Opioid

Peptides:

An Update

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Opioid Peptides: An Update

Editors:

Rao S. Rapaka, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Bhola N. Dhawan, M.D.

Central Drug Research Institute

Lucknow, India

NIDA Research Monograph 87

1988

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402
NIDA Research Monographs are prepared by the research divisions of the

National lnstitute on Drug Abuse and published by its Office of Science The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and

Integrative research reviews. Its dual publication emphasis is rapid and targeted

dissemination to the scientific and professional community.

Editorial Advisors

MARTIN W. ADLER, Ph. D.

Temple University School of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic lnstitute

Troy, New York

RICHARD E. BELLEVILLE, Ph. D.

NE Associates, Health Sciences

RockviIle, 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 Unversity School of

Medicine

Baltimore, Maryland

THEODORE J. CICERO, Ph. D.

Washington University School of

Medicine

St. Louis, Missouri

MARY L. JACOBSON

National Federation of Parents for

Drug Free Youth

Omaha, Nebraska

REESE T. JONES, M. D.

Langley Porter Neuropsychiatric lnstitute

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

THEODORE M. PINKERT, M.D., J.D.

Acting Associate Director for Science, NIDA

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Opioid Peptides: An Update
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the

Indo-U.S. Conference on Recent Progress in the Chemistry and Biology

of Biologically Active Peptides With Emphasis on Opioid Peptides,

held in Lucknow, India, on February 25-27, 1987. The conference was

jointly sponsored by the National Institute on Drug Abuse; the

Department of Science and Technology, Government of India; Central

Drug Research Institute, Lucknow; and the Indian National Science

Academy.

COPYRIGHT STATUS

The figures on pages 28 and 54 are copyrighted and are reproduced

with the permission of the copyright holders. Further reproduction

of this copyrighted material is permitted only as part of a

reprinting of the entire publication or chapter. For any other use,

the copyright holder's permission is required. All other material

in this volume is in the public domain and may be used or reproduced

without permission from the Institute or the authors. Citation of

the source is appreciated.

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)89-1604

Printed 1988

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
Preface

To bring into focus the rapidly expanding areas of research

associated with the opioid peptides, an Indo-U.S. Symposium was

held in February 1987 at the Central Drug Research Institute

(CDRI), Lucknow, India. This symposium was organized by the

National Institute on Drug Abuse (NIDA) and the CDRI and was

jointly funded by NIDA; CDRI; the Department of Science and

Technology, Government of India. and the National Academy of

Sciences, New Delhi.

A number of individuals were responsible for the success of this

conference. We especially acknowledge the help of Dr. Phil

Schambra, Science attache, U.S. Embassy (India). Mrs Linda A.

Vogel, Associate Director for Management and Program Coordination,

Office of International Health; Dr. George V. Coelho, Chief,

International Activities Program, ADAMHA; at NIDA, Dr. James C.

Cooper, Associate Director for Medical and International Affairs;

Dr. Marvin Snyder, Director, Division of Preclinical Research; and

Dr. Richard L. Hawks, Chief, Research Technology Branch; and at

CDRI, Dr. R. Raghubir and Mr. K.L. Gupta.

Selected papers from this symposium have appeared as a research

monograph published by the CDRI, entitled Recent Progress in

Chemistry and Biology of Centrally Acting Peptides. Other

presentations from the symposium, along with contributions from

invited authors, comprise the present monograph.

We are grateful to all the contributors for their cooperation in

preparing this publication. We hope that this volume will serve as

a useful reference source on various aspects of the medicinal

chemistry, pharmacology, and biochemistry of opioid peptides and

will provide new incentives for drug abuse researchers in the

opioid peptide field.

Rao S. Rapaka

Bhola N. Dhawan

v

Contents

Preface . . . . . . . . . . . . . . . . . v

Neuropeptides, A Personalized History

Sidney Udenfriend. . . . . . . . . . . . . 1

Synthesis and Biological Activity of Novel Met-enkephalin

Analogs

Krishna B. Mathur; Balaram J. Dhotre; Shubh D. Sharma;

Ram Raghubir; Gyanendra K. Patnaik; and Bhola N. Dhawan . . 10

Approaches to Studying Structure-Activity Relationships in

Peptide Hormones Through the Expression of Synthetic Genes

John W. Taylor. . . . . . . . . . . . . . . . 20

X-Ray Diffraction Studies of Enkephalins and Opiates

Jane F. Griffin and G. David Smith . . . . . . . . 41

Conformational Analysis of Cyclic Opioid Peptide Analogs

Peter W. Schiller and Brian C. Wilkes . . . . . . . . 60

Conformational Studies of Dermorphin

V. Renugopalakrishnan and Rao S. Rapaka . . . . . . . 74

Use of Molecular Biological Methods to Study Neuropeptides

Michael J. Brownstein . . . . . . . . . . . . . 83

Three Technical Approaches for Cloning Opioid Receptors

Curtis A. Machida; John Salon; David Grandy; James Bunzow;

Paul Albert; Eric Hanneman; and Olivier Civelli . . . . 93

Effects of Opioid Peptides on Human Neuroblastoma Cells

Wolfgang Sadee; Victor C. Yu; and Gunther Hochhaus. .111

Analgesia and Neuropeptides

David J. Mayer. . . . . . . . . . . . . . . .118

Mechanism of Development of Tolerance and Dependence to

Opioids in Neuroblastoma x Glioma Hybrid Cells and Mice

Shail K. Sharma; Madhav Bhatia; and Ranju Ralhan . . . .157

Development of Spinal Substrate for Nociception in Man

Veena Bijlani; Tilat A. Rizvi; and S. Wadhwa. . . . . .167

vii
Differential Effects of Opioid Peptides Administered Intracerebrally in Loci of Self-Stimulation Reward of Lateral

Hypothalamus and Ventral Tegmental Area-Substantia Nigra

Jitendra Singh and T. Desiraju . . . . . . . . . .180

Peptides and Thermoregulation

R. Shukla and Bhola N. Dhawan. . . . . . . . . .192

Endogenous Opioids and Immune Responses: An Experimental

Study

P. K. Mediratta; N. Das; V. S. Gupta; and P. Sen . . . .209

An Update of Selected Topics in the Biology and Chemistry of

Opioid Peptides

Rao S. Rapaka; Bhola N. Dhawan; and V. Renugopalakrishnan .217

List of NIDA Research Monographs . . . . . . . . . .233

viii
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Neuropeptides, A Personalized History

Sidney Udenfriend, Ph.D.

The full significance of the important roles in neurobiology played

by peptides is still emerging. It seems that each day we read of

the discovery and characterization of new peptides in extracts of

neural tissue. In fact the chemical structures of neuropeptldes are

now elucidated before their biological significance becomes

apparent. What is responsible for this rapidly growing field are

the recent advances in microprotein chemistry and molecular

biology. Therefore a brief history of peptide chemistry is

important to understand how we have advanced in so relatively short

a period of time in our ability to isolate and characterize trace

amounts of peptides in biological extracts. In this review, I will

not differentiate between small peptides and large peptides

(proteins). I will also discuss peptides in the nervous system as

well as in the endocrine system because of the interrelationship of

these two systems.

The earliest neuroactive substances to be characterized were the

amlnes and substituted amlnes--first acetylcholine, then adrenaline,

noradrenaline, and serotonln. Although dopamine was known for some

time, it was considered to be an intermediate rather than a

neuroregulator in Its own right until somewhat later. However, in

some of the earliest studies (late forties and early fifties) it was

recognized that other substances were active in nerve and muscle

preparations that were not amlnes and whose activities were

destroyed by proteases.

In 1950 peptide chemistry was still emerging as a discipline. While

synthetic chemists could synthesize small peptides, methods for

their purification, even from synthetic mixtures, were not yet very

efficient. Sequencing of peptides had not yet been introduced. In

fact, there was still controversy as to whether each peptide and

protein was a stoichlometrlc entity (i.e., had a unique and

invariable sequence) or whether a given protein or peptide was

composed of molecules each varying somewhat from the other but the

average being fairly constant for that peptide. The dlpeptldes

carnosine and anserine had been isolated from muscle. Homocarosine,

the GABA analog of carnosine. was found to be uniquely present In

brain. However. the high concentration (mg of peptide per g of

1
tissue) and small size of these compounds required no unusual

methodology. The first findings that specific proteins have an

invariant molecular composition came from end-group analysis when it

was shown that the same protein isolated from highly diverse species

always had the same amino terminus. Not until the late 1950s. after

Fred Sanger had devised a method for elucidating the amino acid

sequence of a protein and applied it to insulin, was it generally

conceded that all proteins and peptides had exact molecular

compositions.

Several years later, Vincent du Vigneaud isolated and characterized

oxytocin and vasopressin from extracts of the posterior pituitary

gland. It should be noted, however, oxytocin and vasopressin are

present in relatively large amounts in the pituitary gland (ca l

Pmole/g). Column chromatography became generally available in the

late fifties, and Edman introduced the manual procedure for

sequencing peptides at about the same time. These procedures were

used in the isolation and characterization of angiotensin and

bradykinin. In the late sixties Roger Guillemin and Andrew Schally

used similar procedures to isolate TRH from hypothalamus. However,

the hormone was so potent that to isolate the amounts needed at the

time for analysis and sequencing (ca 50 nmoles) they had to extract

tons of tissue. This was very costly and time consuming and required

all the patience and skill of these two outstanding scientists. At

about the same time many other biologically active factors that were

apparently peptide in nature were also reported--substance P, many

growth factors, including nerve growth factor, and a host of

lymphokine activities including the interferons. Isolation and

characterization of these had to await newer developments.

Several major discoveries and advances in technology in the late

sixties and seventies, advances that are still continuing today,

completely changed our approach to dealing with biologically active

peptides. High performance liquid chromatography (HLPC) with

ultrasensitive detection systems made it possible to achieve many

thousandfold degrees of purification in a relatively few steps and

with relatively small amounts of material. The continued

improvement in sensitivity and speed of commercial automated

sequenators now make it possible to determine the primary structure

of a modest sized peptide within a day, starting with 100 pmole or

less of pure material. With monoclonal‘antibody techniques, it is

now possible to prepare specific antibodies to a biologically active

peptide while it is still present in a crude mixture. These

monoclonal antibodies can then be used for rapid purification of the

peptide. With monoclonal antibodies it is frequently unnecessary to

actually isolate the biologically active peptide to determine its

sequence. When expression vectors are used for cloning the cDNA

derived from the tissue of interest, monoclonal antibodies can be

used for clone selection. Sequencing is then carried out on

specific cDNA from which the primary structure of the peptide

precursor can be deduced. Having isolated and determined the

sequence of a peptide or its cDNA, the peptide can be readily

synthesized for further study. The synthetic peptide can then be

2
used to prepare site directed polyclonal antibodies, which provide

additional tools for studying the localization and regulation of

that peptide.

I would like to briefly discuss some of the developments in the

chemistry and biology of neuropeptides that took place in the Roche

Institute of Molecular Biology over the past 20 years. Although the

structures of oxytocin and vasopressin were already known, it was

Sachs and Takabatake (1964) who first demonstrated that each of

these was produced as part of a larger polypeptide containing the

neurophysin sequence. This information laid the groundwork for the

subsequent cloning and sequencing of the cDNAs for oxytocinneurophysin and vasopressin-neurophysin (Richter 1987).

The dipeptide carnosine, which was originally considered to be

restricted to muscle, was found to be present in the olfactory bulb

by Margolis (1974). Carnosine is now considered to be a transmitter

with a presynaptic site of action. This peptide along with the

enzyme carnosine synthetase and the degradative enzyme carnosinase

are now used as markers of olfactory nerves and to monitor nerve

regeneration. Margolis has also isolated, sequenced, and cloned a

protein that is uniquely expressed in the olfactory mucosa (Keller

and Margolis 1976).

In my own laboratory (Bohlen et al 1975) we developed HPLC and

introduced the fluorescent reagent, fluorescamine, for high

sensitivity. These procedures made possible many studies that were

subsequently carried out in our Institute and elsewhere. Shortly

after the discovery of the enkephalins, my colleagues and I used the

fluorescamine HPLC procedure in the discovery of the prescursor of

beta-lipotropin (Rubinstein et al. 1978a). Chromatograms of

pituitary extracts revealed a ß-endorphin-containing peptide that

was larger than beta-lipotropin, about 30K. This appeared

simultaneously with the report of Mains et al. (1977), who found

that the 30K protein also contained within it the sequence of ACTH.

We coined the name proopiocortin for this common precursor of

ß-endorphin and ACTH. Subsequently, to emphasize the sequences of

MSH in the 30K protein, it was renamed pro-opiomelanocortin, now

better known as POMC. In their studies in cell culture, Mains and

Eipper found that POMC was produced as a glycosylated protein. For

this reason it was generally believed that all forms of POMC are

glycosylated and that the latter may have something to do with

biological activity. However, we showed that in vivo glycosylation

is highly variable from individual to individual within a given

species. In pituitaries taken from individual camels, we showed

that in some glands isolated POMC was almost entirely in the

glycosylated form while in others the molecule contained little or

no glycosyl residues (Kimura et al. 1979). Apparently in the case

of POMC as in other proteins and peptides the significance of

glycosylation remains to be determined.

It is of interest that Rubinstein, who had isolated POMC,

subsequently used the same procedures in my laboratory in

3
collaboration with Pestka to isolate a-interferon from human

leukocytes (Rubinstein et al. 1978b). This represented the first

purification and characterization of any form of interferon and

quite a feat because the amount of interferon in these cells was

very small. Although interferon was discovered in the early fifties

and many had tried to purify it over the years, it was not until

1978 with the availability of these newer peptide methods that its

purification and characterization were achieved.

Getting back to the enkephalin pentapeptides, many believed that

they were derived from POMC However, we reasoned that POMC could

not give rise to free Leu-enkephalin since it did not contain that

sequence. Furthermore, although the B-endorphin derived from POMC

contained a Met-enkephalin sequence at its amino terminus, there was

no known processing site adjacent to this sequence that could lead

to its processing to Met-enkephalin. For those reasons we decided

to look for other enkephalin-containing compounds that could serve

as precursors. We devised a bioassay that involved homogenizing

tissue, then treating the extract with trypsin followed by

carboxypeptidase B. By analogy with other peptide precursors, we

felt that there was a good possibility that the enkephalin sequence

within its precursor would be bracketed by pairs of Lys and/or Arg

residues, i.e., -Lys-Arg-enkephalin-Lys-Lys. Trypsin cleaves basic

residues at their carboxyl termini and would be expected to cleave

out a hexapeptide. enkephalin-Lys or enkephalin-Arg. The latter

would be converted to free enkephalin by the carboxypeptidase and so

detectable by the binding assay. The first application of the above

procedure to guinea pig and rat striatum revealed many large

peptides from which both Met- and Leu-enkephalin were released on

treatment with the two proteases (Lewis et al. 1978). Following a

report that bovine adrenal medulla is rich in enkephalins

(Schultzberg et al. 1978), we turned to that tissue from which we

isolated and sequenced about ten different enkephalin-containing

peptides, some containing withln them as many as four enkephalin

sequences (Lewis et al. 1980). Others contained both Met- and

Leu-enkephalin. Even before these enkephalin-containing peptides

were characterized, we had demonstrated the presence in brain and

adrenal medulla of a very large peptide which, on treatment with

trypsin and carboxypeptidase B, yielded approximately six

Met-enkephalin residues per Leu-enkephalin residue. This led to the

concept of a multivalent proenkephalin. It appeared that

proenkephalin like POMC was a polyprotein, one containing the

sequences of more than one active peptide, and that these sequences

were designed to be released by processing.

The many enkephalin-containing peptides that we had isolated and

sequenced at the time were like pieces of a jigsaw puzzle. Although

we had most of the pieces, some were still missing and their exact

order was not clear. However, one of the pieces, Peptide F,

contained a sequence that showed no degeneracy at the nucleic acid

level, enabling us to make an excellent cDNA probe. The same probe

was used by us (Gubler et al. 1982) and by Numa and his colleagues

4
(Noda et al. 1982) to clone and sequence bovine proenkephalin. The

latter was indeed shown to contain six [Metlenkephalin and one

[Leu]enkephalin sequence as predicted from the peptides we had

isolated. Two of the sequences were designed to be processed out as

heptapeptides.

With POMC and proenkephalin sequenced, there still remained two

[Leu]enkephalin-containing peptides that were present in the

posterior pituitary and whose sequences did not appear in either of

these precursors. These were a-neoendorphin (Kangawa et al. 1981)

and dynorphin (Goldstein and Ghazarossian 1980). It was already

apparent that a third enkephalin-containing precursor must exist.

Before it was characterized we (Kilpatrick et al. 1982) and Fischli

et al. (1982) independently isolated and characterized a third

[Leu]enkephalin-containing peptide (rimorphin) and showed it to be

present in posterior pituitary extracts in amounts comparable to

a-neoendorphin and dynorphin. Using the sequence information

provided by these peptides, Numa's laboratory cloned and sequenced

their precursor. prodynorphin (Kakidani et al. 1982). The

significance of the redundancy of enkephalin precursors is still not

clear, nor are the exact functions of these neuropeptides known.

Later, Howells in my laboratory cloned and sequenced rat

proenkephalin CDNA (Howells et al. 1984) so that we would be able to

study the biology of the opioid peptides in the species that has

been used most in studies on opiate drugs. We used the cDNA,

antibodies, and HPLC in many different ways. Proenkephalin derived

peptides and the corresponding mRNA were found in several nonneural

tissues including the heart ventricles (Howells et al. 1986) and the

testis and ovary (Kilpatrick et al. 1985) as well. They are also

present in the intestine and pancreas. It is apparent that the

biological functions of proenkephalin derived peptides, like those

of many other neuropeptides. are not limited to the central nervous

system. Adrenal proenkephalin transcription and translation are

markedly increased by denervation (Howells et al. 1984; Lewis et al.

1981) and are apparently under the control of glucocortlcoids

(Yoburn et al. 1987; Mocchetti et al. 1985). Brain proenkephalin

transcription and translation are increased by several centrally

acting drugs (Tang et al. 1983; Romano et al. 1987).

Antibodies to opioid peptides had been used to trace the

proenkephalln innervation in the CNS. However, a more precise and

complete localization of proenkephalin neurons in the brain was

recently carried out with the corresponding rat cDNA. The in situ

hybridization studies were carried out by Howells in my laboratory

in collaboration with Pfaff and his colleagues (Harlan et al.

1987). In situ hybridization methods utilizing cDNA are becoming

more sensitive and may even be more specific than

immunocytochemistry. The latter visualizes nerve bodies as well as

fibers, whereas hybridization visualizes nerve bodies exclusively.

When dealing with antibodies directed to small molecules, such as

the opioid peptides or the catecholamines, diffusion may limit the

precision of localization by immunocytochemistry. Localization of

5
macromolecules, whether precursors, specific enzymes, or specific

mRNA, is generally more precise.

I will conclude with two recent examples of the advances that modern

biotechnology has brought to the neurosciences. In the late sixties

Guillemin had to use tons of beef hypothalami to isolate hundreds of

nanomoles for characterizing the first releasing factor, TRH. More

recently Guillemin's laboratory was able to isolate a few hundred

picomoles of growth hormone releasing factors from several grams of

human tissue (Guillemin et al. 1982). This was sufficient for

sequencing. Shortly thereafter, in collaboration with Roche

scientists they cloned the corresponding cDNA (Gubler et al. 1983).

A fraction of the amount of tissue was used, and purification and

sequencing were accomplished on far less material and in a much

shorter time.

Horecker, while at our Institute, had shown that extracting tissues

with 6M guanidine hydrochloride prevents proteolysis (Hannappel et

al. 1982). Under these conditions most larger proteins are

denatured and precipitate and supernatant solutions contain peptides

that are characteristic of a tissue and are not experimental

artifacts. A brain extract prepared in this manner yields many

peptides on HPLC. Some of these peptides are present in other

tissues and are probably known. Others are unique to brain. When

Morgan applied this extraction procedure along with HPLC to

different portions of the brain, the resulting chromatographic

eluates contained relatively few peptides. Two of the unidentified

minor components in extracts of cerebellum when isolated and

sequenced were shown to be unique molecules and limited to the

cerebellum (Slemmon et al. 1984). These two related peptides, which

he named cerebellins, are localized in Purkinje cells (Slemmon et

al. 1985). The cerebellins are now used as markers for maturation

of these cerebellar cells. No doubt many other "minor peptides"

present in extracts recovered from specific brain areas will prove

to be of interest.

One of the most interesting developments relating to peptides in the

brain concerns oncogene products. Oncogenes were originally

discovered through their association with tumors, and the

translations products of oncogenes are considered to be aberrant

versions of normal regulatory proteins or peptides. Protooncogenes

code for normal proteins the functions of which are still being

determined. Morgan and Curran in our Institute showed that the

c-fos protooncogene is rapidly and transiently induced by

receptor-ligand interaction and by agents that affect voltage

dependent calcium channels (Morgan and Curran 1986). The magnitude

of these effects is modulated by pharmacologic agents. More

recently they applied immunocytochemical methods to c-fos

protooncogene induction as a means of determining the exact cells in

brain where specific drugs act.

With all the advances that have been made in methodology and

6
instrumentation and with our increasing knowledge of how proteins

and peptides are made, there is still a great future for peptide

research in neurobiology.

REFERENCES

Bohlen, P.; Stein, S.; Stone, J.; and Udenfriend, S. Automatic

monitoring of primary amines in preparative column effluents with

fluorescamine. Anal Biochem 67:438-445, 1975.

Fishcli, W.; Goldstein,A.;Hunkapillar, M.W.; and Hood, L.

Isolation and amino acid sequence analysis of a 4,000-dalton

dynorphin from porcine pituitary. Proc Natl Acad Sci USA

79:5435-5437. 1982.

Goldstein. A., and Ghazarossian. V.E. Immunoreactive dynorphin in

pituitary and brain. Pro Natl Acad Sci USA 77:6207-6210, 1980.

Gubler. U.; Monahan. J.J.: Lomedico. P.T.: Bhatt. R.S.: Collier.

K.J.;Hoffman,B.J.;Bohlen,P.;Esch,F.;Ling,N.;Zeytin,F.;

Brazeau,P.; Poonian, M.S.; and Gage, P.L. Cloning and sequence

analysis of cDNA for the precursor of human growth hormonereleasing factor, somatocrinin. Proc Natl Acad Sci USA

80:4311-4314, 1983.

Gubler, J.; Seeburg, P.; Gage, L.P.; and Udenfriend, S. Molecular

cloning establishes proenkephalin as precursor of enkephalincontaining peptides. Nature 295:206-208, 1982.

Guillemin, R.; Brazeau, P.; Bohlen, P.; Esch, F.; Ling, N.; and

Wehrenberg, W.B. Growth hormone-releasing factor from a human

pancreatic tumor that caused acromegaly. Science 218:585-587,

1982.

Hannappel. E .; Davoust, S .; and Horecker, B.L. Isolation of

peptides from calf thymus. Biochem Biophy Res Commun 104:266-271,

1982.

Harlan. R.E .; Shivers, B.D.; Romano. G.J.; Howells. R.D.; and Pfaff,

D.W. Cellular localization of proenkephalin mRNA in the rat

neurazis. J Comp Neurol 258:159-184, 1987.

Howells, R.D.; Kilpatrick, D.L.; Bailey, L.C.; Noe, M.; and

Udenfriend. S. Proenkephalin mRNA in rat heart. Proc Natl Acad

Sci USA 83:1960-1963, 1986.

Howells, R.D.; Kilpatrick, D.L.; Bhatt, R.; Monohan, J.J.; Poonian,

M.; and Udenfriend, S. Molecular cloning and sequence

determination of rat preproenkephalin cDNA: Sensitive probe for

studying transcriptional changes in rat tissues. Proc Natl Acad

Sci USA 81:7651-7655, 1984.

Kakidanl, H.; Furutani, Y.; Takahashi, H.; Noda, H.; Morimoto, Y.;

Hirose, T .; Asai, M.; Inayama, S.; Nakanishi, S.; and Numa, S.

Cloning and sequence analysis of cDNA for porcine betaneoendorphin/dynorphin precursor. Nature 298:245-249, 1982.

Kangawa, K.; Minamino. N.; Chino, N.; Sakakibara, A.; and Matsuo. H.

The complete amino acid sequence of alpha-neo-endorphin. Biochem

Biophys Res Commun 99:871-878. 1981.

Keller, A., and Margolis, F.L. Isolation and characterization of rat

olfactory marker protein. J Biol Chem 251:6232-6237, 1976.

7
Kilpatrick, D.L.; Howells, R.D.; Noe, M.; Gailey, L.C.; and

Udenfriend. S. Expression of orearoenkeohalin-like mRNA and its

peptide products in mammalian testis and ovary. Proc Natl Acad

Sci USA 82:7467-7469, 1985.

Kilpatrick. D.L.; Wahlstrom. A.: Lahm. H.W.; Blacher. R.: and

Udenfriend, S. Rimorphin a unique, naturally occurring

[Leu]enkephalin-containing peptide found in association with

dynorphin and alpha-neo-endorphin. Proc Natl Acad Sci USA

79:6480-6483, 1982.

Kimura, S.; Lewis, R.V.; Gerber, L.D.; Brink, L.; Rubinstein. M.;

Stein, S.; and Udenfriend, S. Purification to homogeneity of

camel pituitary pro-opiocortin, the common precursor of opioid

peptides and corticotropin. Proc Natl Acad Sci USA 76:1756-1759,

1979.

Lewis, R.V.; Stein, S.; Gerber, L.D. Rubinstein, M.; and Udenfriend,

S. High molecular weight opioid-containing proteins in striatum.

Proc Natl Acad Sci USA 75:4021-4023, 1978.

Lewis, R.V.;Stern, A.S.; Kilpatrick, D.L.; Gerber, L.D.; Rossier,

J.; Stein, S.; and Udenfriend, S. Marked increases in large

enkephalin-containing polypeptides in the rt adrenal gland

following denervation. J Neurosci 1:80-82, 1981.

Lewis, R.V.; Stern, A.S.; Kimura, S.; Rossiet-, J.; Stein, S.; and

Udenfriend. S. An about 50,000-dalton protein in adrenal medulla:

A common precursor of [Met]- and [Leu]enkephalin. Science 208:

1459-1461, 1980.

Mains, R.E.; Eipper, B.A.; and Ling, N. Common precursor to

corticotropins and endorphin. Proc Natl Acad Sci USA

74:3014-3018, 1977.

Margolis, F. Carnosine in the primary olfactory pathway.

Science 184:909-911, 1974.

Mocchetti, I.; Guidotti, A.; Schwartz, J.P.; and Costa, E. Reserpine

changes the dynamic state of enkephalin stores in rat striatum and

adrenal medulla by different mechanisms. J Neurosci 5:3379-3385,

1985.

Morgan, J.I., and Curran, R. The role of ion fluxes in the control

of c-fos expression. Nature 322:552-555, 1986.

Noda, M.; Furutani, Y.; Takahashi, H.; Toyosato, M.; Hirose, T.;

Inayama, S.; Nakanishi, S.; and Numa, S. Cloning and sequence

analysis of cDNA for bovine adrenal preproenkephalin. Nature

(London) 295:202-206, 1982.

Romano, G.; Shivers, B.D.; Harlan, R.E.; Howells, R.D.; and Pfaff,

D.W. Haloperidol increases proenkephalin mRNA levels in the

caudate-putamen of the rat: A quantitative study at the cellular

level using in situ hybridization. Molec Brain Res 2:33-41, 1987.

Richter, D. Biochemistry and biology of vasopressin, oxytocin, and

their corresponding neurophysins. In: Udenfriend, S., and

Meienhofer, J., eds. The Peptides, Vol. 8. New York: Academic

Press, 1987. pp. 41-75.

Rubinstein, M.; Stein, S.; and Udenfriend, S. Characterization of

pro-opiocortin. a precursor to opioid peptides and corticotropin.

Proc Natl Acad Sci USA 75:669-671, 1978a.

8
Rubinstein, M.; Rubinstein, S.; Familletti. P.C.; Gross, M.S.;

Miller, R.S.; Waldman, A.A.,; and Pestka, S. Human leukocyte

interferon purified to homogeneity. Science 202:1289-1290, 1978b.

Sachs, H., and Takabatake, Y. Evidence for a precursor in vasopressin biosynthesis. Endocrinology 75:943-948, 1964.

Schultzberg, M.; Lundber, J.M.; Hokfelt, T.; Terenius, L.; Brandt,

J.; Elde, R.P.; and Goldstein, M. Enkephalin-like

immunoreactivity in gland cells and nerve terminals of the adrenal

medulla. Neuroscience 3:1169-1186, 1978.

Slemmon, J.R.; Blacher, R.; Danho. W.; Hempstead, J.; and Morgan,

J.I. Isolation and sequencing of two novel cerebellum-specific

peptides. Proc Natl Acad Sci USA 81:6866-6870, 1984.

Slemmon, J.R.; Danho,W.; Hempstead, J.; and Morgan, J.I.

Cerebellin, a quantifiable marker for Purkinje cell maturation.

Proc Natl Acad Sci USA 82:7145-7148, 1985.

Tang, R.; Costa,E.; and Schwartz, J.P. Increase in proenkephalin

mRNA and enkephalin content of rat striatum after daily injection

of haloperidol for 2 to 3 weeks. Proc Natl Acad Sci USA

80:3841-3844, 1983.

Yoburn, B.C.; Franklin, S.O.; Calvano, S.E.; and Inturissi, C.E.

Regulation of rat adrenal medullary enkephalins by

glucocorticoids. Life Sci 40:2495-2503, 1987.

AUTHOR

Sidney Udenfriend, Ph.D.

Roche Institute of Molecular Biology

Building 102--340 Kingsland Street

Nutley, New Jersey 07110, U.S.A.

9
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Synthesis and Biological Activity

of Novel Met-enkephalin Analogs

Krishna B. Mathur, Ph.D.; Balaram J. Dhotre, Ph.D.;

Shubh D. Sharma, Ph.D.; Ram Raghubir, Ph.D.;

Gyanendra K. Patnaik, Ph.D.; and Bhola N. Dhawan, M.D.

INTRODUCTION

The discovery of enkephalins (Hughes 1975) ushered in a new era in

which peptides emerged as a novel class of potent analgesics. Soon

after their isolation and characterization was reported by Hughes et

al. (1975), the search for other endogenous peptides possessing opioid

activity was further intensified and several of them could be isolated

from different mammalian tissues and fluids within a short span of

time (Rossier 1982). Almost simultaneously, structure-activity relationship

studies on these pentapeptides were undertaken in a number of laboratories, primarily with the object of getting a synthetic peptide that

could be utilized as a pain reliever in clinical practice (Morley 1980,

1983; Hansen and Morgan 1984). Endeavors of this sort were also started

at this Institute, and the interesting results obtained by us are being

briefly reviewed in this communication.

RATIONALE FOR THE DESIGN OF ANALOGS

Since the analgesia evoked by enkephalins was found to be only weak

and transient after they were injected directly into the cerebral ventricles (Chang et al. 1976; Belluzzi et al. 1976), our emphasis was mainly

on the design and synthesis of such analogs of enkephalins that would

elicit profound and long-lasting analgesia even after their administration

by the systemic routes. For designing new congeners, special care was

also taken while introducing novel structural features in the molecules

so that the resulting peptides could be synthesized rather conveniently

even on a large scale when required. At the very outset, we were struck

by the finding that -endorphin, which is a 31 amino acid peptide having

the Met-enkephalin sequence at its N-terminus (Li and Chung 1976;

Bradbury et al. 1976), produces a powerful and long-lasting analgesia

even after intravenous (i.v.) injection (Tseng et al. 1976). It is nearly

100 times more potent than morphine by intracerebroventricular (i.c.v.)

route and 3 to 4 times as potent as morphine when administered intravenously (Li and Chung 1976; Tseng et al. 1976). Based on a comparison

of the analgesic properties of -endorphin and two synthetic congeners

10
of Met-enkephalin whose N- and C- termini were stabilized against

enzymatic degradation (Pert et al. 1976; Bradbury et al. 1977), Bradbury

et al. (1976) and Cox et al. (1976) suggested that the high order of

activity of -endorphin was not merely due to its being impervious

to brain enzymes. According to them, it could be more of a reflection

of its higher binding affinity with the opioid receptors that might be

arising from the sequence of amino acids that extends from its Nterminal pentapeptide (Bradbury et al. 1977). If that was true, derivatization of the terminal carboxy function of Met-enkephalin to get alkylamides could be expected to lead to derivatives that would not only

be resistant to the attack of carboxypeptidases, but would also possess

higher binding affinity for the opioid receptors due to enhanced hydrophobicity at the C-terminus (Beddell et al. 1977). Moreover, the presence

of alkyl chains in these derivatives was also expected to increase their

overall lipophilic character so that their penetration through the bloodbrain barrier would be possible (Belluzzi et al. 1978). To investigate

this hypothesis, a series of alkylamides of Met-enkephalin were synthesized

and examined for their morphinomimetic activity. As reported earlier,

all the newly synthesized compounds of this series exhibited marked

opioid activity (Patnaik et al. 1982). The most active compounds were,

however, the n-propyl and isopropyl amide derivatives. Encouraged

by the initial results, we launched the synthesis of another series of

alkylamides in which the N-terminus of the pentapeptides was also

protected against the attack of brain enzymes (Hambrook et al. 1976).

In this series, the Gly2 residue of enkephalins was replaced by D-Ala

as Pert et al. (1976) and Beddell et al. (1977) had already demonstrated

that such a substitution not only protected the Tyr 1-Gly2 bond against

cleavage by aminopeptidases but also potentiated the receptor-binding

affinity of the peptides. As expected, all the pentapeptides thus obtained

showed more pronounced and sustained antinociceptive activity following

intracerebral (i.c.) administration (Mathur et al. 1979). The most promising compounds here again were the isopropyl- and n-propyl derivatives.

With a view to further optimize the morphine-like activity of these

pentapeptide derivatives, we proceeded in two ways. On the one hand,

a few substituted hydrazides of (D-Ala2 , Met5

)-enkephalin were synthesized

as we found that the hydrazide derivative (11) was twice as potent

an analgesic as the corresponding amide (compd. 10, table 2). On the

other hand, we decided to substitute Phe4 in peptides 3 and 4 (table 1)

with a MePhe residue so that the Gly2 -Phe4 bond would be stabilized

against the action of enkephalinases (Schwartz et al. 1981; Gorenstein

and Snyder 1980) and the hydrophobic site provided by the phenyl ring

of Phe4 would be retained in the molecules for interaction with the

opiate receptors (Corin et al. 1980).

The incorporation of this modification could be expected to yield highly

potent and systemically active peptides (Römer et al 1977; Römer

and Pless 1979). The sulfoxides of derivatives 5 and 6 were also synthesized (compds. 7 and 8, table 1) for studying their activity profile.

11
TABLE 1

Comparative Morphine-Like Activity of New Enkephalin Analogs

S.No. Compound

Relative

Molar Potency

Analgesia GPI

(i.c.)

1.

2.

3.

4.

5.

6.

7.

8.

9.

Tyr-Cly-Gly-Phe-Met-NH.C3H7(n)

C3H7(i)

D-Ala C3H7

(n)

C3H7(i)

MePhe C3H7

(n)

C3H7(i)

Met(0) C3H7(n)

C3H7(i)

Gly- C3H7(i)

Tyr-Gly-Cly-Phe-Met

0.82 0.52

2.88 0.36

2.53 4.55

8.84 5.60

24.17 30.00

716.08 12.95

20.85 24.78

353.62 19.06

706.09 2.98

0.01 2.00

Morphine hydrochloride

ED50 Morphine analgesia in mice(i.c.): 0.11±0.01 μg;

IC50 (10-8M):5.7±0.07mg/ml

1.00 1.00

TABLE 2

Analgesic Activity of (D-Ala2, Met5)-Enkephalin Amide and Hydrazides

in mice (i.c.)

S.No. Compound Relative

Molar Potency

10. Tyr-D-Ala-Cly-Phe-Met-NH2 0.31

11. NH.NH2 0.56

12. NH.NH.C6H5 21.52

Tyr-Gly-Gly-Phe-Met 0.01

Morphine hydrochloride 1.00

ED50 Morphine analgesia: 0.10 ± 0.01μg

1 2
It is evident from the molar. potencies of peptides 6 and 8 (table 1)

that the oxidation of Met residue to Met(0) leads to a drastic reduction

of the intrinsic activity of the parent peptide (Raghubir et al. 1982).

An attempt was, therefore, made to substitute the Met side-chain

of analog 6 by other stereochemically equivalent side-chains by replacement of the thiomethylene group of Met with an amide residue and

several analogous peptides were obtained (Sharma 1983). In addition,

the side-chain of Met5 was altogether deleted in one of the analogs.

As discussed in the subsequent sections, some highly potent systemically

active enkephalin analogs could thus be obtained. Finally, a few analogs

in which Nval, D-Nval and Gly residues were incorporated as the fifth

amino acid and their carboxy functions were derivatized as isopropylamide

or phenyl hydrazide were also synthesized in an effort to get an analog

which may be more selective for the -subtype of opiate receptors.

The synthetic strategies and biological activities of some of the more

promising analogs of enkephalin obtained under this study are being

discussed in the following sections.

SYNTHESIS OF PEPTIDES

All the enkephalin analogs were synthesized in the solution phase by

well-established procedures of peptide synthesis. In general, coupling

of amino acids and peptides2 was achieved by the DCC/HOBt, mixed

anhydride and 2,4,5-trichlorophenyl ester methods. Except in the case

of Met-enkephalin alkylamides, where the carboxy function of Met

was initially protected by p-nitrobenzyl group, methyl or ethyl esters

were employed for protecting the carboxyl groups. The -NH2 functions

of amino acids and intermediate peptides were protected either with

a carbobenzoxy (Z) or t-butyloxycarbonyl (Boc) group. The cleavage

of Boc group was accomplished by treatment of the protected derivatives

with TFA, HCOOH or HCl/dioxane in presence of ethanedithiol and

anisole. Z-group was removed either by hydrogenolysis over Pd/C or

by catalytic transfer hydrogenation using HCOOH as the hydrogen donor.

The synthetic strategy for getting alkylamides of Met-enkephalin involved

sequential peptidation of Met-ONBzl with appropriate Boc-amino acid2,4,5-trichlorophenylesters to get the protected pentapeptide Boc-TyrGly-Gly-Phe-Met-ONBzl. Treatment of this pentapeptide ester with

the required amines followed by cleavage of Boc group from the resulting

amides gave the desired analogs (Dhotre et al. 1984).

The synthesis of alkylamides of (D-Ala2, Met5)-enkephalin was also

carried out in a stepwise manner starting from Met-ONBzl in the same

way as described for Met-enkephalin alkylamides (Dhotre and Mathur

1984). An alternate strategy involving (3+2) fragment condensation

technique was also adopted for the synthesis of peptides of this series

as well as for the n-and isopropylamides of (D-Ala2

 , MePhe4 , Met5)-

enkephalin (figure 1). In this procedure, the C-terminal dipeptide ester

Boc-Phe-Meta-OMe or Boc-MePhe-Met-OMe was synthesized by either

DCC/HOBt or mixed anhydride method and converted into the required

alkylamides via the free peptide acid. The N-terminal tripeptide fragment

was coupled with the appropriate C-terminal dipeptide alkylamides

via the 2-4-5-trichlorophenyl ester for getting (D-Ala2, Met5)-enkephalin

13
alkylamides and via the mixed anhydride for getting (D-Ala2

, MePhe4

,

Met5)-enkephalin propylamides 5 and 6. Synthesis of Tyr-D-Ala-GlyMePhe-Gly-NH.C3H7

(i) (compd.9) was also achieved in the same fashion

by coupling the N-terminal tripeptide with . MePhe-Gly-NH.C3Hr(i).

The sulfoxides 7 and 8 (table 1) could be obtained by direct oxidation

of compounds 5 and 6 using approximately IN H202

in acetic acid

(Raghubir et al. 1982).

FIGURE 1

Synthetic Strategy For Various Enkephalin Analogs

Aaa:Phe or MePhe; Bbb: Met or Gly; R: n-propyl or isopropyl

Various (D-Ala2, Met5)-enkephalin hydrazides (table 2) were synthesized

by a slightly modified procedure. The common intermediate Boc-TyrD-Ala-Gly-Phe-Met-OMe, was obtained first by coupling the mixed

anhydride of Boc-Tyr-D-Ala-Gly with Phe-Met-OMe. Treatment of

the pentapeptide ester with hydrazine hydrate directly gave the corresponding hydrazide which was deblocked with HCl/dioxane to give the

compound 11. For the synthesis of the phenyl hydrazide 12, the pentapeptide ester, mentioned above, was hydrolyzed and the resulting acid

14
coupled with phenylhydrazine by the mixed anhydride procedure (Sharma

1983). Cleavage of the Boc group by treatment with HCOOH gave

the final product. Homogeneity of all the peptides was checked by

chromatography including HPLC.

BIOLOGICAL ACTIVITY

Morphinomimetic activity of all the newly synthesized analogs of enkephalin was examined both in vitro and in vivo. For in vitro tests, the

electrically stimulated myenteric plexus-longitudinal muscle preparation

of guinea pig ileum (GPI) was prepared from adult guinea pigs of either

sex as described by Kocterlitz and Watt (1968). Graded concentrations

of various compounds were added to the bath and agonist activity calculated from inhibition of the contraction (IC50 values). Morphine hydrochloride and Met-enkephalin were used as standard for comparison.

Naloxone antagonism was used to investigate the specificity of action.

For assaying analgesic activity of peptides, the method of Eddy and

Leimbach (1953) was employed using mice of either sex in groups of

10 for each dose. The compounds were administered in graded doses

intracerebrally (i.c.) and the control group received an equal volume

of normal saline by the same route. Percentage of animals showing

analgesia was determined at each dose level and ED50 calculated according to Finney’s Probit analysis (1952). Morphine hydrochloride and Metenkephalin were used as standard drugs and naloxone was used to antagonize the analgesic effect.

The relative molar potencies of some of the more promising analogs

of enkephalin obtained by us in analgesia and GPI tests are presented

in tables 1 and 2. As mentioned earlier, the most active compounds

among the alkylamides of Met-enkephalin were the n-propyl and isopropylamide derivatives, being 80 and 270 times more potent than the parent

pentapeptide in the analgesia test. Lengthening and shortening of the

alkyl chain was found to have an adverse effect on the antinociceptive

activity. However, the most potent compound of this series in the GPI

test was the n-hexyl amide, its activity being 12 times higher than

the isopropylamide (Patnaik et al. 1982).

In the (D-Ala2,Met5)-enkephalin alkylamide series too, the highest

order of analgesic activity was exhibited by the isopropylamide (compd.4)

followed by the n-propylamide (compd.3). It can be seen that replacement

of Gly2 with D-Ala has led to a threefold increase in the activity of

these peptides (Mathur et al. 1979). Peptides 3 and 4 are approximately

240 and 800 times more potent than Met-enkephalin on a molar basis.

Here again, there is no correlation in the in vivo and in vitro activities

of compounds. The most active derivative of this series in the GPI

test was the ethylamide. The hydrazide of (D-Ala2, Met5)-enkephalin

was found to be twice as potent as the corresponding amide. When

the hydrazide nitrogen was substituted with alkyl groups, no significant

change in the activity was observed. In the case of phenyl hydrazide,

however, a substantial enhancement of analgesic activity was achieved

(table 2). It is nearly 2,000 times more active than Met-enkephalin.

15
When Phe4

 was substituted by a MePhe residue in the propylamides

3 and 4, highly potent analogs could be obtained. The antinociceptive

activity of peptides 5 and 6 is nearly 12 and 80 times higher than that

of compounds 3 and 4 respectively. As compared to Met-enkephalin,

(D-Ala2, MePhe4, Met5)-enkephalin isopropylamide is nearly 70,000

times more potent as an analgesic (Raghubir et al. 1982). The corresponding sulfoxide retains only half the activity of the parent peptide. It

has, however, been found to be active even when administered by

systemic routes. This is in conformity with the results obtained by

Römer et al. (1977) and Römer and Pless (1979).The most significant

finding of this study is that the side chain of Met5 in (D-Ala2, MePhe4,

Met5)-enkephalin isopropylamide does not seem to play any role in

the manifestation of the opioid activity of this analog. As such, the

Met residue can

Ala2, MePhe4, Gly5)-Enkephalin isopropylamide is, in fact, a highly potent

be replaced by Gly without any loss of activity. (Danalog and elicits profound and longer-lasting analgesia even after

systemic administration (Raghubir et al. 1984). Both the systemically

active compounds mentioned above have been taken up for detailed

investigation, and their pharmacological profile is discussed elsewhere

(Raghubir et al. 1988). We have also found that the corresponding phenyl

hydrazide, (D-Ala2, MePhe4, Gly5)-enkephalin phenyl hydrazide, is

comparatively more selective for s-subtype of opiate receptor.

CONCLUDING REMARKS

It is evident from this review that incorporation of such structural

modifications that would inhibit metabolic deactivation of enkephalins

and enhance their receptor-binding affinity leads to peptides that show

a high order of antinociceptive activity after systemic administration.

The aromatic side chain of Phe4 in enkephalins plays an important

role in the manifestation of opioid activity. Stabilization of Gly5-Phe4

peptide bond against the action of enkephalinases, so that the hydrophobic

site for receptor interaction provided by the Phe residue is not lost,

enhances the activity of peptides remarkably. Introduction of hydrophobic

chains at the C-terminus of the pentapeptides certainly has a favorable

effect on their activity, but the presence of such groups that would

lead to an optimum hydrophobicity of the solvent facing part of the

molecule is most desirable. The deletion of Met5 side-chain without

causing any loss in the biological activity of the analog (D-Ala2, MePhe4,

Met5)-enkephalin isopropylamide may be viewed in this context.

It is now well established that enkephalins play several important roles

in the CNS in addition to their role in the process of analgesia. They

are known to be involved in learning and behavior, modulation of other

neuropeptides and putative neurotransmitters, modulation of neuroendocrinal activities and central control of autonomic activities. It

will be useful to study the effect of the modified enkephalins on these

central parameters as well. This may provide useful leads for potential

drugs/tools beyond their present significance in analgesia.

16
FOOTNOTES

1Communication no.4192 from Central Drug Research Institute, Lucknow,

India.

2Abbreviations for amino acid and peptide derivatives are according

to IUPAC-IUB Commission on biochemical nomenclature, Biochemistry

11:1726,1972; other abbreviations are: DCC,NN-dicyclohexylcarbodiimide; HOBt, I-hydroxy-benzotriazole; TFA, trifluoroacetic acid.

REFERENCES

Beddell, C.R.; Clark, R.B.; Hardy, C.W.; Lowe, L.A.; Ubatuba, F.B.;

Vane, J.R.; Wilkinson, S.; Chang, K.J.; Cuatrecasas, P.; and Miller,

R.J. Structural requirements for opioid activity of analogues of the

enkephalins. Proc R Soc Lond B198:249-265, 1977.

Belluzzi, J.D.; Grant, N.; Garsky, V.; Sarantakis, D.; Wise, CD.; and

Stein, L. Analgesia induced in vivo by central administration of enkephalin in rat. Nature 260:625-626, 1976.

Belluzzi, J.D.; Stein, L.; Dvonch, W.; Dheer, S.; Gluckman, M.I.; and

McGregor, W.H. Enhanced analgesic activity of D-Ala2 enkephalinamides

following D-isomer substitutions at position five. Life Sci 23:99-104, 1978.

Bradbury, A.F.; Smyth, D.C.; Snell, C.R.; Birdsall, N.J.M.; and Hulme,

E.C.C-fragment of lipotropin has a high affinity for brain opiate

receptors. Nature 260:793-795, 1976.

Bradbury, A.F.; Smyth, D.G.; Snell, C.R.; Deakin, J.F.W.; and Wendlandt,

S. Comparison of the analgesic properties of lipotropin C-fragment

and stabilized enkephalins in the rat. Biochem Biophys Res Commun

74:748-754, 1977.

Chang, J.-K.; Fong, B.T.W.; Pert, A.; and Pert, C.B. Opiate receptor

affinities and behavioral effects of enkephalin: Structure activity

relationship of ten synthetic peptide analogues. Life Sci 18:1473-

1482, 1976.

Cox, B.M.; Goldstein, A.; and Li, C.H. Opioid activity of a peptide,

-lipotropin-(61-91), derived from -lipotropin. Proc Natl Acad Sci

USA 73:1821-1823, 1976.

Dhotre, B.J., and Mathur, K.B. Design and synthesis of enkephalin

analogues: Part II- Synthesis of (D-Ala2, Met5)-enkephalin alkylamides

having morphinomimetic activity. Indian J Chem 238:1231-1236,

1984.

Dhotre, B.J.; Chaturvedi, S.; and Mathur, K.B. Design and synthesis

of enkephalin analogues: Part I-Synthesis of Met-enkephalin alkylamides

with enhanced analgesic potency. Indian J Chem 238:828-833, 1984.

Eddy, N.B., and Leimbach, D. Synthetic analgesics II. Dithienylbutenyl

and dithienylbutylamines. J Pharmacol Exp Ther 107:385-393, 1953.

Finney, D.J. Probit Analysis. Cambridge: University Press, 1952.

Gorenstein, C., and Snyder, S.H. Enkephalinases. Proc R Soc Lond B210:

123-132, 1980.

17
Gorin, F.A.; Balasubramanian, T.M.; Cicero, T.J.; Schwietzer, J.; and

Marshall, G.R. Novel analogues of enkephalin: identification of functional groups required for biological activity. J Med Chem 23:1113-

1122, 1980.

Hambrook, J.M.; Morgan, B.A.; Rance, M.J.; and Smith, C.F.C. Mode

of deactivation of the enkephalins by rat and human plasma and

rat brain homogenates. Nature 262:782-783, 1976.

Hansen, P.E., and Morgan, B.A. Structure-activity relationships in enkephalin peptides. In: Meienhofer, J., and Udenfriend, S., eds. The

Peptides. Vol. 6. New York: Academic Press, 1984. pp.269-321.

Hughes, J. Isolation of an endogenous compound from the brain with

pharmacological properties similar to morphine. Brain Res 88:295-

308, 1975.

Hughes, J.; Smith, T.W.; Kosterlitz, H.W.; Fothergill, L.A.; Morgan,

B.A.; and Moris, H.R. Identification of two related pentapeptides

from the brain with potent opiate agonist activity. Nature 258:577-

579, 1975.

Kosterlitz, H.W., and Watt, A.J. Kinetic parameters of narcotic agonists

and antagonists with particular reference to N-allylnoroxymorphone

(naloxone). Br J Pharmacol 33:266-276, 1968.

Li, C.H., and Chung, D. Isolation and structure of an untriacontapeptide

with opiate activity from camel pituitary glands. Proc Natl Acad

Sci USA 73:1145-1148, 1976.

Mathur, K.B.; Dhotre, B.J.; Raghubir, R.; Patnaik, G.K.; and Dhawan,

B.N. Morphine like activity of some new Met-enkephalin analogues.

Life Sci 25:2023-2028, 1979.

Morley, J.S. Structure-activity relationships of enkephalin-like peptides.

Annu Rev Pharmacol Toxicol 20:81-110, 1980.

MOrley Chemistry of opioid peptides. Br Med Bull 39:5-10, 1983.

Patnaik, G.K.; Raghubir, R.; Mathur, K.B.; Dhotre, B.J.; and Dhawan,

B.N. Opioid activity of some new Met-enkephalin analogues. Indian

J Pharmacol 14:15, 1982.

Pert, C.B.; Pert, A.; Chang:, J.K.; and Fong, B.T.W. (D-Ala2)-Met-enkephalinamide: A potent long-lasting synthetic pentapeptide analgesic.

Science 194:330-332, 1976.

Raghubir R.; Patnaik, G.K.; Sharma, S.D.; Mathur, K.B.; and Dhawan,

B.N. Antinociceptive activity of new Met-enkephalin analoguesImportance of the side chain of fifth amino acid. Indian J Pharmacol

16(1):35, 1984.

Raghubir, R.; Sharma, S.D.; Mathur, K.B.; Patnaik, G.K.; Srimal, R.C;

and Dhawan, B.N. Pharmacological profile of some (D-Ala2, MePhe4,

Met5)-enkephalin-alkylamides, new potent analogues of Met-enkephalin.

In: Dhawan, B.N., ed. Advances in the Biosciences. Vol. 38, Current

Status of Centrally Acting Peptides. Oxford: Pergamon Press, 1982.

pp.61-68.

Raghubir, R.; Patnaik, G.K.; Sharma, S.D.; Mathur, K.B.; and Dhawan,

B.N. Pharmacological profile of two new analogues of Met-enkephalin

In: Dhawan, B.N., and Rapaka, R.S., eds. Recent Progress in Chemistry

and Biology of Centrally Acting Peptides, Central Drug Research

Institute, Lucknow, 1988. pp.167-174.

Römer, D.; Buscher, H.H.; Hill, R.C.; Pless, J.; Bauer, W.; Cardinaux,

F.; Closse, A.; Hauser, D.; and Huguenin, R. A synthetic enkephalin

268:547-549, 1997.

analogue with prolonged parenteral and oral analgesic activity. Nature

1 8
Römer, D.; and Pless, J. Structure activity relationship of orally active

enkephalin analogues as analgesics. Life Sci 24:621-624, 1979.

Rossier, J. Opioid peptides have found their roots. Nature 298:221-222,

1982.

Schwartz, J.C.; Malfroy, B.; and Baume, S.D.L. Biological inactivation

of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase

(enkephalinase) as neuropeptidase. Life Sci 29: 1715-1740, 1981.

Sharma, S.D. Studies in the synthesis of peptides with possible biological

activities. Ph.D dissertation: 196-218, 1983.

Tseng, L.F.; Loh, H.H.; and Li, C.H. -Endorphin as a potent analgesic

by intravenous injection. Nature 263:239-240, 1976.

AUTHORS

Krishna B. Mathur, Ph.D.

Balaram J. Dhotre, Ph.D.

Shubh D. Sharma, Ph.D.

Ram Raghubir, Ph.D.

Gyanendra K. Patnaik, Ph.D.

Bhola N. Dhawan, M.D.

Central Drug Research Institute

Lucknow 226001, India

19
Opioid Peptides: An Update: NIDA A Research Monograph

Rao S.Rapaka, Ph.D., and Bhola M. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Approaches to Studying StructureActivity Relationships in Peptide

Hormones Through the Expression

of Synthetic Genes

John W. Taylor, Ph.D.

INTRODUCTION

The primary structures of a very large number of peptide hormones having diverse

pharmacological properties have now been elucidated. In order to understand the functions of these peptides and to exploit their potential as pharmaceutical agents, it is

essential to study structure-activity relationships in each case. To this end, the techniques of peptide synthesis have commonly been applied, allowing the direct preparation

of deletion analogues and analogues incorporating natural and nonnatural amino acid substitutions. However, many peptide hormones are too large to consider extensive investigations of the role of individual amino-acid residues, as the preparation of multiple analogues in this way would be too time-consuming and expensive, and even the synthesis of

the natural structure alone may represent a significant synthetic achievement or produce

material that is too poorly characterized to be useful (Clark-Lewis et al. 1986). In such

cases, the alternative approach of employing synthetic genes and recombinant DNA

technology is becoming increasingly more attractive as new methods in this area are

developed. The latter approach consists of three main stages: (a) the assembly from

synthetic oligonucleotides of a gene coding for the peptide hormone being studied; (b) the

preparation of mutant genes coding for the desired hormone analogues; (c) the efficient

expression of the original and mutant genes in overproducing cells from which the hormone analogues are recovered and purified. Compared to peptide synthesis, this

approach has the major advantage that all possible deletion analogues or single residue

substitution analogues may be prepared simply and rapidly, at little extra cost, by mutation of the original gene once it has been assembled. Furthermore, the purity of the final

peptide products prepared in this way, and the likelihood that they will be contaminated

with pharmacologically active impurities, is not a function of the size of the hormone or

its amino acid composition, as it is when direct chemical synthesis is employed. The

purpose of this article is to review the methods that are currently available for preparing

peptide hormones by gene expression in Escherichia coli and to discuss how they might be

employed to perform structure-activity analyses.

OLIGONUCLEOTIDE SYNTHESIS AND GENE CONSTRUCTION

Oligomeric DNA must be synthesized in order to construct a gene that codes for the

desired peptide hormone and can be incorporated into a suitable plasmid vector for

expression. In addition, synthetic oligonucleotides are employed in the subsequent sitedirected mutagenesis of that gene and the sequence characterization of all of the gene

constructs produced, as described below. There are two synthetic strategies in common

use, each involving the stepwise addition of protected nucleotides to the 5’ end of the

20
FIGURE 1

Oligonucleotide synthesis by the phosphite method (A) and the phosphate method (B)

Oligonucleotides are built up on a solid phase support through the repetitive application of

these reactions for each base addition. DMT, dimethoxytrityl; MSNT, 1-(mesitylene-2-

sulfonyl)-3-nitro 1,2,4-triazole.

growing oligonucleotide chain which is anchored to a solid support at its 3’ end (reviewed

by ltakura et al. 1984 and Sonveaux 1986). The two methods are distinguished by the

use of either a phosphite analogue of the nucleotide (a phosphoramidite) in the addition

step which is subsequently oxidized to the phosphate form, or the introduction of the

protected nucleotide in the phosphate form directly (figure 1). After completion of the

final synthetic cycle, the base- and phosphate backbone-protecting groups are removed,

and the linkage to the solid support is cleaved to yield the crude oligonucleotide. If an

automated synthesizer is employed under optimal conditions, the synthesis of oligonu21
cleotides as long as 100 bases may be achieved. However, manual syntheses that

employ apparatus as simple as a small syringe are quite adequate for the preparation of

oligonucleotides up to 20 bases long.

Purification of the correct oligonucleotides from crude synthetic mixtures may be

achieved by HPLC on ion-exchange or reversed-phase columns, or by electrophoresis

through polyacrylamide gels under denaturing conditions (Sonveaux 1986). The HPLC

methods are suitable for oligonucleotides shorter than about 20 bases long and can be

used for larger quantities, but often require the assumption that the major product from

the synthesis is the correct one. In contrast, polyacrylamide gel electrophoresis can

readily provide adequate purification of oligonucleotides 10-100 bases long in sufficient

quantities for the uses described here, and can be performed on multiple samples simultaneously in the presence of reliable standards to identify the desired products.

The solid supports that are most commonly used are derivatized silica, which is useful

for preparing relatively large quantities of shorter oligonucleotides, and derivatized controlled-pore glass, which gives higher yields per synthetic cycle and is more useful for the

present purposes (Sonveaux 1986). In addition, however, Helmut Blocker’s group has

demonstrated that paper disks may also be used as a solid support for oligonucleotide

synthesis (Frank et al. 1983). If these disks are used in conjunction with a synthetic

apparatus that incorporates one reaction vessel dedicated to the addition of each of the

four base types, then the simultaneous synthesis of multiple oligonucleotides (as many as

40 is reasonable) having unique base sequences can be achieved by numbering the paper

disks and resorting them between the four reaction vessels after each successive base

addition. Both the phosphite and the phosphate chemistries have been applied to this

process, which can result in considerable savings in time and expensive synthetic

reagents (Frank et al. 1983; Matthes et al. 1984; Ott and Eckstein 1984). In our laboratory, for example, we have successfully employed the phosphoramidite chemistry to the

simultaneous manual synthesis on paper disks of 12 unique oligonucleotides that ranged

from 13 to 19 bases in length. These were then purified in a few hours from the crude

synthetic products by simultaneous electrophoresis on a single polyacrylamide gel in

sufficient quantities and purities for use in oligonucleotide-directed mutagenesis or DNA

sequencing experiments. An analysis of the crude and purified products of these syntheses by gel electrophoresis and autoradiography, after 5'-end labelling with ATP-y-32P

and polynucleotide kinase, is presented in figure 2. Since the preparation of each mutant

gene coding for a new peptide hormone analogue requires a unique oligonucleotide of this

size range, the application of Blocker’s approach should be particularly useful in this

regard.

Several semisynthetic approaches to the assembly of the double-stranded DNA comprising the synthetic gene and its incorporation into a double-stranded plasmid or phage

DNA vector may be considered. Most commonly, both strands of the gene are synthesized in their entirety as oligonucleotide fragments that have overlapping base complementarities. These oligonucleotides are enzymatically phosphorylated at their 5’ ends,

and then annealed together and ligated using T4 DNA ligase and ATP. The resulting

segment of double-stranded DNA is designed to have unpaired “sticky ends” that allow

another enzymatic ligation into vector DNA linearized with a restriction enzyme that

produces complementary “sticky ends.” This approach, illustrated in figure 3, can be

refined by (a) the use of two restriction enzymes producing different “sticky ends” so

that the orientation of the inserted DNA is unambiguous, and (b) using the synthetic DNA

in excess over the vector (about five- to tenfold has given us good results) and leaving

the oligonucleotides that provide the 5’ ends of the synthetic gene unphosphorylated so

that ligation of either the insert or the vector to itself is minimized. Using this approach,

synthetic genes can be efficiently assembled into vectors in segments of 100-300 base

pairs, punctuated by unique restriction enzyme sites (for examples, see Ferretti et al.

22
FIGURE 2

Analysis of the products from a manual synthesis of twelve unique oligonucleotides. The

oligonucleotides were synthesized simultaneously on individual paper disks using the phosphite method. They were then desalted on Sep-Pak C18 cartridges (Waters Co. Ltd.),

and then purified by electrophoresis on a 20% polyacrylamide gel and desalted again, as

before. The crude (left pane/) and gel-purified (right pane/) synthetic products were

analyzed by autoradiography, after 5'end labeling with 32P and electrophoresis on a

15% gel. The oligomers ranged in length from thirteen to nineteen base residues, as

follows (left to right): 18mer; 14mer; 18mer; 17mer; 18mer; 18mer; 19mer; 18mer; 16mer;

19mer; 18mer; 13mer.

23
FIGURE 3

Assembly of a synthetic gene and its incorporation into a vector. For best results, the

oligonucleotides at the 5’-end of each strand (in this case, I and V) are left unphosphorylated, and the “sticky ends” of the synthetic gene are different, so that the vector must

be cut with two restriction enzymes that produce complementary “sticky ends.”

1986 and von Bodman et al. 1986). Thus, a synthetic gene coding for a given peptide

hormone can usually be assembled in a single step. Oligonucleotides about 50 bases long

and overlaps of about 8-12 bases appear to provide the most reliable combination for

obtaining the correct annealing and ligation and the fewest sequence errors after cell

transformation and characterization of the amplified products. We have also found that

it is quite reliable to perform the two ligation steps simultaneously, by adding both the

vector and ligase directly to the annealed oligonucleotides. The use of longer oligonucleotides can result in more sequence errors, since there will inevitably be more impurities

present from their synthesis even after purification, and the use of shorter oligonucleotides and/or overlaps makes the design of unique overlapping segments difficult and

increases the difficulties of obtaining correct annealing. Nevertheless, synthetic genes

have been successfully assembled from shorter oligonucleotides prepared by the paper

disk method of Blocker described above (Grundstrom et al. 1985; Brodin et al. 1986).

Two other notable strategies have been employed. First, the approach of ligating the

synthetic oligonucleotides one strand at a time was successful in a synthesis where the

presence of a repeated codon made direct assembly of the duplex difficult (Smith et al.

1984). In this case, the oligonucleotides comprising one strand only were phosphorylated

with kinase. These were then ligated together after annealing to the unphosphorylated

overlaping oligonucleotides comprising the opposite strand. After denaturing this mixture, the ligated strand was then purified by polyacrylamide gel electrophoresis. This

process was performed simultaneously for the second strand, and the two purified

strands were then annealed and ligated into the vector in a separate step. The second

alternative strategy is one in which oligonucleotide synthesis is minimized by limiting the

synthesis to longer overlapping single-stranded segments of the gene, so that the gaps

between the overlaps can be filled enzymatically using a suitable polymerase and the

24
deoxynucleotide triphosphates (dNTPs), before the segments are ligated. This ap roach

has also been quite successfully employed (see, for example, Bergmann et al. 1986 but is

generally found to be less reliable due to the difficulties of obtaining correct annealing and

strand synthesis.

MUTAGENESIS

The preparation of peptide hormone analogues for structure-function analysis requires

that the synthetic gene coding for the natural structure be mutagenized. This may be

achieved in a random or partially random fashion by a number of chemical or physical

methods, including treatments with sodium bisulfite, nitrous acid, hydrazine or UV light,

that involve damaging the DNA so that its sequence is incorrectly repaired or replicated

upon cell transformation giving rise to mutant progeny (Myers et al. 1985). Alternatively,

restriction enzyme recognition sites purposefully introduced into the synthetic gene at

strategically useful points may be exploited to introduce more specifically chosen mutations. In this case, either the DNA is cleaved in one strand only by restriction enzyme

digestion under suboptimal conditions and then “misrepaired” enzymatically (Shortle et al.

1982), or else two such restriction sites are employed in order to cut out a “cassette” of

double-stranded DNA that can then be replaced by any new “cassette” consisting of

synthetic oligonucleotides (Lo et al. 1984). The most universally applicable methods,

however, involve oligonucleotidedirected mutagenesis (Zoller and Smith 1983). In these

methods, a synthetic oligonucleotide carrying a mismatch near the center of its sequence

to direct the desired mutation is annealed to its partially complementary sequence on a

single-stranded vector carrying the gene or gene fragment to be mutagenized. This

oligonucleotide is then used to prime the synthesis of the entire complementary strand in

vitro, using a DNA polymerase and each of the deoxynucleotide triphosphates (dNTPs).

Competent cells are transformed with the resultant heteroduplex DNA and spread out on

agar plates, giving rise to both forms of the DNA, from which the desired mutant form

must be selected (figure 4a). In principle, all types of mutations of the target DNA

sequence are achievable in this way, including deletions, additions, and substitutions of

one or more bases, and there are no limitations on the sites that are accessible to

change. It is necessary only that the vector carrying the gene can be prepared in the

single-stranded form, and that a suitable mismatched primer oligonucleotide can be

designed and synthesized so that it will anneal specifically to the correct site on the gene.

Since most expression vectors are double-stranded plasmids, the requirement for the

single-stranded form usually necessitates a subcloning step, where the gene or gene

fragment to be mutagenized is cut out by restriction enzyme digestion and transferred to

a single-stranded bacteriophage such as one of the convenient M13 vectors adapted by

Messing and coworkers to contain multiple cloning sites (Messing 1983). Once the mutant

has been prepared, the gene can then be transferred back to the original plasmid for the

purposes of expression. In some cases, however, this process has been simplified either

through the use of phage vectors for expression directly (Wilkinson et al. 1983), or

through the use of plasmid expression vectors of the pEMBL type, which are normally

double-stranded but contain the origin of replication of M13 phage DNA and can readily be

prepared in the single-stranded form upon infection of the plasmid-carrying cells with

phage (Dente et al. 1983). The requirement for specific annealing of the mismatched

oligonucleotide to the desired site usually presents no problems in cases involving the

addition, deletion, or substitution of a single codon. It is usually sufficient to place the

mismatched bases at least eight bases from either end of the oligonucleotide, and the

precise arrangement can be further optimized for specific annealing to the correct site

through the use of commercially available computer programs designed for this purpose.

The major difficulty in oligonucleotide-directed mutagenesis is often the identification,

after ce l transformation with the heteroduplex DNA, of the plaques of phage-infected l

cells that contain the mutant phage. E. coli cells have a DNA mismatch repair system

25
FIGURE 4

Oligonucleotide-directed mutagenesis using M13 vectors. (A) The unmodified procedure

of Zoller and Smith (1983): (1) Mismatched primer extension with the Klenow fragment

of DNA polymerase I and the dNTPs; ligation with T4 DNA ligase and ATP. (2) Transformation of competent cells and colony growth on agar plates. (B) The phosphorothioate method (Sayers and Eckstein 1987): (1) Mismatched primer extension with

Klenow, dATP, dGTP, dTTP and dCTP- -S; ligation with T4 ligase and ATP. (2)

Nicking with Nci I; gapping with exonudease III in the 3’ to 5’ direction, or with A exonuclease or the T7 gene 6 exonuclease in the 5’ to 3’ direction, as appropriate. (3) Repair

with DNA polymerase I, the dNTPs, T4 ligase and ATP. (4) Transformation and

colony growth.

that depends upon the action of the dam methylase, which labels DNA in vivo by methylation of GATC sequences, in order to recognize the original (methylated) viral (+)-

strand and correct mismatches that were introduced into the (-)-strand during synthesis

in vitro (Kramer et al. 1984a). The efficiency of this system is dependent on the type of

mismatch introduced, but it typically reduces the frequency of occurrence of the mutant

phage to about 10% of the plaques, and much lower frequencies are quite common

(Taylor et al. 1985a). Furthermore, most of the plaques containing mutant phage that

were obtained by plating out transformed cells directly are contaminated by the presence

of the nonmutant form and must be replated at least once to ensure that the pure

mutant form is obtained. Since the mutagenesis of a hormone gene in a phage vector

26
carried by E. coli cells will not normally result in a recognizable change in plaque phenotype, the mutant phage obtained by this method must be identified by screening a large

number of plaques with a hybridization assay using the 32P-labelled mismatched oligonucleotide (which is perfectly complementary to the mutant page DNA). This assay

involves transferring the plaques obtained in a mutagenesis experiment onto nitrocellulose

filter paper, denaturing the phage and then hybridizing the radiolabelled oligonucleotide to

the exposed immobilized phage DNA. Mutant and nonmutant phage are then distinguished

by autoradiography after washing the nitrocellulose paper in a high salt buffer at

successively higher temperatures. The radiolabel is washed off the samples of nonmutant phage at lower temperatures (Zoller and Smith 1983).

