsaicin can also reduce thermal injury to the rat's paw. In

this study, edema and protein extravasation was produced by exposure of the anesthetized rat's paw to 48-58°C water for 5 min.

CRF, injected intradermally or intravenously at microgram doses,

dramatically inhibited these indices of thermal injury. By contrast, the effects of opioid peptides and morphine were less

clearcut. These results suggest that pharmacological modification of the activity of sensory nerve endings may have potential

therapeutic benefit for certain forms of skin injury. Supported

in part by grant USPHS NIDA-00091.

647
INCREASED MESOLIMBIC DOPAMINE METABOLISM IN THE RAT

AFTER INTRAVENTRICULAR BETA-ENDGRPHIN. H.S. Kim,

P.L. Wood and S. Ivengar. Neuroscience Research,

Research Dept., Pharmaceuticals Division, Ciba Geigy

Corp., Summit, N.J. 07901, U.S.A.

Intraventricular (ivt) injections of betaendorphin are known to increase nigrostriatal

dopamine metabolism in the rat (Wood et al., J.

Pharmacol. Exp. ther. 215:697-703, 1980). We now

report the effects of beta-endorphin on rat

mesolimbic dopamine metabolism in the N. accumbens

and olfactory tubercle. The levels of

dihydroxyphenylacetic acid (DOPAC) and homovanillic

acid (HVA) were measured via gas chromatography-mass

spectrometry.

Beta-endorphin (0.5 to 20 ug, ivt , 60 min)

produced potent, dose-dependent increases of DOPAC

and HVA, up to 175% and 155% of control in the N.

accumbens and olfactory tubercle, respectively. These

actions were antagonized by both naloxone (5 mg/kg,

ip) and the long acting narcotic antagonist WIN

44,441-3 (5 mg/kg, ip).

These data suggest that mesolimbic dopamine

projections also possess an opioid receptor

modulation. Selective tolerance experiments are still

required, however, to determine if the actions

reported involve a unique epsilon receptor.

648
CHRONIC –ANTAGONIST TREATMENT RETARDS DEVELOPMENT OF

HYPERTENSION IN THE SPONTANENEOUSLY HYPERTENSIVE RAT

(SHR). K. Kraft, J. Diehl and K. O. Stumpe, Medizinische Poliklinik, 5300 Bonn, Wilhelmstr. 35 - 37, FRG.

In general, acute application of -receptor agonists

lowers blood pressure in several species. The involvement of the -receptor in the development of hypertension is still unknown. Therefore male SHR rats at 6

weeks of age and their normotensive controls (n=11 per

group) were implanted with osmotic minipumps chronically delivering the -antagonist MR 2266 (10 μg/h) or

solvent. Systolic blood pressure (SBP) was recorded

twice a week for 4 weeks. In normotensive rats development of SBP was not affected by treatment, in SHR rats

MR 2266 significantly (p<0.01) retarded the increase of

SBP, the effect starting at 8 weeks of age (167.1 ± 3,5

vs. 178.7 ± 2.3 mm Hg (control) x±SEM). At 10 weeks of

age SBP reached 189.1 ± 3.8 and 201.6 ± 3.7 mm Hg respectively. MR 2266 did not influence the body weight

development in all groups.

It is concluded that the increase of SBP in young SHR

is retarded by chronic -antagonist treatment. This indicates a possible influence of -receptors in the development of hypertension in SHR.

649
THE DIFFERING PLASMA ß-ENDQRPHIN AND LEUCINE-ENKEFHALIN

CONCENTRATIONS IN NORMOTENSIVE AND HYPERTENSIVE PATIENTS ARE EQUALIZED BY CLONIDINE. K. Kraft, R. Theobald, K. O. Stumpe, Medizinische Poliklinik, 5300 Bonn,

Wilhelmstr. 35 - 37, FRG.

Cpiate antagonists e.g. naloxone have been shown to

block acutely the antihypertensive effect of clonidine

(C) in hypertensive (H) rats and patients, but not in

normotensive (N) controls. Thus, endogenous opioids may

participate in the antihypertensive action of C in H

patients. To determine whether there are differences in

the secretion pattern of endogenous opioids in H and N

patients, plasma ß-endorphin (ß-E) and leucine-enkephalin (L-Enk) concentrations of N and mild essential H

males (n=7, 22-35 years of age) were measured hourly

from 9 p. m. to 8 a.m. after treatment with placebo or

C (2x150 μg) for 14 days. Blood pressure and heart rate

were significantly reduced after C treatment in both

groups. R-E was significantly lower in the placebo H

group than in the N group (7.7 ± 1.8 vs. 18.7 ± 1.9

pg/ml, ± SEM) from 9 p.m. to 2 a.m., but not-during

the R-E peak (2 - 8 a.m.>. In the placebo H group also

the L-Enk concentration was lower (7.8 ± 0.5 vs. 11.9

± 0.7 pg/ml). After 14 days of C, R-E concentration was

increased in the H patients (18.4 ± 2.8), but not in

the N group (19.9 ± 4.6; 9 p.m. -2a.m.) The shape of the

ß-E peak was not influenced by C in both groups. Plasma

L-Enk was significantly reduced in N patients but not

altered in the H group (9.3 ± 0.8 vs. 9.0 ± 1.3). In

conclusion, ß-E and L-Enk plasma concentrations in

young H males are reduced. Treatment with C does not

only normalize blood pressure, but also equalizes plasma concentrations of ß-E and L-Enk. This indicates a

possible involvement of endogenous opioids in the maintenance of mild essential hypertension.

650
DERMORPHIN BINDING IN RAT BRAIN AND HEART

S. A. Krumins, Department of Neurology, Uniformed

Services University of the Health Sciences, Bethesda,

MD 20814-4799, U.S.A.

Competitive inhibition of [

3

H]diprenorphine (DPN)

binding in rat brain by dermorphin, represented by

multiphasic plots, suggests multi-site interaction

for dermorphin. Detailed displacement studies using

selective tritiated opioid ligands revealed that

dermorphin interacts preferentially with μ receptors.

In competition with [

3

H][D-Ala2, MePhe4, Gly(ol)5]-

enkephalin (DAGO), dermorphin (IC50 = 2 nM) appeared

equipotent with DAGO (IC50 = 3 nM) in Tris buffer.

The μ-preferring characteristics prevailed in competition with [

3

H]ethylketocyclasocine (EKC) in phosphate

buffer. The displacement of [

3

H]EKC by dermorphin fit

a 2-site model with approximate IC50 values of 2 nM

and 1 μM. At the addition of 0.1 μM DAGO, the major

portion of [

3

H] EKC binding was eliminated at very low

concentrations of dermorphin (> 0.1 nM). Additional

portions of [

3

H] EKC binding were displaced by the

presence of 1 μM [D-Ala2, D-Leu5)enkephalin (DADLE) and

large concentrations of dermorphin (>10 μM). However,

a considerable portion of [

3

H]EKC binding, as indicated by the displacement with unlabeled EKC, was not

displaced by large concentrations of dermorphin, indicating that dermorphin interacts very poorly, if at

all, with receptors. In competition with 3H DADLE,

dermorphin displaced the &preferring ligand according

to a 1-site model with IC50 = 75 nM, while the homologous displacement by DADLE was represented by a 2-

site model with an overall IC50 value of 3.5 nM.

Dermorphin displaced [

3

H]diprenorphine binding in both

atria and the right ventricle but overall not as

effectively as unlabeled diprenorphine.

This work was supported by USAMRDC Grant G19228.

651

172-683 O - 87 - 22 : QL 3
IN VITRO PRODUCTION AND RELEASE OF OPIOID PEPTIDES IN THE TOOTH

PULP INDUCED BY BRADYKININ. T. Kudo, M. Kuroi and R. Inoki

Department of Pharmacology, Osaka University Faculty of Dentistry,

1-8, Yamadaoka, Suita, Osaka 565, Japan

It has been suggested in in vivo experiments that bradykinin

(BK) might be a trigger in an increase of met-enkephalin(ME)-like

peptide content in the rat incisor pulp induced by noxious stimulation, and in contrast, ME-like peptides in the pulp might inhibit BK release from the pulp which was noxiously stimulated in a

mode of negative feedback mechanism. In the present study, a

possible relationship between ME-like peptides and BK in the rat

incisor pulp was examined in in vitro experiments using whole

pulp, in order to clarify the suggestion mentioned above. ME-like

peptide content in the pulp was increased by BK at a concentration

of 1 nM, but not in higher concentrations, while the release of

ME-like peptides from the pulp into the incubation medium was increased dose-dependently by BK(1 - 100 nM). These effects of BK

were inhibited by Des-Arg9-[Leu8]-BK, a potent BK-antagonist,

suggesting that the effects of BK were mediated through a specific

BK-receptor, Bl-receptor which might be generated de novo in the

noxiously stimulated tissue, in the pulp. On the other hand,

high K+(40 mM) did not induce any increased release of ME-like

peptides from the pulp and the BK-effects were influenced neither

in Ca++-free medium nor in the presence of ouabain(1 mM). These

results suggested that ME-like peptide releasing effect of BK was

not due to depolarization of the pulp cell membrane and was not

active. In addition, kyotorphin(10 μM), an enkephalin releaser,

the effect of which was Ca++-dependent, could not only elicit a

release of ME-like peptides from the pulp, but also a marked increase of the peptide content in the pulp. However, a combination

of BK(1 μM) and kyotorphin(10 μM) attenuated the effect of BK or

kyotorphin each other. These results suggested that there might

be two kinds of mechanisms of ME-like peptide production in the

pulp, one of which was Ca++-independent and another of which was

Ca++-dependent, and those mechanisms might interfere mutually.

From these findings, a physiological role of ME-like peptides in

the pulp will be discussed.

652
CHARACTERIZATION AND SOLUBILIZATION OF OPIATE RECEPTORS FROM NCB20 CELLS. L. Kushner, M. Eufemio, S.R. Zukin and R.S. Zukin,

Depts. of Neuroscience and Psychiatry", Albert Einstein College of

Medicine, Bronx, NY 10461, U.S.A.

The neuroblastoma x Chinese hamster brain hybrid cell line NCB20 is known to contain the opiate receptor and benzomorphanspecific binding sites (West, et al., 1982). The various opiate

receptors present in this cell line have been further elucidated.

The specific binding of [

3

H]ethylketocyclazocine (EKC) in the

presence of 109 nM D-Ala2, NPhe4, Gly-ol5-enkephalin (OAGO) and

100 nM D-Ala2, D-Leu5

-enkephalin (DADLE) revealed a linear

Scatchard plot, The receptor affinity (Kd) of 5.2 nM and density

(Bmax) of 92 fmol/mg were very similar to the values observed for

rat bain K receptors. The rank order of potency of a series of

opioids and related drugs was determined to be ketocyclazocine>cyclazocine>EKC at this site, whereas phencyclidine (PCP),

TCP, and (+)SKF-10,047 do not compete for binding. The binding

site for the specific ligand [3H]TCP was characterized by a Kd

of 99.7 nM and Bmax of 8863 fmol/mg. The rank order of potency of

a series of opiates, opioid peptides, and related drugs was determined to be TCP>dexoxadrol>(-)SKF-10,047, cyclazocine>(+)SKF10,047>1evoxadrol>PCE, PCP at this site, whereas DAGO, DADLE, and

normorphine do noT compeTe for binding. Binding sites for the

specific ligand [3H]D-Pen2, D-Pen5-enkephalin (DPDPE) have a Kd of

6.5 nM and a Bmax of 167 fmol/mg. Specific binding could not be

obtained using the u-specific ligand DAGO. Thus, NCB-20 cell

membranes appear to have , K, and receptors (no μ receptors

are evident), and would seem to be an excellent source for the

purification of multiple opiate receptors. Solubilization of

opiate receptors from NCB-20 cell membranes was achieved using

glycerol (50%) and the zwitterionic detergent CHAPS (10 nm); a 20%

protein yield and a two-fold enrichment of receptor binding activity in the soluble protein fraction was obtained. The opioid

receptor affinity in the solubilized fraction was very similar to

that observed in membrane preparations of the cells. Thus, the

procedure appears to maintain the native conformation of the

opioid receptor.

653
AUTORADIOGRAPHIC ANALYSIS OF KAPPA (3

H-U-69593) AND MU

(

3

H-dihydromorphine) BINDING IN GUINEA PIG BRAIN SLICES

R.A. Lahti and M.M. Mickelson, CNS Diseases Research,

The Upjohn Company, Kalamazoo, Michigan 49001 USA.

Receptor binding studies have been conducted in a

series of coronal and sagittal sections from Guinea Pig

brain using the highly selective kappa ligand 3

H-U-69593

(Lahti, et al., Eur. J. Pharmacol. 109: 281 (1985)) and

in closely adjacent slices using the selective mu ligand

3

H-dihydromorphine. Use of the two ligands demonstrated

the clear difference in localization of kappa and mu

opioid binding.

Analysis of binding results obtained with 3H-U-69593

demostrate that kappa sites are located in specific

brain areas. Highest concentrations of kappa binding were

noted in cortical areas, nuc. accumbens, olf. tubercles,

and substantia nigra reticulata. On the other hand, mu

binding had its highest concentration of sites in the

striata, piriform cortex, amygdala, hypothalmus and the

substantia nigra.

Autoradiograms of coronal and sagittal sections will

be presented as will quantitative data on selected brain

areas.

654
YOHIMBINE PRODUCES OPIOID WITHDRAWAL-LIKE REACTION IN ADDICTS.

R. R. Lannon, S. Batkl and R.T. Jones, Drug Dependence

Research Center, Dept. of Psychiatry, University of California

Medical Center, San Francisco, CA 94143, U.S.A.

Yohimbine HCI, an alpha2 adrenergic receptor antagonist, or

placebo was administered orally (0.43 mg/kg and 0.21 mg/kg) to

methadone addicts and normal volunteers to test whether opiate

addicts have dysregulated noradrenergic system homeostatic

mechanisms and, specifically, to determine whether antagonism

of the alpha2 feedback system produces an accentuated noradrenergic response. Yohimbine or placebo was given 30 min

after the patients’ usual daily oral methadone dose (38 mg)

under double-blind conditions. Blood pressure, heart rate,

skin temperature, respiration, other signs and symptoms of

opiate withdrawal and changes in mood were measured. After

the high dose of yohimbine, the addicts experienced shakiness, restlessness, loss of appetite, tension, chills and hot

flashes, stomach cramps, urge to urinate, twitching, muscle

cramps, sweating ng, nausea, dlarrhea, difficulty concentrating,

anxiety and irritability. Addicts had greater blood pressure

increase and skin temperature drop after yohimbine compared

to controls. The addicts characterized the symptoms as similar to opiate wfthdrawal. Clonidine, an alpha2 agonist,

suppresses noradrenerglc hyperactivity in opiate withdrawal.

Addicts, even after their usual dose of methadone, demonstrate

symptoms consistent with opiate withdrawal upon exposure to an

alpha2 antagonist. Opiate addicts and patients with affective

disorders (panic disorder, depression) may share similar

defects in locus ceruleus activity Important for malntaining

homeostasis In the noradrenerglc system.

655
DOES NALOXONE ANTAGONIZE THE ANALGESIC EFFECT OF THE

PARTIAL OPIATE AGONIST BUPRENORPHINE IN MAN?

W.P. Lehmann and C. Schmauss, Max Planck Institute for

Psychiatry, Munich F.R.G

In human volunteers the thermal pain threshold was

determined by use of a Marstock thermode through which

heat was applied to the thenar of either hand. The

temperature at the thermode-skin interface was measured

by a thermocouple glued to the thermode surface and

continuously recorded by a pen recorder. Subjects manipulated the stimulus temperature by turning a potentiometer until they experience a "slightly painful" sensation which was referred as thermal pain threshold. The

partial opiate agonist buprenorphine (0.2-0.4 mg s.1.)

significantly elevated the thermal pain threshold.

Naloxone alone administered as i.v. bolus (O-1-10 mg)

did not significantly change pain threshold. Naloxone

pretreatment with 0.1 mg i.v. still completely blocked

the analgesic effect of buprenorphine but a 200 fold

higher dose was required to antagonize the established

analgesic effect of buprenorphine. The antagonism, however, was readily established when suprahigh doses of

naloxone were administerd. These data do not necessarily support the concept of a "slow dissociation" of

buprenorphine from its receptor which is often referred

as reason for buprenorphines resistance to naloxone

antagonism. It is suggested that either a high receptor

occupancy or a "facilitated association" of buprenorphine with its opiate receptor accounts for its relative naloxone resistance.

656
THE EFFECT OF ANALGESIC DOSES OF MORPHINE ON CELLULAR

ACTIVITY IN PAIN RELATED STRUCTURES: A 2-DEOXYGLUCOSE

AUTORADIOGRAPHIC STUDY. R.M. Levy, M.P. Stryker, M.M.

Heinricher and H.L. fields, Deoartments of Neurological

Suroery, Neurology and Physiology, University of

California, San Francisco, CA 94143, U.S.A.

Desoite reports that morohine (MS) diffusely

decreases regional cerebral glucose metabolism (RCGM),

the effect of opioids on RCGM in behaviorally

documented analgesic states has not been investipated.

We studied RCGM using high resolution 2-deoxyglucose

(2-DG) autoradiography in rats during naloxonereversible MS-induced analgesia. Tail flick latency

(TF) to a noxious heat stimulus was tested in 12

ligntly anesthetized adult male rats. TF testings was

repeated 2 min after MS (5 mg/kg IV). All animals had

inital TF of 3-4 sec: after MS, TF increased to cutoff

(> 10 sec). 14C-2-DG. 100 uCi/kg IV, was then administered and arterial glucose and radioactivity measured

at logarithmic intervals. 45 min later, rats received

2 mg/kg naloxone IV and in all cases, TF returned to

baseline. Animals were then sacrificed and standard

autoradiography was oerformed on 20 micron tissue

sections. Sections were analyzed using computer

assisted digital techniques and RCGM calculated from

tissue and plasma curves. Evaluation of RCGM after MS

administration in pain related structures. including

periaqueductal pray, nucleus reticularis paragigantocellularis and nucleus raphe magnus, reveals that while

there is a trend toaward increased RCGM in these

structures. there is no statistically significant

change in RCGM as compared to non-pain related

structures and to saline treated controls. These

results suggest that, within the limits of resolution

of this technique (200 microns), these pain related

structures contain several cell oaoulations with heterogeneous responses to ooiates in doses which produce

analpesia.

