Journal of Neurochemisrry, 1977. Vol. 28, pp. 857-860. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION GABA Receptor binding in frog spinal cord and brain (Received 6 July 1976. Accepted 13 October 1976)
GABA is thought to elicit synaptic inhibition by two dif- delivery and synaptic membranes prepared as previously ferent mechanisms. Presynaptic inhibition appears to described for rat brain (ZUKINet al., 1974), on the assumpdepend on partial depolarization by GABA of sensory tion the frog brain synaptic membranes sediment in a mannerve terminals primarily in the spinal cord and lower ner similar to rat brain. After preparation, the membranes brain stem, causing a diminished release of their neuro- were frozen at -20°C for at least 18 h prior to assay of & PHILLIS,1969; receptor binding. For the receptor binding assay, the transmitter (ECCLESet al., 1963; TEBECIS DAVIDOFF,1972; BARKERet al. 1975a,b). In higher brain frozen membranes were homogenized with a Brinkmann centers GABA is a major transmitter of postsynaptic inhi- Polytron P-10 (setting 6) for 60 s in 0.05 M-Tris-citrate bition, directly eliciting hyperpolarization of cells by an (pH 7.1 at 4°C) at a final concentration of 10 mg proincrease in chloride permeability (CURTIS& JOHNSTON, tein/ml. Triton X-100 was added to a final concentration 1974; KRNJEVIC, 1974). Receptor sites for GABA effects of 0.05% (v/v) and the suspension was incubated at 37°C on presynaptic and postsynaptic inhibition differ in their for 20 min, followed by contrifugation at 48,0009 for sensitivities to various drugs (ECCLESet al., 1963; OBATA 10 min. Under these conditions, rat brain synaptic memet al., 1970; NICOLL,1971; NICOLLet al., 1976). Thus it branes not treated with Triton bind approx 4000 c.p.m./mg would be of interest to compare biochemical properties protein when incubated in the presence of t3H]GABA of GABA receptors in the spinal cord, the major locus alone and about 1800c.p.m./mg protein when incubated of presynaptic inhibition, with GABA receptors in whole on the presence of ['HIGABA and 1mM unlabelled GABA brain, where GABA elicited postsynaptic inhibition pre- (blank), yielding about 2200 c.p.m./mg protein specifically sumably predominates. Several studies of presynaptic inhi- bound C3H]GABA. After Triton treatment, 8000 c.p.m./mg bition have utilized the frog spinal cord in which some protein are bound in the presence of C3H]GABA alone, aspects of GABA transmission appear to differ from mam- with a blank of only lOOOc.p.rn./mg protein, revealing malian species (BARKER et al., 1975a,b; PIXNER, 1974). Con- about 7000 c.p.m./mg protein specifically bound to the ceivably, biochemical characteristics of GABA receptors GABA receptor. In both cases, amino acids and drugs displace the specifically bound C3H]GABA stereospecifically might differ in the frog and in mammals. Recently synaptic GABA receptors have been labelled with relative potencies similar to their neurophysiological in press). The precise mechanby the sodium independent binding of C3H]GABA to CNS activities (ENNA& SNYDER, membranes (ZUKINet al., 1974; ENNA& SNYDER,1975; ism of this Triton enhanced binding is unknown, though it ENNAet al., 1975; ENNA& SNYDER,in press). Evidence appears to be due to an increase in affinity of the GABA that this binding involves physiological GABA receptors in- receptor for the ligand. With frog brain, the ratio of the cludes the close correlation between neurophysiological specific to nonspecific binding using Triton-treated tissue potency of several amino acids and their binding affinities. is about 10, similar to that observed with rat brain. The Neurophysiological antagonism of GABA synaptic effects Triton-treated pellet was resuspended in the same volume by bicuculline is stereospecific with essentially all activity of buffer as above and 2ml portions were incubated, in