In order to avoid the time-consuming hybridization screening step described above and

eliminate the use of high specific activity 32P radiation, a number of modified mutagenesis procedures have been developed that increase the frequency of occurrence of the

mutants to a point where they may be conveniently identified and characterized directly

by DNA sequencing. For this purpose, it is necessary to increase the efficiency of the

mutagenesis to a point where the random selection for sequencing of only two or three

plaques from each experiment will result in a high probability of identifying the desired

mutant in a pure form. The relationship between the mutagenesis efficiency and the

number of plaques that must be screened to achieve a satisfactory 90% probability of

success is graphically illustrated in figure 5 (Kramer et al. 1982). Clearly, any method

that will be useful in this regard needs to achieve reliable efficiencies of mutagenesis that

are higher than about 50%. Only a few methods are adequate in this regard. These

methods depend upon either selecting against the original DNA sequence in the (+)-strand

of the heteroduplex in vivo, after transformation, or eliminating the original DNA

sequence in that strand in vitro, before transformation.

Selection against the (+)-strand in vivo has been successfully achieved in different ways.

Kunkel (1985) has shown that single-stranded Ml 3 vectors carrying cloned genes may be

prepared in host cells deficient in dUTPase (dut - ) and uracil glycosylase (ung - ) to

contain several deoxyuridine residues per molecule in place of the normal thymidine

residues. When this DNA is used as the template to produce heteroduplex DNA in the

normal way, transformation into ung + cells results in glycosylation and excision of the

uracil moieties, creating multiple abasic sites and a consequent strong selection against

the original (+)-strand molecules during the subsequent replication. The result is that

more than 50% of the plaques obtained from these transformed cells contain progeny

phage that are the desired mutants derived from the (-)-strand sequence. In a similar

manner, the amber nonsense mutation or the Eco K restriction site have been exploited

as genetic markers that may be incorporated into the (+)-strand of M13 DNA when it is

prepared in a suitable suppressor-carrying or Eco K

-

host, respectively (Carter et al.

1985). In these approaches, selection against the (+)-strand of the heteroduplex DNA

that is subsequently prepared in vitro requires an additional mismatch in the (-)-strand

opposite the marker, so that transformation into a suppressor-deficient or Eco Krestricting host, as appropriate, will select against progeny derived from the (+)-strand

only. The simplest way to achieve this double mismatch is to use two mismatched

oligonucleotides simultaneously to prime the (-)-strand synthesis in vitro. In this case, the

oligonucleotide annealed to the site of the genetic marker would be identical for all the

mutations directed by the second mismatched primer in the normal way, so that little

extra synthetic effort is required. Other, more difficult approaches involving the preparation of heteroduplex, gapped DNA have also been applied to these selection methods

(Kramer et al. 1984b). Again, the typical mutation efficiencies appear to be about 50%,

although higher efficiencies have been reported, so that all of the preceding methods for in

vivo selection against the nonmutant strand appear to allow the use of DNA sequencing

methods to identify and characterize the mutants generated directly.

27
FIGURE 5

Relationship between the number of phage plaques that must be screened to have a 90%

probability of identifying a mutant and the efficiency of the mutagenesis method used.

(Adapted from Kramer et al. 1982. Copyright 1982, IRL Press, Ltd.)

Most recently, my coworkers and I in Fritz Eckstein’s laboratory have developed a very

efficient method for oligonucleotide-directed mutagenesis that takes the alternative

approach of eliminating the original (+)-strand sequence opposite the mismatched primer

in the heteroduplex DNA, before it is used to transform competent cells (Taylor et al.

1985a; Nakamaye and Eckstein 1986; Sayers and Eckstein 1987). In this approach, the

mismatched oligonucleotide primer is annealed to the (+)-strand of the M13 vector DNA

with its cloned insert in the normal wa y, and then the in vitro synthesis of the complementary (-)-strand is performed with the dCTP in the reaction mixture entirely substituted by its -thiophosphate analogue, dCTP- -S. The a-thiophosphate analogues of

the natural dNTPs are diastereomeric and all of the DNA polymerases that have been

investigated to date readily accept the Sp diastereomer of each dNTP- -S as a substrate, incorporating it into DNA with inversion of configuration about the phosphorus

atoms almost as efficiently and accurately as for the unmodified dNTPs (Burgers and

Eckstein 1979). The product of this in vitro synthesis is, therefore, heteroduplex covalently closed circular DNA that contains in its (-)-strand the desired mutant sequence, as

well as hosphorothioate internucleotidic linkages of the Rp configuration on the 5’ side of

each dCMP base residue (figure 4b). Phosphorothioate-substituted Ml 3 DNA has similar

physicochemical characteristics to the natural DNA and retains its infectivity upon

transformation of competent host cells, but also has the property of being resistant to

linearization by restriction enzymes, particularly those which cleave their recognition

sequences on the 5’ side of the base type that was substituted by its a-thiophosphate

analogue in the synthesis. Indeed, for about one-third of the restriction enzymes that we

have tested, linearization that requires cleavage of a phosphorothioate linkage appears

to be completely blocked (Taylor et al. 1985b). However, these enzymes are still able to

hydrolyse the unsubstituted (+)-strand of the phosphorothioate DNA quite efficiently in

the normal way, to produce double-stranded DNA with a single “nick” placed specifically

in the (+)-strand at the restriction enzyme cleavage site. We have exploited this property of the Nci I restriction enzyme in order to specifically hydrolyse the (+)-strand of the

heteroduplex DNA containing dCMPS-substitutions in the (-)-strand at recognition sites

of the type shown below.

28
(+)-strand: 5’-CC/GGG-3’

(-)-strand: 3’-GGC/CC-5’

Nci I normally cleaves the internucleotidic linkage on the 5’side of the central base residue

in both strands of such sequences, as indicated, but in this case cleavage of the (-)-

strand is blocked. The nicks that are generated in the (+)-strand can then be used as

starting points for its digestion in the 3’ to 5’ direction, using the enzyme exonuclease Ill.

This digestion occurs at a reproducible rate of about 100 bases per minute in all of the

DNA sample simultaneously, provided that the correct buffer conditions and excess of

the exonuclease are used. When digestion has proceeded to a point safely beyond the

desired point of mutagenesis (opposite the mismatched primer oligonucleotide), the gapped

DNA can be repaired by polymerization with dNTPs, using the mutated sequence of the

(-)-strand as the template to produce phosphorothioate DNA having the desired mutant

sequence in both strands (figure 4b). In a number of trial base-substitution mutagenesis

experiments, where mutant a 13 DNA prepared in this way was used to transform competent cells, 80% - 90% of the plaques obtained were consistently found to contain the

desired mutant phage uncontaminated by the unmutated form (Nakamaye and Eckstein

1986).

Based on the above results, the phosphorothioate DNA approach promises to be the

most efficient of the enhanced oligonucleotide-directed mutagenesis methods, and it

requires only a few hours of additional manipulations over the basic protocol and has no

special requirements for unusual host cells or vectors. The dNTP- -S analogues are

commercially available and give satisfactory results when used as the racemic mixtures

directly. Furthermore, all of the potential limitations of this approach appear to present

no additional difficulties and it should be applicable to every mutagenesis problem. For

example, other restriction enzymes besides Nci I may be employed for the nicking reaction (in conjunction with the appropriate thiophosphate nucleotide analogue); the exonuclease Ill digestion and subsequent repair reactions are efficient over a range of several

thousand bases; and gapped DNA may also be prepared by digestion from a nick in the

opposite, 5’ to 3’ direction, using either exonuclease or the T7 gene 6 exonuclease

(Sayers and Eckstein 1987). Thus, one or more appropriate restriction enzyme recognition site present at any position in the vector or cloned gene is probably a sufficient

prerequisite for the method and one that is easily satisfied. In the case of Nci I, the

recognition sequence described above is present in all of the M13 vectors and the

alternative Nci I site, where the (+)-strand sequence is 5-CCCGG-3’, does not seem to

interfere when it is also present, because its cleavage in the (-)-strand is also blocked by

the dCMPS substitution (Nakamaye and Eckstein 1986). Initial experiments (Sayers and

Eckstein 1987) indicate that deletion mutagenesis is also just as efficient using these

methods, despite the expected difficulty of digesting the loop of unpaired bases in the (+)-

strand opposite the mismatched primer during the gapping step. (The third alternative of

insertion mutagenesis, in which the heteroduplex phosphorothioate DNA will have an

unpaired loop in its (-)-strand, is expected to be straightforward.) Finally, we have failed

to detect any unexpected mutations in addition to those that were directed by the mismatched oligonucleotides during our extensive characterization by sequence analysis of

the mutants generated by these procedures. This indicates that the phosphorothioate

DNA in the (-)-strand is a suitable template for accurate DNA synthesis by DNA polymerase I during repair of the gapped DNA in the final step of the in vitro manipulations.

The high efficiency of the phosphorothioate DNA method for mutagenesis suggests that

new approaches to the rapid preparation of large numbers of mutants of a given peptide

or protein might be possible. Nearly all of the DNA that is prepared in vitro contains the

mutant sequence that is determined by the mismatched primer oligonucleotide. Therefore, the use of oligonucleotides that contain degenerate positions would allow the effi29
cient single-step preparation of, for example, mutants of an enzyme in which an active

site residue is replaced by all other possible naturally occurring amino-acid residues. The

subsequent identification and characterization of these mutants would be very rapid,

because it could be performed directly by DNA sequencing. Alternatively, multiple specific mutations at many different sites could be generated very rapidly by combining this

mutagenesis method and direct sequence analysis with the paper disk approach of

Helmut Blocker to synthesize the large number of mismatched oligonucleotides that would

be required. This should be extremely useful in the characterization of structure-activity

relationships in the larger peptide hormones that we are considering here, particularly

when methods are available for the efficient expression and purification of the mutants

generated.

EXPRESSION AND PURIFICATION

The most important aspects of the expression of a synthetic gene in E. coli cells and the

purification of the protein product are the requirements for efficient transcription of the

gene and subsequent translation of the mRNA; the location, structure, and stability of

the peptide product; and the possible methods by which it, and any mutants of interest,

might be separated from the other protein constituents of the host cells. These subjects

have been reviewed extensively elsewhere (Gold et al. 1981; Wetzel and Goeddel 1983;

Marston 1986) and the present discussion will focus on the particular problems that are

associated with the preparation of peptide hormones and their analogues, and the

approaches to solving those problems that have been adopted to date.

Various factors are known to affect the efficiency of transcription and translation,

sometimes dramatically. These include the strength of the promoter, the structure of the

ribosome-binding site and its position relative to the translation start codon (usually

AUG) on the mRNA, the stability of the mRNA, and the presence of transcription attenuators near the end of the open reading frame of the message. There are also strong

correlations between codon usage in E. coli genes and the levels to which the corresponding proteins are expressed, which suggest that expression levels are determined by the

codon-anticodon interaction energies and by tRNA abundancies (Grosjean and Fiers

1982). Unfortunately, there appear to be no absolute rules governing the precise choices

that must be made in order to optimize these many factors for efficient expression. The

approach that most researchers have adopted has been to employ the best natural

systems, either directly or through the design of consensus structures. Thus, trp, lac,

Ipp, PL and pho A promoters and ribosome-binding sites have all been employed

directly, or in synthetic combinations such as the tac hybrid derived from trp and lac (De

Boer et al. 1982) or the rac hybrid derived from rrnB and lac (Boros et al. 1986) which

appear to be stronger than their parent structures. The design of hybrid or consensus

promoters and ribosome-binding sites does not, however, always lead to better expression systems (see, for example, Deuschle et al. 1986). Most of the promoters that are

commonly employed also offer the advantage of being inducible, for example by

temperature shift ( PL) or addition of a metabolite (systems based on trp and lac).

This allows gene expression to be switched on at the most advantageous time, which is

particularly important in cases where the protein produced is detrimental to the cell.

The codons chosen to design a synthetic gene are usually taken from tabulations of the

most abundant tRNAs (Ikemura 1981) or the most abundant codons in highly expressed

genes (Yarus and Folley 1985) although exceptions are often made so that convenient

restriction enzyme recognition sites can be built in. The importance of this consideration

was highlighted by the use of a synthetic gene that had been optimized in this manner to

produce bovine growth hormone (Seeburg et al. 1983). The synthetic gene was expressed at much higher efficiency than the natural mammalian gene had been. Additional

empirical rules governing codon choice have also been suggested (Yarus and Folley 1985).

30
Unfortunately, it is possible that the choices made at this stage of the design of an

expression system may result in quite unpredictable problems that are difficult to identify

or solve: if certain DNA sequences cause the RNA polymerase to slow or stop, this may

result in premature dissociation of the transcription complex and truncation of the message; or, if protein folding occurs concomitant with translation, other sequences may

disrupt this process through their effects on the rate of translation of the mRNA on the

ribosome, and incorrectly folded products may result. Even less information is available

regarding mRNA stability and how it can be enhanced. Although stability might be

related to secondary structure formation throughout the message, as well as, possibly,

its overall length, experiments suggest that the structure of the 5’ end is critical. Again,

the approach to this problem has been to employ natural sequences in the 5’ untranslated

region that are taken from stable systems. In the case of the T4 gene 32 message, for

example, the 5’ sequence has been shown to confer stability on unstable sequences that

are fused downstream to it. This property has been exploited in the design of a plasmid

vector employing the promoter region and start codon of that gene and requiring phage

T4 infection for expression (Duvoisin et al. 1986).

In the expression of foreign proteins in E. coli cells, the stabilit y of the product within the

cells is an especially important consideration. Such expression has been shown to turn on

the production of the Ion protease and other gene products regulated by the htp R gene

product associated with the heat shock response, leading to much higher rates of proteolytic degradation than normal (Goff and Goldberg 1985), and the use of Ion- and htp R

mutant host strains is certainly helpful in this regard (Buell et al. 1985). Whether these

responses are triggered by particular “foreign” amino acid sequences, or by incorrectly

folded structures, or some other mechanism is responsible is not presently known. In

addition, the half-lives of proteins that are endogenous to the cells vary themselves over

a wide range, indicating that there are mechanisms for targetting proteins for degradation that are normally in effect and might act on an overexpressed foreign protein also.

E. coli has a large number of proteolytic enzymes that might participate in these processes, and are vital to the normal functioning of the cell (Swamy and Goldberg 1981).

Empirical correlations suggest a strong relationship between the half-life of a protein in E.

coli and its N-terminal amino acid sequence. In particular, the “PEST” hypothesis suggests that sequences rich in Pro, Glu, Ser, and Thr will be most highly susceptible to

degradation (Rogers et al. 1986). Alternatively, procaryotic cells may have a mechanism for protein turnover that is similar to the ubiquitin-associated mechanism that

appears to operate in eucaryots, in which case an important determinant of the half-life

will be the N-terminal residue itself, with Arg, Lys, Asp, Leu, and Phe directing the most

rapid degradation, and Met, Ser, Ala, Thr, Val, and Gly dictating the greatest resistance to degradation (Bachmair et al. 1986).

Proteolytic degradation of the desired product is, perhaps, the most difficult problem

attendant to the production of smaller peptides such as the peptide hormones. Their

direct expression is usually followed by their rapid degradation, so that large quantities

can never accumulate. This is almost certainly because these peptides are too small to

fold into a stable globular structure, and their extended flexible conformations can readily

fit into the typical active-site groove of a broad-specificity protease. The first successful solution to this problem, and the approach that is still most commonly applied, was to

express the peptides as part of a much larger fusion protein by splicing the corresponding

synthetic gene in frame to the end of the gene coding for a highly expressed protein that

could act as an intracellular carrier. In this way, somatostatin, -endorphin) and both the

A and B chains of insulin were separately produced, each fused to a point near the C

terminus of -galactosidase (Itakura et al. 1977; Shine et al. 1980; Goeddel et al. 1979).

Expression was directed under the control of the strong inducible lac promoter and the

peptides were recovered from the purified fusion protein by proteolytic ( -endorphin) or

31
chemical (somatostatin and the insulin chains) cleavage at specific amino-acid residues

engineered into the structure for this purpose.

The fusion protein approach solves a number of the problems discussed above simultaneously, provided that the carrier protein is normally expressed at a high level in E. coli and

it is being used as the N-terminal portion of the hybrid. At the RNA level, the structure

around the ribosome-binding site should already be optimized for transcription, translation,

and mRNA stability, and codon usage for most of the rest of the message should also be

correct. After translation, the N-terminal amino acids should be consistent with the

requirements in that region for a stable protein in vivo. There are additional advantages

in relation to the purification procedures, First, the production of a larger protein aids in

its direct identification by polyacrylamide gel electrophoresis, since the identification and

visualization of small peptides by these techniques is difficult. This usually eliminates the

need for antibodies directed against the desired peptide in order to follow its expression

and purification. If enzymatic carrier proteins such as -galactosidase or alkaline phosphatase are used, activity can often be assayed directly on agar plates by calorimetric

methods. Second, affinity columns can often be designed that target the carrier portion

of the fusion protein during purification. Such affinity columns are usually constructed

using antibodies to the carrier protein or, if it is an enzyme, inhibitors may be employed

more conveniently. Affinity chromatography based on the carrier is a particularly

important asset when multiple mutants of the peptide portion are being prepared, since

the purification procedures should be identical in each case. Finally, procaryotic proteins

are synthesized in vivo with N-formyl-Met at the N terminus which is usually cleaved

from the mature protein during posttranslational processing. However, overexpressed

foreign proteins are often produced with the N-terminal Met deformylated but still

attached or incompletely removed, and the factors that govern the processing are not

understood. The fusion protein strategy, with the carrier portion at the N terminus,

therefore eliminates this potential problem also.

To date, many small peptides in addition to those mentioned above have been expressed

using the gene fusion strategy. These include antigenic determinants from hepatitis B

fused to -galactosidase and expressed under control of the lac promoter (Charnay et

al. 1980) or fused to -lactamase (trp promoter; Edman et al. 1981); influenza virus

antigens fused to -galactosidase (lac promoter; Davis et al. 1981); hirudin fused to

galactosidase (lac or PL promoters; Bergmann et al. 1986); TGF fused to 17

residues of the trp LE protein and promoter (Winkler et al. 1986); calcitonin fused to

interferon-y ( PL promoter; lvanov et al. 1987); and 27-desamidosecretin fused to

galactosidase (lac promoter; Sumi et al. 1984). Frequently, these fusion proteins are

insoluble within the cellular environment, even when produced at low levels, possibly as a

result of incorrect folding of the carrier. This results in the formation of intracellular

aggregates or inclusion bodies consisting predominantly of the fusion protein. These

structures can often be isolated by centrifugation, and subsequently dispersed under

denaturing conditions, as reviewed recently by Marston (1986), with considerable advantages in the purification strategy.

In a number of cases, the fusion approach is unsatisfactory, either because recovery

from the inclusion bodies is difficult, because a soluble fusion protein is desired (when the

peptide is to be used directly as an antigen) or, very commonly, because proteolyic

degradation of the peptide component still occurs, leading to impure products and a low

yield. In these cases, proteins such as alkaline phosphatase, Omp A or -lactamase,

which have N-terminal signal- or leader-peptide extensions that direct their translocation

across the cytoplasmic membrane and subsequent release into the periplasmic space,

have been useful as alternative carriers. When correct transport and processing by

signal peptidase occurs, large quantities of the fusion protein can accumulate in the

periplasm, where precipitation has not generally been encountered, although proteolytic

32
degradation can still be a problem. Since relatively few E. coli proteins are present in the

periplasm, and its contents can usually be selectively released by osmotic shock, secretion to the periplasm is also advantageous in terms of purification (Marston 1986).

Examples of peptides that have been prepared in this way include human proinsulin, which

was fused to N-terminal fragments of -lactamase (Chan et al. 1981) and the Met27

analogue of human GRF, which was fused to the phosphate-binding protein, pho S (Anba

et al. 1987). However, when -neoendorphin was fused to alkaline phosphatase near its

C terminus, transport or release into the periplasm was unexpectedly blocked (Ohsuye et

al. 1983). On the other hand, it is often possible to dispense with the mature carrier protein entirely and simply connect the synthetic peptide directly to a signal peptide sequence

with its signal peptidase recognition site intact. Thus, for example, human EGF and

hirudin have both been expressed, transported across the cytoplasmic membrane and

released into the periplasm after correct signal peptidase cleavage, through their fusion

to the alkaline phosphatase signal peptide (Oka et al. 1985; Dodt et al. 1986); human

growth hormone was similarly processed under the direction of the alkaline phosphatase

signal peptide or its own (eukaryotic) signal peptide (Gray et al. 1985); and proinsulin was

secreted into the periplasm by fusion to the -lactamase signal peptide (Chan et al.

1981) or through the correct transport and processing of the preproinsulin gene product

(Talmadge et al. 1980). In a small number of cases, the processed peptides have been

found predominantly in the extracellular medium, indicating that they have passed

through the outer cell membrane also. These include -endorphin, which had been fused

to the Omp F signal peptide and an additional 12 amino-acid residues, and was subject to

proteolytic degradation at its C terminus (Nagahari et al. 1985) and human growth

hormone (Kato et al. 1987) and -fibrinogen (Lord 1985) each of which had been fused to

-lactamase signal peptides. In the human growth hormone study, passage across the

outer membrane was promoted by additional weak expression of the kil gene, which

makes the outer cell wall permeable without cell lysis, suggesting that passive diffusion

across this membrane occurred. The large size of -fibrinogen (molecular weight 67,000)

would seem to preclude passive diffusion, indicating that other mechanisms of translocation across the outer membrane may also be important. However, extensive degradation

of this protein was demonstrated, and the method of detection employed was dependent

only on the presence of a short peptide fragment at its N terminus, so that passive

diffusion across the relatively porous outer membrane might explain the extracellular

presence of all three of these peptides. Whatever the mechanism, transport to the

extracellular medium appears to yield smaller quantities of these peptides than can be

obtained from intracellular fractions, and is not necessarily advantageous in terms of

purification.

Unless an endogenous proteolytic cleavage system such as the signal peptide/signal peptidase system described above is accurately exploited, the fusion protein strategy to

peptide production requires that a specific cleavage reaction must be devised for the

release of the target peptide from the fusion protein. This step is performed after cell

lysis and, usually, after some initial purification. Several site-specific methods for such

peptide backbone cleavages have been employed, all of them requiring that a “recognition

site” consisting of one or more residues be engineered into the fusion protein on the Nterminal side of the scissile peptide bond. In most cases, the structural requirements for

these sites are limited to the N-terminal side of that bond only. Since many biologically

active peptides do not tolerate N- or C-terminal modifications to their natural structures,

and given the additional possibility that peptides may be produced in E. coli with an additional Met at their N termini (see above), this situation is ideal for recovery of a precise

peptide structure if that peptide is positioned at the C terminus of a fusion construct.

The cleavage methods can be divided into three categories: chemical, enzymatic at a

single specific residue, and enzymatic at sites defined by a specific sequence of residues.

Three chemical methods have been employed. Cleavage on the C-terminal side of Met

33
residues using cyanogen bromide has often been used to release peptides that contain no

additional Met residues from fusion protein products (Itakura et al. 1977; Goeddel et al.

1979). Occasionally, the target peptide sequence has been modified in order to meet this

requirement, as in the case of [Thr59] insulin-like growth factor I fused to an eight residue

leader peptide (Peters et al. 1985). The cyanogen bromide reaction is usually carried out

in 70% formic acid, and is therefore often useful for cleavages that must be performed

under denaturing conditions such as those involving insoluble products in inclusion bodies,

although additional denaturating agents such as urea or guanidinium thiocyanate are

often added. A second chemical method, used to cleave Trp E-bovine growth hormone

fusion proteins at Asp-Pro bonds, is acid pH treatment (Szoka et al. 1986). Again, the

conditions require 70% formic acid, and often a denaturing agent is added, but elevated

temperatures are also required. Although this method has the advantage of greater

selectivity because it requires a two-residue site, it results in a cleaved peptide with an

N-terminal Pro and may not always be efficient or specific for the Asp-Pro bond. Furthermore, there are reports that some Asp-Pro bonds are quite resistant to acidolysis,

even under forcing conditions where extensive side reactions are occurring (see, for

example, Allen et al. 1985). The third chemical method is similar to the second, consisting

of cleavage at Asn-Gly bonds by hydroxylamine treatment at pH 9 and elevated temperature. This method was recently used to release human insulin-like growth factor

from the C-terminal end of an IgG-binding domain of staphylococcal protein A, after the

intact fusion protein had been purified on an IgG-Sepharose affinity column (Moks et al.

1987). Its general applicability remains to be determined.

Enzymatic cleavages at single-residue sites include the reported use of clostripain to

cleave C-terminal to Arg in an a-chloramphenicol acetyltransferase-calcitonin fusion

protein (see Marston 1986) and trypsin cleavage of -endorphin from -galactosidase,

also C-terminal to Arg, after the fusion protein had been treated with citraconic anhydride to provide temporary protection of the Lys residues in -endorphin from cleavage

(Shine et al. 1980). In this category, the Staphylococcus aureus V8 protease is also

potentially useful in that cleavage by this enzyme can be limited to sites C-terminal to

Glu residues, even in the presence of Asp residues, under specific buffer conditions

(Houmard and Drapeau 1972). This specificity has not yet been exploited, but might well

be applicable to the production of a wide variety of active hormone analogues that have

been engineered to replace any internal Glu residues with Asp. Such substitutions represent very minor changes in peptide structure, and they might often have little effect on

pharmacological activities.

There are, at present, only two enzymes having recognition sites comprised of several

amino-acid residues that have been exploited for the recovery of peptides from a fusion

product. Collagenase cleaves collagen at multiple sites having a consensus structure -

Pro-Xxx-Gly-Pro-Yyy-, with cleavage occurring on the C-terminal side of both of the

unspecified residues. In one example of the early application of this cleavage reaction,

Germino and Bastia (1984) designed a plasmid to express a fusion protein consisting of

-galactosidase at the N terminus, connected to the plasmid R6K initiator protein via a

60 residue segment of chicken pro -2 collagen containing several potential collagenase

cleavage sites. They were then able to purify this protein by affinity chromatography

based on the -galactosidase segment and recover the R6K initiator, which is normally

rapidly degraded in E. coli, after collagenase-catalyzed hydrolysis of the linker at multiple

sites. The final product, however, was probably extended from the N terminus of the

R6K initiator by about 10 residues of the linker, and may have been heterogeneous. More

satisfactory results have been obtained by Nagai and Thøgersen (1984) to recover

human -globin from a hybrid with c-II protein, and by Steven Benner’s group (Nambiar

et al. 1987) to cleave -galactosidase-linked ribonuclease A, using the action of blood

coagulation factor Xa to cleave at the C-terminal end of its consensus linker -lle-Glu-GlyArg-. In this case, the enzyme does not appear to require the three-dimensional structure

34
of its normal substrate for efficient cleavage, which probably contrasts with the requirements of collagenase, and the products obtained appeared to be homogeneous and

devoid of unwanted N-terminal extensions, although in neither case was any sequence

characterization performed. As one final note, it is worth pointing out that the potential

utility of any enzymatic method is limited by the purity of the enzyme preparation, and

contaminating proteolytic activities are often a problem.

The utility of fusion constructs for enhancing the stability of overexpressed peptides in

vivo and for simplifying their purification has stimulated a number of researchers to

design novel systems for these purposes that do not involve natural carrier proteins. In

several cases, the use of multiple fused copies of the target peptide has been shown to

enhance its stability and recovered yield. For example, three copies of the proinsulin gene

were found to be optimal for resistance to proteolysis when they were fused directly to

the tac promoter, and two copies were optimal when fusion to the lac promoter plus a

small fragment of the -galactosidase gene was tested (Shen 1984). The repeated

proinsulin gene segments were connected by linkers coding for the amino acid sequence -

Arg-Arg-Asn-Ser-Met-, with the expectation that the correct proinsulin peptide could be

recovered by cyanogen bromide cleavage followed by proteolytic digestion with trypsin

and carboxypeptidase B. A similar strategy has been employed for the production of

Met-enkephalin (Hostomsky et al. 1985). This opioid peptide was released from a fusion

product consisting of 11 copies of the peptide connected by -Arg-Arg- linkers and fused to

part of the SV40 small-t antigen, after trypsin and carboxypeptidase B digestion, as

indicated by RIA and guinea-pig ileal assays. A substance P analogue having a C-terminal homoserine amide was also successfully prepared from overexpressed fusion proteins

containing 4, 16 and 64 copies of the corresponding C-terminal Met analogue (Kempe et

al. 1985). These were fused together between part of the cro repressor on the Nterminal side and ß-galactosidase on the C-terminal side. In this case, the substance P

analogue was obtained by cyanogen bromide cleavage to release multiple copies of the Cterminal homoserine lactone analogue, which were converted to the desired

[HSe11]substance P with a C-terminal amide, as in the natural hormone, by treatment

with 30% ammonium hydroxide. This last approach is likely to prove useful for the

preparation of many other peptide hormone analogues where a C-terminal amide is

required for activity.

Another approach taken by Sung et al. (1986) to optimize the production of proinsulin was

the addition of different homo-oligomeric peptides at its N terminus. As discussed above,

this tactic is likely to affect the efficiency of expression as a result of modifications to

the 5’ end of the mRNA structure, in addition to affecting the lifetime of the peptide in

vivo as a result of the effects of the N-terminal amino-acid residues on endogenous

mechanisms for targetting protein degradation. In this investigation, hexamers of Ala,

Asn, Cys, Gln, His, Ser, or Thr were found to produce the highest yields, and the yields

for the(Ala)6- and (Ser)6-modified constructs were indeed found to be strongly dependent

on the specific choice of codons used, indicating the importance of the structure of the

ribosome-binding site on the mRNA. The use of poly-Arg fused to the C terminus of an

overexpressed protein has also been explored as an aid to purification (Smith et al.

1984). This method was applied to the production in E. coli of human growth hormone

(urogastrone) as a fused protein with 14 residues of the trp E protein at its N terminus

and a C-terminal tail of 5 Arg residues. After expression, the fusion protein was purified

from the cell lysate by cation-exchange chromatography, where it eluted after the bulk

of the bacterial proteins on a salt gradient. The poly-Arg tail could be removed by

carboxypeptidase B digestion.

In our laboratory, we are attempting to exploit the potential amphiphilic nature of many

peptide hormones and other biologically active peptides, and the general propensity of

these structures to form organized aggregates in aqueous solution (Taylor and Kaiser

35
FIGURE 6

Schematic diagram of the structure of a helix bundle protein consisting of four amphiphilic

a helices. Fusion proteins of this type, designed to incorporate amphiphilic peptide hormones at their C termini (shaded segment), might be resistant to proteolysis in E. coli.

1986) in order to generate fusion proteins that are specifically designed to include the

peptide hormone as an integral part of their globular structure. For example, many of the

intermediate-sized flexible peptide hormones can form amphiphilic -helical segments

(Taylor and Kaiser 1986). These peptides might be incorporated into globular fusion

protein structures consisting of multiple near-parallel helices similar to the four-helix

bundles commonly found in natural proteins (Weber and Salemme 1980) if they were

connected to repeated segments of peptide sequences designed to form idealized

amphiphilic helices (figure 6). Such tailor-made structures are expected to be soluble and

more resistant to proteolytic degradation than the randomly connected fusion proteins

usually are. Furthermore, they could be specifically engineered to allow convenient purification of the peptide hormone by, for example, ion-exchange chromatography. Eventually, these simple modifications of the fusion protein approach are likely to result in very

efficient systems for the production of peptide hormones in bacterial cells in high yield.

Combined with the rapid, multiple oligonucleotide synthesis methods of Blocker’s group,

and the high-efficiency mutagenesis methods that we have developed in the Eckstein laboratory, the exploration of structure-activity relationships in peptide hormones through

the expression of multiple mutant genes in E. coli should often be competitive with the

direct chemical synthesis approach, and may have advantages in terms of time, cost of

reagents, and product purity.

REFERENCES

Allen, G.; Paynter, CA.; and Winther, M.D. Production of epidermal growth factor in

Escherichia coli from a synthetic gene. J Cell Sci (Suppl) 3:29-38, 1985.

Anba, J.; Baty, D.; Lloubes, R.; Pages, J.M.; Joseph-Liauzun, E.; Shire, D.; Roskam, W.;

and Lazdunski, C. Expression vector promoting the synthesis and export of the human

growth-hormone-releasing factor in Escherichia coli. Gene 53:219-226, 1987.

Bachmair, A.; Finley, D.; and Varshavsky, A. In vivo half-life of a protein is a function of

its amino-terminal residue. Science. 234:179-186, 1986.

Bergmann, C.; Dodt, J.; Kohler, S.; Fink, E.; and Gassen, H.G. Chemical synthesis and

expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech

Hirudo medicinalis. Biol Chem Hoppe-Seyler 367:731-740, 1986.

36
Boros, I.; Lukacsovich, T.; Baliko, G.; and Venetianer, P. Expression vectors based on

the rac fusion promoter. Gene 42:97-100,1986.

Brodin, P.; Grundstrom, T.; Hofmann, T.; Drakenberg, T.; Thulin, E.; and Forsen, S.

Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product. Biochemistry 25:5371-5377, 1986.

Buell, T.A.; Schulz, M.-F.; Seizer, G.; Chollet, A.; Mowa, N.R.; Semon, D.; Escanez, S.;

and Kawashima, E. Optimizing the expression in E. coli of a synthetic gene encoding

somatomedin-C (IGF-I). Nucleic Acids Res 13:1923-1938, 1985.

Burgers, P.M.J., and Eckstein, F. A study of the mechanism of DNA polymerase I from

Escherichia coli with diastereomeric phosphorothioate analogs of deoxyadenosine

triphosphate. J BioI Chem 254:6889-6893, 1979.

Carter, P.; Bedouelle, H.; and Winter, G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13:4431-4443, 1985.

Chan, S.J.; Weiss, J.; Konrad, M.; White, T.; Bahl, C.; Yu, S.D.; Mark,.D.;and Steiner,

D.F. Biosynthesis and periplasmic segregation of human proinsulm in Eschericbia coli.

Proc Natl Sci USA 78:5401-5405, 1981.

Charnay, P.; Gervais, M.; Louise, A.; Galibert, F.; and Tiollais, P. Biosynthesis of

hepatitis B virus surface antigen in Escberichia coli. Nature 286:893-895, 1980.

Clark-Lewis, I.; Aebersold, R.; Ziltener, H.; Schrader, J.W.; Hood, L.E.; and Kent, S.B.H.

Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3. Science 231:134-139, 1986.

Davis, A.R.; Nayak, D.P.; Ueda, M.; Hiti, A.L.; Dowbenko, D.; and Kleid, D.G. Expression

of antigenic determinants of the hemagglutinin gene of a human influenza virus in

Eschericbia coli. Proc Nat Acad Sci USA 78:5376-5380, 1981.

De Boer, H.A.; Comstock, L.J.; Yansura, D.G.; and Heyneker, H.L. Construction of a

tandem trp-lac promoter and a hybrid trp-lac promoter for efficient and controlled

expression of the human growth hormone gene in Eschericbia coli. In: Rodriguez, R.L.,

and Chamberlin, M.J., eds. Promoters: Structure and Function. New York: Praeger

Publishers, 1982. pp.462-481.

Dente, L.; Cesareni, G.; and Cortese, R. pEMBL: A new family of single stranded plasmids. Nucleic Acids Res 11:1645-1655, 1983.

Deuschle, U.; Kammerer, W.; Gentz, R.; and Bujard, H. Promoters of Escherichia coli: A

hierarchy of in vivo strength indicates alternate structures, EMBO J 5:2987-2994,

1986.

Dodt, J.; Schmitz, T.; Schafer, T.; and Bergmann, C. Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. FEBS Lett

202:373-377, 1986.

Duvoisin, R.M.; Belin, D.; and Krisch, H.M. A plasmid expression vector that permits

stabilization of both mRNAs and proteins encoded by the cloned genes. Gene 45:193-

201, 1986.

Edman, J.C.; Hallewell, R.A.; Valenzuela, P.; Goodman, H.M.; and Rutter, W.J. Synthesis

of hepatitis B surface and core antigens in E. coli. Nature 291:503-506, 1981.

Ferretti, L.; Karnik, S.S.; Khorana, H.G.; Nassal, M.; and Oprian, D.D. Total synthesis of

a gene for bovine rhodopsin. Proc Natl Acad Sci USA 83:599-603, 1986.

Frank, R.; Heikens, W.; Heisterberg-Moutsis, G.; and Blocker, H. A new general approach

for the chemical synthesis of large numbers of oligonucleotides: Segmental solid

supports. Nucleic Acid Res 11:4365-4377, 1983.

Germrno, J., and Bastia, D. Rapid purification of a cloned gene product by genetic fusion

and site-specific proteolysis. Proc Natl Acad Sci USA 81:4692-4696, 1984.

Goeddel, D.V. Kleid, D.G.; Bolivar, F.; Heyneker, H.L.; Yansura, D.G.; Cre, R.; Hirose, T.;

Kraszewski, A.; Itakura, K.; and Riggs, A.D. Expression in Escherichia coli of

chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 76:106-110,

1979.

Goff, S.A., and Goldberg, A.L. Production of abnormal proteins in E. coli stimulates transcription of Ion and other heat shock genes. Cell 41:587-595, 1985.

37
Gold, L.; Pribnow, D.; Schneider, T.; Shinedling, S.; Singer, B.S.; and Stormo, G. Translation initiation in prokaryots. Annu Rev Microbid 35:365-403, 1981.

Gray, G.L.; Baldridge, J.S.; McKeown, K.S.; Heyneker, H.L.; and Chang, C.N. Periplasmic

production of correctly processed human growth hormone in Escherichia coli: Natural

and bacterial signal sequences are interchangeable. Gene 39:247-254, 1985.

Grosjean, H., and Fiers, W. Preferential codon usage in prokaryotic genes: The optimal

codon-anticodon interaction energy and the selective codon usage in efficiently

expressed genes. Gene 18:199-209, 1982.

Grundstrom, T.; Zenke, W.M.; Wintzerith, M.; Matthes, W.D.; Staub, A.; and Chambon, P.

Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ gene synthesis. Nucleic

Acids Res 13:3305-3316,1985.

Hostomsky, Z.; Smrt, J.; and Paces, V. Cloning and expression in Escherichia coli of the

synthetic proenkephalin analogue gene. Gene 39:269-274, 1985.

Houmard, J., and Drapeau, G.R. Staphylococcal protease: A proteolytic enzyme specific

for glutamoyI bonds. Proc Natl Acad Sci USA 69:3506-3509, 1972.

Ikemura, T. Correlation between the abundance of Escherichia coli transfer RNAs and

the occurrence of the respective codons in its protein genes. J Mol Biol 151:389-409,

1981.

Itakura, K.; Hirosi, T.; Crea, R.; Riggs, A.D.; Heyneker, H.L.; Bolivar, F.; and Boyer, H.W.

Expression in E. coli of a chemically synthesized gene for the hormone somatostatin.

Science 198:1056-1063, 1977.

Itakura, K.; Rossi, J.J.; and Wallace, R.B. Synthesis and use of synthetic oligonucleotides.

Annu Rev Biochem 53:323-356, 1984.

Ivanov, I.; Gigova, L.; and Jay, E. Chemical synthesis and expression in E. coli of a

human Val8-calcitonin gene by fusion to a synthetic human interferon-y gene. FEBS

Lett 210:56-60, 1987.

Kato, C.; Kobayashi, T.; Kudo, T.; Furusato, T.; Murakami, Y.; Tanaka, T.; Baba, H.;

Oishi, T.; Ohtsuka, E.; Ikehara, M.; Yanagida, T.; Kato, H.; Moriyama, S.; and Horikoshi,

K. Construction of an excretion vector and extracellular production of human growth

hormone from Escherichia coli. Gene 54:197-202, 1987.

Kempe, T.; Kent, S.B.H.; Chow, F.; Peterson, SM.; Sundquist, W.I.; L’ltalien, J.J.; Harbrecht, D.; Plunkett, D.; and DeLorbe, W.J. Multiple-copy genes: Production and

modification of monomeric peptides from large multimeric fusion proteins. Gene 39:239-

245, 1985.

Kramer, B.; Kramer, W.; and Fritz, H.-J. Different base/base mismatches are corrected

with different efficiencies by the methyl-directed DNA mismatch-repair system of E.

coli. Cell 38:879-997,1984a.

Kramer, W.; Drutsa, V.; Jansen, H.-W.; Kramer, B.; Pflugfelder, M.; and Fritz, H.-J. The

gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic;

Acids Res 12:9441-9456, 1984b.

Kramer, W.; Schughart, K.; and Fritz, H.-J. Directed mutagenesis of DNA cloned in filamentous phage: Influence of hemimethylated GATC sites on marker recovery from

restriction fragments. Nucleic Acids Res 10:6475-6485, 1982.

Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotype selection.

Proc Natl Acad Sci USA 82:488-492, 1985.

Lo, K.-M.: Jones, S.S.; Hackett, N.R.; and Khorana, H.G. Specific amino acid substitutions

in bacteriodopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci USA 81:2285-

2289, 1984.

Lord, ST. Expression of a cloned human fibrinogen cDNA in Escherichia coli: Synthesis

of an A alpha polypeptide. DNA 4:33-38, 1985.

Marston, F.A.O. The purification of eukaryotic polypeptides synthesized in Escherichia

coli. Biochem J 240: 1-12, 1986.

38
Matthes, H.W.D.; Zenke, W.M.; Grundstrom, T.; Staub, A.; Wintzerith, M.; and Chambon, P.

Simultaneous rapid chemical synthesis of over one hundred oligonucleotides on a microscale. EMBG J 3:801-805, 1984.

Messing, J. New M13 vectors for cloning. Methods Enzymol 101:20-78, 1983.

Moks, T.; Abrahmsen, L.; Holmgren, E.; Bilich, M.; Olsson, A.; Uhlen, M.; Pohl, G.; Sterky,

C.; Hultberg, H.; Josephson, S.; Holmgren, A.; Jornvall, H.; and Nilsson, B. Expression of

human insulin-like growth factor I in bacteria: Use of optimized gene fusion vectors to

facilitate protein purification. Biochemistry 26:5239-5244, 1987.

Myers, R.M.; Lerman, L.S.; and Maniatis, T. A general method for saturation mutagenesis of cloned DNA fragments. Science 229:242-247, 1985.

Nagahari, K.; Kanaya, S.; Munakata, K.; Aoyagi, Y.; and Mizushima, S. Secretion into

the culture medium of a foreign gene product from Escherichia coli: Use of the omp F

gene for secretion of human -endorphin. EMBO J 4:3589-3592, 1985.

Nagai, K .and Thøgersen, H.C. Generation of -globin by sequence-specific proteolysis of

a hybrid protein produced in Escherichia coli. Nature 309:810-812, 1984.

Nakamaye, K., and Eckstein, F. Inhibition of restriction endonuclease Nci I cleavage by

phosphorothioate groups and its application to oligonucleotide-directed mutagenesis,

Nucleic Acids Res 14:9679-9698, 1986.

Nambiar, K.P.; Stackhouse, J.; Presnell, S.R.; and Benner, S.A. Expression of bovine

pancreatic ribonuclease A in Escherichia coli. Eur J Biochem 163:67-71, 1987.

Oka, T.; Sakamoto, S.; Miyoshi, K.; Fuwa, T.; Yoda, K.; Yamasaki, M.; Tamura, G.; and

Miyaki, T. Synthesis and secretion of human epidermal growth factor by Escherichia

coli. Proc Natl Acad Sci USA 82:7212-7216, 1985.

Ohsuye, K.; Nomura, M.; Tanaka, S.; Kubota, I.; Nakazato, H.; Shinagawa, H.; Nakata,

A.; and Noguchi, T. Expression of chemically synthesized a-neo-endorphin gene fused

to E. coli alkaline phosphatase. Nucleic Acid Res 11:1283-1294,1983.

Ott, J., and Eckstein, F. Filter disc supported oligonucleotide synthesis by the phosphite

method. Nucleic Acids Res 12:9137-9142, 1984.

Peters, M.A.; Lau, E.P.; Snitman, D.L.; Van Wyk, J.J.; Underwood, L.E.; Russell, W.E.; and

Svoboda, M.E. Expression of a biologically active analogue of somatomedin-C/insulinlike growth factor I. Gene 35:83-89, 1985.

Rogers, S.; Wells, R.; and Rechsteiner, M. Amino acid sequences common to rapidly

degraded proteins: The PEST hypothesis. Science 234:364-372, 1986.

Sayers, J., and Eckstein, F. Phosphorothioate-based oligonucleotide-directed mutagenesis. In: Setlow, J.K., ed. Genetic Engineering: Principles and Methods. Vol. X. New

York: Plenum Press, 1988. pp. 109-122.

Seeburg, P.H.; Sias, S.S.; Adelman, J.; De Boer, H.A.; Hayflick, J.; Jhurani, P.; Goeddel,

D.V.; and Heyneker, H.L. Efficient bacterial expression of bovine and porcine growth

hormones. DNA 2:37-45, 1983.

Shen, S.-H. Multiple joined genes prevent product degradation in Escherichia coli. Proc

Natl Acad Sci USA 81:4627-4631, 1984.

Shine, J.; Fettes, I.; Lan, N.C.Y.; Roberts, J.L.; and Baxter, J.D. Expression of cloned

endorphin gene sequences by E. coli. Nature 285:456-461,1980.

Shortle, D.; Grisafi, P.; Benkovic, S.J.; and Botstein, D. Gap misrepair mutagenesis:

Efficient site-directed induction of transition, transversion, and frameshift mutations in

vitro. Proc Natl Acad Sci USA 79:1588-1592, 1982.

Smith, J.C.; Derbyshire, R.B.; Cook, E.; Dunthorne, L.; Viney, J.; Brewer, S.J.; Sassenfeld,

H.M.; and Bell, L.D. Chemical synthesis and cloning of a poly(arginine)-coding gene

fragment designed to aid polypeptide purification. Gene 32:321-327, 1984.

Sonveaux, E. The organic chemistry underlying DNA synthesis. Biooraanic Chem

14:274-325, 1986.

Sumi, S.; Nagawa, F.; Hayashi, T.; Amagase, H.; and Suzuki, M. Overproduction of a

gastrointestinal hormone, secretin, in Escherichia coli cells and its chemical

characterization. Gene 29:125-134, 1984.

39
Sung, W.L.; Yao, F.-L.; Zahab, D.M.; and Narang, S.A. Short synthetic oligodeoxyribonucleotiie leader sequences enhance accumulation of human proinsulin synthesized in

Escherichia coli. Proc Natl Acad Sci USA 83:561-565, 1986,

Swamy, K.H.S., and Goldberg, A.L. E. coli contains eight soluble proteolytic activities,

one being ATP dependent. Nature 292:652-654, 1981.

Szoka, P.R.; Schreiber, A.B.; Chan, H.; and Murthy, J. A general method for retrieving

the components of a genetically engineered fusion protein. DNA 5:11-20, 1986.

Talmadge, K.; Kaufman, J.; and Gilbert, W. Bacteria mature preproinsulin to proinsulin.

Proc Natl Acad Sci USA 77:3988-3992, 1980.

Taylor, J.W., and Kaiser, E.T. The structural characterization of B-endorphin and

related peptide hormones and neurotransmitters. Pharmacol Rev 38:291-319, 1986.

Taylor, J.W.; Ott, J.; and Eckstein, F. The rapid generation of oligonucleotidedirected

mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res

13:8765-8785, 1985a.

Taylor, J.W.; Schmidt, W.; Cosstick, R.; Okruszek, A.; and Eckstein, F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.

Nucleic Acids Res 13:8749-8764, 1985b.

Von Bodman, S.B.; Schuler, M.A.; Jollie, D.R.; and Sligar, S.G. Synthesis, bacterial

expression, and mutagenesis of the gene coding for mammalian cytochrome b5. Proc

Natl Acad Sci USA 83:9443-9447, 1986.

Weber, P.C., and Salemme, F.R. Structural and functional diversity in 4- -helical proteins. Nature 287:82-84,1980.

Wetzel, R., and Goeddel, D.V. Synthesis of polypeptides by recombinant DNA methods.

In: Gross, E., and Meienhofer, J., eds. The Peptides. Vol. V. New York: Academic

Press, 1983. pp. 1-64.

Wilkinson, A.J.; Fersht, A.R.; Blow, D.M.; and Winter, G. Site-directed mutagenesis as a

probe of enzyme structure and catalysis: Tyrosyl-tRNA synthetase cysteine-35 to

glycine-35 mutation. Biochemistry 22:3581-3586, 1983.

Winkler, M.E.; Bringman, T.; and Marks, B.J. The purification of full active recombinant

transforming growth factor a produced in Escherichia coli. J Biol Chem 261:13838-

13843, 1986.

Yarus, M., and Folley, L.S. Sense codons are found in specific contexts. J Mol Biol

182:529-540, 1985.

Zoller, M.J., and Smith, M. Oligonucleotidedirected mutagenesis of DNA fragments

cloned into M13 vectors. Methods Enzymol 100:468-500, 1983.

ACKNOWLEDGMENTS

Oligonucleotide synthesis, purification, and analysis was performed by Ms. G.V. Babiera.

Funding support was provided by the National Institute on Drug Abuse through P.H.S.

grant DA 04197.

AUTHOR

John W. Taylor, Ph.D.

Laboratory of Bioorganic Chemistry and Biochemistry

The Rockefeller University

1230 York Avenue

New York, New York 10021, U.S.A.

40
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds

National Institute on Drug Abuse, 1988

X-Ray Diffraction Studies

Enkephalins and Opiates

of

Jane F. Griffin, Ph.D., and G. David Smith, Ph.D.

The conformation or conformations of the enkephalins responsible for

biological activity, that is, the conformation assumed at the receptor and

resulting in the transduction of a message, has been of enormous interest

since the enkephalins were first isolated by Hughes and Kosterlitz (1975).

These pentapeptides with sequence Tyr-Gly-Gly-Phe-Leu(Met) have many

degrees of conformational freedom due to peptide chain flexibility, centered

in the four and five torsional rotations, and side-chain flexibility from the

rotations of the Tyr1, Phe4 and Leu5 or Met5 residues. How does the

opioid receptor(s) recognize a flexible linear pentapeptide, the relatively

inflexible fused ring morphine-like compounds and the other families of

drugs with which it interacts? What are the chemical moieties recognized by

the receptor(s) and how are they arranged in three dimensions? Do the

different opioid receptor subtypes bind different low energy conformations of

the endogenous compounds or induce them into an “active” conformation?

Since 1975 the conformations of enkephalin have been studied by every

available spectroscopic technique in a variety of solvents and in the solid

state, by more traditional biochemical structure/activity studies, by theoretical

methods and by single crystal X-ray diffraction techniques. These studies

have been reviewed in an excellent chapter by Schiller (1984). A more

detailed review of the crystallographic literature appears in a previous

monograph in this series (Camerman and Camerman 1986). This review will

concentrate on the enkephalin structures determined by X-ray diffraction

techniques since that paper was published, and on certain aspects of the

structures which were not highlighted in the previous review. The results of

X-ray diffraction studies on some morphine analogues with interesting opioid

activity profiles will also be discussed.

INFORMATION OBTAINED FROM X-RAY DIFFRACTION STUDIES

The results of an X-ray diffraction study are a set of atomic coordinates, unit

cell constants and space group. Calculations made from the above

information provide not only geometric details on the compound under

41
study, precise bond distances, valency angles and torsion angles, but also

intramolecular and intermolecular hydrogen bond geometry. The interactions

that represent the forces holding molecular crystals together are exactly the

same forces that are responsible for substrate-receptor binding: electrostatic

interactions between charged groups, hydrogen bonds between functional

groups, hydrophobic interactions and dipolar interactions. Thus the bioactive

compound in the crystal lattice is a model of the substrate in a set of

complementary surroundings. Determination of different crystalline forms,

polymorphs and solvates, of the same material show different gross

intermolecular interactions that can be related back to interactions in

solution. Stable patterns of aggregation and even solvation present in

solution persist in the crystalline form. Thus, multiple determinations of the

same compound (either more than one molecule in the asymmetric unit or

different crystalline forms) give information on conformational flexibility,

stable aggregates, patterns of solvation, and intermolecular interactions.

Main-Chain Conformation

There have been three X-ray crystallographic studies of [Leu5] enkephalin

and two studies of the same crystalline form of (Met5]enkephalin. The

crystal data on these structures is given in table 1. The structures labelled

[(4-bromo-Phe4] analogue of [Met5]enkephalin (Doi et al., 1987b) have been

LEE2, MEE2 (Griffin et al., 1985, 1986a), MEE2 and MEBrE2 the

determined since the previous review. Structures MEE2 and MEE2 are two

independent studies on crystals that appear to vary only in the amount of

water present (see table 1). Previously, the crystal structures of the

[(4-bromo-Phe4] analogue of [Leu5]enkephalin and a few other analogues ues

with individual amino acid replacements have also been determined. These

compounds and the main-chain conformations observed in each of these

studies are given in table 2. Two conformations predominate, a turn

centered on Gly2Gly3 and a family of extended conformations.

Solution spectra have for the most part been interpreted as showing that

there are both extended and folded conformations in solution; the folded

conformation most frequently found by interpretation of spectra is a turn

centered on Gly3 Phe4, a conformation not yet observed in the solid state.

Khaled (1986) has pointed out some of the limitations in magnetic resonance

studies of peptides: (1) assignments are complicated in a molecule containing

a recurring amino acid residue such as in enkephalin with two adjacent

glycine residues; (2) a controversy exists regarding the interpretation of the

temperature dependence of NH protons. These assignments are used to

distinguish inter- and intramolecular hydrogen bonds. The presence of the

type I’ -turn conformation of enkephalin, centered on Gly2Gly3 and

characterized by a Phe4 NH to O=C Tyr1 hydrogen bond, observed in the

solid state studies of [Leu5]enkephalin and analogues (table 2) has been

reported in only one spectroscopic study, enkephalin amides complexed with

18-crown-6-ether in chloroform (Beretta et al. 1984).

42
TABLE 1

Crystal data on enkephalins.

[Leu5]enkephalin

LE(B)a LE(E4)b

[Met5]enkephalin

LE(E2)C ME(E2') d,e MEBr(E2)d

LE(E2)c

Space Group

Z

No. independent

mol./ asym. unit

solvent/ asym. unit

Vol. (A3)

No. observations

No. obs. > 2 sigma

a (A)

b (A)

c (A)

alpha (°)

beta (o)

gama (°)

C2 P21 P21

16 8 4

4

6 H2O

13501

8869

5430

24.861

17.084

31.937

95.54 115.9 92.19 91.24 91.23

4 2

8H20, 8C3H7NO, 0.5H2O

8459 2999

10942 3180

5965 3004

18.720 11.549

24.732 15.587

20.311 16.673

P21

4

2

10.6H2O

3448

5056

4725

11.607

17.987

16.519

P21

4

 2

10 H2O

3413

6037

4407

11.592

17.871

16.480

P1

2

2

4.5 H2O

1730

5908

5270

11.619

11.609

12.943

93.92

96.10

86.99

a

 Smith and Griffin 1978; Blundell et al. 1979.

b

 Karle et al. 1983; Camerman et al. 1983.

c

 Griffin et al. 1985, 1986a. Subsequent to this report of structures LE(E2) and ME(E2), Hastropaolo et

al. (1986, 1987) reported the independent structure determination of the same structures using the same

X-ray diffraction data of Blundell. The report that the [Met5]enkephalin crystals were grown from

ethanol/water (Hastropaolo et al. 1986) is incorrect. The experimental details of crystallization and

data collection from Blundell’s laboratory are given in Griffin et al. 1986a.

d

 Doi et al. 1987b.

e

 ME(E2) and ME(E2') are tvo independent studies on crystals that appear to vary only in the amount of

water present. Note the almost identical cell parameters, differing by 34 A

3

 in unit cell volume.
TABLE 2

Conformations of enkephalin and enkephalin analogues

from X-ray crystallographic determinations.

Peptide Conformation

Tyr-Gly-Gly-Phe-Leu- 2H2O 

(Smith and Griffin, 1978)

Tyr-Gly-Gly-Phe-Leu-2H2O,

2C3H7NO,X (Karle et al. 1983)

type I’ ß-bend NPhe to OTyr

(Gly-Gly) NTyr to OPhe

Extended None

antiparallel ß-sheets

Tyr-Gly-Gly-Phe-Leu•0.5H2O

(Griffin et al. 1986a)

Tyr-Gl-Gly-Phe-Met•5.3H2O

(Griffin et al. 1986a) 

Tyr-Gly-Gly-Phe-Met•5.0H2O

(Doi et al. 1987b)

Extended None

antiparallel ß-sheets

Extended None

antiparallel ß-sheets

Extended None

antiparallel ß-sheets

Tyr-Gly-Gly-(4Br)Phe-Leu•2.5H2O type I’ ß-bend

(Ishida et al. 1984) (Gly-Gly)

NPhe to OTyr

NTyr to OPhe

Tyr-Gly-Gly-(4Br)Phe-Met•2.3H2O

(Doi et al. 1987b)

Tyr-D-Nle-Gly-Phe-NleS•2.5H2O,

C2H5OH (Stezowski et al. 1985)

Tyr-Gly-Gly-Phe•DMSO,2H2O

(Foumie-Zaluskie et al. 1977)

Extended

type II’ ß-bend

(D-Nle-Gly)

NPhe to OTyr

NTyr to OPhe

type I’ ß-bend

(Gly-Gly)

Gly-Gly-Phe-Leu•H2O

(Foumie-Zaluskie et al. 1977)

Extended

BOC-Tyr-Gly-Gly-(4Br)Phe-MetOH

(Doi et al. 1984)

Extended None

antiparallel -sheets

BOC-Aib-Aib-Phe-MetNH2•DMSO

(Prasad et al. 1983)

BOC-Gly-Gly-Phe Ethyl Ester

(Ishida et al. 1983)

310 Helix

type I’ -bend

(Gly-Gly)

Intramolec.

H-bonds

None

NPhe to OTyr

None

NPhe to OBOC

NMet to Aib

None

44
Extended Main-Chain Conformations

The six independent observations of [Leu5]enkephalin, 4 molecules in LEE4

and 2 molecules in LEE2, in the extended conformation are superimposed in

figure la. The two independent molecules of [Met5]enkephaIin (MEE2) are

superimposed in figure lb. The peptide chains observed in LEE2, but

particularly in the three (Met5) structures are unusually planar. This is

apparent by examination of the torsion angles for the [Me5]enkephalin

structures and [Leu5]enkephalin (LEE2) versus the extended conformations

seen in LEE4 (see table 3). The main-chain torsion angles of the dimer of

[4-bromo-Phe4,Met5]enkephalin (MEBrE2) are remarkably similar to those in

MEE2 and MEE2, even though the space group and the amount of solvent

water in the crystal are different. The torsion angles for Gly2 and Gly3

in the three [Met5] structures indicate an unusually extended conformation.

all the values lie between ± 160° and ± 180°, The values for the Phe4

residue in the [Met5]enkephalin structures (six observations) are almost

invariant, -154° ± 4°/155o ± 4°, and lie in the ideal -sheet region; the

values in the [Leu5]enkephalin structures show greater variability, especially

in LEE4 The Met residues are in two slightly different conformations,

 -138°/157° and -155°/170°. These results could be interpreted to indicate

less flexibility in the main chain of [Met5]- versus [Leu5]enkephalin, but the

small size of the data base imposes caution in drawing this conclusion.

Side-Chain Conformation

In addition to the torsion angle values, which define main-chain

conformation, table 3 contains the torsion angles, which describe the Tyr,

Phe, Leu, and Met side-chain conformations in the -sheet enkephalin

structures. The values for the Tyr and Phe side chains in the

(Met5]enkephalin structures are almost invariant. both close to +60° (six

observations of each). This is in contrast to the values observed in the

[Leu5]enkephalin structures: Tyr +60° (three observations), 180° (three);

Phe -60o (five), 180° (one).

If the side-chain conformation observed in the extended structures is

compared with that observed in the type I’ -turn [Leu5] structures, LE and

[(4-bromo)Phe4 ,Leu5]enkephalin, the major difference is in of Tyr: -60°

in the -turn forms and +60o and 180° in the extended forms; of Phe is

-60o in the turn structures, similar to the major conformer in the -sheet

[Leu5]enkephalin structures.

In summary, in this admittedly small sample, the side-chain conformations in

[Leu5]- versus (Met5]enkephalin are distinguished by mutually exclusive Phe

values. The [Leu5] structures have of Phe -60°, 180°, while the

[Met5] structures have of Phe values +60o. The extended versus -turn

structures have mutually exclusive values for Tyr -60o ( turn), and

+ 60°, 180° (extended),

INTERMOLECULAR INTERACTIONS

The [Met5]enkephalin structures consist of dimers forming infinite anti45
FIGURE 1

(a) A stereo view of a least-squares superposition of the six

crystallographically observed extended conformations of [Leu5]enkephalin.

The 21 main chain atoms were fit. (b) A stereo view of a least-squares

superposition of the two crystallographically observed extended

conformations of [Met5]enkephahn in MEE2. Each methionine residue is

observed in two distinct conformations; one is dashed.

46
parallel -sheets with varying amounts of water molecules per dimer. The

two crystallographically independent molecules of the dimer are remarkably

similar with respect to both main-chain and tyrosine and phenylalanine sidechain conformations (table 3). In both MEE2, and MEE2 each methionine

side chain is disordered, and the disordered conformations are different in the

two independent molecules (see table 3). The disorder appears to be the

result of close contacts between methionine side chains in adjacent

independent molecules. The amount of solvent in the unit cell appears to

affect the methionine side-chain conformation. In the dimer the nitrogen and

carbonyl of GIy2 and Phe4 of one molecule are hydrogen bonded to the

carbonyl and nitrogen of Phe4 and Gly2,

(figure 2).

respectively, of the other molecule

Additional hydrogen bonds connect adjacent dimers and form

head-to-tail (NTyr to OMet) connections, which loin crystallographically

equivalent molecules related by translation.

The [Leu5]enkephaIin structure LEE2 is made up of planar -sheets arranged

similarly to those in the [Met5] structures. However, the side chains are

oriented differently in the two independent molecules (see table 3). There is

only one molecule of water per dimer of [Leu5]enkephalin versus 10.6 and

10.0 in MEE2 and MEE2 , respectively. The packing is more compact than

is observed in the other two [Leu5] forms. The one water molecule is

tetrahedrally coordinated; it donates two hydrogen bonds to the two

independent leucyl carboxyl groups and accepts two hydrogen bonds from

the hydroxyl groups of two independent tyrosine side chains.

In the [Met5]enkephalin structures MEE2 and MEE2, the distance between

sheets is approximately 9.0 (one-half the b axis), while the value observed

in the [Leu5]enkephahlin structure is 7.8 As a result of this increase in

interplanar spacing, the unit cell of [Met5]enkephalin accommodates

additional water molecules.

Comparison of the -Sheet Forms

The asymmetric unit, that is, repeating unit, is a dimer in structures LEE2

MEE2, MEE2 and MEBrE2 and a tetramer in LEE4 The dimers are similar

in all four structures; two independent molecules form an antiparallel -sheet

with four hydrogen bonds N(Gly2)A to O(Phe4)B N(Phe’)B to O(Gly2)A,

N(Gly2)B to O(Phe4)A, N(Phe4)A to O(Gly2)B, where A and B refer to the

two independant molecules. The dimers are joined in head-to-tail fashion,

with identical dimers related by translation forming infinite ribbons. These

ribbons are joined to adjacent ribbons by antiparallel hydrogen bonds

translated by approximately 1/2 the length of the peptide. Therefore, dimers

adjacent in the plane of the -sheet hydrogen bond across the head-to-tail

connections (see figure 2). The -sheets thus formed are not pleated in the

normal direction, that is, along the peptide chain. Instead the chains are

very flat and extended, and the pleat is perpendicular to the direction of the

peptide chain.

The asymmetric unit in LEE4 is a tetramer; two of the four molecules, A and

B, form a dimer similar to the one described above for the dimeric structures

with respect to hydrogen bonding. The other two molecules of the tetramer.

C and D, are rotated 180o in the plane of the -pleated sheet and form

47
FIGURE 2

Packing pattern of [Met5]enkephalin MEE2 showing the -sheet structure.

This pattern is common to the three [Met5] structures. Water is shown as

large circles. The water structure is not common to the three structures.

FIGURE 3

Comparison of the -sheet formed in [Leu5

]enkephalin, LEE4, left, and LEE2 ,

right. The dimer common to both structures is outlined.

48
hydrogen bonds with one another and with B and the translationally related

A molecules through the peptide C=O and NH groups. The tetramers form

a continuous -pleated sheet along a. These sheets are connected to

translationally related sheets along c, forming an infinite sheet in the ac

plane. The sheets are separated from one another by solvent, much of it

disordered. The distance between the sheets is 12.4 (1/2 b) and there is no

interaction between the side-chain residues of adjacent sheets.

The side-chain residues pack quite differently in the space between adjacent

sheets in the two [Leu5] structures, LEE4 and LEE2 (see figure 3). In LEE4

there is a layer of solvent molecules separating the side-chains of the -sheets

adjacent along b. The structure is a sandwich layered along b, the bottom

slice is a -sheet. followed by a side-chain layer forming hydrogen bonds

(Tyr) and hydrophobic interactions (Leu and Phe) to the next layer, solvent,

which in turn forms hydrogen bonds and hydrophobic interactions to the

side-chain layer from the top -sheet, the final slice. Because in LEE4, the

dimer formed by molecules C and D is rotated 180° in the plane of the sheet

with respect to the AB dimer. all three side-chain residues from the plane at

0.25 and three from the plane at 0.75 pack in the space between

sheets separated by b/2. In LEE2, on the other hand, the residues between

the planes consist of tyrosyl and leucyl side-chains from one sheet and

phenylalanyl residues from the sheet separated by b/2. These form relatively

tight hydrophobic interactions connecting the B-sheets along b.

Packing of the -Turn Form

The asymmetric unit in the -turn form LE is a tetramer (see figure 4). The

two central molecules form a hydrogen- onded dimer, the N-terminus of b

each is hydrogen-bonded to the tyrosyl hydroxyl of the other. Molecules one

and three are connected by three water molecules forming bridging hydrogen

bonds between them. Molecules two and four are almost identical to one

and three. These tetrameric units are repeated in three dimensions by the

symmetry operations of the space group. The interactions between separate

tetramers are mainly hydrophobic; the two-fold axis is surrounded by Phe

and Leu side-chains (see figure 4b).

In the -turn structure there are two intramolecular hydrogen bonds that

connect the Tyr and Phe groups of each of the four independent molecules,

N(Tyr) to O(Phe) 2.79-2.84 and N(Phe) to O(Tyr), 2.99-3.10 . The six

water molecules take part in a variety of interactions. Two water molecules

form a bridge between the Phe4 carbonyl oxygen and the Leu5 carboxy

oxygen of one molecule. In each of the four independent molecules, both

the Gly2 nitrogen and the Gly3 carbonyl oxygen are hydrogen bonded to

water molecules.

There are patterns of solvation common to more than one of the enkephalin

crystal structures; that is, water molecules bind to the same atoms even

though the packing in the structures may vary. The waters common to more

than one structure are given in table 4.

49
TABLE 3
FIGURE 4

(a) Stereo view of the tetramer that makes up the asymmetric unit in LE

Large circles are water molecules. (b) Stereo view of the packing of a unit

cell, 16 molecules, in LE Note the hydrophobic holes formed by tyrosyl,

phenylalanyl, and leucyl residues.

51
TABLE 4

Common patterns of solvation in enkephalin crystal structures:

atoms of enkephalin molecules hydrogen-bonded to water.

LE LEE4 LEE2 MEE2*

CO-(leu5) CO-

(leu5) CO-

(leu5) CO-

(met5)

OH(tyr 1) OH(tyr1

) OH(tyr1

) OH(tyr1

)

N

+

(tyr1) N

+

(tyr1)

C=O(gly2) C=O(gly2

)

C=O(phe4) C=O(phe4

) C=O(phe4

)

*The authors do not have the crystallographic coordinates for MEE2, and

MEBrE2, so the solvent structure cannot be calculated.

Conformation of Enkephalin at the μ and Receptor

Since the demonstration of the existence of multiple forms of opioid

receptors, arguments have been made that the μ and subtypes bind different

con ormations of the endogenous opioids. Soos et al. (1980) suggested this f

on the basis of CD spectra on the enkephalins and enkephalin analogues.

Raman spectra was interpreted as showing more folded conformations of

[Leu5]enkephalin than [Met5]enkephalin in aqueous solution and DMSO

(Renugopalakrishnan et al. 1985). The observed solid state conformations

led to the proposal that [Leu5]enkephalin binds to the μ receptor in a folded

conformation and [Met5]enkephalin to the s in an extended conformation

(lshida et al. 1984). Recently Doi et al. (1987b) suggested that a pair of

bend molecules of enkephalin can mimic the -sheet dimer, placing similar

functional groups in the same regions of three-dimensional space, and bind

to the receptor. In a subsequent paper the same group (Doi et al. 1987a)

suggested that the -turn monomer and the extended dimer could each bind

to both μ and subtypes of the opioid receptor. They based this conclusion

on computer graphics and empirical energy studies of the two conformations,

adjusting the tyrosyl and phenylalanyl side chains on both conformations to

superimpose the analogous residues in the same regions of three-dimensional

space. This hypothesis requires further testing by biochemical techniques.

The -turn structure of [Leu5]enkephalin was initially proposed to be the

biologically active conformation (Smith and Griffin 1978). The conclusion

was based on a least-squares superposition of the -turn conformation of

enkephalin with the structures of morphine, etorphine and PET, 7-(1-phenyl3-hydroxybutyl-3]-endoethenotetrahydrothebaine, the latter two superactive

agonists. In addition, the fact that the torsion angles of Gly2 place the

conformation in a region of conformational space allowed for D-amino acids,

and D-Ala2 substituted enkephalins had been shown to be very active,

supported the proposal. These arguments were made before the existence of

opioid receptor subtypes was demonstrated and the selectivity of the

52
individual subtypes shown experimentally. The activity of etorphine and the

tetrahydrothebaines at both u and s opioid receptor subtypes, however, has

not been accounted for in the suggestion that u and s subtypes bind different

conformations of the enkephalins.

The fully extended conformation of a small peptide can form the maximum

number of intermolecular hydrogen bonds. In going from the extended form

to the type I’ -turn conformation centered on Gly2Gly3

, the enkephalin

molecule decreases the number of hydro en bonding groups available for

receptor interactions and increases the surface hydrophobicity. This may be

relevant to the conformation assumed at the receptor(s), since the morphineand tetrahydrothebaine-like molecules are more hydrophobic than enkephalin.

