National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Molecular

Approaches to

Drug Abuse

Research

Volume I

U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health

111
Molecular Approaches

to Drug Abuse

Research Volume I:

Receptor Cloning,

Neurotransmitter

Expression, and

Molecular Genetics

Editors:

Theresa N.H. Lee, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Research Monograph 111

1991

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, MD 20857

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

Washington, D.C. 20402
ACKNOWLEDGMENT

This monograph is based on the papers and discussion from a technical

review on “Molecular Approaches to Drug Abuse Research” held on August 24-

25, 1989, in Bethesda, MD. The review meeting was sponsored by the National

Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from the

copyright holders to reproduce certain previously published material as noted in

the text. 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

except quoted passages from copyrighted sources 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.

Opinions expressed in this volume are those of the authors and do not

necessarily reflect the opinions or official policy of the National Institute on Drug

Abuse or any other part of the U.S. Department of Health and Human Services.

The US. 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.

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.

DHHS publication number (ADM)91-1798

Printed 1991

i i
Contents

Page

Introduction 1

Theresa N.H. Lee

Brain Nicotinic Receptor Genes 3

Stephen F. Heinemann, Jim Boulter, John Connolly,

Evan Deneris, Robert Duvoisin, Melissa Hartley,

Irm Hermans-Borgmeyer, Michael Hollmann,

Anne O’Shea-Greenfield, Roger Papke,

Scott Rogers, and Jim Patrick

Regulation of Acetylcholine Receptor Gene Expression 24

Alex M. Simon, Emma K. Dutton, and Steven J. Burden

Mutations Affecting Local Anesthetic Block of the Nicotinic

Acetylcholine Receptor Ion Channel 28

Reid J. Leonard, Pierre Charnet, Cesar Labarca,

Nancy J. Vogelaar, Linda Czyzyk, Annie Gouin,

Norman Davidson, and Henry A. Lester

Molecular Biology of the Dopamine D2 Receptor 45

Olivier Civelli, James Bunzow,

Paul Albert, Hubert Van Tol,

and David Grandy

Characterization of Mammalian Potassium Channel Genes 54

James O. Douglass, Macdonald Christie,

John P. Adelman, and R. Alan North

iii
Problems and Approaches in Studylng Membrane

Opioid Receptors 69

Andrew P. Smith and Horace H. Loh

An Approach to the Molecular Biology of Opiate Tolerance:

Identification of Opiate-Regulated Transcripts 85

Mark vonZastrow, Jack D. Barchas,

and James H. Eberwine

Prenatal Expression of Pro-Opiomelanocortin mRNA,

POW-Derived Peptides, and μ-Opiate Receptors in the

Mouse Embryo 96

Y. Peng Loh, R. Adrian Rius, Stela Elkabes,

Wojciech Bern, and Carmine J. Coscia

Regulation of Endorphin Biosynthesis in the Brain: Different

Effects of Morphine Pelleting and Repeated Stress 113

David M. Bronstein, Jeffrey E. Kelsey, and Huda Akil

Transcriptional Regulation of the Rat Prodynorphin Gene 133

James O. Douglass, Michael J. ladarola,

Jau-Shyong Hong, James E. Garrett,

and Cynthia T. McMurray

Mechanisms Underlying Synaptic Regulation of Proenkephalin

Transcription 149

Michael Comb, Steven E. Hyman, Linda Kobierski,

Hung Ming Chu, and Tuong Van Nguyen

Regulation of Carboxypeptidase E (Enkephalin Convertase) 171

Lloyd D. Fricker, Banasree Das, Robyn S. Klein,

David Greene, and Yong-Keun Jung

Use of Site-Directed Mutagenesis To Elucidate the Active

Site of Neutral Endopeptidase 24.11 (Enkephalinase) 188

Louis B. Hersh

Human Cytochrome P450: Possible Roles of Drug-Metabolizing

Enzymes and Polymorphic Drug Oxidation in Addiction 202

Frank J. Gonzalez

List of NIDA Research Monographs 214

iv
Introduction

Theresa N.H. Lee

The extraordinary advances in molecular biology since the early 1970s have

created unprecedented opportunities for progress in every field of biological

research. The sensitivity and specificity of these techniques have enabled

scientists to acquire an enormous amount of knowledge about the genetic

control of the physiological and pathological processes and to resolve these

processes at the molecular level.

The opportunity for progress presented by molecular biology in understanding

the fundamental nature of the addictive process is particularly timely in light of

the national consensus to develop new approaches to the problem of drug

addiction.

Despite advances in many fields, molecular biological approaches remain

underutilized in research related to understanding the actions and

consequences of abused substances. In recent years, extensive progress has

been made on the structure, biosynthesis, modification, degradation, and

regulation of opioid peptides and their precursors. However, similar studies on

the receptors, transporters, and ion channels relevant to many abused

substances are still lacking.

To stimulate more research in these and other areas, the first technical review

by the National Institute on Drug Abuse (NIDA) on “Molecular Aproaches to

Drug Abuse Research” was held on August 24-25, 1989, in Bethesda, MD. The

proceedings of this conference are presented in the following chapters of this

monograph.

All the speakers either have successfully cloned a gene relevant to drug abuse

or have been employing molecular approaches to drug abuse research.

However, due to limited research carried out in some areas, the specific abused

substances studied by the speakers do not necessarily represent NIDA’s

current programmatic preference. For example, because the muscle nicotinic

acetylcholine receptor genes have been cloned and studied for a number of

years, results of experiments manipulating the expression of these subunit

genes instead of the neuronal genes were presented at this meeting.

1
This monograph provides many elegant examples of what researchers can do

with the molecular approaches to drug abuse research. For drug abuse

researchers interested in molecular approaches, the decade to come

undoubtedly will prove to be both challenging and fruitful, with ground-breaking

results contributing to understanding the underlying basis of addiction and the

consequences of long-term drug abuse to generate better strategies for

effective diagnosis, treatment, education, and prevention.

AUTHOR

Theresa N.H. Lee, Ph.D.

Program Administrator

Molecular Biology and Genetics Program

Biomedical Branch

Division of Preclinical Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-31

5600 Fishers Lane

Rockville, MD 20857

2
Brain Nicotinic Receptor Genes

Stephen F. Heinemann, Jim Boulter, John Connolly, Evan

Deneris, Robert Duvoisin, Melissa Hartley, Irm HermansBorgmeyer, Michael Hollmann, Anne O’Shea-Greenfield, Roger

Papke, Scoff Rogers, and Jim Patrick

Nicotine is one of the most widely taken addictive drugs, and the history of the

cultivation of tobacco suggests that early man appreciated the behavioral

effects of nicotine. About one-third of the adult population of the United States

is addicted to nicotine, which is mostly delivered to the body through the

smoking of cigarettes. Nicotine addiction has led to an epidemic of lung cancer

and heart disease in the United States and in many other industrialized

countries. The less developed countries of the world increasingly face a similar

fate. As is the case for chemical addiction in general, the mechanism of

nicotine addiction is not known.

To develop a rational theory of nicotine addiction, it is necessary to understand

how nicotine exerts its effects on the nervous system. With this knowledge, it

might be possible to develop drugs that would help patients overcome their

nicotine addiction. Many drugs that alter brain function or have proven useful

for the treatment of mental disorders are known to affect synaptic transmission.

Thus, the synapse is a plausible place to look for the site of nicotine action in

the brain. During the past decade, the availability of radiolabeled nicotine with

high specific activity has led to the discovery and mapping of nicotine binding

sites in the mammalian brain (Clarke et al. 1985, 1986; for review, see Martin

1986; Clarke 1988). These data suggested that the mammalian brain contains

an important nicotinic receptor system.

To investigate this possibility that the mammalian brain contains a major

nicotinic receptor system, the authors decided to use a molecular genetic

approach to identify genes that code for nicotinic receptors expressed in the

brain. We reasoned that the brain nicotinic receptors might be evolutionarily

related to the Torpedo electric organ and skeletal muscle nicotinic receptors

that we had cloned previously (Ballivet et al. 1982; Claudio et al. 1983; Patrick

et al. 1983; Boulter et al. 1985, 1986a). Based on this assumption, genomic

3
and brain cDNA libraries were screened at low stringency using the muscle

nicotinic receptor cDNAs as probes. The subject of this chapter Is the family of

brain nicotinic acetylcholine receptors that the authors have discovered in the

past few years through the use of this molecular genetic approach (Boulter et al.

1985, 1986a, 1986b, 1987; Goldman et al. 1986, 1987; Deneris et al. 1988,

1989; Wada et al. 1988; Duvoisin et al. 1989).

The authors have identified seven genes in the rat or mouse genome that code

for proteins with homology to the nicotinic acetylcholine receptor. These genes

are expressed in the mammalian brain and in some peripheral neurons. We

have named these genes In our terminology,

the genes coding for the muscle nicotinic acetylcholine receptors are called al,

Thus, the nicotinic acetylcholine receptor family is encoded by at

least 12 genes in the mammalian genome. A similar set of genes has been

identified in chicken (Ballivet et al. 1988; Nef et al. 1988). Recently, the GABA

and glycine receptors also have been cloned and shown to be structurally

related to the nicotinic receptor (Grenningloh et al. 1987; Schofield et al. 1987;

Levitan et al. 1988). Thus, the major inhibitory receptors in the nervous system

are similar In structure to the nicotinic receptor, an excitatory receptor. This was

an unexpected finding and raises the exciting possibility that all ligand-gated

channels are members of one superfamily of related proteins. Thus, it is

reasonable to expect that knowledge about one receptor will be useful in

understanding the role and function of other ligand-gated channel receptors.

The primary structures of the brain nicotinic receptor subunits expressed in the

brain have been deduced from the sequences of the cDNA clones (figures 1

and 2). Analysis of the hydrophobicity profiles of the brain nicotinic receptor

subunits suggests that they fold through the membrane in an identical manner

to the Torpedo fish electric organ nicotinic receptor. Each subunit contains four

stretches of about 20 to 24 amino acids that are hydrophobic. We have

suggested that these hydrophobic stretches are membrane-spanning regions.

We therefore proposed that each subunit spans the membrane four times,

placing the N-terminal and C-terminal outside the cell (Claudio et al. 1983).

This four transmembrane model places about half the protein bulk outside the

cell. This model is also consistent with structural studies of the Torpedo

receptor (Stroud and Finer-Moore 1985; Toyoshima and Unwin 1988). In

addition, this model predicts that the C-terminal is outside the cell, and this

prediction has been confirmed by McCrea and coworkers (1987).

The proposed model (Claudio et al. 1983) also predicts that several potential

phosphorylation sites are cytoplasmic. In one case, phosphorylation at one of

these sites is known to regulate receptor function (Huganir et al. 1986; Safran et

4
NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNITS

FIGURE 1. Amino acid sequence alignment of the subunit with neuronal nAChR subunits. Aligned with the

subunit are the rat Indicated in the figure are the positions of the

predicted leader peptide, potential N-linked glycosylation sites cysteine residues conserved in each

member of the neurotransmitter-gated ion-channel subunit superfamily (*), putative transmembrane

domains (TMD I-IV), and cytoplasmic domain.
FIGURE 2. Amino acid alignment of the rat neuronal -type subunits. Aligned with the ß4 subunit are the ß2

(Deneris et al. 1988) and ß3 (Deneris et al. 1989) sequences. Identical residues in all three subunits

are shown on a black background. Conservative changes are indicated by a gray background.

Putative signal peptides and membrane-spanning regions (MSR) are identified below the sequences.

The region referred to as the extracellular domain is located between the amino terminus and MSR I,

and the putative cytoplasmic domain is located between MSR Ill and MSR IV. The numbering is that of

the precursor ß4 subunit.

SOURCE: Duvoisin et al. 1989, copyright 1989, Cell Press
al. 1986). The brain subunits contain adjacent cysteines at homologous

positions to the Torpedo -subunit cysteines 192 and 193, which are known to

be near the acetylcholine binding site (for review, see Heinemann et al. 1986).

At present, it is not known how many copies of each subunit form a brain

nicotinic receptor. However, because of the observed structural homology

between the ligand-gated channels, we have proposed that the brain receptors

are a pentameric structure, as has been shown to be the case for the Torpedo

fish nicotinic acetylcholine receptor and, recently, the glycine receptor

(Langosch et al. 1988; for review, see Popot and Changeux 1984). The results

of the physiological experiments described below indicate that the brain

nicotinic receptors are composed of two different gene products, an and a

ß-subunit. This conclusion is also compatible with the recent biochemical

analysis of a nicotine binding site isolated from rat brain (Whiting and Lindstrom

1987).

The availability of cDNA clones coding for the brain nicotinic receptors has

made it possible to study their function. Expression studies in Xenopus oocytes

have shown that, in general, two nicotinic receptor gene products—an and a

ß-subunit—are necessary for the formation of a functional nicotinic receptor.

Although the 4-subunit alone produces a weak response to nicotine in

oocytes, 2 and 3 are inactive (figures 3 and 4) (Boulter et al. 1987).

However, when 2, 3, or 4 is combined with ß2, a strong reproducible

response is observed (figures 3 and 4) (Boulter et al. 1987; Wada et al. 1988).

Thus, ß2 is a promiscuous subunit that can combine with three different

subunits to form a functional receptor. This is consistent with its wide

distribution of expression in the brain (figure 5) (Wada et al. 1989; and see

below). A similar result is seen when the ß4-subunit is expressed in the

presence of each of the three subunits (i.e., 2ß4, 3ß4, or 4ß4) (figure 6)

(Duvoisin et al. 1989). At present, we have not been able to demonstrate any

function for the 5 or ß3 subunits. Thus, their designation as nicotinic receptor

subunits remains unproven.

These results demonstrate that, in general, two different subunits—an and

a ß—are required to form a functional brain nicotinic receptor. The brain

a-subunits 2, 3, and 4 contain adjacent cysteines found in all muscle

a-subunits (see above for discussion), while the brain ß-subunits ß2, ß3, and

ß4 are missing these cysteines. We have called the non subunits ß2 and ß4

because they can functionally substitute for the muscle ß1 subunit to form

muscle-type nicotinic receptors (i.e., and ) (Boulter et al. 1987;

Duvoisin et al. 1989). On the basis of these results and by analogy with the

7
FIGURE 3. This figure shows the effect of two different neurotoxins on the

activation by acetylcholine of two neuronal nicotinic acetylcholine

receptor subtypes, 3ß2 and 4ß2. The voltage tracing on the

left shows the response before application of the toxin, and the

voltage tracing on the right shows the response following a brief

washing and a 30-minute incubation in the indicated

concentrations of the two toxins.

SOURCE: Boulter et al. 1987, copyright 1987, National Academy of Sciences

8
FIGURE 4. This figure shows the effect of two different neurotoxins on the

activation by acetylcholine of the neuronal nicotinic acetylcholine

receptor subtype 2ß2. The voltage tracing on the left shows the

response before application of the toxin, and the voltage tracing

on the right shows the response following a brief washing and a

30-minute incubation in the indicated concentrations of the two

toxins.

SOURCE: Wada et al. 1988, copyright 1988, American Association for the

Advancement of Science

we propose that the brain nicotinic receptors are a pentameric structure made

from and ß-subunits in some as yet unknown stochiometry. That we have

shown that the brain ß-subunits can function as part of the muscle nicotinic

receptor, which is known to be pentameric, supports our proposal.

We have used the patch-clamp technique to record from single receptor

molecules to characterize the biophysical properties of individual nicotinic

receptor subtypes. The single unit conductances and channel open times of

four subtypes have been analyzed thus far: 2ß2, 3ß2, 4ß2, and 4ß4.

These data demonstrate that each of the subtypes has unique biophysical

properties that can be used to identify the subtype (Papke et al. 1989). Each of

the six functional combinations of subunits forms a pharmacologically distinct

subtype that is activated by acetylcholine and nicotine and is resistant to

bungarotoxin (figures 3, 4, 6, and 7). The 3ß2 and 4ß2 subtypes are

9
A) ANTISENSE B) SENSE

FIGURE 5. In situ hybridization analysis. Rat forebrain and midbrain sections

were probed with PSI-radiolabeled antisense (A) or sense (B) ß2

RNA transcribed in vitro using a plasmid into which a 571bp Pst1/

EcoR1 fragment of PCX49 was subcloned.

10
Key: DLG=lateral geniculate nucleus (dorsal part); DG-dentate gyrus;

H=Ammon’s horn (hippocampus); IPN=interpeduncular nucleus;

MG=medial geniculate nucleus; MH=medial habenular nucleus;

NC=neocortex; PC=piriform cortex; PVN=paraventricular hypothalamic

nucleus; SON=supraoptic hypothalamic nucleus; SNC=substantia nigra,

pars compacta; SC=superior colliculus; ST=striatum; TH=thalamus;

Tu=olfactory tubercle; VTA=ventral tegmental area; VMH=ventromedial

hypothalamic nucleus

SOURCE: Deneris et al. 1988, copyright 1988, Cell Press

blocked by a toxin isolated from the venom of Bungarus multicinctus, called 3.1

toxin. This result is consistent with a ganglionic nicotinic-type pharmacology

(figure 3). However, the 2ß2 and 3ß4 receptor subtypes are resistant to the

3.1 toxin and, therefore, represent new receptor subtypes with a pharmacology

that has not been observed previously (figures 4 and 7) (Boulter et al. 1987;

Wada et al. 1988; Duvoisin et al. 1989).

One important and unexpected result is that, while the 3ß2 subtype is blocked

by 3.1 toxin, the 3ß4 subtype is resistant to this snake toxin (figure 7). This

was unexpected because it generally has been accepted that the subunit

contains the ligand- and toxin-binding site. This result shows that the ß-subunit,

at least in this case, determines the sensitivity to 3.1 toxin (Duvoisin et al.

1989).

To determine where the individual nicotinic receptors are expressed in the

brain, we have utilized the method of in situ hybridization to visualize the

distribution of expression of the mRNA coding for each nicotinic receptor

subtype. These experiments demonstrated that each of the three subunit

mRNA’s has a unique distribution of expression, consistent with the proposal

that they are part of three independent nicotinic receptor systems (figure 8).

The ß2 transcript is distributed throughout the brain, consistent with the

hypothesis that it is a common subunit used to form at least three different

receptor subtypes (figure 5). On the other hand, the ß4 transcript shows a

much more localized distribution of expression (figure 9). In general, the

distribution of mRNA coding for the nicotinic receptor family parallels the map of

nicotine binding (Boulter et al. 1986b, 1987; Goldman et al. 1986, 1987; Deneris

et al. 1988, 1989; Wada et al. 1988, 1989; Duvoisin et al. 1989).

The finding that these nicotinic receptor genes are expressed widely in the brain

indicates that the nicotinic receptor is a major excitatory system. In the past few

11
FIGURE 6. Electrophysiological recordings of Xenopus oocytes injected with

in vitro synthesized RNA encoding the nAChR’s subunits in the

indicated combinations. Representative responses induced by

acetylcholine and nicotine stimulations at the given concentrations

are shown. Potential measurements were monitored on a digital

voltmeter and recorded on a pen recorder (Gould). Voltage

traces were scanned and prepared for publication using a

personal computer.

SOURCE: Duvoisin et al. 1989, copyright 1989, Cell Press

12
FIGURE 7. Voltage recordings of Xenopus oocytes injected with 3 and

either ß2 or ß4 before and after exposure to an estimated

concentration of 0.1 mM 3.1 toxin. Experiments were performed

as described in figure 6.

SOURCE: Duvoisin et al. 1989, copyright 1989, Cell Press

years, physiologists using recently developed sophisticated techniques, such as

the slice preparation and patch-clamp recording methods, have found extensive

evidence for nicotinic receptor function in the brain, The in situ hybridization

results demonstrated that the ß2, ß3, and ß4 genes are expressed at

high levels in the medial habenula (figures 5, 8, and 9). The presence of

functional nicotinic receptors has been confirmed in the medial habenula by

intracellular recording techniques (McCormick and Prince 1987a). Nicotine also

has been shown to increase glucose utilization in the medial habenula (London

et al. 1988). There is now good evidence for functional nicotinic receptors in

many other regions of the brain that have high levels of nicotinic receptor gene

expression, These areas include the interpeduncular nucleus, the retina, the

lateral and medial geniculate, and the neocortex (Brown et al. 1984; Lipton et

al. 1987; Lipton 1988; McCormick and Prince 1987b; Vidal and Changeux

1989).

13
alpha2 alpha2

alpha3 alpha3

alpha4 alpha4

FIGURE 8. Comparison of the distribution of 2, 3, and 4 transcripts by in

situ hybridization histochemistry. Serial coronal sections through

the medial habenula (A) and the interpeduncular nucleus (B) were

hybridized with the probes for and . In (B) slides

contain sections of the trigeminal ganglion. Tissue preparation

and hybridization were performed with minor modifications.

Briefly, paraformaldehyde-fixed rat brain sections (25mm) were

mounted on poly-L-lysine-coated slides, digested with proteinase

K (10 mg/mL, 37°C, 30 min), acetylated, and dehydrated,

Hybridization with 35S-radiolabeled RNA probe (5-10x106

cpm/mL)

was performed at 55°C for 12 to 18 hours in a solution containing

14
50 percent formamide, 0.3M NaCI, 10mM Tris (pH 8.0), 1mM

EDTA, 0.05 percent tRNA, 10mM DTT, 1xDenhardt’s solution,

and 10 percent dextran sulfate. Because of the high sequence

similarities in the protein coding regions of the cDNA’s, 3’

untranslated sequences were used to make probes. The EcoRI/

3’ end, Ball/3’ end, and Bgll/3' end fragments derived from Cl 83,

PCA48, and 4-2 cDNA clones, respectively, were subcloned into

the plasmid, pSP65 and used to synthesize antisense RNA

probes in vitro. After hybridization, sections were treated with

RNaseA (20mg/mL, 37°C, 30 min) and washed in 0.1xSSC at

55°C. Dehydrated slides were exposed to x-ray films for 3 to 16

days at 4°C. An RNA probe coding the sense strand of C183

clone was used as a control.

Key: C=cortex; IPN=iterpeduncular nucleus; MH=medial habenula; MG=medial

geniculate nucleus; T=Thalamus

SOURCE: Deneris et al. 1988, copyright 1988, Cell Press

The discovery of a family of genes coding for nicotinic acetylcholine receptors

expressed in the brain brings for the first time the full power of molecular biology

to the study of this important receptor system. The existence of multiple

subtypes and the fact that they are expressed throughout the brain suggest that

the nicotinic receptor is a major excitatory system. The anatomical distribution

of these receptors now can be studied using specific antibodies to localize the

receptor subtypes in the brain. In situ hybridization methods can be used to

localize the cell bodies that synthesize the receptor protein. The regulation of

receptor gene expression can be studied under a variety of conditions.

Understanding the promoters of these receptor genes will provide insight into

how the brain builds, regulates, and maintains specific neural networks. At

present, there is little reliable pharmacological data on the properties of the

nicotinic acetylcholine receptor in the brain. The receptors now can be

expressed in the oocyte system where rigorous pharmacology can be

performed without the complications that are inherent in pharmacological

studies in the brain. By engineering mammalian cell lines, it will be possible to

study these receptors in several convenient cell systems to ask specific

questions about their function. The availability of the primary structure of the

brain and muscle receptors makes it possible to build models that relate

structure to pharmacology and function. These models can be tested by sitespecific mutagenesis. At present, the only ligand-gated ion channel protein that

can be isolated in milligram amounts is the Torpedo nicotinic receptor. Thus, it

15
FIGURE 9. Analysis by in situ hybridization histochemistry of the distribution

of ß4-transcripts. Adult rat brain coronal sections (30mm thick)

were hybridized with 35S-radiolabeled sense and antisense

probes derived from Kpnl- and BamHI-linearized pGDD15,

respectively. A. Only sections across the thalamus as the one

shown here gave above background signals by x-ray film

autoradiography. In parallel experiments, a sense probe was

hybridized to adjacent sections and gave background levels of

hybridization (data not shown). B. Dark-field microphotograph of

a medial habenula from an identical section to that shown in A

after emulsion dipping.

SOURCE: Duvoisin et al. 1989, copyright 1989, Cell Press

is the only candidate for structural studies leading to a complete, highresolution, three-dimensional structure. Because of the structural homologies

between the ligand-gated channels, it should be possible to apply the results of

the structural studies of the Torpedo nicotinic receptor to the muscle and brain

nicotinic receptors. It is very likely that these studies can be extended to the

glycine and GABA receptors and even to the glutamate receptor system, which

also may be a member of this ligand-gated channel superfamily.

Interesting mutations can be introduced into the receptors and expressed in

transgenic mice. The mutant mice then can be studied to gain insight into the

function of the nicotinic receptors. The diversity of structure and function in this

receptor system now can be studied with the long-range goal of understanding

the functional roles that the nicotinic receptors play in the nervous system.

16
At present, little is known about the roles of nicotinic receptor systems in the

brain. However, nicotine is one of the most widely consumed and addictive

drugs. Many behavioral effects have been observed, including mood changes,

effects on learning and memory, and weight loss (Wellman et al. 1986; for

review, see Clarke 1988). Under some conditions, nicotine is a relaxant; under

other conditions, nicotine is a stimulant. Many of these effects may be

beneficial, and this raises the possibility of designing better and more specific

nicotinic drugs (Luyten and Heinemann 1987).

Recently, another nicotinic receptor subtype has been identified in the rat

dorsalateral septal nucleus. Electrophysiological experiments indicate that

activation of this receptor leads to a hyperpolarization of the membrane

mediated by an increase in potassium conductance (Wong and Gallagher

1989). It will be interesting to see whether this receptor is a member of the

same family of nicotinic receptors that we have described. One can predict

that, if it is a member of the family, it will have an altered channel region.

The mammalian brain also contains another nicotinic receptor that binds

bungarotoxin (Patrick and Stallcup 1977). The receptors described above

are not blocked by this toxin, and the structure and function of the brain

bungarotoxin sites remain to be elucidated.

The nicotinic receptor system has been implicated in several serious health

problems. Nicotine is a widely consumed and addictive drug and is a major

factor in the present smoking epidemic. Behavioral studies have linked the

nicotinic receptor system to learning and memory, which is intriguing given the

finding that Alzheimer’s patients have a deficiency in memory function and

cortical nicotinic receptors (Perry et al. 1987). The possibility that one or more

of the nicotinic receptor subtypes is depressed in patients with Alzheimer’s

disease now can be explored, That in Alzheimer’s disease the number of

nicotine binding sites is depressed and the fraction of high- and low-affinity sites

is altered suggests that one nicotinic receptor subtype may be specifically

affected by the disease (Nordberg et al. 1988; Whitehouse et al. 1986).

The existence of receptor subtypes and the availability of cDNA clones may

make it possible to design new drugs that are subtype-specific that will prove

useful in the battle against smoking-related illnesses and Alzheimer’s disease.

The known ability of nicotine to affect mood suggests that new nicotinic drugs

also may prove useful in the treatment of various mood disorders.

17
Ballivet, M.; Nef, P.; Couturier, S.; Rungger, D.; Bader, C.R.; Bertrand, D.; and

Cooper, E. Electrophysiology of a chick neuronal nicotinic acetylcholine

receptor expressed in Xenopus oocytes after cDNA injection. Neuron 1:847-

852, 1988.

REFERENCES

Ballivet, M.; Patrick, J.; Lee, J.; and Heinemann, S. Molecular cloning of cDNA

coding for the gamma subunit of the Torpedo acetylcholine receptor. Proc

Natl Acad Sci U S A 79:4466-4470, 1982.

Boulter, J.; Connolly, J.; Deneris, E.; Goldman, D.; Heinemann, S.; and Patrick,

J. Functional expression of two neuronal nicotinic acetylcholine receptors

from cDNA clones identifies a gene family. Proc Natl Acad Sci U S A 84:

7763-7767, 1987.

Boulter, J.; Evans, K.; Goldman, D.; Martin, G.; Treco, D.; Heinemann, S.; and

Patrick, J. Isolation of a cDNA clone coding for a possible neural nicotinic

acetylcholine receptor alpha subunit. Nature 319:368-374, 1986b.

Boulter, J.; Goldman, D.; Evans, K.; Martin, G.; Mason, P.; Stengelin, S.;

Heinemann, S.; and Patrick, J. Isolation and sequence of cDNA clones

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ACKNOWLEDGMENTS

The work reviewed in this chapter was supported by National Institute of

Neurological and Communicative Disorders and Stroke grant 5 R01 NS-11549-

18 (SH and JP) and a grant from the Muscular Dystrophy Association of

America (SH and JP).

21
AUTHORS

Stephen F. Heinemann, Ph.D.

Professor

Jim Boulter, Ph.D.

Senior Research Associate

John Connolly, Ph.D.

Postdoctoral Fellow

Evan Deneris, Ph.D.

Postdoctoral Fellow

Robert Duvoisin, Ph.D.

Postdoctoral Fellow

Melissa Hartley, B.S.

Research Assistant

Irm Hermans-Borgmeyer, Ph.D.

Postdoctoral Fellow

Michael Hollman, Ph.D.

Postdoctoral Fellow

Anne O’Shea-Greenfield

Research Assistant

Roger Papke, Ph.D.

Postdoctoral Fellow

Scott Rogers, Ph.D.

Postdoctoral Fellow

Molecular Neurobiology Laboratory

The Salk Institute

P.O. Box 85800

San Diego, CA 92186-5800

Jim Patrick, Ph.D.

Professor and Head

Division of Neuroscience

22
Baylor College of Medicine

One Baylor Plaza

Houston, TX 77030

23
Regulation of Acetylcholine Receptor

Gene Expression

Alex M. Simon, Emma K. Dutton, and Steven J. Burden

INTRODUCTION

The skeletal muscle acetylcholine receptor (AChR) is a ligand-gated channel

composed of four structurally related subunits that assemble into a pentamer

(Karlin 1980; Anderson 1987). The genes encoding the

subunits are activated coordinately during embryonic development as

myoblasts withdraw from the cell cycle and fuse to form multinucleated

myotubes (Baldwin et al. 1988). Following activation of AChR genes during

myogenesis, expression of these genes is regulated subsequently by

physiological signals that include the pattern and intensity of muscle cell

electrical activity (Merlie and Kornhauser 1989; Tsay and Schmidt 1989). To

understand how AChR genes are regulated by physiological signals in mature

myofibers, knowledge of how these genes are activated initially during

development is required. Thus, the authors’ initial studies have focused on the

mechanisms involved in activation of the AChR delta subunit gene during

myogenesis.

Analysis of the regulation of skeletal muscle genes provides a particularly

attractive system to understand mechanisms that are responsible for cell-type

specific gene expression. In particular, identification of a small family of

myogenic basic-helix-loop-helix proteins (e.g., myoD1) that is capable of

altering the fate of mesodermal cells and activating the myogenic phenotype

provides one of the clearest examples in vertebrates of genes that are critical

for early decisions in cell determination and differentiation (Davis et al. 1987;

Wright et al. 1989; Edmondson and Olson 1989; Weintraub et al. 1991). An

understanding of how these myogenic basic-helix-loop-helix proteins activate

the myogenic program is likely to provide an important framework for

understanding the mechanisms that control cell-type specific gene expression

and cell differentiation.

24
RESULTS AND DISCUSSION

We have characterized the muscle-specific regulatory region of the AChR delta

subunit gene by transfecting gene fusions between the delta subunit gene and

the human growth hormone (hGH) gene into cell lines and assaying expression

of hGH. These studies have shown that ~500-bp of 5’ flanking DNA from the

delta subunit gene (nucleotides -501 through +24) is necessary and sufficient

for muscle-specific gene expression.

Mutational analysis reveals that this cis-acting regulatory region contains three

elements that collectively limit activation of the delta subunit gene to myotubes.

One element, an enhancer, is necessary for maximal gene expression in

muscle cells, but does not confer muscle-specific expression; rather, this

enhancer is similarly active in muscle and nonmuscle cells. The other two

regulatory elements limit enhancer activity to myotubes and are responsible for

muscle-specific gene expression. One of these muscle-specificity elements is

necessary for repressing the delta subunit gene in nonmuscle cells and is not

required for activating the gene in myotubes. The other muscle-specificity

element, a binding site for myogenic basic-helix-loop-helix proteins, is required

both for activating the delta subunit gene in myotubes and for repressing the

gene in other cell types.

Currently, the authors are identifying regulatory elements that control gene

expression by electrical activity, producing transgenic mice harboring a gene

fusion between ~1.8 kbp of 5’ flanking DNA from the delta subunit gene and the

hGH gene, and showing that hGH RNA levels are low in innervated skeletal

muscle and high in denervated muscle. Thus, 1.8 kbp of 5’ flanking DNA from

the delta subunit gene contains the control elements that are necessary to

confer innervation-dependent gene regulation.

We have also established a cell culture system to study electrical activitydependent regulation. Primary rat myotubes, which form in cell culture, are

electrically active, and this spontaneous activity can be abolished by

tetrodotoxin, which blocks action potentials. We have transfected primary

myotubes with delta subunit-hGH gene fusions and have shown that ~1.8 kbp

of 5’ flanking DNA from the delta subunit gene is sufficient to confer electrical

activity-dependent gene expression in this cell culture system. We are

exploiting these two systems further to delineate more precisely the critical cisacting regulatory elements and to characterize the regulatory pathway that

couples changes in the pattern of electrical activity to alterations in gene

expression.

25
REFERENCES

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Regulation of acetylcholine receptor transcript expression during

development in Xenopus laevis. J Cell Biol 106:469-478, 1988.

Davis, R.L.; Weintraub, H.; and Lassar, A.B. Expression of a single transfected

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Edmondson, D.G., and Olson, E.N. A gene with homology to the myc similarity

region of MyoD1 is expressed during myogenesis and is sufficient to activate

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Karlin, A. Molecular properties of nicotinic acetylcholine receptors. In:

Cotman, C.; Post, G.; and Nicolson, G., eds. The Cell Surface and Neuronal

Function. Amsterdam, NY: Elsevier-North Holland, 1980. pp. 191-260.

Merlie, J.P., and Kornhauser, J.M. Neural regulation of gene expression by an

acetylcholine receptor promoter in muscle of transgenic mice. Neuron 2:

1295-1300, 1989.

Tsay, H.J., and Schmidt, J. Skeletal muscle denervatlon activates acetylcholine

receptor genes. J Cell Biol 108:1523-1526, 1989.

Weintraub, H.; Davis, R.; Tapscott, S.; Thayer, M.; Krause, M.; Benezra, R.;

Blackwell, T.K.; Turner, D.; Rupp, R.; Hollenberg, S.; Zhuang, Y.; and

Lassar, A.B. The myoD gene family: Nodal point during specification of the

muscle cell lineage. Science 251:761-766, 1991.

Wright, W.E.; Sassoon, D.A.; and Lin, V.K. Myogenin, a factor regulating

myogenesis, has a domain homologous to MyoD. Cell 56:607-617, 1989.

ACKNOWLEDGMENT

This work was supported by National Institutes of Health grant NS-27963.

AUTHORS

Alex M. Simon, B.S.

Emma K. Dutton, Ph.D.

Postdoctoral Fellow

Steven J. Burden, Ph.D.

Associate Professor

26
Biology Department

Massachusetts Institute of Technology

Building 16, Room 820

Cambridge, MA 02139

27
Mutations Affecting Local Anesthetic

Block of the Nicotinic Acetylcholine

Receptor Ion Channel

Reid J. Leonard, Pierre Charnet, Cesar Labarca, Nancy J.

Vogelaar, Linda Czyzyk, Annie Gouln, Norman Davidson, and

Henry A. Lester

A common goal in molecular pharmacology is to understand how the structure

of a complex transmembrane protein determines its behavior. One such protein

is the nicotinic acetylcholine receptor (AChR), which transduces a chemical

signal (binding of ACh) into an electrical signal (change in the postsynaptic

membrane potential) by acting as a ligand-gated pore for the conduction of ions

across the cell membrane (for review, see Popot and Changeux 1984; Stroud

and Finer-Moore 1985). The functional AChR is an oligomer composed of four

homologous subunits that form a pentamer with stoichiometry (Raftery et

al. 1980). Functional AChR’s are expressed in Xenopus oocytes by injecting

them with a mixture of mRNA transcripts from cDNA’s encoding each of the

subunits. Site-directed mutagenesis (Kunkel et al. 1987) is used to alter the

coding sequence of the subunit cDNA’s, ultimately expressing “mutated”

AChR’s in the plasma membrane of the oocyte. By comparing the

electrophysiological characteristics of the mutated AChR’s with those of the

unaltered channels, inferences can be drawn about the roles that various

portions of the primary sequences play in shaping the behavior of the normal

receptor-channel molecule.

We focused on a region of the primary sequence that had been implicated by

the elegant experiments of Hucho and colleagues (1986) and Giraudat and

coworkers (1986, 1987) as forming part of the ion conduction pathway of

the Torpedo AChR. Those experiments used channel-blocking compounds as

photoaffinity reagents to map the binding site for noncompetitive antagonists.

The drugs were attached covalently to the receptors under conditions that

favored the open channel conformation, and the covalently modified receptors

were subjected to peptide cleavage and microsequencing analysis. The

labeled residues were determined to lie within a proposed membrane-spanning

helix (M2) of the ß, and subunits. The stoichiometry of binding of the

photoaffinity reagents to the AChR was 1:1; however, the label was

28
incorporated into residues occupying homologous positions in at least three of

the four subunit polypeptides. These results supported a hypothesized

structure (Noda et al. 1983a; Claudio et al. 1983; Devillers-Thiery et al. 1983)

for the ion channel of the AChR in which homologous domains (in this case, the

M2 helix) from each of the subunit polypeptides contributed equally to form the

walls of the pore, similar to staves of a barrel.

To test this hypothesis, we altered the amino acid side chains at the

homologous positions in the mouse muscle AChR subunits (figure 1) and

looked for changes in the properties of the channel, measured

electrophysiologically. In particular, we wanted to examine the effects of such

mutations on the interaction between the channel and a local anesthetic analog,

with the hope that the drug could act as a probe for the lumen of the channel.

(For details on experimental methods, see Leonard et al. 1988 and Charnet et

al. 1990.) One advantage of working with AChR’s expressed from mouse

cDNA’s, rather than Torpedo, Is that the effects of local anesthetics on the

native mammalian AChR’s have been studied extensively (Neher and

Steinbach 1978; Neher 1983).

Local anesthetics interact with nicotinic acetylcholine receptors in a

noncompetitive fashion (with respect to agonist binding) to interfere with ion flux

through the channel. A simple model for this interaction is given below:

where R=AChR; Q=blocker; ß is the channel opening rate; a is the channel

closing rate; and G and F are the respective rates for blocking and unblocking.

This model proposes that local anesthetics block current through the channel by

entering the pore and plugging it, much like a stopper in a bottle. Two important

features of this model are that the blocker cannot enter the channel until it

opens and that the channel is not free to close while the blocking molecule

resides in the pore. In the case of the permanently charged quaternary amine

derivative of lidocaine, QX-222, these conditions appear to hold true (Neher

1983). Many other noncompetitive antagonists of the AChR, however, have

actions that are not limited to those proposed for a simple “open channel

blocker” (Changeux et al. 1986).

29
FIGURE 1. Aligned amino acid sequences of the M2 hydrophobic domains of

the mouse skeletal muscle nicotinic acetylcholine receptor

subunits, Residues are numbered 1’ through 19' with 1’ at the

presumed cytoplasmic end of the transmembrane helix and 19’ at

the extracellular end. The boxed residues are hypothesized to

face the lining of the ion channel pore.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

The most compelling evidence for the action of QX-222 on the pore of the

AChR comes from direct observations of the current through individual channel

proteins using the giga-seal “patch clamp” technique. Such recordings (figure

2) show that addition of QX-222 to the extracellular face of the pore transforms

the normal open state of the channel into a burst of rapid transitions between

the conducting and blocked states. Kinetic analysis of channel activity before

and during exposure to QX-222 yields an estimate of the affinity of QX-222 for

the open channel. The forward rate of block (G in equation 1) can be

determined from measurements of mean open time at various concentrations

of blocker, according to the formula
FIGURE 2. Effect of QX-222 on ACh-induced single channel currents.

