INTERNATIONAL REVIEW OF
Neurobiology VOLUME 24
Editorial Board W. Ross ADEY
SEYMOUR KE~V
JCLICS AXELROD
KEII H KI...
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INTERNATIONAL REVIEW OF
Neurobiology VOLUME 24
Editorial Board W. Ross ADEY
SEYMOUR KE~V
JCLICS AXELROD
KEII H KILLAM
Ross RALDESSARINI
CON AN
SIR ROGER B.L\NNISTER
ABELLAITHA
FLOYD BLOOM
BORISL F . B E D ~ V
DANIEL. Boi'E'r
PAVL MANDELL
PHILLIPBRADLEY
HUMPHRY OSMONI)
JOSE
DELGADO
KOR~ETSKY
RODOLFOPAOLETT1
SIRJOHX E(:CI,LS
SOl.OMON SNYDEK
JOEI+ ELKS
STEPHENSZARA
H. J .
JOHN VANE
EYSESCK
KJELL Fort
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Bo HOLMSrEr)r
RICHARDW'I'Ar-r
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INTERNATIONAL REVIEW OF
Neurobiology Edited by JOHN R. SMYTHIES Deportment of Psychiatry ond The Neurosciences Program University of Alabomo Medical Center Birminghorn, Alabomo
RONALD J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birrninghom, Alobomo
VOLUME 24
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovonovich, Publishers
New York London Pork Son Diego Son Francisco SBo Paul0 Sydney Tokyo Toronto
COPYRIGHT @ 1983, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY F O R M OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, I N C . 111 Fifth Avenue, New York. New York 10003
United Kingdom Edition published by ACADEMIC PRESS, I N C . ( L O N D O N ) LTD. 24/28Oval Road, London N W l 7DX
LIBRARY OF
59- 13822
CONGRESS CATALOG CARD NUMBER:
I S B N 0-12-366824-7 PRINTED IN THE UNITED STATES OF AMERICA
83 84 8s 86
9 816 5 4 3 2 1
CONTENTS ..................................................................... CONTRIBUTORS
ix
Antiacetylcholine Receptor Antibodies and Myasthenia Gravis
BERNARD W. FULPIUS 1. Introduction . . . . . . . . . . . . . . 11. Pathogenicity of Circulating Anti-nAcChR Antibodies 111. Assays for Circulating Anti-n
................... 1 ...................... 2 ................... 5
IV. Anti-nAcChR Antibody Concentration in Different Forms of Myasthenia Gravis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Anti-nAcChR Antibodies in the Cerebrospinal Fluid ....................... VI. Antigenic Determinants on nAcChR . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 10
12 13
Pharmacology of Barbiturates: Electrophysiological and Neurochemical Studies
MAX WILLOW
AND
GRAHAMA. R.JOHNSTON
I. Introduction ..... .............................. 11. Neuropharmacolog 111. Biochemical and Neurochemical Studies . .................... 1V. Conclusions ............................................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15 16 34
44 45
lmrnunodetection of Endorphins and Enkephalins: A Search for Reliability
ALEJANDRO BAYON.WILLIAM J. SHOEMAKER, JACQUELINE F. McGiwY. AND FLOYI)BLOOM
........................................................... .............................. .............................. IV. Is Immunodetection Reliable? ............................................ I. Introduction
11. Tissue Processing, Extraction, and Handling 111. Identification, Quantitation, and Localization
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51 53 62 87 88
On the Sacred Disease: The Neurochemistry of Epilepsy
0. CARTERSNEADi n I. Introduction
........................................................... ...................................
11. Epilepsy: The Diversity of the Problem
94 94
vi
CONTENTS
111. Neurophysiology of Seizures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I\'. Models . . . . . . . . . . . . . . . . .................... V. Neurotransmitters and Ot Seizures .......... VI. Developmental Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
95 99 106 150 152 152
Biochemical and Electrophysiological Characteristics of Mammalian GABA Receptors SrZLVATORE J. ENNA A N D JOEL
P.
GALLACHER
I. Introduction . . . . . . 11. Electrophysiological
I l l . Biochemical Studies IV. Summary and Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
181 182 192 204 2O(i
Synaptic Mechanisms and Circuitry Involved in Motoneuron Control during Sleep
MICHAELH. CHASE I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
213
....................
215
IV. Motoneuron Membrane Potential during
akefulness . . . .
during Active Sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1. Central Control Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Concluding Remarks .......................... ......... VIII. Summary Statements . . .............. References . . . . . . . . . . . .............. .....................
232 240 251 257
Recent Developments in the Structure and Function of the Acetylcholine Receptor
F. J .
B.4RRANTES
1. Introduction .... ..................... 11. The AChR Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biosynthesis of the AChR .... ....................... IV. Three-Dimensional Topography of the AChR in the Membrane . . . . . . . . . . . V. In Search of the Functional Role of the Nonreceptor pProteins . . . . . . . . . . . Vl. The Ion-Translocation Function in Membrane-Bound AChR . . . . . . . . . . . . . . VII. Summary and Perspectives . . ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
259 261 272
279 290 301 329 33 1
CONTENTS Characterization of
a,-
vii
and ap-Adrenergic Receptors
DAVID B . BYLUNDAND DAVIDc. U'PRICHARD ............................... ........................ .................. 111. ap-Adrenergic Receptors ......................... IV. Summary and Conclusions .............................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction
11. a,-Adrenergic Receptors
344 354 420 422
Ontogeneis of the Axolemma and Axoglial Relafionships in Myelinated Fibers: Electrophysiological and Freeze-Fracture Correlates of Membrane Plasticity
STEPHENG. WAXMAN. JOEL A. BLACK,AND ROBERTE. FOSTER I. Introduction . . . . . . . . . . .
434 437 440 Development of the Optic Nerve Freeze-Fracture Structure of Myelinated Axons ........................... 449 Freeze-Fracture Studies on Myelin Development in Optic Nerve Axons . . . . . 461 Differentiation of the Axon Membrane in the Absence of Myelin .......... 475 Concluding Comments ................................................. 479 References ............................................................ 48 1
11. Specificity in Myelination
111. IV. V. VI. VII.
..........................................
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
485 49 1
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CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.
F. J. BARRANTES, Max-Planck-Institutfur Biophysikalische Chemie, Gottingen-Nikolausberg, Federal Republic of Germany (259) ALEJANDROBAY ON,^ Departamento de Neurociencias, Centro de Investigaciones en Fisiologza Celular, Universidad Nacional Autonoma de Mexico, Mexico D.F., Mexico (51)
A. BLACK,^ Department of Neurology, Stanford University School of Medicine, and Veterans Administration Medical Center, Palo Alto, California 94304 (433)
JOEL
FLOYDBLOOM,A. V. Davis Center f o r Behavioral Neurobiology, The Salk Institute, San Diego, California 92138 (51) DAVIDB. BYLUND, Department of Pharmacology, School of Medicine, University of Missouri at Columbia, Columbia, Missouri 65212 (343) MICHAELH. CHASE,Brain Research Institute and Departments of Physiology and Anatomy, School of Medicine, University of California, Los Angeles, Los Angeles, California 90024 (213)
SALVATORE J. E N N A ,Departments of Pharmacology and of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77025 (181)
ROBERTE. FOSTER,Neurotoxicology Branch, U S . Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010 (433) BERNARDW. FULPIUS, Department of Biochemistry, University of Geneva, Geneva, Switzerland (1) JOEL P. GALLAGHER,Department of of Texas Medical Branch, Galveston,
Pharmacology and Toxicology, University Texas 77550 (181)
GRAHAM A. R. JOHNSTON, Department of Pharmacology, University of Sydney, New South Wales 2006, Australia (15) 'Present address: Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Apartado Postal 70-228, 04510 Mexico D. F., Mexico. 2Present address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115. ix
X
(:ONTRI BUTORS
JACQI'ELINE E MCCINTE'. A . C'. Dailis Ceiiter f o r Behavioral Neurobiology, The Sulk institute, Sari Diego, Califoritin 92138 (51)
WILLIAM J. SHOEh1.lKF.R. A . .'I Dnsis Cenlerfor Behaz~ioral(Vepi1-obiolqgy,l h e Salk l,isfitute, Sail Diego, Califbrrtia 92138 (51)
0. C : i \ R T t R SXEAI)1 1 1 . Department (fPedintiicr arid Neurology, Unzuersity of Alubama i n Birmingham Sciiool of Medicine, Bi?ini@ani, Alabama 35294 (93)
INTERNATIONAL REVIEW OF
Neurobiology VOLUME 24
This Page Intentionally Left Blank
ANT IAC ETYLCHOLlNE RECEPTOR ANT I BODIES AN D MYASTHENIA GRAVIS By Bernard W. Fulpiur Dopa~tmontof B k h o r n i r t y
Univonity of Gomvo
Gonova, Switurlond
I. Introduction
......................................................... ....................
11. Pathogenicity of Circulating Anti-nAcChR Antibodies
111.
IV. V.
VI.
A. Pathogenic Mechanisms of Free Antibodie B. Pathogenic Mechanisms of Complexed An Assays for Circulating Anti-nAcChR Antibodies ......................... A. Immunoprecipitation Assa-rs ........................................ B. Inhibition Assays ....................................... ..... Anti-nAcChR Antibody Concentration in Different Forms of Myasthenia Gravis .................................................. Anti-nAcChR Antibodies in the Cerebrospinal Fluid ..................... A. Antibody Origin ................................................... B. Cross-Reactivity with Brain nAcChR ................................ C. Antibody Pathogenicity ............................................ Antigenic Determinants on nAcChR ....................... A. Torpedo Fish nAcChR ......... ....................... B. Human Skeletal Muscle nAcChR.. .................................. References ................................... ...............
1 2 3 4
5
6 6
7 10 10 11
I1 12 12 13 13
1. lntrodwtion
Myasthenia gravis is a human muscle disease characterized by weaknes8sand abnormal fatigability of voluntary muscles with recovery of motor power on resting, as well as positive response to treatment with ant icholinesterase drugs. T h e basic defect appears to consist in a reduction of available nicotinic acetylcholine receptors (nAcChR) at neiiromuscular junctions, brought about by an antibody-mediated autoimmune reaction. The elucidation of this defect has followed detailed studies of the molecular organization of nicotinic cholinergic synapses, made possible by the development and the application of a set of tools, the neurotoxins from elapid snake venoms, used to identify specifically nAcChR.' I n this context, one should emphasize two particular neurotoxins of very wide use: ( I ) a-bungarotoxin (a-BuTx) mainly used as an iodinated derivative to label specifically nAcChR because of its extremely slow dissociation rate, and (2) a-cobra, linked to Sepharose beads, mainly used to purify nAcChR by affinity chromatography. 1 INTERNATIONAI. REVIEW O F NEIIROBIOLOGY. VOL. 24
Cnpynght 0 1983 by Acadcmx Press, Inr ,411 righis of rcproduction in any form reserved ISBN 0-12-366824-7
2
BERNARD W. FULPIUS
Obviously both humoral and cellular immunity to nAcChR are implicated in the pathogenesis of myasthenia gravis. There is little evidence about their relative role in the defect of the neuromuscular transmission, although humoral immunity, in the form of anti-nAcChR antibodies alone or in conjunction with complement factors, would be sufficient to cause a reduction of available receptors at neuromuscular junctions. As exhaustive reviews on the subject, including data on experimental autoimmune myasthenia gravis, have been published (Lindstrom, 1979; Vincent, 1980; Drachman, 1981), we shall restrict ourselves to a discussion of some of the basic questions one may ask in an effort to understand the mode of action of anti-nAcChR antibodies in this disease. 1. How can these specific antibodies be pathogenic? This question pertains to the diffusion of immunoglobulins in compartments other than the vascular one, the interaction of immunoglobulins with the antigen, and the consequences of complex formation with the receptor. 2. How can these specific antibodies be detected? This question is related to the heterogeneity of the population of anti-nAcChR antibodies and the limitations of the different methods at disposal to test them. 3. To what extent does the concentration of circulating anti-nAcChR reflect the severity of myasthenia gravis? This question must be raised because a lack of correlation between the measured titers and the observed clinical status has been noticed. 4. Why d o these specific antibodies occur within the cerebrospinal fluid? This question concerns the origin, central or peripheral, of these antibodies, their possible specificity for brain nAcChR, and their pathogenicity. 5 . What is known of the structure of nAcChR? This question refers to the receptor considered as the autoantigen in myasthenia gravis. More specifically, it deals with the existence of different antigenic determinants, involved or not in the primary autoimmune reaction.
il. Pathogenicity of Circulating Anti-nAcChR Antibodies
According to Lefvert et al. (1978), the synthesis of anti-nAcChR antibodies is triggered by antigenic stimuli, and the antibodies are not a primary cause of myasthenia gravis. These authors postulate that the early release of nAcChR (probably from damaged endplates or myoid cells within the thymus) could act as the primary antigenic stimulus. This
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
3
is quite possible, although nothing is known of the factors that would have led to synaptic or cellular damages. As an alternative proposal, one should consider a more complex sequence of events. Namely, the neoplastic development of an anti-nAcChR antibody-producing clone could initiate locally complement activation on the postsynaptic membrane and cause the liberation of nAcChR-containingfragments into the surrounding tissue and circulation. This would then present the receptor as well as asher antigens from skeletal muscle tissue to the immune system. Additional antibody responses would be generated, among others, to normally unexposed or inaccessible nAcChR antigenic determinants? In any case, anti-nAcChR antibodies are present, often in large amounts, in the vascular compartment. A. PATHOGENICMECHANISMS OF FREEANTIBODIES
j[n order to cause synaptic dysfunction, antibodies must leave the vascular compartment, diffuse into the extracellular space, enter the narrow synaptic cleft, and reach the receptor molecules located at the top of the postsynaptic folds. To test the accessibility of nAcChRs located in the neuromuscular junctions, complexes made of 12sI-labeleda-BuTx covidently coupled to unspecific IgG were injected into mice (Zurn and Fullpius, 1976). In such experimental conditions, the region of nerve terminals appeared labeled. This was considered by the authors as a sufficient proof that molecules of about 150,000 MW could indeed enter the synaptic cleft. This result was confirmed by another set of experiments in which 1251-labeledanti-a-cobra toxin antibodies injected into mice that had received sublethal doses of a-cobra toxin were shown to reach the toxin molecules bound to the nerve terminals. Circulating anti-nAcChR antibodies can be pathogenic by interacting in situ with the receptor, hence impairing its specific role in synaptic transmission. According to Engel et al. (1977), the resulting synaptic dysfunction can be caused by three different mechanisms: (1) an alteration of the turnover of nAcChR due to a decrease in the rate of synthesis or an increase in the rate of degradation, (2) a complement-mediated
* This sequence of events might also explain why about one-quarter of myasthenic patients have serum antibodies directed against skeletal muscle tissue determinants other than nAcChR located at the level of the sarcoplasmic reticulum, as well as on the musclelike cells of the thymus. According to Feltkamp (1978), the antibodies directed against these antigens, different from nAcChR, do not seem to contribute to the pathogenesis of myasthenia gravis. They seem to be related more to thymomas, even in absence of myastherda, than to myasthenia gravis itself.
4
BERNARD W. FULPIUS
muscle membrane destruction, or (3) a blockade of the acetylcholine binding site on the receptor, Each of these mechanisms leads to a diminution of available and functional nAcChRs on the postsynaptic membrane. All three mechanisms may be involved in myasthenia gravis, but their relative role in the pathogenesis of the disease is still matter of controversy. By comparison with anti-nAcChR antibodies of the IgG type, much less is known about those of the IgM type. It is even uncertain whether molecules of large size (MW 900,000) enter the synaptic cleft. The only indication in that direction is given by the localization of IgM derivatives within the region of nerve terminals. This is observed in an experiment in which complexes of 12SI-labeleda-BuTx covalently coupled to unspecific IgM were injected in viuo into mice, an experimental condition in which the possible release of small amounts of 1251-labeleda-BuTx through enzymatic hydrolysis cannot be entirely excluded (Zurn and Fulpius, 1976). There is no evidence yet for a pathogenic role for anti-nAcChR antibodies of the IgM type. For example, when tested on muscle cells in culture, these immunoglobulins do not induce an increased rate of receptor degradation (F. Clementi, unpublished observation). The question of the pathogenicity of antibodies of the IgM type is, however, of importance in view of the well-known prevalence of these immunoglobulins in the early phase of an immunization procedure and considering the observation by Lefvert et al. (1978). These authors studied three patients with a relatively short duration of myasthenic symptoms. When the patients were examined for the first time, there were no detectable anti-nAcChR antibodies of the IgG type. There were, however, antibodies of the IgM type detectable in two patients. Later on, in all three patients, IgG anti-nAcChR antibodies appeared, whereas IgM antibodies decreased in concentration. This IgM-IgG pattern was interpreted by the authors as an indication that the synthesis of anti-nAcChR antibody was triggered by antigenic stimuli. B. PATHOGENIC MECHANISMS OF COMPLEXED ANTIBODIES Circulating anti-nAcChR antibodies could be pathogenic in another manner, namely, as immune complexes, because any humoral antibody immune response eventually involves the formation of such complexes. As such, they would cause less specific damages than free-anti-nAcChR antibodies, since receptors for these complexes are known to occur in several anatomic areas leading, in those places, to immune complex de-
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
5
positon and injury. The possible existence of such complexes raises several questions of importance in connection with the pathogenesis of myasthenia gravis. (a) How are they formed? (b) What is their size? (c) To what extent do they activate the complement system? (d) What is their clearing system? These questions must be related to several factors (Williams, 1981): (1) the quality and immunoglobulin class of antibody involved. For example, immune complexes comprising IgM antibodies are larger and more rapidly cleared than those formed from IgG antibodies; (2) the relative quantities of antigen and antibody present. For example, soluble, and hence circulating, immune complexes are formed in situations of antigen excess, whereas increasing precipitation out of complexes occurs when the relative quantities of antigen and antibody approach equivalence. I n addition, immune complexes composed of more than one antigen molecule and cross-linked lattice-wise by several bivalent IgG molecules are often capable of effective complement pathway activation, the same being true for immune complexes composed of IgM antibodies; (3) the reticuloendothelial system; (4)the presence of Fc receptors on a number of circulating blood elements; and ( 5 ) the presence of actual receptors for activated complement components or Fc portions of immunoglobulins in various tissue sites. Unfortunately, very little is known of immune complexes in myasthenia gravis, although very sophisticated methods have been developed for a quantitative estimation, in several pathological conditions, of such complex levels in serum or other body fluids. Their existence has been, however, suggested by reports of anticomplementary and C l q binding activities in myasthenic sera and has been confirmed recently by more elaborated methods (Barkas et al., 1981). One should emphasize that the methods used so far are nonspecific in that all complexes, whatever the antigen involved, will be detected. Methods for the detection of specific immune complexes containing nAcChR-derived material are therefore needed. 111. Assays for Circulating Anti-nAcChR Antibodies
T h e design of assays for anti-nAcChR antibodies is rather complicated since there exist several clones of antibodies directed against nAcChR, each of them being specific for an antigenic determinant, but not all of them being pathogenic. At present, circulating anti-nAcChR antibodies are identified by several different methods, all of which depend on cu-BuTx for their specificity.
6
BERNARD W. FULPIUS
A. IMMUNOPRECIPITATION ASSAYS
Immunoprecipitation assays are the most widely used assays (Appel et al., 1975; Lindstrom, 1977; Monnier and Fulpius, 1977). They require first labeling the receptor protein with 1251-labeleda-BuTx. AntinAcChR antibodies that combine with the toxin-receptor complexes obtained in this manner are precipitated by adding the appropriate anti-human IgC or IgM serum3 and are found in about 90% of patients with myasthenia gravis. This type of assay, however, underestimates the actual amount of anti-nAcChR antibodies that circulate free in the serum because '251-labeled a-BuTx bound to nAcChR may alter or sterically occlude antigenic determinants recognized by two particular subpopulations of anti-nAcChR antibodies: (1) those that cannot bind to the receptor when a-BuTx is already bound and (2)those that block specifically the binding of acetylcholine, the natural ligand, to the receptor (Dwyer P t al., 1979). These two subpopulations are not mutually exclusive since the first one is not necessarily specific for the acetylcholine binding site. Although both subpopulations could induce a reduction of the receptor density, and hence the myasthenic neuromuscular deficiency, by mechanisms which imply binding to the receptor on the postsynaptic membrane followed by receptor internalization and/or complement-mediated membrane destruction, the second subpopulation deserves special attention because it could act also by causing only an immunopharmacologic blockade, a potentially operative mechanism already envisioned for myasthenia gravis (Simpson, 1960). Antibodies of group 1 are revealed by a modification of the precipitation assay. They have been reported to be the only anti-nAcChR antibodies present in one of the myasthenic patients studied by Dwyer et al. (1979).
B. INHIBITION ASSAYS lnhibition assays are used to detect antibodies which block the binding of acetylcholine a n d o r a-BuTx to nAcChR; they consist of a quantitative evaluation of the competition between antibodies and a-BuTx for binding to nAcChR from different sources. Antibodies directed against the binding site of the receptor were first Protein A can also be used for precipitating complexes made of toxin-labeled nAcChR and IgC. In this case, however, one has to remember that IgG of the subclass 3 does not react with protein A and then will escape detection. This is of importance since it is known that, in certain myasthenic patients, a large proportion of anti-nAcChR antibodies belong to that subclass (Lefvert t t 01.. 1981).
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
7
described by Almon et al. (1974) who used detergent-extracted nAcChR from denervated rat muscle. According to the reports of several authors, the proportion of myasthenic patients with antibodies directed against the toxin binding site varies from 7 to 60%. There is no correlation between their concentration and the concentration of antibodies directed against other sites on nAcChR (Bender et al., 1975; Mittag et al., 1976; Lefvert and Bergstrom, 1977, 1978; Vincent and Newsom-Davis, 1979; Lefvert et al., 1981). It is difficult to evaluate quantitatively the inhibition by antibodies of toxin binding to nAcChR because anti-nAcChR antibodies might also inhibit toxin fixation by steric mechanisms (when they are directed against sites adjacent to the toxin binding site) or by allosteric mechanisms (when they are directed against remote portions of the receptor molecule, a situation likely to happen whenever the receptor is solubilized by detergent and hence looses its native conformation). Appropriate inhibition assays must therefore meet the following conditions: (1) The receptor must be in its native environment as is the case in cells or intact membrane fragments, and (2) cross-reactivity with human antinAcChR antibodies should be restricted to the ligand binding site as seems to be the case with Torpedo electric organs (Vincent, 1980) and cultured chicken muscle cells (Fulpius et al., 1980b). Further, difficulties will be encountered while performing inhibition assays, namely, to obtain sufficient amounts of antigen, to reach an adequately high sensitivity, and to purify large amounts of high-titer IgG from myasthenic patients. Finally, as the cholinergic binding site is not equal to the toxin binding site, it is necessary to test, in an additional step, acetylcholine or another small cholinergic ligand in order to assay specifically the nicotinic cholinergic nature of the inhibition by antibodies (Fulpius et al., 1981). It follows that the development of suitable specificimmunodiagnostic methods for detecting antibodies directed against the cholinergic binding site is still needed. In this respect, one should consider of great potential value the recent development of monoclonal antibodies directed against the cholinergic binding site of the receptor (Jameset al., 1890).
IV. Anti-nAcChR Antibody Concentration in Different Forms of Myadhenia Gmvis
The discovery of the existence in myasthenia gravis of autoantibodies pathognomonic for the disease originally raised a great deal of interest among clinicians in consideration of the following question: Would the
8
BERNARD W. FULPIUS
level of measured anti-nAcChR antibodies be related to the patient’s clinical status? This question is of fundamental importance. As a matter of fact, a positive correlation would permit control of the evolution of the disease, predict the occurrence of relapses, and test whether the therapy has been appropriately selected. Unfortunately, most of the data published in this context were disappointing: T h e reported antibody titers corresponded only loosely with the patient’s clinical status (Almon et al., 1974; Appel et at., 1975; Bender et al., 1975; Lindstrom et al., 1976; Mittag et al., 1976; Ito et al., 1978; Bradley et al., 1979; Roses et al., 1981). In particular, many patients who appeared to be in complete clinical remission had titers well within the range of those with active disease (Lefvert et d ,1978), and about 10%of myasthenic patients had no detectable antibody (Lindstrom, 1977). This obvious lack of correlation can be explained in several ways: 1. T h e immunoprecipitation assay used in most of the studies does not detect all kinds of anti-nAcChR antibodies. 2. The anti-nAcChR antibodies that are detected in this manner are not necessarily those which are pathogenic. 3. Usually, the immunoprecipitation assay is carried on to detect class G immunoglobulins. 4. T h e level of circulating antibodies does not necessarily reflect the level existing in the vicinity of the receptor. 5. An uptake of antibodies at affected end plates might significantly deplete the circulation of appreciable amounts of anti-nAcChR antibodies. 6. Anti-nAcChR antibodies circulating as immune complexes escape detection. 7. T h e half-life of class G immunoglobulins shows large variations according to the four subclasses known.4 8. Sera are not always taken from patients according to the same protocol. 9. There are differences in the susceptibility to proteolysis among different antibody subpopulations. IgG sublcasses 1 , 2, 3, and 4 differ in several respects. IgG 3, in particular, has a strong tendency to aggregate and form complexes, a high susceptibility to proteolysis, and a rapid turnover (half-life of 7 days) when compared to that of the other subclasses (halflife of 2 1 days). In this context two reports of Lefvert pf al. (1978, 1981)are of considerable interest. According to these authors, anti-nAcChR antibodies have half-lives shorter than 8 days, and most of those detected in myasthenic patients by both imrnunoprecipitation and inhibition assays belong to either subclass 1 or 3.
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
9
T h e comparison of titers from one patient to another is difficult because of the use, in assays performed in different laboratories, of antigens from different sources. As a matter of fact, the cross-reactivity of myasthenic serum anti-nAcChR antibodies with nAcChR from different mammalian muscle extracts is highly variable. For example, antinAcChR titers against rat extracts are always lower than those against human muscle; in some cases they are even undetectable (Savage Marengo et al., 1979; McAdams and Roses, 1980). This explains why most researchers currently agree that human muscle is the most reliable source of antigen for determining anti-nAcChR in human sera, but even so, there are still difficulties in selecting the source of antigen. This is due mainly to the following two reasons: (1) There are still differences between the various muscles of a same species. For example, there are indications that ocular muscle nAcChR has some determinants distinct from those present on limb muscle nAcChR and vice versa. This finding is of importance in view of the fact that patients with predominant ocular symptoms represent the population with the lowest mean titer of antinAcChR antibodies when the assay is performed with nAcChR extracted from limb muscle (Lindstrom et al., 1976; Ito et al., 1978). (2) There are differences in the reactivity of myasthenia gravis sera toward junctional or extrajunctional receptors? higher titers being obtained with extrajunctional nAcChR (Weinberg and Hall, 1979). It follows that much more information related to the pathogenesis of myasthenia gravis could be obtained from anti-nAcChR antibody determinations, provided that: 1. Some form of standardization be always realized with, for example, sera of well-stablished activity. 2. Myasthenic sera be tested according to more than one assay in order to measure different subpopulations of anti-nAcChR antibodies. In this respect one should mention the report by Lefvert et al. (198 1) of the occurrence of anti-nAcChR antibodies competing for the ligand binding site in 50% of the myasthenic patients studied. All of the patients with these antibodies were severely ill, an indication that such antibodies might have a more disturbing effect on the neuromuscular function than those directed against other sites on the receptor. The junctional receptors are exclusively present in innervated muscles, whereas the extrajunctionalreceptors also present in innervated muscles, happen to exist in especially large amounts in denervated muscles, a preparation often used by biochemists to increase the yield of nAcChR.
10
BERNARD W. FULPIUS
3. An assay specific for pathogenic anti-nAcChR antibodies be developed. 4. Assays with sensitivites higher than those presently used be available. V. Anti-nAcChR Antibodies in the Cerebrospinal Fluid
Antibodies directed against nAcChR from human skeletal muscle and tested by a conventional immunoprecipitation assay were originally detected by Lefvert and Pirskanen (1977) in the cerebrospinal fluid of 9 out of 12 myasthenic patients. This finding raises three intriguing questions: 1. Are these antibodies synthesized locally or does their appearance in the cerebrospinal fluid result from a passive leakage through the blood-brain barrier! 2. Do these antibodies cross-react with central nervous system nAcChR? 3. Do these antibodies alter the synaptic transmission within the central nervous system?
'4.ANTIBODY ORIGIN T h e study of antibody origin implies, in addition to the assay for specific antibodies, the use of an appropriate test to assess the integrity of the blood-brain barrier, because any damage at that level would allow anti-nAcChR antibodies to gain access to the cerebrospinal fluid by passive leakage from the serum. T h e data available on this question bring rather conflicting evidence. On one side, data from Keesey et al. (1978) favor a passive leakage from the serum in view of the observed cerebrospinal fluid :serum ratios for the concentration of albumin and antinAcChR antibodies. On the other side, data from Lefvert et al. (1978) favor a local synthesis of anti-nAcChR antibodies since the cerebrospinal fluid : serum ratio for the concentration of IgG is normal and that for anti-nAcChR antibodies increased. Further studies on this question are expected. In particular, more specific information on the different subpopulations of anti-nAcChR antibodies existing within the cerebrospinal fluid is needed. T h e problem is complicated since the concentration of anti-nAcChR antibodies in the cerebrospinal fluid is lower than that in the serum by a factor of about
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
11
100. This makes it hard to perform tests that are sensitive enough to detect these antibodies and casts some doubt on a pathogenic action for them, at such low concentrations (Keesey et al., 1978).
B. CROSS-REACTIVITY WITH BRAIN nAcChR The possible Occurrence within the central nervous system of a cholinergic receptor of the nicotinic type is usually studied by using a-BuTx as a probe. The data pertaining to that question have been reviewed by Oswald and Freeman (1981). These authors conclude from their analysis that there are a-BuTx binding sites in the mammalian central nervous system which are located on nAcChR proteins similar to those of the muscle and electroplaque. It should be remembered, however, that the amount of information concerning the molecular properties of these a-BuTx binding proteins of neural origin is especially limited. This is especially true of the immunological characterization of the receptor, which is restricted to a few conflicting reports on the crossreactivity between anti-Torpedo sera and mammalian brain a-BuTx binding protein and a sole publication on the respective antigenic properties of muscle and brain nAcChR of human origin (Fontana et al., 1979). According to these authors, the two receptors have different antigenic determinants, a conclusion based on the following observation: A myasthenic serum of high titer against muscle nAcChR shows a much lower titer when tested against brain receptor, whereas three epileptic sera with antibodies against brain n AcChR show lower titers when tested against the muscle receptor. Such a result awaits confirmation. It emphasizes that specific assays must be used to test the relative reactivity toward brain andlor muscle receptor of the anti-nAcChR antibodies found in the cerebrospinal fluid.
C. ANTIBODY PATHOGENICITY Thre is some evidence for an involvement of the central nervous system in myasthenia gravis, namely, alterations of the hypothalamopituitary axis, electroencephalographic abnormalities, psychiatric symptoms, and a reduction of rapid eye movement sleep. According to Papazian (1976), this latter finding might indicate a disturbance of central cholinergic pathways. This is of great interest in view of the existence of anti-nAcChR antibodies of nicotinic specificity in the cerebrospinal fluid and the possibility for them to diffuse to neuroneuronal synapses and
12
BERNARD W. FULPIUS
intereact with the receptors located on the postsynaptic membrane. However, almost nothing is known of an impairment of central cholinergic synapses by these antibodies. The only report on this subject describes the induction of electroencephalographic abnormalities in rabbits by microinjection of human myasthenic serum into the caudate nucleus (Fontana et al., 1978). A comparison of antibody titers within the cerebrospinal fluid with central measurable alterations in man, and the possible effect, in this respect, of an immunosuppressive therapy should bring valuable information. VI. Antigenic Determinants on nAcChR
The nicotinic acetylcholine receptor is a protein complex embedded within the postsynaptic membrane of skeletal muscle cells. It follows that in situ not all antigenic determinants of the receptor are accessible and can interact with circulating immunoglobulins. Conversely, in myasthenia gravis, not all circulating anti-nAcChR antibodies are necessarily pathogenic since the formation of some of them might well have been triggered by parts of nAcChR normally not accessible and released in the course of the destruction of the postsynaptic membrane. Most of our information on nAcChR structure comes from studies performed with nAcChR from fish electric organs. A short review of the present state of knowledge concerning this latter receptor might give an idea of the complexity of the problems regarding the relative role of different antigenic determinants in the pathogeny of myasthenia gravis. A. TORPEDO FISHnAcChR Exhaustive reviews on torpedo fish nAcChR have been published (Fulpius et al., 1980a; Vincent, 1980). The receptor is a pentameric protein complex of about 270,000 daltons embedded within the membrane. Is quaternary structure is a p p y 6 , the two a polypeptide chains being identical. Only a chains bind the cholinergic ligands. Accordingly, there are two agonist or antagonist binding sites on each nAcChR complex of 270,000 daltons. The protein complex does not seem to show any symmetry because each subunit contains oligosaccharides of unknown size and must therefore face the extracellular space. The complex may, however, form dimers which are covalently linked together by a disulfide bridge between 6 chains. According to electron microscopy studies, the receptor protrudes about 50 A from the lipid matrix into the extracellu-
ANTIACETYLCHOLINE RECEPTOR ANTIBODIES
13
lar space. T h e pure receptor protein is readily available in large quantities. This has permitted thorough biochemical studies. For example, it has been shown by Raftery et al. (1980) that the four subunits have distinct but homologous amino acid sequences, in the first 56 N-terminal acids sequenced so far.
SKELETAL MUSCLEnAcChR B. HUMAN Significant differences have been reported between various mammalian muscle nAcChRs and their counterparts in fish electroplaques. There is, however, not much information related to the molecular structure of the receptor from human skeletal muscle. This is mainly due to obvious difficulties encountered in obtaining sufficient quantities of muscle with a minimum of tissue autolysis. T h e problem is further complicated by the variability of motor innervation inherent in lower leg muscles suffering from ischemia, a factor known to be linked to a more or less pronounced proliferation or extrajunctional receptors. Most of our knowledge on human nAcChR comes from the report of Stephenson et al. (198l), according to which the receptor has the following characteristics: (1)a specific activity for a-BuTx similar to that of nAcChRs purified from other sources; (2)a sedimentation coefficient of 9 S but no evidence for the existence of a dimerized 13 S form; (3)a microheterogeneity of the carbohydrate residues; (4) an original subunit pattern with two major protein bands of 42,000 and 66,000 daltons, the acetylcholine binding subunit being of the type common to all nAcChRs. In addition, the authors have observed that immunization of rabbits with this preparation generates low titers of the corresponding anti-nAcChR antibodies and does not cause experimental autoimmune myasthenia gravis. By comparison with the information available on Torpedo nAcChR and experimental autoimmune myasthenia gravis, many more studies are needed to better characterize the antigenic determinants of the human nAcChR in order to progress in the understanding of the immune response in myasthenia gravis. References
Almon, R. R., Andrews, C. G., and Appel, S. H. (1974). Science 186, 55-57. Appel, S. H., Almon, R. R., and Levy, N. (1975). N . Engl. J . Med. 293, 760-761. Barkas, T., Boyle, R. S., and Behan, P. 0. (1981).J. Clzn. Lab. Imrnunot. 5, 27-30. Bender, A. N., Ringel, S. P., Engel, W. K., Daniels, M. P., and Vogel, Z. (1975). Lancet 1, 607-609.