OPIATE DRUGS

Naltrexone Derivatives

FUNALTREXAMINE

- and -Funaltrexamine ( - and -FNA) are naltrexone derivatives differing

only in chirality at C6. Both compounds bind to the μ opioid receptor in

mouse vas deferens (MVD) and guinea pig ileum (GPI) preparations, but

only the -epimer binds irreversibly, presumably by forming a covalent bond

to the receptor. For this reason, -FNA has been used to irreversibly block u

receptors in order to isolate the binding characteristics of receptors in GPI

and MVD preparations, and Sites in brain homogenate preparations.

A two-step recognition process had been proposed to account for the

different binding characteristics of and -FNA for the μ receptor; both

bind in the first recognition step but only the -epimer is in the proper

orientation for the second recognition step which results in alkylation. The

crystal structures of - and -FNA suggested a possible explanation for the

observed differences (Griffin et al. 1986b). The two compounds have almost

identical conformations in the fused rings with the exception of the C ring;

the -epimer has a chair conformation and the -epimer a twist-boat

conformation, resulting in the fumaramate side chain being equatorial to the

C ring in both cases. Analysis of the superposition of the two structures

5 3
FIGURE 5

Stereo view of the superposition of the crystallographically observed

structures of - and -funaltrexamine, with the proposed sites of receptor

interaction. The first recognition step involves the phenol ring. the phenol

hydroxyl, and the charged nitrogen. The second recognition step involves

the fumaramate group and possibly the amino nitrogen at C6 (from Griffin et

al., 1986b, Copyright 1986, American Chemical Society).

5 4
revealed that the fumaramate groups occupied the same region with respect

to the naltrexone frame in both structures, but the conjugated fumaramate

roups were oriented orthogonal to one another. A close oxygen contact (03

from a symmetry related molecule) to the double bond carbon (C23)

observed in the crystal structure was used to model this second recognition

site and explain the failure of the -epimer to irreversibly bind to the

receptor (see figure 5).

The conformation of the C ring of naltrexone derivatives appears to be

sensitive to substitution at C6. The crystal structure of the 6 -amino

derivative of naltrexone, 6 -oxymorphamine (Lever et al. 1985) confirmed

their conclusions based on lH NMR that the C ring in the 6 -amino epimer

is in a twist-boat conformation and in the -epimer is a chair. As in the

FNA structures this places the 6 substituent in the equatorial position in both

epimers. Comparison of the C ring torsion angles in -FNA and 6 -

oxymorphamine shows they have almost identical conformations.

Acylmorphinans

Although the phenolic hydroxyl group is considered essential to activity in

morphine and structurally related analogues, its presence results in rapid

inactivation when given by oral route. Mohacsi et al. (1985) have reported

the crystal structure of a C3-acyl morphinan that retains morphine-like

analgesic activity even when administered orally. The 03 atom could be

serving as a hydrogen bond acceptor, since the phenolic oxygen in morphine

can act as both donor and acceptor of hydrogen bonds. The oxygen to

nitrogen distance in the acylmorphinan structure is 7.94 , whereas in the 30

crystal structures containing the morphine five fused-ring system in the

Cambridge Structural Database (Allen et al. 1979). the value of the distance

ranges from 6.77 to 7.16 , average 7.03(11)

Conformations of the Morphine Fused-Ring System

Brown et al. (1983) reported that the conformation of morphine in the crystal

is different from that in aqueous solution, and that the latter is the one

“available for receptor binding, ” although no evidence or explanation for this

assertion was included in the report. Their study was based on a comparison

of the high resolution 13C NMR spectra of solid morphine sulfate versus

solid morphine free base, solid morphine sulfate versus morphine sulfate

dissolved in D2O, and morphine sulfate dissolved in D2O at 22°C and 70°C.

Their data were interpreted as showing a minor component of the nitrogen

invertoisomer in solution, the nitrogen methyl went from equatorial to axial,

and the inversion was associated with a “greater freedom of motion in the

piperidine ring than previously believed.” They speculated that the

downfield chemical shifts of the spectral lines assigned to C2, C7, C11, C15,

and C16 on going from solution to solid were due to interactions in the solid

state between these atoms and the sulfate group. The authors did not

identify the crystalline polymorph they were examining. Solid state spectral

studies should always verify the identity of the crystals studied by means of

5 5
powder or single crystal X-ray diffraction to determine cell constants and

space group. Without this information, no meaningful comparison can be

made between the spectral studies and crystallographic results. The change

in chemical shifts are difficult to explain from the crystal structure of

morphine, 0.5 SO4, 2.5 H2O (Wongweichintana et al. 1984); although C2,

Cl5, and Cl6 show close contacts to sulfate oxygens in the crystal (3.39 to

3.49 ), C7 and C11 do not, while C3 and the N-methyl carbon also show

short contacts.

The Cambridge Structural Database (Allen et al. 1979) contains 30 crystal

structure determinations of compounds that contain the morphine five fusedring framework. In this data base the methyl substituent on the piperidyl

nitrogen is always observed equatorial and there is little evidence for great

freedom of motion in the piperidine ring especially at C15 and C16. The

piperidyl ring is always a chair, and the torsion angle C13-C15-C16-N only

varies from -47° to -57° in the entire sample, average 51.2 (2.0)°. The main

region of flexibility is ring C, and the changes in ring C conformation can be

correlated with changes in chemical constitution, either a double bond at C6.

C7 or C7, C8. or the substitution of a nitrogen on C6, as discussed above.

There are also changes in the three carbon-nitrogen bonds of the piperidyl

nitrogen correlated with whether the structure is the free base or salt.

SUMMARY

Information on intramolecular geometry, low energy conformations,

hydrogen bonds, both intramolecular and intermolecular, and preferred

intermolecular interactions is obtained from single crystal X-ray diffraction

experiments. Structure determinations of crystals with more than one

molecule in the asymmetric unit and of different crystalline forms provide

information on conformational flexibility and stable aggregation states.

The single crystal X-ray diffraction studies of enkephalin have demonstrated

a number of minimum energy conformations of the main chain and side

chains, a type I’ turn centered on Gly2Gly3 and a number of extended

main-chain conformations. In the studies of native enkephalins, dimers and

tetramers form repeating units, and some patterns of solvation are observed

in more than one form. Certain conformations and interactions proposed on

the basis of solution spectroscopic studies have yet to be observed in the

solid state. Efforts should be made to grow different crystalline forms of the

enkephalins and to determine the solid state structure of enkephalin

analogues that exhibit high selectivity for a particular opioid receptor

subtype.

Crystallographic studies of opiate drugs with selective activity profiles at

different receptor subtypes can give infomration on the features responsible

for active site differenttation between the subtypes of opioid receptor.

Analysis of the crystallographic data base of 30 compounds containing the

five fused-ring morphine moiety indicate that: (1) the methyl group on the

56
piperidyl nitrogen prefers the equatorial position, (2) the conformation of the

piperidyl ring is a relatively invariant chair conformation, (3) C-ring

conformation depends on the chemical constitution of the C-ring and

substitution at C6.

REFERENCES

Allen, F.A.; Bellard, S.; Brice, M.D.; Cartwright, B.A.; Doubleday, A.;

Higgs, H.; Hummelink, T.; Hummelink-Peters, B.G.; Kennard. 0.;

Motherwell, W.D.S.; Rodgers, J.R.; and Watson, D.G. The Cambridge

Crystallographic Data Centre: Computer-based search, retrieval, analysis

and display of information. Acta Cryst B35:233l-2339, 1979.

Beretta. C.A.; Parrilli, M.; Pastore, A.; Tancredi, T.; and Temusi. P.A.

Experimental simulation of the environment of the opioid receptor. A

500 MHz study of enkephalins in CDC13. Biochem Biophys Res

Commun 121:456-462, 1984.

Blundell, T.L.; Hearn, L.; Tickle, I.J.; Palmer, R.A.; Morgan, B.A.; Smith,

G.D.; and Griffin, J.F. Crystal structure of [Leu5]enkephalin. Science

205:220, 1979.

Brown, C.E.; Roerig, S.C.; Fujimoto, J.M.; and Burger, V.T. The structure

of morphine differs between the crystalline state and aqueous solution. J

Chem Soc Chem Commun 1983: 1506-1508, 1983.

Camerman,A.,an Camerman, N. Conformational features of the opioid

peptides in the solid state: A review of X-ray crystallographic research.

In: Rapaka, R.S.; Bamett, G.; and Hawks, R.L., eds. Opioid Peptides:

Medicinal Chemistry. National Institute on Drug Abuse Research

Monograph 69. DHHS Pub. No. (ADM) 86-1454. Washington, D.C.:

Supt Docs., U.S. Govt. Print. Off., 1986, pp. 351-363.

Camerman, A.; Mastropaolo, D.; Karle, I.L.; Karle, J.; and Camerman, N.

Crystal structure of leucine-enkephalin. Nature 306:447-450, 1983.

Doi, M.; Ishida, T.; Inoue, M.; Fujiwara, T.; Tomita, K.-i.; Kimura, T.;

and Sakakibara, S. Three-dimensional structure of monoanionic

methionine-enkephalin: X-ray structure of tert-butyloxycarbonyl-Tyr-GlyGly-(4-bromo)Phe-Met-OH. FEBS Lett 170:229-231, 1984.

Doi, M.; Tanaka, M.; Ishida, T.; and Inoue, M. The three-dimensional

similarity between a dimeric antiparallel extended structure and a -turn

folded form of enkephalin. FEBS Lett 213:265-268, 1987a.

Doi. M.; Tanaka, M.; Ishida,T.; Inoue, M.; Fujiwara, T.; Tomita, K-i.:

Kimura, T.; Sakakibara, S.; and Sheldrick, G.M. Crystal structures

of[Met5] and [(4-bromo)Phe4 , [Met5]enkephalins: formation of a dimeric

antiparallel -structure. J Biochem 101:485-490, 1987b.

Foumie Zaluskie. M-C.; Prange,T.; Pascard,C.; and Roques, B.P.

Enkephalin related fragments. Conformational studies of the

tetrapeptides Tyr-Gly-Gly-Phe and Gly-Gly-Phe-X (X = Leu, Met) by Xray and 1H NMR spectroscopy.

79:1199-1206, 1977.

Biochem Biophys Res Commun

Griffin, J.F.; Langs, D.A.; Smith, G.D.; Blundell, T.L.; Tickle, I.J.; and

57
Bedarkar, S. The crystal structures of [Met5

]enkephaIin and a third form

of [Leu5]enkephalin: Observations of a novel pleated -sheet. Proc Natl

Acad Sci USA 83:3272-3276, 1986a.

Griffin.J.F.;Langs, D.A.; Smith, G.D.: Van Roey, P.V.R.; Blundell, T.L.;

Tickle, I.J.; and Hearn, L. The crystal structures of [Met5]enkephalin

and a third form of [Leu5]enkephalin. Abstracts of the Annual Meeting

of the American Crystallographic Association, Stanford University,

Stanford, California, Series 2, Volume 13, Abstract No. PC36, p. 61,

1985.

Griffin, J.F.; Larson, D.L.; and Portoghese, P.S. Crystal structures of aand -funaltrexamine: conformational requirement of the fumaramate

moiety in the irreversible blockage of u opioid receptors. J Med Chem

29:778-783, 1986b.

Hughes, J.; Smith, T.W.: Kosterlitz, H.W.; Fothergill, L.A.; Morgan, B.A.;

and Morris, H.R. Identification of the two related pentapeptides from

the brain with potent opiate agonist activity. Nature (London)

258:577-579, 1975.

Ishida. T.: Kenmotsu, M.; Mino, Y.; Jnoue. M.; Fujiwara, T.; Tomita,K-i.;

Kimura, T.; and Sakakibara, S. X-ray diffraction studies of enkephalins,

crystal structure of [4’-bromo)Phe4,Leu5]enkephalin. Biochem J

218:677-689, 1984.

Ishida. T.; Tanabe, N.; and Inoue, M. Structure of tertbutoxycarbonylglycylglycyl-L-phenylalanine ethyl ester, C20H29N3O6

.

Acta Cryst C39:110-112, 1983.

Khaled, M.A Conformations of opioid peptides as determined by nuclear . .

magnetic resonance and related spectroscopies. In: Rapaka, R.S.;

Bamett, G.; and Hawks, R.L., eds. Opioid Peptides: Medicinal

Chemistry. National Institute on Drug Abuse Research Monograph 69

DHHs Pub. No. (ADM) 86-1454 Washington, D.C.: Supt. of Docs.,

U.S. Govt. Print. Off., 1986. pp. 266-290.

Karle, J.L.; Karle, J.; Mastropaolo, D.; Camerman, A.; and Camerman, N.

[Leu5]enkephalin: Four cocrystallizing conformers with extended

backbones that form an antiparallel -sheet.

1983.

Acta Cryst B39:625-637,

Lever, O.W.. Jr.: Bhatia, A.V.: and Chang, K.-J. Opioid receptor

interactions and conformations of the 6 and epimers of

oxymorphamine. Solid-state structure of 6 -oxymorphamine. J Med

Chem 28:1652-1656, 1985.

Mastropaolo, D.; Camerman, A.; and Camerman, N. Crystal structure of

methionine-enkephalin. Biochem Biophys Res Commun 134:698-703,

1986.

Mastropaolo. D.; Camerman, A.; Ma, L.Y.Y.; and Camerman, N. Crystal

structure of an extended-conformation leucine-enkephalin dimer

monoydrate. Life Sciences 40:1995-1999, 1987.

Mohacsi, E.; O’Brien, J.; Blount. J.; and Sepinwall, J. Acylmorphinans. A

novel class of potent analgesic agents. J Med Chem 28:1177-1180,

1985.

Prasad, B.V.V.; Sudha, T.S.; and Balaram, P. Molecular structure of BocAib-Aib-Phe-Met-NH2

. DMSO. A fragment of a biologically active

enkephalin analogue. J Chem Soc Perkin I 1983:417-421, 1983.

Renugopalakrishnan, V.; Rapaka, R.S.; Colllete, T.W.; Carriera, L.A.; and

Bhatnagar, R.S. Conformational states of Leu5- and Met5-enkephalins in

solution. Biochem Biophys Res Commun 126:1029-1035, 1985.

5 8
Schiller, P.W. Conformational analysis of enkephalin and conformationactivity relationships. In: Udenfriend, S., and Meienhofer, J., eds. The

Peptides: Analysis, Synthesis, Biology. Vol. 6. New York: Academic

Press, 1984. pp.219-268

Smith, G.D., and Griffin, J.F. Conformation of [Leu5]enkephalin from Xray diffraction: Features important for recognition at opiate receptor.

Science 199:1214-1216, 1978.

Soos, J.; Berzetei, I.; Bajusz, S.; and Ronai, A.Z. Correlation between

circular dichroism data and biological activities of 2,5 substituted

enkephalin analogues. Life Sciences 27:129-133, 1980.

Stezowski, J.J.; Eckle, E.; and Bajusz. S. A crystal structure determination

for Tyr-D-Nle-Gly-Phe-NleS [NleS=MeCH2 CH2 CH2 CH(NH2)SO3H]: An

active synthetic enkephalin analogue. J Chem Soc Chem Commun

1985:681-682, 1985.

Wongweichintana, C.; Holt, E.M.; and Purdie, N. Structures of morphine

methyl iodide monohydrate, (C18 H22 NO3)

+

 .I-.H2O, and di(morphine)

dihydrogensulfate pentahydrate 2(C17H

20NO3

)

+

.SO4

2-.5H2O. Acta Cryst

C40:1486-1490, 1984.

AUTHORS

Jane F. Griffin, Ph.D.

G. David Smith, Ph.D.

Molecular Biophysics Department

Medical Foundation of Buffalo, Inc.

73 High Street

Buffalo, New York 14203, U.S.A.

59
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Conformational Analysis of

Cyclic Opioid Peptide Analogs

Peter W. Schiller, Ph.D., and Brian C. Wilkes, Ph.D.

INTRODUCTION

Extensive conformational studies of the enkephalins [H-Tyr-GlyGly-Phe-Met(or Leu)-OH] by various spectroscopic techniques and

by X-ray diffraction analysis revealed that these linear

pentapeptides are highly flexible molecules capable of assuming a

number of both folded and extended conformations of comparably

low energy (Schiller 1984). In fact, it has been demonstrated

quite convincingly that in solution enkephalin exists in a

conformational equilibrium (Fischman et al. 1978). It has,

therefore, become clear that conformational studies of these

small opioid peptides and of many of their analogs are unlikely

to provide any insight into their bioactive (receptor-bound)

conformation(s). Furthermore, the conformational flexibility of

the enkephalins is most likely the reason for their lack of

specificity toward the different opioid receptor classes

because conformational adaptation to the various

receptor topographies is possible. Thus, even though the

enkephalins preferentially bind to the -receptor, they also

have quite good affinity for the μ-receptor and, therefore, are

only moderately -receptor selective.

CONFORMATIONALLY RESTRICTED ANALOGS OF OPIOID PEPTIDES

The conformational flexibility of opioid peptides can be reduced

through incorporation of conformational constraints. In recent

years conformationally restricted analogs of enkephalin have been

synthesized with two goals in mind. First, such analogs are

more amenable to a meaningful conformational analysis since

because of their relatively rigid structure they are unlikely to

undergo major conformational changes upon binding to the receptor

and, therefore, Information about the bioactive conformation at

the various opioid receptors can be obtained. Second, the

introduction of conformational constraints may result in improved

receptor selectivity, because the conformationally restricted

analog may have good affinity for one receptor type but may no

longer be able to undergo a conformational change necessary to

60
bind to another receptor class. Local conformational

restrictions in enkephalins have been achieved either at

particular peptide backbone positions (e.g., through

methylation or through incorporation of small ring structures) or

in selected side chains (e.g., through substitution of a

dehydroamino acid) (for a review, see Schiller 1984). However,

the most drastic restriction of the overall conformational space

available to the peptide has been realized through the design and

synthesis of cyclic enkephalin analogs.

Three families of biologically active cyclic opioid peptide

analogs have been synthesized to date. A cyclic enkephalin

analog was first prepared through substitution of Ddiaminobutyric acid (A2bu) in position 2 of the peptide sequence

followed by cyclization between the side chain amino group of

A2bu and the C-terminal carboxyl group (DiMaio and Schiller

1980). The resulting analog, H-Tyr-cyclo[-D-A2bu-Gly-Phe-Leu-]

(figure 1, compound 2), containing a fairly rigid 14-membered

ring structure, turned out to be moderately μ-receptor selective

(Schiller and DiMaio 1982). Pharmacologic comparison of 2 with a

corresponding open-chain analog revealed that its μ-receptor

preference is a direct consequence of the conformational

restriction introduced through ring closure and, furthermore,

permitted the fundamental conclusion that μ- and -opioid

receptors differ indeed from one another in their conformational

requirements toward peptide ligands (Schiller and DiMaio 1982).

Homologs of 2 (compounds 1, 3, and 4) were obtained through

variation of the side chain length in position 2 and also were

found to display moderate preference for μ-receptors over sreceptors (DiMaio et al. 1982). On the other hand, a

diastereomer of cyclic peptide 2, H-Tyr-cyclo[-D-A2bu-Gly-Phe-DLeu-], was non selective (Mierke et al. 1987). Several partial

retro-inverso analogs of 2 were found to be very potent and μselective (figure 1, structures 2b and 2c), whereas both

diasteromers of 2d showed very weak activity (Berman et al. 1983;

Richman et al. 1985), presumably because the Gly3 -Phe4 peptide

bond is important for binding to the receptor.

A second class of cyclic enkephalin analogs was obtained through

side chain-to-side chain cyclization between a D-Cys and a D- or

L-cys residue substituted in positions 2 and 5 of the peptide

sequence. respectively (Schiller et al. 1981). The resulting

cystine-containing analogs (5)

were highly potent and showed either no receptor selectivity (X =

NH2) or moderate -receptor selectivity (X = OH) (Schiller et

al. 1985a). The Trp4 analog NH2 (6)

displayed an activity profile similar to that of the

corresponding Phe4 parent compound (Schiller 1983). Replacement

of the half-cystine residues in positions 2 and/or 5 with

penicillamine residues resulted in compounds (7-7c) with markedly

improved -receptor selectivity (Mosberg et al 1983).

The configurational requirements of cyclic analogs 4 and 5 at the

residues in positions 1, 2, 4, and 5 of the peptide-sequence were

61
FIGURE 1

Structural Formulas of Cyclic Opioid Peptide Analogs
found to be the same as those of the corresponding residues in

linear enkephalins (Schiller and DiMaio 1983). This finding as

well as other structure-activity data obtained with these

compounds indicated that corresponding moieties in these cyclic

and linear enkephalin analogs interact with the same subsites on

the receptor. It can thus be concluded that both H-Tyr-cyclo[-DLys-Gly-Phe-Leu-] and NH2 have the same

mode of binding to the receptor as the linear enkephalins.

Another family of side chain-to-side chain cyclized opioid

peptide analogs was obtained through amide bond formation between

the side chain amino and carboxyl groups of appropriately

substituted Orn (or Lys) and Asp (or Glu) residues (Schiller and

Nguyen 1984; Schiller et al. 1985b, 1987). Among the various

cyclic lactam analogs of this type prepared,

NH2 (8) turned out to be potent and highly μ-selective. The

latter analog contains a rather rigid 13-membered ring structure

and a Phe residue in the 3-position as it is the case with the

dermorphins and the -casomorphins. Among several prepared

analogs of 8,

(10) showed the same high μ-selectivity as the parent

NH2 (9) and

compound, whereas (11) was less μselective due to reduced affinity for the μ-receptor. Analogs H-

(11) and (13)

showed very weak activity. Taken together, the structureactivity data obtained with analogs of

indicated that the mode of binding of the latter peptide

identical with that of the dermorphins but different from that of

the casomorphins (Schiller et al. 1987).

CONFORMATIONAL ANALYSIS OF CYCLIC OPIOID PEPTIDE ANALOGS

Side Chain-to-End Group Cyclized Enkephalin Analogs

The conformational behavior of the cyclic prototype analog H-Tyrcyclo[-D-A2bu-Gly-Phe-Leu-] (2) has been investigated by various

groups, using a variety of theoretical and experimental

techniques. In a first theoretical conformational analysis, a

molecular mechanics approach was used to determine low energy

conformers of 2 (Hall and Pavitt 1984a). The lowest energy

conformer obtaized was characterized by a Gly3 -Phe4 type II'

bend, and it was suggested by the authors that the same type of

bend might also be present in the receptor-bound conformation of

linear enkephalins. Furthermore, some of the low energy

conformers found showed a transannular hydrogen bond between the

side chain NH of A2bu and the A2bu carbonyl group. Another

energy minimization study of cyclic analog 2 resulted in a number

of low energy conformers that contained either relatively planar

ring structures stabilized by intramolecular hydrogen bonds

turns or C7 structures) or twisted ring structures devoid of any

hydrogen bonds (Maigret et al. 1986). Furthermore, the authors

postulated that the tyramine portion of Tyr1 and the Gly3

carbonyl group in 2 might correspond to the tyramine portion and

the C-ring hydroxyl group contained in morphine. It was then

63
attempted to impose spatial overlap of these pharmacophoric

moieties with those in the rigid morphine molecule through

conformational adaptation of each one of the low energy

conformers of cyclic peptide 2. This was possible at a

relatively low energy expenditure (6 kcal/mol) with only one of

the low energy conformers and resulted in a tilted structure

that was characterized by intramolecular hydrogen bonds from

Leu5-NH to A2bu-CO bend) and from Gly3-NH to Tyr1-CO (C7

structure). It was suggested that the latter conformation might

represent the bioactive one at the μ-receptor, since the 6

kcal/moL increase in energy as a consequence of the imposed fit

could certainly be compensated for by favorable binding

interactions at the receptor. It must be realized, however, that

this proposed model depends entirely on the authors' somewhat

arbitrary choice of the pharmacophore.

In a third study, both computer simulations and 1HNMR

spectroscopy were employed to determine the conformation(s) of

cyclic analog 2 (Mammi et al. 1985). The performed molecular

dynamics study showed that the ring structure of 2 is not

entirely rigid but fluctuates about a few equilibrium

conformations. The most stable ring conformations were found to

be stabilized by transannular hydrogen bonds implicated in the

formation of C7 structures. The carried out energy minimization

study resulted in one lowest energy conformer characterized by

hydrogen bonds from A2bu(side chain)-NH to A2bu-CO, from Leu5 -NH

to Gly3-CO and from Gly3-NH to Tyr1 -CO. The ring structure of

another obtained low energy conformer also showed a Leu5

Gly3 hydrogen bond, whereas the A2bu side chain NH was hydrogenbonded to the Phe4-CO rather than to the A2bu-CO. Determination

of the temperature dependence of the amide proton chemical shifts

in the 1HNMR study performed with cyclic analog 2 in

[

2H6]DMSO revealed that both the Leu5 NH and the side chain NH of

A

2bu may be involved in hydrogen bond formation. These results

are consistent with the hydrogen bonding patterns obtained in the

energy minimization and suggest that the solution conformation of

2 may indeed be stabilized by the transannular hydrogen bonds

observed in the calculated lowest energy conformers.

It is obvious that the results of these various conformational

studies on cyclic analog 2 have not yet led to a consensus

concerning a possible unique bioactive conformation at the

receptor. Clearly, the most important finding of these

endeavors is the realization that the ring structure in 2 still

retains some flexibility and that the various intramolecular

hydrogen bonds observed are constantly formed, broken, and reformed again, as shown most conclusively in the molecular

dynamics study.

In a recent study, the conformational behavior of H-Tyr-cyclo[-DA2bu-Gly-Phe-Leu-] (2) was compared with that of its diastereomer

with Leu5 in the D-configuration (analog 2a), using again a

combination of computer simulations and 1HNMR in [

2

H6]DMSO

(Mierke et al. 1987). The obtained results showed that, in

64
contrast to the L-Leu5 peptide, the conformation of the D-Leu5

analog is not stabilized by any intramolecular hydrogen bonds.

Furthermore, the NMR data (T1 relaxation time measurements) and

molecular dynamics study indicated that both the peptide backbone

and the side chains in 2a are considerably more flexible than in

2. In particular, the computer simulation carried out for 20

picoseconds revealed that the important intramolecular distance

between the Tyr1 and Phe4 aromatic rings remains constant (~20

in the case of the L-Leu5 analog, whereas considerable variation

of that same distance with time (5-16 is seen in the case of

the D-Leu5 analog. It was concluded that the more rigid

conformation and the relatively large and fixed distance between

the Tyr and Phe4

aromatic rings of analog 2 may be responsible

for its μ-receptor preference, whereas the Lack of receptor

selectivity shown by 2a may be due to its comparatively higher

structural flexibility The same approach was also used to

investigate the conformational behavior of partial retro-inverso

analogs of 2, characterized by a reversed amide bond in three

different positions of the ring structure (figure 1, compounds

2b, 2c, and 2d). Various transannular hydrogen bonds, giving rise

to C7 structures or also C6 and C8 structures, were detected in

the computer simulation study and where found to be compatible 

with the results of the NMR temperature perturbation study, which

permitted the identification of hydrogen-bonded amide protons.

No -turns were observed, as the conformational restriction

introduced through ring closure renders the formation of a

hydrogen bond energetically unfavorable. In all analogs the side

chains of

flexible,

the Phe4 and Leu5

residues were found to be very

whereas the Tyr side chain appeared to exist primarily

in the trans conformation 1 ~ 180°), which was favored by more

than 3 kcal/mol over gauche conformations.

Hall and Pavitt (1985) compared the Low energy conformations of

H-Tyr-cycLo[-D-A2pr-Gly-Phe-Leu-] (1) and H-Tyr-cyclo[-D-Orn-GlyPhe-Leu-] (3) with those of

(2), using the

H-Tyr-cyclo[-D-A2bu-Gly-Phe-Leu-]

same molecular mechanics approach as in their

original study of 2 (Hall and Pavitt 1984a). This investigation

resulted in a number of low energy ring conformations common to

all three analogs, which differed from one another primarily in

the orientation of a particular amide bond.

conformations

Several of these

contained a type II' bend centered on Gly3 -Phe4 ,

and it was speculated that one of these might represent the

bioactive conformation at the receptor. Kessler et al. (1985)

performed a 1H NMR study in [

2

H6]DMSO with H-Tyr-cyclor-DOrn-GLy-Phe-Leu-] (3) and H-Tyr-cyglo[-D-Lys-Gly-Phe-Leu-] (4).

The obtained NMR data indicated that cyclic analog 3 has- a

relatively rigid ring conformation, and the determination of the

temperature dependence of the amide proton chemical shifts

suggested the existence of two transannular hydrogen

bonds (Leu5

-NH OC-Gly3and Orn2OC-Orn2). The

analogous -turn and A2bu OC-A2bu hydrogen bond had also

been observed with cyclic analog 2 in the conformational study

described above (Mammi et al. 1985). In comparison with 3, the

ring conformation in cyclic analog 4 was found to be considerably

65
more flexible. The values and coupling constants

determined with 4 suggested the existence of a -turn centered

on Phe4-Leu5 and stabilized by a hydrogen bond bet een the

group of D-Lys2 and the carbonyl group of Gly3 . The ring

conformation of analog 3 defined by the two hydrogen bonds

detected in this study formed the basis for a recent computermodeling study aimed at determining topological similarities

between this cyclic peptide and 7- -[(1R)-1-hydroxy-1-methyl-3-

phenylpropyl]-6,14-endo-ethenotetrahydrooripavine (PEO) (DiMaio

et al. 1986).

Side Chain-to-Side Chain Cyclized Enkephalin Analogs

Using the molecular mechanics approach, Hall and Pavitt (1984b)

performed a theoretical conformational analysis of the potent,

non selective enkephalin analogs

NH2 (5). A Gly3 -Phe4 type II' bend was indicated by the obtained

results, as it had been the case with the H-Tyr-cyclo[-D-Xxx-GlyPhe-Leu-] analogs analyzed in the same manner by these authors

(see above). However, comparison of the 14-membered ring

structures in these two cyclic peptides with the 14-membered ring

of H-Tyr-cyclo[-D-A2bu-Gly-Phe-Leu-] (2) indicated a higher

structural flexibility in the case of the cystine-containing

analogs, which may explain their lack of receptor preference. In

a conformational study by fluorescence techniques, the tyrosine

fluorescence quantum yield determined with

-NH2 was found to be 2.5 times lower than that of the linear

analog H-Tyr-D-Ala-Gly-Phe-Met-OH (Schiller 1983), presumably due

to the quenching effect of the adjacent disulfide group, which is

known to occur at distances of less than 6 between the Tyr

phenol ring and the -S-S moiety. This observation suggested the

existence of an energetically favorable intramolecular complex

between the phenol ring and the disulfide group, which would

result in an interaction energy of about 1 kcal/mol at an optimal

distance of 3.5 between the plane of the aromatic ring and one

of the sulfur atoms. On the basis of these findings and

arguments, a close interaction between the tyrosine side chain

and the disulfide bridge in -NH2 was

proposed.

ring of Tyr1 and the indole moiety of Trp4 was measured

Singlet-singlet energy transfer between the phenol

in the

analog -NH2 (6). Evaluation of the

determined fluorescence parameters on-the basis of Förster's

equation resulted in an average intramolecular distance of 9.7 ±

0.2 between the two aromatic rings contained in 6. This mean

distance is nearly identical with the average Tyr1-Trp4 distance

(9.5 ± 0.3 in the linear enkephalin analog H-Try-D-Ala-GLyTrp-Met-OH, which was determined by the same technique.

The conformations of the two non selective cystine-containing

analogs -NH2 and

Cys-NH2 (structures 5) were compared with those of the related sselective Pen2-containing analogs -NH2

and -NH2 (structures 7) in a 1HNMR study

carried out in D2O (Mosberg and Schiller 1984), Similar chemical

66
shifts, values, and coupling constants were observed for

corresponding penicillamine and cysteine containing analogs,

indicating similar overall conformations. However, the obtained

NMR data suggested that, in comparison with the corresponding

CYS2 analogs, the Pen2 analogs show higher structural rigidity in

the C-terminal part of the molecule, which may explain their

receptor selectivity. The determined temperature dependences of

the amide proton chemical shifts did not indicate the existence

of intramolecular hydrogen bonds in any of these four cyclic

analogs. The large chemical shift difference observed for the

two penicillamine methyl resonances in both Pen2 analogs are

indicative of a ring current effect caused by the tyrosyl

aromatic moiety and, as in the case of H-Tyr-DNH2, (see above), suggest a close proximity between the Tyr1

aromatic ring and the disulfide moiety.

The same type of NMR analysis was then performed with six

penicillamine-containing cyclic enkephalin analogs (structures

7a, 7b, and 7c), all of which display -receptor selectivity

(Mosberg 1987), Some variation in various NMR parameters was

observed even between compounds that displayed similar potency

and receptor selectivity. These findings were interpreted to

indicate that these analogs must contain the crucial

pharmacophoric elements in a similar spatial disposition,

presumably as a consequence of the observed conformational

flexibility of the Gly3 residue, which allows for conformational

compensation. Measurement of the temperature dependence of the

amide proton chemical shifts indicated that most amide protons in

these analogs were fully exposed to the solvent. The exceptions

were the D-Pen5 amide protons in [D- 2 5]enkephalin and 2 5]enkephalin, which showed low values,

indicating a possible involvement in an intramolecular hydrogen

bond. Interestingly, very good agreement of all NMR parameters

was Observed between [D- 2

Pen5]enkephalin,

5]enkephalin and [D- 2

suggesting very similar conformations of these

two compounds. The latter analog is about six times more

receptor selective than the former as a consequence of its poor

affinity for the μ-receptor.

improved -receptos selectivity of [D- 2 5]enkephalin as

These results indicate that the

compared to [D- 2 5]enkephalin is not due to a different

conformational behavior but rather to the presence of the gem

dimethyl group of the Pen2 residue, which causes more severe

steric interference at the μ-receptor than at the -receptor.

Belleney et al. (1987) performed a comparative 1HNMR study with

H-Tyr-D- -OH and H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH

(DTLET)in[2H6]DMSO In contrast to the data obtained by

Mosberg (1987) with H-Tyr-D- -OH in D2O, low

values were observed for the amide proton chemical shifts of both

L-Pen3 and Gly3 in the case of the cyclic peptide. These data

were interpreted to indicate that the conformation(s) of H-Tyr-D-

-OH4in [

2H6]DMSO might contain two turns either

a -turn around Phe or a II' turn centered on Gly3-Phe4 in the

C-terminal region and a -turn centered on Pen2 in the N-terminal

67
region. In the case of DTLET, the obtained values also

suggested the existence of a -turn and a -turn in the N- and Cterminal region of the peptide, respectively, and, thus, H-Tyr-Dconformations in DMSO. This type of backbone conformation

-OH and DTLET appear to have similar backbone

together with the similar arrangement of the Tyr1 and Phe4 side

chains in both the cyclic and the linear peptide was suggested to

be responsible for the observed preferential interaction with the

-receptor. Furthermore, the authors proposed on the basis of

these models that the two threonine methyl groups in DTLET might

play the same role in the interaction with the receptor as the

gem dimethyl groups of the two Pen residues in the cyclic

peptide.

Cyclic Lactam Analogs Containing the Phenylalanine Residue in the

3-Position

Recently, Wilkes and Schiller (1987) applied a systematic

procedure for the determination of the allowed low energy

conformations of the highly μ-receptor selective cyclic analog HTyr-D- -NH2 (8), using the software package SYBYL

(Tripos Associates, St. Louis, MO). A comprehensive grid search

of the 13-membered ring structure of 8 lacking the exocyclic Tyr1

residue and the Phe3 side chain resulted in only four low energy

conformers. These four conformations showed considerable

similarity, being all fairly round and flat. The three amide

bonds within the ring were generally either perpendicular to the

plane of the ring or slightly tilted toward the ring center, but

no linear transannular hydrogen bonds were observed. The

exocylic Tyr1 residue and Phe3 side chain were then added to

these four low energy conformers and an extensive energy

minimization was carried out with each on

results indicated that the Tyr1 and Phe3 side chains en joy

of them. The obtained

considerable orientational freedom but nevertheless only a

limited number of low energy side chain configurations were

found. The lowest energy conformer obtained by this approach was

characterized by a tilted stacking arrangement of the two

aromatic rings (figure 2, structure 8). However, several other

conformers with different side chain configurations were found to

be only slightly higher in energy (1 kcal/mol or less above the

energy minimum).

This conformational analysis has recently been extended to

several 13-membered ring cyclic analogs related to 8

which show considerable diversity in their μ-receptor affinity

and selectivity (figure 1, compounds 9-13) (Wilkes and Schiller

1988). Again, the analyses performed with the bare ring

structures resulted in no more than four low energy conformers

(within 2 kcal/mol of the lowest energy structure) in all cases.

The gross topological features of all the low energy ring

conformations observed with these analogs were similar to those

of the low energy conformers of the cyclic parent peptide 8.

Thus, in all cases no transannular hydrogen bonds were found.

Only two low energy ring conformations were obtained in the case

of compound 11, indicating that the additional conformational

68
FIGURE 2

Lowest Energy Conformers of Cyclic Opioid Peptide Analogs

8, 10, 12 and 13.

69
constraint introduced by N-methylation of the Phe3 residue led to

a further increase in structural rigidity. After addition of the

exocyclic Tyr1 residue and the Phe3 side chain to the low energy

ring conformations of analogs 9-13 low energy side chain

configurations were again determined by extensive energy

minimization (figure 2). The two potent and μ-selective analogs

9 and 10 showed a tilted stacking arrangement of the two aromatic

rings in their lowest energy conformations similar to that

observed in the lowest energy conformer of 8. Among the analogs

with reduced μ-receptor affinity, compound 11 showed a lowest

energy conformation characterized by a fully stacked parallel

arrangement of the two aromatic rings rather than a tilted

stacking interaction. Analysis of the weak μ-agonist 12 revealed

that chiral inversion at the 2-position precludes a low energy

stacking configuration. The lack of stacking of the aromatic

rings observed in the low energy conformers of the weakly active

analog 13 appears to be due to steric interference of the bulky

N-methyl group at the 3-position residue. Taken together, these

results suggest that a specific tilted stacking interaction of

the aromatic rings in the 1- and 3-position of cyclic opioid

peptide 8 and its analogs may represent an important structural

requirement for binding at the μ-receptor. However, it should be

kept in mind that other side chain configurations were found to

be only slightly higher in energy and that a change in side chain

conformations could occur upon binding to the receptor at an

energy expense, which could be compensated for by part of the

binding energy.

CONCLUSION

Conformational studies of three families of cyclic opioid peptide

analogs carried out to date have revealed that the ring

structures contained in these compounds are not entirely rigid

but, depending on the type and size of the ring, undergo more or

less extensive conformational fluctuations. Nevertheless, the

results of the performed molecular mechanics and molecular

dynamics studies indicate that these structural fluctuations are

relatively minor in the case of 13- or 14-membered rings and that

the various Low energy ring conformers observed for a given

cyclic analog do not differ very much in their overall shape from

one another. As expected, the results various conformationa

analyses indicate that the exocyclic Tyr1 residue and the Phe3

side chain still enjoy considerable orientational freedom. In

order to determine the distinct conformational requirements of

the μ - and the -receptor, it will be necessary to prepare and

characterize cyclic opioid peptide analogs in which the

conformational freedom of these important pharmacophoric moieties

is restricted in various ways.

FOOTNOTES

1

Abbreviations: A2bu, -diaminobutyric acid; A2pr,

diaminopropionic acid; DTLET, H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH;

NMR, nuclear magnetic resonance; Pen, penicillamine; Phe(NMe),

-methylphenylalanine; Phg, phenylglycine.

70
REFERENCES

Belleney, J.; Roques, B.P.; and Fournié-Zaluski, M.-C. Comparison

of conformational properties of Linear and cyclic selective

opioid Ligands DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) and DPLPE

(Tyr-c[D-Pen-Gly-Phe-Pen]) by 1H n.m.r. spectroscopy. Int J

Peptide Protein Res 30:356-364, 1987.

Berman, J.M.; Goodman, M.; Nguyen, T.M.-D.; and Schiller, P.W.

Cyclic and acyclic partial retro-inverso enkephalins: mu

receptor selective enzyme resistant analogs.

Res Commun 115:864-870, 1983.

Biochem Biophys

DiMaio, J., and Schiller, P.W. A cyclic enkephalin analog with

high in vitro opiate activity. Proc Natl Acad Sci USA 77:7162-

7166, 1980.

DiMaio; J.; Nguyen, T.M.-D.; Lemieux, C.; and Schiller, P.W.

Synthesis and pharmacological characterization in vitro of

cyclic enkephalin analogues: Effect of conformational

constraints on opiate receptor selectivity. J Med Chem 25:1432-

1438, 1982.

DiMaio, J.; Bayly, C.I.; Villeneuve, G.; and Michel, A.

Topological similarities between a cyclic enkephalin analogue

and a potent opiate alkaloid: A computer-modeling approach. J

Med Chem 29:1658-1663, 1986.

Fischman, A.J.; Riemen, M.W.; and Cowburn, D. Averaging of 2 and

3 in [5-leucyl]enkephalin. FEBS Lett 94:236-240, 1978.

Hall, D., and Pavitt, N. Conformation of a cyclic tetrapeptide

related to an analog of enkephalin. Biopolymers 23:1441-1455,

1984a.

Hall, D., and Pavitt, N. Conformation of cyclic analogs of

enkephalin. II. Analogs containing a cystine bridge.

Biopolymers 23:2325-2334, 1984b.

Hall. D., and Pavitt, N. Conformation of cyclic analogs of

enkephalin. III. Effect of varying ring size. Biopolymers

24:935-945. 1985.

Kessler, H.; Hölzemann, G.; and Zechel, C. Peptide

conformations. 33. Conformational analysis of cyclic

enkephalin analogs of the type Tyr-cycLo-(-Nw-Xxx-GLy-PheLeu-). Int J Peptide Protein Res 25:267-279, 1985.

Maigret, B.; Fournié-Zaluski, M.-C.; Roques, B.; and Premilat, S.

Proposals for the μ-active conformation of the enkephalin

analog Tyr-cyclo( -D-A2bu-Gly-Phe-Leu-]. Mol Pharmacol

29:314-320, 1986.

Mammi, N.J.; Hassan M.; and Goodman, M.

of cyclic enkephalin analogue by 1HNMR and computer

Copformational analysis

simultions. J Am Chem Soc 107: 4008-4013, 1985.

Mierke, D.F.; Lucietto, P.; Schiller, P.W.; and Goodman, M.

Comparative conformational analysis of two diastereomeric

cyclic enkephalin analogues by 1HNMR and computer simulations.

Biopolymers 26:1573-1586, 1987.

Mosberg. H.I. 1H n.m.r. investigation of conformational features

of cyclic, penicillamine-containing enkephalin analogs. Int J

Peptide Protein Res 29:282-288, 1987

Mosberg, H.I., and Schiller, P.W. 1H n.m.r. investigation of

conformational features of cyclic enkephalinamide analogs. Int

J Peptide Protein Res 23:462-466, 1984.

71
Mosberg, H.I.; Hurst, R.; Hruby, V.J.; Gee, K.; Yamamura, H.I.;

Galligan, J.J.; and Burke, T.F. Bis-penicilLamine enkephalins

possess highly improved specificity toward opioid receptors.

Proc Natl Acad Sci USA 80:5871-5874, 1983 (and references cited

therein)

Richman, S.J.; Goodman, M.; Nguyen, T.M.-D.; and Schiller, P.W.

Synthesis and biological activity of linear and cyclic

enkephalins modified at the Gly3-Phe4 amide bond. Int J

Peptide Protein Res 25:648-662, 1985.

Schiller, P.W. Fluorescence study on the conformation of a

cyclic enkephalin analog in aqueous solution. Biochem Biophys

Res Commun 114:268-274, 1983.

Schiller,P.W. Conformational analysis of enkephalin and

conformation-activity relationships. In: Udenfriend, S., and

Meienhofer, J., eds. The Peptides: Analysis, Synthesis,

Biology. Vol. 6. Orlando: Academic Press, 1984. pp. 219-268.

Schiller, P.W., and DiMaio, J. Opiate receptor subclasses differ

in their conformational requirements. Nature (London) 297:74-

76, 1982.

Schiller, P.W., and DiMaio, J. Aspects of conformational

restriction in biologically active peptides. In: Hruby, V.J.,

and Rich, D.H., eds. Peptides: Structure and Function.

Rockford, Ill.: Pierce Chemical Company, 1983. pp.69-278.

Schiller, P.W., and Nguyen, T.M.-D. Activity profiles of novel

side chain-to-side chain cyclized opioid peptide analogs.

Neuropeptides 5:165-168, 1984.

Schiller, P.W.; Eggimann, B.; DiMaio, J.; Lemieux, C.; and

Nguyen, T.M.-D. Cyclic enkephalin analogs containing a cystine

bridge. Biochem Biophys Res Commun 101:337-343, 1981.

Schiller, P.W.; DiMaio, J.; and Nguyen, T.M.-D. Activity

profiles of conformationally restricted opioid peptide analogs.

In: Ovchinnikov, Y.A., ed. Proc 16th FEBS Meeting. Utrecht, The

Netherlands: VNU Science Press, 1985a. pp. 457-462.

Schiller, P.W.; Nguyen, T.M.-D.; Lemieux, C.; and Maziak, L.A.

Synthesis and activity profiles of novel cyclic opioid peptide

monomers and dimers. J Med Chem 28:1766-1771, 1985b.

Schiller, P.W.; Nguyen, T.M.-D.; Maziak, L.A.; Wilkes, B.C.; and

Lemieux, C. Structure-activity relationships of cyclic opioid

peptide analogues containing a phenylalanine residue in the 3-

position. J Med Chem 30:2094-2099, 1987.

Wilkes, B.C., and Schiller, P.W. Theoretical conformational

analysis of a μ-selective cyclic opioid peptide analog.

Biopolymers 26:1431-1444, 1987.

Wilkes, B.C., and Schiller, P.W. Theoretical conformational

analysis of μ-selective cyclic opioid peptide analogs. In:

Marshall, G.R., ed. Peptides: Chemistry and Biology. Leiden,

The Netherlands: ESCOM Science Publishers, 1988. pp. 619-620.

72
ACKNOWLEDGMENTS

The authors' work described in this chapter was supported by

operating grants from the Medical Research Council of Canada (MT5655), the Quebec Heart Foundation, and the U.S. National

Institute on Drug Abuse (DA-04443-01).

AUTHORS

Peter W. Schiller, Ph.D., Director

Brian C. Wilkes, Ph.D.

Laboratory of Chemical Biology and Peptide Research

Clinical Research Institute of Montreal

110 Pine Avenue West

Montreal, Quebec, Canada H2W 1R7

73
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds

National Institute on Drug, Abuse, 1988

Conformational Studies of

Dermorphin

V. Renugopalakrishnan, Ph.D., and Rao S. Rapaka, Ph.D.

Dermorphin, H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, a D-Alacontaining amidated heptapeptide, is a potent μ-receptor

agonist. It was originally isolated from the skin of a South

American frog of the genus Phyllomedusae by Broccardo et al. (1981)

(for a review, see Feuerstein 1986). and it was subsequently

synthesized (de Castiglione et al. 1981; Montecucchi et al. 1981).

Its occurrence in mammalian tissue has not been demonstrated to

date (Negri et al. 1981). Dermorphin is similar to the other

opioid peptides, especially the enkephalin and dynorphin family of

peptides, in having Tyr residue in the first position of the

sequence. The presence of D-Ala residue in the second position and

the occurrence of Phe and Tyr residues in the third and fifth

positions should be expected to confer unique conformational

preferences and thus dermorphin serves as an excellent model system

for investigating conformation-receptor site selectivity

relationships. We have investigated the conformations of

dermorphin using vibrational spectroscopic studies (which are ideal

for peptides with chromophoric residues possessing unique

electronic transitions), 1D and 2D NMR studies (both at room

temperature and at elevated temperatures), molecular

mechanics-dynamics approach, and CD studies. CD studies of

dermorphin in H20, trifluroethanol (TFE) and methanol, and the

temperature effects in the above solvents provide insight into the

conformation of dermorphin, although chromophoric residues present

in dermorphin complicate the observed CD spectra.

The studies reported here represent our continuing investigations

(Bhatnagar et al. 1985; Pattabiraman et al. 1986), and similar

studies from other laboratories are also discussed. Aqueous

solutions of dermorphin were used in all the spectroscopic studies,

whereas CD studies were performed in H20, trifluroethanol (TFE),

and methanol. While the earlier 1D and 2D NMR studies had utilized

dimethyl sulfoxide (DMSO) (Salvadori et al. 1983; Arlandini et al.

1985; Pastore et al. 1985; Toma et al. 1985) as the solvent, we

employed 90 per cent H20-10 per cent D20 in our 1D and 2D NMR

studies, as we believe aqueous solutions are more relevant than

organic solvents to deduce physiologically relevant conformation(s).

74
Modern spectroscopic techniques utilized here offer the advantage

of suppression of H20 signals, a problem that plagued especially

IR and NMR studies in the 1960s and 1970s.

EXPERIMENTAL STUDIES

CD, FT-IR, and Raman studies were performed as described earlier

(Renugopalakrishnan et al. 1985, 1986; Prescott et al. 1986; Rapaka

et al. 1987). 1D and 2D 1H NMR studies of polypeptides were

performed in 90 per cent H20-10 per cent D20 on a 500 MHz

Bruker AM-500 spectrometer (Renugopalakrishnan et al. 1987, 1988;

Huang et al. 1988). Typical concentrations were in the range of

10-12.4 mM. Proton resonances were assigned based on homonuclear

decoupling experiments and chemical shift data (Wuthrich 1986).

Chemical shifts are reported in ppm relative to the shift of HDO at

4.8 ppm as an internal reference. In 2D NOESY experiments, H20

resonance was irradiated at all times except during the data

acquisition (Huang et al. 1988).

THEORETICAL STUDIES

Molecular mechanics studies employed the program AMBER developed in

the laboratory of Peter Kollman, and the conformations were

displayed visually using the program MIDAS (Weiner and Kollman

1981). The details of the calculations have been reported

previously (Pattabiraman et al. 1986). Recently, we have begun a

reinvestigation of the conformation of dermorphin using the program

CHARMM developed in the laboratory of Martin Karplus (Karplus and

McCammon 1981). The molecular dynamics calculations have been

recently extended up to 15 picoseconds, and the results will be

reported elsewhere (Prabhakaran et al. submitted for publication).

In the molecular mechanics calculations, we considered two B-turn

conformations and an extended conformation to restrict the search

in conformational space. A distance-dependent dielectric constant,

, simulated qualitatively the effect of solvent, but we did not

explicitly include solvation shells in the computations.

GENERAL DISCUSSION OF THE CONFORMATIONAL MODEL OF DERMORPHIN

DERIVED FROM EXPERIMENTAL AND THEORETICAL STUDIES

An energetically stable conformation of dermorphin derived from

molecular mechanics calculations is shown in figure 1. Dermorphin

assumes a type III’ B-turn conformation at the N-terminal segment,

Tyr-D-Ala2-Phe3-Gly4, and a type I B-turn conformation at the

C-terminal segment, Tyr5-Pro6-Ser7-NH2. Total energies of

the ß-turn conformations, (III’-I), (II’-III), and (II’-I), and a

fully extended conformation are presented in table 1. The fully

extended conformation is less stable than the folded

conformations. The three folded conformations, however, differ by

1 kcal/mole from one another and therefore can interconvert on

75
FIGURE 1

Conformation of dermorphin from molecular mechanics

calculations

conventional spectroscopic time scale, although time-resolved FT-IR

and Raman may be able to detect them. The Tyr residues, which

stack with a skew and therefore are not superimposable, probably

provide the major driving force for the stabilization of the folded

conformations. The stability of the folded conformation is,

however, not surprising, considering the presence of D-Ala2 and

Pro6 residues, which are compulsive B-turn promoting residues

(Chou and Fasman 1978). The stacking of Tyr1-Tyr5 side chains

can be observed from figure 1.

The folded conformation is consistent with the bulk of the

experimental spectroscopic data in aqueous solution. Temperature

dependence of CD spectra of dermorphin in TFE solution (not shown

here) manifests an unusual CD pattern with a broad positive band at

=226nm (band II) and somewhat less broad positive band at

=198nm (band I). As the temperature is increased to 45°C, a

dramatic shift in the CD bands occurs. A broad negative band

begins to appear at =218nm, which persists at 60°C with a

shift of the negative trough toward lower wave length region. The

above observation qualitatively indicates that the chromophore

responsible for band II is disrupted by heating. The CD spectra of

dermorphin in TFE, H20, and methanol, representing solvents of

varying polarity, suggest that dermorphin manifests discrete

conformational states in solution phase. The observed CD spectra

are difficult to interpret in terms of the secondary structure but

probably are the composite of two ß-turns (Renugopalakrishnan et

al. submitted for publication).

Raman spectrum of dermorphin in H20 shows an amide I band at 1681

cm-1 and a doublet amide III band at 1253 cm-l and 1265

cm-1. Raman amide I bands beyond the 1668-1678 cm-1 are

76
TABLE 1 Conformational Energies of the seven models of dermorphine

Model -Turn-I -Turn-II Total Energy in kcal/mol

Ill’-I III’

II’-III II'

II'-I II'

III'-III II’

V'-I V’

V'-III V’

Extended Trans

I -70.7

III -69.9

I -69.6

III -57.2

I -56.9

III -46.4

Trans -46.3

believed to originate from ß-turns (Bandekar and Krimm 1979; Tu

1986). The doublet amide III band at 1253 cm-1 and 1265 cm-1

is at best representative of extended/"random"/ß-turn structures.

The alternative explanation for the amide III doublet can be

advanced from the side chain Raman vibrations of the aromatic

residues. To decide between the alternatives, deuterium exchange

studies have been carried out to observe the shift of amide III

bands resulting from exchange of labile hydrogen atoms. After the

dissolution and equilibration of the sample in D20 solution, one

of the amide III bands, the band at 1253 cm-1 is lost, whereas

the 1265 cm-l band shifts toward lower wave numbers to 1261

cm-1. Therefore the 1253 cm-l band is assigned to the peptide

backbone vibrations, and the above frequency is probably a mixed

mode originating from R-turn and probably extended structures. The

amide I' band occurs at 1661 cm-l. Although Raman results are

largely Indicative of R-turn or folded conformation, an admixture

of extended structure cannot be ruled out.

500 MHz 1D 1H NMR spectrum of dermorphin in H20 is shown in

figure 2. Ramachandran angles, , for Tyrl, D-Ala2, Phe3,

Gly4, Tyr5 and Ser7 were derived from a Karplus-like

equation, using the values of the coefficients A, B, and C derived

by Bystrov et al. (1973). From the angles derived, it is

concluded that a fully extended conformation is not compatible with

the observed spin coupling constants, J. From the calculated

Ramachandran angles, , one is led to conclude that either type

II’ or III’ ß-turn occurs at the N-terminal segment, whereas the

C-terminal ß-turn may contain any one of three types of

ß-turns--types I, II, and III. It is difficult to discriminate

between them based only on spin coupling constant data. The

variation of aromatic proton resonances as the temperature is

increased is indicative of the unfolding of the dermorphin from a

folded conformation to an unfolded state in which Tyr residues are

no longer stacked. From the combined results, one cannot but

conclude that dermorphin probably assumes a manifold of folded

conformations, which interconvert in the spectroscopic time scale.

It is possible the heptapeptide contains a partially extended

77
FIGURE 2

500 MHz 1H NMR spectrum of dermorphin in H20

segment at the N-terminal end, but, in any event, such a

conformational state has a low probability of occurrence in the

aqueous solution.

Most of the previous spectroscopic studies of dermorphin were

exclusively performed in dimethyl sulfoxide (Salvadori et al. 1983;

Arlandini et al. 1985; Pastore et al. 1985; Toma et al. 1985), and

NMR studies were utilized to derive the secondary structure of

dermorphin. Toma et al. (1985) investigated the solution

conformation of a series of [Alan] substituted dermorphin analogs

by 500 MHz 1H NMR spectroscopy. Preferred conformations were

calculated using a semi-empirical method. They reached the

conclusion that none of the calculated conformations were able to

satisfy the experimental data. In contrast, both experimental and

theoretical studies could at best be reconciled with a type I

B-turn at the C-terminus. Nevertheless, no consensus could be

reached at the N-terminus, and the question of any preferred

conformation at the N-terminus could not be resolved. However,

Pastore et al. (1985) have reported 1D and 2D-NMR study of

dermorphin in DMSO and reached the conclusion that the heptapeptide

assumes an essentially extended structure. The above investigators

assumed corrections for ring current effects in dermorphin. The

question of interaction between amide protons and DMSO was thought

to be a major factor in the observed conformation in DMSO. An

extended structure of dermorphin in DMSO may be a special

78
situation that is probably not valid in aqueous solutions of

dermorphin. We believe that dermorphin assumes a manifold of

folded conformations in aqueous solution, although an extended

conformation at the N-terminus should also be considered.

Nevertheless, in a llpophilic environment, it is likely that

dermorphin assumes a folded conformation, which is probably the

most relevant at the μ-receptor site (Alford et al. in

preparation). Therefore, two different perspectives of dermorphin

conformation relevant to DMSO and aqueous solution exist in the

literature currently. Due to the relatively small size of

dermorphin, which exhibits rapid molecular tumbling, it is

difficult to provide a clear-cut resolution of the existing

conformational populations.

Numerous μ- and -receptor selective opioid peptide analogs

have been synthesized, and conformational studies have been

conducted on a number of analogs. ß-turns, ß-sheets, and extended

structures have been hypothesized as some of the recognition

features for the opioid receptor types (see Rapaka 1986 for a

review). However, conformation-receptor selectivity relationships

have not yet been established. It is expected that with the

multifaceted research techniques employed, the large number of

analogs available, and a greater understanding of the biochemistry

of the opioid receptors, the critical factors for conformationreceptor selectivity relationships will be better understood,

paving the way for design of highly receptor selective analogs and

safe and potent analogs devoid of undesirable side effects.

REFERENCES

Alford. D.; Duzgunes, N.; Renugopalakrishnan, V.; and Rapaka. R.S.

Circular dichrolsm study of dermorphln encapsulated in liposomes.

Submitted.

Arlandini. E.; Ballabio, M.; de Castiglione, R.; Gloia, B.;

Malnatl, M.L.; Perseo, G.; and Rizzo, V. Spectroscopic

investigations on dermorphin and its (L-Ala2) analog. Int J

Peptide Protein Res 25:33-46, 1985.

Bandekar, J., and Krimm, S. Vibrational analysis of peptldes,

polypeptides, and proteins. 4. Characteristic amide bands of

ß-turns. Proc Natl Acad Sci USA 76:774-777, 1979.

Bhatnagar, R.S.; Pattabiraman, N.; Sorensen, K.R.; Collette, T.W.;

Carreira. L.A.; Renugopalakrlshnan, V.; and Rapaka, R.S.

Conformational studies of dermorphin from FT-IR, Laser Raman, CD,

conformational energy calculations and molecular modeling. In:

Deber, C.M.; Hruby, V.J.; and Kopple, K.D., eds. Peptides:

Structure and Function. Rockford, Illinois: Pierce Chemical

Company, 1985. pp. 525-528.

Broccardo, M.; Erspamer, V.; Falconieri-Erspamer, G.; Improta,

G.; Linari, G.; Melchlori. P.; and Montecucchl, P.C.

Pharmacological data on dermorphlns. A new class of potent opioid

peptldes from amphibian skin. Br J Pharmacol 73:625-631, 1981.

79
Bystrov, V.F.; Portnova. S.L.; Balashova, T.A.; Koz-m. S.A.;

Gavrilov, Yu.D.; and Afanasev, V.A. Some aspects of NMR

techniques for conformational analysis of peptides. Pure and

Appl Chem 36:19-34, 1973.

de Castlglione. R.; Faoro, R.; Perseo. G.; and Piana, S. Synthesis

of dermorphin, a new class of opiate like peptides. Int J

Peptide Protein Res 17:263-272, 1981.

Chou, P.Y., and Fasman. G.D. Empirical predictions of protein

conformation. Annu Rev Biochem 47:251-276, 1978.

Feuerstein, G. Dermorphin: Autonomic pharmacology and

structure-activity relationships. In: Rapaka, R.S.; Hawks, R.L.;

and Barnett, G., eds. Opioid Peptides: Medicinal Chemistry.

National Institute on Drug Abuse Research Monograph 69. DHHS Pub.

No. (ADM)87-1454. Washington, D.C. U.S. Govt. Print. Off., 1986.

pp. 112-127.

Huang, S.-G.; Renugopalakrishnan, V.; and Rapaka, R.S. 2D

NMR study of dynorphin A(1-13) in solution. Biochemistry in

press 1988.

Karplus, M., and McCammon, J.A. The internal dynamics of globular

proteins. CRC Crit Rev (Biochemistry) 9:293-349. 1981.

Montecucchi, P.C.; de Castiglione, R.; Piani, S.; Gozzini, L.; and

Erspamer, G. Amino acid composition and sequence of dermorphln:

A novel opiate like peptide from skin in Phyllomedusa sauvagei.

Int J Peptide Protein Res 17:275-283, 1981.

Negri, L.; Melchiorri, P.; Falconlerri-Erspamer, G.; and

Erspamer, G. Radioimmunoassay of dermorphin-like peptides in

mammalian and non-mammalian tissue. Peptides (suppl) 2:45-49,

1981.

Pastore, A.; Temussi, P.A.; Salvadori, S.; Tomatis. R.; and

Mascagni, P. A conformational study of the opioid peptide

dermorphln by one-dimensional and two-dimensional nuclear

magnetic resonance spectroscopy. Biophys J 48:195-200, 1985.

Pattlbiraman. N.; Sorensen, K.R.; Langridge, R.; Bhatnagar, R.S.;

Renugopalakrishnan, V.; and Rapaka, R.S. Molecular mechanics

studies of dermorphin. Biochem Biophys Res Commun 140:342-349,

1986.

Prabhakaran, M.; Renugopalakrishnan, V.; and Rapaka, R.S. Molecular

dynamics simulation of secondary structure of dermorphin.

Submitted.

Prescott, B.; Renugopalakrishnan, V.; Glimcher, M.J.; Bhushan, A.;

and Thomas, G.J., Jr. A Raman spectroscopic study of hen egg

yolk phosvitin. Biochemistry 25:279-298, 1986.

Rapaka, R.S. Research topics in the medicinal chemistry and

molecular pharmacology of opioid peptides--present and future.

Life Sci 39:1825-1843, 1986.

Rapaka, R.S.; Renugopalakrishnan, V.; Collette. T.W.; Dobbs, J.C.;

Carrelra, L.A.; and Bhatnagar, R.S. Conformational features of

dynorphin A(1-13): Laser Raman spectroscopic studies. Int J

Peptide Protein Res 30:284-287. 1987.

Renugopalakrishian. V.; Horowitz, P.M.; and Glimcher, M.J.

Structural Studies of phosvitin in solution and in the solid

state. J Biol Chem 254:11406-11413. 1985.

80
Renugopalakrishnan, V.; Strawich, E.S.; Horowitz, P.M.; and

Glimcher, M.J. Studies of the secondary structure of amelogenln

from bovine tooth enamel. Biochemistry 25:4879-4887, 1986.

Renugopalakrishnan, V.; Huang, S.-G.; and Rapaka, R.S. A 500 MHz

lH NMR spectroscopic study of Met5-enkephalinamide in aqueous

solution: Ethanol induced conformational changes. Biochem

Biophy Res Commun 143:126-132, 1987.

Renugopalakrishnan, V.; Rapaka, R.S.; Huang, S.-G.; Moore, S.; and

Hutson, T.B. Dynorphin A(1-13) peptide NH groups are solvent

exposed: FT-IR and 500 MHz 1H NMR spectroscopic evidence.

Biochem Biophys Res Commun 151:1220-1225, 1988.

Renugopalakrishnan, V.; Rapaka, R.S.; Balschi, J.A.; Collette,

T.W.; Dobbs, J.C.; Carreira, L.A.; Pattabiraman, N.; Langridge,

R.; Sorensen, K.R.; Bhatnagar. R.S.; Huang. S.-G.; and MacElroy,

R.D. Molecular conformations of dermorphin. Submitted.

Salvadori, S.; Tomatis, R.; Gibbons, W.A.; Tancredi, T.; and

Temussi, P. NMR studies of neutral dermorphin and its synthetic

analogs. In: Hruby, V.J., and Rich, D.H., eds. Peptides:

Structure and Function. Rockford, Illinois: Pierce Chemical

Company, 1983. pp. 785-788.

Toma, F.; Dive, V.; Fermandijian, S.; Darlak, K.; and Grzonka, Z.

Preferred solution and calculated conformations of dermorphin and

analysis of structure-conformation-activity relationships in the

series [Alan]-dermorphin. Biopolymers 24:2417-2431, 1985.

Tu, A.T. Peptide backbone conformation and microenvironment of

side chains. In: Clark, R.J.H., and Hester, R.E., eds.

Spectroscopy of Biological Systems New York: John Wiley and

Sons, 1986. pp. 47-112.

Wuthrich, K. NMR Spectroscopy of Proteins and Nucleic Acids. New

York: John Wiley and Sons, 1986. 292 pp.

Weiner, P.K., and Kollman, P.A. Assisted model building with

energy refinement. A general program for modeling molecules and

their interactions. J Computational Physics 2:287-303, 1981.

ACKNOWLEDGMENT

Dr. R. L. Hawks and Mrs. Rani S. Rapka provided helpful discussions

to the authors during the preparation of the manuscript.

81
AUTHORS

V. Renugopalakrlshnan, Ph. D.

Laboratory for the Study of Skeletal Disorders and Rehabilitation

Department of Orthopaedlc Surgery

Harvard Medical School and Children's Hospital

Enders-1220

300 Longwood Avenue

Boston, Machusetts 02115, USA

Rao S. Rapaka, Ph. D.

10A-13, Natioral Institute on Drug Abuse

5600 Fishers lane

Rockvllle. Maryland 20857, USA

82
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds

National Institute on Drug Abuse, 1988

Use of Molecular Biological

Methods to Study Neuropeptides

Michael J. Brownstein, M.D., Ph.D.

INTRODUCTION

Armed with molecular biological techniques, neurobiologists interested in

biologically active peptides have begun to work on problems that they

could only dream of attacking 10 years ago. The structures of several

peptide precursors have been determined by isolating and sequencing DNAs

complementary to their respective mRNAs, and similarities among peptide

precursors have been cataloged. Based on the structures of the precursors,

inferences about their processing were generated and a number of

processing enzymes have been identified. Two of these have been

purified, and one has itself been cloned.

The availability of cDNA probes that specifically hybridize to mRNAs that

encode peptide precursors and processing enzymes allows the levels of

these mRNAs to be measured and—by means of in situ hybridization

histochemistry—to be visualized in discrete cells. In addition, cDNA

probes can be used to screen genomic libraries to isolate genes that encode

peptide mRNAs (or to determine the cause of genetic defects in peptide

production). The regulatory elements of the genes can ultimately be

identified and the transactivating molecules that mediate tissue-specific

expression of gene products and regulate mRNA levels can be identified.

Examples of the latter include the recently characterized intracellular

receptors for steroids and thyroid hormones.

To date only two neuropeptide receptors have been purified and sequenced.

Advances in expression cloning technology may soon contribute to pushing

aside our ignorance of this class of binding protein.

83
CLONING PEPTlDE PRECURSORS: THE CASE OF GALANIN

Galanin, a 29 amino acid peptide, was isolated from the upper small

intestine of pigs by Tatemoto, Mutt, and their colleagues (1983). Like so

many other peptides, it has a C-terminal amide. This structural feature led

to its discovery. Afterwards, galanin was found to be present in the enteric

nervous system of the gastrointestinal tract (Melander et al. 1985; Rökaeus

et al. 1984), the urogenital tract (Bauer et al. 1986a), the pancreas (Dunning

et al. 1986), the adrenal medulla (Bauer et al. 1986b), and the brain

(Melander et al. 1986). It has several actions, including inhibition of phasic

activity of the small intestine (Fox et al. 1986), production of hypoglycemia

(McDonald et al. 1985), suppression of insulin release, and central

stimulation of growth hormone (Ottlecz et al. 1986).

With few exceptions, DNAs encoding peptide precursors have been isolated

from cDNA libraries by screening them with mixtures of synthetic

oligonucleotides, the compositions of which were based on the amino acid

sequence of the peptides. It was in this way that we isolated cDNA.

encoding the galanin precursor from a pig adrenal medullary library

(Rokaeus and Bownstein 1986). The precursor (preprogalanin,

preproGAL) is ruher typical of this class of proteins. It is 123 amino acids

long and is comprised of a leader (signal) sequence. the 29 amino acids of

galanin, and a 59 amino acid sequence (galanin message associated protein,

GMAP) (see figure 1). Unlike proopiomelanocortin, for example,

preproGAL may encode only one active peptide, galanin itself. The galanin

sequence is flanked on both sides by pairs of lysine and arginine residues.

The C-terminal lysine and arginine are separated from the final alanine in

galanin by a glycine, the donor of the amide moiety.

84
1

Met

Ser

Thr

Lys

Tyr

His

Ala

Pro

Leu

Leu

Arg

Leu

Arg

Gly

Arg

Leu

Gly

Gly

Leu

Ser

Lys

Gly

Leu

Leu

Trp

Gly

Phe

Cys

Ala

Gly

Thr

Pro

His

Ala

Ser

Ser

Leu

His

Asp

Leu

Ala

Pro

Asn

Als

Lys

Leu

Leu

Val

Ser

lle

Tyr

Leu

Ser

Lys

Ala

Asp

Gly

Ala

Ala

Glu

Gly

Asn

Leu

Arg. . . Ser

FIGURE 1. Partial sequence of preprogalanin. A very hydrophobic Nterminal signal sequence (beginning with residue 1, Met) precedes the

(underlined) sequence of galanin (residues 33-61). Pairs of basic amino

acids (Lys-Arg) bracket the galanin sequence. A glycine residue, the donor

of the C-terminal amide, is found in position 62. The 59-residue sequence

of as yet unknown function comprises the amino-terminal half of the

precursor.