Recordings made from a patch of membrane excised (“outsideout”) from a Xenopus oocyte that had been injected 48 hours

earlier with mRNA transcripts of cDNA’s encoding the mouse

skeletal muscle AChR subunits. Channel openings are downward

deflections in the traces. Left: Channel openings in the presence

of 0.5 μm ACh. Right: Same patch after addition of 10 4M QX222 to the bath. Inset: Structure of QX-222.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

which simply states that the reciprocal of the open time is the sum of the rates

for leaving the open state. As stated in equation 1, is the channel closing rate

measured in the absence of QX-222. The unblocking rate can be determined

from the mean blocked time within a burst

31
and is independent of blocker concentration. The apparent equilibrium

dissociation constant of QX-222 for the open channel, therefore, may be

calculated

KQ=F/G. (4)

The effect of QX-222 also can be measured macroscopically, on a cell’s entire

population of AChR channels. At low ACh concentrations, voltage-jump

relaxations (Adams 1977) provide an independent measure of the total lifetime

of the agonist-activated state, including brief nonconducting periods (such as

those induced by QX-222). As mentioned above, the channel is not free to

close while the QX-222 is in the pore. The burst duration is therefore prolonged

in the presence of blocker by an amount equal to the sum of the individual

blocked times. This property of block of AChR’s by QX-222 also was observed

directly from single channel records: Neher and Steinbach (1978) and Neher

(1983) noted that the integral open time during a burst is unaffected by the

presence of the blocker. That is, the sum of the conducting periods within a

burst induced by the rapid blocking and unblocking by QX-222 is the same as

the normal duration of the uninterrupted burst in the absence of blocker. The

“clock” (the conditional probability that the channel will enter the closed state)

only runs when the channel is unblocked. This prolongation of the burst

duration increases the time constant of an exponential voltage-jump relaxation

as follows:

where are the decay time constants of voltage jumps in the presence

and absence of QX-222, respectively. KQ, the apparent dissociation constant

for QX-222, therefore, is obtained easily by comparing the decay constants

before and after addition of blocker. An example of the effect of QX-222 on

AChR voltage-jump relaxations is shown in figure 3.

Using the two complementary approaches outlined above, we examined the

effect of amino acid substitutions in the M2 domain upon the interaction

between the channel and QX-222. For each new mutant construct, we first

used single channel recording to measure the mean blocked times and to verify

the open channel block model of QX-222 as given in equation 1. Following

that, it was much more efficient to use macroscopic recording to measure KQ

32
FIGURE 3. Prolongation of voltage-jump relaxation of ACh-induced currents

by QX-222. Whole-cell recording from a Xenopus oocyte that had

been injected 48 hours earlier with mRNA transcripts of cDNA’s

encoding the mouse skeletal muscle AChR subunits. Upper:

Currents elicited in the presence of 1 μM ACh before and during

addition of QX-222 to the bath. QX-222 reduces the initial

amplitude of the current elicited by the jump to -150 mV and

prolongs the decay phase. Lower: Voltage-clamp protocol used

to elicit the currents.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

from the various mutants. In all cases, the values obtained from single channel

and macroscopic recording were in good agreement.

The first series of mutations were carried out at the position where photoaffinity

reagents bound in the Torpedo sequence. This position (6’ in figure 1) contains

serines in every subunit of Torpedo (Sumikawa et al. 1982; Noda et al. 1982,

1983b; Ballivet et al. 1982; Claudio et al. 1983), but only in the and ß subunits

of the mouse AChR (Boulter et al. 1985, 1986; LaPolla et al. 1984). Our first

mutation was to change the -Ser6, to Ala, substitution from a polar to a

nonpolar residue. The result of this mutation is shown in figure 4.

33
FIGURE 4. Comparison of average residence time of QX-222 in normal vs.

mutated AChR’s expressed in oocytes. Residence time of the

blocker equals the time constant of an exponential fit to the

histogram of nonconducting periods (blocked times) caused by

QX-222.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

34
The residence time of QX-222 for the AChR’s containing the mutation was

decreased by -40 percent relative to nonmutated AChR’s. The apparent

dissociation constant for QX-222 (KQ) derived from voltage jumps to -130 mV

increased from 27 μM to 45 μM. The second mutation, produced an

increase in QX-222 residence time and a decrease in KQ, but the magnitude of

the change was twice as large as that seen for the mutation (KQ=62 μM).

This was interesting, since a mutation in a produces two serine to alanine

substitutions per pentamer, compared with the single substitution achieved with

the mutant. For receptors containing both mutated and mutated together,

the effects were additive (KQ=134 μM for The final mutation

constructed at site 6’ replaced the phenylalanine of the ß subunit with a serine

to produce AChR’s with a total of four serines at position 6’. This

mutant, in contrast to the ones in which the number of serines was reduced,

exhibited an increased residence time for QX-222 and a decrease in the KQ to

23 μM.

The effects of the site 6’ mutations on affinity for QX-222 can be summarized

(figure 5) as follows: Polar to nonpolar substitutions decreased the affinity of

the open channel for QX-222 in a graded fashion, with each successive serine

to alanine substitution producing a lower affinity, while the nonpolar to polar

substitution (Phe to Ser) had the opposite effect. These results indicate that the

serines normally present at site 6’ exert a stabilizing influence on the binding of

QX-222 to the pore, presumably via favorable interactions between the

quaternary ammonium group of the blocker and the polar (and net

electronegative) -OH side chains. Replacement of the hydroxyl side chains by

hydrogen (in the Ser to Ala mutation) therefore renders the channel less

attractive to the blocker. It is important to note that while the change in free

energy of the QX-222 binding produced by the mutations amounts to only 0.2

kcal/mol/serine (much less than even a single hydrogen bond), the aggregate

stabilizing influence of all the residues forming the annulus of the channel at site

6’ is comparable to that exerted by a 50 mV hyperpolarization of the membrane.

Additional evidence that the residues at position 6’ lie within the ion conduction

pathway comes from measurements of the single channel conductance. None

of the single-subunit mutations affected the conductance of the channel, but the

conductance of the channels, in which no polar residues remained

at site 6’, exhibited a dramatic change: The conductance for ions flowing in the

inward direction (extracellular to cytoplasmic) was unaffected, but the

conductance for current in the outward direction was reduced by half (figure 6).

A change such as that observed would be expected from an increased barrier

to permeation (Lauger 1976; Hille 1984) toward the intracellular end of the pore.

Hydroxyl side chains have been shown to facilitate conduction through synthetic

pore-forming peptides (Lear et al. 1988), perhaps because they can interact

35
FIGURE 5. Effect of mutations (Ser->Ala or Phe->Ser) at site 6’ on the

apparent equilibrium binding constant for the interaction between

QX-222 and the open AChR channel. KQ was determined from

macroscopic voltage jumps and single channel recording. The

arrow on the abscissa represents the number of serines normally

present at site 6’. Removal of serines lowers the affinity for QX222.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

with the hydration shells of permeant ions. Removal of the -OH groups at site 6

could then restrict the ability of ions to enter the channel at the intracellular end,

thus producing the observed rectification in channel conductance.

Charged molecules, such as ions or local anesthetics, within ion channel sense

the transmembrane potential. The degree of voltage dependence of block by

QX-222 in muscle AChR’s (Neher and Steinbach 1978) suggests that the

blocker travels approximately two-thirds of the distance across the membrane

field to reach its binding site. The same voltage dependence was observed in

the AChR’s expressed in oocytes, and this value was unaffected by our

mutations. Although it is incorrect to equate electrical distance across the field

with physical distance across the membrane, the lack of change in voltage

36
FIGURE 6. Change in single channel conductance of AChR’s caused by

removal of all serines at position 6’. The amplitude of the current

through ACh-gated channels is plotted against the voltage across

the membrane. Open Circles: Normal mouse AChR’s expressed

in Xenopus oocytes. Closed Squares: Mutated receptors

The selective decrease in the conductance for

outward current (positive limb) implies a change in an energy

barrier at the cytoplasmic end of the pore.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

dependence implies that the mutations affected only the affinity, and not the

site, of the QX-222 binding.

Several lines of evidence therefore converge to support the hypothesis that the

residues of the proposed M2 helix form part of the lining of the ion channel: Site

37
6’ is predicted from hydropathy (Kyte and Doolittle 1982) analysis to lie at the

cytoplasmic end of a transmembrane helix. The voltage dependence of QX222 block places the binding site for the drug toward the cytoplasmic end of the

pore. Photoaffinity labeling of the Torpedo AChR by channel-blocking drugs

occurs at site 6’. Mutations of the mouse AChR subunits at site 6’ change the

apparent affinity of QX-222 for the open channel, suggesting that those

residues form part of the drug’s binding site. Replacement of all the polar

serines at site 6’ by nonpolar alanines reduces the channel conductance for

cations entering the cytoplasmic end of the pore.

To test this hypothesis and extend our studies of the interaction between the

channel and QX-222, we performed a series of mutations at site 10’ (figure 1),

which in an helix should be approximately one full turn away from site 6’ and

therefore would be expected to contribute to the lining of the pore. When we

examined the effects of polar to nonpolar substitutions at site 10’, we observed

changes in QX-222 block that were of the same magnitude as those for the site

6’ mutations, but in the opposite direction. For example, the mutant channel

exhibited a longer residence time for QX-222 block and a lower KQ

value compared with controls. Figure 7 shows the effect of the entire series of

site 10’ mutations on the affinity of the open channel for QX-222. Polar to

nonpolar substitutions increased the affinity of the open channel for QX-222 in a

graded fashion, with each successive serine or threonine to alanine substitution

producing a higher affinity, while the nonpolar to polar substitution (Ala to Ser)

decreased the affinity. Once again, there was no change in the voltage

dependence of the block by QX-222. The single channel conductance was not

altered by any combination of site 10’ mutations tested.

An explanation for the opposite influence of polar to nonpolar substitutions at

positions 6’ vs. 10’ is suggested by the structure of QX-222. As shown in the

inset to figure 2, QX-222 consists of a nonpolar aromatic moiety whose center,

in CPK modeling, is separated by 5-6 from the positively charged quaternary

amine. This spacing (or its integral multiple) between aromatic and amine

moieties is a common structural feature of many anesthetics (for reviews, see

Ritchie and Greengard 1966; Courtney and Strichartz 1987). Since the 5-6

spacing coincides with the repeat distance of one face of an helix, we

propose that QX-222 “partitions” itself within the lumen of the channel as shown

in figure 8, with the quaternary amine interacting with the site 6’ residues and

the aromatic ring in the vicinity of site 10’.

The clinical targets of local anesthetics are the voltage-gated sodium and

potassium channels of heart and nerve. If our model of QX-222 binding is

correct, then at least some of the structural features proposed for the lining of

the AChR pore, such as rings of amino acids with polar side chains, also should

38
FIGURE 7. Effect of mutations (Ser/Thr->Ala or Ala->Ser) at site 10’ on the

apparent equilibrium binding constant for the interaction between

QX-222 and the open AChR channel. KQ was determined from

macroscopic voltage jumps and single channel recording. The

arrow on the abscissa represents the number of serines and

threonines normally present at site 6’. Removal of serines raises

the affinity for QX-222.

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

be found in the lumen of Na+ and K

+

 channels. With the availability of cDNA

clones for several subtypes of these channels becoming available, it will be

possible to study the effects of structural changes on the action of local

anesthetics on these channels as well. Experiments similar to those conducted

on the nicotinic AChR may help to identify structural elements comprising the

lumen of other ion channels. Furthermore, it will be possible to test the

generality of the mechanism of local anesthetic binding proposed here.

39
FIGURE 8. Schematic model of the interaction between QX-222 and adjacent

turns of helical M2 domains of the AChR subunits. The M2

domains from all five subunits of the channel are

presumed to contribute equally to the lining of the pore. Only the

 helices are shown in the drawing. In this model, the

blocker can enter the channel only from the extracellular side and

can go no deeper than the level of the 6’ residues,

SOURCE: Leonard et al. 1991, copyright 1991, Annals of the New York

Academy of Sciences

40
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Stengelin, S.; Martin, D.; Heinemann, A.; and Patrick, J. Isolation of a clone

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Changeux, J.-P.; Pinset, C.; and Ribera, A.B. Effects of chlorpromazine and

phencyclidine on mouse acetylcholine receptor kinetics. J Physiol 378:497-

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Charnet, P.; Labarca, C.G.; Leonard, R.J.; Vogelaar, N.J.; Czyzyk, L.; Gouin,

A.; Davidson, N.; and Lester, H.A. An open-channel blocker interacts with

adjacent turns of -helices in the nicotinic acetylcholine receptor. Neuron

4:87-95, 1990.

Claudio, T.; Ballivet, M.; Patrick, J.; and Heinemann, S. Nucleotide and

deduced amino acid sequences of Torpedo californica acetylcholine receptor

gamma subunit. Proc Natl Acad Sci U S A 80:1111-1115, 1983.

Courtney, K.R., and Strichartz, G.R. Structural elements which determine local

anesthetic activity. In: Strichartz, G.R., ed. Handbook of Experimental

Pharmacology. Vol. 61. local Anesthetics. Berlin: Springer-Verlag, 1987.

pp. 53-94.

Devillers-Thiery, A.; Giraudat, J.; Bentaboulet, M.; and Changeux, J.-P.

Complete mRNA coding sequence of the ACh binding alpha subunit from T.

marmorata AChR. A model for the transmembrane organization of the

polypeptide chain. Proc Natl Acad Sci U S A 80:2067-2071, 1983.

Giraudat, J.; Dennis, M.; Heidmann, T.; Chang, J.Y.; and Changeux, J.P.

Structure of the high-affinity binding site for noncompetitive blockers of the

acetylcholine receptor: Serine-262 of the delta subunit is labeled by

[3H]chlorpromazine. Proc Natl Acad Sci U S A 83:2719-2723, 1986.

Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P.T.; Lederer, F.; and

Changeux, J.-P. Structure of the high-affinity binding site for noncompetitive

blockers of the acetylcholine receptor: [3H]Chlorpromazine labels

homologous residues in the beta and delta chains. Biochemistry 26:2410-

2418, 1987.

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Hille, B. Ionic Channels in Excitable Membranes. Sunderland, MA: Sinauer,

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acetylcholine receptor is formed by the homologous helices M-II of the

receptor subunits. FEBS Lett 205:137-142, 1986.

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Evidence that the M2 membrane-spanning region lines the ion channel pore

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5822, 1982.

AUTHORS

Reid J. Leonard, Ph.D.

Senior Research Biochemist

Department of Membrane Biochemistry and Biophysics

Merck Institute for Therapeutic Research, 80N-31C

P.O. Box 2000

Rahway, NJ 07065

Pierre Charnet, Ph.D.

Research Fellow

Cesar Labarca, Ph.D.

Research Scientist

Linda Czyzyk, M.S.

Research Assistant

Annie Gouin, B.S.

Research Assistant

Norman Davidson, Ph.D.

Professor Emeritus of Chemistry and Biology

Henry A. Lester, Ph.D.

Professor of Biology

43
Department of Biology, 156-29

California Institute of Technology

Pasadena, CA 91125

Nancy J. Vogelaar, Ph.D.

Research Fellow

Department of Chemistry

Princeton University

Princeton, NJ 08554

44
Molecular Biology of the Dopamine

D2 Receptor

Olivier Civelli, James Bunzow, Paul Albert, Hubert Van Tol, and

David Grandy

INTRODUCTION

Dopamine is the dominant catecholamine neurotransmitter in the mammalian

brain. It is found throughout the central nervous system but is predominant in

the nigrostriatal, mesocorticolimbic, and tuberoinfundibular tracts (Creese et al.

1983). Dopamine exerts its effects through binding to two types of receptor, the

D1 and D2 receptors (Kebabian and Caine 1979). Binding of dopamine to its

receptors induces several second messenger systems, most importantly

affecting cAMP levels (Vallar and Meldolesi 1989). Activation of the D1 receptor

stimulates adenylyl cyclase activity, which results in an increase in intracellular

cAMP levels, whereas binding of dopamine to the D2 receptor inhibits the

cyclase activity (Caron et al. 1978).

The importance of the dopamine D2 receptor is evidenced by the number of

physiological activities it modulates, including control of movement,

maintenance of emotional stability, and regulation of prolactin secretion (Hess

and Creese 1987). The D2 receptor has been implicated specifically in the

pathophysiology of most of the dopamine-associated disorders. It has been

proposed that the D2 dopamine receptor is involved in the etiology of

schizophrenia (Seeman 1987), Parkinson’s disease, and Tourette’s syndrome.

In this respect, it is noteworthy that many drugs used in treating mental

disorders have high affinity for the D2 receptor site, making this receptor the

focus of numerous studies (Seeman and Lee 1975). Though progress has

been made in understanding the pharmacology and physiology of the D2

receptor, its biochemical characterization has been difficult. Only recently has

the D2

 dopamine receptor been purified to homogeneity (Senogles et al. 1988).

We discuss here our studies on the molecular cloning and expression of the rat

brain dopamine D2

 receptor.

45
CLONING OF THE RAT DOPAMINE D2

 RECEPTOR

With the cloning of the ß2-adrenergic receptor in 1986 (Dixon et al. 1986), it was

observed that its sequence was very similar to rhodopsin, the retinal receptor.

Since both receptors are transduced by G proteins, a new concept evolved that

proposed that G-protein-coupled receptors might share certain sequence

similarities with each other and thus be part of a large gene family.

Indeed, with the recent cloning of several other G-protein-coupled receptors

(Stevens 1987; Hall 1987; Dohlman et al. 1987; Masu et al. 1987; Julius et al.

1988), it is clear that all these receptors are evolutionarily related. In particular,

it was shown that G-protein-coupled receptors share three structural

characteristics: seven hydrophobic domains, about two dozen conserved

amino acid residues (some of which might be targets of posttranslational

modifications), and a significant degree of sequence similarity at both the

peptide and the nucleotide levels. It should be noted that not all the G-proteincoupled receptors necessarily will share these characteristics; but to date, all

the proteins having these structural characteristics are G-protein-coupled

receptors.

Bunzow and colleagues (1988) embarked on the cloning of G-protein-coupled

receptors by taking advantage of their sequence homology. They used the ß2

-

adrenergic receptor coding sequence as a hybridization probe to screen a rat

genomic library under nonstringency hybridization conditions and were able to

isolate numerous clones. One, RGB-2, was studied in detail.

The RGB-2 genomic fragment was found to hybridize to a 2.8 kb rat brain

mRNA and was used subsequently to screen a rat brain cDNA library. One

clone containing a 2.5 kb insert was sequenced and its corresponding peptide

sequence was determined. This clone encodes a 415 amino acid protein with

all the expected characteristics of a G-protein-coupled receptor: It has seven

hydrophobic domains, the 21 amino acid residues conserved among all cloned

G-protein-coupled receptors and potential glycosylation and phosphorylation

sites, and a significant degree of sequence similarity with the other receptors in

this gene family. Therefore, it appeared that RGB-2 could be a G-proteincoupled receptor.

PHARMACOLOGY OF THE CLONED DOPAMINE D2 RECEPTOR

To determine the ligand specificity of RGB-2, we first analyzed the tissue

distribution of its mRNA to relate RGB-2’s expression to the distribution of a

known receptor. Northern blot analysis showed that RGB-2 mRNA sequences

are expressed throughout the rat brain, with the highest levels found in the

46
striatum. We also determined that RGB-2 mRNA is present in high levels in the

intermediate lobe of the pituitary and at much reduced levels In the anterior

lobe. These data suggested that RGB-2 could encode a dopamine D2 receptor.

The full-length RGB-2 cDNA then was cloned into a plasmid containing the

metallothionein promoter, and this construct was cotransfected with pRSVneo

(a selectable marker conferring resistance to the antibiotic neomycin) into

mouse Ltk- cells. This fibroblast cell line does not express endogenous mouse

RGB-2 mRNA sequences. Stable transfectants expressing RGB-2 were

isolated, and membranes from one of these clones, L-RGB2Zem-1 (also called

LZR-1, Neve et al. 1989) were prepared and analyzed for their ability to bind

dopamine ligands (Bunzow et al. 1988).

L-RBG2Zem-1 membranes bound dopamine D2

 agonists and antagonists with

the same pharmacological profile as do rat striatal membranes. These studies

used the antagonist [

3H]-spiperone whose binding was shown to be saturable

(950 fmol/mg protein) and of high affinity (48 pM). [

3H]-Spiperone binding to LRGB2Zem-1 membranes was displaced by several antagonists with the

stereospecificity expected of a D2

 receptor and with the same K1‘s as

determined in rat striatal membranes. Finally, as determined using compounds

that detect more than one receptor type, the transformed Ltk- cells expressed

only one type of receptor. Therefore, we had demonstrated that RGB-2

encodes a protein that possesses the D2

 receptor binding characteristics. The

next step was to show that this D2 receptor was functional (i.e., that it couples to

a second messenger system).

G-PROTEIN COUPLING AND SECOND MESSENGER INDUCTIONS

D2 receptors are present on lactotroph cells of the anterior pituitary where they

regulate prolactin secretion. The somatomammotroph cell line GH4C1 is

derived from a rat pituitary tumor and is known to secrete prolactin. This cell

line, however, does not bind dopamine and therefore represents an excellent

cell system in which to study exogenously expressed D2 receptor activity.

GH4C1 cells were transfected with the RGB-2 cDNA metallothionein

construction, and several stably transformed cells were cloned and raised.

One, GH4ZR7, was found to express high levels of RGB-2 mRNA. Membranes

prepared from GH4ZR7 cells bound PHI-spiperone with saturable kinetics, a Kd

of 96 pM, and a Bmax of 2.3 pu/mg protein (Albert et al. 1989).

To analyze second messenger coupling, we first showed that dopamine inhibits

PHI-spiperone binding with an IC50 of 49 μM and a Hill coefficient of 0.69. A Hill

coefficient less than unity is suggestive of high- and low-affinity binding sites.

47
Addition of GTP and NaCl increased dopamine IC50 value twofold (109 μM) with

a Hill coefficient approaching unity (0.93). Thus, the presence of GTP and NaCl

converted the dopamine receptor from a population of high- and low-affinity

receptors to the low-affinity state. These results indicated that the cloned

dopamine receptor interacts with G proteins.

We next analyzed the effects of dopamine binding on the levels of intracellular

and extracellular cAMP. Since the D2 receptor is expected to inhibit cAMP

levels, vasointestinal peptide (VIP) was used first to stimulate endogenous

cAMP production. Dopamine inhibited both basal and VIP-stimulated cAMP

levels in media from GH4ZR7

 cells. Furthermore, intracellular CAMP levels were

inhibited, albeit at a less pronounced level, probably due to the lower recovery

of intracellular cAMP. The stereospecificity of these inhibitions was

demonstrated using isomers of sulpiride: The active enantiomer (-)sulpiride

blocked the inhibition while (+) sulpiride had no effect.

To demonstrate that the changes in cAMP levels were the result of an inhibition

of adenylyl cyclase, dopamine was added to membranes of VIP- or forskolinstimulated GH4ZR7

 cells, and adenylyl cyclase activity was measured.

Dopamine inhibited adenylyl cyclase activity by 45 percent. This inhibition was

stereoselective, since the agonist quinpirole was active while its enantiomer

LY181990 did not have any significant effect. Moreover, since receptors that

couple to inhibitory G proteins are known to be sensitive to pertussis toxin,

adenylyl cyclase activity was measured in membranes prepared from GH4ZR7

cells pretreated with pertussis toxin and stimulated with forskolin or VIP.

Pertussis toxin was able to uncouple dopamine-mediated inhibition of adenylyl

cyclase and dopamine-stimulated cAMP accumulation. Therefore, the D2

receptor expressed in GH4ZR7 cells is capable of inhibiting adenylyl cyclase

activity through a pertussis-toxin-sensitive mechanism.

Finally, the inhibition of prolactin (PRL) secretion by dopamine was assayed in

GH4ZR7 cells. VIP and thyrotropin-releasing hormone (TRH) are known to

enhance PRL release by a CAMP-dependent and a CAMP-independent

mechanism, respectively. Dopamine was able to inhibit PRL secretion

stimulated by both hormones. These inhibitions were reversed by the active

antagonist (-)sulpiride but not (+)sulpiride. Therefore, we have demonstrated

that the RGB-2 cDNA encodes a dopamine D2 receptor that is functional, since

it can couple to inhibitory G protein and since this coupling results in an

inhibition of adenylyl cyclase activity as measured by the drop in cAMP levels

and in an inhibition of PRL secretion.

48
GENE LOCALIZATION OF THE HUMAN D2

 RECEPTOR

As mentioned above, several human neurological diseases have been

associated with imbalances in the dopaminergic system, and in some cases, D2

receptor involvement has been proposed. For example, several lines of

evidence have linked the D2 receptor to schizophrenia. First, a linear

relationship exists between the affinity of different neuroleptic drugs for the D2

receptor and their therapeutic dosage (Seeman et al. 1976). Also, the brains of

some schizophrenics have been reported to possess an unusual density of D2

receptors in their striatum (Seeman 1987). In addition, the dopamine D2

receptor has been proposed to be involved in Parkinson’s disease and

Tourette’s syndrome. To study the relationship between human diseases and

the D2 receptor, we have cloned the human gene.

A human genomic library was screened with the rat D2

 cDNA. Several clones

were detected and one, HD2G1, contained a 1.6 kb BamHl fragment, which

was sequenced and found to code for the last 64 amino acids of the D2

receptor. It should be stressed that the D2 receptor gene has introns and that

its entire sequence spans more than 30 kb.

The HD2Gl DNA was hybridized to human metaphase chromosomes and to

DNA prepared from a panel of rodent-human hybrids that have selectively lost

human chromosomes (Grandy et al. 1989a). All the data were consistent with

localizing the dopamine D2

 receptor to the long arm/of chromosome 11 in the

region q22-q23. Moreover, when the 1.6 kb BamHl fragment from HD2Gl was

used to probe human genomic DNA digested with different restriction enzymes,

only single fragments were detected. This result, plus the fact that the total size

of hybridizing bands in the hybrid panel analysis was consistent with the size of

the HD2Gl phage DNA, indicates the human genome contains only one D2

receptor gene. It should be mentioned that, although our studies were done

under stringent hybridization conditions, these conditions would have detected

genes whose sequences are closely related (75-percent identity).

The presence of one dopamine D2

 receptor gene is of pharmacological

importance. The dopamine receptors have been classified into at least two

subtypes, D1 and D2. The D2 receptors themselves have been further

subdivided into autoreceptors and postsynaptic receptors (Carlsson 1975). The

autoreceptors have a D2

-like pharmacological profile but are found on neurons

that synthesize dopamine. In these neurons, upon binding of dopamine, the

autoreceptors regulate dopamine production through a feedback mechanism.

Our data, therefore, indicate that either the diverse D1

 and D2 receptors are

encoded by different genes whose sequences are divergent, at least more than

49
the sequences of the muscarinic receptors, or that these dopamine receptors

are encoded by the same unique gene and that the pharmacological differences

are the results of either alternative posttranscriptional events or diverse

posttranslational modifications. In view of the current knowledge about the

differences between the D1 and D2 receptors, it seems that they may be coded

for by different genes. On the other hand, some recent experiments indicate

that the D2

 autoreceptors are similar in sequence to the postsynaptic D2

receptor and that these two receptors are products of the same gene (MeadorWoodruff et al. 1989).

The human genomic clone, HD2G1, has been used in several laboratories to

investigate the linkage of the D2

 receptor gene and several human genetic

disorders. Thus far, the studies have focused on families afflicted with

schizophrenia (Moises et al. 1989; Byerley et al. 1989), on other kindreds

afflicted with Tourette’s syndrome (Gelernter et al. 1989; lsenberg et al. 1989),

and on families with a high incidence of manic depression (Byerley et al. 1989).

The D2 receptor was not linked directly to disease in any of these cases. It

should be mentioned, however, that more families currently are being studied.

DOPAMINE D2 RECEPTOR SUBTYPES

By using the rat cDNA as a probe, a human dopamine D2 receptor was cloned

and sequenced from a pituitary cDNA library (Grandy et al. 1989b). The

deduced sequence of the human clone revealed a surprising result. The

human pituitary D2 receptor is highly similar to its rat brain counterpart since

they share 96-percent identity in their amino acid sequence. However, we

found that the human pituitary receptor contains a stretch of 29 amino acid

residues that are unaccounted for in the rat receptor. These additional residues

are found in the putative third cytoplasmic loop of the receptor, suggesting that

they might be important for differentiating G-protein couplings. When the

human pituitary receptor is expressed into mouse Ltk- cells, it binds dopamine

ligands with the same pharmacological profile as does the rat brain receptor,

indicating that the 29 amino acid extension does not affect ligand binding

significantly. Furthermore, we have since shown that a rat equivalent of the

human pituitary receptor exists and that it is also possible to find a human D2

receptor that does not contain the 29 amino acid residues. Therefore, presence

of this peptidic extension is not species-specific.

Finally, the human dopamine D2

 receptor gene was isolated and sequenced.

This analysis revealed that the dopamine receptor gene is split into exons and

that the coding sequence of the receptor is carried by seven different exons.

Moreover, we demonstrated that the 29 amino acid extension is encoded by a

specific exon that does not carry sequences of any other part of the molecule,

50
thus strongly indicating that the existence of the two forms of D2

 receptors is the

result of an alternative splicing event occurring during pre-mRNA maturation.

REFERENCES

CONCLUSIONS

We have demonstrated that the G-protein-coupled receptor that we have cloned

has the pharmacological profile and the biological function expected of the

dopamine D2 receptor. Furthermore, we have localized the dopamine D2

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do not affect ligand binding and are the result of an alternative splicing event

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AUTHORS

Olivier Civelli, Ph.D.

Assistant Professor

James Bunzow, M.Sc.

Research Associate

Paul Albert, Ph.D.

Research Associate

Hubert Van Tol, Ph.D.

Research Associate

David Grandy, Ph.D.

Research Associate

Vollum Institute for Advanced Biomedical Research

Oregon Health Sciences University

3181 Southwest Sam Jackson Park Road

Portland, OR 97201-3098

53
Characterization of Mammalian

Potassium Channel Genes

James O. Douglass, Macdonald Christie, John P. Adelman, and

R. Alan North

INTRODUCTION

Potassium channels are transmembrane proteins that form a pore for the

passage of potassium ions through the membrane lipid bilayer. Of the

membrane ion channels characterized to date, potassium channels are the

most widespread and diverse. In endocrine and exocrine cell types, they play

an important role in the regulation of secretion of hormonal factors. In the heart,

they are responsible for repoiarization of myocardial ceils, and they determine

the frequency of firing in Purkinje fibers. In lymphocytes, they are involved with

the development of mature T cells from immature thymocytes and are

necessary for T-lymphocyte activation.

Potassium channels are most prevalent in neuronal cell types, where they play

a central role in regulating the level of neuronal excitability. Some types of

potassium channels are opened by depolarization to limit the duration of single

action potentials (delayed rectifier) or to set the pattern of bursts of action

potentials (transient, or “A,” currents). Other potassium channels are opened or

closed by second messengers, such as intracellular calcium, to mediate the

actions of synaptic transmitters (Ca-activated potassium currents). Additional

types of potassium channels are involved in mediating entry of potassium ions

into neurons (inward rectifiers) and in controlling excitation following activation

of neurotransmitter receptors (neurotransmitter- and second messengerregulated channels). These various types of potassium channels are present in

unique regions and cell types of the mammalian central and peripheral nervous

systems where they serve to imprint on each neuron its particular properties of

plasticity and excitability (for review, see Rudy 1988; Latorre et al. 1984; Hille

1984).

The potassium channel responsible for the generation of the transient outward

current (la current) is present on the majority of neuronal cell types and is a

characteristic of excitable cells. la channels in excitable membranes are

54
essential for a variety of cellular functions, including neuron bursting, the

determination of resting potentials, the duration of action potentials, and the

modulation of cardiac pacemaking functions. They also have been implicated in

some model systems of learning.

The molecular cloning of the Shaker locus of Drosophila melanogaster has

provided the starting point for the molecular analysis of the potassium Achannel. Shaker mutations phenotypically show a loss of A currents without

affecting other potassium currents. Therefore, the Shaker locus was perceived

to encode the structural component of the A-channel. Genomic DNA

rearrangements in five distinct Shaker mutants were genetically mapped to a 60

kb segment of the Drosophila genome (Papazian et al. 1987). Genomic DNA

from this region was isolated and then subcloned, and specific restriction DNA

fragments were used as hybridization probes to screen adult fly head cDNA

libraries (Papazian et al. 1987; Tempel et al. 1987; Schwarz et al. 1988; Pongs

et al. 1988). Isolation and sequence analysis of cDNA clones, followed by

comparison with genomic DNA sequences, revealed that transcription from the

wild-type Shaker genomic locus results in the production of multiple species of

mRNAs, each encoding a potassium channel protein, Distinct Shaker mRNAs

result from alternate splicing of precursor mRNA, and the resulting potassium

channels share a common core sequence with different amino and carboxyl

termini.

Various Shaker cDNA clones have been expressed in Xenopus oocytes and

characterized electrophysiologically (Timpe et al. 1988a, 1988b; lverson et al.

1988). RNA synthesized in vitro from each cDNA clone directs the synthesis of

a voltage-dependent potassium channel. Unique kinetics of inactivation are

seen for each channel type, indicating that the Shaker products may contribute

to kinetic diversity in A-channels of the fly and that sequences in the amino

terminal region may be important for inactivation.

Shaker sequences have been used as hybridization probes to isolate potassium

channel cDNA clones from mouse brain (Tempel et al. 1988) and rat brain

(Baumann et al. 1988; Christie et al. 1989; McKinnon 1989) cDNA libraries,

This chapter presents a short summary of efforts to characterize mammalian

potassium channel genes and their products, A brief discussion of the possible

structure of a typical, mammalian, voltage-gated potassium channel is

presented. Genomic Southern blot analysis suggests that rat and human

voltage-gated potassium channels have been highly conserved during

evolution, and they represent a large gene family. Finally, some

electrophysiological properties of cloned rat potassium channels are described,

using the Xenopus oocyte system to define specific channel properties,

55
AMINO ACID SEQUENCE AND PREDICTED STRUCTURE OF THE RAT

VOLTAGE-GATED POTASSIUM CHANNEL, RBK1

A cDNA library constructed from rat hippocampal polyadenylated mRNA was

screened with two radiolabeled oligonucleotide probes whose sequences were

based on conserved amino acid residues between the Shaker A potassium

channel of Drosophila (Tempel et al. 1987) and the mouse potassium channel

equivalent, MBK1 (Tempel et al. 1988). One of the rat hippocampai cDNA

clones (designated as RBK1), which was hybridization-positive with both

oligonucleotide probes, was purified, and the nucleotide sequence of the 1.7 kb

cDNA insert was determined (Christie et al. 1989). The sequence contains a

single, long open reading frame that encodes a protein of 495 amino acids.

The predicted amino acid sequence is 69 percent homologous to the 453

residues representing the conserved core region of the Shaker A potassium

channel and is 99 percent homologous to the MBK1 channel.

The amino acid sequence and location of predicted transmembrane regions of

the RBK1 potassium channel are diagramed in figure 1. Both amino and

carboxyl terminal domains are located on the cytoplasmic side of the cell

membrane. Six regions of clusters of hydrophobic residues represent putative

transmembrane domains of the channel (designated as S1 to S6). Residues

between Sl/S2, S3/S4, and S5/S6 are presumably extracellular, with residues

between S2/S3 and S4/S5 located on the cytoplasmic side of the membrane.

Conservation of amino acid sequence motifs between Shaker A, MBK1 , and

RBK1 potassium channels reveal specific moieties that are of functional

significance. For example, seven positively charged amino acids (arginine-R1

and lysine-K) that repeat at every third position are in the S4 transmembrane

domain. It is probably this region of the channel that responds to changes in

membrane potential, regulating potassium ion gating. Five possible sites for Nlinked glycosylation are present in the predicted sequence, as well as one

possible A kinase phosphorylation site in the carboxyl terminal intracellular

region.

In contrast to the predicted structure of mammalian voltage-dependent

potassium channels, mammalian voltage-dependent calcium and sodium

channels contain four repeating units, with six putative transmembrane domains

located in each repeat unit (Noda et al. 1984; Trimmer et al. 1989; Tanabe et al.

1987). Thus, by analogy with the latter two types of ion channels, functional

potassium channels in vivo may represent a complex of four individual subunits

(figure 2). This hypothesis is of note in that there exists a variety of functionally

specialized voltage-dependent potassium channels, and some of this diversity

may be due to the formation of potassium channels by assembly of different

subunits (i.e., heteropolymerization).

56
RBK1 Rat brain potassium channel

FIGURE 1. Amino acid sequence and predicted transmembrane domains of the rat voltage-gated potassium

channel, RBK1

NOTE: Amino acid sequence is predicted from the nucleotide sequence of the RBK1 cDNA clone (Christie et al.

1989). Predicted transmembrane domains represent clusters of hydrophobic amino acids. In the fourth

transmembrane domain (S4), positively charged R and K residues are noted as darkened circles.
FIGURE 2. Schematic representation of the possible general structure of a

functional mammalian voltage-gated potassium channel

NOTE: Transmembrane domains are represented as cylinders through the cell

membrane (pseudocylindrical, helical in nature). tines represent

regions separating the transmembrane domains, both extracellular and

intracellular. Individual subunits contain six transmembrane domains.

Different patterns of shading represent the notion that four individual

subunits assemble as a tetramer to form a functional potassium

channel with a central conducting pore (by analogy with the voltagedependent calcium and sodium channel).

MULTIPLE GENES ENCODE MAMMALIAN VOLTAGE-GATED POTASSIUM

CHANNELS

In Drosophila, transcription at the Shaker locus results in the production of

multiple species of mRNAs (via alternate splicing mechanisms) encoding

unique potassium channel proteins. The coding region of the Shaker

transcript(s) encompasses approximately 70,000 bases of genomic DNA, with

58
11 to 12 introns separating exonic sequences (Schwarz et al. 1988). The 10 or

11 exons encoding specific portions of the Shaker potassium channels are from

100 to 450 bases in length.

To determine if a similar situation exists at the genomic locus encoding the

RBK1 potassium channel, a rat genomic DNA library was screened under high

stringency conditions (50 percent formamide, 42°C hybridization) with 32Plabeled RBK1 cDNA. The hybridization positive genomic DNA clone, RGK1 ,

was isolated and then purified via successive rounds of low-density screening;

phage DNA was subjected to Southern blot analysis using the 1.7kb RBK1

cDNA as a hybridization probe. A 1.9kb, RGK1 Hind III restriction fragment was

observed to be hybridization-positive following autoradiography. This

observation suggested that the majority of the RBK1 coding region was present

on a single exonic domain in the corresponding (RGK1) gene. To determine if

this was indeed the case, the 1.9kb Hind III fragment was isolated from the

RGK1 rat genomic DNA clone, restriction mapped, and subcloned into M13 for

dideoxynucleotide sequence analysis. This analysis revealed that the 1.9kb

Hind III fragment contained an uninterrupted sequence representing 250 bases

of RBK1 mRNA 5’ untranslated region, the entire 1,485 bases of RBK1 mRNA

coding region, and 165 bases of RBK1 mRNA 3’ untranslated region. Thus, the

entire series of nucleotides encoding the RBK1 voltage-gated potassium

channel is contained on a single exonic domain-a situation dramatically

different from that seen at the Shaker locus. It follows, then, that alternate

splicing at the RGK1 locus cannot result in the production of multiple species of

mRNAs encoding unique potassium channel products.

At our present level of understanding, it appears that diversity of mammalian

voltage-gated potassium channels is due to the presence of multiple,

structurally related genes in the genome, with each member of the gene family

encoding a distinct potassium channel product. In an effort to begin

characterizing the multitude of mammalian potassium channel genes, Southern

blot analysis of rat and human genomic DNA was performed under different

hybridization stringencies (figures 3 and 4).

