Dopamine Receptors 9780841207813, 9780841210523, 0-8412-0781-X

Table of contents :
Title Page ......Page 1
Half Title Page ......Page 3
Copyright ......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
EDITORS' PREFACE......Page 7
PREFACE......Page 9
Literature Cited......Page 12
PdftkEmptyString......Page 0
1 D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase, cAMP Accumulation, and PTH Release in Dispersed Bovine Parathyroid Cells......Page 13
Dopamine Enhances cAMP Accumulation in Dispersed Bovine Parathyroid Cells......Page 14
Dopamine Stimulates Adenylate Cyclase Activity of Dispersed Bovine Parathyroid Cells......Page 17
Does cAMP Trigger the Release of IR-PTH?......Page 22
Receptors Inhibiting Dopamine-stimulated cAMP Accumulation and PTH Secretion......Page 24
Possible Role of Dopamine in Regulating PTH Secretion In Vivo......Page 26
Relationship of Dopamine Receptors on Bovine Parathyroid Cells to Other Dopamine Receptors: Dopamine Receptors Stimulating Adenylate Cyclase Activity......Page 27
Dopamine Receptors Inhibiting Adenylate Cyclase Activity......Page 29
Literature Cited......Page 31
Commentary: Dopamine-Sensitive Adenylate Cyclase as a Receptor Site......Page 34
Is Dopamine-Sensitive Adenylate Cyclase a Dopamine Receptor ?......Page 35
Is Parathormone Secretion Mediated through a Dopamine D1 Site ?......Page 37
Literature Cited......Page 40
Dr. Brown's Replies to Dr. Laduron's Comments......Page 41
Literature Cited......Page 43
2 The D-2 Dopamine Receptor in the Intermediate Lobe of the Rat Pituitary Gland Physiology, Pharmacology, and Biochemistry......Page 44
Cyclic AMP and the Intermediate Lobe β-adrenoceptor......Page 46
Cyclic AMP and the Intermediate Lobe Dopamine Receptor......Page 52
Concluding Remarks......Page 59
Literature Cited......Page 61
Commentary: The DA_ Dopamine Receptor in the Anterior and Intermediate Lobes of the Pituitary Gland......Page 64
The dopamine receptor in the anterior pituitary gland is negatively coupled to adenylate cyclase......Page 65
Modulation of the anterior pituitary dopamine receptor by gonadal steroids......Page 67
The dopamine receptor in the intermediate lobe of the pituitary gland is negatively coupled to adenylate cyclase......Page 68
Coupling of dopamine receptors with adenylate cyclase: DA+, DA_ and DA0 receptors......Page 71
Corticotropin-releasing factor stimulates adenylate cyclase in the intermediate lobe of the pituitary gland......Page 74
Literature Cited......Page 80
3 The Dopamine Receptor of the Anterior Pituitary Gland Ligand Binding and Solubilization Studies......Page 84
Quantitative Relationship of Agonist and Antagonist Ligand Binding to Porcine Anterior Pituitary Gland Membranes......Page 86
Guanine Nucleotides Modulation of Receptor Affinity for Agonists......Page 87
N-ethylmaleimide and Heat Treatments of Membranes Mimic the Effect of Guanine Nucleotides on Ligand Binding......Page 91
Solubilization of the Dopamine Receptor......Page 95
Conclusions......Page 97
Literature Cited......Page 101
Commentary: The Dopamine Receptor of the Anterior Pituitary Gland......Page 104
Literature Cited......Page 109
4 Differentiation of Dopamine Receptors in the Periphery......Page 111
DA1 receptors......Page 112
DA2 agonists......Page 115
DA2 antagonists......Page 117
Comparison of data obtained in vivo and in vitro......Page 119
Determinations of the DA receptor subtypes responsible for physiological and pharmacological actions......Page 121
Literature Cited......Page 122
Commentary: Utility and Problems in the Classification of Dopamine Receptors......Page 124
Four Major Reasons for Problems in the Classification and Identification of Dopamine Receptors in the Periphery......Page 125
Conclusion......Page 126
5 Dopamine Receptors in the Neostriatum: Biochemical and Physiological Studies......Page 127
A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of Acetylcholine......Page 128
A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of GABA?......Page 135
A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of Glutamate?......Page 136
A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of Peptide Neurotransmitters?......Page 137
A Postsynaptic Dopamine Receptor Regulating Cyclic AMP Formation......Page 138
Autoreceptors Regulating the Turnover of Dopamine......Page 142
Presynaptic Autoreceptor Regulating the Release of Dopamine......Page 144
A Presynaptic Autoreceptor Regulating cyclic AMP Formation?......Page 145
Concluding Remarks......Page 148
Literature Cited......Page 150
6 Potential Therapeutic Uses of Dopamine Receptor Agonists and Antagonists ......Page 156
Dopamine Agonists......Page 157
Dopamine Antagonists......Page 159
Literature Cited......Page 161
Commentary: Potential Therapeutic Uses of Dopamine Receptor Agonists and Antagonists......Page 163
Literature Cited......Page 164
7 Dopaminergic Benzazepines with Divergent Cardiovascular Profiles......Page 165
Benzazepine Chemistry......Page 166
Benzazepine Pharmacology......Page 168
Discussion......Page 174
Literature Cited......Page 175
Commentary: Dilemmas in the Synthesis of Clinically Useful Dopamine Agonists......Page 178
Literature Cited......Page 181
8 Dopamine Agonists and Antagonists in Duodenal Ulcer Disease......Page 182
Structure-activity Relationship of Duodenal Ulcerogens......Page 183
Pharmacologic Modulation with Dopamine-related Drugs......Page 188
Biochemical Studies with Catecholamines......Page 194
Correlations and Implications......Page 195
Literature Cited......Page 200
Commentary: Dopamine Agonists and Antagonists in Duodenal Ulcer Disease......Page 204
9 The Development of Novel Dopamine Agonists......Page 207
Structural Dissection......Page 208
Rigidification......Page 212
Stereochemistry and Absolute Configuration......Page 215
Hydrophobic features of the receptor......Page 216
Importance of nitrogen electron pair orientation......Page 219
Literature Cited......Page 223
Commentary: The Development of Novel Dopamine Agonists......Page 225
Literature Cited......Page 227
10 Stereoisomeric Probes of the Dopamine Receptor......Page 228
Pharmacology of (R) and (S)-I-III......Page 230
Structure-Activity Relationship Considerations......Page 233
Footnotes to Table I.......Page 235
Discussion......Page 238
Literature Cited......Page 246
Commentary: Stereoisomeric Probes of the Dopamine Receptor......Page 252
Literature Cited......Page 255
Structural Requirements......Page 256
A Hypothetical Receptor Model......Page 257
Development of a Pyrroloisoquinoline Antipsychotic (Ro 22-1319)......Page 262
Pharmacology and Biochemistry of Ro 22-1319......Page 265
The Auxiliary Binding Site and Selectivity for D-2 and D-1 Dopamine Receptor's......Page 270
Literature Cited......Page 278
12 Renal Vascular Dopamine Receptor Topography Structure-Activity Relationships That Suggest the Presence of a Ceiling......Page 280
Literature Cited......Page 285
A......Page 286
B ......Page 287
C ......Page 288
D ......Page 289
H ......Page 290
N ......Page 291
P ......Page 292
S ......Page 293
Z ......Page 294

Citation preview

Dopamine Receptors

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Dopamine Receptors Carl Kaiser,

EDITOR

Smith Kline & French Laboratories

John W Kebabian

EDITOR

National

Based on a symposium sponsored by the ACS Division of Medicinal Chemistry at the 184th Meeting of the American Chemical Society Kansas City, Missouri, September 12-17, 1982

ACS

SYMPOSIUM

AMERICAN

CHEMICAL

WASHINGTON,

D.C.

SERIES

SOCIETY 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

224

Library of Congress Cataloging in Publication Data Dopamine receptors. (ACS Symposium series, ISSN 0097-6156; 224) "Based on a symposium sponsored by the ACS Division of Medicinal Chemistry at the 184th meeting of the American Chemical Society, Kansas City, Missouri, September 12-17, 1982." Bibliography: p. Includes index. 1. Dopamine—Receptors—Congresses mine—Agonists—Congresses. I. Kaiser, Carl, 1929. II. Kebabian, J. W . (John W . ) III. American Chemical Society. National Meeting (184th: 1982: Kansas City, Mo.) IV. Series. [ D N L M : 1. Receptors, Dopamine—Congresses. 2. Receptors, Dopamine—Drug effects—Congresses. W L 102.8 D692 1982] QP563.D66D66 1983 612'.015 83-6433 ISBN 0-8412-0781-X

Copyright © 1983 American Chemical Society A l l Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

ACS Symposium Series M . Joa

Advisory Board David L. Allara

Robert Ory

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Theodore Provder

Brian M . Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

Herbert D. Kaesz

Davis L. Temple, Jr.

Marvin Margoshes

Charles S. Tuesday

Donald E. Moreland

C. Grant Willson

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

FOREWORD The A C S

SYMPOSIUM

SERIES

was f o u n d e d i n 1 9 7 4 to p r o v i d e

a m e d i u m for p u b l i s h i n g s y m p o s i a q u i c k l y i n b o o k f o r m . T h e format of the Series parallels that of the c o n t i n u i n g IN

CHEMISTRY

SERIES

ADVANCES

except that i n order to save t i m e

papers are not typeset b u t are r e p r o d u c e d as t h e y are m i t t e d b y the authors i n camera-ready

the sub-

f o r m . Papers are re-

v i e w e d u n d e r the s u p e r v i s i o n of the E d i t o r s w i t h the assistance of the Series A d v i s o r y B o a r d a n d are selected to m a i n t a i n the i n t e g r i t y of the s y m p o s i a ; h o w e v e r , v e r b a t i m r e p r o d u c t i o n s of p r e v i o u s l y p u b l i s h e d papers are not a c c e p t e d . B o t h r e v i e w s a n d reports of research are acceptable since s y m p o s i a m a y e m b r a c e b o t h types of presentation.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

EDITORS' PREFACE

Τ JL H E I N V I T A T I O N B Y J . L . N E U M E Y E R and the Division of Medicinal Chemistry of the American Chemical Society to organize the symposia "Multiple Categories of Dopamine Receptors" and "Modulation of Dopa­ mine Receptors" offered us the opportunity to highlight some of the recent advances in the understandin f dopamin d th drug inter acting with these receptors the "two dopamine receptor hypothesis." This hypothesis is the theme of the first symposium. Similarly, several years ago, Carl Kaiser participated in the discovery of S K & F 38393, a dopaminergic agonist relatively selective for the D - l receptor. The second symposium focuses attention on the development of novel agonists for dopamine receptors and their use as therapeutic agents. Because some of these differentiate between different dopamine receptors, the concept of multiple categories of dopamine receptors is an integral part of the second symposium. The requirement of the American Chemical Society that all material published by the Society be subjected to outside review offered an oppor­ tunity for us to solicit a second (and sometimes conflicting) opinion about the material presented in these symposia. It cannot be denied that the topic of dopamine receptor(s) remains an area of ongoing investigation, controversy, and disagreement. F o r many questions about dopamine re­ ceptors, the "final verdict" is not yet in. Indeed, if these symposia have delineated the areas of disagreement, the readers of this volume form the jury. F o r each disagreement or misunderstanding highlighted in this volume, the reader can review the ideas of the different authors and then decide which observations and interpretations are most helpful to their individual endeavors. The editors are grateful to each of the authors of chapters and to those who contributed commentaries on these chapters. We also acknowl­ edge with gratitude the financial support of Smith Kline & French Laboratories. CARL KAISER

JOHN W. KEBABIAN

Smith Kline & French Laboratories Philadelphia, PA 19101

National Institutes of Health Bethesda, MD 20205

February 1983 ix In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

PREFACE BY LESLIE L. IVERSEN

THE

CHAPTERS

PRESENTED

HERE

REPRESENT

an interesting state-of-the-

art reflection of current trends i n research o n d o p a m i n e receptors. D . C a l n e a n d T . A . L a r s e n are p r o b a b l y accurate i n suggesting that " d o p a m i n e has overtaken acetylcholine a n d n o r e p i n e p h r i n

th

extensivel

investi

gated neurotransmitter i n present a n d potential c l i n i c a l uses of d o p a m i n e agonists a n d

antagonists

indicates that this research effort has indeed l e d to useful new therapeutic agents, i n c l u d i n g those acting o n p e r i p h e r a l o r endocrine targets as w e l l as centrally acting drugs. I n p a r t i c u l a r , the c a r d i o v a s c u l a r a n d endocrine effects of d o p a m i n e have attracted considerable interest, b o t h f r o m

the

basic a n d a p p l i e d research v i e w p o i n t s . F u r t h e r m o r e , S. S z a b o a n d J . L . N e u m e y e r suggest that the actions of d o p a m i n e i n the

gastrointestinal

tract, a n d its possible etiological role i n peptic ulcers, w i l l represent

an

i m p o r t a n t new focus for future w o r k o n the p e r i p h e r a l actions of d o p a m i n e . T h e availability of p e r i p h e r a l m o d e l s offers an i m p o r t a n t means of characterizing the p h a r m a c o l o g i c a l properties of d o p a m i n e receptors. I n m a n y cases it is possible to measure a clear-cut tissue response a n d , thus, to establish the agonist, p a r t i a l agonist, a n d antagonist properties of test c o m p o u n d s . It is perhaps o n l y n o w b e i n g recognized b y

neurochemists

that receptors cannot be characterized fully i n any other w a y . T e n years ago, w i t h the discovery of n e w b i o c h e m i c a l approaches

to the study of

d o p a m i n e receptors i n C N S m a n y of us were doubtless too o p t i m i s t i c i n t h i n k i n g that s u c h approaches w o u l d l e a d to r a p i d progress i n defining the characteristics of d o p a m i n e receptors. T h e fundamental p r o b l e m i n achieving s u c h understanding, however, has been that we d o not k n o w what d o p a m i n e does as neurotransmitter i n the various C N S pathways that c o n t a i n it. F u r t h e r m o r e , there are n o s i m p l e m o d e l systems

that

a l l o w one to measure agonist a n d antagonist effects o n C N S targets. W e cannot k n o w h o w reliable the results of n e u r o c h e m i c a l studies of d o p a m i n e receptors are i f we have n o b i o l o g i c a l response against w h i c h to assess the n e u r o c h e m i c a l data. A recent r e v i e w ( 7 ) listed some 28 different a p p l i c a -

xi In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

tions of r a d i o l i g a n d b i n d i n g methods to the study of d o p a m i n e receptors i n b r a i n , that use m o r e t h a n twenty different agonist o r antagonist r a d i o ­ ligands. R e m a r k a b l y little is said i n the present v o l u m e about r a d i o l i g a n d b i n d i n g assays, a n d I suspect J . C . Stoof caught the m o o d of the meeting i n stating: " B i n d i n g studies, a l t h o u g h easy to p e r f o r m , have y i e l d e d too m a n y data, too m a n y categories o f d o p a m i n e receptors a n d too m a n y controversies." T h e n e u r o c h e m i c a l emphasis has c l e a r l y shifted to " f u n c ­ t i o n a l " assays, i n w h i c h some b i o c h e m i c a l response is measured,

rather

t h a n s i m p l y o c c u p a t i o n of receptor b i n d i n g sites b y ligands. T h e activation o r i n h i b i t i o n of adenylate cyclase has p r o v e d a v a l u a b l e m o d e l i n this sense, a n d other b i o c h e m i c a l responses m a y p r o v e s i m i l a r l y useful (e.g., i n h i b i t i o n of peptide h o r m o n e o r neurotransmitter release i n response to dopamine agonists). I n this v o l u m e a g o o d o p a m i n e receptors i n pituitary. T h i s emphasis seems w e l l justified. T h e clear-cut effects

o f d o p a m i n e i n suppressing p r o l a c t i n secretion

from

anterior l o b e m a m m o t r o p h s , a n d the i n h i b i t o r y effects o n secretion of α - M S H and related secretory products f r o m intermediate l o b e are i m p o r t a n t models for d o p a m i n e receptor studies. I n b o t h cases n e w evidence was put f o r w a r d to support the hypothesis that the actions o f d o p a m i n e o n the secretory cells are mediated b y i n h i b i t i o n of adenylate cyclase. T h i s is far easier to demonstrate i n the intermediate l o b e , w h e r e a l l cells appear to respond to d o p a m i n e , t h a n i n the

anterior lobe, where the

dopa-

mine-sensitive cells p r o b a b l y represent o n l y a s m a l l m i n o r i t y . I n terms of m u l t i p l e receptor categories, the suggestion m a d e b y K e b a b i a n a n d C a l n e ( 2 ) of a d i s t i n c t i o n between D - l a n d D - 2 subtypes, based o n whether the receptors l e a d to s t i m u l a t i o n of adenylate cyclase o r not, has been w i d e l y accepted. It n o w seems that i n m a n y cases (perhaps a l l ) the D - 2 sites are also c o u p l e d to adenylate cyclase, a l t h o u g h i n an i n h i b i t o r y rather t h a n s t i m u l a t o r y manner. A l l of the studies o n pituitary place the d o p a m i n e receptors there clearly i n the D - 2 category. M . C a r o n et a l . , however, report interesting n e w results that indicate that these sites c a n exist i n m o r e than one f o r m — w i t h about h a l f o f the sites i n the resting state i n a f o r m w i t h h i g h affinity for agonists a n d half i n a l o w agonist affinity state. These forms c a n be interconverted, and g u a n y l nucleotide o r N E M treatment shifts the p o p u l a t i o n m a i n l y to the agonist h i g h affinity f o r m . T h i s m a y also help to e x p l a i n the observations of K e b a b i a n et a l . that agonists were far m o r e potent i n intact pituitary c e l l preparations t h a n i n b r o k e n c e l l preparations.

xii In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

T h e r e r e m a i n m a n y difficulties i n further understanding the nature of the different d o p a m i n e receptor categories. W e continue to l a c k suitably selective agonists o r antagonists f o r the D - l a n d D - 2 sites. I n terms of agonists, b r o m o c r i p t i n e a n d related ergoline derivatives are still the most selective D - 2 - s t i m u l a n t s , a n d the series of benzazepines related to S K & F 3 8 3 9 3 are the most p r o m i s i n g D - l - s e l e c t i v e agents ( J . W e i n s t o c k et a l . ) . T h e d i s c o v e r y that benzazepines act i n a stereochemically specific manner, a n d the resolution o f the active and inactive stereoisomeric forms, offers further hope for m o r e selective agonists i n future. D . E . N i c h o l s p r o v i d e s a detailed a n d thoughtful r e v i e w of the m e d i c i n a l chemistry aspects of d o p a m i n e agonist design. T h e r e is still n o D - l - s e l e c t i v e antagonist, a l t h o u g h s u l p i r i d e a n d related benzamides D-2-antagonists. T h e a v a i l a b i l i t

f

are w i d e l y u s e d as selective

leas

tissu

m o d e l fo

D-l

receptors, the stimulator secretion f r o m b o v i n e p a r a t h y r o i d cells, is of considerable i m p o r t a n c e , but we still have n o c o r r e s p o n d i n g m o d e l to elucidate the possible f u n c t i o n of D - l sites i n the C N S . T h e r e is also considerable difficulty i n relating the b i o c h e m i c a l classification

of D - l a n d D - 2 sites to the p h a r m a c o l o g i c a l l y defined d o p a m i n e

receptor subtypes, described b y L . G o l d b e r g a n d J D . K o h l i , o n the basis of their p a i n s t a k i n g analysis o f agonist/antagonist actions o n c a r d i o v a s c u l a r responses. T h e y describe t w o subcategories of d o p a m i n e receptors,

but

these do not c o r r e s p o n d r e a d i l y to D - l a n d D - 2 sites. T h u s , their " D A i " receptors that cause r e l a x a t i o n of vascular s m o o t h m u s c l e have an agonist specificity s i m i l a r to the D - l sites: w i t h a n absolute requirement f o r a catechol g r o u p i n g ; r i g i d catechol analogues

s u c h as A D T N

are

fully

active; ergolines are inactive. T h e responses are b l o c k e d b y neuroleptics, b u t u n l i k e the D - l site, w h i c h is quite unresponsive to s u l p i r i d e , the D A i receptors are potently b l o c k e d b y sulpiride. T h e " D A " receptors, mediat2

i n g presynaptic c o n t r o l of n o r e p i n e p h r i n e release f r o m sympathetic nerve terminals, resemble D - 2 sites i n their specificity, but again the c o m p a r i s o n is not precise. Interestingly, the D A sites c a n be stimulated even b y some 2

m o n o p h e n o l i c agonists. T h i s is i n t r i g u i n g because some of the n e w l y described "autoreceptor agonists" i n C N S , s u c h as 3 - P P P (3)

are m o n o -

p h e n o l i c structures. T h e t o p i c a l question of whether the receptors l o c a t e d o n the surface of d o p a m i n e neurons i n C N S (autoreceptors)

represent a

u n i q u e p h a r m a c o l o g i c a l class does not perhaps receive as m u c h attention i n this v o l u m e as it s h o u l d have, a l t h o u g h the issue is discussed i n some detail b y J . C . Stoof. A l t h o u g h s u c h receptors c l e a r l y resemble the D - 2 class i n m a n y respects, there remains the s u s p i c i o n that there m a y be some subtle differences.

xiii In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

D e s p i t e the large effort already directed to studies o f d o p a m i n e a n d d o p a m i n e receptors it is clear that m a n y questions r e m a i n unanswered. The

area continues to be one of considerable p r o m i s e a n d i n t e l l e c t u a l

v i g o r a n d f r o m this ferment useful new p h a r m a c o l o g i c a l and, possibly, n e w therapeutic tools m a y eventually emerge. Literature

Cited

1. Seeman, P. Pharmac. Rev. 1980, 32, 229-313. 2. Kebabian, J. W.; Calne, D. B. Nature (London) 1979, 277, 93-96. 3. Hjorth, S.; Carlsson, Α.; Wikström, H.; Lindberg, P.; Sanchez, D.; Hacksell, U.; Arvidsson, L. E.; Svensson, U.; Nilsson, J. L. G. Life Sci. 1981, 28, 1225-1238. LESLIE L. IVERSEN

MRC N e u r o c h e m i c a l P h a r m a c o l o g M e d i c a l Research C o u n c i l Centre Hills R o a d C a m b r i d g e CB2 2QH, United K i n g d o m

xiv In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1 D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase, cAMP Accumulation, and PTH Release in Dispersed Bovine Parathyroid Cells EDWARD M. BROWN and BESS DAWSON-HUGHES Brigham and Women's Hospital, Endocrine-Hypertension Unit, Boston, MA 02115 The evidence supportin category o f dopamine receptor on the parenchymal c e l l s of the bovine parathyroid gland and the p o s s i b l e biochemical mechanisms by which dopamine stimulates the release of parathyroid hormone are reviewed. The dopamine receptor on the bovine parathyroid cell i s compared to other dopamine receptors. The parathyroid glands play a major r o l e i n normal calcium homeostasis (±). Calcium i s g e n e r a l l y recognized as the p r i n c i p a l p h y s i o l o g i c a l r e g u l a t o r of the r e l e a s e of parathyroid hormone (PTH) (2.). When the plasma concentration o f i o n i z e d calcium decreases, PTH s e c r e t i o n increases. In turn, t h i s PTH acts to r a i s e plasma calcium by three mechanism: f i r s t , PTH increases r e n a l tubular reabsorption of calcium; second, PTH enhances the release of s k e l e t a l calcium; and t h i r d , PTH increases g a s t r o i n t e s t i n a l absorption o f calcium by s t i m u l a t i n g r e n a l formation of 1,25 dihydroxyvitamin D. These three mechanisms elevate the plasma concentration of ionized calcium and consequently reduce the augmented s e c r e t i o n of PTH, thereby c l o s i n g a negative feedback loop. The i n h i b i t o r y e f f e c t of calcium upon PTH s e c r e t i o n contrasts with the stimulatory e f f e c t of calcium upon most other secretory systems (3.)· In a d d i t i o n to calcium, other f a c t o r s a l s o modify PTH s e c r e t i o n . Many o f these f a c t o r s change c e l l u l a r c y c l i c adenosine 3 5 , monophosphate (cAKP) l e v e l s at the same time that they modify PTH s e c r e t i o n This volume provides a forum i n which i t i s appropriate to d i s c u s s the bovine parathyroid gland and the e f f e c t s of dopamine upon t h i s t i s s u e . When administered intravenously to cows, dopamine r a i s e s the plasma content of immunoreactive PTH (X). This stimulatory e f f e c t of dopamine i s p a r t i a l l y blocked by pimozide, a dopamine antagonist, but i s unaffected by p r o p r a n o l o l , a beta-adrenergic antagonist. An understanding of !

0097-6156/83/0224-0001$06.25/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

!

2

DOPAMINE

RECEPTORS

the c e l l u l a r mechanisms involved i n mediating t h i s dopamine-induced s t i m u l a t i o n of PTH s e c r e t i o n i s l i m i t e d by the c e l l u l a r heterogeneity of the bovine parathyroid gland (2.) as w e l l as by the temporal and s p a t i a l imprecision of intravenous infusions. Nevertheless, the I n v i v o r e s u l t s provide a standard against which i n v i t r o r e s u l t s can be compared. The c e l l u l a r and molecular events involved i n the dopamine-stimulated release of PTH can be c l a r i f i e d i n experiments u t i l i z i n g bovine parathyroid c e l l s dispersed with collagenase and DNase (&). This d i s p e r s i o n procedure y i e l d s parenchymal c e l l s with only a s l i g h t contamination by red blood cells. The parenchymal c e l l s exclude trypan blue and appear normal by l i g h t and e l e c t r o n microscopy (&). These c e l l s r e l e a s e PTH i n a l i n e a r f a s h i o n f o r s e v e r a l hours; the release i s i n h i b i t e d by calcium and stimulated by dopamine and beta-adrenergic agonists at concentrations comparable to those used to e l i c i t p h y s i o l o g i c a Dopamine Enhances PTH Cells

S e c r e t i o n i n Dispersed

Bovine Parathyroid

Dopamine (1 μΜ) causes a t r a n s i e n t 2 to 4 - f o l d increase i n the rate of release of immunoreactive PTH (IR-PTH) from dispersed bovine parathyroid c e l l s (ϋ). The stimulatory e f f e c t of dopamine i s maximal a f t e r 5 minutes exposure and p e r s i s t s f o r approximately 30 minutes (Figure 1). Several compounds mimicking the e f f e c t s of dopamine i n other systems mimic the stimulatory e f f e c t of dopamine on IR-PTH r e l e a s e . Both 2-amino, 6,7-dihydroxy t e t r a l i n (6,7-ADTN) and SKF 38393 increase the r e l e a s e of IR-PTH to the same degree as does dopamine. The release of IR-PTH i s half-maximally stimulated by dopamine, 6,7-ADTN and SKF 38393 at 0.2 μΜ, 0.15 μΗ and 0.3 μΜ, r e s p e c t i v e l y (Figure 2) (1Q_). In c o n t r a s t , other dopaminergic agonists are s u b s t a n t i a l l y l e s s potent than dopamine; both apomorphine and l e r g o t r i l e e l i c i t no more than 25% of the maximal response to dopamine, and l i s u r i d e i s devoid of agonist a c t i v i t y . The dopamine-stimulated r e l e a s e of IR-PTH i s i n h i b i t e d i n a s t e r e o s p e c i f i c manner by the isomers of f l u p e n t h i x o l (£) ; c i s - f l u p e n t h i x o l i s approximately 100-fold more potent than i t s trans-isomer ( c a l c u l a t e d K s of 33 nM and 3,300 nM, r e s p e c t i v e l y ) (Figure 3)· l

i

Dopamine Enhances cAHP Accumulation i n Dispersed Parathyroid C e l l ?

Bovine

Dopamine causes a 20 to 30-fold increase i n the content of cAMP i n dispersed bovine parathyroid c e l l s (Figure 4) (ϋ.). Like the dopamine-stiraulated enhancement of PTH r e l e a s e , the dopamine-stimulated increase i n cAMP content i s maximal a f t e r 5 to 10 minutes of exposure to 10 μΜ dopamine (S.) and

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BROWN

A N D DAWSON-HUGHES

D-l

Mediated

Activation

TIME, MINUTES Figure 1. Stimulation of PTH release from dispersed bovine parathyroid cells by dopamine. Cells were incubated with (M) or without (%) 1 μΜ dopamine, and PTH release was determined by radioimmunoassay.

[AGONIST], M

Figure 2. Stimulation of PTH release from dispersed bovine parathyroid cells by varying concentrations of dopamine (O), 6,7-ADTN SKF 38393 (A), apomorphine or dihydroergocryptine (M)> (Reproduced with permission from Ref. 10. Copyright 1980, American Society for Pharmacology and Experimental Therapeutics.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

4

DOPAMINE

RECEPTORS

10-6 [ANTAGONIST], M

Figure 3.

Inhibition of PTH-release stimulated by 1 μΜ dopamine by a-flupenthixol (Φ), β-flupenthixol (0),or(—) propranolol ( Α λ

0) ο

i ο 5 α οΓ

< ο

ιο­ ί AgonistI, Μ

Figure 4. Stimulation of cAMP accumulation in dispersed bovine parathyroid cells by varying concentrations of dopamine (O), 6,7-ADTN ( Π λ SKF 38393 (A), or apomorphine (Reproduced with permission from Ref. 10. Copyright 1980, American Society for Pharmacology and Experimental Therapeutics.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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A N D DAWSON-HUGHES

D-l Mediated

Activation

subsequently decreases (Figure 5). S i g n i f i c a n t q u a n t i t i e s of cAMP are excreted from the dispersed c e l l s during the f i r s t 15 minutes of exposure to dopamine. As i s the case f o r the dopanine-stimulated enhancement of IR-PTH r e l e a s e , s e v e r a l dopaminergic agonists mimic the a b i l i t y of dopamine to increase cAMP accumulation i n the dispersed bovine parathyroid c e l l s (Table I ) . For each compound tested, the concentration of agonist half-maximally enhancing cAMP accumulation i s approximately the same as the concentration of agonist required to half-maximally stimulate the release of IR-PTH. Dopamine antagonists block the dopamine-stimulated increase i n cAMP (Figure 6) (Table I ) . I n t e r e s t i n g l y , apomorphine, l i s u r i d e , l e r g o t r i l e and bromocriptine each block the dopamine receptor i n t h i s system (Figure 6 ) . Dopamine Stimulates Adenylate Cyclase A c t i v i t y o f Dispersed gpvine Parathyroid C e l l When tested on o s m o t i c a l l y - l y s e d bovine parathyroid c e l l s , dopamine enhances the a c t i v i t y o f adenylate c y c l a s e , the enzyme converting ATP to cAKP (JUL). In comparison with i t s e f f e c t on cAMP accumulation, the e f f e c t of dopamine on adenylate cyclase a c t i v i t y i s r e l a t i v e l y modest, only a 2 - f o l d increase i n enzyme a c t i v i t y (Figure 7 ) . Guanosine 5'-triphosphate (GTP) increases the stimulatory e f f e c t of dopamine; i n the presence of GTP, there i s 3 to 4-fold s t i m u l a t i o n of enzyme a c t i v i t y (JL1). In other c e l l types, guanine nucleotides i n t e r a c t with a guanine nucleotide subunit (G- or N -subunit) to t r a n s l a t e receptor s t i m u l a t i o n i n t o increased adenylate c y c l a s e a c t i v i t y (12.). Cholera t o x i n i n h i b i t s a s p e c i f i c GTPase on t h i s guanine n u c l e o t i d e subunit and thereby increases adenylate cyclase a c t i v i t y (13.)· In dispersed c e l l s from the bovine parathyroid gland, c h o l e r a t o x i n markedly increases cAWP formation and causes a 3 to 10-fold increase i n the apparent a f f i n i t y c f dopamine f o r i t s receptor (as determined by cAMP accumulation or IR-PTH s e c r e t i o n (14). The e f f e c t s of guanine nucleotides and c h o l e r a t o x i n on cAMP accumulation i n parathyroid c e l l s r e s u l t from i n t e r a c t i o n s with the guanine nucleotide subunit i n this c e l l . In e i t h e r the presence or absence of GTP, half-maximal s t i m u l a t i o n of enzyme a c t i v i t y i s achieved with 3 yM dopamine. Both 6,7-ADTN and epinine (N-methyl dopamine) stimulate adenylate cyclase a c t i v i t y to the same degree as does dopamine (Figure 8 ) . In c o n t r a s t , apomorphine i s a p a r t i a l agonist e l i c i t i n g only 30? o f the maximal e f f e c t of dopamine. The dopamine-stimulated adenylate cyclase a c t i v i t y i s s e l e c t i v e l y blocked by c i s - f l u p e n t h i x o l rather than the trans-isomer of t h i s antagonist (11). Among the antagonists tested, the order of potency i s : c i s - f l u p e n t h i x o l = fluphenazine > chlorpromazine > h a l o p e r i d o l > t r a n s - f l u p e n t h i x o l (Table I ) . g

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

5

6

DOPAMINE

15

30

RECEPTORS

60 120

TIME, MINUTES Figure 5. Stimulation of intracellular (%) and extracellular (O) cAMP by 10 μΜ dopamine in dispersed bovine parathyroid cells. cAMP was determined by radio­ immunoassay.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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A N D DAWSON-HUGHES

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7

TABLE I A f f i n i t y of drugs f o r the dopamine receptor i n bovine parat h y r o i d c e l l s determined i n experiments measuring cAMP accumulation, adenylate c y c l a s e , or PTH r e l e a s e . K

a

or K i (yM) Cyclas

Agonists Dopamine ADTN Epinine

0.6 0.5 0.6

3 4 10

0.2 0.15

P a r t i a l Agonists SKF 38393 Apomorphine

1 1

3 3

0.3 1

Antagonists d-Butaclamol a-Flupenthixol Fluphenazine Lisuride Lergotrile Bromoergocryptine YM-09151-2 (+) S u l p i r i d e (-) S u l p i r i d e 20% i n h i b i t i o n at 3 χ 10"

.005 .03 .04 .015 .7 21 15 13 Ï 5

.011 .07 .16 .37 2.1

^

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

.033 .09 .9

DOPAMINE

8

^fo*

io^e

iô^T jô^ë

RECEPTORS

10-5 10-4

ANTAGONIST^ Figure 6. Inhibition of cAMP accumulation stimulated by 1 μΜ dopamine by lisuride (O), a-flupenthixol ([2), cryptine

1200 ι

1000 h c

ε ο 'à

ε < υ "δ

ε α

(Dopamine), Μ

Figure 7. Dopamine-stimulated adenylate cyclase activity in lysates of bovine parathyroid cells in the absence (O) or presence (O) of 100 μΜ guanosine triphos­ phate (GTP). (Reproduced with permission from Ref. 11. Copyright 1980, The Endocrine Society.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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D-l