657
REGULATION OF THE CONCENTRATION OF DYNORPHIN A (1-8) IN THE STRIATONIGRAL PATHWAY BY THE DOPAMINERGIC SYSTEM. S. Li, S. P. Sivam, J. F.

McGinty* and J. S. Hong, LBNT, NIEHS, NIH, Research Triangle Park,

NC, *Dept. Anatomy, East Carolina Univ., Greenville, NC, U.S.A.

It has been reported that the dopaminergic system exerts potent

influence on the metabolism of [Met5]-enkephalin and Substance P in

the basal ganglia. Recent reports indicate that dynorphin-like immunoreactivity (DN-LI) is present in the striato-nigral pathway and

is found in high concentration in substantia nigra. However, the

regulation of dynorphin in this pathway is not known. The purpose

of this study was to explore the dopaminergic control of the striatonigral dynorphin system by measuring the levels of DN-LI after repeated injections of dopaminergic agonist and antagonist. Rats received different doses of apomorphine (0.5 mg-5.0 mg/kg, s.c. twice

a day), haloperidol (1 mg/kg s.c. twice a day) or in combination

for different periods and were decapitated 16 h after the last injection. Concentration of DN-LI was determined by radioimmunoassay

using a specific antiserum against dynorphin A (1-8). Seven daily

injections of apomorphine (0.5, 1.0, 2.5 and 5.0 mg/kg) caused a

dose-related increase of DN-LI in the striatum (26, 34, 63, 85% over

control at each corresponding dose). Similar increases were observed in the substantia nigra (22, 52, 50 and 62% over control).

Immunocytochemical study revealed an increase in DN-LI, but not

[Met5]-enkephalin-like immunoreactivity in cell bodies located in

striatal patches. An increase in fiber staining of DN-LI in the

substantia nigra was also observed. In another experiment, rats

received 5 mg/kg of apomorphine for 1, 3, and 7 days. There was a

time-related increase in DN-LI both in the striatum (37, 50 and 85%

over control at each corresponding period) and in the substantia

nigra (32, 78 and 62%). In order to examine whether apomorphineelicited increase in DN-LI in the basal ganglia is mediated by the

dopaminergic receptors, haloperidol was injected alone or in combination with apomorphine for 7 days. Haloperidol alone failed to

alter the striatal DN-LI, but completely blocked the apomorphineinduced increase in DN-LI. These results suggest that dopamine

exerts potent influence on the metabolism of dynorphin in the basal

ganglia.

658
CLEAVAGE OF PROENKEPHPALIN BY A CHROMAFFIN GRANULE TRYPSIN-LIKE

PROCESSING ENZYME. I.Lindberg and G. Thomas, Dept. Biochem.,

L.S.U.M.C, 1901 Perdido St., New Orleans, LA 70112, and I.A.B.R.,

Oregon Health Sciences University, 3181 S.W. Sam Jackson Park

Rd., Portland, OR 97201 USA.

Proteolytic processing enzymes with trypsin-like specificity

are required for the maturation of prohormones and neuropeptide

precursors into active peptide products. We have been interested

in the use of cDNA expression systems as a means to generate

native prohormones which can then be used to study processing

enzyme candidates. Proenkephalin was produced by infect ion of

African Green Monkey Kidney cells with vaccinia virus containing

cDNA for human proenkephalin. Intact proenkephalin secreted into

the medium was partially purified by passage over a Peptide B

affinity column followed by gel filtration chromatography. This

material was used as the substrate for the chromaff in granule

trypsin-like protease (Lindberg et al, BBRC 106, 186, 1982).

Following incubation at 37°C for varying amounts of time the

reactions were stopped by boiling, and then further incibated

with pancreatic carboxrpeptidase B (in order to remove carboxyl

terminal basic residues), Digestion products were analyzed by

radioimmunoassay using antiserum to met-enk-arg-gly-leu, an

internal sequence in proenkephalin. The production of met-enkarg-gly-leu-immunoreactive peptides from proenkephalin was

dependent on the length of incubation with the trypsin-like

enzyme. In addition, no immunoreactivity was observed in the

absence of additional carboxrpeptidase B digestion. Gel filtration of digestion products revealed the presence of low molecular

weight immunoreactive met-enk, leu-enk, and met-enk-arg-gly-leu

in incubated samples but not in control reactions which were

stopped immediately. HPLC identification of low molecular weight

immunoreacttve products revealed the presence of authentic metenk, met-enk-arg-gly-leu, and their sulfoxides. Taken together,

the results indicate that 1) the chromaffin granule protease

possesses the correct specificity to act as an enkephalingenerating enzyme in vivo; and that 2) cDNA expression systems

represent a useful method for the production of prohormone

substrates for processing enzymes.

659
PARTIAL PURIFICATION OF THE MU OPIOID RECEPTOR IRREVERSIBLY

LABELED WITH [

3

H] FUNALTREXAMINE. L.-Y. Liu-Chen, C. A.

Phillips, S. W. Tam, and L. G. Davis, Central Research and

Development Department and Biomedical Products Department,

E. I. du Pont de Nemours and Company, Experimental Station,

Wilmington, Delaware 19898, U.S.A.

The mu opioid receptor in bovine striatal membranes was

irrevessibly labeled with high specificity by incubation with

5 nM [3H] funaltrexamine ( FNA) at 37°C for 90 minutes in

the presence of 100 mM NaCl. The specific irreversible

binding of [3H] FNA, as defined by that blocked by 1 μM

naloxone, was approximately 60% of total irreversible binding.

The specific irreversible binding was saturable, stereospecific, time-, temperature-, and tissue mass-dependent. Mu

opioid ligands were much more potent than delta or kappa

ligands in inhibiting the specific irreversible labeling. SDS

polyacrylamide gel electrophoresis of solubilized membranes in

the presence of 2-mercaptoethanol yielded a major radiolabeled

broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled

in the presence of excess unLabeled naloxone or FNA. The

glycoprotein nature of the [3H] FNA-labeled opioid receptor

was confirmed by its binding to a wheat germ agglutinin

(WGA)-Sepharose column and its elution with N-acetylglucosamine. Following this lectin column chromatography the mu

opioid receptor became the only protein labeled by [

3

H] FNA

in the absence of naloxone; no labeled protein was observed in

the presence of naloxone. WGA column eluate was digested with

pronase and the resultant mixture was subjected to high

performance liquid chromatography using C18 reverse phase

column. Three radiolabeled peptide peaks were obtained and

purified to apparent homogenity. Works are currently under

way to obtain sufficient material for sequence determination.

660
CONTROL OF PROOPIOMELANOCORTIN (POMC) GENE EXPRESSION

BY CAMP AND CALCIUM. J.Ph. Loeffler, N. Kley C.W.

Pittius and V. Höllt, Department of Neuropharmadology,

Max-Planck-Institut für Psychiatrie, D-8033 Martinsried

and Department of Physiology, University of Munich,

D-8000 Miinchen, F.R.G.

The role of the second messengers CAMP and Ca++ in

the control of POMC gene expression was investigated.

Using hybridization with cloned cDNA probes, we measured POMC mRNA level in primary cultures of intermediate (IL) and anterior (AL) rat pituitary cells maintained in serum free medium.

8 Bromo-cAMP (1 mM) but no 8 bromo-cGMP (1 mM) induced a 2-fold increase in IL POMC mRNA levels. Furthermore, forskolin (1 μM) which directly stimulates the

catalytic unit of the adenylate cyclase (AC), and cholera toxin (100 ng/ml) which activates the stimulatory

subunit (Ns) of the AC, induced an increase in CAMP

levels and a two-fold increase in POMC mRNA levels in

IL and AL cells after two days of treatment.

In another set of experiments, we analyzed the role

of Ca++ in POMC gene expression. When Ca++ entry was

blocked with D600 (50 μM) and nifedipine (100 nM), POMC

mRNA levels decreased to 50% of the control levels.

Similar decrease was obtained when Ca++ free medium was

used. The Ca++channel agonist BAYK 8644 (100 nM) stimulated POMC gene expression and this effect was additive with forskolin. Taken together, these data suggest

that CAMP and Ca++ are implicated in the regulation of

POMC gene expression in both intermediate and anterior

cells of the rat pituitary.

661
EFFECTS OF PHORBOL ESTERS ON OPIOID RECEPTOR ACTIVITY IN

NEUROBLASTOMA X GLIOMA NG108-15 HYBRID CELLS. A. K. Louie,

P. Y. Law and H. H. Loh, Departments of Pharmacology and

Psychiatry, University of California, San Francisco, CA, 94143,

U.S.A.

Incubation of neuroblastana x glioma NG108-15 hybrid cells

with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate

(TPA) decreased opioid inhibition of CAMP accumulation in

intact cells. In contrast, basal and prostaglandin

E1-stimulated cAMP accumulation were unaltered by TPA

treatment. The effect on opioid inhibition was dose- and

time-dependent and TPA abolished up to 4% of opioid activity.

The potency order of phorbol esters and apparent Kact (90nM)

for TPA were consistent with phorbol esters mediating this

attenuation through the stimulation of protein kinase C. TPA

also decreased the inhibition of CAMP accumulation seen with

muscarinic and alpha2-adrenergic agonists.Scatchard analysis

of saturation binding of these membrane receptors demonstrated

that TPA decreased the number of opioid and muscarinic

receptors but not the number of alpha2-adrenergic receptors.

These data suggested that TPA attenuation of receptor activity

might be at the site of the inhibitory-nucleotide-binding

protein (Ni). When Ni was quantified by measuring the

ADP-ribosylation of its 41 kDa alpha subunit by pertussis toxin

and [

32P]NAD+

, TPA treatment decreased the maximal labeling

without a change in the NAD Km. In contrast, ADP-ribosylation

of the 45 kDa alpha subunit of the stimulatory-nucleotidebinding protein (Ns) by cholera toxin was unchanged by TPA

treatment. The functioning of the remaining N. protein was

assessed by measuring their activation by guanine nucleotides.

Studies on the effects of GTP and its stable analogue GppNHp on

membrane adenylate cyclase activity were consistent with TPA

inducing a decrease in Ni activation compared to control. We

conclude that TPA attenuated opioid inhibition of adenylate

cyclase and decreases in the number of opioid receptors and Ni

proteins were identified as potential mechanisms for this

action.

662
AUTORADIOGRAPHIC VISUALIZATION OF SIGMA RECEPTORS WITH

[

3

H]1,3 DI-O-TOLYL-GUANIDINE (DTG). S. McLean, J. F. W. Keana2

and E. Weber3, Laboratory of Neurophysiology, NIMH, Bethesda,

MD 20892, 2Dept. Chemistry, Univ. Oregon, Eugene, Oregon 97403,

and 3

Institute for Advanced Biomedical Research, Oregon Health

Science University, Portland, Oregon 97201, USA.

The existence of sigma receptors, which appear to mediate

the psychotomimetic effects of certain opiates, was proposed

by Martin et al. in 1976. Recently, a new compound, DTG has

been synthesized (Weber et al., this meeting) that exhibits

selectivity and specificity for the sigma receptor.

Using receptor autoradio raphy we have investigated the neuroanatomical distribution of [3

H]DTG binding. Biochemical studies

using slide mounted tissue sections were carried out to establish

binding conditions. Sections of guinea pig and rat brain were

incubated for 45 min at 22o C in 50mM Tris (pH 8.0) containing

1mg/ml BSA and 2nM [

3

H]DTC, washed (4x2min) in ice-cold 10mM

Tris(pH 7.4) with 1mg/ml BSA, blown dry and placed in x-ray

cassettes with 3H sensitive film. Blanks incubated with 10

uM haloperidol indicated specific binding of 90 percent.

[

3

H]DTG binding sites were heterogeneously distributed

throughout the guinea pig and rat brain. In the forebrain, dense

binding was present in the diagonal band of Broca, the paraventricular nucleus of the hypothalamus, and along the border of the

medial and lateral habenula. Moderate densities were present in

olfactory and retrosplenial cortex, pyramidal and granule cell

layers of the hippocampal formation, and throughout the hypothalamus, septum, zona incerta, and midline nuclei of the thalamus.

Low densities of diffuse binding were present in cortex, striatum,

and thalamus. In midbrain a high density of binding was localized

to the subcommissural organ, red nucleus and oculomotor nuclei,

a moderate density in the periaqueductal gray, and low levels

in the colliculi and substantia nigra. In the hindbrain dense

binding was found in the locus coeruleus with moderate levels

in the nucleus of the facial nerve and cerebellum.

These results demonstrate a distinct distribution of sigma

receptors that is different from that of the mu, delta, or kappa

opiate receptor subtypes.

663
INVESTIGATION OF THE INHIBITORY EFFECT OF HIGH FREQUENCY ELECTRICAL

STIMULATION ON THE GUINEA PIG ILEUM. L. R. McMartin, J. E. Olley

and W. Hope, Department of Pharmacology, Monash University, Clayton,

Victoria, Australia, 3168.

High frequency electrical stimulation (> 5Hz) of the isolated

ileum of guinea pig elicits a post-stimulation inhibition of twitch

responses at 0.1 Hz - a major component of which is considered to

be opioid (Puig et al, 1978). Experiments were performed to

investigate this inhibition. Naloxone (10 nmol/l) significantly

reduced the inhibitory response from 76.0% inhibition (s.e.m.= 4.2:

n = 61 to 51.6% (s.e.m.=1.8; n = 6) (P < 0.051. Mr 2266 [(-)2-

(3-furylmethyl)-5,9-diethyl-2'-hydroxy-6,7-benzomorphan]

(10 nmol/ll also significantly reduced the inhibition from 84.5%

(s.e.m. = 3.1; n = 5) to 36.3% (s.e.m. = 8.0; n = 5)(P < 0.05).

These data indicate a possible involvement of both μ opioid

receptor ligands. The purinergic antagonist 8-phenyltheophylline

(10 μmol/l) did not significantly affect the inhibition (n = 5,

P > 0.051. Since the slow offset and resistance to washing

resembled properties of the potent endogenous ligand dynorphin,

the bathing fluid was examined for the presence of dynorphin

following 3 min high frequency stimulation. Preliminary evidence

was found for the presence of dynorphin-(1-13). dynorphin-(1-17)

and a number of other as yet unidentified inhibitory components

using reverse phase liquid chromatography techniques. Spasmogenic

activity was also found. It is suggested that the high frequency

stimulation-induced inhibition comprises several components, some

of which may be the long-acting opioid dynorphins.

REFERENCES

Puig, M.M., Gascon, P. & Musacchio, J.M. (1978). The Journal of

Pharmacology and Experimental Therapeutics, 206, 289-302.

664
MIDBRAIN STIMULATION-PRODUCED ANTINOCICEPTION (SPA)

IN THE FREELY-MOVING RAT: EVIDENCE FOR MEDIATION BY

ß-ENDORPHIN(ß-EP) AND μ-OPIOID RECEPTORS:

M.J. Millan, A.Czlonkowski, M.H.Millan and A.Hers,

Cepartment of Neuropharmacology, Max-Planck-Institute

for Psychiatry, Am Klopferspitz 18a, F.R.G.

Electrical stimulation of the ventral midbrain periaqueductal grey(PAG) of the rat evokes an antinocicention, in the mediation of which opioids play a role.

The particular opioid peptide and receptor type responsible is, however, unknown. We have, thus, addressed this

question employing freely-moving rats.

Unilateral stimulation of the ventral PAG elicited

an antinociception strongly attenuated by a low dose

of naloxone. The locomotor effects (ipsilateral turning) were unaffected. Destruction of central ß-endorphinergic neurones, which project from the hypothalamus

to the PAG, greatly depleted ß-EP from brain and strongly reduced SPA, but not the locomotor effects. Pharmacological blockade of pituitary secretion of ß-EP into

the plasma was ineffective. Chronic perfusion with a

low dose of naloxone selectively blocked the antinociception induced by a μ-agonist (morphine) but not a

agonist (U50,488). Following pump removal, morphine but

not U50,488 antinociception was enhanced, reflecting a

supersensitivity of μ-receptors. As with morphine, SPA

was reduced in the presence of naloxone and potentiated

upon its removal. Recurrent stimulation led to a tolerance of SPA: tolerant rats showed a reduced antinociceptive action of morphine but not U50,488.

We suggest that the antinociception against noxious

pressure and heat evoked by stimulation of the ventral

midbrain PAG is mediated by central ß-EP and μ-opioid

receptors. Whether ß-EP acts directly on μ-receptors

remains to be clarified. In addition, a possible significance of any ' '-receptor, for which ß-EP has been

postulated to be a ligand, remains open.

Supported by the Deutsche Forschungsgemeinschaft.

665
KAPPA-TYPE OPIOID RECEPTOR IN HUMAN PLACENTAL MEMBRANE.

M. Niwa, *S. Ohta, **K. Kamikubo, M. Nozaki, K. Tsurumi and

***H. Fujimura. Departments of Pharmacology, *Anesthesiology and **3rd Internal Medicine, Gifu University School

of Medicine, Gifu 500 and ***Kyoto Pharmaceutical University

Yamashina, Kyoto 607, Japan.

We have reported that kappa opioid receptor in rat brain

possesses a different coupling mechanism from those of mu

and delta receptor between receptor binding site and GTPbinding protein. In human placental membrane, 3

H-EKC bound

to a single class of binding site (Kd:0.75nM, Bmax:60fmol),

and 3H-DHM and 3H-DADLE showed no binding activities. The

EKC binding was strongly displaced by U-50488H, dynorphin A

(1-13) and bremazocine, but not by morphine and DADLE.

