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Specific [3H]γ-aminobutyric acid binding to vestibular membranes of the chick inner ear
Brain Research, 337 (1985) 179-183 Elsevier
179
BRE 20886
Specific [3H]7-aminobutyric acid binding to vestibular membranes of the chick inner ear GRACIELA MEZA 1, MA. TERESA GONZALEZ-VIVEROS1and MARIANO RUIZ2 ]Departarnento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, and 2Departamento de Histologia, Facultad de Medicina, UNAM, M~xico, D. F. (Mexico)
(Accepted January 28th, 1985) Key words: y-aminobutyric acid (GABA) - - GABA-specific binding - - GABA receptor - - inner ear
To support a postulated neurotransmitter character of y-aminobutyric acid (GABA) in the vertebrate vestibule, [3H]GABA binding was measured in a crude membrane preparation of chick inner ear ampullary cristae. In the absence of divalent cations bound [3H]GABA was displaced by unlabeled GABA, muscimol or bicuculline, but it was not displaced by (+)-baclofen. A single population of pH]GABA binding sites with an equilibrium constant of 19.4 nM and a maximum binding capacity of 0.58 pmol/mg protein was found. These results suggest the possible existence of a synaptic GABAA receptor in the chick inner ear membranes and sustain the neurotransmitter role of GABA in the chick vestibule. Recent investigations support the involvement of y-aminobutyric acid ( G A B A ) as the afferent neurotransmitter in the sensory periphery of some statoacoustic organs of vertebrates. G A B A synthesis has been found in the amphibian labyrinth and in the fish vestibule and lateral line 12. Sodium and energy-dependent high-affinity [ 3 H ] G A B A uptake has been described in isolated ampullary cristae of the chick vestibule 22. G A B A synthesis and glutamate decarboxylase with properties similar to those of the brain enzyme were demonstrated in chick vestibular tissue 23,24. Further, a G A B A - l i k e substance was identified in the perilymph of guinea pig cochlea after sound stimulation 6. Electrophysiological experiments have shown that picrotoxin can block spontaneous and stimulated responses of the isolated rat labyrinth12; iontophoretically applied picrotoxin and bicuculline inhibit spontaneous afferent fiber firing of the cat labyrinth 11, thus highly suggesting the presence of a postsynaptic G A B A receptor in vertebrate statoacoustic organs. In vitro specific [ 3 H ] G A B A binding to membranes is recognized as an expression of the interaction between G A B A and its physiological receptor sites2S;
however, this interaction should comply with several criteria. According to these prerequisites, one should be able to demonstrate that the binding of [3H]GAB A to its receptor sites is displaced by an excess of the amino acid itself and by a specific antagonist31; that these interactions are independent of sodium ionsS,15,16,19,25,28,3° and that incubation in the presence of increasing concentrations of the amino acid results in saturable [ 3 H ] G A B A bindingS,9,10,15,17,28,31. In addition, all these criteria should be demonstrated in Triton-treated, extensively washed, frozen and thawed membranes3,15. Since the demonstration of G A B A recognition .sites by biochemical means may give support to the above-mentioned physiological evidences for a G A B A receptor and sustain the neurotransmitter role of G A B A in the vertebrate vestibule, in the present study binding of [ 3 H ] G A B A was examined in a Triton-treated, thoroughly washed, frozen and thawed membrane fraction obtained from isolated chick vestibular cristae. In order to try to characterize the nature of the receptor, unlabeled G A B A , muscimol and bicuculline ( G A B A A receptor agonist and blocker respective-
Correspondence: G. Meza, Departamento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, U.N.A.M., Apartado Postal 70-600, 04510 Mexico, D. F. M6xico.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