residing in the (+)-isomer. (+)-Bicuculline is a potent in- triplicate, at 4°C for 5 min with 9 n~-[~H]y-aminobutyric hibitor of sodium independent C3H]GABA receptor bind- acid (C3H]GABA) (36.7 Ci/mmol, New England Nuclear) ing, while the neurophysiologically inactive ( -)-isomer is alone or in the presence of 1 mM-GABA, 0.1 mM-bicuculin press; ENNAet al., in line or other indicated drugs. Time course experiments inmuch weaker (ENNA& SNYDER, press). Bicuculline stereospecificity is not observed with dicated that C3H]GABA binding is at equilibrium within receptor binding for other neurotransmitters. Regional 5 min under these conditions. After incubation, the reacvariations in C3H]CABA receptor binding parallel the dis- tion was terminated by centrifugation for 10min at tribution of glutamic acid decarboxylase in monkey brain 48,000 g, the supernatant was decanted and the pellet (ENNAet al., 1975), though the correlation is less evident rinsed rapidly and superficially with 5m1, then 10ml of in rat brain (ZUKINet al., 1974; ENNA& SNYDER,1975). ice-cold distilled H 2 0 . Bound radioactivity was extracted In the present study we have characterized the sodium into 1 ml of Protosol (New England Nuclear), 10ml of independent binding of C3H]GABA in frog spinal cord and toluene phosphor were added and radioactivity assayed brain and compared properties of these binding sites with by liquid scintillation spectrometry (Packard Tricarb those of mammalian brain. Model 3385). Greater than 95% of the bound radioactivity was identified as authentic GABA by TLC in 3 solvent systems (ENNA& SNYDER,1975). Specific receptor bound MATERIALS AND METHODS C3H]GABA was determined by subtracting from the total Bullfrogs (Rana catesbiana, 6 8 " body length) were bound radioactivity the amount not displaced by high conobtained from Mogul-ED Corporation of Oshkosh, Wis- centrations of bicuculline (0.1 mM) or GABA (1 mM). Proconsin, the brains and spinal cords were removed upon tein was measured by the method of LOWRYet al. (1951). 857
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858
All compounds used in this study were obtained from commercial suppliers, except for (+) cis- and trans-3aminocyclopentane-4-carboxylic acid which were gifts from Dr. G. A. R. JOHNSTON and muscimol which was a gift from Dr. E. COSTA. RESULTS Sodium independent GABA binding is saturable in frog spinal cord and brain (Fig. 1). In both tissues binding plateaus at about 0.1-0.3 PM C3H]GABA and reaches halfmaximal values at 30-60 IIM. Scatchard analysis suggests only one population of binding sites in frog brain and spinal cord with respective dissociation constants of 58 nM and 33 nM. However, the amount of scatter observed with
A
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-
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SPINAL CORD
02 04 0 6 00 10 BOUND/FREE (pmoles/nmole)
12
FIG. 1. Saturation of specific C3H]GABA binding to frog brain and spinal cord synaptic membranes with increasing concentrations of C3H]GABA. Frog brain and spinal cord synaptic membrane suspensions were incubated in Triscitrate (pH 7.1) with various concentrations of C3H]GABA. Specific binding was obtained by subtracting the amount of radioactivity not displaced by 1 mM-GABA from the total C3H]GABA bound. Values are the means of triplicate determinations which varied less than 10%. The experiment was replicated 4 times. (A) Linear plot. (B) Scatchard analysis.
TABLE1. SUBSTRATE SPECIFICITY
OF C3H]GABA BINDING IN FROG CENTRAL NERVOUS SYSTEM
G
Compound
Brain
0.05 GABA 2.0 ( + )-Bicuculline 0.09 3-Aminopropane sulfonic acid 0.007 Muscimol 2,4-Diaminobutyric acid 600 Picrotoxin >' 500 ( +)-Trans-3-aminocyclopentane0.30 4-carboxylic acid ( +)-Cis-3-Aminocyclopentane-45.0 carboxylic acid me.* Pentobarbital-Na n.e. Pentylenetetrazol
o (PI)
Spinal cord 0.03 3.0 0.10 -
-
-
n.e. n.e.