These data suggest that the EKC-binding site in human

placental membrane is a homogeneouse kappa receptor.

The inhibitory effect of Gpp(NH)p in high concentration

(1mM on the EKC-binding was very weak in the presence or

absence of 100mM NaCl, in a similar manner as kappa receptor

in rat brain. The effects of SH-group brocking reagent and

pertussis toxin on the EKC-binding and post-receptor

mechanisms will be discussed.

666
IN VIVO AUTORADIOGRAPHY: MATING-PRODUCED CHANGES IN

OPIATE RECEPTOR OCCUPANCY ASSESSED BY

[

3

H] DIPRENORPHINE AND [

3

H]CYCLOFOXY BINDING IN

BRAIN. N. L. Ostrowski, K. Rice and A. Pert,

Biological Psychiatry Branch, NIMH and Laboratory of

Chemistry, NIADDK, NIH, 9000 Rockville Pike, Bethesda,

MD 20892, U.S.A.

Opiate receptors are found in hypothalamic regions

that regulate neuroendocrine and reproductive functions

in rats and hamsters. Using the procedure of Seeger et

al., 1984 autoradiographic experiments were conducted

to determine whether mating (a physiological stimulus

Known to affect the hypothalamic-pituitary-gonadal

[H-P-G] axis) produced changes in opiate receptor

occupancy, an index of endogenous opioid release. Male

hamsters were selected for testing because they show

dense opiate receptor binding in the sexually dimorphic

nuclear complex of the hypothalamus and their mating

behavior is reliable, precisely timed and resistant to

disruption by the administration of opiate antagonists.

Males were injected (IV) with [

3

H]cyclofoxy (CF) or

[

3

H]diprenorphine (DP) after: a) the 5th

ejaculation of a mating session, b) exposure to a

receptive female wearing a vaginal mask, or, c)

exposure only to their home cage. Twenty min after the

injection, animals were sacrificed, their brains

removed, rapidly frozen and processed for film

autoradiography. Computer assisted densitometric

analysis of the binding showed a decrease in the

accumulation of both [

3

H]DP and [

3

H]CF in the

cingulate cortex sexually dimorphic nuclear complex

and in the anterior but not caudal striatum of brains

from mated males relative to control-mated males.

Liquid scintillation counting of adjacent brain

sections verifled high brain to cerebellar accumulation

ratios, a lack of difference in cerebellar (i.e.,

non-specific) binding among groups and the ability of

naloxone, when co-administered with the radioligands,

to completely block brain accumulation of labeled drug.

These data suggest that mating produces an Increase in

opiate receptor occupancy, presumably by releasing

endogenous opioids in discrete brain regions. This

technique may prove valuable for investigating the

neuroanatomical loci of opioid peptide-regulation of

the H-P-G axis in living animals.

667
MU AND KAPPA RECEPTORS ARE COUPLED TO Ca++ TRANSPORT IN REGULATING

TEMPERATURE RESPONSE. N. P. Pillai and D. H. Ross, Molecular

Pharmacology Division, University of Texas Health Science Center,

San Antonio, TX 78284-7764, U.S.A.

The effect of opiates on body temperature has been shown to

depend on both dose and route of administration. Additionally, Mu

and Kappa agonists have recently been shown to increase and decrease respectively basal temperature in unrestrained rats (Geller

et al, l993). Opiates in vitro and in vivo have been shown to

alter Ca++ content, binding and transport in brain membranes.

Temperature regulation has previously been shown to be influenced

by Ca++ ion movements in the hypothalamus. We have evaluated two

opiate agonists, morphine (Mu) and U50-488H (Kappa) for their

effect on temperature and Ca++ ATPase in hypothalamus. Morphine

(15 mg/kg s.c.) produced hyperthermia (+ 1.5°C) at 2 hr. Ca++

ATPase in synaptic membranes was inhibited 24%. Hyperthermia and

enzyme inhibition were both reversed by naloxone (2-5 mg/kg).

Ca++ ATPase activity was decreased in P2 fractions of hypothalamus

(18%) and cortex (20%) (p<.05) but not cerebellum. Ca++ channel

antagonists nimodipine (1 mg/kg), verapamil (2.5 mg/kg) or diltiazem (5 mg/kg) reversed hyperthermia and Ca++ ATPase inhibition.

Administration of U50-488H (3-20 mg/kg s.c.) produced a decrease

in basal temperature in unrestrained rats. This hypothermia was

accompanied by an enhancement of Ca++ ATPase in synaptosomal P2

fractions from hypothalamus but not cortex or cerebellum. Naloxone reversed the hypothermia and enhanced enzyme activity while

nimodipine, verapamil and diltiazem potentiated the hypothermic

effects and stimulation of enzymic activity. These results

suggest that opiate Mu and Kappa agonists may increase and decrease respectively

intracellular Ca++

basal temperature by a mechanism which alters

levels. Changes in Ca++ ATPase activity in the

hypothalamus together with blockade of Ca++ influx may contribute

to altered intracellular Ca++. These biochemical changes may be

differentially coupled to Mu and Kappa receptors. Supported in

part by grant from Upjohn Company.

668
BINALTORPHIMINE: A HIGHLY SELECTIVE, KAPPA OPIOID RECEPTOR

ANTAGONIST. P. S. Portoghese,* A. W. Lipkowski,* and A. E.

Takemori, Departments of Medicinal Chemistry* and Pharmacology,

University of Minnesota, Minneapolis, MN 55455, U.S.A.

The concept of multiple opioid receptors has created a need

for selective antagonists for the identification of different

receptor types and for the investigation of agonist selectivity.

Although selective opioid antagonists are available for mu and

delta receptor types, the development of highly selective kappa

antagonists has been elusive. A key problem has been the inability to eliminate the mu antagonist component. Here we report

on the utilization of the "bivalent ligand" approach to designing

an extraordinarily selective kappa antagonist, binaltorphimine

(BNI), that is devoid of significant mu antagonist activity. The

chemical structure of BNI is 17,17'-bis(cyclopropylmethyl)-

6,6',7,7'-tetradehydro-4,5 4',5' -diepoxy-6,6'-(methylimino)

[7,7'-bimorphinan]-3,3',14,14'-tetrol. In the guinea pig myenteric plexus longitudinal muscle preparation (GPI), BNI (20 nM)

was a potent antagonist of the kappa-selective agonist, ethylketazocine (EK) (IC50 ratio, 165 ± 30). No significant antagonism

of the mu-selective agonist, morphine, was observed (IC50 ratio,

1.2 ± 0.3). In the mouse vas deferens preparation, BNI (20 nM)

weakly antagonized the effect of the delta-selective antagonist,

[D-Ala2,D-Leu5]enkephalin (IC50 ratio = 4.5 ± 0.7). In mice,

BNI (20 mg/kg IV) exhibited a selectivity profile similar to that

in the GPI, with sc ED50 ratios (treated/control) for EK,

U50488H, and morphine of 5.9, 14.6, and 0.85, respectively.

These data indicate that BNI possesses no demonstrable mu antagonist activity and is the most highly selective kappa opioid

receptor antagonist reported. Moreover, a comparison of the / μ

selectivity ratio of BNI indicates that its kappa selectivity is

over 800-times greater than that of naltrexone. The fact that

BNI contains two opioid antagonist pharmacophores (a "bivalent

ligand") raises the possibility that its high selectivity and

potency are due to the simultaneous occupation of two vicinal

recognition sites on the kappa receptor system. The significance

of these results and the possible organization of vicinal kappa

recognition sites will be discussed. (supported by NIDA grants)

669
CNS INTERACTION OF OPIOIDS AND STEROIDS IN BLOOD PRFSSURE REGULATION. L.A. Reiser and E.E. Hahn, Rockefeller University, 1230

York Ave., New York, NY 10021, U.S.A.

Considerable experimental evidence has now been obtained that

endoyenous opioids are involved in the regulation of blood pressure. Data from our laboratory have demonstrated that hypertensive rats have higher levels of opiate receptors relative to normotensive controls. This increase, which is dependent on age,

coincides with the appearance of elevated blood pressure. In other studies, we showed that opiate receptors in SHR rats exhibited

an abnormal sensitivity to salt which suggests that the impact of

sodium on blood pressure may be related to its effect on the conformation of the receptor. Evidence has also accumulated that a

CNS component may participate in the biochemical mechanisms by

which steroid hormones regulate blood pressure. This activity may

be mediated by specific hormones which are synthesized in situ in

localized brain regions, or alternatively by those which enter the

CNS by crossing the blood brain barrier. The discovery of an interrelationship between the opioid and endocrine systems led us to

evaluate whether such an interaction in specific central sites may

participate in blood pressure regulation. In studies on the metabolism of androstenedione in the CNS of rats we have shown that

19-hydroxylation of androqens greatly exceeds aromatization, and

that 19-hydroxy and 19-oxo androstenedione, both of which are hypertensinoqenic agents, accumulate in quantities five times

greater than estrogens. Using a radiometric procedure we have

demonstrated that CNS production of these 19-oxygenated androyen

metabolites was greater in SHR compared to age matched WKY rats.

When naloxone or naltrexone was injected subcutaneously (0.3-1.0

mg/kg) in CD or WKY rats an increase in CNS androyen 19-

hydroxylase activity was observed. The impact of opiates on this

biotransformation was reinforced by in vitro studies in which

preincubation of

centrations ranging from 10-9 to 10-6 M inhibited the activity of

hypothalamic preparations with morphine in conthe 19-hydroxylase. In conjunction with our report that administration of opiate antagonists to either WKY or SHR rats increases

blood pressure, the results support the hypothesis that an interaction in the brain between endoyenous opioids and 19-

oxygenated androqen metabolites may be involved in blood pressure

regulation.

670
PURIFICATION OF THE HUMAN ERYTHROCYTE OPIOID RECEPTOR.

D. Rhoads, A. Rezvani, T. Cho, P. Law, H. Loh and

E. Way. Department of Pharmacology, University of

California, San Francisco, CA 94143 U.S.A.

Previous studies from this laboratory demonstrated

that opiates reduce human red blood cell deformability

by a mechanism that can be correlated with the

prevention of Ca++ efflux through inhibition of

Ca++ ATPase. Moreover, the inhibiting effect is

dose-dependent, stereospecific and naloxone

reversible. The rank order of potency of various

opiates and opioids in inhibiting cell deformability

as well as their binding affinities suggest the

involvement of an opiate receptor with kappa-like

properties. In order to isolate this receptor,

erythrocyte ghosts were solubilized in 0.5% Triton

X-100 and purified by application to a wheat germ

lectin-agarose column and elution with

n-acetylglucosamine. When the eluate was applied to a

dynorphin A (1-13)-Sepharose affinity column and

eluted with nalbuphine, one major band was obtained

following SDS polyacrylamide gel electrophoresis.

Further purification by HPLC on Spherogel-TSK columns

yielded a protein peak which after two dimensional gel

electrophoresis exhibited a relative pI of 5.8 and an

apparent molecular weight of 60,000. The calculated

degree of purification was 11,250 fold which is close

to the theoretical value of 11,500. That this

apparently homogeneous material is the receptor

responsible for opioid binding and action was

substantiated by: 1) the lack of change in affinity

for 3H-diprenorphine and 3H-dynorphin(1-8) after

purification, 2) other opioids compete for the

3H-diprenorphine binding with the same affinity, and

3) cross linking 3H-dynorphin(1-8) to the isolated

material with bifunctional reagents labeled a 60,000

Dalton protein.

Supported by NIDA grant DA00037.

671
HALOPERIDOL INCREASES PROENKEPHALIN MRNA LEVELS IN THE CAUDATEPUTAMEN OF THE RAT: A QUANTITATIVE STUDY USING IN SITU HYBRIDIZATION. G.J.Romano, B.D.Shivers, R.E.Harlan, R.D.Howells* and

D.W.Pfaff. The Rockefeller University, New York, N.Y. 10021 and

*The Roche Institute for Molecular Biology, Nutley, N.J. 07110 USA

Chronic haloperidol (HAL) treatment increases the levels of

proenkephalin (PE) peptides and mRNA in the caudate-putamen (CPU)

(Tang, et al. PNAS 80:3841,1983). To determine whether this

increase in PE gene expression is exhibited by all enkephalinergic

neurons of the CPU or only by a subpopulation, we have examined

this effect on PE mRNA levels at the cellular level using in situ

hybridization. Male rats were injected ip with 2mg/kg HAL or

vehicle (VEH) daily for 2 wks,decapitated 24hr after the final

injection, and CPu were dissected and stored at -80°C.A cDNA

probe complementary to rat preproenkephalin, (Howells, et al.

PNAS 81:7651,1984), was nick-translated with [32P] or [3H] nucleotides. In situ hybridization was carried out following the method

of Shivers, et al. (Meth. Enz.V124,1986). Emulsion autoradiograms

of cryostat sections hybridized with a [3H] probe were subjected

to grain counting to quantitate the autoradiographic signal.

Labelled neurons were uniformly distributed throughout the CPu.

HAL treated rats (n=6) had 3.0-fold more grains/labelled neuron

than VEH-treated rats (n=5). The total number of labelled neurons

in the CPu did not increase following HAL treatment. Film autoradiograms of sections which were hybridized with a [32Pl labelled

probe also showed a 3.0-fold increase in the signal from the CPu

after HAL treatment. For dot blot analysis, total RNA was isolated from individual CPu tissue blocks. The autoradiographic

signal intensities of the dots from HAL-treated animals (n=7) were

2.7x greater than the dots from VEH-treated animals (n=5),

concurring with the results of northern blots.(Tang, et.al., ibid.)

These findings suggest that dopamine exerts a tonic inhibitory

effect on PE gene expression in all enkephalinergic neurons of the

CPu. The agreement between the in situ hybridization results

and dot blot analysis supports in situ hybridization as a method

for quantitative studies of gene expression in the brain.

672
TEBULET, A NEW HIGHLY POTENT AND SELECTIVE LIGAND FOR OPIOID

RECEPTORS. B.P. Roques, G. Gacel, C. Seguin, V. Daugé, J. Belleney

and P. Delay-Goyet, Département de Chimie Organique, U 266 INSERM,

UA 498 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 4

avenue de l'observatoire 75006 Paris, France.

Two types of compounds are commonly used for the biological and

the pharmacological characterization of 6 opioid receptors ;

DTLET, a linear hexapeptide (1) and D-Pen2

-Pen5 enkephalin a

cyclic pentapeptide (2). However both ligands are not perfect,

since the former is highly potent (Ki = 1.35 ± 0.15 nM) but its

cross-reactivity with μ sites remains relatively high (Ki / Ki μ

= 0.053), whereas the latter is very selective (Ki / Ki μ =

0.0125) but displays a somewhat low affinity (Ki = 10.9 ± 1.2

nM) (3). From the proposed model (4) of selective recognition of μ

or sites and extensive conformational studies of linear and

cyclic peptides, new ligands belonging to the linear hexapeptide

series were designed in order to increase the selectivity

without loss of affinity. The must efficient compound Tebulet (5)

characterized by a constrained structure exhibits a Ki = 4.9 ±

0.6 nM and a discriminatory factor Ki / Ki μ = 0.0122. This new

proble was triticted and is now proposed as reference ligand for

studies of receptors.

1. Zajac, J.M., Gacel, G., Petit, F., Dodey, P., Rossignol, P.

and Roques, B.P., Biochem. Biophys. Res. Commun. 111, 390-397

(1983).

2. Mosberg, H.T., Hurst, R., Hruby, V.J., Gee, K., Yamamuza,

M.J., Gillican, J.J. and Burks, J.F., Proc. Natl. Acad. Sci.

80, 5871-5874 (1983).

3. Delay-Goyet, P., Zajac, J.M., Rigaudy, P., Foucaud, B. and

Roques, B.P., FEBS Letters 183, 439-443 (1985).

4. Fournié-Zaluski, M-C., Gacel, G., Maignet, B., Prenilat, S.

and Roques, B.P., Mol. Pharmacol. 20, 484-491 (1981).

5. French Patent 80.17.523 ; US Patent 4,407,794.

673
AN EXAMINATION OF THE OPIATE RECEPTOR TYPE5 LABELED

BY THE OPIATE ANTAGONIST, [

3

H]CYCLOFOXY: A LIGAND

SUITABLE FOR POSITRON EMMISION TOMOGRAPHY. R. B.

Rothman1, S. McLean2, V. Bykov1, A. E. Jacobson3,

R.A. Lessor3and K. C. Rice3,

 1Laboratory of

Preclinical Pharmacology, St. Elizabeths Hospital,

NIMH, Washington DC 20032. 2

Laboratotory of

Neurophysiology, NIMH, Bethesda, MD, 20205.

3

Laboratory of Chemistry, NIADDK, Bethesda, MD,

20205. U.S.A.

The major goal of this study was to define the

types of opiate receptors labeled in vitro by the

novel opiate antagonist [

3

H]cycloFOXY synthesized by

Rice and associates. The three approaches used

were: 1. quantitative 1 igand binding studies

utilizing the technique of binding surface analysis;

2. selective site-directed alkylating agents BIT

(mu-selective), FIT (delta-selective), and beta-FNA

(mu-selective) ; 3. receptor autoradiography.

Receptor binding assays were conducted using

frozen lysed-P2 preparations of rat brain. The

assay conditions were 4 to 6 hr incubations at 4° C

in 50 mM TRIS-HCl, 100 mM NaCl, pH 7.4, containing

the protease inhibitors bacitracin, bestatin,

chymostatin, and leupeptin.

Pretreatment of membranes with FIT did not

significantly reduce C[3H]cycloFOXY binding, whereas

pretreatment with BIT or FNA reduced binding by 20

and 25 % respectively. Binding surface analysis of

the interaction of DAGO and dynorphin (1-8) with

[

3

H]cycloFOXY binding resolved into 2 binding sites.