180 ly)5,TMand (+)-baclofen, a specific agonist of GABAB receptor I were used as displacer agents. The sodium dependence and the saturation properties of [3H]GABA binding were also analyzed. The procedure followed and the results obtained are described in the present paper. One-day-old Rhode Island Red male chicks were sacrificed by decapitation and the cristae ampullares were dissected out as earlier reported z3. A crude membrane fraction was prepared by a modification of the procedure described for retinal membranes 9. Cristae removed from both ears of 30 animals (0.6 mg wet weight approximately per organ) were osmotically shocked by thorough homogenization in 100 vol. of triple distilled H20 in a glass-teflon tissue grinder and dispersed in an Ultrasonic Power Unit sonicator for 5 s. The homogenates were frozen and stored overnight at 0 °C, then they were thawed and sonicated for 10 min with cycles of 15 s sonication and 5 s of rest. This preparation was kept in the freezer for 24 h, being then thawed and centrifuged at 65,000 g for 20 min. The final pellet was stored frozen and used after 6-7 weeks time at which maximum specific binding was observed. For the binding assay, the membrane pellets were resuspended before use and homogenized in 100 vol. based on the original weight of 50 mM Tris-citrate buffer pH 7.4 and incubated with 0.05% Triton X-100 (Sigma Co) for 30 min at 37 °C; the membrane suspension was then centrifuged at 65,000 g for 15 min. The supernatant was discarded and the pellet was washed twice by resuspension in the same buffer and repeated sedimentation at 65,000 g for 15 min. The resulting pellet was resuspended in suitable volumes of 50 mM Tris-citrate buffer to yield a final protein concentration of 0.014-0.017 mg/ml. The binding assay was carried out according to the centrifugation method of Enna and Snyder s except that centrifugation took place at 65,000 g instead of at 48,000 g and incubation was performed at room temperature; [3H]GABA (38 Ci/mmol) at a concentration of 8 nM was used as a ligand. To measure bound [3H]GABA, the pellet resulting from the binding assay was digested with 0.2 ml of tissue solubilizer (NCS, Amersham) and counted after 24 h of addition of Triton-based scintillation fluid 13 in a Packard TriCarb Scintillation Spectrometer. Specifically bound ligand was calculated as the difference between the
amount of radioactive ligand associated to the membrane fraction of parallel binding experiments performed in the presence and in the absence of 1 mM unlabeled GABA. The percentage of unspecific binding under these conditions was less than 30%. Sodium dependence of [3H]GABA interactions was explored in parallel experiments in which incubation took place in either 50 mM Tris-citrate buffer pH 7.4 containing 118 mM NaCI or in 50 mM Tris Citrate buffer pH 7.4 with 118 mM choline chloride. In competition studies, 4-6 different concentrations (from 0.01 to 200/~M) of unlabeled GABA (Sigma Co), muscimol (Sigma Co), bicuculline methiodide (Sigma Co), or (_+)-baclofen (a generous gift of Dr. S. J. Enna) were tested for their ability to displace specific binding in the presence of 8 nM [3H]GABA. Incubation was performed for 5 min, when experimentally determined equilibrium was reached. For the saturation experiments [3H]GABA was present in the samples in 14 different concentrations in a range of 0.25-188 nM. Each experiment was performed in triplicate. The protein content was measured by the method of Lowry et al. 21. When [3H]GABA interaction with chick inner ear membranes was assessed at 8 nM GABA in the absence of sodium (substituting NaC1 for choline chloride) a specific [3H]GABA binding of 0.34 pmol/mg protein was obtained. Whereas when assayed in the presence of sodium, a value of 0.35 pmol/mg protein of specifically bound [3H] was obtained. (These are the results of 3 independent triplicate experiments run in parallel with less than 10% variation). Thus essentially the specific binding of [3H]GABA to vestibular membranes of the chick inner ear was not dependent on sodium ions. Therefore, the competition as well as the saturation experiments could be safely performed in the absence of sodium. Results obtained in the competition experiments when unlabeled GABA, muscimol, bicuculline methiodide or (+)-baclofen were used as displacers of bound [3H]GABA to membranes of the chick inner ear are shown in Table I. Unlabeled G A B A was able to displace bound [3H]GABA with an IC50 of 0.033 ~M whereas muscimol and bicuculline methiodide performed the same IC50 for displacement at 0.010 /zM and 105 /zM respectively. In contrast, (+)-baclofen did not have any effect even at concentrations as high as 200/zM. These compounds are tra-