Mediated

Activation

Figure 8. Stimulation of adenylate cyclase in lysates of bovine parathyroid cells by 6,7-ADTN (A), epinine (O), or apomorphine in the presence of 100 μΜ GTP. (Reproduced with permission from Ref. 11. Copyright 1980, The Endocrine Society.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

10

DOPAMINE

RECEPTORS

Does Dopamine I n t e r a c t with Beta- or Alpha-adrenergic receptors? The bovine parathyroid gland possesses a beta-adrenergic receptor. Like dopamine, beta-adrenergic agonists enhance adenylate cyclase a c t i v i t y , cAMP accumulation, and the release of IR-PTH ( 1 5 . ) . The beta-adrenergic receptor, however, can be d i f f e r e n t i a t e d from the receptor f o r dopamine with s e l e c t i v e antagonists (Figure 9 ) · For example, p r o p r a n o l o l , a potent antagonist of the beta-adrenergic receptor, causes a nearly complete i n h i b i t i o n of cAMP accumulation stimulated by i s o p r o t e r e n o l , epinephrine, or norepinephrine. Propranolol, on the other hand, does not diminish dopamine-stimulated cAMP accumulation. Conversely, c i s - f l u p e n t h i x o l blocks the dopamine- and epinine-stiraulated accumulation o f cAMP but does not reduce the i s o p r o t e r e n o l - s t i m u l a t e d accumulation o f cAMP. The bovine parathyroi alpha-adrenergic recepto that an i n t e r a c t i o n between dopamine and the alpha-adrenergic receptor accounts f o r the p h y s i o l o g i c a l and biochemical e f f e c t s of dopamine upon t h i s t i s s u e . Phentolamine, an alpha-adrenergic antagonist, has no e f f e c t on dopamine-stimulated cAMP accumulation at concentrations as high as 10 μΜ, a concentration t o t a l l y blocking alpha-adrenergic e f f e c t s i n t h i s system (JLQ.). Furthermore, u n l i k e the dopaminergic receptor, s t i m u l a t i o n o f the parathyroid alpha-adrenergic receptor i n h i b i t s the agonist-stimulated a c t i v a t i o n o f accumulation o f cAMP and r e l e a s e of IR-PTH (l£L). Several dopaminergic drugs i n t e r a c t not only with the dopamine receptor but a l s o with alpha- and beta-adrenergic receptors i n the bovine parathyroid gland. For example, l i s u r i d e , a potent dopamine agonist upon the a n t e r i o r p i t u i t a r y gland, blocks the alpha-adrenergic receptor, the beta-adrenergic receptor, and the receptor f o r dopamine i n parathyroid c e l l s ( H ) . Does cAM? T r i g g e r the Release of IR-PTH? The dopamine-stimulated formation o f cAMP may i n i t i a t e the dopamine-induced r e l e a s e of IR-PTH. A l i n e a r r e l a t i o n s h i p e x i s t s between the dopamine-induced release o f IR-PTH and the logarithm of the dopamine-induced accumulation of cAMP ( H ) . S i m i l a r l y , other agents i n c r e a s i n g cAMP accumulation and IR-PTH release (e.g. beta-adrenergic agonists, s e c r e t i n and phosphodiesterase i n h i b i t o r s , a l s o d i s p l a y such a l o g - l i n e a r r e l a t i o n s h i p . A d d i t i o n a l support f o r the p o s s i b i l i t y that i n t r a c e l l u l a r cAMP might i n i t i a t e PTH s e c r e t i o n comes from the observations that c h o l e r a t o x i n (14), phosphodiesterase i n h i b i t o r s ( H ) and d i b u t y r y l cAMP (IS.), agents known to increase i n t r a c e l l u l a r cAMP or mimic the biochemical e f f e c t s of cAMP, increase the release of IR-PTH.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BROWN

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(-)ISO

(-)EPI

D-l

Mediated

Activation

(-)NOREPI DOPAMINE EPININE

Figure 9. Specificity of β-adrénergie and dopaminergic stimulation of cAMP accumulation. Dispersed parathyroid cells were incubated with the indicated β-adrenergic or dopaminergic agonists either alone (open bars), with 1 μΜ ( — ) propranolol (solid bars), or with 10 μΜ. a-flupenthixol (stippled bars).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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DOPAMINE

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A cAMP-dependent phosphorylation o f s p e c i f i c c e l l u l a r substrates i s hypothesized to i n i t i a t e t i s s u e - s p e c i f i c p h y s i o l o g i c a l responses. C e r t a i n elements o f t h i s hypothesis can be a p p l i e d to the bovine parathyroid gland. Dispersed bovine parathyroid c e l l s contain predominantly the type 2 isozyme(s) o f cAMP-dependent p r o t e i n kinase (19.). The a c t i v a t i o n s t a t e of t h i s form of the kinase remains r e l a t i v e l y constant when t i s s u e i s extracted i n t o b u f f e r s c o n t a i n i n g high concentrations o f s a l t . This s i t u a t i o n permits an estimation of the drug-induced a c t i v a t i o n of the cAMP-dependent p r o t e i n kinase a c t i v i t y i n i n t a c t c e l l s . In a recent s e r i e s of experiments u t i l i z i n g dispersed bovine parathyroid c e l l s , we compared the dopamine-induced r e l e a s e of IR-PTH with dopamine-induced a l t e r a t i o n s i n the a c t i v i t y r a t i o of the cAMP-dependent p r o t e i n kinase (an estimate of the f r a c t i o n a l a c t i v a t i o n of t h i s enzyme) (20.) · A c l o s e c o r r e l a t i o n e x i s t s between dopamine-induce dopamine-induced IR-PT observation i s c o n s i s t e n t with a mediatory r o l e f o r cAMP-dependent p r o t e i n phosphorylation i n dopamine-stimulated hormonal s e c r e t i o n . A d d i t i o n a l evidence supporting t h i s p o s s i b i l i t y i s the observation that cAMP promotes the phosphorylation o f s e v e r a l endogenous p r o t e i n s i n sonicates of dispersed bovine parathyroid c e l l s (21) and that dopamine stimulates the phosphorylation o f two p r o t e i n s o f molecular weight 15,000 and 19,000 i n i n t a c t bovine parathyroid c e l l s (22.). The biochemical mechanisms by which cAMP-dependent phosphorylation leads to enhanced IR-PTH r e l e a s e remain to be determined. I t i s of i n t e r e s t , however, that i s o p r o t e r e n o l a c t i v a t e s phosphorylation of p r o t e i n s o f s i m i l a r molecular weight i n the r a t p a r o t i d gland (21), while glucagon stimulates phosphorylation o f a p r o t e i n of molecular weight 19,000 i n c a l c i t o n i n - s e c r e t i n g c u l t u r e d c e l l s from a medullary carcinoma of the r a t t h y r o i d (2H) · I t i s conceivable that i n a l l three t i s s u e s , a c t i v a t i o n of exocytosis r e s u l t s from a cAMP-dependent phosphorylation of a c r i t i c a l c e l l u l a r s u b s t r a t e . Receptors I n h i b i t i n g Dopamine-stimulated cAMP Accumulation and

Both alpha-adrenergic agonists (16) and prostaglandin F2a (PGF2a) (25.) d i m i n i s h dopamine-stimulated cAMP accumulation and hormonal s e c r e t i o n . The i n h i b i t o r y e f f e c t s o f these agents on hormonal s e c r e t i o n can be q u a n t i t a t i v e l y accounted f o r by t h e i r i n h i b i t o r y e f f e c t on cAMP accumulation (11). The mechanism(s) by which these compounds lower c e l l u l a r cAMP has not been i n v e s t i g a t e d ; i n other systems alpha-adrenergic agonists i n h i b i t adenylate cyclase through a GTP-dependent mechanism

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Activation

0.75 h

200 0.375 Η ιοο Û.

< ιο"'

ιο"°

10"'

[DOPAMINE] , Μ


100

at 10"^ M.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

1.

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17

c i s - f l u p e n t h i x o l , and l i s u r i d e are most potent. Phenothiazines, such as fluphenazine and chlorpromazine, have K s i n the range of 4 χ 10" -4 χ 10~ M. Most ergots (e.g. l e r g o t r i l e , dihydroergocryptine, and bromocryptine) are of moderate to low potency 1-10 uM). (+) and (-) Butaclamol and c i s - and t r a n s - f l u p e n t h i x o l show marked s t e r e o s p e c i f i c i t y , with the a c t i v e isomer being of high potency. Agents p u t a t i v e l y s p e c i f i c f o r dopamine receptors i n the a n t e r i o r p i t u i t a r y or intermediate lobe of the r a t p i t u i t a r y are e i t h e r weak antagonists [(+)- and ( - ) - s u l p i r i d e , YM-09151-2 ( 3 1 ) ] or have no e f f e c t [LY-141865 ( 3 2 ) ] i n bovine parathyroid c e l l s . Several features of the dopamine receptor i n the bovine parathyroid gland a l s o occur i n other mammalian t i s s u e s as w e l l as i n lower vertebrates and even i n v e r t e b r a t e s (Table I I I ) . These dopamine-sensitive adenylate c y c l a s e systems are c h a r a c t e r i z e d by dopamine, epinine and 6,7-ADTN being f u l l agonists of micromolar agonist, and ergots suc bromocriptine d i s p l a y i n g minimal agonist a c t i v i t y but being antagonists of moderate potency. In the preparations o f i n t a c t c e l l s which have been t e s t e d , dopamine agonists increase cAMP accumulation. This dopamine-stimulated increase i n cAMP can be l i n k e d to a c t i v a t i o n of a cAMP-dependent p r o t e i n kinase ( 3 1 ) , or to phosphorylation of s p e c i f i c c e l l u l a r substrates ( 4 1 ) . In some cases, exogenous cAMP mimics the p h y s i o l o g i c a l e f f e c t s of dopamine ( 1 2 ) . Thus, these dopamine receptors (designated as D-1 receptors, ( 1 3 . ) appear to act, at l e a s t i n part, through e l e v a t i o n of c e l l u l a r cAMP, a c t i v a t i o n o f cAMP-dependent p r o t e i n kinases, and phosphorylation of s p e c i f i c c e l l u l a r substrates. f

7

i

Dopamine Receptors J n f r i f r i t i n g Adenylate

Cyclase A c t i v i t y

C e r t a i n dopamine receptors do not resemble the dopamine receptor i n the bovine parathyroid gland. For example, the dopamine receptor on the mammotroph of the p i t u i t a r y gland i s c h a r a c t e r i z e d by dopamine, apomorphine, and ergot a l k a l o i d s being agonists with nanomolar potency. Butyrophenones are potent antagonists o f t h i s receptor. While t h i s c l a s s of receptor was o r i g i n a l l y c l a s s i f i e d on the b a s i s of the lack of any s t i m u l a t o r y e f f e c t s on cAMP accumulation or adenylate cyclase a c t i v i t y ( 1 3 . ) , i t has become c l e a r that s t i m u l a t i o n of t h i s receptor i n h i b i t s cAMP metabolism ( 1 4 ) · An a d d i t i o n a l example of t h i s second category of receptor (designated as the D-2 receptor) occurs i n the intermediate lobe of the r a t p i t u i t a r y gland. In t h i s l a t t e r system, dopamine i n h i b i t s basal and p a r t i c u l a r l y agonist-stimulated cAMP accumulation and adenylate c y c l a s e a c t i v i t y ( 1 5 L , 1 & ) ; these i n h i b i t o r y e f f e c t s are GTP-dependent ( U ) . The changes i n c e l l u l a r cAHP c o r r e l a t e with the i n h i b i t i o n of hormone release from the IL (45,46 f

Kebabian

a l . , t h i s volume).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

a

40 4-8 67 5 3.9 3 ^50 20

4.8 5

1

Fluphenazine

11 1 0.48

a-Flupenthixol

K i , nM

600

3,000 95 5.32 2002

Spiroperidol

9,10 33 34 35 36 37 38 39 40

Reference

Kj[ f o r h a l o p e r i d o l . S p i r o p e r i d o l and h a l o p e r i d o l are of comparable potency i n s e v e r a l dopamines e n s i t i v e adenylate c y c l a s e systems.

Kj_ f o r chlorpromazine. Fluphenazine i s about 7-10-fold more potent than chlorpromazine i n s e v e r a l dopamine-sensitive adenylate c y c l a s e systems.

3 10 2-10 10 6-10 10 1-2 2 1.7

Dopamine

a

K , yM

f o r dopamine and K i f o r s e v e r a l dopamine antagonists on dopamine-sensitive adenylate c y c l a s e

Bovine Parathyroid C e l l Rat Candate Nucleus Substantia Nigra Rat O l f a c t o r y Tubercle Superior C e r v i c a l Ganglion Guinea P i g Retina Carp Retina S n a i l Nervous System Cockroach B r a i n

Tissue

K

TABLE I I I

1.

BROWN

A N D DAWSON-HUGHES

D-l Mediated

Activation

19

There are d i s t i n c t p a r a l l e l s , t h e r e f o r e , between the D-1 and D-2 receptors. Both i n t e r a c t with adenylate c y c l a s e , one through a stimulatory guanine nucleotide subunit and the other, presumably, through an i n h i b i t o r y subunit (UZ). Both may exert t h e i r b i o l o g i c a l e f f e c t s through changes i n c e l l u l a r cAMP (as was i n i t i a l l y hypothesized by Sutherland and h i s c o l l e a g u e s ) . The r e l a t i o n s h i p between the D-1 and the D-2 receptors i s very analogous to that between the beta- and the o^-adrenergic receptors. These receptors a l s o a c t upon adenylate c y c l a s e through stimulatory and i n h i b i t o r y guanine nucleotide subunits, r e s p e c t i v e l y (J2.,4S.) · The ^ - a d r e n e r g i c receptor, on the other hand, i s thought to a c t through changes i n c e l l u l a r calcium dynamics, without appreciable e f f e c t s on adenylate cyclase (4Q). By analogy, i t may be speculated that an a d d i t i o n a l c l a s s o f dopamine receptor a c t i n g through changes i n c y t o s o l i c calcium (and, t h e r e f o r e , equivalent to the α-j- adrenergic receptor) might e x i s t . of dopamine receptor woul postulated to e x i s t by Meunier and Labrie (50). At present, no example of e i t h e r of these t h e o r e t i c a l c o n s t r u c t s has been identified.

guaaary Dispersed bovine parathyroid c e l l s contain a dopamine receptor which increases c e l l u l a r cAMP through a guanine nucleotide-stimulated a c t i v a t i o n of adenylate c y c l a s e . The dopamine-stimulated increase i n c e l l u l a r cAMP c o r r e l a t e s c l o s e l y with a c t i v a t i o n o f cAMP-dependent p r o t e i n kinase, phosphorylation o f endogenous c e l l u l a r p r o t e i n s , and s e c r e t i o n of IR-PTH. The potency o f various dopaminergic agonists and antagonists i n modifying these b i o l o g i c a l e f f e c t s as w e l l as the r o l e of cAMP i n modifying p h y s i o l o g i c a l f u n c t i o n suggest that the bovine parathyroid dopamine receptor i s a D-1 dopamine receptor. This system may be of use i n studying the i n t e r a c t i o n of r a d i o l a b e l e d l i g a n d s with the D-1 receptor.

The authors g r a t e f u l l y acknowledge the e x c e l l e n t t e c h n i c a l help o f Joseph Thatcher and Edward Watson and s e c r e t a r i a l work of Mrs. Nancy O r g i l l . This work was supported by USPHS Grants AM25910 and AM30028.

Literature Cited 1. 2.

Parsons, J.A. "Endocrinology"; Grune and S t r a t t o n : New York, 1979; V o l 2, p 621. Sherwood, L.M.; Potts, J r . , J.T.; Care, A.D.; Mayer, G.P.; Aurbach, G.D. Nature 1966, 209, 52.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

20

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

RECEPTORS

Douglas, W.W. Ciba Foundation Symposium 1978, 54, 61. Brown, E . M . Mineral Electrolyte Metabolism 1982, 130, 3. Peck, W.A.; Klahr, S. "Advances i n Cyclic Nucleotides Research"; Raven Press: New York, 1979; Vol 11, p 89. Heath, I I I , H. Endocrine Reviews 1980, 1, 319. Blum, J.W.; Kunz, P.; Fischer, J . Α . ; Binswanger, U . ; Lichtensteiger, W.; DaPrada, M. Am. J. Physiol. 1980, 239, E255. Brown, E . M . ; Hurwitz, S.; Aurbach, G.D. Endocrinology 1976, 99, 1582. Brown, E . M . ; C a r r o l l , R.; Aurbach, G.D. Proc. Nat'l. Acad. S c i . USA 1977, 74, 4210. Brown, E . M . ; A t t i e , M . F . ; Reen, S.; Gardner, D.G.; Kebabian, J.; Aurbach, G.D. Molec. Pharmacol 1980, 18, 335. A t t i e , M.F.; Brown, E . M . ; Gardner, D.G.; Spiegel, A . M . ; Aurbach, G.D. Endocrinolog Rodbell, M. Natur Cassel, D.; Selinger, Z. Proc. Nat'l. Acad. S c i . USA 1977, 74, 3307. Brown, E . M . ; Gardner, D.G.; Windeck, R.A.; Aurbach, G.D. Endocrinology 1979, 104, 218. Brown, E . M . ; Hurwitz, S.; Aurbach, G.D. Endocrinology 1977, 100, 1696. Brown, E . M . ; Hurwitz, S.H.; Aurbach, G.D. Endocrinology 1978, 103, 893. Brown, E . M . ; Gardner, D.G.; Windeck, R.A.; Aurbach, G.D. Endocrinology 1978, 101, 2323. Morrissey, J.J.; Cohn, D.V. J. C e l l B i o l . 1979, 83, 521. Thatcher, J . G . ; Gardner, D.G.; Brown, E.M. Endocrinology 1982, 110, 1367. Brown, E . M . ; Thatcher, J.G. Endocrinology 1982, 110, 1374. Brown, E . M . ; Thatcher, J.G. Program and Abstracts. Fourth Annual S c i e n t i f i c Meeting of the American Society for Bone and Mineral Research, San Francisco, CA, 1982, p S-37. Lasker, R.D.; Spiegel, A.M. Clin. Res. 1982, 30, 398A. Baum, B.J.; Freiberg, J . M . ; Ito, H . ; Roth, G.S.; Filburn, C.R. J . B i o l . Chem. 1981, 256, 9731. Gagel, R . F . ; Andrews, K . L . Abstracts of the 4th Annual S c i e n t i f i c Meeting of the American Society for Bone and Mineral Research 1982, p S-18. Gardner, D.G.; Brown, E . M . ; Windeck, R.; Aurbach, G.D. Endocrinology 1979, 104, 1. Jakobs, K . H . ; Saur, W.; Schulz, G. FEBS Letters 1978, 85, 167. Brown, E . M . ; Gardner, D.G.; Aurbach, G.D. Endocrinology 1980, 106, 133. Jacobowitz, D.; Brown, E.M. Experentia 1980, 36, 115. Brown, E . M . ; Aurbach, G.D. Vitamins and Hormones 1980, 38, 205.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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BROWN

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

A N D DAWSON-HUGHES

D-l

Mediated

Activation

Brown, E.M. Endocrinology 1980, 107, 1998. Grewe, G.W.; Frey, E.A.; Cote, T.E.; Kebabian, J.W. Eur. J. Pharmacol. 1982, 81, 149. Tsuruta, K.; Frey, E.A.; Grewe, C.W.; Cote, T.E.; Eskay, R.L.; Kebabian, J.W. Nature, 1981, 292, 463. Kebabian, J.W. Petzgold, G.L.; Greengard, P. Proc. Nat'l. Acad. Sci. 1972, 69, 2145. Phillipson, O.T.; Horn, A.S. Nature 1976, 261, 418. Horn, A.S.; Cuello, A.C.; M i l l e r , R.J. J. Neurochem. 1974, 22, 265. Kebabian, J.W. Greengard, P. Science 1971, 174, 1346. Brown, J . H . ; Makman, M.Η. Proc. Nat'l. Acad. Sci. USA 1972, 69, 539. Watling, K.J.; Dowling, J . E . J. Neurochem, 1981, 36, 559. Osborne, N.N. Experentia 1977, 33, 917. Harmar, A.J.; Horn, A.S. Mol. Pharmacol. 1977, 13, 512. Nestler, E.J.; Greengard 1980, 77, 7479. Greengard, P.; McAfee, D.A.; Kebabian, J.W. Adv. Cyclic. Nucl. Res. 1972, 1, 373. Kebabian, J.W.; Calne, D.B. Nature 1979, 277, 93. Camilli, P.D.; Macconi, D.; Spada, A. Nature (London) 1979, 278, 252. Munemura, M.; Eskay, R.L.; Kebabian, J.W. Endocrinology 1980, 106, 1795. Cote, T.E.; Grewe, C.W.; Kebabian, J.W. Endocrinology 1981, 108, 420. Cote, T . E . ; Grewe, C.W., Tsuruta, K.; Stoof, J . C . ; Eskay, R.L.; Kebabian, J.W. Endocrinology 1982, 110, 812. Brown, E.M.; Aurbach, G.D. "Contemporary Metabolism"; Plenum: New York, 1982; Vol 2, p 247. Exton, J.H. Am. J. Physiol. 1980, Vol., E3. Meunier, H.; Labrie, F. Life Science 1982, 30, 963.

RECEIVED

March 25, 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Commentary: Dopamine-Sensitive Adenylate Cyclase as a Receptor Site PIERRE M . LADURON Janssen Pharmaceutica, Department of Biochemical Pharmacology, B-2340 Beerse, Belgium There i s no doub adenylate c y c l a s e (D1 s i t e ) is not involved i n the a n t i p s y c h o t i c e f f e c t of n e u r o l e p t i c drugs. All the pharmacological and behavioural e f f e c t s elicited by dopamine agonists and antagonists i n the b r a i n can only be explained if such an i n t e r a c t i o n occurs a t the l e v e l of the dopamine receptor (D2 receptor site); the D1 s i t e still remains i n search of a f u n c t i o n . Bovine parathyroid cells were reported to possess dopamine D1 s i t e s which should be involved i n the c o n t r o l of parathormone s e c r e t i o n . However, the very poor pharmacological c h a r a c t e r i z a t i o n and the lack of i n v i v o evidence do not allow to assess the dopaminergic nature of t h i s hormone s e c r e t i o n . Dopamine-sensitive adenylate c y c l a s e is thus not a receptor d i r e c t l y i m p l i c a t e d i n the dopaminergic neurotransmission; it is an enzyme which could have an important r o l e i n the c o n t r o l of long term metabolic e f f e c t s such as the synthesis of neuronal c o n s t i t u e n t s .

In the l a s t decade, the term receptor has been used by so many people i n so many d i f f e r e n t ways that we are, now, f a r from the o r i g i n a l d e f i n i t i o n proposed by Langley (1) i n the e a r l y twentieth century. In f a c t the receptor concept arose from p h y s i o l o g i c a l and pharmacological experiments; t h e r e f o r e , a p h y s i o l o g i c a l response i s one of the most e s s e n t i a l elements d e f i n i n g a receptor. According to Langley, a receptor i s a s i t e of competition f o r agonist and antagonist; the agonist produces a stimulus which leads to a p h y s i o l o g i c a l response and t h i s i s blocked by the antagonist. One can e a s i l y apply such a concept to the dopaminergic system; f i r s t i t i s necessary to c l e a r l y d e f i n e the p h y s i o l o g i c a l e f f e c t s of dopamine. Nausea, emesis, f

0097-6156/83/0224-0022$06.00/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

LADURON

Adenylate

Cyclase as Receptor Site

23

stereotypy, h y p e r m o t i l i t y , decrease o f p r o l a c t i n s e c r e t i o n , stomach r e l a x a t i o n , neurogenic v a s o d i l a t a t i o n , a n t i p a r k i n s o n e f f e c t s , psychosis e t c . . . are the main p h y s i o l o g i c a l or pharmacological responses t o dopamine and i t s a g o n i s t s . Às a r u l e , these e f f e c t s are measured under i n v i v o c o n d i t i o n s : t h i s i s a p r e r e q u i s i t e f o r c a l l i n g those p h y s i o l o g i c a l responses. Sometimes one can detect a p h y s i o l o g i c a l e f f e c t i n v i t r o as on i s o l a t e d organs f o r instance; however to be r e l e v a n t such an e f f e c t must correspond to a process o c c u r r i n g i n the whole body. One of the major sources of confusion around dopamine receptor o r i g i n a t e d with the idea that the increase o f the c y c l i c AMP production by dopamine i s a p h y s i o l o g i c a l e f f e c t o f dopamine {2); s t a r t i n g from t h i s viewpoint, i t i s not necessary to t r y to c o r r e l a t e the data obtained i n v i t r o with p h y s i o l o g i c a l e f f e c t s i n v i v o . In f a c t , the s t i m u l a t i o n of c y c l i c AMP by dopamine i s a biochemical e f f e c t f o r which one needs t o f i n d a p h y s i o l o g i c a enzyme i s stimulated o does not i p s o - f a c t o prove that such a process i s involved i n neurotransmission. As we w i l l see f u r t h e r , numerous c r i t e r i a must be f u l f i l l e d before an enzyme or a binding s i t e may be c a l l e d a receptor s i t e . The e f f e c t s of dopamine quoted above, are antagonized by n e u r o l e p t i c or antiemetic drugs (3yj4) and a l l are mediated through the dopamine D2 receptor s i t e (5,6,7). This D2 subtype (we p r e f e r to c a l l i t the dopamine receptor) i s the binding s i t e l a b e l l e d by dopamine antagonists l i k e h a l o p e r i d o l and spiperone a t nanomolar concentrations and by dopamine agonists a t micromolar concentrations (7) and i s not coupled t o adenylate c y c l a s e . More than 17 pharmacological, behavioural and biochemical parameters r e l a t e d to the e f f e c t s o f dopamine agonists and antagonists n i c e l y c o r r e l a t e with ICsQ-values obtained i n the i n v i t r o binding assay (5,8). How the problem a r i s e s whether or not the dopamines e n s i t i v e adenylate c y c l a s e (D^ s i t e ) ( 2 ) a l s o answers these c r i t e r i a or other c r i t e r i a which j u s t i f y i t being c a l l e d a dopamine receptor l i k e the D2 receptor s i t e (j>-8.). The purpose o f the present paper i s to d i s c u s s t h i s problem e s p e c i a l l y with regard to parathormone s e c r e t i o n . S p e c i a l a t t e n t i o n w i l l be paid to the pharmacological c h a r a c t e r i z a t i o n of t h i s hormone s e c r e t i o n . Is Dopamine-Sensitive Adenylate

Cyclase a Dopamine Receptor ?

As r e c e n t l y quoted by Briggs and McAfee (9): "Rigorous q u a n t i t a t i v e pharmacology i s required i n equating the receptor u t i l i z e d i n s y n a p t i c transmission. Unfortunately the a p p l i c a t i o n of t h i s pharmacology when a v a i l a b l e i s o f t e n superficial . As a r u l e , a too small number of drugs, sometimes even one or two and o f t e n given a t a s i n g l e high c o n c e n t r a t i o n M

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24

DOPAMINE

RECEPTORS

have been used to c h a r a c t e r i z e , pharmacologically, the production of c y c l i c AMP stimulated by neurotransmitters. The dopamine-sensitive adenylate c y c l a s e d i d not escape t h i s r u l e ; when Greengard's group reported the occurrence of dopamine-sensitive adenylate c y c l a s e i n r a t caudate nucleus as a p o s s i b l e t a r g e t f o r a n t i p s y c h o t i c drugs (10,11), i t r a p i d l y became evident that the potent n e u r o l e p t i c drugs l i k e h a l o p e r i d o l and pimozide d i s p l a y e d a too low a f f i n i t y f o r the c y c l a s e with regard to t h e i r high potency i n pharmacological t e s t s and i n the c l i n i c . The discrepancy was most apparent f o r pimozide which was found to be 10 times l e s s a c t i v e than chlorpromazine on the c y c l a s e whereas i t i s known t o be 30 to 50 times more potent than chlorpromazine i n v i v o (3,4). Thereafter c e r t a i n drugs l i k e s u l p i r i d e or domperidone were reported t o be p r a c t i c a l l y i n a c t i v e on the c y c l a s e (5,12). Table I shows c l e a r l y that numerous potent dopamine antagonists are poorly a c t i v e or i n a c t i v e on th binding assay, sometime the phenothiazines and the thioxanthenes are approximatively e q u i a c t i v e i n both t e s t s . I t became obvious that the a n t i p s y c h o t i c e f f e c t s of n e u r o l e p t i c drugs were not mediated through the c y c l a s e (D^ s i t e ) (j>-8) . There was a complete lack of c o r r e l a t i o n between the i n h i b i t i o n of the dopamine-sensitive adenylate c y c l a s e and 17 behavioural biochemical, pharmacological and c l i n i c a l parameters {6,&) . Two other pieces o f evidence i n d i c a t e that the s i t e i s not involved i n dopaminergic neurotransmission i n the b r a i n . First the i n v i v o accumulation of c y c l i c adenosine monophosphate induced by apomorphine i n the striatum was not blocked by s u l p i r i d e and h a l o p e r i d o l whereas the behavioural e f f e c t s were blocked by both drugs (12). Secondly, when l a b e l l e d n e u r o l e p t i c s were i n j e c t e d i n t o r a t s , the r a d i o a c t i v i t y was found i n a s s o c i a t i o n with the D2 receptor, but never on the D 1 s i t e s (13); indeed the dopamine s e n s i t i v e adenylate c y c l a s e and the binding s i t e (D2) possess a completely d i f f e r e n t s u b c e l l u l a r l o c a l i z a t i o n (14) , a f a c t which g i v e s r i s e to the idea that they are two d i f f e r e n t e n t i t i e s not r e l a t e d to each other. From these c o n s i d e r a t i o n s , one may conclude that the D^ s i t e i s not d i r e c t l y implicated i n dopaminergic neurotransmission; t h i s does not exclude a p o s s i b l e f u n c t i o n a l r o l e for the c y c l a s e but h i t h e r t o i t remains unknown. A p o s s i b l e hypothesis i s that the c y c l a s e may c o n t r o l long term metabolic e f f e c t s such as the synthesis of neuronal c o n s t i t u e n t s . One may argue that the pharmacology of the D^ s i t e does not n e c e s s a r i l y have to be the same as that of the D 2 receptor s i t e ; t h i s i s true but the problem i s to get an i n v i v o pharmacology f o r t h i s D^ s i t e which e n t i r e l y f i t s the data obtained i n v i t r o on the c y c l a s e . Up t o now there i s no answer to t h i s problem. I t i s g e n e r a l l y b e l i e v e d that parathormone

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

LADURON

Adenylate

Cyclase as Receptor Site

25

Table I IC5Q-values f o r various drugs on dopamine s e n s i t i v e adenylate c y c l a s e (D^) and % - h a l o p e r i d o l binding ( D 2 ) IC Drug

α-Flupenthixol Chlorpromaz ine (+)-Butaclamol Haloperidol Pimozide Spiperone Sulpiride Halopemide Domperidone

D± s i t e A 2. 3 1. 1 2 7. 5 1. 5 2. 2

10'-8 10'-6 X 10'-7 X 10" -7 X 10" -5 X 10" -6 > 10" -3 > 10" -3 X X

5 0

D

2

(M) receptor s i t e Β 2 1.3 1 3.6 3.2 4.4 8 1

X 10" -8 X 10" -7 X 10" -8 X 10" -9 X 10" -9 X 10- -10 X 10" -8 X 10'-8

Ratio A/B

1.2 8.5 20 208 4.,687 5,,500 > 10.,000 >100 ,000

2. 4

s e c r e t i o n i s mediated v i a the D^ s i t e (2) ; we w i l l now examine the c r i t e r i a v a l i d a t i n g or i n v a l i d a t i n g t h i s hypothesis. Is Parathormone S e c r e t i o n Mediated through a Dopamine D] S i t e ? In 1977, Brown e t a l . (15) reported that dopamine (10~ M) s t i m u l a t e s by 2-4 f o l d the s e c r e t i o n of parathormone from dispersed bovine parathyroid c e l l s . ADTN and other dopamine agonists mimicked t h i s e f f e c t which was antagonized by a - and β-flupenthixol, the α-isomer being 100 times more potent. In a s i m i l a r way, dopamine caused a r a p i d 20-30-fold increase i n c e l l u l a r cAMP i n dispersed bovine parathyroid c e l l s . The potency of a s e r i e s of dopaminergic agonists and antagonists on adenylate c y c l a s e a c t i v i t y p a r a l l e l e d the e f f e c t s of these l i g a n d s on cAMP accumulation and parathormone s e c r e t i o n (16). I t was concluded that bovine parathyroid c e l l s possess dopamine D^ s i t e s which are involved i n the c o n t r o l of parathormone s e c r e t i o n . This needs some comments; f i r s t , s e v e r a l other agents that are not dopaminergic agonists, such as β-adrenergic catecholamines, s e c r e t i n , phosphodiesterase i n h i b i t o r s , histamine, protaglandin ( P G E 2 ) and cholera t o x i n were a l s o found to enhance cAMP accumulation and parathormone s e c r e t i o n i n human and bovine parathyroid c e l l s (17,18,19) ( c f r . Table I I ) . Moreover, calcium has been known f o r a long time, to play an important r o l e i n the parathormone s e c r e t i o n ; therefore the e f f e c t s of dopamine agonists are not s e l e c t i v e or s p e c i f i c f o r a given neurotransmitter. Somewhat s u r p r i s i n g i s the f a c t that dopamine i s i n e f f e c t i v e i n human dispersed parathyroid c e l l s ; t h i s c e r t a i n l y 6

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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RECEPTORS

Table I I Compounds which can regulate (+) or not (-) cAMP production and parathormone s e c r e t i o n i n parathyroid c e l l s