* No effect at 1mM. Inhibition of C3H]GABA binding by various concentrations of the different compounds was determined using the assay procedure described in Fig. 2. IC,, values, the concentration of agents which reduced binding of C3H]GABA by SO%, were calculated by log-probit analysis. brain makes a definitive conclusion about the number of sites difficult. By contrast, for tissue prepared in the same way with Triton, rat brain displays high ( K D = 18 nM) and low affinity ( K D= 200 nM) components of GABA binding (ENNA& SNYDER,in press). Without Triton X-100 treatment rat brain membranes also display only a single binding site with a K D of 200-400n~(ENNA& SNYDER,1975, in press). Spinal cord has only about 20% as many GABA binding sites as frog brain, while rat brain has about the same number of receptors as frog brain. The binding differences observed between spinal cord and brain appears to be due to a difference in the total number of receptor sites rather than affinity since the affinity for C3H]GABA is the same or slightly higher in cord than in brain. The possibility that differences in synaptic membrane sedimentation characteristics may explain this result must also be entertained. The substrate specificity of sodium independent GABA binding in Triton treated tissue is quite similar in frog brain and spinal cord and rat brain (Table 1; Fig. 2) (ENNA & SNYDER, in press). In all three tissues the IC,, values for GABA and 3-aminopropane sulfonic acid are about 0.034.10 PM, corresponding to the similar neurophysiologic potencies of these two agents. Muscimol, which is substantially more active neurophysiologically in mimicking et al., GABA than GABA itself (KROGSGAARD-LARSON 1975), has almost 10 times the affinity of GABA. The geometrical isomers of 3-aminocyclopentane-4-carboxylicacid differ markedly in their neurophysiological mimicry of GABA with much greater activity being displayed by the trans than by the cis isomer (JOHNSTON, 1976). The binding selectivity of these two isomers in frog and rat brain (ENNA & SNYDER, in press) corresponds to their neurophysiological differences; in both tissues the trans isomer is about 10 times as potent as the cis isomer. In rat brain the GABA antagonist bicuculline is a potent inhibitor of GABA receptor binding, while picrotoxin, which is also active as a GABA antagonist, has negligible affinity (ZUKINet al., 1974; ENNA& SNYDER,1975). In frog brain and spinal cord bicuculline's potency in compet-
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FIG.2. Competition of GABA agonists and bicuculline for ['HIGABA binding sites in frog brain synaptic membranes. Increasing concentrations of nonradioactive GABA and other agents were added to tubes containing approx 8 nw['H]GABA and frog brain synaptic membranes. The binding values are the means of triplicate determinations. Samples were incubated for 5 min at 4°C and centrifuged to isolate bound ['HIGABA. The abbreviation ACPC refers to aminocyclopentane-4-carboxylicacid.
DISCUSSION Sodium independent binding of ['HIGABA to spinal cord and brain of the frog closely resembles the characteristics of GABA receptor binding in mammalian CNS. The evidence that binding in the frog involves synaptic receptors derives mainly from the close parallel between binding potencies of various agents and their neurophysiologic actions at GABA synapses. The only clear difference between rat brain and frog brain and spinal cord is the failure to detect distinct high and low affinity components of GABA binding in the frog. It is not altogether clear whether sodium independent ['HIGABA binding is associated exclusively with presynaptic or postsynaptic receptors. Since presynaptic inhibition by GABA predominates in the spinal cord, while postsynaptic inhibition is more prominent in higher centers, the fact that the density of GABA receptors is 5 times greater in brain than spinal cord suggests that binding detected in our studies primarily involves postsynaptic receptors. This conclusion is supported by observations in some studies that picrotoxin is more selective for presynaptic than postsynaptic GABA sites (BARKERet al., 1975a), which goes along with the inability to detect picrotoxin inhibition of GABA binding. An alternative explanation for the inactivity of picrotoxin at GABA binding sites is that it acts primarily by competing at the ionophore rather than at the GABA recognition site, as is suggested by neurophysiological studies indicating that picrotoxin effects are competitive with chloride but not with GABA (TAKEUCHI & TAKEUCHI,1969). Another observation suggesting that C3H]GABA binding does not involve presynaptic receptors is that neurophysiologic influences of pentobarbital and pentylenetetrazol occur predominantly at presynaptic rather than postsynaptic sites, possibly by direct receptor actions (NICOLL& PADJEN,1976; NICQLL,1975a,b) yet these drugs fail to affect GABA receptor binding.