181 TABLE I Inhibition of specific [3H]GABA binding to membranes of the chick inner ear by various model compounds Specific [3H]GABA-bound displacement by each compound used was determined by using 8 nM of [3H]GABA in the presence of 4-6 different concentrations (from 0.01 to 200/~M) of each compound. The concentrations of the compounds inhibiting 50% of specific binding (IC50 values) were calculated by log-probit analysis. Results are mean values of 4-5 experiments run in triplicate. Compound GABA Muscimol Bicuculline methiodide (+)-Baclofen
IC5o value (I~M) 0.033 0.010 105 >200
ditionally used to discriminate a G A B A A (muscimol and bicuculline) from a G A B A B type of receptors ((+)-baclofen). The order of potency displayed (muscimol > G A B A >> bicuculline) indicates the possible presence of a G A B A A receptor although bicuculline was much less potent in displacing [3H]G A B A than has been shown in nervous system membranest0,30, 31. As potency of bicuculline to displace bound G A B A in rat brain and retinal membranes has been augmented by incubating in the presence of chaotropic agents3,aA0,26, experiments with thiocyanate and perchloric ions are needed and will be performed later in our research. O u r result however is comparable with results in chick retinal membranes in which an IC50 of displacement of bound [3H]GABA for bicuculline of around 100/tM was obtained20. Further, an IC50 of 100/~M for bicuculline determined in a physiological preparation was obtained at crustacean neuromuscular junction 27. In contrast, (+)-baclofen, a selective agonist of G A B A B receptors1,2, 29 was ineffective in displacing bound [3H]GABA. This is not surprising, since under the conditions used, namely Tris-citrate buffer in the absence of Ca 2+ or Mg 2÷, G A B A B binding sites cannot be detectedL Therefore, even if G A B A B receptors are present in the chick inner ear membranes, they would have not been demonstrated with the incubation medium used. Additional pharmacological experimentations, using other G A B A agonists such as T H I P , imidazole acetic acid, 3-aminopropane sulfonic acid, etc. are now in progress in our laboratory to completely discard this latter possibility. Fig. 1 shows the results of specific [ 3 H ] G A B A
c
v=
-
il
60-
e 40-
,.,
0.1
0.2
0,3
Bound E'H'] GABA (pmoles/mg
0,4
0,5
0,6
protein)
Fig. 1. Scatchard plot of saturation of specific [3H]GABA binding to a crude membrane preparation of chick inner ear. An equilibrium dissociation constant of 19 nM and a maximum binding capacity of 0.58 pmol/mg protein were calculated from the plot with a PDP-11/34 computer by linear regression and drawn with a Hewlett-Packard 7225B plotter. A linear correlation coefficient of-0.988 was also calculated. The saturation curve is shown in the inset. Data in its ordinates represent specifically bound [3H]GABA expressed as pmol/mg protein plotted against GABA concentrations. Each point in the graph is the mean of 3 independent observations each in triplicate. Data of greater GABA concentrations are not shown but were taken into account for the Scatchard plot calculations. The methodology followed and the calculation of the data are described in the text. binding as measured at increasing concentrations of the ligand. As can be observed, the specific binding of [ 3 H ] G A B A to chick inner ear membranes is saturable (Fig. 1, inset) and the binding data yielded a linear Scatchard plot that is consistent with a single class of binding sites. The values for an apparent equilibrium constant (Kd) of 19.4 nM and a maximum binding capacity (Bmax) of 0.59 pmol/mg protein were calculated by linear regression analysis of the data of the Scatchard plot. The finding, in a