High molecular weight rat and human genomic DNA was incubated with several

restriction enzymes (Eco RI, Bgl II, Pst I, and Hind Ill), electrophoresed on a

1 -percent agarose gel, and transferred to nylon membranes. The Southern

blots were hybridized overnight under stringent conditions (50 percent

formamide/42°C hybridization; figure 3) with 32P-labeled RBK1 cDNA. In all

lanes, a single hybridization signal was observed following autoradiography.

These data are consistent with the observation that the RBK1 coding region is

present on a single exonic domain in the rat genome. (Note the presence of a

1.9kb hybridization signal in the rat genomic DNA sample treated with Hind Ill.)

59
FIGURE 3. Southern blot analysis of rat and human genomic DNA hybridized

with 32P-labeled RBK1 cDNA under stringent conditions

NOTE: Rat and human genomic DNA was treated with the noted restriction

endonucleases (Eco RI, Bgl II, Pst I, and Hind Ill) before

electrophoresis on a 1 -percent agarose gel and subsequent transfer to

nylon membranes. The hybridization probe was a 1.7kb RBK1 cDNA

fragment radiolabeled by random-priming with 32P-dCTP. Hybridization

conditions were 50 percent formamide/42°C. DNA molecular weight

markers are shown to the left of each autoradiograph.

Similar profiles (i.e., single hybridization signals) are observed for human

genomic DNA analyzed under identical conditions. The data suggest that the

human RBK1 gene equivalent is highly homologous to rat RBK1 at the

nucleotide level, that it is present as a single copy gene in the human genome,

and that the majority of the coding region probably also is contained on a single

exonic domain.

The same Southern blots were reprobed with the identical radiolabeled RBK1

cDNA fragment, but under slightly less stringent hybridization conditions (40

percent formamide/42oC). The resulting autoradiographs are shown in figure 4.

In rat and human DNA sample lanes, additional hybridizing bands now are

60
Rat Genomic DNA Human Genomic DNA

FIGURE 4. Southern blot analysis of rat and human genomic DNA hybridized

with 32P-labeled RBK1 under semistringent conditions

NOTE: The experiment is identical to that described in figure 3, only the

hybridization conditions were 40 percent formamide/42oC.

observed. Anywhere from two to six additional bands are observed in each

lane. These data suggest that the additional bands may encode other

potassium channel genes, a result consistent with the hypothesis that the

variety of mammalian potassium channels that have been characterized

electrophysiologically share common structural features and are encoded by

different structural genes.

The recent cloning of additional voltage-gated potassium channels further

suggests that this is indeed the case. Low-stringency hybridization screening of

rat brain cDNA libraries has resulted in the cloning of rat voltage-gated

potassium channels RBK2 (McKinnon 1989), RCK1 (RBK1), RCK3, RCK4, and

RCK5 (Baumann et al. 1988; Christie et al. 1989; Stuhmer et al. 1989). Lowstringency screening of a rat genomic DNA library has afforded the

characterization of the potassium channel RGK5 (Douglass et al. 1990).

Expression cloning also has identified a novel rat brain potassium channel,

DRKl (Frech et al. 1989). Amino acid and nucleotide sequence analysis of

61
these potassium channels confirms that distinct genes encode all the

aforementioned channels.

EXPRESSION OF CLONED RAT POTASSIUM CHANNELS IN XENOPUS

OOCYTES

At least 30 distinguishable types of potassium channels have been

characterized (in vivo) to date, showing diversity in their gating and regulation

(Yellen 1987). They vary remarkably with regard to activation threshold and

kinetics; inactivation rate; and sensitivity to blockers such as 4-aminopyridine

(4-AP), tetraethylammonium (TEA), and toxins (for review, see Rudy 1988). To

attempt to correlate cloned mammalian potassium channels with those

characterized in vivo, it is important to determine the properties of the former

channel types. Expression of cloned channels in Xenopus oocytes has

provided an excellent means of analysis.

Figure 5 shows the potassium currents generated in Xenopus oocytes following

the injection of in vitro synthesized, capped RNA encoding the RBK1 (Baumann

et al. 1988; Christie et al. 1989), RBK2 (McKinnon 1989), and RGK5 (Douglass

et al. 1990; RCK3 [Stuhmer et al. 1989]) rat voltage-gated potassium channels.

Oocytes (Dumont stages V-VI) were harvested from adult Xenopus laevis, and

theca/follicular layers were removed by collagenase treatment. Oocytes were

injected within 10 hours of harvest with the aforementioned RNAs (usually 1 ng

RNA). Recording of membrane currents were made 1 to 3 days after injection

of RNA. Oocytes were voltage-clamped at various potentials using two

microelectrodes.

Currents were evoked by depolarizing voltage-command pulses from a holding

potential of -80 mV (records are single sweeps, leak subtracted). The

depolarizing steps were 50 ms in duration, to the potential (in mV) indicated

beside each trace. Large outward currents were produced in all RNA-injected

oocytes, activating at approximately -40 mV for RBK1, -20 mV for RBK2, and

-30 mV for RGK5. No such currents were observed in uninjected oocytes.

Measurement of RBK1, RBK2, and RGK5 tail current reversal potentials

indicated that the currents were carried exclusively by potassium ions.

These channel activation properties alone are not sufficient to assign a

particular cloned potassium channel to one that has been characterized in vivo.

However, channel inactivation kinetics as well as sensitivity profiles to various

potassium channel blockers can be used to functionally distinguish one cloned

channel from another. For example, the channels described above have

dramatically different profiles of sensitivity to the potassium channel blocker

TEA. The IC50s for the expressed channels are 0.3 mM, RBK1; 10 mM, RGK5;

62
FlGURE 5. Generation of transient outward currents in Xenopus oocytes injected with in vitro synthesized RBK1,

RBK2, and RGK5 potassium channel RNA

NOTE: Currents were evoked by depolarizing voltage command pulses from a holding potential of -80 mV. Test

potentials, in mV, are noted beside each trace. The depolarizing step was 50 ms in duration. Generation of

current is dependent on the injection of potassium channel-encoding RNA (for additional details, see Christie

et al. 1989).
and >= 100 mM, RBK2 (Christie et al. 1990; Stuhmer et al. 1989). Unique

sensitivity profiles to other channel blockers, such as 4-AP, dendrotoxin, and

charybdotoxin, in addition to distinguishable channel inactivation kinetics,

provide these cloned channels with a unique compilation of functional

characteristics (Stuhmer et al. 1989).

In at least one instance, channel properties characterized in the Xenopus

oocyte system, coupled with Northern blot analysis, has allowed for a cloned rat

potassium channel to be correlated with an in vivo-characterized channel

(Douglass et al. 1990). The RGK5 rat potassium channel, described above,

appears to represent the n-type potassium channel, which has been

characterized extensively in mammalian lymphocytes (Cahalan et al. 1987;

DeCoursey et al. 1985; Lewis and Cahalan 1988). The RGK5 sensitivity profile

to channel blockers, kinetics of inactivation, and modulation of inactivation by

divalent cations (as determined in the Xenopus oocyte system) are nearly

identical to the properties of the n-channel, as characterized in murine and

human T-lymphocytes. In addition, the presence of RGK5 mRNA in thymus is

consistent with the notion that the RGK5 channel is the T-lymphocyte

n-channel. Similar studies, no doubt, will serve to provide additional functional

assignments in the future.

CONCLUSIONS

Potassium channels are membrane proteins that are selectively permeable to

potassium ions. They are present in virtually all animal cells but take on

particularly important signaling functions in excitable tissues. Potassium

channels have been classified on the basis of the factors that open (or close)

them; some are opened by changes in the membrane potential, others by the

binding of intracellular ligands to the molecule. The classes do not exclude

each other. Roles for voltage-dependent channels are well developed in

neurons and include maintaining a resting potential, action potential

repolarization, patterning of spike bursts, and limiting synaptic depolarizations.

To understand the molecular mechanisms by which these channels regulate

such a variety of cellular functions, it is necessary to determine the structure of

these proteins and their corresponding genes.

Studies utilizing Drosophila as a model system have allowed for the molecular

characterization of voltage-gated potassium channels to begin. In the fly,

transcription from a huge genomic DNA locus results in the production of

multiple species of mRNAs encoding unique potassium channel proteins.

These multiple mRNAs are produced as a result of alternate splicing of

precursor mRNA. The resulting potassium channels share a common core

sequence, with different amino and carboxyl termini present on the different

64
channel proteins. Different species of Shaker channels have been expressed in

Xenopus oocytes, and unique kinetics of inactivation are seen for each channel

type, indicating that the Shaker products may contribute to kinetic diversity in Achannels of the fly.

Shaker DNA sequences have been used to isolate potassium channel cDNA

clones from rat brain libraries. One such clone encodes the rat brain potassium

channel RBK1, The nucleotide sequence of the clone predicts the generation

of a protein 495 amino acids in length. Computer analysis of the RBK1 amino

acid sequence suggests the presence of six transmembrane domains, with the

fourth domain possibly acting as the channel voltage sensor. Individual

subunits may associate as a tetramer to form functional potassium channels.

Rat cDNA and genomic DNA fragments encoding voltage-gated potassium

channels have been used as templates for the in vitro generation of capped

RNA. This RNA has been injected into Xenopus oocytes to functionally

characterize these cloned channels, Each channel reveals unique kinetics of

activation and inactivation as well as unique profiles of sensitivity to a range of

potassium channel blockers, This analysis has served to demonstrate

functional diversity among the cloned rat voltage-gated potassium channels and

also has led to the correlation of at least one cloned channel (RGK5) with a

potassium channel previously characterized in murine and human Tlymphocytes (the rechannel).

The presence in the mammalian genome of multiple genes encoding unique

species of potassium channels appears to be the major basis for diversity of

mammalian voltage-gated potassium channels. Southern blot analysis of rat

and human genomic DNA reveals single hybridizing bands when a radiolabeled

rat potassium channel-encoding cDNA fragment is used as a hybridization

probe under stringent hybridization conditions. Under slightly less stringent

hybridization conditions, however, multiple hybridizing bands are observed.

Thus, voltage-gated potassium channels in mammals appear to represent a

relatively large gene family. The continued isolation and characterization of

mammalian potassium channel cDNA and genomic clones, along with

functional characterization of the cloned channels in Xenopus oocytes, certainly

will improve our understanding of the molecular basis underlying diversity of this

protein family.

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voltage-dependent potassium channel is highly conserved from Drosophila to

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Cahalan, M.D.; Chandy, K.G.; DeCoursey, T.E.; Gupta, S.; Lewis, R.S.; and

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A.S., eds. Mechanisms of Lymphocyte Activation and Immune Regulation.

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potassium channels expressed from Shaker locus cDNA. Proc Natl Acad Sci

U S A 85:5723-5727, 1988.

Latorre, R.; Coronado, R.; and Vergara, C. K channels gated by voltage and

ions. Annu Rev Physiol 46:485-495, 1984.

Lewis, R.S., and Cahalan, M.D. Subset-specific expression of potassium

channels in developing murine T lymphocytes. Science 239:771-775, 1988.

McKinnon, D. Isolation of a cDNA clone coding for a putative second potassium

channel indicates the existence of a gene family. J Biol Chem 264:8230-

8236, 1989.

Noda, M.; Shimizu, S.; Tanabe, T.; Takai, T.; Kayano, T.; Ikeda, T.; Takahashi,

H.; Nakayama, H.; Kanaoka, Y.; Minamino, N.; Kanagawa, K.; Maysuo, H.;

Raftery, M.M.; Hirose, T.; Inayama, S.; Hayashida, H.; Miyata, T.; and Numa,

S. Primary structure of electrophorous electricus sodium channel deduced

from cDNA sequence. Nature 312:121-127, 1984.

Papazian, D.M.; Schwarz, T.L.; Tempel, B.L.; Jan, Y.N.; and Jan, L.Y. Cloning

of genomic and complementary DNA from Shaker, a putative potassium

channel gene from Drosophila. Science 237:749-753, 1987.

Pongs, 0.; Kecskemethy, N.; Muller, R.; Krah-Jentgens, I.; Baumann, A.; Kiltz,

H.H.; Canal, I.; Llamazares, S.; and Ferus, A. Shaker encodes a family of

putative potassium channel proteins in the nervous system of Drosophila.

EMB0 J 7:1087-1096, 1988.

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Rudy, B. Diversity and ubiquity of K channels. Neuroscience 25:729-749,

1988.

Schwarz, T.L.; Tempel, B.L.; Papazian, D.M.; Jan, Y.N.; and Jan, L.Y. Multiple

potassium-channel components are produced by alternative splicing at the

Shaker locus in Drosophila. Nature 331:137-142, 1988.

Stuhmer, W.; Ruppersberg, J.P.; Schroter, K.H.; Sakmann, B.; Stocker, M.;

Giese, K.P.; Perschke, A.; Baumann, A.; and Pongs, O. Molecular basis of

functional diversity of voltage-gated potassium channels in mammalian brain.

EMB0 J 8:3235-3244, 1989.

Tanabe, T.; Takeshima, H.; Mikami, A.; Flockerzi, V.; Takahashi, H.; Kangawa,

K.; Kojima, M.; Matsuo, H.; Hirose, T.; and Numa, S. Primary structure of

rabbit skeletal muscle dihydropyridine receptor deduced from cDNA

sequence. Nature 328:313-318, 1987.

Tempel, B.L.; Jan, Y.N.; and Jan, L.Y. Cloning of a probable potassium

channel gene from mouse brain. Nature 332:837-839, 1988.

Tempel, B.L.; Papazian, D.M.; Schwarz, T.L.; Jan, Y.N.; and Jan, L.Y.

Sequence of a probable potassium channel component encoded at Shaker

locus of Drosophila. Science 237:770-775, 1987.

Timpe, L.C.; Jan, Y.N.; and Jan, L.Y. Four cDNA clones from Shaker locus of

Drosophila induce kinetically distinct A-type potassium currents in Xenopus

oocytes. Neuron 1:659-667, 1988b.

Timpe, L.C.; Schwarz, T.L.; Tempel, B.L.; Papazian, D.M.; Jan, Y.N.; and Jan,

L.Y. Expression of functional potassium channels from Shaker cDNA in

Xenopus oocytes. Nature 331:143-145, 1988a.

Trimmer, J.S.; Cooperman, S.S.; Tomiko, S.A.; Zhou, J.; Crean, S.M.; Boyle,

M.B.; Kallen, R.G.; Sheng, Z.; Barchi, R.L.; Sigworth, F.J.; Goodman, R.H.;

Agnew, W.S.; and Mandel, G. Primary structure and functional expression of

a mammalian skeletal muscle sodium channel. Neuron 3:33-49, 1989.

Yellen, G. Permeation in potassium channels: Implications for channel structure. Annu Rev Siophys Biophys Chem 16:227-246, 1987.

ACKNOWLEDGMENTS

The studies described were supported by the following U.S. Public Health

Service grants: National Institute on Drug Abuse (NIDA) grant DA-04154

(JOD); National Institute of Child Health and Human Development grant HD24562 (JPA); NIDA grants DA-03160, DA-03161, DK-32979; and National

Institute of Mental Health grant MH-40416 (RAN).

AUTHORS

James O. Douglass, Ph.D.

Assistant Scientist

67
Macdonald Christie, Ph.D.

Postdoctoral Associate

John P. Adelman, Ph.D.

Assistant Scientist

R. Alan North, M.D., Ph.D

Senior Scientist

Vollum Institute for Advanced Biomedical Research

Oregon Health Sciences University

3181 Southwest Sam Jackson Park Road

Portland, OR 97201-3098

68
Problems and Approaches in Studying

Membrane Opioid Receptors

Andrew P. Smith and Horace H. Loh

INTRODUCTION

Opioid receptors were identified first in mammalian brain in the early 1970s

(Pert and Snyder 1973; Simon et al. 1973; Terenius 1973), and their

pharmacological properties have been characterized extensively since that time

(for reviews, see Chang 1984; lwamoto and Martin 1981; Smith and Loh 1981;

Wood 1982). However, full structural and functional characterization of a

receptor requires its purification in a form retaining the ability to bind ligands.

Such a purified preparation can be reconstituted into a membrane environment

and tested for the ability to mediate some biochemical function. Its amino acid

sequence can be determined and its conformation deduced. Antibodies to the

purified receptor can be prepared and used to map its tissue and cellular

localizations, to determine the role of various portions of the protein in ligand

binding and receptor-mediated function, and to prepare affinity columns capable

of purifying more receptor.

The conventional procedure for purifying membrane opioid receptors involves

solubilization, followed by fractionation such as affinity chromatography and gel

filtration. Recently, several laboratories have reported some success using this

procedure. Bidlack and coworkers (1981) solubilized rat brain membranes with

Triton X-100, then isolated opioid-binding material from the solubilized

preparation by means of affinity chromatography using 14-ß bromacetamidomorphine. The resulting preparation bound dihydromorphine and several other

opioids with nM affinity, but the specific binding activity (cpm/mg protein) was at

least an order of magnitude lower than theoretically expected for a pure

receptor. SDS gel analysis revealed three polypeptides of molecular weights 25

to 50 kD.

Simon and colleagues solubilized opioid receptors from toad and, later, from rat

and other mammalian brain preparations with digitonin (Howells et al. 1982).

Using opioid ligand affinity chromatography coupled with lectin affinity

chromatography, they were able to purify an opioid-binding protein from bovine

69
striatum to theoretical homogeneity (Gioannini et al. 1985). The opioid-binding

material had a molecular weight of 300 to 350 kD under nondenaturing

conditions and 65 kD on SDS gels. This material was able to bind the

l-specific ligand DAGO and ß-endorphin.

Maneckjee and coworkers (1985) reported partial purification (500-fold) of a

p-specific opioid receptor from rat brain using 3-(3-cholamidopropyl)

dimethylammonio-1-propanesulfonate (CHAPS) as solubilizer; SDS gel

electrophoresis indicated the presence of three peptides, of molecular weights

94 kD, 42 kD, and 35 kD. Subsequently, they prepared a more highly purified

receptor preparation from bovine striatum (Maneckjee et al. 1987) using a

combination of affinity chromatography and hydroxyapatite chromatography.

Studies with polyclonal antibodies prepared to this protein indicated that the 94

kD component was involved in opioid binding.

Cho and coworkers (1986) reported purification from bovine brain of a protein

selective for opioid alkaloid ligands using a combination of affinity

chromatography, lectin chromatography, and gel filtration. A novel feature of

this protein was that it required acidic lipids possessing unsaturated fatty acids

to manifest binding activity; neither the protein nor the lipids alone possessed

significant opioid binding (Hasegawa et al. 1987). The binding affinities of

ligands to this reconstituted rnaterial were lower than the corresponding values

for binding to brain membranes, but the rank order of binding affinities to the

two preparations were highly correlated (Cho et al. 1986; Hasegawa et al.

1987).

Ueda and coworkers (1987, 1988), using a similar method, also obtained a

protein consisting primarily of a 58 kD band. When this preparation was

reconstituted with the purified G-proteins G1 or G0, a large increase in

displacement of 3H-naloxone binding by the p-agonist DAGO was observed,

and this increase was sensitive to GTP. μ-Agonists, but not or agonists,

also stimulated GTP binding and GTPase activity in this preparation. Thus, this

work suggests that μ-opioid receptors may exert their in vivo effects through a

G-protein,

Many of the solubilized opioid preparations initially reported did not bind

k-ligands. However, Chow and Zukin (1983) reported that CHAPS-solubilized

rat brain membranes contained as well as p-binding species, as assayed by

3H-bremazocine binding. The material eluted as two peaks of 50 and about 250

kD. ltzhak and colleagues (Itzhak et al. 1984; ltzhak and Pasternak 1986)

solubilized guinea pig brain membranes using digitonin in the presence of high

NaCl concentrations and found that -binding was associated with material of

750-875 kD, while binding was associated with material of about 400 kD.

70
DeMoliou-Mason and Barnard (1984), using digitonin extraction in the presence

of 10 mM MgCI2, were able to solubilize material that bound dynorphin-(1-9), a

putative k-ligand, as well as μ- and -selective ligands. Subsequently, they

characterized this binding material by gel filtration (DeMoliou-Mason and

Barnard 1986). -Ligands were found to be associated with material over 500

kD, while both μ-alkaloids and dynorphin-(1-9) were associated with both this

high-molecular-weight material and with lower-molecular-weight material.

Simon and coworkers (1987) purified k-opioid receptors from digitoninsolubilized frog brain membranes, An affinity column consisting of D-ala2

-DIeu5

enkephalin (DADLE) coupled to Sepharose-6B was used to isolate μ, , and

receptors from the solubilized preparation; and k-receptors were separated

from the other two by gel filtration. The extent of purification was over 4,000,

based on pmol of 3H-EKC bound/mg protein. SDS gel analysis revealed two

bands of 65 and 58 kD.

This approach has had limited success with opioid receptors, however, for

several reasons. First, these receptors have proved to be very difficult to

solubilize in a form that retains binding activity. Ligand binding of opioid

receptors is eliminated or greatly reduced by low concentrations of most

nonionic detergents that have been used successfully to solubilize other cell

surface receptors. At least part of this problem may be due to the more

stringent criteria necessary to establish the existence of opioid receptors. The

activity of most cell surface receptors is measured by specific, or displaceable,

binding, which is defined as the difference between the amount of radioactive

ligand bound in the presence and absence of a large amount of the same

unlabeled ligand. A genuine opioid receptor, in contrast, must exhibit not only

specific but also stereospecific binding-that which is selective for the 1-forms

of stereoisomeric agonists such as levorphanol and levallorphan.

Most laboratories that have reported solubilization of opioid receptors have

demonstrated the existence only of specific binding in their preparations.

Stereospecific binding either is very low or is not reported at all. While it is

conceivable that opioid receptors lose stereospecificity upon solubilization,

obviously the inability to demonstrate it casts doubt on the pharmacological

relevance of the preparation.

A second obstacle to purification of opioid receptors is that no simple

biochemical process mediating opioid receptor function has as yet been

identified. Opioid receptors have been classically defined as those promoting

antinociception in mammals or, in some cases, by their effects in certain in vivo

tissue systems. Recent work has indicated an association between opioid

receptors and several other functional molecules in cell membranes, including

71
adenylate cyclase (Law et al. 1981; Sharma et al. 1975) and ion channels

(North and Williams 1983; Werz and MacDonald 1984, 1985; Williams et al.

1982). However, none of these molecules has been demonstrated to play a

role in the process of antinociception. Thus, even if a purified protein has

opioid-binding activity, there is no definitive biochemical test that can confirm its

functional relevance.

Finally, opioid receptors are heterogeneous. At least three different classes are

present in brain: μ, and which differ in their ligand selectivity and in their

pharmacological effects (Lord et al. 1977; Martin et al. 1976). Other receptor

types may exist, in brain or in peripheral tissues (Grevel and Sadee 1983;

Schulz et al. 1981), as well as subtypes for p-receptors (Loew et al. 1986;

Nishimura et al. 1984) and receptors (lyengar et al. 1986). Because most

opioid ligands are not completely selective for a single receptor type, it is

difficult to isolate a single type, which complicates pharmacological

characterization.

For these reasons, some investigators have sought alternative ways of purifying

opioid receptors. This chapter discusses and evaluates some of these

procedures.

PURIFICATION OF COVALENTLY LABELED OPIOID RECEPTORS

Since solubilization of opioid receptors in a form retaining ligand-binding

properties has proved to be so difficult, some investigators have used an

alternative approach in which the receptor is labeled with a radioactive,

covalently bound ligand. The receptor then can be solubilized with a denaturing

detergent, and purification followed by means of the radioactive label. That

many opioid ligands are peptides has made this approach especially attractive,

because, in addition to synthesizing and testing compounds capable of reacting

covalently with opioid receptors, some researchers have covalently attached

opioid peptides to the receptor by means of a variety of bifunctional crosslinking reagents.

Zukin and Kream (1979) first applied the latter technique using DADLE as

ligand. The material so labeled had a molecular weight of 380 kD under

nondenaturing conditions and 35 kD on SDS gels. However, as the only

available group for cross-linking on DADLE is the terminal amino group, which

presumably is involved directly in binding, it could be argued that during

cross-linking DADLE was moved from its binding site.

We cross-linked 3H-ß-endorphin to brain membranes that previously had been

solubilized with a nonionic detergent (Brij 36-T). This opioid peptide contains

several internal amino groups that could serve as the site of covalent

72
attachment without interfering with the binding process. SDS gel analysis of the

solubilized, cross-linked material revealed a broad spectrum of species, from 2

to 200 kD (Smith and Loh 1979).

Howard and coworkers (1985, 1986) covalently labeled opioid receptors in brain

membranes by cross-linking 125l-ß-endorphin with the bifunctional reagent bis[2-

succinimido-oxycarbonyl-oxy-O-ethyl] sulfone. Membranes were incubated with

the radioactive ligand, then centrifuged to remove free ligand, and incubated

with the cross-linking reagent. By carrying out the original incubation with

ß-endorphin in the presence of unlabeled ligands selective for μ-, or

receptors, and analyzing the cross-linked products on SDS gels, they were able

to conclude that ß-endorphin labeled both μ and -receptors and that the latter

could be distinguished by molecular weight. The μ-receptors, identified as

bands cross-linked in the presence of and agonists, were associated with a

major 65 kD band and a minor 38 kD band, while the B-receptors consisted of a

major 53 kD band and a minor 25 kD band. The 53 kD band also was observed

in cross-linked membranes from NG 108-15 hybrid cells, which contain only

opioid receptors.

Bero and coworkers (1988), using a similar technique, found that 125l-ßendorphin labeled three peptides of molecular weight 108, 73, and 49 kD.

However, the relationship of these three species to μ-, and receptors was

not clear. Preincubation with unlabeled ß-endorphin was capable of blocking

much or most of the cross-linking to each of these species, but other ligands

were much less effective. Thus, etorphine, which binds to μ-, and opioid

receptors, blocked 60 percent of the cross-linking to the 73 kD band but less

than 40 percent to either of the other two bands. The μ-selective ligand DAGO

and the selective U-50.488H blocked 30 to 50 percent of the binding to the 10

and 73 kD bands, while the S-selective ligand D-Pen2-D-Pen5-enkephalin

(DPDPE) was ineffective in blocking cross-linking to any of the species.

Other investigators have analyzed opioid receptors labeled with ligands that

directly attach themselves to the binding site. Newman and Barnard (1984)

synthesized DALECK, an enkephalin derivative possessing a chloromethyl

ketone group at its C-terminus. This ligand was capable of binding both

reversibly and irreversibly to opioid receptors, depending on the pH. Reversible

binding occurred to both μ- and -receptors, while the irreversible binding was

highly specific for preceptors. Analysis of the irreversibly bound material on

SDS gels indicated a single species, of molecular weight 58 kD.

Liu-Chen and Phillips (1987) labeled bovine striatal membranes with 3H-ßfunaltrexamine and showed that the irreversible portion of this binding was

specific for μ-opioid receptors. SDS gel analysis indicated that the label was

73
associated with material in the molecular weight range 68 to 97 kD. The diffuse

migration pattern was presumed to be due to the attachment of variable

amounts of carbohydrate, for the labeled material was adsorbed by wheat germ

lectin-Sepharose.

Yeung (1987) prepared a photoreactive enkephalin derivative, which was found

to label a 46 kD protein in rat brain and spinal cord membranes, Photolabeling

was inhibited by μ-, and -ligands, suggesting that the 46 kD band was a

peptide shared by all three receptor types. Bochet and coworkers (1988)

labeled rat brain and NG 108-15 cell membranes with the photoaffinity -ligand

azido-DTLET. In rat brain, the ligand labeled two bands of 44 and 34 kD; while

in NG cells, a 33 kD band was detected.

TARGET ANALYSIS

Another approach to studying membrane receptors is target analysis. In this

technique, the cell membrane is subjected to electron beam irradiation of

various intensities, and the degree of inactivation of the receptor, as measured

by ligand binding or some functional assay, is determined as a function of

degree of inactivation. If one assumes that bombardment by a single electron is

sufficient to inactivate the receptor, then the molecular size of the latter can be

calculated from the irradiation vs. inactivation curve. This procedure has the

advantage that molecular weight is determined in situ without the necessity of

extracting the receptor from its membrane environment.

Ott and coworkers (1986) applied target analysis to opioid receptors in rat,

guinea pig, and frog brain, as well as in NG 108-15 neuroblastoma-glioma

hybrid cells. By determining binding to different ligands, they were able to

estimate the molecular weights of μ-, and -opioid receptors. Furthermore,

they also conducted assays in the presence or absence of Na+, Mg++, and GTP,

which are required for coupling of receptors to G-proteins. They found that all

three opioid receptor types had a molecular weight of 98 kD in the presence of

ions and GTP and a molecular weight of 56 kD in their absence. The latter

value is in close agreement with the 58 to 65 kD values for purified opioid

receptors reported by several other groups (Newman and Barnard 1984;

Gioannini et al. 1985). Moreover, the 44-kD increase observed in the presence

of ions and GTP is consistent with association with the subunit of the

inhibitory G-proteins which is approximately 40 kD in molecular weight.

On the other hand, other groups employing target analysis have estimated

considerably higher values for opioid receptors. McLawhon and coworkers

(1983) obtained a value of 200 kD for opioid receptors in NG 108-15 cells, using

either 3H-DADLE or 3H-nalozone as ligand. Lai and coworkers (1984)

74
estimated a value of 110 kD for both μ- and B-receptors in rat brain. In another

study applying target analysis to rat brain membranes, Tao and coworkers

(1986) assayed opioid binding with 3H-diprenorphine, which binds to μ-, and

-opioid receptors. In the presence of DADLE, NaCI, and Gpp(NH)p, the

estimated size was 165 kD. In the presence of Gpp(NH)p, the size was 217 kD,

while in the presence of Mg++, it was 286 kD.

Tao and coworkers (1986) suggested that the 165 kD value obtained in the

presence of NaCl or NaCl and Gpp(NH)p was the size of the uncoupled

receptor. In the presence of DADLE, NaCI, and Gpp(NH)p, the receptor was

coupled to a GTP-binding component of 96 kD (including not only the subunit,

but also the ß- and y-subunits) and a Na+

 binding component of 52 kD. In the

presence of Gpp(NH)p, it was coupled only to the Na+

 binding component; while

in the presence of Mg++, it was coupled only to the GTP-binding component.

The reason for the discrepancies in molecular weight values reported by these

various groups is not entirely clear. However, as discussed above, many

laboratories have reported that the molecular weight of opioid receptors is

considerably greater in nondenaturing detergents than in denaturing ones,

suggesting that in situ the ligand-binding subunit is associated with other

components. Under the conditions used by three research groups (McLawhon

et al. 1983; Lai et al. 1984; Tao et al. 1986), the receptor was apparently in this

larger form: whereas another group (Ott et al. 1986) appeared to be observing

just the binding subunit of the receptor, with or without association of just the

a-subunit of G1

. Moreover, the study of Tao and coworkers (1986) suggests

that opioid receptors may exist in several associated forms, with Na+

-binding

component involved as well as GTP-binding component.

However, several studies (Lai et al. 1984; Tao et al. 1986; Ott et al. 1986) are

consistent with the notion that, under a given set of conditions, different opioid

receptor types are approximately the same molecular weight. Ott and

coworkers (1986) found identical molecular weights for μ-, and -receptors,

whereas Lai and coworkers (1984) reported identical molecular weights for

μ- and b-receptors. While Tao and coworkers (1986) assayed opioid binding

with 3H-diprenorphine, which should bind to all three receptor types, the plot of

log inactivation against radiation dose was linear under all conditions,

suggesting that the receptors being measured were a single size.

Because opioid receptor types can be inactivated selectively by covalent

ligands under conditions in which some types are protected by irreversible

ligands (James and Goldstein 1984), it often is concluded that they must

represent distinct molecules. However, identical receptor molecules could

behave differently if their ligand selectivity were determined by association with

75
different messenger systems and if, on any particular cell type, the receptor had

access to only one type of second messenger system. Thus, one could

postulate, for example, that type opioid receptors result when the ligandbinding molecule associates with adenylate cyclase, while μ- and -OpiOid

receptors result from association of the same binding molecule with other

second messengers.

ANTIBODIES

In addition to their usefulness in characterizing purified receptors, antibodies

also can be used as a rapid and efficient means of purification when other

approaches are not successful. To prepare them, it is necessary only to have a

partially purified preparation of receptor; moreover, the receptor need not bind

ligand. Antibodies can be made to denatured receptors, such as those

identified by covalent labeling with a tritiated ligand and partially purified by SDS

gel electrophoresis. Once such antibodies are available, they can be screened

efficiently by their ability to inhibit opioid binding in vitro. In addition, because of

their high specificity, antibodies can be prepared to a single opioid receptor

type, if each type is a distinct molecule.

Several recent reports illustrate the power and flexibility of this approach in

purifying opioid receptors. Simonds and coworkers (1985) covalently labeled

-receptors on NG 108-15 neuroblastoma-glioma cells with tritiated FIT, which

served as a marker to follow the receptors in subsequent purification steps.

Then antibodies to protein-conjugated FIT were prepared and used to construct

an affinity column. When detergent-solubilized hybrid cell membranes were

applied to this column, the FIT-receptor complex was preferentially bound, thus

effecting a major purification step. When this step was combined with an

additional lectin affinity column elution, the receptor-FIT complex was purified

essentially to homogeneity. It had a molecular weight of 58 kD.

A somewhat different immunological approach to opioid receptor

characterization was introduced by Roy and coworkers (1988a). These

investigators detected and isolated anti-idiotypic antibodies to ß-endorphin.

Binding of the antibodies to brain membranes was inhibited by opioid ligands.

In addition, the antiidiotype bound to a 60 kD peptide in Western immunoblots.

Several groups now have prepared antibodies to opioid receptor preparations

that previously were purified in their laboratory. As discussed earlier, Bidlack

and coworkers (1981) reported purification of an opioid receptor from Tritonsolubilized rat brain membranes using affinity chromatography. Subsequently,

this group prepared a monoclonal antibody (Mab) to this material, which proved

to be directed against the 35 kD band observed on SDS gels. This Mab was

76
capable of partially inhibiting opioid binding to the solubilized preparation of

Bidlack and Denton (1985) although long incubation periods were required.

Moreover, Fab fragments prepared from the antibody rapidly and completely

inhibited binding of μ- and opioid ligands to brain membranes (Bidlack and

O’Malley 1986).

Maneckjee and coworkers (1987) prepared a polyclonal antibody to the highly

purified opioid-binding protein they had previously isolated from bovine striatum.

This antibody inhibited selectively the binding of -opioids to rat brain

membranes and also selectively precipitated a 94 kD band from detergentsolubilized striatal membranes.

Cho and colleagues (1986) and Schofield and coworkers (1989) recently

purified to homogeneity an opioid-binding protein (OBCAM) from bovine brain

and isolated the cDNA coding for this protein. Both monoclonal and polyclonal

antibodies have been prepared to this protein, as well as polyclonal antibodies

to peptides corresponding to portions of the predicted amino acid sequence of

the cDNA (Roy et al. 1988b; Schofield et al. 1989). The monoclonal antibodies,

or the Fab fragments derived from them, inhibit opioid binding to both the

purified protein and to brain membranes, with binding of μ-, and -ligands all

affected (Roy et al. 1988c). The polyclonal antibodies to the purified protein

also inhibit opioid binding to brain membranes (Roy et al. 1988b).

Although OBCAM shows some selectivity toward p-alkaloid ligands, both the

monoclonal Fab fragments and the polyclonal antibodies inhibited opioid

binding to NG 108-15 cell membranes, which contain exclusively -opioid

receptors, as well as to guinea pig cerebellum membranes, which are enriched

in - -opioid receptors, This suggests that the three major opioid receptor types

in mammalian brain contain common antigenic epitopes. However, Western

blot analysis revealed that the polyclonal antibodies interacted with a 63 kD

band in guinea pig cerebellum and with a 39 kD band as well as a 58 kD band

in NG 108-15 cells. In addition, a polyclonal antibody raised to a peptide

corresponding to a portion of OBCAM’s predicted amino acid sequence

specifically adsorbed proteins of 39 and 58 kD from detergent-solubilized NG

108-15 cell membranes (S. Roy, unpublished data).

To determine whether either of the two bands recognized by polyclonal

antibodies to OBCAM in NG 108-15 cells are involved in opioid receptor

function, the cells were treated with 10 mM DADLE for 12 to 48 hours. This

treatment is known to result in down-regulation, or decrease, of opioid receptors

in these cells (Law et al. 1983). Under these conditions, the 39 kD band, but

not the 58 kD band, was decreased in a time-dependent, nalozone-reversible

fashion.

77
In view of the evidence discussed above, the result was somewhat surprising:

A 58 kD protein binds opioids in NG 108-15 ceils. However, OBCAM is thought

to contain carbohydrate and, in fact, its predicted amino acid sequence has a

molecular weight of just 39 kD (Schofield et al. 1989). Thus, it is conceivable

that the 39 kD protein isolated from NG 108-15 cell is identical in amino acid

sequence to OBCAM but has little or no carbohydrate.

GENE CLONING

Another approach to purification of cell surface receptors is provided by gene

cloning. An ultimate goal of most receptor studies is to clone the receptor in

any case, but a particular advantage of this procedure is that it does not require

a receptor that retains ligand bind activity; a denatured receptor, such as that

obtained from covalent labeling, is sufficient. The amino acid sequence of a

portion of the material is determined and, from this, the corresponding

oligonucleotide probe (or probes) is synthesized. These probes then can be

used to screen a cDNA library prepared from tissue rich in the receptor of

interest. When the corresponding cDNA has been identified, it may be possible

to transfect it into certain cell lines that can synthesize large quantities of it.

As discussed earlier, Cho and coworkers (1986) purified an opioid-binding

protein from bovine brain that was selective for alkaloids. The cDNA coding for

this opioid-binding protein then was cloned using standard procedures

(Schofield et al. 1989). This sequence was determined and translated to give

the amino acid sequence of the opioid-binding protein. The protein consists of

345 amino acids, with a calculated molecular weight of 37.9 kD. This

discrepancy between this value and the molecular weight of the originally

purified opioid-binding protein (58 kD) suggests that the protein might be

glycosylated. This conclusion is supported by two other observations: (1) The

opioid-binding protein binds to lectin affinity columns (Cho et al. 1986), and (2)

there are six potential glycosylation sites in the protein’s amino acid sequence,

consisting of an aspartamine residue close to a serine or threonine (Schofield et

al. 1989).

Although it has not yet been possible to show that transfection of the cDNA into

cells lacking opioid receptors confers opioid-binding activity, other indirect

evidence indicates the pharmacological relevance of this opioid-binding protein.

An antibody to a portion of the predicted amino acid sequence was used to

construct an affinity column. This column was capable of specifically binding

protein from solubilized brain membranes that bound opioids in in vitro assays

(Schofield et al. 1989). Moreover, a monoclonal antibody raised to the purified

protein (Roy et al. 1988c) inhibited opioid binding to this affinity-purified

material. These results indicate that the cloned sequence indeed codes for the

purified opioid-binding protein,.

78
A search of the NBRF-PIR database revealed that this opioid-binding protein

had significant homologies to several proteins, all of which are members of the

immunoglobin superfamily (Schofield et al. 1989). This is a group of proteins

characterized by repeating domains flanked by cystine residues (Williams

1987). The highest degree of homology was to two cell adhesion molecules,

neural cell adhesion molecule (N-CAM) and myelin-associated glycoproteln;

the sequences of both these proteins are about 20 percent homologous with

that of the opioid-binding protein. Somewhat lower significance values, also

suggestive of an evolutionary relationship, were found for several other

adhesion molecules, neural adhesion molecule (L1), and fasciculin, as well as

for receptors for interleukin-6 (IL-6) and platelet-derived growth factor.