It is clear that precursors like preproGAL are cleaved to yield their active

products by a series of enzymes. The first of these, located in the cisternum

of the rough endoplasmic reticulum, removes the signal peptide from the

prepropeptide converting it to a propeptide. Subsequent processing seems

to occur principally in the secretory granule after the propeptide has

traversed the Golgi apparatus. First the propeptide is split by an

endopeptidase. More than one such endopeptidase may exist. A 70,000

dalton paired basic residue specific aspartyl protease (Chang and Loh 1984;

Loh et al. 1985) has been shown to be involved in the processing of

proopiomelanocortin and provasopressin. The pH optimum of this enzyme

is low (four-five) allowing it to function in the acid milieu of the secretory

vesicle. It cleaves precursors between their paired bases or C-terminal to

them.

In addition to the paired basic residue specific cleavage enzyme, there are

single basic residue cleaving enzymes. Such an enzyme has been found

associated with rat brain membranes (Devi and Goldstein 1984). It cleaves

85
dynorphin B1-29 between Thr13 and Arg14 to yield dynorphin B1-13. It

seems to be a neutral thiol protease.

Peptides liberated from their precursors by the action of one of the above

endopeptidases may have basic residues attached to their C- or N-termini.

These need to be trimmed off. The first trimming enzyme to be discovered

(Hook et al. 1982) and purified (Fricker and Snyder 1983) was a

carboxypeptidase. Complementary DNA encoding this enzyme has recently

been isolated. There is also an aminopeptidase that removes basic residues

from peptides’ N-termini when cleavage by the paired base specific

endopeptidase occurs between two basic residues leaving one behind

(Gainer et al. 1984). This enzyme appears to be a metalloprotease

stimulated by Co+2 and Zn+2.

The enzyme responsible for carboxyl-amidation of peptides was first

described by Bradbury et al. (1982). This copper dependent enzyme uses a

C-terminal glycine as the donor of the amide group. Other functional

groups are physically added to peptides by enzymes, notably Nacetyltransferases, kinases, and sulfate transferases.

CLONING PEPTIDE “PRECURSORS”: THE CASE OF VALOSIN

The major structural features of a typical peptide precursor and its mode of

processing were outlined above. Comparison of the structure of a protein

containing the sequence of valosin to other precursors illustrates the power

of cDNA cloning to help in identifying peptides as strong or weak

candidates for biological functions.

Valosin is a 25 amino acid peptide purified from side fractions of an earlier

preparation of porcine peptide HI and secretin (Schmidt et al. 1984).

Following its isolation, valosin was found to release gastrin, to augment

pentagastrin-induced gastric secretion, to stimulate pancreatic protein

secretion, and to suppress migrating myoelectric complexes of the small

bowel (Schmidt et al. 1985).

On screening a porcine adrenal medullary cDNA library with an

oligonucleotide pool constructed on the basis of the amino acid sequence of

86
valosin, we detected several cDNAs (Koller and Brownstein 1987). The

longest of these had a 2,466 base pair open reading frame that encoded a ~

88,660 dalton protein containing the amino acid sequence of valosin (see

figure 2). This protein is unlike other peptide precursors characterized to

date: (1) It has no obvious N-terminal or internal signal sequences and

seems, in fact, to reside in the cytoplasm of cells. (2) Its message is found

in many tissues, neuronal and nonneuronal ones alike. (3) The valosin

sequence is not bracketed by single or paired basic residues. In fact, it

appears to have been released from the valosin-containing protein by the

action of a chymotrypsin-like enzyme. It seems likely, then, that valosin is

not a physiologically active molecule but rather an artifactual product of a

(hitherto unknown) and rather ubiquitous cytoplasmic protein.

481

...Gly Leu Glu Asp Val Lys Arg Glu Leu

Gln Glu Leu Val Gln Tyr Pro Val Glu

His Pro Asp Lys Phe Leu Lys Phe Gly

Met Thr Pro Ser Lys Gly Val Leu Phe

Tyr Gly Pro Pro Gly Cys Gly Lys Thr

Leu Leu Ala

FIGURE 2. The portion of the valosin-containing protein (VCP) that

contains the valosin sequence (underlined). Note that valosin is not

neighbored by canonical processing signals. While VCP does have pairs of

basic residues scattered throughout it, many other proteins that are not

peptide precursors do too. Furthermore, analysis of its entire sequence

shows that VCP has no obvious N-terminal or internal signal sequences.

87
PUTTING CLONING TO WORK

Cloning a cDNA and sequencing it are not ends in themselves. A cDNA

and the structural information derived from it should be used to forge tools

that can be used for biological studies (see table 1).

TABLE 1. Examples of uses to which cloning data can be put.

A. Uses of cDNAs

1. Screening genomic libraries allows specific genes or gene families to

be isolated and sequenced. Putative regulatory elements can be fused to

marker genes for studies of gene expression. New genes can be introduced

into the cells of transgenic animals. Genes from animals with genetic

defects can be analyzed.

2. Full-length cDNAs inserted into appropriate vectors can be used to

manufacture large amounts of proteins for biochemical studies. (In the case

of peptide precursors, substrates for processing enzyme assays can be

prepared this way.)

3. cDNAs, cRNAs, or synthetic oligonucleotides can be used to

measure specific mRNAs in tissues or to visualize mRNAs in discrete cells

by means of Northern blotting and in situ hybridization histochemistry,

respectively.

4. Mutant cDNAs can be expressed in cells in order to determine the

function of the altered protein domains.

B. Uses of peptides synthesized on the basis of cDNA sequences

1. Novel candidates for roles as neuropeptide transmitters can be

synthesized and their biological activities explored.

2. Antibodies against peptides can be used for immunocytochemistry or immunoptecipitation (following pulse-chase experiments,

for example).

88
A WORD ABOUT RECEPTOR CHARACTERIZATION

In principle, there are three ways to clone cDNAs that encode receptors.

One might isolate the receptor, partially sequence it, and screen a cDNA

library with oligonucleotide probes based on the amino acid sequence. To

purify enough of a protein to sequence—50-100 pmoles perhaps—is

nontrivial, and receptors are frequently N-terminally blocked.

Consequently close to a nmole of material might be required, the pure

receptor has to be fragmented and the fragments themselves purified and

sequenced.

Antireceptor antibodies can be used to screen bacterial expression libraries.

Raising such antibodies and proving that they are specific for a particular

receptor is difficult. however, and if one employs an antibody that does not

recognize the receptor of interest or one that is promiscuous, one’s work

will be for naught.

Finally, it should be possible to transfect a negative mammalian cell line

with a eucaryotic expression library and to identify clones of cells that begin

to make surface receptors. This method has not yet been employed

successfully for neurotransmitter receptors, but recent advances in vector

design and transfection procedures should lead to its wider adoption.

CONCLUDING REMARKS

Numerous biologically active peptides have been discovered in the past

decade. Unfortunately, more is often known about the anatomy and

neurochemistry of peptidergic neurons than about their function. It is to be

hoped that use of molecular biological methods will contribute to

overcoming this problem.

89
REFERENCES

Bauer, F.E.; Christofides, N.D.; Hacker, G.W.; Blank, M.A.; Polak,

J.M.; and Bloorn, S.R. Distribution of galanin immunoreactivity in the genitourinary tract of man and rat.

Peptides 7:5-10, 1986a.

Bauer, F.E.; Hacker, G.W.; Terenghi, G.; Adrian, T.E.; Polak, J.M.;

and Bloom, S.R. Localization and molecular forms of

galanin in human adrenals: Elevated levels in

pheochromocytomas. J Clin Endocrino Metab

63:1372-1378, 1986b.

Bradbury, A.F.; Finnie, M.D.A.; and Smyth. D.F. Mechanism of Cterminal amide formation by pituitary enzymes. Nature

298:686-658, 1982.

Chang, T.L., and Loh, Y.P. In vitro processing of proopiocortin

by membrane-associated and soluble converting enzyme

activities from rat intermediate lobe secretory granules.

Endocrinology 114:2092-2099, 1984.

Devi, L., and Goldstein, A. Dynorphin converting enzyme with

unusual specificity from rat brain. Proc Natl Acad Sci

USA 81:1892-1896, 1984.

Dunning, B.E.; Ahren. B.; Veith, R.C.; Böttcher, G.; Sundler, F.; and

Táborsky, G.J., Jr. Galanin: A novel pancreatic

neuropeptide. Am J Physiol 251:E127-E133, 1986.

Fox, J.E.T.; McDonald, T.J.; Kostolanska, F.; and Tatemoto, K.

Galanin: An inhibitory neural peptide of the canine small

intestine. Life Sci 39:103-110, 1986.

Fricker, L.D., and Snyder, S.H. Purification and characterization

of enkephalin convertase, an enkephalin synthesizing

carboxypeptidase. J Biol Chem 258:10950-10955, 1983.

Gainer, H.; Russell, J.T.; and Loh, Y.P. An aminopeptidase

activity in bovine pituitary secretory vesicles that

cleaves the N-terminal arginine from -lipotropin60-65

FEBS Lett 175:135-139, 1984.

Hook, V.Y.H.; Eiden, L.E.; and Brownstein, M.J. A carboxypeptidase processing enzyme for enkephalin precursors.

Nature 295:341-342, 1982.

90
Koller, K.J., and Brownstein, M.J. Use of a cDNA clone to

identify a supposed precursor protein containing valosin.

Nature 325:542-545, 1987.

Loh, Y.P.; Parish, D.C.; and Tuteja, R. Purification and

characterization of a paired basic residue-specific proopiomelanocortin converting enzyme from bovine

pituitary intermediate lobe secretory vesicles.

J Biol Chem 260:7194-7205, 1985.

McDonald, T.J.; Dupre, J.; Tatemoto, K.; Greenburg, G.R.; Radziuk,

J.; and Mutt, V. Galanin inhibits insulin secretion and

induces hyperglycemia in dogs. Diabetes 34:192-196,

1985.

Melander, T.; Höfelt, T.; and Rökaeus, A. Distribution of

galanin-like immunoreactivity in the rat central nervous

system. J Comp Neurol 248:475-517, 1986.

Melander, T.; Hökfelt, T.; Rökaeus, A.; Fahrenkrug, J.; Tatemoto,

K.; and Mutt, V. Distribution of galanin-like immunoreactivity in the gastrointestinal tract of several

mammalian species. Cell Tissue Res 239:253-270, 1985.

Ottlecz, A.; Samson, W.K.; and McCann, S.M. Galanin: Evidence

for a hypothalamic site of action to release growth

hormone. Peptides 7:51-53, 1986.

Rökaeus, A., and Brownstein, M.J. Construction of a porcine

adrenal medullary cDNA library and nucleotide sequence

analysis of two clones encoding a galanin precursor.

Proc Natl Acad Sci USA 83:6287-6291, 1986.

Rökaeus, A.; Melander, T.; Höfelt. T.; Lundberg, J.M.; Tatemoto,

K.; Carlquist, M.; and Mutt, V. A galanin-like peptide in

the central nervous system and intestine of the rat.

Neurosci Lett 47:161-166, 1984.

Schmidt, W.E.; Mutt, V.; Carlquist, M.; Kratzin, H.; Conlon, J.M.;

and Creutzfeldt, W. Valosin: Isolation and characterization of a novel peptide from porcine intestine. FEBS

Lett 191:264-268, 1985.

Schmidt, W.E.; Mutt, V.; Konturek, S.J.; and Creutzfeldt, W.

Peptide VQY: Isolation and characterization of a new

biologically active gastrointestinal peptide. Dig Dis Sti

29:75S, 1984.

91
Tatemoto, K.; Rökaeus, A.; Jörvall, H.; McDonald, T.J.; and Mutt,

V. Galanin—a novel biologically active peptide from

porcine intestine. FEBS Lett 164:124-128, 1983.

AUTHOR

Michael J. Brownstein, M.D., Ph.D.

Laboratory of Cell Biology

National Institute of Mental Health

Bethesda, Maryland 20892 U.S.A.

92
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.d., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Three Technical Approaches for

Cloning Opioid Receptors

Curtis A. Machida, Ph.D.; John Salon, Ph.D.; David

Grandy, Ph.D.; James Bunzow, M.S.; Paul Albert, Ph.D.;

Eric Hanneman, Ph.D.; and Olivier Civelli, Ph.D.

INTRODUCTION

A basic tenet of modern pharmacology is that the biological

activity of a drug is the result of chemical events triggered by

the drug's interaction with specific receptors on responsive

cells. Although the chemical structures of drugs have been

extensively studied, very little information has been obtained

about the molecular structures of drug receptors. The structural

analysis of receptors is fraught with difficulties, which are in

part due to limitations of the techniques used to manipulate

integral membrane proteins. Most membrane proteins are

hydrophobic, and &he removal of the lipid moiety severely limits

or abolishes the solubility of the protein in aqueous media.

Although the techniques of nonaqueous protein chemistry have

advanced over the past few years, certain experimental

constraints often make a direct attack on a protein‘s structure

difficult to manage. In addition, the removal of the membrane

lipid can disrupt the oligomeric structure of the membrane

proteins and can obscure the involvement of associated receptor

subunits. Limiting receptor abundance presents an additional

problem. Depending on the nature of the signal transduction, a

specific receptor can constitute from less than 0.01 percent to

as much as 0.5 percent of the plasma membrane protein mass.

Pharmacologically important receptors generally fall into the

lower end of the abundance spectrum. Thus, attempts to purify

receptors are compromised by the need for large amounts of

starting material. This problem is often exacerbated by the need

to use a tissue that is scarce or difficult to obtain. In

addition, receptor preparations are often very unstable. Both

native and purified receptor preparations can be sensitive to

assorted cations, oxidizing or reducing agents, and a variety of

other materials and methods commonly used for their preparation

and storage. The loss of receptor activity compromises the

ability to assess the integrity and purity of the receptor

preparation. For these reasons, it is likely that the

determination of the primary amino acid structure of many low

93
abundance neuroreceptors will be inferred from the analysis of

their cloned nucleic acid sequences.

This review will discuss the strategies that can be applied to

the molecular cloning of one specific family of neuroreceptors,

the opioid receptors.

THE OPIOID RECEPTORS

The analgesic and euphoric responses to opiates are the result of

a cascade of biochemical events that are triggered by the

interaction of the opiates with specific receptors found on the

cell membranes of nervous system tissues (West and Miller 1983;

Paterson et al. 1984; Holaday 1985). These opioid receptors not

only recognize exogeneous alkaloids such as morphine, but

moreover interact with their endogeneous ligands, the opioid

peptides. The opioid receptors are clearly involved in

physiological phenomena such as pain perception, addiction, and

withdrawal, and are implicated in hormone secretion, response to

injury, and gastrointestinal motility (Holaday 1985). The

molecular basis of the participation of opioid receptors in these

phenomena is unknown.

Martin and coworkers (Martin et al. 1976; Gilbert and Martin

1976) were the first to postulate the existence of different

types of opioid receptors based on differences in the

pharmacological profiles of the different opiates. Although the

precise number of opioid receptor types is still a matter of

conjecture, -it is generally accepted that the opioid receptors

can be subdivided into three major types, (Pasternak

et al. 1983; Paterson et al. 1984). These three receptor types

are distinguished on the basis of four criteria: (1) patterns in

ligand selectivity, (2) anatomical distribution, (3)

physiologica:. and behavioral profiles, and (4) differences in the

cellular response to activation (Dole et al. 1975; Paterson et

al. 1984; Holaday 1985).

Three models can account for the heterogeneity of the opioid

receptors. First, the three opioid receptors are structurally

different and are encoded by different genes; second, the opioid

receptors are encoded by different but homologous genes; or

third, the opioid receptors are encoded by identical genes but

differ in posttranslational modifications or in their

interactions with second messengers. At the present time, there

is no definitive evidence to favor one model over the others.

Using Schwyzer's concept (Schwyzer et al. 1980), one can

formulate the existence of at least two domains in the active

site of the opioid receptor: the message domain, which recognizes

the common amino terminal sequence of the enkephalins, Tyr-GlyGly-Phe, and the address domain recognizing the remainder of the

opioid peptide sequence. This model suggests that at least one

region of the opioid receptor is common to the different receptor

types and that the opioid receptors must therefore be homologous.

94
Furthermore, in view of the recently discovered homology between

the -adrenergic receptor, muscarinic acetylcholine receptor, and

opsin genes (Kubo et al. 1986; Dixon et al. 1986; Bonner et al.

1987; Hall 1987), the possibility that the opioid receptors may

also be encoded by homologous if not identical genes is very

attractive.

Knowledge about the physical characteristics of the opioid

receptor has been obtained directly from receptor purification

experiments. Attempts at opioid receptor purification have been

hampered by the receptor's sensitivity to denaturation and low

abundance (Zulkin and Maneckjee 1986). These receptors constitute

less than 0.005 percent of the total rat brain membrane proteins.

In spite of the extreme difficulties in purifying opioid

receptors, several laboratory groups (Bidlack et al. 1981; Newman

and Barnard 1984; Gioannini et al. 1985; Simonds et al. 1985; Cho

et al. 1986) have succeeded in purifying opioid receptor proteins

to homogeneity. The two common characteristics established for

the ligand binding subunit of the opioid receptor are (1) an

apparent Mr range of 55,000-65,000 and (2) its glycosylated

nature. These two properties, plus ligand selectivity, represent

the important criteria for judging the authenticity of a cloned

opioid receptor.

APPROACHES TO THE MOLECULAR CLONING OF THE OPIOID RECEPTOR

Our hope in understanding the molecular structure and activation

of the opioid receptors lies with the use of recombinant DNA

technology. The cloning of the opioid receptors will allow

detailed examination of receptor structure-function relationships

and receptor gene expression. This approach will therefore help

explain the molecular mechanisms of receptor activation and its

resulting cellular responses.

The opioid receptors are macromolecular components of plasma

membranes, which specifically bind opioid ligands according to

three criteria: (1) high affinity for opioids and opiates, (2)

saturable binding, and (3) binding that is stereospecific and

naloxone reversible (Dole et al. 1975). These three criteria

constitute the accepted functional definition of the opioid

receptor. Therefore, an authentic molecular clone of the opioid

receptor must be able to code for a product that fulfills all

three criteria.

In addition, an opioid receptor clone must encode a product with

the expected characteristics of a membrane protein. Two

structural characteristics of membrane proteins are the presence

of a signal sequence that facilitates the insertion of the

receptor in the membrane, and the presence of hydrophobic

sequences that correspond to potential transmembrane domains of

the receptor, The signal peptide is not an absolute requirement,

since some membrane proteins do not have signal sequences.

Since the distribution of the opioid receptor in the central

nervous system and in peripheral organs is well documented (Pert

95
and Synder 1373; Pfeiffer et al. 1982; Atweh and Kuhar 1983;

Paterson et al. 1984; Mansour et al. 1987), in situ hybridization

analysis using an opioid receptor clone as probe should correlate

sites of receptor mRNA synthesis with sites of receptor protein

localization. However, the distinction between sites of mRNA

synthesis and receptor protein localization may be difficult to

evaluate precisely because of differences in assay sensitivity.

Alternatively, an opioid receptor clone may be used to express

receptor protein in sufficient quantity to serve as an antigen in

the preparation of antibodies. These antibodies can then be used

in immunocytochemical analyses to precisely correlate the

distribution of the receptor encoded by the molecular clone to

the known distribution of the opioid receptor. Finally, based on

physical criteria obtained during receptor purification

experiments, the opioid receptor clone should encode a

glycoprotein with an apparent Mr range of 55,000-65,000.

Three different strategies can be employed in the molecular

cloning of the opioid receptor. The first approach begins with

the isolatior of the receptor in sufficient quantity to permit

amino acid sequencing. This sequence would be reverse translated

into a DNA oligonucleotide probe and this probe would then be

used in the isolation of an opioid receptor cDNA or gene clone.

The second strategy proposes to clone the opioid receptor through

gene expression without prior purification of the receptor

protein. The third strategy assumes some sequence similarity

between opioid receptors and other neuroreceptors and exploits

this feature in hybridization experiments designed to identify

potential opioid receptor clones.

CLONING THE OPIOID RECEPTOR WITH THE USE OF RECEPTOR AMINO ACID

SEQUENCE INFORMATION

The traditional cloning strategy consists of purifying and

sequencing the opioid receptor protein. The amino acid sequence

information would then be used to identify a corresponding

receptor cDNA clone.

This approach requires large-scale protein purification in order

to prepare a sufficient quantity of homogeneous opioid receptor

to permit partial amino acid sequencing. Several laboratories

have succeeded in purifying the opioid receptor to homogeneity

(Bidlack et al. 1981; Newman and Barnard 1984; Gioannini et al.

1985; Simonds et al. 1985; Cho et al. 1986). The objectives of

these laboratories were to purify the ligand binding domain of

the opioid receptor. Two approaches have been employed to purify

opioid receptors: the labeled ligand binding approach and the

affinity chromatography approach.

In the labeled ligand binding approach, radioactive opioid

ligands are bound and covalently cross-linked to opioid receptors

in the membrane; the label serves as a marker in subsequent

purification steps. Since binding of the ligand is performed

with native receptor-membrane complexes, artifacts due to

96
aberrant protein interactions are minimized. One drawback,

however, is that the ligand binding site is irreversibly

occupied, making binding assays or reconstitution experiments

impossible.

The purification of solubilized opioid receptors by affinity

chromatography is dependent on the receptor's ability to

recognize immobilized opioid ligands. The receptor population

isolated by this technique can be tested for binding ability and

can be obtained in sufficient quantity to sequence. The major

drawback to this approach is the difficulty in preserving

biological activity during subsequent purification steps.

Pure receptors are subjected to partial proteolytic digestion.

Peptide fragments are separated by high-performance liquid

chromatography and analyzed using automated high-sensitivity gas

phase amino acid sequencing. Currently, automated peptide

sequencing requires as little as 10 picomoles of purified

peptide. The sequences of at least two peptide fragments are

then reverse translated into their corresponding DNA sequences

and oligonucleotides complementary to these sequences are then

chemically synthesized. The choice of oligonucleotide length and

sequence is critical for successful cloning and has been

discussed (Anderson and Young 1987). In general, oligomers of at

least 17 bases in length containing the least amount of

degeneracy are synthesized. The radiolabeled synthetic oligomers

then serve as hybridization probes to identify receptor cDNAs

(or genes) in appropriate libraries. This isolated clone is then

used as a template for determining the entire sequence of the

opioid receptor.

CLONING THE OPIOID RECEPTOR BY GENE TRANSFER IN EUKARYOTIC CELLS

Gene transfer systems have been used successfully in the

molecular cloning of eukaryotic genes, including receptor genes

(Kuhn et al. 1984; Chao et al. 1986). This approach is based on

the expression of cell surface receptors on eukaryotic cells and

does not rely on the availability of receptor amino acid sequence

information. In this approach, a large population of cDNAs (or

large fragments of genomic DNA) from tissues known to express

opioid receptors are transfected into receptor-deficient

eukaryotic cells. Transfectants that have incorporated

exogeneous DNA are selected according to traditional techniques

(Southern and Berg 1982) and are tested for their ability to

express opioid receptor by binding analyses. In transfections

using genomic DNA fragments, the fragments are purified by

conducting secondary transfections. This step consists of

repeating the transfection procedure using the DNA of the primary

transfectant as the donor. If pools of cDNAs are transfected into

cells, the opioid receptor cDNA can be isolated by gradually

decreasing the size of the analyzed pool to unity. Several

different variations of this procedure can be envisioned; in

particular, cDNA libraries enriched in opioid receptor sequences

can be constructed through mRNA subtraction (Hedrick 1984).

97
There is also the possibility of expressing an opioid receptor

cDNA in prokaryotic cells. However, in view of the requirements

necessary to maintain the biological activity of the receptor, it

seems unlikely that such an expression system would be

successful.

The gene transfer and expression approach was adapted by our

laboratory as one strategy to attempt to clone the human opioid

receptor gene. This strategy is shown in figure 1. Large

fragments of human genomic DNA are cotransfected with plasmid DNA

(pRSVneo), which confers neomycin resistance to opioid

receptor-deficient mouse L cells. Drug resistant colonies are

selected by growth in neomycin analogue G418 and receptor-bearing

colonies are identified using an opioid receptor detection assay.

If receptor antibodies are available, these would be used in

fluorescent-activated cell sorting or in situ rosetting assays to

identify receptor-bearing transfectants. The unavailability of

opioid receptor antibodies that recognize extracellular antigenic

determinants has precluded the use of these conventional

detection assays and prompted the development of new techniques

that allow screening of transfectants with radioactive ligands

(see next section for details). Once receptor-bearing

transfectants have been identified, DNA from these cells is

transferred into new recipient mouse L cells. DNA from secondary

transfectants containing the opioid receptor gene is then used to

prepare a genomic library in either a lambda or cosmid cloning

vector. The genomic library is then screened by filter

hybridization using nick-translated human repetitive sequence DNA

to detect sequences contained in the original human donor, but

not recipient, mouse cell lines. Positive clones containing

human donor DNA are mapped with restriction endonucleases, and

then reintroduced by transfection into recipient L cells to assay

for opioid receptor gene expression.

Development of Tools for the Detection of Neuroreceptor

Expression in Eukaryotic Cells

Eukaryotic cells are ideal gene transfer recipients for the

expression of neurohormone and neurotransmitter receptors. The

eukaryotic cell membrane environment and posttranslational

machinery are necessary for correct neuroreceptor conformation

and are critical determinants in the successful detection of the

receptor by ligand binding. In spite of the ideal host

environment, oukaryotic expression libraries have not been widely

used for the isolation of clones expressing neuroreceptors. One

major obstacle that has hindered the use of eukaryotic expression

libraries has been the lack of rapid and reliable screening

procedures.

To facilitate the screening of large numbers of transfectant

colonies, our laboratory has recently adapted a replica copy

technique originally used for identifying somatic cell mutants

(Raetz et al. 1982). The transfer membrane is a polyester nylon

cloth that permits colony replication with high fidelity and

98
Gene Transfer and Molecular Cloning of The Human Opioid Receptor Gene

FIGURE 1

Molecular cloning of the human opioid receptor by gene transfer

and expression in eukaryotic cells.

99
superior resolution (see fig. 2). Following drug selection of

stable transfectants, one or more sheets of sterilized polyester

nylon are placed on top of the cell colonies and overlaid with a

monolayer of glass beads. The beads retain the filter uniformly

against the bottom of the dish and permit the exchange of growth

medium to the cells without changing the colony pattern. After

5-7 days of growth, the medium is aspirated, the beads are

discarded, and the nylon membranes are removed with sterile

tweezers. Up to five multiple copies of the transfectant

population have been made from a single dish.

The multiple copies allow simultaneous screening of transfectant

colonies with different radioiodinated neurohormone ligands in

either the presence or the absence of nonradioactive blockers

(see fig. 3). Positive signals that appear in duplicate using

the radioactive ligand alone are identified by autoradiography.

The specificity of the binding can be determined with the use of

nonradioactive blockers. If desired, cell colonies that express

neuroreceptocs can then be purified from the master plate of

transfectant;.

To test the level of sensitivity of the in situ binding assay in

detecting opioid receptors, the assay was standardized by using

the mouse neuroblastoma N4TG1, a cell line that expresses

approximately 50,000 opioid receptors per cell (Amano et al.

1972). As shown in fig. 4, 106,105, and 104 N4TG1 or opioid

receptor-negative mouse L cells were mechanically spotted on

nylon cloth; the filters were then subjected to binding with (3-

[

125I]iodotyrosyl27) -endorphin (0.15 nM in 50 mM Tris-HCl pH 7.4,

1 percent BSA, 0.05 percent poly-L-lysine, 50 ug/ml bacitracin,

10 ug/ml leupeptin, and 10 ug/ml trypsin inhibitor, for 1 hr at

room temperature) in the absence or presence of opioid receptor

antagonist, naloxone (100uM). The filters were then washed with

ice cold buff‘er, dried briefly, and placed under X-ray film.

Figure 4 shows the results of an 18 hr autoradiographic exposure

of a typical experiment. Clear signals emanating from as few as

10,000 opioic receptor-positive N4TG1 cells were observed; very

little signal was observed for mouse L cells or for N4TG1 cells

whose receptor sites were blocked with naloxone. Other

experiments have shown that a signal from as few as 1,000 N4TG1

cells can be observed. This represents a detection limit of as

few as 5 X 107 opioid receptors for this assay.

Screening Expression Libraries for Opioid Receptor Expression

Eukaryotic expression libraries were constructed using high

molecular weight human genomic DNA [from the human neuroblastoma

SKN-SH cell line, which expresses 50,000 opioid receptors per

cell (Yu et al. 1986)] as the donor DNA for transfections. This

DNA was cotransfected with neomycin resistance plasmids into

opioid receptor-deficient mouse L cells and stable transfectants

isolated by selection with G418. Assuming that the human genome

contains approximately 3 X 109 bases and that each stable

transfectant incorporates 106 bp of DNA, the entire human genome

100
FIGURE 2

Transfectant colonies were grown on polyester nylon filters for 7

days. Replica filters were carefully removed from the master

dish and colonies fixed with 10 percent trichloroacetic acid (10

minutes) and stained with 0.05 percent Coomassie blue in

methanol:water:acetic acid (45:45:10; 10 minutes). Excess stain

was removed by washing with methanol:water:acetic acid.

FIGURE 3

In situ replica filter binding assay for detection of

transfectants expressing neuroreceptors.

101
DOT ASSAY FOR OPIOID RECEPTOR EXPRESSION

FIGURE 4

N4TG1 or L cells were spotted on nylon cloth and subjected to

binding with (3-[125I]iodotyrosyl27) -endorphin (0.15 nM, room

temperature, 1 hr.) in the absence or presence of the opioid

antagonist, naloxone (100 uM). The filters were then washed with

ice cold buffer, dried briefly, and placed under X-ray film.

Clear signals emanating from as few as 10,000 opioid receptorexpressing N4TG1 cells are observed; very little signal is

observed for L cells or for N4TG1 cells whose receptor sites are

blocked with naloxone. The aberrant signal emanating from 10,000

N4TG1 cells in the presence of blocker is not reproducible and is

believed to be an artifact.

102
can be easily represented by approximately 3,000 independent

transfectant colonies (Kuhn et al. 1984). We have prepared

several transfection libraries and screened these libraries with

(3-[125I]iodotyrosyl27) -endorphin using the detection technique

described in the previous section. Thus far, we have been

unsuccessful in detecting opioid receptor-positive clones. Our

results with the opioid receptor-rich N4TG1 cell line indicate

that an opioid receptor-bearing transfectant colony (1,000-5,000

cells per colony) can be detected if the receptor gene is

expressed at a level of at least 10,000 receptors per cell. If

the human opioid receptor promoter is weak in the heterologous

mouse cell system, our screening assay may not be sensitive

enough to detect transfectants expressing receptor at low levels.

CLONING THE OPIOID RECEPTOR BY THE HOMOLOGY APPROACH

An emerging concept in the field of molecular neurobiology is

that transmembrane signalling neuroreceptors involve three

distinct components. First, there is a specific receptor exposed

on the external surface of cell membranes that recognizes and

interacts with ligands, such as hormones or drugs, or responds to

a sensory stimulus such as light. Second, exposed, at the

cytoplasmic surface are effector enzymes such as adenylate

cyclase that either generate the second messenger CAMP or in some

other way effect an intracellular response. Third, interposed

both functionally and physically between the receptor and its

effectors are transducing or coupling proteins that bind GTP (socalled G-proteins). The available evidence suggests that

signalling systems of this type demonstrate a high degree of

structural, functional, and regulatory homology (Dolhman et al.

1987).

To date, seven pharmacologically or functionally related Gprotein receptors have been cloned and their primary sequences

shown to be similar. These are the ß-adrenergic receptor from

human (Kobilka et al. 1987), hamster (Dixon et al. 1986), and

turkey (Yarden et al. 1986), the muscarinic acetylcholine

receptor from porcine brain and heart (Kubo et al. 1986a, b), the

human and bovine opsin protein (Nathans and Hogness 1984, 1983),

as well as the visual pigments from drosophila (Zuker et al.

1985), the human mas-oncogene (Young et al. 1986), and the yeast

mating factor receptor (Burkholder and Hartwell 1985). All these

proteins display a certain degree of sequence similarity at the

amino acid level, display similar hydropathic profiles, and have

conserved functionally important amino acids or glycosylation

sites. The opioid receptor interacts with the same regulatory Gproteins as these other receptors. It is therefore postulated

that the opioid receptor will possess an amino acid structure

similar to the above mentioned G-protein related receptors. This

postulated relationship is the premise upon which the approach to

cloning the opioid receptor by homology is based.

103
Probe Design

The most often used probes are full-length double-stranded cDNAs,

short synthetic oligonucleotides, or mRNA riboprobes that are

labeled with 32P, 35S, or biotin. The selection of the probe will

depend on the screening strategy. Full-length cDNAs for a

related receptor or short oligonucleotides for a consensus region

are the most appropriate probes for the screening of recombinant

DNA libraries. We shall focus our discussion on these methods.

If two nucleic acid sequences are related, the use of a cDNA

probe complementary to one should identify the other if the probe

is labeled by nick-translation and the regions of similarity are

a significant fraction of the nick-translated lengths.

An alternative to using double-stranded cDNAs is to use singlestranded synthetic oligonucleotides, which are complementary to

putative consensus regions of the receptor. Oligomers, which

typically can range from 14 to 60 or more bases, are easily endlabeled with T4 polynucleotide kinase and [ -

32P]ATP. Since the

probe is labeled at either its free 5'-phosphate or hydroxyl

group, only one radiophosphate is incorporated per strand.

Therefore, this type of probe has a lower specific activity than

nick-translated cDNAs.

Screening of the Library

Screening of recombinant libraries involves the hybridization of

radioactive probes to filter bound nucleic acid targets that

constitute the elements of the library. Duplex formation in

terms of both the number of positives and the intensity of

signals is a function of both the stability of the duplex formed

and the rate of hybridization. Nucleic acid hybridization

depends on the random collision of two complementary sequences.

The time course of the reaction is thus determined by the

concentration of the reassociating species and by a second-order

rate equation. While the quantitative hybridization of two

perfectly matched sequences in solution can be rigorously

described mathematically, the hybridization of a nucleic acid

strand in solution to a filter-immobilized complementary sequence

can only be qualitatively approximated.

An important consideration when identifying new proteins by

cross-hybridization involves the discrimination of homologous

from heterologous matches. When probing with both single-strand

oligomers and denatured double-strand cDNAs, the ratio of the

extent of hybridization of homologous sequences to heterologous

sequences is not constant but varies with time. Hybridization to

similar sequences is maximal early in the reaction and decreases

with increasing time of incubation. Therefore, although the

homologous reaction is faster and will reach completion earlier,

the heterologous reaction will eventually catch up. The

discrimination of related sequences is therefore maximal early in

the reaction out deteriorates very quickly. Thus, for most

cases, maximal discrimination between homologous and heterologous

104
sequences is best accomplished by keeping the incubation time

short.

Nucleic acids hybridize very slowly at low ionic strength. As

the ionic strength of the hybridization solution increases, so

does the rate of duplex formation. This effect is most dramatic

at low salt concentrations (0.15 M Na+), but remains marked up to

1.5 M Na+. High salt concentration stabilizes mismatched

duplexes; so to detect cross-hybridizing species, the salt

concentration of both the hybridization and wash solution must be

kept fairly high, generally at 2-6xSSC (1xSSC is 0.15M NaCl,

0.015 NaCitrate, pH 7.0).

The stability of a nucleic acid duplex can be described by its

melting temperature, Tm. In general, the rate of reassociation

as well as the stability of the duplex is maximal at 20-30°C

below the Tm. The optimal temperature for nucleic acid

reassociation in aqueous salt solution lies in the range from 60-

75°C. However, extended incubation at such temperatures can lead

to a considerable amount of thermal strand scission. Hence, it

is desirable to reduce the temperature while maintaining the

stringency of the nucleic acid interaction. The effective

incubation temperature can be lowered by including formamide in

the hybridization solution. Formamide acts to destabilize

hydrogen-bonding between double-strand nucleic acids (McConoughy

et al. 1969). In addition to affecting the Tm, formamide also

affects the rates of hybridization. This effect is minimal at

formamide concentrations of 30-50 percent; however at

concentrations of 20 percent formamide, the rate is decreased by

one-third, while at 80 percent the rate can drop as much as

threefold for DNA-DNA and as much as twelvefold for DNA-RNA

duplexes. Thus, in practice, the inclusion of formamide in the

hybridization solution can be used to alter the stringency of the

incubation by holding the incubation temperature constant and

varying the formamide concentration. The effective incubation

temperature can be reduced to as much as 50°C below the Tm for

perfect matches. The effects of temperature, base composition,

and formamide can be approximated by an equation that estimates

the Tm of the duplex;

Tm= 69.3 + 0.41(G+C) percent - 650/L

where (G+C) percent is the percentage of G and C residues in the

duplex and L is the average length of the probe (Marmur and Doty

1962; Wetmur and Davidson 1968). Furthermore, taking into

consideration possible mismatches, the Tm of the duplex decreases

1°C with every 1 percent increase in the number of mismatches

(Bonner et al. 1973). and the Tm at differing ionic strengths (ul

and u2) can be related by (Tm)u2 - (Tm)u1 = 18.5 log(u2/u1) (Dove

and Davidson 1962).

After hybridization, washing is carried out to remove

unhybridized probe and to dissociate unstable hybrids. The

temperature and salt concentration of the washing solution

determine which hybrids will be detected. In general, washing

should be done under stringent conditions: at 5-20°C below the

105
Tm for a well-matched hybrids (65-70°C), and at 12-2OoC below the

Tm for cross-reacting species (50-60°C). No absolute rule exists

for the duration of the washing period. In general, several

short (5-10 minutes) nonstringent washes are conducted, prior to

the final stringent washes (15-30 minutes). During the washes,

filter-bound background radioactivity can be monitored with the

use of a hand-held monitor.

In principle, this strategy can lead to the molecular cloning of

any G-protein related receptor. Since the extent of the

similarity between opioid receptor sequences and other G-protein

related sequences can only be approximated, the successful use of

this strategy in the cloning of the opioid receptor will require

the careful determination of the appropriate hybridization

conditions. This determination, although based on theoretical

considerations discussed above, will be primarily empirical. The

molecular clone obtained by the homology approach will then be

expressed in eukaryotic cells to confirm the expression of

authentic opioid receptors.

CONCLUSION

Three different strategies can be employed in the molecular

cloning of the opioid receptor. The first strategy consists of

purifying and sequencing the opioid receptor and utilizing the

amino acid sequence in the isolation of a cDNA clone. This

traditional cloning approach has proven to be difficult in the

cloning of the opioid receptor because of the receptor's low

abundance and high sensitivity to denaturation. The second

strategy proposes to clone the opioid receptor through gene

expression in eukaryotic cells without prior purification of the

receptor protein. The cloning of the opioid receptor by this

approach has remained elusive. The third strategy utilizes the

possible homology between opioid receptors and other G-protein

neuroreceptors as a hybridization tool in identifying potential

opioid receptor clones. This approach, while extremely

attractive in light of the growing number of G-protein related

receptors that have found to be homologous, requires the analyses

of many full-length cDNA clones. Regardless of the approach used

in the cloning of the opioid receptor, authenticity must be

verified by careful examination of the clone's structural and

pharmacological properties.

REFERENCES

Amano, T.; Richelson, E.; and Nirenberg, M. Neurotransmitter

synthesis by neurotransmitter clones. Proc Natl Acad Sci USA

69:258-263, 1972.

Anderson, M., and Young, B.D. Quantitative filter hybridization.

In: Hammes, B.D., and Higgins, S.J., eds. Nucleic Acid

Hybridization. Oxford, U.K.: IRL Press, 1987.

Atweh, S.F., and Kuhar, M.J. Distribution and physiological

significance of opioid receptors in the brain. Br Med Bull

39:47-52, 1983.

106
Bidlack, J.M.; Abood, L.G.; Osei-Guinah, P.; and Archer, S.

Purification of the opiate receptors from rat brain. Proc

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

Bonner, T.I.; Brenner, D.J.; Neufeld, B.R.; and Britten, R.J.

Reduction in the rate of DNA reassociation by sequence

divergence. J Mol Biol 81:123-135, 1973.

Bonner, T.I.; Bucke, N.J.; Young, A.C.; and Brann, M.R.

Identification of a family of muscarinic acetycholine

receptor genes. Science 237:527-532, 1987.

Burkholder, A.C., and Hartwell, L.H. The yeast -factor

receptor: Structural properties deduced from sequence of

STE2 gene. Nucleic Acids Res 13:8463-8475, 1985.

Chao, M.; Bothwell, M.; Ross, A.; Koprowski, H.; Lanahan, A.;

Buck, C.; and Sehgal, A. Gene transfer and molecular cloning

of the human NGF receptor. Science 232:518-521, 1986.

Cho, T.M.; Hasegawa, J.I.; Ge, B.L.; and Loh, H.H. Purification

to apparent homogeneity of a μ-type opioid receptor from rat

brain. Proc Natl Acad Sci USA 83:4138-4142, 1986.

Dixon, R.A.; Koblika, B.K.; Strader, D.J.; Benovic, J.L.;

Dohlman, H.G.; Frielle, T.; Bolanowski, M.A.; Bennett, C.;

Rands, E.; Diehl, R.; Mumford, R.; Slater, E.; Sigal, I.;

Caron, M.; Lefkowwitz, R.; and Strader, C. Cloning of the

gene and cDNA for the mammalian -adrenergic receptor and

homology with rhodopsin. Nature 321:75-79, 1986.

Dole, V.P.; Cuatrecasas, P.; and Goldstein, A. Criteria for

receptors. In: Snyder, S.H, and Matthysee, E., eds. Opiate

Receptor Mechanisms. Cambridge, Massachusetts: MIT Press,

1975. pp. 24-26.

Dolhman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of

receptors coupled to guanine nucleotide regulatory proteins.

Biochemistry 26:2657-2664, 1987.

Dove, W.F., and Davidson, N. Cation effects on the denaturation

of DNA. J Mol Biol 5:467-478, 1962.

Gilbert, P.E., and Martin, W.R. The effects of morphine- and

nalorphine-like drugs in the non-dependent, morphinedependent and cyclazocine-dependent chronic spinal dog. J

Pharmacol Exp Ther 198:66-82, 1976.

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

Purification of an active opioid-binding protein from bovine

striatum. J Biol Chem 260:15117-15121, 1985.

Hall, Z. Three of a kind: The -adrenergic receptor, the

muscarinic acetylcholine receptor, and rhodopsin. Trends in

Neuroscience 10:99-100, 1987.

Hedrick, S.M.; Cohen, D.I.; Nielson, E.A.; and Davis, M.M.

Isolation of cDNA clones encoding T cell-specific membrane

associated proteins. Nature 308:149-153, 1984.

Holaday, J.W. Endogeneous opioids and their receptors. In:

Current Concents. Kalamazoo, Michigan: The Upjohn Co.,

1985. pp. 1-64.

107
Kobilka, B.K.; Dixon, R.A.F.; Friele, T.; Dohlman, H.G.;

Bolanowski, M.; Sigal, I.S.; Yang-Feng, T.L.; Francke, U.

Caron, M.G.; and Lefkowitz, R.J. cDNA for the human

adrenergic receptor: A protein with multiple membrane

spanning domains and encoded by a gene whose chromosomal

location is shared with that of the receptor for platelet

derived growth factor. Proc Natl Acad Sci USA 84:46-50,

1987.

Kubo, T.; Fukuda, K.; Mikami, A.; Maeda, A.; Takahashi, H.;

Mishina, M.; Haga, T.; Haga, K.; Ichiyama, A.; Kanagawa, K.;

Kojima, M.; Matsuo, H.; Hirose. T.; and Numa. S. Cloning,

sequencing, and expression of complementary DNA encoding the

muscarinic acetylcholine receptor. Nature 323:411-416,

1986a.

Kubo, T.; Maeda, A.; Sugimoto, K.; Akiba, S.; Mikami, A.;

Tkahashi, H.; Haga, T.; Ichiyama, A.; Kangawa, K.; Matsuo,

H.; Hiroshi, T.; and Numa, S. Primary structure of porcine

cardiac muscarinic acetylcholine receptor deduced from the

cDNA sequence. FEBS Lett 209:367-372, 1986b.

Kuhn, L.C.; McClelland, A.; and Ruddle, F. Gene transfer,

expression, and molecular cloning of the human transferrin

receptor gene. Cell 37:95-103, 1984.

McConoughy, B.L.; Laird, C.D.; and McCarthy, B.J. Nucleic acid

reassociation in formamide. Biochemistry 8:3289-3295, 1969.

Mansour, A.; Khachaturian, H.; Lewis, M.E.; Akil, H.; and Watson,

S.J. Autcradiographic differentiation of opioid

receptors in the rat forebrain and midbrain. J Neurosci

7:2445-2464, 1987.

Marmur, J., and Doty, P. Determination of the base composition

of deoxyribonucleic acid from its thermal denaturation

temperature. J Mol Biol 5:109-118, 1962.

Martin, W.R.; Eades, C.G.; Thompson, J.A.; Huppler, R.E.; and

Gilbert, P.E. The effects of morphine- and nalorphine-like

drugs in the nondependent and morphine-dependent chronic

spinal dog. J Pharmacol Exp Ther 197:517-532, 1976.

Nathans, J., and Hogness, D.S. Isolation and nucleotide sequence

of gene encoding human rhodopsin. Proc Natl Acad Sci USA

81:4851-4855, 1984.

Nathans, J., and Hogness, D.S. Isolation, sequence analysis, and

intron-exon arrangement of the gene encoding bovine

rhodopsin. Cell 34:807-814, 1983.

Newman, E.L., and Barnard, E.A. Identification of an opioid

receptor subunit carrying the μ binding site. Biochemistry

23:5385-5389, 1984.

Pasternak, G.W.; Gintzler, A.R.; Houghton, R.A.; Ling, G.S.F.;

Goodman, R.R.; Spiegel, K.; Nishimura, S.; Johnson, N.; and

Recht, L.D. Biochemical and pharmacological evidence for

opioid receptor multiplicity in the central nervous system.

Life Sci (Suppl 1) 33:167-173, 1983.

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

receptors. In: Udenfriend, S., and Meienhofer, J., eds. The

Peptides. Vol VI. London: Academic Press, 1984. pp. 147-187.

Pert, C.B., and Synder, S.H. Opiate receptor: Demonstration in

nervous tissue. Science 179:1011-1014, 1973.

108
Pfeiffer, A.; Pasi, A.; Mehraein, P.; and Herz, A. Opiate

receptor binding sites in human brain. Brains Res 248:87-96,

1982.

Raetz, C.; Wermuth, M.; McIntyre, T.; Esko, J.; and Wing, C.

Somatic cell cloning in polyester stacks. Proc Natl Acad Sci

USA 79:3223-3227, 1982.

Schwyzer, R.; Karlaganis, G.; and Lang, U. Hormone-receptor

interactions. A study of the molecular mechaism of receptor

stimulation in isolated fat cells by the partial agonist

corticotropin-(5-24)-icosapeptide. In: Ananchenko, S.N., ed.

Frontiers of Bioorganic Chemistry and Molecular Biology

Oxford, U.K. and New York: Pergamon, 277-283, 1980.

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

W. Purification of the opiate receptor of NG-108-15

neuroblastoma-glioma hybrid cells. Proc Natl Acad Sci USA

82:4974-4978, 1985.

Southern, P.J., and Berg, P. Transformation of mammalian cells

to antibiotic resistance with a bacterial gene under control

of the SV40 early region promoter. J Mol Appl Genet 1:327-

341, 1982.

West, R.E., and Miller, R.J. Opiates, second messengers, and

cell response. Br Med Bull 39:53-58, 1983.

Wetmur, J.G., and Davidson, N. Kinetics of renaturation of DNA.

J Mol Biol 31:349-370, 1968.

Yarden, Y.; Rodriguez, H.; Wong, S.K.F.; Brandt, D.R.; May, D.C.;

Burnier, J.; Harkins, R.N.; Chen, E.X.; Ramachandran, J.; and

Ross, E.M. The avian -adrenergic receptor: Primary

structure and membrane topology. Proc Natl Acad Sci USA

83:6795-6799, 1986.

Young, D.; Watches, G.; Birchmeir, C.; Fasano, O.; and Wigler, M.

Isolation and characterization of a new cellular oncogene

encoding a protein with multiple potential transmembrane

domains. Cell 45:711-719, 1986.

Yu. V.; Richards, M.; and Sadee. W. A human neuroblastoma cell

line expresses and opioid receptor sites. J Biol Chem

261:1065-1070, 1986.

Zuker, C.S.; Cowman, A.F.: and Rubin, G.M. Isolation and

structure of a rhodopsin gene from Drosophila melanogaster.

Cell 40:851-858, 1985.

Zukin, R.S., and Maneckjee, R. Solubilization and

characterization of opiate receptors. Methods Enzymol

124:172-190, 1986.

109
AUTHORS

Curtis A. Machida, Ph.D.

John Salon, Ph.D.

David Grandy, Ph.D.

James Bunzow, M.S.

Paul Albert, Ph.D.

Eric Hannemar., Ph.D.

Olivier Civelli, Ph.D.

Vollum Institute for Advanced Biomedical Research

Oregon Health Sciences University

Portland, Oregon 97201, U.S.A.

110
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Effects of Opioid Peptides on

Human Neuroblastoma Cells

Wolfgang Sadee, Dr. rer. nat.; Victor C. Yu, Ph.D.; and

Günther Hochhaus, Ph.D.

The discovery of multiple opioid receptor types (e.g., ) makes it

necessary to study the molecular mechanisms of opioid action in

transformed cell lines that yield homogenous cell populations under

well-defined conditions in vitro. Much has been learned from the use of

such cell lines, which include the mouse neuroblastoma x rat glioma

hybrid NG 108-15 (Gilbert and Richelson 1983). However, this line, as

well as other neuroblastomas tested, expresses only the receptor.

Therefore, we have screened a series of human neuroblastoma cell lines

for the presence of different opioid receptor types. Of three lines with

10,000 opioid sites or more per cell, all displayed s receptor sites

(Hochhaus et al. 1986); however, one of these human neuroblastomas

also expressed abundant μ. receptor sites (SK-N-SH, ~ 50,000 sites per

cell) in a ratio of = 4.5 (Yu et al. 1986). This cell line has potential as

an excellent in vitro model for studying the effects of opiates and opioid

peptides. However, in this cell line we found only a 20 percent inhibition

of PGE1-stimulated adenylate cyclase activity following treatment with

opioid agonists (Yu et al. 1986). An additional problem is that there are

several interconverting phenotypes of SK-N-SH, which include both a

strongly substrate adherent nonneuronal form and a neuroblast form that

grows more slowly to higher saturation cell densities. Only the latter

appears to express opioid receptors.

AN IN VITRO MODEL TO STUDY OPIOID EFFECT

In order to provide more reproducible cell culture conditions, we selected

a phenotypically stable neuroblast subclone of SK-N-SH, designated

SH-SY5Y (Ross and Biedler 1985). The SH-SY5Y clone carries a

similar number of opioid receptors as the parent line, yet opioid inhibition

of adenylate cyclase remains slight.

In order to increase the opioid response, we tested the effects of

neuronal differentiation on receptor-adenylate cyclase coupling. Three

differentiating agents, retinoic acid, nerve growth factor, and dibutyryl

111
FIGURE 1. Human neuroblastoma cells, clone SH-SY5Y, grown in culture in either

the absence (left) or the presence (right) of 10 uM retinoic acid.
cAMP, caused neurite extensions that are characteristic of neuronal

maturation (fig. 1). Of these three, only retinoic acid (10 μM maintained

over 5-6 days) produced differentiated cells that yield a 10- to 50-fold

increase in PGE1-stimulated adenylate cyclase activity. Moreover, the

ability of opioids to inhibit the PGE1 response was also enhanced by

retinoic acid (45 percent inhibition), while nerve growth factor was less

effective (- 35 percent inhibition). We also tested the ability of opioids to

inhibit stimulation of adenylate cyclase by forskolin (100 μM), which

directly acts on the enzyme rather than a stimulatory receptor. Opioids

were even more effective in inhibiting the forskolin response (65 percent

inhibition in retinoic acid treated cells). This opioid effect was inhibited

by naloxone and pretreatment with pertussis toxin, suggesting the

involvement of opioid receptors via an Ni coupling protein (fig. 2). Hence,

the SH-SY5Y subclone, when differentiated with retinoic acid,

represents a system for quantitative studies of u and possibly receptor

mechanisms that include tolerance and opioid dependence.

FIGURE 2. Inhibition of stimulated cAMP production in intact cells by

opioids. The morphine response was defined as maximum

inhibition (i.e., 100 percent). Both PGE1 (1 μM) and forskolin

(100 μM) were employed as the stimulating agents.

113
EFFECT OF μ AND RECEPTOR SELECTIVE OPIOID PEPTIDES

In order to determine which opioid receptor type is responsible for

inhibition of the PGE1- or forskolin–cAMP response, we tested a series

of opioids for their efficacy. Both morphine and the potent general

agonist etorphine gave similar maximal responses (45 percent of the

PGE1 response and 65 percent of the forskolin response). Since these

agonists are not highly selective for either sites, we then chose

several opioid peptides of high selectivity (fig. 2). It should be noted that

the efficacy of any of these ligands at the u sites has not been previously

established. Both the μ specific agonist morphiceptin and the highly μ

selective agonist DAGO gave the same maximal inhibitory effects as

morphine and etorphine. This result strongly suggests that these

peptides are full agonists and that the inhibitory response is mediated by

the μ sites. Indeed, we confirmed earlier findings that morphiceptin is

inactive at the s sites in NG 108-15 cells. In contrast, the potent and

highly selective DPDPE gave only a small partial response at 30 nM, at

which concentration one might expect near maximal activation of sites.

Maximal response of DPDPE at 10 uM was only ~ 65 percent of that

obtained by the other agonists, and it is likely that this partial response is

largely mediated by cross-reaction with the u sites. The agonist

DADLE, on the other hand, gave full inhibitory effects at rather low

concentrations (< 1 μM). These results support the view that the opioid

receptor inhibition of adenylate cyclase in SH-SY5Y cells is largely

mediated by the μ site. Therefore, this subclone is ideally suited for

studying the efficacy, tolerance, and dependence of the narcotic

analgesics in vitro:

It should be noted that all of the above results were obtained in the

presence of the phosphodiesterase inhibitor IBMX. Therefore, the

conclusions from these studies are limited to the activity of adenylate

cyclase. However, initial experiments in the absence of IBMX suggest

that the opioid agonists may also increase the activity of

phosphodiesterase. This would indicate a second mechanism by which

cellular CAMP levels could be regulated. We are currently investigating

this possibility.

BIOTINYLATED ENDORPHIN

A better understanding of the molecular events involved in the opioid

response in neuroblastoma cells can be attained through the study of

opioid receptors on single cells. Such studies would clarify both the

distribution of opioid receptors throughout a cell population and their

location on the cell. A method for rapid isolation and quantitation of

receptors is desirable. Therefore, we intend to utilize the avidin-biotin

system for opioid receptor analysis (Korpela 1984). Typically, a suitable

ligand is labelled with biotin and allowed to bind to the receptor site.

Next, the biotinyl-tracer receptor complex is incubated with avidin.

114
Avidin has an extremely high binding affinity for biotin Kd 10 -15M).

The avidin molecule is then labelled with fluorescent dyes, colloidal gold,

or enzymes. It is important to employ a biotinylated tracer that retains

high receptor affinity in the presence of avidin. (1-31)-endorphin

reacting with biotinyl N-hydroxysuccinamide, with or without an

intervening -aminocaproyl spacer arm, yields a series of biotinylated

products with one or more substitutions per molecule. Tyr-1 and Lys 9,

19, 24, 28, 29 represent possible reaction sites, as shown in figure 3.

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser1 10

Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn11 20

Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu

21 30

FIGURE 3. Human endorphin.

These derivatives were purified by HPLC and characterized by FABmass spectrometry and trypsin digestion followed by HPLC (fig. 4) and

again FAB-MS. Biotinylation of a lysine residue eliminates one tryptic

cleavage site, which allows one to determine the site of biotinylation.

The Tyr-1 terminal NH2 group was not affected in any of the products

isolated, perhaps because, in the tertiary structure, it could have an

internal location and/or it could be involved in hydrogen binding.

Monobiotinylated fractions with substitution at either Lys-9 (fig. 4), Lys19, or Lys-24,28,29 (Bx1 ) were analyzed for binding to the

opioid receptors in rat brain homogenates and neuroblastomas. All

derivatives retained significant opioid receptor affinity, even in the

presence of avidin. The highest affinity among the derivatives was found

with Bx1 , which was equally potent to native endorphin, even in the

presence of avidin (fig. 5). Therefore, this derivative fulfills all criteria for

application of the biotin-avidin system to the analysis of the opioid

receptor.

The biotinylated endorphin derivative Bx1 also displayed high

affinity to a endorphin specific antibody (Peninsula Labs). We

therefore developed an ELISA method for the quantitation of

endorphin, using 21Bx1 as the tracer and an avidin-alkaline phosphatase

complex as the enzyme indicator. Optimal incubation conditions yielded

a sensitivity of 0.5 femtomole ßh-endorphin per sample, equivalent to the

most sensitive 125l-RIA currently available. Current applications of the

biotin-avidin system to opioid molecular pharmacology are in progress.

115
Replica Transfer of Transfectant Colonies

FIGURE 4. HPLC records (reverse phase) of tryptic digests of

endorphin and Bx1 with biotinylation predominantly at

Lys-19.

Screening Eukaryotic Expression Libraries For Neuropeptide

Receptor Genes Using Novel In Situ Replica Filter Binding Assays

FIGURE 5. Equilibrium binding competition curves between 3H-DAGO

(1 rM) and endorphin or Bx1 in rat brain homogenates

in the presence of avidin.

116
REFERENCES

Gilbert, J.A., and Richelson, E. Function of delta opioid receptors in

cultured cells. Mol Cell Biochem 55:83-91, 1983.

Hochhaus, G., Yu, V.C., and Sadée, W. Delta opioid receptors in human

neuroblastoma cell lines. Brain Res 382:327-331, 1986.

Korpela, J. Avidin, a high affinity biotin-binding protein, as a tool and

subject of biological research. Med BioI 62:5-26, 1984.

Ross, R.A. and Biedler, J.C. Presence and regulation of tyrosinase

activity in human neuroblastoma cells. Cancer Res 45:1628-1632,

1985.

Yu, V.C., Richards, M.L., and Sadée, W. A human neuroblastoma cell

line expresses opioid receptors. J Biol Chem. 261:1065-

1070, 1986.

ACKNOWLEDGMENT

Supported by Public Health Research Grant #DA 01095 and DA 01466

from the National Institute on Drug Abuse.

AUTHORS

Wolfgang Sadée, Dr. rer. nat.

Victor C. Yu, Ph.D.

Gunther Hochhaus, Ph.D.

School of Pharmacy

University of California

San Francisco, California 94143, USA

117
Opioid Peptides An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Analgesia and Neuropeptides

David J. Mayer, Ph.D.

INTRODUCTION

Until quite recently, virtually nothing was known about the neurochemical

basis of pain. The past few years have seen an unprecedented explosion of

information in the neurosciences, neurochemistry the role of peptides in

behavior, and, most important here, the neurobiology of pain and pain

modulation.

As statistics about the epidemiology of pain have surfaced, the biomedical

importance of pain research has become apparent. Thus pain research in

general and research on the involvement of opioid and other eptides in pain

in particular have undergone an impressive expansion in the past decade.

This chapter will review the extensive literature on the mechanisms of pain

modulation by opioid and other neuropeptides.

Commensurate with this increase in knowledge about endogenous peptide

involvement in pain modulation has come a search for the environmental

stimuli that might normally activate these systems. Much interest has focused

on the role of painful and stressful environmental events. This chapter will

examine the mvolvement of these types of events in the activation of

endogenous peptide pain modulatory systems.

THE ROLE OF OPIATES AND OPIOID PEPTIDES IN

PAIN MODULATION

The discovery of the enkephalins (Hughes 1975) and the subsequent

discovery of related opioid peptides has led to a great interest in the role

of these substances in pain modulation. This research has elucidated an

important role for these substances in pain modulation. This section will

review both animal and human studies of the role of endogenous opioids in

pain modulation.

Historical Perspective

A number of critical discoveries about the neurobiology of opiate action

have occurred in the past few years: (1) The demonstration of stereospecific

118
and saturable binding sites for opiates in the central nervous system (Hiller

et al. 1973; Pert and Snyder 1973; Terenius 1973). (2) The associated

demonstration for multiple binding sites [See Martin, (Martin

1983) For an excellent review on this topic). (3) The discovery of endogenous ligands for opiate receptors (Hughes 1975). (4) The concept of using

opiate antagonists to antagonize endogenous behavior repertoirses (Akil et

al. 1972). Things findings, along with the extensive utilization of the

intracerebal microinjection technique to localize site of action of

neurochemicals within the central nervous system (Tsou and Jan 1964),

have, in turn, had an important impact on theoretical and methodological

research strategies utilized for the study of the effects of opiates on behavior.

Methodological Considerations

opiates were catalogued and seen in the light of designing drugs with more

therapeutically desirable and fewer therapeutically undesirable effects for

application to clinical pathology. Although clinical utility certainly remains

an important concern of opiate research, the discoveries described above

Before these discoveries the study of the analgesic effect of opiates (as well

as other peptides) was primarily phenomenological. The analgestic effects of

have resulted in research that is conceptually broader. The current overall

view is that opiates often act on endogenous biological substrates for behaviors. Hence the behavioral pharmacology of opiates now addresses the

organization of the neurobiological substrates of behavior. Much of the

research in this area now proceeds in the following pattern: (1) Can a

behavior elicited by opiates be elicited by exogenous environmental manipulations? For example, can analgesia be elicited by transcutaneous nerve

stimulation? (2) If so, can the behavior, when initiated by nonpharmacological means, be shown to utilize endogenous opiates? This question is

generally approached by examining wheth er the behavior can be antagonized

by opiate antagonists and reduce by the induction of tolerance to opiates

(that is, does cross-tolerance occur?). Additional support for a role of

endogenous opiates is provided by the demonstration of a correlation

between release of endogenous opiates and the occurrence of the behavior

under study. (3) If the behavior is shown to involve endogenous opiates, the

precise neuroanatomical foci underlying the behavior are examined utilizing

the microinjection of opiate agonists and antagonists into restricted central

nervous system loci. (4) The opiate receptor type involved in the behavior

is examined by administering opiate agonists and antagonists at least partially

selective for particular receptor subtypes (mu, kappa, delta, etc.). (5) Ideally,

steps (3) and (4) are combined to determme the anatomical locus of specific

receptor types mvolved in the behavior being studied. (6) An attempt is

made to determine the precise endogenous ligand (beta-endorphin,

Met-enkephalin, dynorphin, etc.) involved in a particular behavior. Again

an attempt is made to determine the anatomical locus of action of the particular ligand.

This is, of course, an idealized outline of the path followed to investigate

opioid involvement in a behavior. More often than not the actual course

of research is less direct than this, and the final conclusions are not usually

simple. Nevertheless, keeping in mind the general approach described above

will aid the reader to see the overall progression of the field.

Until the early 1970s, in spite of evidence to the contrary (Tsou and Jang

119
1964; Dewey et al. 1969), the generally accepted theory of opiate analgesic

action (Lim 1966) considered opiates to produce analgesia by a central nervous system mechanism analogous to the action of local anesthetics on a

peripheral nerve. That is, it was thought that opiates acted by directly

inactivating the afferent transmission of pain in the central nervous system.

Since then, a major advance in our conception of the neural processing of

pain has occured. It has become clear that information about tissue damage

is not passively recieved by the nervous system. Rather, it is filtered, even

at the first synopse, by complex modulatory systems. The discovery of these

central nervous system contains endogenous substances, endorphins, that

possess analgesic properties virtually identical to opiates of plant and synthetic origin. In this section, the development of these concepts is examined.

The existence of opiate and nonopiate central nervous system pain modulatory mechanisms activated by environmental stimuli such as stress is then discussed.

Development of the Concept of Endogenous Pain Control

It has long been recognized that a simple invariant relationship between

stimulus intensity and the magnitude of pain perception is often not present.

Two general classes of observations support the complexity of this relationship. The first is the clinical observation that pain is often present without

any apparent precipitating pathology. This situation represents the clinical

problem of pain treatment. More important for the topic of this section is

the common observation that pain may not be experienced in the presence

of factors that should produce it; that is, under a variety of circumstances,

total or partial analgesia is seen. These observations were explicitly

recognized in earlier models of pain perception in spite of the lack of direct

evidence supporting the theoretical models (Noordenbos 1959; Melzack and

Wall 1965). Thus, the concept that the nervous system possesses intrinsic

pain inhibitory mechanisms was recognized when only indirect evidence was

available.

The earliest work indicating opiates produces analgesia, at least in part,

by activation of endogenous pain inhibitory systems was done by Irwin et al.

(1951). They demonstrated that morphine was not effective in inhibiting the

spinally mediated tail flick response in spinalized rats. They reasoned based

on this result that morphine must activate supraspinal neural circuitry which

has an output to the spinal cord and modulates the processing of nociceptive

information at spinal level. This work was largely ignored until the early

1970's even though it was replicated in the mouse (Dewey et al. 1969).

The first impetus for the detailed study of pain-modulatory circuitry resulted

from the observation that electrical stimulation of the brain could powerfully

suppress the perception of pain (Reynolds 1969; Mayer et al. 1971 Further

investigation of stimulation-produced anal esia provided considerable detail

about the neural circuitry Involved [see ayer and Watkins (1984) for a

detailed review of this topic].

Significantly, at that time, several similarities were recognized between these

observations and information emerging from a concomitant resurgence of

interest in the mechanisms of opiate analgesia (Mayer et al. 1971 The most

important parallel facts revealed by these studies were the fo lowing: (1)

120
Effective loci for both opiate microinjection analgesia (Tsou and Jang 1964)

and stimulation-produced analgesia (Mayer et al. 1971) lie within the

periaqueductal and periventricular gray matter of the brain stem. (2) Opiate

analgesia and stimulation-produce analgesia are both mediated in part by

the activation of a centrifugal control system that exits from the brain and

modulates pain transmission at the level of the spinal cord (Dewey et al.

1969; Irwin et al. 1951). (3) The ultimate inhibition of the transmission of

nociceptive information occurs, at least in part, at the initial processing stages

in the spinal cord dorsal horn and homologous trigeminal nucleus caudalis

by selective inhibition of nociceptive neurons (Satoh and Takagi 1971).

In addition to these correlative observations, studies of stimulation-produced

analgesia provided direct evidence indicating that there were mechanisms

in the central nervous system that depend upon endogenous opiates: (1)

Subanalgesic doses of morphine were shown to synergize with subanalgesic

levels of brain stimulation to produce behavioral analgesia (Samanin and

Valzelli 1971). (2) Tolerance, a phenomenon invariably associated with

repeated administration of opiates, was observed to the analgesic effects of

brain stimulation (Mayer and Hayes 1975). (3) Cross-tolerance between the

analgesic effects of brain stimulation and opiates was demonstrated (Mayer

and Hayes 1975). (4) Stimulation-produce (analgesia could be antagonized

by naloxone, a specific narcotic antagonist (Akil et al. 1972, 1976a). This last

observation, in particular, could be most parsimoniously explained if

electrical stimulation resulted in the release of an endogenous opiate-like

factor. Indeed, naloxone antagonism of stimulation-produced analgesia was

a critical impetus leading to the eventual discovery of such a factor (Hughes

1975).

Coincidental with work on stimulation-produced analgesia, another discovery

of critical importance for our current concepts of en ogenous analgesia systems was made. Several laboratories, almost simultaneously, reported the

existence of stereospecific binding sites for opiates in the central nervous

system (Hiller et al. 1973; Pert and Snyder 973; Terenius 1973. These

“receptor” sites were subsequently shown to be localized to neuronal synaptic

regions (Pert et al. 1974) and to overlap anatomically with loci involved in

the neural processing of pain (Pert et al. 1975). The existence of an opiate

receptor again suggested the likelihood of an endogenous compound with

opiate properties to occupy it.

In 1974, Hughes (1975) and Kosterlitz reported the isolation from neural

tissue of a factor enkephalin) with such properties. An immense amount

of subsequent work has characterized this an other neural and extraneural

compounds with opiate properties. As with the opiate receptor, the

anatomical distribution of endogenous opiate ligands shows overlap with

sites involved in pain processing [see

of these studies].

Akil et al. (1984) for a recent review

To summarize these important historical developments, the existence of an

endogenous opiate analgesia system is suggested by several lines of

evidence. Electrical stimulation of the brain produces analgesia The

anatomical structures and neural mechanisms involved in stimulation-produced analgesia parallel those involved in opiate analgesia. The central

nervous system contains opiate binding sites and endogenous ligands capable of interacting with those sites. These binding sites and ligands are found

121
at anatomical loci consistent with sites at which stimulation-produced

analgesia and opiate microinjection analgesia are elicited.

Neural Circuitry Involved in Analgesia Resulting from the Administration

of Exogenous opiates

This section will review the current data available on the sites and mechanisms involved in the modulation of pain by the administration of exogenous

opiates. Primarily, two lines of experimentation will be examined: (1) The

locations in the central nervous system of sites at which administration of

opiates results in analgesia and the administration of opiate antagonists

blocks analgesia. (2) The locations in the nervous system where lesions block

the action of exogenously administered opiates.

Following the work of Tsou and Jang (1964), it wasn’t until the early 1970’s,

with one exception (Lotti et al. 1965), that opiate microin’ection mapping

studies began. Initially these studies concentrated on the periaueductal- eriventricular regions of the mesencephalon and diencephalon (Jacquet

and Laitha 1973; Pert and Yaksh 1974; Yaksh et al. 1976). Overall, these

and other studies confirmed the importance of the periaqueductal-periventricular region in opiate analgesia and provided an impetus for the

examination of other brain areas.