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Bradley, R. J., Dwyer, D., Morley, B. J., Robinson G., Kemp, G . E., and Oh, S. J. (1979). Prog. Brain Res. 49, 441-448. Drachrnan, D. B. (1981). Annu. Rev. d\’eurosci. 4, 195-225. Dwyer, D. S., Bradley, R. J., Oh, S. J., and Kernp, G. E. (1979). Clin. Exp. Immunol. 37, 448-45 1 . Engel, A. G , , Lambert, E. H., and Howard, F. M. (1977). Mayo Clin. Proc. 52, 267-280. Feltkarnp, T. E. W. (1978).In “Neurology” (W. A. den Hartog Jager, G . W. Bruyn, and A. P. J. Heijstee, eds.), pp. 81-89. Excerpta Medica, Amsterdam. Fontana, A,, Fulpius, B. W., and Grob, P. J. (1978). Doc. Ophthalmol. 17, 35-43. Fontana, A.. Fulpius, B. W., and Cuenoud, S. (1979). Adv. Cytopharmcol. 3, 287-292. Fulpius, B. W., Bersinger, N. A., James, R. W., and Schwendimann, B. (1980a).In “Receptors for Neurotransmitters, Hormones and Pheromones in Insects” (D. B. Satelle, L. M. Hall, and J. G . Hildebrand, eds.), p p 3-15. ElseviedNorth-Holland, Amsterdam. Fulpius, B. W., Miskin, R., and Reich. E. (1980b). Proc. Natl. Acad. Sci. U.S.A. 77, 43264330. Fulpius, B. W., Lefvert, A. K., Cuenoud, S., and Mourey, A. (1981). Ann. N.Y. Acnd. Sri. 377,305-315. Ito, Y., Miledi, R., Molenaar, P. C., Newsorn-Davis, J.. Polak, R. L., and Vincent, A. (1978). I n “The Biochemistry of Myasthenia Gravis and Muscular Dystrophy” (G. G. Lunt and R. M. Marchbanks, eds.), pp. 89- 110. Academic Press, New YorWLondon. James, R. W., Kato, A. C., Rey, M.-J., and Fulpius, B. W. (1980).FEBS Lett. 120, 145-148. Keesey, J. C., Tourtelotte, W. W., Hermann, C., Jr., Andrews, J. M., and Lindstrom, J. (1978). Lancet 1, 777. Lefvert, A. K., and Bergstrom, K. (1977). Eur-.J. Clin. Inirest. 7, 115-119. Lefvert, A . K., and Bergstrom, K. (1978). Srund. J. Immunol. 8, 525-533. Lefvert, A. K., and Pirskanen, R. (1977). Loncef 2, 351-352. Lefvert, A. K., Bergstrom, K., Matell, G., Osterman, P. O., and Pirskanen, R. (1978).J. h’Purol., .Veurosurg. Pqchiatry 41, 394-403. Lefvert, A. K., Cuenoud, S., and Fulpius, B. W. (198l).J. Neuroimrnunol. 1, 125-135. Lindstrom. J. M . (1977). Clin.Immunoi. Immunopnthol. 7 , 36-43. Lindstrom, J. M. (1979).Adv. Immutwl. 27, 1-50. Lindstrorn, J. M., Seybold, M. E., Lennon, V. A., Whittngham, S., and Duane, D. (1976). N e u r d o a 26, 1054-1059. McAdams, M. W., and Roses, A. D. (1980). Ann. Neuroi. 8,61-66. Mittag, T., Kornfeld, P., Tormay, A., and Woo,C. (1976).N. Engl. J . Med. 294, 691-694. Monnier, V. M., and Fulpius, B. W. (1977). C h . Exp. Immuml. 29, 16-22. Oswald, R. E., and Freeman, J. A. (1981). Neuroscience 6, 1- 14. Papazian, 0. (1976). Neuroiogy 26, 311-316. Raftery, M. A., Hunkapiller, M. W., Strader, C. D., and Hood, L. E. (1980). S c i m e 208, 1454-1457. Roses, A. D., Olanow, C. W., McAdams, M. W., and Lane, R. J. M. (1981). N m r o l o o 31, 220-224. Savage Marengo, T., Harrison, R., Lunt, G. G., and Behan, P. 0.(1979). Lalvet 1,442. Sirnpson, J. A. (1960). Srott. ,\Zed. J . 5, 419-436. Stephenson, E A,, Harrison, R., and Lunt, G. (1981). Eur.J. Eiochm. 115, 91-97. Vincent, A. (1980). Physiol. Rev. 60, 756-824. Vincent, A., and Newsom- Davis, J. (1979).Adv. Cytophnnacol. 3, 267-278. Weinberg, C. B., and Hall, Z. W. (1979). Proc. Natl. Acad. Sci. U.S.A. 7 6 , 504-508. Wiliiams, R. C., Jr. (1981). Annu. Rmr. .Wed. 32, 13-28. Zurn, A. D., and Fulpius, B. W. (1976). C h . Exp. Immuiwl. 24, 9-17.
PHARMACOLOGY OF BARBITURATES: ELECTROPHYSIOLOGICAL AND NEUROCHEMICAL STUDIES By Max Willow* and Graham A. R. Johnrtont
* Doporlment of Pharmacology Schaol of Medicine University of Washington
Seattle, Washington and
t
Doporlrnent of Pharmacology Univonity of Sydney
New South Wales, Aurfmlio
I. Introduction ......................................................... 11. Neuropharmacological Studies ......................................... A. General Effects of Barbiturates on Synaptic Transmission ............. B. Effects of Barbiturates on Axonal Conduction ........................ C. F'resynaptic Actions of Barbiturates ................................. D. Effects of Barbiturates on Transmitter Action in Vertebrate Central Neurons ................................................... E. Effects of Barbiturates on Neuronal Membrane Properties.. ........... F. Neuropharmacology of Convulsant Barbiturates ...................... 111. Biochemical and Neurochemical Studies ................................ A. Effects of Barbiturates on Mitochondria1 Respiration .................. B. Effects of Barbiturates on Transmitter Release and Reuptake .......... C. Effects of Barbiturates on the Binding of Neurotransmitters to Receptor -Ionophore Complexes .......... .................... IV. Conclusions .......................................................... References ................. ......................................
15 16 16
21 22 24 32 33 34 34 35 41 44 45
I. Introduction
Barbituric acid was h s t synthesized by Baeyer in 1864, and this date marks the birth of an era that has witnessed the production of over 2500 derivatives. The first barbiturate introduced into clinical medicine (1903) was barbital, a long-acting sedative-hypnotic agent. Phenobarbital was marketed in 1912 for use in the treatment of certain forms of epilepsy. The use of ultra-short-acting barbiturates as intravenous anesthetics began in the early 1930s, and thiopental, in particular, gained rapid popularity following its introduction in 1935. While many of the sedative-hypnotic barbiturates have been superseded following 15 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 24
Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366824-7
16
MAX WILLOW A N D GRAHAM A. R. JOHNSTON
TABLE I MOLECCLAR STRUCTURE OF SOME C-5 SUBSTITUTED BARHITURATES
Name
Principal action
R'
RZ ~
Methohexitone Thiopentone Butobarbitone Secobarbitone Amylobarbitone Pentobarbitone Phenobarbitone Barbitone CHEB 3M2B
Anesthetic Anesthetic Sedativehypnotic Sedativehypnotic Sedativelhypnotic Sedativelhypnotic Sedative/anticonvulsant Sedativelanticonvulsant Convulsant Convulsant
Ally1 Ethyl Ethyl Ally1 Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl
l-Methyl-2-pentynyl 1-Methyl butyl 1-Methyl propyl I-Methyl butyl 3-Methyl butyl 1-Methyl butyl Phenyl Ethyl 2-Cyclohexylidene 3-Methylbut-2-enyl
X ~
0 S 0 0 0 0 0 0 0 0
the discovery of the benzodiazepines, barbiturates still maintain an important role in therapeutics, especially in their use as anesthetics and antiepileptics. The molecular structure of many of the barbiturates discussed in this article is shown in Table I. In general, only the effects of barbiturates on neuronal systems in vertebrate species will be discussed here. Detailed accounts of the pharmacological actions of barbiturates on invertebrate neurons can be found elsewhere (Barker, 1975a,b,c; Prichard, 1980; Wilson et al., 1980). \I. NeumpharmMobgical Studies
A. GENERAL EFFECTSOF BARBITURATES ON SYNAPTIC TRANSMISSION
The most detailed studies on the effects of barbiturates on synaptic transmission have been performed on the spinal monosynaptic reflex. Eccles ( 1946) demonstrated that the excitatory postsynaptic potential (EPSP) recorded from ventral roots in decerebrate cats was depressed by pentobarbital (40 mg/kg, iv) and concluded that the block of the monosynaptic reflex was largely due to an increased stability of the soma membrane. Brooks and Eccles (1947) showed that pentobarbital (30 mg/kg, iv) depressed the orthodromically induced focal potential in the
PHARMACOLOGY OF BARBITURATES
17
spinal cord of decerebrate cats. With increasing concentrations, the synaptic potential was progressively depressed, until finally the monosynpatic pathway was completely blocked. Although they observed that pentobarbital (in high doses) was capable of depressing propagation in the afferent presynaptic terminals, blockage of synaptic transmission was thought to be due to a stabilization of the soma membrane which prevented the discharge of impulses initiated by normally effective synaptic potentials. Shapovalov (1963) showed that pentobarbital (20-30 mg/kg, iv) and hexobarbital ( 15-20 mg/kg, iv) depressed EPSPs recorded intracellularly from cat spinal motoneurons without altering resting membrane potential and axonal impulse conduction. A contemporary study by Somjen and Gill (1963) demonstrated that thiopental (30-50 mg/kg, iv) blocked the transmission of the monosynaptic reflex in the cat and the rat, as seen by a depression of the EPSP without alteration of the resting membrane potential. In a parallel study, Somjen (1963) showed that when EPSPs were reduced to 10% of control amplitude following the administration of thiopental (65 mg/kg, iv), impulse conduction in presynaptic terminals was unimpaired. He concluded that the most probable explanation for the depression of synaptic potentials was a nonspecific stabilization of the soma membrane or a decrease in the amount of transmitter released per presynaptic impulse. Ldyning et al. (1964) examined the effects of the short-acting barbiturate thiamylal sodium on the monosynaptic reflex in lightly anesthetized cats. Using intracellular recording techniques, they showed that intravenous administration of this drug (10 mg/kg) decreased the EPSP, without altering the spike potential, resting potential, and accommodation of the motoneurons. When potentials evoked by a volley applied to afferent nerves were recorded extracellularly at the dorsal root entry in the motor nucleus, and from the ventral root, it was found that thiamyal sodium reduced (a) the initial negative spike recorded in the motor nucleus, (b) the focal synaptic potential, and (c) the ventral root potential. It was concluded that the reduced EPSP was due mainly to thiamylal acting on different nerve terminals resulting in a decrease in transmitter release or to a reduced sensitivity of postsynaptic membrane receptor sites to the transmitter. T h e actions of pentobarbital and thiopental on monosynaptic EPSPs in cat spinal motoneurons were examined by Weakly (1969). He showed that these drugs, when administered intravenously (10 mg/ kg), significantly depressed the monosynaptic reflex discharge of triceps sural motoneurons. Both drugs reduced the mean quantum content by about 25% without altering the average amplitude of the unit EPSP. It was concluded in agreement with Ldyning et al. (1964) that depression of monosynaptic reflex transmission by thiopental and pentobarbital was
18
MAX WILLOW AND GRAHAM A. R. JOHNSTON
due to a reduction in the average amount of transmitter released by group l a afferent impulses. A reduction of excitatory synaptic transmission has been demonstrated at a number of other sites in the CNS. Galindo (1969) examined the effects of pentobarbital on synaptic transmission in the cuneate nucleus of decerebrate cats. Cuneate neurons were excited (a) by various stimuli including a jet of air applied to hairs, a weight applied on the skin, or the movement of a joint; (b) electrical pulses applied to peripheral nerves; and (c) stimulation of nerve endings in the dorsal column. I n each case, pentobarbital (2-30 mg/kg, iv) reduced synaptic transmission in response to these stimuli. Microelectrophoretic administration (40200 nA) of pentobarbital (0.2 M, pH 9.5) produced similar effects. I n addition, pentobarbital administered electrophoretically (40 nA) significantly reduced the firing rate of cuneate neurons excited by glutamate (60 nA). Nicoll (1972) examined the effects of pentobarbital, hexobarbital, and other anesthetic agents on synaptic excitation and inhibition in the olfactory bulb of the rabbit. Pentobarbital (3-30 mg/kg, iv) prolonged the granule cell inhibition of mitral cells while having little effect on synaptic excitation of granule cells and antidromic field potentials invading mitral cell dendrites. Relatively large doses of pentobarbital (40-70 mg/kg, iv) were needed to significantly depress synaptic excitation of granule cells and antidromic invasion of mitral cell dendrites. Richards ( 1972) showed that pentobarbital (0.05-0.25 mM) depressed the EPSP component of the evoked field potential recorded in an in nitro preparation of guinea pig olfactory cortex. T h e population spikes that were superimposed on the EPSP were reduced in amplitude and frequency with these concentrations of pentobarbital, indicating a failure of transmission through the cortical relay. It was concluded that pentobarbital produces its effects (a) by reducing the amount of transmitter released from presynaptic nerve terminals in response to the afferent volley or (b) by reducing the sensitivity of the postsynaptic membrane to the released transmitter. Gordon et nl. (1973) have demonstrated that synaptic transmission in the mossy fiber pathway of the cat cerebellum is depressed by thiopental (0.5-8.0 mg/kg, iv), whereas transmission in the climbing pathway is enhanced. T h e depression of' excitatory synaptic transmission is exemplified by the abolition of the axon discharge of granule cells, and as a consequence, Purkinje cells are unable to respond to the tibia1 nerve stimulation via the mossy fibers. Barbiturates have also been shown to exert depressant effects on excitatory synaptic transmission at various sites other than the CNS. Larrabee and Posternak (1952) have shown that concentrations of pentobarbital (0.2-0.5 miM) that depress postsynaptic responses in cat stellate ganglia following stimulation of the preganglionic fiber, have no
PHARMACOLOGY OF BARBITURATES
19
effect on impulse conduction in postsynaptic fibers. A similar selective action of barbiturates on synaptic transmission in the superior cervical ganglion from a number of species has been observed by Quillam and his collaborators (Brown and Quillam, 1964a,b; Elliott and Quillam, 1964; Quillam and Shand, 1964). Synaptic transmission at the vertebrate neuromuscular junction is also impaired by barbiturates. Thesleff (1956) demonstrated that pentobarbital (0.6 mM) blocked neuromuscular transmission in the sartorius nerve-muscle preparation of the frog. This effect was characterized by an increase in the electrical threshold of the muscle membrane and a reduced action potential. While the dose of pentobarbital required to produce these effects was five times greater than the mean hypnotic dose for pentobarbital in the frog, Thesleff (1956) concluded that the anesthetic activity of pentobarbital may be due to a reduction of the sodium conductance change in the cell membrane of certain neurons in the central nervous system. Adams ( I 976) examined in detail the effects of amylobarbital, methohexital, and thiopental on the physiology of voltage-clamped end plates of frog sartorius muscles. In the presence of barbiturates (80 p M ) , the conductance change evoked by electrophoretic carbachol was reduced by a prepulse of carbachol. This desensitization disappeared exponentially with a time constant of 150-200 msec. All barbiturates tested (0.4-1.2 mM) produced an increased rate of decay of nerve-evoked end-plate currents. In addition, thiopental, in a dose-dependent manner, depressed conductance changes produced by bath-applied agonists (choline, carbachol, and tetramethylammonium bromide). Adams ( 1976) also observed that the concentrations of barbiturates required to depress the bath agonist response are much greater than the kinetically determined dissociation constant for binding to active receptor-channel complexes. It was concluded that the depressant effects of barbiturates on synaptic transmission at the frog end plate were mainly mediated by a blockage of open end-plate receptor channels. While emphasis has been placed on the depressant effects of barbiturates on excitatory synaptic transmission, these compounds also enhance synaptic inhibition at a number of sites. Larson and Major (1970) showed that hexobarbital (10 mg/kg, iv) markedly prolonged the time course of the recurrent inhibitory postsynaptic potential (IPSP) in cat spinal motoneurons. This effect is not attributable to a prolonged Renshaw cell discharge since equivalent closes have been shown to shorten the Renshaw cell discharge (Eccles et ul., 1956). As mentioned above, Nicoll (1972) demonstrated that doses of barbiturates that are without effect on the EPSP in the olfactory bulb markedly prolong postsynaptic inhibition. Nicoll et al. (1975) demonstrated that pentobarbital (10-33 mg/kg, iv) markedly prolonged the time course of the IPSP recorded in cat hip-
20
MAX WILLOW AND GRAHAM A. R. JOHNSTON
pocampal pyramidal neurons. In addition, pentobarbital (10 mg/kg, iv) prolonged both the evoked and spontaneous unitary IPSPs. Barbiturates appear to enhance postsynaptic inhibition at a number of other sites in the CNS. Bloedel and Roberts (1969) examined various aspects of cerebellar physiology before and after the administration of pentobarbital in decerebrated and spinalized cats. Pentobarbital (15 mg/kg, iv) enhanced the postsynaptic inhibition of Purkinje neurons by basket cells. This result was confirmed by Eccles et al. (1971) using thiosecobarbital(l0-40 mg/kg, iv). It was concluded that in the unanesthetized cat cerebellum there is a higher level of Purkinje cell excitability, probably due to a domination of the excitatory action of parallel fibers over the inhibitory action of basket cells. Scholfield (1977) demonstrated that pentobarbital (100 p M ) prolonged the evoked IPSP recorded in guinea pig olfactory slices in vitro. The evoked EPSP was depressed with higher concentrations of pentobarbital. Barbiturates also enhance the recurrent inhibition of cortical pyramidal tract neurons (Veselyuneneet al., 1971; Steriade et al., 1974) and the recurrent inhibition of thalamic relay neurons (Bremer, 1970).All of the inhibitory pathways referred to above are thought to release y-aminobutyric acid (GABA), with the exception of recurrent inhibition of spinal motoneurons, which is mediated by glycine (Curtis and Johnston, 1974; KrnjeviC, 1974). Eccles and Malcolm ( 1946) first demonstrated that pentobarbital greatly prolongs the decay of the dorsal root potential in the isolated frog spinal cord. This observation has been confirmed in many studies both in the frog (Schmidt, 1963, 1964; Grinnell, 1966; Richens, 1969; Nicoll, 1975a) and in the cat (Lloyd, 1952; Eccles et al., 1963). The synaptic depolarization of primary afferents, which underlies the dorsal root potential, is considered to result in reduced release of excitatory transmitter from the primary afferents (Eccles, 1964), and GABA is thought to be the depolarizing transmitter responsible for this presynaptic inhibitory process (Levy, 1977). Eccles et al. (1963) demonstrated that presynaptic inhibition of monosynaptic reflexes in the cat is prolonged to the same extent as the dorsal root potential by pentobarbital and thiamylal. In addition, picrotoxin (0.3 mg/kg, iv) antagonized the actions of pentobarbital (10 mg/kg, iv) in increasing and prolonging the presynaptic inhibition of the monosynaptic reflex. Presynaptic inhibition is also enhanced by phenobarbital (Miyahara et al., 1966). Nicoll (1975a) demonstrated that concentrations of pentobarbital as low as 5 pM could prolong the dorsal root potential in the isolated frog spinal cord preparation. In addition, pentobarbital (40 p M ) depolarized primary afferent fibers. This depolarizing action was approximately equipotent to that of GABA, and was blocked by picrotoxin and bicuculline. These actions of
PHARMACOLOGY OF BARBITURATES
21
pentobarbital were also seen with amylobarbital, thiopental, and barbital. Pentobarbital (10- 15 mg/kg, iv) enhanced surface potentials (similar to dorsal root potentials) recorded on the surface of the cuneate nucleus of the cat (Banna and Jabbur, 1969). This was associated with an increased excitability of presynaptic terminals, although a depression of excitability was observed with higher doses (25 mg/kg, iv). Rudomin (1966) demonstrated that pentobarbital (10 mg/kg, iv). enhanced primary afferent depolarization in the solitary nucleus of the cat. In general, the studies cited above suggest that excitatory synaptic transmission is depressed by barbiturates, whereas inhibitory synaptic transmission, especially that mediated by GABA, is enhanced. OF BARBITURATES ON AXONAL CONDUCTION B. EFFECTS
It is now generally accepted that barbiturates do not greatly affect the conduction of impulses along axons at concentrations likely to be present during anesthesia (approximately 100-200 p M for pentobarbital; see Fisher et al., 1948; Jori et al., 1970; Richards, 1972; Saubermann et al., 1974). Heinbecker and Bartley (1940) and Schoepfle (1957) demonstrated a local anesthetic-like action of barbiturates in blocking excitation in peripheral nerves. Later studies using voltage-clamp techniques showed that millimolar concentrations of pentobarbital and thiopental decreased and prolonged sodium conductance in lobster giant axons (Blaustein, 1968) and squid giant axons (Narahashi et al., 1969). The potassium conductance is reduced but not significantly prolonged by barbiturates in these preparations. Barbiturates have a more rapid onset of action and greater potency in producing these effects when they are applied intracellularly as opposed to external application (Narahashi et al., 197 1; Frazier et al., 1975). It has been suggested that the un-ionized form of the barbiturate molecule is responsible for the block in conduction (Krupp et al., 1969; Narahashi et al., 197 1). Of interest is the finding that there is no apparent difference in the time course of action between clinically short-acting and long-acting barbiturates in blocking axonal conduction (Frazier et al., 1975). While barbiturates exert local anesthetic actions on nerve fibers of large diameter at only relatively high concentrations, smaller myelinated and nonmyelinated fibers may be more susceptible to lower concentrations of these agents. This proposal was first suggested by Frank and Sanders (1963) and later by Seeman (1972). Staiman and Seeman (1974) have provided some experimental confirmation of this hypothesis. They demonstrated that pentobarbital produced a 50% block in conduction in
22
M A X WILLOW AND GRAHAM A. R. .JOHNSTON
phrenic nerves at 400 pX1, whereas a concentration of 800 g M was required to produce similar effects on large sciatic nerve fibers. Thus, a slight reduction in the amplitude of the action potential (which could possibly occur at anesthetic concentrations) in small-diameter fibers may be of importance in altering the amount of transmitter released from nerve terminals in the presence of barbiturates.
c. PRESYSAPrIC: ACTIOSSOF BARBITURATES Brooks and Eccles ( 1947) demonstrated that barbiturates depressed the focally recorded presynaptic volley associated with the spinal monosynaptic reflex, but dismissed this as a primary site of action because it was seen only with high doses. However, in later studies, Ldyning ut “1. (1964) and Richens (1969) suggested that this action on the primary afferent terminals was the only effect that could adequately account for the depression of the EPSP. Weakly (1 969) established a selective presynaptic action by demonstrating that subanesthetic doses of thiopental and pentobarbital (10 mgkg, iv) reduced the mean quantum content of the unitary EPSP in spinal motoneurons by about 2576, without altering the average amplitude of the unitary EPSP. I n addition, these concentrations of barbiturates did not alter the input resistance of the motoneuron or the strength-duration relationship. Nicoll (1980) has suggested that barbiturates may exert their presynaptic action by (a) decreasing the size and/or blocking terminal invasion of the action potential or (b) directly interfering with the transmitter release mechanism, possibly through an action on calcium fluxes or metabolism. Ldyning Pf nl. (1964) showed that barbiturates reduced the amplitude of the action potential invading primary afferent terminals and suggested that barbiturates were acting like local anesthetics. Such an action would be expected to depress terminal excitability. Galindo (1969) reported a reduction of the excitability of primary afferent terminals on the cuneate nucleus. Nicoll (1975a) demonstrated that the barbiturate depolarization of primary afferent terminals in the isolated frog spinal cord is associated with an increase in terminal excitability. This result does not exclude a local anesthetic action of pentobarbital which may be masked by its action on primary afferent depolarization. Indeed, with higher concentrations (> 1 mM), the local anesthetic action of pentobarbitai predominates (Nicoll, 1975a). At synapses other than those involved with the monosynaptic reflex pathway, the presynaptic actions of barbiturates are variable. Richards ( 1 972) found no change in the size of the presynaptic spike or in the
PHARMACOLOGY OF BARBITURATES
23
excitability of presynaptic fibers with concentrations of pentobarbital (0.25 mM) that depress excitatory transmission in the olfactory cortex. Scholfield and Harvey (1975) demonstrated that pentobarbital exerted a selective depressant action on synaptic potentials compared to action potentials in isolated guinea pig olfactory slices, with rather high concentrations (>1 mM) required to cause a 50% depression of the amplitude of the action potential. Nicoll(l972) has suggested that the depression of the olfactory bulb EPSP by hexobarbital(40-90 mg/kg, iv) may be due in part to a presynaptic action, since the size of the presynaptic dendritic response is concomitantly reduced. The vertebrate neuromuscular junction has been another site where the effects of barbiturates on transmitter release have been examined. Barbiturates have been shown to increase the quantal content of the end-plate potential (EPP) following nerve stimulation (Quastel et al., 1972; Thomson and Turkanis, 1973; Seyama and Narahashi, 1975; Proctor and Weakly, 1976). This increase in release has been attributed to the prolongation of the presynaptic potential since the amplitude of the presynaptic spike is unaffected or even reduced by barbiturates (Thomson and Turkanis, 1973). Barbiturates also increase the frequency of spontaneous miniature end-plate potentials (MEPP) at the vertebrate neuromuscular junction (Quastel et al., 1971, 1972; Westmoreland et al., 1971; Thomson and Turkanis, 1973). On the other hand, barbiturates depress the frequency of MEPPs at the crustacean neuromuscular junction (Iravani, 1965). A recent study by Pincus and Insler (1981) suggests that the effects of barbiturates on transmitter release at the frog neuromuscular junction may depend largely on the calcium content of the bathing medium during periods of evoked release. Both phenobarbital and the convulsant barbiturate 5-ethyl-5-(2’-cyclohexylidene-ethyl)-barbituricacid (CHEB) increased the quantal content of the EPP and the amplitude of the EPP in Ca2+-deficientRinger’s solution. I n contrast, both drugs depressed the amplitude of the EPP without altering quantal content when normal Ringer’s media (Ca2+concentration = 1.8 mM) was employed. T h e variable effects of barbiturates on transmitter release at different synapses may be attributable to differences in the release mechanism (e.g., degree of calcium dependency) in addition to anatomical factors such as the presence of presynaptic inhibitory inputs on nerve terminals (e.g., primary afferent terminals). It may be more relevant to study the effects of barbiturates on transmitter release at central synapses rather than peripheral synapses, despite the technical difficulties involved in measuring release from CNS neurons. T h e puzzling finding that CHEB and phenobarbital exert simi-
24
MAX WILLOW A N D GRAHAM A. R. JOHNSTON
lar actions on release at the neuromuscular junction (Pincus and Insler, 1981)suggest that these actions may be unrelated to the pharmacological effect of these drugs observed in ztivo.
D. EFFECTS OF BARBITURATES ON TRANSMITTER ACTIONI N VERTEBRATE NEURONS CENTRAL 1 . Efects .f Barbiturates on Responses Evoked by Excitatory Transmittn Substnnces Most of the studies examining the interaction of barbiturates with putative excitatory transmitters in the CNS have utilized electrophoretic techniques in which the firing rate of neurons has been recorded extracellularly . Krnjevic and Phillis ( 1 963) reported that systemic administration of barbiturates produced a substantial and prolonged reduction in the firing of cat cerebral cortical neurons by acetylcholine (ACh). T h e cells examined (Betz cells) appeared to have a population of muscarinic cholinergic receptors upon them. Thiopental and hexobarbital (0.5- 10 mg/kg, iv) abolished the responses of cat caudate neurons to electrophoretically ejected ACh (Bloom et al., 1965). Curtis and Ryall (1966) showed that systemically administered pentobarbital reduced the frequency of firing of cat Renshaw cells in response to ACh, n-butyrylcholine, nicotine, and acetyl-P-methylcholine. Pericruciate cortical neurons stimulated by electrophoretic ACh are also sensitive to various barbiturates, including pentobarbital, diallylbarbital, and methylthioethyl-2-pentyl-thiobarbiturate(Crawford and Curtis, 1966; Crawford, 1970). Catchlove et al. (1972) demonstrated a depression of ACh-evoked responses of deep pericruciate neurons by methohexital(3 mg/kg, iv) and suggested that barbiturates, like dinitrophenol, act by inhibiting aerobic mitochondria1 metabolism. T h e firing of rat brainstem neurons by ACh was depressed by systemic or electrophoretic administration of pentobarbital (Bradley and Dray, 1973). Duggan et nl. (1974) concluded that the nicotinic response of cells in the paramedian reticular nucleus of the cat was more sensitive to the depressant actions of barbiturates than was the muscarinic response. Adams (1976) has shown that barbiturates have more potent effects in depressing the response to exogenously applied ACh at the neuromuscular junction than their action on the rise phase of the end-plate current (EPC). Since the opening of ionic channels during the EPC is very fast, it was proposed that barbiturates do not have sufficient time to enter and block during the rising phase of the EPC. O n the other hand, the rising phase of the response to
PHARMACOLOGY OF BARBITURATES
25
exogenous ACh follows a much slower time course, and it was suggested that barbiturates could block open channels during this period, resulting in a diminished response. I n addition, barbiturates have been shown to depress the excitatory effects of acidic amino acids on the firing rate of various neurons. Crawford and Curtis (1966) demonstrated that the firing rate of deep pyramidal cells (of the cat pericruciate cortex) by electrophoretic DLhomocysteic acid is depressed by systemic and electrophoretic administration of barbiturates. Phillis and Tebecis (1967) showed that pentobarbital (2 mg/kg, iv) reduced the responses of cat thalamic neurons to L-glutamate (discharge frequency reduced to about two-thirds of the control magnitude). Pentobarbital, when applied electrophoretically or administered systemically (20 mg/kg, iv), depressed the firing rate of cat cuneate nucleus cells in response to L-glutamate (Galindo, 1969). Of interest is the fmding that doses of barbiturates that depressed the Achevoked firing of rat brainstem neurons had little effect on glutamateevoked responses (Bradley and Dray, 1973). Nicoll (1 975b) has shown that pentobarbital (20- 100 p M ) depressed the depolarization of frog motoneurons by glutamate. Barbiturates have also been shown to depress the glutamate-evoked depolarization of mouse spinal neurons grown in tissue culture (MacDonald and Barker, 1979; Ransom and Barker, 1975). I n addition, barbiturates also depressed the glutamateinduced conductance increases in spinal motoneurons (MacDonald and Barker, 1979). Richards and Smaje (1976) observed that pentobarbital (0.1-0.3 mM) consistently depressed the excitatory actions of L-glutamate on prepiriform cortical cells in vztro. Responses of various neurons, excited by serotonin (5-HT), have also been shown to be sensitive to barbiturates. Roberts and Straughan (1967) have shown that the systemic injection of small quantities of thiopental selectively and reversibly reduced the sensitivity of cat cortical neurons to excitation by 5-HT when at the same time the response to glutamate was unaffected. Johnson et al. (1969) similarly demonstrated a depression of 5-HT and norepinephrine-evoked firing of cortical neurons in the cat. In addition, it was noted that the number of cells excited by norepinephrine in barbiturate anesthetized animals was markedly less than the number of cells excited in unanesthetized or N20-halothane anesthetized animals.
2. The Effects of Barbiturates an Responses Evoked by Inhibitory Transmitter Substances T h e inhibitory effects of 5-HT (Roberts and Straughan, 1967; Johnson et al., 1969; Tebecis and DiMaria, 1972), norepinephrine
26
MAX WILLOW AND GRAHAM A. R. JOHNSTON
(Bloom P t nl., 1965; Johnson et nl., 1969), dopamine (Bloom et al., 1965; Tebecis and DiMaria, 1972), and ACh (Bloom et ai., 1965) are, in general, little affected by moderate amounts of barbiturates. On the other hand, a great deal of attention has recently been focused on the effects of barbiturates on responses to exogenously applied GABA in a variety of i n uiiw and in vitro preparations. Nicoll (1975b) showed that pentobarbital (20 p M ) increased the amplitude and duration of the GABA-mediated hyperpolarization of frog motoneurons. At higher concentrations (200 pLzI),pentobarbital caused a direct hyperpolarization of frog motoneurons. At these concentrations the amplitude of the GABA response was depressed and markedly prolonged. Bowery and Dray (1976) demonstrated a reversal by barbiturates of the bicuculline methochloride (BMC) antagonism of the GABAmediated depolarization of the isolated superior cervical ganglion of the rat and the inhibition by GABA of the firing of medullary neurons. Of particular interest was the finding that pentobarbital did not potentiate these responses to GABA in the absence of BMC. This finding was questioned by Curtis and Lodge (1977), who unequivocally demonstrated that the response of doral horn interneurons in the cat to electrophoretic GABA was enhanced by pentobarbital (also administered electrophoretically in cats anesthetized by a-chloralose or urethane). In agreement with Bowery and Dray (1976), they showed that pentobarbital partially reversed the antagonism by BMC of the inhibition of cell firing by GABA. In a later study, Lodge and Curtis (1978) showed that in the unanesthetized decerebrate cat, pentobarbital (15 mg/kg, iv) increased the time course of recovery of dorsal horn interneurons stimulated by GABA, without altering the time course of recovery of cell firing by glycine. Evans (1979) showed that pentobarbital (10-80 p M ) enhanced the depolarization of immature rat dorsal root fibers by GABA, a process which appeared to be bicuculline insensitive. On the other hand, higher concentrations o f pentobarbital (50- 160 p M ) produced a bicucullinesensitive depolarization of dorsal root fibers, characterized by a slower onset and offset than that produced by GABA. It was suggested that bicuculline could antagonize the GABA-like actions of pentobarbital but could not antagonize the enhancement of GABA by pentobarbital. Nicoll could enhance the GABA(1978) showed that pentobarbital (100 pFLI\I) mediated depolarization of frog sympathetic ganglion cells, but the effects of bicuculline were not investigated in this study. Recently, Connors (198 1) has examined the actions of pentobarbital on neurons of dorsal root ganglia from adult rats. Pentobarbital (40-200 FM, bath applied) enhanced the GABA-mediated transient inward current into ganglion cells voltage clamped at their resting potential. In cells which were not
PHARMACOLOGY OF BARBITURATES
27
voltage clamped, concentrations of 40 and 200 p M pentobarbital enhanced the GABA-induced depolarization of such cells, and in addition, enhanced the increase in conductance due to GABA. In all of these experiments, GABA was bath applied. Increasing the concentration of pentobarbital to 1 mM resulted in an attenuation of GABA responses. Concentrations of pentobarbital that enhanced GABA responses (40200 p M ) did not alter the resting membrane potential or conductance. However, at 1 mM, pentobarbital produced a small depolarization (go%. The radioimmunoassay profiles of these chromatograms were comparable to those in (A). (Taken from Bayon et al., 1978.)
‘TABLE V I RIA SPECIFICITY OF SOMEANTISERA RAISED A G A I N RENKEPHAI.INS~ I,
-.J
Fs
Peptide (antigen)*
Animal species and conjugateC
Met-enk
Rabbit succinyl Met-en k + succinyl hemocyanin to polylysine; CDI Rabbit hemocyanin; CDI Rabbit hemocyanin; glutaraldehyde Rabbit hemocyanin; CDI
Leu-enk Met-enk
Leu-enk
Met-enk
Rabbit ovalbumin; CDI
Tracer antigen and labeling methodd [3 H ]Met-enk
Cross-reactivity with’
Sensitivity
Other enkephalins (‘% )
Endorphins
p
pmol
10
a and
[3H]Leu-enk
5 2 pmol
10
a a n d p ()
[3H]Met-enk
5 10 pmol
70%) of receptors with a-bungarotoxin in order to gain access to the time regime of the quenched-flow technique. The considerable improvement of the quenched-flow method over the conventional integrated flux measurements becomes apparent when one compares the preliminary dose-response curves so far obtained. Values of 600 p M (Neubig and Cohen, 1980) characterize the [L],,, for carbamoylcholine in quenched-flow experiments, whereas values of 15-30 p M result from the slow flux measurements (Popot et al., 1976; Miller et al., 1978; Moore et al., 1979b; Neubig and Cohen, 1980). T h e maximal rate of Na+ that could be measured with the quenched-flow technique is 37 sec-' (Aoshima et al., 1981), 84 sec-' (Aoshima et al., 1980), 65 sec-' (Neubig and Cohen, 1980),and more recently, 310 sec-' (Hess et nl., 1982). The advantages of simultaneous mixing and spectroscopic detection in terms of time resolution should be noted. Rates of 1500 sec-' have been measured with this technique (Moore and Raftery, 1980) in native membranes, and rates of 487 sec-' have been measured in reconstituted vesicles (Wu et al., 1981) at saturating Carb concentrations. I n the case of the quenched-flow experiments, and under the assumption that all a toxin sites are involved in ion transport, the flux rates obtained indicate that there are about 3500 Na ions flowing per a-toxin site per sec, (7000 ions/AChR monomer), that is, only 8% of the maximal rates for the same ion in chick muscle (Catterall, 1975) and even less than this value if the transport expected from single-channel measurements in Tmpedo (Schindler and Quast, 1980; Boheim et al., 1981) is employed. Some of the factors considered above may intensify the underestimation, but it is obvious that values of tIl2 of 10 msec for ssRb+ flux at l-mM Carb (Hess et al., 1982) leave little room for maneuvre. C. FLUXDOSE-RESPONSE CURVES, BINDING EQUILIBRIA, A N D DESENSITIZATION Dose-response curves furnished from rapid-flux measurements are still scarce, but general trends are already emerging. They confirm the
306
F. J. BARRANTES
need to revise the significance of the rather extensive data accumulated on the apparent equilibrium binding parameters of various cholinergic drugs (Table IV). T h e most widely used assay, the inhibition of a-toxin rate of association, usually yields two extreme values (designated t + 0 and t + = in Table IV) for the time-dependent inhibition of toxin rates. T h e interpretation of the apparent [L],., values is of course modeldependent, but intuitively the coincidence of the t -+ 3fl parameter obtained by this method and the corresponding values resulting from equilibrium dialysis or the like is clear: Prolonged exposure to agonists stabilizes the AChR in a state(s) D characterized by its (their) high affinity for the ligand, about two orders of magnitude higher than the corresponding resting state(s)R (Table IV). Whatever the exact interpretation of the nonequilibrium [~5],,~ values (t + 0) is, their assignation to a low-affinity form of the AChR remains unaltered. T h e contribution of the more recent rapid-flux measurements in the context of binding mechanisms is that they challenge the tenability of interpretations ascribing this particular low-affinity form of the AChR to the form leading to channel activation. This stems mainly from the discrepancy between the [L],,, (t + 0) values and the corresponding [L],, figures obtained in the fast-flux measurements. Neubig and Cohen (1980), for instance, report [ L ] , , ( t + 0) values of 0.6 mhf for Carb and 0.2 mM for phenyltrimethylammonium, a partial agonist. Inspection of Table IV clearly indicates that these values depart substantially from the aforementioned [[L],, assigned to the low-affinity state of the AChR. A further difficulty arises when attempting to correlate flux data with fractional occupancy of binding sites in the light of reaction mechanisms currently accepted for the action of agonists in uizro. T h e number of agonist molecules involved in the physiological gating phenomenon appears to be close to two or larger [see Colquhoun (1979), Gage (1976), Steinbach, (1980), and Adams (198 I), for discussion of the subject]. T h e Hill coefficient for Carb found in rapid-flux experiments is close to two (Neubig and Cohen, 1980), and the need to consider a reaction mechanism involving a biliganded, low-affinity state of the AChR preceding the final activated (open) state appears justified. T h e number of unknown variables still present in models like the one shown in Fig. 7 is large, and one should exercise caution in using such multiparameter models even in their provisional character of useful working hypotheses. Simpler reaction mechanisms based on the Katz and Thesleff (1957) type of cyclic scheme (Weiland P t al., 1977; Barrantes, 1978; Boyd and Cohen, 1980) have been used to account for the kinetics of binding of Carb, ACh, or suberyldicholine, and the in ~ ~ i t rinactivation o processes ascribed to the desensitization phenomenon. Although the cyclic mechanisms involving
DEVELOPMENTS IN STRUCTURE AND FUNCTION
307
FIG. 7. Multistate model of AChR-agonist interactions, including (1) binding of two agonist molecules (A) to AChR in resting state, R (preexisting ligand binding and associated with closed channel), or desensitized state, D, and (2) isomerizations between the two states and between the closed-channel and activated, open-channel conformation R*. The new intermediate low-afFinity state postulated by Neubig and Cohen (1980), R,, is included. The model is a development of previous work (Weiland et al., 1977; Barrantes, 1978; Boyd and Cohen, 1980; Heidmann and Changeux, 1979; see also Barrantes, 1979) based, in turn, on early schemes (Changeux et al., 1976; Katz and Thesleff, 1957).
only monoliganded AChR states or even simpler versions can be satisfactorily modified by the addition of a rapid isomerization step to include the channel activation (Bonner et al., 1976; Barrantes, 1978), and yield parameters for the activation step compatible with those observed in vivo, such mechanisms are less tenable when biliganded receptor forms are incorporated and the in vitro rapid-flux data are considered. Fractional occupancy of binding sites by competitive antagonists has been used as a means to determine the number of agonist molecules needed to activate ionic flux. The results are still contradictory. Lindstrom et al. (1980a) and Anholt et al. (1981) observed a linear diminution of activable sites upon titration of AChR vesicles with a toxin and interpreted this result to indicate that monoliganded receptor (AR in Fig. 7) controls channel opening. If the view currently accepted by electrophysiologists were correct (see, e.g., Adams, 1981; Colquhoun, 1979; Steinbach, 1980),and biliganded AChR (A2R) determined gating, the concentration dependence of toxin inhibition would be hyperbolic instead of linear. This is precisely what the results of Sine and Taylor (1980, 1981) show. Reduction and subsequent alkylation of the AChR “freezes” the receptor in a very low-affinity state whose [L],, values for agonists mimic those found with electrophysiological techniques (Walker et al., 198la; Barrantes, 1980). Walker et al. (1981a) further proposed that the reduction reaction could involve attack of thiol groups in the AChR channel itself. But aside from these special cases, the low-affinity state of the AChR detected in binding experiments (Table IV) does not appear to correspond in a direct manner to the one leading to channel activation.