Sequence analysis therefore is consistent with the notion that this protein

functions as a neuropeptide receptor, while also suggesting that it could play a

role in cell adhesion. Accordingly, it has been named OBCAM, or opioidbinding cell adhesion molecule. A possible role of this molecule in cell adhesion

is also consistent with recent evidence that opioids can modulate cell-cell

interactions of immune cells (Stefano et al. 1989).

We also have made an attempt to clone the -opioid receptor of NG 108-15

hybrid cells without beginning with purified receptor. Total mRNA was isolated

from NG 108-15 cells and used to synthesize a “library” of cDNA, that is, DNA

containing all of the genomic sequences found in NG 108-15 cells. This cDNA

library was fractionated by size, then inserted by means of a plasmid vector into

a glioma cell line that expresses no detectable opioid binding.

A key to the success of these studies was the construction of the subtraction

probes, which were used to enrich or “prescreen” the initial colonies for opioid

receptor cDNA. To make these probes, we took advantage of the fact that

several treatments can selectively alter the number of opioid receptors in clonal

cells. Prolonged treatment of NG 108-15 hybrid cells with opioid agonist, such

as 100 nM DADLE for 24 hours, results in a decrease, or down-regulation, of as

much as 80 percent in receptor number (Law et al. 1983). In addition, treatment

of the PC cell subtype PC12h, which ordinarily contains undetectable levels of

opioid receptors, with nerve growth factor for 14 days results in the appearance

of high levels of opioid receptors.

Accordingly, mRNA was prepared from these cells under both high receptor

(normal NG 108-15 cells, or NGF-treated PC12h cells) and low receptor (downregulated NG 108-15 hybrid cells or normal PC12h cells). The mRNA from the

high-receptor cells was used to make cDNA’s, which then were hybridized with

nRNA from low-receptor cells. This hybridization in effect “subtracts” all the

common mRNA from the high- and low-receptor conditions and, in theory,

leaves cDNA highly enriched in opioid receptor DNA.

79
After such screening, the colonies that tested positively for both subtraction

probes were screened for opioid binding. Several candidate cDNA’s that

bestowed both opioid binding and opioid-mediated adenylate cyclase inhibition

on these cells are currently under investigation.

SUMMARY AND CONCLUSIONS

Considerable progress has been made in recent years in understanding opioid

receptors at the molecular level. Several laboratories have reported purification

of opioid receptors by standard procedures involving solubilization of brain

membranes in nondenaturing detergent, followed by affinity chromatography

and other fractionation methods. In addition, several alternative approaches

have been used with some degree of success. Several investigators have

labeled receptors with covalent ligands, then characterized them under

denaturing conditions such as SDS gel electrophoresis. Antibodies have been

prepared to several pure or partially pure opioid receptor preparations that can

be used to (1) map the brain regional distribution of opioid receptors, (2)

determine the role of specific regions of the receptor molecules in ligand binding

and in interaction with other functional molecules, (3) compare the structural

features of different opioid receptor types, and (4) purify receptors further. One

group also has reported cloning of the cDNA for an opioid-binding protein using

antibodies as affinity ligands to purify receptors from solubiiized material and

the application of gene cloning techniques.

The successful use of these varied approaches ensures that future progress in

this field will be rapid. The purification, reconstitution, and detailed structural

characterization of different opioid receptor types appears feasible within a few

years, The next challenge in the molecular understanding of these receptors

will be to identify and characterize the second messenger systems in the cell

membranes that mediate their pharmacological functions.

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ACKNOWLEDGMENT

The authors were supported by National Institute on Drug Abuse grants DA00564 and DA-01583 and Research Scientist Award 5-K05-DA-70554.

AUTHORS

Andrew P. Smith, Ph.D.

Research Associate

Horace H. Loh, Ph.D.

Professor

Department Head

Department of Pharmacology

University of Minnesota Medical School

3-249 Millard Hall

Minneapolis, MN 55455

84
An Approach to the Molecular Biology

of Opiate Tolerance: Identification of

Opiate-Regulated Transcripts

Mark vonZastrow, Jack D. Barchas, and James H. Eberwine

INTRODUCTION

Over the past two decades, much has been learned about cellular adaptation to

opiate exposure. Refinement of receptor-binding methodologies and

development of highly specific receptor ligands, characterization of opiateresponsive cell lines as model systems, and elucidation of receptor-coupled

second messenger systems have greatly facilitated progress in understanding

the function and adaptation of opiate receptor-mediated signal transduction.

Most current knowledge, however, is restricted to understanding adaptation

events that occur at proximal stages in the signal transduction pathway (Clark

1986; Law et al. 1983). These events include opiate-induced changes in

number (down-regulation) and affinity of opiate receptors and, at a slightly distal

level, opiate-induced changes in coupling of receptor to second messenger

system (desensitization). Beyond these stages, little is known about opiate

receptor-mediated signal transduction pathways or their adaptation to chronic

stimulation.

In some systems, it has become apparent that later events in cellular response

to external stimuli involve selective modulation of gene expression (Chang and

Bourne 1987). Although such modulation has not been described in opiate

receptor-mediated systems, clues that it may occur include observations that

the reversal of opiate-induced down-regulation requires new protein synthesis

(Law et al. 1985) and that opiates influence the growth properties of certain

cells (Berry and Haynes 1989). We have begun to test the hypothesis that

cellular adaptation to opiate exposure involves regulation of gene expression by

the application of molecular biologic techniques to two model systems, the

NG108-15 cell line and the rat brain. This chapter summarizes briefly the

current status of our emerging appreciation of opiate-dependent genetic

regulation, derived primarily from study of NG108 cells. The data reviewed in

this chapter (1) establish that opiate-dependent modulation of gene expression

85
does occur and identify several classes of opiate-regulated transcript (ORT), (2)

indicate that this modulation is limited to a small subset of genes expressed in

the cell, and (3) suggest that opiate-dependent modulation of gene expression

may be functionally important to the development of the opiate-tolerant

phenotype. Following this review, our current focus is described, which is to

explore the occurrence of ORTs in rat brain. In view of the structural complexity

of brain, a major consideration in these efforts is the development of

methodologies that are capable of detecting gene regulation in specified brain

regions and from limited amounts of tissue.

IDENTIFICATION OF OPIATE-RELATED TRANSCRIPTS IN NG108-15

CELLS

The NG108-15 neuroblastoma-glioma cell line expresses delta-subtype opiate

receptor coupled to adenylate cyclase via G1—the GTP-binding protein with

inhibitory effect on adenylate cyclase-and to calcium channels, probably via

G0—the GTP-binding protein present in large amounts in brain and currently

thought to be involved in regulating ion channels (Simonds 1988: Hescheler et

al. 1987). This cell line has been characterized extensively and used by a

variety of investigators to study opiate-dependent modulation of receptor

number and affinity as well as desensitization of opiate-dependent inhibition of

adenylate cyclase activity (Law et al. 1983). We have pursued two general

approaches to explore opiate-modulated gene regulation in this cell line. First,

regulation of specific genes ‘was tested directly using oligonucleotide probes

complementary to specific “candidate” genes. Second, we searched more

generally for regulated transcripts by use of differential screening and

subtractive hybridization.

The candidate gene approach was taken with various G protein alpha-subunits

(for more detail, see Evans et al. 1988; vonZastrow et al., in preparation). The

choice of these molecules was motivated by several considerations, including

the pivotal role played by G proteins in mediating signal transduction, the

availability of extensive nucleotide sequence information, and the indirect

evidence that opiate tolerance may involve alteration of relative activities of G5

and G1, GTP-binding proteins with opposing effects on adenylate cyclase

activity (see below). No opiate-dependent change in G1 alpha mRNA level was

noted, but surprisingly, an increase in G5 alpha mRNA level of approximately 30 to

50 percent was induced by opiate agonist (morphine or etorphine). This

increase was slow in onset (detected only after hours of opiate exposure), was

blocked by naloxone, and was reversible after withdrawal of opiate agonist

(figure 1). Attempts to measure changes in G0 alpha levels have been

unsuccessful due to very low levels of hybridization signal detected with

oligonucleotide probe. Reprobing Northern blots with actin cDNA revealed no

86
FIGURE 1. Modulation of Gs alpha mRNA level by opiate agonists

NOTE: Total RNA, prepared by CsCl density centrifugation from NG1O8-15

cells treated for 24 hours in the presence or absence of opiate drugs,

was electrophoresed on 1.2 percent agarose/formaldehyde gels and

transferred to nitrocellulose. Transfers were probed with

oligonucleotide labeled at the 5’-terminus by gamma-32P-ATP catalyzed

by polynucleotide kinase (Maniatis et al. 1982). Two Gs alpha probes

were designed, one 42-mer (Gs) that is homologous to regions

conserved among all murine Gs alpha sequences reported to date and

one (Gs52) that is homologous to the 42 nt sequence unique to the

mRNA encoding the M, 52 k variant cloned from bovine and murine

tissues. Blots were stripped and probed sequentially with Gs

 and Gs52

oligonucleotides, followed by beta-actin cDNA (cDNA provided by L.

Kedes) labeled by random priming with alpha-32P-dCTP (Maniatis et al.

1982). The left panel shows increased Gs alpha signals in response to

24 hours of treatment with 25 nM etorphine. Actin reprobing indicates

no regulation of this mRNA, which verifies equivalent loading of the gel

lanes transferred. The right panel shows that addition of 100 μM

naloxone blocked the increase in Gs alpha mRNA level induced by either

25 μM etorphine or 25 μM morphine.

87
detectable opiate-dependent change in actin mRNA level. These data show

that prolonged opiate exposure causes a selective and moderate increase in

Gs alpha mRNA level. Therefore, Gs alpha represents an ORT. The functional

importance of this regulation has not been established, but it is intriguing to note

that previous observation of “sensitization” of Gs-stimulated adenylate cyclase

activity during opiate withdrawal suggests differential regulation of G protein

activity levels (Griffin et al. 1985), consistent with the present findings of

modulation of Gs alpha mRNA levels.

To approach the problem of identifying ORTs more generally, we have used

conventional techniques of “+/-” screening of a cDNA library constructed in

lambda-zap II vector from NG108 poly A

+

 RNA. Alpha-32P-labeled cDNA,

synthesized from poly A+ RNA purified from control NG108 cells and from

etorphine-treated NG108 cells, was then used to screen replicate plaque lifts.

Following autoradiography of washed filters, visual comparison of 200,000

plaques screened with both probes indicated 15 plaques whose hybridization

intensities were substantially (approximately fivefold) different between the two

(control and etorphine-treated) screens. Ten of these “positives” were

persistent on second-round screening: Eight showed increased hybridization

signal when screened with control cDNA, and two showed increased

hybridization signal with etorphine cDNA. The limitations of this technique, with

regard to both abundance level detectable and extent of change in abundance

necessary to score a definitive “positive,” are well known, so the present finding

of 10 regulated clones should be taken as a lower estimate. We have not yet

fully purified or characterized these opiate-regulated clones; however, it is clear

(1) that they are distinct from Gs alpha according to relative change in

hybridization and abundance level in the labeled cDNA preparation and (2) that

they represent at least two classes of ORT: those that increase and those that

decrease in abundance following opiate exposure.

Because of the limited sensitivity inherent in +/- library screening, we have

searched for the less abundant ORTs by subtractive hybridization techniques.

Single-stranded cDNA probe, purified from hybridization reactions by

hydroxylapatite chromatography, has been used to screen for regulated

species. First-round screening of the NG108 phage library indicates numerous

positives, but we have not as yet confirmed or purified these clones.

In summary, current results of candidate gene screening and +/- library

screening tentatively identify several classes of ORT according to relative

abundance level, magnitude, and direction (increase or decrease) of opiateinduced abundance change. While these findings establish clearly the

existence of ORTs in NG108-15 cells, the number of ORTs identified may

88
significantly underestimate the number expressed in the cell due to the limited

sensitivity of our methods. Subtractive hybridization may allow the identification

of additional ORTs expressed in lower abundance.

IDENTIFICATION OF OPIATE-REGULATED TRANSCRIPTS IN RAT BRAIN

The NG108 cell line provides a convenient model system for the examination of

cellular adaptation to opiates, but our ultimate goal is to understand the

molecular biology of opiate tolerance in the intact nervous system. In addition

to allowing us to identify ORTs that are regulated in neurons analogously to

those in NG108 cells, studying the intact nervous system allows the exploration

of gene regulation events that may require interconnected systems of neurons.

Toward this goal, we have invested considerable effort in developing tools for

the identification of ORTs in rat brain.

A principal limitation associated with studying transcripts in brain results from

the tremendous cellular heterogeneity within the central nervous system,

making it difficult to examine restricted cell populations independently. In

addition, the small amount of tissue available within limited brain regions of

interest presents another technical barrier. To approach these limitations, two

techniques have been developed in our laboratory over the past several years.

First, in situ transcription (IST) allows synthesis of cDNA from limited brain

regions isolated on a tissue section (Tecott et al. 1988). Second, a novel RNA

amplification method allows the synthesis of a population of amplified, antisense

RNA (aRNA) that represents a broad population of cDNA molecules in

increased copy number (Van Gelder et al. 1990). Furthermore, the aRNA

produced is antisense relative to the starting RNA population used to generate

the intermediate cDNA. Hence, the aRNA can be used directly as a probe

without prior cloning. By using aRNA synthesis in combination with ET, we

hope to examine gene expression in restricted brain regions, obtaining enough

nucleic acid to be used for subsequent probing and cloning operations.

Analogous to our approach to ORTs in NG108 cells, we intend to examine

regulation of candidate transcripts and to search for unknown transcripts by

differential screening or subtractive methodologies.

Candidate transcripts include Gs alpha as well as the other ORTs identified in

NG108 cells. So far, we have applied the IST-aRNA technology to rat brain

sections and have been able to obtain detectable hybridization to Gs alpha, actin,

and cyclophillin sequences using aRNA synthesized from several IST sections.

However, the hybridization signal detected remains quite low: therefore, it is not

clear whether the technique is yet able to detect, with sufficient reliability,

regulation of low-abundance mRNA’s. We anticipate that further optimization

will allow reliable detection of these species and that the technique is currently

89
able to detect transcripts expressed in somewhat higher abundance, such as

those identified in NG108 cells by differential screening.

We have begun to pursue the identification of unknown ORTs in rat brain by

using IST-aRNA for +/- library screening. Brain sections prepared from either

control (placebo-treated) or opiate (morphine-treated) male Sprague-Dawley

rats were used for cDNA synthesis by IST (figure 2). Unlabeled cDNA, pooled

from five tissue sections, was used for synthesis of alpha-32P-labeled aRNA

(figure 3). This aRNA, when used to probe replicate lifts of the NG108 cDNA

library, hybridized specifically to a number of clones; first-round screening

indicates multiple positives that are detected in either higher or lower amount

with IST-aRNA synthesized from brain sections of morphine-treated relative to

placebo-treated animals (figure 4); confirmation and purification of these

putative ORTs from rat brain is in progress.

FIGURE 2. IST of rat brain sections

NOTE: Brain sections from placebo- or morphine-treated rats were subjected to

IST with the T757 primer (Van Gelder et al. 1990) in the presence of

alpha-32P-dCTP (Tecott et al. 1988) to allow subsequent localization of

transcribed cDNA by autoradiography. A representative

autoradiograph is shown in the figure.

90
FIGURE 3. Preparation of aRNA probe following IST

NOTE: Brain sections from placebo- or morphine-treated rats were subjected to

IST with T757 oligonucleotide primer but in the absence of radiolabeled

nucleotide. cDNA was eluted from the tissue sections, second-strand

synthesis was completed, and aRNA was transcribed with T7 RNA

polymerase in the presence of alpha-32P-CTP to provide radiolabeled

aRNA probe (Van Gelder et al. 1990). The figure shows

electrophoretic analysis of aRNA produced (1.2 percent agarose/

formaldehyde gel electrophoresis followed by autoradiography).

91
Placebo treatment Morphine treatment

FIGURE 4. Identification of putative ORTs by hybridization of IST-aRNA

NOTE: aRNA produced following IST of brain sections from placebo- or

morphine-treated brain sections was used to screen replicate plaque

lifts of the NG108 cDNA library. Comparison of hybridization intensities

of individual recombinants indicated putative ORTs that are

represented in substantially different amount between IST-aRNA

produced from brain sections subjected to the two treatment protocols.

The left panel shows a representative hybridization of IST-aRNA from

placebo brain sections to a nitrocellulose filter representing 10,000

recombinants. The right panel shows a replicate filter screened with

IST-aRNA produced from morphine brain sections. Arrows point out

recombinant plaques that differ substantially in hybridization intensity

with the two probes and, therefore, represent putative ORTs.

DISCUSSION

The results summarized in this chapter provide direct evidence that opiates

cause modulation in the abundance of a limited subset of cellular genes, which

we term ORTs. Several classes of ORT can be identified in NG108 cells

according to apparent abundance, magnitude, and direction of opiate-induced

variation. Preliminary results from subtractive hybridization experiments

suggest that the number of ORTs expressed in NG108 cells may exceed that

identified so far, due to the limited sensitivity of +/- library screening. By

92
combining IST and aRNA methodologies, we have begun to examine opiateregulated gene expression in rat brain at the level of individual tissue sections,

and initial results suggest the occurrence of multiple ORTs in brain. Further

refinement should allow such analysis to be accomplished with increasingly

limited brain regions, resulting in the identification and spatial resolution of

ORTs involved in specific molecular biologic changes associated with the

opiate-tolerant phenotype.

The functional roles played by various ORTs in opiate tolerance remain to be

determined. In the case of Gs alpha, we suggest that its modulation may serve to

balance signal transduction in response to opiate-induced perturbation of G1-

mediated pathways. In addition to the receptor-based events of downregulation and desensitization, then, selective modulation of G protein

abundance may contribute to the maintenance of cellular homeostasis. The

interdependence of different G proteins in controlling cellular responses is well

known; furthermore, Gs may be expressed in limiting amounts (Gilman 1987))

thereby making the adjustment of its abundance a reasonable control point.

This view of Gs as a control point suggests that modulation of Gs alpha may be

involved in cellular adaptation to other perturbations. In this regard, it is

interesting to note that ethanol, among multiple other cellular effects, causes a

decrease in G,a,ph. mRNA level of similar magnitude to the increase observed in

response to opiates (Mochly-Rosen et al. 1988; Charness et al. 1988).

By studying opiate-dependent modulation of gene expression, we aim to

elucidate regulation events that occur at more distal stages of receptormediated signal transduction pathways. In this way, we hope to shed light on

previously unknown aspects of opiate tolerance and addiction. In addition, by

identifying control points shared by multiple signal transduction systems, we

may be able to devise pharmacologic or even behavioral manipulations that will

influence the development of drug tolerance, addiction, and withdrawal.

REFERENCES

Berry, S., and Haynes, L.W. The opiomelanocortin peptide family: Neuronal

expression and modulation of neural cellular development and regeneration

in the central nervous system. Comp Biochem Physiol 93:267-272, 1989.

Chang, F.-H., and Bourne, H. Dexamethasone increases adenylyl cyclase

activity and expression of the subunit of Gs in GH3. Endocrinology

121:1711-1715, 1987.

Charness, M.E.; Querimet, L.A.; and Henteleff, M. Ethanol differentially

regulates G proteins in neural cells. Biochem Siophys Res Commun

155:138-143. 1988.

93
Clark, R.B. Desensitization of hormonal stimuli coupled to regulation of cyclic

AMP levels. Adv Cycl Nuc Prot Phos Res 20:151-159, 1986.

Evans, C.; vonZastrow, M.; Eberwine, J.; and Barchas, J.D. Morphine induced

alterations in G-protein expression. (Abstract.) J Cell Biol 107:505A, 1988.

Gilman, A.G. G-proteins: Transducers of receptor-generated signals. Annu

Rev Biochem 56:615-649, 1987.

Griffin, M.T.; Law, P.-Y.; and Loh, H.H. Involvement of both inhibitory and

stimulatory guanine nucleotide binding proteins in the expression of chronic

opiate regulation of adenylate cyclase activity in NG108-15 cells.

J Neurochem 45:1585-1589, 1985.

Hescheler, J.; Rosenthal, W.; Trautwein, W.; and Schultz, G. The GTP-binding

protein, G,, regulates neuronal calcium channels. Nature 325:445-447,

1987.

Law, P.-Y.; Horn, D.S.; and Loh, H.H. Opiate receptor down-regulation and

desensitization in neuroblastoma X glioma NG108-15 hybrid cells are two

separate cellular adaptation processes. Mol Pharmacol 24:413-424, 1983.

Law, P.-Y.; Unger, H.K.; Horn, D.S.; and Loh, H.H. Effects of cycloheximide

and tunicamycin on opiate receptor activities in neuroblastoma X glioma

NG108-15 hybrid cells. Biochem Pharmacol 34:9-17, 1985.

Maniatis, T.; Fritsch, E.F.; and Sambrook, J. Molecular Cloning: A Laboratory

Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1982.

Mochly-Rosen, D.; Chang, F.H.; Cheever, L.; Kim, M.; Diamond, I.; and Gordon,

AS. Chronic ethanol causes heterologous desensitization of receptors by

reducing a, messenger RNA. Nature 333:848, 1988.

Simonds, W.F. The molecular basis of opioid receptor function. Endocr Rev

9:200-212, 1988.

Tecott, L.T.; Barchas, J.D.; and Eberwine, J.H. In situ transcription: Specific

synthesis of cDNA in fixed tissue sections. Science 240:1661-1664, 1988.

Van Gelder, R.N.; vonZastrow, M.; Yool, A.; Dement, W.C.; Barchas, J.D.; and

Eberwine, J.H. Amplified RNA (aRNA) synthesized from limited quantities of

heterogeneous cDNA. Proc Natl Acad Sci U S A 87:1663-1667, 1990.

ACKNOWLEDGMENTS

Russel N. Van Gelder assisted in the preparation of aRNA probes. The

research discussed in this chapter was supported by National Institute on Drug

Abuse grant DA-0500 (JDB) and a Giannini Foundation Medical Research

Fellowship (MvZ).

AUTHORS

Mark vonZastrow, M.D., Ph.D.

Resident

94
Psychiatry Department

Stanford University School of Medicine

Stanford, CA 94305

Jack D. Barchas, M.D.

Associate Dean of Neurosciences

University of California, Los Angeles School of Medicine

12-138 CHS

10833 Le Conte Avenue

Los Angeles, CA 90024-1722

James H. Eberwine, Ph.D.

Assistant Professor

Department of Pharmacology

University of Pennsylvania School of Medicine

36th Street and Hamilton Walk

Philadelphia, PA 19104-6084

95
Prenatal Expression of ProOpiomelanocortin mRNA,

POMC-Derived Peptides, and

μ-Opiate Receptors in the Mouse

Embryo

Y. Peng Loh, R. Adrian Rius, Stela Eikabes, Wojciech Bern, and

Carmine J. Coscia

INTRODUCTION

Pro-opiomelanocortin (POMC) is a Mr=30,000 prohormone that is synthesized

primarily in brain and the anterior and intermediate lobes of the pituitary (Liotta

and Krieger 1983). Small amounts are also present in adrenals, testes, ovaries,

placenta, and pancreas (Liotta and Krieger 1983). This prohormone is

processed differentially in brain and the two lobes of the pituitary to yield at least

five biologically active peptides (figure 1). In the adult rodent brain, POMC is

processed to des-acetyl melanocyte stimulating hormone (MSH),

ß-endorphrn, MSH, and N-POMC1-49 (Emeson and Eipper 1986; Loh et al.

1980). Similar products are formed in the intermediate pituitary, except that

ß-endorphin is acetylated and much of the -MSH is diacetylated (Liotta and

Krieger 1983; Rudman et al. 1979; Eipper et al. 1983; Zakarian and Smyth

1982). In contrast, in the anterior pituitary, N-POMC1-74 adrenocorticotropin

(ACTH)1-39 and ß-lipotropin (LPH) are the major products, with a small amount

of ß-endorphin also present (Seger and Bennett 1986; Eipper and Mains 1978).

ß-Endorphin has been shown to have opioid-like activity (Loh et al. 1976) and

-MSH and MSH have been demonstrated to have effects on the central

nervous system (CNS) (van Ree et al. 1981; van Wimersma-Greidanus et al.

1981). ACTH stimulates steroidogenesis, which is potentiated by tryptic

fragments of the N-terminal Mr=16,000 glycopeptide (Pederson and Brownie

1980). In addition to these effects studied in adult rodent tissue, recent data

indicate that some of these peptides are mitogenic and have influences on

cellular differentiation, suggesting that they may play a role in embryonic

development. For example, administration of naloxone, an opiate antagonist, in

infant rats resulted in an increase in cellular proliferation in germinal cell layers

96
in the CNS (Zagon and McLaughlin 1987). In sympathetic ganglia and spinal

cord cells, ß-endorphin was shown to stimulate neurite outgrowth (Ilyinsky et al.

1987). More convincing is the in vivo inhibition of neonatal brain DNA synthesis

by morphine (Kornblum et al. 1987a) and the inhibition of brain ornithine

decarboxylase activity by intracisternally administered ß-endorphin (Bartolome

et al. 1986). ACTH1-39 has been shown to be a specific mitogen for fetal and

adult mammalian myogenic cells; MSH increased the state of commitment of

neural crest cells to melanogenic differentiation in vitro (Satoh and Ide 1987)

and stimulated embryonic growth in vivo (Swaab and Martin 1981).

N-POMC1-49 has been shown to be a mitogen for adrenal cells in culture

(Estivariz et al. 1988). Other effects of the POMC-derived peptides that may be

relevant to embryogenesis and CNS development have been reviewed

extensively elsewhere (Berry and Haynes 1989).

FIGURE 1. Diagrammatic representation of mouse POMC. The pairs of basic

amino acid residues (open bars=Arg; hatched bars=Lys; solid

bars=Gly), which are cleaved to yield various peptide products,

are indicated.

Until recently, little was known about the distribution of POMC mRNA, the

pattern of processing of the prohormone, and the subtypes of opioid receptors

present during early embryonic development. Knowledge of the distribution of

different POMC-derived peptide forms and their receptors in the embryo would

facilitate in assaying for a functional role of these peptides in development. We

have used in situ hybridization histochemistry and immunocytochemistry to map

the first onset of expression of POMC mRNA and POMC in developing mouse

embryos. The prenatal processing pattern of POMC in the brain and pituitary

also has been determined. Finally, the embryonic expression of μ-opioid

receptors, a receptor for ß-endorphin, was investigated to determine the time at

which the POMC opioid system becomes functional during development. The

characterization of opioid subtypes during prenatal development would facilitate

the understanding of abnormal fetal development arising from maternal drug

addiction.

97
TEMPORAL-SPATIAL DISTRIBUTION OF POMC mRNA AND POMC

IMMUNOREACTIVITY DURING FETAL DEVELOPMENT

The temporal-spatial distribution of POMC mRNA was studied in the mouse

embryo by in situ hybridization using a 48 mer POMC oligonucleotide probe

directed at the MSH region of the POMC molecule as described previously

(Elkabes et al. 1989). To determine if there is a lag time between the

expression of the POMC mRNA and its translation, the distribution of POMC

immunoreactivity was analyzed by immunocytochemistry using an ACTH

antibody, DP4, that cross-reacts with POMC and ACTH. Figure 2, panel b,

shows the first detection of POMC mRNA at embryonic day 10.5 (E10.5) in the

ventral diencephalon, in the region of the presumptive arcuate nucleus (figure 2,

panel a). At this stage, POMC immunoreactivity also was detected in the same

region, indicating that translation occurred soon after expression of the POMC

mRNA (figure 2, panel c). At E11.5, there was a significant increase in the

expression of POMC mRNA and POMC immunoreactivity in the region between

the infundibulum and the optic recess in the ventral diencephalon (figure 3,

panels a and b). On E12.5, the distribution, POMC mRNA content, and

intensity of immunostaining of the POMC neurons in the presumptive arcuate

nucleus were similar to that seen on E11.5 (data not shown). From E13.5 to

El 4.5, POMC-containing neurons occupied a large area due to the rapid growth

of the diencephalon/presumptive arcuate nucleus (figure 3, panels c and d).

POMC mRNA first was expressed in the pituitary gland on E12.5 in the

embryonic area that corresponds to the pars distalis (anterior pituitary, figure 4,

panel a). POMC immunoreactivity also was observed at E12.5 in the region

furthest away from the infundibulum process (figure 4, panel b). On E13.5, the

number of POMC-expressing cells increased (data not shown). By E14.5 there

was a great increase in the number of heavily labeled and intensely

immunostained cells located in the edges of the pars distalis, forming a narrow

band, while other immunopositive cells extended toward the center of the lobe

(figure 4, panels c and d). Also at E14.5, the first POMC-expressing cells were

observed in the pars intermedia (intermediate lobe, figure 4, panels c and d).

lmmunostained POMC fibers were detected as early as E10.5. Axons arising

from the ventral diencephalon extended toward the lateral surface of the

diencephalon where they formed fine fiber tracts and continued toward the

dorsal diencephalon. On E11.5, a dense network of thick fibers with varicosities

coursed along the roof of the diencephalon and extended to the

mesencephalon. On E12.5, these fibers descended into the dorsal

metencephalon, and fiber bundles also were seen in the floor of the

myencephalon. A considerable number of processes continued around the

pontine flexure (figure 5). Growth-cone-like structures were seen at the end of

98
FIGURE 2. Expression of POMC in the diencephalon at E10.5. (Panel a)

Schematic diagram showing the region of the presumptive

arcuate nucleus (arrows) where POMC-expressing cells are

located. (Panel b) Low-magnification, bright-field

photomicrograph of a sagittal section in the region containing the

presumptive arcuate nucleus. This section was hybridized with a

synthetic probe for POMC mRNA. Arrows point to some cells

with autoradiographic silver grains (or=optic recess; dc=diocoel;

bar=8 μm). (Panel c) Sagittal section of an E10.5 embryo

immunostained for POMC/ACTH using an antiserum DP4

, which

recognizes POMC and ACTH.

SOURCE: Elkabes et al. 1989, copyright 1989, Elsevier Science Publishers

99
FIGURE 3. Sagittal sections of 11.5 and 14.5 day-old embryos hybridized

with a synthetic probe to POMC mRNA and immunostained for

POMC/ACTH in the region containing the presumptive arcuate

nucleus. (Panel a) Bright-field photomicrograph of a frozen

section of an embryo at 11.5 hybridized with a 35S-labeled

oligodeoxyribonucleotide POMC probe. (Panel b) 11.5 day-old

embryo immunostained for POMC/ACTH. tmmunoreactive cells

are clustered in the ventral diencephalon but also are present in

the region close to the infundibulum. Note the increase in

autoradiographic silver grains and immunostaining compared with

E10.5. (Panels c and d) E14.5 showing adult-like pattern of

distribution of oligonucleotide-labeled (Panel c) and

immunoreactive cells (Panel d).

100
FIGURE 4. Rathke’s pouch of 12.5- and 14.5-day-old embryos hybridized

with a 35S-labeled POMC probe and immunostained for POMC/

ACTH. Autoradiographic silver grains (Panel a arrows) and

immunostaining (Panel b arrows) in the embryonic area

corresponding to the pars distalis of the pituitary gland at E12.5.

labeled (Panel c) and immunostained (Panel d) cells in the pars

intermedia (black arrows) and distalis (white arrows) of a 14.5-

day-old embryo. Note the increase in POMC-expressing cells in

the pars distalis.

SOURCE: Elkabes et al. 1989, copyright 1989, Elsevier Science Publishers

101
FIGURE 5. POMC fibers with growth-cone-like structures (arrows) along the

pontine flexure of a 12.5-day-old embryo

these fibers. By E14.5, the fiber distribution and appearance changed to the

adult-like pattern.

The prenatal expression of POMC mRNA also has been studied in the rat, but

only in the pituitary gland (Lugo et al. 1989). In that study, it was reported that

POMC mRNA expression first was detected in the ventral aspect of the

Rathke’s pouch at E13 and in the intermediate lobe at E15. This is consistent

with our observations because there is a 1- to 2-day shorter gestation period in

the mouse vs. the rat.

PROCESSING OF POMC DURING FETAL DEVELOPMENT

Mouse embryos were analyzed for POMC and for processed products at El 0.5,

When the POMC immunoreactivity first appeared. Sodium dodecyl sulfate

(SDS)-polyacrylamide gel electrophoresis separation of embryo extracts from

E10.5, followed by radioimmunoassay using the ACTH antibody DP4, revealed

the presence of POMC, but no ACTH processed products, at this stage of

development (figure 6). However, 1 day later, at E11.5, only processed

products were visible, and the POMC peak had disappeared (figure 6),

102
FIGURE 6. SDS-polyacrylamide gel electrophoresis of 10.5 and 11.5-day-old

embryo extracts followed by radioimmunoassay using an antibody

DP4,which recognizes POMC and ACTH1-39, Only POMC, the

first translated product, was seen at E10.5; posttranslational

cleavage of the prohormone occurred at E11.5.

SOURCE: Rius et al. 1991 a, copyright 1991, Elsevier Science Publishers

indicating that the processing machinery was activated at this point. Analysis of

the processed products by HPLC and radioimmunoassay revealed the

presence of two ACTH-related peptide forms: des-acetyl- -MSH, which was

the major form, and ACTH1-39 as well as immunoreactive ß-endorphin (table 1)

at both E11.5 and E12.5 (Rius et al. 1991a). At E14.5, there was approximately

a fourfold Increase in ACTH1-39 corresponding to the massive proliferation of

anterior pituitary cells. Similarly, ß-endorphin also increased fourfold at E14.5

No acetylated -endorphin was observed. The presence of these peptides

indicates that, during early development, the first POMC peptides that appear

are the smaller forms (figure 1) characteristic of proteolytic processing in adult

brain and intermediate pituitary. Moreover, although amldation appears to be

an early event in development, giving rise to des-acetyl- -MSH (ACTH1-13NH2

)

acetylation occurs later in development, primarily in the intermediate lobe

(Leenders et al. 1986). Anterior pituitary POMC processing also undergoes a

change, from producing ß-endorphin during early fetal development to

103
TABLE 1. Developmental expression of ACTH/MSH-related peptides and

ß-endorphin between E11.5 and 14.5

fmoles/embryo

Des-acetyl- -MSH ACTH1-39 ß-endorphin

E11.5 17.28±5.8 18.81±2.4 41.81±9.0

E12.5 50.13±4.5 18.93±0.6 50.52±1.0

E14.5 41.43±4.4 72.77±9.2 159.93±6.3

NOTE: Values shown are the mean±SEM of three experiments. Ten embryos

were pooled for each determination at the different embryonic ages.

producing ±-LPH postnatally (Roberts et al. 1978). Similar results also have

been obtained for anterior pituitary POMC processing in rat fetuses and

neonates (Pintar and Lugo 1987; Sato and Mains 1985, 1987; Allen et al.

1984). In the rat, it also was observed that there was a greater production of

des-acetyl- -MSH in the fetal vs. adult anterior pituitary. The change in pattern

of processing of POMC in the fetal anterior pituitary appears to be gradual and

extends through postnatal development. Moreover, in the rat, the curtailment of

processing of ß-LPH to ß-endorphin appears to lag behind the switch-over of

processing from des-acetyl- -MSH to ACTH1-39. Studies on human anterior

pituitary also indicate a greater production of des-acetyl- -MSH in fetuses than

in adults (Coates et al. 1989). The production of smaller, nonacetylated forms

of peptides, such as des-acetyl- -MSH and ß-endorphin in early embryos,

suggests that these peptides may be important in embryogenesis and CNS

development.

ONTOGENY OF MAMMALIAN BRAIN OPIOID RECEPTORS

The ontogeny of opioid binding sites has been studied extensively in rat brain

(Clendeninn et al. 1976; Coyle and Pert 1976; Garcin and Coyle 1976).

Antagonists (

3H-naltrexone and 3H-naloxone) were the first radioligands used to

determine specific binding and binding parameter estimates in membranes. In

the prenatal period, specific binding, regardless of whether expressed in relation

to total brain or to protein, increased with age. Although the absolute number of

opioid binding sites increased until adulthood, their concentration, following a

quiescent period a few days after birth, increased at a slower rate in the

postnatal period than in the prenatal period. Similarly, Kirby (1981) found that

3H-diprenorphine binding to rat spinal cord opioid sites increased with prenatal

and postnatal development but reached a maximal concentration at P15, which

104
is well before that in brain. Because diprenorphine binds to

receptors, it provides a better assessment of total opioid binding than the μselective antagonists, naloxone or naltrexone (Barg et al. 1989).

Receptor autoradiography permitted a refinement in anatomical detail of opioid

binding in fetal and postnatal rat brain (Kent et al. 1982; Unnerstall et al. 1983;

Edley and Herkenham 1984; van der Kooy 1984; Recht et al. 1985; Kornblum

et al. 1987b). Although most embryonic brain regions demonstrated diffuse

distributions of opioid receptors, in some (e.g., striatum and telencephalon)

subsequent dense labeling was observed just before birth. In the postnatal

period, the same dense regions reorganize to form a heterogeneous mosaic of

patches. It is clear from the autoradiography that there are brain regions from

which receptor labeling seems to disappear with time. In light of the difficulties

in reproducing sequential sectioning and in quantitation of receptor

autoradiography noted by Recht and colleagues (1985), the significance of this

transient expression is difficult to assess. Nevertheless, it may be indicative of

specific alterations of opioid receptor density and transient expression of opioid

sites in brain regions such as cerebellum, which also has been detected using

standard membrane binding assays (Tsang et al. 1982; Barg and Simantov

1989).

With the recognition of multiple opioid binding sites, differences in the

developmental profiles were observed for high- and low-affinity naloxone (Koch

et al. 1980; Tsang et al. 1982) and morphine (Pasternak et al. 1980) binding

sites. More recently, the introduction of subtype-specific radioligands facilitated

the examination of the differential prenatal and postnatal development of

and receptors (Wohltmann et al. 1982; Leslie et al. 1982; Spain et al. 1985;

Tavani et al. 1985; Dunlop et al. 1986; Szijcs et al. 1986, 1987; Petrillo et al.

1987; McDowell and Kitchen 1986, 1987; Milligan et al. 1987; Barg et al. 1989;

Barg and Simantov 1989; Magnan and Tiberi 1989). Studies in prenatal (rat),

perinatal (human), and postnatal (rat and mouse) developing species revealed

that brain p-sites were the first to appear, as early as El 5 (Barg et al. 1989);

sites were always the last to appear. In rat, sites emerged about 5 days after

birth in the forebrain (Sziics and Coscia 1990). Autoradiography with selective

ligands revealed a myriad of different regional developmental patterns for opioid

subtypes in postnatal rat brain, including frequent apparent transient expression

(Kornblum et al. 1987b).

Although most of the rat studies were done during the postnatal period, we

have focused on the early prenatal developmental expression of p-binding sites

in fetal mouse brain, beginning from El 1.5 to E18, using the specific

μ-radiolabeled ligand [

3H] D-Ala2

-Me-Phe4-Gly-ol5-enkephalin (DAMGE).

μ-Sites first were detected at E12.5, a day after the processing of POMC had

begun (figure 7). Subsequent to E12.5, ligand binding increased in parallel with

105
FIGURE 7. Prenatal development of specific binding of [

3H]-DAMGE and

membrane protein in mouse brain. Mouse brains of various

embryonic age were pooled and used to prepare cell-free

homogenates from which 20,000 g sediments were obtained.

Binding assays were performed with these membranes in the

presence of 1 nM [

3H]-DAMGE as previously described (Spain et

al. 1985).

Values are the means±SEM of three experiments carried out in duplicate.

the increase in protein (figure 7) and ß-endorphin content (table 1). The

embryonic μ-binding sites at E14.5 have a Kd of 2.9±0.8 nM similar to that of

the adult. Displacement studies with ß-endorphin showed that the [3H]DAMGE

binding was displaced by this peptide, indicating that the embryonic μ-receptors

present will bind ß-endorphin (Rius et al. 1991b). These findings indicate that

by E12.5, the POMC system is fully functional, because the processed peptides

and at least one of the receptors (μ-receptor) are present at this time.