A second brain area that has proved to be of considerable importance for

opiate action is the anatormcally complex re ion of the ventromedial

medulla. This region consists of at least three distinct nuclei: the medially

located nucleus raphe magmus (NRM), more laterally situated nucleus

reticularis paragigantocelluaris (NRP), and the dorsolaterall located nucleus

reticularis gigantocellularis (NRG). Based on retrograde labeling criteria,

Watkins et al. (1980) have proposed the term nucleus raphe alatus (NRA) for

the combined cell groups in NRM and NRP. Takagi et al. 1976) were the

first group to map this region for analgesia resulting

microinjection. Overall (Ta agi k

from morphine

1980), this group found the NRP to be

approximately 20 times more sensitive to mo

found microinjection of morphine into the NRM to be ineffective in the

hine than the NRG. They

production of analgesia. This point is controversial, since other groups

Azami et al. 1982; Zorman et al. 1982) have reported

microinjection into NRM. Axami et al. (1982) did f!ind, however, that the

analgesia from

NRM was less sensitive than the NRPG.

A number of other brain areas including the amygdala (Rodgers 1977, 1978),

the medial lemniscus (VanRee 1977, nucleus medialis dorsalis of the

thalamus (VanRee 1977), the mesence

Mittleman 1978; Pert and Yaksh 1974), and the nucleus of the solitary tract

halic reticular formation (Haigler and

(Oley et al. 1982) have been reported to produce analgesia when injected

with opiates. However, the work on these areas is scant compared with those

discussed above, and the relative potency of injections into these areas has

not been explored.

A final but crucial point to be made in this section concerns the analgesic

effects of microinjections of opiates directly into the intrathecal space of the

spinal cord. Although Tsou and Jang (1964) reported no analgesia from

Direct spinal application of morphine subsequent work has consistently

demonstrated relatively potent effects of intrathecal morphine microinjection

122
(Yaksh and Rudy 1976, 1977).

From this line of evidence it appears, then, that at least three general areas

of the central nervous system are involved in opiate analgesia: the periaqueductal-periventricularray matter, the ventromedial medulla, and the

spinal cord. This observation indicates that the analgesic effects of a systemitally administered opiate may produce analgesia by acting at any, all, or

some combination of these distinct regions.

The utilization of the microinjection of narcotic antagonists has provided at

least a partial answer to this question. A number of early studies concluded

that supraspinal sites of opiate action are the effective ones, since analgesia

from systemically administered opiates was antagonized by either intracranioventricular or intracerebral microinjection of narcotic antagonists

Albus et al 1970; Jacquet and Lajtha 1974; Tsou, 1963; Vigouret et al

1973). Later work, however, demonstrated that naloxone administered

intrathecally could antagonize the analgesia resulting from even relatively

high doses of systemically administered opiates (Yaksh and Rudy 1977).

Thus, these stud ies lead to the paradoxical conclusion that both supraspinal

sites and spinal sites of opiate action are the critical ones involved in

analgesia.

This seeming paradox was resolved in a series of complex but unusually

important studies by Yeung and Rudy (1980a,b). They demonstrated that by

simultaneously administering various doses of morphine intrathecally (into

the spinal cord) and intraventricularly (into the brain) a multiplicative dose

response function was observed. That is, simultaneous spinal and supraspinal

morphine resulted in greater analgesia than the same total dose administered

at either location alone. The effect is quite large, with the multiplicative

factor being as much as 45 under certain circumstances (Yeung and Rudy

1980a). This type of multiplicative interaction, as will be seen below, may

turn out to be a confounding factor in many pharmacological and physiological analyses of the involvement of opiates in various behaviors. Thus, the

reader should keep in mind, when evaluating the literature on this and other

topics, the potential for complex interactions between the same or various

neurotransmitters and/or neuromodulators.

Although the experiments just described elucidate the contribution of spinal

vs. supraspinal sites to opiate analgesia, the relative contribution of the

various supraspinal sites at which opiates act to produce analgesia is Iess

clear. The only work to examine this issue utilizing microinjection of narcotic antagonists was done by Azami et al. (1982). They found, as did the

work of Takagi’s group, that the NRP was more sensitive to morphine than

the NRM for the elicitation of analgesia. Surprisingly, however, they found,

when analgesia was produced by systemically administered morphine,

naloxone injection into the NRM antagonized anal esia more effectively

than injection into more lateral medullary regions including the NRP. They

concluded that NRP does not make a significant contribution to the analgesia resulting from systemic administration of morphine. The relative

contribution of medullary vs. more rostral mesencephalic sites has not been

examined.

An approach similar to the one just described for dissectin the neural

circuitry participating in opiate analgesia utilizes the selective destruction of

123
nuclei and pathways suspected of being involved in opiate analgesia. Opiates

are administered systemically or at discrete sites in the nervous system and

the effects of particular lessons are examined. An overview of this work

supports the conclusion reached above utilizing injection of antagonists. It

appears that several brain areas including the periaqueductal gray matter,

NRM, and NRP need to be intact for the full expression of opiate analgesia.

Environmental Activation of Endogenous Analgesia Systems

The demonstration that opiates activate well-defined neural systems capable

of potently blocking pain transmission suggests, but by no means proves, that

the function of this stem is to dynamically modulate the perceived intensity

of noxious stimuli. If, in fact, this system as such a physiological role, then

one might expect that the level of activity within the system would be

influenced by impinging environmental stimuli. If environmental situations

that produce analgesia could be identified, it would give credibility to the

idea that invasive procedures, such as bram stimulation or narcotic drugs,

inhibit pain by mimicking the natural activity within these pathways.

TABLE 1

REFERENCES FOR TABLE 1

1. Akil and Liebeskind 1975 2. Akil et al. 1976b

124
3. Akil et al. 1972

4. Akil et al. 1978b

5. Augstinsson et al 1977

6. Belenky et al. 1983

7. Bodnar et al. 1978

8. Bodnar et al. 1978

9. Cannon et al. 1982

10. Cannon et al. 1984

11. Chance et al. 1978

12. Chapman et al. 1980

13. Chapman et al. 1976

14. Chesher and Chan 1977

15. Cobelli et al. 1980

16. Coderre and Rollman 1984

17. Das et al. 1984

18. Drugan et al. 1981

19. Fanselow 1979

20. Faris et al. 1983

21. Freeman et al. 1983

22. Frenk and Yitzhaky 1981

23. Galligan et al. 1983

24. Geller 1970

25. Gintzler 1980

26. Girardot and Holloway 1984

27. Girardot and Holloway 1984

28. Grau et al. 1981

29. Grevert et al. 1983

30. Griffiths et al. 1983

31. Hall and Stewart 1983b

32. Hall and Stewart 1983a

33. Han and Xie 1984

34. Han et al .1984

35. Han et al. 1983

36. Hart et al. 1983

37. Hayes et al. 1978

38. Hill and Ayliffe 1981

39. Holaday et al. 1986

40. Hosobuchi et al. 1977

41. Hosobuchi et al. 1979

42. Hyson et al. 1982

43. Janal et al. 1984

44. Jorgensen et al. 1984

45. Juma 1980

46.

47.

Kajander et al. 1984

Kayser and Guilbaud 1981

48. Kraus et al. 1981

49. Kulkami SK 1980

50. Lewis et al. 1981

51. Madden et al. 1977

52. Mah et al. 1980

53. Maier et al. 1980

54.

55.

Mayer and Hayes 1975

Mayer and Murfin 1976

56.

57.

Mayer et al. 1976

Mcgivem et al. 1979

58.

59.

Meyerson et al. 1978

Miczek and Thomson 1984

60. Miczek et al. 1982

61. O’Conner and Chipkin 1984

62. Panerai et al. 1984

63. Pertovaara et al. 1982b

64. Richardson and Akil 1977

65. Ross and Randich 1984

66. Rossier et al. 1977

67. Rossier et al. 1978

68. Saavedra 1981

69. Schlen and Bentley 1980

70. Sessle et al. 1981

71. Shimizu et al. 1981

72.

73.

Shyu et al. 1982

Snow and Dewey 1983

74. Szechtman et al. 1981

75. Terman et al. 1983

76. Teskey and Kavaliers 1984

77. Teskey et al. 1984

78. Urca et al. 1982

79. Urca et al. 1981

80. Watkins et al. 1982d

81. Watkins Cobelli et al. 1982

82. Watkins Young et al. 1983

83. Willer et al. 1982b

84. Willer and Albe-Fessard 1980

85. Willer et al. 1981

86. Willer et al. 1982b

87. Woolf and Wall 1983

88. Zamir et al. 1980

89. Zorman et al. 1982

TABLE 1 - Summary of representative studies on endogenous opiate analgesia

systems. Each number in each cell of the matrix indicates a study utilizing a

particular opiate manipulation to implicate endogenous Opiates in various forms

of environmentaliy produced analgesia. Citation numbers are matched with

authors at the bottom of the table. The various opiate manipulations studied are

indicated in the vertical columns and the environmental manipulations in the

horizontal rows. Abbreviations: CCA = classically conditioned analgesia;

COND = conditioned; CNS = central nervous system; DEP = deprivation;

125
DNIC = diffuse noxious inhibitory controls; ECS = electroconvulsivehock;

FREQ = frequency; FPFS = front paw footshock; INT = intensity; INTER =

intermittent; IT = intrathecal; LO = low; MIN = minutes; NRM = region of

the nucleus raphe magnus; PAG = periaqueductal gray matter; SPA = stimulation produced analgesia,- SPON = spontaneous; TNS = transcutaneous nerve

stimulation; TS = tail shock; 4PFS = four paw footshock

A systematic search for environmental stimuli that activate pain inhibitory

systems was begun by Hayes et al. (1978ab). They observed that potent

analgesia could be produced by such diverse stimuli as brief footshock,

centrifugal rotation, and injection of intraperitoneal saline. These effects

appeared to be specific to pain perception insofar as normal motor behavior,

righting and corneal reflexes, vocalization, startle responses, and response to

touch remain unimpaired (Hayes et al. 1978b). Two important additional

concepts emerged from this work. First was the conclusion that exposure to

“stress” was not sufficient to produce analgesia. Although almost all environmental stimuli studied to date that produce analgesia are stressors (cf.

table 1), the failure of classical stressors, such as ether vapors and horizontal

oscillation, to produce pain inhibition indicated that “stress” was not the

critical variable responsible. Second was the rather unexpected finding that

the opiate antagonist, naloxone, did not block all environmentally induced

analgesias (Hayes et al. 1978b). Therefore, it appeared that non-opiate

systems must exist in addition to the system activated by opiates described

earlier.

Although the stimuli studied by Hayes et al. (1978a,b) did not appear to

activate an opiate system, subsequent investigations found clues that

endogenous opioids might be involved in at least some types of environmentally induced analgesias. Akil et al. (1976b) studied the analgesic effects of

prolonged footshock. In contrast to the results of Hayes et al. (1978a,b),

naloxone did partially antagonize the analgesia. This initial indication of

opiate involvement led Akil and coworkers to look for biochemical evidence

that footshock caused brain opiates to be released. They found that changes

in brain opiate levels did indeed parallel the development of footshockinduced analgesia (Akil et al., 1976b). When tolerance developed to the

analgesic effects of footshock, brain opiate levels returned to control values.

The controversy over the involvement of opiates in footshock induced

analgesia was resolved, in art, by Lewis et al. (1980). They noted that the

duration of footshock used by Hayes et al. (1978b; 1978a) and Akil et al.

(1976b differed greatly and exammed whether this variable might explain

the difference in their results. By comparing the effects of naloxone on

analgesia produced by brief (3 min) vs. prolonged (30 min) footshock, Lewis

et al. (1980) showed that only the latter could be blocked by naloxone. This

suggested that different analgesia systems become active as the duration of

footshock increases.

Concurrent with this work of Lewis et al. (1980), Watkins et al.( 1982d) made

the observation that brief shock restricted to the front paws produced

analgesia that was antagonized by low doses (0.1 mg/kg) of naloxone. In

contrast, even high doses (20 mg/kg) of naloxone failed to reduce analgesia

produced by hind paw shock. In addition, they showed that animals made

tolerant to morphine showed cross-tolerance to front paw but not hind paw

126
footshock analgesia.

endogenous opiate anal gesia system while hind paw shock activates an indeThus it appears that front paw shock activates an

pendent nonopiate analgesia system. In addition, this work showed again

that stress is not a sufficient factor to activate opiate analgesia systems, since

identical shock parameters were used for hind paw and front paw shock

(Watkins et al. 1982d).

Additional work has revealed the following important facts about front paw

and hind paw footshock-induced analgesias (FSIA): (1) Front aw FSIA is

mediated by central nervous system opioids, since elimination of extraneural

opiates by hypophysectomy, adrenalectomy, or sympathetic blockade does not

block the effects (Watkins et al. 1982c). (2) Front paw FSIA involves a

neural circuit that ascends to the brain and then descends by way of the

dorsolateral funiculus (DLF) to block pain transmission at the spinal level

(Watkins et al. 1982a). This descending DLF athway originates in the

nucleus raphe alatus (NRA)(Watkins et al. 1983a). (3) The complete

circuitry for the effect is caudal to the mesencephalon since decerebration

does not affect the analgesia (Watkins et al. 1983b) (4) The critical opiate

synapse for the system is situated in the spinal cord (see figure 1) at the

segment of nociceptive input, since intrathecal injection of naloxone in the

lumbosacral but not thoracic spinal cord blocks the effect (Watkins and

Mayer 1982). (5) Once the system is activated, continued opiate release is

not needed, since naloxone only blocks the effect when given before

footshock but not after (Watkins and Mayer 1982). (6) The integri of

spinal cord serotonin is critical for the expression of front paw FSIA

(Watkins et al. 1984a). (7) Front paw FSIA is blocked by small systemic or

intrathecal doses of the peptide CCK-8 (Faris et al. 1983) and potentiated by

the putative CCK antagonists proglumide and benzotript (Watkins et al.

1984b) (see section below on CCK for a more detail discussion of this

point).

Hind paw FSIA is also a CNS-mediated phenomenon (Watkins et al. 1982c).

However, this manipulation activates both intraspinal and suraspinal pain

inhibitory systems (Watkins et al. 1982a). The brain centers or hind paw

FSIA differ from those for front paw FSIA since NRA lesions do not

eliminate the analgesia Watkins et al 1983a

hind paw FSIA also differ from front paw FSIA: (1) CCK, serotonin, and

. The neurochemical bases of

norepinephrine do not appear to be involved in hind paw FSIA (Watkins et

al. 1984a). (2) Brain, but not spinal cord, acetylcholine is necessary for the

expression of hind paw FSIA but does not appear to be involved in ront paw

FS IA (Watkins et al. 1984c).

involved in front paw FSIA is activated, since conditioned analgesia is

eliminated by (1) systemic and intrathecal naloxone, (2) morphine tolerance,

(3) DLF lesions, and (4) NRA lesions and is unaffected by hypophysectomy,

a enalectomy, and sympathetic blockade (Watkins et al. 1982c) . In addition,

as would be expected, higher structures are involved in the conditioned

analgesia since it is eliminated by decerebration and reduced by periaqueductal gray (PAG) lesions (Watkins et al. 1983b).

Of considerable interest is that both hind paw and front paw FSIA can be

classically conditioned by repeated pairings of a CS with footshock (Watkins

et al. 1982b). Regardless of whether hind paw or front paw shock is used as

127
Figure 1. Neural circuitry of opiate and nonopiate analgesia induced by front

paw and hind paw shock Front paw shock activates the nucleus raphe alatus

(NRA) within the ventral medulla This nucleus sends descending projection

through the DLF to the dorsal horn of the spinal cord A serotonergic pathway

lying outside of the DLF (non-DLF) is recruited as well In turn, endogenous

opioids are released, inhibiting pain transmission neurons (PTN). Activation of

endogenous opiates stimulates a negative feedback loop, which utilizes CCK to

reduce activity of endogenous opioid systems. Hind paw shock inhibits PTN via

two nonopioid pathways: an intraspinal pathway and a descending DLF

pathway. The latter originates from the NRA and from some other yet

unidentified medullary area(s). Classical conditioned (opioid) analgesia seems

to result from activation of the same DLF output path way as from paw (opioid)

FSIA. After conditioning trials in which the conditioned stimulus is paired with

either front paw or hind paw shock (the unconditioned stimulus), the conditioned stimulus becomes capable of activating rostral centers in the brain which,

in turn, activate the periaqueductal gray (PAG) and subsequently the nucleus

raphe alatus. This results, via a descending DLF pathway, in the release of

endogenous opiods within the dorsal horn, producing analgesia.

128
A circuit diagram of these systems is shown in figure 1. A point that should

be emphasized is that the involvement of other neurotransmitters or

neuromodulators at the spinal cord level may be quite complex. For exam le, as shown in figure 1 and discussed in detail below, cholecystokinin

(CCK) appears to modulate endogenous opioid systems. Intrathecal

application of CCK antagonizes anagesia from application of exogenous

opiates as well as analgesia elicited by activation of endogenous opiates

(Faris et al. 1983). Also, CCK antagonists applied intrathecally potentiate

these analgesias as well as reversing opiate tolerance Watkins et al. 1984b).

These findings suggest that other transmitters and/or modulators may

interact with opiates to form complex circuits.

In addition to the work done by Liebeskind’s group [see Terman et al. (1984)

for a review and our own, a number of other laboratories have now

demonstrated that numerous environmental variables can be critical in

determining the particular pain modulatory circuitry activated. Table 1

summarizes the environmental events now known to influence the transmission of pain utilizing endogenous opioid peptides. It is clear that numerous

environmental manipulations result in modulation of pain transmission. The

involvement of endogenous opioids in pain modulation is now beyond

question. On the other hand, man questions remain unanswered. For

example, it is generally not known where a particular endogenous opioid is

released by a particular environmental manipulation nor is the endogenous

ligand or the receptor type involved usually known. Nevertheless, the

techniques and general strategies for answering these questions are available,

and progress in this area should be forthcommg. It should also be pointed

out that, in addition to systems that modulated nociceptive information

utilizing endogenous opioids, there are nonopiate systems as well. For

example, Maier’s group has shown that inescapable tail shock results in

opiate analgesia, but analgesia resulting from tail shocks with identical

temporal and intensity characteristics is nonopiate if the shocks are escapable

(Maier et al. 1982). In sum, a review of the animal data provides strong

evidence for the existence of multiple pain modulatory systems. Our

knowledge of endogenous opiate analgesia systems probably represents the

most detailed description of any opioid behavioral system available. It is,

however, less clear what the critical characteristics of the environmental

stimuli activating these systems are. Although many of the events activating

the systems are clearly stressful, not all are. Also, a number of known

stressors are ineffective.

Evidence for Endogenous Opiate Analgesia in Man

At this point, some parallels will be made between the work described above

and experimental and clinical studies in humans. This will be done in order

to high ight the potential relevance of this work to the very difficult problem

of treating pain syndromes in man. Throughout this discussion, it will be

important to bear in mind that a number of distinct modulatory systems have

been identified under controlled laboratory conditions. In the more

naturalistic circumstances of clinical research, it is likely that more than one

of these systems may be active at any given time, which may account for the

variability and controversy in the clinical literature.

Research on the involvement of endogenous opioids in pain modulation in

129
man has examined a number of environmental manipulations known to have

some degree of efficacy for the reduction of clinical and experimental pain.

Most of these procedures were developed before the recent explosion of

information about endogenous pain control systems. Indeed, many of them

evolved from theoretical approaches that are now outdated or incorrect.

Nevertheless, the procedures are efficacious and have inspired a considerable

body of research aimed at determining the involvement of endogenous

opioids in pain modulation.

There are at least two situations available for study in which endogenous

pain modulatory systems may be active in man. The first involves the basal,

tonic activity within these systems and allows the experimenter to assess

whether pain inhibition occurs continuously, at least to some degree. The

second involve:; clinical manipulations that attempt to activate pain inhibitory

systems.

Attempts have been made to determine whether pain modulatory systems

are tonically active. The assumption made by these studies has been that

administration of opiate antagonists should alter the perception of pain if

opiate systems are tonically active. This change in pain perception would

be recorded either as a decreased pain threshold or an increased level of

ongoing pain. In general, naloxone has failed to affect pain thresholds of

normal human volunteers (Grevert et al. 1978; El-Sobky et al. 1976). On the

other hand, Buchsbaum et al. (1977) found that naloxone lowered the

thresholds of subjects with naturally high pain thresholds, yet had no effect

in subjects with low pain thresholds. This observation is consistent with

reports that the high pain thresholds seen in some cases of congenital

insensitivity to pain can lowered by naloxone (Cesselin et al. 1984; Dehan

et al. 1977; Dehen et al. 1978. It should be pointed out that demonstrating

analgesic or hyperalgesic effects of drugs in human subjects is diffcult.

These studies, taken together, suggest that endogenous opiate pain modulatory circuits may not always be tonically active in people. Such a

conclusion is supported by the report that naloxone decreases the higher pain

thresholds seen in man in the morning as compared with those in the

afternoon (Davis et al. 1978).

Naloxone a pears to be more consistently effective when delivered to

experimental subjects who are experiencing some level of clinical pain. In

this regard, these results are consistent with the animal studies described

above in which pain was observed to be a powerful activator of endogenous

analgesia systems. Thus, Lasagna (1965), Levine et al. (1979), and Gracely

et al. (1983) report that naloxone can increase the reported intensity of

posto erative pain. It is important to note that two of these studies reported

cmplex dose-response interactions between naloxone and pain. Levine et

al. found that while naloxone produced hyperalgesia at high doses, low doses

produced analgesia. The Lasagna study is somewhat more difficult to

interpret because no placebo control group was included, but the results are

consistent with a conclusion that naloxone has both analgesic and hyperalgesic effects. These reports, in turn, are consistent with reports in the animal

literature (Girardot and Holloway 1984a; Grau 1984; Mcgivem et al. 1983)

indicating that certain environmental manipulations produce hyperalgesia as

well as analgesia, and some of these “hyperalgesia systems” may be opiate in

nature since the hyperalgesia can sometimes be antagonized by naloxone.

This is a complex but important observation because it indicates that studies

130
of the effects of naloxone on pain levels must be interpreted with extreme

caution. In conclusion, it appears that, under normal circumstances, endogenous opiate pain inhibitory systems have little spontaneous activity.

However, when some level of pain is present, these systems seem to be

activated.

A second line of research on the involvement of endogenous opioids in pain

modulation in man has examined a number of environmental manipulations

known to have some degree of efficacy for the reduction of clinical and

experimental pain. This research has utilized two primary experimental strategies. The first reasons that if a particular environmental manipulation

induces anal

antagonized by a narcotic antagonist (usually naloxone). The second strate

esia by utilizing endogenous opioids, that analgesia should be

reasons that if endogenous opioids are mvolved in an environmentally

elicited analgesia, then changes should be observed in the levels of these

compounds in plasma or the central nervous system.

Stimulation-produced analgesia in man

Perhaps the most dramatic outcome of the basic science research on

endogenous opioids has been the rapid and effective clinical application to

the treatment of chronic pain syndromes in man. As early as 1973,

Richardson and Akil (1977) reported the use of periventrtcular gray

stimulation to treat pain syndromes. Since then there have been more than

20 reports in the literature describing various studies of this technique. In

a recent review of this literature Young et al. (1984) conclude that “the

method is reasonably effective in properly selected patients and, importantly,

safe.”

Several lines of evidence indicate a likely but not unequivocal role for

endogenous opiates in stimulation produce analgesia (SPA) in man. Table

2 summarizes the evidence for the involvement of endogenous opioids in

SPA in man. Opiate antagonists are reported to reduce SPA, tolerance

develops to SPA, and dependence upon SPA has been reported. A more

controversial literature exists with regard to the release of endogenous opioids, primarily Beta-endorphin, by electrical stimulation of the periaqueductal gray matter in man. It can be seen from table 2 that generally increased

levels of endogenous opioids have been found to result from this stimulation. However, the possibility that these results are due to an artifact of

using a contrast medium for electrode placement has been raised Dionne

et al. 1984; Fessler et al. 1984). This criticism has been convincingly disputed

for at least for some circumstances (Akil and Richardson 1985). At this

point, it seems likely that endogenous opioids mediate, at least in part, the

analgesia elicited by periaqueductal gray stimulation in man. The particular

endogenous opioid and its site an mechanism of action have not been

established.

Counter-irritation analgesia in man

The belief that an acute painful stimulus can be used to alleviate ongoing

pain has been held since antiquity and is known as counter- irritation. This

procedure has a great deal in common with acupuncture and TNS. All use

the application of somatic stimuli, either noxious or innocuous, to obtain

relief from pain. The site of treatment in relation to the painful area is

131
highly variable, ranging from the painful dermatome itself to a theoretically

unpredictable constellation of points in classical Chinese acupuncture. Lastly,

the duration of treatment varies from less than a minute to hours. All of

these factors, as we have seen, are important determinants of the effects

produced by footshock in animals. Thus, the highly variable effects observed

in the clinic would be predicted from animal research. Nevertheless, human

data suggest the involvement of the same systems described above.

TABLE 2

EVIDENCE FOR THE INVOLMENT OF OPIOID PEFTIDES

IN STIMULATION-PRODUCED ANALGESIA IN MAN

NALOXONE TOL/DEP ENDORPHIN LEVELS

B-ENDORPHIN ENKEPHALIN

PLASMA CSF PLASMA CSF

4< 15 2> 5> 4>

10< 1 4 6= 3>

l3< 8 7= ll>

l< 1 2 9> 15<

REFERENCES FOR TABLE 2

1. Adams 1976 9. Hosobwhi 1981

2 Akil and Richardson 1985 10. Hosobuchi et al. 1977

3. Akil et al. 1978a 11. Hosobuchi et al. 1979

4. Akil et al. 1978b 12. Hosobuchi and Wemmer 1977

5. Amano et al. 1980 13. Richardson and Akill 1977

6. Dianne et al. 1984 14. Schmidt et al. 1981

7. Fessler et al. 1984 15. Tsubokawa et al. 1984

8. Hosobuchi 1978

TABLE 2 - The effect of various opiate manipulations on analgesia and

endorphin levels resulting from electrical stimulation of the brain Symbols “< ”

= attenuates analgesia; “= ” = no effect on endorphin level; “>” = increases

endorphin levels. Numbers in the table refer to numbered citations listed

alphabetically below the table. Abbreviation: CSF = cerebrospinal fluid;

TOL/DEP = tolerance or dependence

Twelve studies have measured the effect of naloxone on clinical or experimental analgesia produced by acupuncture. Of these, eight reported

that naloxone reduce the analgesia while four found no effect. In two of

the studies failing to find analoxone effect (Chapman et al. 1980, 1983) the

negative interetation of the results has been called into uestion (Miglen

and Szekely 985; Mayer and Price 1981). The third of the four negative

studies examined lon -term

(Lenhard and Waite 1983) and thus does not fit into the general paradigm

effects of acupuncture on migraine headache

of the other studies addressed here. The final ne

1983) utilized a dose of naloxone (0.4 mg.), which is on the low end of the

ative study (Kenyon et al

132
TABLE 3

(11)==

(8)= (2)<

(29)<

(21)=

EVIDENCE FOR THE INVOLVMENT OF OPIOID PEPTIDES

IN COUNTERRITATION ANALGESIA IN MAN

A

ACUPUNCTURE ANALGESIA

NALOXONE ENDORPHIN LEVELS

B-ENDORPHIN ENKEPHALIN

PLASMA CSF PLASMA CSF

(9)< (27)< (6)>

(14)= (1)<

(24)= (13)> (9)>

(28)= (23)> (6)=

(12)= (22)<

(4)= (3)<

(25)=

(29)< (18)<

(13)=

(26)=

(5)= (17)< (16)>

B

TRANSCUTANEOUS NERVE STIMULATION

NALOXONE B-ENDORPHIN LEVELS

HI FREQ LOW FREQ HIGH FREQ LOW FREQ

PLASMA CSF PLASMA

(15)=

(19)=

(8)=

(2)=

(29)=

(20)=

(22)=

(15)<

(19)=

(2)<

(29)<

(21)=

(22)<

(19)=

(7)>

(10)>

(11)= (19)=

(10)>

REFERENCES FOR TABLE 3

1. Boureau et al. 1979

2. Casale et al. 1983

3. Chapman 1978

4. Chapman et al. 1983

5. Chapman et al. 1980

6. Clement-Jones et al. 1980

7. Facchinetti et al. 1984

8. Freeman et al. 1983

9. He and Do 1983

10. Hughes et al. 1984

11. Johanson et al. 1980

12. Kenyon et al. 1983

13. Kisher et al. 1983

14. Lenhard and Waite 1983

15. Lundberg et al. 1985

16. Masala et al. 1983

17. Mayer et al. 1977

18. Mayer et al. 1976

19. O’Brien et al. 1984

20. Pertovaara and Kermppainen 1981

21. Pertovaara et al. 1982b

22. Sjolund and Eriksson 1979

23. Sjolund et al. 1977

24. Szczudlik and Kwasucki 1984

25. Szczudlik and Lypka 1983

26. Szczudlik and Lypka 1983

27. Tsunoda et al. 1980

28. Umimo et al. 1984

29. Willer et al. 1982b

TABLE 3 - The effect of various opiate manipulations on analgesia and

endorphin levels resulting from transcutaneous nerve stimulation and acupuncture. Symbols:"<"= attenuates endorphin or analgesia level; "=" = no effect

on analgesia or endorphin level; ">" = increase in endorphin levels. Numbers in

the table refer to numbered refrences. Abbreations CSF= cerebrospinal fluid

range of effective doses. Thus, it seems clear that naloxone, at least under

most circumstances, appears to antagonize acupuncture analgesia.

133
Table 3-A summarizes the studies that have examined the effect of naloxone

on “acupuncture” analgesia as well as those studies which have measured

“acupuncture’‘-induced changes in plasma or CSF Beta-endorphin or

enkephalin levels. It is important to note that “acupuncture” is not a

well-defined procedure. The only criterion for including a study

was that the authors call the procedure “acupuncture.” Many of the

in this table

procedures discussed below under transcutaneous electrical nerve stimulation (TNS) are similar or identical to those defined as “acupuncture” here.

The effects of acupuncture on CSF and plasma endorphin levels resent a

somewhat less consistent picture, but this is not surprising considering the

complexities of these types of data Considering that one could question

the entire concept of plasma endorphin levels, since they are indicative of

CNS level in only very indirect ways, a nevertheless somewhat consistent

picture emerges. As can be seen in table 3-A, five studies have reported

endorphin increases while six have reported no effects. Such results should

be interpreted with extreme caution since (1) the meaning of increases in

plasma endorphin levels are entirely unclear, and

levels are likely to be ambiguous since the site of endorphin release probably

2) even CSF endorphin

varies with the particular type of acupuncture stimulation. Nevertheless, an

overview of these data is consistent with an involvement of endogenous

opioids in at least some forms of acupuncture analgesia.

are apparent in the literature. While only 4 of 13 studies of TNS analgesia

have reported naloxone antagonism, all 4 studies utilized low-frequency

TNS. On the other hand none of 6 studies utilizing high-frequen TNS

found a naloxone antagonism (see table 3-B for references). The effects of

TNS on endorphin levels have been less well studied. As seen in table 3-B,

3 of the 6 reported studies have found an increase in endorphin levels, while

the remainder have found no effects. Such results should be interpreted with

the caveats discussed above in mind. Overall, these results are strikingly

consistent with reports in the animal literature (e.g., Han et al. 1984) and

suggest the posseibility that certain types of sensory stimulation either

inactivate opiate systems or activate opiate hyperalgesia systems as discussed

above. Nevertheless, these results are consistent with an emerging picture

Eriksson 1979) invokes endogenous opioid mechanisms. Such studies, taken

together, probaably provide the most convincing evidence available that

endogenous opioids can function to modulate pain transmission in man.

The literature concerning the involvement of endogenous opioids in TNS

analgesia is considerably more complex than that associated with acupuncture analgesia This is likely to result from a greater variability in the

intensity, frerquency, duration, location, and ot er parameters of TNS.

Despite this diversity in experimental paradigm, some general consistencies

that low-freguency, high-intensity acupuncture-like TNS (cf. Sjolund and

In conclusion, acupuncture and transcutaneous nerve stimulation appear to

be forms of counterirritation that activate both opiate and nonopiate

systems. The variable clinical outcomes observed following these treatments

probably result from differential recruitment of segmental, extrasegmental,

opiate, and nonopiate pain inhibitory systems, all of which are now known

to be activated by these types of stimulation in animals.

Stress analgesia in man

A manipulation related to, but not identical with, counterirritation analgesia

is a phenomenon most generally referred to as “stress analgesia.” This issue

134
was discussed above in relation to animal studies, and similar caveats should

be taken under consideration in human studies. These studies have utilized

environmental manipulations tht are either severe physical or psychological

stressors. The stressors catalogued in this section include surgery, labor, and

childbirth, application of overtly painful stimuli such as cold pressor am or

ischemic pain, chronic pain anticipation of pain, and chronic stressfu states

such as life threatening disease.

TABLE 4

EVIDENCE FOR THE INVOLVEMENT OF OPIOID PEPTIDES

IN STRESS ANALGESIA IN MAN

NALOXONE ENDORPHIN LEVELS

B-ENDORPHIN ENKEPHALIN

PLASMA CSF PLASMA CSF

(6) < (8) > (7) > (11) = (12) <

(9) < (3) > (10) <

(15) <

(5) <

(11) >

(1) >

(14) <

(13) < (2) >

(4) <

REFERENCES FOR TABLE 4

1. Atkinson et al. 1983 9. Pertovaara et al. 1982a

2. Cohen et al. 1982

3. Delke et al. 1985

10. Puig et al. 1982

11. Smith et al. 1985

4. Frid and Sin er

5. Frid et al. 1981

1979

6. Jungkinz et al. 1983

7. Katz et al. 1982

8. Pancheri et al. 1985

12. Szczudlik and Lypka 1983

13. Willer and Albe-Fessard 1980

14. Willer et al. 1981

15. Willer et al. 1982a

TABLE 4 - The effect of various opiate manipulations on analgesia and

endorphin levels resulting from stress. Symbols: "<" = attenuates analgesia or

endorphin level; "=" = no effect on analgesia or endorphin level; ">" =

increases endorphin levels. Numbers in the table refer to numbered citations

listed alphabetically below the table. Abbreviation: CSF = cerebrospinal fluid.

Examination of table 4 reveals a strikingly consistent outcome for studies of

this sort. It can be seen that in eight of eight studies in which a “stressful”

manipulation increased nociceptive thresholds, naloxone at least partially

reversed this increase. Another study (Janal et al. 1984

exercise, at least a possible stressor, to increase pain threshold and also

utilized vigorous

showed naloxone reversibility. In addition, and not surprisingly, since

beta-endorphin is coreleased with ACTH, stress resulted in an increase in

plasma beta-endorphin levels in five of five studies. Although such a result

is not convincing alone, it is certainly consistent with the notion that

endogenous opioids may underlie changes in pain threshold produced by

stress. In addition, such results are consistent with the results of counterirritation studies discussed above with respect to the observation that naloxone

135
reversibility is more likely to occur with hi intensity peripheral stimulation

(see table 3) In conclusion, considering the diversirty of procedures used in

counterirration studies and stress studies, a generally convincing picture of

opioid involvement in pain modulation in man emerges. Many important

questions about the nature of this involvement remain unanswered. The

most important of these are the particular endogenous opiate involved and

its site of action. Answers to such questions are unlikely to come from

human studies, since invasive procedures are probably necessary to acquire

such information. The consistency of the animal and human studies,

however, indicates the likelihood that such questions may be studied with

animal models and verified in man.

Placebo analgesia in man

A placebo manipulation may well be considered a form of stress, at least

under some circumstances. Naloxone has also been used to examine whether

endogenous opiates are involved in placebo analgesia. Levine and coworkers

(1978) reported that naloxone antagonized placebo analgesia in postsurgical

patients. Although this conclusion as been questioned on technical rounds

(Goldstein and Grevert 1978; Korczyn 1978; Skrabanek 1978), little

conflicting data have been published. Gracely et al. (1983) have reported

that naloxone results in an increase in pain levels experienced by post

surgical patients that is independent of placebo effects.On the other hand,

Levine and Gordon (1984). in a study at rectified the technical problems

of their earlier one Levine et al. 1978), found convincing evidence for at

least a partial reversal of placebo analgesia by naloxone. A difficulty with all

of these studies is that surgical stress itself is a powerful activator of

endogenous opioid systems (see above and may influence the experimental

results. A carefully controlled study utilizing experimentally induced ischemic

pain (Grevert et al. 1983) indicates that a artial antagonism of placebo

analgesia can occur in the absence of any effects of naloxone on baseline

pain responsivity. At this point, it seems most likely that endogenous opioids

mediate at least some components of placebo analgesia. It should be kept

in mind, however, that placebos, like other analgesic manipulations discussed

above, are multidimensional manipulations likely to ativate multiple pain

inhibitory and possibly pain facilitatory systems. Thus, hypnosis, a manipulation that has at least a superficial resemblance to placebo procedures,

consistently has failed to be antagonized by naloxone (Barber and Mayer

1977; Goldstein and Hilgard 1975; Mayer et al. 1976; Spiegel and Albert

1983). The possibility that opiates are involved in some aspect of placebo

analgesia appears articularly reasonable considering the fact that footshock

analgesia can be classically conditioned in rats. Placebo analgesia can easily

be conceived of as a classical conditioning paradigm wherein the placebo

manipulation (e.g., injections, pills) serves as the conditioned stimulus and

prior medication or treatment serves as the unconditioned stimulus. The

observation by Grevert et al. (1983) that placebo effects tend to extinguish

with repeated trials supports such a conceptualization.

Although explanations of this sort are clearly speculative, they are indicative

of the wealth of concepts from experimental pain research now available for

clinical evaluation. Our increasing knowledge of pain modulatory systems

has the potential not only of providing explanations of current therapies but

of suggesting new approaches for the control of pain. The preponderance of

current pain therapies involve either the surgical destruction of neural tissue

or the use of addictive drug. Such procedures offer great difficulties for the

prolonged treatment of chronic pain. If multiple pain inhibitory systems

136
could be activated pharmacologically or otherwise in an alternating sequence,

the problems of tissue destruction and addiction could be circumvented.

OTHER PEPTIDES

In addition to the endogenous opioids. other peptides have been reported to

be involved in endogenous analgesia-producing mechanisms in various ways.

Thus, neurotensin and substance P produce analgesia upon central administration by themselves; CCK, on the other hand, has been demonstrated

to modulate analgesia produced by other manipulations.

Substance P

In addition to the numerous papers that have proposed substance P (SP) as

a transmitter of primary afferent neurons in the spinal cord and medulla, it

has been demonstrated that SP possesses analgesic properties, as well.

Systemic and intracerebroventricular (ICV) admmistration of SP produce

analgesia in mice (Stewart et al. 1976; Starr et al. 1978; Hall and Stewart

1983a; Meszaros et al 1981; Oehme et al. 1980) and in rats following both

systemic (Mohrland and Gebhart 1979) and ICV (Malick and Goldstein 1978;

Meszaros et al. 1981) injections. It should be noted that this analgesic effect

when compared with that produced by opiates is very small (Frederickson et

at all, particularly following systemic administration, by others Goldstein and

al. 1978; Frederickson and Gesellchen 1980) and has failed to be observed

Malick 1979; Growcott and Shaw 1979; Hayes and yers 1979; Inoki et al.

1977).

The analgesic action of SP is antagonized by naloxone

1978; Frederickson and Gesellchen 1980; Malick and Goldstein 1978; Stewart

Frederickson et al

et al 1976). Since SP does not bind to opiate receptors (Teremus 1975;

Szreniawsky et al. 1980) SP a arently releases opioid peptides. Both the

periaqueductal central gray (PAG) and the spinal cord are likely substrates

for this SP effect because (1) SP has been demonstrated to produce analgesia

both after administration into the PAG (Mohrland and Gebhart 1979 and

following intrathecal administration (Doi and Juma 1981, 1982b , (2) SP

causes a release of opioid peptides in both the PAG (del Rio et al 1983) and

the spinal cord (Tang et al. 1983), and 3) tolerance to and opiate cross-tolerance with the analgesic action of SP has been demonstrated following

intraventricular injections (Naranjo et al. 1984).

The apparent paradox that intrahecal injection of SP may induce both

analgesia and "hyperalgesia" (Akerman et al. 1982; Lembeck et al. 1981;

Matsumra et al. 1985; Yashpal and Henry 1983; Yasphal et al. 1982) [but

see Piercey et al. (1981) for absence of any change in nociceptive threshold]

has been partially resolved by Yashpal and coworkers. They showed that the

SP-induced analgesia occurs after 10-15 min, when "hyperalgesia" disappears.

Although these findings are consistent with results of others, they are in

conflict with those obtained by Han et al. (1984). The latter administered SP

antibodies into the PAG and intrahecally prior to inducing electroacupuncture analgesia. Whereas the SP antibodies reduced electroacupuncture

analgesia following intracerebral administration, confirming the importance

of the analgesic effects of SP at this site in this behavior, they potentiated

electroacupuncture analgesia upon intrahecal administration. In view of the

fact that the hyperalgesic effects of SP have been shown to be generally

short-lived [up to 10 minutes (Yasphal and Henry 1983; Yasphal et al. 1982)]

137
while the analgesic effect has lasted in some studies for over 30 minutes

(e.g. Doi and Juma 1981, 1982b; Yashpal and Henry, 1983), the predominant

hyperalgesic action of spinal SP on electroacupuncture is surprising.

Although the analgesic effect of SP has been reliably observed and its

mechanism understood, the very existence of SP-induced analgesia remains

enigmatic. First, it seems to negate a role for SP as a transmitter of pain, as

the analgesic effects clearly abolish its presumably algesic ones. Second,

since it has been proposed that the presumed hyperalgesia results from spinal

convulsive-like excitation (Bossut et al. 1986), the physiological relevance of

the analgesic action is to be doubted.

Neurotensin

NT has been administered to mouse and rat via three different routes:

intracerebroventricularly (ICV), intracistemally (IC), and intrathecally (IT).

As NT affected the pain threshold in a different way depending on the route

of administration, the literature is reviewed accordingly.

IC administration. The analgesic effect of NT has been observed in mice

Whereas NT analgesia in mice was dose-related and could be observed on

(Clineschmidt and McGuffin 1977) and rats Clineschmidt et al. 1979).

the hot plate test, writhing test Clineschmidt and McGuffin 1977; Davis et

al. 1983; Osbahr et al. 1981), and tail immersion test (Nemeroff et al. 1979,

it could be demonstrated only on the hot plate test and not on the tail-flick

test in rats (Clineschmidt et al. 1979). Naloxone (1-5 mg/kg failed to

antagonize NT-induced analgesia in both species (Clineschmidt and

McGuffin 1977; Clineschmidt et al. 1979. Osbahr et al 1981). however the

observation that repeated pretreatment with morphine reduced the analgesic

effect of NT (Luttmger et al. 1983) could be viewed as cross-tolerance, thus

suggesting some involvement of opiate mechanisms.

NT analgesia is reduced by thyrotropin releasing hormone (Osbahr et al.

1981) and its structural analogs Hemandez et al. 1984). The mechanism of

this antagonism is, as yet, not fully understood.

IT administration. The behavioral effects of a drug injected IC may be

caused at both spinal and supraspinal sites of action. One way to test

whether spinal sites are involved in NT-induced analgesia is administering

the drug IT.

The results of IT administration of NT are somewhat confusing. In rats NT

in doses up to 90 micrograms [3,000 times the dose producmg hot plate

anal esia in this species following IC administration Clineschmidt et al.

1979)], failed to produce hot plate analgesia when administered IT

(Clineschmidt et al. 1982; Martin and Naruse 1982

indicate that the site of action for NT-induced analgesia is supraspinal.

. These results seem to

However, Yaksh et al. (1982) reported that doses of 1-100 micrograms of

neurotensin injected IT produce analgesia on the hot plate, and even some

analgesia on the tail flick test. Moreover, this grou reported that this

analgesia was fully reversible by naloxone (10 or 15 mg/kg). Consistent with .

this report are the finding that IT-administered NT produced naloxone (2

mg/kg) reversible analgesia in the mouse Hylden and Wilcox 1983).

However, this analgesia was not observed on the tail flick reflex but rather

138
ICV administration When NT is administered ICV to mice, it readily

produces analgesia on the writhing test, and this analgesia is not reversible

by naloxone (5 mg/kg) (Nicolaides et al. 1985). However, results obtained

in the rat are more equivocal. Thus, while 20 micrograms of NT ICV in the

latter species induced analgesia in one stud (Martin et al. 1981), 5 micrograms did not in others (Kalivas et al. 1982; Parolaro et al. 1983). Although

these observations seem consistent with the view that these negative findings

result from an insufficient dose of NT, hot plate analgesia from the cisterna

magna was reported following 25 ng of NT (Clineschmidt and McGuffin

1977). It seems, therefore, hardly likely that the anatomical substrate for NT

analgesia is located in the immediate vicinity of the lateral ventricles;

structures in the brainstem in the immediate proximity of the cisterna seem

better candidates. This conclusion was not confirmed by a mapping study,

in which both periventricular structures (medial preoptic area, amygdala),

mesencephalic (rostral periventricular gray), and rhombencephalic (medial

pars of the reticular formation were equally effective injection sites for

inducing hot plate analgesia with NT in rats (Kalivas et al. 1982).

Conclusion NT undoubtedly has analgesic properties, but neither its

mechanism nor its site of action are understood. Thus, it is possible that NT

induces analgesia upon IT administration by releasing endogenous opioids.

This would explain some of the results using this paradigm (Hylden and

Wilcox 1983; Yaksh et al. 1982) but not the lack of analgesia obtained by

others (Clineschmidt et al. 1982; Martin and Naruse 1982).

The increase in latency to response reported from discrete injection of NT

into supraspinal areas (Kalivas et al. 1982) may well reflect true analgesia in

some cases, but not all. Thus, although it is not known whether NT excites

neurons in amygdala or PAG upon iontophoretic application, in view of its

effects in spinal cord and cortex this is a likely possibility. If NT causes

general neuronal excitation, it could well cause analgesia in the PAG, as do

electrical stimulation (Mayer et al. 1971) and injection of glutamate

1977 in forebrain produce analgesia. Further work is needed in order to

Behbehani and Fields 1978; Urca et al. 1980). Also, electrical stimulation

Abbott and Melzack 1978) and morphine (Rodgers 1977, 1978; VanRee

decide these issues.

CCK

The earliest experiments with CCK in systemic doses of over 50 micrograms/kg in the mouse (Zetler 1980, 1983) and intracerebral or intrathecal

doses of 10-40 ng in the rat (Juma and Zetler 1981) demonstrated the

analgesic potential of this peptide. The observation that this anal esia was

reversible by naloxone made these authors suggest that it was mediated by

endogenous opioids. However, at these and higher doses it failed, unlike

morphine, to depress ascending A-delta and C fiber activation (Doi and

Jurna 1982a), suggesting a different mode or site of action.

(Itoh et al. 1985; Han et al. 1985). The antagonistic effect of CCK fo lows an

inverted “U” dose response curve with doses lower and higher than the

aforementioned producing less effect.

More recent experiments indicate that lower doses of CCK, although playing

a role in opiate analgesia, have effects opposite to the one imtially described. Thus, 3 micrograms/kg of systemic and 3.6 ng of intrathecal CCK

antagonize the analgesia produced by morphine and opiate mediated foot

shock analgesia (Faris et al. 1983). Similar effects were observed by others

139
has similar effects. Also, morphine analgesia in rats immunized a ainst CCK

is potentiated (Faris 1985). Proglumide (Suberg et al. 198 or CCK

antibodies (Suberg et al. 1985) potentiate the morphine inducea

reversible suppression of nociceptive spinal cord dorsal horn neurons

, naloxone

indicating that the interaction between C

same site.

CK and morphine occurs at the

Subsequent work with proglumide, a CCK antagonist, showed that this

compound, after both systemic and intrathecal administration, significantly

otentiated analgesia induced by morphine, endogenous opioids, and various

behavioral manipulations that release endogenous opioids in rats (Watkins

et al. 1984b, 1985; Tang et al. 1984) Anothe CCK antagonist, benzotript,

The antagonist action of CCK on opiate action and roglumide’s potentiation seems not to be limited to analgesia. Itoh and Katsuura (1981) found

that CCK significantly

traventricularly injected beta-endorphin in rats, whereas proglumide potenreduced the duration of catalepsy following intiated it Itoh et al. 1985; Katsuura and Itoh 1985). Similarly, proglumide

was found to potentiate morphine’s effect on open field motility in the rat

(Ben-Horin et al. 1984).

The interaction between CCK and opiate action has some very interesting

clinical implications. Thus, it has been demonstrated in rats that proglumide prevents or reduces tolerance induced by repeated morphine administration (Watkins et al. 1984b; Tang et al. 1984). Together with the recent

demonstration that

well (Price et al. 1985),. these findings promise new perspectives both for the

proglumide potentiates morphine analgesia in humans as

use of opiates in treating pain and for treating opiate addicts.

REFERENCES

Abbott, F.V., and Melzack, R. Analgesia produced by stimulation of limbic structures and its

relation to epileptiform after-discharges. Exp Neural 62:720-734, 1978.

Adams, J.E. Naloxone reversal of analgesia produced by brain stimulation in the human. Pain

2:161-166, 1976.

Akerman, B.; Rosell S.; and Folkers, K. (D-Pro2,D-Trp7,9)-SP elicits hypoalgesia and motor

blockade in the rat and antagonizes noxious responses induced by substance P. Acta Physiol

Scand 114:631-633, 1982.

Akil, H., and Liebeskind, J.C. Monoaminergic mechanisms of stimulation-produced analgesia.

Brain Res 94:279-296, 1975.

Akil, H., and Richardson, D.E. Contrast medium cause the apparent increase in beta-endorphin levels in human CSF following brain stimulation. Pain 23:301-304, 1985.

Akil, H.; Mayer, D., and Liebeskind, J. Comparaison chez le rat entre l’analgesie induite par

stimulation de la substance grise periaqueducale et l’analgcsie morphinique. C R Acad Sci

274:3603-3605, 1972.

Akil, H.; Mayer, D.J.; and Liebeskind, J.C. Antagonism of stimulation-produced analgesia by

the narcotic antagonist, naloxone. Science 191:961-962, 1976a.

140
Akil, H.; Madden, J.; Patrick, R.L.; and Barchars, J.D. Stress-induced increase in endogenous

opiate peptides: Concurrent analgesia and its partial reversal by naloxone. In: Kosterlitz, H.W.,

ed. Opiates and Endogenous Opioid Peptids Amsterdam: Elsevier, 1976b. pp. 6367.

Akil, H.; Richardson, D.E.; Hughes, DJ.; and Barchas, J.D. Enkephalin-like material elevated

in ventricular cerebrospinal fluid of pain patients after analgetic focal stimulation. Science

201:463-465, 1978a.

Akil, H.; Richardson, D.E.; Barchas, J.D.; and Li, C.H. Appearance of beta-eadorphin-like

immunoreactivity in human ventricular cerebrospinal fluid upon analgesic electrical stimulation.

Proc Natl Acad Sci USA 75:5170-5172, 1978b.

Akil, H.; Watson, SJ.; Young, E.; Lewis, M.E.; Khachaturian, H.; and Walker, J.M.

Endogenous opioids: Biology and function. Ann Rev Neurosci 73:223-256, 1984.

Albus, K.; Schott, M.; and Herz, A. Interaction between morphine and morphine antagonists

after systemic and intraventricular application. Eur J Pharmacol 12:53-64, 1970.

Amano, K.; Kitamura, K.; Kawamura, H.; Tanikawa, T.; Kawabatake,H.; Notani, M.; Iseki,

H.; Shiwaku, T.; Suda T.,; and Demura, H. Alterations of immunoreactive beta-endorphin in

the third ventricular fluid in response to electrical stimulation of the human periaqueductal

gray matter. Appl Neurophysiol 43:150-158, 1980.

Atkinson, J.H.; Kremer, E.F.; Risch, S.C.; Morgan, C.D.; and Azad, R.F. Plasma measures

of beta-endorphin/beta-Lipotropin-like immunoreactivity in chronic pain syndrome and

psychiatric subjects. Psvchiatry Res 9:319-327, 1983.

Augustinsson, L.-.E.; Bohlin, P.; Bundsen, P.; Carlsson, C.-A.; and Forssman, L. Pain relief

during delivery by trancutaneous electrical nerve stimulation. Pain 4:59-66, 1977.

Azami, J.; Llewelyn, M.B.; and Roberts, M.H.T. The contribution of nucleus reticularis

paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique. Pain 12:229-246, 1982.

Barber, J., and Mayer, D. Evaluation of the efficacy and neural mechanism of a hypnotic

analgesia procedure in experimental and clinical dental pain. Pain 4:41-48, 1977.

Behbehani, M.M., and Fields, H.L. Analgesic effect of glutamate injected into the

periaqueductal gray (PAG) of the rat: Evidence for excitatory interaction between the PAG

and nucleus raphe magnus (nrm). Soc Neurosci Abstr 4:457, 1978.

Belenky, G.L.; Gelinas-Sorelf, D.; Kenner, J.R.; and Holaday, J.W. Evidence for delta-receptor

involvement in the post-ictal antinociceptive responses to electroconvulsive shock in rats. Life

Sci 33 Suppl 1:583-585, 1983.

Ben-Horin, N.; Ben-Horin, N.; and Frenk, H. The effects of proglumide on morphine induced

motility changes. Psychopharmacology 84:541-543, 1984.

Bodnar, R.J.; Kelly, D.D.; Spiaggia, A.; Ehrenberg, C.; and Glusman, M. Dose-dependent

reductions by naloxone of analgesia induced by cold- water stress. Pharmacol Biochem Behav

8:667-672, 1978a.

Bodnar, R.J.; Kelly, D.D.; Spiaggia, A.; and Glusman, M. Stress-induced analgesia: Adaptation

following chronic cold water swims. Bull Psych Soc 11:337-340, 1978b

Bossut, D.; Frenk, H.; and Mayer, D.J. Substance P does not appear to be a primary afferent

141
transmitter of pains in the spinal cord. Soc Neurosci Abstr 12:228, 1986.

Boureau, F.; Willer, J.C.; and Yamaguchi, Y. Abolition by naloxone of the inhibitory effect

of peripheral electrical stimulation on the blink reflex. Electroencephalogr Clin Neurophysiol

47:322-328, 1979.

Buchsbaum, M.S.; Davis, G.C.; and Bunney W.E., Jr. Naloxone alters pain perception and

somatosensory evoked potentials in normal subjects. Nature 270:620-621, 1977.

Cannon, J.T.; Prieto, G.J.; Lee, A.; and Liebeskind, J.C. Evidence for opioid and non-opioid

forms of stimulation-produced analgesia in the rat. Brain Res 243:315-321, 1982.

Cannon, J.T.; Tennan, G.W.; Lewis, J.W.; and Liebeskind, J.C. Body region shocked need not

critically defme the neurochemical basis of stress analgesia. Brain Res 323:316-319, 1984.

Casale, R.; Zelaschi, F.; Guamaschehi, C.; and Bazzini, G. Electroanalgesia by transcutaneous

stimulation (TNS). Response to the naloxone test. Minerv Med 74:941-946, 1983.

Cesselin, F.; Bourgoin, S.; Hamon, M.; Artaud, F.; Testut, M.F.; and Rascal, A. Normal CSF

levels of Met-enkephalin-like material in a case of naloxone-reversible congenital insensitivity

to pain. Neuropeptides 4:217-226, 1984.

Chance, W.T.; White, A.C.; Krynock, G.M.; and Rosecrans, J.A. Conditional fear-induced

antinociception and decreased binding of [3H]N-Leu-enkephalin to rat brain. Brain Res

141:371-374, 1978.

Chapman, C.R. Modulation of experimental dental pain in man with acupuncture and by

transcutaneous electric stimulation. Ann Anesthesiol Fr 19:427-433, 1978.

Chapman, C.R.; Wilson, M.E.; and Gehrig, J.D. Comparative effects of acupuncture and

transcutaneous stimulation on the perception of painful dental stimuli. Pain 2:265-284, 1976.

Chapman, C.R.; Colpitts, Y.M.; Benedetti, C.; Kitaeff, R.; and Gehrig, J.D. Evoked potential

assessment of acupunctural analgesia: Attempted reversal with naloxone. Pain 9:183-198, 1980.

Chapman, C.R.; Benedetti, C.; Colpitts, Y.H.; and Gerlach, R. Naloxone fails to reverse pain

thresholds elevated by acupuncture: Acupuncture analgesia reconsidered. Pain 16:13-32, 1983.

Chesher, G.B., and Chan, B. Footshock induced analgesia in mice - its reversal by naloxone

and cross tolerance with morphine. Life Sci 21:l569-1574, 1977.

Clement-Jones, V.; McLoughlin, L.; Tomlin, S.; Besser, G.M.; Rees, L.H.; and Wen, H.L.

Increased beta-endorphin but not Met-enkephalin levels in human cerebrospinal fluid after

acupuncture for recurrent pain. Lancet 2:946-949, 1980.

Clineschmidt, B.V., and McGuffin, J.C. Neurotensin administered intracisternally inhibits

responsiveness of mice to noxious stimuli. Eur J Pharmacol 46:395-396, 1977.

Clineschmidt, B.V.; McGuffin J.C.; and Bunting, P.B. Neurotensin: Antinocisponsive action

in rodents. Eur J Pharmacol 54:129-139, 1979.

Clineschmidt, B.V.; Martin, G.E.; and Veber, D.F. Antinocisponsive effects of neurotensin and

neurotensin-related peptides. Ann NY Acad Sci 400:283-304, 1982.

Cobelli, DA.; Watkins, L.R.; and Mayer, DJ. Dissociation of opiate and non-opiate footshock

produced analgesia. Soc Neurosci Abstr 6:247, 1980.

142
Coderre, TJ., and Rollman, G.B. Stress analgesia: Effects of PCPA, yohimbine, and naloxone.

Pharmacol Biochem Behav 21:681-686, 1984.

Cohen, M.R.; Pickar, D.; Dubois, M.; and Bunney, W.E. Stress-induced plasma beta-endorphin immunoreactivity may predict postoperative morphine usage. Psychiatry Res 6:7-12, 1982.

Das, S.; Chatterjee, T.K., Ganguly, A.; and Ghosh, J.J. Role of adrenal steroids on

electroacupuncture analgesia and on antagonizing potency of naloxone. Pain 18:135-144, 1984.

Davis, G.C.; Buchsbaum, M.S.; and Bunney, W.E. Naloxone decreases diurnal variation in

pain sensitivity and somatosensory evoked potentials. Life Sci 23:1449-1460, 1978.

Davis, K.R.; Hernandez, D.E.; and Wolfenden, R. Leucinal inhibits brain aminopeptidase

activity and potentiates analgesia induced by Leu-enkephalin. Pharmacol Biochem Behav

19:791-794, 1983.

Dehan, H.; Willer, J.C.; Boureau, F.; and Cambier, J. Congenital insensitivity to pain, and

endogenous morphine-like substances. Lancet 2:293-294, 1977.

Dehen, H.; Willer, J.C.; Prier, S.; Boureau, F.; Gonce, M.; and Cambier, J. Insensitivity to

pain: Electrophysiological study of the nociceptive reflex. Influence of naloxone. Rev Neurol

134:255-262, 1978.

del Rio, J.; Naranjo, J.R.; Yang, H.Y.T.; and Costa, E. Substance P-induced release of

Met5-enkephalin from striatal and periaqueductal gray slices. Brain Res 279:121-126, 1983.

Delke, I.; Minkoff, H.; and Grunebaum, A. Effect of Lamaze childbirth preparation on

maternal plasma beta-endorphin immunoreactivity. Am J Perinatol 2:317-319, 1985.

Dewey, W.L.; Snyder, J.W.; Harris, L.S.; and Howes, J.F. The effect of narcotics and narcotic

antagonists on the tail-flick response in spinal mice. J Pharm Pharmacol 21:548-550, 1969.

Dionne, R.A.; Mueller, G.P.; Young, R.F.; Greenberg, R.P.; Hargreaves, K.M.; and Dubner,

R. Contrast medium causes the apparent increase in beta-endorphin levels in human cerebrospinal fluid following brain stimulation. Pain 20:313-321, 1984.

Doi, T., and Jurna, I. Intrathecal substance P depresses the tail-flick response-antagonism by

naloxone. Naunyn Schmied Arch Pharmacol 317:135-139, 1981.

Doi, T., and Jurna, I. Analgesic effect of intrathecal morphine demonstrated in ascending

nociceptive activity in the rat spinal cord and ineffectiveness of caerulein and cholecystokinin

octapeptide. Brain Res 234:399-487, 1982a

Doi, T., and Jurna, I. Intrathecal substance P depresses spinal motor and sensory responses

to stimulation of nociceptive afferents - Antagonism by naloxone. Naunyn Schmied Arch

Pharmacol 319:154-160, 1982b.

Drugan, R.C.; Grau, J.W.; Maier, S.F.; Madden J, IV, and Barchas, J.D. Cross tolerance

between morphine and the long-term analgesic reaction to inescapable shock. Pharmacol

Biochem Behav 14:677-682, 1981.

El-Sobky, A.; Dostrovnky, J.O.; and Wall, P.D. Lack of effect of naloxone on pain perception

in humans. Nature 263:783-784, 1976.

Facchinetti, F.; Sandrini, G.; Petraglia, F.; Alfonsi, E.; Nappi, G.; and Genazzani, A.R.

143
Concomitant increase in nociceptive flexion reflex threshold and plasma opioids following

trancutaneous nerve stimulation. Pain 19:295-304, 1984.

Fanselow, M.S. Naloxone attenuates rat’s preference for signaled shock. Physiol Psychol

7:70-74, 1979.

Faris, P.L. Opiate antagonistic function of cholecystokinin in analgesia and energy balance

systems. Ann NY Acad Sci 448:437-447, 1985.

Faris, P.; Komisaruk, B.; Watkins, L.; and Mayer, DJ. Evidence for the neuropeptide

cholecystokinin as an antagonist of opiate analgesia. Science 219:310-312, 1983.

Fessler, R.G.; Brown, ED.; Rachlin, J.R.; Mullan, S.; and Fang, V.S. Elevated beta-endorphin

in cerebrospinal fluid after electrical brain stimulation: Artifact of contrast infusion? Science

224:1017-1019, 1984.

Frederickson, R.C.A., and Gesellchen, P. Analgesic and other activities of substance P and

fragments. Neuropeptides and Neural Transmission. New York: Raven Press, 1980. pp.

111-120.

Frederickson, R.C.A.; Burgis, V.; Harrell, C.E.; and Edwards, J.D. Dual actions of substance

P on nociception Possible role of endogenous opioids. Science 199:1359-1362, 1978.

Freeman, T.B.; Campbell J.N.; and Long, D.M. Naloxone does not affect pain relief induced

by electrical stimulation in man. Pain 17:189-196, 1983.

Frenk, H., and Yitzhaky, J. Effects of amygdaloid kindling on the pain threshold of the rat.

Exp Neurol 71:487-496, 1981.

Frid, M., and Singer, G. Hypnotic analgesia in conditions of stress is partially reversed by

naloxone. Psychopharmacology 63:211-215, 1979.

Frid, M.; Singer, G.; Oei, T.; and Rana, C. Reactions to ischemic pain interactions between

individual situational and naloxone effects. Psychopharmacology 73:116-120, 1981.

Galligan, J.J.; Porreca, F.; and Burks, T.F. Dissociation of analgesic and gastrointestinal

effects of electroconvulive shock-released opioids. Brain Res 271:354-357, 1983.

Geller, I. Effect of punishment on lever pressing maintained by food reward of brain

stimulation. Physiol Behav 5:203-206, 1970.

Gintzler, A.R. Endorphin-mediated increases in pain threshold during pregnancy. Science

210:193-195, 1980.

Girardot, M.N., and Holloway, FA. Intermittent cold water stress-analgesia in rats:

Cross-tolerance to morphine. Pharmacol Biochem Behav 20:631-634, 1984a.

Girardot, M.N., and Holloway, FA. Cold water stress analgesia in rats: Differential effects

of naltrexone. Physiol Behav 32:547-555, 1984b.

Goldstein, A., and Grevert, P. Placebo analgesia, endorphins and naloxone. Lancet 2:1385,

1978.

Goldstein, A., and Hilgard, E.R. Failure of the opiate antagonist naloxone to modify hypnotic

analgesia. Proc Natl Acad Sci USA 72:2041-2043, 1975.

144
Goldstein, J.M., and Malick, J.B. Lack of analgesic activity of substance P following

intraperitoneal administration. Life Sci 25:431-436, 1979.

Gracely, R.H.; Dubner, R.; Wolskee, PJ.; and Deeter, W.R. Placebo and naloxone can alter

post-surgical pain by separate mechanisms. Nature 306:264, 1983.

Grau J.W. Influence of naloxone on shock-induced freezing and analgesia. Behav Neurosci

98:278-292, 1984.

Grau, J.W.; Hyson, R.L.; Maier, S.F.; Madden J, IV; and Barchas., J.D. Long-term

stress-induced analgesia and activation of the opiate system. Science 213:1409-1411, 1981.

Grevert, P.; Baizman, E.R.; and Goldstein, A. Naloxone effects on a nociceptive response of

hypophysectomized and adrenalectomized mice. Life Sci 23:723-728, 1978.

Grevert, P.; Albert, L.H.; and Goldstein, A. Partial antagonism of placebo analgesia by

naloxone. Pain 16:129-144, 1983.

Griffiths, E.C.; Longson, D.; and Whittam, A. Biotransformation of dynorphin (1-17) by rat

brain peptidases. J Physiol (London) 342:339P, 1983.

Growcott, J., and Shaw, J. Failure of substance P to produce analgesia in the mouse. Br J

Pharmacol 66:129P, 1979.

Haigler, H.J., and Mittleman, R.S. Analgesia produced by direct injection of morphine into

the mesencephalic reticular formation. Brain Res Bull 3:655-662, 1978.

Hall M.E., and Stewart, J.M. Substance P and antinociception. Peptides 4:31-36, 1983a.

Hall M.E., and Stewart, J.M. Prevention of stress-induced analgesia by substance P. Behav

Brain Res 10:375-382, 1983b.

Han, J.S., and Xie, G.X. Dynorphim Important mediator for electroacupuncture analgesia in

the spinal cord of the rabbit. Pain 18:367-376, 1984.

Han, J.S.; Zhou Z.F.; and Xuan, Y.T. Acupuncture has an analgesic effect in rabbits. Pain

15:83-92, 1983.

Han, J.S.; Xie, GX.; and Zhou Z.F. Acupuncture mechanisms in rabbits studied with

microinjection of antibodies against beta-endorphin, enkephalin and substance P.

Neuropharmacology 23:1-5, 1984.

Han, J.S.; Ding X.Z.; and Fan, S.G. Is cholecystokinin octapeptide (CCK-8) a candidate for

endogenous anti-opioid substrates? Neuropeptides 5:399-405, 1985.

Hart, S.L.; Slusarczyk, H.; and Smith, T.W. The involvement of delta-receptors in stress

induced antinociception in mice. Eur J Pharmacol 95:283-286, 1983.

Hayes, A., and Tyers, M. Effect of intratheral and intracerebroventricular injection of

substance P on nociception in the rat and mouse. Br J Pharmacol 66:488P, 1979.

Hayes, R.L.; Price, D.D.; Bennett, G. J.; Wilcox G.L.; and Mayer, D.J. Differential effects of

spinal cord lesions on narcotic and non-narcotic suppression of nociceptive reflexes: Further

evidence for the physiologic multiplicity of pain modulation. Brain Res 155:91-101, 1978a.

Hayes, R.L.; Bennett, G.J.; Newlon, P.G.; and Mayer, DJ. Behavioral and physiological

145
studies on non-narcotic analgesia in the rat elicited by certain environmental stimuli. Brain Res

155:69-90, 1978b.

He, L.F., and Dong W.Q. Activity of opioid peptidergic system in acupuncture analgesia.

Acupunct Electrother Res 8:257-266, 1983.

Hernandez, D.E.; Nemeroff, C.B.; Valderrama, M.H.; and Prange, AJ., Jr. Neurotensin-induced antinociception and hypothermia in mice: Antagonism by TRH and structural

analogs of TRH. Regul Peptides 8:41-49, 1984.

Hill, R.G., and Ayliffe, SJ. The antinociceptive effect of vaginal stimulation in the rat is

reduced by naloxone. Pharmacol Biochem Behav 14:631-633, 1981.

Hiller, J.M.; Pearson, J.; and Simon, EJ. Distribution of stereospecific binding of the potent

narcotic analgesic etorphine in the human brain: Predominance in the limbic system. Res

Commun Chem Pathol Pharmacol 6:1052-1062, 1973.

Holaday, J.W.; Tortella, F.C.; Meyerhoff, J.L.; Belenky, G.L.; and Hitzemann, RJ.

Electroconvulsive shock activates endogenous opioid systems: Behavioral and biochemical

correlates. Ann NY Acad Sci 467:249-255, 1986.

Hosobuchi,Y. Tryptophan reversal of tolerance to analgesia induced by central grey

stimulation. Lancet 2:47, 1978.

Hosobuchi, Y. Periaqueductal gray stimulation in humans produces analgesia accompanied

by elevation of beta-endorphin and ACTH in ventricular CSF. Mol Probl Pharmacopsychiat

17:109-122, 1981.

Hosobuchi, Y., and Wemmer, J. Disulfiram inhibition of development of tolerance to

analgesia induced by central gray stimulation in humans. EurI J Pharmacol 43:385-388, 1977.

Hosobuchi, Y.; Adams, J.E.; and Linchitz, R. Pain relief by electrical stimulation of the

central gray matter in humans and its reversal by naloxone. Science 196:183-186, 1977.

Hosobuchi, Y.; Rossier, J.; Bloom, F.E.; and Guillemin, R. Stiiulation of human periaqueductal gray forr pain relief increases immuno-reactive beta-endorphin in ventricular fluid.