Epinephrine Dopamine Histamine Secretin Prostaglandin Phosphodiesterase Choieratoxin

inhibitors

Bovine parathyroid

Human parathyroid

+ +

+

-+

-+ -+

-? +

+

l i m i t s the importance o e s p e c i a l l y as the present r e s u l t s do not allow the p o s s i b i l i t y of a non s p e c i f i c e f f e c t of dopamine on parathormone s e c r e t i o n to be excluded. One could assume, f o r instance that dopamine l i k e the other agonists can increase the membrane p e r m e a b i l i t y so that more hormone can be released a f t e r a d d i t i o n of these compounds. Compatible with t h i s hypothesis i s the f a c t that the increase of parathormone s e c r e t i o n e l i c i t e d by dopamine i s a r e l a t i v e l y slow process and that i t can l a s t as long as 60 minutes. As a r u l e the presence of agonists on a receptor s i t e for a long p e r i o d of time leads to a d e s e n s i t i z a t i o n phenomenon; t h i s i s not the case here. More i n t r i g u i n g i s the f a c t that the parathormone s e c r e t i o n occurs i n the absence of dopamine; what dopamine i s doing, i s t o enhance a phenomenon already present; t h i s i s a l s o a t variance with the normal p h y s i o l o g i c a l response to a neurotransmitter which i s g e n e r a l l y an a l l or none process. In f a c t the most important p o i n t concerns the very poor pharmacological c h a r a c t e r i z a t i o n of the cAMP formation enhanced by dopamine and of the parathormone s e c r e t i o n . F i r s t l y , apomorphine i s much l e s s potent than dopamine, a f a c t which i s not compatible with what we know from pharmacological, behavioural and even biochemical s t u d i e s (3,4^,2) · ^ b e l i e v e d that apomorphine i s a p a r t i a l antagonist, but t h i s has never been found i n i n v i v o c o n d i t i o n s . The higher potency o f apomorphine i s a l s o r e f l e c t e d by i t s high a f f i n i t y i n H - h a l o p e r i d o l and H-spiperone b i n d i n g . Secondly ergot d e r i v a t i v e s which r e v e a l a c l e a r c u t a g o n i s t i c a c t i v i t y on p r o l a c t i n s e c r e t i o n and as antiparkinson agents (20) were i n a c t i v e on the c y c l a s e . S u r p r i s i n g l y , l i s u r i d e and l e r g o t r i l e were found to be weak antagonists of dopamine stimulated cAMP accumulation, but they could a l s o antagonize the cAMP production stimulated by i s o p r o t e r e n o l ; as I f c

3

s

3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Cyclase as Receptor Site

27

both compounds are completely i n a c t i v e on the β-receptors, t h i s f i n d i n g c o n s t i t u t e s a strong argument a g a i n s t the s p e c i f i c i t y o f the s o - c a l l e d dopaminergic and β-adrenergic s i t e s i n v o l v e d i n the cAMP accumulation i n the bovine p a r a t h y r o i d c e l l s . Another point concerns the K i ' s o f 1- and d-butaclamol on dopamine s e n s i t i v e adenylate c y c l a s e which are 1 /uM and 2.5 juM respectively. I t i s well-known that the d-form i s the a c t i v e enantiomer and 1- the i n a c t i v e one; t h e r e f o r e i f d-butaclamol i s l e s s a c t i v e than 1-butaclamol, and a l s o that i t i s l e s s a c t i v e than β-flupenthixol, again an i n a c t i v e enantiomer, the e f f e c t s of these drugs on the c y c l a s e are thus i r r e l e v a n t p h y s i o l o g i c a l l y . In f a c t t o assess the dopaminergic nature f o r the c o n t r o l o f parathormone s e c r e t i o n , the f o l l o w i n g c r i t e r i a should be f u l f i l l e d : 1) a l a r g e number o f dopamine antagonists should be t e s t e d i n c l u d i n g compounds belonging t o d i f f e r e n t chemical c l a s s e s such as phenothiazines diphenylbutylamines, domperidone 2) drugs belonging t o other pharmacological c l a s s e s should be tested (a and β-adrenergic, a n t i s e r o t o n e r g i c , a n t i h i s t a m i n e , a n t i c h o l i n e r g i c , and lysosomotropic drugs l i k e c l o r o q u i n e f o r instance; 3) the a f f i n i t y o f dopamine agonists should be compared t o that of non dopaminergic a g o n i s t s ; 4) a good c o r r e l a t i o n should be found between the i n h i b i t i o n o f the parathormone s e c r e t i o n measured under i n v i t r o as w e l l as i n v i v o c o n d i t i o n s and the decrease i n cAMP and the i n h i b i t i o n o f dopamine-sensitive adenylate c y c l a s e ; i n t h i s regard, a l a r g e number o f drugs with a broad range o f a c t i v i t y should be t e s t e d ; 5) the increase o f cAMP production e l i c i t e d by dopamine antagonists and i t s i n h i b i t i o n by dopamine antagonists should be examined i n the p a r a t h y r o i d under i n v i v o c o n d i t i o n s . Only such an a n a l y t i c a l approach can decide whether the c o n t r o l o f the parathormone s e c r e t i o n i n v o l v e s a dopaminergic receptor. In my o p i n i o n , one may assume a p r i o r i that these c r i t e r i a w i l l not be f u l f i l l e d ; f i r s t , i f the dopamine-sensitive adenylate c y c l a s e was r e a l l y involved i n the parathormone s e c r e t i o n , the p a t i e n t s treated with n e u r o l e p t i c s and e s p e c i a l l y with the most potent drugs on the s i t e s (phenothiazine and thioxanthene d e r i v a t i v e s ) would have normally revealed marked changes i n t h e i r parathormone s e c r e t i o n , j u s t l i k e as i s the case f o r the p r o l a c t i n s e c r e t i o n ; i n f a c t such changes have never been observed; secondly, a recent report c l e a r l y i n d i c a t e s that the i n j e c t i o n o f dopamine i n man does not modify parathormone s e c r e t i o n although a marked decrease i n p r o l a c t i n was observed (21) . There i s no receptor without p h y s i o l o g i c a l response; the study o f receptor r e q u i r e s a m u l t i d i s c i p l i n a r y approach. H i t h e r t o , i t has not been proved t h a t the ϋχ s i t e i s

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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r e a l l y involved i n parathormone s e c r e t i o n ; the D^ s i t e i s thus an enzyme but not a receptor s i t e s i n c e a p h y s i o l o g i c a l r o l e has not been demonstrated. Acknowledgments Part o f t h i s work was supported by I.W.O.N.L. I thank David Ashton f o r h i s h e l p i n preparing the manuscript.

Literature

Cited

1. Langley, J.N. P r o c . Roy. S o c . S e r . Β 1906, 78, 107-194. 2. Kebabian, J . W . ; Calne, D . B . Nature 1979, 277, 93-96. 3. Niemegeers, C.J.E.; Janssen, P.A.J. L i f e Sci. 1979, 24, 2201-2216. 4. Costall, B.; N a y l o r R.J. L i f e Sci. 1981 28 215-229 5. Laduron, P . Trend 6. Laduron, P . "Advance Pergamon, 1982, Vol. 37; p. 71. 7. Seeman, P . Pharmacol. Rev. 1980, 32, 229-313. 8. Laduron, P . "Apomorphine and Other Dopaminomimetics" Gessa and Corsini, E d s . ; Raven P r e s s , 1981, Vol. 1; p . 85. 9. B r i g g s , C.A.; McAfee, D . A . Trends Pharmacol. Sci. 1982, 3, 241-244. 10. Kebabian, J . W . ; P e t z o l d , G.L.; Greengard, P . P r o c . Natl. Acad. Sci. USA 1972, 69, 2145-2149. 11. Clement-Cormier, Y.C.; Kebabian, J . W . ; P e t z o l d , G.L.; Greengard, P . Proc. Natl. Acad. Sci. USA , 71, 1113-1117. 12. T r a b u c c h i , M.; Longoni, R . ; Fresia, P.; Spano, P . F . L i f e Sci. 1975, , 1551-1556. 13. Laduron, P . M . ; Janssen, P.F.M.; Leysen, J.E. Biochem. Pharmacol. 1978, 27, 323-328. 14. Leysen, J.E.; Laduron, P . L i f e Sci. 1977, 20, 281-288. 15. Brown, E.M.; Carroll, R . ; Aurbach, G.D. P r o c . Natl. Acad. Sci. USA 1977, 74, 4210-4213. 16. Brown, E.M.; Attie, M.F.; Reen, S . ; Gardner, D . G . ; Kebabian, J.; Aurbach, G . D . M o l . Pharmacol. 1980, 18, 335-340. 17. Brown, E.M.; H u r w i t z , S . , Aurbach, G.D. Endocrinology 1977, 100, 1696-1702. 18. Brown, E.M.; Gardner, D . G . ; Windeck, R.A.; Aurbach, G . D . Endocrinology 1979, 104, 218-224. 19. Brown, E.M.; Gardner, D . G . ; Windeck, R . A . ; H u r w i t z , S . ; Brennan, M.F.; Aurbach, G.D. J. Clin. E n d o c r i n o l . Metab. 1979, 48, 618-626. 20. Schachter, M.; B é d a r d , P.; Debono, A.G.; Jenner, P.; Marsden, C.D.; P r i c e , P.; Parkes, J.D.; Keenan, J.; Smith, B.; Rosenthaler, J.; Horowski, R . ; Dorow, R. Nature 1980, 286, 157-159. 21. B a n s a l , S . ; Woolf, P . D . ; F i s c h e r , J.A.; Caro, J.F. J. Clin. E n d o c r i n o l . Metab. 1982, 54, 651-652. R E C E I V E D February 18, 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

D r . Brown's Replies to D r . Laduron's Comments D r . L a d u r o n raises a number of issues w h i c h r e q u i r e c o m m e n t . H e r e f e r s t o the w o r k of L a n g l e y (1) t o p r o v i d e support for a p r e d o m i n a n t l y i n v i v o d e f i n i t i o n of r e c e p t o r s . B y n e c e s s i t y , o f c o u r s e , these e a r l y e x p e r i m e n t s w e r e based s o l e l y on p h y s i o l o g i c a l responses, such as m u s c l e c o n t r a c t i o n . It is of i n t e r e s t , h o w e v e r , t h a t L a n g l e y was p r e s c i e n t i n p o s t u l a t i n g t h a t i n t e r a c t i o n of agonists w i t h a " r e c e p t i v e substance", w h i c h r e c e i v e s and t r a n s m i t s i n f o r m a t i o n , leads t o a change i n i n t r a c e l l u l a r substances (?second messengers) and u l t i m a t e l y to a change in c e l l u l a r f u n c t i o n . M o d e l systems such as b o v i n e p a r a t h y r o i d c e l l s and c e l l s of the i n t e r m e d i a t e lobe of the r a t p i t u i t a r y g l a n d have a l l o w e d for a d i r e c t d e m o n s t r a t i o n of such a s e q u e n c e . D r . L a d u r o n is c o r r e c t t h a t a p h y s i o l o g i c a l r o l e for D - l d o p a m i n e r g i ha bee d e m o n s t r a t e d i n the b r a i n f u n c t i o n does not e x i s t y respons dopamin d e m o n s t r a t e d i n v i v o i n the c o w (2) w h i c h was not b l o c k e d by p r o p r a n o l o l . It should also be p o i n t e d out t h a t m u c h of t h e a c c u m u l a t e d k n o w l e d g e about w e l l - d e f i n e d r e c e p t o r systems such as the betaa d r e n e r g i c r e c e p t o r have c o m e f r o m models l i k e the t u r k e y or the f r o g e r y t h r o c y t e , i n w h i c h the p h y s i o l o g i c a l r o l e of the r e c e p t o r is u n k n o w n . B y D r . L a d u r o n ' s d e f i n i t i o n , these s y s t e m s do not have a b e t a r e c e p t o r . T h i s " s h o r t c o m i n g " has c e r t a i n l y not lessened t h e u t i l i t y of these s y s t e m s for c a r r y i n g out d e t a i l e d b i o c h e m i c a l c h a r a c t e r i z a t i o n of the b e t a r e c e p t o r . F i n a l l y , a major weakness o f a p u r e l y p h a r m a c o l o g i c a p p r o a c h or e v e n one w h i c h c o r r e l a t e s binding s i t e s w i t h p h y s i o l o g i c a l responses is t h a t i t c a n be o n l y c o r r e l a t i v e . W i t h such an a p p r o a c h , i t is a l w a y s possible t h a t i f a d d i t i o n a l drugs w e r e t e s t e d , e x c e p t i o n s w o u l d a r i s e w h i c h w o u l d not support the p o s t u l a t e d r e c e p t o r - m e d i a t e d l i n k a g e t o a p h y s i o l o g i c a l response. A c a u s a l r e l a t i o n s h i p b e t w e e n the i n t e r a c t i o n of an agonist w i t h a r e c e p t o r and a p h y s i o l o g i c a l response c a n o n l y be e s t a b l i s h e d by w o r k i n g out the d e t a i l e d m o l e c u l a r m e c h a n i s m s by w h i c h the r e c e p t o r - m e d i a t e d changes i n c e l l u l a r f u n c t i o n t a k e p l a c e . D r . L a d u r o n also feels t h a t the d o p a m i n e - s t i m u l a t e d i n c r e a s e i n c y c l i c A M P a c c u m u l a t i o n and P T H s e c r e t i o n m a y be " n o n - s p e c i f i c " and has not been shown to be m e d i a t e d by a d o p a m i n e ( D - l ) r e c e p t o r . We w o u l d s i m p l y l i k e t o r e i t e r a t e the e v i d e n c e w e p r e s e n t e d p r e v i o u s l y and t o point out a d d i t i o n a l e v i d e n c e for a s p e c i f i c r e c e p t o r - m e d i a t e d p r o c e s s . The e f f e c t s o f d o p a m i n e a r e r a p i d (less t h a n a m i n u t e i n v i v o and n e a r l y as fast i n v i t r o ) and are b l o c k e d s p e c i f i c a l l y by d o p a m i n e r g i c antagonists of the p h e n o t h i a z i n e , t h i o x a n t h i n e , and butyrophenone classes as w e l l as by d - b u t a c l a m o l (see b e l o w ) . T h e y are t o t a l l y u n a f f e c t e d , o n the o t h e r h a n d , by the b e t a - a d r e n e r g i c a n t a g o n i s t p r o p r a n o l o l and the a l p h a a d r e n e r g i c b l o c k e r p h e n t o l a m i n e at c o n c e n t r a t i o n s w h i c h t o t a l y i n h i b i t responses due to beta-adrenergic or alpha-adrenergic agonists, r e s p e c t i v e l y . In a d d i t i o n , i n p i l o t studies we h a v e found t h a t h i s t a m i n e , s e r o t o n i n , o c t o p a m i n e and c a r b a m y l c h o l i n e h a v e not c o n s i s t e n t e f f e c t on c y c l i c A M P a n d / o r P T H r e l e a s e . F i n a l l y , t h e e f f e c t s o f P G E are not b l o c k e d by p r o p r a n o l o l , a l p h a - f l u p e n t h i x o l , or p h e n t o l a m i n e (3), the i n h i b i t o r y e f f e c t s of p r o s t a g l a n d i n F a r e not i n h i b i t e d by p h e n t o l a m i n e 29 In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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(4), and the s t i m u l a t o r y e f f e c t s of s e c r e t i n are not i n h i b i t e d by propranolol, alpha-flupenthixol, or phentolamine (5). Since m e t h y l i s o b u t y l x a n t h i n e and other phosphodiesterase i n h i b i t o r s as w e l l as c h o l e r a t o x i n a c t i n t r a c e l l u l a r ^ through a n o n - r e c e p t o r m e d i a t e d p r o c e s s , w e do not f e e l i t l i k e l y t h a t these agents a c t t h r o u g h any of the r e c e p t o r s n o t e d a b o v e . We f e e l , t h e r e f o r e , t h a t the s p e c i f i c i t y of the d o p a m i n e r g i c response of b o v i n e p a r a t h y r o i d c e l l s speaks for i t s e l f . We agree w i t h D r . L a d u r o n , h o w e v e r , t h a t the use of further drugs t o t e s t s p e c i f i c i t y w o u l d s t r e n g t h e n these d a t a e v e n f u r t h e r . It should be p o i n t e d out t h a t the response o f a c e l l t o o n l y a single c l a s s o f agonists w o u l d be the e x c e p t i o n r a t h e r t h a n the r u l e . In the c e n t r a l nervous s y s t e m , i t is b e c o m i n g i n c r e a s i n g l y c l e a r t h a t the i n t e r p l a y of a number o f p h a r m a c o l o g i c i n f l u e n c e s m a y d e t e r m i n e the i n t e g r a t e d response o f g i v e n c e l l t y p e . T o a v o i d c o n f u s i o n o n the p a r t of the r e a d e r , s e v e r a l i n a c c u r a c i e s o n the p a r t of D r . L a d u r o n should be p o i n t e d o u t . (1) It is i n c o r r e c t t o s t a t e t h a t d e s e n s i t i z a t i o n does not o c c u r i n the d o p a m i n e r g i cells. We d i d not D e s e n s i t i z a t i o n , h o w e v e r , probably a c t u a l l y does o c c u r a t m o r e t h a n one l o c u s i n this s y s t e m . F i r s t , the secretory response o f the p a r a t h y r o i d c e l l r a p i d l y b e c o m e s r e f r a c t o r y t o agents such as d o p a m i n e and i s o p r o t e r e n o l w h i c h produce l a r g e e l e v a t i o n s i n c y c l i c A M P (see r e f . 2). S e c o n d l y , t h e r e is a p r o g r e s s i v e d e c r e a s e i n c e l l u l a r c y c l i c A M P despite the c o n t i n u e d presence of dopamine (see F i g u r e 5 i n our m a n u s c r i p t ) possibly due t o d e s e n s i t i z a t i o n of the r e c e p t o r adenylate cyclase compex. (2) D r . L a d u r o n uses the r e l a t i v e p o t e n c y of d - and 1b u t a c l a m o l as a major a r g u m e n t against the s p e c i f i c i t y of t h e p a r a t h y r o i d dopamine r e c e p t o r . We a p o l o g i z e for the confusion w h i c h we a p p a r e n t l y caused h i m i n t h i s r e g a r d . In our o r i g i n a l m a n u s c r i p t , the s y m b o l w h i c h D r . L a d u r o n took t o indicate a potency of 1 m i c r o m o l a r for 1-butaclamol a c t u a l l y r e f e r r e d to F o o t n o t e 1 w h i c h s t a t e d t h a t 1 - b u t a c l a m o l had no e f f e c t a t 100 m i c r o m o l a r . In a d d i t i o n , b e c a u s e of the r e l a t i v e l y l o w p o t e n c y o f d - b u t a c l a m o l i n our o r i g i n a l studies (k. = 2.5 m i c r o m o l a r ) , w e c a r r i e d out a d d i t i o n a l studies w i t h fresh samples o f d - a n d 1 - b u t a c l a m o l . The p o t e n c y of t h e s e n e w e r samples are r e f l e c t e d i n T a b l e I o f the present m a n u s c r i p t (k for d - b u t a c l a m o l 5 = n a n o m o l a r ; 1 - b u t a c l a m o l a g a i n had no e f f e c t at 10 m o l a r ) . S i n c e we do not have d a t a on the e f f e c t s o f the newer samples o n a d e n y l a t e c y c l a s e , w e h a v e not i n c l u d e d d a t a for the e f f e c t s of b u t a c l a m o l o n c y c l a s e i n the newer m a n u s c r i p t . (3) D r . L a d u r o n s t a t e s t h a t b o t h l i s u r i d e and l e r g o t r i l e are a n t a g o n i s t s a t the b e t a - r e c e p t o r i n b o v i n e p a r a t h y r o i d cells. H e a g a i n uses this p i e c e o f e v i d e n c e as a s t r o n g a r g u m e n t against the s p e c i f i c i t y o f the d o p a m i n e r e c e p t o r i n t h i s s y s t e m . We k n o w of no e v i d e n c e , h o w e v e r , e i t h e r i n our w o r k or t h a t of o t h e r s t h a t l e r g o t r i l e has such a n e f f e c t o n

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Site

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p a r a t h y r o i d c e l l s . M o r e o v e r , the k. f o r l i s u r i d e at the b e t a r e c e p t o r d i f f e r s b y 3 t o 4 - f o l d f r o m hhat for i t s e f f e c t s on the dopamine r e c e p t o r , suggesting t h a t i t i s , i n f a c t , a c t i n g at t w o different receptors. (4) In T a b l e II o f his c o m m e n t s , D r . L a d u r o n points out d i f f e r e n c e s b e t w e e n b o v i n e and h u m a n p a r a t h y r o i d c e l l s . S e v e r a l of these are i n e r r o r . Prostaglandins affect both h u m a n and b o v i n e p a r a t h y r o i d c e l l s . Phosphodiesterase i n h i b i t o r s have not been t e s t e d d i r e c t l y i n h u m a n p a r a t h y r o i d c e l l s , although d i b u t y r y l c y c l i c A M P , which may act i n part by i n h i b i t i n g phosphodiesterase, stimulated P T H release in f r a g m e n t s o f h u m a n p a r a t h y r o i d glands (6). (5) It is v e r y l i k e l y an i n a c c u r a c y to c a l l the D - l dopamine receptor an e n z y m e . B e c a u s e of the e f f e c t s o f guanine nucleotides on dopamine-stimulated adenylate c y c l a s e , i t is l i k e l y by a n a l o g y w i t h o t h e r r e c e p t o r s (i.e. the 3 r e c e p t o r ) t h a t the w h i c h is c o u p l e d t o b i n d i n g subunit. P r o o f o f this p o i n t , o f c o u r s e , w i l l r e q u i r e p h y s i c a l s e p a r a t i o n of these e n t i t i e s . (6) D r . L a d u r o n c a t e g o r i c a l l y s t a t e s t h a t the D - 2 r e c e p t o r is not c o u p l e d t o a d e n y l a t e c y c l a s e . R e c e n t w o r k , h o w e v e r , u t i l i z i n g b o t h the e f f e c t s o f d o p a m i n e on the m a m m o t r o p h as w e l l as o n dispersed c e l l s o f the i n t e r m e d i a t e l o b e of the r a t p i t u i t a r y gland (the f o r m e r o f w h i c h D r . L a d u r o n appears t o f e e l i t is an e x a m p l e of t h e D - 2 r e c e p t o r ) suggests t h a t the D - 2 r e c e p t o r is, i n f a c t , c o u p l e d t o a d e n y l a t e c y c l a s e i n a n e g a t i v e w a y . It m a y w e l l t u r n o u t , t h e r e f o r e , t h a t , l i k e the alpha-2 a d r e n e r g i c r e c e p t o r , the D - 2 r e c e p t o r is c o u p l e d t o a d e n y l a t e c y c l a s e through a n i n h i b i t o r y guanine n u c l e o t i d e subunit. W h e t h e r or not o t h e r D - 2 r e c e p t o r s i n the c e n t r a l nervous s y s t e m are l i n k e d t o the c y c l a s e i n t h i s fashion r e m a i n s t o be d e t e r m i n e d .

Literature Cited 1. 2. 3. 4. 5. 6.

L a n g l e y , J.N. Proc. R o y . S o c . Ser. B . 1906, 78, 170-194. B l u m , J . W . ; K u n z , P . ; F i s c h e r , J.A.; B i n s w a n g e r , U.; L i c h t e n s t e i g e r , W . ; D a P r a d a , M. Am. J. P h y s i o l . 1980, 239, E 2 5 5 . Gardner, D . B . ; Brown, E . M . ; Windeck, R . ; Aurbach, G . D . E n d o c r i n o l o g y 1978, 103, 577. Gardner, D . B . ; Brown, E.M.; Windeck, R . ; Aurbach, G . D . E n d o c r i n o l o g y 1979, 104, 1. Windeck, R . ; Brown, E.M.; Gardner, D . B . ; Aurbach, G . D . E n d o c r i n o l o g y 1978, 103, 2020. D i e t e l , M.; D o r n , G.; M o n t z , R . ; A l t e n a k r , E. Acta E n d o c r i n o l o g i c a (Kbh) 1977, 8 5 , 5 4 1 .

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2 The D-2 Dopamine Receptor in the Intermediate Lobe of the Rat Pituitary Gland Physiology, Pharmacology, and Biochemistry J. W . K E B A B I A N , M . B E A U L I E U , T . E . C O T E , R. L . E S K A Y , E . A . F R E Y , M . E. G O L D M A N , C . W. G R E W E , M . M U N E M U R A , J. C . S T O O F , and K . T S U R U T A National Institutes of Health, Experimental Therapeutics Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20205

Dopaminergic neuron cells of the p i t u i t a r y gland. Dopamine decreases the c a p a c i t y of the IL c e l l s to synthesize c y c l i c AMP and i n h i b i t s the r e l e a s e o f αMSH and other peptides from t h i s t i s s u e . The presence o f a D-2 receptor accounts f o r both o f these phenomena. This D-2 dopamine receptor can be studied i n a binding assay using [3H]-spiroperidol, a dopamine antagonist. These observations support the two dopamine receptor hypothesis. The hypothesis suggesting the existence o f two c a t e g o r i e s of dopamine receptor (1_, Figure 1 ) arose from the observation that l e r g o t r i l e blocks the dopamine-induced enhancement o f s t r i a t a l adenylate c y c l a s e a c t i v i t y (2, see a l s o Brown and Dawson-Hughes, t h i s volume) but mimicks the a c t i o n o f dopamine upon the mammotrophs o f the a n t e r i o r p i t u i t a r y gland (_3). When the hypothesis was i n i t i a l l y put forward, the dopamine receptor upon the mammotrophs was taken as the p r o t o t y p i c example o f a D-2 r e c e p t o r . According t o t h i s hypothesis, s t i m u l a t i o n o f the D-2 dopamine receptor does "not i n v o l v e e i t h e r the s t i m u l a t i o n o f adenylate c y c l a s e or the accumulation of intracellular c y c l i c AMP" (1_, 4-7)· Although the mammotroph provided an example o f a D-2 dopamine r e c e p t o r , the c e l l u l a r heterogeneity of the a n t e r i o r p i t u i t a r y gland l i m i t e d the p r e c i s i o n o f t h i s biochemical model o f the D-2 dopamine receptor (8; see a l s o Labrie et a l . , this volume). Indeed, many biochemical i n v e s t i g a t i o n s o f the a n t e r i o r p i t u i t a r y dopamine receptor make the untestable assumption that a biochemical s i g n a l not l i n k e d to p r o l a c t i n i s generated by the mammotrophs and none o f the other c e l l types i n the gland. The intermediate lobe (IL) o f the r a t p i t u i t a r y gland possesses a D-2 dopamine receptor amenable t o experimental i n v e s t i g a t i o n . The IL c o n s i s t s o f an homogeneous population o f c e l l s (£) possessing both a βadrenoceptor (U)) and a dopamine receptor (11_, 1_2). An This chapter not subject to U.S. copyright. Published 1983, American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

Name

D-l

Cyclase linkage Location of proto­ type receptor Dopamine Apomorphine Dopaminergic ergots

Selective antagonist Radiolabelled ligand

Figure 1.

Agonist ^molar potency) Partial agonist or antagonist Potent antagonist (nmolar potency) Weak agonist ^molar potency) None known as yet c/s-flupenthixol

RECEPTORS

D-2

anterior pituitary Agonist (nmolar potency) Agonist (nmolar potency) Agonist (nmolar potency)

Metoclopramide sulpiride Dihydroergocryptine

Criteria for the classification of dopamine receptors.

Radiolabelled cis-fiupenthixol can be used as a ligand specific for the dopamine receptor linked to adenylyl cyclase in the rat striatum (64). Its affinity for the dopamine receptor in the anterior pituitary has not been measured. (Previously (65) the two categories of dopamine receptors were designated as "α-dopaminergic" and "β-dopaminergic." This has led to confusion with the a and β adrenoreceptors. The new designations should prevent further confusion.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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35

u n d e r s t a n d i n g o f the b i o c h e m i c a l consequences o f s t i m u l a t i o n o f t h e D - 2 dopamine r e c e p t o r h a s come from i n v e s t i g a t i o n s o f the I L dopamine r e c e p t o r . The C e l l B i o l o g y o f t h e I n t e r m e d i a t e

Lobe

The I L o f t h e r a t p i t u i t a r y g l a n d c o n s i s t s o f a n a r r o w band o f c e l l s a d h e r i n g t o t h e s l i g h t l y l a r g e r n e u r a l l o b e . The p a r e n c h y m a l c e l l s o f t h e I L s y n t h e s i z e p r o o p i o m e l a n o c o r t i n and then c l e a v e t h i s l a r g e molecule i n t o s m a l l e r fragments which a r e c o n v e r t e d i n t o t h e hormones o f t h e I L ( 1_4 ) . In rodents, t h e s e hormones a f f e c t c o a t c o l o r by s t i m u l a t i n g t h e d e r m a l m e l a n o c y t e s t o s y n t h e s i z e and s e c r e t e m e l a n i n (1_5, 1_6). The most w i d e l y s t u d i e d o f t h e m e l a n o t r o p i c I L hormones i s a l p h a m e l a n o c y t e - s t i m u l a t i n g hormone ( N - a c e t y l ACTH1-13 a m i d e , oMSH). However, r e c e n t r e p o r t s document t h a t N - , O - d i a c e t y l oMSH i s the predominant m o l e c u l a et a l . , submitted). I color i n rodents, t h e r e l e a s e o f hormones from t h e r a t I L provides a convenient, physiologically relevant sign of a c t i v i t y i n the I L . The hormones r e l e a s e d from t h e I L c a n be q u a n t i f i e d by e i t h e r b i o a s s a y o r by radioimmunoassay. Several o M S H - l i k e m o l e c u l e s a r e r e l e a s e d from t h e I L ; h o w e v e r , i n t h e v a s t m a j o r i t y o f s t u d i e s , t h e amount o f m e l a n o t r o p h i c hormone o r i m m u n o r e a c t i v e , otMSH-like m a t e r i a l (IR-otMSH) i s q u a n t i f i e d i n a s s a y s u s i n g otMSH as a s t a n d a r d . C y c l i c AMP and t h e I n t e r m e d i a t e

Lobe

β-adrenoceptor

A β - a d r e n o c e p t o r o c c u r s upon t h e p a r e n c h y m a l c a l l s o f t h e r a t I L and c a n be s t u d i e d w i t h b i o c h e m i c a l p r o c e d u r e s . The β adrenoceptor itself can be identified with [1251]monoiodohydroxybenzylpindolol (IHYP) (19). Using IHYP to i d e n t i f y b i n d i n g s i t e s and p r o p r a n o l o l t o d e f i n e ' n o n - s p e c i f i c b i n d i n g * , s p e c i f i c b i n d i n g s i t e s f o r the r a d i o l a b e l e d l i g a n d c a n be d e t e c t e d . The a f f i n i t y o f t h e s p e c i f i c b i n d i n g s i t e f o r a n o n - r a d i o a c t i v e d r u g c a n be as d e t e r m i n e d by p e r m i t t i n g t h e d r u g t o compete w i t h IHYP f o r o c c u p a n c y o f t h e s p e c i f i c b i n d i n g sites. I n t h e I L ( a s i n many o t h e r t i s s u e s ) t h e β - a d r e n o c e p t o r regulates adenylate cyclase, the enzyme catalyzing the c o n v e r s i o n o f ATP i n t o cAMP ( 1_9 ) . The a d e n y l a t e cyclase activity of cell-free homogenates of the IL i s markedly stimulted (up t o 7 - f o l d ) by c a t e c h o l a m i n e s ; t h e s t i m u l a t o r y e f f e c t o f c a t e c h o l a m i n e s r e q u i r e s t h e p r e s e n c e o f GTP and c a n be b l o c k e d by s e v e r a l β - a d r e n e r g i c a n t a g o n i s t s ( F i g u r e 2 , l e f t ; V9, 2 0 ) ) . B e c a u s e t h e IHYP b i n d i n g a s s a y and t h e adenylate c y c l a s e assay u t i l i z e s i m i l a r assay c o n d i t i o n s , a comparison between the affinities obtained i n the two a s s a y s seems justified. The a p p r o x i m a t e agreement o f t h e a f f i n i t i e s from t h e two a s s a y s s u g g e s t s t h a t some o r a l l o f t h e b i n d i n g s i t e s are the receptors enhancing adenylate c y c l a s e a c t i v i t y i n the

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. I

1

1(H GTP (M)

I

10-5

I 0

L L y /

1

10-7

1

GTP (M)

10-6

1

10-5

L _

104

Guanosine 5'-triphosphate participation in the functioning of the IL β-adrenoceptor the D-2 dopamine receptor in the IL of the rat pituitary gland.

10-7

and

Key: left, effects of GTP on basal and isoproterenol (i)-stimulated adenylate cyclase activity in particulate material from a homogenate of fresh IL tissue; at the indicated concentrations, GTP was tested in the absence of drugs «>) or in the presence of 3 μΜ isoproterenol (φ) (data are from Reference 20); and right, effect of GTP upon adenylate cyclase activity in a homogenate of cholera toxin-treated (30 nM, 2 h) IL tissue; at the indicated concentrations, GTP was tested alone (0) or in combination with either 3 μΜ isoproterenol (+) or 10 μΜ apo­ morphine ( Δ ) (data are from Reference 22).

Figure 2.

0

2.

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ET AL.

D-2 Dopamine

37

Receptor

c e l l - f r e e homogenates. The β - a d r e n o c e p t o r i n t h e I L i s a β a d r e n o c e p t o r (1_9, 21). C y c l i c AMP formed i n r e s p o n s e t o s t i m u l a t i o n o f t h e β a d r e n o c e p t o r may i n i t i a t e t h e i n t r a c e l l u l a r e v e n t s u l t i m a t e l y expressed as an enhanced release of OMSH. β-Adrenergic agonists ( F i g u r e 3) a s w e l l a s o t h e r a g e n t s i n c r e a s i n g cAMP content (e.g. theophylline, 3-isobuty-1 -methylxanthine or cholera toxin) increase t h e r e l e a s e o f otMSH (1_2, 2 2 , 23). Furthermore, c y c l i c n u c l e o t i d e analogues ( e . g . d i b u t y r y l cAMP o r 8 B r cAMP) a l s o i n c r e a s e t h e r e l e a s e o f oMSH (1_2, 2 4 ) . A "working hypothesis" accounting f o r the e f f e c t s o f β - a d r e n e r g i c a g o n i s t s and o t h e r d r u g s upon t h e I L i s a s f o l l o w s : occupancy o f t h e β - a d r e n o c e p t o r by an a g o n i s t e n h a n c e s a d e n y l a t e c y c l a s e a c t i v i t y and t h e r e b y i n c r e a s e s the i n t r a c e l l u l a r content of cAMP. I n t u r n , cAMP i n i t i a t e s t h e i n t r a c e l l u l a r e v e n t s l e a d i n g t o enhanced hormone s e c r e t i o n (Figure 4). The p r e s e n c e o f c a l c i u m ions i n the e x t r a c e l l u l a stimulated release o i n t r a c e l l u l a r s i t e ( s ) where c a l c i u m and cAMP i m p i n g e upon t h e r e l e a s e p r o c e s s i s unknown. The e v i d e n c e from t h e I L , t o g e t h e r w i t h t h e e v i d e n c e f r o m many o t h e r b i o l o g i c a l s y s t e m s , supports the h y p o t h e s i s that cAMP m e d i a t e s t h e e f f e c t s o f β - a d r e n e r g i c a g o n i s t s upon t h e I L . H o w e v e r , t h e r e a r e a number o f unanswered q u e s t i o n s a b o u t how this receptor, cAMP and c a l c i u m i o n s 'work* i n t h e I L . For example, isoproterenol enhances the release of otMSH from dispersed IL c e l l s at concentrations s u b s t a n t i a l l y lower than the concentrations required to either occupy the specific b i n d i n g s i t e i d e n t i f i e d w i t h IHYP, s t i m u l a t e adenylate c y c l a s e a c t i v i t y i n c e l l - f r e e homogenates o f t h e I L o r enhance cAMP f o r m a t i o n by i n t a c t I L c e l l s ( F i g u r e 5 , 19). At present no e x p e r i m e n t a l o b s e r v a t i o n upon I L t i s s u e e x p l a i n s t h i s a p p a r e n t discrepancy. However, t h e r e a r e a number o f i n d i c a t i o n s t h a t the I L g i v e s a maximal p h y s i o l o g i c a l response w h i l e u t i l i z i n g o n l y a f r a c t i o n o f i t s c a p a c i t y t o s y n t h e s i z e cAMP. Thus, m a x i m a l i s o p r o t e r e n o l - s t i m u l a t e d hormone r e l e a s e i s a c c o m p a n i e d by a n a c c u m u l a t i o n o f cAMP w h i c h i s o n l y 3% o f t h e cyclic n u c l e o t i d e a c c u m u l a t i o n e l i c i t e d by a c o m b i n a t i o n o f c h o l e r a t o x i n and a p h o s p h o d i e s t e r a s e i n h i b i t o r ( 2 3 ) . Furthermore, the I L c e l l s e x c r e t e cAMP i n t o t h e e x t r a c e l l u l a r medium so t h a t a f t e r a few m i n u t e s o f s t i m u l a t i o n o f t h e β - a d r e n o c e p t o r , more cAMP i s f o u n d o u t s i d e t h e c e l l s t h a n i s i n s i d e t h e m . This e v i d e n c e s u g g e s t s t h a t a m i n i m a l change i n i n t r a c e l l u l a r cAMP i s needed t o i n i t i a t e s e c r e t i o n and p o i n t s t o t h e need f o r a greater understanding of calcium-dependent secretion, in general, before significant progress can be made in understanding the β - a d r e n e r g i c s t i m u l a t i o n o f s e c r e t i o n from the I L , i n p a r t i c u l a r . 2

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

38

DOPAMINE

0

1

2

RECEPTORS

3

TIME (hours) Figure 3. Effect of certain drugs on lR-aMSH release from rat cells. Dispersed rat IL cells were exposed to isoproterenol, lisuride, or no drug (control) for the indicated periods of time. Isoproterenol enhances the release of IR-aMSH while lisuride inhibits the release of IR-aMSH. (Data are from Reference 12.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

KEBABIAN

E T AL.