Triton-treated, extensively washed, frozen and thawed membrane preparation of the chick inner ear, of a single population of binding sites for [ 3 H ] G A B A as indicated by the linear Scatchard plot (Fig. 1) differs from the two populations of binding sites for G A B A consistently found in nervous tissue 3,9,10,15,17. However, it resembles the encountering of only one high-affinity site in human anterior pituitary 14. Notwithstanding, the K d values and number of binding sites found in the chick inner ear membranes for [ 3 H ] G A B A are very similar to what has been shown for the high-affinity G A B A binding site
182 TABLE II
high-affinity, saturable, s o d i u m - i n d e p e n d e n t G A B A
Comparison of [3H]GABA binding data found in several nervous tissue membrane preparations with [3H]GABA binding values obtained in chick vestibular membranes
binding system of the same magnitude, kinetic con-
Preparation
Ka (nM)
Bmax (pmol/mg protein)
Reference
vous system m e m b r a n e fractions, strongly suggests the possible presence of a G A B A A receptor in our
Chick inner ear Human anterior pituitarya Rat retina b Rat brainb Rat brainb Rat cerebellumb Rat brainb,c
19.4"
0.59
Our work
40 18 16 32 32 20
0.65 0.60 0.60 1.17 1.4 0.48
Grandison et a1.14 Enna and Snyder9 Enna and Snyder1° Greenlee et a1.15 Browner et al. 3 Jordan et al. 17
stants and properties in front of agonists and antagonists obtained for G A B A receptors in various ner-
preparation and may well represent a physiological
Only one high-affinity site was encountered. b Although two sites were present, data refer only to the highaffinity one. ¢ Highly purified synaptic plasma membrane fraction. a
one. Studies in which the coincidence of the presence of a G A B A postsynaptic site and of glutamate decarboxylase together with a high-affinity G A B A transport system in discrete regions of m a m m a l i a n brain have been used in support for the participation of G A B A as n e u r o t r a n s m i t t e r in those regions 7. As all these parameters have been d e m o n s t r a t e d in isolated chick vestibular cristae, a n e u r o t r a n s m i t t e r role for GABA
may be postulated in these organs.
Our thanks to Dr. S. J. E n n a for a generous gift of in the formerly m e n t i o n e d nervous tissue preparations3,9,10,15A7. Table II compares the results ob-
(___)-baclofen; to Ing. Federico Fern~indez Cancino for help with the computer-based calculations and to
tained in the chick i n n e r ear m e m b r a n e s with those
Mrs. Virginia G o d i n e z and Miss Yoland Diaz de Castro for secretarial assistance. Supported in part by
data. In conclusion: the presence in a crude m e m b r a n e preparation of isolated chick vestibular cristae, of a
1 Bowery, N. G., Doble, A., Hill, D. R., Hudson, A. L., Show, J. S. and Turnbull, M. J., Baclofen: a selective agonist for a novel type GABA receptor, Brit. J. Pharmacol., 67 (1979) 444-445. 2 Bowery, N. G., Hill, D. R. and Hudson, A. L., Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes, Brit. J. Pharmacol., 78 (1983) 191-206. 3 Browner, M., Ferkany, J. W. and Enna, S. J., Biochemical identification of pharmacologically and functionally distinct GABA receptors in rat brain, J. Neurosci., 1 (1981) 514-518. 4 Churchill, L. and Redburn, D. A., Effects of chaotropic anions on bicuculline inhibition of high affinity y-aminobutyric acid binding in bovine retina and rat brain membranes, Neurochem. Int., 5 (1983) 221-226. 5 Curtis, D. R., Duggan, A. W., Felix, D. and Johnston, G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222-1224. 6 Drescher, M. J., Drescher, D. G. and Medina, J. E., Effect of sound stimulation at several levels on concentrations of primary amines, including neurotransmitter candidates, in perilymph of the guinea pig inner ear, J. Neurochem., 41 (1983) 309-320. 7 Enna, S. J., Kuhar, M. J. and Snyder, S. H., Regional distribution of postsynaptic receptor binding of gamma-aminobutyric acid (GABA) in monkey brain, Brain Research, 93 (1975) 168-174.
Grant P C C B B N A 020897 from Consejo Nacional de Ciencia y Tecnologia ( C O N A C Y T ) , M6xico.