Science 203:279-281, 1979.

Hughes, J. Search for the endogenous ligand of the opiate receptor. Neurosci Res Prog Bull

13:55-58, 1975.

Hughes, G.S.; Lichstein, P.R.; Whitlock, D.; and Harker, C. Response of plasma beta-endorphins to transcutaneous electrical nerve stimulation in healthy subjects. Phys Ther

64:1062-1066, 1984.

Hylden, J.L.K., and Wilcox, G.L. Antinociceptive action of intrathecal neurotensin in mice.

Peptides 4:517-520, 1983.

Hyson, R.L.; Ashcraft, L.J.; Drugan, R.C.; Grau, J.W.; and Maier, S.F. Extent and control of

shock affects naltrexone sensitivity of stress-induced analgesia and reactivity to morphine.

Pharmacol Biochem Behav 17:1019-1025, 1982.

Inoki, R.; Matsurooto, K.; Oka, M.; Kotami, Y.; and Kudo, T. Algesiogenic activity of

synthetic substance P. Jpn J Pharmacol 27:75, 1977.

Irwin, S.; Houde, R.W.; Bennett, D.R.; Hendershot, L.C.; and Seevers, M.H. The effects of

146
morphine, methadone and meperidine on some reflex responses of spinal animals to

nociceptive stimulation. J Pharmacol Exp Ther 101:132-143, 1951.

Itoh, S., and Katsuura, G. Suppressive effect of cholecystokinin and its related peptides in

beta-endorphin-induced catalepsy in rats. Eur J Pharmacol 74:381-385, 1981.

Itoh, S.; Katsuura, G.; Yoshikawa, K; and Rehfeld, J.F. Potentiation of beta-endorphin effects

by cholecystokinin antiserum in rats Can J Physiol Pharmacol 63:81-83, 1985.

Jaquet, Y.F., and Lajtha, A. Morphine action at central nervous system sites in rat: Analgesia

or hyperalgesia depending on site and dose. Science 182:490-491, 1973.

Jaquet, Y.F., and Lajtha, A. Paradoxical effects after microinjection of morphine in the

periaqueductal gray matter in the rat. Science 185:1055-1057, 1974.

Janal, M.N.; Colt, E.W.D.; Clark, W.C.; and Glusman, M. Pain sensitivity, mood and plasma

endocrine levels in man following long-distance running: Effects of naloxone. Pain 19:13-26,

1984.

Johansson, F.; Almay, B.G.L.; Von Knorring, L.; and Terenius, L. Predictors for the outcome

of treatment with high frequency transcutaneous electrical nerve stimulation in patients with

chronic pain. Pain 9:55-65 1980.

Jorgensen, HA.; Fasmer, O.B.; Berge, O.G.; Tveiten, L.; and Hole, K. Immobilization-induced analgesia: Possible involvement of a non-opioid circulating substance. Pharmacol

Biochem Behav 20:289-292, 1984.

Jungkinz, G.; Engel, R.R.; King, U.G.; and Kuss, H.J. Endogenous opiates increase pain

tolerance after stress in humans. Psychiatry Res 8:13-18, 1983.

Jurna, I. Effect of stimulation in the periaqueductal grey matter on activity in ascending axons

of the rat spinal cord: Selective inhibition of activity evoked by afferent A-delta and C fibre

stimulation and failure of naloxone to reduce inhibition. Brain Res 196:33-42, 1980.

Jurna, I., and Zetler, G. Antinociceptive effect of centrally administered caerulein and

cholecystokinin octapeptide (CCK-8). Eur J Pharmacol 73:323-331, 1981.

Kajander, KC.; Ebner, TJ.; and Bloedel, J.R. Effects of periaqueductal gray and raphe

magnus stimulation on the responses of spinocervical and other ascending projection neurons

to non-noxious inputs. Brain Res 291:9-37, 1984.

Kalivas, P.W.; Jennes, L.; Nemeroff, C.B.; and Prange A.J, Jr. Neurotensin: Topographical

distribution of brain sites involved in hypothermia and antinociception. J Comp Neural

210:225-238, 1982.

Katsuura, G., and Itoh, S. Potentiation of beta-endorphin effects by proglumide in rats. Eur

J Pharmacol 107:363-366, 1985.

Katz, E.R.; Sharp, B.; Kellerman, J.; Marston, A.R.; Hershman, J.M.; and Siegel, S.E.

Beta-endorphin immunoreactivity and acute behavioral distress in children with leukemia. J

Nerv Ment Dis 170:72-77, 1982.

Kayser, V., and Guilbaud, G. Dose-dependent analgesic and hyperalgesic effects of systemic

naloxone in arthritic rats. Brain Res 226:344-348, 1981.

Kenyon, J.N.; Knight, CJ.; and Wells, C. Centre for the study of alternative therapies.

147
Acupunct Elect Other Res 8:17-24, 1983

Kiser, R.S.; Khatami, M.J.; GatcheI, R.J.; Huang, X.Y.; and Bhatia, K. Acupuncture relief of

chronic pain syndrome correlates with increased plasma met-enkephalin concentrations. Lancet

2:1394-1396, 1983.

Korczyn, A. Mechanism of placebo anaIgesia. Lancet 2:1304-1305, 1978.

Kraus, E.; LeBars, D.; and Besson, J.M. Behavioral confirmation of diffuse noxious inhibitory

controls (DNIC) and evidence for a role of endogenous opiates. Brain Res 206:495-595, 1981.

Kulkarni, S.K. Heat and other physiological stress-induced analgesia; catecholamine mediated

and naloxone reversible responses. Life Sci 27:185-189, 1980.

Lasagna, L. Drug interaction in the field of analgesic drugs, Proc Roy Soc Med 58:978-983,

1965.

Lembeck, F.; Donnerer, J.; and Colpaert, F.C. Increase in substance P in primary afferent

neurons during chronic pain. Neuropeptides 1:175-180, 1981.

Lenhard, L., and Waite, P.M. Acupuncture in the prophylactic treatment of migraine

headaches: Pilot study. NZ Med J 96:663-666, 1983.

Levine, J.D., and Gordon, N.C. Influence of the method of drug administration on analgesic

response. Nature 312:755-756, 1984.

Levine, J.D.; Gordon, N.C.; and Fields, H.L. The mechanism of placebo analgesia. Lancet

2:654-657, 1978.

Levine, J.D.; Gordon, N.C.; Bornstein, J.C.; and Fields, H.L. Role of pain in placebo

analgesia. Proc Natl Acad Sci USA 76:3528-3531, 1979.

Lewis, J.W.; Cannon, J.T.; and Liebeskind, J.C. Opioid and nonopioid mechanisms of stress

analgesia. Science 208:623-625, 1980.

Lewis, J.W.; Cannon, J.T.; Chudler, E.H.; and Liebeskind, J.C. Effects of naloxone and

hypophysectomy on electroconvulsive shock-induced analgesia. Brain Res 208:230-233, 1981.

Lim, R.K.S. A revised concept of the mechanism of analgesia and pain. In: Knighton, R.S. and

Dumke, P.R., eds. Pain. Boston: Little, Brown and Company, 1966, 117-154.

Lotti, VJ.; Lomax P.; and George, R. Temperature responses in the rat following

intracerebral microinjection of morphine. J Pharmacol Exp Ther 150:135-139, 1965.

Lundberg, T.; Bondesson, L.; and Lundstrom, V. Relief of primary dysmenorrhea by

transcutaneous electrical nerve stimulation. Acta Obstet Gynecol Scand 64:491-497, 1985.

Luttinger, D.; Burgess, S.K.; Nemeroff, C.B.; and Prange, AJ., Jr. The effects of chronic

morphine treatment on neurotensin-induced antinociception. Psychopharmacology 81:10-13,

1983.

Madden, J. IV, AkiI, H.; Patrick, R.L.; and Barchas, J.D. Stress-induced parallel changes in

central opioid levels and pain responsiveness in the rat. Nature 265:358-360, 1977.

Mah, C.; Suissa, A.; and Anisman, H. Diiation of antinociception and escape deficits

induced by stress in mice. J Comp Physiol Psych 94:1160-1171, 1980.

148
Maier, S.F.; Davies, S.; Grau, J.W.; Jackson, R.L.; Morrison, D.H.; Moye, T.; and Barchas,

J.D. Opiate antagonists and long-term analgesic reaction induced by inescapable shock in rats.

J Comp Physiol Psychol 194:1172-1183, 1980.

Maier, S.F.; Drugan, R.C.; and Grau, J.W. Controlability, coping behavior, and stress-induced

analgesia in the rat. Pain 12:47-56, 1982.

Malick, J., and Goldstein, J. Analgesic activity of substance P following intracerebral

administration in rats. Life Sci 23:835-844, 1978.

Martin, W.R. Pharmacology of opioids. 4. Pharmcol Rev 35:283-383, 1983.

Martin, G.E., and Naruse, T. Differences in the pharmacological actions of intrathecally

administered neurotensin and morphine. Regul Peptides 3:97-103, 1982.

Martin, G.E.; Naruse, T.; and Papp, N.L. Antinociceptive and hypothermic actions of

neurotensin administered centrally in the rat. Neuropeptides 1:447-454, 1981.

Masala, A.; Satta, G.; Alagna, S.; Zolo, TA.; Rovasio, PP.; and Rassu, S. Suppression of

electroacupuncture (EA)-induced beta-endorphin and ACTH release by hydrocortisone in

man. Absence of effects on EA-induced anaesthesia. Acta Endocrinol 103:469-472, 1983.

Matsumura, H.; Sakurada, T.; Hara, A.; Sakurada, S.; and Kisara, K. Characterization of the

hyperalgesic effect induced by intrathecal injection of substance P. Neuropharmacology

24:421-426, 1985.

Mayer, DJ., and Hayes, R. Stimulation-produced analgesia: Development of tolerance and

cross-tolerance to morphine. Science 188:941-943, 1975.

Mayer, DJ., and Murfin, R. Stimulation-produced analgesia (SPA) and morphine analgesia

(MA): Cross-tolerance from application at the same brain site. Fed Proc 35:385, 1976.

Mayer, DJ., and Price, D.D. Naloxone reversal of acupuncture analgesia. Pain 11:273-275,

1981.

Mayer, DJ., and Watkins, L.R. Multiple endogenous opiate and nonopiate analgesia systems.

In: Kruger, L., and Liebeskind, J.C., eds. Advances in Pain Research and Therapy. New York:

Raven Press, 1984 pp. 253-276.

Mayer, DJ.; Wolfle, T.L.; Akil, H.; Carder, B.; and Liebeskind, J.C. Analgesia from electrical

stimulation in the brainstem of the rat. Science 174:1351-1354, 1971.

Mayer, DJ.; Price, D.D.; Rafii A.; and Barber, J. Acupuncture hypalgesia: Evidence for

activation of a central control system as a mechanism of action. In: Bonica, J.J., and

Albe-Fessard D.G., eds. Advances in Pain Research and Therapy. New York Raven Press,

1976. pp. 751-754.

Mayer, D.J.; Price, D.D.; and Rafii, A. Antagonism of acupuncture analgesia in man by the

narcotic antagonist naloxone. Brain Res 121:368-372, 1977.

Mcgivern, R.; Berka, C.; Bernston, G.G.; Walker, J.M.; and Sandman, CA. Effect of naloxone

on analgesia induced by food deprivation. Life Sci 25:885-889, 1979.

Mcgivern, R.F.; Mousa, S.; Courti, D.; and Bernston, G.G. Prolonged intermittent footshock

stress decreases Met and Leu enkephalin levels in brain with concomitant decreases in pain

149
threshold. Life Sci 33:47-54, 1983.

Melzack, R., and Wall, P.D. Pain mechanisms: A new theory. Science 150:971-979, 1965.

Meszaros, J.B.; Tarchalska, B.; Gajewska, S.; Janicki, J.; Duriasz, H.; and Substance P,

hexapeptide pGlu6(SP6-11), analgesia and serotonin depletion. Pharmacol Biochem Behav

14:11-15, 1981.

Meyerson, BA.; Boethius, J.; and Carlsson, A.M. Percutaneous central gray stimulation for

cancer pain. Appl Neurophysiol 41:57-65, 1978.

Miczek, KA., and Thompson, M.L. Analgesia resulting from defeat in a social confrontation

- the role of endogenous opioids in brain. In: Bandler, R., ed. Modulation of Sensorimotor

Activity During Alterations in Behavioral States. New York: Alan R. Liss, 1984. pp. 431-456.

Miczek, K.A.; Thompson, M.L.; and Shuster, L. Opioid-like analgesia in defeated mice.

Science 215:1520-1522, 1982.

Miglecz E., and Szekely, J.I. Intracerebroventricular saline treatment elevates the pain

threshold. Is this phenomenon mediated by peripheral opiate receptors? Pharmacol Res

Commun 17:177-187, 1985.

Mohrland, J.S., and Gebhart, G.F. Substance P-induced analgesia in the rat. Brain Res

171:556-559, 1979.

Naranjo, J.R.; Fernandez-Tome, M.P.; and del Rio, J. Analgesic activity of substance P in rats:

Tolerance and cross-tolerance with enkephalin. Eur J Pharmacol 98:l33-136, 1984.

Nemeroff, C.B.; Osbahr III, A.J.; Manberg, P.J.; Ervin, G.N.; and Prange, A.J., Jr. Alterations

in nociception and body temperature after intra- cisternal administration of neurotensin,

beta-endorphin, other endogenous peptides and morphine. Proc Natl Acad Sci USA

76:5368-5371, 1979.

Nicolaides, E.D.; Lunney, EA.; Kaltenbronn, J.S.; Wiley, J.N.; and Downs, DA. Anti-writhing

activity of some peptides related to neurotensin and tuftsin. J Pept Prot Res 25:435-441, 1985.

Noordenbos, W. Pain. Amsterdam: Elsevier, 1959.

O’Brien, WJ.; Rutan, F.M.; Sanborn, C.; and Omer, G.E. Effect of transcutaneous electrical

nerve stimulation on human blood beta-endorphin levels. Phys Ther 64:1367-1374, 1984.

O’Conner, P., and Chipkin, R.E. Comparisons between warm and cold water swim stress in

mice. Life Sci 35:631-640, 1984.

Oehme, P.; Hecht, K.; Pieche, L.; Hilse., H.; Morgenstern, E.; and Poppei, M. Substance P

as a modulator of physiological and pathological processes. In: Marssan, C.A., and Traczyk,

W.Z., eds. Neuropeptides and Nueral Transmission. New York: Raven Press, 1980.

Oley, N.; Cordova, C.; Kelly, M.L.; and Bronzino, J.D. Morphine administration to the region

of the solitary tract nucleus produces analgesia in rats. Brain Res 236:511-515, 1982.

Osbahr III, A.J.; Nemeroff, C.B.; Luttinger, D.; Mason, GA.; and Prange, AJ., Jr.

Neurotensin-induced antinociception in mice: Antagonism by thyrotropin-releasing hormone.

J Pharmacol Exp Ther 217:645-651, 1981.

Pancheri, P.; Zichella, L.; Fraioli, F.; Carilli, L.; Perrone, G.; Biondi, M.; Fabbri, A.; Santoro,

150
A.; and Moretti, C. ACTH, beta-endorphin and Met-enkephalin Peripheral modifications

during the stress of human labor. Psychoneutoendocrinology 10:289-301, 1985.

Panerai, A.E.; Martini, A.; Sacerdote, P.; and Mantegazza, P. Kappa-receptor antagonist

reverse ‘non-opioid’ stress-induced analgesia. Brain Res 304:153-156, 1984.

Parolaro, D.; Sala, M.; Crema, G.; Spazi, L.; and Gori, E. Effect on intestinal transit of

neurotensin administered intracerebroventricularly to rats. Life Sci 33:485-488, 1983.

Pertovaara, A., and Kemppainen, P. The influence of naloxone on dental pain threshold

elevation produced by peripheral conditioning stimulation at high frequency. Brain Res

215:426-429, 1981.

Pertovaara, A.; Kemppainen, P.; Johansson, G.; and Karonen, S.L. Ischemic pain nonsegmentally produces a predominant reduction of pain and thermal sensitivity in man: A selective role

for endogenous opioids. Brain Res 251:83-92, 1982a.

Pertovaara, A.; Kemppainen, P.; Johansson, G.; and Karonen, S.-.L. Dental analgesia

produced by non-painful, low-frequency stimulation is not influenced by stress or reversed by

naloxone. Pain 13:379-384, 1982b.

Pert, C.B., and Snyder, S.H. Opiate receptor: Demonstration in nervous tissue. Science

179:1011-1013, 1973.

Pert, A., and Yaksh T. Sites of morphine induced analgesia in the primate brain: Relation

to pain pathways. Brain Res 80:135-140, 1974.

Pert, C.B.; Snowman, A.M.; and Snyder, S.H. Localization of opiate receptor binding in

synaptic membranes of rat brain. Brain Res 70:184-188, 1974.

Pert, C.B.; Kuhar, M.J.; and Snyder, S.H. Autoradiographic localization of the opiate receptor

in rat brain. Life Sci 16:1849-1854, 1975.

Piercey, M.F.; Dobry, P.J.K; Schroeder, LA.; and Einspahr, J. Behavioral evidence that

substance P may be a spinal cord sensory neurotransmitter. Brain Res 210:407-412, 1981.

Price, D.D.; von der Gruen, A.; Miller, J.; Rafii, A.; and Price, C. Potentiation of systemic

morphine analgesia in humans by proglumide, a cholecystokinin antagonist. Anesthesia Analges

64:801-806, 1985.

Puig, M.M.; Laorden, M.L.; Miralles, F.S.; and Olaso, M. J. Endorphin levels in cerebrospinal

fluid of patients with postoperative and chronic pain. Anesthesiology 57:1-4, 1982.

Reynolds, D.V. Surgery in the rat during electrical analgesia induced by focal brain

stimulation Science 164:444-445, 1969.

Richardson, DE., and Akil, H. Pain reduction by electrical brain stimulation in man. Part I:

Acute administration in periaqueductal and periventricular sites. J Neurosurg 47:178-183, 1977.

Rodgers, RJ. Elevation of aversive threshold in rats by intra-amygdaloid injection of

morphine sulphate. Pharmacol Biochem Behav 6:385-390, 1977.

Rodgers, RJ. influence of intra-amygdaloid opiate injections on shock thresholds, tail-flick

latencies and open field behaviour in rats. Brain Res 153:211-216, 1978.

Rossier, J.; French, E.D.; Rivier, C.; Ling, N.; Guillemin, R.; and Bloom, F.E. Foot-shock

stress increases beta-endorphin levels in blood but not brain. Nature 270:618-619, 1977.

151
Rossier, J.; Guillemin, R.; and Bloom, F. Foot shock induced stress decreases Leu5-enkephalin immunoreactivity in rat hypothalamus. Eur J Pharmacol 48:465-466, 1978.

Ross, R.T., and Randich, A. Unconditioned stress-induced analgesia following exposure to

brief footshock. J Exp Psychol Anim Behav Proc 10:127-137, 1984.

Saavedra, J.M. Naloxone reversible decrease in pain sensitivity in young and adult

spontaneously hypertensive rats. Brain Res 209:245-250, 1981.

Samanin, R., and VaIzelli, L. Increase of morphine-induced analgesia by stimulation of the

nucleus raphe dorasalis comment. Eur J Pharmacol 16:298-382, 1971.

Satoh, M., and Takagi, H. Effect of morphine on the pre- and postsynaptic inhibitions in the

spinal cord. Eur J Pharmacol 14:150-154, 1971.

Schlen, H., and Bentley, GA. Tbe possibility that a component of morphine-induced analgesia

is contributed indirectly via the release of endogenous opioids. Pain 9:73-84, 1980.

Schmidt, B.; Richter, R.G.; and Thoden, U. Addiction-like behavior with continuous

self-stimulation of the mediothalamic system. Arch Psychiatr Nervenkr 230:55-61, 1981.

Sessle, B.J.; Hu, J.W.; Dubner, R.; and Lucier, G.E. Functional properties of neurons in cat

trigeminal subnucleus caudalis (medullary dorsal born). II. Modulation of responses to noxious

and nonnoxious simuli by PAG, n. raphe magnus, cerebral cortex, and afferent influences and

effect of naloxone comment. J Neurophysiol 45:193-207, 1981.

Shimizu, T.; Koja, T.; Fujisaki, T.; and Fukuda, T. Effects of methysergide and naloxone on

analgesia induced by the peripheral electric stimulation in mice. Brain Res 208:463-467, 1981.

Shyu, B.-.C.; Andersson, SA.; and Thoren, P. Endorphin mediated increase in pain threshold

induced by long-lasting exercise in rats. Life Sci 30:833-841, 1982.

Sjolund, B.H., and Eriksson, M.B.E. Tbe influence of naloxone on analgesia produced by

peripheral conditioning stimulation. Brain Res 173:295-382, 1979.

Sjolund, B.; Terenius, L.; and Erikson, M. Increased cerebrospinal fluid levels of endorphins

after electro-acupuncture. Acta Physiol Scand 100:382-384, 1977.

Skrabanek, P. Naloxone and placebo. Lancet 2:791, 1978.

Smith, R.; Besser, G.M.; and Rees, L.H. The effect of surgery on plasma beta-endorpbin and

methionine-enkephalin. Neurosci Lett 55:17-21, 1985.

Snow, A.E., and Dewey, W.L. A comparison of antinociception induced by foot shock and

morphine. J Pharmacol Exp Ther 227:42-50, 1983.

Spiegel, D., and Albert, L.H. Naloxone fails to reverse hypnotic alleviation of chronic pain.

Psychopharmacology 81:140-143, 1983.

Starr, M.; James, ‘I’.; and Gaytten, D. Behavioral depressant and antinociceptive properties

of substance P in the mouse: Possible implication of brain monoamines. Eur J Pharmacol

48:203-212, 1978.

Stewart, J.; Getto, C.; Neldner, K.; Reeve, E.; Krivoy, W.; and Zimmermann, E. Substance

P and analgesia. Nature 262:784-785, 1976.

152
Suberg, S.N.; Watkins, L.R.; Culhane, E.S.; and Carstens, E. Morphine-induced suppression

of spinal transmission is potentiated by proglumide in a naloxone reversible manner. Soc

Neurosci Abstr 10:106, 1984.

Suberg, S.N.; Culhane, E.S.; Rosenquist, G.; Carstens, E.; and Watkins, L.R. Effect of

anti-cholecystokinin antibody (AB) on morphine (MOR) induced suppression of spinal

nociceptive transmission. Soc Neurosci Abstr 11:285, 1985.

Szczudlik, A., and Kwasucki, J. Beta endorphin-like immunoreactivity in the blood of patients

with chronic pain treated by pinpoint receptor stimulation (acupuncture). Neurol Neurochir

Pol 18:415-420, 1984.

Szczudlik, A., and Lypka, A. Plasma concentration of immunoreactive beta-endorphinin

healthy persons due to pinpoint stimulation of receptors (acupuncture). Neruol Neurochir Pol

17:535-540, 1983a.

Szczudlik, A., and Lypka, A. Plasma immunoreactive beta-endorphin and enkephalin

concentration in healthy subjects before and after electroacupuncture. Acupunct Electrother

Res 8:127-137, 1983b.

Szechtman, H.; Simantov, R.; and Hershkowitz, M. Sexual behavior decreases pain sensitivity

and stimulates endogenous opioids in male rats. Eur J Pharmacol 70:279-286, 1981.

Szreniawsky, A.; Czlonkowski A.; Janicki, P.; Libich, J.; and Gumulka, S. Substance P: Pain

transmission and analgesia. In: Ajmon, C., ed. Neuropeptides and Neural Transmission. New

York: Raven Press, 1980 pp. 121-130.

Takagi, H. The nucleus reticularis paragigantocellularis as a site of analgesic action of

morphine and enkephahlin. Trends Pharmacol Sci 1:182-184, 1980.

Takagi, H.; Doi, T.; and Akaike, A. Microinjection of morphine into the medial part of the

bulbar reticular formation in rabbit and rat: Inhibitory effects on lamina V cells of spinal

dorsal horn and behavioral analgesia. In: Kosterlitz, H.W., ed. Opiates and Endogenous Opioid

Peptides. Amsterdam: N. Holland, 1976. pp. 191-198.

Tang, J.; Chou, J.; Yang, H.-.Y.T.; and Costa, E. Substance P stimulates the release of

met5-enkephalin-arg6-phe7 and met5-enkephalin from rat spinal cord Neuropharmacol

22:1147-1150, 1983.

Tang, J.; Chou, J.; Iadarola, M.; Yang, H.Y.T.; and Costa, E. Proglumide prevents and curtails

acute tolerance to morphine in rats. Neuropharmacol 23:715-718, 1984.

Terenius, L. Stereospecific interaction between narcotic analgesics and a synaptic plasma

membrane fraction of rat cerebral cortex. Acta Pharmacol Toxicol 32:317-320, 1973.

Terenius, L. Effect of peptides and aminoacids on dihydromorphine binding to the opiate

receptor. J Pharm Pharmacol 27:450-453, 1975.

Terman, G.W.; Lewis, J.W.; and Liebeskind, J.C. The sensitivity of opioid mediated stress

analgesia to narcotic antagonists. Proc W Pharmacol Soc 26:49-52, 1983.

Terman, G.W.; Sbavit, Y.; Lewis, J.W.; Cannon, J.T.; and Liebeskind, J.C. Intrinsic

mechanisms of pain inhibition: Activation by stress. Science 226:1270-1277, 1984.

Teskey, G.C.; and Kavaliers, M. Ionizing radiation induces opioid-mediated analgesia in male

153
mice. Life Sci 35:1547-1552, 1984

Teskey, G.C.; Kavaliers, M.; and Hirst, M. Social conflict activates opioid analgesic and

ingestive behaviors in male mice. Life Sci 35:303-316, 1984.

Tsou, K. Antagonism of morphine analgesia by the intracerebral microinjection of nalorphine.

Acta Physiol Sin 26:332-337, 1963.

Tsou, K., and Jang, C.S. Studies on the site of analgesic action of morphine by intracerebral

micro-injection. Sci Sinica 13:1099-1109, 1964.

Tsubokawa, T.; Yamamoto, T.; Katayama, Y.; Hirayama, T.; and Sibuya, H. Thalamic relay

nucleus stimulation for relief of intractable pain. Clincall results and beta-endorphin immunoreactivity in the cerebrospinai fluid. Pain 18:115-126, 1984.

Tsunoda, Y.; Sakahira, K.; Nakano, S.; Matsumoto, I.; Yoshida, T.; Nagayama, K.; and

Ikezono, E. Antagonism of acupuncture analgesia by naloxone in unconscious man. Bull

Tokyo Med Dent Univ 27:89-94, 1980.

Umimo, M.; Shimada, M.; and Kubota, Y. Effects of acupuncture anesthesia on the pituitary

gland. Bull Tokyo Med Dent Univ 31:93-98,1984.

Urca, G.; Nahin R.L.; and Liebeskind, J.C. Glutamate-induced analgesia: Blockade and

potentiation by naloxone. Brain Res 192:5-530, 1981.

Urca, G.; Yitzhaly, J.; and Frenk, H. Different opioid systems may participate in post-electroconvulsive shocks (ECS) analgesia and catalepsy. Brain Res 219:385-397, 1981.

Urca G.; Harouni, A.; and Sarne, Y. Electroconvulsive shock (ECS) and h-endorphin-induced

analgesia: Unconventional interactions with naloxone. Eur J Pharmacol 81:237-243, 1982.

VanRee, J.M. Multiple brain sites invoIved in morphine antinociception. J Pharm Pharmacol

29:765-766, 1977.

Vigouret, J.; Teschemacher, H.J.; Albus, K.; and Herz, A. Differentiation between spinal and

supraspinal sites of action of morphine when inhibiting the hindleg flexor reflex in rabbits.

Neuropharmacology 12:111-121, 1973.

Watkins, L.R., and Mayer, DJ. Involvement of spinal opioid systems in footshock-induced

analgesia: Antagonism by naloxone is possible only before induction of analgesia. Brain Res

242:309-316, 1982.

Watkins, L.R.; Griffin, G.; Leichnetz, G.R.; and Mayer, D.J. The somatotopic organization

of the nucleus raphe magnus and surrounding brainstem structures as revealed by HRP

slow-release gels. Brain Res 181:1-15, 1980.

Watkins, L.R.; Cobelli, DA.; and Mayer, DJ. Opiate vs non-opiate footshock induced

analgesia (FSIA): Descending and intraspinal components. Brain Res 245:97-106, 1982a.

Watkins, L.R.; CobelIi, D.A.; and Mayer, D.J. classical conditioning of front paw and hind

paw footshock induced analgesia (FSIA): Naloxone reversibility and descending pathways,

Brain Res 243:119-132 1982b.

Watkins, L.R.; Cobelli, D.A.; Newsome, H.H.; and Mayer, D.J. Footshock induced analgesia

is dependent neither on pituitary nor sympathetic activation. Brain Res 245:81-96, 1982c.

154
Watkins, L.R.; Cobelli, DA.; Faris, P.; Aceto, M.D.; and Mayer, DJ. Opiate vs non-opiate

footshock-induced analgesia: The body region shocked is a critical factor. Brain Res

242:299-308, 1982d.

Watkins, L.R.; Young, E.G.; Kinscheck, I.B.; and Mayer, DJ. The neural basis of footshock

analgesia: The role of specific ventral medullary nuclei. Brain Res 276:305-315, 1983a.

Watkins, L.R.; Kinscheck, I.B.; and Mayer, DJ. The neural basis of footshock analgesia: The

effect of periaqueductal gray lesions and decerebration. Brain Res 276:317-324, 1983b.

Watkins, L.R.; Johannessen, J.N.; Kinscheck I.B.; and Mayer, DJ. The neurochemical basis

of footshock analgesia: The role of spinal cord serotonin and norepinephrine. Brain Res

290:107-117, 1984a.

Watkins, L.R.; Kinscheck, I.B.; and Mayer, DJ. Potentiation of opiate analgesia and apparent

reversal of morphine tolerance by proglumide. Science 224:395-396, 1984b.

Watkins, L.R.; Katayama, Y.; Kinscheck, I.B.; Mayer, DJ.; and Hayes, R.L. Muscarinic

cholinergic mediation of opiate and nonopiate environmentally induced analgesias. Brain Res

300:231-242, 1984c.

Watkins, L.R.; Kinscheck, I.B.; Kaufman, E.F.; Miller, J.; Frenk, H.; and Mayer, DJ.

Cholecystokinin antagonists selectively potentiate analgesia induced by endogenous opiates.

Brain Res 327:181-190, 1985.

Willer, J.C., and Albe-Fessard, D. Electrophysiological evidence for a release of endogenous

opiates in stress-induced analgesia in man. Brain Res 198:419-426, 1980.

Willer, J.C.; Dehen, H.; and Cambier, J. Stress-induced analgesia in humans: Endogenous

opioids and naloxone-reversible depression of pain reflexes. Science 212:689-690, 1981.

Willer, J.C.; Roby, A.; Gerard, A.; and Maulet, C. Electrophysiological evidence for a possible

serotonergic involvement in some endogenous opiate activity in humans. Eur J Pharmacol

78:117-121, 1982a.

Wiier, J.C.; Roby, A.; Boulu, P.; and Boureau, F. Comparative effects of electroacupuncture

and transcutaneous nerve stimulation on the human blink reflex. Pain 14:267-278, 1982b.

Woolf, C.J., and Wall, P.D. Endogenous opioids and pain mechanisms- a complex

relationship. Nature 306:739, 1983.

Yaksh T.L., and Rudy, TA. Chronic catheterization of the spinal subarachnoid space. Physiol

Behav 17:1031-1036, 1976.

Yaksh T.L., and Rudy, TA. Studies on the direct spinal actin of narcotics in the production

of analgesia in the rat. J Pharmacol Exp Ther 202:411-428, 1977.

Yaksh, T.L.; Yeung, J.C.; and Rudy, TA. Systematic examination in the rat of brain sites

sensitive to the direct application of morphine: Observation of differential effects within the

periaqueductal gray. Brain Res 114:83-104, 1976.

Yaksh T.L.; Schmauss, C.; Micevych, P.E.; Abay, E.O.; and Go, V.L.W. Pharmacological

studies on the application, disposition, and release of neurotensin in the spinal cord. Ann NY

Acad Sci 400:228-242, 1982.

Yaksh, T.L.; Terenius, L.; Nyberg, F.; Jhamandas, K.; and Wang, J.-.Y. Studies on the release

155
by somatic stimulation from rat and cat spinal cord of active materials which displace dihydromorphine in an opiate-biding assay. Brain Res 268:119-128, 1983.

Yashpal, K., and Henry, J.L. Endorphins mediate overshot of substance P-induced facilitation

of spinal nociceptive reflex. Can J Physiol Pharmacol 61:303-307, 1983.

Yasphal, K.; Wright, D.M.; and Henry, J.L. Substance P reduces tail-flick latency Implications

for chronic pain syndromes. Pain 14:155-167, 1982.

Yeung, J.C., and Rudy, TA. Multiplicative interaction between narcotic agonisms expressed

at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal

and intracerebroventricuiar injections of morphine. J Pharmacol Exp Ther 215:633-642, 1980a.

Yeung, J.C., and Rudy, TA. Sites of antinociceptive action of systemically injected morphineInvolvement of supraspinal loci as revealed by intracerebroventricuiar injection of naloxone.

J Pharmacol Exp Ther 215:626-632 1980b.

Young, R.F.; Feldman, RA.; Kroening, R.; Fulton, W.; and Morris, J. Electrical stimulation

of the brain in the treatment of chronic pain in man. In: Kruger, L. and Liebeskind, J.C., eds.

Advances in Pain Research and Therapy. New York.: Raven Press, 1984. pp. 289-303.

Zamir, N.; Simantov, R.; and Segal, M. Pain sensitivity and opioid activity in genetically and

experimentally hypertensive rats. Brain Res 184:299-310, 1980.

Zetler, G. Analgesia and ptosis caused by caerulein and cholecystokinin octapeptide (CCK-8).

Neuropharmacology 19:415-422, 1980.

Zetler, G. Cholecystokinin (CCK): Ascension from gut to brain - behavioral pharmacology

of CCK and analogs. Psychopharmacol Bull 19:347-351, 1983.

Zorman, G.; Belcher, G.; Adams, J.E.; and Fields, H.L. Lumbar intrathecal naloxone blocks

analgesia produced by microstimulation of the ventromedial medulla in the rat. Brain Res

236:77-89, 1982.

ACKNOWLEDGMENT

Portions of the work reported in this chapter were supported by P.H.S. Grant

FS-00576.

AUTHOR

David J. Mayer, Ph.D.

Department of Physiology

Medical College of Virginia

Virginia Comrnonwealth University

Richmond, Virginia 23298, U.S.A.

156
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan M.D., eds.

National Institute on Drug Abuse, 1988

Mechanism of Development of

Tolerance and Dependence to

Opioids in Neuroblastoma x Glioma

Hybrid Cells and Mice

Shail K. Sharma, Ph.D.; Madhav Bhatia, M.Sc.; and

Ranju Ralhan, Ph.D.

Neuroblastoma x glioma (NG108-15) hybrid cells are

enriched with opioid receptors (Klee and Nirenberg

1974; Chang and Cuatrecasas 1979). In these cells

opiates and opioid peptides regulate the synthesis of

cyclic AMP at two different levels. A rapid receptormediated inhibition of adenylate Cyclase is followed

by a delayed and long-lived compensatory increase in

its activity. It has been suggested that the latter

effect may be responsible for narcotic dependence and

tolerance (Sharma et al. 1975a,b 1977; Traber et al.

1975; Lampert et al. 1976). Ascorbic acid suppresses

the etorphine-induced compensatory increase in the

levels of cAMP with little or no effect on the shortterm response of NG108-15 hybrid cells to the

effector agents (Sharma and Khanna 1982). These

studies suggest that ascorbic acid may play a role in

the Prevention of the development of tolerance in

therapeutic applications of narcotics as analgesics.

The mechanism by which ascorbic acid prevents the

development of tolerance to opiates is still obscure.

Effect of Ascorbate on Etorphine-Induced Alterations

in Cyclic AMP Levels

Results reported earlier from this laboratory show

that ascorbic acid suppresses the etorphine-induced

compensatory increase in the levels of CAMP in NG108-

15 hybrid cells (Sharma and Khanna 1982) It was of

interest to study the effect of variation of

ascorbate concentration on etorphine-induced

157
compensatory increase in basal and PGE1-stimulated

cAMP levels. Results in tl show that in the absence

of ascorbate, etorphine led to a compensatory

increase of 80 percent in the basal and 65 percent in

the PGE1-stimulated CAMP levels. A progressive

decrease in the compensatory response is seen as the

concentration of ascorbate is raised to 10mM.

Initially, at lower concentrations of ascorbate the

decrease is more pronounced for PGE1-stimulated CAMP

levels.

Preexposure of NG108-15 hybrid cells toascorbate did

not abolish stimulatory or inhibitory effects of

drugs on cAMP levels (Sharma and Khanna 1982).

indicating that receptors for opiates and PGE1 were

functionally coupled to adenylate cyclase. The effect

of preincubation of NG108-15 hybrid cells with

ascorbate on etorphine-induced compensatory increase

in the levels of CAMP was studied. In this experiment

cells were preexposed to ascorbate for 4 hours. After

washing, the second incubation was with and without

etorphine for an additional 4 hours. The monolayer

cultures were washed free of ascorbate and used to

determine basal and PGE1-stimulated CAMP levels as in

t1. Results in t2 show that etorphine-induced basal

and PGE1-stimulated compensatory increase in CAMP

levels observed is of the same order of magnitude in

the cells preincubated with or without ascorbate. The

data from tl and t2 clearly show that simultaneous

presence of ascorbate and etorphine is necessary to

suppress the etorphine-induced compensatory increase

in basal and PGE1-stimulated CAMP levels. These

studies have been carried out in a cultured cell line

of neuronal origin. Therefore, we studied the effect

of ascorbic acid on development of tolerance and

dependence in vivo in mice.

The in vivo effects of ascorbate on morphine-induced

analgesia, physical dependence, and tolerance were

studied. In mice ascorbate, when administered along

with morphine, suppresses the development of

tolerance and physical dependence on the drug,

without significantly affecting its analgesic

properties. Mice receiving morphine along with

ascorbate became less tolerant to and dependent on

the drug. The duration of morphine-induced analgesia,

however, is progressively reduced with an increase in

the amount of ascorbate. However, ascorbate does not

alter the intensity of morphine-induced analgesia

(Khanna and Sharma 1983). Ascorbate at lg/kg body

weight did not alter the pH of blood and did not have

an effect on the levels of lipid-peroxides in blood

and brain. These results suggest that action of

ascorbate is not mediated by its oxidation and lipid

peroxide formation.

158
TABLE 1

Suppression of opiate-induced compensatory increase

in c-AMP levels by ascorbate

NG108-15 hybrid cells (1.2 x 106, passage 20) were

plated in (Medium A) OMEM (Flow Labs U.K.)

supplemented with 0.1 mM-hypoxanthine, 1μMaminopterin, 16 μM-thymidine, and 10 percent of fetal

bovine serum (Flow Labs) in 60 mm Falcon petridishes. When 60-80 percent confluent, the culture

plates were divided in five groups: (i) control with

no effector agents; (ii) etorphine (1.6 μM); (iii)

etorphine 1.6 μM and sodium ascorbate (1 mM); (iv)

etorphine 1.6 μM and sodium ascorbate (5 mM); (v)

etorphine 1.6 μM and sodium ascorbate (10 mM). The

cells were incubated for 4 hours at 37°C, washed with

medium B [DMEM with 25 mM Hepes (4-(2-hydroxyethyl)-

1-piperazine-ethanesulfonic acid)]. Incubations were

carried out in 3 ml of medium B with 0.5 mM Ro20-1724

[4-(3-butoxy-4-methoxybenzyl) imidazolidine-2-one].

Amounts of intracellular levels of c-AMP were

determined over a period of 15 minutes with and

without PGE1 (10 μM) by the method of Gilman (1970).

Protein in each petri dish was estimated by the

method of Lowry et al. (1951). Values are the

average of three individual experiments (each

experiment had four plates in a group).

Additions during cell cAMP (Pmole/mg protein)

growth for 4 h Basal PGE1

328 3800

Etorphine 584 6263

Etorphine + ascorbate 1 mM 535 3954

Etorphine + ascorbate 5 mM 308 4400

Etorphine + ascorbate 1OmM 192 3542

159
TABLE 2

Compensatory increase in CAMP levels after

preincubation of cells with ascorbate

NG108-15 hybrid cells (1.4x106, passage 20) were

plated in medium A in 60 mm petri dishes. When 60-80

percent confluent the culture plates were divided in

two groups (eight plates in each group): (i) control

with no effector agents; (ii) sodium-ascorbate

(5mM). The cells were incubated for 4 hours at 37°C,

washed with 4 x 3 ml medium A, and each set of

culture plates was divided into two groups: (ia)

control with no effector agents; (ib) etorphine 1.6

μM; (iia) control with no effector agents; (iib)

etorphine 1.6 μM. Incubations were carried out in 4

ml medium A for 4 hours at 37°C and washed with

medium B. 3ml medium B with 0.5 mM Ro20-1724 was

added, and the plates were incubated in presence of

naloxone with and without PGE1 (10 μM) for 15 min.

Intracellular CAMP levels were determined as in tl.

Each value is the average of three individual

experiments (each experiment had four plates in a

group).

Additions during cell growth CAMP (pmole/mg protein

Preincubation 1 Preincubation 2

(4 h) (4 h) Basal PGE1

Ascorbate

Ascorbate

Etorphine

Etorphine

433±49 5821±254

782±36 8063±422

371±19 5216±397

679±42 7976±510

160
Mechanism of Ascorbate Action

Ascorbic acid may exert its effect at the level

either of binding of opiates to the receptor or of

coupling of the receptor to other components of the

adenylate cyclase system, i.e., inhibitory guanine

nucleotide regulatory protein (Ni) and catalytic unit

of adenylate cyclase.

Effect of Ascorbate on (

3

H) Etorphine and (

3

H) DADLE

Binding

Effect of ascorbate on binding of (15,16-3H)

etorphine to intact NG108-15 hybrid cells was studied

as a function of ascorbate concentration. Ascorbate

at 1 and 5 mM concentration inhibited the specific

binding of etorphine by 28 percent and 56 percent

(t3). Similar effect of sodium ascorbate on D-Ala2-

(tyrosyl-3,5-3H) enkephalin (5-D-leucine) binding was

observed. Specific binding was inhibited by 24

percent, 56 percent, and 68 percent, respectively, in

the presence of 1 mM, 5 mM, and 10 mM ascorbate (t4).

These studies are compatible with those of Dunlap et

al. (1979).

In order to ascertain that ascorbate does not destroy

opiate receptors, NG108-15 hybrid cells in monolayer

were preincubated with 1 and 5 mM ascorbate, washed

and assayed for (

3

H) etorphine binding. There was no

significant change in specific (

3

H) etorphine binding

in the cells preincubated with ascorbate as compared

to that in controls (t5). These results show that

ascorbic acid does not destroy opiate receptors.

Effect of Ascot-bate on GTPase Activity

Klee et al. (1984) proposed that opiate inhibition of

adenylate cyclase in NG108-15 hybrid cells is the

direct result of stimulation of a low Km GTPase

component associated with Ni protein of the adenylate

cyclase complex. Adenylate cyclase shuttles between

two states, an active one with bound GTP and an

inactive one with bound GDP. Stimulatory receptors

facilitate exchange of GTP for bound GDP, whereas

inhibitory receptors (including opiate receptors)

stimulate low Km GTPase activity, which converts GTP

bound to the Ni complex to GDP (Ross and Gilman 1980;

Katada and Ui 1979, 1982a,b; Koski et al. 1982).

We have studied the short- and long-term effects of

sodium ascorbate and etorphine on low Km GTPase

activity and thus Ni in NG108-15 hybrid cells.

Etorphine stimulates low Km GTPase activity of NG108-

15 hybrid cells by 54 percent. This acute stimulatory

161
effect of etorphine is reversed by opiate antagonist

naloxone (t6).

TABLE 3

Effect of sodium ascorbate on binding of (

3

H)

etorphine to monolayer cultures of NG1O8-15

hybrid cells

NG108-15 hybrid cells (2x104, passage 20) were plated

in medium P in 24 well Falcon culture plates. When 80

percent confluent, the wells were washed with medium

Incubations were carried out in 2 ml of medium B

containing 2 nM (15,16-3H) etorphine at 37°C in a

water bath. Specific binding was determined by

subtracting the nonspecific binding measured in the

presence of 2.5 μM naloxone from total binding at

each concentration of sodium ascorbate. Each value is

the average of four individual determinations and is

expressed as Mean ± S.D.

Addition (

3

H) etorphine binding

CPM/mg protein

None 750 ± 101

Sodium ascorbate 0.1 mM 770 ± 58

Sodium ascorbate 0.5 mM 697 ± 47

Sodium ascorbate 1.0 mM 537 ± 56

Sodium ascorbate 5.0 mM 327 ± 20

162
TABLE 4

Effect of sodium ascorbate on binding of (

3

H) DADLE

to monolayer cultures of NG108-15 hybrid cells

Conditions of the experiment were the same as in t3.

Specific binding of 5nM D-Ala2-(tyrosyl-3,5-3H)

enkephalin (5-D-leucine) was measured.

Addition (

3

H) DADLE binding

CPM/mg protein

None 295 ± 14

Sodium ascorbate 1 mM 225 ± 18

Sodium ascorbate 5 mM 131 ± 11

Sodium ascorbate 10 mM 92 ± 5

TABLE 5

Binding of (

3

H) etorphine to NG108-15 hybrid

cells, preincubated with ascorbate

NG108-15 hybrid cells were plated in 60 mm Falcon

petri dishes as described in tl. When 60-80 percent

confluent, the monolayer of cells was washed with 5 x

3 ml of medium B, and plates were divided in three

sets: (i) control with no effector agents; (ii)

sodium ascorbate (1 mM); (iii) sodium ascorbate 5mM.

Incubations were carried out in 3 mlof medium B, for

60 min at 37°C. washed with 5 x 3 ml of medium B, and

incubated in 3 ml of medium B containing 2 nM (15,16-

H) etorphine at 37°C in a water bath. Specific

binding was determined as described in t3.

Preincubation (

3

H) etorphine binding

60 min CPM/mg protein

None 1085 ± 134

Sodium ascorbate 1 mM 1243 ± 78

Sodium ascorbate 5 mM 1153 ± 133

163
TABLE 6

Effect of etorphine and sodium ascorbate on

GTPase activity in membranes prepared from

NG108-15 hybrid cells

Membranes were prepared and used for GTPase assay as

described by Koski and Klee (1981). All experimental

conditions were the same except that membranes were

collected by centrifugation at 48,000 g for 50 min.

Concentration of effector agents were naloxone, 2.5

mM; etorphine, 1 μM; sodium ascorbate, 5 mM; PGE1, 10

μM.

Each value is the difference of GTPase activity

obtained in the absence and presence of 50 μM GTP

determined in triplicates and is expressed as Mean ±

S.D.

Additions GTPase activity (+ PGE1)

pmole of Pi formed/min/mg protein

35.3 ± 1.3

Naloxone 35.5 ± 3.8

Etorphine 54.6 ± 0.89

Etorphine + naloxone 33.5 ± 1.11

These results are consistent with the earlier

observations of Koski and Klee (1981) on opiate

stimulation of low Km GTPase activity. Recently we

have shown that NG108-15 hybrid cells grown with

etorphine or ascorbic acid for 4 hours had decreased

low Km GTPase activity. Inhibition of the low Km

GTPase by ascorbic acid may be the mechanism by which

ascorbic acid inhibits the etorphine-induced

compensatory increase in CAMP formation (manuscript

under preparation).

164
References

Chang, K. J., and Cuatrecasas, P. Multiple opiate

receptors-enkephalins and morphine bind to receptors

of different specificity. J Biol Chem 254:2610-2618,

1979.

Dunlap, C.E.; Leslie, F.M.;

Ascorbate destruction of

binding in guinea pig brain

Pharmacol 16:105-119, 1979.

Gilman, A.G. A protein bind

3':5'-cyclic monophosphate.

67:305-312, 1970.

Rado, M.; and Cox, B.M.

opiate stereospecific

homogenate. Mol

ing assay for adenosine

Proc Nat1 Acad Sci USA

Katada, T., and Ui, M. Islet-activating protein:

Enhanced insulin secretion and cyclic AMP

accumulation in pancreatic islets due to activation

of native calcium ionophores. J Biol Chem 254:469-

479, 1979.

Katada T., and Ui, M. ADP-ribosylation of the

specific membrane protein of C6 cells by isletactivating protein associated with modification of

adenylate-cyclase activity. J Biol Chem 257:7210-

7216, 1982a.

Katada, T., and Ui, M. Direct modification of the

membrane adenylate cyclase system by isletactivating protein due to ADP-ribosylation of a

membrane protein. Proc Nat1 Acad Sci USA 79:3129-

3133, 1982b.

Khanna, N.C., and Sharma, S.K. Megadoses of vitamin

C prevent the development of tolerance and physical

dependence on morphine in mice. Life Sci 33:401-404,

1983.

Klee, W.A., and Nirenberg, M. A neuroblastoma X

glioma hybrid cell line with morphine receptors.

Proc Nat1 Acad Sci USA 71:3474-3477, 1974.

Klee, W.A.; Koski, G.; Tocque, 8.; and Simonds, W.F.

On the mechanism of receptor-mediated inhibition of

adenylate cyclase. Adv Cyclic Nucleotide Phosphoprotein Res 17:153-159, 1984.

Koski, G., and Klee W.A. Opiates inhibit adenylate

cyclase by stimulating GTP hydrolysis. Proc Natl

Acad Sci USA 78:4185-4189, 1981.

Koski, G.; Streaty, R.A.; and Klee, W.A. Modulation

of sodium-sensitive GTPase by partial opiate agonist:

An explanation for the dual requirement for Na+

165
and GTP in inhibitory regulation of adenylate

cyclase. J Biol Chem 257:14035-14040, 1982.

Lampert, A.; Nirenberg, M.; and Klee, W.A. Tolerance

and dependence evoked by an endogenous opiate

peptide. Proc Nat1 Acad Sci USA 73:3165-3167, 1976.

Lowry, O.H.; Rosenbrough, N.J.; Farr, A.L.; and

Randall, R.J. Protein measurements with the folin

phenol reagent. J Biol Chem 193:265-275, 1951.

Ross, E.M., and Gilman, A.G. Biochemical properties

of hormone-sensitive adenylate cyclase. Annu Rev

Biochem 49:533-564, 1980.

Sharma, S.K.; Nirenberg, M.; and Klee, W.A. Morphine

receptors as regulators of adenylate cvclase

activity. Proc Natl Acad Sci USA 72:590-594, 1975a.

Sharma, S.K.; Klee, W.A.; and Nirenberg, M. Dual

regulation of adenylate cyclase accounts for

narcotic dependence and tolerance. Proc Natl Acad

Sci USA 72:3092-3096, 1975b.

Sharma, S.K.; Klee, W.A.; and Nirenberg, M. Opiatedependent modulation of adenylate cyclase. Proc Natl

Acad Sci USA 74:3365-3369, 1977.

Sharma, S.K., and Khanna, N.C. Ascorbate suppresses

the opiate-induced compensatory increase in cyclic

AMP in neuroblastoma X glioma hybrid cells.

Biochem J 208:43-46, 1982.

Traber, G.; Gullis, R.; and Hamprecht, B. Influence

of opiates on the levels of adenosine 3':5' cyclic

monophosphate in neuroblastoma X glioma hybrid

cells. Life Sci 16:1863-1868. 1975.

Acknowledgments

This work is supported by grant No. 5/3-1 (8)-BMSII

from Indian Council of Medical Research to Dr.

Sharma. Mr. Bhatia is a recepient of the K.D. Birla

Smarak Kosh Scholarship. Or. Ralhan is a Lecturer in

the Department of Biochemistry.

Authors

Shail K. Sharma, Ph.D.

Madhav Bhatia, M.Sc.

Ranju Ralhan, Ph.D.

Department of Biochemistry

All-India Institute of Medical Sciences

New Delhi-110 029, India

166
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Development of Spinal Substrate

for Nociception in Man

Veena Bijlani, Ph.D.; Tilat A. Rizvi, M.Sc.; and

S. Wadhwa, MS.

The dorsal horn of the spinal cord is the first site

of interaction between primary afferents and intrinsic

neurons. There is considerable evidence in literature

about the existence of a variety of descending fibers

taking part in the spinal neuronal circuitry. The

small diameter nociceptive fibers have been shown to

establish synaptic contacts with the neurons of lamina

I, II, and III(Kerr 1975; Bishop 1980; Christensen

and Perl 1970; Willis and Coggeshall 1978). Spinothalamic tract neurons are dispersed in lamina I, IV,

V, and VI (Trevino et al. 1978; Kerr 1975; Carsten

and Trevino 1978). The small neurons of lamina II and

III do not appear to project for long distances

(Trevino and Carsten 1975). Kerr (1975) has suggested

these to be inhibitory neurons. The precise understanding of function of neuronal circuitry requires

that the afferent and efferent synaptic connections

and the transmitters of its constituent be identified.

Studies were done on the dorsal gray of human fetal

spinal cord ranging-in age from 8 to 37 weeks of

intrauterine life with a view to identify neuronal

circuits that modify/modulate the afferent inputs.

MATERIAL AND METHODS

Present study was conducted on 30 human embryos and

fetuses ranging in age from 8 to 37 weeks of intrauterine life obtained by hysterotomy and still birth

autopsies. The fetal age was calculated on the basis

of last menstrual period, crown rump length, biparietal

diameter, and external features of the fetuses, while

that of premature infant was assessed on the basis of

crown heel and biparietal length. Lower cervical

segments of spinal cord were processed for various

techniques.

The neurons and afferent fibers were identified in the

167
Golgi preparation (100 μ thick celloidin sections),

and circuitry was reconstructed using camera lucida.

One Golgi impregnated neuron was processed for electron

microscopy using the method of Fairen et al. (1977).

1 μrn semitnin sections were cut to identify different

zones for ultrastructural studies, and electron

microscopy was done to study synaptogenesis.

Immunohistochemical localization of substance P,

enkephalin, and serotonin at different age periods

was carried out with the help of avidin biotin

peroxidase complex technique using monoclonal antibody (Sera Lab). GABA was localized using polyclonal

antibody (Sera Lab) with PAP technique of Sternberger

(1974).

RESULTS

Morphologically, on the basis of axonal projections

and dendritic arborization, different types of neurons

could be identified in different zones of dorsal horn.

Interneurons (encased of Lima and Coimbra 1983) and

projection neurons in marginal zone (Bijlani et al.

1986), islet, stalked, bipolar and multipolar neurons

in substantia gelatinosa, and multipolar and antenna

neurons in deeper zone were typed in Golgi preparation

at all age periods (unpublished observation).

One interneuron at 13 weeks (encased) after Golgi

impregnation was processed for electron microscopy.

Different types of axodendritic and axosomatic

synapses with clear spherical, dense core and

pleomorphic vesicles were found on the dendrite and

soma of this neuron (figs. 1c, 1d).

Mild substance P immunoreactivity was localized at the

age of 8 weeks in the tangential plexus of fibers in

superficial dorsal horn and in laterally curving

fibers. Immunostained ventrally curving fibers could

also be seen in the region of substantia gelatinosa

(figs. 2a, 2d). Intensity of immunoreactivity increased

with the age (fig. 3).

Serotonin inmunoreactivity was also found to be of the

same pattern as that of substance P at 12 weeks in the

superficial dorsal horn. Eoth medially and laterally

curving fibers picked up intense reaction. Intermediate horn was found to have network of immunostained

fibers (fig. 2b). Immunoreactivity again intensifies

with the increasing age.

Enkephalin was localized at the age of 12 weeks of

gestation in the laterally curving fibers and in

intermediate gray. At 14 weeks onward mild immunoreactive positive cells were found in marginal zone

and substantia gelatinosa. At the same age superficial

dorsal gray: i.e., the region capping the dorsal horn,

168
FIGURE 1

Electron micrographs showing (la) an axodendritic asymmetrical

synapse (arrow) with clear spherical and dense core vesicles

at 18 weeks of intrauterine life in marginal zone. X 35640.

(1b) serial types of synapses from deeper zone at 25 weeks of

age. X 35640.

(1c) Golgi impregnated neuron from marginal zone after gold

toning at 13 weeks of age. X 6385.

(1d) asymnetric synapse (arrow) on the soma of gold toned

neuron(c) with clear spherical vesicles. X 46827.

169
FIGURE 2

Photomicrographs showing immunoreactivity in the dorsal horn

of spinal cord at the age of 16 weeks of gestation.

2a - Substance P X 75.

2b - Serotonin X 75.

2c - Enkephalin X 75.

2d - Substance P immunoreactive fibres under high power X 750.

170
FIGURE 3

Comprehensive chart illustrates the appearance of various neurotransmitters in

the dorsal gray at different age periods.
and laterally curving fibers were also immunopositive

(fig. 2c).

GABA was first localized in terminals on dorsal side

of superficial dorsal gray at 8 weeks, while at 10

and 12 weeks immunostained cells were seen in the

marginal zone, substantia gelatinosa, and intermediate

gray (unpublished observation).

At ultrastructural level dense core vesicles were

seen in axonal terminals at 8 weeks. From 12 weeks

onward axosomatic synapses with plemorphic vesicles

were identified (Rizvi et al. 1986). These vesicles

could contain either GABA or serotonin or even

enkephalin. Asymmetric synapses were seen at 18 weeks

(fig. la), while axoaxonic synapse was visible at 20

weeks (Rizvi et al. 1986). Serial types of synapses

could be visualized at 25 weeks (fig. 1b).

DISCUSSION

Gobel (1978a) stated that all neurons in the marginal

zone are projection neurons. Our studies are not in

agreement with Gobel, as we have identified both

projection (Waldeyer 1888) and interneurons at 13

weeks in that marginal zone on the basis of cell size,

location, and dendritic morphology (Bijlani et al.

1986). We could also identify "encased neuron",

specifically described by Lima and Coimbra (1983) in

rat spinal cord as having a prominent dorsally

directed dendrite situated dorsolaterally. Encased

neuron picked up from Golgi section reembedded in

araldite, was sectioned for electron microscopy (fig.

1c) and was found to have axosomatic (fig.1d) as well

as axodendritic synapses. This neuron appears to be

contacted by descending supraspinal fibers and could

thus be involved in modulation mechanisms (Lima and

Coimbra 1983). We also found this neuron situated in

close proximity of GABAergic terminals at 8 weeks.

No GABA positive cells were seen in dorsal horn at

this age period (unpublished observations). The

punctate appearance of GABA positive terminals in the

region of Lissauer tract suggests the presence of GABA

in the bulbcspinal fibers.

Cajal (1909) and Gobel (1978b) have reported central

and limiting cells, islet, arboreal, and lamina II-III

border cells in substantia gelatinosa, respectively.

In our Golgi studies at 13 to 14 weeks we could find

primitive stalk cell with axon projecting to lamina I.

According to Gobel (1978b), these neurons are

equivalent to the limiting cells of Cajal (1909) and

may be excitatory interneurons, but according to

Bennett et al. (1982) they contain enkephalin, an

inhibitory neurotransmitter.

172
We could also find islet cell with axon in its

vicinity in the same lamina (Bijlani et al. 1985).

According to Gobel (1978b), these neurons may be

inhibitory interneuron of Golgi type II class and

central cells of Cajal (1909).

In deeper zone we could find antenna neurons with

dendrites directed toward substantia gelatinosa.

According to Willis et al. (1979), some spinothalamic

tract cells in monkey do send dorsally directed

dendrites through lamina II to lamina I so it may

correspond to spinothalamic tract cell.

Substance P is putative neurotransmitter for dorsal

sensory neurons (Otsuka and Konishi 1977; Nicoll

et al. 1980). Recently Bossut et al. (1986) stated

that substance P does not appear to be a primary

afferent transmitter for pain in the spinal cord.

Present investigations have suggested that substance

P appears very early in ontogeny, as it was visible

at 8 weeks embryonic spinal cord in our study (Mehra

and Bijlani 1986). The neuropeptide has earlier been

reported in the dorsal horn of human spinal cord at

12 weeks (Charnay et al. 1983) with immunofluorescent

technique. Our immunohistochemical studies corroborate

with Charnay et al. (1983) but with earlier ontogenic

appearance (figs. 2a,2d).

The role of descending fibers in the modulation of

sensory pathways at spinal level is well illustrated

(Fields and Basbaum 1978; Mayer and Price 1976).

Activation of serotoninergic pathways has been

described to inhibit nociceptive responses of

spinothalamic neurons (Messing and Lytle 1977). In

dorsal horn serotoninergic axons have been described

by Dahlstrom and Fuxe (1965) that originate from brain

stem cells containing 5 HT. In our study serotonin

positive immunoreactivity was localized at the age of

12 weeks using monoclonal antibodies. Serotonin

positive neurons could not be identified in the spinal

cord, thus supporting supraspinal origin of these

fibers. Serotoninergic terminals gave an appearance

of honey comb network around unstained neurons in

substantia gelatinosa, supporting close proximity of

gelatinosa neurons to supraspinal serotoninergic

terminals.

At ultrastructural level visualization of axosomatic

and axodendritic synapses with pleomorphic vesicles

may correspond to serotoninergic terminals (Rizvi et

al. 1986). Ruda and Gobel (1980) have described in

detail the mode of action of descending serotoninergic

fibers on islet, stalked, and projection neurons and

173
have stressed the modulatory role in nociception.

Our observations indicate the existence of a

morphological substrate corroborating Ruda and Gobel's

(1980) findings.

Neuromodulatory role of enkephalin in the pain sensory

system was supported by physiological (Basbaum and

Fields 1978; Mountcastle 1980) and anatomical

(Hokfelt et al. 1977; Glazer and Basbaum 1981)

findings. Enkephalin has been localized immunohistochemically in the spinal cord of man (DeLanerolle

and LaMotte 1983) and monkey (Aronin et al. 1981) and

in human fetuses and infants (Charnay et al. 1984) by

immunofluorescent technique. Enkephalin immunoreactive

fibers in these studies were predominant in the

marginal layer and substantia gelatinosa (Elde et al.

1976; Sar et al. 1978), while enkephalin containing

cell bodies were located in the substantia gelatinosa

(Ditirro and Ho 1980). Opiate receptors have been

described to be located in the superficial dorsal horn

on primary afferent fibers (Atweh and Kuhar 1977),

thereby indicating the formation of axoaxonic synapses

(Jessel et al. 1979) and causing presynaptic inhibition

by enkephalinergic neurons of substantia gelatinosa

(Mudge et al. 1979).

In contrast, physiological studies have shown that

enkephalin acts at postsynaptic sites in dorsal horn

(Henry et al. 1980). At electron microscopic levels

in rat dorsal horn enkephalin has been localized in

axon terminals (Pelletier and Leclere 1979) and found

to form synapses with aendrites and soma (Hunt et al..

(1980). For the first time, enkephalin positive

neurons were found in lamina I, II, III, and V at ultrastructural level (Aronin et al. 1981). According to

these authors, enkephalin binds mostly to opioid

receptors contained on dendrites and soma within the

spinal cord, while direct synapses by enkephalinergic

axons with primary afferent fibers form a secondary

and not so common site of interaction. Therefore,

earlier concept that opiate receptors are on primary

afferent terminals and form axoaxonic synapses for

presynaptic inhibition appears to be contradicted.

In present investigation on developing dorsal gray,

enkephalin immunoreactivity was localized in the

dorsal gray at the age of 12 weeks in fibers entering

to intermediate dorsal horn through lateral funiculus.

From 14 weeks, immunoreactive fibers were found in

superficial dorsal gray, i.e., marginal zone and

substantia gelatinosa (fig. 2c). Laterally curving

fibers also were found to show immunoreactivity. Our

immunohistochemical findings corroborate with the

immunofluorescent finding of Charnay et al. (1984).

174
At 17 weeks, in addition to immunoreactive fibers,

some cells positive for enkephalin could also be

visualized, suggesting the presence of enkephalin in

substantia gelatinosa neurons. The intensity of

reaction in fibers and cells increased from 12 weeks

to 25 weeks (the specimen used for immunohistochemical

localization).

Curtis and Johnston (1974) and Levy (1977) have

described GABA as a major neurotransmitter involved

in mediation of presynaptic and postsynaptic

inhibition and presynaptic felicitation. Wolf (1981)

has described relationship between GABA positive

neurons and development of excitatory synapses in

developing visual cortex and has specified that in

prenatal phase GABA has extra synaptic function.

Light et al. (1979) and Kumazawa and Perl (1978) have

stressed the roles of GABAergic synapses in the

integration of afferent information.

In the present investigations on human fetal material,

GABA was localized using polyclonal antibodies. At 8

weeks of intrauterine life, immunopositive terminals

were identified in dorsal most-regions of the spinal

cord. These appear to be in the region of Lissauer

tract and could be the terminals of bulbospinal

pathways. At 12 weeks of intrauterine life, GABA

positive cells were found in the marginal zone,

substantia gelatinosa, as well as in the deeper zones.

These findings support the results of Barber et al.

(1982) in the rat spinal cord. These authors,

however, identified GAD positive cell bodies.

At ultrastructural level presence of various types

of synapses with morphologically different types of

vesicles, namely, clear spherical (fig. 1b), dense

core (fig. 1a), and pleomorphic vesicles (Rizvi et

al. 1986), suggest the presence of different

transmitter, e.g., substance P, serotonin, GABA, and

enkephalin.

CONCLUSIONS

In conclusion, present investigations suggest

that among various neurotransmitters that have been

described in the literature to play a role of

mechanism of nociception, substance P of dorsal root

origin appears at 8 weeks and at the same age GABA

in the bulbospinal fibers is present. Serotonin and

enkephalin neurotransmitters are present at 12 weeks

in the fibers while enkephalinergic cell bodies are

visualized at 14 weeks onward. The presence of these

transmitters indicates the possibility of the

existence of various components of morphological

175
substrate for nociception in spinal cord at very early

stages of human ontogeny. All the same, possibility

of neurotrophic role of these various transmitters

cannot be ruled out.

REFERENCES

Aronin, N.; Di Figlia, M.; Liotta, A.S.; and Martin,

J.B. Ultrastructural localization and biochemical

features of immunoreactive leuenkephalin in monkey

dorsal horn. J Neurosci 1:561-577, 1981.

Atweh. S.E., and Kuhar, M. Autoradiographic

localization of opiates and opiate-receptors in rat

brain. I. Spinal-cord and medulla. Brain Res

124:53-67, 1977.

Barber, R.P.; Vaughn, J.E.; and Roberts, E. The

cytoarchitecture of GABAergic neurons in rat spinal

cord. Brain Res 238:305-328, 1982.

Basbaum, A.I., and Fields, H.L. Endogenous pain control

mechanisms: Review and hypothesis. Ann Neurol

4:451-452, 1978.

Bennett, G.J.; Ruda, M.A.; Gobel, S.; and Dubner, R.

Enkephalin immunoreactive stalked cells and lamina

II b islet cells in cat substantia gelatinosa.

Brain Res 240:162-166, 1982.

Bijlani, V.; Balakrishna, S.; Rizvi, T.A.; Wadhwa, S.;

and Mehra R.D. Substrate of pain in dorsal gray.

In: Sharma, K.N., and U.Nayyar, eds. Current Trends

in Pain Research and Therapy. Basic Mechanisms and

Clinical Applications. Vol.I. New Delhi: Indian

Society for Pain Research and Therapy, 1985.pp. 19-30.

Bijlani, V.; Rizvi, T.A.; Wadhwa, S.; and Mehra, R.D.

Development of neuronal circuitry in marginal zone

of dorsal spinal gray in man. In: Sharma, K.N., and

Nayyar, U., eds. Current Trends in Pain Research and

Therapy. Stimulus Produced Analgesia. Vol. II.

New Delhi: Indian Society for Pain Research and

Therapy, 1986. pp. 1-15.

Bishop, B. Pain:

management. Phys Ther 60:13-37, 1980.

Its physiology and rationale for

Bossut, D., H.; and David, J.M. Substance P

does not appear to be a primary afferent transmitter

of pain in the spinal cord. Soc Neurosci Abs

12(1):228, 1986.

Cajal, R.Y. Histologie du systeme Nerveux de l'Homme

et des Vertebres. Vol. I. Paris; Maloine, 1909 --

(quoted by Rexed 1952; Beal and Fox 1976; Willis and

Coggeshall 1978; Cervero and Iggo 1980)

Carsten, S.E., and Trevino, D.L. Laminar origins of

spinothalamic projections in the cat as determined

by the retrograde transport of horseradish peroxidase.

J Comp Neurol 182:151-166, 1978.

Charnay, Y.; Paulin, C.; Chayvialle, J.A.; and Dubois,

P.M. Distribution of substance P like immunoreactivity

in the spinal cord and dorsal root ganglion of the

human fetus and infant. Neuroscience 10:41-55, 1983.

176
Charnay, Y.; Paulin, C.; Dray, P.; and Dubois, P.M.

Distribution of enkephalin in human fetus and infant

spinal cord: An immunofluorescence study. J Comp

Neurol 223:415-423, 1984.

Christensen, B.N., and Perl, E.R. Spinal neurons

specifically excited by noxious or thermal stimuli:

Marginal zone of the dorsal horn. J Neurophysiol

33:293-307, 1970.

Curtis, D.R., and Johnston, G.A.R. Aminoacid

transmitters in the mammalian central nervous system.

Ergeb Physiol 69:97-188, 1974.

Dahlstrom A.; and Fuxe, K. Experimentally induced

changes in the intraneuronal amine levels of

bulbospinal neuronal systems. Acta Physiol Stand

64(Suppl):247, 1965.

De Lanerolle, N.C., and LaMotte, C. Ultrastructure

of chemically defined neuron systems in the dorsal

horn of the monkey. I. Substance P immunoreactivity.

Brain Res 274:31-49, 1983.

Ditirro, F.J.; and Ho, R.H. Distribution of substance

P (SP), somatostatin (SOM) and methionin enkephalin

(ENK) in spinal cord of domestic rabbit. Anat Rec

196:47A, 1980.