ing for GABA receptor binding resembles the drug's effects in rat brain, while picrotoxin is inactive in frog brain as in rat brain. 2,4, Diaminobutyric acid, with high affinity 1975) but negligfor GABA neuronal uptake sites (IVERSEN, ible neurophysiological activity at GABA receptors, is also very weak in competing for GABA binding in frog and rat brain. Pentobarbital sodium and pentylenetetrazol have prominent influences on presynaptic inhibition, acting in a fashion which could reflect a direct influence on the GABA receptors which mediate presynaptic inhibition (NICOLL& PADIEN, 1976; NICOLL,1975a,b). Neither of these drugs have any influence on GABA receptor binding Acknowledgements-We thank Dr. R. NICOLLfor helpful in frog brain and spinal cord or rat brain. The striking for technical assimilarities in affinity, total number of binding sites and suggestions and Ms. CINDYKAUFMANN drug specificity between rat and frog brain tissue suggests sistance. that, under the conditions of these experiments, ['HIGABA labels the same site in both species, presum- Departments of Pharmacology and S. J. E N N A ~ ably the synaptic receptor site for this neurotransmitter Experimental Therapeutics and S. H. SNYDER amino acid. Psychiatry and Behuvioural Sciences, Receptor binding for other neurotransmitters, such as Johns Hopkins University School of Medicine, the a-adrenergic, muscarinic cholinergic, dopamine and Baltimore, MD 21205, serotonin receptors, differs for agonists and antagonists U.S.A. (SNYDER et el., 1975; BURTet al., 1976; GREENBERG et al., 1976; BENNETT& SNYDER,1976; SNYDER& BENNETT, 1976). While Hill coefficients for displacement of a REFERENCES 'H-agonist by other agonists or for 'H-antagonists by other antagonists are about 1, Hill coefficients of less than BARKER J. L., NICOLLR. A. & PADJEN A. (1975a) J. Physiol. 1 are associated with displacement of 'H-agonists by an245, 521-536. tagonists and vice versa. Accordingly, we examined in BARKERJ. L., NICOLLR. A. & PADJEN A. (1975b) J. Physiol. detail the displacement curves in frog brain of ['HIGABA 245, 537-548. by GABA itself, by cis and trans isomers of 3-aminocyclo- BENNETT J. P. & SNYDERS. H. (1976) Motec. Pharmac. pentane-Ccarboxylic acid and by the antagonist bicucul12, 373-389. line. Reduction of ['HIGABA binding by the antagonist BURTD. R., CREESEI. & SNYDERS. H. (1976) Molec. Pharbicuculline, by GABA and by the agonists cis and trans-3mac. 12, 8 W 8 1 2 . aminocyclopentane-4-carboxylic acid display parallel displacement curves. Hill plots of these data reveal Hill coeffiPresent address: Department of Neurobiology and cients of 1.0, indicating the absence of positive or negative cooperativity for any of these agents. These data resemble Anatomy, University of Texas Medical School at Houston, previous results in the rat (ENNA& SNYDER, 1975, in press). P.O. Box 20708, Houston, TX 77025, U.S.A.
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NICOLLR. A. (1971) Bruin Res. 35, 137-149. NICOLLR. A. (1975a) Proc. Natn. Acad. Sci., U.S.A. 12, 1460-1463. NICOLLR. A. (1975b) Brain Res. 96, 119-123. NICOLLR. A. & PADJENA. (1976) Neuropharmacology 15, 69-73. press. NICOLLR. A., PADJEN A. & BARKER J. L. (1976) NeuropharENNAS . J. & SNYDER S. H. (1975) Brain Res. 100, 81-97. macology 15, 45-54. ENNAS. J., KUHARM. J. & SNYDERS. H. (1975) Brain OBATAK., TAKEDA K. & SHINOZAKI J. (1970) Expl Bruin Res. 11, 327-342. Res. 93, 168-174. ENNAS. J. & SNYDER S. H. Molec. pharmac. in press. PIXNERD. B. (1974) Br. J . Pharmac. 52, 3S39. D., U'PRICHARD D. & SNYDERS. H. (1976) SNYDER S. H., CHANGK. J., KUHARM. J. & YAMAMURA GREENBERG Life Sci. 19, 69-76. H . I . (1975) Fedn Proc. Fedn Am. Socs. exp B i d . 34, IVERSEN L. L. (1975) in Handbook of Psychopharmacology 1915-1921. (IVERSEN L. L., IVERSEN S. & SNYDER S. H., eds) Vol. SNYDER S. H. & BENNETT J. P. (1976) Ann. Rev. Physiol. 13, pp. 381-442. Plenum Press, New York. 38, 153-175. JOFINSTONG. A. R. (1976) in GABA in Neruous System TAKEUCHI A. & TAKEUCHI N. J. (1969) J. Physiol., Lond. Firncrion (ROBERTS E., CHASET. & TOWERD. B., eds.) 205, 377-391. pp. 395441. Raven Press, New York. TEBECIS A. K. & PHILLISJ. W. (1969) Comp. Biochem. PhyKRNJEVlC K. (1974) Physiol. Reo. 54, 418-540. id. 28, 1303-1315. KROGSGAARD-LARSEN P., JOHNSTON G. A. R., CURTISD. ZUKINS. R., YOUNG A. B. & SNYDER S. H. (1974) Proc. R., GAME C. J. A. & MCCULLOCH R. M. (1975) J. NeuroNatn. Acad. Sci., U S A . 71, 4802-4807. chem. 25, 803-809. LOWRY 0.H., ROSEBROUGH N. H., FARRA. L. & RANDALL R. J. (1951) J. bid. Chem. 245, 447450.