106
CONCLUSION

The findings reviewed in this chapter strongly support a role of the POMCderived peptides in embryonic development. First, in the mouse, POMC mRNA

and POMC-derived peptides are present by E11.5, and ß-endorphin receptors

were detected in the CNS by E12.5, at the beginning of neurogenesis. In

addition, adrenal blastemas are evident at El 1.5. Because N-POMC1-49 is a

potent mitogen for adrenal cells (Estivariz et al. 1988), the CNS source of this

peptide may be involved in adrenal development and perhaps the

organogenesis of other tissues as well. Second, analysis of the processing of

POMC in early embryos reveals that the major peptides present were desacetyl- -MSH and ß-endorphin in several species. Acetylation of these

peptides appears to occur primarily in the intermediate lobe during the perinatal

period and continues postnatally (Leenders et al. 1986). In the anterior lobe,

there was a change in the proteolytic cleavage of POMC from producing the

embryonic peptides, des-acetyl- -MSH and ß-endorphin, to the larger peptides,

ACTH1-39 and ß-LPH, further indicating that the smaller embryonic peptides may

be functionally more important during embryogenesis. Finally, the prenatal

appearance of opiate p-receptors before (Rius et al. 1991b) and -receptors

in the brain suggests that the POMC-ß-endorphin system is the opioid system

likely to play a role in early developmental events. Future studies using

transgenic mice with altered POMC gene expression, as well as in vitro assays

for effects of POMC peptides on various embryonic tissue cultured cells, should

shed light on the mechanisms of action of POMC peptides in fetal CNS

development. Such knowledge will enhance the understanding of the

pharmacopathology associated with fetuses born to mothers addicted to opiate

drugs.

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ACKNOWLEDGMENT

This work was supported in part by National Institutes of Health grant DA-05412

to CJC.

AUTHORS

Y. Peng Loh, Ph.D.

Section Chief

R. Adrian Rius, M.D., Ph.D.

Visiting Associate

Section on Cellular Neurobiology

Laboratory of Developmental Neurobiology

National Institute of Child Health and Human Development

111
National Institutes of Health

Building 36, Room 2A-21

9000 Rockville Pike

Bethesda, MD 20892

Stela Elkabes, Ph.D.

Instructor

Division of Developmental Neurology

Cornell University Medical College

545 East 61st Street

New York, NY 10021

Wojciech Bern, M.D., Ph.D.

On leave from the institute of Biostructure

Warsaw

POLAND

Carmine J. Coscia, Ph.D.

Professor

Department of Biochemistry and Molecular Biology

St. Louis University Medical Center

1402 South Grand Boulevard

St. Louis, MO 63104

112
Regulation of ß-Endorphin

Biosynthesis in the Brain: Different

Effects of Morphine Pelleting and

Repeated Stress

David M. Bronstein, Jeffrey E. Kelsey, and Huda Akil

INTRODUCTION

In the past 15 years, there has been a tremendous growth in understanding the

anatomy and biochemistry of endogenous opioid peptides, both in endocrine

and in neural tissues. However, regulatory questions about peptide systems,

particularly in the brain, have been difficult to address: thus, much less currently

is known about this aspect of opioid peptide physiology. In fact, understanding

Of the regulation Of peptide biosynthesis derives primarily from studies of

pituitary tissue. An important regulatory principle learned from these studies is

that biosynthetic activity usually is coupled fairly tightly to the rate of secretion.

Thus, stimuli that cause the release of a peptide product from a cell generally

induce biochemical mechanisms that lead to compensatory increases in peptide

biosynthesis, whereas decreased peptide release causes biosynthesis to

decline. Although there are obvious major differences between endocrine and

neural systems, information about how peptide biosynthesis is regulated in the

pituitary gland lays a groundwork for conceptualizing how these systems may

be regulated in the brain. The usual strategy of regulatory studies in the brain

has been to measure the possible changes in steady-state peptide

concentrations following exposure to an experimental manipulation. It is now

clear that all opioid peptides have a number of immunologically related forms,

each with its own spectrum of biological properties. It is important to measure

the concentrations of each of the biologically relevant peptide forms following an

experimental treatment because alterations in total immunoreactive

concentrations may not necessarily be related to behavioral or physiological

changes. (Conversely, physiologically relevant changes may occur even

though the total amount of immunoreactive peptides does not change.) Another

point to consider when studying peptide regulation is that steady-state

concentrations provide no information about the “turnover rate” of the peptide of

interest. It is now clear that additional biochemical parameters are needed to

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help discriminate whether alterations in peptide levels are due to changes in

peptide release or biosynthesis. For example, a manipulation that causes

peptide levels to rise may result from increased peptide biosynthesis induced by

increased peptide release or may be due to an intracellular accumulation of

peptide subsequent to reduced release. Now, thanks to recent technological

advances in molecular biology that allow the measurement of low levels of

specific mRNAs in discrete brain regions, and in conjunction with the knowledge

gained from regulatory studies in the pituitary gland, it is feasible to begin to

examine how peptide systems in the brain are regulated. This chapter

describes the results of brain studies examining the potent endogenous opioid

peptide, ß-endorphin (ßE). The goal of these studies was to determine some of

the regulatory mechanisms employed by ßE-containing neurons in response to

behavioral and pharmacological treatments that alter peptide release and

biosynthesis.

PRO-OPIOMELANOCORTIN STRUCTURE, ANATOMY, AND

BIOSYNTHESIS

The biosynthesis of ßE begins with the transcription of the gene coding for the

precursor protein pro-opiomelanocortin (POMC) (for a general review of peptide

biosynthesis, see Sherman et al. 1989). Within the nucleus, introns of the

primary transcript (or heteronuclear RNA) are spliced out, and mature mRNA is

transported to the cytoplasm. Translation of the nucleic acid message into its

corresponding amino acid sequence occurs as mRNA molecules interact with a

ribosomal complex of RNA and protein molecules. The pre-POMC protein

contains at its amino terminus a signal peptide sequence that is critical for

directing the nascent protein toward the endoplasmic reticulum. The translation

process is temporarily arrested as the newly synthesized POMC chain is

translocated to the inside of the endoplasmic reticulum, Where the signal

peptide is cleaved off. With the resumption of translation, synthesis of the

precursor, including its glycosylation, is completed. Prohormone molecules

then are shuttled to the Goigi apparatus and packaged into vesicles along with

several enzymes important for precursor protein cleavage and processing. In

addition to ßE, other biologically important peptides contained within the amino

acid structure of POMC include adrenocorticotrophic hormone (ACTH);

ß-lipotropin (ß-LPH); and , ß-, and -melanocyte-stimulating hormones.

Peptides are liberated from the precursor molecule by enzymatic cleavage at

pairs of dibasic amino acids (i.e., lysine and arginine), which are found at the

amino- and the carboxy-termini of all these peptide products. That there are

tissue-specific differences in the POMC-derived peptides produced in ceils

indicates that enzymatic cleavage of the POMC precursor is not uniform across

all tissues. However, because little is known about the precise nature of the

enzyme(s) that cleave at the dibasic sites, it is not clear whether some tissues

114
lack the proper enzyme(s) for fully processing POMC or whether other factors

are responsible for the tissue-specific distribution of POMC peptides. In

addition to the trypsin-like enzymes that cleave POMC at paired dibasic sites,

vesicles often contain other enzymes that can further trim (e.g.,

carboxypeptidases) or chemically modify (e.g., phosphorylate, acetylate, or

amidate) specific peptide products. Post-translational modifications of POMCderived peptides can dramatically alter the biological characteristics of the

original peptide. For example, carboxy-terminal cleavage or N-terminal

acetylation of ßE1-31, abolishes its analgesic properties and greatly reduces its

ability to displace opiate binding (Akil et al. 1961 a; Deakin et al. 1980). More

dramatically, there is also evidence to suggest that ßE1-27, is antagonistic to the

opiate effects of ßE1-31 (Bals-Kubik et al. 1988; Nicolas et al. 1964). Tissue--

specific (and perhaps vesicle-specific) distribution of these cleaving and

modifying enzymes may account for the different peptide products detected in

different tissues. Following appropriate stimulation, the peptide contents of

vesicles are released from the cell, usually in a Ca2+-dependent manner. When

considering the question of how POMC cells respond and adapt to incoming

stlmuli, one should keep in mind that every step in the pathway from gene

transcription through protein translation and processing to peptide release, in

theory, may be regulatable. The potential to regulate discrete steps

differentially in the biosynthetic pathway thereby provides cells with a rangeof

possible responses when challenged.

Given the large number of possibilities for cleavage and posttranslational

modifications, it is not surprising that the precise composition of POMC peptides

synthesized in a cel is variable, depending on the tissue examined and on the

physiological status of the cell (i.e., whether it is in a “resting” basal state or has

been recently activated or inhibited). The highest concentrations of ßEimmunoreactive (ßE-ir) peptides are found in the pituitary gland. In the rat

anterior pituitary gland (AP), ß-LPH and full-length ßE (i.e., ßE) are theprimary peptide products derived from the carboxy-terminal third of POMC. On

the other hand, in the intermediate lobe of the pituitary (IP), virtually no ß-LPH is

detected because it is all processed to ßE1-31 most ßE1

-31 is then further

cleaved at its carboxy terminus to produce ßE1

.27 or BE1-26 in addition, the

majority of all ßE-ir species in the IP is acetylated at the amino termini. In the

central nervous system (CNS), all ßE-ir is derived from one of two clusters of

POMC cell bodies. The major POMC cell group, situated in the arcuate nucleus

of the hypothalamus, has axonal projections that terminate in a diverse number

of forebrain and midbrain structures—including various hypothalamic nuclei, the

periventricular thalamic nucleus, the periaqueductal gray (PAG) through the

diencephalon and midbrain-and such limbic structures as the amygdala and

septum (Bloom et al. 1978; Joseph 1980; Khachaturian et al. 1985; Watson et

al. 1977). A much smaller and more diffuse cluster of POMC cell bodies has

115
been localized in the nucleus of the solitary tract (NTS) and nucleus

commisuralis in the caudal medulla (Khachaturian et al. 1983; Schwartzberg

and Nakane 1983). POMC fiber projections from the medullar cell group have

not been definitively described. Although they do not appear to project

extensively to rostral structures (Palkovits et al. 1987), it is possible that NTS

and arcuate POMC neurons share some common terminal fields. In all rostral

brain regions studied, ßE1-31, is the major ßE-ir species found, with smaller

amounts of ßE1-27, and little or no ß-LPH present. In areas innervated primarily

by the arcuate cell group (i.e., forebrain and midbrain structures), the great

majority of BE-ir peptides is not acetylated, although a substantial degree of

acetylation appears to occur in the NTS region (Dares et al. 1986).

REGULATION OF POMC BIOSYNTHESIS IN THE PITUITARY GLAND

The best illustration of how POMC biosynthesis is regulated at multiple levels is

provided by studies of pituitary tissue. A variety of stressors induce the release

of ßE-ir peptides and ACTH from corticotrophs of the anterior pituitary. Results

from pulse labeling studies indicate that one of the earliest cellular responses to

the increased secretion of POMC products is an increase in the rate at which

POMC precursor is translated and in the rate at which the precursor protein is

converted into peptide products (Shiomi et al. 1986). Thus, Under conditions of

acute stress, increases in POMC translation and processing allow pools of ßE-ir

peptideS and ACTH to be replenished much faster than normal. If animals are

stressed daily for an extended period (i.e., 1 to 2 weeks), changes in other

POMC biochemical parameters are observed: There are small but reliable

increases in POMC mRNA and larger (fourfold to fivefold) increases in ßE-ir

concentrations in the AP (Shiomi et al. 1986). Thus, corticotroph cells adapt to

chronic stimulation by increasing the amounts of stored peptide products as well

as increasing their overall biosynthetic capacity by producing more POMC

mRNA. Interestingly, rates of POMC translation and processing, which

increase following acute stress, return to near normal levels in repeatedly

stressed animals and do not change even when chronically stressed animals

are acutely rechallenged. Increasing translation and processing rates

apparently are only short-term mechanisms for increasing peptide products in

anterior lobe corticotrophs and are no longer required once more long-term

mechanisms are invoked (e.g., increased mRNA).

In addition to regulating discrete steps in the POMC biosynthetic pathway, there

is also evidence that corticotrophs possess the capacity to differentially regulate

the release of specific ßE-ir peptides. For example, whereas ß-LPH and ßE1-31

are stored in the anterior pituitary gland in a ratio of 2:1, stress or a

corticotropin-releasing hormone (CRH) challenge causes these peptides to be

secreted into the plasma in a ratio of 1:2 (Watson et al. 1988; Young et al.

116
1986). This implies that ßE1-31 is roughly fourfold more likely than ß-LPH to be

released from corticotrophs. On the other hand, adrenalectomy, which removes

steroid inhibition of POMC production in the AP and causes total ßE-ir in the

plasma to increase dramatically, results in sixfold higher ratios of ß-LPH:ßE in

plasma relative to normal levels (Young 1989). Thus, while ßE is preferentially

secreted following acute stress, ß-LPH appears to be preferentially released

following chronic activation by adrenalectomy. These data support the notion

that which peptide products are released may depend on the length of time they

remain in the vesicle, increasingly susceptible to further enzymatic

modifications. For example, when secretion from corticotrophs is chronically

stimulated by adrenalectomy, it is likely that POMC-derived peptides are stored

in vesicles for shorter periods, and one sees a relative increase in the release of

the less processed product, ß-LPH. At the present time, it is not known whether

the mechanisms by which different peptide products from the same precursor

are differentially released from the AP might be related to heterogeneity in

corticotroph ceils, to intracellular differences in storage vesicles, or to other

factors. Similar results to those described for the AP also have been observed

in studies of the POMC-containing melanotrophs in the IP (Akil et al. 1985;

Meador-Woodruff et al. 1990). Taken together, these results clearly illustrate

the multiplicity and complexity of mechanisms that POMC cells in the pituitary

gland use to regulate ßE biosynthesis and release in response to changes in

cellular activiation. We were interested in whether some of the regulatory

mechanisms found in pituitary POMC ceils also might be utilized by POMC cells

in the brain. In the remainder of this chapter, we present data describing

several changes in POMC biosynthesis that take place following exposure to

either stress or morphine, treatments that we believe activate or inhibit,

respectively, central ßE-containing cells.

EFFECTS OF REPEATED STRESS ON POMC BIOSYNTHESIS IN BRAIN

The effects of stress on the release and regulation of POMC-derived peptides in

the pituitary, both in the anterior and the intermediate lobes, have been

characterized in detail (for example, see Akil et al. 1988). In addition to its wellknown effects on the pituitary, there is also evidence that stress activates

POMC systems in the brain. A variety of stressors (e.g,, footshock, cold-swim,

learned helplessness) have been shown to elicit analgesia that appears to be at

least partially opioid-mediated (Akil et al. 1976, 1984). That the endogenous

opioid mediating this effect might be ßE is supported by data showing that

hypothalamic levels of ßE-ir declined significantly following acute footshock

stress (Akil et al. 1981b; Millan et al. 1981). More recent data also support the

possibility that stress might affect hypothalamic POMC neurons in a manner

similar to its effects on anterior lobe corticotrophs. CRH and arginine

vasopressin, both of which are well established as mediating stress-induced

117
secretion of ßE and ACTH from the anterior lobe, recently have been shown to

stimulate ßE-ir peptide release from the hypothalamus in vivo (Nikolarakis et al.

1988) and In vitro (Bronstein and Akil 1990; Burns et al. 1989; Sweep and

Wiegant 1989). Therefore, it was reasonable to examine the effects of acute

and repeated exposure to a stressor on POMC peptide and mRNA levels in the

brain in an attempt to uncover some aspects of ßE biosynthetic regulation.

The stress paradigm involved the use of intermittent footshock (1.5 mA, 1 sec

duration, every 5 sec) for a 30-minute period. Four different experimental

groups were employed: (1) Control (Con)—animals were unhandled; (2) Acute

Stress (AS)—animals received a single, 30-minute stress session immediately

before sacrifice; (3) Chronic Stress/Rested (CS/R)animals were stressed for

30 minutes a day for 14 days and rested for 24 hours prior to sacrifice; (4)

Chronic Stress/Acute Stress (CS/AS)—animals were stressed daily for 14 days

and received a final 30-minute stress session on the 15th day immediately prior

to sacrifice. The inclusion of the two chronic stress groups provided us with the

opportunity not only to measure peptide and mRNA changes following repeated

challenges but also to detect possible peptide effects of acute stress in

chronically stressed animals, something difficult to do in previously unstressed

animals. Two components of the arcuate nucleus POMC group were

examined: (1) a hypothalamic region containing POMC cell bodies as well as

ßE-ir nerve terminals and fibers of passage and (2) a midbrain region, including

the PAG, which is rich in ßE-ir terminals derived primarily, if not exclusively,

from the arcuate nucleus (Joseph 1980; Millan et al. 1984).

The effects of the different stress paradigms on ßE-ir peptide levels in the

hypothalamus and midbrain are shown in table 1. In the hypothalamus, acute

stress caused ßE-ir content to increase in both naive (AS) and repeatedly

stressed (CS/AS) animals, whereas CS/R animals showed only a slight

increase in hypothalamic peptide stores compared to unstressed controls.

Although these data are consistent with our previous report on the effects of

acute stress on total opiate-like factors in brain (Madden et al. 1977) and with

increased occupancy of opioid receptors following acute stress (Christie et al.

1981), they do not agree with earlier results from this (Akil et al. 1981 b) and

other (Millan et al. 1981) laboratories. Decreases in peptide content following

acute manipulations generally have been interpreted to be the result of peptide

release. It is difficult to understand how a stimulus that purportedly induces

transmitter/hormone release could acutely cause tissue levels of that substance

to increase. One explanation for the paradoxical acute increase in ßE-ir content

in the hypothalamus may be related to this tissue containing both POMC cell

bodies and nerve terminals. If POMC translation and processing in the arcuate

nucleus increased following acute stress, as it does in the pituitary gland, and

118
ßE were degraded at a slow rate following release from nerve terminals, then a

mixed cell body/nerve terminal brain region might be expected to exhibit

increased ßE-ir content following acute activation,

TABLE 1. Effects of acute and chronic stress on ßE-ir peptides in brain

Con AS CS/R CS/AS

Hypothalamus 4.42±.28 5.61±.32 4.84±.25 5.34±.39

(100%) (127%) (109%) (120%)

Midbrain 0.81±.08 0.99±.12 1.24±.21 0.87±.44

(100%) (122%) (154%) (108%)

KEY: Con-animals were unhandled; AS-animals received 30 minutes of

electric footshock immediately before sacrifice; CS/R—animals were

stressed once a day for 14 days and sacrificed on the 15th day; CS/AS—

identical to CS/R group except that animals received a final stress

session immediately prior to sacrifice on the 15th day.

NOTE: Values represent the means±SEM of total ßE-ir (pmoles/tissue)

measured in different brain regions. Numbers in parentheses represent

ßE-ir values expressed as a percent of control levels.

More pronounced effects of stress on total ßE-ir content were observed in the

midbrain (table 1). In naive animals, acute stress tended to increase ßE-ir

levels in the midbrain, as it did in the hypothalamus, although this effect was not

statistically significant. On the other hand, there was a dramatic decline in ßE-ir

peptide levels immediately following acute stress of chronically stressed

animals. Furthermore, in chronically stressed animals that were allowed to rest

24 hours before sacrifice, ßE-ir content in the midbrain increased by

approximately 50 percent over control levels. The changes in midbrain ßE-ir

content in chronically stressed animals are compatible with the hypothesis that

stress stimulates release and biosynthesis in central ßE-containing neurons.

Chromatographic analyses of midbrain extracts revealed two majorßE-ir peaks,

corresponding to ßE1-31 and ßE1-27/ßEI

-26 (which could not be resolved from

each other under these chromatographic conditions). These two ßE-ir forms

were found in relative concentrations of 1:0.6 in control animals (figure 1).

Acutely stressed animals showed no changes in the relative amounts of the

different ßE-ir forms. In chronic stress/rested animals, however, the proportion

of smaller size ßE-ir peptides was greatly increased such that the ratio of

119
FIGURE 1. Changes in the relative amounts of ßE1-31/ß1

.26 and ßE1-31

peptides following acute and repeated footshock stress.

ßE1-27/ßE1-26 concentrations are expressed as a ratio of the ßE1-31

concentration. Pooled aliquots from the four experimental groups

(Con; AS; CS/R; and (CS/AS, see table 1 legend) were

chromatographed on a Sephadex G-50 column and collected

fractions were assayed for ßE-ir. Total immunoreactivity was

determined in the peaks that co-eluted with authentic ßE1-31 and

ßE1-27 standards (since ßE1-26 and ßE1-27 could not be resolved on

this column, the ßE1-27 peak includes both of these peptides).

ßE1-31:ßE1-27/ßE1-26 was 1:15. The amounts of ßE1-31 in control and chronically

stressed/rested animals were comparable, indicating that the increase in total

ßE-ir in the chronically stressed animal was due to a selective increase in

carboxy-terminal cleaved products of ßE1-31. In chronic/acute stress animals,

the ßE1-31:ßE/ßE1-26 ratio returns to near normal levels (i.e., 1:0.5),

suggesting that more processed ßE-ir forms may have been released more

readily than full-length ßE1-31 This implies that less opioid active material is

liberated by a stress challenge in animals with a history of chronic stress

compared to acutely stressed naive animals. This conclusion is supported by

behavioral data showing that chronically stressed animals that are rechallenged

acutely do not display the analgesic responses that are apparent in acutely

stressed naive animals; that is, they have developed tolerance (Akil et al. 1976).

Keeping in mind that ßE1-27/ßEI.26 are much less potent opioids than or may be

antagonistic to ßE1-31 (Akil et al. 1981a; Bals-Kubik et al. 1988; Deakin et al.

1980; Nicolas et al. 1984), the analgesic tolerance observed following repeated

120
stress may have resulted from a shift in the ßE-ir products released, with ßE1-31

being released following the Initial stress exposure and ßE1-27/ßE1.26 being

released following repeated stress exposures.

As mentioned earlier, one cannot determine a priori whether the chronic stressinduced increases in midbrain ßE-ir result from an increase or decrease in

neural activity and secretion. Hence, a time course of the effects of repeated

stress on POMC mRNA levels was carried out. Animals were stressed for 30

minutes a day for 0 (controls), 3, 5, 8, 15, or 22 days and were sacrificed

immediately after the final stress session. Total hypothalamic RNA was

extracted by a guanidium isothiocyanate/CsCl method, separated on 1.4

percent agarose gels, and transferred to nylon membranes. The membranes

were hybridized overnight with a 420-base radiolabeled riboprobe

complementary to a mouse POMC cDNA sequence (courtesy of Dr. James

Roberts). Following treatment with RNase A (to digest unhybridired riboprobe),

the membrane was washed, air dried, and exposed to x-ray film. As shown in

figure 2, POMC mRNA levels were elevated following 3 to 5 days of stress but

returned toward control levels after 8 to 22 days. Densitometric quantitation of

the labeled bands revealed that the maximal effects at 3 and 5 days were 196

percent and 158 percent, respectively, of control values, suggesting elevated

POMC biosynthetic capacity at these times. If one assumes that changes in

brain levels of mRNA are linked to changes in secretion, as they are in the

pituitary gland, then one may conclude that stress activates ßE-ir secretion from

brain POMC cells. Interestingly, POMC mRNA levels return to control levels

during the second and third weeks of stress exposure. Perhaps it is no longer

necessary for cells to maintain elevated message levels once they accumulate

sufficiently high peptide pools. Alternatively, animals may have adapted to the

stressor with repeated exposure, making it progressively less stressful with

subsequent exposures.

At this point, the regulatory responses of POMC neurons can be compared with

those of corticotrophic and melanotrophic cells of the pituitary. Both types of

POMC cells alter the rate and/or extent of POMC precursor processing to

produce different mixtures of ßE-ir products under appropriate conditions.

Following repeated activation, there are compensatory increases in ßE-ir

peptide and POMC mRNA levels, thereby providing cells with a ready reserve

of peptide transmitter and an increased biosynthetic capacity with which to

respond to future challenges. There is also evidence that, under certain

circumstances, endocrine and central POMC cells have the capacity to

differentially regulate the release of the various ßE-ir peptides, in effect altering

the precise signal emitted into the synapse or blood. Taken together, it seems

fair to say that there are several similarities in POMC regulation in the brain and

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FIGURE 2. Northern analysis of the effects of repeated stress on POMC

mRNA in the hypothalamus. Animals were footshock stressed for

30 minutes a day for 3, 5, 8, 15, or 22 days (unstressed controls

are designated by 0). Total hypothalamic RNA was separated on

a 1.5 percent araose gel, transferred to a nylon membrane, and

hybridized with a radiolabeled riboprobe complementary to a

mouse POMC cDNA sequence. The membrane then was treated

with RNase, washed, air dried, and exposed to x-ray film.

pituitary gland following acute or repeated stimulation, although the onset and

duration of these responses may differ widely. In the next section, we will

examine some of the changes in POMC biosynthesis that occur when ßE

neurons are chronically inhibited.

EFFECTS OF MORPHINE TREATMENT ON POMC BIOSYNTHESIS IN THE

BRAIN

One of the most obvious questions raised after the enkephalins were first

discovered was whether the phenomenon of opiate tolerance and/or

dependence might be related to alterations in endogenous opioid systems in

the brain, Kosterlitz and Hughes (1975) hypothesized that chronic morphine

treatment may result in agonist-induced feedback inhibition of endogenous

opioid biosynthesis. However, to date, no changes in the steady-state levels of

any of the opioid peptides have been detected following relatively short-term

morphine treatments (3 to 10 days), which produce morphine tolerance and

dependence (Berglund et al. 1989; Childers et al. 1977; Fratta et al. 1977; Hollt

et al. 1978; Wesche et al. 1977). More recently, there have been conflicting

reports about whether chronic morphine pelleting (5 to 7 days) affects POMC

mRNA levels In the hypothalamus, with Costa and colleagues reporting

morphine-induced decreases in POMC message (Mocchetti and Costa 1986;

Mocchetti et al. 1989), while Hollt and coworkers (1989) found no effect. We

reexamined the question of morphine’s effects on POMC biosynthesis

(Bronstein et al. 1990), attempting in these studies to obtain measures that

might reflect the dynamics of ßE biosynthesis. Hence, in addition to steady

122
skate levels of total ßE-ir, concentrations of different ßE-ir species (i.e., ß-LPH,

ßE1-31 and ßE1.27/ßE1.28) in different brain regions as well as POMC mRNA

levels in the arcuate nucleus were measured at several time points during the

course of 7 days of morphine treatment.

Male Sprague-Dawley rats were implanted with morphine (75 mg/pellet) or

placebo pellets (provided courtesy of the National Institute on Drug Abuse).

Some animals received one pellet on day 1 and were killed 24 or 72 hours later.

Other animals were implanted with one pellet on day 1 and three additional

pellets on day 4 and were killed on day 7. As in the stress experiments, we

examined a POMC cell body/nerve terminal region in the hypothalamus and an

exclusively nerve terminal midbrain region, containing the PAG.

In accordance with previous reports, we found that 7 days of morphine

treatment had no effect on ßE-ir concentrations in the hypothalamus or

midbrain, Gel chromatographic analyses then were carried out, followed by

radioimmunoassay to measure different size ßE-ir species. Although morphine

had no effect on total ßE-ir, it was conceivable that it produced changes in

POMC processing that may have altered the relative concentrations of the

different ßE-ir species. Our results with repeated stress had revealed that

tolerance to stress-induced analgesia was associated with a selective increase

in ßE1-27/ßE1-26, peptides that have much less opiate activity than or may be

antagonistic to ßE1-31. It was attractive to consider the possibility that a similar

mechanism might mediate the tolerance induced by chronic opiate

administration. Chromatographic analyses indicated that the relative amounts

of ßE1-31 and ßE1-27/ßE1-26 -size material in the hypothalamus or midbrain were

similar in placebo- and morphine-pelleted animals; in addition, morphinepelleted animals injected with naloxone (to produce precipitated withdrawal)

showed no changes in their chromatographic ßE-ir profiles (table 2). These

data, indicating that brain concentrations of different ßE-ir peptides are not

altered by 7 days of morphine pelleting, suggest that changes in POMC

processing may not play a significant role in mechanisms of opiate tolerance or

dependence.

Although opiate treatment does not appear to affect steady-state concentrations

of different ßE-ir peptides, there have been conflicting reports on whether this

treatment alters POMC mRNA levels in the hypothalamus. Hence, we

assessed the effects of morphine pelleting on POMC mRNA levels using the

sensitive solution phase hybridization/RNase protection technique. Total

nucleic acids were extracted from hypothalami in LET buffer (Lithium dodecyl

sulfate, EDTA, Tris) and hybridized with a radiolabeled riboprobe

complementary to a coding region of the POMC mRNA sequence.

Nonhybridized single-stranded RNAs then were digested by RNase

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TABLE 2. Effect of morphine pelleting and precipitated withdrawal on ßE-ir

peptides in brain

Plac-Sal Plac-Nal Mor-Sal Mor-Nal

Hypothalamus

ßE1-31

ßE1-27/ßE1-26

4.41 4.65 4.69 4.70

3.32 3.8 3.41 3.41

Midbrain

ßE1-31

ßE1-27ßE1-26

3.39 3.11 2.75 3.26

1.35 1.34 1.14 1.16

KEY: Plac-Sal—animals were implanted with four placebo pellets over a 7-day

period and received an injection of saline vehicle 30 minutes before

sacrifice; Plac-Nal—placebo-pelleted animals received an injection of

naloxone (2.5 mg/kg) 30 minutes before sacrifice; Mor-Sal-animals

were implanted with four morphine pellets (75 mg) over a 7-day period

and were injected with saline vehicle 30 minutes before sacrifice; MorNal-morphine-pelleted animals received an injection of naloxone 30

minutes before sacrifice.

NOTE: Values represent total immunoreactivity associated with the ßE1-31 and

ßE1-27/ßE1-26 peaks of chromatographed pools of hypothalamus or

midbrain.

treatment, leaving intact the double-stranded hybrids of POMC mRNA and

complementary riboprobe. Protected hybrids were fractionated on

polyacrylamide gels, visualized by autoradiography, and quantitated by

densitometry. In an initial study, using a mouse POMC cRNA that was not

completely homologous to the rat sequence, we found a significant 30- to

40-percent decrease in POMC mRNA levels following 7 days of morphine

treatment. In a subsequent study, using the homologous rat POMC riboprobe

(courtesy of Dr. Jim Eberwine), a decrease in POMC message also was

observed following morphine treatment. However, the magnitude of the

decrease (about 20 percent) was smaller than that observed in the initial study,

and POMC mRNA levels in morphine- and placebo-treated animals in the

second study were not statistically different, No changes in POMC mRNA

levels were detected following 3 days of morphine pelleting. The reasons for

obtaining a significant morphine-induced decrease in POMC message in one

study but not in the other may be related to the use of different riboprobes or,

perhaps, to our failure to control other factors consistently (e.g., stress, which

124
previously has been shown to affect central POMC systems). Although

variable, these data tend to support those of Mochetti and coworkers (Mocchetti

and Costa 1986; Mocchetti et al. 1989) and suggest that 7 days of morphine

pelleting apparently causes an overall decrease in ßE biosynthetic capacity by

reducing levels of POMC message.

If one extends the notion derived from studies in the pituitary to the brain, the

observed decrease in POMC biosynthetic capacity would be the result of

decreased ßE-ir secretion. We reasoned that if morphine does in fact inhibit

ßE-ir release from nerve terminals, then at some time point during the 7-day

morphine treatment we might be able to detect an accumulation of these

peptides in tissue. Hence, we carried out a time-course study in which we

compared animals implanted with morphine or placebo pellets for 1, 3, or 7

days. Morphine treatment had no effect on total ßE-ir in the hypothalamus or

midbrain following 1 or 7 days of pelleting. However, 3 days after morphine

pellet implantation, there was a significant increase in ßE-ir in the hypothalamus

(figure 3). This result is consistent with the hypothesis that morphine inhibits

the release of ßE-ir peptides, causing the gradual accumulation of these

peptides intraneuronally. This conclusion also is supported by chromatographic

analyses that revealed that the increase in ßE-ir at 3 days was primarily due to

a selective increase in more processed ßE-ir forms (i.e., ßE1-27/ßE1-26) with little

effect on the concentrations of full-length ßE1-31 (Bronstein et al. 1990). It is

generally believed that peptide maturation is a time-dependent process; thus,

posttranslational modifications that occur later in processing (such as the

COOH-terminal cleavage of ßE1-31) may not proceed fully to completion (for

example, see Akil et al. 1985). Hence, the elevated levels of ßE1-27/ßE1-26 are

likely the result of increased storage time in granules, as a result of decreased

release, leading to more complete processing. Given that opiate tolerance and

dependence have developed following 3 days of morphine pelleting, it is

tempting to speculate that the selective accumulation of carboxy-terminalcleaved products of ßE1-31 has some role in the development or expression of

these phenomena (see discussion below).

The data described above illustrate the importance of measuring several

parameters of biosynthetic activity at multiple time points to detect possible

regulatory changes in peptide biosynthesis. If only ßE-ir peptide concentrations

were measured, one would have concluded that opiate administration had no

effect on POMC biosynthesis. By measuring POMC mRNA, peptide levels, and

peptide forms at several different times during the morphine treatment, we

observed changes in hypothalamic POMC neurons that have led us to suggest

a sequence of cellular events that unfold as a result of chronic exposure to

morphine. We propose that the initial effect of morphine is to reduce neural

125
FIGURE 3. Effect of chronic morphine pelleting on total ßE-ir in the

hypothalamus. Rats were implanted subcutaneously with either

placebo or morphine pellets for 1, 3, or 7 days, and total ßE-ir in

hypothalamic extracts was determined by radioimmunoassay.

Data represent the means±SEM of morphine-treated animals

expressed as a percent of placebo control values.

activity and ßE release from POMC neurons. During the initial period of

morphine pelleting, POMC formation and processing in hypothalamic cells

continue at a relatively normal rate despite the reduced rate of ßE-ir peptide

release, thereby causing an eventual accumulation of ßE-ir after several days.

As the peptides remain longer in secretory granules, they undergo the relatively

slow process of conversion from ßE1-31 to the opiate antagonist ßE1-27. (This

conversion step has been shown to be slow in the IP by Seizinger and Hollt

[1980] and may be rate-limiting under some circumstances, such as stress [Akil

et al. 1985]). This leads to the observed shift in ratio toward the ßE1-27/ßE1-26

forms following 3 days of pelleting. With continued exposure to the opiate,

neurons exhibit a reduction in the amount of POMC message present, as cells

apparently strive to reduce overall ßE-ir biosynthetic capacity. (The decrease in

message levels could be due to either a decrease in the transcription rate of the

POMC gene or to an increase in the degradative rate of its mRNA.)

Presumably, homeostatic pressures would continue to reduce POMC mRNA

levels until the rate of ßE biosynthesis once again matches the rate of ßE

release. Thus, following 7 days of treatment, the new equilibrium between

synthesis and secretion results in placebo- and morphine-treated animals

126
having similar steady-state peptide levels. Preliminary results from in vitro

release studies, indicating that basal and stimulated release of ßE-ir were

reduced in morphine-pelleted animals compared with placebo-pelleted controls,

further support the hypothesis that morphine inhibits release from ßE-containing

neurons (Bronstein and Akil, unpublished observation).

If morphine treatment did cause the sequelae of molecular events in POMC

biosynthesis just described, it could have profound effects when the opiate is

removed (i.e., withdrawal). At earlier stages of morphine treatment, when there

is a relative increase in the levels of ßE1-27/ßE1.26, removal of the exogenous

opiate would leave the organism with relatively lower levels of endogenous

opiolds as well as increased amounts of opiate inactive or antagonistic

materials. At later stages of morphine pelleting, opiate withdrawal still would

result in decreased ß-endorphinergic tone, as overall biosynthesis and release

both appear to be reduced. From these data, it is not unreasonable to suggest

that the alterations in POMC biosynthesis, processing, and release that occur

following chronic morphine treatment may play a role in mediating some of the

effects of opiate withdrawal.

Obviously, the intricacies involved in the regulation of opioid peptide systems in

the brain are just beginning to be understood. Although several techniques

exist that permit the expansion of understanding of regulatory processes in the

pituitary gland (e.g., pulse labeling, transcription run-on assays, heteronuclear

RNA quantitation), there are some limitations on the kinds of regulatory

questions that can be asked about neural systems since these systems are

usually quantitatively (and qualitatively) more difficult than their endocrine

counterparts. Several of these biochemical and molecular biological techniques

need to be adapted to studying the CNS. In addition, the anatomical complexity

of this tissue needs to be respected, along with the range of timeframes in

which it operates (from nanoseconds to hours, days, and months). By studying

cellular regulation-from gene transcription to secretion-in the context of

known circuits and in conjunction with specific behavioral manifestations, we

can begin to understand the intrinsic function of endogenous opioids and their

modulation by drugs of abuse.

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ACKNOWLEDGMENTS

This research was supported by a National Institute on Drug Abuse grant DA02265 (HA), a National Institutes of Health-funded pilot project granted through

the University of Michigan Gastrointestinal Peptide Research Center, and

postdoctoral fellowships from the Medical Research Council of Canada and the

National Institutes of Health training grant in Endocrinology, Metabolism, and

Aging (DMB).

AUTHORS

David M. Bronstein, Ph.D.

Postdoctoral Research Fellow

Huda Akil, Ph.D.

Professor of Psychiatry and Research Scientist

Mental Health Research Institute

University of Michigan

205 Washtenaw Place

Ann Arbor, Ml 48109-0720

Jeffrey E. Kelsey, Ph.D., M.D.

Department of Psychiatry and Behavioral Sciences

131
Stanford University Medical Center

Room TD114

Stanford, CA 94305

132
Transcriptional Regulation of the Rat

Prodynorphin Gene

James O. Douglass, Michael J. ladarola, Jau-Shyong Hong,

James E. Garrett, and Cynthia T. McMurray

INTRODUCTION

cDNA cloning techniques have been used to characterize a variety of

neuroendocrine peptide precursor molecules encoding small biologically active

peptides that act as neurotransmitters and neuromodulators in the central

nervous system (CNS) and hormones in the endocrine system (Douglass et al.

1984). One such precursor, prodynorphin, encodes the dynorphin family of

opioid peptides (Kakidani et al. 1982; Horikawa et al. 1983; Civelli et al. 1985).

The prodynorphin precursor contains three leucine-enkephalin moieties, with at

least six additional biologically active opioids (Dyn A 1-8, Dyn A 1-17,

neoendorphin, ß-reoendorphin, Dyn B 1-13, and Dyn B 1-28) representing

carboxy-terminal extended forms of the leucine-enkephalin sequences.

Northern blot and in situ histochemical techniques have been used to determine

the rat tissues and cell types in which the prodynorphin gene is transcriptionally

active (Civelli et al. 1985; Akil et al. 1984). Such regions include numerous

brain structures (e.g., the hypothalamus, striatum, hippocampus, midbrain, brain

stem, and cerebral cortex), as well as spinal cord neurons. In endocrine

tissues, the prodynorphin gene is transcriptionally active in the anterior pituitary,

adrenal gland, testis, ovary, and uterus (Civelli et al. 1985; Douglass et al.

1987). Numerous studies have suggested that the prodynorphin opioids serve

to regulate a variety of physiological and behavioral responses such as pain

perception, intestinal peristalsis, motor function, and reproductive function(s). A

role for dynorphin peptides in narcotic tolerance mechanisms also has been

suggested (Lee 1984).