D-2 Dopamine

ISOPROTERENOL

Receptor

DOPAMINE

Figure 4. A representation of the "working hypothesis" of the dual regulation of adenylate cyclase activity and hormone release by a β-adrenoceptor and a D-2 dopamine receptor in the IL of the rat pituitary gland. A β-adrenergic agonist (isoproterenol) occupies a β-adrenoceptor (β) to interact with a stimulatory guanyl nucleotide site (Ns); GTP interacts with Ν s to stimulate adenylate cyclase (cyclase) activity. A dopaminergic agonist (dopamine) occupies a D-2 dopamine receptor (D-2) to interact with the hypothetical inhibitory guanyl nucleotide component (Ni); GTP interacts with Ni to inhibit adenylate cyclase activity. GTP activates Ns only in the presence of α βadrenergic agonist and activates Ni only in the presence of a dopaminergic agonist. Cholera toxin selectively acts upon Ns. Increased formation of cAMP results in the enhanced release of IR-aMSH via an unknown mechanism(s). Newly formed cAMP can be hydrolyzed by intracellular phosphodiesterase(s) (PDE) to become 5'-AMP, or can be excreted into the extra­ cellular milieu.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

RECEPTORS

τ

I-ISOPROTERENOL (log M)

Figure 5. Comparison of the potency of isoproterenol in eliciting physiological and biochemical responses from the rat IL. Substantially lower concentrations of iso­ proterenol stimulate the release of IR-aMSH (aMSH) than are required to enhance cAMP accumulation by intact IL cells (cAMP), stimulate adenylate cyclase activity in cell-free homogenates of IL tissue (cyclase), or occupy the specific binding sites defined with IHYP (binding) (19).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2.

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ET

AL.

D-2 Dopamine

C y c l i c AMP and t h e I n t e r m e d i a t e

Receptor

4.

Lobe Dopamine R e c e p t o r

The r a t I L i s i n n e r v a t e d b y d o p a m i n e r g i c n e u r o n s . The somata o f t h e s e n e u r o n s a r e l o c a t e d i n t h e a r c u a t e n u c l e u s ; t h e i r a x o n s l e a v e t h e b r a i n v i a t h e p i t u i t a r y s t a l k and p r o j e c t t o t h e i n t e r m e d i a t e l o b e where t h e y t e r m i n a t e i n t h e v i c i n i t y o f the parenchymal I L c e l l s ( 2 6 ) . Baumgarten e t a l . d e s c r i b e 'synapse-like' connections between the dopaminergic nerve t e r m i n a l s and t h e p a r e n c h y m a l c e l l s o f t h e I L ( 2 7 ) . Several lines of evidence support the view that dopaminergic n e u r o t r a n s m i s s i o n o c c u r s w i t h i n t h e I L . F i r s t , dopamine i s t h e predominant catecholamine w i t h i n the I L (28, Saavedra, p e r s o n a l communication). S e c o n d , r a d i o l a b e l l e d dopamine i s t a k e n up and s t o r e d i n t h e I L ; s u b s e q u e n t l y , t h i s n e w l y s t o r e d dopamine c a n be r e l e a s e d from t h e n e r v e t e r m i n a l s ( 2 9 , 3 0 ) . The a v a i l a b l e evidence (which u n f o r t u n a t e l y does not include electrical r e c o r d i n g from t h e d o p a m i n e r g i t h a t i n v i v o the dopaminergi (30). Dopaminergic I n h i b i t i o n of Intermediate Lobe A d e n y l a t e Cyclase. The IL dopamine receptor can be studied with biochemical procedures. The r e c e p t o r i t s e l f c a n be i d e n t i f i e d in binding studies (31-33)* Using [3H]-spiroperidol, a dopamine a n t a g o n i s t from t h e b u t y r o p h e n o n e s e r i e s , t o i d e n t i f y binding sites and fluphenazine to define 'non-specific b i n d i n g ' , s p e c i f i c b i n d i n g s i t e s f o r the r a d i o l a b e l e d l i g a n d c a n be d e t e c t e d i n t h e i n t e r m e d i a t e l o b e ( F i g u r e 6 ) . The r a t I L c o n t a i n s 2 0 . 2 f m o l e o f h i g h a f f i n i t y ( K d = 0 . 3 nM) s p e c i f i c spiroperidol binding sites (33). The affinity of non­ radioactive dopaminergic agonists or antagonists can be i n f e r r e d from t h e i r a b i l i t y t o compete w i t h [ 3 H ] - s p i r o p e r i d o l f o r occupancy o f the s p e c i f i c b i n d i n g s i t e s ( e . g . see F i g u r e 7). L i s u r i d e i s t h e most p o t e n t o f t h e dopamine agonists t e s t e d and s p i r o p e r i d o l i s t h e most p o t e n t o f t h e dopamine antagonists tested. The dopamine receptor i n the IL regulates adenylate cyclase a c t i v i t y . S t i m u l a t i o n o f t h e I L dopamine receptor decreases the c a p a c i t y o f the parenchymal c e l l s to s y n t h e s i z e cAMP ( 1 2 , 1 9 - 2 2 , 3 7 ) . T h i s e f f e c t o f dopamine i s e s p e c i a l l y pronounced (and therefore amenable to experimental i n v e s t i g a t i o n ) when t h e I L a d e n y l a t e c y c l a s e a c t i v i t y h a s been increased with either isoproterenol or cholera t o x i n . The c h o l e r a t o x i n - t r e a t e d I L h a s p r o v e n t o be a n e s p e c i a l l y u s e f u l t i s s u e f o r i n v e s t i g a t i n g dopaminergic drugs s i n c e t h e i r e f f e c t upon t h e dopamine r e c e p t o r c a n be s e g r e g a t e d from any p o s s i b l e e f f e c t upon t h e β - a d r e n o c e p t o r ( F i g u r e 8 ) . Demonstration o f the dopaminergic i n h i b i t i o n o f I L adenylate c y c l a s e a c t i v i t y r e q u i r e s t h e p r e s e n c e o f GTP ( F i g u r e 2 , r i g h t ) .

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

RECEPTORS

3

[ H]-SPIROPERIDOL (nM)

Figure 6.

Binding of [3H]-spiroperidol to cell-free homogenates of the neurointermediate lobe of the rat pituitary gland.

Key: A, total (%) and nonspecific binding (i.e., binding in the presence of 2 μΜ fluphenazine) (O) was determined in the presence of the indicated concentrations of [3H]-spiroperidol; and B, specifically bound [3H]-spiroperidol (i.e., the difference between total and nonspecific bind­ ing) is shown as a function of the concentration of [3H]-spiroperidol. Left inset a plot of the data in Β according to Rosenthal's method', yields an apparent Kd of 0.2 nM and a maximal concentration of specific binding sites of 94.1 fmol/mg protein (this is equivalent to 20.2 fmol/NIL) (33).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2.

KEBABIAN

ET AL.

Ο

D-2 Dopamine

6

10"

Receptor

5

10"

43

4

3

10"

10"

DOPAMINE (M)

Figure 7. Competition between [3H]-spiroperidol and dopamine for occupancy of binding sites in the NIL of the rat pituitary gland. The amount of [3H]-spiro­ peridol bound to NIL tissue was determined in the presence of 2 μΜ fluphenazine (0), 2 μΜ fluphenazine and 100 μΜ GTP (4) or dopamine (at the indicated concentrations) in the absence (O) or presence (Φ) of 100 Μ GTP (33). μ

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

0 10-6

10-5

10-4

(-)SULPIRIDE(M)

Figure 8.

10-6

10-5

RECEPTORS

10-4

APOMORPHINE (M)

Competitive interaction between apomorphine and (—)-sulpiride in regulating adenylate cyclase activity in the IL (33,).

IL tissue was treated with cholera toxin and adenylate cyclase activity was determined. Enzyme activity was determined in the presence of the indicated concentrations of (-)-sulpiride alone (O) or in combination with apomorphine (3 μΜ, φ; 10 μΜ, Δ ; or 30 μΜ, A) (left). In a separate experiment, enzyme activity was determined in the presence of the indicated concentrations of apomorphine alone (O) or in combination with (-)-sulpiride (3 μΜ, Δ»" 30 μΜ, A) (right). In both experiments, data represent the mean ± SE (n = 3).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2.

KEBABIAN

ET

AL.

D-2 Dopamine

Receptor

45

r3Hl-Spiroperidol Identified the Intermediate Lobe Dopamine R e c e p t o r . B e c a u s e t h e s p i r o p e r i d o l b i n d i n g a s s a y and the adenylate c y c l a s e assay are performed under equivalent a s s a y c o n d i t i o n s , a c o m p a r i s o n between t h e a f f i n i t i e s o f d r u g i n t h e two a s s a y s y s t e m s seems a p p r o p r i a t e . F i g u r e 9 shows t h e a p p r o x i m a t e agreement between t h e r e s u l t s o b t a i n e d from t h e two experimental protocols. The similarity between the values o b t a i n e d from t h e two a s s a y s y s t e m s s u p p o r t s t h e hypothesis t h a t some o r a l l o f t h e s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g s i t e s a r e t h e dopamine r e c e p t o r s w h i c h when s t i m u l a t e d i n h i b i t adenylate cyclase a c t i v i t y . A "working hypothesis" o f the b i o c h e m i c a l o r g a n i z a t i o n o f t h e D - 2 dopamine r e c e p t o r i n t h e I L i s as f o l l o w s : t h e dopamine r e c e p t o r on t h e e x t e r i o r s u r f a c e o f a n I L c e l l i s c o u p l e d t o a d e n y l a t e c y c l a s e by an i n h i b i t o r y guanyl n u c l e o t i d e r e g u l a t o r y p r o t e i n ( N i ) , occupancy o f the r e c e p t o r by an a g o n i s t ( i . e s t i m u l a t i o n ) a l t e r s the p r o p e r t i e s o f N i so t h a t i n t r a c e l l u l a t h e r e b y i n h i b i t t h e enzym Dopamine a f f e c t s t h r e e p h y s i o l o g i c a l p r o c e s s e s i n t h e I L w h i c h , w i t h v a r y i n g d e g r e e s o f c e r t a i n t y , may be l i n k e d t o t h e a b i l i t y o f dopamine t o i n h i b i t t h e c a p a c i t y o f t h e I L t o s y n t h e s i z e cAMP. F i r s t the parenchymal c e l l s o f the r a t I L display spontaneous propagated e l e c t r i c a l spikes (38, 39). Dopamine d i m i n i s h e s t h e f r e q u e n c y , b u t n o t t h e a m p l i t u d e , o f t h e s e e l e c t r i c a l d i s c h a r g e s a p p a r e n t l y by s t i m u l a t i n g a D - 2 dopamine r e c e p t o r ( 4 0 ) . The p o s s i b l e i n v o l v e m e n t o f c y c l i c AMP i n t h i s i n h i b i t o r y p r o c e s s has not been i n v e s t i g a t e d . Second, dopamine i n h i b i t s t h e s p o n t a n e o u s r e l e a s e o f IR-aMSH ( F i g u r e 3) o r m e l a n o t r o p h i c hormones f r o m t h e I L ( 1 0 ) . The p r e s e n c e o f a r e c e p t o r f o r dopamine i n t h e I L o f many v e r t e b r a t e s p e c i e s h a s been i n f e r r e d from t h e a b i l i t y o f dopamine t o i n h i b i t the r e l e a s e o f m e l a n o t r o p h i c hormones o r IR-otMSH and t h e a b i l i t y o f dopaminergic antagonists to abolish t h i s effect o f dopamine (11 , 1 2 , 3 4 ) . The d o p a m i n e - i n d u c e d i n h i b i t i o n o f a d e n y l a t e cyclase activity and the dopamine-induced inhibition of electrical activity may participate in the dopaminergic i n h i b i t i o n o f the r e l e a s e o f IR-aMSH, T h i r d dopamine w i l l d i m i n i s h t h e a b i l i t y o f β - a d r e n e r g i c a g o n i s t s t o enhance e i t h e r a d e n y l a t e c y c l a s e a c t i v i t y o r t h e r e l e a s e o f IR-aMSH (1_2, _35, 37). The dopamine-induced decrease in the β-adrenergic enhancement o f cAMP s y n t h e s i s seems l i k e l y t o be i n v o l v e d i n t h i s phenomenon ( 3 8 ) . When the potency of dopaminergic agonists upon p r e p a r a t i o n s o f i n t a c t I L c e l l s i s compared t o t h e p o t e n c y o f t h e same compounds i n c e l l - f r e e a s s a y s y s t e m s ( i . e . a d e n y l a t e c y c l a s e o r s p i r o p e r i d o l b i n d i n g , a s t r i k i n g d i s c r e p a n c y emerges (Figure 10). F o r each o f the a g o n i s t s t e s t e d , the i n t a c t c e l l s respond to approximately 1/100th the c o n c e n t r a t i o n o f drug t h a t i s r e q u i r e d t o e l i c i t a comparable e f f e c t from the c e l l - f r e e homogenate. At the present time the hypothesis that the pituitary dopamine r e c e p t o r can e x i s t i n two " s t a t e s " of

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

-3

I ζ

-4

Ω Ζ

m

-5

RECEPTORS

1. Spiroperidol 2. Fluphenazine 3. Cis-Flupenthixol 4. Lisuride 5. Bromocriptine 6. Trans-Flupenthixol 7. ( - )-Sulpinde 8. Apomorphine 9. Dopamine 10. (+)-Sulpiride 11. LY 141865 12. Norepinephrine

—Ι

Ο g oc LU Ω­

- 6 h

Ο

-7

Ο

ce

-10

- 9

- 8

- 7

- 6

- 5

AFFINITY FROM CYCLASE (log M)

Figure 9. Apparent affinity constants of agonists and antagonists for the dopamine receptor in the neurointermediate lobe of the rat pituitary gland. In studies of [3H]-spiroperidol binding, the apparent affinity constant of an agonist or antagonist was determined on the basis of the ability of the compound to compete with [3H]-spiroperidol for occupancy of the specific binding site (IL contributes 86% of the specific [3H]-spiroperidol binding sites in the NIL) and assuming a competitive interaction between the radiolabelled ligand and the nonradioactive compound. In studies of adenylate cyclase activity the apparent affinity constant of an agonist was the concentration of agonist producing half of its maximal inhibition of the enzyme activity in cholera toxin treated IL tissue. The apparent affinity constant of each antagonist was determined on the basis of the ability of the compound to reverse the apomorphine-induced inhibition of adenylate cyclase activity, by assuming a competitive interaction between apomorphine and each antagonist. Each value is the mean of determinations from three or more separate experiments (33).

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2.

KEBABIAN

E T A L .

D-2 Dopamine

Receptor

47

APOMORPHINE (LOG M)

Figure 10.

Comparison of the potency of apomorphine as a dopaminergic agonist upon intact IL cells or cell-free homogenate of IL tissue.

For each response examined, inhibition of isoproterenol-stimulated cAMP accumulation by intact cells ( • ); inhibition of basal release of IR-aMSH by intact cells ( φ ); occupancy of specific [3H]-spiroperidol binding sites in a cell-free homogenate (O); and inhibition of isoproterenol-stimulated adenylate cyclase activity in a cell-free homogenate (M), the effect achieved with the indicated concentration of apomorphine is expressed as a percentage of the maximal effect of apomorphine (33J.

American Chemical Society Library 1155 13th St. N. w. Washington. 0. C. 20036 In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

48

DOPAMINE

RECEPTORS

differing affinity f o r agonists i s the most attractive explanation f o r t h i s discrepancy (4j_, 4 2 ; see a l s o Caron, t h i s volume). Concluding Remarks The IL o f the r a t p i t u i t a r y gland possesses a dopamine receptor amenable to experimental investigation. Two interesting points have emerged from the combined p h y s i o l o g i c a l , pharmacological and biochemical approach towards this tissue. F i r s t , the p h y s i o l o g i c a l a c t i o n s o f dopamine upon the IL "seem not to i n v o l v e e i t h e r the s t i m u l a t i o n o f an a d e n y l y l c y c l a s e or the accumulation of i n t r a c e l l u l a r c y c l i c AMP (1_)." Furthermore, the IL dopamine receptor i s stimulated by ergots (e.g. l i s u r i d e or bromocriptine) and blocked by the s u b s t i t u t e d benzamides (e.g. ( - ) - s u l p i r i d e ) . Therefore, the IL dopamine receptor ressembles the D-2 dopamine receptor i n the c l a s s i f i c a t i o n schema o IL has become a u s e f u designed to i d e n t i f y s e l e c t i v e antagonists o f e i t h e r the D-1 or the D-2 r e c e p t o r . The a b i l i t y o f LY 141865 ( f o r s t r u c t u r e see F i g u r e 11) to s e l e c t i v e l y stimulate the D-2 receptor (36) and the a b i l i t y o f YM-09151-2 (Figure 11) t o s e l e c t i v e l y block t h i s receptor (43) were discovered i n experiments u t i l i z i n g the IL dopamine receptor as the model D-2 receptor and the g o l d f i s h r e t i n a as the model D-1 r e c e p t o r . These compounds together with the s e l e c t i v e D-2 a g o n i s t s , RU 24213 and RU 2 4 9 2 6 , and the s e l e c t i v e D-2 antagonist, domperidone, ( f o r s t r u c t u r e s see Figure 11 ) provide u s e f u l pharmacological t o o l s f o r i d e n t i f y i n g other D-2 receptors ( 4 4 - 4 8 ) . Two categories of dopamine receptor exist i n the cardiovascular system. These two receptors have been designated as the DA-1 and the DA-2 receptors ( 4 9 , see Goldberg and K o h l i , t h i s volume). The D-2 and the DA-2 receptors have very s i m i l a r pharmacological properties. However, the DA-1 receptor and the D-1 receptor are perceived by Goldberg and h i s colleagues as being d i s t i n c t e n t i t i e s . In p a r t i c u l a r , the DA-1 receptor i s blocked by ( + ) - s u l p i r i d e ; ( + ) - s u l p i r i d e i s the most potent and most s e l e c t i v e antagonist o f the DA-1 receptor ( 4 9 ) . In contrast, the D-1 receptor i s a t best only weakly antagonized by t h i s compound ( 4 6 , 4 7 ) . Although ( + ) - s u l p i r i d e i s the most potent and most s e l e c t i v e antagonist o f the DA-1 receptor i n i n v i t r o experiments, when i t i s t e s t e d i n v i t r o upon s t r i p s o f i s o l a t e d canine mesenteric a r t e r y , i t i s a weak dopamine antagonist ( 5 0 ) . S i m i l a r l y , ( + ) - s u l p i r i d e i s a weak antagonist o f the D-1 receptor i n the bovine p a r a t h y r o i d gland (see Brown and Dawson-Hughes, t h i s volulme). In the f u t u r e , i t w i l l be important to i d e n t i f y the b a s i s f o r the d i f f e r e n c e between the i n v i v o and i n v i t r o potencies o f s u l p i r i d e . However, i n the context o f the present d i s c u s s i o n about the number and p r o p e r t i e s o f the c a r d i o v a s c u l a r dopamine r e c e p t o r s ,

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

KEBABIAN

Figure IL

ET AL.

D-2 Dopamine

Receptor

Structures of agonists (top) and antagonists (bottom) of the D-2 receptor.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

50

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i t seems worthwhile to consider four s i m i l a r i t i e s between the DA-1 and the D-1 receptors. F i r s t , (+)-bulbocapnine i s an antagonist capable o f b l o c k i n g e i t h e r the D-1 or the DA-1 receptor; t h i s compound i s s u b s t a n t i a l l y l e s s potent as an antagonist o f the DA-2 receptor and i s devoid of antagonist a c t i v i t y upon the D-2 receptor (51, 52, Itoh, Goldman and Kebabian, unpublished o b s e r v a t i o n s ) . Second, SKF 38393 i s an agonist upon the D-1 and the DA-1 receptor, yet i t does not e l i c i t the p h y s i o l o g i c a l responses o c c u r r i n g as a consequence of s t i m u l a t i o n of e i t h e r the D-2 or the DA-2 receptor (53) * T h i r d , LY 141865, a s e l e c t i v e D-2 agonist (36), f a i l s to stimulate the DA-1 receptor i n the r e n a l v a s c u l a t u r e of the r a t (54). Fourth, domperidone i s an antagonist o f e i t h e r the D-2 or the DA-2 receptor which does not block e i t h e r the D-1 or the DA-1 receptors (45, 4B). These four s i m i l a r i t i e s between the D-1 and the DA-1 receptor suggest that they are extremely s i m i l a r i f not i d e n t i c a properties. Clearly, i b a s i s f o r the d i f f e r e n c e i n the i n v i v o and i n v i t r o potencies o f the s u b s t i t u t e d benzamides. However, at present i t may be premature to discount the s t r i k i n g s i m i l a r i t i e s between the D-1 and the DA-1 r e c e p t o r s . The IL also serves as a model for dopaminergic neurotransmission i n the b r a i n ; the concepts f i r s t e l u c i d a t e d i n the IL can be a p p l i e d to the b r a i n . For example, i n the neostriatum the i n t e r a c t i o n between dopamine and a D-2 receptor i n h i b i t s the stimulated e f f l u x of cAMP (and by i n f e r e n c e the enhancement of adenylate c y c l a s e a c t i v i t y ) o c c u r r i n g as a consequence of s t i m u l a t i o n of the D-1 receptor 55, 56). The types o f dopamine receptors i n the CNS and t h e i r p h y s i o l o g i c a l f u n c t i o n are discussed i n t h i s volume by Stoof.

Literature Cited 1. 2. 3.

4.

5. 6. 7.

Kebabian, J . W.; Calne, D. B. Nature 1979, 277, 93-96. Kebabian, J . W.; Calne, D. B.; Kebabian, P. R. Commun. i n Psychopharmacol. 1977, 1, 311-318. Kebabian, J. W. "Dopamine, Advances in Biochemical Psychopharmacology Volume 19"; Raven Press: New York, 1978; pp 131-154. Borgeat, P.; Chavancy, G.; Dupont, Α.; Labrie, F.; Arimura, Α.; S c h a l l y , Α. V. Proc. N a t l . Acad. S c i . USA 1972, 69, 2677-2681. D e C a m i l l i , P.; Macconi, D.; Spada, A. Nature 1979, 278, 252-254. G i a n n a t t a s i o , G.; De F e r r a r i , M. E.; Spada, A. L i f e S c i . 1981, 28, 1605-1612. Thorner, M. O.; Hackett, J . T.; Murad, F.; MacLeod, R. M. Neuroendocrinology 1980, 31, 309-402.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26.

27. 28. 29. 30. 31.

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AL.

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51

Baker, B. "Handbook o f Physiology: S e c t i o n 7, Volume 4, Part I , The Pituitary Gland and I t s Neuroendocrine Control"; American Physiological Society: Bethesda, Maryland, 1974; pp 45-80. Dube, D.; L i s s i t z k y , J . C.; L e c l e r c , R.; P e l l e t i e r , G. Endocrinology 1978, 102, 1283-1291. Bower, Α.; Hadley, M. E.; Hruby, V. J . Science 1974, 184, 70-72. Morgan, C. M.; Hadley, M. E. Neuroendocrinology 1976, 21, 10-19. Munemura, M.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1980, 106, 1795-1803. Baker, Β. I . J. E n d o c r i n o l . 1974, 63, 533-538. E i p e r , Β. Α.; Mains, R. E. Endocrine Rev. 1980, 1, 1-27. S i l v e r s , W. K. "The Coat Colors o f Mice"; S p r i n g e r - V e r l a g : New York, 1979; pp 1-5 Geschwind, I . I . Horm. Res. 1972, Rudman, D.; Chawla, R. K.; H o l l i n s , B. M. J . Biol. Chem. 1979, 254, 10102-10108. Browne, C. Α.; Bennett, H. P. J . ; Solomon, S. Biochemistry 1981, 20, 4538-4546. Cote, T.; Munemura, M.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1980, 107, 108-116. Cote, T. E.; Grewe, C. W.; Kebabian, J . W. Endocrinology 1982, 110, 805-811. Muniere, H. and L a b r i e , F. Eur. J. Pharmacol. 1982, 81, 411-420. Cote, T. E.; Grewe, C. W.; Tsuruta, K.; Stoof, J . C.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1982, 110, 812-819. Tsuruta, K.; Grewe, C. W.; Cote, T. E.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1982, 110, 1133-1139. Baker, B.I. J . E n d o c r i n o l . 1974, 63, 533-538. Tomiko, S. Α.; Taraskevich, P. S.; Douglas, W. W. Neuroscience 1981, 6, 2259-2267. L i n d v a l l , O.; Bjorklund, A. "Handbook o f Experimental Pharmacology, Volume 9"; Plenum Press: New York, 1978; pp 139-231. Baumgarten, H. G.; Bjorklund, Α.; H o l s t e i n , A. F.; Nobin, A. Z. Z e l l f o r s c h . 1972, 126, 483-517. Saavedra, J . M.; P a l k o v i t s , M.; K i z e r , J . S.; Brownstein, M.; Z i v i n , J . J. Neurochem. 1975, 25, 257-260. Tilders, F. J . H.; Mulder, A. H.; Smelik, P. G. Neuroendocrinology 1975, 18, 125-130. T i l d e r s , F. J . H.; Van Der Woude, Η. Α.; Swaab, D. F.; Mulder, A. H. B r a i n Res. 1979, 171, 425-435. S i b l e y , D. R.; Creese, I . Endocrinology 1980, 107, 14051419.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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52 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50.

51.

52. 53. 54. 55. 56.

RECEIVED

RECEPTORS

Stefanini, E.; Devoto, P.; Marchisio A . M.; V e r n a l e o n e , A . M.; Collu, R . Life Sci. 1 9 8 0 , 2 6 , 5 8 3 - 5 8 7 . F r e y , Ε . Α . ; C o t e , T . E.; Grewe, C . W . ; K e b a b i a n , J. W. Endocrinology 1 9 8 2 , 110, 1 8 9 7 - 1 9 0 4 . Munemura, M.; C o t e , T . E.; Tsuruta, K.; Eskay, R. L.; K e b a b i a n , J. W. Endocrinology 1980, 1 9 7 , 1676-1683. C o t e , T . E.; Grewe, C . W . ; K e b a b i a n , J. W. E n d o c r i n o l o g y 1981, 108, 420-426. Tsuruta, K.; F r e y , Ε . Α . ; Grewe, C . W . ; C o t e , T . E.; E s k a y , R . L.; K e b a b i a n , J. W. N a t u r e 1 9 8 1 , 2 9 2 , 4 6 3 - 4 6 5 . M u n i e r e , H.; Labrie, F. Life Sci. 1 9 8 2 , 3 0 , 9 6 3 - 9 6 8 . D o u g l a s , W. W . ; Taraskevich, P. S. J. Physiol. (London) 1978, 285, 171-184. D o u g l a s , W. W . ; Taraskevich, P. S. J. Physiol. (London) 1980, 209, 623-630. D o u g l a s , W. W . ; Taraskevich, P . S. J. Physiol. (London) 1 9 8 2 , 326, 2 0 1 - 2 1 1 De L e a n , Α . ; Kilpatrick 1 9 8 2 , 110, 1 0 6 4 - 1 0 6 6 . S i b l e y , D . R . ; De L e a n , Α . ; C r e e s e , I. J. Biol. Chem. 1982, 257, 6351-6361. G r e w e , C . W . ; F r e y , Ε . Α . ; C o t e , T . E.; K e b a b i a n , J. W. Eur. J. P h a r m a c o l . 1 9 8 2 , 81, 1 4 9 - 1 5 2 . E u v a r d , C; Ferland, L.; Dipaolo, T.; Beaulieu, M.; Labrie, F.; Oberlander, C.; Raynaud, J.P.; Boissier, J.R. Neuropharmacology, 1980, 19, 379. Denef, C.; Follebouckt, J.-J. Life Sci. 1978, 23, 4 3 1 . W a t l i n g , K.J.; D o w l i n g , J.E.; Iversen, L.L. Nature, 1979, 281, 578. W a t l i n g , K.J.; D o w l i n g , J.E. J. Neurochem. 1 9 8 1 , 3 5 , 5 5 9 . Kohli, J.D.; Glock, D.; Goldberg, L.I. Eur. J. Pharmacol. 1983, in press. Goldberg, L.I.; Kohli, J.D. Commun. Psychopharmacol. 1 9 7 9 , 3, 4 4 7 - 4 5 6 . Kohli, J.D.; Takeda, H.; Ozaki, N.; Goldberg, L.I. "Advances in the Biosciences: Volume 20, Peripheral Dopaminergic Receptors"; Pergamon P r e s s : Oxford, U.K., 1 9 7 9 ; pp 1 4 3 - 1 4 9 . Goldberg, L.I.; Musgrave, G.E.; Kohli, J.D. "Sulpiride and O t h e r B e n z a m i d e s " ; Italian Brain Research Foundation Press: Milan, Italy 1 9 7 9 ; pp 7 3 - 8 1 . Shepperson, N.B.; D u v a l , N.; M a s s i n g h a m , R . ; L a n g e r , S . Ζ . J. P h a r m a c o l . E x p . T h e r . 1 9 8 0 , 2 2 1 , 7 5 3 - 7 6 1 . Setler, P.E.; Sarau, H.M.; Zirkle, C.L.; Saunders, H.L. Eur. J. P h a r m a c o l . 1 9 7 8 , 5 0 , 4 1 9 . H a h n , R.A.; M c D o n a l d , B.; Martin, M. J. Pharmacol. Exp. T h e r . 1983, 224, 206-214. S t o o f , J. C.; K e b a b i a n , J. W. N a t u r e 1 9 8 1 , 2 9 4 , 3 6 6 - 3 6 8 . S t o o f , J. C.; K e b a b i a n , J. W. Brain R e s . 1 9 8 2 , 2 5 0 , 2 6 3 270. November 4, 1982

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Commentary: The D A _ Dopamine Receptor in the Anterior and Intermediate Lobes of the Pituitary Gland F . L A B R I E , H . M E U N I E R , M . G O D B O U T , R. V E I L L E U X , V . G I G U E R E , L . P R O U L X - F E R L A N D , T . DI P A O L O , and V . R A Y M O N D Le Centre Hospitalier de l'Université Laval, Department of Molecular Endocrinology, Quebec G 1 V 4G2, Canada

Dopamine release d i b u l a r neurons is the main f a c t o r of hypothalamic o r i g i n which exerts a d i r e c t i n h i b i t o r y e f f e c t on p r o l a c t i n s e c r e t i o n in the a n t e r i o r p i t u i t a r y gland. The dopamine receptor on p i t u i t a r y mammotrophs is n e g a t i v e l y coupled to adenylate c y c l a s e . Estrogens i n h i b i t while androgens and progestins as w e l l as androgens facilitated the activity of dopamine on p r o l a c t i n s e c r e t i o n by an a c t i o n at a post-receptor site. The p e p t i d i c c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) i s a potent s t i m u l a t o r of adenylate c y c l a s e activity and peptide s e c r e t i o n in pars intermedia cells. The intermediate lobe i s thus c o n t r o l l e d by two stimulatory r e c e p t o r s , namely CRF and β-adrenerg i c while it is i n h i b i t e d by a dopaminergic receptor negatively coupled to adenylate c y c l a s e . The nomen­ c l a t u r e DΑ+, DA_ and DA0 is proposed f o r dopamine receptors which are p o s i t i v e l y , n e g a t i v e l y or un­ coupled, r e s p e c t i v e l y , to adenylate c y c l a s e . The dopamine receptors i n both the a n t e r i o r and interme­ d i a t e lobes of the p i t u i t a r y gland are n e g a t i v e l y coupled to adenylate cyclase and are thus t y p i c a l of the DA_ type.