G. A. R. (1974) Ergebn. Physiol. CURTIS D. R. & JOHNSTON 69, 97-188. DAVIDOFF R. A. (1972) Science 175, 331-333. ECCLESJ. C., SCHMIDT R. F. & WILLISW. D. (1963) J. Physiol. 168, 5W530. ENNA S. J., COLLINS J. F. & SNYDER S. H. Brain Res. in
SHORT COMMUNICATION GABA Receptor binding in frog spinal cord and brain (Received 6 July 1976. Accepted 13 October 1976)
GABA is thought to elicit synaptic inhibition by two dif- delivery and synaptic membranes prepared as previously ferent mechanisms. Presynaptic inhibition appears to described for rat brain (ZUKINet al., 1974), on the assumpdepend on partial depolarization by GABA of sensory tion the frog brain synaptic membranes sediment in a mannerve terminals primarily in the spinal cord and lower ner similar to rat brain. After preparation, the membranes brain stem, causing a diminished release of their neuro- were frozen at -20°C for at least 18 h prior to assay of & PHILLIS,1969; receptor binding. For the receptor binding assay, the transmitter (ECCLESet al., 1963; TEBECIS DAVIDOFF,1972; BARKERet al. 1975a,b). In higher brain frozen membranes were homogenized with a Brinkmann centers GABA is a major transmitter of postsynaptic inhi- Polytron P-10 (setting 6) for 60 s in 0.05 M-Tris-citrate bition, directly eliciting hyperpolarization of cells by an (pH 7.1 at 4°C) at a final concentration of 10 mg proincrease in chloride permeability (CURTIS& JOHNSTON, tein/ml. Triton X-100 was added to a final concentration 1974; KRNJEVIC, 1974). Receptor sites for GABA effects of 0.05% (v/v) and the suspension was incubated at 37°C on presynaptic and postsynaptic inhibition differ in their for 20 min, followed by contrifugation at 48,0009 for sensitivities to various drugs (ECCLESet al., 1963; OBATA 10 min. Under these conditions, rat brain synaptic memet al., 1970; NICOLL,1971; NICOLLet al., 1976). Thus it branes not treated with Triton bind approx 4000 c.p.m./mg would be of interest to compare biochemical properties protein when incubated in the presence of t3H]GABA of GABA receptors in the spinal cord, the major locus alone and about 1800c.p.m./mg protein when incubated of presynaptic inhibition, with GABA receptors in whole on the presence of ['HIGABA and 1mM unlabelled GABA brain, where GABA elicited postsynaptic inhibition pre- (blank), yielding about 2200 c.p.m./mg protein specifically sumably predominates. Several studies of presynaptic inhi- bound C3H]GABA. After Triton treatment, 8000 c.p.m./mg bition have utilized the frog spinal cord in which some protein are bound in the presence of C3H]GABA alone, aspects of GABA transmission appear to differ from mam- with a blank of only lOOOc.p.rn./mg protein, revealing malian species (BARKER et al., 1975a,b; PIXNER, 1974). Con- about 7000 c.p.m./mg protein specifically bound to the ceivably, biochemical characteristics of GABA receptors GABA receptor. In both cases, amino acids and drugs displace the specifically bound C3H]GABA stereospecifically might differ in the frog and in mammals. Recently synaptic GABA receptors have been labelled with relative potencies similar to their neurophysiological in press). The precise mechanby the sodium independent binding of C3H]GABA to CNS activities (ENNA& SNYDER, membranes (ZUKINet al., 1974; ENNA& SNYDER,1975; ism of this Triton enhanced binding is unknown, though it ENNAet al., 1975; ENNA& SNYDER,in press). Evidence appears to be due to an increase in affinity of the GABA that this binding involves physiological GABA receptors in- receptor for the ligand. With frog brain, the ratio of the cludes the close correlation between neurophysiological specific to nonspecific binding using Triton-treated tissue potency of several amino acids and their binding affinities. is about 