Cloning of rat prodynorphin cDNA and genomic DNA (Civelli et al. 1985;

Douglass et al. 1989) has afforded the opportunity to study regulated patterns

of prodynorphin mRNA levels in specific neuronal cell types following surgical or

pharmacological manipulations. Two such studies described here represent

those in which the most dramatic alterations in prodynorphin mRNA levels have

been observed to date. Proenkephalin mRNA levels, which also were

133
measured in these studies, exhibited regulatory profiles distinct from

prodynorphin mRNA. The isolation and characterization of the rat prodynorphin

gene has enabled us to begin dissecting the regions of genomic DNA involved

in the regulated production of prodynorphin transcripts, Both transcriptional and

posttranscriptional events may regulate the ultimate production of prodynorphinderived peptides. In the testis, alternate processing of prodynorphin (and

proenkephalin) precursor mRNA results in the generation of mature transcripts

containing unique 5’ untranslated regions, perhaps affecting the translational

status of the mRNA. The characterization of the rat prodynorphin promoter

region has allowed us to initiate prelude studies aimed at determining the

cis-acting nucleotide sequence elements controlling the production of

prodynorphin mRNA.

IN VIVO REGULATION OF PRODYNORPHIN mRNA LEVELS

Prodynorphln Gene Regulation In the Rat Spinal Cord

The prodynorphin gene is transcriptionally active in the mammalian spinal cord.

In situ hybridization histochemistry/immunohistochemistry has localized the

majority of rat spinal cord prodynorphin transcripts and peptides to laminae I-II

and V-VI (Ruda et al. 1988). Other studies have localized proenkephalinderived peptides to similar regions of the rat spinal cord (Basbaum and Fields

1984). The localization of both prodynorphin- and proenkephalin-derived opioid

peptides to these specific regions of the spinal cord suggests that these

peptides play a role in the endogenous pain recognition/control system.

Numerous experimental paradigms have served to strengthen this belief

(ladarola et al. 1988: Ruda et al. 1988; Millan et al. 1985, 1987).

In the study described here, prodynorphin mRNA and peptide (Dyn A 1-8) levels

were measured in the rat lumbar spinal cord at various times following

inflammation of the hindpaw (ladarola et al. 1988). An experimental model of

unilateral inflammation of the hindlimb (induced by a single, intraplantar

injection of Freund’s adjuvant) produced hyperalgesia to both mechanical and

radiant thermal stimulation, which was rapid in onset. During this period,

prodynorphin biosynthesis was substantially elevated in the region of the spinal

cord receiving sensory input from the affected limb (figure 1, panel A).

Prodynorphin mRNA levels rose substantially within the first 24-hour period, and

maximal stimulation (eightfold to ninefold increase) was observed between 3 to

5 days after injection. By day 14, prodynorphin mRNA levels approached

control values. This time course of induction and subsequent decline closely

paralleled that of hindpaw edema and hyperalgesia. Spinal cord Dyn A 1-8

levels also rose approximately threefold during the inflammatory period. This

pattern is consistent with an increase in both the rate of synthesis and the

134
FIGURE 1. Differential regulation of spinal cord prodynorphin and proenkephalin mRNA and peptide levels following

the induction of unilateral peripheral inflammation

NOTE: Prodynorphin mRNA/Dyn A 1-8 peptide levels (panel A), and proenkephalin mRNA/MERGL peptide levels

(panel B) in the rat lumbar spinal cord were measured at various times after the administration of Freund’s

adjuvant in a rear footpad. Values are represented as percent of control (saline-injected animals). (For

specific details of the procedure, see ladarola et al. 1988.)
release of dynorphin peptides from spinal cord neurons. In contrast, the spinal

cord proenkephalin system was relatively unresponsive (figure 1, panel B).

Proenkephalin mRNA levels rose a modest twofold, with met-enkephalin-ArgGly-Leu (MERGL) levels remaining relatively unchanged. These data suggest

the active participation of dynorphin-containing spinal cord neurons in the

modulation of sensory afferent input during peripheral inflammatory pain states.

The study is also of interest in that both the prodynorphin and the proenkephalin

genes are transcriptionally active in similar regions of the spinal cord; yet, only

the prodynorphin gene is transcriptionally responsive to peripheral inflammation.

Prodynorphln Gene Regulation In the Rat Hippocampus

Opioid peptides derived from the prodynorphin and proenkephalin precursors

are present at high levels in the rodent hippocampus. Enkephalin

immunoreactivity is observed mainly in the mossy fiber axons of the dentate

gyrus granule cells, with staining also observed in axons bordering the stratum

radiatum and the lacunosum of the stratum moleculare of CA1, and in perforant

path axons that arise from the lateral entorhinal/perirhinaI cortices (Gall et al.

1981). Dynorphin immunoreactivity exhibits a more restricted distribution, being

localized to the mossy fiber axonal system (McGinty et al. 1983). Thus, both

prodynorphin- and proenkephalin-derived peptides are present in the granule

cells of the hippocampal dentate gyrus.

The hippocampus is a highly excitable structure, unusually susceptible to

seizure activity. Numerous studies have documented specific alterations in the

levels of hippocampal opioid peptides and opioid peptide mRNAs following the

induction of seizure activity by electroconvulsive shock (Yoshikawa et al. 1985;

Kanamatsu et al. 1986a; Xie et al. 1989a) or kindling (ladarola et al. 1986; Xie

et al. 1989b). Kainic acid (KA) is an excitatory amino acid that also elicits the

induction of hippocampal epileptiform activity and motor seizures. Over the

past decade, a variety of studies have utilized this potent neurotoxin as a model

of temporal lobe epilepsy (Ben-Ari 1985). Administration of KA results in the

onset of a range of epileptiform behaviors, which also depends on the dose and

route of administration of the drug. The hippocampal proenkephalin system is

stimulated by intrastriatal administration of KA, with increases in both peptide

and mRNA levels observed in a time-dependent manner (Kanamatsu et al.

1986b). In the study described here, hippocampal prodynorphin and

proenkephalin mRNA and peptide levels were measured at various times

following a single subcutaneous injection of KA (8.0 mg/kg).

Prodynorphin mRNA levels rose remarkably quickly, with a 13 to 14-fold

induction observed 3 hours after KA administration (figure 2, panel A).

Prodynorphin mRNA levels began to decline at 12 hours, and by 48 hours, the

138
FIGURE 2. Differential regulation of hippocampas prodynorphin and proenkephalin mRNA and peptide levels

following subcutaneouss administration of KA

NOTE: Prodynorphin mRNA/Dyn A 1-8 peptide levels (panel A) and proenkephalin mRNA/met-enkephalin peptide

levels (panel B) in the rat hippocampus were measured at various times after the administration of KA (s.c., 8

rng/kg). Values are represented as percent of control (saline-injected animals).
levels were at or below control values. Even though mRNA levels were

dramatically stimulated, hippocampal Dyn A 1-8 levels remained at values

below control at all time points monitored. This observation suggests that KA

treatment also results in a prolonged stimulation of release of dynorphin

peptides from hippocampal neurons.

The hippocampal proenkephalin system also responded in dramatic fashion to

KA administration (figure 2, panel B). Proenkephalin mRNA levels began to rise

within 3 hours, showing a fourfold elevation, and at 24 hours, a maximal 11- to

1 P-fold induction was observed. At the 48-hour time point, proenkephalin

mRNA values were still elevated (twofold to threefold) but had returned to

control values by 72 hours, Hippocampal met-enkephalin peptide levels also

were stimulated following KA administration. Levels remained close to control

values for the first 12 hours, then proceeded to increase steadily throughout the

24-to 72-hour period, eventually reaching values 3.0-to 3.5-fold above control

levels. This observation is consistent with a KA-induced increase in both

synthesis and release of hippocampal met-enkephalin.

Once again, the prodynorphin and proenkephalin systems appear to be

regulated differentially. Prodynorphin mRNA levels rose quickly and had

returned to control values by 24 hours. Conversely, proenkephalin mRNA

levels were stimulated maximally at the 24-hour time point and were still

elevated at 48 hours. Both species of opioid peptide mRNAs were present in

the granule cells of the hippocampal dentate gyrus, suggesting that the

prodynorphin and proenkephalin systems are differentially regulated not only in

the same brain region but also in the same neural network.

CHARACTERIZATION OF THE RAT PRODYNORPHIN GENE

The studies described above document relatively dramatic changes in

prodynorphin mRNA levels in neuronal cell types. These changes are

presumably the result of alterations in the rate of transcription of the

prodynorphin gene. We have isolated and characterized the rat prodynorphin

gene to begin to understand the molecular mechanisms that mediate

transcriptional regulatory events. Such analysis has led to the identification of

the rat prodynorphin mRNA cap site, promoter element, and possible regulatory

sequences. In addition, RNase protection studies, using various gene

sequences as complementary probes, have enabled us to characterize the

alternately spliced forms of both rat prodynorphin and proenkephalin mRNA that

are present in the testis, These studies are described in the following section.

138
Alternate Spliclng of Opioid Peptide mRNAs

Prodynorphin transcripts are present in both rat brain and testis (Civelli et al.

1985; Douglass et al. 1987; Garrett et al. 1989). Northern blot analysis has

determined that the 2,400-base transcript present in brain is approximately 100

bases larger than the 2,300-base transcript present in adult testis. Different

sized transcripts encoding the proenkephalin precursor also are seen in these

tissues (Douglass et al. 1987; Garrett et al. 1989). In this case, however, the

1,400-base transcript seen in brain is approximately 350 bases smaller than the

1,750-base transcript present in adult testis. In the rat, single copy genes

encode both prodynorphin and proenkephalin; therefore, the tissue-specific

differences in transcript size must arise from alternative sites of transcription

initiation and termination, alternative mRNA splicing patterns, or differing

lengths of poly(A) tails, RNase protection mapping studies were undertaken to

define precisely the basis for the observed size differences. The results are

diagramed in figure 3.

For prodynorphin mRNA, the testicular transcript is smaller than the brain

transcript due to the absence of exon 2 in the former species of mRNA. Exon 2

encodes 65 bases of 5’ untranslated region, with 15 bases of additional 5’

untranslated sequence present on exon 3. Thus, the 5’ untranslated region

beginning 15 bases from the translational start site is unique in the two species

of prodynorphin mRNA. Sucrose gradient poiysome analysis of striatal and

testicular prodynorphin mRNA reveals that both species of mRNA are present

on high-molecular-weight polysomes, suggesting that the mRNA is translated

efficiently in both tissues (for data presentation, see Garrett et al. 1989). The

functional significance of any alternate splicing event involving prodynorphin

mRNA is currently unknown.

For proenkephalin mRNA, the testicular transcript is larger than the brain

transcript due to the retention of approximately 350 bases of intron 1 sequence

in the former species of mRNA. Exon 2 contains 3 bases of 5’ untranslated

sequence; again, the nucleotide sequence immediately adjacent to the

translational start codon is unique to each species of mRNA. Furthermore, the

intron sequence present in the testicular transcript contains three open reading

frames (ORFs) varying from 9 to 156 bases in length (Garrett et al. 1989).

Evidence from experimental systems and from naturally occurring examples

indicates that the presence of methionine-initiated ORFs 5’ to a protein-coding

region will severely inhibit translation of the downstream region. Presumably,

ribosomes load onto the mRNA at the 5’ cap and scan downstream until the first

AUG codon in a context favorable for translation initiation is reached. On most

mRNA’s, subsequent reinitiation at any downstream AUG does not appear to

occur at any appreciable frequency. The sequence around the initiation codon

139
FIGURE 3. Alternate splicing of rat prodynorphin and proenkephalin

precursor mRNA to generate different forms of mature mRNA in

the brain and testis

NOTE: For prodynorphin, exons 1-4 are spliced together to generate a 2,400-

base transcript present in brain. In the testis, exon 2 is spliced out of

the mature prodynorphin transcript. For proenkephalin, exons 1-3 are

spliced to generate a 1,400-base brain transcript. In the testis,

approximately 350 nucleotides of intron 1 remain in the mature

proenkephalin transcript.

140
of the first intron-encoded ORF of the testicular proenkephalin transcript

conforms to the consensus sequence of a strong translation initiation site and

therefore may inhibit efficient translation of the mRNA. Sucrose gradient

polysome analysis does reveal that the majority of the testicular proenkephalin

transcript is unassociated with poiysomes or is present on monosomes or lowmolecular-weight poiysomes (Garrett et al. 1989). In contrast, striatal

proenkephalin mRNA is present on high-molecular-weight polysomes,

suggesting that the mRNA is translated efficiently. Thus, the additional

nucieotide sequence present on the testicular form of proenkephalin mRNA

may play a role in determining the translational status of the transcript in this

tissue.

Characterization of the Rat Prodynorphin Promoter Region

In vivo studies have shown that rat prodynorphin mRNA levels in specific

neuronai ceil types can be dramatically altered following surgical or

pharmacological manipulations. These changes presumably reflect alterations

in the rate of prodynorphin mRNA synthesis. Thus, signals received by

prodynorphin-expressing neurons must be relayed to the cell nucleus, resulting

in altered levels of transcription of the prodynorphin gene.

In many cases, chemical signals received at the neuronal cell membrane are

transduced through the plasma membrane via cell surface receptors coupled to

G-proteins (for review, see Johnson and Dhanasekaran 1989). Activation of

these G-proteins regulates numerous cellular events, including the production of

second messenger molecules and the activity of a variety of protein kinases.

Second messengers, such as cyclic AMP and diacylglycerol, act to regulate the

activity of kinases, including cAMP-dependent protein kinase and protein kinase

C, respectively (for review, see Ross 1989). it is believed that one class of

protein factors whose activity is regulated at the level of phosphorylation by the

aforementioned protein kinases are DNA binding proteins that control the

transcriptional activity of specific genes in a tissue- and cell type-specific

manner (Mitchell and Tjian 1989; Jones et al. 1988). Thus, the regulation of

neuronal gene expression by signals received at the postsynaptic membrane is

an important mechanism by which neurons control the synthesis of peptide

neurotransmitters specific to that particular cell. Moreover, the presence of a

variety of postsynaptic receptors on any given ceil body provides an additional

degree of complexity to the phenomenon of transsynaptic regulation of neuronai

gene expression. As a first step in characterizing the unique compilation of cisacting nucieotide sequence elements regulating the production of prodynorphin

mRNA, we have used gene transfer techniques to begin to functionally

characterize the rat prodynorphin promoter region.

141
Specific regions of rat prodynorphin genomic DNA, containing the mapped

mRNA cap site and a consensus RNA poiymerase II promoter sequence

(TATAAA), have been ligated 5’ to the bacterial chloramphenicol acetyl

transferase (CAT) gene (figure 4). One such piasmid contains prodynorphin

promoter sequences from -122 to +88 (with the cap site at position +1

representing the point of reference). A second plasmid contains nucleotides

from -510 to +88 linked to the CAT structural gene. These plasmids have been

introduced via CaPO4

 precipitation into three different eukaryotic cell lines

(Douglass et al. 1989). The NG 108 cell line is of neural origin and represents a

mouse neuroblastoma x rat C6 giioma hybrid. No detectable levels of

prodynorphin mRNA are observed in this line. CV1 cells are derived from an

African green monkey kidney tumor and do not contain detectable levels of

prodynorphin mRNA. R2C cells are derived from a rat testicular Leydig cell

tumor and do contain detectable, albeit low, levels of prodynorphin mRNA

(McMurray et al. 1989).

The promoter-reporter plasmids were introduced into the various cell lines, and

24 hours later, cellular extracts were assayed for CAT activity (figure 4). CAT

activity is represented as percent conversion of 14C-chloramphenicol to the

mono- and di-acetylated forms (McMurray et al. 1989). CAT activity levels are

dependent on the cellular levels of CAT mRNA, which presumably are related

directly to the relative strength of the upstream promoter element(s). A control

piasmid (containing the constitutive Rous sarcoma virus long terminal repeat

promoter region linked to the bacterial ß-galactosidase structural gene) was

cotransfected into the various ceil lines to ensure that all transfection reactions

proceeded with approximately similar efficiencies. Plasmids containing rat

prodynorphin 5’ flanking DNA were transcriptionally active, resulting in the

production of CAT activity. The -122 to +88 construct produced the highest

levels of CAT activity, and percent CAT conversion ranged from 14.5 percent in

NG 108 cells to only 1.0 percent in R2C cells. This observation is consistent

with the presence of a consensus TATAAA element and mapped cap site 5’ to

the CAT structural gene. The -510 to +88 construct also was transcriptionally

active, although CAT levels were an average of fivefold lower than those

observed with the -122 to +88 construct. This observation suggests that

nucieotide sequences between -510 and -122 may be serving to inhibit

transcription from the promoter located at approximately -30. Also, there was

no correlation between transient promoter activity and expression of the

endogenous prodynorphin gene in the cell lines tested.

In the R2C ceil line, endogenous prodynorphin mRNA levels are elevated

approximately fourfold following an 8-hour treatment with 8-bromo-cAMP

(McMurray et al. 1989). To determine if this increase in mRNA levels correlates

with induction of prodynorphin promoter activity, the -122 to +88 CAT construct

142
Rat Prodynorphin Promoter

Cell Lines Transfected

CAT Constructs NG108 CV1 R2C R2C+8Br-cAMP

-122/+88 CAT 14.5±6.0 2.0±1.0 1.0±0.5 3.0±0.5 %conversion CAT

(1.10) (0.72) (0.42)

-510 /+80 CAT 2.0±0.2 0.5±0.3 0.3±0.2

(0.80) (0.57) (0.36)

FIGURE 4. Characterization of the ability of rat prodynorphin genomic DNA

fragments to serve as transient transcriptional promoters

NOTE: Nucleotides are shown representing 5’ and 3’ ends of rat prodynorphin

genomic DNA fragments (containing the mapped cap site at +1 and the

promoter element TATAAA at -28) ligated proximal to the CAT

structural gene. The resulting plasmids were transfected into the NG

108, CV1 , and R2C cells lines, and cellular CAT activity was

measured. RSV-ß-gal was cotransfected to monitor transfection

efficiency, and cellular levels of ß-gal were determined. Some

transfected R2C cells were treated with 1 mM 8-Br-cAMP for 8 hours

before determination of CAT activity.

was transfected into the R2C cell line, followed by an 8-hour period of treatment

with 1.0 mM 8-Br-cAMP. Cellular CAT activity levels were increased

approximately threefold following this treatment. Thus, increases in cellular

R2C prodynorphin mRNA levels by cAMP analogs are more than likely due to

enhanced promoter activity. Furthermore, at least some of the nucleotide

sequences involved in mediating this form of regulation appear to be present in

the prodynorphin promoter region from -122 to +88.

143
CONCLUSIONS

Neurons are constantly receiving and assimilating information represented by

chemical messengers; environmental stimuli, internal biological clocks, and

signals from endocrine tissues all serve to modulate the biochemical properties

that define each neuronal cell type. The introduction of drugs of abuse also

dramatically affects neuronal cellular events. The molecular mechanisms

underlying narcotic dependence, withdrawal, and tolerance associated with the

compulsive use of drugs are poorly understood. The problem of physical

dependence and withdrawal is highly complex and probably involves a complex

cascade of events that begins with the diverse and widely distributed drug

receptor systems and extends to complex arrays of autonomic and

sensorimotor neural networks (Abood 1984). These events also are influenced

by genetic, species, and environmental factors, which only serve to complicate

scientific ventures aimed at characterizing the molecular basis of these narcoticrelated syndromes, It is reasonable to assume that the introduction of

exogenous drugs results in the activation of specific neuronal G-protein coupled

receptors, This activation then alters numerous cellular events, including

changes at the genomic DNA level regarding the transcriptional activity of

specific sets of genes. Thus, to gain additional insight into the molecular events

associated with substance abuse, researchers must understand the basic

mechanisms that regulate the expression of transcriptionally active genes in the

CNS.

We have been studying specific aspects of opioid peptide gene expression,

focusing on the regulated expression of the prodynorphin gene. In vivo studies

have documented that prodynorphin-expressing neurons are capable of

dramatic transcriptional responses, resulting in profound increases in the

cellular levels of prodynorphin mRNA. In prodynorphin-expressing spinal cord

neurons, inflammation of a peripheral limb results in marked increases in both

prodynorphin mRNA and peptide levels. Furthermore, these increases occur in

the spinal cord segments that receive sensory imput from the affected limb.

The rapid onset of behavioral hyperalgesia may act as a trigger for the

activation of dynorphin biosynthesis, with such neurotransmitters as substance

P or calcitonin gene-related peptide mediating the response (ladarola et al.

1988). In hippocampal granule cells of the dentate gyrus, prodynorphin mRNA

levels are dramatically, positively regulated following the subcutaneous

administration of the excitatory amino acid KA. The prodynorphin biosynthetic

system responds rapidly to KA administration, with maximal stimulation of

prodynorphin mRNA levels seen within 3 hours. In contrast, proenkephalin

mRNA levels rise at a much slower rate, with maximal stimulation observed 24

hours after drug administration. Thus, the hippocampal prodynorphin and

144
proenkephalin systems, presumably active in overlapping sets of neurons,

respond differentially to stimulation by KA administration.

Thus far, in Investigations of the molecular events controlling the production of

prodynorphin mRNA, the rat prodynorphin gene has been isolated and

characterized. The intron/exon architecture has been determined, the mRNA

cap site mapped, and the promoter region functionally characterized. Alternate

splicing of precursor mRNA results in the generation of different forms of

prodynorphin mRNA in the brain and testis. The two species of mRNA contain

different 5’ untranslated regions beginning 15 nucleotides from the translational

start site that are apparently translated at similar efficiencies. Any functional

significance resulting from this pattern of alternate mRNA splicing is currently

unknown.

Transfection of DNA into various eukaryotic cell lines has allowed for a

preliminary characterization of the rat prodynorphin promoter region. The

consensus TATAAA promoter element at position -30 appears to be functionally

active, with nucleotide sequences between -510 and -122 perhaps serving to

negatively regulate transcription from the aforementioned promoter. In

addition, a CAMP-responsive element may be present in the region from -122 to

+88. Additional studies are needed to define more precisely the sequence

elements mediating the controlled production of prodynorphin mRNA in

neuronal cell types. Such studies will increase understanding of the nuclear

events underlying transsynaptic regulation of neuronally active genes.

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Xie, C.W.; Lee, P.H.K.; Takeuchi, K.; Owyang, V.; Li, S.J.; Douglass, J.; and

Hong, J.S. Single or repeated electroconvulsive shocks alter the levels of

prodynorphin and proenkephalin mRNAs in rat brain. Mol Brain Res 6: 11-19,

1989a.

Yoshikawa, K.; Hong, J.-S.; and Sabol, S. Electroconvulsive shock increases

preproenkephalin messenger RNA abundance in rat hypothalamus. Proc

Natl Acad Sci U S A 82:589-593, 1985.

ACKNOWLEDGMENTS

The studies described in this chapter were supported in part by National

Institute on Drug Abuse grant DA-04154 (JOD), National Research Service

Award fellowship NS-08068 (JEG), and American Cancer Society postdoctoral

fellowship PF-2997 (CTM).

147
AUTHORS

James O. Douglass, Ph.D.

Assistant Scientist

James E. Garrett, Ph.D.

Postdoctoral Research Associate

Cynthia T. McMurray, Ph.D.

Postdoctoral Research Associate

Vollum Institute for Advanced Biomedical Research

Oregon Health Sciences University

3181 Southwest Sam Jackson Park Road

Portland, OR 97201-3098

Michael J. ladarola, Ph.D.

Scientist

Neurobiology and Anesthesiology Branch

National Institute of Dental Research

National Institutes of Health

Bethesda, MD 20892

Jau-Shyong Hong, Ph.D.

Scientist

Laboratory of Behavioral and Neurological Toxicology

National Institute of Environmental Health Sciences

National Institutes of Health

P.O. Box 12233

Research Triangle Park, NC 27709

148
Mechanisms Underlying Synaptic

Regulation of Proenkephalin

Transcription

Michael Comb, Steven E. Hyman, Linda Kobierski, Hung Ming

Chu, and Tuong Van Nguyen

INTRODUCTION

The molecular mechanisms underlying learning, memory, and other stable/

adaptive changes in the nervous system are largely unknown. With the

discovery that neurons transduce environmental inputs into changes in gene

expression, the hypothesis has emerged that transsynaptic regulation of gene

expression may induce the biochemical changes underlying many forms of

neural plasticity. To understand these processes, it is necessary to identify both

the pathways and the mechanisms mediating the effects of neurotransmitters,

psychoactive drugs, learning, and other synaptically mediated processes on

gene expression. Neural regulation of the opioid precursor, proenkephalin, is

an excellent model system to study mechanisms underlying synaptic regulation

of gene expression because (1) its transcription is both activated and repressed

by synaptic signals; (2) in the brain, such events as electrical stimulation that

induce long-term potentiation (LTP) also induce dramatic and long-lasting

changes in proenkephalin gene expression; and (3) the gene products,

enkephalins, are important signaling molecules regulating diverse neural

pathways in the brain and peripheral nervous system. This chapter discusses

approaches and methodologies to define how synaptic activity, intracellular

signaling pathways, and nuclear events regulate expression of the human

proenkephalin gene.

NEURAL PLASTICITY AND REGULATION OF PROENKEPHALIN GENE

EXPRESSION: A MODEL SYSTEM TO EXPLORE MECHANISMS

Over the past 20 years, a variety of neurotransmitters have been identified that

carry out and regulate cell-to-cell communication in the nervous system.

Peptide neurotransmitters differ from conventional small molecule transmitters

in their extraordinary potency, prolonged actions, and presumed effects on

149
neural plasticity. The endogenous opioids met- and leu-enkephalin mediate

neurotransmission at the opiate receptor, which is also the site of narcotic drug

action. These pentapeptides function as important neurotransmitters/

neuromodulators, regulating diverse neural pathways, including analgesia,

reward, motivation, and hormone release. Both the synthesis and the release

of enkephalins are regulated by synaptic events involving activation of

neurotransmitter receptors, second messenger pathways, and membrane

depolarization. Regulation of proenkephalin gene expression (mRNA levels)

has been studied in a variety of systems, including primary cultures of bovine

chromaffin cells (Eiden et al. 1984; Quach et al. 1984; Kiey et al. 1987), tissue

culture cells (Yoshikawa and Sabol 1988; Schwartz 1988; Boarder et al. 1986),

rat adrenal (Kilpatrick et al. 1984; La Gamma et al. 1985), and various brain

regions (Tang et al. 1983; Sabol et al. 1983; Sivam et al. 1986; Shivers et al,

1986).

The influences of synaptic activity and second messengers have been

examined in the greatest detail using primary cultures of bovine chromaffin

cells. Chromaffin cells have been used to show that agents that increase

intracellular cyclic AMP (cAMP) levels, such as forskolin, or stable CAMP

analogs increase proenkephalin mRNA levels (Eiden et al. 1984; Quach et al.

1984). In addition, agents that cause membrane depolarization or Cat+ entry,

such as nicotine, potassium, veratridine, and barium, all have been shown to

increase proenkephalin mRNA levels in bovine chromaffin cells (Kley et al.

1987). Activation of protein kinase C by phorbol esters also will induce

proenkephalin mRNA levels in these cells (Kley et al. 1988). In vivo

experiments have shown that proenkephalin mRNA levels are dramatically

elevated by increased impulse intensity of the rat splanchnic nerve (Kanamatsu

et al. 1986; Colbrie et al. 1988).

In situ hybridization has allowed visualization of changes, at the single cell level,

in striatial proenkephalin mRNA levels resulting from neuroleptic drug

treatments and specific lesions (Romano et al. 1987; Young et al. 1986). These

results suggest that dopamine negatively regulates proenkephalin gene

expression in the caudate-putamen. In situ hybridization and RNA blot

techniques also have been used to study the regulation of opioid gene

expression in rat hippocampus, a region in which long-term changes in synaptic

efficacy have been readily demonstrated and may play a role in the molecular

basis of memory (White et al. 1987; Morris et al. 1988). Granule cells of the rat

dentate gyrus contain undetectable levels of proenkephalin mRNA and very

high levels of prodynorphin mRNA. Electrical stimulation of this region in vivo,

using stimulation parameters identical to those that can induce LTP, produces

dramatic increases in proenkephalin mRNA levels and decreases prodynorphin

mRNA levels (Morris et al. 1988), Enkephalin biosynthesis in the granule cell/

150
mossy fiber system of the mouse also has been investigated following the

induction of seizure induced by placement of a small unilateral lesion within the

hilus of the dentate gyrus (Gall 1988; White and Gall 1987). Marked increase in

both the enkephalin peptides (14-fold) and the proenkephalin mRNA levels (18-

fold) were observed in granule cells 24-hours postseizure induction. These

data indicate that recurrent seizure activity stimulates a large increase in

proenkephalin biosynthesis in the dentate gyrus cell/mossy fibers of the rodent

hippocampus (Gall 1988).

RECONSTITUTING REGULATION AND DEFINING CONTROL REGIONS

To study the mechanisms underlying synaptic regulation of the proenkephalin

gene, a plasmid, pENKAT-12, was constructed in which human proenkephalin

control regions were fused to the bacterial chloramphenicol acetyl transferase

(CAT) gene (figure 1) (Comb et al. 1986). Expression of this reporter gene can

be easily monitored after transfection into tissue culture cells (figure 2). After

introduction and expression of this fusion gene in tissue culture cells, we have

shown the gene to be regulated by CAMP, TPA, and Ca++ in a fashion similar

to the endogenous gene (Comb et al. 1986; Nguyen et al. 1990). Deletion and

transfer mapping studies of the 5’-flanking region indicate that regulation by the

cAMP, TPA, and Ca++ intracellular signaling pathways is controlled by a short

50-base pair region of proenkephalin DNA that acts as a second messengerInducible DNA enhancer (Comb et al. 1986,1988). Effects of TPA and Ca++

are dependent on and synergistic with simultaneous or prior stimulation of the

CAMP pathway, indicating that these pathways interact in both a conditional and

a synergistic fashion to regulate proenkephalin gene expression (Comb et al.

1986, 1987; Nguyen et al. 1990). The mechanisms underlying these effects are

not as yet understood and require further study.

TRANSCRIPTION IS RAPIDLY ACTIVATED AND RAPIDLY DESENSITIZED

TO cAMP

Because proenkephalin mRNA is quite stable in C6-glioma cells, with a half-life

of at least 24 hours (Yoshikawa and Sabol 1986), while the RNA transcript

produced from the proenkephalin/CAT fusion gene is relatively unstable,

analysis of CAT mRNA is a much more sensitive indicator of transcriptional

events than is the endogenous gene. To determine the time course of mRNA

activation and its dependence on new protein synthesis, RNA isolated from a

stable clonal line of C6-glioma cells (C6-D2), which express the proenkephalin/

CAT fusion gene stably integrated into their chromosomal genome, was

analyzed at various times after treatment with forskolin and the

phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) by S1 or primer

151
FIGURE 1. Structure of the human proenkephalin/CAT fusion gene. This

plasmid directs expression of CAT enzyme under the control of

the human proenkephalin promoter and poly (A) regions (Comb et

al. 1986). After transfection into tissue culture cells, CAT enzyme

activity is proportional to proenkephalin promoter activity, Open

box is CAT coding sequence; large black boxes represent

proenkephalin noncoding exons; and smaller stippled regions

represent proenkephalin 5’-flanking sequence, intron A, and 3’-

untranslated sequences.

extension analysis. Correctly initiated RNA is detected within 15 minutes of

forskolin treatment, peaks by 90 to 120 minutes, and rapidly declines to

baseline by 8 hours following treatment. Further addition of the stable cAMP

analog CPT-CAMP at several times following the peak in mRNA produced no

further increase in steady-state mRNA levels, suggesting that transcription had

become uncoupled or “desensitized” to intracellular levels of CAMP. The rapid

152
FIGURE 2. Synergistic and conditional effects of CAMP, phorbol esters, and

K+ on CAT expression directed by the human proenkephalin

promoter after treatment of a stable rat PC-12 cell line D2 that

expresses the proenkephalin/CAT fusion plasmid pENKAT-72.

Treatments were for 6 hours with 25 μM forskolin, 50 nM TPA,

500 μM IBMX, and 40 μM KCl.

CAMP-dependent increase in mRNA is not affected by the addition protein

synthesis inhibitors cyclohexamide (100 μM) or anisomycin (10 μM) 30 minutes

before forskolin addition, consistent with activation being caused by a rapid

posttranslational modification of preexisting proteins. In sharp contrast,

anisomycin blocked mRNA decline during the refractory phase and caused

mRNA levels to accumulate, resulting in superinduction. Neither inhibitor

caused induction in the absence of CAMP.

153
These results suggest that transcriptional activation is due to a posttranslational

modification of preexisting factors and that new protein synthesis is required for

the “refractory phase” that rapidly follows transcriptional activation. It seems

likely that sustained stimulation of the cAMP pathway leads to rapid repression

of proenkephalin transcription by mechanisms that require new protein

synthesis. Hence, proenkephalin transcription is regulated both positively and

negatively by second messenger pathways, resulting in a brief pulse of mRNA

synthesis. Desensitization of transcription to CAMP is remarkably similar to

neurotransmitter receptor desensitization and may share similar mechanistic

features.

The Second Messenger-lnducible Enhancer Consists of Multiple DNA

Elements That Act In a Highly Synergistic Fashion

To further resolve the inducible enhancer into individual elements and to

provide regulatory mutations necessary to correlate binding of transcription

factors with in vivo enhancer function, a series of single-base substitution (SBS)

mutants was generated throughout the enhancer region (figure 3). The effect of

these point mutations on cAMP-inducible enhancer function was determined by

examining transient CAT expression after transfection into CV-1 cells or by

examining RNA transcripts from pooled, stably expressing C6 glioma cells

(Comb et al. 1986, 1988). Two regions that are sensitive to SBS mutations

have been defined: One, called ENKCRE-1, is located between -104 and -98

and spans the sequence TGGCGTA; a second domain, called ENKCRE-2,

extends from -93 to -86 and spans the sequence TGCGTCA (Comb et al.

1988). A third region that is sensitive to SBSs has been identified adjacent and

downstream from the ENKCRE-2 element and has been identified as an AP-2

element (Hyman et al. 1989). Inactivation of either the ENKCRE-1 or the

ENKCRE-2 element by mutation or by alteration of their stereospecific

alignment eliminates inducible enhancer activity (Comb et al. 1988).

Inactivation of the AP-2 element by point substitutions results in a fivefold

reduction of CAMP- and TPA-inducible transcription (Hyman et al. 1989). This

analysis clearly demonstrates that each of the three proenkephalin-inducible

elements acts in a highly cooperative or synergistic fashion to induce

transcription in the presence of either CAMP or TPA. In addition, these results

suggest that assembly of a transcription complex, involving proteins bound at

the ENKCRE-1 and ENKCRE-2 elements, is required for the transcriptional

effects of signals transmitted from cell surface receptors.

THE ROLE OF EACH ELEMENT IN ISOLATION

The functional activity of each individual element in isolation from the rest of the

enhancer was examined to determine which elements respond individually to

154
FIGURE 3. The human proenkephalin promoter contains a short CAMP-,

phorbol ester-, and Ca++-inducible enhancer located between

-104 and -65 base pairs upstream from the start site of

transcription. The enhancer consists of multiple DNA elements

that act cooperatively to regulate transcription in response to

intracellular signaling pathways.

CAMP and TPA (figure 4). Individually responsive elements are candidates for

direct interactions with the signal transduction pathways. Multiple copies of

oligonucleotides containing individual factor binding sites (ENKTF-1, AP-1, AP-4

(Mermod and Tjian 1988], and AP-2) were inserted upstream of a proenkephalin

promoter truncated at position -72 that deletes the entire inducible enhancer.

The ability of these single-element promoters to respond to cAMP and TPA was

measured after transfection into several different cell types. Multiple copies of

the ENKCRE-1 element result in small (twofold to threefold inductions) to either

CAMP or TPA. Multiple copies of either the AP-2 or the AP-4 element did not

confer significant induction, indicating that a promoter consisting solely of

multiple copies of these elements is not capable of responding to signals

propagated through either the CAMP or the TPA intracellular signaling pathway.

In contrast, lmagawa and colleagues (1987) previously reported that multiple

copies of an AP-2 element when fused to the B-globin promoter conferred both

CAMP- and TPA-inducible expression to this promoter. We do not yet

155
FIGURE 4a. Analysis of individual elements comprising the proenkephalin

second messenger-inducible DNA enhancer. Multiple copies of

each element were introduced into the enhancerless plasmid

PENKAT-72. Each plasmid was introduced into C6-glioma cells,

and the ability of individual elements to respond to intracellular

signaling pathways was examined. pENKAT-12 is the wild-type

promoter/enhancer, and ENKCRE1+2 is a plasmid containing

six copies of the ENKCRE-1 and ENKCRE-2 elements.

understand these differences, but they may be attributed to different AP-2

proteins or to differences between the proenkephalin and B-globin promoters.

Multiple copies of the ENKCRE-2 element induce transcription in response to

cAMP and TPA, although at levels significantly lower than the wild-type

promoter. Multiple copies of the AP-4 element increase basal but not inducible

transcription, suggesting that this element may play an important role in basal

expression.

ENKCRE-2 CAN RECEIVE SIGNALS FROM THE CAMP, TPA, AND CA++

PATHWAYS

To further characterize the ability of the ENKCRE-2 element to respond to

intracellular signaling pathways, we isolated stably expressing cell lines in which

156
FIGURE 4b. Effects of individual elements

the plasmid is stably introduced into C6-glioma cell chromosomal DNA. Five

independent lines were tested, and a typical response is shown in figure 5.

Surprisingly, the multiple copy ENKCRE-2 plasmid exhibited the entire

spectrum of responses exhibited by the wild-type enhancer (pENKAT-12). This

plasmid is responsive to cAMP, TPA, and depolarizing concentrations of K+ (40

mM). In addition, the additive effects of cAMP and TPA and of CAMP and K+

are reproduced by the ENKCRE-2 element, suggesting that much of the

observed regulation may be mediated through this element.

It is important to remember that a single copy of the ENKCRE-2 element is not

sufficient, since point mutations that inactivate the ENKCRE-1 element and

leave the ENKCRE-2 element intact are unable to regulate transcription in

response to activation of second messenger pathways. Therefore, the

presence of tandem ENKCRE-2 binding sites is able to compensate for the

absence of ENKCRE-1 and AP-2 elements. This suggests that the protein/

protein interactions between proteins bound at the ENKCRE-1 and ENKCRE-2

elements may be replaced by a single protein (complex) bound at multiple

ENKCRE-2 elements.

157
FIGURE 5. Multiple copies of the ENKCRE-2 element reconstitute regulation by cAMP, TPA and Ca++. The

plasmid pENKCRE2#2 was cotransfected together with pRSVNE0 into C6-glioma cells and a clonal

cell line, C6-D11, was isolated and analyzed. In addition, four other independent lines were tested

and gave results similar to those shown above for the C6-D11 line. Cells were treated for 6 hours

with 50 nM TPA (T), 25 μM forskolin (F), and 40 mM potassium chloride (K), and CAT activities were

determined.
PROTEINS BINDING THE PROENKEPHALIN SECOND MESSENGERINDUCIBLE ENHANCER

Using DNAase I footprinting protection assay and DNA recognition site affinity

chromatography, we have identified and purified four different factors present in

Hela cell and bovine brain nuclear extracts that interact with the human

proenkephalin enhancer region (figure 6). ENKTF-1, a novel DNA binding

protein, binds to the enhancer at the promoter distal ENKCRE-1 element (Chu

et al. 1991). The transcription factors AP-1 (Lee et al. 1987; Angel et al. 1987)

and AP-4 (Mermod et al. 1988) bind to overlapping sites within the enhancer

region spanning ENKCRE-2; and a fourth transcription factor, AP-2 (Mitchell et

al. 1987), binds to a site immediately downstream of the ENKCRE-2 element

FIGURE 6. Proteins that bind the proenkephalin cAMP-, TPA-, and Ca++-

inducible enhancer. ENKTF-1, a family of at least three different

proteins purified from total brain, binds the ENKCRE-1 element;

AP-1, AP-4, and, possibly, CREB/ATF proteins interact with the

ENKCRE-2 element; and AP-2 binds the AP-2 element (Comb et

al. 7988; Hyman et al. 1988).