The Kd’s (nM) of DAGO and dynorphin were: 7.1 and

480 for DAGO, and 2912 and 141 for dynorphin.

Autoradiographic studies are in progress.

The data presently available suggests that

[

3

H]cycloFOXY labels mu and kappa binding sites in

rat brain.

674
UNEXPECTEDLY HIGH LEVELS OF OPIOID FEPTIDES IN RAT

BRAIN MEMBRANES. R. B. Rothman,1

J.A. Danks1and M.

Iadarola1.

 1Laboratory of Preclinical Pharmacology,

St. Elizabeths Hospital, NIMH, Washington DC 20032,

U.S.A.

An important experimental paradigm is the

measurement of neurotransmitter receptors in vitro

following in vivo manipulations of the animal. The

possibility that observed changes reflect changes in

receptor occupancy by endogenous ligands must be

carefully ruled out.

These considerations led us to directly measure

using radioimmunassay the levels of dynorphin (1-8),

CCK-8, and MERGL in lysed P2 membranes (group I),

membranes incubated 60 min at 25o C (group II) in 50

mM TRIS-HCl at pH 7.4, and membranes incubated 60

min at 25° C in TRIS buffer containing 0.4 M NaCl

(group III).

pmol/mg protein

GROUP DYN(1-8) CCK-8 MERGL

I .253+.031 1.727±.221 .983±.081

II .108_+.009 1.015±.224 .585±.090

III .106_+.029 .179+±.007 .240±.060

Similar results were obtained when substance-P was

measured. Additional experiments indicated that

membranes rapidly degraded MERGL added to the

homogenate. Freincubation of membranes with 0.4 M

NaCl failed to increase the levels of [

3

H]etorphine

binding, indicating that frozen lysed-P2 membranes

do not have endogenous ligands occupying opiate

receptors. Detailed comparisons of the two binding

sites labeled by [

3

H]naloxone revealed changes in

binding parameters induced by preincubation with

NaCl. Additional studies are required to determine

the significance of apparently sequestered

neuropeptides in this preparation of rat brain

membranes.

675
Purified mu-opioid receptor exhibits phosphatase activity. S.Roy,

N.M. Lee and H.H. Loh. Deparment of Pharmacology,University of

California, San Francisco, CA. 94143.

A mu-opioid receptor protein has been purified by this

laboratory (Cho et al., 1986). The purified receptor is a

glycoprotein with a molecular weight of 58,000 as determined by

SDS-polyacrylamide gel electrophoresis. Preliminary

investigations show that endogenous phosphatase-like activity is

assosciated with this protein. Further studies show that the pH

optimum for the enzymatic activity was at pH 9 with a Km= 9uM

when para-nitro phenol phosphate was used as the substrate.

Analysis of gel slices following SDS-gel electrophoresis show

that the major peak of phosphatase activity is assosciated with a

band of apparent molecular weight 60,000, which corresponds to

the molecular size of the purified mu-opioid receptor protein.

Our results also show that the enzymatic activity is activated by

Mg2 + , Mn2 + and Ca2 +. Further characterization of the enzymatic

activity will be presented.

676
HUMAN NEUROBLASTOMA SK-N-SH CELLS: PHENOTYPIC VARIATION OF μ AND

OPIOID RECEPTORS, MUSCARINIC CHOLINERGIC RECEPTOR AND THE UPTAKE1

SYSTEM. W. Sadée1

, V.C. Yu1, M.L. Richards1, F.M. Brodsky2 and

J.L. Biedler3.

 1School of Pharmacy, University of California San

Francisco, CA 94143, 2Becton Dickinson Immunocytometry Systems,

Mountain View, CA 94039, and 3

Memorial Sloan-Kettering Cancer

Center, New York, NY 10021, U.S.A.

Neuroblastoma cells grown in culture can undergo phenotypic

changes between epitheloid and neuroblast forms. We have

established the presence of μ and opioid receptors, muscarinic

cholinergic receptors and an effective uptake1 transport sytem for

norepinephrine in the human SK-N-SH cell line.In order to

address the question of the expression of these systems during

phenotypic modulation, phenotypically stable subclones of SK-N-SH

were used that exist either in the neuroblast state (SY-5Y), an

intermediate state (IN), or the epitheloid state (EP). While both

the neuroblast SY-5Y and the intermediate IN subclones expressed

all three systems to similar degrees,epitheloid cells were devoid

of these systems. This result documents that the coordinate

expression of multiple receptor systems correlates with the

phenotypic switch from epitheloid to neuroblast status. In

contrast there was no change of the expression of clathrin heavy

and light chain subunits with the different phenotypes. Further,

the light chain clathrin subunit was of the peripheral tissue-type

rather than the brain-type, which is consistent with the

peripheral source of neuroblastoma tissue samples. Supported by

DA 01095 from NIDA, Bethesda, MD.

677
ACTIVATION OF MEDULLARY OPIOID-PEPTIDERGIC SYSTEMS BY

FORMALIN-INDUCED PERSISTENT PAIN AND ITS PHYSIOLOGICAL

ROLE. M. Satoh, M. Suqimoto, Y. Kuraishi and H.

Takaqi, Department of Pharmacology, Faculty of

Pharmaceutical Sciences, Kyoto University, Sakyo-ku,

Kyoto 606, Japan.

The nucleus reticularis gigantocellularis (NRGC)

including nucleus reticularis paragigantocellularis is

known to be an important site of analgesic actions of

opioids. Kuraishi et al. have demonstrated an increased

release of immunoreactive Met-enkephalin (iME) from the

nucleus following injection of formalin to the hind-paw

in rats. In the present experiments, we further studied

the physiological role of opioid-peptidergic system and

the content of iME in the rat NRGC. 1) Durations of

licking response were measured at periods of 0-10 min

(P-A), 10-30 min (P-B), 30-60 min (P-C) and 60-120 min

(P-D) after formalin injection into the hind-paw of the

rat. Naloxone injected systemically (using miniosmotic

pumps) or into the 4th ventricle (30 nmol/rat), but not

intrathecally (up to 300 nmol/rat), significantly

increased the duration at P-C. On the contrary,

injections of enkephalin-degrading enzyme inhibitors

(thiorphan plus bestatin; 50 μg each/rat) into the 4th

ventricle significantly suppressed the duration at P-C.

2) The region of NRGC was punched out from the freezed

slices of lower brain stem of rats. The content of iME

in the NRGC increased after treatment with thiorphan

plus bestatin. This increase was augmented by formalin

injection into the hind-paws, while treatment with

cycloheximide blocked such an augmentation. The iME

content measured after digestion of NRGC extracts with

trypsin and carboxypeptidase B was elevated by formalin

injection in rats given thiorphan plus bestatin.

These findings suggest that formalin-induced persistent

pain stimulates the biosynthesis of ME in the NRGC and

is inversely attenuated by the stimulated MEergic

system.

678
THE ROLE OF SPINAL KAPPA-OPIATE RECEPTORS IN ANALGESIA.

C. Schmauss and A. Herz, Max Planck Institute for

Psychiatry, Munich, F.R.G.

Male Spraque Dawley rats were implanted with a

chronic intrathecal catheder extending into the lumbar

subarachnoid space. The antinociceptive potency of

intrathecally administered morphine and the putative

kappa-opiate receptor ligands bremazocine, Dyn 1-17,

Dyn 1-13, U50488H and trifluadom was estimated in the

tail flick (TF) and tail pressure (TP) nociceptive

tests. Morphine equipotently inhibited both TF and TP

nociceptive responses. The kappa-ligands Dyn 1-17,

Dyn 1-13, trifluadom and U50488H blocked TP but not TF

nociceptive responses at doses which did not impair

motor function. Only bremazocine was more potent in

blocking TF than TP nociceptive responses. Morphinetolerant rats did not exhibit tolerance to the analgesic effects of U50488H and trifluadom on the TP response, indicating that these ligands act upon a

spinal opiate receptor which is distinguishable from

the receptor through which morphine exerts its analgesic effect. In line with these results is the observation that the effects of bremazocine on the TF, but

not on the TP nociceptive response, show cross-tolerance to morphine. These data suggest that the spinal

kappa-opiate receptor is involved in the mediation of

TP analgesia. Further support is obtained for the suggestion that spinal opiate receptor subtypes differentially associate with different nociceptive input.

679
A MONOCLONAL ANTI-IDIOTYPIC OPIOID RECEPTOR ANTIBODY. R.Schulz1

and C. Gramsch, Department of Neuropharmacology, Max-PlanckInstitut für Psychiatrie, Martinsried; 1

 Institut für Pharmakologie, Toxikologie und Pharmazie, Universität München, FRG.

The 3-E7 monoclonal antibody, which recognizes the N-terminal sequence of endogenous opioid peptides, has been employed

to raise monoclonal antibodies against opioid receptors via an

anti-idiotypic route. Culture supernatants were screened for

immunoglobulins that interacted with F(ab)2 -fragments of the

3-E7 antibody coated to the wells. Their presence was identified by an anti-IgM-Fc -peroxidase test(ELISA). A second screening procedure involved binding to opioid receptors in rat

brain membranes, using 125I-ß-endorphin as tracer. Positive

master wells were cloned by limiting dilution. Supernatants of

posltive clones were tested in the radio receptor assay

(

125I-ß- endorphin as tracer). Positive samples were then

purified by anion-exchange chromatography and the purified

material was subjected to SDS-gel electrophoresis. This

technique revealed the presence of an immunoglobulin, showing

characteristics of a reference mouse-IgM. Furthermore,

purified antibodies were submitted to affinity-chromatography

(3-E7 linked to Sepharose). The gel retained the active

principle responsible for the interaction with opioid

receptors. The highly purified antibodies were employed for

detailed receptor binding studies on rat brain membranes. The

antibody was most powerful in competing with 125I-ß-engorphin, followed by 3

H-D-Ala2,-D-Leu5-enkephalin and

3

H-ethylketazocine; it showed a much lower competition with

3

H-dihydromorphine. In contrast, the antibody failed to

interfere with the benzodiazepin 3H-flunitrazepam. The data

suggest the successful generation of a monoclonal anti-idiotypic antibody of the IgM-type with affinity for both the 3-E7

monoclonal antibody (antigen) and membrane-bound opioid

receptors.

680
MECHANISM BY WHICH ASCORBIC ACID ABOLISHES THE ETORPHINE

INDUCED COMPENSATORY INCREASE IN INTRACELLULAR CYCLIC AMP

LEVELS IN NG108-15 HYBRID CELLS

Shail K. Sharma, Sasanka Chakraborti and Madhav Bhatia,

Department of Biochemistry,All India Institute of Medical

Sciences, New Delhi-110029, INDIA.

In NG108-15 hybrid cells ascorbic acid suppressed

the delayed etorphine induced compensatory increase in the

levels of cyclic AMP. It had however, no effect on the

early response of the cells to etrophine, as manifested in

2 transient decrease in the levels of cyclic AMP (1,2).

The mechanism of action of ascorbic acid is not understood.

In this communication we report on the following

observations. (1) The effect of ascorbic acid on transient

inhibition of adenylate cyclase activity in the homogenates

of NG hybrid cells. (2) Binding of (15,16-3H) etrophine to

monolayer cultures of NG hybrid cells in the presence or

absence of ascorbic acid. (3) Adenylate cyclase and

guanine nucleotide regulatory protein activity of homogenates

of NG hybrid cells which were grown with etrophine in the

presence or absence of ascorbic acid.

(1) Shail K. Sharma and Navin C. Khanna. Biochem. J. 208,

43-46 (1982).

(2) Navin C. Khanna and Shail K. Sharma, Life Sciences,

33, 401-404 (1983).

681
BETA-ENDORPHIN ACUTELY STIMULATES SUPEROXIDE PRODUCTION BY HUMAN

POLYMORPHONUCLEAR LEUKOCYTES: COMPARISONS TO MORPHINE AND FMLP.

B. Sharp, D. Tsukayama, W.F. Keane and P.K. Peterson, Dept. of

Medicine, Hennepin County Medical Center and University of

Minnesota, Minneapolis, MN 55415, U.S.A.

We have recently shown that -endorphin ( -END), dynorphin

and morphine rapidly elicit chemiluminescense from human

polymorphonuclear leukocytes (PMN) in vitro due to the production of superoxide (O2

-

). The present work evaluated the opiate

receptor involved in the acute PMN response to -END. Using a

ferricytochrome C reduction assay to detect

significantly stimulated O2

-

 production by PMN from normal

donors (N = 5) at concentrations between 10-14-10-8M. The peak

response, 14.3 ± 1.3 (mean ± SE) nM2O2

-

/mg protein, occurred at

a physiological concentration (10 . N-acetyl- -END did not

affect O2

-

abolished the stimulation by -END 10-12M, whereas (+)-naloxone

production and (-)-naloxone 10-12M completely

showed minimal inhibition (<16%). (-)-Morphine sulfate (MS)

produced a similar dose-response profile to -endorphin,

although peak PMN O2

-

accumulation was significantly greater

with -endorphin (p<.O5). Unlike -endorphin, MS was ineffective

at 10 M. N-formyl-methionyl-leucyl phenylalanine (FMLP), a

peptide which stimulates PMN chemotactic activity and O2

-

 production, was tested for comparison. FMLP-stimulated O2

-

 accumulation

was first detectable at 10-12M; in contrast to -END and MS, the

response to FMLP increased directly with concentration. Both

(+) and (-)-naloxone were equipotent against FMLP 10-8M,

resulting in only 30% inhibition which was not significant.

H2O2, the immediate product of O2

-

 metabolism, was also

measured. Significant HzO2 release by PMN followed stimulation

by endorphin 10-12M. This was markedly reduced (92%) by

nitroprusside, an inhibitor of superoxide dismutase. We conclude

that: a) -endorphin acutely stimulates O2

-

 and H2C2 production

by PMN at peptide concentrations present in the systemic

circulation; b) a stereoselective, μ -like opiate receptor

mediates the action of -endorphin. Thus, stress-peptides and

exogenous opiates may modulate host defense.

682
QUATERNARY OPIOID ANTAGONISTS : AN APPRAISAL, J.S. Shaw, N.J.W.

Russell and R. James, ICI Pharmaceuticals Division, Alderley Park,

Macclesfield, Cheshire. SK10 4TG, U.K.

There have been several recent reports on the action of

opioids at receptor sites outwith the blood-brain barrier. Such

studies have relied on the use of standard opioids quaternised on

the basic nitrogen which are believed to have limited access

to the CNS.

In the present study, receptor affinities have been determined

for five quaternary antagonists and their parent compounds

(naloxone, naltrexone, nalorphine and levallorphan). This has

been related to their ability to reverse μ and k agonist effects

in an abdominal constriction test modified to demonstrate

peripheral opioid actions (Bentley, Newton and Starr, Brit. J.

Pharm. 73, 325, 1981) and in conventional antinociceptive assays.

In all cases quaternisation reduced the affinity for both μ

and k-receptors and the extent of this reduction varied

considerably between compounds. Taking this into account in

subsequent in-vivo studies, N-methyl-naloxone showed no peripheral

selectivity. In contrast, the potencies of N-methyl-levallorphan,

N-allyl-levallorphan, N-methyl-nalorphine and N-methyl-naltrexone

were substantially lower (10-100 fold) in hot plate and

conventional abdominal constriction tests than could be accounted

for by loss of affinity. This indicates limited CNS penetration.

However, attempts to demonstrate peripheral antagonism with these

compounds in the Bentley, Newton and Starr test were only

successful with N-methyl-nalorphine and N-methyl-naltrexone. The

two levallorphan analogues were ineffective at doses up to 30mg/kg

S.C.

These results highlight the need for caution in the use of

quaternary opioid antagonists as research tools.

683
BIPHASIC MOTIVATIONAL PROPERTIES OF PARTIAL OPIOID AGONISTS:

μ-RECEPT0RS MEDIATE THEIR REINFORCING EFFECTS. T.S. Shippenberg,

R.F. Mucha and A. Herz, Dept. of Neuroparmacoloqy, Max-PlanckInstitut für Psychiatrie, Am Klopferspitz 18a, D-8033 Martinsried,

F.R.G. and Addiction Research Foundation of Ontario, 33 Russell

Street, Ontario, Canada.

The positive reinforcing effects of opioids have been attributed to agonist actions at μ-receptors, whereas activation of

receptors may underlie their dysphoric or aversive effects. The

motivational properties of the opioid agonist-antagonist analgesics are little understood and the nature of their interactions

with particular opioid receptor subtypes remains ill-defined. The

present study employed a place conditioning procedure to characterize the reinforcing/aversive properties of these druqs and the

receptor mechanisms mediating their effects. Rats were exposed to

one of two distinctive environments following injections of drug

or vehicle. After six such training sessions, preference for a

particular environment was assessed by allowing rats free access

to both settings and measuring the time spent on each. Pure p-agonists such as morphine produced clear preferences for the drugpaired place, while the selective -agonists, U-69593 or U-50488,

produced dose-related aversions. Of the partial agonists tested,

pentazocine was reinforcing at low doses while nalorphine was

aversive. Nalbuphine and buprenorphine produced biphasic motivational effects. Preferences were observed with low doses while

higher doses resulted in significant aversions for the drug-paired

place. Constant infusion of naloxone via minipumps during training, atadose sufficient to blocky-receptors but not -receptors, antagonized the preferences induced by morphine and the partial agonists, but was ineffective in modifying their aversive

properties or those of U-69593. These data strongly indicate that

the partial agonists pentazocine, nalbuphine and buprenorphine,

in addition to their purported -agonist and μ-antagonist like

actions, can function as agonists at the μ-opioid receptor and

that activation of this receptor underlies their reinforcing

effects.

Supported by Bundesgesundheit Berlin, F.R.G.

684
CELLULAR LOCALIZATION OF PROENKEPHALIN mRNA-CONTAINING NEURONS IN

RAT BRAINSTEM AND SPINAL CORD. B.D. Shivers, R.E. Harlan, G.J.