10, similar to that observed with rat brain. The Neurophysiological antagonism of GABA synaptic effects Triton-treated pellet was resuspended in the same volume by bicuculline is stereospecific with essentially all activity of buffer as above and 2ml portions were incubated, in residing in the (+)-isomer. (+)-Bicuculline is a potent in- triplicate, at 4°C for 5 min with 9 n~-[~H]y-aminobutyric hibitor of sodium independent C3H]GABA receptor bind- acid (C3H]GABA) (36.7 Ci/mmol, New England Nuclear) ing, while the neurophysiologically inactive ( -)-isomer is alone or in the presence of 1 mM-GABA, 0.1 mM-bicuculin press; ENNAet al., in line or other indicated drugs. Time course experiments inmuch weaker (ENNA& SNYDER, press). Bicuculline stereospecificity is not observed with dicated that C3H]GABA binding is at equilibrium within receptor binding for other neurotransmitters. Regional 5 min under these conditions. After incubation, the reacvariations in C3H]CABA receptor binding parallel the dis- tion was terminated by centrifugation for 10min at tribution of glutamic acid decarboxylase in monkey brain 48,000 g, the supernatant was decanted and the pellet (ENNAet al., 1975), though the correlation is less evident rinsed rapidly and superficially with 5m1, then 10ml of in rat brain (ZUKINet al., 1974; ENNA& SNYDER,1975). ice-cold distilled H 2 0 . Bound radioactivity was extracted In the present study we have characterized the sodium into 1 ml of Protosol (New England Nuclear), 10ml of independent binding of C3H]GABA in frog spinal cord and toluene phosphor were added and radioactivity assayed brain and compared properties of these binding sites with by liquid scintillation spectrometry (Packard Tricarb those of mammalian brain. Model 3385). Greater than 95% of the bound radioactivity was identified as authentic GABA by TLC in 3 solvent systems (ENNA& SNYDER,1975). Specific receptor bound MATERIALS AND METHODS C3H]GABA was determined by subtracting from the total Bullfrogs (Rana catesbiana, 6 8 " body length) were bound radioactivity the amount not displaced by high conobtained from Mogul-ED Corporation of Oshkosh, Wis- centrations of bicuculline (0.1 mM) or GABA (1 mM). Proconsin, the brains and spinal cords were removed upon tein was measured by the method of LOWRYet al. (1951). 857
Short communication
858
All compounds used in this study were obtained from commercial suppliers, except for (+) cis- and trans-3aminocyclopentane-4-carboxylic acid which were gifts from Dr. G. A. R. JOHNSTON and muscimol which was a gift from Dr. E. COSTA. RESULTS Sodium independent GABA binding is saturable in frog spinal cord and brain (Fig. 1). In both tissues binding plateaus at about 0.1-0.3 PM C3H]GABA and reaches halfmaximal values at 30-60 IIM. Scatchard analysis suggests only one population of binding sites in frog brain and spinal cord with respective dissociation constants of 58 nM and 33 nM. However, the amount of scatter observed with
A
SPINAL CORD
r
1
,I
.2 .3 .4 [3H] GABA ( p M )
.5
140k
I'"
B
-L
O Z 3
80 *
0
\,TIN
m
a 60-
I 6 p m o l e s / m g protein
m
-
-58nM TOTAL BINDING SITES
4
(3
I
40.
SPINAL CORD
02 04 0 6 00 10 BOUND/FREE (pmoles/nmole)
12
FIG. 1. Saturation of specific C3H]GABA binding to frog brain and spinal cord synaptic membranes with increasing concentrations of C3H]GABA. Frog brain and spinal cord synaptic membrane suspensions were incubated in Triscitrate (pH 7.1) with various concentrations of C3H]GABA. Specific binding was obtained by subtracting the amount of radioactivity not displaced by 1 mM-GABA from the total C3H]GABA bound. Values are the means of triplicate determinations which varied less than 10%. The experiment was replicated 4 times. (A) Linear plot. (B) Scatchard analysis.