ZP'O LP'O 8000 20 52-6'5
POO'O 6ZO'O (.'?I)Z'O P("Y) 7110.0
WOO 800'0
P20.0 ED'0-lO'O
190
S'Z 91'0 O'Z60'0
0'1
ROO'O 0' F 800.0
co 0 m
15.0
PO00 PIO'O 0'2
ozno 890'0
unon wn 50.0 S'O SO'O W'O S' I
W V
Carb
24 20 40 22 1.4 1.9 1.5
30-50 20 20 20 8
35-121 70- 148
0.5 0.4 5 50 60 0.1 0.05-0.12 0.5 0.5 0.5 0.02 58-170 3.5 6.3 2.3 3.0 11-36 18-40
Electroph Elerhoph Electrophonrs Electmphonrs Electmphm Electroph Torpedo mannorata Torpedo mamorafa Torpedo marmordu Torpedo marmorata Torpedo mannoraka Turpedo mannoraka Tapedo mamorata Torpedo califmica Torpedo colifmnua Torpedo califmua Torpedo m m o r a f a Torpedo caliiornica Torpedo californua Rat diaphragm (denervated) Rat diaphragm (denervated) Rat diaphragm Cat leg muscle (denervated) BC3H-1 cell line BC H-1 cell line
Membrane Membrane Membrane Membrane Purified Purified Membrane Membrane Membrane Membranes Triton extract Membranes Membranes Membranes Membranes Membranes Membranes Membranes Purified Crude homogenate Triton extract Homogenate Membranes Cultured cells Cultured cells
Elerhoph Electrophonrs
Membranes Membranes Membranes Membranes Purified Membranes Membranes Membranes Cultured cells Homogenate Triton extract Membranes Membranes
1 (a-BuTX)
I (a-Bum) I (a-BuTX) I (a-BuTX) 1 ([aJHICT) I (a-BuTX) I (a-Bum) R (a-Bum) R (a.BuTX) R (a-BuTX) R (a-BuTX) 1 (Nala u toxin) I ( N a p a toxin)
Bulger and Hess (1973) Fu ef al. (1974) Weher and Changeux (1974) Kasai and Changeux (1971h) Meunier el d.(1974) Meunier and Changeux (1973) Cohen et al. (1974) Weber and Changeux (1974) Griinhagen and Changeux (1976) Franklin and Potter (1972) Eldefrawi and Eldefrawi (1973a,b) Bonner el al. (1976) Neubig and Cohen ( I 979) Quast rt al. (1978) Blanchard el al. (1979) Weiland el 01. (1977) Barrantes (1980) Walkerrf 01. (1981a) Raftery et al. (1976) Colquhoun and Rang (1976) Colquhoun and Rang (1976) Colquhoun and Rang (1976) Barnard ef al. (1977) Sine and Taylor (1979) Sine and Taylor (1980)
I (a-BuTX) E (WID=+) 1 ( N a p a toxin) E (PHlDeW E (rHIDeca) I (Naja a toxin) E (PHIDe4 F (quinacrine) I (Nala a toxin) 1 (a-BuTX) I (a-Bum) I (a-BuTX) I (PHI-a-toxin)
Bulger and Hess (1973) Fu rf al. (1974) Weber and Cbangeux (1974) Kasai and Changeux (1971b) Meunier and Changeux (1973) Weber and Changeux (1974) Weber and Changeux (1974) Criinhagen and Changeux (1976) Sine and Taylor (1979) Colquhoun and Rang (1976) Colquhoun and Rang (1976) Barnard el 01. (1977) Barrantes (1980)
I (rH1Deca) I (Naja a toxin) I (PHID-) I (PHIDeca) I (PHlDeca) F (dansyl-C,-choline) I (Naja a toxin) F (quinacrine)
F (intrinsic)
K (PHICarh)
Deca
34-50
0.7
8 0.9 0.8 1.3 0.02 1 0.8 0.74 0.6 22 2.1 8.6 3.0 50
Eltcfroph
Elerhophmus Elerhoph Torpedo Topedo mannorata Torpedo mannoraka BC3H-1 cell Line Rat diaphragm (denervated) Rat diaphragm (denervated) Cat leg muscle (denervated) Torpedo manmala
(continued)
PZ'O 8F'O 22.0
vo 2'0 p(".Y) I ' O - F E O O EZ'O LI'O LI n
(1.Y)S'I-FO
6E'O
zo LI'O Z (1 3.L-P 0%
2
m
6X HI1
06 09 Ot z9 19
SubCh
0.5-0.8 0.31 7.7 2 4.6 27.5 I 0.4-0.97 0.13
01 w
0.04 0.045
Cat leg muscle (denervated) Rat diaphragm
0.55
Rat diaphragm
0.4-0.7 0.38 0.033 -c 0.006 0.3 0.01 0.15-0.24 0.0004-0.004 0.17 0.01 (0.005. 0.025)
c.
PTA
35 111
1.6 26.2
64 427
0.2-2.5 81.7
BC3H-I cell line Tmprdo rnlifrrnim Tmpfdo mammala
BC3H-1 cell line Torpedo mammala BC3H-1 cell line Torpedo cahfmira BC3H-I cell line Tmpede mannoratn T. caiif0rnUa T. mbii. Tmppdo califmnjca BC3H-1 cell line
Membrane Purified (junctional) Purified (extrajunctional) Cultured ceUs Membranes Membranes Cultured cells Membranes Cultured cells Membranes Cultured cells Membranes
I (a-BuTX) I (a-BuTX)
Barnard e/ a/. (1977) Brockes and Hall (1975h)
I (a-BuTX)
Brockes and Hall (1975b)
1 ( N a p a toxin)
I (Naja a toxin) E ([aH]d-TC) 1 (Naja a toxin) I (a-CT) 1 (Naja a toxin) I ( N a p a toxin) I (NQ@ a toxin) I (Naja a toxin)
Sine and Taylor (1979) Weiland and Taylor (1979) Neubig and Cohen (1979) Sine and Taylor (1981) Barrantes (1976, 1978) Sine and Taylor (1979) Weiland and Taylor (1979) Sine and Taylor (1980) Neubig and Cohen (1979)
Membranes Cultured cells
I (Naja a toxin) I (Naja a toxin)
Weiland and Taylor (1979) Sine and Taylor (1979)
Membranes Cultured cells
I (Naja a toxin) 1 (Na@ a toxin)
Weiland and Taylor (1979) Sine and Taylor (1979)
Nicotine Torpedo calt/nrnira
BC3H-I cell line
The term equilibrium dissociation constant is reserved for determinations employing equilibrium measurements. [Lb.,are the half-concentrations for 1 -+ 0 and t + co, respectively (sometimes referred to as K , , , "protection constant"). Abbreviations used are: ACh, acetylcholine; C a h , carbamoylcholine; D e a , decamethonium; Hexa, hexamethonium; I-TC, d-tubocurarine; BuTX, a-bungarotoxin; Naja a toxin, a-neurotoxin from Naja naja siomeruis; a-CT, a-cobrotoxin (N.n.atra). SubCh, suberyldicholine; PTA, phenyltrimethylarnmonium. Methcds: 1, inhibition of binding of given ligand; E, direct determination of equilibrium dissociation constant; F, fluorescence of specified ligand; K , indirect determination of equilibrium constant from kinetic parameters. Gibson (1976) fitted his data to an allosteric model and calculated the equilibrium dissociation constant for the two states, T and R.
312
F. J. BARRANTES
Neubig and Cohen ( 1980) ascribed the “conventional” low-affinity state to a biliganded form, A,R,, different from the native resting (activable) biliganded receptor, A2R,which ought to precede channel gating (A2R* in Fig. 7). I have included the corresponding monoliganded species in the scheme in Fig. 7 not because of symmetry considerations but attending to current electrophysiological data to be discussed below. One interesting property of Neubig and Cohen’s (1980) low-affinity R, state is that it will contribute to the apparent inactivation (desensitization) processes (Fig. 7). When only the biliganded AChR is considered (AR,is not allowed to exist), desensitization ought to be apparent only at high agonist concentrations, at which the maximal response will be diminished. Under such conditions desensitization would show u p as a biphasic phenomenon. Responses at low ligand concentration should not be affected. If on the other hand the monoliganded form of this lowaffinity AChR state (AR,) manifests itself, two phases of desensitization should be apparent at all ligand concentrations. Only one desensitization phase was originally observed in similar measurements undertaken in EIectrophorus membranes (Aoshima et al., 1981), but in more recent studies (Walkeret al., 1981b; Hess et al., 1982) two inactivation processes were found in Tmpedo membranes. T h e rapid inactivation process proceeds with a rate of 2 sec-’, and the slow inactivation step superceeds it with a rate of 0.12 sec-’ in the presence of Carb. Bonner et nl. (1976) first characterized this slow phase of desensitization with rapid kinetic methods and reported values of 0.16 and 0.37 sec-* for this process with ACh and Carb in Torpedo membranes. Later, Barrantes (1978) found values of 1.2- 1.5 sec-’ with suberyldicholine, which may correspond to the faster desensitization process. These values also show close resemblance to those described more recently by Sakmann et al. (1980) (to be discussed later) in their study of desensitization by patch-clamp techniques. Only one very slow desensitization phase is apparent in reduced Tmpedo AChR (Walker et al., 1981a). Recovery from desensitization is a slower process occurring over seconds to minutes, depending on various parameters (see Adams, 1981; Cohen and Boyd, 1979; Boyd and Cohen, 1980). In the absence of agonist, the majority of receptors should exist in the resting, activable conformation R (Fig. 7). At equilibrium, the R c, D balance is characterized by a net predominance of R over D. T h e R/D equilibrium constant measured in various systems indicates values between 10% (Weiland and Taylor, 1979) and 20-25% (Heidmann and Changeux, 1978; Barrantes, 1978; Boyd and Cohen, 1980) desensitized AChR preexisting ligand binding.
DEVELOPMENTS I N STRUCTURE AND FUNCTION
D. NEWVIEWSON
THE
313
BEHAVIOR OF ACH-OPERATED CHANNELS
T h e patch-clamp technique has allowed the direct observation of the currents flowing through individual ionic channels. Since its first application to ACh-activated extrasynaptic channels (Neher and Sakmann, 1976a), glutamate (Patlak et al., 1979; Cull-Candy and Parker, 1982), GABA (Bormann et al., 1981), Na+ (Sigworth and Neher, 1980; Horn et al., 1981), Ca2+-activatedchannels (Marty, 1981; Hamill, 1981; Pallota et al., 1981; Colquhoun et al., 1981), K+ channels of the anomalous (inward current) rectifier type (Ohmori et al., 1981; Fukushima, 1981), and Ca+ channels (Lux and Nagy, 1981; Lux et al., 1981; Fenwick et al., 1982) have been explored with this technique. Many other neurotransmitter, hormonal, ionic, or voltage-sensitive systems are certain candidates for the immediate future. It is possible to distinguish three periods in the evolution of findings from the patch-clamp methodology. The initial period, which extends to 1979, appeared to provide experimental confirmation of many of the hypotheses on AChR-channel properties implicit in the theory behind noise or perturbation techniques, transforming essential assumptions of the macroscopic methods into observable variables. The picture which emerged from all available techniques was convergent on a simple view of the ion gating process: Channels behaved as two-state switchlike devices, open or closed. The second period (the present one) coincides with the availability of several methodological refinements (Neher et al., 1978; Sigworth and Neher, 1980; Horne and Patlak, 1980; Neher, 1982; Hamill et al., 1981), enabling the observation of new phenomena in AChR channels. The simplicity of the all-or-none phenomenon gives way to a more complicated, subtle structure underlying the “apparent” single-channel event. I n fact, what was believed not long ago to be the unitary channel response is now recognized as a composite of open and closed states. Additional discrete open states have also been found. All these findings question the tenability of some of the classical views on the AChR gating. A brief description of the third and future stage in this evolution will follow after presentation of the characteristic elements of AChR channel activity. 1. Quantitative Kinetic Parameters Associated with Single-Current Recordings Let us start with a brief phenomenological description of patch-clamp recordings. A typical trace usually appears as rapidly flickering pulselike current events (Fig. 8). The silent intervals during which channels are closed can be easily distinguished from those in which one or a few are
314
F. J. BARRANTES
gaps
40 rnsec Flc.. 8. Patch-clamp recording from a rat muscle cell in culture (“myoball”) in the presence of 5-p.if i\Ch at -70 mV membrane potential. A “classical” single-current event of the all-or-none open-close type is exemplified by the rightmost current trace. The beginning of the trace shows several short closures (gaps) within the apparent open period, which is redefined as an N-burst (see text), a composite of open and closed states. Colquhoun and Sakmann (1981) found at least three time constants of gap durations (rt 45 psec, representing 77% of the events; T , 350 psec representing 21%;and T~ < I msec representing 3%). Notice the higher noise in the open-channel state, a feature characterized by Sigworth (1982). The original record was provided by Dr. 0. P. Hamill.
-
-
open when the number of activated channels is kept low. This is accomplished in practice by working in the low agonist-concentration domain and ( ( 1 ) by choosing a membrane area with low receptor density, as in extrasynaptic regions (Neher and Sakmann, 19’76a,b), (b) by using quasi-irreversible antagonists to eliminate most of the receptors, or (c) by exploring high concentration domains, but it is then necessary to allow agonist-induced desensitization to occur before individual channel gating can be resolved (Sakmann rt al., 1980). When the number of contributing channels is small, opening and closure of a given channel will take place most likely before any other individual channel opens. On probabilistic grounds the distribution of the conducting and silent intervals can then be attributed to open and closed states of a single unit, whose gating is rare and apparently random. Although the stochastic model of chemical reactions specifies that the transition from a given state to another state is determined by the transition probability per unit time,ptr,iridr/mdeiitly of the number of molecules which happen to be in either state I or j (see, e g . , Zwolinski and Eyring, 1947), the technical difficulties arising when the number of active units is large still impede the analysis of traces from many contributing channels. Assuming the
DEVELOPMENTS IN STRUCTURE AND FUNCTION
315
independence of channels, the transitions between the nonconducting and the conducting states are treated as Markov processes. The duration of each state (dwell time) is exponentially distributed in time. The inherent advantages of dealing with a single operating unit are made apparent in the fact that independent and direct estimates of the individual rate constants for channel opening and closing can be obtained from such distributions. For comparison, in a “macroscopic” measurement (e.g., noise or relaxation experiment), and in the simplest case of a two-state first-order (A c, B) reaction, the relaxation rate at which the system strives toward equilibrium is given by
that is, a composite of the individual probabilities determining the rate constants that obtain. The dwell times displayed repeatedly and sequentially by a single channel reproduce in their random behavior that of an assembly of many channels simultaneously driven out from equilibrium; the comparison drawn by Ehrenstein et al. (1974) with a radioactive decay curve is a useful analogy. Under certain circumstances the individual current events are grouped into bursts whose average duration depends on agonist concentration (Fig. 8). This type of burst will henceforth be called N-burst2 because it occurs under apparently normal circumstances (Nelson and Sachs, 1979; Colquhoun and Sakmann, 1981) and is to be distinguished from B-bursts ( B for blockers, following Colquhoun and Sakmann, 1981) and D-bursts, observed with desensitizing agonist concentrations (Sakmann et al., 1980). The significance of the different types of bursts will be clarified below. The square-like pulse events (“single channels”) are
* Burst is a term coined by Neher and Steinbach (1978) when describing the modification of the usual squarelike shape of single-current pulses occurring in the presence of local anesthetic substances. TheN-burst is defined as a group of openings (believed to arise from activation of a single AChR channel) separated by interruptions (gaps) whose duration is short relative to the total length of the burst. N - is meant to imply normal; an alternative philological root is found in Narhschlag, the German word for backlash, as first used by Neher and Sakmann, to describe the multiple gating phenomenon. Nelson and Sachs (1979) first reported the occurrence of this phenomenon in embryonic muscle cells (myoballs) in culture. The term Nachschlag was used only subsequently (e.g., Adams, 198 1; Lester, 1982) and referred to Nelson and Sachs but without clarification of its origins or significance. How normal and widespread the phenomenon is awaits further experimentation. It also appears to occur with acetylcholine (in addition to suberyldicholine) and in the junctional region of adult muscle (in addition to myoballs) (B. Sakmann, personal communica tion).
316
F. J. BARRANTES
“compressed” in the slow records and appear as spikes. Bursts, in turn, can be seen to be grouped into a higher order association-clusters of bursts-when long strings of records are examined (see Fig. 1 1 ) . T h e separation of bursts and interburst intervals within a cluster and the distances between clusters and intercluster intervals also vary with the agonist concentration (Sakmann et al., 1980). Quantitative parameters can be defined for these “aggregates” in a manner analogous to those used for the single-channel traces; tb is the burst duration; t i the interburst interval (see Fig. 1 1 ) ; accordingly, cb is the cluster duration and c , the intercluster interval. Typical time scales of busts and clusters are a few hundred milliseconds and seconds, respectively. These parameters have been been used to study the kinetics of channel blockage and desensitization, as will be illustrated. Recently, short interruptions within an individual single-channel pulse could also be resolved (Colquhoun and Sakmann, 1981). These silent periods within the apparent open state of the channel are termed gaps and have average durations of tens to hundreds of microseconds (Fig. 8). T h e gaps complete the presently available repertoire of channel phenomena amenable to observation with improved patch-clamp instrumentation (Hamill et al., 198 1). Having named the individual features distinguishable to date in the microsecond-second time domains, we can now illustrate the way the corresponding average kinetic parameters are derived and some of the information to be gained from these. T h e basic assumption here is that the single-current events are exponentially distributed random variables. Thus analysis of a large number of events (gaps, bursts, etc.), or their corresponding interevent durations can most conveniently be done in the form of the distribution of their dwell times as a function of the number of events. In view of the stochastic nature of the events, exponential fitting of the distributions will yield mean (average) time constants of such distributions, as expected for Poisson processes. We shall also see that in some cases the analysis yields a sum of exponential terms; a collection of Poisson phenomena is then assumed. the more extensively characterized of these time (decay) constants are T, (mean channel-open time) and 7, (mean channel-closed time, Fig. 9), but analogous mean times can be defined for the other channel-associated parameters. T h e relationship between the experimentally determined T, and T, and the rate constants for channel closing and opening, respectively (a and p), is of course model-dependent. For a two-state reaction scheme of the type
317
DEVELOPMENTS IN STRUCTURE AND FUNCTION
u)
c C
$
0
50
0
loo
100
50
to (rnsec)
5
tc (rnsec)
20
50
[AChl PM
FIG.9. Distribution of the durations of open (to) and closed (te)states of current pulses during D-bursts and dependence of their reciprocals on agonist concentration. (a,b) Sequences of D-bursts occurring in the presence of 10-pM ACh (12"C, -130 mV) were analyzed for dwell times in the open and closed states. The distribution of intervals in each state was fitted with single exponential functions (. . .) yielding mean open T, and mean closed T~ times of 10.6 and 18 msec, respectively. (c) Double-logarithmic plot of the reciprocal of mean open (open symbols), T,, and close (closed symbols), T,, time constants as a function of agonist concentration. T , is noticeably concentration dependent, whereas 7" is not. (From Sakmann et a/.,1980.)
the mean dwell time in state 0 is given by ( L 1 ) - l , and the mean dwell time in state C is (kJ1. That is, the mean period spent in a given state is given by the elementary rate constant leading away from that state. Generalization of this to other more complicated reaction mechanisms is possible (Colquhoun and Hawkes, 1977; Neher and Steinbach, 1978); the reciprocal of the mean dwell time in a given state is the sum of all reaction rates depopulating (i.e., leaving) that state: kin
= T~ =
(5)
n
I n the case of the binding-isomerization models which are currently taken as the most plausible ones, such as the two-binding two-isomerization scheme below (see also Fig. 7): K,
R
+ A-
ki k-1
KZ
RA
A k-a
RA,
.B
a
R+A,
(6)
the reciprocal of the mean open or close times are related to the rate constants for channel opening and closing by obviously more compli-
318
F. J . BARRANTES
cated relationships than in the simpler two-state model above:
a (7) (8 ) I h , = p‘ = {[A]k,/([A]k, + k 1 ) } , p (assuming k l = 2 k 2 , k-, = 2 k 1 , and a + p e k, + kl). Sakmann et nl. (1980) studied the behavior of AChR channels exposed to desensitizing agonist concentrations, and within a limited ACh concentration domain they could follow the behavior of mean open and closed times as illustrated in Fig. 9c. T h e intersection of the two reciprocal time constants T~ and I, occurred at about 22-pM (-90-mV holding potential) and 17-pM (- 130-mV) ACh. This intersection can be defined as the concentration of agonist, at a given membrane potential, at which the apparent rates of channel opening and closing are equal, that is, an “apparent” equilibrium isomerization constant results (which is less prone to depend on modeling assumptions). ( ~ “ ) - l= a’is independent of agonist concentration and becomes slower with membrane hyperpolarization; ( ~ ~ ) -=l p’ depends on agonist concentration, this dependence being described in a first approximation by a power law with an exponent of 1.4. This has been interpreted as being consistent with reaction schemes involving the binding of two agonist molecules preceding channel opening. U T , , = a’ =
2. Fin r Structurr .f f l i p “Siriglr”-CzLrmit Pulse: The Multiplr Gcitiiig Piirnomenon
Improved instrumental resolution has recently enabled the observation of short closure (gaps) occurring within the apparent open channel state in the sole presence of agonist, even at low agonist concentration. There are various types of gaps, attending to their mean time distributions. The shorter gaps so far observed have a mean duration of about 50 psec. In addition, Colquhoun and Sakmann (1981) observed gaps of about 10 times longer duration in muscle endplates, which presumably correspond to the longer gaps described by Nelson and Sachs (1979) in myoballs. These constitute the intermediate population (7, 0.7 msec) of the former authors. T h e briefness of the “short” gaps led Colquhoun and Sakmann (1981) to infer that they are unlikely to be associated with t w o independent channel activations. Instead, the suggestion was made that they could arise from rapid open-close transitions of the channel (multiple gating) occurring during the time for which the AChR remains occupied by the agonist, as previously considered on statistical grounds by Colquhoun and Hawkes (1977). Three other interpretations of the presence of gaps were considered by Colquhoun and Sakmann (1981): ( a ) blocking by the agonist, as previously observed by Adams and Sakmann (1978) in the case of decamethonium; (6) blocking by an unknown L-
DEVELOPMENTS IN STRUCTURE AND FUNCTION
319
endogenous substance; and ( c ) blocking by a mechanism related to ion permeation. Hypothesis (a) was rejected and the others not considered further. Channel-blocking mechanisms will be dealt with later. T h e occurrence of the intermediate type of gaps was not elaborated. Gaps are electrically silent events. Their significance remains to be evaluated, but one can already advance that these transient closures within the apparent open state will not be a mere addition to the phenomenological repertoire of channel parameters. Instead, these substructures will probably constitute manifestations of an electrically silent AChR state apparently different from the resting state. Observation of the gaps forces a redefinition o’f the parameters previously used to derive the channel lifetime: What was previously considered to be the channel open state now becomes a composite of open and closed states. Thus, the apparent length of the “open” channel state previously used to derive the rate constant for channel closing ti in a macroscopic measurement (e.g., noise or relaxation experiment) now becomes a mean N-burst duration, which includes both open and “closed within the open” phenomena [the open state($ + gaps].
3 . Heterogeneous Distribution .f Mean Open Times: The N-Burst The first level of supraorganization of the individual current pulses is the burst. T h e heterogeneity of N-burst lengths manifests itself in the histogram of apparent open times (Fig. 3 of Colquhoun and Sakmann, 198 l ) , which needs to be fitted with at least two exponentials. An N-burst is defined as a series of openings interrupted by gaps that are less than some critical duration (0.5-3 msec). Since this length is very much smaller than the mean intervals betweefi current pulses assumed to be independent, the N-burst duration is an operationally well-defined quantity. The two time constants fitting the N-burst distributions were rf = 0.15 msec (25%)and T, = 10 msec (75%)with 20 nM suberyldicholine in the perisynaptic region of frog neuromuscular region (Colquhoun and Sakmann, 1981). The slow time constant is the one which would be apparent in noise or relaxation analysis as a single component of about 10 msec, from which false values for the mean channel lifetime would be derived. The true channel open time is clearly shorter, after taking into account the temporarily closed periods (gaps), for the mean open time is the mean number of openings per N-burst ( i i ) multiplied by the mean length of an opening plus (ii - 1) times the mean gap duration (&). Applying this notion to the sequential scheme [Eq. (S)]the mean number of openings per N-burst is given by
320
F. J. BARRANTES
where the mean length of a gap is
and the resulting “true” mean open time is given by
T h e actual open lifetime is of course inaccessible as yet, since one cannot exclude the possibility of openings lasting less than the currently available resolution of the technique (a few microseconds). In any event, using the figures of Colquhoun and Sakmann (1981) mentioned above, the average N-burst consists of roughly four openings in quick succession, so the mean channel-open time would be about one fourth of the observed 10 msec (i.e., only 2.5 msec). Estimates of the rate constants for channel opening (p) and for ligand dissociation ( k - 2 ) can be obtained from the measurement of the mean gap duration and from the mean number of gaps per N-burst as given above. Values of /3 = 10,00015,000 sec-’ and k2 = 2000 sec-’ have been derived with suberyldicholine in adult muscle (Colquhoun and Sakmann, 1981). It is of particular interest to analyze these new findings in the light of theoretical considerations (Colquhoun, 1979; Colquhoun and Hawkes, 1977, 1981) in relation to the stochastic properties of channel behavior. One of the above properties concerns the equilibrium distribution of states, here listed for the two-agonist binding scheme [Eq. (6)]:
where [A] is the agonist concentration and the K’s are equilibrium dissociation constants [Eq. (6)]. Using currently accepted values for the various rate constants of agonist action obtained from macroscopic measurements, Colquhoun and Hawkes (1981) were able to express these equilibrium state probabilities in actual lifetimes spent in each state, from which it became apparent that, for independent binding steps with l and at ligand concentrations apparent K, values of about 30 ~ h (ACh), below this value (but still reasonably high to open about 5% of the channels) the channel spends most of the time in the biliganded activated (open) state R*A2 (see Fig. 7). However, 97% of all single occupancies would not be followed by channel opening via R*Az (though there is a
DEVELOPMENTS IN STRUCTURE AND FUNCTION
32 1
0.7% chance of opening via R*A). This is because dissociation of the agonist occurs before a second ligand has a chance to bind: a closed AChR channel with only one agonist bound has a higher probability (97%)of loosing the ligand than of reaching the biliganded state. There is only a roughly 2.5% chance that monoliganded receptors become doubly liganded (which does not mean that they necessarily open thereafter). However, once the biliganded state is reached, channel opening has almost the same probability as the dissociation of the ligand. This implies that doubly occupied channels may continue to open several times after the ligand concentration has fallen to zero. I n addition, whenever the states RA or RA2 are reached (in the presence of finite agonist concentrations) there is the possibility of any number (0 to m) of RA + RA2 oscillations (state transitions) before leaving to either R or R*A2. Here one finds a possible explanation of the N-burst (the multiple gating, Nachschlag) phenomenon. Although the idea is tempting, it is still premature to make it of general applicability. 4. Implications of the Assignation of the Rate-Limiting Step(s)
An outcome of the above finding (which is of course modeldependent) is its contradiction of the widely accepted assumption that agonist binding steps are faster than the close-open isomerization. If the multiple gating hypothesis were correct, the possibility should be entertained that the initial reaction steps in Eq. (6) proceed slower than the channel gating kinetics, constituting the rate-limiting steps in the activation pathway. A possible advantage of such constraints was discussed by Barrantes (1980) in relation to the affinity states of the AChR. In fact, two sets of experimental data in which the association rates of agonist binding have been measured in vitro with fast kinetic techniques yield similar on-rates of 1-2 X lo7 M-' sec-'. In one case (Neumann and Chang, 1976) AChR binding to T. califmica purified AChR was measured by absorption techniques, using murexide as an indicator of ACh binding. In a second case (Barrantes, 1978) the intrinsic fluorescence of membrane-bound AChR from T. marmorata was monitored to follow SubCh binding. These figures probably represent an overall, complex rate parameter involving some faster elementary steps. Off-rate constants were on the order of 10-3000 sec-'. These figures contrast with those obtained with a fluorescent agonist, NBD-choline, with which rates of 1.2-40 x lo* M-' sec-' have been reported (Prinz et al., 1980). The latter values would certainly make the rate-limiting character of the binding step(s) untenable. It is interesting to note that using essentially different types of arguments and experimental observations Lands et al.
322
F. J. BARRANTES
(1981) have recently calculated binding rates of 2-5 x lo7i t V 1 sec-' for ACh at the living end plate. 5. Chntinel Hi&wg.eiieity in Embl-rotiic n ~ i dDenerz1nted Muscle Denervation hypersensitivity is a well-known phenomenon that follows the sectioning of the motor innervation. One manifestation of this pathological situation is the spread of sensitivity to agonists, normally localized at the end-plate region, to the whole of the sarcolemmal surface. This phenomenon is accompanied by the appearance of newly synthesized AChR molecules at the surface membrane, and the normal density of AChRs of extrajunctional areas increases dramatically (200%) within a few days, depending on the species (see Fambrough, 1979). The gating properties of these AChR channels have been characterized by fluctuation analysis (Katz and Miledi, 1972; Neher and Sakmann, 1976b; Dryer et nl., 1976). Basically, the extrajunctional type of channel appear to have smaller conductance and slower kinetics (longer mean open time) than the corresponding mature junctional type (Gage and Hamill, 1980). The norrti(iI ontogenetic development of AChR channel gating properties in rat muscle cells appears to recapitulate the pathological situation found in the denervation of adult muscle. T h e analogy extends to the distributional habits of the AChR, initially diffusely located, mobile and with high turnover, and localized, relatively immobile and metabolically stable at subsequent stages of development. Diffusely located AChRs in embryonic muscle cells display initially the gating characteristics of the extrajunctional channels in adult tissue (Michler and Sakmann, 1980). Neonatal muscle cells still show the extrajunctional type of gating during the initial stages of synapse formation, but subsequently, a progressive transformation to the junctional type occurs (Sakmann and Brenner, 1978; Steinbach et nl., 1979; Michler and Sakmann, 1980). Both types of channels coexist at intermediate stages of development. Recent single-current recordings from embryonic rat muscle cells (Hamill a1 01.. 1981; Hamill and Sakmann, 1981) and from denervated adult frog muscle (Sakmann et d.,1980) have extended the description of the two types of channels occurring before innervation and after denervation, respectively. Most electrical properties of the two channel types are essentially similar (reversal potential, cation selectivity, and pharmacological specificity). T h e crucial question as to what leads to the conversion and stabilization of the junctional type or the reappearance of the extrajunctional type remains unanswered (for discussion of this subject see Fambrough, 1979). Figure 10 shows current traces obtained with the patch-clamp technique from the sarcolemma of 14-day-old rat
323
DEVELOPMENTS IN STRUCTURE AND FUNCTION
y
l.l-T T S W7rVf -
4.2 2.8
100
msec
FIG. 10. Two types of AChR channels coexist in embryonic muscle: the junctional type (J), with a main conductance level of about 50 pS and a short lifetime, and the extrajunc-
tional type (E), of longer duration and smaller conductance (-35 pS). A discrete sublevel (S) occurs in bothjunctional and extrajunctionalchannels. Notice the nonintegral nature of the sublevel S in comparison to the main levels J or E. (From Hamill and Sakmann, 1981.)