Elde, R.T.; Hokfelt, 0.; Johansson.; and Terenius, L.

Immunohistochemical studies using antibodies to

Leucine-enkephalin. Initial observations on the

nervous system of the rat. Neuroscience 1:349-351,

1976.

Fairen, A.; Peters, A.; and Saldhana, J. A new

procedure for examining Golgi impregnated neurons by

light and electron microscopy. J Neurocytol 6:311-337,

1977.

Fields, H.L., and Basbaum, A.I. Brain stem control

of spinal pain transmission neurons. Annu Rev

Physiol 40:217-248, 1978.

Glazer, E.J., and Basbaum, A.I. Immunohistochemical

localization of leucin enkephalin in spinal cord of

cat: Enkephalin containing marginal neurons and pain

modulation. J Comp Neurol 196:377, 1981.

Gobel, S. Golgi studies of the neurons in layer I of

the dorsal horn of the medulla (trigeminal nucleus

caudalis). J Comp Neurol 160:395-414, 1978a.

Gobel, S. Golgi studies of the neurons in layer II of

the dorsal horn of medulla (trigeminal nucleus

caudalis). J Comp Neurol 180:395-414, 1978b.

Henry, J.L.; Sessle, B.J.; Lucier, G.E.; and Hu, J.W.

Effects of substance P on nociceptive and nonnociceptive trigeminal brain stem neurons. Pain

8133-45, 1980.

Hokfelt, T.; Johansson, 0.; Terenius, L.; and Stein, L.

The distribution of enkephalin immunoreactive cell

bodies in rat central nervous system. Neurosci Lett

5:25, 1977.

177
Hunt, S.P.; Kelly, J.S.; and Emson, P.C. The electron

microscopic localization of methionin enkephalin

within the superficial layers (I & II) of the spinal

cord. Neuroscience 5:1871-1890, 1980.

Kerr, F.W.L. Neuroanatomical substrates of nociception

in the spinal cord. Pain 1:325-356, 1975.

Kumazawa, T., and Perl E.R. Excitation of marginal

and substantia gelatinosa neurons in the primate

spinal cord: Indications of their place in dorsal

horn functional organization. J Comp Neurol 177:417-

434, 1978.

Levy, R.A, The role of GABA in primary afferent

depolarization. Prog Neurobiol 9:211-267, 1977.

Light, A.R.; Trevino, D.L.; and Perl, E.R. Morphological features of functionally defined neurons in

the marginal zone and substantia gelatinosa of the

spinal dorsal horn. J Comp Neurol 186:151-172, 1979.

Lima, D., and Coimbra, A. The neuronal population of

the marginal zone (Lamina I of the rat spinal cord).

A study based on reconstruction of serially sectioned

cells. Anat Embryol 167:273-288, 1983.

Mayer, D.J., and Price, D.D. Central nervous

mechanisms of analgesia. Pain 2:379-404, 1976.

Mehra, R.D., and Bijlani, V. Substance P like immunoreactivity along the lateral side of the dorsal horn

of human spinal cord. In: Barold, C., and Slavkin,

H.C., eds. Progress in Developmental Biology Part B.,

New York: Alan R. Liss Inc., 1986. pp 149-151.

blessing, R.B., and Lytle, L.D. Serotonin containing

neurons: Their possible role in pain and analgesia.

Pain 4:1-21, 1977.

Mountcastle, V.B. Pain and temperature sensibilities.

In: Mountcastle, V.B., ed. Medical Physiology. St.

Louis: The C.V.Mosby Company, 1980. pp. 391-416.

kludge, A.W.; Leeman, S.E.; and Fischbach, G.D.

Enkephalin inhibits release of substance P from

sensory neurons in culture and decrease action

potential duration. Proc Natl Acad Sci USA 76:526-

530, 1979.

Nicoll, R.A.; Schenker, C.; and Leeman, S.E. Substance

P as a neurotransmitter candidate. Ann Rev Neurosci

3:227-268 1980.

Otsuka, M. and Konishi, S. Electrophysiological and

neurochemical evidence for substance P as a

transmitter of primary sensory neurons. In: Euler

Von, U.S., and Pernow, B., eds. Substance P. New York:

Raven Press, 1977. pp. 207-213.

Pelletier, G., and Leclere, R. Localization of

leuenkephalin in dense core vesicles of axon terminals.

Neurosci Lett 12:159-163, 1979.

Rizvi, T.A. Wadhwa, S.; Mehra, R.D.; and Bijlani, V.

Ultrastructure of marginal zone during prenatal

development of human spinal cord. Exp Brain Res

64:463-490, 1986.

178
Ruda, M.A., and Gobel, S. Ultrastructural

characterization of axonal endings in the substantia

gelatinosa which take up (

3

h) serotonin. Brain Res

184:57-83, 1980.

Sar, M.W.; Stumpf, E.; Miller, R.J.; Chang, K.J.; and

Cuatre-cabab, P. Immunohistochemical localization of

enkephalin in rat brain and spinal cord. J Comp Neurol

181:17-38, 1978.

Sternberger, L.A. Immunocytochemistry. Englewood Cliffs,

N.J.: Prentice Hall Inc., 1974. p.246.

Trevino, D.L.; Maunz, R.A.; Bryan, R.N.; and Willis,

W.D. Location of cells of origin of the spinothalamic

tract in the lumbar enlargement of cat. Exp Neurol

34164-77, 1972.

Trevino, D.L., and Carsten, E. Confirmation of the

location of spinothalamic neurons in the cat and

monkey by retrograde transport of horseradish

peroxidase. Brain Res 98:177-182, 1975.

Waldeyer, W. Dass Gorilla-Ruckenmark. Abhandlungen

der Konighlichen Akademie der Wissen chaften. 1888.

pp.1-147 (quoted by Willis

Willis, W.D.; Kenshalo, D.R. Jr.; and Leonard, R.B.

and Coggeshall 1978).

The cells of origin of the primate spinothalamic

tract. J Comp Neurol 188:534-574, 1979.

Wolf, J.R. Some morphogenetic aspects of the

development of the central nervous system. In:

Immelmann, K.; Barolow, G.W.; Petrinovitch, L.; and

Main, M. eds. The Bielefeld Interdisciplinary Project.

New York: Cambridge University Press, 1981. pp. 164-

190.

AUTHORS

Professor Veena Bijlani, M.B.B.S., M.S., Ph.D., F.A.M.S.

Tilat A. Rizvi, M.Sc.

S.Wadhwa, M.B.B.S., M.S., M.A.M.S.

Department of Anatomy

All-India Institute of Medical Sciences

New Delhi-110 029, India

179
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Differential Effects of Opioid Peptides

Administered lntracerebrally in Loci of SelfStimulation Reward of Lateral Hypothalamus

and Ventral Tegmental Area-Substantia Nigra

Jitendra Singh, Ph.D., and T. Desiraju, Ph.D.

INTRODUCTION

In recent years the knowledge on the wide distributions of the opioid

neurons, fibre terminals, and their receptors has been growing (Atweh

and Kuhar 1983; Akil et al. 1984; Snyder 1984, 1986). The opioid

peptide neuronal and receptor mechanisms are considered to participate

in a range of homeostatic integrations in the different subsystems of

the brain, including the stress response and coping, pain control, analgesia, neuro-endocrine, cardiovascular, and other visceral regulations,

and the mechanisms of reward, mood, and psyche. The present state

of knowledge generally indicates that the beta-endorphin neurons

and the epsilon, mu, and delta receptor mechanisms might be predominantly involved in the visceral and neuro-endocrine regulations, the

enkephalins and the delta receptors involved primarily in the psychic

and other limbic neuronal mechanisms, the dynorphins and the kappa

receptors primarily involved in the analgesic and sedative neuronal

mechanisms, and with several overlapping roles also in the functioning

of these neurons and receptors in different subsystems.

The enkephalins were shown to have the reward properties in intraventricular self-administration experiments (Stein and Belluzzi 1979),

the leu-enkephalin was more self-administered than the met-enkephalin

or the morphine. The self-administration of morphine and of D-ala2-

mets-enkephalinamide (and its blockade by naloxone) into the selfstimulation sites of lateral hypothalamus was also reported (Olds

1979; Olds and Williams 1980), but these results were argued to be

artifacts by Bozarth (1983), who stated that the morphine selfadministration property was not present in lateral hypothalamus, but

present only in the ventral tegmental area (Bozarth and Wise 1981).

Shaw et al. (1984) reported that while the intra-ventricularly injected

morphine caused a reduction of spontaneous motor activity, the electrical self-stimulation (SS) of medial prefrontal cortex (MPC) was

facilitated. Similar results were also observed by them with the intraventricular injection of the enkephalin analogue BW 180 (tyr-D-alagly-phe-D-leu), but these effects were indirect according to them

as direct injections of morphine or naloxone into MPC caused no change

180
in the SS of MPC. Naloxone (intraperitoneal) has also been shown

to cause a small but significant reduction in the SS of lateral hypothalamus (Ichitani et al. 1985). Broekkamp et al. (1976) reported that

the SS of LH was inhibited by injecting morphine into central grey.

In contrast, microinjections of morphine or of D-ala2-met5-enkephalinamide administered into ventral tegmental area facilitated the SS

of lateral hypothalamus and the effect of morphine was antagonized

by systemic naloxone (Phillips et al. 1983). They also reported rewarding

effects in the conditioned place preference paradigm and that such

a facilitatory effect was attenuated by haloperidol or by lesions

of dopamine pathways (Phillips et al. 1983).

The above studies indicate the need for investigations of the role of

the opioid peptides administered into the different regions of SS to

understand the intrinsic and interlinked components in the mechanisms

of the brain reward neuronal systems. The present study was designed

to investigate the effects of enkephalins and dynorphins administered

directly into the loci of self-stimulation of ventral tegmental areasubstantia nigra (SN-VTA) and of lateral hypothalamus (MFB-LH)

on the self-stimulation operant rates of the same loci. Such studies

have not appeared in the literature so far.

METHODS

Wistar rats weighing 260-320 g were implanted with bipolar SS electrodes made up of 28 g stainless steel wire, and bipolar cannula-cumwire electrode made up of the same type of wire joining to 22 g cannula

needle. The cannula-wire electrode (both insulated except for tips)

served either as bipolar SS electrode or for microinjections of chemicals

through the cannula into the SS site. In each rat one bipolar wire

electrode and one bipolar cannula electrode were implanted, one in

MFB-LH and the second in ipsilateral SN-VTA . In different rats

the placement of the two types of electrodes was interchanged between

the regions. After a few days of post-operative recovery, the selfstimulation rates of each of these sites were shaped and stabilized

reproduceably in a few sessions. The stimuli used were sine waves

of 50 Hz, 0.25 sec train duration for each pedal press, and current

intensities ranging between 75 μA and 200 μA as required to get the

maximum possible SS without apparent side effects. The duration

of a control or a test session was 15 min. At least three test sessions

(with drug) and three control sessions (with vehicle) were made for

each dose, and all repeated in at least three rats. 33 rats were used

in the present study. All intra-cerebral injections (drug or vehicle

solutions) were made through the cannula slowly in about 60 seconds

in a constant volume of I μl. At the end of the experiment, the placements of the electrodes were noted from frozen sections cut at 60 μ

stained with cresyl violet, and marked on the charts of atlas of Paxinos

and Watson (1982). Data were processed by using the F test (Anova)

to test the homogeneity of variance and by Student’s ‘t’ test to know

the levels of significance of the effects.

The chemicals used were: leu5-enkephalin; met5-enkephalin; leu5-

enkephalinamide; dynorphin-A[1-13] (all these of Sigma, USA); D-ala2-

D-leu5-enkephalin (BW 180C of Wellcome Research Laboratories,

181
UK); naloxone-HCI (Endo Laboratories, USA).

RESULTS

The leu5-enkephalin (leu-enk) was administered (500 ng) into SN-VTA

and one hour and two hours later, the SS operants were recorded.

It was observed that the SS of the site was reduced by 48% at 1 hr

and by 70% at 2 hrs from the level of the control value obtained

under the vehicle injection (Fig. 1), the SS of the other site (MFB-LH)

was not changed. In the reverse experiment, administration of the

leu-enk in MFB-LH has not changed the SS of the same site but has

caused a facilitation of the SS of the SN-VTA (Fig. 1). A similar pattern

of response was obtained with the leu5-enkephalinamide also (Fig. 2).

The D-ala2-D-leu5-enkephalin (ala-leu-enk or BW 180c) caused, contrary

to the above opioids, a facilitatory effect on the SS of SN-VTA when

it was admiristered either in the SN-VTA or in the MFB-LH (Fig. 3).

This opioid also has not influenced the SS of the MFB-LH. Naloxone

hydrochloride (30 mg/kg ip) has brought down the facilitated effect

of SN-VTA back to about the normal level (Fig. 3).

The met5-enkephalin (met-enk) administered in doses of 500 ng or

2 μg either into SN-VTA or into the MFB-LH has not been found to

influence the SS of either region (Fig. 4).

Dynorphin-A[1-13](dyn) administered in doses of 500 ng or 1 μg into

SN-VTA has caused facilitation of its SS. Administration of the same

into the MFB-LH has not influenced either its SS or that of the

SN-VTA (Fig. 5).

In summary, the leu-enk or its amide have caused reduction in the

SS of SN-VTA when administered into the same site. When these opioids

were administered into the MFB-LH its own SS was not influenced,

but caused indirect facilitatory effects on the SS of SN-VTA. Met-enk

caused no changes of SS in both these regions. Contrary to leu-enk,

dynorphin-A[1-13] caused facilitatory effect in the SN-VTA, and also

no effect through MFB-LH. Ala-leu-enk caused both types of facilitatory effects on the SS of SN-VTA, when it was administered into

it and also into the MFB-LH, without changing the SS of the

MFB-LH.

DISCUSSION

There seems to be no study made previously to find the effects of

the above cited opioid peptides on the SS of SN-VTA and of MFB-LH,

after administering them into the same SS sites.

It was previously reported (Shaw et al. 1984) that only the intraventricular administrations of BW 180 (ala-leu-enk) up to 40 μg doses,

or morphine up to 20 μg, caused facilitation of the SS of the medial

prefrontal cortex (MPC), but no effects were observed when morphine

or naloxone was administered into the MPC. As the ventricular drug

could act at many sites in the brain, the authors interpreted the effects

as indirect ones on the MPC. These authors have also reported that

182
FIGURE 1

Effects of slow microinjection (1 μ1 in 60 sec) of leuenkephalin either

into SN-VTA (A) or into MFB-LH (B) on the electrical self-stimulation

of the injected site and of the other site. Cannula site is open circle,

filled circle is wire. Typical placement sites of histology indicated

in the atlas drawings of hypothalamus and midbrain. A’, one hour

or two hours (A”) after the drug is administered in other site; B,

before the drug is administered in the other site; D’, one hour or

two hours (D”) after the drug is administered in the same site; V,

after the vehicle (saline) is administered in the same site. ‘n’ indicates

the data of number of rats and the number of sessions conducted

on each of them. * = P < 0.005; ** = P < 0.001.

183
FIGURE 2

Effects of leu-enkephalinamide in two doses represented by the two

sets of bars (1,2). Self-stimulation sessions were conducted one hour

after the drug (D) or the vehicle (V) administration. After testing

the injected site, the other site was tested. Other symbols as in Figure 1.

184
FIGURE 3

Effects of D-ala2-D-leu5-enkephalin, and of intraperitoneal (ip) naloxone

hydrochloride administered during the enkephalin effect. Naloxone

effect studied from 15 min after its administration. Symbols as in

Figure 2.

185
FIGURE 4

Effects of met-enkephalin in two doses represented by the two sets

of bars (1,2). Symbols as in Figure 2.

186
FIGURE 5

Effects of dynorphin-A[1-13] in two doses represented by the two

sets of bars (1,2). Symbols as in Figure 2.

187
the intraventricular BW 180 caused a reduction of the spontaneous

motility, particularly in the dose range of 20-40μg. In our local study

on SN-VTA and MFB-LH, only a dose of 2 μg of the ala-leu-enk has

been used and the effects on their self-stimulation have been very

significant. Further, we have also observed that the facilitatory effect

was relieved by administration of naloxone hydrochloride intraperitoneally. Naloxone has not significantly affected the normal SS of SN-VTA

or of MFB-LH in our study, in contrast to the report of Ichitani et

al. (1985) who observed a small but significant reduction of the SS

of lateral hypothalamus under naloxone (5mg/kg ip). The effects of

naloxone hydrochloride on the SS could depend on the regional differences and on the conditions of reinforcement, the FR-30 schedules being

most affected and the CR schedules least affected. Schaefer and

Michael (1985) observed the SS (FR-30) of the periventricular grey

to decrease in a dose-dependent manner, by about 70% at a naloxorie

dose of 30 mg/kg given sub-cutaneously. At this dose, they have also

observed slight reduction (about 15%) in the ambulatory activity.

Phillips et al. (1983) reviewed the observations to show that morphine

or ala-met-enk administration into SN-VTA caused facilitation of

the SS of lateral hypothalamus, whereas our results with the ala-leu-enk

administration into the SN-VTA have not caused any change in the

SS of MFB-LH. Probably the difference in the opioids used and the

receptors upon which they act could be the reason for the opposite

patterns of the results. Our present study with the leu-enk, met-enk,

ala-leu-enk, and dyn has also disclosed such differences in the patterns of the effects on SS of the SN-VTA and the MFB-LH. In the

studies of Phillips et al. (1983) the effect of morphine or of ala-metenk on the _SS of the injected site (SN-VTA) was not studied, whereas

we have found that with the ala-leu-enk the SS of the injected site

(SN-VTA) was facilitated. We have also observed the converse, i.e.,

administration of the ala-leu-enk into the MFB-LH has not changed

the SS of the injected site and yet led to a change of the SS of the

SN-VTA. This could imply that at the MFB-LH the ala-leu-enk could

have acted on an additional interlinked substrate besides on the SS

substrate (intrinsic) of MFB-LH and that the additional substrate

could have been linked to the SN-VTA intrinsic substrate of SS. In

other words, the opioid effects probably differentiated in the MFB-LH

the intrinsic SS substrate from a separate substrate present there

and interlinked to the SN-VTA mechanism of SS.

The mode of synaptic transmission actions of opioids and the details

about differerces in the proportions of the opioid receptor types present

on the neurons of the MFB-LH and the SN-VTA are not adequately

available to enable to infer the possible mechanisms underlying the

effects seen in the present study. A review of the previous studies

(North and Williams 1983) indicates that morphine would enhance K

+

conductance and cause inhibition of noradrenergic neuronal firing.

These effects could widely differ according to the types of the neurons

and the sites in the brain. There was also an opposite type of observation that morphine when administered iontophoretically led probably

indirectly to an increase in the firing of dopaminergic neurons at

SN-VTA and that iontophoressed naloxone had not reversed this effect,

188
whereas the non-DA neurons in the vicinity were suppressed, which

could be reversed by naloxone (Gysling and Wang 1983). It has been

reported that the leu-enk has primarily the delta receptor activity

(Akil et al. 1984). Dynorphin-A [1-13] is known to be very potent

at both mu and kappa receptors, and its fibre terminals are present

only in SN and not in LH, which has the cell bodies (Akil et al. 1984).

Ala-leu-enk also labels preferentially the delta sites. Hence, the

opioid peptides used in the present study would be primarily acting

upon the delta, mu, and kappa receptors of the non-opioid and the

opioid neurons and axon terminals in the MFB-LH and SN-VTA. However,

the differences in the proportions of these receptor types present

in the two regions, and the organization of the types of neurons and

terminals in the linkages of circuits in which these receptors are

located in the two regions, are unknown. The mu receptors are probably more predominant in the MFB-LH (Akil et al. 1984), whereas

the delta and kappa may be relatively more in the SN-VTA substrates

of SS. It is hypothesized from the present results that the leu-enk

in the MFB-LH could be inhibiting a projection mechanism inhibitory

to the SS system of SN-VTA, thereby releasing the facilitation of

the SN-VTA. The opposite type of effects of the leu-enk and the

ala-leu-enk on the SS of SN-VTA, when these are administered in the

SN-VTA, is probably due to their differences in actions on the receptor types present in the SN-VTA. The dynorphin-A actions suggest

that the kappa receptors seem to be involved in the SS mechanism

of SN-VTA and not in that of MFB-LH.

The differential effects of the above opioids on the SS of the SN-VTA

and the MFB-LH emphasize the differences in the neural mechanisms

of the SS of the two regions. Our previous study (Singh and Desiraju

1986) of the SS of the two regions with local administrations of haloperidol and apomorphine also disclosed that the two regions have

separate synaptic types of intrinsic mechanisms of SS, besides having

an inter-linkage mechanism. As the opioids have shown either inhibitory

or facilitatory effects on the SN-VTA, and little such effects on the

MFB-LH, the possibility arises whether this striking difference could

be due to the dopaminergic neurons and receptors present in the SN-VTA.

This difference in effects could also as well be due to the opiate

receptors present on non-dopaminergic neurons and terminals located

in the SN-VTA.

SUMMARY

Leu-enkephalin, leu-enkephalinamide, ala-leu-enkephalin, met-enkephalin

and dynorphin-A[1-13] were administered in microinjection into one

of the self-stimulation sites of SN-VTA or MFB-LH and the electrical

self-stimulation (SS) of the injected site and of the second site was

recorded. The study revealed that the leu-enkephalin and the leuenkephalinamide inhibited the SS of SN-VTA and produced no effect

on the SS of MFB-LH, when administered into these sites. The MFB-LH

injection, however, facilitated the SS of SN-VTA. The effect of alaleu-enkephalin injection in MFB-LH was similar to the above, but

the effect of the injection in SN-VTA was different in that it caused

189
the facilitation of its SS and not the depression as seen with leuenkephalin. Met-enkephalin injections in the two regions caused no

direct or indirect changes of the SS of the regions. Dynorphin injection

in SN-VTA facilitated its SS, like the injection of ala-leu-enk, but

dynorphin injections in MFB-LH produced no effects. The results essentially demonstrate the differences in the effects of the different

opioids in the reward system of the SN-VTA, and it is discussed that

these differences are probably due to the preferences in the types

of the receptors upon which these opioids act in the SN-VTA neuronal

organisation. The results also demonstrate the major difference in

the organisation of the reward substrate of the MFB-LH from that

of the SN-VTA, as the effects of the opioids in the MFB-LH are markedly differert or none compared to the effects in the SN-VTA.

REFERENCES

Akil, H.; Watson, S.J.; Young, E.; Lewis, M.E.; Khachaturian, H.; and

Walker, J.M. Endogenous opioids: biology and function. Ann Rev NeuroSci 7: 223-255, 1984.

Atweh, S.D., and Kuhar, M.J. Distribution and physiological significance

of opioid receptors in the brain. Br Med Bull 39:47-52,-1983.

Bozarth, M.A. Opiate reward mechanisms mapped by intracranial selfadministration. in: Smith, J.E., and Lane, J.D., eds; The Neurobiology

of Opiate Reward Processes. Amsterdam: Elsevier Biomedical, 1983.

pp.331-359.

Bozarth, M.A., and Wise, R.A. Intracranial self-administration of

morphine into the ventral tegmental area in rats. Life Sci 28:551-

555, 1981.

Broekkamp, C.L.; Van Den Bogaard, J.H.; Heynen, H.J.; Rops, H.;

Coofs, A.R.; and Van Rossum, J.M. Separation of inhibiting and stimulating effects of morphine on self-stimulation behaviour by intracerebral

microinjections. Eur J Pharmac 36:443-446, 1976.

Gysling, K., and Wang, R.Y. Morphine-induced activation of A10 dopamine neurons in the rat. Brain Research 277:119-127, 1983.

Ichitani, Y.; Iwasaki, T.; and Satoh, T. Effects of naloxone and chlordiazepoxide on lateral hypothalamic self-stimulation in rats. Physiol

Behav 34:779-782, 1985.

North, A.R., and Williams, J.T. Neurophysiology of opiates and opioid

peptides. In: Smith, J.E., and Lane, J.D., eds. The Neurobiology of

Opiate Reward Processes. Amsterdam, Elsevier Biomedical, 1983.

pp. 89-105.

Ofds, M.E. Hypothalamic substrate for the positive reinforcing properties

of morphine in the rat. Brain Research 168:351-360, 1979.

Ofds, M.E., and Williams, K.N. Self-administration of D-Ala2-Metenkephalinamide at hypothalamic self-stimulation sites. Brain Research

194:155-170, 1980.

Paxinos, G., and Watson, C. The Rat Brain in Stereotaxic Coordinates.

New York: Academic Press, 1982. 71 pp.

Phillips, A.C.; Broekkamp, C.L.; and Fibiger, H.C. Strategies for studying the neurochemical substrates of drug reinforcement in rodents.

Prog Neuro-Psychopharmacol & Biol Psychiat 7:585-590, 1983.

190
Schaefer, G.J., and Michael, R.P. Effects of opioid antagonists and

their quaternary derivatives on locomotor activity and fixed ratio

responding for brain self-stimulation in rats. Pharmacol Biochem

Behav 23:797-802, 1985.

Shaw, S.G.; Vives, F.; and Mora, F. Opioid peptides and self-stimulation

of the medial prefrontal cortex. Psychopharmacology 83:288-292,

1984.

Singh, J., and Desiraju, T. Differences in synaptic receptor types

primarily involved in self-stimulation reward behaviour. Ind J Physiol

Pharmacol 30(5):43-44, 1986.

Snyder, S.H. Drug and neurotransmitter receptors in the brain. Science

224:22-34, 1984.

Snyder, S.H. Neuronol receptors. Ann Rev Physiol 48:461-471, 1986.

Stein, L., and Belluzzi, J.D. Brain endorphins: possible role in reward

and memory formation. Federation Proc 38:2468-2472, 1979.

ACKNOWLEDGEMENT

The general support of the Indian Council of Medical ‘Research, New

Delhi, to this Department research.

AUTHORS

Jitendra Singh

Research Scholar

Department of Neurophysiology

National Institute of Mental Health and Neuro Sciences

Bangalore 560 029, INDIA

T. Desiraju

Professor & Head

Department of Neurophysiology

National Institute of Mental Health and Neuro Sciences

Bangalore 560 029, INDIA

191
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Peptides and Thermoregulation

Rakesh Shukla, Ph.D., and Bhola N. Dhawan, M.D.

Many neurotransmitters are involved in the central regulation of

body temperature under normal conditions, in fever, and during

exercise and heat and cold stress. Emphasis on amines and

prostaglandins has declined to some extent, and a major focus in

recent years has been on the peptides that might participate in

central nervous system (CNS) mediation of temperature control.

Interest in -the neurobiology of peptides has developed coincident

with their identification and chemical characterization in mammalian

brain. Several of these peptides were originally isolated from

pituitary tissue or the alimentary canal, but they also occur in

various regions of the CNS. Although the central roles of most of

the peptides are still not adequately understood, many neuropeptides

are known to have profound effects on regulation of body

temperature, and the available data on the neuropeptides will be

briefly reviewed.

1. OPIOIDS

The endogenous opioids are unique in that their ability to alter

thermoregulatory mechanisms is shared by nonpeptide opioids. While

evidence is accumulating that opioid ligands and opioid receptors

play a functional role in thermoregulation, an exhaustive study in

different species has not yet been undertaken for opioid peptides.

Much less information is available on the opioid peptides than on

morphine.

A. Morphine

Most studies have been conducted with morphine, although some

investigations have also been carried out with other opioids.

Several reviews have appeared on the effects of morphine on body

temperature, the most comprehensive being those by Clark (1979).

Clark and Lipton (1983, 1985), and a recent review by Adler et al.

(1988). The effects of morphine are species-dependent. A

hypothermic response is predominant in the dog, rabbit, and pigeon;

a hyperthermic response in cat, cattle, goat, and horse; and a dual

response (low--dose hyperthermia and high-dose hypothermia) in rat,

mouse, and primates (Clark and Lipton 1985). A biphasic response,

192
hypothermia followed by hyperthermia, in rat and mouse is also

reported (Rosow et al. 1980). All these responses to morphine are

blocked and antagonized by naloxone. There is also some interaction

with calcium channel antagonists (Pillai and Ross 1986b). Morphine

responses are dependent on ambient temperature: a hypothermic

response is predominant at ambient temperature below

thermoneutrality; biphasic responses appear as the ambient

temperature increases, and ultimately hyperthermia predominates at

the highest ambient temperatures (Adler et al. 1988). Recent

studies have demonstrated that morphine responses are also dependent

on the route of administration (Adler et al. 1984; Geller et al.

1986), and route is a critical determinant not only of quantitative

effects but of qualitative effects as well. The exact mechanisms

involved in the body temperature changes produced by morphine are

still not known with certainty. The effects may result primarily

from the actions on oxygen consumption, however. Morphine-induced

hypothermia is associated with decrease in oxygen consumption and

metabolic heat production in a variety of species (Lotti et al.

1966; Lin et al. 1980). Recent studies by Adler and his colleagues

(1986, 1988) suggest that body temperature responses to morphine are

due to the change in thermal set point in hypothalamus. In dog, an

increased heat loss after morphine administration has been

demonstrated (see Clark and Lipton 1985 for a review and

references). Some interaction of morphine with dopaminergic and

cholinergic systems in thermoregulation has been shown by Cox et al.

(1975) and Adler et al. (1986).

B. Naloxone

Naloxone has been used by several investigators to establish

evidence for involvement of opioid receptors. Naloxone, per se,

however, does not have an effect on thermoregulation in rat (Pillai

and Ross 1986a) or man (Fuenmayor and Cubeddu 1986) though in

morphine-dependent rats naloxone and related compounds reduced

rectal temperature by a central action since quarternary derivatives

were inactive (Katovich et al. 1986). The drug interactions are not

always well defined. Thus naloxone only partially blocked ethanol

hypothermia, which was completely blocked by kappa antagonist MR

2266 (Pillai and Ross 1986a), whereas it completely antagonized

microwave blockade of amphetamine hyperthermia (Lai et al. 1986).

The blphasic temperature changes with phencyclidlne are antagonized

by naloxone, by the E and K antagonist SKF 10,007 as well as by

the pure k antagonist MR2266 (Hiramatsu et al. 1986).

C. Endogenous Opioids

Identification of endogenous peptides and their precursor molecules

in the brain encouraged intense research efforts to determine both

the in vivo and the in vitro effects of these substances and the

possible functional roles played by the opiold

receptors. Recent data utilizing more selective ligands in rat

suggest that μ opioid receptors play a role in hyperthermic

response. Morphine, a prototypic μ ligand, and DAGO, a μ

selective receptor agonist, produce hyperthermla in several species

193
of animals (Adler et al. 1988) and prompt rats to seek warmer

temperatures. On the other hand, receptors seem to be involved

in hypothermia, as U50488H, a receptor agonist, produces

dose-related hypothermia. A role for receptors in

thermoregulation cannot be ruled out completely.

a. ß-Endorphin. If nonpeptide opioids act at receptors that are

stimulated by oploid peptides, the thermoregulatory effects of

endogenous opioids and of morphine and related agonists ought to be

alike. When ß-endorphin is injected centrally to a variety of

species, it induces patterns of temperature change that closely

resemble those associated with central or peripheral administration

of morphine (Clark and Lipton 1985). Relatively low doses of

morphine and ß-endorphin raise the level at which temperature is

regulated in the cat, rat, mouse, and rabbit (Rezvani et al. 1982),

primate (Murphy and Lipton 1983), and fish (Kavaliers 1982).

Evidence from studies on rabbits indicates that this change in

regulation may be due to impaired input from peripheral warmth

sensors (Rezvaini and Heath 1984). Such an effect would be of

greater significance in a warmer environment and could account for

the greater increase in body temperature at usual ambient

temperatures than in a cold environment after injection of a given

dose of ß-endorphin to cats, rabbits, etc. In contrast to the above

species, which have been used most often for the study of

opioid-induced temperature changes, ß-endorphin lowers the

temperature of chickens and hamsters (Clark and Lipton 1983).

In those species in which small doses of ß-endorphin induce

hyperthermia. naloxone and similar antagonists ought to lower

temperature if' ß-endorphin contributes to maintenance or elevation

of body temperature. Both naloxone and naltrenone prevented a rise

in the temperature of rats secondary to restraint or handling, or

lowered temperature when stress was minimized by habituation of the

animals to the experimental procedures (Clark and Lipton 1983; Pae

et al. 1985). Except for such reports, evidence is not available to

indicate that reasonable doses of opioid antagonists appreciably

affect temperature. So it is unlikely that ß-endorphin, which has

been readily antagonized in most studies, plays a major role in

normal thermoregulation, even in hot or cold environments (Clark et

al. 1983). Likewise, naloxone has not altered responses to pyrogen

in the cat, rat (Clark and Lipton 1983), guinea pig (Kandasamy and

Williams 1983), and sheep (Duranton and Bueno 1984). So a role for

ß-endorphin in fever is unlikely. This view is also supported by

the fact that antipyretics failed to antagonize the hyperthermic

action of opioid peptides in the cat, mouse (Clark and Lipton 1983),

guinea pig (Kaidasamy and Williams 1983), and rabbit (Kandasamy and

Williams 1983; Rezvani and Heath 1984). It is likely that the

endorphin effects are mediated via a mechanism independent of the

arachidonic acid cascade.

194
b. Enkephalins. Very little information is available on the

potential thermoregulatory actions of enkephalins. Met-enkephalin

is a relatively weak agonist in altering body temperature (Clark and

Lipton 1985), probably because of its rapid inactivation (Miller

1981). To obtain measurable temperature changes, larger doses

(0.1 mg to 10 mg centrally) of the enkephalins (as compared to

ß-endorphin) have been required. The usual response at room

temperature has been hyperthermia (Clark and Lipton 1985), although

there have been exceptions (Sakurada et al. 1983). Shukla et al.

(1982) have demonstrated that Met-enkephalin induced hyperthermia

varied inversely with ambient temperature over a temperature range

of 10° to 33°C In the desert rat Mastomys natalensis.

A major difference between Met-enkephalin and ß-endorphin is that

the hyperthermic response to the former is less readily antagonized

by naloxone (Clark and Lipton 1983). Hence the lack of appreciable

effect of naloxone on temperature does not rule out the mediation of

thermoregulatlon by enkephalins. Assessment of this possibility has

been hampered by the lack of a selective enkephalln antagonist. A

number of relatively stable and more potent enkephalin analogs have

been tested (see 1985 review by Clark and Lipton). Unfortunately,

these have generally been more readily antagonized by naloxone, and

act more like ß-endorphin than like the parent enkephallns, probably

because the structural modifications that increase stability tend to

shift sensitivity away from naloxone-insensitive types of receptors.

Recently Shukla et al. (1988) suggested an enkephalinergic and

serotonergic interaction in the thermoregulation of the desert rat.

c. Other Opioid Peptides. Dermorphin produces a naloxone-reversible

hypothermia which is unaffected by antagonists of other putative

neurotransmitters such as serotonin, catecholamines, GABA,

acetylchollne, etc. (Tatara et al. 1986).

Currently available data do not permit a definitive statement

regarding the role of opioid peptides in thermoregulatlon. It is,

however, abundantly clear from data reviewed above that multiple

oploid receptors are involved. It appears likely that several types

of transmitters/modulators are involved in regulation of body

temperature in health and disease and that their relative importance

varies in various situations. It is necessary to study the effect

of specific agonists and antagonists at various opioid receptors in

several species, not only at different ambient temperatures but also

during pyrexia induced by different agents to assess their roles.

It is also necessary to study their interaction with other putative

neurotransmitters and to analyze in depth their effect on heat

production and heat loss mechanisms. There is evidence to indicate

that some of the peptides may be acting at sites such as the septum

(see below). Such sites have not been investigated for the effect

of oploid peptides.

195
II. ANTIPYRETIC PEPTIDES

A. ACTH/MSH

ACTH and MSH, which were first isolated from pituitary tissue, are

widely distributed within the brain (Krieger and Martin 1981). They

were first shown to reduce temperature when given centrally to

rabbits (Lipton et al. 1984). Subsequently, ACTH and MSH were found

to be antipyretic when administered centrally or peripherally in

doses that had no effect on normal temperature (Murphy et al.

1983). The parent molecule ACTH (1-39) was actually the first

peptide with documented antipyretic activity. Most of the recent

research, however, has been focused on MSH. This peptide is about

25,000 times more potent than acetaminophen as an antipyretic

(Murphy et al. 1983). MSH reduces fever whether administered

i.c.v., i.v., or into the stomach of rabbits (Murphy and Lipton

1982). Its central site of action may be the septum, as injections

of MSH into the septum are also antipyretic. ICV administration of

ACTH reduces endotoxin-induced fever in squirrel monkeys, and MSH is

likewise antipyretic when given i.v. to these primates (Lipton and

Shih 1985). Corticotropin-releasing factor (CRF), which releases

ACTH/MSH from the pituitary, also reduces fever in a dose-related

fashion when given i.c.v. to rabbits.

The precise mechanism of action of MSH in reducing fever has not

been determined. However, it is unlikely that antipyretic doses

directly inhibit central heat production and conservation pathways,

since MSH has no effect on the temperature of a febrile rabbit

exposed to cold (Richards and Lipton 1984). The doses required to

induce the antipyretic and hypothermic effects of peptides differ.

These effects appear to represent distinct actions within the CNS.

Studies on hyperthermic responses to arachidonates suggest that

central release of MSH could mediate the antipyretic effect of

acetaminophen

B. Arginine Vasopressin (AVP)

The role in thermoregulation of arginine vasopressin, the

antidiuretic hormone in man, has been studied in the past. Most of

the supportive data have been obtained recently, however. Studies

of temperature-induced changes in plasma vasopressin levels or

diuresis have produced conflicting data. Concentration of AVP did

not change in the pig during exposure to warm, neutral, or cold

environments (Forsling et al. 1976). Cooling of the rostral

hypothalamus in cold and thermoneutral environments, or heating in

thermoneutral and warm environments, likewise did not affect

vasopressin levels. However, increasing core temperature to 42°C

caused a rise in plasma vasopressln that could be completely

suppressed by reducing preoptic/anterior hypothalamic temperature.

Brisk water diuresis was observed with preoptic/anterior

196
hypothalamic cooling in rhesus macaques, presumably due to

inhibition of vasopressin release (Hayward and Baker 1968). These

findings suggest a relation between vasopressin and central

temperature control mechanisms. They give no indication, however,

that this relationship is important under normal circumstances. The

evidence for arginine vasopressin as an endogenous antipyretic is

strong but not yet conclusive. It includes direct suppression of

fever by the application of AVP in a sensitive, dose-dependent

manner by push pull perfusion into the region of the septum in

animals given intravenous pyrogens, and the appearance of AVP in the

perfusate after perfusion of the septal region with an artificial

interstitial fluid during fever. The vasopressin concentration is

inversely related to the magnitude of fever.

Hemorrhage, a well-known stimulus for the release of vasopressin

into the circulation, was found to be without effect on normal body

temperature but to suppress the fever elicited from intravenous

pyrogen. The application of an antibody to AVP in the septal region

led to a markedly greater rise in body temperature than would

normally occur from a fixed dose of endotoxin given intravenously

(Cooper 1987). AVP itself is without effect on normal body

temperature in the sheep when perfused through the ventral septal

area. Zeisberger et al. (1980) and Merker et al. (1983) have

demonstrated that the pregnant guinea pig close to term and several

hours post partum is refractory to endotoxin. In the nonpregnant

guinea pig during fever, AVP reactivity was increased in fibers

projecting from the hypothalamus to the septum and amygdala

(Zeisberger et al. 1983).

Arginine vasopressin suppressed pyrexia caused by intraventricular

injection of PGE when the AVP was perfused through the rostral

diencephallc loci at which PGs caused fever (Ruwe et al. 1985).

Evidence of the endogenous antipyretic action of AVP has been found

in rats (Kovacs and De Wied 1983). Banet and Wleland (1985) found

that AVP infused into the lateral septum of the rat suppressed the

heat production caused by hypothalamic cooling but did not affect

vasomotor tone in the skin. The reduction in thermoregulatory

increase in heat production that led to a fall of body temperature

during cold exposure might explain the antipyretic action of AVP.

Wilkinson and Kasting (1986) psesented evidence to indicate that

vasopressin reduces the set point for body temperature in febrile

rats.

Only one study (Lee et al. 1985) has evaluated the role of AVP in a

subhuman primate, macaque monkeys, where intraventricular AVP had no

effect on the normal body temperature or fever, except at a dose of

65 ng, where the mean fall of body temperature of 0.5°C appeared

to be due to a larger response in one animal. AVP has also been

shown to cause hyperthermia in rabbit (Lipton and Glyn-Ballinger

1980) but is without effect in cats and causes hypothermia or

hyperthermia when microinjected into the preoptic area in the rat

(Naylor et al. 1986).

197
Arginine vasopressin might be a mediator of fever within the

anterior hypothalamus/preoptic anterior hypothalamus and an

antipyretlc within the ventral septal area. Such a dual function

would be consistent with a negative feedback system to modulate the

magnitude of' fever, a useful function if fever is a beneficial

accompanimert of disease. A role for AVP as a fever mediator as

well as an endogenous antipyretic is, however, far from conclusive.

III. OTHER PEPTIDES

A. Thyrotropin-Releasing Hormone (TRH)

TRH is the most extensively studied peptide, and it usually induces

a brief increase in temperature (Clark 1979). Cats respond with

hypothermia (Metcalf 1974; Myers et al. 1977), however, as do rats

(Lin et al. 1980) and nonhibernating ground squirrels (Stanton et

al. 1981). TRH-induced temperature changes are independent of its

action on the thyroid. Effects of TRH on body temperature were

determined over a range of ambient temperatures, but the pattern of

body temperature change in the majority of reports is consistent

with increase in the level of regulation. TRH has frequently been

observed to have a general excitatory action that would tend to

raise temperature by increasing heat production. It can enhance

locomotor and other behavioral activities alone and can reverse

behavioral depression, hypnosis, and anesthesia induced by other

agents (Clark and Lipton 1983).

The hyperthermic effect of TRH administered into the

preoptic/anterior hypothalamus of rats was unaffected by

pretreatment with aspirin, but was reduced by adrenergic antagonists

(Chi and Lin 1983). However, based on resistance to inhibition by

adrenergic antagonists, other investigators have concluded that the

noradrenerglc system is not essential for this response in mice

given TRH by intraperitoneal injection (Pawlowski and Kwiatek

1983). In another study, TRH given intraperitoneally to mice caused

hyperthermia that was associated with increased plasma

noreplnephrlne and eplnephrine concentration (Boschi et al. 1983).

Simultaneous injection of these catecholamlnes increased

hyperthermia. Adrenal demedullation suppressed both the increase in

plasma catecholamines and TRH-induced hyperthermia, but not the

hyperthermic response to TRH plus catecholamines. Hypophysectomy

also reduced --RH hyperthermia, in contrast to earlier reports, but

not the increase of plasma catecholamines or the effect of

combinations with exogenous catecholamines. These authors concluded

that both the adrenal and pituitary have essential roles in

TRH-induced hyperthermia. When injected into the hypothalamus of

pigeons, TRH induced hyperthermia, shivering, and vasodilation that

was probably compensatory (Lathi et al. 1983). Evidence that TRH

might be involved in the maintenance of body temperature has been

presented by Prasad et al. (1978, 1980). In this study, a

198
dose-related decrease in temperature occurred when rabbit TRH

antiserum was injected into the lateral ventricle of rats. and this

response was prevented if the antiserum was neutralized with TRH.

B. Neurotensin (NT)

Neurotensin was initially isolated from bovine hypothalamus

(Carraway and Leeman 1973). The primary temperature response to

central administration of NT in a variety of species is hypothermia,

which is enhanced in cold environments. Neurotensin has been

reported to have less than one-thousandth the potency of bombesin in

rats exposed to cold (Brown et al. 1977), whereas under similar

conditions in mice both of these peptides were equipotent (Mason et

al. 1980). Injections of NT into 223 brain sites in rat (Kalivas et

al. 1982) indicated that regions responsive to its hypothermic

action are distinct from sites at which it inhibits nociception.

Administration of NT into the periaqueductal gray had no clear

effect on normal body temperature (Kalivas et al. 1982). The

N-terminal fragments of NT were inactive, but a C-terminal fragment

(8-13) did lower temperature (Widdowson et al. 1983). Neurotensin

also lowered the temperature of guinea pigs and hamsters only in a

cold environment and did not change the temperature of fish, frogs,

lizards, pigeons, ground squirrels, woodchucks, and rabbits (Prange

et al. 1979).

The research on the mechanism of action of NT has been limited to

studies on interactions with other substances. Intracisternally

administered NT potentiated the hypothermic response of ethanol

(Luttinger et al. 1981) and of dopamine agonists (Jolicoeur et al.

1983), but inconsistent effects were obtained with pentobarbitalinduced hypothermia (Nemeroff et al. 1977). The hypothermic

response to NT in rat was antagonized by i.c.v. administration of

prostaglandin E2 and by calcium chelator EGTA (Lee et al. 1983) but

not by TRH. somatostatin, or naloxone. The hypothermic response was

also antagonized by amine and cholinergic antagonists (Morley et al.

1982) and was enhanced by thyroidectomy (Nemeroff et al. 1980).

Since central administration of EGTA and PGE2 alone causes

hyperthermia, a nonspecific antagonism may be responsible for

preventing the hypothermic effect of NT at thermoneutral

environment. Mason et al. (1982) suggested that release of central

prostaglandlns may oppose the hypothermic response of NT in cold

environment. Lee and Myers (1983) concluded that NT renders the

animals poikilothermic, because NT caused hypothermia in rats in

thermoneutral and cold environments but not in hot (34°-4O°C)

environment.

C. Bombesin

Bornbesin was originally isolated from frog skin (Anastasi et al.

1972, 19751. The central administration of bombesin caused

hypothermia in rat and mouse at normal laboratory temperatures. The

magnitude of the fall in the temperature has generally been dose

199
related. However, systemically given bombesin did not affect the

temperature of rats in the cold (Brown et al. 1977; Brown and Vale

1980). It was subsequently found that injections of bombesin into

the PO/AH of rats increased locomotor activity and enhanced both

heat reinforcement and heat escape behavior (Hawkins and Avery

1983). It was concluded that the effects on behavior are secondary

to the change in activity and do not demonstrate that bombesin

lowers the regulated temperature. Avery and Calisher (1982) showed

that in a thermoneutral environment i.c.v. bombesin caused

dose-related decrease in body temperature and food intake of

food-deprived rats.

D. Somatostatin

Nemeroff et al. (1979) found that intracisternal administration of

somatostatin in mice caused hyperthermia at normal laboratory

temperature. Brown et al. (1981) did not find any change in body

temperature of rat after intracisternal administration of

somatostatin in cold environment. Lateral ventricular

administration of somatostatin also did not alter the temperature of

rabbits (Lipton and Glyn 1980). In only one study somatostatin

caused hyperthermia at high ambient temperature and hypothermia at

low ambient temperature (Chandra et al. 1981). The pattern of

associated effector changes was unusual in that the hyperthermic

response at ambient temperature was associated with vasoconstriction

and an increase in metabolic rate, whereas the hypothermic response

at lower ambient temperature was associated with vasodilation and

decreased metabolism. Morley et al. (1982) elicited a small

decrease in temperature of rats with i.c.v. injection of

somatostatin (10 μg). which was even less if given with TRH.

Somatostatin did not appreciably affect changes in temperature

induced by bombesin or neurotensin. Intracisternal administration

of somatostatln 10 min after pentobarbital did not alter the

hypothermic response to the barbiturate (Breese et al. 1975).

However, a slight enhancement by i.p. somatostatin of

pentobarbital-induced hypothermia has been reported in the mouse

(Prange et al. 1975).

E. Angiotensin II

The major response to central administration of angiotensin in most

of the species has been a fall in temperature; the monkey, when

experiments were conducted in a hot environment, was an exception

(Clark 1979). Later, Lin and coworkers (1980) reported that central

injection of angiotensin to rats did not alter body temperature when

the ambient tenperature was 30°C but caused comparable

dose-related hypothermia at lower ambient temperatures. The

hypothermic response was inhibited by pretreatment with propranolol

or by depletion of whole brain catecholamines with

6-hydroxydopamine. Depletion of brain serotonin by

5,6-dihydroxytyptamine did not inhibit the response to angiotensin,

nor did prior administration of atropine or phentolamine. They

200
concluded that the hypothermic action of angiotensin II is due to

its effects on the mechanisms that promote reduced metabolism and

increased heat loss. The heat loss effecters vary in different

species.

IV. KININS

Small hyperthermic responses, which were associated with increased

muscle tone, ear vessel vasodilation, and piloerection were

inhibited by pretreatment with indomethacin and acetaminophen.

These responses were recorded after lateral ventricular

administration of bradykinin to rabbit (Almeida et al. 1978).

However, i.c.v. administration of bradykinln to rat (Francesconi

and Mager 1981) and to mouse (Nemeroff et al. 1979) did not alter

body temperature. Similarly, substance P caused a small rise in

temperature of rabbit when given in the third ventricle. This

hyperthermic response was reduced by pretreatment with indomethacin,

phenobarbital, or phentolamine but not by acetazolamide, atropine,

capsaicin, or propranolol (Krupin et al. 1982). In the rat,

substance P was consistently ineffective in altering temperature

whether given i.c.v., i.v., or intracisternally at any ambient

temperature.

V. CONCLUDING REMARKS

Attention has been focused on the role of neuropeptldes in

thermoregulation, because many of them are found within

neuroanatomical sites important to temperature control and exert

profound effects on body temperature. As with several other classes

of putative neurotransmitters, the effect seen is dependent on

species, ambient temperature, degree of restraint imposed on the

subject, route of drug administration, and several other factors.

For neuropeptides also the effect observed is dependent on several

factors. A limiting factor has been the unavailability of selective

pharmacological antagonists in many classes. Specific antisera to

many peptides have, however, become available in recent years. The

physiological relevance of a peptide to temperature control could be

studied by inactivation of the endogenous peptide through local

treatment with specific antibodies. There is evidence that certain

peptldes such as bombesin, which disrupt thermoregulation, have no

physiological significance in temperature control (Clark and Lipton

1983). For many of the peptides, the data available are not yet

adequate to determine their roles as mediators in central

thermoregulatlon. As noted above, however, convincing data exist to

support an antipyretic role for certain peptides, such as ACTH/MSH.

To understand the physiological role of central peptldes in

thermoregulation. several methodological factors should be

considered in future studies. The effect of peptides should be

tested in animals exposed to cold, hot, and thermoneutral

environments. This will make it possible to assess the functional

nature of the peptide effect. Past research clearly reveals that

201
species differences do exist for the temperature responses obtained

by the central administration of peptides. These may reflect either

real differences in the utilization of specific peptides in

temperature control or artifactual differences due to physical

factors such as variation in spread of peptide to active sites after

intracerebral injections. Many systems of the brain are involved in

thermoregulation, and multiple contributions of a given neuropeptide

to thermoregulatory control may lead to conflicting results when the

peptide is injected into different brain regions. It is already

known that endogenous peptide levels are affected by stress, and it

has been shown that opiate-induced temperature responses are

reversed by restraint stress. Stress may be reduced by avoiding

restraint; all all studies, of course, appropriate use of control

animals is essential to minimize misinterpretation of data, since

stress can naver be eliminated from the experiments with conscious

animals. The route of administration also plays an important role.

Thus it has been shown that intracerebroventricular administration

of most agonists, including agonists, produced

hyperthermia. possibly because mostly in receptors are reached by

that route (Geller et al. 1986).

It is also important to recall that the thermoregulatory apparatus

utilizes other major body systems (respiratory, cardiovascular, and

others). Thus, for example, a centrally acting peptide that

primarily affects the distribution of peripheral blood flow could

secondarily alter body temperature and be thought erroneously to act

directly on thermoregulatory controls. Direct actions on

thermoeffectcr pathways can generally be discovered in experiments

carried out in disparate thermal environments. These considerations

underline one obvious conclusion: only through triangulation of

information from experiments using different methods can the

importance of a neuropeptide to thermoregulation be established.

Techniques that are relatively new in this field, such as assay of

peptides in brain samples taken from febrile, hypothermic, and

hyperthermic animals, and immunoneutralization coupled with pyrogen

administration or direct thermal challenge, can provide convincing

evidence about the role of specific peptides in physiological

control of body temperature. There are technical difficulties and

other problems with these approaches, but there is little doubt

about the advantages they provide.

A second major question in research on neuropeptides is whether they

may eventually have some clinical application. In the case of

thermoregulation, as with several other functions, it is too early

to predict. There is considerable hope, however, that applications

will be found once the roles of central neuropeptides are

understood. We speculate that the mechanism of action of

temperature-altering agents such as pyrogens may be explainable in

terms of alterations in central peptides. Naturally occurring

changes in these peptides with aging, metabolic diseases, etc., may

contribute to the increased incidence of dysthermia in certain

populations. Further, the discovery of the temperature-altering

202
effects of peptides may lead to new therapeutic compounds for

antipyresis. for induction of surgical hypothermia, and for

treatment of dysthermias such as heatstroke and accidental

hypothermia.

REFERENCES

Adler. M.W., and Geller. E.B. The opioid svstem and temperature

regulation. Annu Rev Pharmacol Toxicol 28:429-449, 1988.

Adler. M.W.; Geller. E.B.; Rowan, C.H.: and Pressman, N. Profound

reversible hypothermia induced by the interaction of a

kappa-agonist opioid and chlorpromazine. In: Cooper, K.; Lomax,

P.; Schonbaum. E.; and and Veale, W., eds. Homeostasis and Thermal

Stress. Basel: Karger. 1986. pp. 160-162.

Adler, W.; Rowan, C.H.; and Geller, E.B. Intracerebroventricular

vs subcutaneous drug administration: Apples and oranqes?

Neuropeptides 5:73-76, 1984.

Almeida, E.; Sllva, T.C.; and Pela, I.R. Changes in rectal

temperature of the rabbit by intracerebroventricular injection of

brabykinin and related kinins. Agents Actions 8:102-107, 1978.

Anastasl. A.: Erspamer. V.; and Bucci. M. Isolation and amino acid

sequences of alytesin and bombesin, two analogous active

tetrapeptides from the skin of European discoglossid frogs. Arch

Biochem Biophys 148:443-446. 1972.

Anastasi, A.; Erspamer. V.; and Endean, R. Aminoacid composition and

sequence of litorin, a bombesin-like nonapeptide from the skin of

the Australian leptodadactylid frog Litoria aurea. Experientia

31:510-511, 1975.

Avery, D.D., and Calisher, S.B. The effects of injections of

bombesin into the cerebral ventricles on food intake and body

temperature in food deprived rats. Neuropharmacology 21:1059-

1063, 1982.

Banet, M., and Wieland, U. The effect of intraseptally applied

vasopressin on thermoregulation in the rat. Brain Res 14:

113-116, 1985.

Boschi, G.; Nomoto, T.; and Rips, R. Thyrotropin releasing

hormone-induced hyperthermia in mice: Possible involvement of

adrenal and pituitary glands. Br J Pharmacol 80:229-233, 1983.

Breese, G.R.; Cott, J.M.; Cooper, B.R.; Prange, A.J.; Lipton, M.A.;

and Plotnikoff, N.P. Effects of thyrotropin releasing hormone

(TRH) on the actions of pentobarbital and other centrally acting

drugs. J Pharmacol Exp Ther 193:11-22, 1975.

Brown, M.; Ling, N.; and Rivier, J.. Somatostatin-28,

somatostatin-l4 and somatostatin analogs: Effects on

thermoregulation. Brain Res 214:127-135, 1981.

Brown, M.; Rivier, J.; and Vale, W. Bombesin: Potent effects on

thermoregulation in the rat. Science 196:998-1000. 1977.

Brown, M., and Vale, W. Peptides and thermoregulation. In: Cox,B.;

Lomax, P.; Milton, A.S.; and Schonbaum, E., eds. Thermoregulatory

Mechanisms and Their Therapeutic Implications. Basel: Karger,

1980. pp. 186-194.

203
Carraway, R., and Leeman, S.E. The isolation of a new hypotensive

peptide. neurotensin, from bovine hypothalami. J Biol Chem

248:6854-6861,1973.

Chandra, A.; Chou, H.C.; Chang, C.; and Lin, M.T. Effects of

intraventricular administration of neurotensin and somatostatin on

thermoregulation in the rat. Neuropharmacol 20:715-718, 1981.

Chi, M.L., and Lin, M.T. Involvement of adrenergic receptor

mechanisms within hypothalamus in the fever induced by amphetamine

and thyrotropin releasing hormone in the rat. J Neural Transm

58:213-222, 1983.

Clark, W.G. Changes in body temperature after administration of

amino acids, peptides. dopamine. neuroleptics and related agents.

Neurosci Biobehav Rev 3:179-231, 1979.

Clark, W.G., and Lipton, J.M. Brain and pituitary peptides in

thermoregulation. Pharmacol Ther 22:249-297, 1983.

Clark, W.G., and Lipton, J.M. Changes in body temperature after

administration of amino acids, peptides, dopamine, neuroleptics

and related agents-II. Neurosci Biobehav Rev 9:211-216, 1985.

Clark, W.G.; Pang. I.H.; and Bernardini, G.L. Evidence against

involvement of beta endorphin in thermoregulation in the cat.

Pharmacol Biochem Behav 18:741-745, 1983.

Cooper, K.E. The neurobiology of fever: Thoughts on recent

developments. Annu Rev Neurosci 10:297-324, 1987.

Cox, B.; Ary, M.; and Lomax, P. Dopaminergic mechanisms in

withdrawal hypothermia in morphine dependent rats. Sci Life

17:41-42, 1975.

Duranton. A., and Buena, L. Central opiate mechanism involved in

gastrointestinal motor disturbances induced by E. coli endotoxin

in sheep. Life SCi 34:1795-1799, 1984.

Forsling, M.L.; Ingram. D.L.; and Stanier, M.W. Effects of

various ambient temperatures and of heating and cooling the

hypothalamus and cervical spinal cord on antidiuretic hormone

secretion and urinary osmolality in pigs. J Physiol (Lond.)

257:673-686, 1976.

Francesconi. R., and Mager, M. Thermoregulatory effects of

centrally administered bombesin, bradykinin. and Met enkephalln.

Brain Res Bull 7:63-68, 1981.

Fuenmayor, N., and Cubeddu, L. Cardiovascular and endocrine effects

of naloxone compared in normotensive and hypertensive patients.

Eur J Pharmacol 126:189-197, 1986.

Geller, E.B.; Rowan, C.H.; and Adler, M.W. Body temperature effects

of opioids in rats: Intracerebroventricular administration.

Pharmacol Biochem Behav 24:1761-1765, 1986.

Hawkins, M.F., and Avery, D.D. Effects of centrally administered

bombesin and adrenalectomy on behavioral thermoregulation and

locomotor activity. Neuropharmacology 22:1249-1255, 1983.

Hayward, J.N., and Baker, M.A. Diuretic and thermoregulatory

responses to preoptic cooling in the monkey. Am J Physiol

214:843-850, 1968.

Hiramatsu. M.; Nabeshima, T.; and Kamayama, T. Involvement of

opioid receptors in hypo- and hyperthermlc effects induced by

phencyclidine in mice. J Pharmacobiodyn 9:466-472, 1986.

204
Jolicoeur, F.B.; De Michele. G.; Barbeau, A.; and St-Pierre, S.

Neurotensln affects hyperactivity but not stereotype induced

by pre and post synaptic dopaminergic stimulation:. Neurosci

Biobehav Rev 7:385-390, 1983.

Kallvas, P.W.; Jennes, L.; Nemeroff, C.B.; and Prange, A.J.

Neurotensin: Topographical distribution of brain sites

involved in hypothermia and antlnoclception. J Comp Neurol

210:225-238, 1982.

Kandasamy, S.B., and Williams, B.A. Hyperthermic responses to

central injections of some peptide and nonpeptide opioids in

the guinea pig. Neuropharmacology 22:621-628, 1983.

Katovlch, M.J.; Simpklns, J.W.; and O'Meara, J. Effects of opioid

antagonists and their quarternary analogs on temperature changes

in morphine-dependent rats. Life Sci 39:1845-1854, 1986.

Kavaliers. M. Pineal mediation of the thermoregulatory and

behavioral activating effects of beta endorphin. Peptides

3:679-685. 1982.

Kovacs, G.L., and De Wled, D. Hormonally active argininevasopressin suppresses endotoxin-induced fever in rats: Lack of

effect of oxytocin and a behaviorally active vasopressin fragment.

Neuroendocrinology 37:258-261, 1983.

Krieger, D.T., and Martln, J.B. Brain peptides. New Engl J

Med 304:876-885, 1981.

Krupin, T.; Koloms, B.A.; Klutho, L.; Webb, G.; and Becker, B.

Increased intraocular pressure and hyperthermia following

administration of substance P into rabbit third ventricle.

EXp Eye Res 34:319-324, 1982.

Lai, H.; Horita, A.; Chou, C.K.; and Guy, A.W. Effects of low-level

microwave irradiation on amphetamine hyperthermia are blockable by

naloxone and classically conditionable. Psychopharmacol 88:

354-361, 1986.

Lathi, H.; Koskinen. M.; Pyornila, A.; and Hissa, R. Hyperthermia

after intrahypothalamic injections of thyrotropin releasing

hormone (TRH) in the pigeon. Experientia 39:1338-1340, 1983.

Lee, T.F.; Helper, J.R.; and Myers, R.D. Evaluation of

neurotensin's thermolytlc action by icv infusion with receptor

antagonists and a calcium chelator. Pharmacol Biochem Behav

19:477-481, 1983.

Lee, T.F.; Wora, F.; and Myers, R.D. The effect of intracerebroventricular vasopressin on body temperature and endotoxin

fever of macaque monkey. Am J Physiol 248:R674-R678, 1985.

Lee, T.F., and Myers, R.D. Analysis of the thermolytic action of

icv neurotensln in the rat at different ambient temperatures.

Brain Res Bull 10:661-665, 1983.

Lin, M.T.; Chandra, A.; and Jou, J.J. Angiotensin II inhibits both

heat production and heat loss mechanisms in the rat. Can J

Physiol Pharmacol 58:909-914, 1980.

Lipton, J.H., and Glyn-Ballinger, J.R. Central administration of

peptides alters thermoregulation in the rabbit. Peptides 1:15-18,

1980.

205
Lipton, J.M.; Glyn-Ballinger, J.R.; Murphy, M.T.; Zimmer, J.A.;

Bernardlne, G.; and Samson, W.K. The central neuropeptides ACTH

and MSH in fever control. J Therm Biol 9:139-143, 1984.

Lipton, J.M., and Shih, S. Intravenous MSH reduces fever in

squirrel monkeys. Fed Proc 44:1196, 1985.

Lotti, V.J.; Lomax, P.; and George, R. Heat production and heat

loss in the rat following intracerebral and systemic

administration of morphine. Int J Neuropharmacol 5:75-83, 1966.

Luttlnser, D.; Nemeroff, C.B.; Mason, G.A.; Frye, G.D.; Breese,

G.R., and Prange, A.J. Enhancement of ethanol induced sedation

and hypothermia by centrally administered neurotensin, beta

endorphin and bombesin. Neuropharmacology 20:305-309, 1981.

Mason, G.A.; Hernandez, D.E.; Nemeroff, C.B.; Adcock, J.W.;

Hatley, O.L.; and Prange, A.J. Interaction of neurotensin with

prostaglandin E2 and prostaglandln synthesis inhibitors: Effects

on colonlc temperatures in mice. Regulatory Peptides 4:285-292,

1982.

Mason, G.A.; Nemeroff, C.B.; Luttinger, D.; Hatley, O.L.; and

Prange, A.J. Neurotensin and bombesin: Differential effects on

body temperature of mice after intracisternal administration.

Regulatory Peptides 1:53-60, 1980.

Merker, G.; Zeisberger, E.; Blahser, S.; and Kraunig, M.

Immunocytochemical reaction of vasopressin containing neurons

during deve'opment of fever in the guinea pig. NaunynSchmiedeberg's Arch Pharmacol 322(suppl.):R81, 1983.

Metcalf, G. TRH: A possible mediator of thermoregulatlon. Nature

(London) 252:310-311, 1974.

Miller, R.J. Peptides as neurotransmitters: Focus on the

enkephalins and endorphins. Pharmacol Ther 12:73-108, 1981.

Morley, J.E.; Levine, A.S.; Oken, M.M.; Grace, M.; and Kneip, J.

Neuropeptides and thermoregulation: The interactions of

bombesln. neurotensin, TRH, somatostatin, naloxone and

prostaglandlns. Peptides 3:1-6, 1982.

Murphy, M.T., and Lipton, J.M. Peripheral administration of MSH

reduces fever in older and younger rabbits. Peptides 3:775-

779, 1982.

Murphy, M.T., and Lipton, J.M. Beta endorphin: Effect on

thermoregulatlon in aged monkeys. Neurobiol Aging 4:187-190,

1983.

Murphy, M.T.; Richard, D.B.; and Lipton, J.M. Antipyretic potency

of centrally administered alpha melanocyte stimulating hormone.

Science 221:192-193, 1983.

Myers, R.D.; Metcalf, G.; and Rice, J.C. Identification by microinjection of TRH-sensitive sites in the cat's brain stem that

mediate respiratory, temperature,and other autonomic changes.

Brain Res 125:105-115, 1977.

Naylor. A.M.; Ruwe, W.D.; Kohut, A.F.; and Veale, W.L. Perfusion of

vasopressln within the ventral septum of the rabbit suppresses

endotoxin fever. Brain Res Bull 15:209-213, 1986.

206
Nemeroff, C.B.; Blssette, G.; Manberg, P.J.; Osbahr, A.J.;

Breese, R.; and Prange, A.J. Neurotensin-induced hypothermia:

Evidence for an interaction with dopaminergic systems and the

hypothalamic-pituitary-thyroid axis. Brain Res 195:69-84, 1980.

Nemeroff, C.B.; Bissette, G.; Prange, A.J.; Loosen, P.T.; Barlow,

T.S.; and Lipton, M.A. Neurotensin; central nervous system

effects of a hypothalamic peptide. Brain Res 128:485-496, 1977.

Nemeroff, C.B.; Osbahr, A.J.; Manberg, P.J.;Ervin, G.N.; and

Prange, A.J. Alterations in nociception and body temperature

after lntracisternal administration of neurotensin, beta

endorphin, other endogenous peptides, and morphine. Proc

Nat1 Acad Sci USA 76:5368-5371, 1979.

Pae, Y.S.; Lai, H.; and Horita, A. Hyperthermia in the rat from

handling stress blocked by naltrexone injected into the

preoptic-anterior hypothalamus. Pharmacol Biochem Behav

22:337-339, 1985.

Pawlowski, L., and Kwiatek, H. The effect of highly selective

inhibitors of the uptake of noradrenaline or 5-hydroxytryptamlne on TRH-induced hyperthermia in mice. Psychopharmacology 81:48-53, 1983.

Pillai. N.P.. and Ross, D.H. Ethanol-induced hypothermia in rats:

Possible involvement of opiate kappa receptors. Alcohol 3:

249-323, 1986a.

Pillal, N.P.; and Ross, D.H. Opiate receptor mediated hyperthermic

response in rat following Ca++ channel antagonists. Pharmacol

Biochem Behav 25:555-560, 1986b.

Prange, A.J.; Breese, G.R.; Jahnke, G.D.; Martin, B.R.; Cooper,

B.R.; Cott, J.M.; Wilson, I.C.; Alltop, L.B.; Lipton, M.A.;

Bissette, G.; Nemeroff, G.; and Loosen, P.T. Modification of

pentobarbital effects by natural and synthetic polypeptides:

Dissociation of brain and pituitary effects. Life Sci 16:

1907-1913. 1975.

Prange, A.J.; Nemeroff, C.B.; Bissette, G.; Manberg, P.J.; Osbahr,

A.J.; Burnett, G.B.; Loosen, P.T.; and Kraemer, G.W. Neurotensin:

Distribution of hvpothermic response in mammalian and submammalian

vertebrates. Pharmacol Biochem Behav 11:473-477, 1979.

Prasad. C.; Jacobs. J.J.; and Wilber, J.F. Immunological blockade

of endogenous thyrotropln releasing hormone produces hypothermia

in rats. Brain Res 193:580-583, 1980.

Prasad, C.; Matsui, T.; Williams, J.; and Peterkofsky, A. Thermoregulation in rats: Opposing effects of thyrotropln releasing

hormone and its metabollte histidyl-proline diketopiperazine.

Biochem Biophys Res Commun 85:1582-1587, 1978.

Rezvanl, A.H.; Gordon, C.J.; and Heath, J.E. Actions of preoptic

injections of beta endorphin on temperature regulation in

rabbits. Am J Physiol 243:R104-R11, 1982.

Rezvanl, A.H., and Heath, J.E. Reduced thermal sensitivity in the

rabbit by beta endorphin injection into the preoptic/anterior

hypothalamus. Brain Res 292:297-302, 1984.

207
Richards, D.B., and Lipton, J.M. Antipyretic doses of MSH do not

alter afebrile body temperature in the cold. J Therm Biol

9:299-301, 1984.

Rosow, C.E.; Miller, J.M.; Pelkin, E.W.; and Cochin, J. Opiates and

thermoregulation in mice. I. Agonists. J Pharmacol Exp

Ther 213:273-283, 1980.

Ruwe, W.D.; Naylor, A.M.; and Veale, W.L. Perfusion of vasopressin

within the rat brain supresses prostaglandin E hyperthermia.

Brain Res 338:219-224, 1985.

Sakurada, T.; Sakurada. S.; Watanbe, S.; Mastumura, H.; and Kisara,

K. Actions of intracerebroventricular administration of

kyotorphin and an analog on thermoregulation in the mouse.

Peptides 4:859-863, 1983.

Shukla, R.; Srimal, R.C.; and Dhawan, B.N. Centrally mediated

effects of Met-enkephalin and morphine on the body temperature

of Mastomys natalensis. In: Dhawan, B.N., ed. Current Status of

Centrally Acting Peptides. Oxford: Pergamon, 1982. pp. 85-91.