Romano, *R.D. Howells and D.W. Pfaff, The Rockefeller University,

New York, NY 10021 and *Roche Institute of Molecular Biology,

Nutley, NJ 07110, U.S.A.

Previously, we reported the location of proenkephalin

mRNA-containing neurons in rat forebrain (Harlan et al., Society

for Neuroscience 11:143, 1985), and we have now extended the

description of the location of these neurons to brainstem and

spinal levels. We performed in situ hybridization on frozen,

paraformaldehyde-fixed tissue sections probed with a tritiated 435

b.p. cDNA complementary to rat proenkephalin mRNA encoding amino

acids 56-200 (Howells et al., PNAS 81:7651, 1984).Autoradiograms

were exposed 40 days. Hybridizations following RNase

pretreatment, or with probe diluent alone yielded no labeled

cells. Competition with a 25-fold excess of unlabeled probe

greatly reduced the autoradiographic signal. Sections probed with

tritiated rat pro-opiomelanocortin cRNA (gift of B. Schachter)

yielded a different labeling pattern than that observed with

proenkephalin cDNA. Labeled cells were found in the lateral

geniculate nucleus, periaqueductal gray, midbrain reticular

formation, ventral tegmental area of Tsai. inferior colliculus,

dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral

parabrachial nuclei, pontine and medullary reticular formation,

some of the raphe nuclei, nucleus of the solitary tract, nucleus

of the spinal trigeminal tract, ventral and dorsal cochlear

nuclei, medial and spinal vestibular nuclei, cuneate and external

cuneate nuclei, gracile nucleus, superior olive, nucleus of the

trapezoid body, some deep cerebellar nuclei, Golgi II neurons of

the cerebellum, and most laminae of the spinal cord, especially I

and II. Generally, there was good agreement between regions shown

previously to contain immunoreactive enkephalin neurons and those

regions which we found to have neurons containing proenkephalin

mRNA; however, in many of these regions, more neurons were found

to contain proenkephalin mRNA than were shown previously to

contain immunoreactive enkephalins. The results are consistent

with the concept that opioid peptides derived from proenkephalin

participate in the integration of motor and sensory information.

685
ISOLATION AND SEQUENCING OF OPIOID PEPTIDES FROM PORCINE

BRAIN EXTRACTS USING A [

3

H]-DAGO BINDING ASSAY TO FOLLOW

THE PURIFICATION. Mark Sonders, Bruce Campbell, Charles

Jimenez, Marjorie Shih, and Eckard Weber. Institute for

Advanced Biomedical Research, The Oregon Health Sciences

University, Portland, Oregon 97201, U.S.A.

One of the current projects in our laboratory concerns

the isolation of a putative endogenous sigma receptor

ligand using a binding assay to follow the purification

(see our presentation elsewhere in this meeting). Binding

assays have rarely been used to isolate and characterize

endogenous neurotransmitter or neuromodulator candidates

and it is therefore unclear whether the use of binding

assays to purify endogenous receptor ligands will yield

substances of physiological relevance. In order to

determine whether binding assays can be used to follow the

purification of endogenous receptor ligands yielding

physiologically meaningful compounds, we used an opioid

receptor binding assay to follow the purification of

putative opioid active peaks in chromatography fractions

from separated acid acetone extracts of 5 porcine brains

(513 g). The mu-opioid receptor-specific radioligand

[

3

H]-DAGO was used in the binding assay. At least eight

major peaks of [

3

H]-DAGO displacement activity were

observed in porcine brain extracts that had been separated

on a preparative reverse phase C-18 column. Five of the

peaks were purified to homogeneity using various reverse

phase HPLC separation systems. The homogenous peaks were

subjected to gas phase sequencing and/or amino acid

composition analysis using microanalysis methods

established in our laboratory. The five compounds were

unambiguously identified as known opioid peptides from the

proenkephalin and prodynorphin precursors.

[Met5]-enkephalin and alpha-neoendorphin represented the

largest peaks of [

3

H]-DAGO displacement activity. These

studies demonstrate that radioligand binding assays can be

used to follow the purification of endogenous

neurotransmitter or neuromodulator candidates and that this

method can yield physiologically meaningful endogenous

receptor ligands.

Supported by NIMH grant MH40303.

686
IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN

ENDOGENOUS COMPOUND THAT COMPETITIVELY AND REVERSIBLY

INHIBITS [

3

H]-DTG FROM BINDING TO THE

HALOPERIDOL-SENSITIVE SIGMA RECEPTOR. Mark Sonders, Bruce

Campbell, and Eckard Weber, Institute for Advanced

Biomedical Research, The Oregon Health Sciences University,

Portland, Oregon 97201, U.S.A.

The development of [

3

H]-DTG as a novel, selective

ligand for the haloperidol-sensitive sigma receptor (see

our presentation elsewhere in this meeting) has enabled us

to screen brain extracts for the presence of endogenous

material that can displace [

3

H]-DTG from its brain

membrane binding sites. Acid extracts from whole cow

brains were purified by cation exchange chromatography.

These partially purified extracts contain a small molecular

weight compound which competitively and reversibly inhibits

the binding of [

3

H]-DTG to the haloperidol-sensitive

sigma receptor in guinea pig brain membrane preparations.

The substance is not a peptide. Its physicochemical

properties [chromatographic behavior in various systems,

solubility properties in polar and nonpolar solvents etc.]

will be presented. Whether or not this compound is a

physiologically relevant, endogenous ligand for the

haloperidol sensitive sigma receptor can only be determined

after the compound has been purified to homogeneity and its

chemical structure has been determined. These studies are

underway.

Supported by NIMH grant MH40303.

687
EVIDENCE FOR THE EXISTENCE OF ENDOGENOUS LIGANDS FOR SIGMA

OPIOID RECEPTORS IN THE BRAIN ("SIGMAPHIN"): STUDIES FROM

BINDING ASSAYS. T.-P. Su and S. Y. Yeh, National Institute on

Drug Abuse, Addiction Research Center, Baltimore, Maryland

21224, U.S.A.

SKF-10047 was the prototypic drug for the sigma opioid

receptors which was purported to mediate the psychotomimetic

effects of certain opioids and other drugs.

receptors were later identified in tHe guinea-pig brain

Sigma opioid

homogenates in a binding assay using [

3

H]SKF-10047 (Su, J.

Pharmacol. Exp. Ther. 223:284, 1982). Ligand selectivities of

the sigma opioid receptors indicated that sigma opioid receptors

do not represent any other known receptor systems in the brain

and may therefore represent a new psychotomimetic pathway. The

unique ligand selectivities of the sigma opioid receptors and

the fact that its prototypic ligand SKF-10047 is psychotomimetic

in humans suggest a possibility of the existence of endogenous

ligand for this receptor. This report presents evidence that

endogenous ligands for sigma opioid receptors ("SIGMAPHIN") may

exist in the brain. Two endogenous ligands which interacted

preferentially with the sigma opioid receptors were identified

from the guinea-pig extract in a Sephadex G-50 fractionation.

These two ligands were more potent in the sigma receptor assay

than in the mu, kappa and delta receptor assays. In the

phencyclidine receptor assay, two ligands were almost

inactive. Incubation of those ligands with trypsin destroyed at

least 50% of the activities of those ligands in the sigma

receptor assay. Both ligands inhibited sigma binding in a dose

response manner. The inhibitions were competitive. The

inhibitions could be eliminated when the ligands were removed

from assay media by extensive washings. It is concluded

therefore that sigma opioid receptors are not phencyclidine

receptors and that endogenous ligands for sigma opioid receptors

may exist in the brain.

688
MOOULATION BY ENKEPHALIN ANALOGS AND NEUROLEPTICS OF APOMORPHINE-INDUCED STEREOTYPY AND TURNING BEHAVIOR IN RATS.

J.I. Székely, I. Mate, K. Polgár and M. Till, Institute

for Drug Research, H-1325 Budapest, POB 82. HUNGARY

Some of the most characteristic behavioral effects of

classical opiates and opioid peptides are similar to those

elicitable by neuroleptics (Bloom et al., Science, 194, 630,

1976; Jacquet and Marks, idem 194, 632, 1976). Thus the question arises whether also opioids inhit the central dopaminergic tone. The neuroleptics are known to antagonize the motor effects of apomorphine (A) at specific low dose levels.

In our previous study (Sineger et al., Eur., J. Pharmacol.,

80, 359, 1982) S.C. injected morphine (M) and a potent enkephalin analog (2-D-Met,5-Pro-enkephalinamide, EA) only

partially inhibited the A-induced stereotypy (S) and turning

behavior (TB) in rats with unilateral nigral lesion. Nevertheless, the inhibition by EA of A-induced S was statistically significant already at subanalgesic dose levels. The

aim of the present investigations was to examine that which

brain areas might mediate the anti-A effect of opioids. As

the first step of these examinations the substances under

study were injected either i.c.v. or into the nucleus accumbens (NA) i.e. a mesolimbic region rich in dopamine. Substances examined were M a μ-selective opiate, 2-D-Ala,5-Nle-

-enkephalin sulfonic acid (ES) a delta-selective opioid, EA a

highly potent non-selective opioid peptide, furthermore,

haloperidol (H) and chlorpromazine (C). As for the inhibition

of A-induced TB the order of potency was: EA>H>M>ES C if

injecting the substances i.c.v. but EA>M>H ES C when administering them into the NA. Examining the suppression of

A-induced S the order of potency was EA H M>ES C upon

i.c.v. administration and EA H>M ES>C upon injecting them

into the NA. The data show that certain endogenous opioids

might inhibit the brain dopamine activity via the NA.

689
KYOTORPHIN AS A NEUROREGULATOR; EVIDENCE FOR ITS

BIOSYNTHESIS, RELEASE, RECEPTOR AND POST RECEPTOR

MECHANISM H. Takagi, H. Ueda and Y. Yoshihara,

Department of Pharmacology, Faculty of Pharmaceutical

Sciences, Kyoto University, - Kyoto 606, Japan

Kyotorphin (Tyr-Arg) is distributed unevenly

throughout the brain regions and concentrated in the

synaptosomes. Following studies have demonstrated

that kyotorphin produces an analgesia via a release of

met-enkephalin from the brain. In the course of

studying the mechanism of met-enkephalin release, we

found

C a 2 +

the facilitatory effects of kyotorphin on the

entry into synaptosomes. Thus it has been

postulated that kyotorphin plays a physiologically

significant role as a neuroactive peptide. In the

present study, we reports the further evidence

indicating the role of kyotorphin as a neuroregulator.

BIOSYNTHESIS Kyotorphin is formed from tyrosine and

arginine in the brain synaptosomes. The partially

purified kyotorphin synthetase was characterized.

UPTAKE AND-RELEASE Kyotorphin was actively taken up

into P2 (crude synaptosomes). Kyotorphin preloaded

into synaptosomes was released by high KCl stimulation

in a Ca2+ dependent manner. KYOTORPHIN SPECIFIC

RECEPTOR 3H-kyotorphin specific binding activities

were obtained using the brain membrane preparations.

Leu-Arg showed a potent inhibition of the

POST RECEPTOR MECHANISM We observed the

binding.

kyotorphininduced mobilization of the plasma membrane-bound Ca2+

into cytosolic space, determined with the measurement

of the fluorescence of Quin-II and chlortetracycline,

and 45Ca2+ entry into synaptosomes.

induced increase of the 45Ca2+ entry

The kyotorphinwas completely

abolished by Leu-Arg (a putative kyotorphin

antagonist). ANALGESIA Kyotorphin (i.cisternal)-

induced analgesia in mice was significantly attenuated

by co-administration of Leu-Arg.

690
THE EFFECT OF HALOPERIDOL ON PRO-DYNORPHIN END PRODUCTS IN THE RAT

STRIATUM AND SUBSTANTIA NIGRA. R. Tandon, R. Day, J.E. Kelsey,

S.J. Watson and H. Akil, Mental Health Research Institute,

University of Michigan, Ann Arbor, MI 48109-0720, U.S.A.

doses of 0.25, 0.5, 1.0 or 2.0 mg/kg/day. The STR and SN were

collected and extracted in methanol:0.1N HCl (1:1 v/v). Dyn A 1-8

and Dyn A 1-17 content was measured using specific RIAs. In both

regions, Dyn A 1-8 levels increased at the lower doses of HAL

treatment (0.25 and 0.50 mg/kg), but returned to control values or

lower than control values at the higher doses of HAL. However Dyn

A 1-17 content in STR and SN progressively decreased from control

values with increasing doses of HAL. A comparison of the Dyn A

1-17:Dyn A 1-8 ratio shows an increase in the conversion of Dyn A

1-17 to Dyn A 1-8 in STR and SN in rats treated with HAL. The sum

of these observations may be explained by alterations in rates of

biosynthesis, processing and/or release of Pro-Dyn end products.

Our results demonstrate the regulation of the striatonigral

dynorphin system by dopamine. In turn, the changes in Pro-Dyn end

product ratios could subsequently modify nigrostriatal

dopaminergic function. This work was supported by the T. Raphael

Research Fund and NIMH Grant # MH39717.

While the nigro-striato-nigral loop has been extensively

studied in the context of interactions between dopamine,

acetylcholine and GABA, little is known about the role of the

recently discovered striatonigral-dynorphin pathway.

Pro-dynorphin (Pro-Dyn) perikarya are localized in the

caudate-putamen and some of these neurons are thought to be the

source of dynorphin innervation to the pars reticulata of the

substantia nigra (SN). On the other hand, dopaminergic cell

groups within the SN give rise to ascending projections to the

striatum (STR) via the nigrostriatal pathway. Pharmacological

manipulation of this system using the dopamine receptor antagonist

haloperidol (HAL) was used to examine Dyn-dopamine interactions.

Male Sprague Dawley rats were injected i.p. over 4 consecutive

days, and sacrificed on the fifth, with either saline or HAL at

691
DETERMINATION OF SIZES OF THE OPIOID RECEPTORS IN THEIR

MEMBRANE ENVIRONMENT BY RADIATION INACTIVATION.

Pao-Luh Tao, Li-Ren Chang, Hsheng-Kai Lee and Horace H.

Loh. Department of Pharmacology, National Defense

Medical Center, Taipei, Taiwan, Republic of China and

Department of Pharmacology, University of California,

San Francisco, San Francisco, California 94143, U.S.A.

Opioids, like other drugs, are thought to initiate

their effects by associating with their specific

receptors. However, very little is known about the

opioid receptor as a molecular entity. The binding

components have been solubilized in detergent and

purified by different approaches, but the molecular

size of soluble opioid receptor complexes reported by

different groups varied from 23,000 to 750,000. In this

study, the technique of radiation inactivation by

gamma ray was used to investigate the apparent size of

the opioid receptor in the rat brain membranes under

different conditions. The molecular sizes of opioid

receptor complexes were egstimated as 313,000 in the

presence of [D-Ala2,D-Leu5] enkephalin, NaCl

and Gpp(NH)p; as 165,000 in the presence of NaCl only

or of both NaCl and Gpp(NH)p; as 217,000 in the

presence of Gpp(NH)p only: and as 286,000 in the

presence of MgCl2 only. A simple model has been

proposed to explain these different apparent target

sizes of opioid receptor obtained under different

conditions.

692
ROLE OF THROMBOXANE A, IN THE MORPHINE INDUCED POTENTIATION OF

THE GASTROULCEROGENIC EFFECTS OF INDOMETHACIN IN RATS.

M. Tariq, N. S. Parmar and A.M. Ageel, Research Centre and

Department of Pharmacology, College of Pharmacy, King Saud University, P. O. Box 2457, Riyadh - 11451, Saudi Arabia.

Morphine has been shown to potentiate the ulcerogenic activity

of indomethacin in a dose dependent manner following subcutaneous

administration in rats. This potentiation is blocked partially

by anticholinergics and cimetidine and completely by naloxone

thus showing the involvement of opiate receptors in its aetiology.

We have further studied the effect of OKY-046, a specific thromboxane A2 synthesis inhibitor; BM 13177, a thromboxane A2 receptor antagonist and sodium cromoglycate, a mast cell stabilizing

agent on the morphine induced potentiation of the gastroulcerogenic activity of orally administered indomethacin in rats. Our

results show that thromboxane A2 is significantly involved in

this action, whereas the involvement of histamine released through

the disruption of mast cells is completely ruled out as pretreatment with sodium cromoglycate does not affect the potentiating

action of morphine. It would be interesting to study as to how

thromboxane A2 is involved in the opiate receptor mediated potentiation of indomethacin's ulcerogenic effect.

693
THE ROLE OF μ- AND RECEPTORS IN GASTROINTESTINAL PROPULSION IN

RATS. A. Tavani, M. Sbacchi, A. La Regina, P. Petrillo, Istituto

di Ricerche Farmacologiche "Mario Negri", via Eritrea 62 - 20157

Milan, Italy.

To study the role of μ- and receptors in gastrointestinal

propulsion, overnight fasted rats received i.p. injections of

[D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or morphine or [D-Ala2,

D-Leu5]enkephalin (DADLE) respectively 1, 5 and 5 min before a

charcoal meal and the percentage of small intestine traversed by

charcoal was measured 5 min after the meal. The doses inhibiting

gastrointestinal transit by 50% compared to controls (ED50), obtained from the dose-response curve, were 6.20, 15.19 and 23.03

μg/kg respectively for DAGO, morphine and DADLE. In the presence

of naloxone (50 μg/kg i.p. 5 min before DAGO and 1 min before morphine or DADLE), the dose-response curves of DAGO and morphine were

parallel but shifted to the right of the respective curve in the

absence of the antagonist; the ED50 were respectively 153 and 239

μg/kg. Conversely, the dose-response curve of DADLE with the same

dose of naloxone was flat and doses from 15 to 1000 μg/kg of DADLE

produced about 50% of the maximal effect. In the presence of the

selective antagonist ICI 174,864 (1 mg/kg i.p. 5 min before DAGO

and 1 min before morphine or DADLE), the dose response curves of

DAGO and morphine were the same as in the absence of the antagonist.