“myoballs” (i.e., myotubes in cell culture 4 days after treatment with colchicine). Two types of AChR channels coexist within a very small radius of noninnervated sarcolemmal membrane (the one from which the patch pipette records): Ajunctional J type, by analogy to the one seen in adult end-plate region, and an extrajunctional E type. T h e J type (Fig. 10) is characterized by a main conductance of about 50 pS and a short lifetime; the E type has a longer duration but a smaller conductance (35 pS) Hamill and Sakmann, 1981). 6 . Additional Alien Phenomena That Challenge the Open-Close Switch: The Sublevels
The preceding paragraphs document the recently available information questioning the tenability of some of the assumptions of noise, relaxation, and patch-clamp data in terms of a simple two-state, open-shut mechanism. The channel heterogeneity observed in embryonic muscle does not contradict accepted dogma: Different channels coexisting spatially could each show gating modalities characteristic of extrasynaptic and synaptic type of receptors. This finding is reasonable enough; but Hamill and Sakmann (198 1) showed that in addition to these two gating habits about 10% of the channels display a more complex behavior. Aside from the main current levels of J- and E-type channels, both types share a sublevel of lower conductance (Fig. 10). Some characteristics of the main and substates have been summarized by Hamill (1982): (i) the conductance of all states follows Gaussian distributions, (ii) all states display ohmic behavior, (iii) conductances for all states have a weak temperature dependence (Qlo 1.2) whereas the average lifetimes are more strongly temperature-dependent (Qlo 3 3), and (iv) all states display similar ionic selectivity for alkali cations. The probability of the open channel adopting the sublevel is almost negligible under normal conditions. It increases with membrane hyper-
-
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F. J. BARRANTES
polarization and upon decreasing the temperature. When the AChR channel has reached the sublevel, the probabilities of switching to the resting or to the main level are approximately equal, an experimental observation reminiscent of the statistical property of biliganded AChR in a two-binding, two isomerization scheme (Colquhoun and Hawkes, 1981). Hamill and Sakmann (1981) offered two alternative explanations for the multiple conductance states: subunit rearrangement or different aggregational forms of the AChR subunits. 7. Eloqiieiit Sileme: Deserwitization ,tlcmured by Loiig-Li-oed Electrically S i l m t I ti terunls The amount of membrane depolarization elicited by an agonist at the cholinergic synapse depends on the nature of such a ligand and its concentration at the synaptic cleft. Upon prolonged exposure to the agonist, however, the depolarization is not maintained, but a slower and spontaneous repolarization supervenes. This phenomenon, termed “desensitization” by Thesleff (1955), implies that in spite of the persistent presence of the agonist, the average number of channels in the open state decreases. T h e kinetics of this phenomenon is different from those of the open-close transitions of the individual receptor-controlled channels (see Gage, 1976). T h e first attempts to formalize the desensitization phenomenon within the context of AChR-agonst interactions date to the work of Katz and Thesleff (1957). They postulated the conversion of the active agonist-receptor complex into an inactive, desensitized state via a comparatively slow transition of the active complex involving dissociation of the bound agonist. Many more complex reaction mechanisms have since been invoked, the now-classical cyclic scheme of Katz and Thesleff (1957) having provided a useful frame of reference on which much of the current in uitro work has developed (Weiland et al., 1977; Barrantes, 1978; Neubig and Cohen, 1980; Cohen and Boyd, 1979; Boyd and Cohen, 1980; Heidemann and Changeux, 1979). As we have seen, a variety of AChR states leading to the activated AChR-channel complex are associated with the shut-channel conformations (e.g., R, RA, RA2in Fig. 7). These states are electrically silent and at first glance are indistinguishable from one another in a patch-clamp recording. T h e complicating addition of other states associated with closed channels would appear to make the situation worse, since their contribution could not be distinguished from that of the other electrically silent forms (i.e., having the same resting current value) in a patchclamp recording. Sakmann et nl. (1980)had only one clue for interrogating these silent domains of agonist action: the time ranges in which the desensitization
DEVELOPMENTS IN STRUCTURE AND FUNCTION
325
D
1
1 sec
ti
I ”
tb
D BURS1
CLUSTER OF BURSTS
FIG. 1 1 . Supraorganization of current events into D-bursts and clusters in the presence of desensitizing agonist concentrations. Immediately after contacting the cell surface, hyperactivity is manifested by gating of several AChR channels [at least four levels are detected (upper trace)]. After a few seconds, desensitization occurs (D). The lower trace shows bursts of current (D-bursts) hypothetically arising from resensitization of AChR in the biliganded, desensitized state, AID (see Fig. 7) to reach the activated state, A,R*, for the duration of the burst, tb. Termination of the D burst is assumed to be caused by a rapid desensitization, whose rate is measured by the distribution of interburst intervals, ti. D-bursts, in turn, are grouped into clusters, whose time distribution yields information on a second, slower desensitization process (see text and Sakmann et al., 1980).
phenomenon manifests itself in “macroscopic” recordings. They first observed that when the extrajunctional area of frog denervated muscle was exposed to high agonist concentration in the patch pipette an initial hyperactivity of open channels could be recorded (up to four current levels were resolved, as shown in Fig. 11). The activity faded within seconds, as the desensitization onset progressed. Subsequently, bursts of activity reappeared at irregular intervals (Fig. 11). Elementary currents within these bursts appear to have the same characteristics as those recorded under nondesensitizing agonist concentrations, as if the openclose transition were independent of the desensitization phenomenon (Fig. 9). This bears a relationship to hypotheses linking desensitization with channel blockage by the agonist (e.g., Adams, 1975a). Sakmann et al. (1980) specifically rejected a channel blocking mechanism, and analyzed the duration of bursts (which we have defined above as D-bursts) and of the intervals between bursts of single current traces. They also measured the duration of the supraorganization of D-bursts, the clusters, and the mean duration of the intercluster intervals (ci). The reciprocal of the mean durations of D-bursts [(tb)-l]and clusters [(ti)-’]increase as a function of agonist concentration, that is, they become shorter as the rate of desensitization augments. Assuming that the sequence of D-bursts and D-burst-intervals represents the conversion to and from a rapidly desensitizing state, rates of 2
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and 5 sec-’ were calculated, respectively, from data obtained at 20 p M ACh. At the same concentration, the ci and c, values were used to derive rate constants leading to and from a second, slowly attainable desensitized state (0.2 and (0.03 sec-I). The latter figures are strikingly similar to those found by Bonner et nl. (1976), Barrantes (1 978), Heidmann and Changeux (1978, 1979), and Quast ~f nl. (1978) in rapid kinetic studies of agonist-induced state transitions measured by fluorescence spectroscopy. It should be stressed that the interpretation of burst-cluster phenomena in terms of the desensitization process(es) is the most plausible and interesting hypothesis, but data covering more than one agonist concentration are needed to make the generalization valid. 8. The B-Butst Silent (nonconducting) states other than the resting (R) or desensitized state (D) of the AChR can be defined when attempting to interpret the action of some local anesthetic drugs on channel kinetics. T h e most straightforward hypothesis (Adams, 1975c, 1976, 1977) is that in addition to the open-close transitions, occlusion (plugging) of the open channel occurs in the presence of barbiturates, procaine, lidocaine derivatives, or quinacrine. This hypothesis is not universally accepted (Katz and Miledi, 1980) but it still offers the simplest explanation of most, if not all, the experimentally observed alterations of channel kinetics, consisting of the appearance of an extra relaxation time constant in noise or voltage jump experiments (Steinbach, 1968a,b; Kordai, 1970) together with a modification of the preexisting time constant. T h e effects of the lidocaine derivative QX-222 have been studied with the patch-clamp technique (Neher and Steinbach, 1878), and a channel-plugging mechanism also emerged as the most plausible one. In the presence of QX-222 channel activity is abnormal; B-bursts characterize the pathological blocked state. A sequential scheme of the type
QX-222
A I
1
PL
fL
ff
b
C-0-B
was postulated by Neher and Steinbach (1978) to describe the blocking reaction. T h e open channel state 0 has two possible routes to decay; a is the “normal” route, [see Eq. (4)], in which the AChR reverses to the closed conformation C. The additional route consists of the plugging of the open channnel by the blocker, leading to the B state via f. This
DEVELOPMENTS IN STRUCTURE AND FUNCTION
32’1
forward rate for blocking is proportional to blocker concentration, [B]. Applying Eq. (5) yields
t, = (a +f[BI)
(14)
where i0 is the (apparent) mean dwell time of the open-channel state. Its reciprocal is linearly proportional to [B]. Two closed intervals are also observed. One corresponds to the “true” closed dwell time (the time spent in the C state by a normal channel), and the other, which is much faster (for QX-222), is the blocked interval. Fast voltage-dependent channel blockers like QX-222 or procaine produce biphasic MEPC decays, and voltage-jump relaxation or noise analysis equally show altered, multiphasic kinetics. Substances like quinacrine produce slowly reversible block of the AChR channel (Adams and Feltz, 1977, 1980a,b; Tsai et ul., 1979), similar to that seen with barbiturates (Adams, 1976). Slowly reversible blockers give rise to bursts that do not resemble those observed with QX-222; the kinetics within the B-burst are apparently normal, and the interburst interval is l/Nb seconds long (N being the number of channels in the patch). The different blocking modalities reflect underlying differences in ufinities of the substances for the channel. Thus, the mean gap duration in a B-burst is given by
ig = (([Blf)/a}b-’
= ([B]/K,)
(Y-’
(15)
where K, is the equilibrium dissociation constant blf of the blocker for the open-channel state. Furthermore, the mean number of openings per B-burst is
(1
+ ([Blf/a)){a + iBlf1-l
(16)
Combining Eqs. (15) and (16) yields the mean B-burst duration in the case of a blocker showing resolvable gaps:
I n all above cases Eq. (13) is applied and [B] is the blocker concentration. An interesting offspring of the more extensive work on local anesthetic blockage of AChR channelsis the series of studies on the effect of permeant ions in channel kinetics. The basic issue of these studies concerns the possibility that the same ions that permeate the channel are capable of impeding channel closure upon binding to sites inside the
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channel (Van Helden el al., 1977; Ascher et al., 1978; Gage and Van Helden, 1979; Marchais and Marty, 1979; Adamset al., 1981).T h e problem is similar to that of channel blockage by local anesthetics in that the a h i t y of the ion determines the dwell time on its "binding" site. One outcome of this spreading hypothesis is that channel gating is not independent of the nature of the permeant cation (Adams et al., 1981). 9. Chanwl Behnziior as a Diagnostic Tool of Reaction hiiechanisms It has been stressed throughout that the recently observed gating phenomena call for a redefinition of some views of AChR channel kinetics. Already the qualitative observation of some of the AChR gating modes in patch-clamp records makes the need for a reappraisal evident. T h e observation of N-bursts in the presence of low agonist concentrations, for instance, indicates that at least three AChR states are involved in the gating reaction. The two-state mechanism is automatically excluded, though the number of states indicated by the N-burst is not dependent on a particular model. When association of bursts is apparent, then at least four states can be postulated. More generally, the number of open states must be as large as the number of states observed. Since the dwell time of a given state is the reciprocal of the sum of rate constants leading away from that state, if only one open state is observed in a control experiment and addition of a drug decreases the lifetime of this state, it is possible to infer that the drug in question depopulates that state (e.g., Neher and Steinbach, 1978). On the other hand, if the openstate lifetime is found to be independent of drug concentration, then channel closing cannot be associated with binding of that ligand. This was the case, for instance, with the observations of Sakmann et al. (1980) on the relative insensitivity of i, on agonist concentration, leading to the postulation that the D-bursts were not the result of channel blockage but of desensitization, a phenomenon attributed to isomm'zntions of the AChR and not to binding steps (see Fig. 7). Exploring the action of blocking substances, further diagnostic elements concerning the affinity of the drug for the AChR channel can be deduced from inspection of patch-clamp recordings. Thus 1 . Drugs having relatively high affinities result in long-lived blocked states [B in Eq. (13)Jrelative to the open state 0: Channel currents will display faster kinetics, but the channel conductance remains unaltered. 2. If the blocked state is in a time range comparable to that of the open channel lifetime, B-bursts result. Channel conductance is not affected. Affinities of the blockers are not particularly high in this case. 3. Low affinity blocking drugs yield brief blocked states, manifested
DEVELOPMENTS I N STRUCTURE AND FUNCTION
329
in a longer apparent channel lifetime in patch-clamp records with a smaller conductance than corresponding controls; the apparent conductance is a composite of the blocked and unblocked open states. 4. Permeating ions are a category apart; they constitute ultrafast blocking ligands, the dwell times of the blocked state being in the lower microsecond time domain; hence they will pass undetected in (present) patch-clamp records.
VII. Summary and Perspectives
A. THECOUPLING BETWEEN ELECTROPHYSIOLOGICAL A N D BIOCHEMICAL TECHNIQUES The description of the AChR gating function is no longer restricted to electrophysiological measurements, but has recently entered a new phase by the successful reconstitution of the channel in planar bilayers (Schindler and Quast, 1980; Nelson et al., 1980; Boheim et al., 1981). T h e reader is referred to the work of Montal et al. (198 l), Anholt (198l), and Briley and Changeux (1978) for reviews on reconstitution of the AChR. T h e merit of the above preliminary phenomenological descriptions of channel gating properties in reconstituted systems should be judged in the light of the unavailability of such information in the living electrolyte. It appears that the Torpedo AChR channel is voltageinsensitive and that it responds to cholinergic agonists in a manner similar to the neuromuscular junction. The agonist concentrations eliciting responses are extremely low, and probably different experimental conditions will be called for in order to characterize fully the dose-response curve. T h e strategy of Schindler and Quast (1980) offers the possibility of correlating channel behavior with physical properties amenable to characterization in the intermediate, monolayer state such as cohesive energy of the planar bilayer (“surface pressure”). Thus, the physical state of the final bilayer can be adjusted to that of the monolayer and ultimately to that of the parental AChR vesicle, thus matching the thermodynamic state of the host membrane. The method introduced by Schindler, from which all others have derived, also offers the unique advantage of being able to control the amount of receptor (at the monolayer stage) which will finally be incorporated in the planar bilayer. I n Nelson et al. (1980) the possibility of combining flux measurements in reconstituted vesicles and electrical properties in the bilayer is suggested. This complementation might alleviate the limitations of the integrated flux measurements, especially in relation to the time resolution problems
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F. J. BARRANTES
of the latter. T h e work of Boheim et 01. (1981) constitutes the first attempt to study the AChR gating behavior in a defined lipid environment. A pure synthetic lipid, having well-defined chemical and physical properties was employed. Furthermore, AChR preparations of increasing degree of purity were compared, monomers and dimers were found not to differ in gating properties, and the lack of influence of the nonreceptor v proteins (see above) on channel activity was shown. The study of Boheim ef d.(198 1) also provided the first comparison of gating modalities of muscle and electric organ AChR channels. Channel behavior in planar bilayers resembled in some instances the burst and cluster phenomena observed with the patch-clamp technique. These strategies define the trends that might prove more fruitful in the future for investigating the contribution of the AChR polypeptide components, nonreceptor peptides, lipid classes, and environmental factors to AChR channel properties. Given the remarkable technical advantages of patch-clamp recordings, making now possible the excision of small membrane fragments from the intact cell, the control of the sidedness of this membrane patch, and the ability to rapidly modify the environment of the membrane by superfusion of any of its t w o faces (Hamill rt a/., 1981), the major contribution of bilayer measurements should reside in their successful combination with adequate chemical dissection of AChR building blocks and other individual molecular constituents of the postsynaptic membrane. Furthermore, it could well be that in some instances planar bilayer experiments offer a more apt framework than the corresponding electrophysiological measurements for the formulation of certain problems, given the greater ability to control a complex set of multivariate parameters and detailed knowledge of geometrical constraints, diffusion barriers, exact composition of reaction partners, and state of aggregation of the AChR, etc, under defined in ziitr-o conditions. Knowledge of these parameters in the membrane area under patchclamp or even in the excised membrane patch is still difficult to obtain. In attempting to resolve some of these problems, the art of reconstitution may be on its way to achieving the restitutio ad integrum of the AChR system.
B. STRUCTURAL COUSTERPARTS OF GATING A N D STATEOF LIGATION The supramolecular arrangement of the AChR in the synapse, a subject of theoretical considerations in the past (see Changeux et al., 1967a,b; Changeux and Podleski, 1968; Levitzki, 1974), makes it unlikely that the activation of any one AChR could pass undetected or have
DEVELOPMENTS IN STRUCTURE AND FUNCTION
33 1
no consequences on the adjacent units. A major challenge of future studies (the third stage in the evolution of patch-clamp) will be to attempt the simultaneous description of the individual channel behavior, identifying microscopic states and the kinetics of their transitions, and the population behavior resulting from the recruitment of many such individual channels upon agonist action. An important link between structure and function would thus be established. The observation of discrete conductance states of the AChR other than the main open state (the “sublevels,” Fig. 10) raises hopes of being able to identify structural forms associated with a given kinetic state. Aside from the possibility of different subunit arrangements to account for the occurrence of conductance sublevels (Hamill and Sakmann, 198l), different oligomm’c states of the AChR might underlie this phenomenon. This is the type of hypothesis which may be more easily tested in the planar lipid bilayers. The conventional flux experiments do not appear adequate for this purpose. Whereas the prospective line of research considered above puts weight on the structural counterparts .f gating, another important avenue for the future concerns the correlation between the latter function and the state of ligation of the AChR. The possibility that the fast opening events ( T ~ 150 psec) detected in addition to the main longer openings ( T ~ 10 msec) correspond to the monoliganded AR* state (see Fig. 7) has been considered (Colquhoun and Sakmann, 1981). Although almost negligible in probabilistic terms, one could hope that all conducting receptor species R* will become apparent and adequately characterized under appropriate conditions (e.g., driving force leading away from equilibrium in perturbation experiments). Finally, the heterogeneity of gap lifetimes within an N-burst (Colquhoun and Sakmann, 1981) also merits further investigation, since characterization of these short-lived events might lead to identification of the otherwise silent, closed AChR states that are normally the daily bread of the biochemist.
-
-
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CHARACTERIZATION OF ( ~ 1 -AND (~2-ADRENERGIC RECE PTORS By D a v i d B. Bylund Deportment of Pharmacology School of Medicine University of Missouri Columbia, Missouri and David
C. U’Prichard
Department of Pharmacology School of Medicine Northwestern University Chicago, Illinois and Department of Neurobiology and Physiology College of Arts and Sciences Northwestern university Evonrton, Illinois
I. Introduction ......................................................... A. Receptors for Epinephrine and Norepinephrine ...................... B. Pharmacological Subdivision of a-Adrenergic Receptors ............... C. Radioligand Binding Studies ....................................... 11. a,-Adrenergic Receptors ................... ......... A. Characterization by Radioligand Binding ............................ B. Effector Systems Coupled to a,-Adrenergic Receptors ................ C. Regulation of a,-Adrenergic Receptors .............................. D. Solubilization of a,-Adrenergic Receptors ............................ 111. a,-Adrenergic Receptors .............................................. ................ A. Characterization by Radioligand Binding B. Effector Systems Coupled to a,-Adrenergic Receptors ................ C. Comparison of Agonist and Antagonist Binding: Toward a Kinetic Model of a,-Receptor Function ........................................... D. Regulation of a,-Adrenergic Receptors .............................. E. Localization of a,-Adrenergic Receptors ............................. F. Solubilization of a,-Adrenergic Receptors ............................ IV. Summary and Conclusions ............................................. References ...........................................................
344 344 345 349 354 3 54 364 368 376 376 377 398 404 410 417 419 420 422
343 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 24
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366824-7
344
DAVID
B. BYLUND
AND DAVID
c.
U’PRICHARD
1. Introduction
A. RECEPTORS FOR EPINEPHRINE A N D NOREPINEPHRINE The catecholamines are important regulatory compounds in the body and produce a variety of physiological effects. T h e actions of norepinephrine, a peripheral sympathetic and central neurotransmitter, and epinephrine, an adrenal hormone and putative central neurotransmitter, are mediated through adrenergic receptors. A large number of drugs also produce their effects by interacting with these receptors. T h e concept of adrenergic receptors originated with Langley (1905). Extrapolating from his work on the action of nicotine and curare on muscle, he suggested that epinephrine also exerts its effects by interacting with “receptive substances.” Dale (1906) made use of this concept to explain the differential effects of the ergot alkaloids on smooth muscle. He raised the possibility that the receptors at myoneural junctions could be of t w o types: one mediating the excitatory (motor) actions of norepinephrine and the other mediating the inhibitory actions of epinephrine. This functional receptor subclassification, based on the idea that adrenergic receptors could be considered to be of two classesthose whose actions result in excitation of the effector cells and those whose actions result in inhibition of the effector cells-competed with an alternative suggestion that two opposing transmitter substances (Sympathin E and Sympathin I) competed antagonistically at the same receptive site (Cannon and Rosenblueth, 1937). Then Ahlquist (1948) made a major advance by suggesting that the subtypes of adrenergic receptors could be differentiated on the basis of their pharmacology and not on the basis of their function. He studied the effects of five catecholamines (norepinephrine, methylnorepinephrine, epinephrine, methylepinephrine, and isoproterenol) on eight different physiological functions (nasal vasoconstriction, contraction of the uterus and the nictitating membrane, dilation of the pupil, relaxation of the gut, nasal vasodilation, relaxation of the uterus, and myocardial stimulation) and clearly showed that the order of potency for the catecholamines for the first five functions was markedly different from the order of potency for the remaining three functions. He attributed this difference to an actual difference in the receptors involved and suggested that they be called a-and P-adrenergic receptors, respectively. Inherent in these data is the concept that a single tissue, such as vascular smooth muscle, could contain both subtypes of adrenergic receptors which would mediate different functions. At the time of Ahlquist’s experiments the known adrenergic antagonists, such as phenoxybenzamine, phentolamine, and various ergot
CHARACTERIZATION OF a1- AND ff2-ADRENERGIC RECEPTORS
345
derivatives, appeared to block only a-adrenergic receptor responses, whereas specific p-receptor antagonists such as dichloroisoproterenol and propranolol were not developed for another 10 years. More recently Lands et al. (1967) proposed the subdivision of P-adrenergic receptors into P1 and P2 subtypes. This was also a pharmacological differentiation based on a comparison of the relative potencies of 12 agonists in several isolated organ systems. This subclassification of /3 receptors has been substantiated both by the development of subtype selective antagonists and by direct binding studies (Minneman et al., 1981).
B. PHARMACOLOGICAL SUBDIVISION OF a-ADRENERGIC RECEPTORS In contrast to the simple subclassification of P-adrenergic receptors by pharmacological criteria, attempts to correctly subdivide the a-adrenergic receptors have followed a more circuitous route. The initial subclassification was based on the presumed anatomic or topographic localization of the receptor. The idea that the terminals of noradrenergic axons contain functionally important a-adrenergic receptors stemmed from early work of Brown and Gillespie (1957) who reported that phenoxybenzamine, an a-adrenergic antagonist, increased the overflow of norepinephrine elicited by nerve stimulation in the perfused cat spleen. Although this data was originally misinterpreted, as early as 1959 Furchgott suggested the possibility that these results were due to an increase in neurotransmitter release from the nerve ending. Langer et al. ( 197 1) and Starke (197 1) suggested that presynaptic a-adrenergic receptors were responsible for the increase in norepinephrine overflow seen with a-adrenergic blockers and for the decrease seen with a-adrenergic agonists. The existence of these presynaptic or autoreceptors for norepinephrine which modulate its release is now widely accepted (Starke, 1977; Langer, 1977), although a presynaptic location for the receptors which mediate this function has not yet been conclusively proved. The observation that phenoxybenzamine was 30 times more potent in antagonizing autoreceptor function as compared to the classical postsynaptic receptor led Langer (1974) to suggest that autoreceptors and postsynaptic receptors are not identical and that they should be referred to as a2and a l ,respectively. This anatomic subdivision of a-adrenergic receptors (a,-receptors on presynaptic terminals) has not proved particularly useful because of considerable evidence suggesting that receptors having similar pharmacological properties to the autoreceptors are not located presynaptically. Inhibition of adenylate cyclase activity and subsequent physiological responses have been shown to be mediated
346
DAVID B. BYLUND AND DAVID
c. U’PRICHARD
through a-adrenergic receptors, which would be classified pharmacologically as azreceptors in both human platelets (Grant and Scrutton, 1979; Lasch and Jakobs, 1979; Hsu et al., 1979) and human adipocytes (Burns et al., 1981; Lafontan and Berlan, 1980; Kather and Simon, 1981). The human adipocyte also contains a receptors which would be pharmacologically classified as a1 receptors and can be differentiated from the az receptors by both functional and radioligand binding studies (Burns P t nl., 1981). Similarly, vascular smooth muscle, in addition to containing the classical postsynaptic a l receptor, also appears to contain postsynaptic receptors which are pharmacologically similar to az receptors (Drew and Whiting, 1979; Hamilton and Reid, 1980; Ruffolo et al., 1980; Timmermans et al., 1979). I n the rat submandibular gland azadrenergic receptors appear following reserpine treatment or denervation (Bylund and Martinez, 1980), and these receptors are localized postsynaptically (Bylund and Martinez, 1981). Nonpresynaptic azadrenergic receptors also have been characterized in pancreatic islets (Nakaki P t a!., 1981) and undifferentiated neuroblastoma X glioma hybrid cells (Sabol and Nirenberg, 1979a; Kahn et al., 1982). Even before most of the above data were available, Berthelsen and Pettinger ( 1977) realized the limitations of the anatomic subdivision of a-adrenergic receptors and suggested instead that they be classified on the basis of their function. According to this classification scheme, aladrenergic receptors are excitatory and az-adrenergic receptors are inhibitory. While this classification scheme certainly has merit and does tend to subdivide a receptors into groups that have similar pharmacological characteristics, it also has a number of drawbacks. As these authors pointed out in their paper, “the designation of a receptor as inhibitory or excitatory is an arbitrary one which is limited by our ignorance of the more complex molecular mechanism of each receptor’s actions” (Berthelsen and Pettinger, 1977). For example, several a2 responses could be considered excitatory, rather than inhibitory. The inhibition by a-adrenergic agonists of the release of catecholamines from adrenergic terminals may be due to activation of the sodium pump, which results in enhancement of calcium efflux (Cohen et al., 1980) and/or activation of guanylate cyclase (Pelayo et al., 1978; O’Dea and Zatz, 1976). Thus, the functional or physiological subclassification of subtypes of a-adrenergic receptors has been replaced by a more useful pharmacologic definition, just as the original subdivision of adrenergic receptors into excitatory and inhibitory types was replaced by the pharmacologic subdivision into a and /3 receptors. The third attempt to subclassify a-adrenergic receptors was based on what might be thought of as a biochemical approach. Wikberg (1979)
CHARACTERIZATION OF a1- AND a2-ADRENERGIC RECEPTORS
347
and Fain and Garcia-Sainz (1980) suggested that the at receptors mediate effects secondary to an elevation in intracellular calcium and involve an increased turnover of phosphatidylinositol, while a2-adrenergic effects are mediated by the inhibition of adenylate cyclase. While a subclassification based on biochemical mechanisms may eventually prove to be useful, at the present time the attempt to do this is limited by our relative ignorance of the actual mechanisms for a-adrenergic receptors in most tissues. Table I summarizes some of the biochemical responses that have been observed following a-adrenergic receptor stimulation. It would appear that there are a variety of biochemical mechanisms inTABLE I BIOCHEMICAL RESPONSES MEDIATEDBY Q-ADRENERGIC RECEPTORS Biochemical responses
Tissue
References
Q1
Phosphatidylinosital turnover (increase in cytosol Ca*+?) Phosphatidylinosital turnover Phosphorylase activation Potassium release Potassium release
Adipocyte
Burns et al. (198 1)
Aorta Liver Submandibular gland Parotid gland
Vilalobos-Molina et al. (1982) El-Refai et al. ( 1979) Bylund et al. (1982a) It0 et al. (1982)
Kidney cells (MDCK)
Levine and Moskowitz (1979)
Pineal gland Submandibular gland
Pelayo et al. (1978) ODea and Zatz (1976) Cohen et al. (1980)
Platelet
Grant and Scrutton (1979)
Adipocyte
Burns et al. (1981)
Pancreatic islets
Nakaki et al. (1981) Sabol and Nirenberg (1979a)
Adenylate cyclase inhibition Adenylate cyclase inhibition
Neuroblastorna X glial hybrid Liver Thyroid gland
Adenylate cyclase inhibition Intestinal secretion inhibitionb
Renal cortex Jejunum
4,) Promotes phospholipid deacylation Presynaptid" Guanylate cyclase activation (inhibits norepinephrine release) Na,K-ATPase activation (promotes calcium efflux, inhibits norepinephrine release) Q2
Adenylate cyclase inhibition (promotes aggregation) Adenylate cyclase inhibition (inhibits lypolysis) Adenylate cyclase inhibition (inhibits insulin release) Adenylate cyclase inhibition
Jard et al. (1981) Desmedt (1980) Yamashita et al. (1980) Woodcock et al. (1980) Nakaki et al. (1982)
Some evidence indicating the receptor is of the a2subtype. Some evidence indicating that this response is not mediated by cyclic AMP.
348
DAVID B. BYLUND AND DAVID
c . U’PRICHARD
volved. This conclusion is consistent with recent evidence that P-adrenergic receptors may have biochemical functions or mechanisms that are independent of the activation of adenylate cyclase (Maguire and Erdos, 1980; Hirata et al., 1979). For example, the mechanism of a-adrenergic inhibition of neurotransmitter release is not known, but one of the suggested hypotheses involves calcium. T h e biochemical subclassification would tend to indicate an a1 subtype rather than the pharmacologic a2 subtype (Cohen et al., 1980). T h e ultimate subclassification of adrenergic receptors will probably be based on the primary structure of the macromolecules involved. However, until that information is available it would appear prudent to use a pharmacologic subclassification scheme to subdivide a-adrenergic receptors. This is consistent with the primary division of adrenergic receptors into a and p types, as well as the p, versus P2 subclassification, both of which are pharmacologic schemes. The validity of a pharmacologic subclassification scheme can be determined by comparing the potencies of a variety of a-adrenergic agonists and antagonists in a variety of systems. A summary of some of these data is given in Tables I1 and 111. These tables compare the potencies or affinities of several a-adrenergic drugs in mediating or inhibiting physiologic responses, or in inhibiting the binding of selective a-adrenergic radioligands. The data have been normalized by dividing the potency of each drug by the potency of norepinephrine for agonists and that of phentolamine for antagonists. A ratio less than 1.0 means the drug is more potent (lower K , ) than the reference drug, whereas a ratio greater than 1.O means that it is less potent (higher K , ) . It is important to note that essentially the same conclusions are drawn from both the physiologic studies and radioligand binding studies. In both cases epinephrine tends to be slightly more potent than norepinephrine and is nonselective for the two subtypes. Phenylephrine is at selective, while clonidine is a2 selective as can be seen from the ratio of clonidine to phenylephrine, which tends to be about 1 for a1systems, but much less than 1 for a2systems. For the antagonists, prazosin is a , selective, while yohimbine is somewhat a2selective. T h e ratio of yohimbine to prazosin clearly differentiates the two subtypes and is a much better indicator than the clonidine : phenylephrine ratio. Rauwolscine may be even more a2 selective than yohimbine (Tanaka and Starke, 1980). I n addition to these physiologic and binding studies, there are several tissues such as the adipocytes in which two different a-adrenergic biochemical responses can be measured. These studies also support the pharmacological selectivity of clonidine and yohimbine as a,-selective agents and prazosin as an a,-selective agent (Burns et al., 1981).
CHARACTERIZATION OF (Y1- AND ff2-ADRENERGIC RECEPTORS
349
C. RADIOLIGAND BINDINGSTUDIES The successful labeling of a-adrenergic receptors using the radioligand binding technique, in which a drug of high specific radioactivity binds in a reversible manner to the receptor recognition site, was developed subsequent to labeling of /3-adrenergic receptors. In 1976 Lefkowitz took advantage of the fact that hydrogenation of naturally occurring ergot alkaloids results in compounds which are highly potent and selective a-adrenergic antagonists. The product of the catalytic reduction of a-ergocryptine with tritium gas, PHIdihydroergocryptine (DHEC), specifically labeled a-adrenergic receptors in the rabbit uterus (Williams and Lefkowitz, 1976). Independently, Snyder and collaborators used [3H]clonidine and rH]WB4 101 [2-[(2’,6’-dimethoxy) phenoxyethanolamino]methylbenzodioxan]to label a-adrenergic receptors in the brain (Greenberget al., 1976). These investigators felt that the agonist [3 Hlclonidine would preferentially label presynaptic receptors on noradrenergic nerve terminals in the brain. However, it was found that [3H]clonidine binding in brains from animals treated with the neurotoxin, 6-hydroxydopamine, was slightly increased rather than decreased as compared to control brains (U’Prichardet al., 1977a).The binding of the antagonist [3H]WB4101 was also increased slightly. These data were interpreted as an indication of denervation supersensitivity and that both ligands were labeling postsynaptic receptors. Since there were differences in the absolute affinities of agonists and antagonists at the binding sites labeled by the two ligands, it was felt that brain a-adrenergic receptors existed in two distinct but interchangeable conformational states (Greenberget al., 1976; U’Prichard et al., 1977a; Greenberg and Snyder, 1978). However, subsequent investigations in several laboratories have led to the conclusion that [3H]WB4101 selectively labels central aladrenergic receptors and rH]clonidine selectively labels a2 receptors (U’Prichard et al., 1979b; U’Prichard and Snyder, 1979; Tanaka and Starke, 1980). After an initial negative report (Davis et al., 1978) rH]DHEC was also shown to label a-adrenergic receptors in the brain. It became apparent that PHIDHEC labels both al-and az-adrenergic receptors with equal affinity in the brain (Peroutka et al., 1978; U’Prichard et al., 1978b, Miach et al., 1978). This general relationship of differential a-ligand binding to a1and az receptors has also been shown to apply to many peripheral tissues as well. A number of other radioligands have been used to label a-adrenergic receptors. These include [3H]epinephrine and [3H]norepinephrine in the brain (U’Prichard and Snyder, 1977a) and liver (El-Refai et al., 1979). [3H]Dihydroazapetine has been used in the rat vas deferens (Ruffolo et al., 1976). More recently, [3H]-
Agonist affinity ratio'
'Tissue
Epinephrine
Phenylephrine
&-Methyl norepinephrine
Clonidine
Oxymetazoline
Clonidine : phenylephrine
Reference
a, Receptors
w in 0
Physiologic studies Guinea pig aorta Rabbit pulmonary artery Rabbit aorta Guinea pig ileum Rabbit jejunum Rat kidney Radioligand binding studies Rat brain ([SH]WB4101)
0.83 0.22 1.4 0.14 0.5 0.72
8.3 4.3 5.0 0.25 6.2 1.5
4.0 12.4
0.59
2.6
6.8
0.43
0.024
0.17
Rat brain ([3H]prazosin)
0.37
2.2
2.7
0.30
0.036
0.14
Rat lung (PHlWB4101)
1.8
68
4.4
0.45
0.28
Rat lung (rHJprazosin) Rat submandibular gland Hlprazosin)
0.48
5.2
27
0.34
0.38
0.07
U'Prichard rt al. (1977a) Perry and U'Prichard (1982) Latifpour and Bylund (1981) Latifpour (1981)
0.52
2.3
1.22
0.021
0.53
Bylund rt al. (1982a)
(r
16
4.3 5.6
-
5.1
62 6.7 25 5 40 9.4
5.0 1.5 0.2 0.03 0.01 -
7.5 1.6 5.0 20 6.5 6.3
Wikberg (1979) Starke rt nf. (1975) Wikberg (1979) Wikberg (1979) Boudier ~t a/. (1975) Schmitz rt al. (1981)
Rat submandibular gland (PHlWB4 101) Rat kidney (rH]prazosin) Rat liver (PHIprazosin) Rat liver ([3H]norepinephrine) at Receptors Physiologic studies Guinea pig ileum Rabbit pulmonary artery Radioligand binding studies Rat brain ([3H]clonidine) Bovine brain (PH]rauwolscine)
W u1 c1
6.4 1.o 2.0
0.47 0.88 0.62
-
1.10 0.72 0.1
0.085 -
0.17 0.72 0.05
Bylund et al. (l982a) Schmitz et al. (1981) Geynet et al. (1981)
-
0.11
Geynet et al. (1981)
1.2
28
-
3.2
0.29 0.16
112 140
0.31 0.67
0.1 1 0.83
0.59 0.26
0.0010 0.0059
Wikberg (1979) Starke et al. (1975)
0.35
16
0.94
0.34
0.11
0.021
U’Prichard et al. (1977a)
0.32
0.57
0.81
0.013
0.003
0.023
Perry and UPrichard (1982)
Human platelet (PHIyohimbine) Human platelet (PHIp-aminoclonidine)
0.2 1
2.6
1.7
0. I4
0.017
0.054
Daiguji et al. 71981a)
0.37
6.0
-
0.47
0.41
0.068
UPrichard et al. (1982a)
Rat submandibular gland ( [3H]clonidine)
1.1
13.8
0.50
0.17
0.050
Bylund and Martinez ( 1980)
Rat lung [(neonatal) [3H]yohimbine]
1.2
0.11
1.o
0.0069
Latifpour et al. (1982)
10
16 ~~
a
5.6 20
Affinity ( K , ) of drug/a&ity
(K,) of norepinephrine.
0.82
Antagonist affinity ratio" Tissue
Prazosin
Yohimbine
Yohimbine : prazosin
Reference
Q1
Physiologic studies Rat anococcugeus muscle Rabbit aorta Rat kidney Radioligand binding studies Rat brain (rHlWB4101) Rat lung (FHIprazosin) Rat lung (['H]WB4101) Human lung (PHIprazosin) Rat submandibular gland ([3H]prazosin) Rat submandibular gland ([3H]WB4101) Rat kidney (rH1prazosin)
0.32 0.83
20 45
62 54
0.20
87
440
42 80 462 152 480 1130 36
470 1700 1500 12,000 2400 3600 1600
0.089 0.046 0.31 0.012 0.20 0.31 0.023
Doxey et al. (1977) Sheys and Green (1972); Cavero rt a/. (1978) Schmitz et a/. (198 1) U'Prichard et al. ( 1 9 7 8 ~ ) Latifpour and Bylund (1981) Latifpour and Bylund (1981) Barnes et a/. (1980) Bylund et al. (1982a) Bylund et a/. (1982a) Schmitz et al. (1981)
ff2
01
01 01
Physiologic studies Rat vas deferens Radioligand binding studies Human platelet (PHIclonidine) Human platelet (PHIyohimbine) Human adipocyte (PHIyohimbine) Bovine brain (PHIepinephrine) Bovine brain (PH]rauwolscine) Rat submandibular gland ([SHIclonidine) Rat kidney ( [3H]yohimbine) Rat lung (neonatal ([3H]yohimbine) Neuroblastoma (PHIyohimbine) Rat brain (cortex) (rHlyohimbine) Rat brain (striatum) (PHIyohimbine) Rat brain (cortex) (rH1clonidine) Rat brain (striatum) ([3H]clonidine) a
Affinity (K,) drug/affinity (K,) phentolamine.
>60 142 100 350 4000 125 1440 5.0 1.54 1.08 3.8 0.91 352 142
1.6 0.23 0.22 0.83 11 0.63 17 0.67 0.28 0.20 0.50 0.12 15 7.1
(0.03 0.0016 0.0022 0.0024 0.0027 0.0050 0.012 0.13 0.18 0.19 0.13 0.13 0.043 0.050
Doxey et al. (1977) Shattil et al. (198 1) Daiguji et al. (1981a) T h a r p et al. (1981) Perry and U'Prichard (1982) Perry and U'Prichard (1982) Bylund and Martinez (1980) Schmitz et al. (1981) Latifpour et al. (1982) Kahn et al. (1982) D. B. Bylund (unpublished) D. B. Bylund (unpublished) D. B. Bylund (unpublished) D. B. Bylund (unpublished)
354
DAVID B. BYLUND AND DAVID
c.
U'PRICHARD
prazosin, [3H]p-aminoclonidine (PAC), [3H]phentolamine, VHIyohimbine, [3H]rauwolscine, and [1251]BE-2254(HEAT) also have been used to label a-adrenergic receptors. It is the purpose of this article to review recent contributions to the characterization of a,- and a,-adrenergic receptors. Several previous reviews are available which deal in part with various areas of this subject (Lefkowitz, 1978; Kunos, 1978; Hoffman and Lefkowitz, 1980a,b; U'Prichard, 1981; Wood et al., 1979a). We have focused on those studies which deal with a 1 -and a,-adrenergic receptor subtypes and have emphasized the regulation and the relationship of binding and function.
II. a,-Adrenergic Receptors
A.