Shukla, R.; Srimal, R.C.; and Dhawan, B.N. Central interaction

between classical neurotransmitters and Met enkephalin on the

core temperature of Mastomys natalensis. In: Dhawan, B.N., and

Rapaka, R.S., eds. Recent Progress in Chemistry and Biology of

Centrally Acting Peptides. Lucknow, India: CDRI, 1988. pp. 201-208.

Stanton, T.L.; Beckman, A.L.; and Winokur, A. Thyrotropinreleasing hormone effects in the central nervous system:

Dependence on arousal state. Science 214:678-681, 1981.

Tartara, A.; Maurelli. M.; and Machioni, E. Autonomic effects of

centrally administered dermorphin in conscious rabbits. Pharmaco

41:214-224, 1986.

Widdowson, P.S.; Griffiths, E.C.; Slater, P.; and Yajima, H.

Effects of mammalian and avian neurotensins and neurotensin

fragments on wet-dog shaking and body temperature in the rat.

Regulatory Peptides 7:357-365. 1983.

Wilkinson, M.F., and Kasting, N.W. Centrally applied vasopressin

is antipyretic due to its effects on febrile set point. Proc

IUPS 30th Congr 16(119.07):60, 1986.

Zeisberger, E.; Merker, G.; and Blahser, S. Fever response in the

guinea pig before and after parturition and its relationship to

the antipyretic reaction of the pregnant sheep. Brain Res

212:379-39, 1980.

Zeisberger. E.; Merker, G.; Blahser, S.; and Krannig, M. Changes in

activity of vasopressin neurons during fever in the guinea

pig. Neurosci Lett (supp1.)14:S414, 1983.

AUTHORS

Rakesh Shukla, Ph.D., Scientist

Bhola N. Dhawan, M.D, F.N.A, F.A.M.S., Director

Central Drug Research Institute

Lucknow 226001, India

208
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

Endogenous Opioids and Immune

Responses: An Experimental

study

P. K. Mediratta, M.D.; N. Das, Ph.D.; V. S. Gupta, B.Sc.;

and P. Sen, M.D., Ph.D.

ABSTRACT

Possible involvementofendogenousopioids inhumoral immune responses

has been explored in the experimental animals. Opioid agonists like

morphine and leu-enkephalin significantly enhanced antigen-induced

histamine release from the peritoneal mast cells of sensitised rats in

vitro; this was effectively antagonised by naloxone. Naloxone itself

inhibited antigen-induced histamine release. Animals were effectively

protected against anaphylactic shock by naloxone which also antagonised

morphine-induced increase in anaphylactic mortality. Naloxone reduced

haemagglutination titre to sheep red blood cells and IgE antibody titre

as measured by passive cutaneous anaphylaxis. Thus, endogenous opioids

appear to be involved in the mediation of humoral immune responses.

They seem to act at various steps in the immune mechanism viz (i)

antibody production and (ii) release of mediators of hypersensitivity

reactions.

INTRODUCTION

In the cities we live in, we are surrounded all over by a polluted

atmosphere containing billions of heterogenous substances which are

foreign to our system. Although most of us can withstand an onslaught by

these agents, a considerable number of individuals respond violently

and develop various types of immunological disorders. It is, however,

not known why only some and not all persons respond violently to these

offending substances. It has been reported that many psychosocial

situations and stressful conditions can modify host resistance to a

variety of illnesses including allergic and infectious disorders and

influence the immune processes of an individual (Solomon and Amkraut

1981). Hypothalamus is known to be a centre for correlation and coordination of the autonomic and endocrinal activities with the

emotions of an individual. Stein et al. (1976) observed that in

sensitised animals lesion of the anterior hypothalamus produced

significant protection against anaphylaxis, lowered the titre of

the circulating antibodies, and also depressed the delayed type of

209
hypersensitivity reactions. Hypothalamus is rich in neurohormones

and neurotransmitters, which may affect immune functions. Opioid

peptides are one such group of hypothalamic neuropeptides. A number of reports indicate that administration of opioids or opioid

antagonists can modulate the immune competence (Lancet 1984).

Hence, it was considered worthwhile to explore the possible role

of endogenous opioids in immune responses.

MATERIALS AND METHODS

The experiments were carried out mainly on Wistar rats (100-150 gms)

and Swiss mice (20-30 gms) of either sex. They were obtained from the

local source and fed on Hind Lever Laboratory animal feed with water

available ad lib. Animals were sensitised using egg albumin (EA) or

sheep red blood cells (SRBC) as antigens. Freund's complete adjuvant

and B. pertussis were used as adjuvants. The results were statistically

analysed by Chi2 test or t test as appropriate.

Acute Systemic Anaphylaxis In Mice

Four groups of mice (15-25 in each group) were sensitised by injecting

subcutaneously 0.2ml EA(25mg/ml) along with 0.5ml of triple antigen

(containing 20,000x 10°B. pertussis organisms) and 0.2ml of Freund's

complete adjuvant. On the 12th day following sensitisation, control

group received 0.2 ml of normal saline; one group was given naloxone

10mg/kgi.p., second group receivedmorphine in a dose of 10mg/kg i.p

and third group was administered naloxone (10mg/kg) along with

morphine (10mg/kg). Thirty minutes after the drug pretreatments all

the groups were challenged with 0.2 ml EA i.v., and mortality was

recorded after two hours.

Histamine Release From The Peritoneal Mast Cells Of Sensitised Rats In

Vitro

Histamine release from the peritoneal mast cells was studied by the

method of Chakravarty et al. (1973). Rats were sensitised by injecting

subcutaneously 0.5 ml (25 mg/ml) along with 0.5 ml triple antigen

(containing 20,000 x 10°B. pertussis organisms) and 0.5 ml Freund's

complete adjuvant. Rats were sacrificed by exsanguination on the 14th

day; peritoneal cell suspensions were prepared and divided into 10-12

samples. Cells were usually pooled from two sensitised rats. Samples

were treated with various drugs under investigation (opioid agonists

and antagonist) and incubated at 37°C under gentle shaking for 10

minutes. The antigen (0.5 mg/ml EA) was added to the cell suspension

and the incubation, thereafter, continued for another ten minutes. The

reaction was terminated by placing the samples in ice cold water.

Adequate controls using agonists or antagonist alone were included.

Histamine content of different samples was estimated by biological

assay using isolated guinea pig ileum. Control histamine release as

well as spontaneous release values were detected from the results.

210
Haemagglutination Titre To Sheep Red Blood Cells (SRBC)

Twogroups of 6 mice were immunized by injecting 1 x 109SRBC i.p. One

group was given naloxone 5 mg/kg twice daily from day 1 to 5 and the

control group received normal saline in the same schedule. Animalswere

bled on the 8th day and sera separated. Two fold dilutins (0.025ml) of

the seraweremade inthemicrotitre plates with normal saline. To each

well 0.025 ml of 1% (V/V) SRBC was added. The plates were incubatedat

37°C for one hour and then observed for haemagglutination.

Passive Cutaneous Anaphylaxis

This was studied in two groups of 6 rats sensitised with 0.5 ml EA

(25mg/ml) with 0.5 ml triple antigen (containing 20,000 x 106B.

pertussis organisms) and 0.5 ml of Freund's complete adjuvant

subcutaneously. The test group received naloxone 5 mg/kg twice daily

from day 1 to 5 while the control group received normal saline. Animals

were bled on the 6th day and sera collected. Serial dilutions of the

sera were made,and O.1ml of different dilutions was injected into the

shaven dorsal skin of normal rats. 72 hours later i.v. challenge was

given with EA (0.5ml) and Evan's blue (1%,0.2ml) and blueing reaction

at test site was observed after one hour.

RESULTS

Effect On Acute Systemic Anaphylaxis

Pretreatment with naloxone (1Omg/kg) significantly protected animals

against anaphylactic shock; mortality was reduced from 56% in the

control group to 32% in the naloxone pretreated group (p<0.01).

Morphine pretreatment (10mg/kg) enhanced anaphylactic mortality which

was effectively antagonised by naloxone (p<O.001; Table 1).

Effect On Antigen Induced Histamine Release

Opioid agonists like morphine (100 μM) and leu-enkephalin (10 μM)

significantly enhanced antigen induced histamine release from the

peritoneal mast cells of sensitised rats in vitro; this enhancement was

effectively antagonised by naloxone (Table 2(i) and 2(ii)). Naloxone

(1 μM) pretreatment itself significantly inhibited antigen induced

histamine release (p<0.01, Table 3).

Effect On Haemagglutination Titre To SRBC

In the control group in all the 6 i.e., 100% animals the detectable

level of haemagglutination titre was between 1/128 and 1/2048.

Pretreatment with naloxone reduced the detectable level of

haemagglutination titre to 1/2 - 1/16 in all the 6(100%) animals (Table

4).

211
TABLE 1. Effect of Naloxone and Morphine on Acute Systemic

Anaphylactic Mortality in Mice

Group No. of Senitization (Day 12) %

Animals (Day 0) Drug Pre- Chal- Mortality P

treatment lenge

1 25 EA+DPT+FA Normal Saline EA 56.00

2 25 EA+DPT+FA Naloxone EA 32.00 <0.01

10mg/kg I.P.

3 23 EA+DPT+FA Morphine EA 78.26

10mg/kg I.P.

<0.01

4 15 EA+DPT+FA Morphine

10mg/kg I.P.

EA 33.33

Naloxone

<0.001

10mg/kg I. P.

EA- Egg albumin 0.2ml(25mg/ml)

DPT- Triple antigen 0.5 ml (containing 20,000,X 106B. pertussis

organisms)

FA- Freund's complete adjuvant 0.2 ml

TABLE 2(i) Effect of Morphine and its Modification by Naloxone on

the Antigen Induced Histamine Release from Peritoneal

Mast Cells of Sensitized Rats in Vitro

Expt. % Histamine Release

No. Egg Albumin (EA) EA+Morphine

(0.5mg/ml) (100 μM)

EA+Morphine+Naloxone

(1 μM)

A B C

1. 16.0 31.6 4.4

2. 10.5 27.7 12.4

3. 17.4 32.4 7.3

4. 31.7 39.4 21.7

The differences between columns A and B (p<0.01) and B and C

(p<0.01 are significant by t test for paired comparison.

212
TABLE 2(ii) Effect of Leu-enkephalin and its Modification by Naloxone

on the Antigen Induced Histamine Release from Peritoneal

Mast Cells of Sensitized Rats in Vitro

Expt. % Histamine Release

No. Egg Albumin(EA) EA+Leu-enkephalin EA+Leu-enkephalin+

(0.5mg/ml) (10 μM) Naloxone (1 μM)

A B C

1. 17.4 34.1 5.5

2. 10.5 24.0 12.0

3. 16.0 34.0 4.0

4. 31.7 49.6 6.7

The differences between columns A and B (p<0.001) and B and C

(p<O.05) are significant by t test for paired comparison.

TABLE 3. Effect of Naloxone on the Antigen Induced Histamine

Release from the Peritoneal Mast Cells of Sensitized Rats

in Vitro

Expt. % Histamine Release

No. Egg Albumin (EA) EA+Naloxone

(0.5 mg/ml) (1 μM)

A B

1. 11.9 4.3

2. 33.3 3.7

3. 16.0 5.0

4. 10.5 0.5

5. 17.4 6.7

6. 31.7 10.6

The difference between columns A and B is significant (p<0.01) by

t test for paired comparison.

213
Table 4. Effect of Naloxone on Haemagglutination Titre to Sheep Red

Blood Cells (SRBC) in Mice

Group Pretreatment No. of Antibody Detectable Level of HaeAnimals Not Det- magglutination titre

ectable 1/2-1/64 1/128-1/2048

1. SRBC 6 0 0 6

(1x109I.P.) (100%)

+

Normal Saline

(from day 1 to 5)

2. SRBC

+

Naloxone 6 0 6 0

(5mg/kg BD- (100%)

from day 1 to 5

Animals wre bled on 8th day after the sensitization with SRBC.

Effect on Passive Cutaneous Anaphylaxis

The detectable level of IgE antibody in the control animals was between

1/1 - 1/10 in 33.33% and up to 1/100 in 66.67% of the animals. In the

naloxone treated group, on the other hand, in 50% of the animals the

antibodies could not be detected, while in 16.67% cases it ranged

between 1/1 - 1/10 and up to 1/100 in 33.33% cases (Table 5).

Discussion

Hypothalamus and other areas of the brain have several

neurotransmitters including endogenous opioids. Recently it has been

reported that hypothalamus is activated by the immune system which by a

feed back mechanism may modulate the immune responses (Besedovsky and

Sorkin 1977; Stein et al. 1976). Opioid agonists and antagonists have

been shown to modulate the immune competence (Lancet 1984). An agent

may affect the immune system by acting atone or more than one step in

the immune mechanism. Our experimental studies were confined to

finding out the roleofendogenous opioids in humoral immune responses

in albino rats and mice using egg albumin and sheep red blood cells as

antigens and Freund's complete adjuvant and B.pertussisas adjuvants.

In the initial experiments, role of opioids in acute systemic

anaphylaxis was studied. Naloxone, an opioid antagonist when given in

a dose of 10mg/kg i.p., significantly protected animals against

anaphylactic shock. This corroborates the findings of Amir (1982) who

showed that administration of naloxone and naltrexone provided a

214
TABLE 5. Effect of Naloxone on Passive Cutaneous Anaphylaxis in

Rats

Group Pretreatment No. of Antibody Detectable Level of IgE

Animals NOt Det- Antibody

ectable 1/1-1/10 1/100

1. EA+DPT+FA 6 0 2 6

+ (33.33%) (66.67%)

Normal Saline

(from day 1 to 5)

2. EA+DPT+FA

+

Naloxone 6 3 1

(mg/kg BD2

from day 1 to 5)

(50%) (16.67%) (33.33%)

EA- Egg albumin 0.5 ml (25mg/ml)

DPT- Triple antigen 0.5 ml (containing 20,000 x 106

B. pertussis

organisms)

FA- Freud's complete adjuvant 0.5 ml

significant protection against anaphylactic shock. Morphine, an

opioid agonist, however, enhanced anaphylactic mortality which was

effectively antagonised by naloxone (Table 1). The present findings

that apparent blockade of opioid receptors protects against

anaphylactic shock suggest a role for endogenous opioids in the

pathophysiology of systemic anaphylaxis, a condition associated with

antigen antibody interaction.

To further elucidate the involvement of endogenous opioids in immune

mechanisms, experiments were done to study the effect of opioids on

histamine release, the latter being an important mediator of antigenantibody reaction. Opioid agonists like morphine (a μ receptor

agonist) and leu-enkephalin (a delta receptor agonist) produced a

significantincreaseintheantigen induced histamine release from the

peritoneal mast cells of sensitised rats in vitro. This increase was

effectivelyantagonised bynaloxone (Table 2(i) and 2(ii)) suggesting

thereby that the potentiating effect of opioid agonists on antigen

induced histamine release is mediated by opioid receptors and both μ

and delta receptors appear to be involved. Involvement of μ and delta

opioid receptors has also been reported for IgE mediated serotonin

release from rat mast cells (Yamasaki et al. 1982). Naloxone 'perse'

also significantly inhibited antigen induced histamine release from

peritoneal mast cells of sensitised rats in vitro (Table 3) indicating

a role of endogenous opioids in the mediation of antigen induced

histamine release. The protective effect of naloxone against

anaphylactic shock observed in the present study (Table 1) could be

because of its inhibitory effect on the mediator release.

215
Experiments were also done to find out the role of endogenous opioids on

antibody production. Naloxone administration effectively reduced

haemagglutination titre to SRBC in mice (Table 4), suggesting an effect

on IgG and/IgM antibodies. Naloxone pretreatment also suppressed IgE

antibody production as measured by passive cutaneous anaphylaxis in

the present study. In the naloxone treated group antibody production

was completely inhibited in 50% of the animals (Table 5). Thus, it is

observed that apparent blockade of opioid receptors by naloxone

suppressed formation of anti SRBC and IgE antibodies,i.e.,endogenous

opioids, appear to be involved in the synthesis of these

immunoglobulins.

In conclusion, the present findings suggest that endogenous opioids

appear to be involved in the mediation of humoral immune responses.

They seem to act at various steps in the immune mechanism viz(i)

antibody production and (ii) release of mediators of hypersensitivity

reactions.

REFERENCES

Amir, S. Opiate antagonists improve survival in anaphylactic

shock. Eur J Pharmacol 80:161-162, 1982.

Besedovsky, F.O., and Sorkin, E. Network of immune-neuroendocrine

interactions. Clin Exp Immunol 27:1-12, 1977.

Chakravarty, N.; Goth, A.; and Sen, P. Potentiation of dextraninduced histamine release from rat mast cells by phophotidyl

serine. Acta Physiol Stand 88:469-480, 1973.

Opiates, opioid peptides and immunity. Lancet 1:774-775, 1984.

Solomon, G.F., and Amkraut, A.A. Psychoneuroendocrinological

effects on the immune response. Annu Rev Microbiol 35:155-

184, 1981.

Stein, M.; Schiavi, R.C.; and Camerino, M. Influence of brain

and behaviour on the immune system. Science 191:435-440, 1976.

Yamasaki, Y.; Shimamura, 0.; Kizu, A.; Nakagawa, M.; and Ijichi,H.

IgE-mediated C-serotonin release from rat mast cells modulated by morphine and endorphins. Life Sci 31:471-478, 1982.

AUTHORS

P. K. Mediratta, M.D.

N. Das, Ph.D.

V. S. Gupta, B.Sc.

P. Sen, M.D., Ph.D.

Department of Pharmacology

University College of Medical Sciences

New Delhi - 110 029, India

216
Opioid Peptides: An Update: NIDA Research Monograph 87

Rao S. Rapaka, Ph.D., and Bhola N. Dhawan, M.D., eds.

National Institute on Drug Abuse, 1988

An Update of Selected Topics in

the Biology and Chemistry of

Opioid Peptides

Rao S. Rapaka, Ph.D.; Bhola N. Dhawan, M.D.; and

V. Renugopalakrishnan, Ph.D.

The discovery of enkephalins by Kosterlitz and colleagues (Hughes

et al. 1975) was immediately followed by an era of synthesis of a

large number of opioid peptides. At present, a great deal of

attention is being paid to conformational analysis, receptor

biochemistry, mechanism(s) of action, addiction mechanisms, and

physiological actions of opioids. The chemistry and biology of

opioids have been extensively reviewed (Udenfriend and Meienhofer

1984; Rapaka 1986; Rapaka and Hawks 1986; Rapaka et al. 1986; Olson

et al. 1987; Dhawan and Rapaka 1988). This article is intended to

focus on selected topics only and is not a thorough review. More

detailed information is available from the above reviews. In this

article, the importance of using pure peptides and the need for

extensive characterization of these peptides is emphasized. In

addition, some of the recent advances in synthetic methodology,

rationales for some of the newly synthesized

specific ligands, conformational properties of opioids, the role of

lipids in conformational induction of llgands, and a few selected

biological aspects (such as immunomodulation by opioids) are

briefly discussed.

Numerous opioid peptides have recently been synthesized and

evaluated for various biological activities. In order to obtain

dependable data, a pure compound is essential and purity of the

compound cannot be overemphasized. Much more rigorous analytical

testing should be undertaken for commercial samples and for those

compounds that are synthesized by solid phase peptide synthesis.

As there is no way of establishing absolute purity, as many

criteria as possible should be employed to establish the purity of

the product. If the peptide is synthesized in-house, the material

is likely to be checked for purity at each stage and the end

product is therefore likely to be pure. Due to the ready

availability of new products from several commercial suppliers, and

the facilities for custom synthesis, a large number of studies now

utilize commercial samples. Recently. Brown et al. (1986) reported

on the problems associated with commercial peptides. Samples of

the tachykinin-related peptides were analyzed from several

commercial sources. Some of the problems encountered were

217
these: (1) peptides had incorrect sequences; peptides might have an

incorrect residue, or material supplied might be a mixture of the

peptides; (2:) peptide content varied as much as three- to sixfold;

(3) for radiolabeled compounds, radioactivity might not be

associated with the authentic substance. These findings indicate

that results obtained with these compounds will make the data

worthless and adequate quality control checks must be made for

these compounds prior to their use. Amino acid analysis,

sequencing data, TLC data in several systems, HPLC analysis in at

least two solvent systems, elemental analysis, and any other

pertinent physicochemical data should be obtained prior to their

use. In addition, optical purity and radiochemical purity (in the

case of using labeled products) of the peptides is also critical.

A measure of optical rotation alone will not guarantee an optically

pure product. Enzyme digestions (Rapaka et al. 1976, 1977) to

establish the optical purity, along with utilization of synthetic

methods that yield optically pure products, might be helpful.

Radiochemical purity of the labeled product should be determined,

and since some of the labeled products are unstable, chemical

purity should also be determined prior to use.

Batch-to-batch variation should always be monitored. This problem

may be much more important with solid phase peptide synthesis than

with synthetic material obtained from conventional methods. All

batches should be compared with a reference standard batch.

Recently, Taylor et al. (1986) reported that anomalous binding of

DPDPE resulted due to batch variability, and the results from the

impure batch indicated a two receptor model instead of a single

receptor model. HPLC purification of the material yielded

chromatographically pure compound and gave the predicted results.

Hence, even a minor component can potentially lead to erroneous

conclusions.

The chemical complexity of peptides makes the analysis of peptides

very difficult and demands utilization of innovative, extremely

specific, and sensitive methods. In spite of the recent advances

(Desiderio et al. 1986; Fridland and Desiderio 1987; Sternson 1987)

in analytical techniques for the analysis of peptides, adequate

methodology for the analysis of extremely small amounts of opioid

peptides, their precursors and metabolites, is not currently

available. FAB-MS and LC analysis is increasingly being utilized

successfully in the area of peptide analysis (Canova-Davis et al.

1988).

The developmert of solid phase peptide synthesis (SPPS) was

instrumental in providing a large number of peptides for

structure-activity studies. Following the development of solid

phase peptide synthesis by Merrifield (1963), many other methods

have been explored to simplify and to automate the synthesis. None

of them have really been widely accepted. Recently, Houghten and

colleagues have reported a modified solid phase method that is

simple (Houghten 1985; Houghten et al. 1986a,b. 1988). This method

permits simultaneous synthesis of up to 100 or more peptides

218
of up to 15 residues in length. The synthesis is fast, and these

analogs can be synthesized within a month. No special equipment is

needed, and the procedure is not labor-intensive. This method is

referred to as the simultaneous multiple peptide synthesis (SMPS),

and it involves placing the solid phase resin in a porous

polypropylene packet. Several such packets (up to 100 or so)

containing the protected amino acid resin can be deprotected

together in a Nalgene bottle. Coupling steps are performed in

different containers. After the coupling step, the resin packets

are returned to the common reaction bottle and the synthesis is

continued. At the end of the desired synthesis, the peptide can be

cleaved utilizing an apparatus that permits simultaneous cleavage

of several samples (up to 24). After the peptides are liberated

from the resin, they are purified by routine methods. Thus SMPS

permits the synthesis of a large number of peptides with reduced

time and cost, and it appears to be suitable for the synthesis and

evaluation of a large number of analogs for structure-activity

studies.

Another interesting approach to obtain complex and difficult-tosynthesize peptides for structure-activity studies is through the

expression of synthetic genes (for a review, see Taylor, this

volume). Standard methods of peptide synthesis, either

conventional or solid phase methods, are too cumbersome for the

preparation of large numbers of peptides, their deletion analogs,

and analogs incorporating natural and unnatural amino acid

residues. The approach of utilizing synthetic genes and

recombinant DNA technology is an attractive alternative to

conventional peptide synthesis. This involves synthesis of the

desired oligonucleotide sequence and preparation of mutant gene

coding for the desired analogs followed by expression. Mutagenesis

of the synthetic gene code of the natural structure is the critical

step in the generation of the analogs. Mutagenesis may be induced

in either a random or a more controlled fashion. Chemicals such as

hydrazine, nitrous acid, and sodium bisulfite have been used, as

well as physical methods, such as UV irradiation. Other methods

include purposeful introduction of one or more restriction enzyme

recognition sites to achieve chosen mutations and oligonucleotide

directed mutagenesis. Oligotide directed mutagenesis appears to be

the most commonly used, and the phosphorothiate DNA method for

mutagenesis is one of the most efficient methods to achieve an

oligonucleotide directed mutagenesis. Methodologies for the

expression of the synthetic gene and purification of the product

have been reviewed extensively (see Marston 1986).

Numerous selective opioid analogs have been

synthesized, and several reviews have already appeared (Morley

1980; Hansen and Morgan 1984; Morley and Dutta 1986; Udenfriend and

Meienhofer 1984). Here, some of the recently synthesized analogs

are discussed briefly. One of the usual approaches was

"conformational restriction" by cyclization. Synthetic analogs,

H-Tyr-Cyclo [-D-A2bu-Gly-Phe-Leu-] (DiMaio and Schiller 1980;

DiMaio et al. 1982), homologs containing amino acid residues of

219
varying chain lengths in position 2 (Schiller et al. 1985; Schiller

1986), and related retro-inverso homologs (Berman et alN 1983) were

μ-selective compounds. Later, a cyclic tetrapeptide

H-Tyr-D-Orn-Phe-Asp-NH2

was designed by Schiller et al. (1985). and pharmacological

characterization revealed that it was one of the most μ-selective

compounds.

Another approach to the development of P-receptor specific

peptides was utilized by Hruby and colleagues (1988). In this

approach, a synthetic analog related to the somatostatin fragment,

D-Phe-Cys-Phs-D-Trp-Lys-Thr-Cys-Thr(ol), which interacts weakly

with opioid receptors as well, was utilized as a template to the

development of opioid ligands of high receptor affinity. Following

a detailed structure-activity study, a number of μ-receptor

selective analogs, such as

H-D--Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP),

were developed. Some of these analogs exhibited decreased affinity

for somatostatin binding and a high specificity. This

template was further extended to analogs containing

tetrahydroisoquinoline moieties. This represents a successful

manipulation for modulation of one major physiological activity

into another activity. Conformational studies on these analogs

might shed light on conformation-receptor selectivity

relationships.

Recently, novel μ-selective analogs with antagonist activity were

reported by Judd et al. (1987). The analogs were obtained by

modifications of the Tyrl residues of D-Ala2-Met5-

enkephalinamide, and the modified tyrosine residues were m-Tyr,

ß-methyl-m-Tyr, , ß-dimethyl-Tyr and N-phenethyl-m-Tyr.

Biological evaluation of these analogs revealed that (1) shifting

of phenolic-OH from the ortho to meta position enhanced relative

antagonist activity, (2) addition of ß-CH3 to m-Tyr enhanced

P-selectivity. (3) analogs with P-selectivity had a common

energy access ble 2-3 B 11' turn, (4) , ß-dimethyl-m-OH,

Tyr1-analog had a low affinity and an N-phenylethyl analog was a

pure antagonist. A rationale for design of peptides based on

structural comparison to an opiate compound [3-(m-OH

phenyl)-piperidine] was presented.

One of the earliest successful syntheses of receptor-selective

ligands was that of a s-receptor specific compound, D-Pen2,

D-Pen5-enkephalin. Two other examples of -selective analogs

are linear herapeptides, DTLET and DSLET. and these analogs have

been extensively used as -receptor probes. Recently, Roques and

colleagues reported the synthesis of analogs of

220
H-Tyr-Ser-Gly-Phe-Leu-Thr-OH and H-Tyr-Thr-Gly-Phe-Leu-Thr-OH

(Delay-Goyet et al. 1988). The three analogs reported are

[D-Ser2(0-t-butyl), Leu5] enkephalinyl-Thr6, DSTBULET;

[D-Ser2(O-t-butyl), Leu5] enkephalinyl-Thr6 (o-t-butl),

BUBU; and [D-Thr2(0-t-butyl), Leu5]-enkephalinyl-Thr6

DTTBULET. DSTBULET displays a higher affinity for rat brain

membrane preparation than DSLET and [D-Pen2, Pen5] enkephalin.

DSTBULET has a high specific binding, and it is resistant to

degradation by peptidases present in the crude rat brain membrane

preparation (a t1/2 of 1 hr, compared to t 1/2 of 10 min for

Met-enkephalin). BUBU is an even more selective -receptor

ligand. However, DTTBULET is an inactive ligand. These ligands

are apparently influenced by the large conformational changes

induced by the bulky t-butyl groups. It was suggested that these

analogs are better probes, as the other two -selective analogs,

DPDPE and DPLPE, although highly -receptor specific, have

variable affinity depending on the preparation, toxic effects are

exhibited at higher concentrations, and more important, have

comparatively lower affinity for the receptor site.

Although a large number of μ- and -selective peptides have

already been reported, very few -receptor selective peptide

ligands have been reported to date. Recently, Schiller et al.

(1988) reported conformationally restricted dynorphin analogs such

as [D-Orn2 Asp5]-dynorphin A-(1-8). However, these peptides

are no long -receptor selective, but μ-receptor selective.

This led the authors to hypothesize that these cyclizations

resulted in overall folded conformations, which are incompatible

with the conformational requirements of the -receptor.

Conformational aspects of the peptides are being actively

investigated to help design potent and receptor specific compounds,

and to understand conformation-receptor selectivity relationships.

1

H NMR studies were performed on a number of -selective

penicillamine-containing enkephalin analogs by Mosberg and

colleagues (Mosberg 1987; Mosberg et al. 1987). Discrepancies of

conformation-dependent NMR parameters between DPDPE and DPLPE

(which have similar -selectivities) were observed. It appears

that the Gly residue is flexible and as such plays a crucial role,

altering its local conformation to allow similar spatial

relationships between key groups in different analogs leading to

-affinity. From NMR data, conformational features of DPDPE and

DCDPE appear very similar. However, DPDPE is much more

-selective and less potent as compared with DCDPE. This

suggests that gem-dimethyl groups of Pen2 in DPDPE lead to

adverse reaction. The adverse steric reaction may be much greater

at μ-sites than at -receptor sites. Studies on the ring

expanded analogs of DPDPE and DPLDPE revealed that increasing the

ring size led to a reduction in -receptor selectivity and that

expanded conformations favored μ-receptor binding, whereas

compact conformations are favorable for -receptor binding.

Analogs containing bulky t-Leu residue(s) revealed that bulky

substituents at residue 2 decrease affinity to -receptors and

even more so to μ-receptors.

221
In a related study by Belleney et al. (1987), conformational

properties of DTLET, (H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH) and

D-Pen2, L-Pen5-enkephalin (DPLPE) were determined by 1H NMR

spectroscopy. The results, surprisingly, indicated similarities in

the overall structure for the two-folded backbone conformations for

the constrained DPLPE and the flexible peptide DTLET. Relative

orientations of the Tyr and Phe residues were found to be similar.

Interestingly, the Tyr-Phe distance was 10 A° for both the

peptides, which reflects a quasilinear backbone orientation. This

extended conformation may be associated with -receptor

selectivity. From these studies it was hypothesized that

incorporation of sterically bulky residues at positions 2 and/or 6

in DSLET and DTLET would reinforce structural analogy to DPDPE-like

peptides, thus leading to inhibition of μreceptor binding.

However, this should still permit -receptor affinity due to the

inherent flexibility of the linear hexapeptide. The above

hypothesized analogs have been recently synthesized and were shown

to have high affinity as well as high selectivity for the

s-receptor (Delay-Goyet et al. 1988). In spite of a large number

of studies, conformation-receptor selectivity relationships are not

adequately understood.

The role of the lipid phase of the membrane for the facilitation of

ligand-receptor interaction has been addressed earlier (Schwyzer et

al. 1982; Gysin and Schwyzer 1983; Erne et al. 1985; Sargent and

Schwyzer 1986; Schwyzer 1986). Erne et al. (1985) demonstrated

that interaction of dynorphln A-(1-13) with the lipid membrane

results in the formation of an -helix at the N-terminal (from

1-9, and helix formation beyond the 9th residue is inhibited by Pro

10). The N-terminal message portion is perpendicularly inserted

into the hydrophilic membrane. Recently conformational features of

dynorphin A-(1-13) and interaction of dynorphin A-(1-13) with

lipids were investigated by Renugopalakrishnan and colleagues

(Rapaka et al. 1987; Huang et al. 1988; Renugopalakrishnan et al.

1988a,b; Renugopalakrishnan and Rapaka 1988) utilizing NMR. CD,

FT-IR and Raman spectroscopy. These studies indicated that in

aqueous solution dynorphin has an open structure, and the segment

Arg6-Arg7-Ile-Argg- manifests considerable side chain

mobility on the NMR time scale and N-terminal segment may be

disordered. In the presence of lipid, a steep increase in molar

ellipticity value starting at 240nm indicated a large

conformational change. This is consistent with the observations of

Erne et al. (1985). Recently, Schwyzer (1986) estimated the

conformation, orientation, and accumulation of dynorphin A-(1-13)

on the surface of lipid membrane. Schwyzer used -helical

conformation of dynorphln as the most stable conformation

interacting with the hydrophilic phase. He calculated the Gibbs

free energy, G for the transfer of increasing number, m, of

n-terminal residues from "random" coil conformation in water to

-helical conformation in the hydrophobic phase to predict the

preferred conformation of dynorphin and its orientation and

accumulation on the surface of neutral lipid membrane. A model of

dynorphin was derived in which the hydrophobic segments of the

222
lipid were in contact with the a-helical segment and the "random"

coil segment was protruding into the aqueous phase. This ingenious

study presents a plausible explanation of the events leading to the

observed interaction of dynorphln A-(1-13).

The role of neuropeptldes in general, and of oplold peptides in

particular, is being increasingly studied in the regulation of the

immune system, as the neuropeptides appear to be agents of

communication between the immune system and the brain (for reviews,

see Morley et al. 1987; Plotnikoff et al. 1986a). ACTH suppresses

-interferon production (Johnson et al. 1984). VIP inhibits the

egress of small lymphocytes (Moore 1984). substance P is

chemotactic and cross-reacts with the chemotactic receptor of

rabbit neutrophlls (Marasco et al. 1981; Ruff et al. 1985; Payan et

al. 1983), and somatostatln inhibits the proliferation of

T-lymphocytes (Payan et al. 1984). Thus, the studies to date

indicate that the neuropeptides may play an important role in

immunomodulation and that such modulation may involve the central

nervous system, the endocrine system, and the autonomic nervous

system (Plotnikoff et al. 1985; Morley et al. 1987; Wybran 1985;

Donohoe et al. 1985; Blalock and Smith 1985).

Two interesting and independent observations, one by Wybran et al.

(1979) demonstrating the presence of morphine-like and

Met5-enkephalin-like receptors on blood lymphocytes, and the

other by Hazum et al. (1979) demonstrating the presence of

ß-endorphln receptors on lymphoblastold cells, generated a great

deal of interest on the role of oplold peptldes in

immunoregulation. Human blood T cells have receptors for morphine

and enkephallns, whereas B cells have receptors for ß-endorphin.

and T and B cell involvement may be a critical factor in generating

an immune response. For researchers in the drug abuse area, study

of the role of morphine and other opiates might yield clues to the

higher Incidence of bacterial and viral infections in opiate

addicts. It has been shown that morphine decreases the abillty to

eradicate infections (Tubaro et al. 1983; for a minireview, see

Yahya and Watson 1987). Morphine injections into lateral

ventricles suppress natural klller cell activity (Shavit et al.

1986), and heroin addicts have decreased T-lymphocyte activity

(Singh et al. 1980), thus suggesting that prolonged abuse of

opiates impairs the immune system. On the other hand, enkephallns

have been shown to prolong the survival time of tumor-bearing mice,

enhance lymphocyte blastogenesis in mice, Increase size of thymus

in mice. and, in humans, to show significant increase in active T

cell rosettes and increase natural killer (NK) cell activity

(Plotnikoff et al. 1985). Similarly, ß-endorphin enhances NK

activity and active T cell rosettes. The enhancement of NK cell

activity is important for defense against bacterial and viral

infections. Here it is interesting to note that the NK cell

stimulating ablllty appears to be associated with the N-terminal

sequence of ß-endorphin (Kay et al. 1983). In addition, both

Met5-enkephalln and ß-endorphin enhance interferon production

(Mandler et al. 1986).

223
Morphine depresses T cell E-rosetting (Donohoe et al. 1985), and it

was shown by Tubaro et al. (1983) that polymorphonuclear cell

activity for ohagocytosis and superoxide radical production by

polymorphonuclear cells are depressed. In contrast, ß-endorphin

and dynorphin enhance superoxide radical formation (Sharp et al.

1985). It is very interesting that the opiates and opioid peptides

act similarly at the analgesic receptors, where they appear to

mediate diffe-ent immunologic responses. It is worthwhile that a

large number of opiates and oploid peptide analogs be tested for

their actions on the immune system and to develop SAR relationships

that will lead to the design of therapeutically useful peptide

drugs. This is particularly interesting in terms of initial

encouraging results obtained with methionine enkephalin in the

treatment of AIDS patients (Plotnikoff et al. 1986b). It appears

that there is an exciting possibility of obtaining new leads for

management of this condition from centrally acting peptides.

Awareness of the important role opioid peptides may be playing in

regulation/modulation of physiological activities/behavior has been

increasing. There also appears to be a close interaction between

the enkephalirs and other naturally occurring peptides and other

putative neurctransmitters like dopamine (Radhakishun et al. 1988)

and 5-HT (Foster and Roberts 1988). It should be rewarding to

study the interaction with other neurotransmitters, particularly

the biogenic amines, in greater detail. Among the central

autonomic activities possibly regulated by the opioid peptides, the

effects on cardiovascular regulation (Said 1987) and

thermoregulation (Shukla and Dhawan, this volume) appear to be

particularly significant. The role in reaction to stress has been

extensively studied, the initial studies having been undertaken

with morphine. The recent work has been reviewed by Olson et al.

(1987).

There is evidence that some of the central effects of clonidine and

central antihypertensives like centhaquin may be mediated via the

opioid receptors (Srimal et al. 1988). Phencyclidine may be

exerting some of its effects Via μ- or -receptors,

particularly its effects on body temperature (Hiramatsu et al.

1986).

In the past few years, considerable data have been generated on the

role of opioid peptides in the control of eating, which is

stimulated by opioid agonists (Majeed et al. 1986) in several

species (Kavaliers and Hirst 1986; Yirmiya et al. 1986), as well as

that of fluid intake, perhaps through a homeostatic control of

water balance (Spencer et al. 1986). It is interesting to note

that their effects may depend on the taste. Thus agonists were

reported to enhance eating of palatable foods (Jackson and Cooper

1986). This behavior could be suppressed by naltrexone (Kirkham et

al. 1986), which also lowered pleasure rating for sweet food and

selected odors (Fantino et al. 1986). The possible role of opioids

in pathogenesis of several functional disorders of gut has been

reviewed by Dhawan et al. (1988).

224
Recent evidence indicates an important role of opioid systems in

the regulation of sexual behavior in both sexes. Initial reports

suggest an inhibitory effect of opioid peptides on male sexual

behavior via the medial preoptic area (Band et al. 1986), but if

the male sex drive is low they may mediate rewards of sex (Miller

and Baum 1986). They also mediate sexual behavior in the female,

particularly the proceptive behavior (Wiesner et al. 1986). and

interaction with 5 HT in such regulatory effects has been suggested

(Allen et al. 1986).

There are several neurological disorders in whose etiology opioid

peptides may be involved (Dhawan et al. 1988). More definitive

data need to be generated in these disorders, but a major limiting

factor is the nonavailability of suitable animal models for

conditions such as Alzheimer's disease. There is an urgent need

also to develop suitable models for neurodegenerative disorders,

nerve injury, and other neurological conditions (Taylor et al.

1987). It will be useful to develop sensitive in vitro bioassay

systems involving critical biochemical parameters having predictive

capabilities.

With the increased availability of data in species other than the

rat and mouse, it has become abundantly clear that significant

species differences do exist in the effects of opioid agonists, and

these help in better understanding of their possible physiological

roles. Several investigators have in recent years included

multiple species in their studies (Davis et al. 1986; Fanselow and

Sigmundi 1986) to assess the central roles of peptides. Recently,

studies have been extended, not only to other species of laboratory

animals and nonhuman primates, but also to insects, fish, and some

unnatural ones like the bat, deer mice, and ground squirrel (Shukla

et al. 1988; see Olson et al. 1987 for additional references).

It is difficult to explain some of the results obtained with the

opioids based on the currently available data, but more comparative

studies are indicated. It is also necessary to extend studies to

different age groups of animals, since age-related changes in

modification of stress responses (Iny et al. 1986) as well as

analgesia (Hoskins et al. 1986) have been reported. It is

therefore important to carry out carefully controlled studies

before drawing firm conclusions regarding various regulatory roles

being assigned to opioid peptides.

A great deal of research work has been carried out during the past

decade on opioids, and yet the mechanisms of their various

biological actions are far from clear. It is hoped that a greater

understanding of their role will finally result in the development

of therapeutically useful drugs and help in the alleviation of pain

and human misery.

225
REFERENCES

Allen, D.L.; Frankfurt, M.; and Luine. V.N. Opioid-serotonergic

interactions in the control of female sexual behavior. Soc

Neurosci Abstr 12:840, 1986.

Band, L.R.; Warner, R.; Pehek, E.; Bitran, D.; Holmes

Hull, E. Opiate regulation of male sex behavior

Effects of morphine and naloxone infused into the

area. Soc Neurosci Abstr 12:536, 1986.

Berman, J.M.; Goodman, M; Nguyen, T.M.-D.; and Schiller, P.W.

Cyclic and acyclic partial retro-inverse enkephalins:

mu-receptor selective enzyme resistant analogues.

Biophys Res Commun 115:864-870, 1983.

Blalock. J.E., and Smith. E.M. A complete loop between the immune

and neuroendocrine systems. Fed Proc 44:108-111, 1985.

Belleney, J.; Roques, B.P.; and Fourni-Zaluski, M-C. Comparison of

conformational properties of linear and cyclic -selective

opioid ligands DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) and DPLPE

(Tyr-c[D-Pen-Gly-Phe-Pen]) by 1H n.m.r. spectroscopy. Int J

Peptide Protein Res 30:356-364, 1987.

Brown, J.R.; Hunter, J.C.; Jordan, C.C.; Tyers, M.B.; Ward, P.; and

Whittington, A.R. Problems with peptides--all that glitters is

not gold. Trend Neurosci 9:100-102, 1986.

Canova-Davis, E.; Chloupek, R.C.; Baldonado, L.P.; Battersby, J.E.;

Spellman, M.W.; Basa, L.J.; O'Connor, B.; Perman, R.; Quan. C.;

Chakel, J.A.; Stults, J.T.; and Hancock, W.S. Analysis by FAB MS

and LC of proteins produced by either biosynthetic or chemical

techniques. Biotech Lab 6:8-17, 1988.

Davis, T.P.; Han, A.; and Yousef, M.K. Central metabolism of

beta-endorphin in different species of temperature acclimated

rodents. Comp Biochem Physiol 84:105-111, 1986.

Delay-Goyet, P.; Seguin, C.; Gacel, G.; and Roques, B.P.

[

3

H][D-Ser2(O-tert-butyl) Leu5] Enkephalyl-Thr6 and

[D-Ser2(O-tert-butyl, Leu5]enkephalyl-Thr6

(O-tert-butyl). Two new enkephalin analogs with both a good

selectivity and a high affinity toward -opioid binding sites.

J Biol Chem 263:4124-4130, 1988.

Desiderio, D.M.; Takeshita. H.; Onishi, H.; Fridland, G.; Tanzer,

F.S.; Wakelyn, C.; and Dass, C. The analysis of endogenous opioid

peptides with HPLC, radioreceptor assay, radioimmunoassay and

mass spectrometry. In: Rapaka; R.S., and Hawks, R.L. eds: Opioid

Peptides: Molecular Pharmacology, Biosynthesis, And Analysis.

National Institute on Drug Abuse Research Monograph 70. DHHS Pub.

No. (ADM)87-1455. Washington, D.C.: Supt. of Docs., U.S. Govt.

Print. Off., 1986. pp. 192-208.

Dhawan, B.N.; Rapaka, R.S.; and Raghubir, R. Opioids and drug

development. In: Dhawan, B.N., and Rapaka, R.S., eds. Recent

Progress in the Chemistry and Biology of Centrally Acting

Peptides. Lucknow: CDRI, 1988. pp. 317-329.

DiMaio, J., and Schiller, P.W. A cyclic enkephalin analog with a

high in vitro activity. Proc Natl Acad Sci USA 77:716217166, 1980.

226
DiMaio, J.; Nguyen, T.M.-D.; Lemieux, C.; and Schiller, P.W.

Synthesis and in vitro pharmacological characterization of cyclic

enkephalin analogues: Effect of conformational constraints on

opiate receptor selectivity. J Med Chem 25:1432-1438, 1982.

Donohoe. R.M.; Madden, J.J.; Hollingsworth, F.; Shafer, D.; and

Falek, A. Morphine depression of T cell E-rosetting: Definition

of the process. Fed Proc 44:95-99, 1985.

Erne, D.; Sargent, D.F.; and Schwyzer, R. Preferred conformation,

orientation, and accumulation of dynorphin

A-(1-13)-tridecapeptide on the surface of neutral lipid

membranes. Biochemistry 24:4261-4263, 1985.

Fanselow, M.S., and Sigmundi. Species-specific danger signals,

endogenous opioid analgesia, and defensive behavior. J Exp

Psychol 12:301-309, 1986.

Fantino, M; Hosotte, J.; and Applebaum, M. An opioid antagonist,

naltrexone, reduces preference for sucrose in humans. Am J

Physiol 251:R91-R96, 1986.

Foster, G.A.; and Roberts, M.H.T. Anatomical and physiological

correlates of 5HT and coexistent peptidergic neurotransmitters.

In: Dhawan, B.N.; and Rapaka, R.S., eds. Recent Progress in the

Chemistry and Biology of Centrally Acting Peptides. Lucknow,

India: CDRI 1988. pp. 317-329.

Fridland, G.H., and Desiderio, D.M. Measurement of opioid peptides

with combinations of high performance liquid chromatography,

radioimmunoassay, radioreceptor assay and mass spectrometry. Life

Sci 41:809-812, 1987.

Gysin, B., and Schwyzer. R. Head group and structure-specific

interaction of enkephalins and dynorphin liposomes: Investigation

by hydrophobic photolabeling. Arch Biochem Biophys 225:467-474,

1983.

Hazum, E.; Chang, K.J.; and Cuatrecasas, P. Specific non-opiate

receptors for ß-endorphins. Science 205:1033-1035. 1979.

Hansen, P.E., and Morgan, B.M. Structure-activity relationships In

enkephalin peptides. In: Udenfriend, S., and Meienhofer, J..

eds. The Peptides. Analysis, Synthesis, Biology. Vol. 6, Opioid

Peptides: Biology, Chemistrv and Genetics. Orlando, FL: Academic

Press, 1984. pp. 269-321.

Hiramatsu, M.T.; Nabeshima, T.; and Kameyama, T. Involvement of

opioid receptors in hypo- and hyper-thermic effects induced by

phencyclidine in mice. J. Pharmacobiodyn 9:446-472, 1986.

Hoskins, B.; Burton, C.K.; and Ho, I.K. Differences in

morphine-induced antlnociception and locomotor activity in mature

adult and aged mice. Pharmacol Biochem Behav 25:599-605, 1986.

Houghten, R.A. General method for solid-phase synthesis

of large numbers of peptides: Specificity of antigen-antibody

interaction at the level of individual amino acids. Proc Nat1

Acad Sci USA 82:5131-5135, 1985.

Houghten, R.A.; Bray, M.K.; DeGraw, S.T.; and Kirby, C.J.

Simplified procedure for carrying out simultaneous multiple

hydrogen fluoride cleavages of protected peptide resins. Int J

Peptide Protein Res 27:673-678, 1986a.

227
Houghten, R.A.; DeGraw, S.T.; Bray, M.K.; Hoffmann, S.R.; and

Frizzell, N.D. Simultaneous multiple peptide synthesis: The

rapid preparation of large numbers of discrete peptides for

biological, immunological and methodological studies.

Biotechniques 4:522-528, 1986b.

Houqhten. R.A.; Cuervo. J.H.; Ostresh. J.M.; Bray, M.K.: and

Frizzell, N.D.; Simuitaneous multipie peptide synthesis: The

rapid preparation of large numbers of discrete peptides for

biological, immunological, and methodological studies. In:

Marshall, G.R., ed. Peptides: Chemistry and Biology. Leiden,

Netherlands: Escom Science Publishers, 1988. pp. 166-172.

Hruby, V.J.; Kazmierski, W.; Pelton, J.T.; Porreca, F.; Shook,

J.E.; Knapp, R.; Lui, G.; Yamamura, H.I.; and Burks, T.F.

Conformaticnal and structural considerations in the development

of cyclic peptides with extraordinary μ opioid receptor

selectivity and antagonist activities. In: Dhawan, B.N., and

Rapaka. R.S. eds. Recent Progress in the Chemistry and Biology

of Centrally Acting Peptides. Lucknow, India: CDRI, 1988. pp.

53-69.

Huang, S.-G.; Renugopalakrishnan, V.; and Rapaka, R.S. 500 MHz

Proton NMR studies of dynorphin A(1-13) in aqueous solution:

Assignments of proton resonances and conformational implications.

Biochemistry in press, 1988. .

Hughes, J.; Smith, T.W.; Kosterlitz, H.N.; Fothergill, L.A.;

Morgan, B.A.; and Morris, R.H. Identification of the two related

pentapeptides from the brain with potent opiate agonist

activity. Nature 258:577-579, 1975.

Iny, L.J.; Aitken, D.H.; Gianoulakis, C.; Palmour, R.; and Meaney,

M.J. The beta-endorphin and monoaminergic response to stress in

the developing rat pup. Soc Neurosci Abstr 12:1484, 1986.

Jackson, A., and Cooper, S.J. An observational analysis of the

effect of the selective kappa opioid agonist, U-50-488H, on

feeding and related behaviours in the rat. Psychopharmacology

(Berlin) 90:217-221, 1986.

Johnson, H.M.; Torres, B.A.; Smith, E.M.; Dion, L.D.; and Blalock,

J.E. Regulation of lymphokine (y-interferon) production by

corticotropin. J Immunol 132:246-250, 1984.

Judd, A.K.; Lawson, J.A.; Olsen, C.M.; Toll, L.R.; Polgar, W.E.;

Uyeno, E.T.; Keys, C.J.; and Loew, G. Novel P-selective

Met-enkephalinamide analogs with antagonist activity. Int J

Peptide Protein Res 30:299-317, 1987.

Kavaliers, M., and Hirst, M. Naloxone-reversible stress induced

feeding and analgesia in the slug Limax maximus. Life Sci

38:203-209, 1986.

Kay, N.; Morley, J.E.; and Van Ree, J.M. Enhancement of human

lymphocyte natural killing function by non-opioid fragment of

ß-endorphin. Life Sci 40:1083-1087, 1987.

Kirkham, T.C.; Barber, D.J.; Heath, R.W.; and Cooper, S.J.

Differential effects of CGS 8216 and naltrexone on inaestional

behaviour. Pharmacol Biochem Behav 26:145-151, 1986. 

Majeed, N.H.; Przewlocka, B.; Wedzony, K.; and Prezewlocki, R.

Stimulation of food intake following opioid microinjection Into

the nucleus accumbens septi in rats. Peptides 7:711-716, 1986.

228
Mandler, R.N.; Biddison, W.E.; Mandler. R.; and Serrate, S.A.

ß-endorphin augments the cytotoxic activity and interferon

production of natural killer cells. J Immunol 136:934-939, 1986.

Marasco, W.A.; Showell, H.J.; and Becker, E.L. Substance P binds

to the formylpeptide chemotaxis receptor on the rabbit

neutrophil. Biochem Biophys Res Commun 99:1065-1072, 1981.

Marston, F.A.O. The purification of eukaryotic polypeptides

synthesized in escherichia coli. Biochem J 240:1-12, 1986.

Merrifield, R.B. Solid phase peptidesynthesis. I. The synthesis

of a tetrapeptide. J Amer Chem Soc 85:2149-2154, 1963.

Miller, R.L., and Baum, M.J. Naloxone inhibits mating and

conditioned place preference for an estrous female in male rats

soon after castration. Soc Neurosci Abstr 12:674, 1986.

Moore, T.C. Modification of lymphocyte traffic by vasoactive

neurotransmitter substances. Immunology 52:511-518. 1984.

Morley, J.E.; Kay, N.E.; Soloman. G.F.; and Plotnikiff, N.P.

Neuropeptides: Conductors of the immune orchestra. Life Sci

41:527-544, 1987.

Morley, J.S. Structure-activity relationships of enkephalin-like

peptides. Annu Rev Pharmacol Toxicol 20:81-110. 1980.

Morley, J.S., and Dutta, A.S. Structure-activity relationships of

opioid peptidea. In: Rapaka, R.S.; Barnett. G.: and Hawks, R.L.,

eds. Opipid Peptides: Medicinal Chemistry. National Institute

on Drug Abuse Research Monograph 69. DHHS Pub No. (ADM) 87-1454.

Washington, D.C.: Supt. of Docs., U.S. Govt. Print. Off., 1986.

pp. 42-64.

Mosberg, H.I. 1N.M.R. investigation of conformational features of

cyclic, penicillamine-containing enkephalin analogs. Int J

Peptide Protein Res 29:282-288, 1987.

Mosberg, H.I.; Omnass, J.R.; and Goldstein, A. Structural

requirements for L-receptor binding. Med Pharmacol 31:599-602,

1987.

Olson, G.A.; Olson, R.D.; and Kastin, A.J. Review. Endogenous

opiates: 1986. Peptides 8:1135-1164, 1987.

Payan, D.G.; Brewster, D.R.; and Goetzl, E.J. Specific stimulation

of human T lymphocytes by substance P. J Immunol 131:1613-1615,

1983.

Payan, D.A.; Levine, J.D.; and Goetzl, E.J. Modulation of immunity

and hypersensitivity by sensory neuropeptides. J Immunol

132:1601-1604. 1984.

Plotnikoff, N.P.; Murgo, A.J.; Miller, G.C.; Corder, C.N.; and

Faith, R.E. Enkephalins: Immunomodulators. Fed Proc 44:

118-122, 1985.

Plotnikoff. N.P.; Faith, R.E.; Murgo, A.J.; and Good, R.A.

Enkephalin and Endorphins. Stress and the Immune System. New

York: Plenum Press, 1986a. 447 pp.

Plotnikoff, N.P.; Wybran, J.; Nimeh, N.F.; and Millet, G.C.

Methionine enkephalin: Enhancement of T-cells in patients with

Kaposi's sarcoma (AIDS). Psychopharmacol Bull 22:695-697. 1986b.

Radhakishun, F.S.; Horn, A.S.; and Van Ree, J.N. Desenkephalin

endorphin and neuroleptics counteract the DP-7-TN-induced

hypomotility after intra-accumbens treatment. Eur J Pharmacol

147:137-140, 1988.

229
Rapaka, R.S. Research topics in the medicinal chemistry and

molecular pharmacology of opioid peptides--present and future.

Life Sci 39:1825-1843. 1986.

Rapaka, R.S., and Hawks, R.L., eds. Opioid Peptides: Molecular

Pharmacology, Biosynthesis, and Analysis. National Institute on

Drug Abuse Research Monograph 70. DHHS Publication No.

(ADM)87-1455 Washington, D.C.: Supt. of Docs., U.S. Govt Print.

Off., 1986. pp. 414.

Rapaka, R.S.; Bhatnagar. R.S.; and Nitecki, D.E. Racemization in

the synthesis of sequential polypetides using N-hydroxysuccimide

esters. Biopolymers 15:1585-1592, 1976.

Rapaka, R.S.. Nitecki, D.E.; and Bhatnagar, R.S. Strategies in the

racemizatlon free synthesis of polytripeptides related to

Collagen. Adv Exp Med Biol 86A:473-490, 1977.

Rapaka, R.S.: Hawks, R.L.; and Barnett, G. Opioid Peptides:

Medicinal Chemistry. National Institute on Drug Abuse Research

Monograph 69. DHHS Pub. No. (ADM)87-1454. Washington, D.C.: Supt.

of Docs., U.S. Govt. Print. Off., 1986, 376 pp.

Rapaka, R.S.; Renugopalakrishnan, V.; Collette, T.W.; Dobbs, J.C.;

Carreira, L.A.; and Bhatnagar, R.S. Conformational features of

dynorphin A(1-13): Laser Raman spectroscopic studies. Int J

Peptide Protein Res 30:284-287, 1987.

Renugopalamshnan, V., and Rapaka, R.S. Lipid modulation of the

secondary structure of dynorphin A(1-13). Is: Makriyannis, A.,

and Prous, J.R., eds. New Methods In Drug Research, Vol.3.

Barcelona, Spain: In Press, 1988.

Renugopalakrishnan, V.; Rapaka, R.S.; Moore, S.; and Hutson, T.B.

Dynorphin A-(1-13) peptide NH groups are solvent exposed: FT-IR

spectroscopic evidence. Biochem Biophys Res Commun

151:1220-1225, 1988a.

Renugopalakrishnan, V.; Rapaka, R.S.; and Huang, S.-G. Secondary

structural studies of dynorphin. In: Dhawan, B.N., and Rapaka,

R.S., eds. Recent Progress in the Chemistry and Biology of

Centrally Acting peptides. Lucknow, India: CDRI, 1988b. pp.

71-85.

Ruff, M.R.; Wahl, S.M.; and Pert, C.B. Substance P receptormediated chemotaxis of human monocytes. Peptides 6:107-111, 1985.

Said, S.I. Neuropeptides in cardiovascular regulation. In:

Buckley, J.P., and Ferrarario, C.M., eds. Brain Peptides and

Catecholamires in Cardiovascular Regulation. New York: Raven

Press, 1987. pp. 93-107.

Sargent, D.F., and Schwyzer, R. Membrane lipid phase as catalyst

for peptide-receptor interactions. Proc Natl Acad Sci USA

83:5774-5778, 1986.

Schiller, P.W. Conformational analysis of opioid peptides and the

use of conformational restriction in the design of selective

analoss. In: Rapaka. R.S.: Barnett. G.: and Hawks. R.L., eds.

Opioid Peptides: Medicinal Chemistry. National Institute on

Drug Abuse Research Monograph 69. DHHS Pub. No. (ADM) 87-1454.

Washington, D.C.: Supt. of Docs.. U.S. Govt. Print. Off., 1986.

pp. 291-311.

Schiller, P.W.; Nguyen, T.M.D.; Maziak, L.; and Lemieux, C.A.

Novel cyclic opioid peptide analog showing high preference for

p-receptors. Biochem Biophys Res Commun 127:558-564, 1985.

230
Schiller, P.W.; Nguyen, T.M.-D.; and Lemieux, C. Synthesis and

opioid activity: Profiles of cylic dynorphin analogs.

Tetrahedron 44:733-743, 1988.

Schwyzer, A. Estimated conformation, orientation, and accumulation

of dynorphin A-(1-13) tridecapeptide on the surface of neutral

lipid membrane. Biochemistry 23:4281-4286, 1986.

Schwyzer, R.; Gremllch, H.-D.; Gysin, B.; and Fringeli, U.P.

Interactions of lipid bilayers with corticotropin, dynorphin and

enkephalln peptides. Peptides 3:55-71, 1982.

Sharp, B.M.; Keane, W.F.; Suh, H.J.; Gekker, G.; Tsukayama, D.; and

Peterson, P.K. Opioid peptides rapidly stimulate superoxide

production by human polymorphonuclear leukocytes and macrophages.

Endocrinology 117:793-795 1985.

Shavit, Y.; Depaulis, A.; Martin, F.C.; Terman, G.W.; Pechnick,

R.N.; Zane, C.J.; Gale, P.P.; and Leiberkind, J.C. Involvement

of brain opiate receptors in the immune-suppressive effect of

morphine. Proc Natl Acad Sci USA 83:7114-7117, 1986.

Shukla, R., and Dhawan, B.N. Peptides and thermoregulatlon. This

volume.

Shukla, R.; Srimal, R.C.; and Dhawan, B.N. Central interaction of

classical neurotransmitters and Met-enkephalin on the core

temperature of matomys natalensis. In: Dhawan, B.N., and

Rapaka, R.S., eds. Recent Progress in the Chemistry and Biology

of Centrally Acting Peptides. Lucknow, India: CDRI, 1988. pp.

201-207.

Singh, V.K.; Jakubovic, A.; and Thomas, D.A. Suppressive effects

of methadone on human blood by lymphocytes. Immuno Lett

2:177-180, 1980.

Spencer, R.L.; Deupree, D.; Hslao, S.; Mosberg, H.I.; Hruby, T.;

Burks, F.; and Porreca, F. Centrally-administered oploid

selective agonists inhibit drinking in the rat. Pharmacol

Biochem Behav 25:77-82, 1986.

Srimal, R.C.;Raghubir, R.; Gulati, K.; Agarwal, S.K.; and Dhawan,

B.N. Possible involvement of endogenous opioids in the mediation

of cardiovascular effects of centhaquin. In: Dhawan, B.N., and

Rapaka, R.S. eds. Recent Progress in the Chemistry and Biology

of Centrally Acting Peptides. Lucknow, India: CDRI, 1988. pp.

191-199.

Sternson, L.A. Analytical methodology to support the

pharmaceutical development of peptides and proteins. Ann N Y

Acad Sci 507:52-63, 1987.

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

binding of DPDPE as a result of batch variability. In: Holaday,

J.W.; Law, P-Y.: and Herz. A., eds. Progress in Opioid

Research. National Institute on Drug Abuse Research Monograph

75. DHHS Pub. No. (ADM) 87-1507. Washington, D.C.: Supt. of

Docs., U.S. Govt Print. Off., 1986. pp 201-204.

Taylor, J.E.; Moreau, J.P.; and DeFeudis, F.-V. Small peptides and

nerve growth: Therapeutic implications. Drug Develop Res

11:75-86, 1987.

Taylor, J.W. Approaches to studying structure-activity

relationships in peptide hormones through the expression of

synthetic genes. This volume.

231
Tubaro, E.; Borelli, G.; Croce, C.; Cavallo, G.; and Santianqeli,

C. Effect of morphine on resistance to infection. J Infect Dis

148:656-666, 1983.

Udenfriend, S., and Meienhofer, J. The Peptides. Analysis,

Synthesis, Biology. Vol. 6. Opioid Peptides: Biology, Chemistry

and Genetics. Orlando, FL: Academic Press, 1984. pp. 410.

Wiesner, J.B.; Dudley. C.A.; and Moss, R.L. Effect of ß-endorphin

on sociosexual proclivity in a choice paradigm. Pharmacol

Biochem Behav 24:507-511, 1986.

Wybran, J.; Appelboom, T.; and Famaey, J.P. Suggestive evidence

for receptors for morphine methionine-enkephalin on normal and

human blood T lymphocytes. J Immunol 123:1068-1070, 1979.

Wybran. J. Enkephalins and endorphins modifiers of the immune

system : Present and future. Fed Proc 44:92-94. 1985.

Yahya, M.D., and Watson, R.R. Minireview: Immunomodulation by

morphine and marijuana. Life Sci 41:2503-2510, 1987.

Yirmiya, R.; Holder, M.D.; and Garcia, J. Effects of naloxone and

cholecystotinin on food and water intake in the Brattleboro rat.

Soc Neurosci Abstr 12:1491, 1986.

AUTHORS

Rao S. Rapaka, Ph.D.

1OA-13, National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857 U.S.A.

Bhola N. Dhawan, M.D.

Director, Certral Drug Research Institute

Chattar Manzil

P.O. Box 173

Lucknow 226OC1, India

V. Renugopalakrishnan, Ph.D.

Laboratory for the Study of Skeletal Disorders and Rehabilitation

Department of Orthopaedic Surgery

Harvard Medical School and Children's Hospital

ENDERS-1220

300 Longwood Avenue

Boston, Massachusetts 02115 U.S.A.

232
While limited supplies last, single copies of the monographs may be

obtained free of charge from the National Clearinghouse for Alcohol

and Drug Information (NCADI). Please contact NCADI also for

information about availability of coming issues and other

publications of the National Institute on Drug Abuse relevant to

drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service

(NTIS) as indicated. NTIS prices are for paper copy. Add $3.00

handling charge for each order. Microfiche copies, at $6.95, are

also available from NTIS. Prices from either source are subject to

change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville, Maryland 20852

GPO

Superintendent of Documents

U.S. Government Printing Office

Washington, D.C. 20402

NTIS

National Technical Information

Service

U.S. Department of Commerce

Springfield, Virginia 22161

For information on availability of NIDA Research Monographs 1-24

(1975-1979) and others not listed, write to NIDA Office for Research

Communications, Room 1OA-54, 5600 Fishers Lane, Rockville, MD 20857.

25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman A.

Krasnegor, Ph.D., ed. NCADI out of stock

GPO out of stock NTIS PB #80-112428/AS $28.95

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. (Reprint from 1979 Surgeon General's Report on Smoking and

Health.)

GPO out of stock NTIS PB #80-118755/AS $21.95

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

J. Lettieri, Ph.D.; Mollie Sayers; and Helen W. Pearson, eds.

GPO Stock #017-024-00997-1 $10 NCADI out of stock

Not available from NTIS

233
31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D., ed.

GPO out of stock NTIS PB #80-215171/AS $28.95

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz.

Ph.D.; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz.

GPO out of stock NTIS PB #81-133746/AS $28.95

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF

MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y.

Ng, M.D., ed.

GPO out of stock NTIS PB #81-240913/AS $28.95

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D.. and Chris E. Johanson, Ph.D.. eds.

NCADI out of stock

GPO out of stock NTIS PB #82-136961/AS $36.95

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri, Ph.D.,

and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO out of stock NCADI out of stock

NTIS PB #82-148198/AS $21.95

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin, M.D., Ann Pollinger, Ph.D., Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. NCADI out of stock

GPO out of stock NTIS PB #82-1331171AS $21.95

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed.

GPO out of stock NTIS PB #83-136044/AS $21.95

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude, Ph.D., and Jacqueline P. Ludford, M.S., eds. A

RAUS Review Report. NCADI out of stock

GPO out of stock NTIS PB #85-150563/AS $21.95

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Pinkert. M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report. NCADI out of stock

GPO out of stock NTIS PB #84-184670/AS $15.95

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D., and Jack E.

Henningfield, Ph.D., eds. NCADI out of stock

GPO out of stock NTIS PB #84-184688/AS $21.95

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford. M.S., eds. A RAUS Review Report.

GPO Stock #O17-024-01180-1 $5.50 NTIS PB #85-159663/AS $28.95

234
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 NCADI out of stock

NTIS PB #84-145184/AS $21.95

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed.

GPO Stock #017-024-01214-9 $4 NTIS PB #85-150381/AS $21.95

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-024-01218-1 $4.50 NTIS PB #85-150365/AS $21.95

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #017-024-01204-1 $4.25 NTIS PB #85-150373/AS $21.95

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

Ph.D., and Sharon M. Hall, Ph.D.. eds. NCADI out of stock

GPO Stock #017-024-01266-1 $3.50 NTIS PB #89-123186/AS $21.95

54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed.

GPO out of stock NTIS PB #89-103279/AS $36.95

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. NCADI out of stock

GPO out of stock NTIS PB #89-123194/AS $42.95

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 NTIS PB #89-1231601/AS $28.95

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 NTIS PB #88-248083/AS $21.95

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 NTIS PB # to be assigned

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 NTIS PB # to be assigned

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 NTIS PB # to be assigned

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 NTIS PB #131866/AS $28.95

235
76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF THE 48TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-1316-1 $16 NTIS PB #88-208111/AS $44.95

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT RESEARCH.

Elizabeth R. Rahdert, Ph.D., and John Grabowski, Ph.D., eds.

GPO Stock #017-024-1348-O $4 NTIS PB #89-125488/AS $21.95

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO DRUGS

David P. Friedman, Ph.D., and Doris H. Clouet, Ph.D., eds.

GPO Stock #017-024-01330-7 $6 NTIS PB #88-245683/AS $25.95

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE CANNABINOIDS

Rao S. Rapaka, Ph.D., and Alexandros Makriyannis, Ph.D., eds.

GPO Stock #017-024-01331-5 $6 NTIS PB #89 109201/AS $25.95

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS: NATIONAL AND

INTERNATIONAL PERSPECTIVES. Robert J. Battjes, D.S.W., and Roy W.

Pickens, Ph.D., eds.

GPO Stock #017-024-01345-5 $5.50 NTIS PB #88-236138/AS $25.95

81 PROBLEMS OF DRUG DEPENDENCE, 1987. PROCEEDINGS OF THE 49TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01354-4 $17 NTIS PB $89 109227/AS $50.95

82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

David P. Friedman, Ph.D. eds.

GPO Stock #017-024-01344-7 $4.25 NTIS PB #88-245691/AS $19.95

83 HEALTH HAZARDS OF NITRITE INHALANTS. Harry W. Haverkos, M.D.,

and John A. Dougherty, Ph.D., eds.

GPO Stock #017-024-01351-O $3.25 NTIS PB #89-125496/AS $21.95

84 LEARNING FACTORS IN SUBSTANCE ABUSE. Barbara A. Ray, Ph.D., ed.

GPO Stock #017-024-01353-6 $6 NTIS PB #89-125504/AS $28.95

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE. Raquel A. Crider,

Ph.D., and Beatrice A. Rouse, Ph.D., eds.

GPO Stock #017-024-01360-9 $5.50 NTIS PB #89-123178/AS $28.95

86 COMPULSORY TREATMENT OF DRUG ABUSE: RESEARCH AND CLINICAL

PRACTICE. Carl G. Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #017-024-01352-8 $7.50 NTIS PB # to be assigned

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H. Clouet,

Ph.D., Khursheed Asghar, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO Stock #Ol7-024-01359-5 $11 NTIS PB #89-125512/AS $36.95

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE. Roy W. Pickens, Ph.D.,

and Dace S. Svikis, B.A., eds.

GPO Stock #017-022-01054-4 $5 NTIS PB #89-125520/AS $21.95

237
DHHS Publication No. (ADM)89-1604

Alcohol, Drug, and Mental Health Administration

Printed 1988