8 Enna, S. J. and Snyder, S. H., Properties of 7-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fraction, Brain Research, 100 (1975) 81-97. 9 Enna, S. J. and Snyder, S. H., Gamma-aminobutyric acid (GABA) receptor binding in mammalian retina, Brain Research, 115 (1976) 174-179. 10 Enna, S. J. and Snyder, S. H., Influences of ions, enzymes and detergents on ~,-aminobutyricacid receptor binding in synaptic membranes of rat brain, Mol. Pharmacol., 13 (1977) 442-453. 11 Felix, D. and Ehrenberger, K., The action of putative neurotransmitter substances in the cat labyrinth, Acta otolaryngol., 93 (1982) 101-105. 12 Flock, A. and Lam, D. M. K., Neurotransmitter synthesis in inner ear and lateral line sense organs, Nature (Lond.), 249 (1974) 142-144. 13 Fricke, V., Tritosol: a new scintillation cocktail based on Triton X-100, Anal. Biochem., 63 (1975) 555-558. 14 Grandison, L., Cavagnini, F., Schmid, R., Invitti, C. and Guidotti, A., 7-Aminobutyric acid and benzodiazepine binding sites in human anterior pituitary tissue, J. clin. Endocrinol. Metab., 54 (1982) 597-601. 15 Greenlee, D. V., Van Ness, P. C. and Olsen, R. W., Endogenous inhibitor of GABA binding in mammalian brain, Life Sci., 22 (1978) 1653-1662. 16 Greenlee, D. V., Van Ness, P. C. and Olsen, R. W., Gamma-aminobutyric acid binding in mammalian brain: receptor-like specificity of sodium-independent sites, J. Neuro-
183 chem., 31 (1978) 933-938. 17 Jordan, C. C., Matus, A. I., Piotrowki, W. and Wilkinson, D., Binding of [3H]y-aminobutyric acid and [aH]muscimol in purified rat brain synaptic plasma membranes and the effects of bicuculline, J. Neurochem., 39 (1982)52-58. 18 Krogsgaard-Larsen, P., Johnston, G. A. R., Curtis, D. R., Game, C. J. A. and McCulloch, R. M., Structure and biological activity of a series of conformationally restricted analogues of GABA, J. Neurochem., 25 (1975) 803-809. 19 Lester, B. R. and Peck, E. J., Jr., Kinetic and pharmacologic characterization of gamma-aminobutyric acid receptive sites from mammalian brain, Brain Research, 161 (1979) 79-97. 20 L6pez-Colom6, A. M. and Pasantes-Morales, H., Taurine interactions with chick retinal membranes, J. Neurochem., 34 (1980) 1047-1052. 21 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 22 Meza, G., Hern~indez, C. and Ruiz, M., [3H]-GABA uptake in isolated vestibulary cristae of chick inner ear, Soc. Neurosci. Abstr., 7 (1981) 147. 23 Meza, G., C~rabez, A. and Ruiz, M., G A B A synthesis in isolated vestibulary tissue of chick inner ear, Brain Research, 241 (1982) 157-161. 24 Meza, G., Some characteristics of glutamic acid decarbox-
25
26
27
28
29
30
31
ylase of chick ampuilary cristae, J. Neurochem., 43 (1984) 634-639. Peck, E. J., Jr., Schaeffer, J. M. and Clark, J. H., 7-Aminobutyric acid, bicuculline and postsynaptic binding sites, Biochem. Biophys. Res. Comm., 52 (1973) 394-400. Redburn, D. A. and Mitchell, C. K., GABA receptor binding in bovine retina: effects of Triton X-100 and perchloric acid, Life Sci., 28 (1981) 541-549. Takeuchi, A. and Onodera, K., Effect of bicuculline on the GABA receptor of the crayfish neuromuscular junction, Nature (Lond.), 236 (1972) 55-56. Toffano, G., Guidotti, A. and Costa, E., Purification of an endogenous protein inhibitor of the high affinity binding of 7-aminobutyric acid to synaptic membranes of rat brain, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4024-4028. Turner, A. J. and Whittle, S. R., Biochemical dissection of the gamma aminobutyrate synapse, Biochern. J., 209 (1983) 29-41. Wong, D. T. and Horng, J. S., Na+-independent binding of GABA to the Triton-X-100 treated synaptic membranes from cerebellum of rat brain, Life Sci., 20 (1977) 445-451. Zukin, S. R., Young, A. B. and Snyder, S. H., Gammaaminobutyric acid binding to receptor sites in the rat central nervous system, Proc. nat. Acad Sci. U.S.A., 7l (1974) 4802-4807.