C H A K A C I'EKI/ATION
I31 R A D I O L I G 4 N D
BINDING
1. A n t a g o m t RadiolzgandT a. [3H]Ij'BBJIOl and r H ] p r a z o ~ n .The potent a,-adrenergic antagonists, WB4101 and prazosin, have been used as radioligands to label receptor binding sites in both the brain (Table IV) and in a variety of peripheral tissues (Table V). T h e K , values obtained in various laboratories for both CJHlWB4101 and VHIprazosin binding in the brain are remarkably consistent and, with a few exceptions, range between 0.14 and 0.31 n'tf. T h e K , values reported for peripheral tissues are also relatively consistent, although the variation is somewhat larger. T h e majority of the values lie between 0.1 and 0.9 niM. T h e reportedB,,, values for the various tissues are generally in the range of about 20-400 pmol/ gm prot and are similar to other catecholamine receptors such as the P-adrenergic and dopamine receptors (Maguire et al., 1977; Seeman, 1980). In contrast to the relatively good agreement among tissues and laboratoriey for K,, and B,,, values for a,-receptor binding, the K, for inhibitors that have been reported in the literature are much more variable. Table VI presents data for the inhibition of rHlWB4101 binding by various a-adrenergic drugs in four tissues from the rat. Although the K , values vary by as much as 10-fold or more, in each of the four tissues the pharmacological profile of the labeled receptor is characteristic of an a,-receptor subtype. It is generally accepted that rHlWB4lOl selectively labels a, receptors in CNS tissue, but its selectivity in peripheral tissues has been questioned. For example, in the rat uterus, [3H]WB4101 appears to bind to both a1and a , receptors with similar a h i t i e s (Hoffman
CHARACTERIZATION OF 0 1 - AND (Y2-ADRENERGIC RECEPTORS
355
and Lefkowitz, 1980d). In the human platelet, which lacks detectable a , receptors, [3H]WB4101 appears to bind potently to a2 receptors, although the K , value is still about 10-fold greater than in tissues where it labelsa, receptors (Daigujiet al., 1981a). In the platelet the& values for prazosin and yohimbine in inhibiting rHIWB4 101 binding are about 3000 and 5 nM, respectively, confirming the a2 characteristics of the binding (D. B. Bylund, unpublished). In the bovine retina, rHlWB4101 also appears to label a2 receptors (Bittiger et al., 1980). Due partially to this lack of specificity of [3H]WB4101in at least some tissues, rH]prazosin is now generally used for the study of a,-adrenergic receptors. The Ki values summarized in Table VII clearly indicate that t Hlprazosin labels an a,-receptor binding site. However, as was the case with [3H]WB4101, there is a considerable variation in the Ki values among tissues and species. Although it is generally assumed that rHlWB4101 and THIprazosin label the same population of al-adrenergic receptors, this has not yet been demonstrated due to the lack of studies which have compared the binding of both ligands under identical conditions. In the rat submandibular gland, lung, and cortex where this comparison was made, we have found that the Ki values for drugs in inhibiting the binding of the two radioligands were similar but not identical (Tables I1 and 111). In the submandibular gland, the B,,, value for rH]prazosin was 61% higher than that for THlWB4101 (Table V). Furthermore, in the rat lung the B,,,, for [3H]prazosinwas more than double that found for rHlWB4101 (Table V). The importance of these discrepancies is not yet clear. In addition to the human platelet and the bovine retina, several other tissues apparently lack a,-adrenergic receptors. These tissues include neuroblastoma glioma X hybrid cells (Haga and Haga, 1981; Kahn et al., 1982), possibly human subcutaneous adipose tissue (Burns et al., 1981; Tharp et al., 1981), and rat and pig cerebral microvessels (Harik et al., 1980). On the other hand, a,-adrenergic receptor binding was detected in the bovine cerebral microvessels (Peroutka et al., 1980). The monovalent cations, such as lithium and sodium, and the guanine nucleotides appear to have little effect on the binding of p Hlantagonists to a,-adrenergic receptors in most tissues, although these agents markedly affect the binding of radioligands to a2receptors. In the calf and rat cerebral cortex, the specific binding of [3H]WB4101 and rH]prazosin, respectively, was not significantly altered by guanine nucleotides at concentrations up to 1 mM (U’Prichard and Snyder, 1978a; Perry and U’Prichard, 1983). Sodium ,lithium, or potassium ions at concentrations up to 150 mM did not alter rHlWB4101 binding in the calf cortex (Greenberg et al., 1978). In addition, no effect of guanine nuc-
TABLE IV RAI>IOLIGANL) BINUING TO CENTRAL (Y~-AI)HENEKGI(: RECEPTOKS" Bm,,
W
01 Q,
Brain region
Species
Radioligand
pmol gm prot
100 82 t 17 77 rfr 3
Whole brain Whole brainb*= Whole brainb Whole brainb
Rat Rat Rat Rat
WB4101 DHEC Prazosin Prazosin
Whole brainb Whole braind Cerebral cortex Cerebral cortex Cerebral cortex Hypothalamus Hypothalamuse Thalamus
Rat Rat Rat Rat Rat Rat Rat Rat
Prazosin WB4101 WB4101 BE2254g BE2254' WB4101 DHEC WB4101
pmol gm tissue
11
80 rfr 75 rfr 84 rfr 210 180 rfr
*
2 9 9 26
20 3.2 4.1
85 t 4
h.,,
(nM) 0.48 4.7 rfr 1.3 0.26 t 0.08 0.28 rfr 0.04 0.26 rfr 0.08 0.16 rfr 0.02 0.14 rfr 0.01 0.08 rfr 0.014 0.10 rfr 0.03 0.28 1.07 0.32 rfr 0.06
Reference U'Prichard PI al. (1977a) Miach el al. (1978) Miach rt al. (1980) Greengrass and Bremner (1979) Morns et al. (1980) U'Prichard P I al. ( 1979a) U'Prichard rt al. (1979a) Engel and Hoyer (1981) Glossmann rt al. (1981) Neethling et al. (1981) Neethling ~t al. (1981) U'Pnchard et al. (1980b)
Midbrain Hippocampal gyrus' Dentate gyrus' Cerebral cortex Cerebral cortex Hippocampus Corpus striatum Cerebral cortex Frontal cortex Caudate nucleus Pons
Rat Rat Rat Mouse Mouse Mouse Mouse Calf Calf Calf Calf
WB4101 WB4101 WB4101 WB4101 WB4101 WB4101 WB4101 WB4101 WB4101 WB4101 WB4101
58 169? 14 269 ? 7 66 230 48 3 10
0.17
Gheyouche et al. (1980) Crutcher and Davis (1980) Crutcher and Davis (1980) Rehavi et al. (1980a,b) Rehavi et al. (1980a,b) Rehavi et al. (1980a,b) Rehavi et al. (1980a,b) Lyon and Randall (1980) U'Prichard et al. (1977b) U'Prichard et al. (1977b) U'Prichard et al. (1977b)
3
6.5 5.2 4.9 2.9
k
3 0.25 2.9 0.27 4.3 0.26 f 0.03 0.3 1 0.3 1 0.29
1.0
~
~
~
~
~
Unless otherwise noted the assay temperature was 23-25"C, the tissue preparation was a crude particulate fraction, and tritium was the isotope. Values given are means f SEM. Less cerebellum. In the presence of 0.1 p M yohimbine. Less cortex. Nonspecific binding defined by 0.1 p M prazosin. 'Assays temperature 30°C. le51 used as isotope. a
nm,,
Ds
w?
m
pmol gm prot
~~
Tissue
Species
Tritiated ligand
Adipocyte Heart, ventricle Heart, left ventricle Heart Aorta Aorta Aorta Irisb Livee Livef Livef Lung Lung
Human Rat Rat Guinea pig Rabbit
WB4101 WB4101 WB4101 Prazosin WB4101 WB4101 Prazosin WB4101 Prazosin Norepinephrine Prazosin Prazosin Prazosin
cow cow
Rabbit Rat Rat Rat Human Guinea pig
303 f 46 52 2 4 28 ? 1 65 ? 26 162 ? 6 134 ? 5 730 436 If: 75 340 ? 70 760 ? 40 600 47 ? 7
pmol gm tissue
2.57
?
0.14
2.0
?
0.6
ti,, (11
nr)
0.86 ? 0.07 1.2 f 0.1 0.18 0.53 f 0.17 0.84 ? 0.07 1.67 t 0.33 0.66 ? 0.16 2.3 0.05 138 ? 60 0.15 ? 0.02 1.75 0.20 ? 0.05
Reference Burns rt (11. (1981) Torda rt d.( 1981) Yamada rt 01. (1980b) Karliner ef nl. (1979) Fuder r / nl. (1981) Rosendorff ct a/. (1981) Rosendorff rt nl. (1981) Taft rt ul. (1980) Hoffman rt al. (1981a) Geynet rt nl. (1981) Ceynet P / al. (1981) Barnes et al. (1980) Barnes rt a/. (1979)
Lung Lung Lung Lung Lung Sublingual gland Submandibular gland Submandibular gland Parotid gland Uteruse Uterus Kidney cw
g
Rat Rat Rat Rat Dog Rat Rat
WB4101 WB4101 DHEC Prazosin Prazosin Prazosin Prazosin
57 f 2 51 f 3 60 f 7 126 f 2 22 57 ? 11
Rat
WB4101
Rat Rabbit Rabbit Rat
Prazosin DHEC Prazosin Prazosin
3.2 t 0.6 8.7 h 0.06
0.33 f 0.03 1.2 f 0.1 1.7 f 0.3 0.11 +. 0.01 0.48 0.43 ? 0.16 0.43 h 0.06
Latifpour and Bylund (1981) Torda et al. (198 1) Latifpour and Bylund (1981) Latifpour (1981) Hasegawa and Townley (1981) Martinez et al. (198213) Bylund et al. (1982a)
96 f 10
5.3
0.37 f 0.04
Bylund et al. (1982a)
13 f 1 25 29 ? 7 57 f 6
0.43
0.78 rt 0.32 4.7 0.5 ? 0.15 0.85 ? 0.05
Ito el al. (1982) Hoffman and Lefkowitz (1980~) Lavin et al. ( I98 1) Schmitz et al. (1981)
rt
0.7
Unless otherwise noted the assay temperature was 23-25°C and the tissue preparation was a crude particular fraction. Values given are mean f SEM. * Microsomal fraction. Plasma membranes. Chronic obstructive airway disease. 17% of total DHEC binding.
360
DAVID B. BYLUND A N D D A V I D
c. U’PRICHARD
TABLE VI I S H I ~ I T I OOF N [3H]WB4101 BINDING I N RAT TISSUES
Drug Agonists (-)-Epinephrine (-)-Norepinephrine Oxymetazoline ( - f-Phenylephrine (+)-Norepinephrine Clonidine Antagonists Phentolamine Prazosin WB4101 Yohi mbi ne
Brain“
Heartb
590 1000
90 723
200
1170
1800
369
8500 480
24 2600 67,000 430 3.6 0.49 0.6 480
Lung
110
50
5.3 0.18 0.20 48 1
1.6 0.5 740
Submandihular glandd
64 136 10 764 13,500 150
0.71 0.22 0.23 800
U’Prichard ut ai. (1977a). (1980h). Latifpour and Bylund (1981). Bylund et rrl. (1982a).
* Yamada P I 01.
leotides was found on the binding affinities of agonists for rat liver and brain a l receptors labeled with THIDHEC, rHlWB4101, or [3H]prazosin (Hoffman Pt nl., 1980a; U’Prichard and Snyder, 1978a; Perry and U’Prichard, 1983). However, it has been reported that guanine nucleotides decrease the affinity of epinephrine at rat cardiac a 1 receptors labeled by [3H]WB4101 (Yamada ff nl., 1980~) and decrease the binding of [3H]norepinephrine to rat hepatic a1receptors (Geynet el al., 1981). The binding of [3H]prazosin to rat brain membranes was not altered by the presence of 150 nM NaCl, although sodium did alter the affinity of agonists in inhibiting [3H]prazosin binding (Glossmann and Hornung, 1980a). It appears that sodium may decrease the affinity of a1 agonists but may increase the affinity of agagonists at the receptor site labeled by [3H]prazosin (Glossmann and Hornung, 1980a). [3H]WB4101 appears to be useful in characterizing receptors using the autoradiographic technique. In the rat brain, HlWB4 101 binding to slide-mounted tissue sections had all the characteristics associated with a, receptors, and thus was used for light microscopic autoradiographic localization of these receptors (Young and Kuhar, 1980). [3 HIPhentolamine was used in autoradiographic study of the urinary bladder of the rat, although specific binding to a-adrenergic receptors was not demonstrated (Jonaset al., 1980).
TABLE V I I INHIBITION OF rH]PRAZOSIN BINDING
Drug Agonists (-)-Epinephrine (-)-Norepinephrine Oxymetazoline ( -)-Phenylephrine (+)-Norepinephrine Clonidine Antagonists Phentolamine Prazosin WB4101 Yohimbine a
Miach et al. (1980).
Rat brain"
1440 5615
193,000 2315 304 0.3 3.8 8900
* Greengrass and Bremner (1979). Bylund et al. (1982a). Latifpour (1981). Barnes et al. (1980). Barnes ~t al. (1979).
Rat brainb
600 900 23 1400 43,000 340
0.1 1.o 1000
Rat submandibular gland'
40 77 1.6 174 2200 94 0.98 0.32 0.17 450
Rat lungd
262 400 327 2085
Human lunge
460 590
690 1400
46,000
110,000 110,000 2,200
365 9 0.38 750
Guinea pig lung'
27 0.33 4100
6.6 0.13 0.80 3100
362
DAVID B . BYLUND A N D DAVID c . U'PRICHARD
a- 1-Adrenergic receptor binding studies have also proved useful in understanding the side effects of some psychoactive drugs. T h e affinities of the tricyclic antidepressant drugs for a ,-adrenergic receptor sites labeled by [3H]WB4101 in the brain correlate well with the capacity of these drugs to relieve psychomotor agitation and to induce sedation and hypotension (U'Prichard et a(., 1978a). Similarly, the relative affinity of neuroleptic drugs for rHIWB4101 binding sites provides an index of the relative propensities of these drugs for eliciting autonomic side effects such as orthostatic hypotension and sedation (Peroutka rt al., 1977). In a study of the metabolites of neuroleptic drugs, there was also an excellent correlation between the potencies of the metabolites at the a 1 receptor as determined by binding studies and their potency as established by clinical and animal studies (Bylund, 1981). h. ['251]BE-225-f].In binding studies where the yield of receptorcontaining tissue is necessarily limited, for example, using cell and tissue cultures, i t is especially useful to have a radioiodinated ligand. [Iz5I]BE2254, 2-[~-(4-hydroxphenyl)ethylaminomethyl]tetralone, also called [1251]HEAT,an aminotetralone derivative has recently been synthesized with a specific activity of 2000 Cdmmol and shown to label brain a 1 receptors quite specifically with a K , in the 0.1 nM range (Engel and Hover, 1981; Glossmann el ul., 1981). r . [3H]Dili~~~ro~).gocr~ptitie. As a radioligand for the specific study of a,-adrenergic- receptors, HIDHEC suffers from the disadvantage of labeling a , and a2receptors with about equal affinity. Several techniques have been developed to overcome this difficulty and permit the selective study of the a-receptor subtypes. One method involves the use of complex computational techniques with computer modeling of inhibition curves of prazosin against rH]DHEC (Hoffman et al., 1979). Using this method the percentage of a 1 and a , receptors in half of a given tissue sample is determined from inhibition studies, and the total number of a receptors is determined by HIDHEC saturation analysis on the other half of the tissue sample. T h e density of, and inhibitor affinity at, the two receptor subtypes is then calculated. The other method has several variations, but basically involves the use of a subtype-selective drug at a specified concentration. T h e concentration is chosen so that essentially all of radioligand binding at one of the subtypes will be inhibited, while binding at the other subtype will not be affected (Miach P t nl., 1978). In the best docutnented example, B,,,, and K,)values in rabbit uterine membranes were obtained in the presence and absence of 100-nM prazosin. The B,,,, in the absence of prazosin was taken to represent the total adrenergic receptor population, whereas that in the presence of prazosin was defined as a,-adrenergic receptors. T h e contribution of
r
CHARACTERIZATION OF (Y1- AND (Y2-ADRENERGIC RECEPTORS
363
al-adrenergic receptors was then calculated by the difference (Hoffman and Lefkowitz, 1980~). Using this method, the density of a , sites labeled by [3H]DHEC was 38 fmoVmg prot, which is in fair agreement with 29 fmoVmg prot of a1sites labeled by [3H]prazosin(Lavin et al., 1981; there is a typographical error in this paper which gives the density of pH]prazosin sites as 19 fmoVmg prot rather than the correct value of 29). The estimate of the density of a2receptors using rH]DHEC was 80% higher (129 fmoumg prot) than that obtained from rHlyohimbine binding (72 fmoVmg prot). The reason for this rather large difference is not presently understood. Using a similar approach, the total a-, al-, and a2adrenergic receptor sites in the rat hypothalamus were assayed using 100 n M phentolamine, prazosin, and clonidine, respectively, to define specific binding (Neethling et al., 1981). The values obtained in this manner agreed reasonably well with the values obtained using [3 HlWB4 101 and p Hlclonidine to assay a , and a2 receptors separately (Haga and Haga, 1980). Thus, while THIDHEC can be used to study a , receptors, it is not clear that the use of these techniques has any advantages over the more direct approach using subtype specific radioligands, such as THlprazosin or [1251]BE-2254. d. Multiple Afinity States of Alpha-1 Receptms. In contrast to the a2and P-adrenergic receptor systems, where there is strong evidence for the existence of high- and low-affinity agonist states, there is thus far little evidence that a,-adrenergic receptors have multiple affinity states. The inability to demonstrate 3H-labeled agonist binding to a , receptors at low ligand concentrations suggests the absence of a high-affinity agonist state. The lack of an effect of guanine nucleotides on agonist inhibition of 3H-labeled antagonist binding in almost all a1systems studied might also be taken as evidence of a high-affinity agonist state, although it is probably more properly interpreted as a result of a , receptors not being coupled to adenylate cyclase. There is, however, a recent report of [3H]norepinephrine binding to putative a , receptors in rat liver plasma membranes which is decreased by GTP (Geynetet al., 1981). The reports of regulation by sodium ions of agonist affinity in inhibiting central [3 Hlprazosin and [1251]BE-2254binding indicate that in some ionic media multiple affinity states do exist, although no attempt has yet been made to fit them to a kinetic model of a,-receptor function. The Rosenthal analyses of p HlWB4 101 and THlprazosin saturation data are generally monophasic indicating a single class site, although in a few instances biphasic Rosenthal plots have been reported (U’Prichard et al., 1979a; Lyon and Randall, 1980; Weinreich et al., 1980; Rehavi et al., 1980b). In some of these cases the low-affinity component may be due to the binding of [3H]WB4101to a2 receptors (see above) or to calcium ion
364
DAVID B. BYLUND A N D DAVID
c. U'PRICHARD
channels (Atlas and Adler, 1981) rather than to a low-affinity state of the a , receptor.
2. Agonist RadioligaridA To date, H-labeled agonist binding to a,-adrenergic receptors has been reported only in a single tissue and only by one laboratory (Geynet et al., 1981). I n general, VHIepinephrine and [3H]norepinephrine appear to bind specifically to a2-adrenergic receptors (see below), which can be interpreted as indicating that the a , receptors lack a high-affinity agonist conformation. Thus, the binding of these agonists to a , receptors is of too low affinity to be observed with the present techniques. It is certainly reasonable to think that eventually a, agonists with sufficiently high affinity will be found which can be used as radioligands.
B . EFFECTOR SYSTEMS COUPLED TO a,-ADRENERGIC RECEPTORS 1. Adipocytes During the past several years, the adipocyte has emerged as one of the better systems for the study of a-adrenergic receptor binding and function. Adipocytes from both the human and hamster appear to have a modest level of a,-adrenergic receptor binding (Table VIII), although one laboratory has been unable to observe &,-receptor binding in human subcutaneous adipose tissue. T h e density of a,-adrenergic receptors appears to be somewhat variable among subjects or among animals, although generally it is in the range of 50 to 300 fmol/mg prot. TABLE V I I I CI~-ADRENERGIC RECEPTOR BINDING I N ADIPOSETISSUE
Species ~~
Tissue type
Tritated ligand
Bin,, (fmoVmg prot)
Properitoneal Subcutaneous Subcutaneous White fat White fat
WB4101 Prazosin DHEC DHEC DHEC
300" 290 lod 2off
Reference
~
Human Human Human Hamster Hamster
67d
Burns et al. (1981) Wrightut 01. (1981) Tharp t t al. (1981) Pecquery and Giudicelli (1980) Garcia-Sainz et al. (1980b)
Range was 175-450 fmoUmg tissue.
* N o binding to al receptors was detectable. 15-25% of total DHEC binding was estimated to be to a, receptors. 0-30% of total DHEC binding was estimated to he to a1receptors.
CHARACTERIZATION OF (Y1- AND Q2-ADRENERGIC RECEPTORS
365
Simulation of a,-adrenergic receptors in the adipocyte appears to be coupled to increased turnover of the phosphatidylinositol and phosphatidic acid. It has been proposed that the increased turnover of these phospholipids in many tissues results in an increase in cytoplasmic calcium ion concentration due to the uptake of extracellular calcium and the release of bound calcium (Michell, 1975; Fain and Garcia-Sainz, 1980). The evidence for the a1nature of the increased turnover of these phospholipids is based on the selective inhibition of the epinephrinestimulated 32Pincorporation. The potency order of prazosin and yohimbine in reducing 32 P incorporation into phosphatidylinositol and phosphatidic acid is clearly that of an a,-mediated process (Burnsetal., 1981). Thus, while the adipocyte appears to have both a receptor binding site that can be labeled by radioligands and a biochemical function that has a specificity for a ,-receptor processes) this does not yet constitute proof that the receptors which are labeled in binding studies are identical to those receptors responsible for the biochemical effect. At the present time, neither the molecular mechanism whereby a,receptor stimulation results in an increase in the turnover of phospholipids nor the link between the increased turnover and calcium ion movement is known. In the rat adipocyte a,-adrenergic stimulation results both in an increase in phosphatidylinositol turnover and in the inactivation of glycogen synthase (Garcia-Sainz et al., 1980a). The divalent cation ionophore A23 187 can also inactivate glycogen synthase (Lawrence and Larner, 1977, 1978). These and other data suggest that turnover of phosphatidylinositol is involved in some fashion in the gating or mobilization of calcium. It has, in fact, been suggested that phosphatidic acid itself is a calcium ionophore (Putney et al., 1980; Harris et al., 1981). However, a causal relationship between the a,-mediated increase in the turnover of phosphatidylinositol and the elevation of cytosolic calcium is not yet established.
2. Salivary Glands The rat salivary glands have also proved to be a useful tissue for the investigation of relationship between receptor binding and function. The rat has three salivary glands. The parotid is a serous gland which releases amylase followingP-adrenergic stimulation. The submandibular is a mixed serous and mucous gland to which is attached the mucous sublingual gland. Both the parotid (Batzri et al., 1973) and the submandibular glands (Martinez et al., 1976))but not the sublingual gland (Martinez et al., 1982b), release potassium in response to an a-adrenergic stimulation. As would be expected, but contrary to a previous report (Arnett and Davis, 1979), the al-adrenergic receptor subtype mediates
366
DAVID B. BYLUND A N D DAVID c . U’PRICHARD
this potassium release response in the submandibular glands (Bylund et al., 1982a). Both norepinephrine and the a1 agonist methoxamine stimulate the release of potassium, while prazosin, but not yohimbine, is able to block the norepinephrine-stimulated release. Similarly, in the rat parotid gland, the K , values for a series of eight adrenergic drugs in inhibiting pH]prazosin binding correlated significantly ( r = 0.85, p < 0.01) with the potency to stimulate or inhibit potassium (Itoet al., 1982). Several groups have studied the binding of HIDHEC to various preparations of rat salivary glands. [3 HIDihydroergocryptine binding has been characterized in both the parotid (Strittmatter et al., 1977) and the submandibular glands (Pointon and Banerjee, 1979), although in neither study was a differentiation attempted between a 1 and ag subtypes. In another study of rH]DHEC binding to submandibular glands, it was concluded that the a receptors labeled by the radioligands were of the az subtype (Arnett and Davis, 1979). This conclusion is difficult to reconcile with the finding that WB4101 was equally potent with yohimbine in inhibiting the binding of the radioligand and that prazosin was only eightfold less potent than these antagonists. Furthermore, significant binding of neither [3H]clonidine nor [3H]yohimbine has been demonstrated in adult rat submandibular gland (Bylund and Martinez, 1980; Pimoule et al., 1980; Bylund et al., 1982a). Thus, the delineation of the receptor subtype(s) to which [3H]DHEC binds in the submandibular glands is not yet clear, and the resolution of the question will await the application of the techniques described above for the study of subtype specific receptor binding using VHIDHEC. Alpha- 1-adrenergic receptors in the rat submandibular and sublingual glands have been studied using [3H]WB4101 and rH]prazosin. As can be seen from Tables VI and VII, the submandibular gland shows 1500-3000-fold specificity of prazosin as compared to yohimbine for these radioligands. T h e density of binding sites in the sublingual gland is only about one-third of that found in the submandibular gland (Table V), which is consistent with the lack of measurable norepinephrinestimulated potassium release in the sublingual gland slices (Martinez ct al., 1982b). Unilateral ligation of the main excretory duct of the rat submandibular gland results in a progressive decrease in gland weight due mainly to acinar cell atrophy. Based on [3H]WB4101 binding, the density of a l receptor binding sites in the ligated glands were approximately onethird that found in the contralateral control glands (Martinez rt al., 1982a). These data suggest that the majority of the al-adrenergic receptors in the submandibular gland are located on acinar cells.
r
CHARACTERIZATION OF (Xi- AND a2-ADRENERGIC RECEPTORS
367
3. Liver
Several groups have studied the binding of radioligands of a-adrenergic receptors in the rat liver, as compared to the effect of a-adrenergic drugs on glycogen phosphorylase activity. It is generally agreed that HIDHEC labels predominantly a ,-adrenergic receptor sites in this tissue and that the activation of glycogen phosphorylase is an a,-mediated process. Two laboratories obtained a good correlation between the potencies of a-adrenergic agonists and antagonists in inhibiting rH]DHEC binding in plasma membranes and the potency of agonists (antagonists) in activating (inhibiting) glycogen phosphorylase (Hoffman et al., 1980b; Aggerbeck et al., 1980). Another group found a better correlation between PHlepinephrine binding and phosphorylase activity than between [3H]DHEC binding and phosphorylase activity (El-Refai et al., 1979) and in addition, presented limited evidence that [3H]epinephrinewas binding to at receptors (El-Refai and Exton, 1980). Geynet et al. (1981) have presented evidence suggesting that [3H]prazosin and [3 Hlnorepinephrine label distinct a,-receptor binding sites, whereas rH]DHEC labels both sites. They speculate that the site labeled by rHJnorepinephrine, which has a higher af€inity for agonists, is the physiologically active form of the receptor. On the other hand, Hoffman et al. (198lb) found that [3H]epinephrine at low concentrations binds selectively to a2 receptors. The discrepancy appears to lie partly in the different concentrations of Hlcatecholamine used in the experiments, although an adequate resolution of these conflicting data is not obvious.
r
r
4. Central N m o u s System In some CNS tissues a,-receptor activation appears to be coupled to an increase in the production or levels of cyclic nucleotides. In both the brain and the spinal cord, a,-adrenergic agonists increased the tissue content of cyclic AMP, an effect which appears to be dependent upon calcium (Schwabeet al., 1978). However, few studies have attempted to correlate binding characteristics of a ,-receptors with brain adenylate cyclase activity or cyclic AMP levels. In the rat cerebral cortex a significant potency correlation ( r = 0.87) was found between rHlWB4101 sites and a-adrenergic-mediated cyclic AMP accumulation in brain slices (Davis et al., 1978). These data suggest that [3H]WB4101may bind to the membrane receptor sites mediating the adrenergic accumulation of cyclic AMP in this tissue. Other evidence suggests that this a, response is probably mediated by the interaction of calcium with calmodulindependent adenylate cyclase and by analogy with the peripheral system
368
DAVID B. BYLUND AND DAVID
c. U’PRICHARD
cited above, may result from an increased phosphatidylinositol metabolism to generate phosphatidic acid and elevate intracellular calcium levels. C. REGULATION OF CY,-ADRENERGIC RECEPTORS Physiological and pharmacological regulation of receptor density as measured by the number of receptor binding sites is emerging as an important area of study. I n at least some instances a change in receptor density has been strongly implicated as the important factor in supersensitive and subsensitive responses to hormones and neurotransmitters. T h e levels of adrenergic receptors can be altered by a number of stimuli (Bylund, 1979) and are often inversely related to the effective concentration of norepinephrine in the synapse. For example, up-regulation of receptor number which is frequently associated with functional supersensitivity appears to result from a decrease in the level of norepinephrine or from chronic receptor blockade by appropriate antagonists. Conversely, a down-regulation in receptor number and functional subsensitivity is often associated with an increase in the level of norepinephrine or with chronic administration of an adrenergic agonist. This type of regulation is termed homologous regulation because it seems to be the direct result of changes in the amount of neurotransmitter or hormone available for binding to the receptor. By contrast, regulation of adrenergic receptors and their function by other hormones or regulatory agents is termed heterologous regulation. Adrenergic receptors also undergo changes during alterations in physiological conditions such as development, as well as in some pathological states. In order to assess the significance of changes in receptor number, it is critical that biochemical and physiological determinants be studied in addition to receptor binding. For instance, in the rat submandibular gland surgical denervation or reserpine treatment results in a doubling of &receptor number which is associated with a marked increase in the accumulation of cyclic AMP in response to isoproterenol (Bylund et al., 1981). However, the isoproterenol-stimulated adenylate cyclase activity is not altered, and the increased cyclic AMP levels are actually due to a decrease in the activity of phosphodiesterase. Thus, the change in receptor number does not appear to be physiologically important. Although considerable progress has been made in the past several years in the area of a,-adrenergic regulation, most of this work has been limited to receptor binding studies, and in the future much more emphasis must be placed on the corre-
CHARACTERIZATION OF CX1- AND a2-ADRENERGIC RECEPTORS
369
lation of receptor binding data with physiological and biochemical alterations. 1. Up-Regulatim In several systems the number of a,-adrenergic receptors (as labeled by [3H]WB4101) is increased following drug treatments or procedures designed to lower the effective concentration of norepinephrine at the synapse. A summary of these data is given in Table IX. Chemical sympathectomy in the central nervous system of the rat with the neurotoxin 6-hydroxydopamine drastically reduces norepinephrine levels in certain brain regions due to a destruction of the nerve terminals. The drug reserpine also markedly lowers norepinephrine levels. This reduction in neurotransmitter is accompanied by, and may result in, a modest increase in the levels of a,-adrenergic receptors. Chronic treatment with amitriptyline, a potent a ,-adrenergic antagonist, causes a similar increase in the density of receptors. In the submandibular gland of the rat an up-regulation in a-adrenergic receptors is seen following a surgical denervation or norepinephrine depletion following chronic reserpine treatment. None of the studies referenced in Table X found a significant change in the K , value for [3H]WB4101. It is of interest that in these experiments, the magnitude of the changes in a,-receptor levels appears to be generally less than that for either p- or a2-adrenergic receptors. For example, in the submandibular gland, norepinephrine depletion results in approximately a 10-fold increase in a2-adrenergic receptors (Bylund and Martinez, 1980, 1981). In the brain neonatal 6-hydroxydopamine treatment results in a 16% increase in the density of a , receptors, but causes a 40% increase in the number of P-adrenergic receptors as determined by [3H]dihydroalprenolol binding (D. B. Bylund and w. 0. Shekim, unpublished). However, in general the changes are similar in magnitude. It also should be pointed out that there are multiple examples of the lack of change of a , receptors following treatments similar to those in Table IX. I n the salivary gland initial attempts have been made to correlate changes in receptor binding with the release of potassium induced by a1 agonists. I n one study the density of a,-adrenergic receptors increased 55% following 7 days of reserpine treatment, while the release of potassium increased 56 and 37% in response to norepinephrine and methoxamine, respectively (Bylund et al., 1981). Thus, in this case the increase in receptor binding and the biochemical effect were approximately equal, suggesting a possible causal relationship. However, other workers have failed to fmd an increase in potassium release following
Tissue“
Treatment
H,,,,,for [9H]WB4101 (percentage of control)* 124
Submandibular Submandibular Forebrain Forebrain Forebrain (mouse) Cerebral cortex Cerebral cortex
Canglionectomy Reserpine Reserpine Amitriptyline Amitriptyline Neonatal 6-hydroxydopa
116
Midbrain
Neonatal 6-hydroxydopa
109
Frontal cortex Thalamus
Dorsal bundle lesion Dorsal bundle lesion
7
146 151
129 149
6-W ydroxydopamine
~
180 177
155
Norepinephrine (percentage of control)
17
174 120
3 21
Reference Bylund rf nl. (1981) Bylund ct nl. (1981) U’Prichard and Snyder (1978b) U’Prichard rt (11. (197813) Rehavi uf al. (1980a) U’Prichard et a/. ( 1979a) D. B. Bylund and W. 0. Shekim (unpublished) D. B. Bylund and W. 0.Shekim (unpublished) U’Prichard et (11. ( 1980b) U’Prichard rf al. (1980b)
~~~~
Tissues were from the rat unless otherwise indicated. * B,,,, values were calculated from saturation experiments. In all cases, the value for treated animals was significantly different from control (p < 0.05). The first column is percentage calculated from the data expressed as pmoVgm tissue and the second column from the data expressed as pmol/gm protein. a
37 1
CHARACTERIZATION OF al- AND QI~-ADRENERGICRECEPTORS
TABLE X BINDING CHARACTERISTICS OF P H ] P R A Z O S I N T O R A T LUNGMEMBRANES AT DIFFERENT TIMES OF THE DAY
Time
No. of animals
B,,, (fmoYmg prot)
K D (nM)
132 f 3 125 f 4 119* 2 129 6
0.105 f 0.004 0.109 0.012 0.110 f 0.012 0.130 f 0.011
126 2 2
0.114 2 0.006
2 A.M. 2
P.M.
8
P.M.
6 6 6 5
Mean
23
8 A.M.
*
*
surgical denervation of the submandibular gland (Arnett and Davis, 1979) and the parotid gland (de Peusner et al., 1979). In contrast to findings (see below) in which an increased level of the agonist decreases a,-adrenergic response and receptor binding, it appears that under certain conditions a brief pretreatment with an a agonist can increase the response and density of receptors (Hata et al., 1980a,b).In the rat vas deferens, the contractile response to epinephrine was significantly enhanced by preexposing the tissue to 25 p M epinephrine for 10 min. This treatment increased the maximal contractile response by 39%, but did not affect the EDm.The number of al receptors, as measured by [3H]WB4101,increased 27% following the epinephrine treatment. The increased contractile response was blocked by phentolamine but not by propranolol, suggesting a specific a-adrenergic phenomenon. There is preliminary evidence that chronic lithium treatment increases a,-adrenergic receptor binding. Rats that had been on a lithium diet for 3 or 5 weeks had 28 and 17% increases, respectively, in the binding of rHlWB4101 when assayed at a single concentration. This increase appeared to be due to a B,,, change with little change in K , (Rosenblatt et al., 1979).
2 . Down-Regulation The term desensitization is used to describe the phenomenon of reduced cellular responsiveness to an agonist following a previous exposure to that agonist. Down-regulation of receptor number often accompanies desensitization and in at least some systems seems to be an important mechanism for producing the desensitized state. Desensitization of a-adrenergic receptors has been studied in rat parotid acinar cells. In these experiments the release of potassium in response to epinephrine in cells that had been preexposed to epinephrine was reduced compared to
372
DAVID B. BYLUND AND DAVID c . U’PRICHARD
control cells. Since there was concurrent reduction in rH]DHEC binding to membranes prepared from these cells, it was concluded that the a-adrenergic desensitization was mediated, at least in part, by a downregulation in receptor number (Strittmatter et al., 1977; Davis et al., 1980). These studies are hard to interpret in the context of the present article since the binding of E3H]DHEC to a , and a 2 receptors was not differentiated. Indirect evidence in support of the ability of a 1 receptors to be down-regulated includes observations of reductions in rat cerebellar rH]WB4 101 sites after dorsal bundle lesion, which elevates cerebellar norepinephrine by 50% (U’Prichard et al., 1980b), and reductions in rat heart and vas deferens rHlWB4 101 binding after chronic immobilization stress, which accelerates norepinephrine turnover and release (U’Prichard and Kvetnansky, 1980).
3. Phjsiologxal Regulation a. Ontogmy T h e ontogeny of a,-adrenergic receptors has been described in four tissues: heart, brain, submandibular gland, and lung. Figure 1 illustrates the change in the density of a,-adrenergic receptors during early postnatal development. In addition, norepinephrine levels are given over the same time period for all four tissues. For the submandibular gland the development of the biochemical effect (potassium release) is also given. There were no significant changes in K , ) values noted in any of the four tissues during development. In the mouse heart, a , receptors labeled by [3H]WB4101 are significantly higher during the first 2 weeks of life than they are in either the fetus or the adult (Yamada et al., 1980b). These data were interpreted to suggest that the receptors mature prior to the development of the sympathetic innervation and then decrease markedly with the innervation of the tissue. It is of interest to note that between 2 and 3 weeks, when there is the greatest increase in norepinephrine levels, the number of a,adrenergic receptors is decreasing. This may indicate that the receptors are down-regulating in response to the increased norepinephrine levels. T h e levels of a,-adrenergic receptors Hlprazosin binding) in the rat brain increase relatively uniformly to a peak at 3 weeks of age and then drop off slightly to adult levels. T h e concentration of norepinephrine increases at a somewhat slower rate and even by 3 weeks is only about 75% of adult levels. However, a direct comparison of norepinephrine levels and receptor binding in whole brain is of doubtful significance since most of the norepinephrine is in the hypothalamus, while the a,-adrenergic receptor binding reflects the predominance of cerebral cortical tissue.