The predominant r o l e of dopamine i n normal b r a i n f u n c t i o n s as w e l l as i n diseases such as Parkinson's disease and schizophrenia i s a strong stimulus f o r research on the dopamine receptor and i t s mechanism of a c t i o n . Since dopamine appears to be the main i f not the e x c l u s i v e f a c t o r of hypothalamic o r i g i n a c t i n g as i n h i b i t o r of prolactin secretion 2) and changes of p r o l a c t i n s e c r e t i o n can be measured with p r e c i s i o n i n vivo and i n v i t r o , the a n t e r i o r p i t u i t a r y gland i s an a t t r a c t i v e model f o r a dopaminergic recep­ t o r . Another model of great i n t e r e s t i s the intermediate lobe of the p i t u i t a r y gland, a pure population of dopamine-sensitive c e l l s 0097-6156/83/0224-005 3 $06.00/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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s p e c i a l i z e d i n the s e c r e t i o n of proopiomelanocortin-related p e p t i ­ des, s p e c i a l l y α-MSH (a-raelanotropin) and 3-endorphin ( 3 ) . Both mammotrophs of the r a t a n t e r i o r p i t u i t a r y gland and pars interme­ d i a c e l l s can e a s i l y be grown i n monolayer c u l t u r e where changes of dopamine receptors can be c o r r e l a t e d with biochemical parame­ ters such as changes i n c y c l i c AMP l e v e l s and peptide s e c r e t i o n i n t o the medium. Kebabian et a l . ( t h i s volume) present a c l e a r and p r e c i s e r e ­ view of the l a r g e body of biochemical data obtained by t h e i r group on the c o n t r o l of pars intermedia c e l l a c t i v i t y by 3-adrenergic agents and dopamine during the l a s t years (4-13). Since the p e p t i d i c c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) i s a potent s t i m u l a t o r of pars intermedia c e l l a c t i v i t y (14^ 15, 16), we w i l l complement t h i s review by summarizing recent data on the mechanism of a c t i o n of CRF i n the intermediate lobe as w e l l as i t s i n t e r a c t i o n with 3adrenergic and dopaminergic agents Data w i l l a l s o be presented on the coupling of the t r o l l i n g prolactin secretio the marked i n t e r a c t i o n s of t h i s receptor with gonadal s t e r o i d s . F i n a l l y , taking i n t o account a l l a v a i l a b l e evidence, we w i l l pro­ pose to use the nomenclature DA+, DA^ and DA f o r dopamine r e ­ ceptors coupled p o s i t i v e l y , n e g a t i v e l y or uncoupled, r e s p e c t i v e l y , to adenylate c y c l a s e . The dopamine receptors i n the a n t e r i o r and intermediate lobes of the p i t u i t a r y gland are examples of dopamine receptors n e g a t i v e l y coupled to adenylate c y c l a s e (DA_ t y p e ) . Q

The dopamine receptor i n the a n t e r i o r p i t u i t a r y gland i s negative­ l y coupled to adenylate c y c l a s e The r o l e of c y c l i c AMP as modulator of p r o l a c t i n s e c r e t i o n was f i r s t suggested by the f i n d i n g of a s t i m u l a t o r y e f f e c t of c y c l i c AMP d e r i v a t i v e s (17-22) and i n h i b i t o r s of c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y such as t h e o p h y l l i n e and IBMX (22-26) on the s e c r e t i o n of t h i s hormone. More convincing evidence supporting a r o l e of c y c l i c AMP i n the a c t i o n of dopamine on pro­ l a c t i n s e c r e t i o n had to be obtained, however, by measurement of adenohypophysial adenylate c y c l a s e a c t i v i t y or c y c l i c AMP accumu­ l a t i o n under the i n f l u e n c e of the catecholamine. As i l l u s t r a t e d i n F i g . 1, a d d i t i o n of 100 nM dopamine to male r a t h e m i p i t u i t a r i e s led to a rapid i n h i b i t i o n of c y c l i c AMP accumulation, a maximal e f f e c t (30% i n h i b i t i o n ) being already obtained 5 min a f t e r a d d i ­ t i o n of the catecholamine. Thus, while dopamine i s w e l l known t o stimulate adenylate cyclase a c t i v i t y i n the striatum (27, 28), i t s e f f e c t at the adenohypophysial l e v e l i n i n t a c t c e l l s i s i n h i b i t o ­ r y . Dopamine has also been found to exert p a r a l l e l i n h i b i t o r y e f f e c t s on c y c l i c AMP l e v e l s and p r o l a c t i n r e l e a s e i n ovine adeno­ hypophysial c e l l s i n c u l t u r e (29) and p u r i f i e d r a t mammotrophs (30). Using paired h e m i p i t u i t a r i e s obtained from female r a t s , Ray and W a l l i s (22) have found a r a p i d i n h i b i t o r y e f f e c t of dopamine on c y c l i c AMP accumulation to approximately 75% of c o n t r o l .

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Figure 1. Time course of the effect of dopamine (100 nM) on cyclic AMP accumulation in male rat anterior pituitaries (54). Key: O — O, control; and ·—·, with dopamine.

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As much as higher concentrations of dopamine are required to stimulate s t r i a t a l adenylate cyclase a c t i v i t y as compared to the a f f i n i t y of the drug f o r the a g o n i s t i c s i t e s of the receptor, the s e n s i t i v i t y of the a n t e r i o r p i t u i t a r y adenylate cyclase to dopamine agonists i n both human prolactinoma (26) and r a t adenohypophys i a l homogenate (31) i s lower than the potency of the compounds to i n h i b i t p r o l a c t i n s e c r e t i o n i n i n t a c t c e l l s ( 2 ) . This v a r i a b l e loss of s e n s i t i v i t y upon c e l l homogeneization i s a phenomenon frequently observed i n other systems (32). The reports that DA d i d not show s i g n i f i c a n t changes of cyc l i c AMP l e v e l s i n the adenohypophysis (33, 34, 35) have l e d to the suggestion that the p i t u i t a r y DA receptor i s a prototype of D receptors (D ) not coupled to adenylate cyclase (36). These negat i v e r e s u l t s were however obtained using a very heterogeneous t i s s u e , the t o t a l a n t e r i o r p i t u i t a r y gland. The background of c y c l i c AMP contributed by the f i v e other c e l l types could e a s i l y mask the changes i n c y c l i trophs under the i n f l u e n c te obtained from female r a t s containing a higher proportion of mammotrophs than the t i s s u e used i n previous studies (33, 34), Giannattasio et a l . (31) could demonstrate a c l e a r GTP-dependent i n h i b i t i o n of adenylate cyclase a c t i v i t y . Since c y c l i c AMP d e r i v a t i v e s and i n h i b i t o r s of c y c l i c nucleot i d e phosphodiesterase stimulate p r o l a c t i n r e l e a s e (17, 37, 38) and dopamine i s a potent i n h i b i t o r of p r o l a c t i n s e c r e t i o n (1_, 2> 39), i t i s not s u r p r i s i n g that the catecholamine does not s t i m u l a te the adenylate cyclase system. On the contrary, the data summar i z e d above show that the p i t u i t a r y DA receptor i s n e g a t i v e l y coupled to adenylate c y c l a s e . The p i t u i t a r y DA receptor i s thus a t y p i c a l DA_-receptor (40, 41). In view of the m u l t i p l i c i t y of f a c t o r s involved i n the c o n t r o l of p r o l a c t i n s e c r e t i o n , i n c l u d i n g sex s t e r o i d s , i t i s l i k e l y that mechanisms other than c y c l i c AMP are involved (39, 42)· It does however appear that i n h i b i t i o n of c y c l i c AMP formation by dopamine i s a key element i n a m u l t i f a c t o r i a l c o n t r o l system responsible f o r the f i n e tuning of p r o l a c t i n secretion.

2

2

Modulation of the a n t e r i o r p i t u i t a r y dopamine receptor by gonadal steroids Administration of estrogens i s w e l l known to cause an i n c r e a se i n plasma p r o l a c t i n (PRL) l e v e l s i n man (43, 44 ) as w e l l as i n the r a t (45-48). This stimulatory e f f e c t of estrogens i s a l s o observed i n v i t r o i n a n t e r i o r p i t u i t a r y gland expiants (49), tumor a l adenohypophysial c e l l s (50) and normal r a t a n t e r i o r p i t u i t a r y c e l l s i n primary c u l t u r e (39, 40, 42, 51). Seventeen-3-estradiol ( E ) does not only stimulate basal and TRH-induced PRL s e c r e t i o n i n r a t a n t e r i o r p i t u i t a r y c e l l s i n c u l t u r e but i t can also reverse almost completely the i n h i b i t o r y e f f e c t of dopamine (DA) agonists on PRL release (40). 2

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Our data have shown that the non-aromatisable androgen DHT and progestins can act d i r e c t l y at the p i t u i t a r y l e v e l at p h y s i o l o g i c a l concentrations to i n h i b i t spontaneous PRL s e c r e t i o n and reverse the well-known s t i m u l a t o r y e f f e c t of E (3^9, 40_, 4_2, 51). In a d d i t i o n , the e f f e c t of E , DHT and progestins i s observed, not only on spontaneous and TRH-induced PRL s e c r e t i o n , but a l s o i n the presence of IBMX. This l a s t observation suggests that the marked modulatory e f f e c t s of sex s t e r o i d s are exerted at a step f o l l o w i n g c y c l i c AMP formation. Ihese r e s u l t s prompted us to suggest a model f o r the s i t e of the modulatory r o l e of sex s t e r o i d s on PRL s e c r e t i o n i n the female r a t ( F i g . 2). TRH probably acts through a c t i v a t i o n of adenylate cyclase, the e f f e c t of TRH being mimicked by c y c l i c AMP d e r i v a t i ves and i n h i b i t o r s of c y c l i c n u c l e o t i d e phosphodiesterase (17, 52, 53) ( F i g . 2) while DA receptors are n e g a t i v e l y coupled to adenylate c y c l a s e (26, 54). Estrogens exert t h e i r stimulatory a c t i o n on TRH receptors as w e l l a androgens and progestin post-receptor step. This schematic r e p r e s e n t a t i o n does not exclude other elements which are probably involved i n the c o n t r o l of the mammotroph c e l l but i t only i n d i c a t e s the main s i t e s of s t e r o i d a c t i o n using the p r e s e n t l y a v a i l a b l e evidence. C a and the phosp h a t i d y l i n o s i t o l response are also i n t i m a t e l y involved i n the s e c r e t i o n mechanisms i n mammotrophs. The present data i n d i c a t e that besides p r o v i d i n g a b e t t e r knowledge of the c o n t r o l of p r o l a c t i n s e c r e t i o n , the tuberoinfund i b u l a r system can a l s o be used as a model f o r other l e s s a c c e s s i b l e dopaminergic systems i n the c e n t r a l nervous system. The i n t e rest of such studies i n the r a t on the i n t e r a c t i o n of sex s t e r o i d s with b r a i n catecholaminergic systems i s strengthened by the observ a t i o n that estrogen a d m i n i s t r a t i o n to male or female p a t i e n t s r e c e i v i n g n e u r o l e p t i c s can f a c i l i t a t e the appearance of parkinsonian symptoms (55). Moreover, treatment with estrogens has been found to improve the symptoms of t a r d i v e dyskinesias induced by chronic treatment with L-Dopa or n e u r o l e p t i c s (56). The abovementioned e f f e c t s of estrogen treatment i n the human are compatible with an antidopaminergic a c t i o n and open the p o s s i b i l i t y of c l i n i c a l a p p l i c a t i o n s of sex hormones i n the treatment of neurolog i c a l as w e l l as p s y c h i a t r i c d i s e a s e s . 2

2

2 +

The dopamine receptor i n the intermediate lobe of the p i t u i t a r y gland i s n e g a t i v e l y coupled to adenylate c y c l a s e As i l l u s t r a t e d i n F i g . 3A, dopamine leads to a 30% (p < 0.01) i n h i b i t i o n of basal c y c l i c AMP l e v e l s i n pars intermedia c e l l s at an E D value of 5.0 nM. An almost i d e n t i c a l potency of dopamine i s observed on the elevated c y c l i c AMP concentration induced by simultaneous incubation with 30 nM ( - ) i s o p r o t e r e n o l ( F i g . 3B). S i m i l a r i n h i b i t o r y e f f e c t s of dopamine are observed i n the presence of a phosphodiesterase i n h i b i t o r , isobutylmethylxanthine, thus 5 Q

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Figure 2. Representation of the site of interaction of sex steroids, TRH, and dopamine on PRL secretion in the anterior pituitary gland according to the data obtained in the female rat.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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B

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DOPAMINE (LOG M)

Figure 3. Effect of increasing concentrations of dopamine on basal (A) and (—)isoproterenol-induced (B) cyclic AMP levels in rat pars intermedia cells in culture. Cells were incubated for 30 min in DM EM containing 5 mM HEPES, 100 μΜ ascorbic acid, and the indicated concentrations of dopamine alone (A). In B, 30 nM ( — isoproterenol was present during the last 4 min of incubation. Data are expressed as percent of control (in the absence of dopamine). Control cyclic AMP levels were 0.96 ± 0.10 and 5.29 ± 0.13 pmol/2 χ 10 cells in the absence (A) or presence (B) of 30 nM (—isoproterenol, respectively (4\). 5

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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i n d i c a t i n g that the changes i n c y c l i c AMP l e v e l s are due to p a r a l l e l i n h i b i t i o n of adenylate cyclase a c t i v i t y rather than to stimul a t i o n of c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y . Apomorphine, the prototype of dopaminergic agonists ( 5 7 ) , i s approximat e l y 3 times more potent than dopamine i n i n h i b i t i n g pars i n t e r media c y c l i c AMP l e v e l s at a K value of 1.5 nM ( 4 1 ) . Ergot a l k a l o i d s which act as potent dopaminergic agonists on p r o l a c t i n r e l e a s e i n the a n t e r i o r p i t u i t a r y gland ( 2 ) a l s o show potent agon i s t i c a c t i v i t y i n the intermediate lobe: l i s u r i d e , 2-bromo-cte r g o c r y p t i n e , p e r g o l i d e , dihydroergocryptine and l e r g o t r i l e i n h i b i t b a s a l c y c l i c AMP l e v e l s at KQ values of 0.05, 0.20, 0 . 2 7 , 0.50 and 2.7 nM, r e s p e c t i v e l y . (-)Norepinephrine and ( ^ e p i n e p h r i ne are l e s s potent, half-maximal i n h i b i t o r y e f f e c t s on c y c l i c AMP l e v e l s being measured at 3 0 and 5 0 nM, r e s p e c t i v e l y . P r o p r a n o l o l ( 1 0 0 nM) was present during i n c u b a t i o n with (-)epinephrine and (-)norepinephrine i n order to block t h e i r i n t e r a c t i o n with the 3adrenergic receptor whic t i o n i n pars intermedi The s p e c i f i c i t y of the dopamine receptor was f u r t h e r studied with a s e r i e s of dopaminergic antagonists of w e l l known pharmacological activity. The 3 0 - 4 0 % i n h i b i t o r y e f f e c t of 10 nM dopamine was completely reversed by the a d d i t i o n of i n c r e a s i n g concentrat i o n s of the potent n e u r o l e p t i c s (+)butaclamol ( K = 1.5 nM) and ( - ) s u l p i r i d e ( K = 0.5 nM) while t h e i r pharmacologically weak enantiomers (-)butaclamol and ( + ) s u l p i r i d e were 86 and 167 times l e s s potent, r e s p e c t i v e l y . The n e u r o l e p t i c s s p i r o p e r i d o l , t h i o properazine, domperidone, h a l o p e r i d o l , fluphenazine and pimozide completely reversed the i n h i b i t o r y e f f e c t of dopamine at low K values ranging from 0.02 to 0.8 nM ( 4 1 ) . D

N

N

N

Coupling o f dopamine receptors with adenylate c y c l a s e : DA+, DA_ and PA receptors Q

Dopamine can thus be added to the l i s t of hormones and neurot r a n s m i t t e r s which can stimulate or i n h i b i t c y c l i c AMP formation, depending upon t h e i r t i s s u e of a c t i o n . Thus, while dopamine stimul a t e s c y c l i c AMP formation i n parathyroid c e l l s , s u p e r i o r c e r v i c a l g a n g l i a , r e t i n a and s t r i a t a l t i s s u e ( 2 7 , 5 8 - 6 1 ) , i t i n h i b i t s the accumulation of the c y c l i c n u c l e o t i d e i n c e l l s of the intermediate and a n t e r i o r lobes of the p i t u i t a r y gland. Opposite e f f e c t s on the c y c l i c AMP system are a l s o found with LHRH which stimulates and i n h i b i t s c y c l i c AMP l e v e l s i n the a n t e r i o r p i t u i t a r y gland ( 6 2 ) and ovary ( 6 3 ) , r e s p e c t i v e l y . S i m i l a r l y , alpha-adrenergic agents show opposite e f f e c t s on c y c l i c AMP formation i n b r a i n ( 6 4 ) and p l a t e l e t s ( 6 5 ) . PGE, stimulates c y c l i c AMP formation i n the anter i o r p i t u i t a r y gland ( 6 2 ) while i t i n h i b i t s the same parameter i n fat cells ( 6 6 ) . There i s much ambiguity i n the c u r r e n t l y used c l a s s i f i c a t i o n s of dopamine r e c e p t o r s . This complexity i s p a r t l y due to the combined use of a c l a s s i f i c a t i o n based on binding data ( 6 7 , 6 8 ) and

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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another one based on f u n c t i o n of the dopamine receptor (36, 41, 54). U n t i l more p r e c i s e data are a v a i l a b l e i n a l a r g e v a r i e t y of t i s s u e s (using homogeneous c e l l populations, i f p o s s i b l e ) , c l a s s i ­ f i c a t i o n s of dopamine receptors based on the binding c h a r a c t e r i s ­ t i c s of various ligands and c l a s s i f i c a t i o n s d e s c r i b i n g f u n c t i o n a l coupling to various c e l l u l a r a c t i v i t i e s such as a c t i v a t i o n of adenylate c y c l a s e , should be kept separate. Although c l i n i c a l i n t e r e s t has stimulated the synthesis of long s e r i e s of dopamine agonists and antagonists, there i s a r e l a t i v e l y small number of model systems where the binding c h a r a c t e r i s t i c s of dopaminergic agents can be c o r r e l a t e d with a t y p i c a l p r o f i l e of c e l l u l a r func­ t i o n . In f a c t , p r e c i s e parameters of i n t a c t c e l l u l a r a c t i v i t y under dopaminergic c o n t r o l can be measured with p r e c i s i o n only i n a few dopaminergic systems, namely p r o l a c t i n - s e c r e t i n g ( 2 ) , pars intermedia (4) and parathyroid (60) c e l l s . Unfortunately, turning and s t e r e o t y p i c behavior i n r a t s p s y c h i a t r i c and n e u r o l o g i c a l symptoms i n man and othe nervous system are r e l a t i v e l While the c l a s s i f i c a t i o n of α-adrenergic receptors i n t o o^and ou (69, 70) and 3-adrenergic receptors i n t o 3 and 3 -subtypes (71, 72) i s based on the rank order of potency of a s e r i e s of catecholamines and analogs to e l i c i t a l a r g e s e r i e s of b i o l o g i c a l responses, there i s no such l a r g e s c a l e basis f o r c l a s s i f i c a t i o n of dopamine r e c e p t o r s . One of the e a r l i e s t biochemical a c t i o n s of dopamine to have been described i s the s t i m u l a t i o n of s t r i a t a l adenylate cyclase a c t i v i t y . Although i t has the disadvantage of a h i g h l y heterogenous c e l l population and i t s assay i s performed i n a broken c e l l preparation, s t r i a t a l dopamine-induced a d e n y l y l c y c l a s e has been much u s e f u l f o r c h a r a c t e r i z a t i o n of the dopamine receptor (27, 58, 73). However, not a l l dopaminergic responses appear to be mediated by c y c l i c AMP (74, 75, 76). This has l e d to the proposal by Kebabian and Calne (36) of two categories of dopa­ mine receptors, namely D, ( l i n k e d to adenylate cyclase) and D (not l i n k e d to cyclase) ( Table 1, 36). The parathyroid and the p i t u i t a r y mammotroph were taken as examples of Dl and D2 recep­ t o r s , r e s p e c t i v e l y . While few data were then a v a i l a b l e on the coupling of the a n t e r i o r p i t u i t a r y dopamine receptor to adenylate cyclase (26^ 29), i t i s now quite c l e a r that the D1-D2 terminology can no longer adequately describe the f u n c t i o n a l coupling between the dopamine receptors and adenylate cyclase i n various t i s s u e s . It has i n f a c t been convincingly demonstrated that the ante­ r i o r p i t u i t a r y dopamine receptor i s n e g a t i v e l y coupled to adenyla­ te c y c l a s e i n both tumoral (26), and normal (54 ) adenohypophysial t i s s u e . Thus, the adenohypophysis cannot be taken, as o r i g i n a l l y proposed (36), as example of a receptor not coupled to adenylate c y c l a s e . Moreover, i t i s w e l l known that some dopaminergic respon­ ses appear independent of c y c l i c AMP (74, 75, 76)· In order to take i n t o account a l l the a v a i l a b l e data, i t appears p r e f e r a b l e to use the terminology DA+, DA- and DA^ (54, F i g . 4 ) . Other advan­ tages of t h i s terminology are i t s c l e a r separation from the nomen2

2

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

0

+

Figure 4. Representation of the classification of the dopamine receptor based on its coupling with adenylate cyclase activity. DA receptors (left) are coupled to adenylate cyclase through the Νs GTP-binding protein (9\) with secondary activation of adenylate cyclase. DA. receptors (middle) are coupled through the Ni GTP-binding protein, thus resulting in inhibition of cyclic AMP for­ mation. DA receptors (right) are those uncoupled to cyclic AMP formation, the example being possibly some autoreceptors on nigrostriatal dopaminergic neurons.

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c l a t u r e D to based on binding data (68), the use of the w e l l accepted acronym DA f o r dopamine and the l a c k of p o s s i b l e confu­ s i o n with the vitamin Ό receptor. 1

C o r t i c o t r o p i n - r e l e a s i n g f a c t o r stimulates adenylate c y c l a s e i n the intermediate lobe of the p i t u i t a r y gland Although the f i r s t evidence suggesting the presence of hypo­ thalamic substances c o n t r o l l i n g p i t u i t a r y gland a c t i v i t y was that of a c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) (77_, 78), i t i s only r e c e n t l y that the s t r u c t u r e of ovine CRF could be e l u c i d a t e d (79, 80). The 41-amino a c i d peptide i s a potent stimulator of adrenoc o r t i c o t r o p i n (ACTH) s e c r e t i o n i n vivo i n the r a t as w e l l as i n adenohypophysial c e l l s i n c u l t u r e (79, 81, 82, 83)· Somewhat unex­ pectedly, we have r e c e n t l y found that the a d m i n i s t r a t i o n of CRF leads to a p a r a l l e l increase i n plasma l e v e l s not only of ACTH a peptide secreted mainl of α-melanocyte-stimulatin product o r i g i n a t i n g almost e x c l u s i v e l y from the intermediate lobe of the p i t u i t a r y gland O , 84)· In order to obtain d i r e c t evidence that CRF i s a c t i n g d i r e c t l y on c e l l s of the pars intermedia rather than i n d i r e c t l y at the hypothalamic l e v e l , we have studied the e f f e c t of CRF on α-MSH s e c r e t i o n i n pars intermedia c e l l s i n c u l ­ ture. A f t e r a 6-h incubation with pars intermedia c e l l s i n c u l t u r e , a maximal concentration of ovine CRF (300 nM) causes a 2 - f o l d s t i m u l a t i o n of α-MSH release at an E D value of 1 nM ( F i g . 5B). It can also be seen that preincubation with the potent glucocor­ t i c o i d dexamethasone under conditions which lead to an almost complete i n h i b i t i o n of ACTH s e c r e t i o n i n c o r t i c o t r o p h s of the a n t e r i o r p i t u i t a r y gland (85), has no i n h i b i t o r y e f f e c t on e i t h e r spontaneous or CRF-induced α-MSH s e c r e t i o n . In r a t pars intermedia c e l l s , the rate of s e c r e t i o n of the proopiomelanocortin-derived peptides (α-MSH being the major secre­ t o r y product) (3_, 85) was so f a r known to r e s u l t from a balance between the stimulatory e f f e c t of 3-adrenergic agents and the i n h i b i t o r y i n f l u e n c e of dopaminergic substances (14, 41, 86-89). The present data c l e a r l y demonstrate that i n a d d i t i o n to 3-adre­ n e r g i c agents, a second substance, namely CRF, could w e l l be involved as p h y s i o l o g i c a l stimulator of the a c t i v i t y of pars intermedia c e l l s . Since the adenylate cyclase system has been w e l l demonstrated to play a mediatory r o l e i n c o n t r o l l i n g the a c t i o n of 3-adrenergic and dopaminergic agents i n pars intermedia c e l l s (5-13, 41), we have studied the p o s s i b i l i t y of a s i m i l a r r o l e of c y c l i c AMP i n CRF a c t i o n . A f t e r a 10-min incubation with i n c r e a s i n g CRF concen­ t r a t i o n s , an approximately 6-fold increase i n c y c l i c AMP content i s measured at an E D value of 6 nM ( F i g . 5A). A maximal stimu­ l a t o r y e f f e c t of CRF on c y c l i c AMP accumulation i s observed 2 min a f t e r a d d i t i o n of CRF. As observed on α-MSH s e c r e t i o n , preincuba5 Q

5 Q

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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t i o n with dexamethasone has no i n h i b i t o r y e f f e c t on b a s a l or CRFinduced c y c l i c AMP accumulation. The above-described data show that CRF added to c e l l s of the rat intermediate lobe i n c u l t u r e causes a r a p i d s t i m u l a t i o n of ot­ MSH r e l e a s e and c y c l i c AMP accumulation, thus demonstrating a d i r e c t a c t i o n of the peptide on pars intermedia c e l l s (15). I t i s however d i f f i c u l t , using i n t a c t c e l l s , to d i s s o c i a t e between increases i n c y c l i c AMP l e v e l s due to s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y or to i n h i b i t i o n of c y c l i c n u c l e o t i d e phospho­ d i e s t e r a s e or to a combination of both e f f e c t s . D e f i n i t i v e proof of the r o l e of adenylate c y c l a s e i n the a c t i o n of CRF i n the intermediate lobe of the p i t u i t a r y gland i s provided by the f o l ­ lowing f i n d i n g s of a CRF-induced s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y i n homogenate of r a t and bovine pars intermedia c e l l s . As i l l u s t r a t e d i n F i g . 6, maximal concentrations of s y n t h e t i c ovine CRF cause a 100% s t i m u l a t i o n of adenylate cyclase a c t i v i t y i n bovine pars intermedi 150 nM. Since guanyl n u c l e o t i d e c e n t r a l r o l e i n mediating the a c t i v a t i o n of adenylate c y c l a s e by many hormones (90, 91), we next i n v e s t i g a t e d the p o s s i b i l i t y of such a r o l e of GTP i n CRF a c t i o n . We thus studied the i n t e r a c t i o n of CRF with GTP and the two p r e v i o u s l y known r e g u l a t o r s of pars intermedia c e l l a c t i v i t y using the 3-adrenergic agonist (-)isopro­ t e r e n o l and dopamine. As i l l u s t r a t e d i n F i g . 7, 3 μΜ CRF and 1 uM ( - ) i s o p r o t e r e n o l cause a 190 and 110% s t i m u l a t i o n of adenylate cyclase a c t i v i t y i n r a t pars intermedia p a r t i c u l a t e f r a c t i o n , r e s p e c t i v e l y . An a d d i ­ t i v e e f f e c t i s observed when both s t i m u l a t o r y agents are present. Dopamine (30 μΜ), on the other hand, has no s i g n i f i c a n t e f f e c t alone. However, i n the presence of GTP, the catecholamine causes a 40 to 60% i n h i b i t i o n of adenylate c y c l a s e a c t i v i t y stimulated by CRF, ISO or CRF + ISO. It can also be seen that while 0.3 mM GTP alone causes a 100% increase i n basal adenylate c y c l a s e a c t i v i t y , i t leads to a marked p o t e n t i a t i o n of the e f f e c t of ISO and CRF on [ P ] c y c l i c AMP accumulation. I t should be n o t i c e d that i n the absence of the guanyl n u c l e o t i d e , dopamine has no i n h i b i t o r y e f ­ f e c t on adenylate c y c l a s e a c t i v i t y i n any of the groups s t u d i e d . The present data c l e a r l y demonstrate that the 41-amino a c i d ovine CRF i s a potent s t i m u l a t o r of adenylate cyclase a c t i v i t y i n r a t and bovine pars intermedia t i s s u e . Our previous data have shown that CRF causes a r a p i d and marked s t i m u l a t i o n of c y c l i c AMP accumulation i n r a t pars intermedia c e l l s i n c u l t u r e (15). The f i n a l proof of the r o l e of adenylate cyclase i n the observed chan­ ges of c y c l i c AMP l e v e l s had to be obtained by d i r e c t measurement of adenylate cyclase a c t i v i t y . As mentioned e a r l i e r , guanyl n u c l e o t i d e s have been found to play an important r o l e i n the a c t i v a t i o n of adenylate c y c l a s e a c t i v i t y by many hormones (90, 91). The present observations show that i n pars intermedia t i s s u e , GTP causes an almost doubling of the s t i m u l a t o r y e f f e c t of CRF while that of the 3-adrenergic ago32

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7 0 0 -ι

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Figure 6.

Effect of increasing concentrations of CRF on adenylate cyclase activity in bovine pars intermedia pituitary homogenate. ζ LU

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). More r e c e n t l y , i t has been p o s s i b l e t o c o r r e l a t e , i n the p i t u i t a r y gland, occupancy

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

3.

CARON

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75

of the receptor with i n h i b i t i o n of the enzyme adenylate c y c l a s e a c t i v i t y (10) o r c y c l i c AMP l e v e l s (11). In the b r a i n where these s i t e s had been i d e n t i f i e d p r e v i o u s l y using n e u r o l e p t i c s no such c o r r e l a t i o n had been p o s s i b l e . Because o f the complex r e l a t i o n s h i p between the binding o f the numerous agonist and antagonist l i g a n d s f o r the dopamine receptor and the d i f f e r e n c e s i n c h a r a c t e r i s t i c s and numbers o f the s i t e s l a b e l l e d by these l i g a n d s , we set out t o examine the p r o p e r t i e s o f the dopamine receptor o f the porcine a n t e r i o r p i t u i t a r y gland using both an agonist r a d i o l i g a n d , [ H]n-propylapomorphine, and an a n t a g o n i s t , [ H j s p i r o p e r i d o l . Q u a n t i t a t i v e a n a l y s i s o f these data suggest t h a t both ligands i n t e r a c t with a s i n g l e dopamine receptor i n the a n t e r i o r p i t u i t a r y gland. This r e c e p t o r , however, appears t o e x i s t i n two d i s t i n c t a f f i n i t y forms r e c i p r o c a l l y favored by agonists and a n t a g o n i s t s . One o f the two forms o f the r e c e p t o r the agonist high a f f i n i t y / a n t a g o n i s from the s t a b l e a s s o c i a t i o n u c l e o t i d e binding p r o t e i n . Furthermore, t h i s form o f the receptor can be s o l u b i l i z e d with d i g i t o n i n ; however only upon p r e l a b e l l i n g o f the receptor s i t e with the l a b e l l e d agonist can n u c l e o t i d e - s e n s i t i v i t y be returned. This paper w i l l summarize the evidence supporting the f o r m u l a t i o n that a s i n g l e dopamine receptor l a b e l l e d by [ H ] s p i r o p e r i d o l e x i s t s i n two d i s t i n c t a f f i n i t y forms i n porcine a n t e r i o r p i t u i t a r y gland (12,13). This f o r m u l a t i o n appears as an a t t r a c t i v e a l t e r n a t i v e to the proposal o f Seeman and coworkers (8) o f two d i s t i n c t subtypes designated and f o r t h i s receptor. Q u a n t i t a t i v e R e l a t i o n s h i p o f Agonist and Antagonist Ligand Binding t o Porcine A n t e r i o r P i t u i t a r y Gland Membranes In p a r t i c u l a t e preparations o f porcine a n t e r i o r p i t u i t a r y gland prepared by d i f f e r e n t i a l c e n t r i f u g a t i o g (12), the agonist [ H]n-propylapomorphine and the antagonist [ H ] s p i r o p e r i d o l bind with the same appropriate dopaminergic s p e c i f i c i t y (Figure 1A and B) ( i . e . η-propylapomorphine^ apomorphine > ADTN > dopamine) (ADTN not shown f o r the [ H]NPA curves). I t should be noted that the agonist competition curves f o r [ H ] s p i r o p e r i d o l binding are complex o r m u l t i p h a s i c with slope f a c t o r s o f l e s s than 1. Q u a n t i t a t i v e a n a l y s i s o f these data by l i n e a r l e a s t square f i t t i n g (_14) i n d i c a t e t h a t the agonists compete f o r the s i t e s l a b e l l e d by [ H ] s p i r o p e r i d o l with two d i f f e r e n t a f f i n i t i e s ; roughly 50% o f the t o t a l [ H ] s p i r o p e r i d o l s i t e s e x h i b i t high a f f i n i t y f o r the agonists and the remaining s i t e s have lower a f f i n i t y f o r the same a g o n i s t s . The r a t i o o f the two agonist a f f i n i t i e s (Κ,,/Κ,) vary from 30 i n the case o f apomorphine (Κ = 4.7 ± 2.4 nM, μ

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K. = 160 ± 61 nM) t o 200 f o r η-propylapomorphine (Κ = 0 . 0 8 1 ± 0.022 ήΜ, K, = 18 ± 3 nM). On the other hand, agonist competition curves f o r [ H]n-propylapomorphine are monophasic with slope f a c t o r s around u n i t y (Figure IB) suggesting t h a t both the l a b e l l e d agonist and the competing agonist i n t e r a c t with a s i n g l e form of the r e c e p t o r . The IC values c a l c u l a t e d from the agonist competition o f [ H]n-propylapomorphine correspond c l o s e l y t o those obtained from the higher o f the two a f f i n i t y forms evidenced i n the agonist competition curves o f [ H ] s p i r o p e r i d o l (e.g. f o r n-propylapomorphine: K „ , 3 = 0.081 ± 0.022 nM vs. K , 3 " ! A ™ ^ ^ ± 0.022 nM). J -P °Py' PO P ) These data suggest t h a t whereas [ H ] s p i r o p e r i d o l l a b e l s the e n t i r e population o f the dopamine r e c e p t o r , only a p o r t i o n of these same s i t e s are l a b e l l e d with [ H]n-propylapomorphine This i s confirmed by f i n d i n g show that i n a given membran morphine l a b e l s with high a f f i n i t y ( K = 0.26 nM) only one-half as many s i t e s as [ H ] s p i r o p e r i d o l . The remaining [ H ] s p i r o p e r i d o l s i t e s possess a f f i n i t y that i s too low f o r agonists ( K = 18-20 nM f o r n-prooylapomorphine) t o be l a b e l l e d by d i r e c t binding with [ H]n-propylapomorphine with the concentrations of l i g a n d normally used t o perform s a t u r a t i o n isotherms (10 DM to 1 nM). Therefore, i t appears t h a t by d i r e c t b i n d i n g , [H]n-propylapomorphine l a b e l s only the agonist high a f f i n i t y form o f the receptor population l a b e l l e d by [ H ] s p i r o p e r i d o l . μ