TABLE1. SUBSTRATE SPECIFICITY
OF C3H]GABA BINDING IN FROG CENTRAL NERVOUS SYSTEM
G
Compound
Brain
0.05 GABA 2.0 ( + )-Bicuculline 0.09 3-Aminopropane sulfonic acid 0.007 Muscimol 2,4-Diaminobutyric acid 600 Picrotoxin >' 500 ( +)-Trans-3-aminocyclopentane0.30 4-carboxylic acid ( +)-Cis-3-Aminocyclopentane-45.0 carboxylic acid me.* Pentobarbital-Na n.e. Pentylenetetrazol
o (PI)
Spinal cord 0.03 3.0 0.10 -
-
-
n.e. n.e.
* No effect at 1mM. Inhibition of C3H]GABA binding by various concentrations of the different compounds was determined using the assay procedure described in Fig. 2. IC,, values, the concentration of agents which reduced binding of C3H]GABA by SO%, were calculated by log-probit analysis. brain makes a definitive conclusion about the number of sites difficult. By contrast, for tissue prepared in the same way with Triton, rat brain displays high ( K D = 18 nM) and low affinity ( K D= 200 nM) components of GABA binding (ENNA& SNYDER,in press). Without Triton X-100 treatment rat brain membranes also display only a single binding site with a K D of 200-400n~(ENNA& SNYDER,1975, in press). Spinal cord has only about 20% as many GABA binding sites as frog brain, while rat brain has about the same number of receptors as frog brain. The binding differences observed between spinal cord and brain appears to be due to a difference in the total number of receptor sites rather than affinity since the affinity for C3H]GABA is the same or slightly higher in cord than in brain. The possibility that differences in synaptic membrane sedimentation characteristics may explain this result must also be entertained. The substrate specificity of sodium independent GABA binding in Triton treated tissue is quite similar in frog brain and spinal cord and rat brain (Table 1; Fig. 2) (ENNA & SNYDER, in press). In all three tissues the IC,, values for GABA and 3-aminopropane sulfonic acid are about 0.034.10 PM, corresponding to the similar neurophysiologic potencies of these two agents. Muscimol, which is substantially more active neurophysiologically in mimicking et al., GABA than GABA itself (KROGSGAARD-LARSON 1975), has almost 10 times the affinity of GABA. The geometrical isomers of 3-aminocyclopentane-4-carboxylicacid differ markedly in their neurophysiological mimicry of GABA with much greater activity being displayed by the trans than by the cis isomer (JOHNSTON, 1976). The binding selectivity of these two isomers in frog and rat brain (ENNA & SNYDER, in press) corresponds to their neurophysiological differences; in both tissues the trans isomer is about 10 times as potent as the cis isomer. In rat brain the GABA antagonist bicuculline is a potent inhibitor of GABA receptor binding, while picrotoxin, which is also active as a GABA antagonist, has negligible affinity (ZUKINet al., 1974; ENNA& SNYDER,1975). In frog brain and spinal cord bicuculline's potency in compet-
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FIG.2. Competition of GABA agonists and bicuculline for ['HIGABA binding sites in frog brain synaptic membranes. Increasing concentrations of nonradioactive GABA and other agents were added to tubes containing approx 8 nw['H]GABA and frog brain synaptic membranes. The binding values are the means of triplicate determinations. Samples were incubated for 5 min at 4°C and centrifuged to isolate bound ['HIGABA. The abbreviation ACPC refers to aminocyclopentane-4-carboxylicacid.