(r
CHARACTERIZATION OF (Y1- AND CQ-ADRENERGIC RECEPTORS
373
-I
W
I
>
W
-I
W
z
X
CI
U I m
E
I
a
W
z E
W K 0
z
AGE (days) FIG. 1. Comparison of a,-adrenergic receptor density (BmaX)and norepinephrine levels in four tissues during development. For the rat submandibulargland, a comparison with epinephrine-stimulated K+ release is also included as an indication of al-receptor function.
374
DAVID B. BYLUND AND DAVID
c.
U’PRICHARD
T h e a ,-adrenergic receptors ([3 Hlprazosin binding) in the rat submandibular gland are similar to those in the rat brain in that they are barely detectable at birth and show the greatest increase between 1 and 3 weeks of age (Bylund rt nl., 1982b). T h e al-stimulated release of potassium is also very low at birth, but then increases to adult levels between 1 and 3 weeks of age (Martinez and Camden, 1982). Similarly, the greatest increase in norepinephrine levels also occurs between 1 and 3 weeks of age (Kuzuyact al., 1980). Thus, in this tissue there is a remarkably good correlation between the innervation as measured by norepinephrine levels, the development of receptor binding sites, and the development of the biochemical response. In the rat lung, the density of a,-adrenergic receptor binding sites ([3H]prazosin binding) is moderately high at birth and then slowly increases to a peak at 2 1 days which is approximately 50% higher than the level at birth (Latifpour, 1981). Norepinephrine levels in the rat lung also peak at about 3 weeks at a level approximately three times that at birth (Gardey-Levassort et nl., 1981). T h e comparison of the development of a,-adrenergic receptors in these four tissues fails to reveal a consistent pattern, but rather indicates that each tissue has a unique developmental pattern which is presumably consistent with the function of the cells and tissues involved. b. Circcicliu?i Rhythm. T h e possibility of a circadian variation in a l adrenergic receptor binding has been investigated in the rat brain and lung. A circadian rhythm was found in the rat brain with a variation of 30-40% in binding of [3H]WB4101 over a 24-hr period (Kafka et ul., 1981). Saturation analyses conducted at the time of maximal and minimal binding indicated the change in receptor binding was due to a change in B,,,, and not in K,. In addition, both the wave-form of the circadian rhythm and the time of maximal binding were dependent on the month in which the assays were conducted, suggesting that a seasonal rhythm may also be present. If confirmed, these observations have serious implications for design and conduct of experiments intended to investigate changes in receptor levels. As a minimum, the experiments must be conducted at the same time each day. However, even this may not be sufficient since chronic drug treatment appears to modify the circadian rhythm in the rat brain (Wirz-Justice et al., 1980). T h e physiological significance of this circadian rhythm cannot be ascertained, since there were no concomitant studies on the functional effects of the receptor-agonist interaction. By contrast, no circadian variations were found in the binding of pH]prazosin to rat lung membranes (Latifpour, 1981). In these experiments rats were sacrificed over a 3-day period every 6 hr, and the K , , and
CHARACTERIZATION OF a1- AND a2-ADRENERGIC RECEPTORS
375
B,,, were determined by saturation analyses. The maximum deviation of B,,, at any time point from the mean of all time points was only 5 % . These results suggest that rat lung a,-adrenergic receptors do not undergo any circadian changes; these results also illustrate that highly reproducible data can be obtained in carefully controlled experiments (Table X). 4 . Pathological Regulation
There is increasing evidence that variations in receptor characteristics may be the result of, result in, or be associated with certain disease states. For the a,-adrenergic receptor, the pathological state that has received the most attention is hypertension. The results of these studies are summarized in Table XI. In the heart of the deoxycorticosterone/salt hypertensive rat, the density of a ,-adrenergic receptors as determined by rHlWB4101 was significantly reduced in the ventricles but not in the atria (Yamada et al., 1980d). Cardiac a-adrenergic receptors were also found to be decreased using the subtype nonselective ligand HIDHEC (Woodcock and Johnston 1980).The density of a,-adrenergic receptors was similarly reduced in the kidney using both the spontaneously hyperactive rat and the deoxycorticalstarone/saltmodels of hypertension (Yamada et al., 1980d; U’Prichard et al., 1979b).In the brain the levels of a1 adrenergic receptors are generally increased (U’Prichard et al., 1979b), although this is dependent upon the particular brain region studied. For instance, significant increases in B,,, were found in both the cerebral cortex (Yamada et al., 1980d) and the midbrain (Gheyouche et al., 1980),although no change was found in other brain regions includ-
r
ALTERATIONS IN a
l
-
TABLE XI A RECEPTORS ~ ~ IN~ ANIMAL ~ ~ MODELS ~ OF~ HYPERTENSION ~ Bmax
Tissue
Hypertensive model
Heart Kidney Kidney Cerebral cortex Brain Midbrain
Deoxycorticosteronehalt Deoxycorticosteronekalt Spontaneously hypertensive Deoxycorticosteronehalt Spontaneously hypertensive Spontaneously hypertensive
for
[3H]WB4101 (percentage of control)’ 67 71 74
62 75 125
119 135
Reference Yamada et al. ( 1980d) Yamada et al. (1980d) U’Prichard et al. (1978b) Yamada et al. (1980d) U’Prichard et al. (1978b) Gheyouche et al. (1980)
B,,, values were calculated from saturation experiments. The first column is percentage calculated from the data expressed as pmol/gm tissue and the second column from the data expressed as pmoVmg protein. (I
376
DAVID B. BYLUND AND DAVID
c . U’PRICHARD
ing the hippocampus, hypothalamus/thalamus, cerebellum, and brainstem (Yamadaet ul., 1980d; Cantor et al., 1981). While these results suggest that a ,-adrenergic receptors may have a role in hypertension, it is not clear even in the animal models whether the alterations in receptor number are involved in the etiology of the hypertension or are only a consequence of other compensatory changes. D.
SOLUBILIZATION OF
QI I-ADRENERGIC
RECEPTORS
The solubilization and purification of the a,-adrenergic receptor will ultimately be necessary in order to have an accurate understanding of its characteristics and function. There are two reports of a,-adrenergic receptors that have been solubilized in the active form. Using the detergent digitonin a- and P-adrenergic receptors were solubilized from hepatic plasma membrane and were then identified by the binding of [3H]WB4101 and ~H]dihydroalprenololand characterized (Wood P t nl., 1979b). These workers were then able to separate the solubilized receptors by affinity chromatography and concluded that the active a- and P-adrenergic binding sites d o not simultaneously reside on the same macromolecule. Unfortunately, only 3 to 10% of the receptors present in particulate preparation were solubilized, and those solubilized receptors had a 2- to 8-fold decrease in affinity for adrenergic drugs. Solubilized hepatic a 1 receptors have been purified about 500-fold using affinity chromatography (Graham et al., 1982). T h e specificity of binding to these receptors is similar to that of the membrane-bound receptors as determined by the inhibition of PHlprazosin binding. However, the K , for [3H]prazosin (16 n,M) in saturation experiments is about 30-fold higher than in membranes. Taking a different approach, Guellaen et al. (1979, 1982) first prelabeled the receptor with the irreversible a antagonist [3H]phenoxybenzamine, and then solubilized with 2% SDS or 0.5% Lubrol PX. These workers concluded that the rat liver a 1receptor has a molecular weight of 96,000 and is composed of at least two subunits. The binding site (at least for [3H]phenoxybenzamine) is on a 44,800-dalton subunit.
111. cuZ-AdrenergicReceptors
In general, the a2receptor and its relation to the coupled response of adenylate cyclase inhibition has been much better characterized than the
CHARACTERIZATION OF (Y1- AND (Y2-ADRENERGIC RECEPTORS
377
receptor, especially in well-defined homogeneous cell populations such as human platelets, adipocytes, and rodent neural cell lines in culture. The a , receptor appears to occur in two or more states, primarily differentiated with respect to their affinity for agonists. According to the model of a,-receptor function proposed by Lefkowitz and co-workers (Hoffman et al., 1980a), antagonists recognize both states of the receptor with equally high affinity, whereas agonists have high affinity for only one of the states. Thus, in typical experimental situations, labeled agonists bind to a subset of the total a,-receptor population which is determined by labeled antagonist binding. The data are generally consistent with the Lefkowitz model, but recurrent inconsistencies are illustrated which tend to suggest that the model is an oversimplification. This section of the article will deal with the characteristics of antagonist and agonist binding sites representing a, receptors, the comparison of agonist and antagonist binding within the framework of regulation of different affinity states of the receptor, and the relationship of this regulation to transduction of the a,-receptor response. Regulation, localization, and solubilization of a2 receptors will then be considered.
a
A. CHARACTERIZATION BY RADIOLIGAND BINDING 1. Antagonist Radioligands a. [3H]Dihydro~g~cTyptine. The first tissue in which VHIDHEC binding to a receptors was demonstrated was rabbit uterus (Williams and Lefkowitz, 1976), which has been estimated to contain predominantly (80%) a , receptors (Hoffman and Lefkowitz, 1980b). Subsequently, [3 HIDHEC binding was characterized in human and rabbit platelets (Kafka et al., 1977; Newman et al., 1978; Tsai and Lefkowitz, 1978), which appear to contain exclusively a, receptors, and in rat and bovine brain membranes (Greenberg and Snyder, 1978; Peroutka et al., 1978), which have roughly equal amounts of a1 and a, receptors depending upon the specific brain region. Although the K Dof brain [3H]DHECbinding was reported to be about 1.0 nM (Greenberg and Snyder, 1978), other early studies of VHIDHEC binding gave generally higher and quite variable KD values, ranging from 3-35 nM (L. T. Williams and Lefkowitz, 1976; Kafka et al., 1977; R. S. Williams and Lefkowitz, 1978). To some extent, K , values appeared elevated because receptor concentrations in small assay volumes were quite high (Williams and Lefkowitz, 1976; Kafka et al., 1977). More recent studies of VHIDHEC binding to a receptors in many different tissues have employed lower receptor concentrations and demonstrate considerable agreement for the affinity of
378
DAVID
B. BYLUND
A N D DAVID
c:. U’PRICHAKD
[3H]DHEC at a-receptor binding sites, with K, values in the range of 1-3 n2W. [3 H]Dihydroergocryptine labels both a , and a,-receptors. In several studies, the number of PHIDHEC sites was found to be approximately equal to the sum of sites labeled by ligands more specific for the a 1and a , receptors, respectively, such as PHIWB-4101 and [3H]clonidine in rat brain and peripheral tissues (U’Prichard and Snyder, 1979; Neethling et ul., 1981). I n retrospect, these studies can be criticized because the a,receptor ligand used, [3H]clonidine, is an (partial) agonist and as such labels only a subpopulation of the a,-receptor sites (rather than the total a,-receptor pool), thereby underestimating a,-receptor density. In a more recent study (Lavin et ul., 1981),the number of a , receptors labeled by E3H]DHEC in the rabbit uterus was 80% higher than the number labeled by Hlyohimbine (see Section II,A,l ,b). Similarly, in platelets the number of PHIDHEC sites (all az)is 65% higher than PHIyohimbine sites (Hoffman et ul., 1982). On the other hand, in the rabbit uterus good agreement was found between the affinities of various antagonists at a2 sites labeled by [3H]yohimbine and by PHIDHEC. In order to determine reliable affinities for the a , and a, subtypes using PHIDHEC, a 100-fold selectivity of a competitor is required along with sophisticated computer analysis of the data (Lavin et ul., 1981). Furthermore, it is assumed that [3H]DHEC labels a1 and a , receptors with equal affinity. In almost all tissues studied, Rosenthal plots of [3 HIDHEC saturation data appear linear, which is consistent with this assumption. However, the data generated with this ligand are generally quite scatter prone, and few studies have attempted rigorously to demonstrate that in the same membrane preparation PHIDHEC has equal affinity for a 1 and a2 receptors. One early examination of [3 HIDHEC saturation in bovine cortex membranes in the presence of receptor-saturating concentrations of unlabeled a1and a , competitors did suggest that PHIDHEC is equipotent at the two receptors (Peroutka rt ul., 1978). Another, as yet unreplicated, analysis of saturation of rat heart [3H]DHEC binding in the absence and presence of the a,-specific antagonist competitor ARC 239 indicated that [3 HIDHEC exhibited “positive homotropic cooperativity” of binding at myocardial a,, but not a l , receptors (Guicheney lit nl., 1978). Despite the limitations of PHIDHEC as a radioligand, such as its nonselectivitv and its poor binding to a receptors on intact cells such as human platelets (Alexander et al., 1978), and the current availability of yohimbine isomers as more selective a,-receptor antagonist radioligands (Motulsky and Insel, 1982), PHIDHEC has been a useful tool in the analysis of a,-receptor function in human platelets, which contain a pure
r
CHARACTERIZATION OF CY1- AND ff2-ADRENERGIC RECEPTORS
379
population of a, receptors. Platelet [3 HIDHEC binding, extensively studied by Lefkowitz and colleagues, is homogeneous with respect to antagonist competitors, but heterogeneous with respect to agonists. That is, the pseudo-Hill coefficients (nH)for antagonists are about 1.0, but the n H values for agonists are significantly less than 1.0 (Tsai and Lefkowitz, 1979). The favored explanation was that [3 HIDHEC binds to distinct conformations of the az receptor which recognized agonists, but not antagonists, with different affinity, rather than that [3H]DHEC exhibits negative cooperative interactions with the receptor. Sodium affects the interactions of agonists at the platelet rH]DHEC site by reducing their apparent affinity (right shift of curve). T h e extent of this right shift correlates with the known intrinsic activities of a series of a-receptor agents in a fashion reminiscent of the “sodium shift” at opiate receptors (Tsai and Lefkowitz, 1978). Alpha receptor antagonists interacting at the platelet rH]DHEC site are unaffected by Na+. Divalent cations such as Mg2+ selectively increase agonist affinities at these sites. These data are analogous to the existence of conformations of the P-adrenergic receptor with high and low affinity for agonists, whose equilibrium is modulated by guanosine di- and triphosphates (Maguire et al., 1977). T h e platelet az receptor, like the P receptor, is coupled to adenylate cyclase, but in an inhibitory manner. GTP and the nonhydrolyzable analog guanyl-5’-yl imidodiphosphate [Gpp(NH)por GMP-PNP] shift competition curves to the right for agonists but not antagonists at platelet THIDHEC sites, whereas corresponding adenine nucleotides are ineffective. In addition, in the presence of guanine nucleotides, agonist n H values approach 1.0, suggesting increased homogeneity of agonist interactions at the receptor (Tsai and Lefkowitz, 1979). In more recent studies, the techniques of computer modeling of platelet rH]DHEC competition curves indicate that agonist interactions at this site are best fit to a two-affinity-state model, designated az(H) and a,(L) (high and low affinity for agonists, respectively). Hoffman et al. (1980a) demonstrated that antagonists such as phentolamine have equal affinity for aZ(H)and a,(L). Relatively more a2(H) are found in the presence of the full agonist (-)-epinephrine than the partial agonist methoxamine. Guanine nucleotides at saturating concentrations (100 p M ) shift all the receptors into the az(L)state. The inability of partial agonists to generate as many a , ( H ) explains the apparent lesser effect of guanine nucleotides on partial agonist competition curves compared to those of full agonists. In somewhat less elaborate studies, the azcomponent of [3H]DHEC binding to rat liver membranes was also shown to be GTP-sensitive (Hoffman et al., 1980a). These experiments strongly indicate that the platelet a , receptor is coupled to adenylate cyclase via an intermediate membrane protein(s) with a site of at-
380
DAVID B . BYLUND A N D DAVID c . U’PRICHARD
tachment for GTP, in a manner analogous to /3 receptors that stimulate adenylate cyclase activity. HIDihydroergocryptine has also been shown to label a2-receptor sites on membranes from a neuroblastoma X glioma hybrid clonal cell line NG 108-15 with high affinity ( K , = 1.8 nM). At these sites, agonist affinities are affected by GTP in the same manner as in platelets (Haga and Haga, 1981). 6. [3H]Ebhim6ineand ~ H ] R a u w o l s c i n T ~ .h e alkaloid yohimbine is an a antagonist with selective potency for agreceptors in pharmacological experiments (Hedler et al., 1981). [3H]Yohimbine binding to a2receptors has been demonstrated in membranes from a variety of tissues (Table XII). In saturation studies, the KD of PHIyohimbine ranges from about 0.3 to 11 nil4 depending on species, tissue, buffer, and assay conditions. The affinity increases about threefold in most tissues when glycylglycine buffer is used rather than Tris-HC1 or Tris-NaC1 buffers. In glyclyglycine buffer, the KD for human and pig tissues is about 0.35 nM, while in rat and guinea pig tissues it is around 1.5 nM. For most a2 receptors, the data obtained using P Hlyohimbine are much superior to the data with PHIDHEC. There is good concurrence about the basic properties of PHIyohimbine binding to human platelet membranes in five different laboratories (Daiguji et al., 1981a; Motulskyrt al., 1980; Mukherjee, 1981; Smith and Limbird, 1981; Garcia-Sevilla et al., 198lb). Both kinetic and equilibrium experiments performed at 25 or 37°C indicate that rHlyohimbine binds to a single population of sites with the same affnity over a 0.1-10 nM concentration range. T h e pharmacological properties of platelet P Hlyohimbine sites determined from competition analyses are very similar to those of platelet [3 HIDHEC sites and indicate an a2-receptor interaction, with a catecholamine potency order (-)-epinephrine > (-)-norepinephrine % (-)-isoproterenol, selectively high affinity of (-)catecholamine isomers, and yohimbine generally about 500- 1000 times more potent than prazosin (see Table XIII). As with platelet PHIDHEC sites, agonist competition curves have nHvalues significantly less than 1.0. However, competition curves for imidazoline partial agonists are somewhat steeper and antagonists interact in a homogeneous manner at platelet [3H]yohimbine sites with n H of about 1.0. Guanine nucleotides right shift and Mg2+ left shift agonist and partial agonist competition curves, so that the apparent effect of GTP is more pronounced in the presence of Mg2+ (Fig. 2). Hoffman et al. (1982) have suggested that, as for platelet [3 HIDHEC binding, agonist competition at platelet PHIyohimbine sites best fits a two-site model, and that in the absence of nucleotide, both catecholamine full agonists and imidazoline partial agonists (intrinsic activity determined using the response of inhibition of
TABLE XI1 [3H]YOHIMBINEBINDING TO aZ RECEPTORS ~~
Tissue"
Species
pmoVgm protein
Platelet Platelet Platelet Platelet Platelet Platelet Platelet Platelef Adipocytes" Adipocytes' Cerebral cortex Cerebellum Uterus Cerebral cortex Cerebral cortex Liver Lung (neonatal) Kidney Kidney Cerebral cortex Cerebellum Corpus striatum Neuroblastoma X glioma
Human Human Human Human Human Human Human Human Human Human Human Human Rabbit Pig Guinea pig Rat Rat Rat Rat Rat Rat Rat
182 ? 29 334 ? 161 191 f 23 188 2 12 138 -+ 13 422 f 22 265 2 12
Rat-mouse
pmollgm tissue
K , (nM)
Buffefl
Reference
1 1 1 1 1 2 1 1 2 1 2 2 1 2 2
2
Daiguji et al. (1981a) Motulsky et al. (1980) Mukherjee (1981) Garcia-Sevilla et al. (198lb) Hoffman et al. (1982) D. B. Bylund (unpublished) Smith and Limbird (1981) Motulsky et al. (1980) Burns et al. (1982a) Tharp et al. (1981) D. B. Bylund (unpublished) D. B. Bylund (unpublished) Lavin et al. (1981) Harris et al. (1983) D. B. Bylund (unpublished) Hoffman et al. (1981a) Latifpour (1981) Yamada et al. (1980a) Schmitz et al. (1981) D. B. Bylund (unpublished) D. B. Bylund (unpublished) D. B. Bylund (unpublished)
1
Kahn et al. (1982)
170 f 10 120 2 16 37 t 4 106 f 6
7.5 f 1.0 2.5 I0.3 6.8 f 0.6
1.25 2 0.10 2.8 2 0.9 1.7 2 0.2 3.0 f 0.1 1.5 f 0.1 0.38 k 0.01 5.7 f 0.4 2.7 f 0.7 0.39 2 0.02 3.9 2 2.4 0.46 ? 0.05 0.33 2 0.03 llf5 0.27 f 0.02 1.7 f 0.3 5 1.53 f 0.11 0.83 f 0.08 7.4g 2.2 f 0.3 1.1 f 0.1 1.3 rL- 0.1
258 f 83
22,600 f 5,OOod
9.1 2 1.1
*
543 99 145 f 34 201 f 18 54 f 5 72 f 19 167 ? 21 95 f 9 110 f 21 304 2 28
207 f 41d
122 1 3.3 2 0.3 8.3 ? 1.0 5.5 2 0.2
4.8 f 0.2
Membrane preparation, unless otherwise noted. Buffers: 1, Tris; 2, glycylglycine; 3, sodium potassium phosphate. Intact platelets. Number of sites per cell. Properitoneal tissue. Subcutaneous tissue. Determined by kinetic analysis.
2 3 3 2
2
382
DAVID B. BYLUND A N D DAVID A
2
c. U'PRICHARD
Conhol
eo-
E
j
60-
4020-
n
0-
-
100
2
:!I [40
P
#
20 -I
O 1 0
9
8
7
6 -log [PENTOCAMNI (Mi
FIL. 2. Inhibition of [3H]yohimbine binding (0.2 n N ) to platelet membranes at 25°C (60 min) by (-)-epinephrine, p-aminoclonidine, and phentolamine in the presence or absence of GTP and MgC12. Points represent mean data from four to six experiments.
platelet adenylate cyclase) cause 60-70% of the receptors to be in the az(H) state. These authors found a correlation between intrinsic activity
and the ratio of affinities of agonists for a 2 ( H ) and a 2 ( L ) ,so that the K , , values for (-)-epinephrine were 11 nM (H) and 520 nM (L) with a ratio of 47.3, whereas the corresponding values for clonidine (59% intrinsic activity) were 9 nM (H) and 110 nM (L) with a ratio of 12.2. Unlike the
CHARACTERIZATION OF al- AND Q2-ADRENERGIC RECEPTORS
383
corresponding analysis of frog erythrocyte /3 receptors (Kent et al., 1980), intrinsic activity of agonists at platelet a2 receptors labeled with [3 Hlyohimbine did not correlate with the number of receptors in the a2(H) state. Similarly, submaximal concentrations of Gpp(NH)p appeared to reduce the afhity of (-)-epinephrine at a2(H)sites, rather than simply convert a2(H)to a2(L),as was found for the corresponding p-receptor analysis (Kent et al., 1980). In some respects, platelet p Hlyohimbine and ["HIDHEC sites differ. As noted earlier (Section III,A,l ,a), p HIDHEC consistently labels more platelet sites than PHlyohimbine (Daiguji et al., 1981a; Motulsky and Insel, 1982; Hoffman et al., 1982),a fkding that would not be predicted from the Hoffman and Lefkowitz (1980a) model, since both drugs, as antagonists, should label equally well both a2(H)and a 2 ( L )states of the receptor. The excess rH]DHEC binding does not appear to be to a1 receptors or serotonin transport sites. Earlier studies had shown that neither metal cations nor guanine nucleotides affected platelet THIDHEC binding. However, Mg2+concentrations as low as 1.0 mM increased the KD of p Hlyohimbine without apparently changing the B,,, (Daiguji et al., 1981a), and 50- to 100-mM Na+ decreased the KD and increased the B,,, of [3H]yohimbinebinding to solubilized membranes (L. E. Limbird, personal communication). Although these results cannot be explained at the present time, there is the possibility of some selectivity in the potency of the antagonist [3H]yohimbinefor different states of the a2 receptor (see Section 111,C). Another cell type that lacks a1 receptors but contains a2 receptors and the associated response of adenylate cyclase inhibition is the cultured hybrid neural cell NG 108-15 (Kahn et al., 1982). THlYohimbine binds in an apparently monophasic fashion to NG 108-15 membrane sites that have the pharmacological properties of at receptors and closely resemble platelet at receptors (Table XIII) except that yohimbine itself is about eightfold less potent in saturation and inhibition experiments, and the yohimbine/prazosin potency ratio is higher at NG 108-15 compared to human platelet sites by two orders of magnitude (Table 111). THlYohimbine labels about 30,000 a2 receptors per NG 108-15 cell, which is about the same density of receptors per platelet (c.a. 100-200 receptors per cell) when the calculations are made on the basis of receptors per unit surface area of cell plasma membrane. As with platelets, PHIyohimbine sites on NG 108-15 membranes exhibit heterogeneity with respect to agonist competitors (nH = 0.5-0.7) but not antagonists, and GTP and Gpp(NH)p at high concentrations (50 p M ) right shift and steepen agonist competition curves, supporting the general concept of the existence of (H) and (L) states of the a2receptor on these neural cells
384
DAVID B. BYLUND AND DAVID
I S H I R I T I O S OF
TABLE XI11 3H-LABEI,ED ALOSISTBINDING TO (I-ADRENERGIL RECEPTORS"
r3 HlEpinephrine (ICm, n.tf)
Drug Agonists (-)-Epinephrine (-)-Norepinephrine ( +)-Norepinephrine ( -)-lsoproterenol Imidazolines p-Aminoclonidine Clonidine Antagonists Phentolamine Yohimbine WB4101
Prazosin
c. U'PRICHARD
Platelet
3.3
14 410 1,500
3.3 3.8 11 19 20,000
NG-108
P H ] p Aminoclonidine (lCm,ni\J) Platelet
NG-108
[3H]Yohimbine V C W ,nM) Platelet
NG-108
11 6.1 32 270
2.4 6.5 250 800
5.4 3.0 90 60
87 420 6,800 29,000
250 390 1,900 29,000
4.0 32
3.4 3.1
1.3 30
34 60
52 48
65
140 3 50 5500
2.9 17 26 29,000
22 36 120 2,100
4.3 1.o
2.8 430
39 8 16 42
" Assays were performed on membranes from outdated platelets at 25°C. 1.0-mM MgCI, was present in EPI and PAC assays. Ligand concentrations were VHIepinephrine 1.0 nhl; [3H]p-aminoclonidine 0.6 nM; [3H]yohimbine 0.2 nJf . IC,, values are the mean of four to eight experiments.
(Kahn ei nl., 1982). Interestingly low (1.0-10 p L b f )concentrations of nucleotides seem to increase agonist affinities somewhat at [3 Hlyohimbine sites in membranes when residual endogenous divalent cations are not removed by treatment with a chelator. The imidazolines, clonidine and p-aminoclonidine, are partial agonists with respect to inhibiting NG 108-15 adenylate cyclase (Kahn et a/., 1982; Atlas and Sabol, 1981), showing, as in platelets, about 50% of the efficacy of full (catecholamine) agonists. T h e potencies of agonists and antagonists in general corresponded well at NG 108-15 r3H]yohimbine sites and the cyclase response. The bindinglcyclase potency ratio for agonists, K,Jk', , may be an index of the efficiency of coupling of the NG 108-15 a2 receptor to adenylate cyclase, by analogy to the K D / K a c ratio t for p receptors (Maguire et al., 1977). T h e K,IKi ratio was 1.0 for full agonists, but only 0.1 for the partial agonists clonidine and p-aminoclonidine (Kahn et al., 1982). Although these values are meaningful in a relative sense, as absolute indices they are suspect because both binding and cyclase assays w e r e performed in a cell-free system. A truer estimate of efficiency of coupling would involve determinations of
CHARACTERIZATION OF al- AND (Y~-ADRENERGICRECEPTORS
385
agonist potencies in intact cells at [3H]yohimbine binding sites and in decreasing cellular CAMPlevels. It is of interest that the pharmacologic characteristics of the human and rat a2receptors labeled by [3 Hlyohimbine are different, particularly with respect to the potencies of yohimbine and prazosin. I n human tissues, yohimbine is much more potent than prazosin, with yohimbine/ prazosin ratios about 0.002, whereas in rat tissues including NG 108-15 cells, yohimbine is only slightly more potent than prazosin, with ratios about 0.15. In glycylglycine buffer, the KD for PHIyohimbine binding is 0.32 to 0.46 nM in human tissues, whereas it is between 1.1 and 2.2 nM in rat. I n the fist report of PHlyohimbine binding in a rat tissue, the authors were concerned about the relatively high potency of prazosin and therefore did not claim that it represented binding to at receptors (Yamada et al., 1980a). However, according to the current definition of a2 receptors (see Section l,B and Table 111) and more extensive pharmacologic studies (Latifpour, 1981), it is correct to refer to rH]yohimbine binding in the rat and neuroblastoma NG 108-15 as a2.Whether this pharmacologic difference represents more than a species difference is not yet known. Rauwolscine is a diastereoisomer of yohimbine that is equipotent with yohimbine at a2 receptors in both functional (Hedler et al., 1981) and binding (Tanaka and Starke, 1980) studies, but is about 50 times less potent than yohimbine as an a,-receptor antagonist (Hedler et al., 1981). Thus, rauwolscine exhibits much greater selectivity toward the a2receptor than yohimbine, and potentially would be a more useful ligand than yohimbine in tissues containing a mixed population of a1 and a2 receptors since yohimbine only shows about a 10-fold preference for a preceptors in many tissues (Hedler et al., 1981; U’Prichard et al., 1977a). Two other yohimbine isomers, corynanthine and ajalmacine, are selectively potent at a1 receptors (Tanaka and Starke, 1980). [3H]Rauwolscine labels a2 receptors in human platelets (Motulsky and Insel, 1982) and rat and bovine cerebral cortex (Perry and U’Prichard, 1981, 1983). In platelets, [3H]rauwolscine and rH]yohimbine label the same number of a2 receptors, but the K, and nonspecific binding of [3 H]rauwolscine are somewhat greater, indicating that in this tissue it is no improvement over THIyohimbine. In bovine cortex membranes, [3 H]rauwolscine binding exhibits higher a h i t y in Na+containing buffers and in Na+/K+ phosphate buffer labels a single population of sites over a 0.1-15 nM concentration range with a KD of 1-2 nM from kinetic, saturation, or competition studies. The pharmacological properties of [3H]rauwolscine binding are those of an a2 receptor, with a yohimbine/prazosin potency ratio of about 0.003 (yohimbine Ki of
386
DAVID B . BYLUND AND DAVID
c. U'PRICHARD
4 n'\f). As with other a,-receptor systems, only agonists interact at the cortex rH]rauwolscine site in a heterogeneous manner (nH = 0.5-0.7), and as in platelets, both Na+ (60 m'tf) and GTP (100 p M ) right shift agonist competition curves, but had no effect on antagonist interactions. These data strongly indicate that the brain a , receptor also exists in (H) and (L) states, differentiated primarily with respect to agonists (Perry and U'Prichard, 1983). Some effects of cations and nucleotides on ~H]rauwolscinebinding are inconsistent with the hypothesis that antagonists have invariant affinity at all a,-receptor states (Perry and U'Prichard, 1983). T h e addition of NaCl increases [3 H]rauwolscine binding with a maximum effect at 50 m,\f (ED50= 5.0 mL\f),while higher Na+ concentrations bring binding back down to control levels. Chloride salts of other monovalent cations only decrease [3H]rauwolscine binding. GTP at low concentrations (0.1 nLllto 0.1 pc\I) also increases [3H]rauwolscine binding, even in the presence of 60 m.\l Na+. This effect is reproduced by Gpp(NH)p and GDP, but not adenine nucleotides and higher guanine nucleotide concentrations which inhibit p H]rauwolscine binding. Divalent cations, which appear to favor the formation of a z ( H ) decreased the binding of [3H]rauwolscine, with Mn2+being more potent than Mg2+or Ca2+.GTP at low concentrations produces a decrease in the ability of (-)epinephrine, and an increase in the ability of yohimbine, to inhibit [3 H]rauwolscine binding. These data suggest that [3 H]rauwolscine and other antagonists could have somewhat different affinities for different states of the a z receptor, and these differences might be detected in the presence of agents which alter the equilibrium between different receptor states (Perry and U'Prichard, 1983). Indeed, when [3H]rauwolscinebinding isotherms in phosphate buffer are extended to a ligand concentration of 50 nL\f,two sites are labeled with K , values of about 1 and 30 n,\l, respectively. The higher KIl value corresponds quite closely to the K , of rauwolscine at cortex sites labeled with [3 Hlagonists. c. [3H]L~\~~rzd~>. The ergoline derivative lisuride has a high affinity at brain a , receptors labeled with [3H]rauwolscine (Perry and U'Prichard, 1983), and it appears to have sufficient selectivity for the a, receptor compared to the a , receptor so that [3H]lisuride in appropriate conditions will specifically identify rat cortex a2 receptors (Battaglia and Titeler, 1980). T h e pharmacological profile of P Hllisuride binding indicates that the ligand, which is probably an antagonist at this receptor, labels both a 2 ( H )and a,(L). As with other antagonist ligands, GTP selectively lowers the affinities of agonist competitors. The usefulness of this ligand seems limited because, in common with other ergots, it also interacts with high affinity at other brain monoamine receptors.
CHARACTERIZATION OF
a1- AND
(Y~-ADRENERGICRECEPTORS
387
d. Intact-Cell Binding. I n homogeneous cell systems, it is advantageous to be able to label receptors on intact cells in order to relate binding to response in a more physiological situation. I n intact cell-binding studies, rHlyohimbine appears to label a2 receptors much better than rH]DHEC. The K , and B,,, of [3H]yohimbine are the same in intact platelet cells and platelet membrane preparations (Motulsky et al., 1980). In competition studies, antagonists had roughly the same potency in both preparations, but agonists were much weaker in inhibiting binding to intact cells and exhibited much steeper competition curves, with n H values approaching 1.O (Motulsky et al., 1980). Furthermore, the potency of (-)-epinephrine at Hlyohimbine sites on intact cells is equivalent to its potency at the membrane sites when assayed in a physiological (Hank’s) buffer (M. Daiguji and D. C. U’Prichard, unpublished). Like other adenylate cyclase-coupled receptors, a2 receptors would be expected to appear predominantly in a low-affiity state when assayed in intact-cell preparations because intracellular GTP (established to be about 10 p M ) would very rapidly destabilize high-affinity (complexed) states of the receptor (see below). In other words, the turnover of the high-affinity state of the receptor is exceedingly rapid under normal physiological conditions, but cell lysis and elimination of endogenous GTP “freezes” the a2 receptor in the proportion of (H) to (L) states existing in the membrane at that time. Thereafter, equilibrium between (H) and (L) states can be manipulated by exposing the membrane to receptor and N-site (nucleotide-binding regulatory protein) ligands, including guanine nucleotides, Mg2+, and more speculatively, Na+. Although the agonist epinephrine has the same potency at [3 Hlyohimbine sites on platelet membranes (in Hank’s buffer) and intact cells, GTP will only further reduce the potency of epinephrine in the membrane preparation, indicating that GTP modulat.es agonist affinities at the receptor by acting at a site on the inner surface of the plasma membrane (M. Daiguji and D. C . U’Prichard, unpublished).
2. Agonist Radioligands a. [3H]Epinephrine and [3H]Norepinephrine. Despite earlier difficulties in establishing receptor specificity of [3H]catecholamine binding, these radioligands have in the past few years proved to be viable and important probes for examining a,-receptor function. According to Lefkowitz, one would expect a [3 Hlcatecholamine at low concentrations to label selectively the (H) state of the a2 receptor and with increasing concentrations to identify an increasing amount of a2(L).Thus, Rosenthal transformations of [3H]catecholaminesaturation isotherms would be
388
DAVID B. BYLUND AND DAVID c . U’PRICHARD
expected to be curvilinear over a wide enough range of ligand concentration. Over a more restricted concentration range, however, Rosenthal plots would appear more linear, and the B,,, value derived from these plots would be more an approximation of the number of a2(H)than of the total number of a2 receptors in a tissue. Initial studies utilized racemic (t)-[3H]epinephrineof low specific activity (10-15 Ci/mmol), as well as (-)-PHInorepinephrine (20-40 Ci/mmol) (U’Prichard and Snyder, 1977a). More recently, the active isomer (-)-f Hlepinephrine at high specific activity (80- 120 Ci/mmol) has become available. Data obtained in the same tissue with these different [3 Hlcatecholamine preparations have been generally quite similar (U’Prichard et al., 1980a). Because labeled and unlabeled (Table 11) norepinephrine exhibit lower affinity than epinephrine for a2 receptors in most tissues, the use of [3H]norepinephrine has been very limited compared to [3H]epinephrine. Because of expense and technical difficulties, many fewer az-receptor-containing tissues have been examined with [3H]catecholamines compared to [3H]imidazolines. Care must be taken to prevent interference through binding of the ligand to p receptors since under typical experimental conditions [3H]epinephrine has selectively high potency for the p2 subtype (U’Prichard and Snyder, 1977b; U’Prichard et al., 1978~). On the other hand, at low ~H]catecholamineconcentrations, interference from a ,-receptor interactions is generally insignificant, apparently because the a,receptor does not usually occur in a high-affinity state (see Section II,A,2). Important precautions must be taken to minimize oxidation of the ligand and to include a large excess of pyrocatechol (0.1-3.0 m;M) to inhibit as much catechol-directed nonspecific binding as possible. [3H]Catecholamines have been used to label a2receptors in rat and bovine brain regions, human platelets, rat liver, and NG 108-15 cells (U’Prichard and Snyder, 1977a, 1978a; U’Prichard et al., 1983; Hoffman et nl., 1980b; Smith and Limbird, 1981; Kahn et nl., 1982). The characteristics of binding of [3H]epinephrine in these tissues is very similar. Generally, monophasic Rosenthal plots of saturation data are observed, giving equilibrium K , values of 1- 10 nM, although dissociation in the absence of nucleotides is biphasic at several incubation temperatures (U’Prichard and Snyder, 197713; U’Prichard et al., 1983). An exception is the dissociation of [3H]epinephrine from NG 108-15 membrane a2 receptors, which appears monophasic (Kahn et al., 1982). In human platelet and NG 108-15 cell membranes, the B,,,, for [3H]epinephrine binding is only 30-60% of the B,,, for an a2antagonist radioligand, which would be expected if at the fairly low concentrations used E3H]epinephrine was selectively labeling a2(H).