3

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Guanine n u c l e o t i d e s have been i m p l i c a t e d i n several receptor systems as important modulators o f the a f f i n i t y o f receptors f o r t h e i r s p e c i f i c hormones o r agonists (_15). The e f f e c t s are u s u a l l y observed as a decrease i n the a f f i n i t y o f the receptor f o r agonists (_16). For example, i n the beta-adrenergic receptor system, guanine n u c l e o t i d e s have been shown t o reduce the a b i l i t y o f agonists t o compete f o r antagonist binding t o the receptor ( J 7 ) . In the betaadrenergic system, agonists promote the formation of a t e r n a r y complex (18) composed o f the hormone, receptor and a n u c l e o t i d e binding p r o t e i n (19). This ternary complex i s d e s t a b i l i z e d or i t s formation inhiïïited i n the presence o f guanine n u c l e o t i d e s (20). In dopaminergic receptor systems, s i m i l a r e f f e c t s o f guanine n u c l e o t i d e s on agonist potency a t the receptor have been reported (reviewed i n (8) and c f . r e f s . (19-24) c i t e d i n ( 1 2 ) ) . In order t o probe the r e l a t i o n s h i p o f the two d i f f e r e n t a f f i n i t y forms o f the dopamine r e c e p t o r , i t was o f i n t e r e s t t o

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

CARON

Anterior

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Pituitary

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Competition curves of a series of dopaminergic agonists for the direct binding of [ H] agonist and antagonist ligands. 3

Porcine anterior pituitary gland membranes prepared as described previously (12), were incubated with [ H]spiroperidol (~ 140 pM) (A) or [ H]n-propylapomorphine (~ 150 pM) (B) and increasing concentrations of the various agonists as shown. Membranes suspended in 50 mM Tris-HCl, 6 mM MgCh, 1 mM EDTA, 100 mM NaCl, 0.1% ascorbate, 10 μΜ pargyline, pH 7.4, at 25 °C were incubated for 60 min at 25 °C in a total volume of 1 mL containing 0.3 mg and 0.6 mg of membrane protein/assay for [ H]spiroperidol and [ H]npropylapomorphine, respectively. Binding was initiated by the addition of membranes, the incubations were terminated by adding 5 mL of cold 25 mM Tris-HCl, 2 mM MgCU (pH 7.4) (4 °C) and rapid vacuum filtration on GF/C or GF/B glass fiber filters with four additional 5 mL washings. Each agonist drug competed for binding to the level of nonspecific binding that was determined in the presence of 1 μΜ (-\-)butaclamol. Under routine conditions specific binding accounted for 80-90% of total binding for [ H]spiroperidol and 50-70% for [ H]n-propylapomorphine. In the experiment shown, 100% [ Η]spiroperidol binding corre­ sponded to 32 pM or ~ 107 fmol/mg whereas 100% [ H]n-propylapomorphine was 33 pM or 55 fmol/mg. The points represent experimental data, and the lines represent the best fit of the data according to a model for one or two binding sites analyzed as described previously (12). The experiment shown was performed in duplicates and is representative of three (A) and two (B) such experiments. (Reproduced with permission from Ref. 12. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3

3

3

3

3

3

3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

3

78

DOPAMINE

RECEPTORS

examine the e f f e c t of guanine nucleotides on dopaminergic l i g a n d binding to porcine a n t e r i o r p i t u i t a r y membranes. As shown i n Figure 2, the guanine nucleotide Gpp(NH)p markedly decreases the apparent potency of an agonist such as n-propylapomorphine i n competing f o r the binding of the antagonist [ H j s p i r o p e r i d o l . The c o n t r o l competition curve which i s b i p h a s i c and shallow i s s h i f t e d to lower apparent potency and steepened i n the presence of guanine n u c l e o t i d e s . In a d d i t i o n , as w i l l be discussed below, an apparent increase i n [ H j s p i r o p e r i d o l binding was observed i n the presence of guanine n u c l e o t i d e s . Q u a n t i t a t i v e a n a l y s i s of the c o n t r o l competition curve i n d i c a t e d that 50% of the [ H j s p i r o p e r i d o l s i t e s d i s p l a y high a f f i n i t y f o r the agonist (Km = 0.29 ± 0.13 nM) and 50% of the s i t e s d i s p l a y e d a lower a f f T n i t y (K. = 8.3 ± 2.0 nM). In the presence of n u c l e o t i d e , however, the proportion of the agonist high a f f i n i t y form i s reduced to only 10 to 15% with no s i g n i f i c a n 0.35 nM). The remainin a f f i n i t y f o r the agonist i n d i s t i n g u i s h a b l e from the lower agonist a f f i n i t y of the c o n t r o l curve (8.3 ± 2.0 nM vs. 12.0 ± 1.9 nM). Thus, guanine nucleotides appear to decrease the proportion of the receptor e x h i b i t i n g high a f f i n i t y f o r agonists (12). This e f f e c t can be t e s t e d d i r e c t l y by examining the e f f e c t s of nucleotides on d i r e c t agonist b i n d i n g . As shown i n Figure 3 i n the presence of i n c r e a s i n g concentrations of GTP, there i s a progressive l o s s of the number of agonist high a f f i n i t y form of the receptor detectable by d i r e c t [ Hjnpropylapomorphine binding ( c o n t r o l = 26.7 ± 2.1 pM; + 10 yM GTP = 19.6 ± 2.4 pM and + 1 mM GTP = 6.0 ± 1.4 pM). The a f f i n i t y of the remaining s i t e s l a b e l l e d by [ H]n-propylapomorphine remains e s s e n t i a l l y the same f o r the l i g a n d . The e f f e c t of guanine nucleotides on t h i s system, much l i k e i n other receptor systems (18), i s to decrease the proportion of the receptor e x i s t i n g i n an agonist high a f f i n i t y form. This e f f e c t appears as e i t h e r an apparent decrease i n the a b i l i t y of agonists to compete f o r antagonist r a d i o l i g a n d binding or an actual decrease i n the number of s i t e s l a b e l l e d with high a f f i n i t y by a l a b e l l e d agonist. This l a t t e r e f f e c t i s due to the f a c t that the agonist lower a f f i n i t y form of the receptor u s u a l l y cannot be l a b e l l e d by d i r e c t agonist l i g a n d b i n d i n g . Thus, these r e s u l t s are c o n s i s t e n t with the t h e s i s that the dopamine receptor i n the porcine a n t e r i o r p i t u i t a r y gland can e x i s t i n two d i f f e r e n t a f f i n i t y forms w i t h respect to agonists and that these two s t a t e s can be i n t e r c o n v e r t e d by guanine n u c l e o t i d e s . These s p e c i f i c e f f e c t s of guanine n u c l e o t i d e s s t r o n g l y suggest that high a f f i n i t y i n t e r a c t i o n s of the receptor with agonists i n v o l v e another component which mediates the e f f e c t s of n u c l e o t i d e s . 3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

CARON

Anterior

ET AL.

Pituitary

Gland's Dopamine

Receptor

30 25

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CONTROL

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15

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Effect of the guanine nucleotide Gpp(NH)p on competition of the agonist n-propylapomorphine for [ H]spiroperidol binding. 3

3

Membranes were incubated as described in Figure 1 with [ Hjspiroperidol (~ 160 pM) and increasing concentrations of n-propylapomorphine in the presence and absence of 100 μΜ Gpp(NH)p. The lines are the best fit obtained with a model for two affinity forms of the receptor as described previously (14). The experiment shown is representative of 10-12 experi­ ments. (Reproduced with permission from Ref. 12. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.)

3

[ H] NPA ADDED (pM)

Figure 3. Effects of guanine nucleotides on the direct binding of apomorphine.

3

[ Η]n-propyl­

Porcine anterior pituitary membranes were incubated with increasing concentrations (10 pM900 pM) of [ H]n-propylapomorphine in the presence of 10 μΜ and 1 mM GTP and in the absence of GTP as the control. Nonspecific binding was determined in the presence of 1 μΜ (-\-)butaclamol and was the same with or without the addition of GTP. Data were analyzed as described in Figure 1. The number of sites labelled by [ H]n-propylapomorphine was 26.7 pM (absence), 19.6 pM in the presence of 10 μΜ GTP and 6.0 pM in the presence of 1 mM GTP and dissociation constants for the ligand were 280 and 210 pM. The experiment was performed in duplicate and is representative of three such experiments. 3

3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

DOPAMINE

80

Guanine Nucleotides Modulation Antagonist?

RECEPTORS

o f Receptor A f f i n i t y f o r

As can be observed from competition experiments o f η-propylapomorphine f o r [ H j s p i r o p e r i d o l binding (Figure 2 ) the presence o f guanine n u c l e o t i d e s y i e l d s an apparent increase i n the binding o f [ H j s p i r o p e r i d o l . This e f f e c t i s q u i t e d i f f e r e n t from the usual e f f e c t o f n u c l e o t i d e s observed on agonist binding (JL5,L8). However, e f f e c t s o f guanine n u c l e o t i d e s on antagonist binding have been described f o r muscarinic receptor systems (22^,23) and more r e c e n t l y f o r the beta-adrenergic receptor system Jzk). In order to explore f u r t h e r t h i s e f f e c t d e t a i l e d s a t u r a t i o n binding isotherms o f [ H j s p i r o p e r i d o l binding were performed i n the presence and absence o f a guanine n u c l e o t i d e . As shown i n Figure 4 , the c o n t r o l s a t u r a t i o n curve as represented i n the Scatchard p l o t coordinates i s s l i g h t l presence o f 1 mM GTP. Quantitativ square f i t t i n g i n d i c a t e s t h a t two K values (45 and 415 pM) can be significantly derived f o r [ Hjspiroperidol binding i n the absence o f GTP and a single value o f 56 pM (not s i g n i f i c a n t l y d i f f e r e n t than 45 pM) i n the presence o f GTP. The two a f f i n i t y forms which are present i n roughly equal proportions i n the c o n t r o l curve are converted to a s i n g l e high a f f i n i t y form i n the presence o f GTP w i t h no change i n the t o t a l number o f s i t e s f o r [ H j s p i r o p e r i d o l ( 2 5 ) . GTP d i s p l a y s the same dose response (EC™ = 32 yMj f o r i t s e f f e c t on L H j s p i r o p e r i d o l binding as the e r f e c t on agonist binding (EC = 17 yM). Thus, i t appears that the same two a f f i n i t y fortfis o f the receptor which are d i s c r i m i n a t e d by agonists may a l s o be d i s c r i m i n a t e d by antagonists although i n a r e c i p r o c a l f a s h i o n . The d i s c r i m i n a t i n g power o f [ H j s p i r o p e r i d o l f o r the two forms o f the receptor i s , however, much weaker than f o r agonists ( 2 - 1 0 f o l d f o r [ ^ s p i r o p e r i d o l and 30-200 f o l d f o r a g o n i s t s ) . Thus, i t would appear t h a t i n the presence o f n u c l e o t i d e s the agonist high a f f i n i t y form/antagonist low a f f i n i t y form i s converted t o the agonist low a f f i n i t y / antagonist high a f f i n i t y form of the receptor. The p o s s i b l e p h y s i o l o g i c a l relevance o f these f i n d i n g s w i l l be discussed l a t e r i n t h i s chapter. D

5 Q

N-ethylmaleimide and Heat Treatments of Membranes Mimic the E f f e c t o f Guanine Nucleotides on Ligand Binding A l l the data presented above point to the involvement of a p u t a t i v e guanine n u c l e o t i d e binding p r o t e i n i n the i n t e r a c t i o n of agonists with the dopamine receptor i n the porcine a n t e r i o r p i t u i t a r y gland. We, t h e r e f o r e , wanted to obtain more compelling evidence f o r the i n t e r a c t i o n o f a n u c l e o t i d e binding

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

CARON

Anterior

E T AL.

Ό

Pituitary

10

Gland's Dopamine

20

SPECIFIC [3H]SPIR0

Figure 4.

30

Receptor

40

BINDING (pM)

Effects of GTP on the binding isotherms of the antagonist peridol.

3

[ H]spiro­

Porcine anterior pituitary membranes were incubated as described in Figure 1 with ^Hjspiro­ peridol (10-1100 pM). Nonspecific binding was determined in the presence of 1 μΜ (+)butaclamol. Data were analyzed by curve fitting with a model for the binding of the radioligand to one or two classes of sites (14). Data are presented in the Scatchard plot coordinates. The experiment was performed in duplicate and is similar to several other experiments with the same results but it represents an extreme case of the ability of the [ H] antagonist to discriminate between the two forms of the receptor. The two dissociation constants calculated for the control curve are different by about 10-fold. (Reproduced with permission from Ref. 25. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

82

DOPAMINE

RECEPTORS

e n t i t y with the receptor. N-ethylmaleimide (NEM), a s u l h y d r y l a l k y l a t i n g agent, mimics the e f f e c t s o f n u c l e o t i d e s on agonist binding a t the w e l l - c h a r a c t e r i z e d beta-adrenergic receptor o f the f r o g e r y t h r o c y t e (26). In a manner s i m i l a r t o the e f f e c t of guanine nucleotides i n t h a t system, NEM prevents the formation o f the agonist high a f f i n i t y form o f receptor with no change i n t o t a l number of receptors. Moreover, i t i s apparent that the guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n a c t i v i t y of the beta-adrenergic receptor demonstrates e x q u i s i t e s e n s i t i v i t y to temperature. Ross e t a l . (27) showed that incubation o f S-49 lymphoma c e l l membranes a t 50° i n a c t i v a t e s the r e g u l a t o r y p r o t e i n a c t i v i t y with a t i o f 2-8 minutes as assayed by r e c o n s t i t u t i o n s t u d i e s . The r e g u l a t o r y p r o t e i n a c t i v i t y assayed by r e c o n s t i t u t i o n i s the same f u n c t i o n a l e n t i t y which i n t e r a c t s with the beta-adrenergic receptor t o form the high a f f i n i t y agonist ternary complex (19). Therefore, i t i s reasonable t o assume tha the r e c e p t o r - a f f i n i t y modulatin n u c l e o t i d e r e g u l a t o r y p r o t e i n i n t h a t system. Treatment o f porcine a n t e r i o r p i t u i t a r y gland membranes with e i t h e r NEM at concentrations between 1-100 yM o r heat (53° f o r 4 min.) q u a l i t a t i v e l y and q u a n t i t a t i v e l y mimics the e f f e c t s of guanine nucleotides on dopaminergic l i g a n d b i n d i n g . As shown i n Figures 5A and B- competition curves o f the agonist η-propylapomorphine f o r [ H j s p i r o p e r i d o l binding are s h i f t e d to the r i g h t and steepened. These e f f e c t s are i d e n t i c a l to the a b i l i t y of guanine nucleotides t o decrease the proportion of agonist high a f f i n i t y form of the receptor. S i m i l a r l y , i n c r e a s i n g concentrations of NEM from 1 yM t o 100 yM cause a progressive decrease i n the d i r e c t binding of the agonist [ Hjn-propylapomorphine (Γ3). Moreover, i t can be observed that both NEM and heat treatments seem t o a f f e c t [ H j s p i r o p e r i d o l binding i n a manner s i m i l a r to guanine n u c l e o t i d e s since an apparent increase i n binding of [ H j s p i r o p e r i d o l can be observed (Figures 5A and B). Heat treatment of membranes does not increase [ H j s p i r o p e r i d o l binding~as much as GTP because under these c o n d i t i o n s (53°for 4 min.) [ H j s p i r o p e r i d o l binding i s s l i g h t l y i n h i b i t e d (15-20%) (not shown) (13). Thus, these r e s u l t s suggest that the e f f e c t s o f heat and NEM treatments mimic the modulation o f dopaminergic l i g a n d binding by guanine nucleotides and are very s i m i l a r to t h e i r e f f e c t s on beta-adrenergic systems. The only exception appears to be the r e l a t i v e s e n s i t i v i t y of the dopaminergic system t o NEM (13,26). Whereas, on dopaminergic b i n d i n g , NEM e l i c i t s i t s e f f e c t s with an EC™ = 6 yM, concentration i n the m i l l i m o l a r range are required Vor the same e f f e c t s on beta-adrenergic agonist b i n d i n g . By analogy with the beta-adrenergic receptor system, these data support an i n t e r a c t i o n of a guanine

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

CARON

Anterior

E T A L .

Έ

25

Q

20

Pituitary

Gland's Dopamine

Receptor

83

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Figure 5. Comparison of the effects of NEM and heat treatments and the presence of GTP on the ability of n-propylapomorphine (agonist) to compete for the binding of the antagonist [ H]spiroperidol. 3

3

Membranes were incubated with [ H]spiroperidol (~ 109 pM) for 1 h at 25 ° C and increasing concentrations of n-propylapomorphine (A). Key to A: · , control membranes; • , membranes treated in the presence of 100 μΜ NEM as described below; and A, membranes incubated in the presence of 1 mM GTP. The curves represent computer drawn lines that best fit the data points. NEM treatment was performed as follows: Membranes were incubated with the indicated concentration of NEM for 30 min at 25 °C prior to addition of the radioligand. The same results were obtained whether or not the membranes were washed free of residual NEM prior to the binding assay. All three curves are from a single experiment that is representative of three experiments. Control membranes and membranes treated at 53 °C for 4 min as described below were incubated with [ Hjspiroperidol (190 pM) in the presence of increasing concen­ trations of n-propylapomorphine. One set of membranes were also incubated in the presence of 100 μΜ GTP. Data were analyzed by computer-based methods as described in Figure 1 with the lines representing the best fit to the data (14). The experiment was performed and is representative of two such experiments. (Reproduced with permission from Ref. 13. Copyright 1982, American Society for Pharmacology and Experimental Therapeutics.) 3

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

84

DOPAMINE

RECEPTORS

n u c l e o t i d e r e g u l a t o r y p r o t e i n with the dopamine receptor i n the a n t e r i o r p i t u i t a r y gland. S o l u b i l i z a t i o n of the Dopamine Receptor One of the p r e r e q u i s i t e s f o r the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a membrane-bound hormone receptor i s i t s s o l u b i l i z a t i o n i n an a c t i v e form, i . e . with r e t e n t i o n of the a b i l i t y of the receptor to i n t e r a c t with s p e c i f i c l i g a n d s . Several reports have appeared already documenting the s o l u b i l i z a t i o n of dopaminergic binding s i t e s from b r a i n t i s s u e (28-31) using detergents such as d i g i t o n i n and 3'-[(3 cholamidopropyldimethyl ami no)dimethyl-amonio]-1-propanesulfonate (CHAPS) or chaotropic agents. In the porcine a n t e r i o r p i t u i t a r y gland we report here that the dopamine receptor can be s o l u b i l i z e d using the detergent d i g i t o n i n . Treatment of membran i n 100 mM NaCl, 10 mM T r i s - H C membrane-binding s i t e s i n a s o l u b l e form that cannot be sedimented at 100,000 xg f o r 60 min. S a t u r a t i o n isotherms of [ H j s p i r o p e r i d o l as measured by a Sephadex G-50 assay (32) f o r separation of bound from f r e e l i g a n d reveal a d i s s o c i a t i o n constant ( K ) of 0.5 nM f o r s p i r o p e r i d o l (not shown). Although, t n i s value i s somewhat lower than the K of 40-70 pM obtained f o r [ H j s p i r o p e r i d o l binding to p a r t i c u l a t e p r e p a r a t i o n s , the s p e c i f i c i t y of binding of the antagonist to the s o l u b i l i z e d s i t e s i s s i m i l a r to t h a t i n the membrane f r a c t i o n (n-propylapomorphine > ADTN > dopamine) (not shown) (12). As mentioned p r e v i o u s l y , agonist competition curves f o r [ H j s p i r o p e r i d o l binding i n p a r t i c u l a t e preparations are b i p h a s i c and high and low a f f i n i t y forms of the receptor f o r agonists can be documented; i n s o l u b i l i z e d preparations a s i n g l e low a f f i n i t y can be detected f o r the competition of agonists f o r s o l u b l e [ H j s p i r o p e r i d o l b i n d i n g . By comparison to the K values (0.080 and 18 nM) c a l c u l a t e d f o r n-propylapomorphine competition of p a r t i c u l a t e [ H j s p i r o p e r i d o l b i n d i n g , the value obtained from s o l u b i l i z e d preparations i s 35 nM. Moreover, the presence of guanine n u c l e o t i d e s i n the assays does not f u r t h e r reduce the a b i l i t y of the agonists to compete f o r [ H j s p i r o p e r i d o l binding (not shown). Therefore, i t appears t h a t s o l u b i l i z a t i o n i n t e r f e r e s with the a b i l i t y of agonists to i n t e r a c t with high a f f i n i t y with the receptor. This i s a common f i n d i n g i n several s o l u b i l i z e d receptor systems (33). D

D

D

However, i f the agonist high a f f i n i t y form of the receptor i s l a b e l l e d p r i o r to s o l u b i l i z a t i o n by i n c u b a t i o n of membranes with [ H]n-propylapomorphine then the agonist high a f f i n i t y form of the receptor appears to be s t a b l e to s o l u b i l i z a t i o n (Table I ) . Shown i n Table I are the r e s u l t s of three

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

3.

CARON

Anterior

ET AL.

Pituitary

Gland's Dopamine

Receptor

85

Table I S o l u b i l i z a t i o n by D i g i t o n i n o f Agonist and Antagonist P r e l a b e l l e d Dopamine Receptor from A n t e r i o r P i t u i t a r y Gland Membranes 3

[ H ] Ligand

Experiment

J

[ H]NPA fmol 63 55 35.9

H]Spir +1 nM NPA fmol 110 90

H]Spir fmol 155 133 60

affinity % 41 42 59

sites

Membranes were incubated f o r 60 minutes a t 25° as described i n the legend t o Figure 1 w i t h 800 pM [ H]n-propylapomorphine (NPA) and 700 pM [ H ] s p i r o p e r i d o l ( S p i r o ) and [ H ] s p i r o p e r i d o l i n the presence o f 1 nM unlabel l e d NPA, a c o n c e n t r a t i o n s u f f i c i e n t t o saturate the agonist high a f f i n i t y form o f the receptor. A f t e r the i n c u b a t i o n , membranes were c e n t r i f u g e d and s o l u b i l i z e d by resuspending the membranes i n 1% d i g i t o n i n , 100 mM NaCl, 25 mM Tris-HCl 2 mM M g C l , 0.1% ascorbate pH 7.4 a t 4°C, s t i r r i n g on i c e f o r 30 minutes and c e n t r i f u g i n g a t 40,000 χ g f o r 45 minutes. S p e c i f i c binding f o r both ligands was determined by Sephadex G-50 chromatography o f the s o l u b i l i z e d samples from incubation with l i g a n d s alone o r l i g a n d s with 10" M (+)butaclamol. Results are expressed i n fmol o f s p e c i f i c binding i n 1 ml o f s o l u b i l i z e d preparation i n the r e s p e c t i v e experiments. % agonist high a f f i n i t y s i t e s i s expressed as the percent o f [ H3NPA s i t e s compared t o the number o f s i t e s l a b e l l e d with [ H ] s p i r o p e r i d o l . J

J

?

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

86

DOPAMINE

RECEPTORS

experiments i n which membrane preparations were~incubated with ["H]n-propylapomorphine, [ H j s p i r o p e r i d o l and [ H j s p i r o p e r i d o l i n the presence of 1 nM u n l a b e l l e d n-propylapomorphine p r i o r t o s o l u b i l i z a t i o n with d i g i t o n i n . Under these c o n d i t i o n s , the r e s u l t s i n d i c a t e t h a t about the same proportion o f agonist high a f f i n i t y form of the receptor can be s o l u b i l i z e d as that which can be evidenced i n membrane preparations ( i . e . about 50%). In the presence o f 1 nM n-propylapomorphine, a concentration s u f f i c i e n t to saturate the agonist high a f f i n i t y form of the receptor i n the membrane, the amount of [ H j s p i r o p e r i d o l binding s o l u b i l i z e d i s reduced by approximately the amount of [ H]n-propylapomorphine s i t e s s o l u b i l i z e d . In a d d i t i o n , the data i n Table I I i n d i c a t e that the s o l u b l i z e d agonist high a f f i n i t y form of the receptor remains s e n s i t i v e t o the e f f e c t of n u c l e o t i d e s . [ H]n-Propylapomorphine b i n d i n g , assayed i n the membranes i n the presence of Gpp(NH)p o r t r e a t e d with Gpp(NH)p a f t e r s o l u b i l i z a t i o n extent. As shown i n Figure i n the number of binding s i t e s f o r [H]n-propylapomorphine i n the presence of guanine n u c l e o t i d e s i n both membranes (Figure 6A) and s o l u b i l i z e d preparations (Figure 6B), can be a t t r i b u t e d to a decrease i n the a f f i n i t y o f the receptor f o r the a g o n i s t . This apparent decrease i n a f f i n i t y can be accounted by the increased d i s s o c i a t i o n rate o f [ H]n-propylapomorphine i n both preparations i n the presence of Gpp(NH)p. The r e s u l t s presented above are c o n s i s t e n t with the formulation postulated p r e v i o u s l y , that the receptor can e x i s t as two a f f i n i t y forms, one of which i s h i g h l y prefered or s t a b i l i z e d i n the presence of an agonist. In a d d i t i o n , these data i n d i c a t e that whereas the receptor can be s o l u b i l i z e d with apparent r e t e n t i o n o f i t s binding s p e c i f i c i t y , high a f f i n i t y i n t e r a c t i o n s of the receptor with agonists are not present i n s o l u b i l i z e d preparations unless s t a b i l i z e d p r i o r to s o l u b i l i z a t i o n by the presence o f an a g o n i s t . These r e s u l t s are e n t i r e l y analogous to the beta-adrenergic receptor system (34) where the component required f o r high a f f i n i t y agonist i n t e r a c t i o n s with the receptor has been documented to be a guanine n u c l e o t i d e binding p r o t e i n . Conclusions The d i s c r i m i n a t i o n i n the membrane preparations by agonists o f two a f f i n i t y forms o f the receptor allows the comparison of the potency o f these agonists with t h e i r a b i l i t y to e l i c i t i n h i b i t i o n p r o l a c t i n r e l e a s e . I t can be observed, that the K values of agonists observed f o r the high a f f i n i t y form of the receptor (Κ„) c o r r e l a t e very c l o s e l y with the a b i l i t y (EC™) o f these same agents to i n h i b i t p r o l a c t i n r e l e a s e (9) \ - 66 nM vs. E C = 35 Q

5

( d o p a m i n e )

5 Q

( d o p a m i n e )

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

3.

CARON

ET AL.

Anterior

Pituitary

Gland's Dopamine

Receptor

87

Table I I E f f e c t o f Guanine Nucleotides on High A f f i n i t y Agonist Binding to Membrane and S o l u b i l i z e d Receptor Preparations

Experiment 1 2

Membrane preparations Control +l00yMGpp(NH)p 193 132

19 (86)

S o l u b i l i z e d preparations Control +100yMGpp(NH)p 40 44

8.6 (79) 13.0 (71)

Membranes were incubated as described i n Table I w i t h 800 pM [ H]n-propylapomorphine ( c o n t r o l ) and i n the presence o f 100 yM Gpp(NH)p. S p e c i f i c binding was determined as described i n the legend t o Figure 1. For data w i t h s o l u b i l i z e d p r e p a r a t i o n s , membranes were incubated with 800 pM [ H]n-propylapomorphine f o r 60 minutes a t 25°C then s o l u b i l i z e d as described i n Table I. The s o l u b i l i z e d samples were then incubated f o r 15 hours a t 4° i n the presence o r absence o f 100 yM Gpp(NH)p and s p e c i f i c binding determined by Sephadex G-50 chromatography from the d i f f e r e n c e between the above samples and samples which had been incubated with 10" M (+)butaclamol from the i n i t i a l i n c u b a t i o n . The numbers i n parenthesis i n d i c a t e the percent decrease i n agonist binding i n the presence o f Gpp(NH)p.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

20

40

60

80

100

ι I 15

ι 20

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Gpp(NH)p

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Gpp(NH)p

10

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40

60

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Ll

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80

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100 < °°

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TIME

20

I

(min)

30

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ο

No

3

3

Effects of guanine nucleotides on the dissociation rate of [ Η]n-propylapomorphine binding from membrane-bound and solubilized receptor preparations.

TIME (min)

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ο

No

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5

40

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Membranes were incubated as described in Figure 1 with 500 pM [ H]n-propylapomorphine for 60 min at 25 °C to achieve equilibrium in the presence of 0.1% ascorbate (A). Dissociation was initiated by adding at time 0 an excess of (-{-)butaclamol (10~ M) or (-\-)butaclamol, plus 10 and 100 μΜ Gpp(NH)p. Samples were filtered on GF/B filters at time intervals shown. Nonspecific binding was determined by parallel incubations containing (-\-)butaclamol from the beginning of the incubation. The experiment performed in duplicate is representative of three such experiments. Membranes were also incubated as above with [ H]n-propylapomorphine to achieve equilibrium (B). After the incubation, membranes were centrifugea and resuspended in 1% digitonin 25 mM Tris-HCl, 100 mM NaCl, 2 mM MgCh, 0.1% ascorbate, pH 7.4, at 4 °C, stirred for 30 min on ice, and centri­ fugea at 40,000 χ g for 45 min to obtain the solubilized labelled receptor preparation. Dissociation of the ligand was started by adding at time 0 excess (-\-)butaclamol (10~ M) or (-\-)butaclamol and 100 μΜ Gpp(NH)p. Sam­ ples were incubated at 4 °C for the time intervals indicated, and binding was determined by Sephadex G-50 chromatography. The results are representative of two similar experiments.

Figure 6.

Ν

ο

Α

INDI

3.

CARON

ET A L .

Anterior

Pituitary

Gland's Dopamine

Receptor

89

nM). ions are much c l overa11 r e l a t i v e potency of an agonist f o r competing f o r l i g a n d binding i s compared with the p h y s i o l o g i c a l response (9). It is t h e r e f o r e reasonable t o p o s t u l a t e t h a t the p h y s i o l o g i c a l response t o dopamine, i . e . i n h i b i t i o n o f p r o l a c t i n r e l e a s e , i s a r e f l e c t i o n o f the high a f f i n i t y i n t e r a c t i o n o f dopaminergic agonists o r dopamine i t s e l f with the receptor i n the a n t e r i o r p i t u i t a r y gland. The d i s c r i m i n a t i o n o f the same two forms o f the receptor by antagonists i n a r e c i p r o c a l f a s h i o n t o agonists i s much more s u b t l e . F i r s t , the d i f f e r e n c e s i n a f f i n i t y observed f o r the antagonist [ H j s p i r o p e r i d o l are small ( 2 - 1 0 f o l d ) as compared to âgonits ( 3 0 - 2 0 0 f o l d ) , hence more d i f f i c u l t t o demonstrate unless extremely d e t a i l e d s a t u r a t i o n isotherms are performed. Moreover, the d i s c r i m i n a t i o n o f the two a f f i n i t y forms o f the receptor by an antagonis [ H j s p i r o p e r i d o l only examined. The f a c t t h a t antagonists i n several receptor systems can s l i g h t l y d i s t i n g u i s h two forms o f a receptor probably has no major p h y s i o l o g i c a l s i g n i f i c a n c e w i t h respect to the a c t i o n s o f antagonists. Nonetheless, the q u a n t i t a t i v e aspect o f the binding o f [ H j s p i r o p e r i d o l t o the two forms o f the receptor and t h e i r modulation by guanine n u c l e o t i d e s can be taken as f u r t h e r support f o r the e x i s t e n c e o f these two d i f f e r e n t i n t e r c o n v e r t i b l e forms o f the receptor which appear more important i n the mechanism o f a c t i o n o f a g o n i s t s . In the beta adrenergic receptor system o f the f r o g e r y t h r o c y t e ( 1 8 ) the amount o f agonist high a f f i n i t y s t a t e (Rn) o f the receptor induced by an agonist and the r a t i o o f K . / K m were found t o c o r r e l a t e c l o s e l y wtih the i n t r i n s i c actiOity o f the agonist i n s t i m u l a t i n g adenylate c y c l a s e . In the alpha^-adrenergic receptor system o f the human p l a t e l e t s , R d i d not c o r r e l a t e with the i n t r i n s i c a c t i v i t y o f agonists f o r t h e i r a b i l i t y t o attenuate adenylate c y c l a s e s t i m u l a t i o n ( 3 6 ) . I n t h a t system, only the r a t i o o f K . / K appeared t o c o r r e l a t e with the i n t r i n s i c a c t i v i t y o f the alpha-adrenergic a g o n i s t s . Here, i n a n t e r i o r p i t u i t a r y membranes constant proportions o f both receptor s t a t e s were evidenced but r a t i o s of 3 0 - 2 0 0 were found f o r K , / K for a s e r i e s o f dopaminergic agonists despite the f a c t t h a t these agonists a l l appear t o display f u l l i n t r i n s i c a c t i v i t y in their a b i l i t y to inhibit p r o l a c t i n s e c r e t i o n from p i t u i t a r y c e l l s . In a d d i t i o n t o the e f f e c t s o f guanine n u c l e o t i d e s on the d i r e c t binding o f agonists and a n t a g o n i s t s , several f a c t o r s p o i n t toward the i n t e r a c t i o n of a p u t a t i v e n u c l e o t i d e binding p r o t e i n i n the mechanism of l i g a n d binding t o the dopamine receptor of a n t e r i o r p i t u i t a r y gland. F i r s t , both heat and NEM treatment of membranes mimic the q u a l i t a t i v e and q u a n t i t a t i v e M

M

M

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

90

DOPAMINE

RECEPTORS

e f f e c t s of n u c l e o t i d e s on the binding of agonists and a n t a g o n i s t s . These treatments have been shown i n other systems to i n a c t i v a t e guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n f u n c t i o n . Second, upon s o l u b i l i z a t i o n the a b i l i t y of the receptor to i n t e r a c t with high a f f i n i t y with agonists i s l o s t . However, l a b e l l i n g of the receptor i n membranes with the agonist [ H]n-propylapomorphine p r i o r to s o l u b i l i z a t i o n produces a s t a b l e a g o n i s t - r e c e p t o r complex s e n s i t i v e to guanine n u c l e o t i d e s . These data s t r o n g l y suggest the i n t e r a c t i o n of a guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n i n the formation or s t a b i l i z a t i o n of the agonist high a f f i n i t y form of the receptor. We envisage t h a t the f o r m u l a t i o n proposed here of the same receptor s i t e e x i s t i n g i n two d i s t i n c t a f f i n i t y forms could p o s s i b l y represent the same e n t i t y as the Dp and D- subtypes of receptors proposed by Seeman ( 8 ) However much more work on the biochemical c h a r a c t e r i z a t i o p i t u i t a r y gland and th conclusion can be reached. The dopamine receptor i n the p i t u i t a r y gland has the pharmacological c h a r a c t e r i s t i c s of the D« subtype of receptor. Recently, dopamine has been found to decrease c y c l i c AMP accumulation or i n h i b i t adenylate c y c l a s e i n the intermediate lobe and the a n t e r i o r p i t u i t a r y gland (2^,4,10,11,35). Thus, t h i s dopamine receptor may be l i n k e d physToTogiciTly to an i n h i b i t i o n of adenylate c y c l a s e s i m i l a r l y to the alpha^adrenergic receptor. Therefore, the high a f f i n i t y i n t e r a c t i o n of the receptor with agonists and i t s modulation by guanine n u c l e o t i d e s must be i m p l i c a t e d i n the biochemical t r a n s l a t i o n of agonist occupancy of the receptor to the e f f e c t o r system, adenylate c y c l a s e . I t has been suggested (IS) that a putative n u c l e o t i d e binding p r o t e i n d i f f e r e n t from that i m p l i c a t e d i n hormone s t i m u l a t i o n of adenylate c y c l a s e may be involved with a t t e n u a t i n g systems. Further s t u d i e s w i l l be r e q u i r e d , however, i n an attempt to demonstrate a d i r e c t p h y s i c a l i n t e r a c t i o n of the receptor with such a guanine n u c l e o t i d e binding p r o t e i n and i t s r e l a t i o n s h i p to that i m p l i c a t e d i n s t i m u l a t o r y systems. Literature Cited 1. Kebabian, J.W.; P e t z o l d , G.L.; Greengard, P. Proc. N a t l . Acad. Sci. USA 1972, 69, 2145-2149. 2. Cote, T.E.; Grewe, G.W.; Kebabian, J.W. Endocrinology 1982, 110, 805-811. 3. Meunier, H.; L a b r i e , F. L i f e Sci. 1982, 30, 963-968. 4. G i a n n a t t a s i o , G.; D e F e r r a r i , M.E.; Spada, A. L i f e Sci. 1981, 28, 1605-1612.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

ET

AL.