Conversely, the dose-response curve of DADLE in presence of ICI

174,864 was different, although the antagonist shifted it only very

weakly. It thus appears that μ-receptors may be involved in the

inhibition of gastrointestinal propulsion induced by DAGO and morphine and both μ- and receptors in the inhibition induced by DADLE.

The effects of the selective -compound [D-Pen2

, D-Pen5] enkephalin

are discussed too.

694
μ, & K OPIOID RECEPTOR DISTRIBUTION IN RAT BRAIN USING IN

VITRO LIGHT MICROSCOPY AUTORADIOGRAPHY. A. Tempel

and R. S. Zukin, Dept. of Neuroscience, Albert Einstein College

of Medicine, Bronx, NY 10461, U.S.A.

We have used quantitative in vitro autoradiography at the

level of the light microscope to determine the precise neuroanatomical distribution of the μ, , and opioid receptors.

Adjacent sectons of frozen rat brain were thaw-mounted on glass

slides, incubated with radiolabelled opioid in the presence or

absence of nonlabelled ligand in order to control for nonspecific binding of the radioligand, and apposed to tritium-sensitive film. The neuroanatomical pattern of μ receptors was

determined using D-Ala2,N-Me-Phe4, Gly-ol5-enkephalin (DAGO μspecific opioid). At the level of the anterior commissure,

discrete patches of densely-labeled opiate receptors were observed, surrounded by areas of diffusely-organized receptors.

At the level of the diencephalon dense labeling of μ receptors

was seen to overlie the molecular layers of the hippocampus,

particularly the pyramidal field. Dense μ receptor labeling

was also observed in specific thalamic nuclei, layers I and

III of the neocortex and the locus coeruleus of the pons. In

contrast, receptors labeled by 3H[D-Pen2, D-Pen5]enkephalin

(DPDPE specific ligand) exhibited a diffuse distribution in the

striatum with few visible patches. In the neocortex, selected

localization was observed in layers I & II and V & VI. Nuclei

of the thalamus, hypothalamus midrain and brainstem were

notably lacking in receptors. receptors (labeled using

3

H-ethylketocyclazocine in the presence of μ and blockers)

exhibited a distribution similar to that of μ receptors in the

striatum and hippocampus, but differed markedly in midbrain and

hindbrain areas. High receptor density was observed in the

midline thalamic nuclei, central gray area, substantia nigra

reticulata, interpeduncular nucleus, the locus coeruleus and

cerebellum. Thus, pronounced differences in the neuroanatomical distributions of the μ, and receptors may account in

part for the diverse pharmacological profiles, of μ, and

opioids. [Supported by NIH grants DA 01843 & DA 00069 (to

R.S.Z.) and NS 21973 (to A. T.)].

695
STRUCTURAL REQUIREMENTS LEADING TO AGONIST AND ANTAGONIST ACTIVITY IN

4-ALKYL-4-m-(HYROXY)-PHENYLPIPERIDINES. L. Toll, J. A. Lawson, W.

Polgar, E. Uyeno, G. Frenking, G. H. Loew, Life Sciences, SRI

International, 333 Ravenswood Ave., Menlo Park, CA 94025, U.S.A.

The family of 4-(m-OH)-phenylpiperidines is a flexible fragment

of the morphine/benzomorphan fused-ring opioid family. Analogs in

this family were synthesized with varying 4-alkyl substituents,

increasing in bulk from H, through methyl and n-propyl, to

t-butyl, each with the three N-substituents, methyl, allyl, and

phenethyl. These twelve compounds were evaluated for analgetic

agonism and antagonism in mice and for binding affinities using

five 3H-labelled ligands. Energy conformation calculations on

these structures were also carried out in order to better under

stand how the 4-alkyl substituents modulate receptor binding

affinities and agonist/antagonist potency ratios through

equatorial/axial conformers. The results obtained show that the

4-alkyl-4-(m -OH)-phenylpiperidines vary as agonists with a range

of activity from weak to morphine-like and that only a few analogs

have antagonist activity. Combined results of energy conformation

calculations and in vivo and in vitro analysis suggest that

binding in a phenyl axial conformation will lead to agonist

activity, while phenyl equatorial binding will lead to antagonist

activity. Furthermore, specific interactions with 4-n -propyl and

N-phenethyl moieties can induce phenyl axial and equatorial

interactions, respectively. None of the analogs, showed fusedring-like N-substituent modulation of agonist/antagonist activity,

even though the 4-t-butyl analogs exist essentially completely in

the phenyl axial conformation. These compounds are the first

simple 4-alkyl substituted 4-phenylpiperidines to be reported with

a strongly preferred phenyl-axial conformation.

696
IMMUNOCYTOCHEMICAL LOCALIZATION OF PRO-OPIOMELANOCORTIN

-DERIVED PEPTIDES IN THE ADULT RAT SPINAL CORD.

K. Tsou*. H. Khachaturian, H. Akil and S. J. Watson

Mental Health Research Institute, University of

Michigan, Ann Arbor, MI 48109, U. S. A. and *Shanghai

Institute of Materia Medica, Academia Sinica, Shanghai,

200031, China.

A dispersed descending pro-opiomelanocortin (POMC)

fiber system has been demonstrated by PAP immunocytochemistry in the adult rat spinal cord. ( -endorphin,

ACTH, -MSH and 16K immunoreactive fibers exist in

the spinal cord from cervical down to sacral level.

Descending fibers running in parallel in the dorsolateral and lateral funiculus send collaterals ventromedially or medially to terminate in the gray matter

surrounding the central canal, where nociceptive

neurons have recently been located, in addition to

those nociceptive cells in the dorsal horn. After

spinal transection at lower thoracic level, POMC

peptide immunoreactivities disappeared below the

lesion. Moreover, no POMC cell bodies were found in

the spinal cord. Therefore, the descending fibers are

most likely of supraspinal origins.

697
THE ANIMAL PHARMACOLOGY OF A NOVEL OPIOID ANALGESIC DRUG. XOHPHANOL

M.B. Tyers, A.G. Hayes, and M.J. Sheehan, Dept. Neuropharmacology, Glaxo

Group Research Ltd., Ware, Herts., England.

Xorphanol is a novel morphinan opioid drug, which has been shown to

have analgesic activity in man(1). In vitro experiments suggest

that xorphanol is a potent opioid partial agonist, with good agonist

selectivity for the -receptor. Thus in the field-stimulated,

isolated, guinea-pig ileum, it showed the low maximum agonist

activity (Emax 40%; IC20 = 0.2nM) which is typical of a partial

agonist. It was not antagonised by the μ-selective antagonist

funaltrexamine; but its dose-response curve showed a large

rightwards shift after effective -receptor antagonism with the

irreversible antagonist -chlornaltrexamine, in the presence of μreceptor protection(2)

. Xorphanol behaved as an antagonist in both

the rabbit vas deferens ( -receptors; pA2 for antagonism of

ethylketocyclazocine = 9.04) and rat vas eferens (μ-receptors; pA2

for antagonisn of D-Ala2, MePhe4, gly(ol)5

 enkephalin = 8.44) which,

together with the guinea-pig ileum data , suggests partial agonist

activity at the -receptor and antagonist activity at the μreceptor. Xorphanol produced potent antinociceptive activity in the

mouse abdominal constriction and guinea-pig paw pressure tests (ED50

values = 0.05mg/kg S.C. and 0.035mg/kg S.C. respectively), but was

inactive in the mouse hotplate test and only weakly active in the

rat paw pressure test. This profile is characteristic of a low

efficacy -agonist(3). Xorphanol, 0.1-0.3mg/kg s.c., also showed

potent and long-lasting antinociceptive activity in the dog

toothpulp stimulation test. As expected for a partial -agonist,

xorphanol produced only low maximum respiratory depression and

inhibition of gastrointestinal motility in the mouse (Emax = 20% and

46% respectively at 3mg/kg s.c.) and produced no significant

sedation in the rotarod test in the mouse at 0.1-9mg/kg S.C. Some

short duration sedation was seen with xorphanol, 0.1-3mg/kg s.c., in

the marmoset. These animal data predict that xorphanol should be a

potent and efficacious analgesic in man, with very low side-effect

potential.

(1) Bloomfield, S S et al (1984) IUPHAR 9th International Congress

of Pharmacology, Abst. 1737P. (2) Sheehan M J et al (1985),

Br.J.Pharmac; 84, 68P. (3) Hayes A G et al (1985), Br.J.Pharmac; 86,

703P.

We would like to acknowledge H.G. Pars, Pharma.Labs.Inc., for

supplies of xorphanol mesylate.

698
EVIDENCE FOR THE PRESENCE OF PRESYNAPTIC AUTOREGULATION OF MET-ENKEPHALIN RELEASE IN THE BRAIN AND

-EFFECTS OF KAPPA OPIOID ANTAGONISTS THEREON. H. Ueda,

N. Fukushima and H. TAKAGI Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto

University, Kyoto 606, Japan.

Presynaptic regulation of met-enkephalin release

from slices of rat midbrain, pons plus medulla

oblongata and its physiological role were studied.

Amounts of met-enkephalin released from slices were

measured by radioimmunoassay. The effects of several

subtypes of opioids on Ca2+-dependent and high K

+

 (30

mM)-evoked release of met-enkephalin in the presence

of tetrodotoxin (0.3 μM) were determined. The IC 20

of the high K

+

-evoked release by μ-type opioid

agonists, morphine and DAGO were 1.2 and 0.9 μM,

-type agonists, DTLET and DPLPE 1.5 and 0.7 μM and

-agonists, U-50488, bremazocine, dynorphin (1-17)

0.06, 0.32 and 0.15 μM, respectively. On the other

hand, opioid -antagonists (0.1-10μM) facilitated the

evoked release. Effects of -opioid anatgonists on

the nociceptive responses were observed in vivo. When

very small doses (0.2-20ng/kg s.c.) of Mr2266 were

administered into mice, it produced a significant

analgesic effect with the tail flick test when

administered intracisternally. To summarize, opioid

-type antagonist produces an analgesic effect via

blocking the autoreceptor on the nerve terminals of

met-enkephalin neurons.

699

172-683 O - 87 - 24 : QL 3
THE INVOLVEMENT OF ENHANCED Ca2+ CHANNEL ACTIVATION IN

THE NERVE TERMINALS OF MORPHINE WITHDRAWAL ABSTINENCE

IN MICE. H. Ueda, S. Tamura, M. Satoh and H. Takaqi,

Department of Pharmacology, Faculty of Pharmaceutical

Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan

When naloxone was administered intrathecally

(i.th.) 24hr after the implantation of a morphine

pellet into mice, withdrawal jumping behavior was

precipitated and lasted for 5 min. Repeated (i.th.)

injection of naloxone produced no more jumping

behavior. The intrathecal pretreatment with IAP (5

μg) 24 hr before morphine pellet implantation

completely abolished the naloxone-precipitated

withdrawal jumping. Furthermore, intrathecal

pretreatment with substance P antagonist,

(D-Pro4,D-Trp7'

9

'

10)-stbstance P(4-11) 5 min before

the naloxone treatment, also completely abolished the

precipitated jumping. High K

+

-evoked 45Ca entry into

the synaptosomes of the spinal cord of the morphine

dependent mice was enhanced by naloxone (10-4M).

Naloxone (10-4M) enhanced maitotoxin (a Ca channel

agonist)-induced 45Ca entry into the spinal

synaptosomes of dependent mice, but not of the intact

mice. Naloxone at 10-4M increased the c-AMP content

in the synaptosomes of the spinal cord of dependent

mice, but not of intact mice. Taking the previous

report that intracellular c-AMP enhances the Ca2 +

entry into synaptosomes of rats, the present findings

Ca2+ channel may be activated via excessively

suggest that in the morphine abstinent state, the

formed

c-AMP and then neurotransmitter release enhanced. In

the withdrawal jumping of mice, excessive release of

substance P from the spinal cord is possibly involved.

700
CHARACTERIZATION OF OPIOIO RECEPTORS IN RAT BRAIN FOLLOWING

LESIONS OF THE NUCLEUS ACCUMBENS. E.M. Unterwald, R.S. Zukin, and

G.F. Koob*, Depts. of Neuroscience and Biochemistry, Albert

Einstein College of Medicine, Bronx, NY, 10461 and *Res. Inst.

Scripps Clinic, La Jolla, CA, 92037, U.S.A.

Autoradiographic studies have identified dense patches of mu

and kappa opioid receptors and less dense patches of delta opioid

receptors in the rat nucleus accumbens. In addition, the nucleus

accumbens is rich in dopaminergic fibers as it receives extensive

innervation from the mesolimbic dopamine tract. Anatomical and

functional relationships between the opioidergic and dopaminergic

circuitry of the brain are well established and considerable

evidence supports the concept that opioid peptides act on presynaptic dopaminergic neurons to modulate dopamine release from their

axon terminals. The present study investigates the topographical

organization of opioid receptor types in the nucleus accumbens

with respect to their pre- and postsynaptic localization and their

relationship to dopaminergic nerve fibers.

Dopaminergic terminal fields in the nucleus accumbens of rats

were destroyed by unilateral injection of 6-hydroxydopamine (2μl,

8μg/μl) into the nucleus accumbens. Fourteen days after the

lesions, the fraction of each receptor type lost in the nucleus

accumbens was determined using highly specific in vitro binding

assays. Aliquots of homogenate from 6-hydroxydopamine-lesioned,

sham-lesioned, and non-lesioned nucleus accumbens (.25 ml, 0.16-

0.39 mg/ml protein) were incubated with selective [3H]-ligands in

the presence and absence of 10 μM levorphanol. The specific ligands used were [

3

H]-D-Ala2,N-Me-Phe4.Gly-ol5-enkephalin (DAGO) for

mu receptor assays, [

3

H]-D-Penicillamine2,D-Penicillamine5-

enkephalin for the delta receptor assays, and [

3

H]-ethylketocyclazocine in the presence of DAGO (100nM) and D-Ala2

, D-Leu5-

enkephalin (100nM) for kappa receptor assays. Results indicate . .

substantial changes in opioid receptor binding, particularly a

dramatic loss of mu receptors, in the nucleus accumbens following

these lesions. These results suggest a neuromodulatory role for

mu opioid peptides on dopamine release in the nucleus accumbens.

(Supported in part by NIH grant DA 01843 and DA 00069 (R.S.Z.)).

701
THE OPIOID RECEPTOR-GTPase-ADENYLATE CYCLASE SYSTEM IN NG108-15

CELLS: DISSOCIATION BETWEEN DESENTIZATION AND DOWN-REGULATION. L.

Vachon, T. Costa and A. Herz, Department of Neuropharmacology,

Max-Planck-Institut für Psychiatrie, D-8033 Martinsried, F.R.G.

Opioid receptor binding, low Km GTPase and adenylate cyclase

were studied in membranes derived from NG 108-15 cells pre-treated

with [D-Ala2, D-Leu5]enkephalin (DADLE) or morphine. Pre-treatment

with DADLE results in a rapid loss of responsiveness of the opioiddependent GTPase: this desensitization occurs as two time-dependent

processes with t 1/2 of 10 min and 8 hr. Opioid binding sites, as

assessed using [3H]diprenorphine, also decay as two time-dependent

processes upon exposure to DADLE. However, in the presence of Na+

and GTP, only the slow component is observed. The inhibition of

adenylate cyclase by opioids is also lost tine-dependently followinq pre-treatment with DADLE. However, significant responsiveness

of the enzyme is still observed even after desensitization of

GFPase has occurred. While the desensitization of GTPase is due to

a pure reduction in the maximal effect of DADLE, desensitization of

adenylate cyclase occurs throuqh both a loss of maximal effect and

a decrease in the potency of DADLE. In contrast to DADLE, morphine

induces only a minimal desensitization of the opioid-GTPase-adenylate cyclase system, and can antagonize the fast component of the

desensitization induced by DADLE. These findings suggest that 1)

desensitization of the opioid receptor-GTPase-adenylate cyclase

system results, as a primary event, from an uncoupling between the

receptor and the nucleotide-binding regulatory protein (Ni), and

occurs before down-regulation of the receptor; and 2) the differential potencies of DADLE and morphine in desensitizing the opioidGTPase-adenylate cyclase system in NG 108-15 cells cannot be accounted for by a mere difference in receptor affinity, but rather

appears to be related to their respective full- and partial-aqonistic properties. (Supported by the Deutsche Forschunqsqemeinschaft,

Bonn, L.V. is postdoctoral fells of the Fond de la recherche en

Sante du Québec, Québec, Canada).

702
METABOLISM OF [MET]ENKEPHALIN IN THE RAT SPINAL CORD: RESPONSE

TO PEPTIDASE INHIBITORS. K.I. Van Alstyne and K. Jhamandas,

Department of Pharmacology and Toxicology, Queen’s University,

Kingston, Ontario, Canada, K7L 3N6.

Enkephalin metabolism in the brain is mainly a result of the

activities of aminopeptidase and enkephalinase, and inhibition

of these enzymes by pharmacological agents leads to analgesia.

At present, little is known about the metabolism of enkephalins

by the spinal cord, a source of significant levels of enkephalins and an important site of opioid analgesia. To study the

relationship between analgesia and pharmacological inhibition

of enkephalin metabolism in the same region, a system was

developed to study the metabolism of [Metlenkephalin (ME) in the

isolated rat spinal cord. The separation of ME from its putative

metabolites, tyrosine (T), tyrosine-glycine-glycine (TGG) and

des[Met]enkephalin, was performed by reverse-phase gradient

HPLC using a C18 column and 0.01 M sodium acetate (pH 4.0)

and acetonitrile as the mobile phase.Coronal slices of rat

spinal cord (400 μM) were incubated with 3H-ME for various time

intervals over a 60-min period. The radiolabelled metabolites

were separated by HPLC. The metabolism of 3

H-ME by spinal cord

tissue yielded mainly 3H-T and 3H-TGG, reflecting the activities

of aminopeptidase and enkephalinase or angiotensin-converting

enzyme respectively. In the absence of peptidase inhibitors,

cervical, thoracic and lumbar regions of the cord showed similar

metabolic profiles. Metabolism of 3H-ME by dorsal and ventral

regions of the lumbar cord also yielded similar metabolic

profiles. Bestatin, an aminopeptidase inhibitor (1 x 10-7 -

2 x 10-5 M), inhibited the formation of “H-T and augmented the

formation of 3H-TGG from 3H-ME. Thiorphan, an enkephalinase

inhibitor (1 x 10-8 - 1 x 10-6 M), did not appear to inhibit

the formation of 3H-TGG from 3H-ME. This model could be useful

in making correlations between inhibition of ME metabolism and

production of analgesia by peptidase inhibitors.