CHARACTERIZATION OF (Y1- AND (Y2-ADRENERGIC RECEPTORS
r
389
Analysis of Hlepinephrine competition in these tissues (Table XIII) shows that the ligand labels a, receptors, with yohimbine 100-1000 times more potent than prazosin. Competition studies also show that Hlepinephrine (in the 1-5 nM range) selectively labels az(H). Catecholamines compete in an a,-receptor potency order with (-)epinephrine and (-)-norepinephrine usually having Ki values in the 1- 10 nM range. Imidazoline partial agonists are also about 10-fold more potent competitors at Hlepinephrine sites than at [3 Hlantagonist sites (Hoffman et al., 1980b; U’Prichard et al., 1983), except in cerebral cortex membranes where these drugs have equally high potency (U’Prichard, 1980; Perry and U’Prichard, 1983). On the other hand, some antagonists such as phentolamine have equal potency at Hlepinephrine and [3 Hlantagonist sites, while other antagonists, including yohimbine, WB4101, piperoxan, and prazosin are 10-50 times less potent at [3H]epinephrine sites (U’Prichard et al., 1983). At Hlepinephrine sites, unlike [3 Hlantagonist sites, agonist and antagonist competitors are not discriminated in terms of nHvalues. In brain tissue and NG 108-15 cells, all drugs competed at az-receptor sites labeled with [3H]epinephrine, with n Hvalues of about 1.O (U’Prichard and Snyder, 1977a; Kahn et al., 1982), indicating that at the concentration used Hlepinephrine was almost exclusively labeling aZ(H)sites. However, in platelet membranes the n H values of agonists and antagonists are less than 1.0 at rH1epinephrine sites (U’Prichard et al., 1983). Binding of [3 Hlepinephrine and Hlnorepinephrine to a, receptors has been directly compared only in bovine cortex and rat liver (U’Prichard and Snyder, 1977a; El-Refai et al., 1979). In these tissues, both catecholamines appear to label an identical population of sites, although ( -)-[3H]norepinephrine had only two-thirds the affinity of (*)Hlepinephrine in bovine cortex in saturation experiments (U’Prichard and Snyder, 1977a). A detailed examination of the thermodynamic aspects of agonist and antagonist interactions with the a, receptor has not been undertaken in the same manner as for the p receptor (Weiland et al., 1979). However, the limited information available suggests that for brain az receptors at least, agonist and antagonist interactions can be discriminated on the basis of temperature effects. When equilibrium binding of [3H]epinephrine and [3H]norepinephrine to bovine cortex membranes was measured at 37,25, and 4”C, a decrease in the K , (increasing affinity) of the agonist ligands was observed with decreasing temperature, along with a decrease in the Ki values of agonist competitors and an increase in antagonist Ki values at these sites (U’Prichard and Snyder, 1977a). The number of sites labeled by Hlepinephrine and [3 Hlnorepinephrine was
r
r
r
r
r
r
r
390
DAVID B. BYLUND A N D DAVID c . U’PRICHARD
not affected. On the other hand, the A’,) of the antagonist [3H]rauwolscine at cy2 receptors in the same tissue preparation was identical at 4 and 25”C, whether derived from kinetic or equilibrium experiments (Perry and U’Prichard, 1983). In &receptor systems such as the turkey erythrocyte, agonist interactions appear to be entropy-driven, whereas antagonist interactions are enthalpy-driven (Weiland et nl., 1979). Similar considerations may well apply to a2 receptors. Monovalent cations inhibit [3H]epinephrine and [3H]norepinephrine binding to brain a2 receptors (Greenberg et al., 1978), as would be expected from their ability to reduce agonist potencies at [3H]DHEC sites (Tsai and Lefkowitz, 1978; U’Prichard and Snyder, 1978b). Sodium and lithium are equally active in this regard, whereas larger ions such as K+ and Cs+ are less effective. T h e a,-receptor model proposed by Lefkowitz and co-workers would also predict that guanine nucleotides directly inhibit [3H]catecholamine binding to a2 receptors if these ligands are predominantly labeling a2(H).Nucleotides inhibit steady-state binding of [3H]epinephrine and [3 Hlnorepinephrine at bovine cortex a2 receptors (U’Prichard and Snyder, 1978b) and also [3H]epinephrine binding at a 2 receptors in rat cortex (U’Prichard and Snyder, 1980), NG 108-15 cell membranes (Kahn ot cil., 1982), and human platelets (U’Prichard ct ol., 1983). The potency order of nucleotides is quite consistent in different tissues: Gpp(NH)p 2 GTP = GDP > ITP > ATP = CTP = ATP > GMP (CT’Prichard ot 01.. 1983). For PHIepinephrine, the apparent ED,, of Cpp(NH)p and GTP is 1- 10 p.11 in neural tissue, and nucleotides seem more potent in bovine as compared to rat cortex membranes (U’Prichard and Snyder, 1980). Gpp(NH)p and GTP are potent inhibitors of [”]epinephrine binding to platelet a2 receptors with EDso values about 0.1 pL21 (U’Prichard ~t nl., 1983). In brain membranes, GTP increases the apparent K,)of [3H]epinephrine with no change inB,,, and accelerates both association and dissociation when added at the onset of labeling, or after steady state was achieved (U’Prichard and Snyder, 1978b). These effects of GTP on agonist binding parallel results obtained with radioagonist binding to a variety of other cyclase-coupled receptors (Lin P t nl., 1977; Williams and Lefkowitz, 1977a; Blume, 1978). However, the observed effects of GTP are not completely consistent with those predicted by the model. If PHIepinephrine labels a 2 ( H )and GTP simply alters the equilibrium between a,(H) and a2(L),then one would predict that in saturation experiments, GTP would reduce the B,,,, of [3Hlepinephrine binding without changing the K , of Hlepinephrine at residual (H) states being labeled. Likewise, if the two phases of [3H]epinephrine dissociation were taken to represent interactions with
CHARACTERIZATION OF
al-AND
(~2-ADRENERGICRECEPTORS
39 1
az(H)and a Z ( L )sites, the presence of GTP during labeling of the receptor should alter the relative amounts of the rapidly and slowly dissociating components of [3H]epinephrine binding, but not change the k - , values for each phase of dissociation. In both cases, the observed effects were opposite to those predicted by the theory. The interactive effects of guanine nucleotides and divalent cations on [3H]epinephrine binding to a2 receptors are complex and appear to be somewhat different for brain and platelet a2 receptors, although these differences may be simply explained by the different capacity of membranes from different cells to sequester cations. In platelet membranes, which are prepared by routine lysis in an EDTA-containing buffer, Mg2+ increases [3H]epinephrine binding (U’Prichard et al., 1983), an effect which parallels the left shift in the epinephrine competition curve at [3H]yohimbinesites in the presence of Mg2+(Fig. 3). On the other hand, Mg2+does not increase Hlepinephrine binding to cortex membranes prepared in the absence of EDTA (U’Prichard and Snyder, 1980),or to NG 108-15 cell membranes prepared in the presence or absence of
\ 40
- ‘* -
:
-30-
< 01
\* \
\
*\
FIG. 3. Effect of 1.0 mM MgCl, on the saturation characteristics of (-)rH]epinephrine binding to platelet membrane a) receptors (Rosenthal plot). (0),N o MgC1,; ( O ) , 1 mM MgCl,.
392
DAVID B . BYLUND AND DAVID
c . U’PRICHARD
EDTA (D. J. Kahn and D. C. U’Prichard, unpublished). Whereas in platelet membranes the ability of GTP and Gpp(NH)p to inhibit PHIepinephrine binding is enhanced in the presence of Mg2+, in brain membranes Mg2+and Ca2+not only reduce the ability of GTP to inhibit binding, but in the presence of these ions (1.O mM), low concentrations of GTP actually increase Hlepinephrine binding (U’Prichard and Snyder, 1980). Similarly, in NG 108-15 membranes prepared in the absence of EDTA, GTP and GDP (0.1-10 p M ) enhance [3H]epinephrine binding before reducing binding at higher concentrations (Kahn ~t nl., 1982). However, in NG 108-15 membranes prepared in the presence of EDTA, GTP and GDP only inhibit [3H]epinephrine binding (D. J. Kahn and D. C. U’Prichard, unpublished). These data indicate that in brain and NG 108-15 membranes, unlike platelet membranes, divalent cations antagonize the effects of GTP and GDP on agonist interactions with the az receptor. A more detailed analysis of PHIepinephrine binding to platelet aZ receptors indicates that there may be two high-affinity states of the receptor at which rH]epinephrine interacts,\ as well as the a 2 ( L ) state (U’Prichard uf nl., 1983; Mitrius and U’Prichard, 1983). In the presence of 1.0-mhf MgC12, the Rosenthal plot of [3H]epinephrine binding appears slightly curvilinear over the concentration range (up to 40 nM) of [3H)epinephrine used (Fig. 3). T h e K , of the linear portion of the curve is approximately 6 nltf corresponding to the K, value (11 nM) for the a z ( H )site derived from analysis of epinephrine inhibition of rH]yohirnbine binding data (Hoffman et GI., 1982). However, the apparent deviation from linearity cannot be due to [3H]epinephrine binding to the a,(L) state ( K D= 520 niV, Hoffman et nl., 1982) since theoretical calculations show a significant (10%)deviation from linearity only at concentrations of PHIepinephrine in excess of 150 nLU,which is well above the 40 n‘bf used. These saturation data (Fig. 3) are too scattered at the higher radioligand concentrations to estimate a K, value for the lower affinity site. However,K, values can be calculated from kinetic data. T h e association of VHIepinephrine to platelet a 2 receptors is monophasic, but the dissociation is biphasic. If one assumes that the biphasic dissociation corresponds to two affinity states [however other models, such as one based on bivalent ligand hypothesis (Minton, 1981) are just as reasonable], K , values of 2 and 35 nltf can be derived. Omission of Mg2+ reduced the number of [3 Hlepinephrine sites, reduced the overall affinity of [3H]epinephrine by a factor of 2, and eliminated the curvilinearity of the Rosenthal plot, such that observable [3 Hlepinephrine binding was to a single population of receptors with a K, of 12.8 nM (Fig. 3). Similarly, with the addition of 10 F M GTP in the presence of 1.0 mA4 Mgz+, the Rosenthal plot of PHlepinephrine binding was linear, with a some-
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CHARACTERIZATION OF ff 1- AND ff2-ADRENERGIC RECEPTORS
393
what reduced B,,, and a K , of 30 nM (U’Prichard et al., 1983). The omission of Mg2+or the presence of GTP reduced the absolute amount of the slowly dissociating component of Hlepinephrine binding, but did not change the k-, values for the two components. U’Prichard and co-workers suggest that [3 Hlepinephrine, in addition to labeling in platelet membranes a2(H) ( K D = 30-40 nM) and perhaps a small portion of a2(L)( K , = 300-500 nM), may label another state that, by analogy to agonist interactions with muscarinic cholinergic receptors (Birdsall et al., 1978; Ehlert et al., 1980), can be called the “super high” state of the receptor [a2(SH)with KD values in the range of 2-6 nM]. T h e “a2(H)” conformation of the platelet receptor ascertained from computer modeling of [3H]yohimbine competition curves (Hoffman et al., 1982) could be a composite of a2(SH) and a2(H), as it is doubtful whether such analysis of competition curves in this system could significantly differentiate a three-state from a two-state receptor model. Unfortunately, [3H]epinephrine binding data at NG 108-15 a2receptors is also not accurate enough to resolve putative a 2 ( S H ) and a2(H)states in that system. b. [3H]Clmidine and [3H]p-Aminoclmidine.Although the imidazoline ligands have been used much more extensively than [3H]epinephrine, these ligands label sites representing high-affinity state(s) of the a2 receptor whose properties are very similar to those sites labeled by Hlepinephrine. [3 HIImidazoline binding will be considered, therefore, in less detail. In common with most other imidazolines, clonidine and p-aminoclonidine exhibit partial agonist activity in several assays of a2receptor function (Starke et al., 1974; Kahn et al., 1982; Atlas and Sabol, 198 1). [3 HIClonidine and H]p-aminoclonidine have been widely used to label a2 receptor sites (Table XIV). Interestingly, the presence of a2 receptors in some classical sympathetically innervated tissues such as rat heart and vas deferens has not yet been conclusively demonstrated using [3 Hlclonidine or H]p-aminoclonidine, although this has been a goal in many laboratories. There is a fair degree of consistency as to the characteristics of [3H]imidazoline binding in all these tissues throughout many laboratories (Table XIV). [3 HIImidazoline-specific binding is saturable with K , values usually in the 1-3 nM range and B,,, values from 20 to 460 pmol/gm prot. Rosenthal plots are generally reported as monophasic, but in some tissues there are increasing reports of curvilinearity (Atlas and Sabol, 198 l ; Garcia-Sevillaet al., 1981b; U’Prichard et al., 1983; Braunwalder et al., 1981). [3H]Imidazoline appears to bind to high-afiity states of the a2 receptor since the K i of prazosin is generally 100-1000 times greater than the K i of yohimbine, and (-)catecholamines have K i values in the 1-10 nM range. Furthermore, the binding is generally directly inhibitable by guanine nucleotides.
r
r
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13 ,,,a\ '
'Tissue Whole hrain" Cerebral cortex Cerebral cortex Cerebellum Corpus striatum Spinal cord Submandibular Submandibular Submandibular Submandibular Sublingual Neuroblastonia X glioma Neuroblastoma X glioma Kidney Ileum Cerebral cortex Retina Cerebral cortex Platelets Platelets Adipocytes Adipocytes a
Species
fritiated ligantl
K,,
pnioYgm protein
Ra t Rat
Clonid i n r
Rat
K dt
Clonidine Clonidine Clonidine Clonidine Clonidine Clonicline Clonidine PAC Clonidine
266 t 37 235 t 19 66 t 7 72 2 5 205 1 27 2 3 150 t 25 378 t 2 460 t 33 287 t 69
Rat- mouse
Clonidine
81; 155
Rat-mouse Guinea pig Guinea pig Pig Bovine Bovine Human Human Human Human
PA c Clonidine Clonidine PAC Clonidine Clonidine Clonidine Clonidine Clonidine PAC
105 t 42
Rat Rat Rat R dt Rat?
Ratd Rat"
P'4 C'
Minus cerebellum.
* Sites per cell.
Chronic reserpine administration.
34 85 t 7 34 1
pmoligm tissue
( I1'\f )
14 13 t 2 14 t 1 4.5 2 0.6 4.6 f 0.5
5.8 0.87 t 0.25 1.8 t 0.1 1.9 2 0.2 1.6 t 0.8 2.1 t 0.2 2.4 t 0.2 1.9 t 0.3 3.7 t 0.5 2.8 t 0.2 2.1 t 0.4
U'Prichard 1.t i l l . (1977a) Rouot and Snyder (1979) D. B. Bylund (unpublished) D. B. Bylund (unpublished) D. B. Bylund (unpublished) Jones vt d. (1982) Pimoule PI ci/. (1980) Bylund and Martinez (1980) Bylund PI rrl. (1982a) Bylund P / I ] / . (1982a) Martinez I,/ I ] / . (1982b)
1.7; 33
Atlas and Sabol ( 1 98 1)
6.2 t 0.9 17 t 3 202 1 9.9 t 1.3
8600 t 23OOb 22 t 2 4.2 +- 0.3 8
35 t 3 64 t 4 348 t 10 166 t 26
Two-weeks old. p-Aminoclonidine.
1.8 ? 0.6 9.0 t 0.8 2.1 1.2 t 0.1 0.32 1.o 5.0 t 0.5 24 t 2 3.9 t 0.2 0.49 t 0.04
Reference
Kahn rt i l l . (1982) Summers (1980) Tanaka and Starke (1979) Harris ct nl. (1983) Bittiger PI nl. (1980) U'Prichard and Snyder (1979) Garcia-Sevilla rt a/. ( 198 1b) Shattil r / 01. (1981) Berlan and Lafontan (1980) Burns et al. (1981)
CHARACTERIZATION OF (Y1- AND a2-ADRENERGIC RECEPTORS
395
In the few tissues where [3H]catecholamine and [3 Hlimidazoline binding to a2 receptors have been directly compared, such as bovine cortex (U’Prichard and Snyder, 1977a), rat cortex (U’Prichard et al., 1979a), human platelets (U’Prichard et al., 1983), and NG 108-15 cells (Kahn et al., 1982), it appears that [3H]clonidine and r H ] p aminoclonidine over restricted concentration ranges label the same high-affinity sites of the a2 receptor as PHIepinephrine, but generally bind to a smaller proportion of these sites. This might suggest that intrinsic activity of agonists at the a2 receptor can be related to the extent of formation of high-affinity states, as is the case for /3 receptors (Kent et al., 1980). However, the analysis of H-labeled antagonist-agonist interactions by Hoffman et al. (1982) indicates that there is no such correlation. In EDTA-treated platelet membranes, [3 H]p-aminoclonidine saturation in the presence of 1.0 mM M 8 + is more markedly biphasic than that of Hlepinephrine. If the curvilinear Rosenthal plot is interpreted to represent two binding sites, KD values for the two sites can be estimated. Approximate KD values of 0.3-0.7 nM and 3-10 nM were obtained by drawing straight lines through the linear portions of the curve (U’Prichard et al., 1983). By this type of analysis, neither site seems equivalent to the KD of p-aminoclonidine at the a 2 ( L ) state as determined by [3H]antagonist binding which is 80-100 nM (Fig. 2). However, this method of determining KD values underestimates the KD for the low-affinity (high KD) component (Minneman et al., 1979). A more careful analysis using the graphical technique of limiting slope coupled with replotting of derived curves suggests that the data from several experiments are best fit to two sites having KD values of 0.3-0.5 nM and 10-17 nM. On the other hand, [3H]p-aminoclonidine dissociation, like rHIepinephrine, is biphasic, and if the two-site model is assumed, KD values from kinetic experiments are 0.2 nM and 2-3 nM (Mitrius and U’Prichard, 1983). Thus, while there is some evidence from [3 H]p-aminoclonidine binding to platelet membranes which can be interpreted to indicate SH and H states of the a2 receptor, much more experimental evidence is needed to establish the model. I n the absence of M 8 + , or in the presence of 10 p M GTP, the Rosenthal plot of [3H]p-aminoclonidine binding is linear, with a KD value of 3-10 nM. Neither treatment alters the kl values of each component of rH]paminoclonidine binding. The number of sites labeled by ?HIP-aminoclonidine is equivalent to 50% of the a,-receptor sites labeled by [3 Hlyohimbine, whereas the full agonist [3H]epinephrine labels 80% of the rH]yohimbine sites (Mitrius and U’Prichard, 1983). Competition studies show that H]p-aminoclonidine labels the same
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DAVID
B. BYLUND
A N D DAVID
c.
U’PRICHARD
r
high-affinity sites of the platelet a2 receptor as Hlepinephrine (Table XIII). For both ligands, the i z H values of all competitors are less than 1.0, indicating that even at the low ligand concentrations used, [3H]epinephrine and [3H]p-aminoclonidine may be labeling more than one state of the receptor. In the presence of 1.0 p M GTP, IC,, values for agonists at ~H]p-aminoclonidineand VHIepinephrine sites increase two- to threefold, but IC,,, values for the antagonist yohimbine decrease two- to threefold. In neither case is there a significant change in n H values. PHJClonidine binding to the platelet a2 receptor has not yet been studied in quite as much detail, but the available evidence is that it binds to high-affinity a,-receptor sites, which constitute 40-60% of the total receptor population measured with antagonist radioligands. These sites are also nucleotide-sensitive (Garcia-Sevilla et al., 198lb). In NG 108-15 cell membranes, [3 H]p-aminoclonidine appears also to label the same high-ahity a2-receptor sites as VHIepinephrine (Table XIII), but as with [3H]epinephrine binding in this system, putative SH and H states cannot be resolved. At low concentrations, [3H]paminoclonidine saturation and dissociation are monophasic (Kahn et nl., 1982). Guanine nucleotide influences are similar for both ligands, with a bimodal effect of GTP and GDP that was converted to unimodel inhibition of binding after pretreatment of membranes with EDTA, whereas Gpp(NH)p in both conditions only inhibited binding (Kahn et al., 1982; D . J. Kahn and D. C. U’Prichard, unpublished). The tissue wherein rH]clonidine binding to a2 receptors has been most extensively examined has been the rat brain, especially the cerebral cortex (U’Prichard et al., 1979a; Glossmann and Presek, 1979; Glossmann and Hornung, 1980b; Summers et al., 1980; Braunwalder et ~ l . ,1981). Early studies of Hlclonidine binding used low-specificactivity radioligand and could only demonstrate monophasic saturation and dissociation. This represents high-affinity a,-receptor sites (catecholamine K , values 5-20 nlM), but was not recognized as such (U’Prichard et nl., 1977a). With the advent of higher specific activity ligand, saturation and dissociation were seen to be biphasic (U’Prichard et nl., 1979a; Vetulani et ol., 1979). In competition studies, agonists were more potent at the higher affinity component, while some antagonists were less potent (U’Prichard et al., 1979a). These data were interpreted as possibly representing different populations of a2receptors on the basis of different regional distribution throughout the rat brain, and difTerent regulation in 6-hydroxydopamine-treated animals (U’Prichard et al., 1979a). At present, a more likely interpretation of these data is that [3H]clonidine in the brain labels two states of the same receptor population, but the complexity of brain tissue and other evidence suggesting
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CHARACTERIZATION OF a1- AND Q2-ADRENERGIC RECEPTORS
397
the existence of both pre- and postsynaptic a2 receptors (which may be coupled to different effectors, or to the same effector with varying efficiency) will make interpretation of a2-agonist-binding data difficult for some time to come. Glossmann and colleagues have identified four or five affinity states of the rat cortex a, receptor labeled with [3H]clonidine on the basis of different thermal stability and Mg2+ sensitivity (Glossmann and Presek, 1979; Glossmann and Hornung, 1980b). The more slowly dissociating phase of cortical [3 Hlclonidine binding is selectively inhibited by guanine nucleotides by reducing the number of sites without altering the K , of Hlclonidine. Guanine nucleotides do not appear to affect the other component of binding, and the K-, values for the two phases of [3H]clonidine binding are not changed by the presence of nucleotides and divalent cations (Rouot et al., 1980). However, Mg2+increases [3H]clonidinebinding to the highest affinity state of the receptor in EDTA-treated membranes (Glossmann and Presek, 1979; Glossmann and Hornung, 198Oc),and more recent evidence indicates that 10 mM M 8 + will increase the number of putative (SH) states labeled by Hlclonidine in cortical membranes not pretreated with EDTA (Salama et al., 1982). It is clear from these and similar data that the extent of clearance of endogenous ligands for the a2receptor and its associated N site from the membranes prior to binding assay plays a critical role in the type and extent of interaction of ions and nucleotides with 3H-labeled agonist a2 receptor sites. In the presence of 1.0 mM divalent cations, GTP increases [3 Hlclonidine binding in cortex (U’Prichard and Snyder, 1980; Rouot et al., 1980), while in the presence of chelating agents, GTP becomes more potent in inhibiting pH]clonidine binding (U’Prichard and Snyder, 1980). Sodium, unlike GTP, reduced rH]clonidine binding in rat cortex (Rouot et al., 1980). In summary, if we compare the characteristics of Hlclonidine binding itself, and of full agonist (catecholamine)competition at sites labeled by rHIclonidine (U’Prichard et al., 1979a) and H]rauwolscine (Perry and U’Prichard, 1983),it seems probable that the cerebral cortex a2receptor exists in multiple affinity states, but the distribution of these states among different cortical a,-receptor populations is still a matter for conjecture. Glossmann and Hornung (1980b) noted that the antagonist prazosin had a lower affinity for the highest affinity state of the rat cortex a2 receptor labeled with Hlclonidine and that prazosin interactions with Hlclonidine sites were apparently noncompetitive in nature. More recently, 10 mM Mg2+ was observed to reduce the affinity of some antagonists such as piperoxan, yohimbine, WB410 1, and prazosin as much as sixfold at rat cortex rHIclonidine binding sites, whereas the affinity of other antagonists such as phentolamine was unaffected (Salama et al.,
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DAVID B . BYLUND AND DAVID
c . U’PRICHARD
1982). hssuming that Mg2+was increasing the ratio of putative SH to H components of [3H]clonidine binding, these data were taken as evidence that the former group of antagonists exhibited preferential affinity for a 2 ( H )compared to a2(SH),whereas phentolamine had equal affinity at both states. a2 Receptors on membranes from human adipocytes from properitorieal adipose tissue have also been characterized using [3 HIPaminoclonidine (Burns rt d.,1981, 1982a). In these studies, [3H]paminoclonidine exhibited high affinity ( K E )of 0.5 to 1.2 nM), and the number of az receptors labeled by [3 H]p-aminoclonidine was approximately 50% of the az receptors labeled by VHIyohimbine. Adipocytes from this source exhibit responses to catecholamines of stimulation of adenylate cyclase activity and glycerol production, inhibition of adenylate cyclase and glycerol production, and stimulation of phosphatidylinositol turnover, which were shown to be associated with p , a z , and a , receptors, respectively (Burns et al., 1981). In other studies concerning adipocyte receptors, analysis of competition of [3 HIDHEC binding by subtype-selective antagonists indicated that the great majority of a receptors were of the a2 subtype in adipocytes from human subcutaneous adipose tissue (Hoffman rt ol., 1979, 1980a) and in hamster adipocytes (Garcia-Sainz P t nl., 1980b). r . [ 3 H ] G ~ i a n f u c u ~Tr .h e tritium-labeled a 2agonist and antihypertensive drug guanfacine has also been used in radioligand binding studies. [3H]Guaiifacine bound to rat brain membranes with a K , of 4 nM and with properties characteristic of a2receptor binding sites (Timmermans P f d., 1982). However, its usefulness may be limited by a relatively high amount of nonspecific binding.
The initial cellular response to activation of aZ receptors in all cell systems s o far examined is inhibition of plasma membrane adenylate cyclase and a consequent decrease in intracellular CAMPlevels. Thus, the a2 receptor falls into the recently recognized large class of receptors that are coupled to adenylate cyclase in an inhibitory manner, including muscarinic cholinergic, opiate, adenosine A1 and dopamine D2 receptors, and receptors on adipocytes for nicotinic acid and prostaglandins of the E series (Jakobs, 1979). T h e mechanisms of coupling of these receptors to adenylate cyclase have recently been discussed by Jakobs ( 1979) and Limbird (1981), and many authors have compared the effects of nucleotides and other agents on the receptor-mediated response of inhibition and stimulation of adenylate cyclase. T h e sequence of events relat-
CHARACTERIZATION OF a1- AND (Y2-ADRENERGIC RECEPTORS
399
ing receptors to adenylate cyclase stimulation in relatively pure cell membranes (e.g., p receptors in frog and turkey erythrocytes and mouse S49 lymphoma cells, and glucagon receptors in rat hepatocytes) has been well described. I n these systems, receptor-mediated stimulation of the enzyme is dependent on the presence of GTP acting at intermediary N protein(s). Stimulation of cyclase is “turned off’ by the hydrolysis of GTP to GDP which is catalyzed by GTPase activity associated with N. Thus, GTP can be viewed as the primary cellular ligand for adenylate cyclase stimulation, and receptor agonists activate cyclase in a GTP-dependent manner by accelerating the GTP-GDP-GTP cycle (Ross and Gillman, 1980). Nonhydrolyzable analogs of GTP, for example, Gpp(NH)p, Gpp(NH,)p, and GTPyS, activate adenylate cyclase in an apparently irreversible manner (Londos ~t al., 1977), behaving similarly to GTP in the presence of cholera toxin, which inhibits GTPase activity and catalyzes the ADP-ribosylation of N proteins (Moss and Vaughan, 1977; Gill and Meren, 1978). These GTP analogs stimulate the enzyme after an initial lag period which is thought to be the time needed for release of inactive GDP from N; this lag period is decreased in the presence of agonists that stimulate cyclase (Birnbaumer et al., 1980). Beta agonists also stimulate GTPase activity in membranes probably by both directly increasing the V,,, and by accelerating GDP-GTP exchange (Limbird, 1981). T h e basic characteristic of receptors (including a,-adrenergic receptors) that are negatively coupled to adenylate cyclase is that stimulation of these receptors causes a decrease in the V,,, of the enzyme without changing the K , for the Mg . ATP substrate or for free Mg2+. This response, like stimulation of cyclase, requires GTP, but in higher concentrations (> 1.0 p M ) and is either dependent on, or merely facilitated by, Na+. It has been suggested that inhibition of cyclase by activation of these receptors may be due directly to increases in GTPase activity (Limbird, 1981; Koski and Klee, 1981). Rodbell and co-workers made the fundamentally important observation that in adipocytes and other tissues GTP itself has a bimodal effect on adenylate cyclase activity, stimulating at low (10- 100 nM) and inhibiting at high (1.0- 10 p M ) concentrations. T h e effects of inhibitory receptors also require GTP to be in the high concentration range (Londos et al., 1978). Rodbell (1980) has suggested that coupling proteins for inhibitory receptor systems ( N i )are physically different from the coupling proteins for stimulatory systems (Ns). I n cells such as adipocytes, where a bimodal GTP function has been demonstrated, agents such as cholera toxin, mercurials, divalent cations,, and trypsin selectively abolish one or the other phase of GTP function, and GTP has very different potencies at each phase (Cooper et al., 1979).
400
DAVID B . BYLUND AND DAVID c . U'PRICHARD
Inhibition of adenylate cyclase by a2-receptor agonists has been shown to occur in plat'elets (Jakobs et al., 1978a,b; Jakobs and Schultz, 1979), human and hamster adipocytes (Burns and Langley, 1975; Aktories et al., 1980, 1981; Jakobs and Aktories, 1981; Garcia-Sainz et al., 1980b), rat hepatocytes (Jard et nl., 1981), and cultured rodent neural cells such as the NG 108-15 hybrid (Sabol and Nirenberg, 1979a). I n other tissues such as arterial smooth muscle (Anderson, 1973; Buonassisi and Venter, 1976), rat myocytes (Watanabe et a/., 1977) and glial cultures (van Calker et nl., 1980; McCarthy and deVellis, 1978), parathyroid cells (Brown et d.,1978), and pancreatic islet /3 cells (Nakaki et ul., 1981), there is also evidence, albeit less substantial, that a2 receptors mediate inhibition of adenylate cyclase. T h e classical pharmacological response associated with a2receptors is inhibition of the release of norepinephrine into central adrenergic and peripheral sympathetic synapses (Langer, 1974). Although the pharmacological characteristics of this response are very similar to those of the cyclase inhibitory response (Starke, 1981), there is as yet no evidence to show that in nerve terminals, inhibition of adenylate cyclase causes decreased release of norepinephrine. However, the Occurrence of a lag time between a2-receptor occupation and altered norepinephrine release might support the existence of a second messenger for this response (Story Pt al., 1981). a,-Receptor function in the brain and autonomic ganglia has also been defined electrophysiologically. a2 Receptors mediate hyperpolarization and decreased firing of norepinephrine-containing cells in the locus coeruleus (Svensson et nl., 1975) and hyperpolarization of postganglionic cells causing slow IPSP (Tokimasa et al., 1981). In many cells, a2-receptor activation causes an increase in cGMP levels (Anderson, 1973) that is presumably mediated via increased free intracellular calcium. T h e relationship between this response and inhibition of adenylate cyclase is unclear. a-2-Receptor-mediated inhibition of adenylate cyclase has not yet been conclusively demonstrated in brain membranes, probably because brain monoamine receptors generally are very inefficiently coupled to cyclase in cell-free preparations (Maguire et nl., 1977). In other cells, differences can be detected in a2 receptor coupling to cyclase depending on whether activity is measured in intact cells or lysates. Stimulation of a2 receptors in intact platelets or NG 108-15 cells reduces CAMPlevels by as much as 90% (Lichtstein et al., 1979), but in cell lysates adenylate cyclase activity is rarely reduced by at agonists by more than 50% at maximally effective concentrations (Jakobs, 1979). I n lysates from cells containing a2 receptors, imidazolines such as clonidine are partial agonists, with the exception of platelet membranes where clonidine has no efficacy uakobs,
CHARACTERIZATION OF
al-AND
GQ-ADRENERGIC RECEPTORS
40 1
1978). However, in intact platelets, clonidine will reduce cAMP levels elevated by PGIz, which indicates that it exhibits some efficacy in a more efficiently coupled system (Lenox et al., 1980). Some further characteristics of the a2-receptor response in three well-studied systems are discussed below. 1. Human Platelets Activation of human platelet a2receptors stimulates the aggregation response (BartheI and Markwardt, 1974), lowers cAMP levels in intact cells (Kafka et al., 1977), and decreases adenylate cyclase activity in membrane preparations (Salzman and Neri, 1969). Jakobs and colleagues (1978a) found that inhibition of basal platelet cyclase activity, or activity stimulated by fluoride, PGE, , or adenosine, is GTP dependent in membranes. A concentration of GTP of 1.0 p M or more appears to be necessary (Jakobs and Schultz, 1979). While epinephrine could inhibit the twofold increase in cyclase activity caused by GTP in cholera toxintreated platelet membranes, no az-agonist inhibition of Gpp(NH)p- or GTPyS-stimulated cyclase was observed, and therefore the authors suggested that activation of az receptors might decrease cyclase activity by directly stimulating GTPase (Jakobs and Schultz, 1979). Steer and Wood (1979) found that in platelet membranes purified by the method of Barber and Jamieson (1970), epinephrine or GTP alone could only inhibit cyclase activity very slightly, whereas epinephrine plus GTP had a synergistic effect to inhibit activity by a maximum of 50%. Guanosine triphosphate has a bimodal effect on adenylate cyclase, with concentrations below 1.O pM stimulating and higher concentrations inhibiting. On the other hand, stimulation of cyclase by PGE appeared to be GTP independent. PGE, reduces the lag time before the onset of Gpp(NH)p stimulation. In the presence of Gpp(NH)p, no a2response is observed, but if membranes are preactivated with Gpp(NH)p, both GTP and epinephrine could inhibit the enhanced cyclase activity. These data together suggest the occurrence of distinct Ni and N, sites in platelets (Steer and Wood, 1979). I n further studies (Steer and Wood, 1981), Na+ and other monovalent cations in purified membranes reduced basal and PGE-stimulated cyclase (Na" > Li+ > K+),but did notchange the K , for Mg * ATP or Mg2+. a-2-Receptor-mediated inhibition of adenylate cyclase in this preparation was not Na+ dependent, in that the ion did not alter the maximal fractional inhibition caused by epinephrine. However, Na+ did increase the K , of epinephrine and K , of PGE, suggesting that here it may have a generalized uncoupling effect. As in adipocytes (Jakobs and Aktories, 1981), low Mn2+ was found to preferentially un-
402
DAVID B. BYLUND AND DAVID
c. U’PRICHARD
couple inhibitory receptors and thus 1.0 m,Lf MnZ+ attenuated azreceptor inhibition of cyclase without affecting PGE stimulation. Since 1.0 mL\l Mn2+did not, when compared with 6.0 miM Mg2+ (which supports the inhibitory response), alter the characteristics of epinephrine or Gpp(NH)p interactions at platelet a2-receptor sites labeled with PHlyohimbine, it was concluded that Mn2+ uncouples the system by affecting N-C, and not R-N, interactions (Hoffman et nl., 1981b). 2.