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Pituitary

Gland's Dopamine

Receptor

Kebabian, J.W.; Calne, D.B. Nature 1979, 277, 93-96. T i t e l e r , M . ; List, S.; Seeman, P. Psychopharmacology 1979, 3, 411-420. T i t e l e r , M . ; Seeman, P. Eur. J . Pharmacol. 1979, 56, 291-292. Seeman, P. Pharmacol. Rev. 1980, 32, 229-313. Caron, M.G.; Beaulieu, M . ; Raymond, J.; Gagne, B . ; Drouin, J.; Lefkowitz, R.J.; Labrie, F. J. B i o l . Chem. 1978, 253, 2244-2253. Munenura, M . ; Cote, T . E . ; Tsuruta, K . ; Eskay, R . L . ; Kebabian, J.W. Endocrinology 1980, 106, 1676-1683. Labrie, F . ; Borgeat, T.; Barden, N . ; Godbout, M . ; Beaulieu, M . ; Ferland, L. "Polypeptide Hormones" (Beers, R.J., J r . and Bassett, E . G . , eds.); Raven Press: New York, 1980; pp. 235-251. De Lean, Α.; K i l p a t r i c k B . F . ; Caron M.G Mol Pharmacol 1982, 22, 290-297 Kilpatrick, B.F. 1982, 22, 298-303. De Lean, Α.; Hancock, Α.Α.; Lefkowitz, R . J . Mol. Pharmacol. 1982, 21, 5-16. Rodbell, M. Nature 1980, 284, 17-22. Rodbell, M . ; Krans, H.M.J.; Pohl, S . L . ; Birnbaumer, L. J. B i o l . Chem. 1971, 246, 1872-1876. Lefkowitz, R.J.; M u l l i k i n , D.; Caron, M.G. J. B i o l . Chem. 1976, 251, 4686-4692. De Lean, Α.; Stadel, J . M . ; Lefkowitz, R . J . J. B i o l . Chem. 1980, 255, 7108-7117. Stadel, J . M . ; Shorr, R.G.L.; Limbird, L.E.; Lefkowitz, R.J. J . B i o l . Chem. 1981, 256, 8718-8723. Kent, R . S . ; De Lean, Α.; Lefkowitz, R . J . Mol. Pharmacol. 1980, 17, 14-23. Stadel, J . M . ; De Lean, Α.; Lefkowitz, R . J . Adv. Enzymol. 1982, 53, 1-43. Ehlert, F.J.; Roeske, W.R.; Yamamura, H . I . J. Supramol. Struct. 1980, 14, 149-155. Burgisser, E . ; De Lean, Α.; Lefkowitz, R . J . Proc. Natl. Acad. S c i . USA 1982, 79, 1732-1736. Wolfe, B . B . ; Harden, T.K. J. Cyclic Nucleotide Res. 1981, 7, 303-312. De Lean, Α.; K i l p a t r i c k , B . F . ; Caron, M.G. Endocrinology 1982, 1064-1066. Stadel, J . M . ; Lefkowitz, R . J . Mol. Pharmacol. 1979, 16, 709-718. Ross, E.M.; Howlett, A . C . ; Ferguson, K.M.; Gilman, A.G. Biol. Chem. 1978, 253, 6406-6412. Gorissen, H . ; Laduron, P. Nature 1979, 279, 72-74. Davis, Α.; Madras, B . K . ; Seeman, P. Eur. J. Pharmacol. 1981, 70, 321-323.

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J.

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RECEPTORS

30.

Lew, J.Y.; Fong, J.C.; Goldstein, M.A. Eur. J . Pharmacol.

31.

Clement-Cormier, Y . C . ; Meyerson, L . R . ; McIascc, A. Biochem. Pharmacol. 1 9 8 0 , 2 9 , 2 0 9 - 2 1 6 . Caron, M.G.; Lefkowitz, R.J. J. B i o l . Chem. 1 9 7 6 , 2 5 1 ,

1981, 7 2 ,

32.

403-405.

2374-2384.

33.

Caron, M.G.; Limbird, L.E.; Lefkowitz, R . J . Mol. C e l l .

34. 35.

Limbird, L . E . Biochem. J. 1 9 8 1 , 195, 1-13. Onali, P . ; Schwartz, J . P . ; Costa, E. Proc. Natl. Acad.

36.

Hoffman, B . B . ; Michel, T.; Brenneman, T . B . ; Lefkowitz, R . J . Endocrinology 1 9 8 2 , 110, 9 2 6 - 9 3 2 .

Biochem. 1 9 7 9 , 2 8 , 4 5 - 6 7 .

Sci.

USA 1 9 8 1 , 7 8 ,

6531-6534.

RECEIVED January 7, 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Commentary: The Dopamine Receptor of the Anterior Pituitary Gland S U S A N R. G E O R G E , M A S A Y U K I W A T A N A B E , and P H I L I P S E E M A N University of Toronto, Department of Pharmacology, Toronto, Canada

There is much evidenc dopamine receptor subtype p i t u i t a r y (AP) contains a pure population of postsynaptic D2 dopamine r e c e p t o r s , so that u n l i k e s t u d i e s i n b r a i n it is p o s s i ­ b l e in AP to directly c o r r e l a t e receptor binding parameters to f u n c t i o n a l , p h y s i o l o g i c a l dopaminergic events such as the inhi­ bition of p r o l a c t i n s e c r e t i o n and the a t t e n u a t i o n of adenylate cyclase a c t i v i t y . The D2 receptor appears to e x i s t i n two s t a t e s , one with high and the other lower affinity f o r dopamine agonists. Both forms appear to have h i g h - a f f i n i t y f o r dopamine antagonists. In general, agonist competition curves o f 3H-agonist binding are monophasic with high Hill c o e f f i c i e n t s (~1), and agonist competi­ t i o n curves of 3H-antagonist binding are b i p h a s i c i n AP with low H i l l c o e f f i c i e n t s (50

10" 7

Sultopride

2.9

X

io-

Bulbocapnine

4.7

X

10"

8

>50

^8

*Minimal dose a t t e n u a t i n g v a s o d i l a t i n g responses to bradykinin or i s o p r o t e r e n o l d i v i d e d by minimal dose a t t e n u a t i n g v a s o d i l a t i n g responses to dopamine.

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

4.

GOLDBERG

AND

KOHLI

Differentiation

in the

Periphery

107

of sympathetic nervous system a c t i v i t y since i t i s e l i m i n a t e d by c u t t i n g the sympathetic nerves or by a d m i n i s t e r i n g a gangl i o n i c blocking agent (22J. The v a s o d i l a t i o n i s s p e c i f i c a l l y antagonized by DA antagonists i n doses which do not attenuate v a s o d i l a t i o n produced by i s o p r o t e r e n o l or b r a d y k i n i n . The advantage of u t i l i z i n g femoral a r t e r y v a s o d i l a t i o n in the i n t a c t dog as an endpoint i s that the r e l a t i v e a c t i v i t y of compounds a c t i v e on DAi and DA receptors can be assessed under s i m i l a r c o n d i t i o n s i n the anesthetized dog. The disadvantage of t h i s technique i s that compounds with potent alpha-adrene r g i c v a s o c o n s t r i c t o r a c t i v i t y or n o n - s p e c i f i c vascular a c t i v i t y cannot be s t u d i e d . However, compounds with alpha-adrenergic a c t i v i t y can be compared with standard agonists i n the canine preparation of Long and a s s o c i a t e s (23_). In t h i s method the p o s t g a n g l i o n i c sympathetic nerves are e l e c t r i c a l l y stimulated and the reduction i n t a c h y c a r d i a induced by e l e c t r i c a l s t i m u l a t i o n of the nerve i s taken a s p e c i f i c DA antagonist ate the e f f e c t s of DA from alpha^-adrenergic agonists. In c o n t r a s t to tne r e l a t i v e l y short l i s t of compounds a c t i v e as DA, agonists (Table I ) , a l a r g e number of compounds have been reported to be a c t i v e on DA receptors (2,11,24,25). Table I I I presents a s e l e c t i v e l i s t of these compounds. In a d d i t i o n to the demonstration that more compounds are a c t i v e on the DA r e c e p t o r s , pronounced d i f f e r e n c e s i n potency s e r i e s have been demonstrated i n agonists which are a c t i v e on both r e c e p t o r s . For example, apomorphine i s a weak p a r t i a l agonist of DAi recept o r s , but i s a potent f u l l agonist of DA receptors (2 2z). Furthermore, DPDA, propyl b u t y l DA, propylpentyl DA, p r o p y l i s o b u t y l DA, propylphenethyl DA, and p r o p y l e t h y l DA e x h i b i t d i f f e r e n t potencies as DAi a g o n i s t s , and a l l of them are much l e s s potent than DA as DAi agonists. However, a l l these compounds are equipotent and strong agonists of the DA receptor (21). As with most DA, a g o n i s t s , the m a j o r i t y of DA agonists are a c t i v e on other r e c e p t o r s . In p a r t i c u l a r , many ergot d e r i v a t i v e s e x h i b i t alpha-adrenergic and serotonin a c t i v i t y (26.). In c o n t r a s t , Bach et a l (27.) reported that the pyrazole d e r i v a t i v e (LY 141865) i s devoid of alpha and serotonin agonist and antagonist e f f e c t s . P r e l i m i n a r y s t u d i e s i n our l a b o r a t o r i e s demonstrated t h a t LY 141865 i s a potent DA agonist without alpha-adrenergic or DAj a c t i v i t y . 2

2

2

2

2

2

2

9

2

2

2

DAQ

antagonists

A l l compounds studied thus f a r as DAi antagonists also antagonize DA r e c e p t o r s . We r e c e n t l y reported that domperidone i s an e x t r a o r d i n a r i l y s e l e c t i v e DA receptor antagonist (28). In a range of 0.5-5 jjg/kg i t producesdose r e l a t e d antagonism of DPDA induced femoral v a s o d i l a t i o n without a f f e c t i n g the vasodil a t i o n produced by i s o p r o t e r e n o l or b r a d y k i n i n . In a dose of 2

2

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108

DOPAMINE

RECEPTORS

Table I I I Selected l i s t o f DA

2

agonists with r e l a t i v e DAj a c t i v i t y Relative

activity

Series

Compound

DAo

DA,

Dopamine (DA)

dimethyl DA

active

inactive

dipropyl DA propyl butyl DA propylphenethyl DA propy

all equipotent and

weak and graded*

Aporphine

apomorphine

potent f u l l agonist

weak p a r t i a l agonist

6,7-Dihydroxy-2aminotetralin (ADTN)

dimethyl ADTN

active

inactive

primary amine

active

inactive

N-N-dipropyl derivative

active

active

bromocriptine

active

inactive

pergolide

active

inactive

lisuride

active

inactive

lergotrile

active

inactive

active

inactive

5,6-Dihydroxy-2aminotetralin

Ergot derivatives

Piribedil

*For r e l a t i v e a c t i v i t i e s , see Table I .

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5 mg/kg i n t r a v e n o u s l y , domperidone does not attenuate DAi-induced renal v a s o d i l a t i o n . Domperidone i s i n e f f e c t i v e as an antagonist of alpha^-adrenergic receptors as determined by lack of antagonism of bradycardic e f f e c t s of c l o n i d i n e i n the canine c a r d i a c postg a n g l i o n i c nerve p r e p a r a t i o n . Q u a l i t a t i v e d i f f e r e n c e s have a l s o been observed i n r e l a t i v e DA, and DA a c t i v i t y of several antagonists. The d i f f e r e n c e s are s t r i k i n g l y demonstrated with the enantiomers of s u l p i r i d e . ( S ) - s u l p i r i d e i s much more a c t i v e than ( R ) - s u l p i r i d e as a DA antagonist; whereas, ( R ) - s u l p i r i d e i s s l i g h t l y more a c t i v e tnan ( S ) - s u l p i r i d e as a DAj antagonist (11). 2

2

Spectrum of receptor a c t i v i t y of DA agonists The determination o f the r e l a t i v e a c t i v i t y of DA agonists on DA and other receptors i s of c r i t i c a l importance i n the e l u c i dation of the s t r u c t u r a DA, and DA a g o n i s t s . Fo compare compounds with mixed receptor a c t i v i t y with the s e l e c t i v e DA agonist SK&F 82526 and the r e l a t i v e l y s e l e c t i v e DAo a g o n i s t , LY 141865 t o e l u c i d a t e the chemical requirements f o r tne d i f f e r e n t i a l receptor a c t i v i t y (Figure 1). Determination of r e l a t i v e receptor a c t i v i t y i s a l s o important f o r developing new DA agonists f o r c a r d i o v a s c u l a r and renal therapy. F o r t u n a t e l y , the r e s u l t s obtained i n the anesthetized dog are r e l e v a n t f o r human s t u d i e s . Indeed, the c l i n i c a l s t u d i e s of DA (4) and p r o p y l b u t y l DA (29) were d i r e c t extensions of canine i n v e s t i g a t i o n s . We compare the r e l a t i v e a c t i v i t y of DAi agonists on the f o l l o w i n g r e c e p t o r s : beta, (both d i r e c t l y and i n d i r e c t l y by r e l e a s e of norepinephrine from myocardial storage s i t e s ) , beta^, alpha, and DA^. Table IV describes the spectrum of a c t i v i t y of several compounds studied i n the p e n t o b a r b i t a l anesthetized dog. The methods used have been described i n d e t a i l (21,30). 2

1

Comparison of data obtained i n v i v o and i n v i t r o A comprehensive reivew of studies of the a c t i o n of DA, agonists and antagonists i n i s o l a t e d blood vessels and organs was r e c e n t l y published (_31). ^ g e n e r a l , there i s good c o r r e l a t i o n between i n v i v o and i n v i t r o data with i s o l a t e d canine blood v e s s e l s . However, exceptions have been reported with i s o l a t e d preparations of other species. The major d i f f e r e n c e s are that s u l p i r i d e and i t s enantiomers appear t o be much more potent i n v i v o than i n v i t r o . Another discrepancy i s that bromoc r i p t i n e , which i s i n a c t i v e on DAi receptors i n v i v o , has been shown t o cause DA-like v a s o d i l a t i o n of the i s o l a t e d perfused r a t kidney and r e l a x a t i o n of r a b b i t mesenteric a r t e r y s t r i p s . These data suggest the p o s s i b i l i t y t h a t both DAi and DA receptors may occur on vascular t i s s u e i n some species. However, other n

2

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110

DOPAMINE RECEPTORS

CI

OH SK 8 F 8 2 5 2 6 Figure 1.

L Y 141865

The DA agonist—SK&F

82526—and the DA agonist—LY

t

2

141865.

Table IV Spectrum o f c a r d i o v a s c u l a r receptor a c t i v i t y o f s e l e c t e d DA agonists

β ^Indirect*

α

DAj

DA

Dopamine (DA)

++

50%

0

++

+++

++

Epinine

+++

25%

+

+++

+++

++

A-6,7-DTN

++

100%

0

++++

+++

++

0

0

++

2

A-5,6-DTN

0

0

+++

Di propyl A-5,6-DTN

0

0

0

++++

+++

+++

Dipropyl DA

0

0

0

+

++

++

Propylphenethyl DA

0

0

0

+

+

++

SK&F 38393

0

0

0

0

++++

0

SK&F 82526

0

0

0

0

++++

0

* 0 j a c t i v i t y caused by release o f norepinephrine storage s i t e s .

from

myocardial

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p o s s i b l e mechanisms w i l l have t o be r u l e d out. The major problem i n comparing DAp receptors studied by the d i f f e r e n t techniques i s that alpha -adrenergic a c t i v i t y , which produces s i m i l a r i n h i b i t i o n of norepinephrine r e l e a s e as a c t i v a t i o n of DA r e c e p t o r s , has not been c l e a r l y r u l e d out i n many of the s t u d i e s . 2

2

Determinations o f the DA receptor subtypes r e s p o n s i b l e f o r p h y s i o l o g i c a l and pharmacological a c t i o n s In 1978 we compared the s t r u c t u r e a c t i v i t y requirements f o r the DA, (vascular DA) receptor with other p u t a t i v e DA recept o r s (1,4). The l i m i t e d SAR data a v a i l a b l e at that time supported the existence of at l e a s t two d i f f e r e n t DA r e c e p t o r s . DA-induced responses were d i v i d e d i n t o two categories on the basis of d i f f e r ences i n agonist a c t i v i t y and potency s e r i e s . The f o l l o w i n g phenomena appeared t o be due t o a c t i v a t i o n of DA r e c e p t o r s : i n h i b i t i o n of norepinephrin sympathetic nerve; i n h i b i t i o t i o n o f p r o l a c t i n r e l e a s e ; and emesis. Review o f the current l i t e r a t u r e has not revealed exceptions t o these c o r r e l a t i o n s . In c o n t r a s t , only v a s o d i l a t i o n has been c l e a r l y r e l a t e d t o DA. r e c e p t o r s . I n t e r e s t i n g l y , the potency s e r i e s of agonists f o r s t i m u l a t i o n of adenylate c y c l a s e and renal v a s o d i l a t i o n i s s i m i l a r (2!). Furthermore, SK&F 82526 i s a c t i v e i n both models (15). However, no c o r r e l a t i o n was found i n the r e l a t i v e actions of antagonists ( 8 ) . F i r s t , s u l p i r i d e , which i s a potent antagon i s t of DA-induced renal v a s o d i l a t i o n , i s i n a c t i v e as an antagon i s t of DA-induced s t i m u l a t i o n of adenylate c y c l a s e (32). Second, ergot d e r i v a t i v e s , which are i n a c t i v e as DA^ a g o n i s t s , are antagon i s t s of DA-induced s t i m u l a t i o n of adenylate c y c l a s e (33). These exceptions prevent us from using the Di and Do s u b d i v i s i o n of Kebabian and Calne (34). We have been unable t o r e l a t e the DAi and DA subtypes to s u b d i v i s i o n s proposed by r a d i o l i g a n d binding assays. This i s an extremely c o n t r o v e r s i a l area: one t o four d i f f e r e n t recept o r subtypes have been postulated by binding assays. The problem with binding assays appears t o be r e l a t e d i n part t o lack of s e l e c t i v i t y of the ligands used. For example, spiperone has been shown t o bind t o serotonin and n o n - s p e c i f i c binding s i t e s (36). In c o n t r a s t , when the s e l e c t i v e DA antagonist, domperidone, i s used as the l i g a n d , a better r e l a t i o n s h i p with DA receptors can be demonstrated (36). A question t o be answered by f u t u r e research i s whether use o f s e l e c t i v e DAi ligands w i l l reveal a potency s e r i e s of agonists s i m i l a r t o the DAi subtype. Both DA, and DA~ agonists cause behavioral changes ana thus demonstration of DAi receptors i n c e n t r a l nervous system binding assays should be p o s s i b l e i f t h i s method i s v a l i d (15,16,37). 2

2

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F i n a l l y , behavioral models cannot be used to e s t a b l i s h q u a n t i t a t i v e potency s e r i e s of DA agonists and antagonists because of d i f f e r e n c e s i n blood-brain b a r r i e r p e n e t r a t i o n , r e g i o n a l d i s t r i b u t i o n , and spectra of receptor and non-receptor actions (38). H o p e f u l l y , better c o r r e l a t i o n of p e r i p h e r a l and c e n t r a l receptors w i l l occur as behavioral models improve and more s e l e c t i v e agonists and antagonists become a v a i l a b l e . Acknowledgments We wish to g r a t e f u l l y acknowledge the cooperation of many chemists who provided us with compounds f o r study. We would a l s o l i k e to thank Ms Dana Glock f o r t e c h n i c a l a s s i s t a n c e and Ms P a t r i c i a Gomben f o r s e c r e t a r i a l a s s i s t a n c e . This work was suppoted by NIH grant GM-22220. Literature

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

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Goldberg, L . I . ; Kohli, J.D. Commun. Psychopharmacol. 1979, 3, 447-56. Goldberg, L . I . ; Volkman, P.H.; K o h l i , J.D. Ann. Rev. Pharmacol. T o x i c o l . 1978, 18, 57-79. McNay, J.L.; Goldberg, L . I . J . Pharmacol. Exp. Ther. 1966, 151, 23-31. Goldberg, L . I . Pharmacol. Rev. 1972, 24, 1-29. Goldberg, L . I . ; S o n n e v i l l e , P.F.; McNay, J.L. J . Pharmacol. Exp. Ther. 1968, 163, 188-97. Creese, I . ; S i b l e y , D.R. Biochem. Pharmacol. 1982, 31, 2568-9. Dolak, T.M.; Goldberg, L . I . Ann. Reports Medicinal Chemistry 1981, Chpt. 11, 103-11. Goldberg, L . I . ; Kohli, J.D. "Advances i n the B i o s c i e n c e s " ; Pergamon Press, New York, 1982; p. 41-9. K o h l i , J.D.; Goldberg, L . I . J. Pharmacy Pharmacol. 1980, 32, 225-6. K o h l i , J.D.; Goldberg, L . I . ; McDermed, J.D. Eur. J . Pharmacol. 1982, 81, 293-9. Goldberg, L . I . ; Kohli, J.D.; L i s t i n s k y , J . J . ; McDermed, J.D. "Catecholamines: Basic and Clinical F r o n t i e r s " ; Pergamon Press, New York, 1979; p. 447-9. Pendleton, R.G.; Samler, L.; K a i s e r , C.; R i d l e y , P.R. Eur. J . Pharmacol. 1978, 51, 19-28. Hahn, R.A.; W a r d e l l , J.R., J r . J . Cardiovasc. Pharmacol. 1980, 2, 583-93. Weinstock, J . ; Wilson, J.W.; Ladd, D.L.; Brush, C.K.; Pfeiffer F.R.; Kuo, G.Y.; Holden, K.G.; Yim, N.C.F.; Hahn, R.A.; W a r d e l l , J.R., Jr.; Tobia, A.J.; S e t l e r , P.E.; Sarau, H.M.; R i d l e y , P.T. J . Med. Chem. 1980, 23, 973-5.

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28. 29. 30. 31. 32.

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Hahn, R.A.; W a r d e l l , J.R., Jr.; Sarau, H.M.; R i d l e y , P.T. J . Pharmacol. Exper. Therap. 1982 (in p r e s s ) . K a i s e r , C.; Dandridge, P.Α.; Garvey, E.; Hahn, R.A.; Sarau, H.M.; S e t l e r , P.E.; Bass, L.S.; C l a r d y , J . J . Med. Chem. 1982, 25, 697-703. K o h l i , J.D.; Glock, D.; Goldberg, L . I . "The Benzamides: Pharmacology, Neurobiology, and Clinical Aspects"; Raven P r e s s , New York, 1982; p. 97. Glock, D.; Goldberg, L . I . ; Kohli, J.D. Fed. Proc. 1981, 40, 290 #318. Hahn, R.A.; W a r d e l l , J.R., J r . Arch. Pharmacol. 1980, 314, 177-182. Shepperson, N.B.; Duval, N.; Massingham, R; Langer, S.Ζ. J . Pharmacol. Exp. Ther. 1982, 221, 753-61. K o h l i , J.D.; Weder, A.B.; Goldberg, L . I . ; Ginos, J.Z. J . Pharmacol. Exp Ther 1980 213 370-4 B u y l a e r t , W.A.; Willems Pharmacol. 1978, 30 Long, J.P.; H e i n t z , S.; Cannon, J.G.; Kim, J . J . Pharmacol. Exp. Ther. 1975, 192, 336-42. Lokhandwala, M.F.; T a d e p a l l i , A.S.; Jandhyala, B.S. J. Pharmacol. Exp. Ther. 1979, 211, 620-5. B a r r e t t , R.J.; Lokhandwala, M.F. Eur. J . Pharmacol. 1982, 77, 79-83. Berde, B.; Schild, H.O. "Ergot A l k a l o i d s and Related Com­ pounds"; S p r i n g e r - V e r l a g , New York, 1978. Bach, N.J.; K o r n f e l d , E.C.; Jones, N.D.; Chaney, M.O.; Dorman, D.E.; Paschal, J.W.; Clemens, J.A.; S m a l s t i g , E.B. J . Med. Chem. 1980, 23, 481-91. Glock, D.; Kohli, J.D.; Goldberg, L . I . Fed. Proc. 1982, 41, 1651 #8077. F e n n e l l , W.; T a y l o r , Α.; Brandon, T.; Goldberg, L.; Ginos, J.; Mitchell, J . ; Miller, R. C l i n . Res. 1980, 28, 469A. Meyer, M.B.; Goldberg, L . I . C a r d i o l o g i a 1966, 49, 1-10. Brodde, O.E. L i f e S c i . 1982, 31, 289-306. Spano, P.F.; S t e f a n i n i , E.; Trabucchi, M.; F r e s i a , P. " S u l p i r i d e and other Benzamides"; I t a l i a n Brain Research Foundation Pres, M i l a n , Italy, 1979; pp. 11-31. Kebabian, J.W.; Calne, D.B.; Kebabian, P.R. Commun. Psychopharmacol. 1977, 1, 311-8. Kebabian, J.W.; Calne, D.B. Nature 1979, 277, 93-6. Seeman, P. Biochem. Pharmacol. 1982, 31, 2563-8. Lazareno, S.; Nahorski, S.R. Eur.J.Pharmacol. 1982, 81, 273-85. C o s t a l l , B.; Naylor, R.J.

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R E C E I V E D February 8, 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Commentary: Utility and Problems in the Classification of Dopamine Receptors B A R R Y A. BERKOWITZ Smith Kline & French Laboratories, Department of Pharmacology, Research & Development Division, Philadelphia, P A 19101

Dopamine and dopamin i n t r i g u i n g and usefu b i o l o g i c a l , and therapeutic endeavors. T h i s , when coupled with advances i n p h y s i o l o g i c a l and receptor technologies, has l e d to a number o f viewpoints on the classification o f dopamine receptors. The b r a i n and c a r d i o v a s c u l a r / r e n a l systems have been the areas where the most work has been accomplished. Dopamine receptor s t u d i e s i n the c e n t r a l nervous system have been p r i m a r i l y b i o -chemical, whereas those i n the periphery have been primarily physiological. In t h e i r review Goldberg and Kohli have well summarized the present state of the a r t f o r p h y s i o l o g i c and pharmacologic a n a l y s i s of peripheral dopamine receptors and provide a reasonable classification designated DA1 and D A 2 to i d e n t i f y the two major receptor subtypes. This commentary addresses the utility and problems of dopamine receptor classification.

The studies of Goldberg and colleagues have been and remain pioneering i n not one but at l e a s t two f r o n t s . F i r s t , the concepts and demonstration of vascular dopamine receptors has allowed and stimulated d e t a i l e d studies o f the l o c a t i o n , funct i o n , mechanism, and r o l e of peripheral dopamine and dopamine receptors. Second, t h e i r work l e d to and a c c e l e r a t e d the u t i l i z a t i o n o f dopamine agonists i n c l i n i c a l medicine with the use of intravenously administered dopamine f o r shock and heart f a i l u r e being the best example. In t h e i r communication the evidence i s summarized suggesting two d i s t i n c t types of dopamine receptors i n the p e r i p h e r y , designated DA] and D A 2 . Goldberg and K o h l i ' s c l a s s i f i c a t i o n i s based p r i m a r i l y on i n vivo r e s u l t s using the vasculature and flow of blood to the r e n a l , femoral and other s e l e c t e d 0097-6156/83/0224-0114$06.00/0 © 1983 American Chemical Society

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Utility and Problems

in

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p e r i p h e r a l v a s c u l a r beds. Most o f the other c l a s s i f i c a t i o n s f o r dopamine receptors have been based on r e s u l t s obtained with the b r a i n and cannot be assumed t o apply t o the vasculature and periphery. I n t e r e s t i n g l y , i t i s u s u a l l y not the o r i g i n a t o r s o f any s p e c i f i c c l a s s i f i c a t i o n system who misuse o r misapply i t but f r e q u e n t l y others who t r y t o apply i t t o d i f f e r e n t areas and f i n d exceptions. Why c h a r a c t e r i z e dopamine receptors? There are a number o f reasons t o t r y t o c h a r a c t e r i z e dopamine receptor i n a d d i t i o n t o the f a c t t h a t i t i s f a s h i o n ­ a b l e . Two o f the major reasons w e l l described by Goldberg and K o h l i are (1) the importance o f dopamine receptors subtypes r e s p o n s i b l e f o r p h y s i o l o g i c a l and pharmacological a c t i o n s and (2) the u t i l i z a t i o n o f the concept o f s e l e c t i v e dopamine r e ­ ceptors i n the design an c a r d i o v a s c u l a r and rena Four Major Reasons f o r Problems i n the C l a s s i f i c a t i o n and I d e n t i f i c a t i o n o f Dopamine Receptors i n the Periphery Heterogeniety and s t r u c t u r e o f the v a s c u l a t u r e . The multitude o f f u n c t i o n s o f the v a s c u l a t u r e , i n c l u d i n g conduit r e s i s t a n c e and f i l t r a t i o n v e s s e l s , may u n f o r t u n a t e l y be sub­ served by an e q u a l l y complex heterogeniety o f receptors and receptor l o c a t i o n s . This obviously i n c l u d e s dopamine r e c e p t o r s . Unfortunately (or f o r t u n a t e l y , depending on one's p o i n t o f view) the v a s c u l a t u r e i s not "mushy" l i k e the b r a i n . The c o l ­ lagen and connective t i s s u e content o f blood v e s s e l s does not a l l o w the gentle homogenization and t i s s u e d i s r u p t i o n which has allowed extensive biochemical a n a l y s i s and binding s t u d i e s o f the b r a i n dopamine r e c e p t o r ( s ) . Thus, there i s a huge v o i d i n the biochemical analyses o f the p e r i p h e r a l dopamine r e c e p t o r ( s ) and mechanisms. There i s c l e a r l y much t o be done i n t h i s area f o r the f u t u r e . Dopamine has m u l t i p l e a c t i o n s on the c a r d i o v a s c u l a r system. Unfortunately dopamine i s not a s e l e c t i v e drug f o r dopamine r e c e p t o r s . Whereas we may s t r i v e t o define a c t i o n s o f drugs o r neurotransmitters as dopaminergic, these end p o i n t s have been f r e q u e n t l y defined under l e s s than i d e a l c o n d i t i o n s . Dopamine a l s o has prominent e f f e c t s on a - andfc-adrenor e c e p t o r s . Thus, i n order t o define any type o f dopamine receptor o r subtype, most i n v i v o o r i n v i t r o s t u d i e s o f the periphery and c a r d i o v a s c u l a r system must u t i l i z e a v e r i t a b l e pharmacopeia o f drugs t o block other receptors o r mediator. In v i v o , s t u d i e s with dopamine agonists must g e n e r a l l y be performed using phenoxybenzamine o r other α-adrenoreceptor b l o c k i n g agents. In v i t r o , not only must α - b l o c k i n g drugs be

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used, but f r e q u e n t l y , 3 - r e c e p t o r s b l o c k e r as well as c y c l o oxygenase i n h i b i t o r s . While t h i s approach has been necessary,and i s by no means unique f o r dopaminergic pharmacology, i t has y i e l d e d data which w i l l r e q u i r e confirmation once we obtain more s e l e c t i v e drugs. The f a c t that most i n v i v o p r o t o c o l s d i f f e r s u b s t a n t i a l l y i n t h e i r use of drugs from i n v i t r o p r o t o c o l s may well e x p l a i n many of the c o n f l i c t s i n comparing dopaminergic a c t i o n s i n vivo and i n v i t r o . Lack of s e l e c t i v e drugs a c t i v e a t dopamine r e c e p t o r s . O b v i o u s l y , there i s a need f o r s e l e c t i v e agonist and antagonists of dopamine r e c e p t o r s . Almost by d e f i n i t i o n one cannot accur a t e l y describe a receptor without a s e l e c t i v e agonist or a n t a gonist. T h i s has been the d i f f i c u l t case we face with dopamine receptor s c i e n c e . Lack of In V i t r o Mode of i n v i t r o receptor model has been l e s s progress on well accepted models f o r dopamine receptors. T i s s u e c u l t u r e may well be used i n c r e a s i n g l y i n the future as a source of receptor m a t e r i a l . In a d d i t i o n , there needs to be continued work on the use of i s o l a t e d v a s c u l a r t i s sue to probe dopamine r e c e p t o r s . Conclusion The tendency to examine one's data with a c a l c u l a t o r i n one hand and a Greek d i c t i o n a r y i n the other with the i n v e s t i g a t o r poised to name a new receptor need not be o v e r l y encouraged. Where p o s s i b l e , receptor c l a s s i f i c a t i o n and concepts based upon b i o c h e m i c a l , p h y s i o l o g i c a l and pharmacological evidence would seem to serve us b e s t . Goldberg and Kohli have well summarized the present s t a t e of the a r t f o r the p h y s i o l o g i c and pharmac o l o g i c a n a l y s i s of p e r i p h e r a l dopamine r e c e p t o r s . They are to be commended f o r t h e i r care and c o n t r i b u t i o n i n d e f i n i n g dopamine r e c e p t o r s . T h e i r work stands as a reasonable and secure base f o r future s t u d i e s . R E C E I V E D January 7, 1983

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

5 Dopamine Receptors in the Neostriatum: Biochemical and Physiological Studies J. C. STOOF Free University, Department of Neurology, Medical Faculty, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Dopamine induce e f f e c t s i n the mammalian neostriatum. The occur rence of a D-1 dopamine receptor ( i n the classific a t i o n scheme o f Kebabian and Calne) accounts f o r the ability of dopamine to enhance cyclic AMP f o r mation. The occurrence o f a D-2 dopamine receptor accounts f o r the ability of dopamine to i n h i b i t c y c l i c AMP formation brought about by s t i m u l a t i o n of a D-1 dopamine r e c e p t o r . Dopamine receptors mediate the r e g u l a t i o n of (1) the r e l e a s e or t u r n over of a c e t y l c h o l i n e (postsynaptic dopamine r e ceptor) and (2) the release or turnover o f dopamine (presynaptic a u t o r e c e p t o r ) . Both receptors can be classified as D-2 dopamine r e c e p t o r s . I n d i cations f o r the occurrence of dopamine receptors a f f e c t i n g the r e l e a s e o r turnover of GABA, g l u t a mate, s e r o t o n i n and s e v e r a l neuropeptides are evaluated.