DISCUSSION Sodium independent binding of ['HIGABA to spinal cord and brain of the frog closely resembles the characteristics of GABA receptor binding in mammalian CNS. The evidence that binding in the frog involves synaptic receptors derives mainly from the close parallel between binding potencies of various agents and their neurophysiologic actions at GABA synapses. The only clear difference between rat brain and frog brain and spinal cord is the failure to detect distinct high and low affinity components of GABA binding in the frog. It is not altogether clear whether sodium independent ['HIGABA binding is associated exclusively with presynaptic or postsynaptic receptors. Since presynaptic inhibition by GABA predominates in the spinal cord, while postsynaptic inhibition is more prominent in higher centers, the fact that the density of GABA receptors is 5 times greater in brain than spinal cord suggests that binding detected in our studies primarily involves postsynaptic receptors. This conclusion is supported by observations in some studies that picrotoxin is more selective for presynaptic than postsynaptic GABA sites (BARKERet al., 1975a), which goes along with the inability to detect picrotoxin inhibition of GABA binding. An alternative explanation for the inactivity of picrotoxin at GABA binding sites is that it acts primarily by competing at the ionophore rather than at the GABA recognition site, as is suggested by neurophysiological studies indicating that picrotoxin effects are competitive with chloride but not with GABA (TAKEUCHI & TAKEUCHI,1969). Another observation suggesting that C3H]GABA binding does not involve presynaptic receptors is that neurophysiologic influences of pentobarbital and pentylenetetrazol occur predominantly at presynaptic rather than postsynaptic sites, possibly by direct receptor actions (NICOLL& PADJEN,1976; NICQLL,1975a,b) yet these drugs fail to affect GABA receptor binding.
ing for GABA receptor binding resembles the drug's effects in rat brain, while picrotoxin is inactive in frog brain as in rat brain. 2,4, Diaminobutyric acid, with high affinity 1975) but negligfor GABA neuronal uptake sites (IVERSEN, ible neurophysiological activity at GABA receptors, is also very weak in competing for GABA binding in frog and rat brain. Pentobarbital sodium and pentylenetetrazol have prominent influences on presynaptic inhibition, acting in a fashion which could reflect a direct influence on the GABA receptors which mediate presynaptic inhibition (NICOLL& PADIEN, 1976; NICOLL,1975a,b). Neither of these drugs have any influence on GABA receptor binding Acknowledgements-We thank Dr. R. NICOLLfor helpful in frog brain and spinal cord or rat brain. The striking for technical assimilarities in affinity, total number of binding sites and suggestions and Ms. CINDYKAUFMANN drug specificity between rat and frog brain tissue suggests sistance. that, under the conditions of these experiments, ['HIGABA labels the same site in both species, presum- Departments of Pharmacology and S. J. E N N A ~ ably the synaptic receptor site for this neurotransmitter Experimental Therapeutics and S. H. SNYDER amino acid. Psychiatry and Behuvioural Sciences, Receptor binding for other neurotransmitters, such as Johns Hopkins University School of Medicine, the a-adrenergic, muscarinic cholinergic, dopamine and Baltimore, MD 21205, serotonin receptors, differs for agonists and antagonists U.S.A. (SNYDER et el., 1975; BURTet al., 1976; GREENBERG et al., 1976; BENNETT& SNYDER,1976; SNYDER& BENNETT, 1976). While Hill coefficients for displacement of a REFERENCES 'H-agonist by other agonists or for 'H-antagonists by other antagonists are about 1, Hill coefficients of less than BARKER J. L., NICOLLR. A. & PADJEN A. (1975a) J. Physiol. 1 are associated with displacement of 'H-agonists by an245, 521-536. tagonists and vice versa. Accordingly, we examined in BARKERJ. L., NICOLLR. A. & PADJEN A. (1975b) J. Physiol. detail the displacement curves in frog brain of ['HIGABA 245, 537-548. by GABA itself, by cis and trans isomers of 3-aminocyclo- BENNETT J. P. & SNYDERS. H. (1976) Motec. Pharmac. pentane-Ccarboxylic acid and by the antagonist bicucul12, 373-389. line. Reduction of ['HIGABA binding by the antagonist BURTD. R., CREESEI. & SNYDERS. H. (1976) Molec. Pharbicuculline, by GABA and by the agonists cis and trans-3mac. 12, 8 W 8 1 2 . aminocyclopentane-4-carboxylic acid display parallel displacement curves. Hill plots of these data reveal Hill coeffiPresent address: Department of Neurobiology and cients of 1.0, indicating the absence of positive or negative cooperativity for any of these agents. These data resemble Anatomy, University of Texas Medical School at Houston, previous results in the rat (ENNA& SNYDER, 1975, in press). P.O. Box 20708, Houston, TX 77025, U.S.A.
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