(Supported by the Medical Research Council of Canada)

703
INCREASE IN MORPHINE ANALGESIA BY CORTICOSTERONE: A CENTRAL EFFECT ?

C. Vidal, J.J.C. Jacob, Unit of Pharmacology, Pasteur Institute,

F 75724 Paris Cedex 15, France.

Evidence has accumulated that opioids influence the secretion

of adrenal steroids. Conversely, little is known about the modulation of the opioid system by glucocorticoids. According to Holaday

et al., corticosteroids modulate opioid effects through a mechanism peripheral to the neuroaxis. In a recent study, we have shown

that corticosterone (CT) potentiates the effect of enkephaline in

the hippocampus in vitro, thus indicating an interaction at a

central level. The possible behavioral correlates of such interaction was investigated in the present work. Mice received either

saline or morphine hydrochloride (1 or 3 mg/kg s.c.); they were

then immediately injected intracerebroventricularly with either

CT (10-4 M or 10-5 M; 20 μl/animal) or its vehicle (ethanol 0.5%

or 2%). Groups of 16 animals were used. Licking latencies (sec)

were measured 30 min after ICV injection (hot plate test; To of

the plate 55°C). Licking latencies were as follows(vehicle was in

Exp. I and II ethanol 0.5%, in Exp. III ethanol 2%)

Exp.I Exp.II Exp.III

Vehicle 12.25 ± 0.7 15.1 ± 1.5 14.5 ± 1.4

CT 10-4 M 13 ± 1.1 11 ± 1.1 12.8 ± 1.4

Mo 1 mg 16.5 ± 1.7

Mo 3 mg 17.8 ± 1.74 14.4 ± 1.1

CT 10-4 M -

+ Mo 1 mg 19.5 ± 2.45

+ Mo 3 mg 22.25 ± 2.7 18.4 ± 2.1

CT alone has no significant effect; morphine I or 3 mg/kg s.c. had

no or slight effects. The associations CT 10-4 + Mo had always

greater effects than those produced by Mo and vehicle. The differences were hardly significant when considereing each experiment

separately but they were significant (for p < 0.05) when all

results were gathered. Further work is in progress.

704
KAPPA OPIATE AGONISTS INHIBIT ADENYLATE CYCLASE AND PRODUCE

HETEROLOGOUS DESENSITIZATION IN RAT SPINAL CORD CELLS. Z. Vogel

and B. Attali, Neurobiology Department, Weizmann Institute of

Science, Rehovot 76100, Israel.

As with other receptor ligands which regulate adenylate cyclase activity, opiate agonist binding in spinal cord is regulated

by GTP and Na+, suggesting a coupling to the adenylate cyclase

system. The present communication investigates the exact conditions in which opiate inhibition of the spinal adenylate cyclase

can be observed. Furthermore, the opioid regulation of the enzyme

has been studied in rat spinal cord primary cultures following

chronic agonist treatment. Using crude P2 preparations of rat

spinal cord, we were able to show that μ (DAGO 10μM) and (DADL

10μM) agonists did not affect the basal cyclase activity (3892±11

pmoles cAMP/15min/mg protein, n=3), whereas the K selective opiate agonist U50488 significantly and dose-dependently inhibited

this activity (30% inhibition at 10μM, p<0.02), Levorphanol at 10μM

inhibited by 20% the basal enzyme levels. The regulatory action

was stereospecific since dextrorphan was ineffective. The K selective antagonist MR2266 (10μM) fully antagonized the K agonist

response. In all cases GTP(10μM) and NaCl(100mM) were required for

the expression of the agonist inhibitory action. The inhibitory

effect of K agonists was also observed when the cyclase activity

was stimulated by forskolin (100μM). These data suggest that in

the spinal cord, opiate receptors of the Kappa type are coupled to

adenylate cyclase. Following chronic exposure to etorphine (1μM

for 4 days without wash) of rat spinal cord primary cultures, the

K agonist U50488 lost its ability to inhibit the cyclase, the basal as well as the forskolin stimulated activities remaining identical to those of paired control cultures. Furthermore, this desensitization process seems to be heterologous, since the 2 adrenergic agonist, norepinephrine, and the muscarinic agonist, carbachol,

exhibit significant lower potency for inhibiting the enzyme activity following exposure to etorphine, suggesting that the site of

tolerance induced by opiates in the spinal cord is related to post

receptor regulatory components.

705
[

3

H]1,3-Di-O-TOLYL-GUANIDINE ([3H]-DTG) , A NOVEL,

SELECTIVE LIGAND FOR THE HAMPERIDOL SENSITIVE SIGMA

RECEPTOR. Eckard Weber. Mark Sonders, Merrit Quarum,

Sovitji Pou*, John F. W. Keana*, Institute for Advanced

Biomedical Research, The Oregon Health Sciences University,

Portland, Oregon 97201 and *Department of Chemistry,

University of Oregon, Eugene, Oregon 97403, U.S.A..

Sigma receptors are thought to mediate the

hallucinogenic effects of benzomorphan-type opiate drugs.

The characterization of the sigma receptor by biochemical

methods has been difficult because of the lack of selective

and potent ligands. During a systematic search for

potential sigma receptor active drugs we discovered that

symmetrically substituted guanidines interact with the

sjgma receptor as judged by their capability to displace

[

3

H](+)SKF 10,047

sites.

from guinea pjg brain membrane binding

We have synthesized a [

3

H]-labeled derivative of

the most potent one of these symmetrically substituted

guanidines. This compound has the following structural

formula:

Radioligand binding assay experiments have shown that this

novel, [

3

H]-labeled compound binds with high affinity

max = 80 pmol/gram brain tissue] in guinea pig brain

[KD = 30 nM] to a single population of binding sites [B

membrane preparations. The drug selectivity profile of the

[

3

H]-DTG binding site was identical to that described for

the haloperidol-sensitive sigma receptor (1,2). The

potential applications of this novel sigma receptor ligand

to the characterization of the sigma receptor in

biochemical and pharmacological experiments will be

discussed.

1.) Largent, B.L. et al., PNAS 81, 4983 (1984).

2.) Tam, S.W. and Cook, L., PNAS 81, 5618 (1984).

Supported by NIMH grant MH40303 to EW.

706
EVIDENCE THAT THE MAJOR IMMUNOREACTIVE MORPHINANS IN MAMMALIAN

BRAIN ARE MORPHINE AND CODEINE. C. J. Weitz, L. I. Lowney,

R. W. Barrett and A. Goldstein, Addiction Research Foundation,

Palo Alto, CA 94304, U.S.A.

Partially purified extracts of bovine hypothalamus and rat

brain contain two major compounds recognized by antisera to

morphine and resolved by reversed-phase high pressure liquid

chromatography (HPLC). Both are found consistently in bovine

hypothalamus and rat brain, but the pattern and concentrations

are highly variable in bovine adrenal. In addition, a third minor

immunoreactive (ir) morphinan is present in hypothalamus.

Extractions and purification procedures were carried out in

sequestered glassware. A "tissueless" blank was run through the

entire system prior to each tissue work-up and shown to be free

of significant immunoreactivity on HPLC analysis.

The two major ir-morphinans in bovine hypothalamus, rat brain,

and (variably) bovine adrenal appear to be morp ine and codeine

by the following criteria: 1) co-lution with 3

H-morphine and

3

H-codeine in two different reversed-phase HPLC systems;

2) crossreactivity with two antimorphine antisera of contrasting

specificities; and 3) absolute crossreactivity in a quantitative

immune-removal system (bovine hypothalamus).

These results support previous work reported from our

laboratory (A Goldstein et al, Proc Natl Acad Sci USA 82: 5203,

1985) in that ir-morphinans (one of which is morphine) are

detected consistently in mammalian brain. In addition, we can

now tentatively identify our previously-reported "peak 4" as

codeine; the minor ir-morphinan in hypothalamus was reported

previously as "peak 5". These results differ from our previous

work in that “peaks 2, 3, and 6" are not detected: we have been

able to identify a source of laboratory contamination that

probably accounts for them.

Supported by DA-1199 from the National Institute on Drug

Abuse.

707
CALCITONIN AND CALCITONIN GENE-RELATED PEPTIDE INDUCED MODULATION

OF CALCIUM FLUXES: CORRELATION TO ANALGESIC EFFECTS AND

MODULATION OF OPIATES. S. P. Welch, C. Cooper and W. L. Dewey,

Dept. of Pharmacology and Toxicology, Medical College of

Virginia, Richmond, VA 23298 and University of Texas Medical

Branch, Galveston, TX 77550 U.S.A.

Salmon calcitonin (CT) and Calcitonin Gene-Related Peptide

(CGRP) were evaluated for analgesic activity in the tailflick,

hotpleate, and p-phenylquinone writhing (PPQ) test in mice

following intraventricular (ivt.) administration. Both CT and

CGRP produce antinociception, however, CGRP is less potent than

CT (ED50=20ug and .36ug respectively in the PPQ test). CT

produced peak analgesic effects at 2 hr., while the maximal

effects of CGRP occurred at 1 hr. Naloxone reversed the

analgesic effects of CT in the tailflick and hot plate tests, but

not in the PPQ test. Naloxone reversed CGRP analgesic effects in

the PPQ test. At 5 min post administration ivt. both CT and CGRP

produced antagonism of subcutaneously administered (s.c.)

morphine. However, CT produced a significant 2 fold shift in the

morphine ED50, while CGRP-induced shifts were not significant,

In the electrically stimulated guinea pig ileum (GPI) assay both

CT and CGRP produced non-dose responsive, naloxone reversible

inhibition of the ileum (25 and 51% respectively). 5 min.

pretreatment of the ileum with CGRP produced an effect additive

with that of morphine, while 5 min. pretreatment with CT

antagonized morphine. However, pretreatment of the ileum for 1

hr. with CT slightly potentiated morphine. CT and CGRP both

produced time-dependent alterations in calcium uptake to whole

brain synaptosomes. Both CT and CGRP increased calcium uptake

when incubated with synaptosomes for 5 min. However both

decreased calcium uptake with 1 hr (CGRP) and 2 hr. (CT)

incubations. Both CT and CGRP appear to have intrinsic

agonist/antagonist effect which correlate well to the modulation

of calcium fluxes in the central nervous system. (Supported by

USPHS Grants DA01647 and T32DA07027).

708
TREATMENT OF PAIN IN CANCER SUBJECTS BY INTRATHECAL

ADMINISTRATION OF DYNORPHIN

H.L. Wen1

, Z.D. Mehali1, B.H. Ong2 and W.K.K. Ho3,

 1Neurosurgical

Unit, Kwong Wah Hospital, Kowloon; 2Surgical Department, United

Christian Hospital, Kwun Tong, Kowloon; 3Department of

Biochemistry, The Chinese University of Hong Kong, Shatin, N.T.,

Hong Kong

Dynorphin, an opioid peptide originally isolated from the pig

pituitary, has been shown to have potent activity in the guinea

pig ileum bioassay. In contrast to -endorphin, this peptide

when administered intracereberally to experimental animals showed

weak to no analgesic activity. Anatomical studies have shown

that the spinal cord contains a high concentration of

inmunoreactive dynorphin and it has been rationalized that this

organ may be the primary site of action for this peptide. In

contrast to the early studies at the brain level, several groups

have shown that the intrathecal administration of dynorphin can

induce a long lasting analgesic effect in rats. In order to

evaluate the potential application of this peptide in the

treatment of pain in human subjects we have performed some

preliminary experiments by administering very small doses of this

peptide intrathecally into six cancer patients. Compared with

the injection of saline, dynorphin 1-13 was able to reduce pain

score by at least 150% between the dose of 7.5 to 60 μg. The

mean duration of relief after the administration of dynorphin 1-

13 was 4.3, 5.8, 4.5 and 5.3 hours at doses of 7.5, 15, 30 and 60

μg, respectively. The mean time of relief after saline was only

0.35 hour. Throughout the study only one to two cases of minor

side effects such as headache and dizziness were recorded.

Dynorphin 1-10 essentiallyproduced a very similar profile of

response. Thus, the structural active canponent of the dynorphin

molecule appears in this case to reside between amino acid 1 to

10.

This work was supported by a grant from the Lee Foundation of

Singapore.

709
ENKEPHALIN BIOSYNTHESIS AND QENE EXPRESSION ARE INCREASED IN

HIPPOCAMPAL MOSSY FIBERS FOLLOWING SEIZURES. J.D. White, C.M.

Gall and J.F. McKelvy, Dept. of Neurobiology and Behavior , SUNY, Stony Brook, NY

11794; *Dept.of Anatomy, Univ. California, Irvine, CA 92717, USA

The biosynthesis and post-translational proccessing of proenkephalin and the level

of preproenkephalin mRNA were investigated in the mossy fiber system of the

granule cells of the hippocampus in the presence or absence of a unilateral lesion of

the hilus, a procedure which produces bilateral hippccampal seizures. In the mossy

fibers contralateral to the lesion, increases in enkephalin immunoreactivity were

detected by both immunocytochemistry and radioimmunoassay. By the technique of

in vivo radiolabeling and high performance liquid chromatographic purification of

identified radiolabeled peptides, we observed a 14-fold increase in incorporation of

radiolabeled methionine into Met5-enkephalin in lesioned vs. control animals, when

Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the

post-translatinal proteolytic processing of proenkephalin, the biosynthesis of five

additional Met2-enkephalin-containing peptides was also examined. We determined

that the post-translational processing of proenkephalin did not yield exclusively

penta-, hepta- and octa-peptides but larger opioid peptides as well in both control

and lesioned animals; however, the ratio of the enkephalin peptides was not altered

following the lesion Measurement of preproenkephalin messenger RNA levels in the

granule cells by Northern analysis revealed a marked increase (24-fold) following

the lesion. These date support the hypothesis that enkephalin biosynthesis in the

granule cells is stimuleted as a consequence of seizure activity and that the mossy

fiber system offers a model system to study the modulation of opiate peptide

synthesis by neuronal activity.

710
ALTERED METABOLISM OF -ENDORPHIN AND DEVIANT BIOLOGICAL ACTIVITY

OF -ENDORPHIN IN POST MORTEM BRAIN OF SCHIZOPHRENIC PATIENTS.

V.M. Wiegant, O. Gaffori, J. Verhoef, J.P.H. Burbach, G.L. Kovács

and D. De Wied, Rudolf Magnus Institute for Pharmacology, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

The - and -endorphins ( E , E) are endogenous neuropeptides,

enzymatically generated in the brain from -endorphin ( E). In a

variety of behavioral test systems E and E produce opposite

effects, not related to their opiate like activity. The actions

of E resemble those of psychostimulant drugs, whereas E displays neuroleptic-like properties. In view of the neurolepticlike activity of -type endorphins, it was hypothesized that a

reduced availability of E-type activity in the brain is an

ethiological factor in psychopathologies for which neuroleptics

are beneficial, such as schizophrenia.

We have pursued this hypothesis, and investigated the concentration of E, E and E in post mortem hypothalamic tissue of

schizophrenic patients with combined HPLC and RIA techniques. In

addition, we determined the activity of E fractions from brain

and pituitary tissue of such patients on the passive avoidance

response in rats. In both lines of experimentation post mortem

tissue of subjects without psychopathology was taken as control.

These results are suggestive of an altered metabolism of E in

brain tissue of schizophrenic patients, although it remains to be

established whether these alterations are consequence of ante

mortem drug treatment of the patients or are connected to the

psychopathology itself. Moreover, our results indicate that E in

schizophrenics possesses deviant biological properties.

The concentration of E and E was found to be significantly

higher in the hypothalamus of schizophrenic patients than in that

of control subjects. No difference was found in the E levels. In

the passive avoidance test, E fractions from hypothalamic or

pituitary tissue of schizophrenic patients displayed an activity

different from control and synthetic E; they were inactive or

induced the opposite response.

711
ENKEPHALIN DEGRADATION IN LOCUS COERULEUS MEASURED

ELECTROPHYSIOLOGICALLY. J.T. Williams, M.J. Christie, R.A. North

and B. Roques*. Neuropharmacology Laboratory, M.I.T., Cambridge MA

02139, U.S.A. and *University Rene Descartes, Paris 75270, France.

The increase in potassium conductance caused by μ-opioid

agonists was measured by intracellular electrodes in single locus

coeruleus neurons in slices cut from rat brain. [Met5

] enkephalin

(ME) was applied by superfusion for periods of 2-5 min, which was

sufficient for its effect to reach steady-state. The effect

measured was membrane hyperpolarization or, more commonly, the

outward current under voltage clamp at -60 mV holding potential.

Kelatorphan (up to 60 μM) had no effect on the LC neurons, but the

amplitude and duration of the response to ME was increased when

kelatorphan was in the superfusing solution; this potentiation was

observed in all cells. The concentration-effect curve for ME was

shifted to the left 3-30 fold by kelatorphan, the degree of shift

being dependent upon the kelatorphan concentration (2-20 μM);

further increase in kelatorphan caused no further increase in the

response to ME. The minimum effective concentration of ME was

reduced from about 300 134 to 30 nM by kelatorphan. The time

course of action of kelatorphan was studied by applying the ME for

longer periods of time (20-30 min) and during this period changing

the superfusion solution to one which contained both ME and

kelatorphan. The increase in the effect of ME occurred within 2

min and reversed 10-20 min after the kelatorphan was removed.