159
(Comb et al. 1988; Hyman et al. 1989). The role of the observed in vitro binding

of these factors to the in vivo functioning of the enhancer was assessed by

correlating the binding of factors to a series of SBS mutations of known in vivo

enhancer activity. ENKTF-1 correlates perfectly with the in vivo enhancer

activity of each mutant, suggesting that binding this factor to the ENKCRE-1

element is the critical regulatory event altered by the various point mutations.

Overlapping binding of purified AP-1 and AP-4 to the ENKCRE-2 element

suggests a complex regulatory scheme at this site that may involve binding of

each factor under different conditions.

The sequence of ENKCRE-2 appears to be a hybrid between the proposed

TRE and CRE binding sites that interact with the fos/jun and CREB families of

transcription factors (Angel et al. 1987; Bohmann et al. 1987; Montminy and

Belezikjian 1987). To examine the in vivo role of AP-1 in CAMP- and TPAinducible enhancer function, we tested the binding of AP-1 to a series of SBS

mutants of known activity. Binding correlates perfectly with inducible enhancer

activity at 5 of 7 substitutions. However, two different SBS mutations, -92 and

-90, severely reduce CAMP- and TPA-inducible enhancer function, yet bind

AP-1 with wild-type affinity (-92) or better (-90). This result may be resolved by

the observation that AP-1 binds as a c-jun/c-fos heterodimer. Since affinity

purified preps contain mostly c-jun with little c-fos (as visualized by silver

stains), it is possible that -92, -91, -90 bases interact with c-fos and not c-jun.

Alternatively, it is possible that other jun molecules (jun-B or jun-D) and not cjun mediate regulation at this site in vivo.

PROTEINS BOUND AT ADJACENT DNA ELEMENT: POSITIVE AND

NEGATIVE COOPERATIVITY

Synergistic transcriptional effects between elements could result from (1)

cooperative DNA binding interactions (binding of one protein helps the binding

of a second protein) between factors bound at each element, (2) cooperative

protein/protein interactions, or (3) recruitment of a third protein essential for

transcriptional activation. Using DNA recognition site affinity chromatography,

four different proteins binding the proenkephalin enhancer—ENKTF-1, AP-1,

AP-4, and AP-2—have been highly purified (figure 7). Each of these

preparations gives clean footprints over its respective DNA binding elements

and is highly purified as determined by gel electrophoresis and silver staining.

The first question was, Which proteins can simultaneously bind the enhancer to

form a stable complex? ENKTF-1, AP-4, and AP-2 bind the enhancer

simultaneously to form a stable protein/DNA complex as the sum of their

individual footprints are observed. Cooperative DNA binding is not observed

between ENKTF-1 and AP-4 or AP-2. However, cooperative DNA binding is

160
FIGURE 7. In vitro binding studies indicate that ENKTF-1, AP-4, and AP-2

bind the enhancer simultaneously to form a stable complex.

Conversely, the binding of AP-1 to the ENKCRE-2 element

inhibits the binding of ENKTF-1 to the CRE-1 element, AP-4 to

the ENKCRE-2 element, and AP-2 to the AP-2 element. Hence,

in vitro binding studies suggest the formation of two mutually

exclusive complexes: an AP-1 complex and a complex consisting

of ENKTF-1, AP-4, and AP-2. The functional role of these

complexes in the regulation of proenkephalin transcription is

currently unknown.

observed between AP-2 and AP-4. The physiological significance of this

interaction is not yet understood. In contrast, AP-1 preparations inhibit the

binding of ENKTF-1, AP-4, and AP-2 to their recognition sites. We believe that

this displacement is due to (1) the higher binding affinity of AP-1 for the

ENKCRE-2 site and (2) the fact that AP-1 may bind DNA as a large protein

complex, which can occlude the binding of nearby factors. This hypothesis is

supported by our observation that the repressive effect of AP-1 on the binding

of ENKTF-1, AP-4, and AP-2 is completely reversed by competing away AP-1

binding using an ENKCRE-2 oligo. The AP-1 footprint disappears and

formation of ENKTF-1, AP-4, AP-2 complex occurs. These results suggest that

complex DNA binding interactions at the ENKCRE-2 element are likely to occur

in vivo and indicate the need for functional in vivo assays to determine whether

these complexes act to regulate transcription in a positive or negative fashion.

161
PROENKEPHALIN AS A NEURAL TARGET GENE REGULATED BY FOS/

JUN NUCLEOPROTEIN COMPLEXES

Because the proenkephalin gene contains a binding site for the transcription

factor AP-1 (which consists of fos/jun heterodimers) and because this binding

site has been shown to play a critical role in mediating second messenger

regulation, this gene represents an excellent model system to explore regulation

by fos/jun complexes, In fact, this gene represents the first in vivo target of the

fos/jun complex recognized in the nervous system.

Transcription of both fos and jun proteins is rapidly activated in vivo by

excitation and the activation of neurotransmitter receptors (Greenberg et al.

1986; Hunt et al. 1987; Bartel et al. 1989; Szekely et al. 1988). Newly

synthesized fos/jun proteins are thought to act as “third messengers,” rapidly

activating or repressing transcription of target genes in response to signals

transmitted from the cell surface (Morgan et al. 1987). White and Gall (1987)

have investigated the regulation of both proenkephalin and c-fos mRNA levels

in the hippocampus after dentate gyrus hilus lesion, Shortly after induction of

seizures, c-fos mRNA induction peaked at 3 hours followed by a slow decline to

control levels by 48 hours, whereas proenkephalin mRNA levels peaked at 18

hours and remained elevated out to 48 hours postlesion. The rapid increase in

c-fos RNA observed in granule cells of the dentate gyrus after electrical

stimulation or seizure induction suggests that the c-fos/jun nucleoprotein

complex may play a role in the subsequent induction of proenkephalin

biosynthesis (figure 8).

COTRANSFECTION STUDIES USING FOS-, JUN-, AND CREBEXPRESSING PLASMIDS

Cotransfection of a v-jun expression plasmid (cDNA under the control of the

CMV promoter) with a proenkephalin/CAT fusion plasmid strongly

transactivates CAT expression in the embryonal carcinoma cell line EC-P19. In

contrast, cotransfection with c-fos-expressing plasmids produces very little

effect (usually a small inhibition is observed) of CAT expression in EC-P19

cells. When c-fos and v-jun expressing plasmids were cotransfected together

with pENKAT-12 Into EC-P19 cells, a strong synergistic activation of the

proenkephalin enhancer was observed. These results are consistent with a fos/

jun heterodimer strongly activating proenkephalin expression in EC-P19 cells,

Cotransfection with a CREB-expressing plasmid also produced strong

transactivation of the proenkephalin enhancer. To determine which enhancer

elements mediate the effects of jun, fos, and CREB, experiments similar to

those described above were performed using the multicopy ENKCRE-2 plasmid

as indicator instead of ENKAT-12 (figure 9). These experiments showed that a

162
FIGURE 8. Transactivation of the human proenkephalin promoter in vivo by

AP-1 and CREB proteins. Cotransfection of embronyal

carcinoma cell line EC-P19 with pENKAT-12 (2.5 μg) and

plasmids expressing c-jun, c-fos, CREB (10 μg), pRSV-BGAL (10

μg) and pUC-19 to a total of 30 μg DNA/calcium phosphate

precipitate. Note that c-fos acts synergistically with c-jun and

CREB to promote transcription from the human proenkephalin

promoter/enhancer.

plasmid containing only the ENKCRE-2 with all other elements deleted was able

to respond in a fashion very similar to the wild-type enhancer, suggesting that

this element alone mediates the above effects of fos, jun, and CREB

expression.

163
FIGURE 9. Transactivation by c-jun, c-fos, and CREB map to the ENKCRE-2

element. The plasmid pENKCRE2#2, which contains only the

ENKCRE-2 element in the absence of all other elements, was

cotransfected with c-jun, c-fos, and CREB to determine if this

element could mediate the effects of transcription factor

cotransfection. As shown, this element alone appears capable,

when present in multiple copies, to mediate the effects of c-fos, cjun, and CREB.

SUMMARY AND CONCLUSIONS

It is now apparent that neurotransmitters and drugs that act at neurotransmitter

receptors are important regulators of gene expression in the nervous system.

These findings suggest that alterations in neural gene expression induced by

neurotransmitters or drugs may underlie many stable/adaptive changes in

neural signaling. Therefore, it is important to develop an understanding of how

neural signaling and synaptic activity influence gene expression and of the role

these processes play in normal physiology. The large changes in

proenkephalin mRNA levels in both the granule cells of the dentate gyrus and

the chromaffin cells of the adrenal medulla in response to electrical or neural

stimulation suggest that these responses may have profound effects on neural

signaling, on opioid physiology, and ultimately on behavior. Although much has

been learned using RNA blotting and in situ hybridization techniques to study

164
changes in mRNA levels in response to physiologic stimuli, these studies are

descriptive by nature and cannot address the mechanisms that underlie the

observed changes. Investigation of mechanism requires analysis of the

intracellular signaling pathways, the responsive DNA regulatory elements, and

the proteins transducing synaptic signals into gene regulation (figure 10). In the

studies described above, we have defined three different intracellular signaling

FIGURE 10. Synaptic regulation of proenkephalin gene expression and

neural plasticity. Short-term effects of a neurotransmitter binding

its cell surface receptor include activation of second messenger

systems and protein kinases, regulation of ion fiux via channel

modification, and modulation of neurotransmitter release. Longterm effects include second messenger- and protein kinasemediated effects on gene expression. These effects serve to

regulate the biosynthesis of neurotransmitters and may underly

many enviromental and drug-induced stable/adaptive changes in

neural physiology.

165
pathways that, when stimulated, activate transcription of the human

proenkephalin gene in a conditional and synergistic fashion. We demonstrate

that these three distinct intracellular signaling pathways converge to regulate

transcription of the proenkephalin gene via a short DNA enhancer. The protein

factors that bind to the enhancer and mediate regulation appear to be complex.

At least four different proteins interact with these elements: ENKTF-1, AP-1,

AP-4, and AP-2. The recent findings that AP-1 consists of a fos/jun

heterodimer; that fos, jun, and CREB all share a common leucine zipper; and

that fos, jun, and CREB each exist as a family of proteins further expands the

number of potential heterodimers and emphasizes the need to identify family

members mediating regulation.

The above complexities suggest a divergence of signals transmitted through a

small number of pathways to a large number of regulatory factors

(heterodimeric permutations of fos, jun, and CREB proteins) that may

differentially regulate and coordinate the neurons’ response to stimulation.

Because the transcription factors described above are activated by both

neurotransmitters and pharmacologic agents, they can be considered as

important targets of drug action. Identification and characterization of these

factors and determination of how their activities are regulated by synaptic

stimulation may lead to the production of specific drugs to activate or block their

actions.

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ACKNOWLEDGMENT

This work was supported in part by National Institute on Drug Abuse grant DA05706.

AUTHORS

Michael Comb, Ph.D.

Director

Laboratory of Molecular Neurobiology

Steven E. Hyman, M.D.

Director of Psychiatric Research

Department of Psychiatry

169
Linda Kobierski, B.S.

Program in Neuroscience

Harvard Medical School

Hung Ming Chu, B.S.

Program in Neuroscience

Harvard Medical School

Tuong Van Nguyen, M.D., Ph.D.

Research Fellow

Department of Psychiatry

Massachusetts General Hospital East

Building 149

13th Street

Charlestown, MA 02129

170
Regulation of Carboxypeptidase E

(Enkephalin Convertase)

Lloyd D. Fricker, Banasree Das, Robyn S. Klein, David Greene,

and Yong-Keun Jung

INTRODUCTION

Many peptide hormones and neurotransmitters are produced initially as large

precursors in which the bioactlve peptide is flanked by basic amino acids

(Docherty and Steiner 1982). Enzymatic processing by trypsin-like

endopeptidases generates intermediates with C-terminal basic residues. These

residues then are removed by a carboxypeptidase with a specificity for basic

residues. Carboxypeptidase E (CPE) originally was identified as the

carboxypeptidase responsible for the formation of met- and leu-enkephalins in

the bovine adrenal medulla (Fricker and Snyder 1982; Fricker 1988a). This

enzyme initially was designated enkephalin convertase (Fricker and Snyder

1982) and is also known as carboxypeptidase H [EC 3.4.17.10) (Webb 1986).

A CPE-like enzyme is present in high levels in several neural and endocrine

tissues, such as brain (Flicker and Snyder 1982), pituitary (Fricker et al. 1982),

and endocrine pancreas (Docherty and Hutton 1983); CPE mRNA is found in

brain, pituitary, and many endocrine tissues (Fricker et al. 1986, 1989;

Rodriguez et al. 1989). In adrenal medulla, pituitary, and an insulin-producing

tumor, CPE is the only carboxypeptidase activity localized to the peptidecontaining secretory granule fraction (Fricker and Snyder 1982; Docherty and

Hutton 1983; Hook and Loh 1984). Purified CPE can remove the basic Cterminal residue for a large number of peptide intermediates (Fricker and

Snyder 1982; Hook and Loh 1984; Davidson and Hutton 1987; Mackin and Noe

1987; Smyth et al. 1989). These results suggest that CPE is involved in the

biosynthesis of numerous peptide hormones and neurotransmitters.

REGULATION OF CPE ACTIVITY BY pH

The processing of peptide hormones is thought to begin either in the trans- Golgi

apparatus or in the newly formed secretory granules, depending on the

hormone (Orci et al. 1987; Schnabel et al. 1989). Since the pH of these

compartments is lower than the neutral pH of the endoplasmic reticulum or

171
other regions of the Golgi (Anderson and Pathak 1985), it is possible that pH

controls the processing enzymes. To investigate whether CPE activity is

regulated by pH, we determined Kcat and Km values for dansyl-Phe-Ala-Arg

hydrolysis by purified CPE at a variety of pH values. The optimum activity

(Kcat/Km) is around pH 4.8-5.6 (figure 1), which is consistent with previous

studies that measured enzyme activity with only a single substrate

concentration (Fricker and Snyder 1982; Docherty and Hutton 1983).

By examining the KcatKm it is possible to infer the number of ionizing groups

that are responsible for the observed pH optimum (Fersht 1983). Typically, a

FIGURE 1. Effect of pH on Kcat/Km for dansyl-Phe-Ala-Arg hydrolysis by

purified bovine CPE, prepared as described by Fricker and

Snyder (1983). The carboxypeptidase assay was performed as

described by Fricker and Snyder (1983), using 100 mM sodium

acetate at the indicated pH. Substrate concentrations for the

kinetic analysis were 200, 100, 50, 25, and 12.5 μM, each

assayed in triplicate for all pH values. Variation within each

triplicate determination was typically less than 10 percent. Kcat

and Km were calculated from Lineweaver-Burke plots.

172
change in the ionization of a single amino acid is sufficient to block enzymatic

activity, providing that residue is involved with the substrate binding or the

catalytic activity. For single ionizations, a plot of log Kcat/Kmvs. pH shows a

slope of l , which is the case for the ionization of CPE that occurs with a pKa of

-5 (figure 1). If two groups are ionizing with similar pKa values and both groups

are critically involved with the enzymatic activity, the slope of the plot will be ±2

This is found with CPE for pH values above 5.8 (figure 1). The enzymatic

activity of CPE is twice as sensitive to pH values above 5.8, compared with its

sensitivity to pH values below 5. Thus, the pH of the environment has a

dramatic effect on the activity of CPE.

MULTIPLE FORMS OF CPE

Early studies on CPE activity in bovine adrenal medulla chromaffin granules

detected both soluble and membrane-associated forms of this enzyme (Fricker

and Snyder 1982). Subsequent studies detected soluble and membrane-bound

forms of CPE in bovine brain and pituitary (Supattapone et al. 1984); in

anglerfish endocrine pancreas (Mackin and Noe 1987); and in neural tissue

from Xenopus, shark, and Aplysia (Fricker and Herbert 1988). In these tissues,

one form of CPE activity is soluble at acidic pH values (pH 5-6). Of the

membrane-bound CPE activity, 10 to 30 percent can be extracted with 1 M

NaCl (Supattapone et al. 1984; Hook 1984; Fricker 1988b). Repeated

extractions of the membranes with hlgh salt (at pH 5-6) do not solubilize the

majority of the membrane-associated CPE activity. This suggests that at least

two distinct membrane-associated forms of CPE are present in these tissues in

addition to the soluble form of CPE (S-CPE). One membrane form (M1-CPE) is

an extrinsic membrane protein that is released by high salt washes. The other

membrane form of CPE (M2-CPE) can be extracted in high yield by a

combination of 1 percent Triton X-100 and 1 M NaCI, at pH 5.5 (Supattapone et

al. 1984). Detergent alone does not solubilize either membrane form

(Supattapone et al. 1984). Interestingly, the membrane-bound forms of CPE

are extracted by neutral pH buffers (Mackin and Noe 1987; Fricker 1988b).

Since the common mechanism for the attachment of proteins to membranes

(such as hydrophobic transmembrane-spanning sequences or

phosphatidylinosltol groups) is not known to be dependent on pH in the 5-7

range, it is likely that a novel mechanism is responsible for the membrane

association of CPE.

The soluble and membrane forms have been purified to homogeneity using

identical chromatographic steps (Supattapone et al. 1984) indicating similar

physical properties. The enzymatic properties of the soluble and membraneassociated forms of CPE are also very similar (Supattapone et al. 1984). All

forms contain the same partial N-terminal amino acid sequence, and tryptic

173
maps are almost identical (Fricker et al. 1986). The only reported difference

between the forms is the apparent molecular weight-approximately 50 kDa for

S-CPE and 52 kDa for M2-CPE (Supattapone et al. 1984).

Results from genomic Southern blot analysis, Northern blot analysis, and

nucleotide sequence analysis of bovine and rat CPE cDNA clones suggest that

the soluble and membrane-associated forms arise from posttranslational

processing of a single precursor and not from different genes or mRNA splicing.

The CPE precursor (preproCPE), deduced from the nucleotide sequence of

cDNA clones, is 475 (bovine) or 476 (rat) amino acids in length (Fricker 1988c;

Fricker et al. 1989; Rodriguez et al. 1989). The precursor contains a short Nterminal peptide 41 amino acids in length that is not present on the forms of

CPE that had been partially sequenced (figure 2). The first 20 to 25 residues of

the precursor resemble a signal peptide (“pre” sequence). The remainder of the

N-terminal extension (“pro” sequence) is presumably removed by a trypsin-like

endopeptidase. There are five adjacent Arg residues immediately preceding

the known N-terminus of the active forms of CPE. The conversion of proCPE

into CPE appears to be fairly rapid since tissue levels of proCPE are extremely

low (Fricker and Das, unpublished observation).

Analysis of the predicted sequence of CPE does not reveal any regions of

sufficient hydrophobicity and length to form a membrane-spanning helical

structure. However, analysis of the C-terminal portion of CPE indicates a

propensity of this region to form an amphiphilic helix with eight hydrophobic

residues protruding from one face of an helix (Fricker et al. 1986). Preceding

this potential amphiphilic helix is a highly charged region containing nine

adjacent basic and acidic amino acids, Antisera raised against C-terminal

peptides are able to distinguish between the soluble and membrane-associated

forms of CPE (Fricker et al. 1990a, 1990b, 1990c), leading to the model shown

in figure 2. The membrane-associated form of CPE that is extractable with a

combination of NaCl and detergent (M2-CPE) contains a full-length C-terminus,

and it is held to the membrane by a combination of electrostatic and

hydrophobic interactions. The membrane form of CPE that can be extracted

with NaCl alone (M1-CPE) contains the highly charged portion of the C-terminal

region but not the potential amphiphilic region. This form of CPE is held to the

membrane primarily through ionic interactions with an unidentified intrinsic

membrane protein (or proteins). The soluble form (S-CPE) does not contain

either the highly charged region or the potential amphiphilic region. The

solubilization of both membrane-associated forms of CPE by neutral pH buffers

is presumably the result of a pH-induced conformational change that disrupts

the structure of the C-terminal region,

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FIGURE 2. Schematic representation of the CPE precursor (top) and the

postulated structure of various forms of CPE (bottom). The amino

acid sequence of preproCPE was deduced from the nucleotide

sequence of a bovine cDNA clone (Fricker et al. 1986): Rat

preproCPE has >90 percent amino acid identity, and all the

indicated features are the same for the two species (Fricker et al.

1989). All sequences of two or more adjacent basic amino acids

are indicated (K=Lys, R=Arg). The regions showing the 20-

percent amino acid identity with carboxypeptidases A (CPA) and

B (CPB) and the 50-percent amino acid identity with

carboxypeptidases M (CPM) and N (CPN) are indicated. The Nterminus of the forms of CPE that had been purified and partially

sequenced (S-CPE and M2-CPE) also is indicated. The

hypothetical structures of the soluble form (S-CPE), of the

membrane-associated form that is released by high salt washes

(M1-CPE), and of the membrane-associated form that is released

by detergent in the presence of high salt (M2-CPE) are based on

the three-dimensional structure of carboxypeptidase B (Schmid

and Herriott 7976). A region of CPE without homology to CPA or

CPB is indicated by a “?": The length of this region is 100 amino

acids (AA). Another "?” indicates a hypothetical membrane

protein that binds the M1 and M2 forms of CPE. The approximate

length of the C-terminal portion of the various forms of CPE was

determined with antisera directed against several C-terminal

peptides (Fricker et al. 1990a).

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EFFECT OF MEMBRANE ASSOCIATION ON THE ACTIVITY OF CPE

The activation state of M2-CPE is influenced by its association with membranes

(Hook 1985; Fricker 1988b). Upon extraction with either 1 M NaCl and 1

percent Triton X-100 at pH 5.5 or neutral pH buffers without detergent or salt,

the amount of CPE activity recovered in the supernatant and pellet fractions is

typically two to three times higher than the amount of CPE activity present

before extraction (figure 3, lanes 1-3). Control extractions with 0.1 M NaAc (pH

5.5) do not release any of the membrane-bound CPE activity, nor is the amount

of CPE activity altered (figure 3, lane 4). The high pH-induced solubilization

and activation of the membrane-associated CPE is partially reversible: If the

membranes are treated at high pH but then brought to pH <6 before

centrifugation, much less CPE is solubilized and the total amount of CPE

activity is reduced (figure 3, lane 5) compared with the samples centrifuged at

the high pH (lane 3).

Kinetic analysis of the membrane fractions before and after the extraction

revealed that the observed activation is due to both a higher Vmax and a lower

Km for substrate hydrolysis (Fricker 1988b). It is not likely that the activation

results from the conversion of catalytically inactive molecules to active ones

since pretreatment of the membranes with an active site-directed irreversible

inhibitor of CPE (bromoacetyl-D-arginine) completely blocks the activation

(Fricker 1988b). Once M2-CPE is solubilized and purified to homogeneity, the

Kcat and Km for substrate hydrolysis are similar to the values with purified SCPE (Supattapone et al. 1984). Furthermore, the treatment of purified S-CPE

or M2-CPE with high pH buffers or 1 percent Triton/1 M NaCl at pH 5.5 does not

influence the enzymatic activity (measured at pH 5.5), suggesting that the

activation is not a result of intrinsic changes within the CPE molecule (Fricker

1988b).

These results indicate that the association of CPE with membranes lowers the

enzymatic activity. The extent of the association with membranes is determined

by the ratio of the various forms of CPE (figure 2), as well as the pH. At neutral

pH, the majority of CPE is soluble, although there is little enzymatic activity at

this pH due to the ionlzation state of sensitive groups. As the pH of the

environment drops in the trans-Golgi network or in newly formed secretory

vesicles, the ionization of these groups changes, thus activating CPE.

However, some of the M1-CPE and M2-CPE will associate with the membranes

as the pH drops below 7, and this membrane binding decreases their enzymatic

activity. This hypothesis predicts that the membrane forms of CPE will have a

broader pH optimum than will S-CPE. To test this, we compared the effect of

pH on the CPE activity in soluble and membrane fractions of bovine pituitary

(figure 4). The CPE activity in the 0.1 M NaAc, pH 5.5 extracts (S-CPE) shows

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FIGURE 3. Extraction and activation of membrane-bound CPE from bovine

pituitary membranes. The amount of CPE activity, determined

with dansyl-Phe-Ala-Arg, in the soluble extract (open bars) or

remaining in the pellet fraction (cross-hatched bars) is shown

relative to the amount of CPE activity initially present in the

membranes. The experiment was performed as described by

Fricker (1988b), using 50 mM NaAc (pH 5.5, 50 mM sodium

phosphate (pH 7.5), or 50 mM sodium bicarbonate (pH 9.0).

a rapid drop in enzymatic activity as the pH is increased above 5.5. In contrast,

membrane homogenates containing Ml-CPE and M2-CPE are less sensitive to

pH values between 6 and 7 and are approximately three to four times more

active than the soluble fraction at pH 6.5 and 7.0. Thus, the activity of CPE can

be controlled by the pH and by the extent of the processing of proCPE into the

soluble and membrane forms.

DISTRIBUTION OF SOLUBLE AND MEMBRANE-ASSOCIATED FORMS OF

CPE IN RAT TISSUES

Previous studies found CPE activity in rat brain and pituitary but not in many

other tissues of the rat (Fricker et al. 1982). In the present study, we have used

a substrate that is 20-fold more selective for CPE. As previously found, high

177
0 0

8 0

6 0

4 0

2 0

0

4.0 5.0 6.0 7.0 8.0

pH of CPE assay

FIGURE 4. Effect of pH on CPE activity in bovine pituitary fractions. One

bovine pituitary was homogenized (Polytron) in 20 mL of 0.7 M

NaAc, pH 5.5, and then centrifuged for 1 hour at 50,000 x g. The

pellet was washed once with the same buffer, and then CPE

activity in either the supernatant fraction or the membrane

homogenate was determined in triplicate with dansyl-Phe-Ala-Arg

in 0.7 M Tris acetate buffers at the indicated pH.

levels of CPE are present in rat pituitary, with moderately high levels in brain.

Moderate levels of CPE are present in adrenal and intestine, and low levels are

detectable in heart and eye. In most of the tissues examined, approximately

two-thirds of the total CPE activity is soluble (table 1). Less of the CPE activity

in brain and heart is soluble, with values of 35 percent and 45 percent,

respectively. The tissue-specific variations in the amount of CPE that is soluble

is presumably due to differences in the posttranslational processing of CPE.

The level of CPE activity in many, but not all, fetal (E17) rat tissues is similar to

the level in the adult (table 1). A large difference is found in the adrenal, where

CPE activity is approximately 10-fold higher in adult tissue. There is also a

difference in the relative amounts of the soluble and membrane-bound forms of

CPE for the two age groups (table 1). In all tissues except brain, the fraction of

CPE that is soluble increases substantially between E17 and adult rats. In

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TABLE 1. Comparison of levels of CPE in adult and fetal rat tissues

Tissue

Adult Fetal (El 7)

CPE Activity CPE Activity

nmoles/min/mg % Soluble nmoles/min/mg % Soluble

Pituitary 62 ±27 66±13

Brain 10 ± 2 35± 7 6.4 ± 2.3 56±9

Adrenal 5.3± 1.2 62± 8 0.55 20

Intestine 4.0± 1.6 64± 2 6.2 ± 3.3 39± 6

Heart 1.2± 0.4 45±10 1.2 ± 0.6 28±10

Eye 1.1± 0.2 61 ±10 1.5 ± 0.5 35± 9

NOTE: Data are the average of three rats±standard error (except fetal adrenal:

tissue from three animals was pooled). Carboxypeptidase activity was

determined with the substrate dansyl-Phe-Ala-Arg (Fricker and Snyder

1983); CPE, expressed in nmole/min per mg protein, is defined as the

difference in carboxypeptidase activity measured in the presence of 1

mM CoCI2

 compared with the activity in the presence of 1 μM GEMSA

(Stack et al. 1984). E17=embryonic day 17.

brain, a larger fraction of CPE activity is soluble in the fetal rats than in the adult

animals. These findings suggest that both the total amount of CPE and the

relative amounts of the soluble and membrane-bound forms are

developmentally regulated in a tissue-specific manner.

The tissue distribution of CPE activity generally is consistent with previous

studies examining the localization of this enzyme in various tissues using a

radiolabeled active site-directed inhibitor, [

3H]-GEMSA (Strittmatter et al. 1984,

1986), and with the levels of CPE mRNA (Fricker et al. 1989; Rodriguez et al.

1989). The major difference in the results of the various studies is in the levels

of CPE in the intestine. This tissue has moderate amounts of CPE activity and

[

3H]-GEMSA binding (Strittmatter et al. 1984; Lynch et al. 1987) but extremely

low levels of CPE mRNA (Fricker et al. 1989). The reason for this discrepancy

is not known.

REGULATION OF CPE SECRETION

CPE activity is secreted from a variety of peptide hormone-producing cells,

including a mouse pituitary corticotrophic cell line (AtT-20 cells), a rat anterior

pituitary-derived cell line (GH4C1 cells), and primary cultures of bovine adrenal

179
chromaffin cells. The secretion of both CPE and prooplomelanocottin (POMC)-

derived peptides from the AtT-20 cell line can be stimulated by corticotrophicreleasing hormone (CRH) and inhibited by dexamethasone (Mains and Eipper

1984). The secretion of CPE from primary cultures of bovine adrenal

chromaffin cells is stimulated by nicotine and inhibited by nicotinic antagonists

(Hook and Eiden 1985). CPE secretion from GH4C,

 cells is stimulated by either

KCI or thyrotropin releasing hormone (Fricker et al. 1990b). These observations

are consistent with the localization of CPE to the secretory granules of rat

pituitary and bovine adrenal medulla (Fricker and Snyder 1982; Docherty and

Hutton 1983). The release of CPE is presumably a consequence of the

secretion of the peptide, although it is possible that secreted CPE performs

some unknown function.

Recently, CPE has been found to be present in type I astrocytes that were

cultured from E17 rat hypothalami (Vilijn et al. 1989). The level of CPE activity

in the cultures of astrocytes is approximately 10-fold lower than the level of CPE

activity in rat hypothalamic neuronal cultures (table 2). However, the amount of

CPE activity secreted from the neurons is only fourfold to fivefold higher,

indicating that the basal rate of secretion is different for these two cell types.

The regulation of the secretion also differs: KCI stimulates the secretion of CPE

from the neuronal cultures but does not alter the amount of CPE secreted from

the cultured astrocytes (table 2). The level of CPE mRNA is fourfold higher in

astrocytes than in neurons, suggesting that the production of CPE protein in

astrocytes is higher than predicted from the low cellular levels of enzymatic

activity (table 2).

TABLE 2. CPE activity and mRNA levels in primary cultures of hypothalamic

neurons (neonatal) and type I astrocytes (E 17)

Cell

Specific Activity

Cellular CPE.

Secreted CPEb

CPE

Basal KCI Stimulation mRNAC

Neurons 120±20 35 103 1.0

Astrocytes 11± 2 8 8.8 4.4

aCPE was measured with the substrate dansyl-Phe-Ala-Arg (Fricker and Snyder

1983) and is expressed in pmoles/min-106

 cells±standard error of the mean for

triplicate determinations (Vilijn et al. 1989).

b

pmoles/min secreted from 106 cells per hour.

CRelative densitometric units/μg total RNA (Vilijn et al. 1989).

180
PHARMACOLOGICAL REGULATION OF CPE IN TISSUES AND CELL

CULTURE

The regulation of CPE has been examined in a variety of systems. Initial

experiments focused on the levels of CPE activity in rat and mouse brain upon

addiction to morphine. The results of these studies were inconclusive: In some

experiments, high doses of morphine caused a 50-percent decrease in the level

of S-CPE activity but had no effect on the level of membrane-associated CPE

activity (Fricker, Supattapone, and Snyder, unpublished observation). However,

this effect was not consistently reproducible. Since CPE is present in many cell

types within the brain, it is possible that changes in a small number of cells are

not detected by measuring CPE activity in whole brain.

Recent studies on the in vivo regulation of CPE have focused on the rat adrenal

medulla since there is only a single type of peptide-producing cell in this tissue.

In the rat adrenal medulla, reflex splanchnic stimulation caused by insulininduced hypoglycemic shock increased the level of enkephalin mRNA

approximately 16-fold and increased the level of enkephalin peptides nearly

150-fold (Viveros et al. 1987). This treatment also caused a onefold increase in

the levels of both CPE mRNA and enzymatic activity within several days of

treatment (Fricker et al. 1990c). Although this degree of change was

substantially smaller than the changes in enkephalin mRNA and peptide levels,

the direction of the change was the same (table 3).

The regulation of CPE also has been examined in a variety of cell culture

systems. Primary cultures of bovine adrenal chromaffin cells produced both

enkephalin-containing peptides of high molecular weight and enkephalin

peptides of lower molecular weight. Reserpine treatment increased the levels

of the low molecular weight species and decreased the amount of the larger

peptides, suggesting that the processing rate is stimulated (Wilson et al. 1980;

Eiden et al. 1984; Lindberg 1986; Viveros et al. 1987). The effect of reserpine

on the level of enkephalin mRNA is not consistent between different studies:

Eiden and colleagues (1984) found a decrease in enkephalin mRNA levels

upon reserpine treatment, but Viveros and colleagues (1987) found no change.

The effect of reserpine treatments on CPE activity also differs between studies.

Hook and colleagues (1985) found that CPE activity was stimulated by

treatment of the cells with reserpine and that this stimulation was due to a

change in the Km for substrate hydrolysis. We have also found that reserpine

stimulates CPE activity in chromaffin cells, but this treatment does not change

the Km for substrate hydrolysis (Fricker, Das, and Lindberg, unpublished

observation). Instead, we found that the apparent Vmax was increased twofold

to threefold by the treatment, which could be due either to a change in the

turnover rate of the enzyme or to an increase in the number of CPE molecules.

181
TABLE 3. Regulation of CPE activity in cell culture and in tissue

This kinetic change does not appear to be the result of the redistribution of CPE

from the membrane-associated form to the soluble form: The amount of CPE

that is soluble is not affected by reserpine treatment (Fricker, Das, and

Lindberg, unpublished observation).

The GH4C1 cell line, derived from rat anterior pituitary, produces both prolactin

and growth hormone (Tashjian et al. 1968; Tashjian 1979). The treatment of

these cells with 1 nM estradiol, 300 nM insulin, and 10 nM epidermal growth

factor increases both the number of secretory granules and the intracellular

level of prolactin approximately 30- to 50-fold (Scammell et al. 1986), Prolactin

mRNA is elevated approximately 10-fold by the hormonal treatment, but CPE

mRNA is not altered by more than 10 percent (Fricker et al. 1990b). The level

of CPE activity is increased approximately onefold by the treatment, which is

due to an increase in the apparent Vmax, with no effect on the Km.

The regulation of CPE activity and mRNA levels also has been examined in the

AtT-20/D16v cell line. This mouse anterior pituitary-derived cell line produces

high levels of POMC-derived peptides, such as adrenocorticotropic hormone

(ACTH) (Eipper and Mains 1980). Treatment of these cells with

182
dexamethasone causes a decrease in POMC mRNA levels and ACTH levels

(Eipper and Mains 1980). CRH increases both POMC mRNA and ACTH levels

in this cell line. in addition, the level of peptidyl-glycine- -amidating

monooxygenase, the enzyme responsible for the amidation of the C-terminus of

many peptide hormones, also is lowered by the dexamethasone treatment and

is elevated by the CRH treatment (Eipper et al. 1987). These treatments do not

appear to alter the level of CPE activity (Mains and Eipper 1984; Thiele and

Fricker 1988) or the level of CPE mRNA (Thiele and Fricker 1988), although

another study reported changes in CPE mRNA that are comparable to the

changes in POMC mRNA (Rodriguez et al. 1989). The treatment paradigm and

culturing conditions differed slightly between the two studies, and it is possible

that these differences influenced the results.

These studies indicate that CPE can be regulated in vivo and in cell culture.

In general, the changes in CPE activity and/or CPE mRNA levels are in the

same direction but of a smaller magnitude than the changes in bioactive peptide

and/or mRNA levels. This is consistent with the proposal that CPE is not a ratelimiting enzyme in the production of most peptide hormones. However, purified

CPE has recently been found to slowly convert neoendorphin to

ß-neoendorphin, and porcine ß-endorphin 1-27 to ß-endorphin 1-26, in vitro

(Smyth et al. 1989). Since tissue levels of neoendorphin and ß-endorphin

1-27 are often comparable to the levels of the C-terminally shortened peptides,

it is likely that CPE is the rate-limiting enzyme in the production of

ß-neoendorphin and ß-endorphin 1-26. Small changes in CPE activity

presumably would have a significant effect on the production of these peptides.

SUMMARY

CPE, EC 3.4.17.10, is involved in the production of numerous peptide

hormones and neurotransmitters. To investigate whether the regulation of this

enzyme may play a role in the control of peptide hormone biosynthesis, we

have examined potential mechanisms by which CPE activity can be regulated.

These studies have involved kinetic analysis of purified enzyme and of tissue

extracts, as well as the determination of enzyme activity and mRNA levels in a

variety of tissues and cell lines. The major findings are that (1) pH has a

significant influence on the activity of purified CPE; (2) pH also has an effect on

the binding of CPE to membranes, and the membrane binding influences CPE

activity; (3) the levels of CPE activity and the degree of membrane binding vary

between tissues and developmental state; and (4) in several cell lines and

tissues, CPE activity is increased by treatments that increase the levels of

peptide hormones, although the changes in CPE levels are typically much

smaller than the corresponding change in peptide levels.

183
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ACKNOWLEDGMENTS

This work was supported by National Institute on Drug Abuse grant DA-04494

(LDF), by an Alfred P. Sloan research fellowship (LDF), and by National

Institute of Arthritis, Diabetes, and Digestive Kidney Diseases (now called

National Institute of Diabetes and Digestive and Kidney Diseases) training grant

T32-AM07513 (RSK).

AUTHORS

Lloyd D. Fricker, Ph.D.

Assistant Professor

Departments of Molecular Pharmacology and Neuroscience

Banasree Das, M.S.

Research Technician

Department of Molecular Pharmacology

Robyn S. Klein, MS.

Department of Neuroscience

David Greene

Research Technician

Department of Molecular Pharmacology

Yong-Keun Jung, M.S.

Department of Molecular Pharmacology

Albert Einstein College of Medicine

1300 Morris Park Avenue

Bronx, NY 10461

187
Use of Site-Directed Mutagenesis To

Elucidate the Active Site of Neutral

Endopeptidase 24.11 (Enkephalinase)

Louis B. Hersh

It is a reasonably well-accepted hypothesis that the physiological actions of the

enkephalins and probably other opioid peptides are terminated by peptidase

action. Thus, these peptidases play a key role in regulating the action of opioid

peptides. Neutral endopeptidase 24.11 (NEP) is one such peptidase. The

enzyme is a 750-amino acid integral membrane protein composed of a 23-

amino acid cytoplasmic domain; a 24-amino acid hydrophobic segment that

functions as a transmembrane region and a signal peptide; and a 699-amino

acid extracellular domain, which contains the active site (Malfroy et al. 1987;

Devault et al. 1987; Shipp et al. 1988). Hydrolysis by NEP appears to represent

the major pathway by which enkephalins are inactivated at enkephalinergic

synapses. Based on this function, NEP has been given the trivial name

“enkephalinase” (Malfroy et al. 1978). In addition to the enkephalins, there are

other opioid peptides that have been shown to be substrates for NEP in vitro.

These include met-enkephalin-Arg-Phe, gamma endorphin, and beta lipotropin

61-68 (Hersh 1984). However, the relative importance of the enzyme in

inactivating these opioid peptides in vivo has not been investigated thoroughly.