Despite the recent burst of i n t e r e s t i n the p e r i p h e r a l actions of dopamine, t h i s catecholamine i s s t i l l best known as a neurotransmitter i n the c e n t r a l nervous system. This chapter discusses the biochemical and p h y s i o l o g i c a l actions of dopamine i n the neostriatum and the s u b s t a n t i a n i g r a , the regions cont a i n i n g most of the dopamine i n the b r a i n . The goal of t h i s chapter i s to show that the concepts derived from the simple p e r i p h e r a l systems c o n t a i n i n g a s i n g l e category of dopamine r e ceptor 2, 3) can be a p p l i e d to the more complex CNS. T h i s chapter i s not intended to be an a l l i n c l u s i v e compendium o f every technique used to study c e n t r a l dopamine r e c e p t o r s . A l though dopamine receptors can be s t u d i e d i n b i n d i n g assays o r by b e h a v i o r a l p r o t o c o l s , I w i l l not focus a t t e n t i o n on e i t h e r o f these methodologies. Binding studies of dopamine receptors, a l though easy to perform, have y i e l d e d too many data, too many 0097-6156/83/0224-0117$08.50/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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c a t e g o r i e s o f dopamine r e c e p t o r s a n d t o o many c o n t r o v e r s i e s (4^ s e e a l s o C a r o n , t h i s v o l u m e ) . I n p a r t , t h i s s i t u a t i o n may be a consequence o f t h e s e n s i t i v i t y o f b i n d i n g assays t o minor changes i n a s s a y c o n d i t i o n s C5). The i n t e r p r e t a t i o n o f b e h a v i o r a l s t u d i e s i s hampered by t h e c u r r e n t i g n o r a n c e o f t h e n e u r o a n a t o m y and n e u r o p h y s i o l o g y as t o how s t i m u l a t i o n o f a dopamine r e c e p tor i s t r a n s l a t e d i n t o an observable behavior. This chapter w i l l f o c u s a t t e n t i o n upon t h e dopamine r e c e p t o r s i n t h e n i g r o - n e o s t r i a t a l axis which e l i c i t e i t h e r biochemical o r p h y s i o l o g i c a l e v e n t s amenable t o i n v i t r o e x p e r i m e n t a l i n v e s t i g a t i o n . I n d i s c u s s i n g t h e p h a r m a c o l o g y o f e a c h s y s t e m , I w i l l u s e t h e two dopamine r e c e p t o r h y p o t h e s i s as t h e b a s i s f o r my c o n s i d e r a t i o n o f r e c e p t o r p h a r m a c o l o g y ( s e e Brown a n d Dawson-Hughes & K e b a b i a n e t a l . , t h i s volume). The

N i g r o - N e o s t r i a t a l D o p a m i n e r g i c Neurons

Some 3500 d o p a m i n e r g i of t h e s u b s t a n t i a n i g r a i n n e r v a t e the e n t i r e neostriatum ( 6 , 7 ) . W i t h i n t h e n e o s t r i a t u m , t h e axons o f t h e s e n e u r o n s f o r m a n e x t r e m e l y dense t e r m i n a l a r b o r i z a t i o n t h a t c a n be v i s u a l i z e d w i t h f l u o r e s c e n c e h i s t o c h e m i s t r y and i m m u n o c y t o c h e m i c a l t e c h n i q u e s (8^, 9). T h i s t e r m i n a l a r b o r i z a t i o n c o n t a i n s approximately 1 b i l l i o n d o p a m i n e r g i c v a r i c o s i t i e s a n d forms a b o u t 2 0 % o f a l l t h e v a r i c o s i t i e s (10) p r e s e n t i n t h e n e o s t r i a t u m . P o s t s y n a p t i c Dopamine R e c e p t o r s The m a j o r i t y o f n e u r o n s i n t h e n e o s t r i a t u m a r e i n t e r n e u r o n s . The n e o s t r i a t u m r e c e i v e s afférents f r o m d i v e r s e a r e a s l i k e the thalamus, t h e d o r s a l raphe n u c l e i , the c e r e b r a l c o r t e x and t h e s u b s t a n t i a n i g r a . E f f e r e n t s f r o m t h e n e o s t r i a t u m i n n e r v a t e t h e g l o b u s p a l l i d u s and t h e s u b s t a n t i a n i g r a ( 1 1 ) . The n e o s t r i a t u m c o n t a i n s many n e u r o t r a n s m i t t e r s a n d p u t a t i v e n e u r o t r a n s m i t t e r s i n c l u d i n g : a c e t y l c h o l i n e , d o p a m i n e , γ-aminobutyric a c i d , g l u t a m a t e , s e r o t o n i n and t h e p e p t i d e s c h o l e c y s t o k i n i n , enkephalin and s u b s t a n c e Ρ (12, 1 3 ) . T h e o r e t i c a l l y , t h e d o p a m i n e r g i c n e u r o n s c o u l d communicate v i a d o p a m i n e r g i c s y n a p s e s and p o s t s y n a p t i c dopamine r e c e p t o r s w i t h n e u r o n s c o n t a i n i n g e a c h o f t h e s e n e u r o t r a n s m i t t e r s . C o n s e q u e n t l y , changes i n e i t h e r t h e r e l e a s e o r t h e t u r n o v e r o f any o f t h e s e n e u r o t r a n s m i t t e r s c o u l d p r o v i d e e v i d e n c e o f a c t i v i t y a t a p o s t s y n a p t i c dopamine receptor. A P o s t s y n a p t i c Dopamine R e c e p t o r R e g u l a t i n g or T u r n o v e r o f A c e t y l c h o l i n e

the Release

A c e t y l c h o l i n e i s a m a j o r n e u r o t r a n s m i t t e r i n t h e neo­ s t r i a t u m . W i t h i n the neostriatum, both the content of a c e t y l ­ c h o l i n e and t h e s p e c i f i c a c t i v i t y o f c h o l i n e a c e t y l t r a n s f e r a s e ,

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the enzyme s y n t h e s i z i n g a c e t y l c h o l i n e , are extremely high (14) . Most of the n e o s t r i a t a l a c e t y l c h o l i n e occurs w i t h i n i n t e r neurons; only a small amount of n e o s t r i a t a l a c e t y l c h o l i n e can be associated with a f f e r e n t neurons o r i g i n a t i n g i n the center median-parafascicular complex of the thalamus (11). The dopaminergic n i g r o - n e o s t r i a t a l neurons make synaptic contacts with the c h o l i n e r g i c interneurons (Γ5, F i g u r e 1). The content of a c e t y l c h o l i n e i n the neostriatum i s increased by dopamine receptor agonists and decreased by dopamine receptor antagonists 0 6 , _1_7, J_8) . Apomorphine and L-DOPA i n h i b i t the i n vivo r e l e a s e of a c e t y l c h o l i n e from cat neostriatum; t h i s e f f e c t i s blocked by n e u r o l e p t i c s ( 19) . RU 24926 (chemical s t r u c t u r e depicted i n F i g u r e 2), a s e l e c t i v e D-2 agonist, i n ­ creases the content of n e o s t r i a t a l a c e t y l c h o l i n e (20). Together, these data from i n v i v o experiments are c o n s i s t e n t with the hypothesis t h a t s t i m u l a t i o n of a D-2 receptor upon the c h o l i n ­ e r g i c interneurons increase c h o l i n e by b l o c k i n g i t In vivo experiments studying e i t h e r the content or the r e l e a s e of n e o s t r i a t a l a c e t y l c h o l i n e are time consuming, tech­ n i c a l l y d i f f i c u l t and not always easy to i n t e r p r e t . Conversely, i n v i t r o studies o f a c e t y l c h o l i n e r e l e a s e with a s u p e r f u s i o n technique are less time consuming, t e c h n i c a l l y e a s i e r to perform and e a s i e r to i n t e r p r e t . With t h i s i n v i t r o technique, s l i c e s of neostriatum are incubated with r a d i o l a b e l e d c h o l i n e , a precursor of a c e t y l c h o l i n e , i n order to l a b e l the pool of newly synthe­ s i z e d a c e t y l c h o l i n e . The t i s s u e i s t r a n s f e r r e d to a s u p e r f u s i o n apparatus and the calciurn-dependent r e l e a s e of r a d i o l a b e l e d a c e t y l c h o l i n e i s evoked e l e c t r i c a l l y , with v e r a t r i d i n e or with elevated potassium concentrations (21). The s u p e r f u s i o n tech­ nique i s a simple procedure f o r i n v e s t i g a t i n g the r e l e a s e of a c e t y l c h o l i n e (or other n e u r o t r a n s m i t t e r s ) . However, there are some l i m i t a t i o n s to the technique. For example: dopaminergic agonists e l i c i t no more than a 50% to 60% i n h i b i t i o n of a c e t y l ­ c h o l i n e r e l e a s e from r a t neostriatum (e.g. F i g u r e 3). I t i s not c l e a r i f this implies that 40% of the c h o l i n e r g i c interneurons i n r a t neostriatum do not possess dopamine r e c e p t o r s . A l t e r ­ n a t i v e l y , because d i f f e r e n t species ( r a t versus c a t or r a b b i t ) and d i f f e r e n t techniques (potassium-evoked r e l e a s e versus e l e c ­ t r i c a l l y stimulated r e l e a s e ) give q u a n t i t a t i v e l y d i f f e r e n t r e s u l t s (22-27), i t remains p o s s i b l e that there are t e c h n i c a l l i m i t a t i o n s to the p r e c i s i o n of t h i s technique. A more d e t a i l e d d e s c r i p t i o n of t h i s method i s presented elsewhere (26, 27). The r e s u l t s obtained from in v i t r o s up erf us i o n experiments are i n accord w i t h the c o n c l u s i o n that the c h o l i n e r g i c i n t e r ­ neurons possess a D-2 dopamine r e c e p t o r . Dopamine i n h i b i t s the r e l e a s e of [3H]-acetylcholine from n e o s t r i a t a l t i s s u e ; however, concentrations greater than 1 μ Μ are required to achieve maximal i n h i b i t i o n . Because dopamine i s removed from the e x t r a c e l l u l a r space by the dopaminergic nerve terminals, i t i s d i f f i c u l t to

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Figure 1. Degenerating nerve ending (^>) in guinea pig neostriatum following 6-OH-dopamine administration making asymmetrical synaptic contact with dendritic spine positively staining for cholineacetyltransferase Bar indicates 0.125 μτη. (Reproduced with permission from Ref. 15. Copyright 1976, Elsevier Biomedical Press.)

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S K & F 38393

Figure 2. Chemical structures of LY 141865, RU 24926, and SKF 38393.

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120

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Figure 3. D-2 receptor agonists, RU 24926 and LY 141865, inhibit the K stimulated release of [ Η]-acetylcholine from blocks of rat neostriatum. (Repro­ duced with permission from Ref. 96. Copyright, Elsevier Biomedical Press.) 3

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r e l i a b l y estimate the concentration of dopamine causing maximal i n h i b i t i o n . Apomorphine and a m i n o t e t r a l i n d e r i v a t i v e s i n h i b i t i n a dose-dependent manner the release o f [3H]-acetylcholine from n e o s t r i a t a l t i s s u e (22-27) . The s e l e c t i v e D-2 agonists, LY 141865 and RU 24926 (28, 29 ; chemical s t r u c t u r e s depicted i n Figure 2) i n h i b i t the r e l e a s e o f [3H]-acetyl c h o l i n e from neos t r i a t a l t i s s u e s l i c e s (Figure 3). The maximal e f f e c t o f e i t h e r drug i s an approximate 50% r e d u c t i o n o f the f r a c t i o n a l rate o f r e l e a s e of [3H]-acetylcholine. LY 141865 i s half-maximally a c t i v e a t a c o n c e n t r a t i o n of 70 nM; t h i s i s very s i m i l a r to i t s potency upon the D-2 receptor i n the intermediate lobe o f the r a t p i t u i t a r y gland (28). RU 24926 i s half-maximally a c t i v e a t a c o n c e n t r a t i o n of 7 nM; t h i s i s very s i m i l a r to i t s potency upon the D-2 dopamine r e c e p t o r on the mammotrophs of the anter i o r p i t u i t a r y gland (29). Likewise ( - ) - s u l p i r i d e , a s e l e c t i v e D-2 antagonist (3, 30 31) reverses the i n h i b i t i o n of L3H]-acetylcholine r e l e a s RU 24926 (Figure 4 ) . Furthermore agonist (32, 33, 34; chemical s t r u c t u r e depicted i n Figure 2 ) , does not i n h i b i t (and a t high concentrations s l i g h t l y stimul a t e s ) the r e l e a s e of [3H]-acetylcholine (Figure 3). Other n e u r o l e p t i c drugs antagonize the i n h i b i t o r y e f f e c t o f dopamine and dopaminergic agonists on the r e l e a s e o f [3H]- a c e t y l c h o l i n e (22, 26, 27). The i n v i t r o e f f e c t s of dopaminergic agonists and antagonists upon the r e l e a s e o f [3Hj-acetylcholine r e i n f o r c e the c o n c l u s i o n drawn from i n vivo studies that a D-2 receptor regul a t e s the r e l e a s e of n e o s t r i a t a l a c e t y l c h o l i n e . Despite the t e c h n i c a l or methodological l i m i t a t i o n s of experiments determining the release of a c e t y l c h o l i n e from the neostriatum, this experimental parameter i s an extremely v a l u able model f o r studying a postsynaptic CNS dopamine receptor. A c e t y l c h o l i n e release i s one of the few p h y s i o l o g i c a l parameters regulated by a dopamine receptor that can be q u a n t i f i e d w i t h i n v i t r o techniques. Furthermore, dopaminergic r e g u l a t i o n of a c e t y l c h o l i n e release i s a matter of some p r a c t i c a l i n t e r e s t . For example, Parkinson's disease i s a n e o s t r i a t a l dopamine d e f i ciency syndrome (35). The loss of n e o s t r i a t a l dopamine d i s r u p t s the balance between the n e o s t r i a t a l dopaminergic and c h o l i n e r g i c systems that i s thought to regulate normal a c t i v i t y i n the neos t r i a t u m (36) . The dopaminergic agonists p r e s e n t l y used i n the treatment of Parkinsonism (37, 38) may achieve some of t h e i r therapeutic e f f e c t by d i r e c t l y s t i m u l a t i n g dopamine receptors ; however some o f t h e i r therapeutic e f f e c t may be a consequence of s t i m u l a t i n g the dopamine receptor upon the c h o l i n e r g i c i n t e r neuron and thereby r e s t o r i n g the balance between the dopamine r g i c and c h o l i n e r g i c systems i n the neostriatum. P r i o r to the advent o f L-DOPA therapy f o r Parkinsonism, c h o l i n e r g i c antagonists were widely used to a l l e v i a t e the symptoms o f t h i s disease (39).

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3

μ

Figure 4. Reversal by (-)-sulpiride of either the RU 24926 (left) 0.1 Μ or the LY 141865 (right) (1.0 iM)-inhibited release of [ H]-acetylcholine (K*-stimulated) from blocks of rat neostri­ atum. The release of [ H]-acetylcholine was determined without drugs (Q) or with one of the D-2 receptor agonists in the presence of (-)-sulpiride(O). (Reproduced with permission from Ref. 96. Copyright, Elsevier Biomedical Press.)

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A Postsynaptic Dopamine Receptor Regulating o r Turnover o f GABA?

the Release

Both GABA and glutamic a c i d decarboxylase (GAD) , a marker enzyme f o r GABA-containing nerve terminals, occur i n the neostriatum, globus p a l l i d u s and s u b s t a n t i a n i g r a (12) . The somata o f GABA-containing neurons occur predominantly i n the globus p a l l i d u s : these neurons p r o j e c t to the zona r e t i c u l a t a of the s u b s t a n t i a n i g r a ; t h e i r recurrent c o l l a t e r a l s p r o j e c t to the neostriatum.GABA-containing interneurons and g l i a a l s o occur i n the neostriatum (40-43). In vivo s t u d i e s i n d i c a t e that dopamine may i n f l u e n c e the GABA-containing c e l l s i n the neostriatum and s u b s t a n t i a n i g r a . Thus, apomorphine increases the content o f n e o s t r i a t a l GABA (44) ; conversely, h a l o p e r i d o l decreases the content (45) and turnover (46) of n e o s t r i a t a l GABA Chronic (8 weeks) treatment with e i t h e r h a l o p e r i d o GAD a c t i v i t y but does neostriatum. D e s t r u c t i o n of the n i g r o - n e o s t r i a t a l dopaminergic neurons also increases n e o s t r i a t a l GAD a c t i v i t y (47). I n e x p e r i ments using a push-pull canula, e i t h e r apomorphine or dopamine i n h i b i t s the r e l e a s e o f endogenous n e o s t r i a t a l GA.BA (48) . In the s u b s t a n t i a n i g r a , h a l o p e r i d o l decreases the content of GABA (45) , while e i t h e r apomorphine o r dopamine i n h i b i t the r e l e a s e of endogenous GABA (49). The e f f e c t s o f dopaminergic drugs upon the s y n t h e s i s , storage and r e l e a s e o f GABA are i n accord w i t h the p o s s i b i l i t y that a dopamine receptor might regulate the a c t i v i t y o f GABAc o n t a i n i n g neurons. However, this p o s s i b i l i t y can be accepted w i t h only l i m i t e d enthusiasm. Extremely high doses of dopamine r g i c drugs are r e q u i r e d to e l i c i t e f f e c t s i n the n e o s t r i a t a l GABA system. Although apomorphine a t a dose o f 0.05 mg/kg w i l l s t i m u l a t e dopamine receptors (50), apomorphine must be used a t a dose of 20 mg/kg to induce a 25% increase i n n e o s t r i a t a l GABA (44). Likewise, h a l o p e r i d o l must be used a t 10 mg/kg to induce a 37% decrease i n the content of n e o s t r i a t a l GABA (45) . Such a massive dosis of h a l o p e r i d o l r e s u l t s i n b r a i n concentrations so high that h a l o p e r i d o l can block not only dopamine receptors b u t a l s o a-1 and a-2 adrenoceptors, H-1 and H-2 histamine receptors and s e r o t o n i n receptors (5 1) . Therefore, i t appears o p t i m i s t i c to conclude from such i n vivo experiments t h a t the observed e f f e c t s of dopaminergic drugs upon the n e o s t r i a t a l GABA system r e s u l t from an i n t e r a c t i o n with a s p e c i f i c dopamine r e c e p t o r . This negative c o n c l u s i o n i s i n accord with the data o f Pycock e t a l . (52) who could not demonstrate that s u b t l e manipulations o f c e n t r a l dopaminergic systems a l t e r e d the c o n c e n t r a t i o n o f GABA. The p o s s i b i l i t y that dopamine regulates n e o s t r i a t a l GABAe r g i c f u n c t i o n receives no support from i n v i t r o s u p e r f u s i o n experiments determining the release o f [3H]-GABA from s l i c e s o f r a t neostriatum (53) . The D-2 receptor agonist RU 24926 i s

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i n e f f e c t i v e i n modulating the potassium-stimulated r e l e a s e o f [3H]-GABA a t concentrations maximally i n h i b i t i n g a c e t y l c h o l i n e r e l e a s e (Figure 3). Also the D-l receptor agonist SKF 38393 does not cause a s i g n i f i c a n t change i n the release o f [J3H_-GABA from n e o s t r i a t a l t i s s u e . Therefore, the reported e f f e c t s o f 100 μ Μ apomorphine (48, 49, 54) must be explained as a consequence o f an i n t e r a c t i o n w i t h an e n t i t y other than a D-1 o r a D-2 dopamine receptor. As noted e a r l i e r , GABA appears to be a neurotransmitter i n the s u b s t a n t i a n i g r a . F o l l o w i n g chronic blockade o f dopamine receptors w i t h h a l o p e r i d o l , the turnover r a t e o f n i g r a l GABA i s depressed; acute h a l o p e r i d o l i s without e f f e c t (47). The e f f e c t s of dopamine upon the r e l e a s e o f Q3H]-GABA from the s u b s t a n t i a n i g r a are c o n t r o v e r s i a l . Reubi e t a l . (55) r e p o r t that dopamine, a t high concentrations, stimulates the r e l e a s e of [3H]-GABA from the n i g r a i n v i t r o . D i b u t y r y l cAMP mimicks t h i s e f f e c t o f dopa­ mine, thereby r a i s i n g ates t h i s e f f e c t . However produce these observations and concluded that dopamine does not modulate the spontaneous or stimulus-evoked r e l e a s e o f GABA i n the s u b s t a n t i a n i g r a . Therefore, i t seems premature to accept the p o s s i b i l i t y that dopamine a f f e c t s GABAergic f u n c t i o n i n the s u b s t a n t i a n i g r a v i a receptors l o c a t e d on GABAergic neurons. A Postsynaptic Dopamine Receptor Regulating Turnover o f Glutamate?

the Release o r

The neostriatum receives a massive neuronal input from the i p s i l a t e r a l c e r e b r a l cortex (57). These f i b e r s d i s t r i b u t e , i n an organized way, to a l l parts o f the neostriatum. The h e a v i e s t p r o j e c t i o n comes from the sensorimotor c o r t e x . Spencer (58) i n i ­ t i a l l y suggested that ( p a r t of) these c o r t i c o s t r i a t a l f i b e r s u t i l i z e glutamate as a neurotransmitter. Evidence i m p l i c a t i n g glutamate as the major neurotransmitter used by these f i b e r s i s gradually accumulating (43). C o r t i c a l a b l a t i o n has been found to r e s u l t i n 40-50% r e d u c t i o n of high a f f i n i t y uptake o f l a b e l e d glutamate i n n e o s t r i a t a l t i s s u e (59, 60) . S t r i a t a l neurons are e x c i t e d by e i t h e r d i r e c t c o r t i c a l s t i m u l a t i o n o r by i o n t o p h o r e t i c a p p l i c a t i o n of glutamate; these e f f e c t s are blocked by L-glutamate d i e t h y l e s t e r , a glutamate antagonist (58). E l e c t r i ­ c a l s t i m u l a t i o n of the f r o n t a l c o r t e x induces s p e c i f i c r e l e a s e of l a b e l e d glutamic a c i d from r a t neostriatum (61). Furthermore, endogenous glutamate i s r e l e a s e d , i n a calcium-dependent manner, by elevated concentrations o f potassium ions (62). Dopamine receptors may occur on the terminals of the glutamatergic afférents to the neostriatum (63). F o l l o w i n g c o r t i c a l a b l a t i o n , the number o f |J3H]-haloperidol b i n d i n g s i t e s i n the s t r i a t u m i s reduced by 32% (63) . I n an i n v i t r o superf u s i o n system, dopamine, apomorphine, amino t e t r a l i n d e r i v a t i v e s o r bromocriptine i n h i b i t the d e p o l a r i z a t i o n - i n d u c e d r e l e a s e o f

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[3H]-gluta ma te (64, 65). However, very high concentrations of these drugs are needed to e l i c i t t h i s i n h i b i t o r y e f f e c t . In one of these studies d i b u t y r y l cAMP d i d not mimic the e f f e c t s of the dopaminergic agonists (65) ; t h e r e f o r e , i t i s very u n l i k e l y that a D-l r e c e p t o r mediates t h i s modulation of glutamate r e l e a s e . In a recent study (53), n e i t h e r the D-l a g o n i s t SKF 38 39 3, nor the D-2 agonist RU 24926, a l t e r e d the d e p o l a r i z a t i o n - i n d u c e d r e l e a s e of [3H]-glutamate (even when tested a t concentrations maximally a c t i v e on t h e i r r e s p e c t i v e r e c e p t o r s ) . Thus, i t appears that neither a D-l nor a D-2 receptor modulates the r e l e a s e of glutamate. A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of Serotonin? Serotonin-containing neurons p r o j e c t from the raphe n u c l e i to the neostriatum (66 projection originates In a d d i t i o n , a major s e r o t o n i n e r g i c pathway from the medial and d o r s a l raphe n u c l e i (69) p r o j e c t s to the s u b s t a n t i a n i g r a . In preparing t h i s review, I d i d not encounter any reports suggest­ ing that the turnover or r e l e a s e of s e r o t o n i n i n the neostriatum i s d i r e c t l y i n f l u e n c e d by drugs s t i m u l a t i n g or b l o c k i n g dopamine r e c e p t o r s . Furthermore, according to Hassler (43) and Pasik (70), there are not many axo-axonal contacts i n the neostriatum. This makes the occurrence of d i r e c t s y n a p t i c contacts between dopaminergic and s e r o t o n i n e r g i c nerve terminals u n l i k e l y * Obviously, i t i s s t i l l p o s s i b l e that both neuronal systems communicate v i a mechanisms other than s y n a p t i c c o n t a c t s . A few studies describe i n t e r a c t i o n s between the dopamin­ e r g i c and the s e r o t o n i n e r g i c systems i n the s u b s t a n t i a n i g r a , thereby suggesting a r o l e f o r n i g r a l dopamine r e c e p t o r s . Dopamine (0.1 μ Μ ) i n h i b i t s the r e l e a s e o f [3H]-serotonin (71), while apomorphine (50 μ Μ ) stimulates the r e l e a s e o f [3H]-serot o n i n (72). These apparently c o n f l i c t i n g observations preclude any d e f i n i t i v e conclusions being drawn about the involvement of dopamine receptors i n r e g u l a t i n g the turnover or r e l e a s e of s e r o t o n i n i n the s u b s t a n t i a n i g r a . A Postsynaptic Dopamine Receptor Regulating the Release or Turnover of Peptide Neurotransmitters? The neostriatum contains many of the peptides which are c u r r e n t l y fashionable research e n t i t i e s ( f o r a review see 73). In t h i s s e c t i o n , I w i l l mention the peptides which have been implicated as being under dopaminergic c o n t r o l . Substance Ρ was among the f i r s t peptides d i s c o v e r e d i n the n i g r o - n e o s t r i a t a l a x i s . This peptide occurs i n both interneurons and i n neurons p r o j e c t i n g to the s u b s t a n t i a n i g r a (74) . Several pieces o f c i r c u m s t a n t i a l evidence p o i n t to a dopaminergic e f f e c t upon

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substance P. Chronic treatment with h a l o p e r i d o l reduces the c o n c e n t r a t i o n of substance Ρ i n the s u b s t a n t i a n i g r a (75) . However, treatment with apomorphine a l s o lowers n i g r a l substance Ρ c o n c e n t r a t i o n ; h a l o p e r i d o l blocks t h i s e f f e c t (76). Likewise, amphetamine causes a dose-dependent r e d u c t i o n i n substance P - l i k e immunoreactive m a t e r i a l w i t h i n the neostriatum (but not i n the s u b s t a n t i a n i g r a ) ; h a l o p e r i d o l blocks t h i s e f f e c t (77) . The neostriatum has a high content of enkephalin and these e n k e p h a l i n - l i k e peptides occur i n n e o s t r i a t a l interneurons (78). Chronic treatment with h a l o p e r i d o l increases both the content and the r e l e a s e of n e o s t r i a t a l enkephalin (79) . However, i n v i t r o s t u d i e s f a i l to demonstrate any dopaminergic r e g u l a t i o n of enkephalin r e l e a s e (79). C h o l e c y s t o k i n i n (CCK)-like immunoreactive m a t e r i a l occurs i n the CNS and the caudate has the h i g h e s t c o n c e n t r a t i o n of any b r a i n r e g i o n (13, 80). However, dopaminergic r e g u l a t i o n of neo­ s t r i a t a l CCK r e l e a s e ha i n g l y , CCK-like p e p t i d e ( s dopaminergic neurons (73) . This r a i s e s the p o s s i b i l i t y that CCK might r e g u l a t e dopaminergic a c t i v i t y with a novel, but at present unknown, mechanism. In summary, i t i s d i f f i c u l t to g e n e r a l i z e about dopamin­ e r g i c c o n t r o l of p e p t i d e r g i c f u n c t i o n i n the neostriatum. This circumstance i s a consequence of the ignorance of the physio­ l o g i c a l functions regulated by the peptides and the l i m i t e d number of i n v e s t i g a t i o n s d i r e c t e d towards n e o s t r i a t a l p e p t i d e s . A Postsynaptic Dopamine Receptor Regulating C y c l i c AMP

Formation

A postsynaptic dopamine receptor i n the neostriatum can be c h a r a c t e r i z e d w i t h biochemical procedures. The b a s i s f o r t h i s c h a r a c t e r i z a t i o n i s the a b i l i t y of dopamine to s t i m u l a t e adenylate c y c l a s e a c t i v i t y i n c e l l - f r e e homogenates of neo­ s t r i a t a l t i s s u e . E a r l i e r i n this volume, Brown and Dawson-Hughes d i s c u s s the p r o p e r t i e s of this receρtor-enzyme system i n the bovine p a r a t h y r o i d gland and summarize the evidence that a dopamine-stimulated formation of c y c l i c AMP t r i g g e r s the r e l e a s e of parathyroid hormone from t h i s bovine t i s s u e . However, the r o l e of this enzyme i n e i t h e r the neostriatum or the s u b s t a n t i a n i g r a i s unknown. In the s u b s t a n t i a n i g r a , dopamine was reported to s t i m u l a t e the r e l e a s e of [3H]-GABA, and this e f f e c t was mimicked by d i b u t y r y l c y c l i c AMP (55) . However, as noted e a r l i e r i n t h i s chapter, these observations could not be reproduced by another group (56) . Thus, i n both the neostriatum and the sub­ s t a n t i a n i g r a , the dopamine-sensitive adenylate c y c l a s e i s a receptor i n search of a f u n c t i o n . At l e a s t a major p a r t of the n e o s t r i a t a l dopamine-sensitive adenylate c y c l a s e a c t i v i t y i s not a s s o c i a t e d with the terminals of the dopamine-containing n i g r o - s t r i a t a l neurons. I n t r a n i g r a l i n j e c t i o n s of 6-OH-dopamine which destroy these dopamine-

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c o n t a i n i n g neurons do not cause a loss of s t r i a t a l dopamines e n s i t i v e adenylate c y c l a s e a c t i v i t y (82). However, i n t r a s t r i a t a l i n j e c t i o n s of k a i n i c a c i d which destroy neuronal soma t a cause a s u b s t a n t i a l loss of t h i s dopamine-sensitive enzyme a c t i v i t y (83, 84). In s t u d i e s of the s u b c e l l u l a r d i s t r i b u t i o n of t h i s enzyme a c t i v i t y , the h i g h e s t s p e c i f i c enzyme a c t i v i t y i s found i n submitochondrial f r a c t i o n s enriched with nerve endings (85). These observations are compatable w i t h a post­ synaptic l o c a t i o n of the enzyme a c t i v i t y upon interneurons or r e c u r r e n t c o l l a t e r a l s of n e o s t r i a t a l e f f e r e n t s . A dopamines e n s t i v e adenylate c y c l a s e a c t i v i t y a l s o occurs i n the sub­ s t a n t i a n i g r a (86, 87, 88). This n i g r a l enzyme i s not a s s o c i a t e d w i t h the dopamine-containing neurons . The pharmacological p r o p e r t i e s of the dopamine-sensitive adenylate c y c l a s e a c t i v i t y i n e i t h e r the bovine p a r a t h y r o i d gland or the neostriatum are summarized by Brown and DawsonHughes e a r l i e r i n t h i (89, 90). Dopamine, i n a d d i t i o n to s t i m u l a t i n g the formation of c y c l i c AMP, i s a l s o able to i n h i b i t the formation of t h i s c y c l i c n u c l e o t i d e . This i n h i b i t o r y e f f e c t of dopamine can be c l e a r l y demonstrated i n e i t h e r the mammotrophs of the a n t e r i o r p i t u i t a r y gland (91-94) or the melanotrophs of the intermediate lobe of the p i t u i t a r y gland (see Kebabian e t a l . , this volume). Both the stimulatory and the i n h i b i t o r y e f f e c t of dopamine under c y c l i c AMP formation can be demonstrated i n the neostriatum u s i n g i n v i t r o s u p e r f u s i o n . E i t h e r dopamine or SKF 38 393 stimulates the e f f l u x of c y c l i c AMP from s l i c e s o f r a t n e o s t r i a tum(Figures 5 and 6,95^, 96) ; this i s i n accord with the a b i l i t y of e i t h e r of these drugs to s t i m u l a t e adenylate c y c l a s e a c t i v i t y i n c e l l - f r e e homogenates o f the neostriatum (37). LY 141865, the s e l e c t i v e agonist upon the D-2 dopamine r e c e p t o r (28), reduced the magnitude of the SKF 38 393-induced e f f l u x of c y c l i c AMP but d i d not change the c o n c e n t r a t i o n of a g o n i s t g i v i n g half-maximal e f f l u x . The i n h i b i t o r y e f f e c t of LY 141865 occurs even i n the absence of c a l c i u m ions from the s u p e r f u s i o n medium. Since i t i s commonly accepted that calcium ions are e s s e n t i a l f o r the r e l e a s e of neurotransmitter (21) this l a t t e r o b s e r v a t i o n suggests that neurotransmitter r e l e a s e i s not required f o r the dopaminergic i n h i b i t i o n of c y c l i c AMP r e l e a s e (96). In a d d i t i o n , ( - ) - s u l p i r i d e , an antagonist of the D-2 dopamine receptor, markedly p o t e n t i a t e s the dopamine-stimulated e f f l u x of c y c l i c AMP but does not appreciably change the molar potency of dopamine (Figure 6). I t must be s t r e s s e d here that i n the experiment depicted i n Figure 6 an unusually high c o n c e n t r a t i o n of ( - ) - s u l p i r i d e has been used (50 μ Μ ) . However, one has to r e a l i z e that approximately 100 μ M dopamine i s r e q u i r e d to maximally a c t i v a t e the D-l dopamine r e c e p t o r . To b l o c k the e f f e c t s of 100 μ M dopamine on the D-2 dopamine receptor high concentrations of ( - ) - s u l p i r i d e are needed. F i g u r e 7 presents a

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10"

7

10"

6

10~

10- 5

S K F 38393 (M) Figure 5. Inhibition by LY 141865 of the SKF 38393-stimulated efflux of cAMP from blocks of rat neostriatum. The efflux of cAMP from neostriatal tissue, stimu­ lated with the indicated concentrations of SKF 38393, was estimated in the absence (O) or presence (M) of 5 μΜ LY 141865. (Reproduced with permission from Ref. 96. Copyright, Elsevier Biomedical Press.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

5.

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ο"ιο-

β

in the

i