Outward currents evoked by Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) and

Tyr-D-Ala-Gly-Phe-D-Leu (DADLE)(which act on the same p-receptors)

or by noradrenaline (which acts on 2 receptors but opens the same

potassium channels) were unaffected by kelatorphan. Noradrenaline

responses were potentiated 10-30 fold by cocaine (which did not

affect responses to ME); acetylcholine responses were potentiated

10-30 fold by physostigmine. The results indicate that a single

brain slice (containing 300-500 LC neurones) avidly destroys a

large proportion of ME superfusing it, that the action of

kelatorphan is readily reversible, and that the degree of

potentiation of response observed is very similar to that seen

with catecholamines and acetylcholine.

712
THE ENDOGENOUS KAPPA AGONIST, DYNORPHIN(1-13) DOES

NOT ALTER NIGROSTRIATAL OR MESOLIMBIC DOPAMINE

METABOLISM IN THE RAT. P.L. Wood. H.S. Kim and S.

Iyengar. Neuroscience Research, Research Dept.,

Pharmaceuticals Division, Ciba-Geigy Corp., summit,

N.J., 07901, U.S.A.

Dopaminergic pathways in the rat brain possess

both mu and delta opioid regulatory inputs. In

contrast, previous studies with kappa agonists have

reported that these pathways appear to lack kappa

receptor regulation. We therefore examined the

actions of the endogenous kappa agonist, dynorphin(1-

13) on nigrostriatal (striatum) and mesolimbic (N.

accumbens dopamine metabolism in the rat. As a

control, plasma corticosterone levels were also

measured in all animals to assess the bioavailability

of the test drugs. This is a useful control since

serum levels of this hormone are under central mu,

delta and kappa opioid control.

While parenteral morphine elevated both plasma

corticosterone and dopamine metabolism (both striatum

and N. accumbens), neither parenteral U 50488H nor

intraventricular dynorphin(1-13) altered dopamine

metabolism. However, both kappa agonists potently

elevated plasma corticosterone levels indicating

their bioavailability to the CNS.

In summary, our data support previous reports that

mesolimbic and nigrostriatal dopaminergic neurons

lack kappa opioid regulatory inputs in the rat.

713
CHARACTERIZATION OF OPIOID RECEPTOR IN HAMSTER BRAIN.

S. -W. Wu, W. -Q. Jin and Z. -Q. Chi.

Shanghai Institute of Materia Medica, Chinese Academy

of Sciences, Shanghai 200031, China.

The distribution density of subtype of opioid receptor differs markedly in brain of different species

. The nature of opioid receptor in hamster brain has

been studied in this paper. The mu binding site was

determined with the highly selective mu ligand, 3HDAGO. For the delta site, 3

H-DADLE was used in the

presence of unlabelled 30 nM morphine to suppress binding to the mu site. For the estimation of kappa binding, the 3

H-etorphine was used in the presence of unlabelled 30 nM morphine and 100 nM DADLE to suppress

bindings to the mu and delta sites. The non-Specific

binding was determined with 10 μM levorphanol. The

results of the experiments indicated that specific

binding of the three 3H-ligands is concentrationdependent and saturable. Scatchard analysis showed

that the Kd and Bmax for mu binding were 1.3 nM and

17.8 pmol/g protein, respectively. For delta binding

0.88 nM and 11.6 pmol/g protein, respectively. For

kappa binding 0.52 nM and 33.8 pmol/g protein, respectively. Competition binding studies indicated that

cold mu ligand, ohmefentanyl easily displaced the binding of 3

H-DAGO (KI=26 pM). Relative selective

ligand, DADLE readily inhibited the binding of 3H-DADL

(KI=1.0 nM). However, selective kap

competed effectively the binding of 3H-etorphine to

a ligand, U-50488

kappa site (KI=5.9 nM). Based in our experiments, we

estimate that the hamster brain conteins 54% kappa sites, 28% mu sites and 18% delta sites. These results

suggested that the binding of the high affinity to the

kappa site is predominantly presented in the hamster

brain. It markedly differs from the brain of rat or

guinea-pig.

714
STRESS-INDUCED CHANGES IN DIFFERENT MOLECULAR WEIGHT FORMS OF

DYNORPHIN PEPTIDES IN RAT BRAIN AND SPINAL CORD. G. X. Xie and

A. Goldstein, Addiction Research Foundation and Department of

Pharmacology, Stanford University, Palo Alto, CA 94304, U.S.A.

The immunoreactivities of three peptide products of the

preprodynorphin gene -- dynorphin A, dynorphin B and -neoendorphin -- were measured in single rat brains and spinal cords

using specific antisera after Sephadex G-50 filtration. Three

peaks of Mr about 7, 4 and 2 kDa for ir-dyn A and ir-dyn B, and

one single peak of Mr less than 2 kDa for a-neo-endorphin were

found both in the brain and in the spinal cord. The 7 kDa peak

was recognized by a specific antiserum against the carboxyl

terminus of dyn B, indicating it terminates in threonine-13 of

dyn B. The relative amounts of 7 and 4 kDa materials to 2 kDa

material were greater in spinal cord than in the brain,

suggesting a different rate of processing or release in different

regions of the central nervous system.

Swimming in 10°C cold water for 10 min produced a

naloxone-blocked increase of tail flick latency, and also caused

a striking reduction of 7 and 4 kDa dynorphins in spinal cord

but little or no change in the small M, products of the

preprodynorphin gene. The processing and release of dynorphin

peptides in the spinal cord may be accelerated by stress.

Supported by BNS-84-16617 from the National Science

Foundation.

715
CONVERSION OF -ENDORPHIN TO -ENDORPHIN IN RAT PLASMA. Elizabeth A.

Young and Huda Akil, Mental Health Research Institute. University of Michigan,

Ann Arbor MI 48109 U.S.A.

With a number of stressors -endorphin is released into plasma. The rate of

disappearance of this released -endorphin is rapid, with estimates of the half

life in the 10 to 15 minute range. it is unclear if -endorphin is coverted into

any other biologically active products, nor is it clear if the rate or pathways of

degredation is changed during chronic stress. To explore these issues, we

incubated [

3H] -endorphinh labelled in position 1 and 27 with plasma from

normal and chronically footshocked rats and measured the rate of conversion of

the label from -endorphin size material to smaller size material. Initial

separations were done using a G-50 molecular sieving column. with subsequent

characterization and identification on BPLC. Using these methods, we see a time

dependent formation of only one radioactive peak with G-50 column sieving.

The size estimate of this peak is 16 or 17 amino acids. Since the [

3H] -endorphin

was labelled on both ends of the molecule, two radiolabelled fragments should be

formed. This suggested that the molecule may be cleaved near the middle of the

molecule resulting in two fragments of similar size. To confirm this hypothesis

we used an HPLC separation of the pre-sieved fractions from the G-50 column,

and looked for the co-migration of label with either o r endorphin as well

as other unidentified peaks. We found two radioactive peaks on HPLC, one of

which co-migrated with -endorphin. and the other peak which is unidentified

currently but may well be -endorphin 18-31. Interestingly. this enzymatic

activity is induced by chronic stress so that the labelled -endorphin shows a 2-

3 fold faster conversion to this product. Whether the -endorphin remains

stable after formation is less clear. but fragments of -endorphin such as destyrosine -endorphin and des-enbphalin -endorphin have been reported to

be behaviorally active. We conclude that post-release conversion of

endorphin into other biologically active fragments can occur, and that

endorphin is one of the fragments formed in plasma. Similar data have been

reported in brain synaptasomes, and a common enzyme may be present in both

tissues.

716
EFFECTS OF DIFFERENTIATION ON CELLULAR AND BIOCHEMICAL PROPERTIES

OF A μ RECEPTOR CONTAINING NEUROBLASTOMA CELL LINE. V.C. Yu,

G. Hochhaus, M.L. Richards and W. Sadée. School of Pharmacy,

University of California, San Francisco, San Francisco, CA 94143,

U.S.A.

We have recently identified a human neuroblastoma cell line,

SK-N-SH, that expresses μ and opioid receptors.

Morphologically, SK-N-SH cells appear to consist of two distinct

cell types (epithelial-like and neuroblast-like) that can be

interconverted to one another. SH-SY5Y is a thrice-cloned cell

line of SK-N-SH which displays an essentially homogenous

population of neuroblast-like cells and can be grown to high cell

density. The SH-SYSY clone, like the parent SK-N-SH cells,

expresses both μ and opioid receptors, muscarinic cholinergic

receptors, and a catechol uptake1 system. The cells can be

stimulated to release inositol triphosphate by acetycholine but

not by opioids, bradykinin, ATP and Sub P. Six days treatment of

SH-SY5Y cells with either nerve growth factor (50 ng/ml), dBcAMP

(1 mM) or retinoic acid (20 μM) induces differentiation of the

cells marked by extensive neurite outgrowth. The various receptor

systems were differentially affected by the agents used to induce

neuronal maturation on receptor expression and the cellular

properties. Most strikingly, nerve growth factor and retinoic

acid increased the opioid receptor density (on calculated per mg

protein) by as much as two-fold, while dBcAMP decreased it by

approximately 40%. The effects of the differentiating agents were

less pronounced on the ACh receptor, while retinoic acid

selectively increased the uptake system by three-fold. Supported

by DA 01095 from NIDA, Bethesda, MD.

717
NEONATAL TYR-MIF- ALTERS NOCICEPTIVE EFFECTS OF NEONATAL

BETA-ENDORPHIN AND MORPHICEPTIN DURING DEVELOPMENT AND IN

ADULTHOOD. James E. Zadina, Abba J. Kastin, Penelope K. Manasco,

and Kent L. Nastiuk, Veterans Administration Medical Center and

Tulane University School of Medicine, New Orleans, LA 70146, USA.

Male rats were injected SC once daily during the first week of

life with beta-endorphin (BE), morphiceptin (a mu opiate

receptor-selective peptide), a putative endogenous antiopiate

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), or one of the two opiate peptides

in combination with Tyr-MIF-1. They were then tested during early

development and in adulthood (4.5 months) for their responsiveness

to thermal pain. Pups treated with neonatal BE removed their tails

from a series of increasingly hot water baths significantly faster

than control animals on day 9, confirming our earlier studies. In

rats injected with both Tyr-MIF-1 and BE, however, the latency to

remove the tail was significantly longer than in the group given BE

alone, and was not different from control animals, indicating that

Tyr-MIF-1 blocked the effect of BE. Randomly chosen subgroups of

animals, which were statistically shown to adequately represent the

groups from which they were drawn, were retested in adulthood for

responsiveness to thermal pain using the hot plate.

a 50°C surface,

When placed on

the rank order of the treatment groups in their

latency to lick a hindpaw was similar to their ranking in the

tail-flick test on day 9. The latencies were significantly longer

in each of the 3 groups given Tyr-MIF-1 than in groups given BE

alone, morphiceptin alone, or the control vehicle. None of the

neonatal peptide treatments significantly affected the degree of

analgesia shown when animals were exposed to footshock and retested

on the hot plate. The mean tail-flick latencies of the animals on

day 9 correlated well with adult baseline scores on the hot plate

test, indicating that the effects of the peptides persisted into

adulthood. These results support the role of Tyr-MIF-1 as an

antiopiate and further illustrate the long-term effects of

neonatally administered peptides.

718
PHENCYCLIDINE RECEPTOR IN BLOOD VESSELS. H. Zhu,

A.Z. Zhang, X.R. Xu* and W.L. Ye, *Dept. of Neurobiology,

Shanghai Medical University, Shanqhai Institute of

Materia Medica*, Shanghai, 200032, China

Binding studies of (3H) Phenclidine (PCP) to rabbit

mesenteric blood vessel detected a specific, saturable

and stereoselective binding site of which Kd=31.9±3.9

nM, Bmax=4.96±0.29 pmol/mg pr. IC50 of a series of

ligands are: PCP 0.39±0.04, dextrosin 3.01±0.19,

levorphanol 18.2 ± 1.2, dl-ketamine 0.17±0.02, dl-pentazocine 0.14±0.03, d-SKF10,047 9.77±0.48, 1-SKF10,047

85.1±2.5, d-cyclazocine 2.57±0.11 and l-cyclazocine

4.07±0.17 uM. Naloxone and etorphine failed to inhibit

(3H) PCP binding at concentrations as high as 200 uM.

In vitro bioassay showed that PCP markedly enhanced

the electrical field stimulated contraction (ESC) of

rabbit central ear artery with a minimum eftective conc.

of 0.1 uM, which was naloxone irreversible. ED50

were: PCP 7.52±0.94, d-SKF10,047 1.52±0.23, pentazocine

6.44 and ketamine 1.43±0.14 uM. Such effect could be

abolished by 1 uM of phentolamine. HPLC detected a

significant increase of NE content (p<0.01) in the bath

medium after PCP administration.

Our finding of PCP and siqma opioid receptors in the

blood vessel and their vascular function may at least

partially explain the significant pressor effect of

these drugs, and also propose a dual system, i.e. kappa

(Neuropeptides, 1985,5;595) and PCP/sigma opioid receptors which cooperatively modulate NE release and the

function of blood vessels.

719
INVOLVEMENT OF ENDOGENOUS OPIOID PEPTIDES IN BURNINDUCED SHOCK. Y. X. Zhu, C. H. Wang and B. C. Lin,

Department of Physiology, Second Military Medical

College, Shanghai 200433, China

192 male Sprague-Dawley rats weighing 200-250g were

given III burn injury of 20% body surface area, and

equally divided into 6 groups: control, naloxone, anti-

-endorphin, anti-dynorphin A, anti-met-enkephalin, and

anti-leu-enkephalin. Each animal of these groups immediately postburn was intravenously administrated 50μl

of normal rabbit serum, naloxone solution(1mg/kg),

antisera against -endorphin, antisera against dynorphin

a, antisera against met-enkephalin and antisera against

leu-enkephalin, respectively. There is no significant

difference(P>0.05) of survival rate at 6 hours postburn

between the control(21%) and anti-met-enkephalin(31%) or

anti-leu-enkephalin(2%) group. However, the survival

rate of the anti-dynorphin A(61%) group at the same time

postburn was much hi her, and those of the naloxone(42%)

and anti -endorphin (44%) group were slightly higher

than that of the control group(3<0.05). The onset of

hypotension, bradycardia, hypothermia and dyspnea were

greatly delayed in the naloxone group, as compared with

the control group. Blood pressure showed a 62% reduction

at 180 minutes postburn in the naloxone group, while a

62% reduction was already seen at 90 minutes postburn in

the control group. It is suggested that endogenous

opioid peptides, especially dynorphin A and -endorphin,

might be involved in burn-induced shock, and naloxone

might to be effective in the treatment of burn-induced

shock.

720
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1 FINDINGS OF DRUG ABUSE RESEARCH. NCDAI out of stock

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7 CANNABINOID ASSAYS IN HUMANS. Robert Willette, Ph.D., ed.

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14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

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15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm. Sharp,

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16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

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17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D., et

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18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

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19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

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20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

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21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

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22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

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26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

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27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

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31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

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32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

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Thomas J. Glynn, Ph.D .; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01180-1 $5.50 NTIS PB #85-1596631AS $24.95

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

John Grabowski, Ph.D., and Catherine S. Bell, M.S., eds.

GPO Stock #017-024-01181-9 $4.50 NTIS PB 84-145-184 $18.95

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $36.95

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed.

GPO Stock #017-020-01214-9 $4.50 NTIS PB 85-150381/AS $18.95

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $18.95

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #D17-024-0204-1 $4.25 NTIS PB 85-150373/AS $18.95

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

Ph.D., and Sharon M. Hall, Ph.D., eds.

GPO Stock #017-024-01266-1 $3.50

725
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01213-1 $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #017-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S., and Louise G. Richards, Ph.D., eds.

GPO Stock #017-024-01246-7 $4.25

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W., ed.

GPO Stock #017-024-01247-5 $4.25

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert, M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang,

Ph.D., and Charles C. Lee, Ph.D., eds.

GPO Stock #017-024-01257-2 $3.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL PERSPECTIVES.

Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S., eds.

GPO Stock #017-024-01258-1 $5

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M. Brown,

Ph.D., and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01260-2 $3.50

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

ADOLESCENTS. Catherine S. Bell, M.S., and Robert Battjes,

D.S.W., eds.

GPO Stock #017-024-01263-7 $5.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

GPO Stock #017-024-01281-5 $6.50

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A. Ray, Ph.D.,

and Monique C. Braude, Ph.D., eds.

GPO Stock #017-024-01283-1 $3.25

726
66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND ALCOHOLISM.

Monique C. Braude, Ph.D., and Helen M. Chao, Ph.D. eds.

GPO Stock #017-024-01291-2 $3.50

67 PROBLEMS OF DRUG DEPENDENCE, 1985. PROCEEDINGS OF THE 47TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01292-1 $18

68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS OF ABUSE.

Monique C. Braude, Ph.D., and Harold M. Ginzburg, M.D., J.D., M.P.H.,

eds.

GPO Stock #017-024-01296-3 $6.50

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

Gene Barnett, Ph.D.; and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1297-1 $11

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY, BIOSYNTHESIS, AND

ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-1298-0 $12

71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M. Brown,

Ph.D.; Doris H. Clouet, Ph.D.; and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01303-0 $6

72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims, Ph.D., and

Carl G. Leukefeld, D.S.W., eds.

GPO Stock #017-024-01302-1 $6

73 URINE TESTING FOR DRUGS OF ABUSE. Richard L. Hawks, Ph.D., and

C. Nora Chiang, Ph.D., eds.

GPO Stock #017-024-1313-7 $3.75

IN PRESS

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE. Stephen I.

Szara, M.D., D.Sc., ed.

76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF THE 48TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, ed.

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