Adiporite\
In hamster adipocytes, catecholamines inhibit CAMP and glycerol production via an a,-receptor interaction, and in this system clonidine is a potent and efficacious agonist (Garcia-Sainz et nl., 1980a). Jakobs and colleagues have extensively characterized the a2 and other inhibitory responses in membranes from hamster adipocyte ghosts. Basal adenylate cyclase activity is inhibited by az agonists [ K , = 3.0 p M for (-)epinephrine and 10 pL\l for (-)-norepinephrine], E series prostaglandins, and nicotinic acid, all of which are antilipolytics, while cyclase is stimulated b) ACTH and p agonists. Inhibition is GTP dependent in the range 1- 10 pL\I GTP, and GTP alone inhibits activity. Sodium stimulates the GTP-dependent component of activity eightfold (unlike platelets, see above), and expression of inhibitory receptor activity was also Na+ dependent in this system. Inhibitory receptor responses were abolished partially by fluoride (10 mill) and completely by Gpp(NH)p (Aktories ~t nl., 1980). As in platelets, increasing Mn2+concentration from 0.05 to 1.O mdLlcompletely abolished the inhibitory effects of GTP and a2 agonists on hamster adipocyte cyclase, and the ED, for Mn2+in uncoupling the inhibitory response (again suggested to involve N-C, not R-N, interactions) was 0.1-0.2 mA\d,whereas Mg2+uncoupled at 10-20-mM concentrations (Jakobs and Aktories, 1981). T h e potency order of monovalent cations in increasing GTP (10 pM)-inhibited enzyme, or in decreasing @agonist or ACTH-stimulated enzyme, was Na+ > Li+ > K+. As in platelets, Na’ appeared to increase the K , of the stimulatory hormone ACTH. Guanyl-5’-yl imidodiphosphate, like GTP, lowered cyclase activity, but did not support a2-receptor inhibition, and in the presence of Gpp(NH)p, cation stimulation was less pronounced (Aktories et nl., 1981). The authors reached the important conclusion that monovalent cations interact at the N site(s) to accentuate N,-mediated, and impair N,-mediated, effects. T h e same group has recently shown that in purified rat liver membranes, cations support GTP-stimulated cyclase (Li+ > Na+ > K+), and this activity is inhibited via a2-receptor interactions (Jard ut ~ 1 . .198 1). In human adipocytes catecholamines have historically been known to
CHARACTERIZATION OF (Y1- A N D (Y2-ADRENERGIC RECEPTORS
403
inhibit cAMP production and lipolysis through an a receptor as well as stimulate cAMP production and lipolysis through a p receptor (Robison et al., 1972). Epinephrine, which activates both a 2 and /3 receptors, increases cAMP levels only about eightfold above basal. Stimulation of only p receptors (either by isoproterenol or epinephrine plus yohimbine) increases cAMP levels about 100-fold, whereas a2receptor activation (clonidine or epinephrine plus propranolol) decreases basal cAMP levels 40-50% (Burns et al., 1981, 1982a). Thus the @stimulated activity is more effectively inhibited by a2 receptors than is the basal activity. Prazosin at concentrations as high as 10 p M is ineffective in inhibiting the a2 response. Similar effects of a2inhibition are seen on adenylate cyclase activity and lipolysis. Other investigators have found that a2 inhibition of membrane basal or PTH-stimulated adenylate cyclase achieves a maximum effect of 30-50% decrease (Kather et al., 1980), with Ki values for (-)-epinephrine and (-)a-methylnorepinephrine of 2.0 and 7.0 p M , respectively. Forskolin has been shown to activate adenylate cyclase in membrane preparations and to increase cyclic AMP levels in intact cells of a variety of tissues (Seamon et al., 1981). The effects of forskolin appear not to be mediated by N, (Seamon and Daly, 1981), although the precise locus of action of forskolin remains unclear (Forte et al., 1982). Forskolin caused a dose-dependent 100-fold increase in the intracellular concentration of cyclic AMP and a 6-fold increase in glycerol release in the human adipocyte. Alpha-2-adrenergic activation (epinephrine plus propranolol) significantly inhibited forskolin-stimulated cyclic AMP accumulation and glycerol release, shifting the dose-response curves approximately 8-fold and 5-fold to the right, respectively (Burns et al., 1982b). It appears that forskolin will be a useful tool in elucidating the mechanism of action of the a,-adrenergic receptor. 3. NG 108-15 Cells Mouse neuroblastoma X rat glioma hybrid cells NG 108-15 ( 108CC15) in culture have “cholinergic” characteristics in that they take
u p choline and synthesize and release acetylcholine (dibutyryl CAMPdifferentiated cells) when stimulated (Hamprecht, 1977). These cells contain receptors for PGEl, adenosine (A2), secretin, and glucagon that stimulate adenylate cyclase, and a2-adrenergic, &opiate, muscarinic, and somatostatin receptors that inhibit cyclase (Propst et al., 1979). Activation of a2 receptors in NG 108-15 membranes reduces basal and PGE,stimulated cyclase activity, and the inhibition occurs without any observable lag time. a2, opiate, and muscarinic inhibition of cyclase in NG 108-15 membranes is GTP dependent (Blume et al., 1979; Kahn et al.,
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1982). The K, values for (-)-epinephrine and (-)-norepinephrine are 0.2-0.5 pXI, similar to other a2-receptor systems discussed above. Recently, a bimodal effect of Gpp(NH)p on cyclase activity in NG 108-15 membranes has been seen, with very low Gpp(NH)p concentrations inhibiting the activity (Propst and Hamprecht, 1981). This has also been found with cyclase activity in rat hippocampal membranes (Girardot et al., 1983). Alpha-:! agonists also inhibit cholera toxin-stimulated cyclase. These findings are adduced to support the existence of separate N, and N, in NG 108-15 membranes (Propst and Hamprecht, 1981). Analysis of the coupling of a2 and other receptors to adenylate cyclase may be inherently more difficult in neural tissue than in platelets or adipocytes because there is abundant evidence that the Ca-calmodulin complex is an essential component of receptor-N-cyclase coupling in nerve cells, and that nucleotide activation (Brostrom et al., 1978, 1981; Partington et a/., 1980; Toscano et al., 1979; Wilkening et al., 1980; Brandt et al., 1980) and inhibition (Girardot et nl., 1983) of adenylate cyclase is calmodulin dependent. Calcium has a bimodal effect on NG 108-15 adenyiate cyclase. Low ( K) and stabilize R-N (L > M). An antagonist may have the same affinity for R and R-N (K’ + K) and neither stabilize nor destabilize R-N ( L = M) (e.g., phentolamine), or it may bind with higher affinity (K‘ < K) to R than to R-N, thus destabilizing R-N ( L < M) (e.g., yohimbine, rauwolscine). A third finding which is at variance with the proposed model is that in some tissues, only [3H]agonist binding can be demonstrated under a variety of incubation conditions (changes in buffer and GTP and Mg2+ concentration). For example, in the rat submandibular gland (reserpinized or denervated), there is good rHIclonidine and pH]paminoclonidine binding, but no [3 Hlyohimbine binding (Bylund and
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Martinez, 1980, 1981; Bylund et al., 1982a), whereas in the neonatal rat lung, VHlyohimbine and ~Hlrauwolscinelabel a large number of sites, but [3H]clonidine and HIP-aminoclonidine binding could not be observed (Latifpourrt al., 1982). It could be argued that in the lung, almost all of the receptors are in the (L) state and thus binding of 3H-labeled agonists would not be expected. If this were the case, then 3H-labeled agonist binding should have been observed in the presence of Mg2+, which would stabilize the (H) state. Furthermore, in the neonatal lung, Gpp(NH)p shifts to the right the curve of norepinephrine inhibition of [3 Hlyohimbine, indicating that [3H]yohimbine is labeling at least to some extent the proposed (H) state of the receptor (Latifpour et al., 1982). A fourth unresolved issue concerns the differences in divalent cation-guanine nucleotide interactions at platelet and brain a2-receptor sites. At the former, Mg2+ facilitates the inhibition of high-affinity agonist binding by nucleotides (Fig. 2), whereas at the latter, Mg2+ and other divalent cations antagonize nucleotide effects on agonist binding, especially those of hydrolyzable nucleotides. T h e antagonism can be partially explained by the ability of divalent cations to accelerate nucleotide metabolism in the presence of brain membranes (Mallat and Hamon, 1982; Hamon et al., 1982), but some of these brain-specific interactions, such as increased agonist binding with low concentrations of GTP and Mg2+ present, may result from phosphorylation reactions specific to brain membranes, or may have to do with calmodulin influences on the receptor-cyclase system in neural tissue.
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D. REGULATION OF (Yz-ADRENERGICRECEPTORS
1. Homologou~Regulation a . L'p-Regttlntiou. In common with most other hormone and neurotransmitter receptors, a2receptors appear to be regulated in such a manner as to compensate for changes in agonist concentration or presynaptic input. For a2 receptors, up-regulation is, in general, operationally defined as an increase in binding site number indicating an increase in receptor density. Only rarely have concomitant increases in receptor function been measured. Up-regulation has been shown to occur either as a result of diminished presynaptic function (e.g., depletion of norepinephrine with reserpine treatment, or chemical or surgical denervation) or chronic occupation of the receptor by an antagonist. An important caveat concerning a2 receptor up-regulation studies is that many of them have utilized only H-labeled agonist ligands and thus
CHARACTERIZATION OF 0 1- AND ff2-ADRENERGIC RECEPTORS
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increases in the number of 3H-labeledagonist sites can be interpreted as shifts in equilibrium toward high-affinity states of the receptor without necessarily involving any change in the total receptor population. Early studies indicated that chemical denervation of rat brain noradrenergic neurones with intracerebroventricular 6-hydroxydopamine (6-OHDA) increased the number of brain a,-receptor sites labeled with [3H]clonidine(U’Prichardet al., 1977a). It was later shown that the same treatment increased the number of higher affinity pH]clonidine-labeled sites in 15 brain areas, with no change in the number of lower afhity p Hlclonidine sites (U’Prichard et al., 1979a). Chronic reserpine treatment also increased with the number of rat cortex a2 receptors labeled by [3H]epinephrine, with no change in [3H]epinephrine affinity (U’Prichard and Snyder, 1978a),as did 6-OHDA treatment (U’Prichard et al., 1979a). A specific lesion of the ascending dorsal noradrenergic bundle in rats also increased p H]clonidine binding in several forebrain areas (U’Prichard et al., 1980b). The rat submandibular gland has been a useful tissue for the study of a2 up-regulation, since no significant [3 Hlclonidine or Hlyohimbine binding is observed in membranes from normal glands. Chronic reserpine treatment or surgical denervation induces the appearance of a2 receptors in membranes from this gland (Bylund and Martinez, 1980, 1981; Pimoule et al., 1980; Bylund et al., 1982a). These a2receptors are labeled by [3H]clonidineand [3 H]p-aminoclonidine and are located postsynaptically. This phenomenon may represent a true de novo generation of postsynaptic a2 receptors in the tissue as a result of interruption of neuronal input. Interestingly, the adjoining sublingual gland, which receives very limited sympathetic input (the submandibular, by contrast is richly innervated), normally has a high level of a2receptors, and the level is further increased by reserpine treatment (Martinez et al., 1982b). Significant increases in [3H]clonidine binding in the rat submandibular gland occur within a few hours after the onset of reserpine administration and then decrease rapidly following termination of reserpine treatment (Fig. 6), indicating that expression of a2 receptors in this tissue is particularly adaptable (Bylund et al., 1982a). Although chronic occupation of a2receptors with a potent antagonist such as mianserin leads to an increase of a2-receptor function (Cerrito and Raiteri, 1981), few data showing a concomitant increase in a 2 receptor number are yet available. Treatment with yohimbine for 3 days reportedly increases the B,,, of pH]clonidine in rat cortical membranes (Johnson et al., 1980),and 7 days of yohimbine administration results in a modest increase in a2 receptors in the rat submandibular gland (Bylund et al., 1982a).
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6.0-
(9)
,
2 4 6 DAYS OF RESERPINE TREATMENT
I
*
4
DAYS AFTER END OF TREATMENT
FIG. 6. The effect of the duration of reserpine treatment on the apparent density of a,-adrenergic receptor binding sites in submandibular gland. Rats were treated daily with reserpine (0.5 mglkg, i.p.) and then sacrificed 24 hr after the last injection (except for the first two time points which were 6 and 12 hr following a single injection). Other rats were treated with reserpine for 7 days, and sacrificed 2, 3, or 5 days after the last injection. Values given are means ? SEM.
6. DoziwRegulation. Receptor down-regulation is believed to be a regulatory response of the cell when it is exposed over a period of time to higher than normal concentrations of agonist. Loss of responsiveness to the agonist can be due to two events which may be associated: uncoupling of the receptor from its effector; and loss of receptors from the cell membrane concomitant with receptor internalization. Perkins and colleagues have shown for p receptors that there is a concomitant reduction in receptor number and receptor-related response during exposure to agonist. However, the loss of response precedes loss of receptors, because the first phase of desensitization involves uncoupling of the receptor from adenylate cyclase [also evidenced by conversion of (H) to (L) states of the receptor] before there is any change in receptor number (Harden rt al., 1979; Su ~t al., 1980). Approaches toward examining the mechanisms of a,-receptor down-regulation involve the exposure to agonists of a,-receptor-containing cells such as platelets, NG 108- 15 cells, and adipocytes, and examining the effects on a,-receptor function and number following treatment with antidepressants, which increase agonist concentration in the synapse by inhibiting norepinephrine reuptake. An early study indicated that incubation of intact human platelets with 100 pM (-)-epinephrine gradually reduced by 50% the number of
CHARACTERIZATION OF
AND fX2-ADRENERGIC RECEPTORS
413
rH]DHEC a2-receptor sites over a 4-hr period (Cooper et al., 1978), suggesting agonist-induced down-regulation. Over the time of exposure, epinephrine gradually induced total refractoriness of the aggregating response to catecholamines. However, more recently, Insel and colleagues have found that decreases in [3H]yohimbinebinding after a 4-hr exposure to 100 p M (-)-epinephrine are due to competition at the [3 Hlyohimbine sites by epinephrine taken up by platelets and retained even after extensive membrane washing. Thus in membranes from epinephrine-exposed platelets, the K, of [3 Hlyohimbine was increased with no change in B,,,, and the assay of these membranes in the presence of Na+ and GTP completely restored platelet Hlyohimbine binding (Karliner et al., 1982). A major problem with the human platelet as a model system for examining a2-receptor down-regulation is that activation of the platelet a2 receptor alters the characteristics of platelets in fundamental ways by stimulating the aggregation response. Regulation of NG 108-15 cell a2 receptors as a function of exposure to (-)-epinephrine can be studied in intact cells, grown in a serum-free defined medium at 37°C (Kahn and U’Prichard, 1983). Within the first 30 min of exposure to 10 p M (-)-epinephrine, there appears to be a shift toward high-affinity states of the receptor (increased B,,, of [3H]epinephrine and [3H]p-aminoclonidine, and decreased IC50 of epinephrine and p-aminoclonidine at [3H]rauwolscine sites) with no change in the total receptor population (B,,, of rH]rauwolscine) in Gpp(NH)p-treated membranes from exposed cells. More prolonged exposure of the cells to 10 pM (-)-epinephrine causes apparently parallel decreases in the B,,, of rH]rauwolscine, [3H]epinephrine, and H]paminoclonidine. However, although epinephrine exposure does not change the KD of [3H]rauwolscine at residual sites, it causes a timedependent increase in the KD of THlepinephrine and rH]paminoclonidine and an increase in the ICJo of epinephrine and p-aminoclonidine competing at H]rauwolscine sites. Thus prolonged exposure to epinephrine causes a selective loss of high-afhity states of the NG 108-15 a2receptor, as is observed for /3 receptors (Suet al., 1980; Kent et al., 1980). The ED50 for (-)-epinephrine in causing a reduction in [3H]rauwolscinesites after 8 hr exposure was 1.0 nM, similar to the potency of epinephrine at high-affinity states of the receptor. The initial increase in 3H-labeled agonist sites at shorter exposure times may represent uncoupling of the a2 receptor from adenylate cyclase, with the induction of more (SH) states (Kahn and U’Prichard, 1983). Unlike @receptor systems, exposure to agonist does not alter the maximum a2-receptor response [inhibition of adenylate cyclase activity in washed membranes with 100 pM (-)-epinephrine] but causes a twofold reduc-
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tion in the apparent potency of (-)-epinephrine in inhibiting cyclase, indicating that N G 108-15 cells have “spare” a2 receptors in addition to “spare” opiate receptors (Fantozzi at al., 1981). Another phenomenon associated with exposure of NG 108-15 cells to agonists for negatively coupled receptors, but not seen with P-receptor systems, is a gradual increase in basal adenylate cyclase activity (Sabol and Nirenberg, 1979b), which may result from decreased ability of inhibitory N proteins to couple to C. Our experiments indicate that epinephrine exposure can increase basal cyclase at early exposure times (Kahn and U’Prichard, 1983). Similar a,-receptor desensitization experiments have been performed with isolated human properitoneal adipocytes by incubation for 3 hr at 37°C with 10 pLVf(-)-epinephrine (in the presence of propranolol to block p receptors). Although exposure to (-)-epinephrine reduced the B,,, of [3H]p-aminoclonidine by 4356, neither the B,,,, of [3H]yohimbine nor the ability of 10 p.M epinephrine (plus propranolol) to lower CAMPlevels in the cells was affected (Burns at af., 1982a). It is possible that adipocytes also have spare a2 receptors, and that for these cells a 3-hr exposure was not long enough to observe the second phase of desensitization, that is, a decrease in total receptor number. When rats under mild restraint were chronically infused with a-methylDOPA (which is metabolized to the at agonist amethylnorepinephrine) via the jugular vein for 72 hr, binding of both [3H]rauwolscine and [3H]p-aminoclonidine to cerebral cortex membranes was decreased by 30 and 50%, respectively. Infusion of the partial az-agonist clonidine for 72 hr had a bimodal effect on rat cortex [3H]p-aminoclonidine sites, with lowest concentrations of clonidine increasing ? H jp-aminoclonidine binding 50% above control, whereas higher doses decreased ? H]p-aminoclonidine binding 60 5% below control values. On the other hand, rat cortex [3H]rauwolscine binding was not affected at any clonidine concentration (U’Prichard et al., 1981). This dose-dependent regulation of a2-receptor high-affinity states with no change in total receptor population following a fixed time of agonist exposure may parallel the time-dependent bimodal regulation of agonist binding with a fixed concentration of agonist seen in N G 108-15 cells. Chronic treatment of rats with tricyclic and other antidepressant drugs, in addition to down-regulating brain PI receptors and the associated adenylate cyclase response, causes a functional desensitization of central and peripheral a2receptors (Crews and Smith, 1978; McMillen et 01.. 1980; Spyraki and Fibiger, 1980), as well as a functional a,-receptor supersensitivity (Menkes and Aghajanian, 1981). Binding studies are somewhat contradictory. Antidepressant treatment reduced the B,,, of
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415
[3H]clonidine binding in brain regions other than cortex (Smith et al., 198l), whereas abbreviated (3 days) treatment with amphetamine, iprindole, or desmethylimipramine increased the number of higher affinity a2 sites in rat cortex membranes labeled with ?HIP-aminoclonidine or [3H]clonidine(Johnson et al., 1980; Reisine et al., 1980).In the absence of concomitant assessment of brain a2-receptor function, it is difficult to determine the significance of these binding changes. Chronic administration of various tricyclic antidepressant drugs to human patients appears to down-regulate platelet a2 receptors (decreased B,,, of ?HIclonidine) (Garcia-Sevilla et al., 198la,c). Chronic immobilization stress, which accelerates turnover of brain norepinephrine, down-regulates rat brain p receptors and increases the number of [3 Hlclonidine a2-receptor sites in cerebral cortex membranes, but decreases the affinity of [3H]clonidine and the number of ?HIclonidine sites in other brain areas (U’Prichard and Kvetnansky, 1980). 2. Heterologous Regulation
Numerous studies have now demonstrated that a2-receptor number can be influenced by other hormones. Estrogen treatment of immature female rabbits causes a three- to fourfold increase in the number of a2 receptors in uterine membranes, compared to tissues from progesterone-dominant animals, and in estrogen-primed tissue the a-receptor (contraction) response predominates over the &receptor response (relaxation) (Williamsand Lefkowitz, 1977b; Roberts et al., 1977). Estrogen treatment also decreases the contractile ED5, for norepinephrine compared to uteri from untreated animals, suggesting the presence of spare a2 receptors (Roberts et al., 1981). After 24 hr in organ culture, rabbit myometrium is more sensitive to a-adrenergic stimulation in vitro, and membranes from cultured myometrium have a threefold increase in a-receptor number, but no change in p receptors, suggesting that uterine a2receptors are under tonic inhibitory control in vivo (Cornett et al., 1981). Estrogen treatment has the opposite effect on rabbit platelet a2receptors, decreasing the density of PHIDHEC sites by 40-50% and diminishing the aggregation response to epinephrine (Roberts et al., 1979; Elliott et al., 1980). On the other hand, platelet membranes enriched or depleted in cholesterol, a membrane-stabilizingagent, exhibited increased or reduced responses to (-)-epinephrine, respectively, which was unaccompanied by any change in the number of a2 receptors labeled by the antagonist CJHIDHEC (Insel et al., 1978). Unfortunately, in this study a2-agonist binding parameters were not examined.
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Activation of brain p receptors appears to lead to an increase in the number of high-affinity sites of the a 2 receptor in cerebral cortical membranes, either in vitro, when rat cortical slices were incubated with isoproterenol (Maggiet al., 1980),or in vivo, when isoproterenol is constantly infused intracerebroventricularlyfor up to 7 days via an Alzet minipump (Wang and U’Prichard, 1980). In the former studies, coincubation with the p antagonist sotalol prevented the isoproterenol-induced increase in H]p-aminoclonidine binding, whereas in the latter experiments, the increase in [3H]p-aminoclonidine sites was dependent on the time of infusion and amount of isoproterenol infused (Wang and U’Prichard, 1980). These data suggest that in membranes of rat cortical neurons, p and a2 receptors may be reciprocally regulated under some conditions.
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3. Physiological Regulation The regulation of a2-receptor binding in response to normal physiologic changes has not been given much attention. Preliminary studies suggest that the number of sites labeled by [3H]yohimbine on human platelets varies during the normal female menstrual cycle (D. B. Bylund, unpublished). On the other hand, factors such as age, gender, and season do not appear to alter the density of platelet a2 receptors to any great extent. Many additional studies are needed in this area, at least as controls for the numerous studies on the regulation of human platelet a2 receptors in various pathologic states (Section III,D,4). The ontogeny of a2 receptors has been investigated in rat submandibular gland and brain. In the submandibular gland, the level of a2 receptors as determined by Hlclonidine and [3H]p-aminoclonidine binding at birth is relatively high (7 pmoVgm tissue) and then increases markedly during the first 2 weeks of life (Bylund et al., 1982b). Thereafter, the binding decreases such that at 6 weeks it approaches the very low level observed in glands from adult animals. By comparison, the level of [3H]clonidine binding in rat brain is low at birth, increases severalfold during the first 3 weeks, and then decreases slightly to adult levels (Morris et al., 1980).
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4. Patholopal Regulation
The existence of a2receptor on human platelets, an easily obtainable tissue, has led to investigations of possible abnormal number, function, or regulation of a2 receptors in pathological states. Thus, platelets from patients with essential thrombocytopenia, which fail to aggregate or release serotonin in response to epinephrine, contained less than 50% of the normal complement of a2-receptor sites as measured with rH]DHEC (Kaywin et al., 1978).
CHARACTERIZATION OF
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(Y~-ADRENERGICRECEPTORS
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The hypofunction of central noradrenergic systems, believed to underlie the pathology of endogenous depression, may be associated with an abnormal increase in a,-receptor function, which in turn may be reflected as an increase in total receptor number or an enhanced coupling to adenylate cyclase. If this abnormality had a genetic basis, it could be expressed also in platelets as well as brain tissue. Although the results are conflicting, several laboratories have examined platelet a , receptors in unmedicated depressed patients. Smith and colleagues observed an increase in platelet [3H]clonidinebinding in a small group of depressed patients (Garcia-Sevilla et al., 198lc), whereas Meltzer and co-workers have observed no changes in the K , or B,,, of THIyohimbine binding to a , receptors in platelet membranes from unipoiar or bipolar depressives (Daiguji et al., 1981b). Basal platelet CAMPproduction was reported to be reduced in samples from male schizophrenic patients, but no change in platelet a,-receptor number or function (percentage inhibition of adenylate cyclase) was observed (Kafka et al., 1979). However, no change in p Hlyohimbine binding was found in unmedicated schizophrenics,but there was an increase in B,,, in a small group of patients termed schizoaffective (U’Prichard et al., 1982). In another study, no alteration in platelet THIyohimbine binding was found when five patients with Parkinson’s disease were compared to appropriate controls (D. Bylund, unpublished). Since centrally acting antihypertensive drugs such as clonidine and a-methylnorepinephrine (formed from a-methylDOPA) are potent a,receptor agonists, animal models of hypertension have been examined for possible a,-receptor changes. In genetically hypertensive (SHR) rats, a 35% increase in the number of a,-receptor sites labeled with pH] clonidine in hypothalamic membranes has been reported (Morris et al., 1981). However, THIyohimbine binding is unaltered in most tissues of the deoxycorticosterone/salthypertensive rat (Yamada et al., 1980a).
E. LOCALIZATION OF (Y2-ADRENERGIC RECEPTORS a, Receptors were originally defined in terms of their capacity to modulate norepinephrine release and were presupposed to occur on presynaptic terminal membranes. However, the Occurrence of presynaptic a , receptors in brain and peripheral tissues by means of binding studies has not yet been demonstrated, due perhaps in large part to two complicating factors. First, if presynaptic and postsynaptic a , receptors occur in the same tissue and are pharmacologically indistinguishable, attempts to establish the presynaptic location of receptors involving de-
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nervation of the noradrenergic input will be bedevilled by the probability of postsynaptic receptor supersensitivity masking the loss of presynaptic receptors. T h e issue is compounded by the necessity of waiting at least 2 to 4 weeks after denervation to allow complete phagocytic removal of presynaptic membranes which could, of course, still be labeled with receptor ligands even though presynaptic function has long been lost. Second, studies published so far have almost exclusively involved the use of as-agonist radioligands, although changes in the number of 3H-labeled agonist sites do not necessarily reflect changes in the az-receptor density of the tissue. T h e equation of at receptors with a presynaptic location has become steadily weakened in recent years with the discovery of a 2 receptors on noninnervated cells such as platelets and isolated adipocytes, and by more recent pharmacological evidence that adrenergic vasoconstriction involves vascular postjunctional a 2 , as well as a l , receptors (Timmermans and van Zwieten, 1980; Kobinger and Pichler, 1981; Ruffolort al., 1980). Alphae receptors have been identified on bovine aorta membranes (Rosendorff et al., 1981). Some authors have gone so far as to suggest that at adrenergic neuroeffector junctions, postsynaptic (possibly a e )receptors may have trans-synaptic effects on terminal norepinephrine uptake and release (Manukhin and Volina, 1979). With the above caveats in mind, VHIclonidine binding studies indicate that rat brain a2 receptors are predominantly “postsynaptic,” in that they are not located on noradrenergic terminals (U’Prichard et al., 1977a, 1979a). After a dorsal noradrenergic bundle lesion, however, rH]clonidine binding was decreased in amygdala and septum, suggesting that in these brain areas there might be a greater prevalence of presynaptic receptors (U’Prichard et al., 1980b). Similarly, az-receptor sites when present, appear to be postsynaptic in rat submandibular gland (Bylund and Martinez, 1981) and kidney (McPherson and Summers, 1982). There has been some controversy over the occurrence and location of rat heart a 2receptors. Guicheney and co-workers obtained evidence for an a,-receptor component of tH]DHEC binding to rat heart membranes and referred to this component as “presynaptic,” but did not demonstrate its location (Guicheney et a/., 1978). Langer and colleagues observed that ventricular HIDHEC binding was decreased 60% after chemical sympathectomy, but a pharmacological differentiation of a 1 and a2 components of rH]DHEC binding was not performed (Briley et nl., 1979; Story rt a/., 1979). In contrast, several laboratories have found no evidence of a2-receptor binding to rat heart membranes using r H ] clonidine (U’Prichard and Snyder, 1979), rH]yohimbine, or VHIDHEC (R. J. Lefkowitz, personal communication).
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CHARACTERIZATION OF al- AND (Y~-ADRENERGICRECEPTORS
4 19
Another approach to the localization of a2receptors in the brain has been the labeling of slide-mounted brain tissue sections with rH]paminoclonidine, and subsequent processing for autoradiography. Tritiated p-aminoclonidine binding in these conditions was shown to be saturable, a2 receptor specific, and highly concentrated in certain brain regions such as locus coeruleus and nucleus tracti solitarii where a2 receptors have been demonstrated electrophysiologically (Young and Kuhar, 1979, 1980). The autoradiographic distribution of az-and opiate receptor sites was found to be very similar in many brain regions (Young and Kuhar, 1980), which is of interest in view of the coexistence of a2 and &opiate receptors in many neuroblastoma cell lines (Hamprecht, 1977; D. J. Kahn and D. E. U'Prichard, unpublished). An intriguing finding in this connection is that intravenous clonidine infusion in rats causes biphasic alterations in the binding to cortex membranes of the &opiate agonist [3H]d-Alad-Leu-enkephalin, which exactly parallel the changes in H]p-aminoclonidine binding (U'Prichard et d.,198 1).
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F. SOLUBILIZATION OF QZ-ADRENERGJC RECEPTORS
Human platelet a2 receptors have been successfully solubilized by Smith and Limbird (1981) using the detergent digitonin. The characteristics of [3H]yohimbinebinding ( K DandB,,,) are the same for membranes and solubilized preparations. The solubilized a2receptors labeled with [3H]yohimbineappear to be in the low-affinity state, with ICsOvalues for agonist competitors equal to the IC,, values in membrane preparations in the presence of 100 p M Gpp(NH)p. Thus, solubilization of platelet membranes with digitonin appears to dissociate R-N complexes. On the other hand, prelabeling membrane-bound receptors with the agonist VHIepinephrine confers some stability on the ternary complex during solubilization, since high-affinity, guanine nucleotide-dissociable [3 Hlepinephrine binding was retained after digitonin treatment. [3H]Epinephrine-labeled receptors sedimented more rapidly through a continuous sucrose gradient after solubilization than did [3H]yohimbine-labeledreceptors, suggesting that the agonist-receptor complex had a larger protein mass. These results support the notion that high-affinity agonist binding represents the ternary H-R-N complex, whereas after solubilization antagonist binding is to the free R species. Essentially, similar results have been obtained by Lefkowitz and colleagues (Michel et d.,1981). Recently, clonidinep-isothiocyanatehas been used as an affinity label for platelet a2-adrenergic receptors (Atlas and Steer, 1982). Vipoxin, a
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protein from Russell’s viper venom, appears to have a high affinity for a,-receptor sites, and the binding is essentially irreversible (Freedman and Snyder, 1981). These two probes, particularly in their radioactive form, should be very useful in the further study of a , receptors in areas such as regulation and purification.
IV. Summary and Conclusions
Within the short period of 5 years, the availability of a variety of specific radioligands has allowed the resolution of a1- and a , -adrenergic receptor populations in many different tissues and enabled researchers to begin investigations of the mechanisms of regulation and coupling of a l and a, receptors to their different cellular effector systems. Binding data have demonstrated that the pharmacological properties of each type of a receptor are, in general, similar across tissues and species, although there are some differences in the relative affinities of antagonist drugs. Further attempts to subclassify a1 and a , receptors may be expected in the future. T h e historical development of the interpretation of pH]clonidine binding is of interest in this regard. [3H]Clonidine was proposed to label the “agonist state” of the a receptor, and then to label a2 receptors. It is now thought that it labels the agonist state of a, receptors. Might it actually label a subpopulation of a , receptors or just the agonist state of that subpopulation? Alpha-1 receptors by and large appear to occur in a single-affinity state with respect to both agonists and antagonists. By comparison, a , receptors may exist in multiple-afbnity states reflecting the ability of the a, binding site protein to complex to additional membrane proteins which themselves are receptors for the physiological substrates GTP, Na+, Mgz+, and possibly Ca2+-calmodulin. Binding studies have also strongly indicated that a, receptors in most, if not all, tissues are probably coupled in an inhibitory manner to adenylate cyclase, as has been demonstrated in platelets, adipocytes, and NG 108-15 cells. Clearly the present status of a-receptor research has left many questions unresolved. We still have no idea what membrane effector system and associated second messenger is coupled to the a 1 receptor. T h e prevailing belief is that Ca2+ and the membrane Ca2+ channel fulfill these roles. However, others have suggested that phosphoinositide turnover represents the proximal receptor response, and indeed a membrane-bound phospholipase C may play an analogous role to adenylate cyclase for other adrenergic receptors (Putney et al., 1980). There is,
CHARACTERIZATION OF
a1-AND
a2-ADRENERGIC RECEPTORS
42 1
however, some evidence that in some situations a1receptors may directly stimulate adenylate cyclase, and guanine nucleotide modulation of agonist affinities at a1-receptor sites has been reported. The significance of these data and reported modulatory effects of Na+ at a1 receptors (Glossmann and Presek, 1979; Glossmann et al., 1981) is still to be resolved. Nothing is known about the mechanisms of regulation of al receptors, although both up- and down-regulation of a1 receptor have been demonstrated. In this regard, the ability to label and study a1 receptors on cells in culture would be particularly useful. With regard to a2receptors, it is still not clear how many affinity states exist and what their role is in terms of the kinetics of a2-receptor coupling to adenylate cyclase. In particular, the interaction of the proposed (SH) state of the receptor with the catalytic enzyme moiety is unresolved. Some questions concerning a2-receptor coupling and function are common to all inhibitory receptors: 1. What is the protein subunit composition of the regulatory proteins (Ni) associated with inhibitory receptors (Rodbell, 1980)? Are they analogous to the subunits of N, (Northup et al., 1980)? 2. What are the similarities and differences between Ni and N,? 3. How does activation of inhibitory receptors result in decreased cyclase activity?
Other questions related to a2 receptors concern whether there are indeed sequential phases of desensitization involving uncoupling and loss of receptor protein. For neural a , receptors, future studies must determine what the role of calmodulin is in the coupling and regulation of a , receptors. More generally, it is unclear at what membrane site Na+ interacts to regulate agonist affinities at the receptor and in particular if the site is associated with the Ni complex. Clearly, the ability to measure a,-receptor populations and the associated response in homogeneous cell populations has been particularly advantageous. By analogy to the mouse lymphoma S49 cell @-receptorsystem, the development of selection pressures to produce mutants of a,-receptor-containing cultured neural cells deficient in one or other aspect of the receptor-effector system would lead to great advances. Indeed, receptor-deficient variants of NG 108-15 cells are currently being developed in several laboratories. For these and other studies, the development of radio-iodinated a2receptor probes would prove very beneficial, particularly to resolve decisively the issue of the existence of nerve terminal “autoreceptors,” whose density would be expected to be very low.
422
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The successful solubilization of platelet az receptors is sure to be followed by solubilization of az receptors in other tissues. Over the next few years, it is reasonable to expect both a 1 and az receptors to be purified at least to the extent that specific receptor antibodies can be produced by monoclonal techniques. One would also predict that reconstitution studies along the lines developed by Gilman and colleagues for p receptors (Ross and Gilman, 1980) will resolve the issue of the uniqueness of Ni for inhibitory receptors. These investigations and others concerning the mechanisms of coupling and regulation of a-adrenergic receptors have great therapeutic relevance in view of the widespread use of &-receptor agents such as prazosin, clonidine, and a-methyIDOPA in the treatment of hypertension, together with the likely development of new therapies for endogenous depression based on az-adrenergic receptors.
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ONTOGENESIS OF THE AXOLEMMA AND AXOGLIAL 0NS H IPS IN M YE LINATE D F IBERS: RE LAT1 E LECTROPHYSIOLOGICAL AND FREEZE-FRACTURE CORRELATES OF MEMBRANE PLASTICITY By Stephen G. Waxman and Joel A. Black* Department of Neurology Stanford University School of Medicine and Veterans Administration Medical Center Palo AI~O,Ca.lifornia
and Robert E. Foster Neurotoxicology and Experimental Therapeutics Branch
U.S. A m y Medical Research Institute Aberdeen Proving Ground, Maryland
I. Introduction ......................................................... 11. Specificity in Myelination
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111. Development of the A. Introduction ... B. Electrophysiology IV. Freeze-Fracture Structure of Myelinated Axons . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction to Freeze-Fracture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Freeze-Fracture of Myelinated Axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Freeze-Fracture of Adult Rat Retinal V.. Freeze-Fracture Studies on Myelin Devel A. Introduction to Myelin Development . . B. Premyelinated Axolemma . . . . . . . . . . . .. . . . . . .. . . . .. .. . . . . . . . . . .. . . . C. Axolemmal Changes Associated with Glial Ensheathment . . . . . . . . . . D. Myelinated Axolemma . . . . . . . . . . . . . . . E. Aberrant Axoglial Association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Summary of Developmental Changes in Axon Membrane.. . . . . . . . . . . . VI. Differentiation of the Axon Membrane in the Absence of VII. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . ........................... .......... .
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* Present address: Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 601 15. 433 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 24
Copyright 6 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366824-7
434
STEPHEN G. WAXMAN
et al.
1. Introduction
Although it is well known that myelinated nerve fibers are differentiated, at the light microscopic level, into regions covered by myelin (internodes) and regions devoid of myelin (the nodes of Ranvier), it has only been in the past few years that details of axon membrane differentiation itself have been studied. It is now becoming apparent that the axon membrane (axolemma) exhibits an elegant differentiation in terms of its macromolecular architecture, and that nodal and internodal regions of this membrane can be shown to be different by morphological, electrophysiological, and pharmacological techniques. This differentiation of the mature axolemma into nodal and internodal domains with distinct properties has been recently reviewed in a number of articles (Waxman and Foster, 1980; Ritchie and Chiu, 1981; Rosenbluth, 1981a). A previous article (Waxman and Foster, 1980) reviewed cytochemical aspects of the plasticity of developing axon membranes. T h e present article reviews studies from our laboratory dealing with electrophysiological and freeze-fracture aspects of axon membrane reorganization during ontogenesis of the trunk of the mammalian myelinated fiber. It is the purpose of this article to discuss the development of the axon membrane of the myelinated fiber, and to demonstrate that the axolemma exhibits a high degree of plasticity during development, with significant changes in axon membrane structure being related to association with glial cells. Action potentials in myelinated fibers are generally considered as being conducted in a saltatory manner, with the impulse traveling discontinuously along the axon. T h e distribution of ionic channels in the axon membrane of mammalian myelinated fibers has been shown to be nonuniform, with markedly different densities of sodium and potassium channels in the node and internode, respectively. Voltage-clamp (Conti et ul., 1976) and PHIsaxitoxin binding studies (Ritchie and Rogart, 1977) suggest a high density (5,000- 12,000/pmZ)of sodium channels in the mammalian nodal axolemma. In contrast, the density of sodium channels in the internodal axolemma (beneath the myelin sheath) is quite low (