NEP also has been implicated as being involved in the regulation of blood

pressure. It is the major degradative enzyme for the potent vasso active

peptides: atrial natriuretic peptide (Koehn et al. 1987; Sonnenberg et al. 1988)

and endothelin (Vijayaraghavan et al. 1990). Altogether, there are more than

20 physiologically active peptides that are reported to be cleaved by the

enzyme (table 1).

It has been demonstrated recently that NEP is identical to the tumor antigen

CALLA (common acute lymphoblastic leukemia antigen) (Shipp et al. 1989;

LeTarte et al. 1988) and that CALLA represents active NEP on these cells

(table 2). CALLA is expressed by early lymphoid progenitors that are as yet

uncommitted to B- or T-cell lineage or are at the earliest stages of B-cell

differentiation. CALLA is barely detectable on mature B cells, suggesting that it

may play a role in the early stages of lymphoid differentiation. In addition,

188
TABLE 1. Physiologically active peptides cleaved by NEP

Met-enkephalin Bradykinin

Met-enkephalin-Arg-Phe Gamma-endorphin

Angiotensin I Chemotactic peptide

Angiotensin II Beta lipotropin 61-69

Angiotensin Ill Leu-enkephalin

Dynorphin 1-9 Substance P

Dynorphin 1-13 Oxytocin

Physalaemin Neurotensin

Neurokinin A

Neurokinin B

lnterleukin I

Somatostatin

CCK-8

ANF

Endothelin

TABLE 2. Identification of active NEP on CALLA+ leukemic and glioma cell

lines

Specific Activity (pmols/min/106 cells)

Cell Line - Phosphoramidon + Phosphoramidon

Leukemic cells

Nalm-6 (CALLA+)

Nall-1 (CALLA+)

SMS (CALLA-)

80 2.8

42 2.6

3.6 3.5

Gliomas

#71

#229

#427

32 2.1

14 1.6

10 1.1

NOTE: Enzyme assays were conducted on whole cell suspensions in 25 mM

HEPES buffer, pH 7.4, using 0.1 mM glutaryl-Ala-Ala-Phe-4-methoxy-2-

naphthylamide as substrate.

CALLA/NEP has been detected on several gliomas (Carrel et al. 1982). As

shown in table 2, this activity is comparable to that found on leukemic cells.

It has been demonstrated that NEP produces a naloxone reversible analgesia

in experimental animals (Roques et al. 1980; Chipkin et al. 1982; Greenberg

and O’Keefe 1982; Lecomte et al. 1986), and several NEP inhibitors currently

are being evaluated clinically for their analgesic properties. A more detailed

knowledge of the active site of NEP would be useful for the design and

synthesis of new inhibitors of the enzyme of potential clinical use.

189
The specificity of NEP is directed toward cleavage of substrates on the amino

side of hydrophobic amino acids (Hersh and Morihara 1986; Pozsgay et al.

1986; Hersh 1982). In this respect, NEP resembles the well-characterized

bacterial neutral endopeptidases of which thermolysin is the best characterized.

Comparison of the specificity of thermolysin and NEP with synthetic substrates

indicates that the enzymes are similar although not identical (Hersh and

Morihara 1986; Pozsgay et al. 1986). This is illustrated in table 3. As can be

seen in the table, the relative order of reactivity between the two enzymes is

similar with the three methoxynaphthylamide (4MeO2NA) substrates; however,

with a nitrophenylalanine-containing substrate, activity can be detected only

with NEP. It is believed that the substrate binding site in NEP is larger and less

hydrophobic than in the bacterial endopeptidases, but these differences are

relatively modest when one considers the similarities in the overall specificity of

NEP and the bacterial endopeptidases.

TABLE 3. Comparison of NEP and thermolysin with synthetic substrates

NEP Thermolysin

Substrate

glut-Ala-Ala-Phe-4MeO-2NA 0.07 23.00 0.55 101.00

glut-Gly-Gly-Phe-4MeO-2NA 0.09 0.14 0.55 0.70

glut-Ala-Ala-Pro-Phe-4MeO-2NA 0.27 6.40 0.95 1.40

dansyl-D-Ala-Gly-(NO,)Phe-Gly 0.08 3.00 (0.10)a<)0.005

aNo reactivity was detected at a substrate concentration of 0.1 mM.

NOTE: glut=glutaryl

Both NEP and the bacterial endopeptidases are inhibited specifically by the

dipeptide analog phosphoramidon, a property that has been used to distinguish

these enzymes from other peptidases. The similarity between NEP and the

bacterial endopeptidases extends beyond substrate and inhibitor specificity.

There are two regions of amino acid sequence that are conserved between the

bacterial and mammalian enzymes (table 4). One of these is a helical segment

containing (1) two histidines that, in thermolysin, serve as zinc ligands and (2) a

glutamic acid that, in thermolysin, is believed to act as an acid-base catalyst.

The other region contains a histidine that, in thermolysin, is proposed to

stabilize a tetrahedral reaction intermediate but appears nonfunctional in NEP

(Erdos and Skidgel 1989).

190
TABLE 4. Sequence homology between NEP and bacterial endopeptidases

NEP

Thermolysin

B. cereus

B. subtilis

NEP

Thermolysin

B. cereus

B. subtilis

NEP does differ, however, from the bacterial endopeptidases in that NEP

contains an active site arginine that is proposed to interact with the C-terminal

carboxylate of its substrates (Jackson and Hersh 1986; Malfroy and Schwartz

1982; Beaumont and Roques 1986). The presence of this active site arginine

results in the preferential binding of substrates containing a free C-terminal

carboxylate as compared to their corresponding amide (table 5), and it leads to

the preferential cleavage of C-terminal carboxylate containing substrates near

their C-terminus. For example, angiotensin I (Asp-Arg-Val-Tyr-lle-His-Pro-PheHis-Leu) contains four potential cleavage sites, which are located on the amino

side of valine3, tyrosine4, isoleucine5, and phenylalanine8 (shown in bold); yet,

NEP cleavage of this peptide occurs preferentially at the Pro-Phe bond (Gafford

et al. 1983). Based on this observation and the sequence homology between

the carboxy-terminal region of NEP and the N-terminal domain of

carboxypeptidase A, it has been suggested that NEP exhibits properties similar

to those of the carboxypeptidases (Malfroy et al. 1987).

TABLE 5. Comparison of binding of enkephalin free acid and amide to NEP

and thermolysin

Peptide

Ki

(μM)

NEP Thermolysin

Ratio

Ki(amide)/Ki(acid)

NEP Thermolysin

Met-enkephalin 13 186 9.1 1.5

Met-enkephalinamide 119 278

Leu-enkephalin 47 313 7.9 2.9

Leu-enkephalinamide 373 900

191
Studies in this laboratory have been initiated to elucidate the active site of NEP

using a combined approach of chemical modification of the enzyme to identify

potential active site residues and site-directed mutagenesis to evaluate the role

of these residues in catalysis. To conduct studies involving site-directed

mutagenesis, a full-length cDNA for the enzyme was required. This was

accomplished by the isolation of two partial NEP cDNA clones that, when joined

together, encompassed the entire coding sequence for the enzyme (Bateman et

al. 1969). The first clone, designated NE-1, was isolated from a bacteriophage

lambda gtl 1 cDNA library constructed from rat kidney mRNA by screening with

an antibody generated against rat kidney NEP. The insert in this clone starts at

the codon for Lys360 and extends into the 3’ untranslated region. This clone

was used to rescreen the library from which a second overlapping clone, NE-2,

was isolated. NE-2 starts 78 nucleotides into the 5’ untranslated region and

overlaps NE-1 by 363 nucleotides. The two clones were linked together

through an overlapping Bgl II site, yielding a full-length cDNA. Subcloning of

this cDNA into the vector pCMV permitted transient expression of NEP activity

in COS cells. The recombinant enzyme, like the native enzyme, is glycosylated

and integrated into the plasma membrane with its catalytic domain extracellular.

It exhibits the same kinetic properties as the enzyme derived from rat tissue.

The arginine-specific reagents butanedione and phenylglyoxal were used to

demonstrate that NEP contains a reactive active site arginine (Jackson and

Hersh 1986) (figure 1). The modification of this arginine results in an increase

in the Km of the substrate with a smaller effect on Vmax (table 6). The reason

for the apparent loss in enzyme activity seen in figure 1 was that the substrate

concentration used in the enzyme assays was below its Km. Since these

reagents incorporate relatively bulky groups into the enzyme, their effect could

result from steric interactions rather than direct modification of an active site

residue. Therefore, it was crucial to identify the reactive arginine and to

examine its function through site-directed mutagenesis.

14C-Phenylglyoxal was used to radiolabel the enzyme and was found to

incorporate approximately 2 mols of reagent/mol enzyme (Bateman et al. 1989).

However, incorporation of only one of the two phenylglyoxal molecules was

blocked by substrates and inhibitors, indicating only one active site arginine. To

identify this active site arginine, NEP was radiolabeled with phenylglyoxal in the

presence and absence of inhibitors. The radiolabeled enzyme was separated

from free phenylglyoxal by molecular sieve chromatography and then digested

with trypsin. The resultant peptides were separated by reverse-phase HPLC

and, after further digestion with pepsin, the 14C-labeled active site peptide was

isolated (figure 2). The isolated peptide was subjected to automated Edman

192
FIGURE 1. Inactivation of NEP by arginine reagents

NOTE: Purified NEP was incubated with phenylglyoxal; with butanedione; or, in

the absence of any additions (control), in 50 mM triethanolamine/50 mM

sodium borate buffer, pH 7.8. At the periods indicated, aliquots were

removed and assayed for residual enzyme activity with 0.1 mM dansylD-Ala-Gly-(NO2

)Phe-Gly as substrate. Data are plotted as pseudo first

order reactions.

degradation, yielding the following amino acid sequence: Asn-Val-lle-Pro-GluThr-Ser-Ser-Arg-Tyr-Ser-Asn-Phe 106.

This sequence tentatively identified arginine 102 as the active site arginine. To

confirm the functionality of arginine 102, site-directed mutagenesis was used to

193
TABLE 6. Kinetic properties of NEP modified with arginine reagents

Treatment Km (mM) Kcat (s-1)

None 0.25 46.4

Phenylglyoxal 1.05 37.1

Butanedionone 1.30 49.6

NOTE: Purified NEP was pretreated with the arginine reagents until no further

decrease in activity was detected using 0.1 mM dansyl-D-Ala-Gly-

(NO2)Phe-Gly as a substrate. Due to the insolubility of this substrate,

the kinetic properties of the treated enzyme then were determined with

Cbz-Ala-Gly-Leu-Ala as substrate.

generate an enzyme form in which this arginine was converted to glutamine.

This was accomplished using the mutagenesis procedure of Zoller and Smith

(1982) as modified by Craig (1985). The codon for arginine was changed to the

codon for glutamine by a single base change. Mutants were identified by

differential hybridization and confirmed by nucleotide sequencing. The wildtype and mutant enzymes were transiently expressed in COS cells. The

amount of NEP recovered from COS cells was determined by quantitative

Western blot analysis, as illustrated in figure 3.

The R102Q mutant enzyme reacted equally well as the wild-type enzyme with a

substrate amide but exhibited low activity with a substrate containing a free Cterminal carboxyl group (figure 4). The wild-type enzyme exhibits a 10-fold

greater affinity for free acids compared to the corresponding amide, and this

ability to discriminate between an acid and an amide is essentially lost in the

mutant enzyme (table 7). This results from a greater than 15-fold increase in

the Km for the free acid substrate in the mutant enzyme, with less than a 3-fold

increase in the Km for the amide substrate, These changes in the binding

properties of the mutant enzyme provide strong evidence for the involvement of

arginine 102 in the binding of the C-terminal carboxylate of substrates.

Modeling studies based on the x-ray crystallographic structure of thermolysininhibitor complexes indicates that the enzyme contains an active site valine

residue (valine 137 in table 4) within the conserved helical segment common

between NEP and the bacterial endopeptidases. This valine forms a part of the

substrate binding pocket of thermolysin and makes a hydrophobic contact with

the amino acid side chain in the S’1 subsite. In NEP, a valine residue occupies

this same relative position (valine 573 of NEP in table 4). It was therefore of

interest to determine whether valine 573 in NEP has a similar binding function.

194
FIGURE 2. Isolation of phenylglyoxal modified peptide

NOTE: NEP radiolabeled with 14C-phenylglyoxal initially was digested with

trypsin and the resultant peptides separated by HPLC. A radiolabeled

peptide was digested further with pepsin and chromatographed on a C8 reverse-phase column using a 0.02 percent trifluoroacetic acid/

acetonitrile gradient system. Shown is the radioactive content of the

various fractions eluted from the HPLC column.

To examine this possibility, a mutant enzyme was prepared in which this valine

residue was changed to a leucine. By adding an additional methylene group to

the amino acid side chain in this position, it was anticipated that the size of the

binding pocket might be decreased and result in a lowering of the affinity of the

enzyme for bulky side chains in the P’1 position.

A series of peptides of the general structure N-[1-(RS)-carboxy-2-phenylethyl-XpAB, where X represents various amino acids and pAB is para-aminobenzoic

acid (Pozsgay et al. 1988), were used to probe the binding characteristics of the

S’1 site of the wild type and valine to leucine mutant. As shown in table 8,

when the P’1 residue is the small alanine side chain, the wild-type and mutant

195
FIGURE 3. Western blot of NEP expressed in COS cells and comparison to

enzyme from rat kidney

NOTE: Samples: lane 1, wild-type NEP expressed in COS cells, 50 μg of

protein; lane 2, R102Q mutant expressed in COS cells, 50 μg of

protein; lane 3, COS cell extract from cells transfected with NEP cDNA

in the reverse orientation, 54 μg of protein; lane 4, purified NEP papain

solubilized from rat kidneys, 2 μg of protein; lane 5, rat kidney

membranes solubilized with Triton X-100, 40 μg of protein.

enzymes exhibit the same binding characteristics. However, when the size of

the P’1 residue is increased, there is decreased binding of the inhibitor to the

mutant enzyme as compared to the wild type. This decrease in the affinity of

bulky P’1 residues to the mutant enzyme is consistent with the hypothesis that

valine 573 forms a part of the S’1 subsite of NEP.

Although the expression of NEP in COS cells provides a useful system for

evaluating mutant forms of the enzyme, it has the drawback that the enzyme is

integrated into the plasma membrane and thus must be solubilized with

detergents prior to study. It is known that NEP can be converted to a soluble

196
FIGURE 4. Comparison of the activities of wild-type and R102Q forms of NEP

NOTE: The activity of the wild-type and R102Q mutant forms of the enzyme was

determined with either 0.1 mM dansyl-D-Ala-Gly-(NO2)Phe-Gly as

substrate (A) or 0.1 mM glutaryl-Ala-Ala-Phe-4MeO-ZNA (B) as

substrate in 100 mM MES buffer at pH 8.5. The enzyme concentration

was determined by its reactions with an anti-NEP antisera and thus is

expressed in terms of immunoreactive material. Open symbols represent

the wild-type enzyme; closed symbols represent the R102Q mutant.

TABLE 7. Binding properties of wild-type and R102Q mutant with a substrate

free acid and its amide

Substrate

Km (μM)

Wild Type Mutant

D-Ala2

-Met5-enkephalin

D-Ala2

-Met5-enkephalinamide

Ratio Km enkephalinamide

Km enkephalin

22

370

385

1,260

16.8 3.3

197
TABLE 8. Binding of inhibitors with variable P'1 residues to wild-type and

V573L mutant forms of NEP

N-[1-(RS)-carboxy-2-phenylethyl-X-pAB Ki (μM)

X Wild Type Mutant

Ala

Leu

Phe

34 34

0.6 >50

0.05 9

Trp

Tyr

5 51

0.7 11

form by digestion with papain; however, this requires initial extraction of the

membrane proteins into deoxycholate, and the yield of NEP is relatively low.

Therefore, site-directed mutagenesis was used to delete the first 52 amino

acids from the enzyme, which includes both the cytoplasmic and the

hydrophobic membrane-spanning domains. Both domains were replaced with a

cleavable hemagglutinin leader sequence. Expression of this NEP fusion

protein in COS cells led to the secretion of soluble active enzyme into the media

(table 9). This secreted enzyme form retained the catalytic properties of the

native enzyme; however, analysis by SDS-PAGE indicated a molecular weight

slightly larger than the papain-solubilized enzyme, indicating that it is

glycosylated differently. This system represents one in which mutant enzyme

forms can be prepared in a secreted form and recovered in a highly purified

state in the culture media. It should prove very useful for the expression and

analysis of other membrane proteins,

TABLE 9. Secretion of NEP activity from transfected COS cells

Sample

Activity in Media (munits/mL)

Hours After Transfection

Fusion protein

Wild type

Cultured cells

0-24 24-48 49-72

0.02 0.12 0.17

ND ND >0.01

ND ND ND

NOTE: ND=not detectable

198
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Boileau, G. Amino acid sequence of rabbit kidney neutral endopeptidase

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“enkephalinase,” a neutral metalloendopeptidase that cleaves active

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Greenberg, R., and O’Keefe, E.H. Thiorphan potentiation of stress-induced

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Hersh, L.B. The reaction of opioid peptides with neutral endopeptidase

(“enkephalinase”). J Neurochem 43:487-493, 1984.

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mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial

neutral endopeptidase thermolysin. J Biol Chem 261:6433-6437, 1986.

Jackson, D.J., and Hersh, L.B. The reaction of arginine reagents with

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Koehn, J.A.; Norman, J.A.; Jones, B.N.; LeSeur, L.; Sakane, Y.; and Ghai, R.D.

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Llorens-Cartes, C.; Leboyer, M.; and Schwartz, J.C. Pharmacological

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Jongeneel, C.V.; and Mclnnis, R.R. Common acute lymphocytic leukemia

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antinociceptive activity in mice. Nature (London) 288:286-288, 1980.

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200
ACKNOWLEDGMENTS

This research was supported in part by grant DA-02243 from the National

Institute on Drug Abuse and by grant l-391 from the Robert A. Welch

Foundation, Houston, TX. Glioma cell lines cited in table 2 were provided by

Dr. N. De Tribolet.

AUTHOR

Louis B. Hersh, Ph.D.

Professor

Department of Biochemistry

University of Texas Southwestern Medical Center

5323 Harry Hines Boulevard

Dallas, TX 75235

201
Human Cytochrome P450: Possible

Roles of Drug-Metabolizing Enzymes

and Polymorphic Drug Oxidation in

Addiction

Frank J. Gonzalez

INTRODUCTION

When a drug or other foreign chemical enters the body, it usually is

metabolized by one or more of a group of proteins collectively known as the

drug-metabolizing enzymes. In general, these enzymes convert nonpolar

compounds into water soluble derivatives that can be easily eliminated from the

body via either the urine or the bile. A chemical, however, does not have to be

hydrophobic to be metabolized. Practically, drug metabolism usually results in

the therapeutic inactivation of drugs, although, as discussed below, a drug also

can be converted from an inactive to an active form.

Metabolism of many drugs occurs in two steps termed “phase I” and “phase II”

reactions. The principal enzymes involved in the first step are the cytochrome

P450s (P450s). These enzymes or monooxygenases serve to functionalize the

chemical through the introduction of a hydroxyl group or an epoxide. The

hydroxyl group can be added to carbon, nitrogen, or sulfur atoms. In many

cases, unstable intermediates can rearrange themselves in the enzyme-active

site resulting in other biotransformations, including dealkylation, dearylation,

dehalogenation, deamination, and reduction (Ortiz de Montellano 1989).

Another enzyme, the flavin-containing monooxygenase, can perform oxidations

at sulfur and nitrogen groups. Subsequent to addition of a functional group into

a substrate by phase I enzymes, the phase II enzymes can use this group for

conjugation of such compounds as glucuronic acid, glutathione, cysteine,

glycine, and sulfate. The phase II enzymes include glutathione transferases,

uridine diphosphate (UDP) glucuronosyltransferases, sulfotransferases, and

others. The end product is a rather hydrophilic compound that can be excreted

easily. Other drug metabolism enzymes, which are not necessarily classified as

phase I or II, also exist; these include epoxide hydratatases, numerous

exterases, N-acetyltransferase, dihydrodiol dehydrogenases, DT-diaphorase,

202
aldehyde dehydrogenases, glycosidases, and glucosidases. A large number of

different enzymes, therefore, exist to metabolize drugs and other foreign

compounds (table 1). It is believed that several of these enzymes are present

in multiple forms, thus accounting for their large substrate specificities.

However, a single form sometimes is capable of metabolizing numerous

structurally diverse chemicals. In addition, several drug-metabolizing enzymes

are expressed polymorphically in both rodents and humans: based on these

findings and other considerations, it is believed that many of them evolved to

metabolize the numerous toxins found in plant life (see below).

Most of the drug-metabolizing enzymes are expressed in liver, which serves as

the main “clearinghouse” for foreign chemicals. Once blood passes through the

liver, a large percentage of a particular drug can be metabolized. Other tissues

also contain drug-metabolizing enzymes-the most notable being the lung and

intestine, organs that are in direct contact with the environment.

This chapter focuses on the cytochrome P450s, the major class of drugmetabolizing enzymes, and discusses polymorphisms in these enzymes and

the possible role of genetic differences in P450 in drug addiction.

CYTOCHROME P450s

These enzymes exist as a gene superfamily composed of 10 families in

vertebrates (Gonzalez 1988; Nebert et al. 1991). P450s within six of these

families are involved in steroidogenic pathways and in cholesterol-bile acid

synthesis. One family, the P450 4 family, encodes fatty acid hydroxylases,

while P450s within families 1, 2, and 3 encode the drug-metabolizing enzymes.

The cytochrome P450s are the most common and well-studied enzymes of drug

metabolism. These enzymes contain a noncovalently bound heme, in the form

of protoporphyrin IX, and receive electrons from the flavoprotein NADPH-P450

oxidoreductase. Those P450s involved in drug metabolism are embedded in

the intracellular endoplasmic reticulum network, a membrane location that is

ideally suited for interaction of P450s with hydrophobic substrates. It is

generally believed that these enzymes are bound to the lipid bilayer via a

hydrophobic amino terminus, with the bulk of the enzyme facing the cytoplasmic

face of the endoplasmic reticulum (Nelson and Strobel 1988).

Evolution of Cytochrome P450s

One of the most fascinating aspects of P450s is the large degree of species

differences in their regulation and their substrate specificities (Gonzalez 1988).

For example, rats, mice, rabbits, and humans can differ widely in their

203
TABLE 1. List of drug-metabolizing enzymes and their reactions and

multiplicities

Multiplicity Polymorphisms Reactions

Phase I Enzymes

Cytochrome P450s

Flavin-containing

monooxygenase

Phase II Enzymes

UDP-glucuronosyltransferase

Sulfotransferase

Glutathione transferase

N-Acetyltransferaee

Other Drug Metabolizing

Carboxyleaterase

Aldehyde dehydrogenase

Epoxide hydrolase

(microeomal)

No

Epoxide hydrolase

(cytosolic)

Unknown

Dihydrodlol dehydrogenase

NAD(P)H-quinone

oxidoreductase

Unknown

N o

Drug Metabolism Support

Enzymes

NADPH-P450

oxidoreductase

No

Cytochrome b6 No

9 gene families

Yes

Yes

Unknown

4 gene families

Yes

Yes

Yes

Yes C-hydroxyiatlon

N-hydroxylatlon

Deamination

Dehalogenation

Sulfoxidatlon

N-dealkylation

S-dealkylatlon

(others)

Yes S-oxldation

N-oxldation

Yes Glucuronate

conjugatlon

Unknown Sulfate

conjugatlon

Yes Glutathione

conjugation

Yes Acetate

conjugatlon

Unknown Carboxyester

hydrolysis

Yes Aldehyde

oxidation

Unknown Epoxlde

hydrolysis

Unknown Epoxyde

hydrolysis

Unknown

Unknown Quinone

reduction

Unknown P450 reduction

Unknown P450 reduction

204
metabolism of drugs. This property, which has been recognized for over 40

years (Williams 1974), Is of utmost importance when attempting to extrapolate

rodent pharmacological data to humans.

The molecular basis for species differences has become more evident in recent

years, with a large amount of data from studies on purified P450s, cDNA, and

genes. It has become evident that P450s have evolved quite differently in

rodents and humans. Even between rats and mice, which split from a common

ancestor about 17 million years ago, many P450s and their genes have evolved

species-unique properties (Gonzalez and Nebert 1990). For example, certain

mouse genes are expressed in males but not females. Genes within the same

subfamily in rats, on the other hand, are expressed in both sexes. Likewise,

certain rat genes may be expressed in females, but genes in the same

subfamily of mice are under different regulation. Interestingly, no sex-specific

P450s have been detected in humans. P450s within a single subfamily of mice

also may have the capacity to metabolize androgens, whereas P450s within the

same subfamily of rats may not be able to catalyze androgen hydroxylation

(Gonzalez 1988; Gonzalez and Nebert 1990). Again, these findings illustrate

the fact that P450s have evolved in a species-specific manner.

It is generally believed that the basis for the species-specific evolution of

mammalian P450s is the environment-more specifically, the consumption of

plants (Nebert and Gonzalez 1985; Nelson and Strobel 1987; Gonzalez and

Nebert 1990). As a defense against animal consumption, plants have

developed a large arsenal of toxins and stress metabolites or phytoalexins. As

each animal species developed among a population of plants producing toxins

and phytoalexins, specific P450s were required for detoxification and survival.

The appearance of a new species or subspecies of plant within an animal’s

environment would necessitate the involvement of P450-catalyzed

detoxification. Therefore, two species evolving in different environments of

vegetation would have their own group of P450 enzymes. The role of plants in

P450 evolution also is supported by the fact that a large explosion and

diversification of P450 genes occurred about 400 million years ago, soon after

animals began to emerge from an aquatic environment to a terrestrial habitat

that had been populated for several million years by plants having unique toxins

previously not seen by animals (Nelson and Strobel 1987). In any case, it is

likely that P450s evolved to metabolize plant chemicals, many of which are

similar to, and were used as the basis for, drugs. Illicit drugs, such as opiates,

cannabidiol, and cocaine, are probably plant toxins developed as a defense

against animals.

Another important feature of the P450s is the high degree of polymorphisms

found in rodents and humans. In the context of drug metabolism, a

205
polymorphism can be defined as a common genetic defect in the ability to carry

out a certain drug oxidation reaction, By definition, the deficiency giving rise to

a polymorphic trait Is so common (generally >1 percent) that it is not maintained

by recurrent mutation. Among the most well-studied polymorphisms in rodents

are the mouse “Ah receptor” polymorphism in which certain strains of mice have

a receptor with weak affinity for the inducing ligand (Nebert 1986). Therefore,

the P450 responsible for oxidizing a compound such as benzo(a)pyrene is

never induced because benzo(a)pyrene is incapable of interacting with the

receptor that causes an increase in transcription of the P450 gene. Another

well-studied polymorphism is the Dark Agouti rat deficiency in debrisoquine

metabolism (Al-Dabbagh et al. 1980). This deficiency is due to a defective

P450 gene (Matsunaga et al. 1989).

Polymorphisms in drug metabolism also have been described in humans. Two

polymorphisms involving P450s were found and were termed the ‘mephenytoin

polymorphism” and ‘debrisoquine polymorphism.” These were uncovered by

screening groups of people for their abilities to metabolize drugs. From 1 to 5

percent of all Caucasians are unable to carry out S-mephenytoin 4’-

hydroxylation (Kupfer and Preisig 1984; lnaba et al. 1986). The frequencies of

this polymorphism in Japanese and Chinese subjects reaches 23 percent and

17 percent, respectively (Horai et al. 1989), indicating a large degree of ethnic

differences in polymorphic drug metabolism. The P450 gene responsible for

the mephenytoin polymorphism has not been clearly identified as yet, although

it is believed to reside within the 2C subfamily (Gonzalez 1988).

The most well-characterized P450 polymorphism is that associated with

debrisoquine metabolism. The earliest clue to the existence of this deficiency

came from clinical studies in which the antihypertensive agent debrisoquine

(Declinax) was found to produce exaggerated effects in a large number of

patients. This toxicity was determined to be due to a genetic deficiency that is

inherited as an autosomal recessive trait (Idle and Smith 1979). The P450

involved in debrisoquine oxidation, designated 2D6, also is capable of

metabolizing numerous other drugs (figure 1). This defect was found to be the

result of mutant alleles for a single gene, designated CYP2D6 (encoding

P450llD6), that is located on chromosome 22 (Gonzalez et al. 1988a, 1988b).

In some cases, defective CYP2D6 alleles can be detected in lymphocyte DNA

by Southern blotting analysis (Skoda et al. 1988). However, a noninvasive test

in which all mutant P450 gene alleles can be diagnosed has not been

developed.

It should be emphasized that individuals who lack a particular P450, such as

the debrisoquine 4-hydroxylase and Smephenytoin hydroxylase, are otherwise

normal. Based on this fact, it appears that some P450s are not required for

206
FIGURE 1. Substrates for CYP2D6. The arrows indicate the position of

hydroxyation of each substrate.

SOURCE: Meyer et al. 1990, copyright, 1990, Academic Press

normal physiological function. Indeed, this is compatible with an exclusive role

of P450s in metabolism and elimination of foreign compounds. It is likely,

therefore, that humans may not need particular P450s for survival and the P450

no longer renders a selective advantage. This is probably because humans are

now capable of freely choosing what they eat, and a poisonous substance

simply can be avoided and a substitute found-a luxury that was not afforded

our ancestors. This might be the reason for P450 loss and for the high

frequency of drug metabolism polymorphisms in humans. Rodents, on the

other hand, are more generally victims of their environment; in fact, there does

seem to be a trend that mice, rats, and rabbits have more active P450s than do

humans. Finally, it should be noted that polymorphisms in other drugmetabolizing enzymes exist in humans. Among the most well-studied are the

N-acetyltransferase polymorphism (Weber 1987) and a glutathione transferase

polymorphism (Seidegard et al. 1988).

207
CONSEQUENCE OF P450 POLYMORPHISMS

The most serious consequence of P450 polymorphism is in drug therapy. As

indicated above, the ß-blocking agent debrisoquine was found to be toxic in

about 10 percent of all patients and, hence, is no longer prescribed. The

debrisoquine 4-hydroxylase P450 is also capable of metabolizing numerous

drugs (figure 1). In this connection, other drugs, such as perhexiline, are

capable of causing toxicities and even death due to this polymorphism (Shah et

al. 1982). Recently, certain cardiac antiarythmic drugs have caused problems

during clinical trials because of a P450 deficiency (Ruskin 1989). The ability to

diagnose mutant P450s prior to treatment with drugs should greatly aid in

avoiding toxic consequences.

The unknown consequence of P450 polymorphism is the association of the

presence or absence of a P450 with cancer or chronic toxicity. For example,

smokers who develop lung cancer are much more likely to actively metabolize

debrisoquine, indicating that individuals who have a normal P450 gene are at

higher risk than those who do not (Ayesh et al. 1984; Caporaso et al. 1989).

Many P450s are capable of activating carcinogens to DNA-binding and

mutagenic metabolites; however, convenient assays for expression of these

forms in humans have not been developed. When assays are available, it may

be possible to conduct molecular epidemiology studies, both retrospective and

prospective, to determine if P450 phenotypes are risk factors in human cancer.

ROLE OF P450 POLYMORPHISMS IN ILLICIT DRUG USE

Rodent P450s are capable of metabolizing cannabidiol and -tetrahydrocannabinol (Watanabe et al. 1987); these compounds have dramatic effects on

total hepatic P450 content, which decreases following acute administration

(Bornheim and Correia 1989a). However, when cannabidiol is administered

chronically, a specific P450 is highly induced in mouse that is capable of

metabolizing -tetrahydrocannabinol (Bornheim and Correia 1989b). The

effects of metabolites generated by P450s in humans is unknown, although it

has been established that they have pharmacologic activity (Christensen et al.

1971). P450s are also able to metabolize and inactivate cocaine, and this

metabolism can produce hepatotoxic metabolites in rodents (Shuster et al.

1988).

Recently, it was discovered that the debrisoquine 4-hydroxylase CYP2D6

enzyme can convert codeine to morphine by a demethylation reaction (figure 2)

(Dayer et al. 1988). This has been confirmed by studying the metabolism of

debrisoquine and codeine in a population of subjects in which lack of codeine

O-demethylation was found to cosegregate with the debrisoquine hydroxylation

208
deficiency (Yue et al. 1989). In this case, codeine is converted by a P450 to its

analgesically active form, morphine. Individuals who do not have this P450 are

unable to respond to codeine treatment.

It is tempting to speculate that oxidation polymorphisms might play a role in

abuse of certain illicit drugs. For example, certain individuals having or lacking

a P450 may not respond favorably to drug ingestion in a way similar to that of

early patients who received debrisoquine. Either an individual cannot

metabolize a drug to its active component or, conversely, the drug is

metabolized too rapidly and, hence, is ineffective; or a drug may be toxic. Any

of these possibilities might play roles in who does or does not become addicted.

CODEINE MORPHINE

FIGURE 2. Conversion of codeine to morphine by human hepatic P450

FUTURE EXPERIMENTATION

Future areas of drug abuse research could focus on the metabolism of illicit drugs

by human P450s and other drug-metabolizing enzymes. These studies can be

performed on liver tissue samples in vitro. Human liver can be obtained from

organ donor transplant units and various agencies, such as the National Disease

Research Interchange. Although it is difficult to purify P450s from limited sources

of human tissues, it is possible to isolate cDNAs and genes encoding a large

number of these enzymes using rodent antibodies and probes. These cDNAs

can be expressed into active enzymes using various cDNA expression systems,

such as vaccinia virus (Aoyama et al. 1989); the expressed enzymes can be

used to screen for metabolism of illicit drugs. A list of known human P450s in

which cDNAs are available and some of their substrates is shown in table 2.

Knowledge of which of these P450 forms inactivate or activate specific drugs,

and whether these forms are polymorphic, might aid in prediction of individuals

209
who are susceptible to drug abuse. In addition, these data might also prove

useful in strategies for effective therapies for addicts.

TABLE 2. Human P450s and some of their substratesa

1A1

1A2

2A3

2B7

2C8

2C9

2D6

2E1

2F1

3A4

4B1

Benzo(a)pyrene, ethoxyresorufin, ethoxycoumarin

Acetaminophen, acetylaminofluorine, aflatoxin, ethoxyresorufin,

heterocyclic arylamines, phenacetin, theophyline

Coumarin, nitrosamines

Etoxycoumarin

R-Mephenytoin, tolbutamide

R-Mephenytoin, tienilic acid, tolbutamide, warfarin

Bufuralol, debrisoquine, dextrometorphan, metoprolol, nortryptiline,

propranolol, sparteine

Acetoacetate, acetol, acetaminophen, acetone, benzene, ethanol,

halothane, nitrosamines, pyridine

7-Ethoxycoumarin, testosterone

Cortisol, cyclosporine, erythromycin, lidocaine mephenytoin, midazolam,

nifedipine, progesterone, testosterone, quinidine, warfarin

Testosterone

a

These lists do not represent every known substrate and every known human

P450; for example, at least four P450s are related to 3A4.

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Dayer, P.; Desmeules, J.; Leeman, T.; and Stibernl, R. Bioactivation of the

narcotic drug codeine in human liver is mediated by the polymorphic

monooxygenase catalyzing debrisoquine 4-hydroxylation (cytochrome P450db1/buf1). Biochem Biophys Res Commun 152:411-416, 1988.

Gonzalez, F.J. The molecular biology of cytochrome P450s. Pharmacol Rev

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D.W.; Gelboin, H.V.; Hardwick, J.P.; and Meyer, U.A. Characterization of the

common genetic defect in humans deficient in debrisoquine metabolism.

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Gonzalez, F.J.; Vilbois, F.; Hardwick, J.P.; McBride, O.W.; Nebert, D.W.;

Gelboin, H.V.; and Meyer, U.A. Human debrisoquine 4-hydroxylase

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AUTHOR

Frank J. Gonzalez, Ph.D.

Chief

Nucleic Acid Section

Laboratory of Molecular Carcinogenesis

National Cancer Institute

National Institutes of Health

Building 37, Room 3E24

Bethesda, MD 20892

213
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74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

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75 PROGRESS IN OPIOID RESEARCH. PROCEEDINGS OF THE 1986

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80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

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RELATED DESIGNER DRUGS, Khursheed Asghar, Ph.D., and Errol De

Souza, Ph.D., eds.

GPO Stock #017-024-01386-2 $11 NTIS PB #90-148958/AS $39

95 PROBLEMS OF DRUG DEPENDENCE, 1989: PROCEEDINGS OF THE

51ST ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS

OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01399-4 $21 NTIS PB #90-237660/AS $67

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY,

AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D., eds.

GPO Stock #017-024-01403-8 $8 NTIS PB #90-237678/AS $31

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY. Lynda

Erinoff, Ph.D., ed.

GPO Stock #017-024-01404-4 $8 NTIS PB #90-237686/AS $31

98 THE COLLECTION AND INTERPRETATION OF DATA FROM HIDDEN

POPULATIONS. Elizabeth Y. Lambert, M.S., ed.

GPO Stock #017-024-01407-9 $4.75 NTIS PB #90-237694/AS $23

99 RESEARCH FINDINGS ON SMOKING OF ABUSED SUBSTANCES,

C. Nora Chiang, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-01412-5 $5 NTIS PB #91-141119 $23

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100 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION DATA.

VOL. II. Steven W. Gust, Ph.D., and J. Michael Walsh, Ph.D., eds.

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR. John W.

Spencer, Ph.D., and John J. Boren, Ph.D., eds.

GPO Stock #017-024-01426-7 $6 NTIS PB #91-172858/AS $31

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and Lynda Erinoff,

Ph.D., eds.

GPO Stock #017-024-01425-7 $8 NTIS PB #91-172866/AS $31

103 DRUGS AND VIOLENCE: CAUSES, CORRELATES, AND

CONSEQUENCES. Mario De La Rosa, Ph.D.; Elizabeth Y. Lambert, M.S.;

and Bernard Gropper, Ph.D., eds.

GPO Stock #017-024-01427-3 $9 NTIS PB #91-172841/AS $31

104 PSYCHOTHERAPY AND COUNSELING IN THE TREATMENT OF

DRUG ABUSE. Lisa Simon Onken, Ph.D., and Jack D. Blaine, M.D., eds.

GPO Stock #017-024-01429-0 $4 NTIS PB #91-172874/AS $23

105 PROBLEMS OF DRUG DEPENDENCE, 1990: PROCEEDINGS OF THE

52ND ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS

OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01435-4 $22

106 IMPROVING DRUG ABUSE TREATMENT. Roy W. Pickens, Ph.D.; Carl

G. Leukefeld, D.S.W.; and Charles R. Schuster, Ph.D., eds.

GPO Stock #017-024-01439-7 $12

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and William J.

Bukoski, Ph.D., eds.

GPO Stock #017-024-01441-9 $9

108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING

MECHANISMS. Pushpa V. Thadani, Ph.D., ed.

GPO Stock #017-024-01446-0

109 LONGITUDINAL STUDIES OF HIV INFECTION IN INTRAVENOUS

DRUG USERS: METHODOLOGICAL ISSUES IN NATURAL HISTORY

RESEARCH. Peter Hartsock, Dr.P.H., and Sander G. Genser, M.D., M.P.H.,

eds.

GPO Stock #017-024-01445-1

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110 THE EPIDEMIOLOGY OF COCAINE USE AND ABUSE. Susan Schober,

Ph.D., and Charles Schade, M.D., M.P.H., eds.

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH:

VOLUME I. Theresa N.H. Lee, Ph.D., ed.

112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG

ABUSE RESEARCH. Rao S. Rapaka, Ph.D.; Alexandros Makriyannis, Ph.D.;

and Michael J. Kuhar, Ph.D., eds.

113 ECONOMIC COSTS, COST-EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT. William S. Cartwright, Ph.D.,

and James M. Kaple, Ph.D., eds.

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DHHS Publication No. (ADM) 92-1798

Alcohol, Drug Abuse, end Mental Health Administration

Printed 1991