179
BRE 20886
Specific [3H]7-aminobutyric acid binding to vestibular membranes of the chick inner ear GRACIELA MEZA 1, MA. TERESA GONZALEZ-VIVEROS1and MARIANO RUIZ2 ]Departarnento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, and 2Departamento de Histologia, Facultad de Medicina, UNAM, M~xico, D. F. (Mexico)
(Accepted January 28th, 1985) Key words: y-aminobutyric acid (GABA) - - GABA-specific binding - - GABA receptor - - inner ear
To support a postulated neurotransmitter character of y-aminobutyric acid (GABA) in the vertebrate vestibule, [3H]GABA binding was measured in a crude membrane preparation of chick inner ear ampullary cristae. In the absence of divalent cations bound [3H]GABA was displaced by unlabeled GABA, muscimol or bicuculline, but it was not displaced by (+)-baclofen. A single population of pH]GABA binding sites with an equilibrium constant of 19.4 nM and a maximum binding capacity of 0.58 pmol/mg protein was found. These results suggest the possible existence of a synaptic GABAA receptor in the chick inner ear membranes and sustain the neurotransmitter role of GABA in the chick vestibule. Recent investigations support the involvement of y-aminobutyric acid ( G A B A ) as the afferent neurotransmitter in the sensory periphery of some statoacoustic organs of vertebrates. G A B A synthesis has been found in the amphibian labyrinth and in the fish vestibule and lateral line 12. Sodium and energy-dependent high-affinity [ 3 H ] G A B A uptake has been described in isolated ampullary cristae of the chick vestibule 22. G A B A synthesis and glutamate decarboxylase with properties similar to those of the brain enzyme were demonstrated in chick vestibular tissue 23,24. Further, a G A B A - l i k e substance was identified in the perilymph of guinea pig cochlea after sound stimulation 6. Electrophysiological experiments have shown that picrotoxin can block spontaneous and stimulated responses of the isolated rat labyrinth12; iontophoretically applied picrotoxin and bicuculline inhibit spontaneous afferent fiber firing of the cat labyrinth 11, thus highly suggesting the presence of a postsynaptic G A B A receptor in vertebrate statoacoustic organs. In vitro specific [ 3 H ] G A B A binding to membranes is recognized as an expression of the interaction between G A B A and its physiological receptor sites2S;
however, this interaction should comply with several criteria. According to these prerequisites, one should be able to demonstrate that the binding of [3H]GAB A to its receptor sites is displaced by an excess of the amino acid itself and by a specific antagonist31; that these interactions are independent of sodium ionsS,15,16,19,25,28,3° and that incubation in the presence of increasing concentrations of the amino acid results in saturable [ 3 H ] G A B A bindingS,9,10,15,17,28,31. In addition, all these criteria should be demonstrated in Triton-treated, extensively washed, frozen and thawed membranes3,15. Since the demonstration of G A B A recognition .sites by biochemical means may give support to the above-mentioned physiological evidences for a G A B A receptor and sustain the neurotransmitter role of G A B A in the vertebrate vestibule, in the present study binding of [ 3 H ] G A B A was examined in a Triton-treated, thoroughly washed, frozen and thawed membrane fraction obtained from isolated chick vestibular cristae. In order to try to characterize the nature of the receptor, unlabeled G A B A , muscimol and bicuculline ( G A B A A receptor agonist and blocker respective-
Correspondence: G. Meza, Departamento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, U.N.A.M., Apartado Postal 70-600, 04510 Mexico, D. F. M6xico.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
180 ly)5,TMand (+)-baclofen, a specific agonist of GABAB receptor I were used as displacer agents. The sodium dependence and the saturation properties of [3H]GABA binding were also analyzed. The procedure followed and the results obtained are described in the present paper. One-day-old Rhode Island Red male chicks were sacrificed by decapitation and the cristae ampullares were dissected out as earlier reported z3. A crude membrane fraction was prepared by a modification of the procedure described for retinal membranes 9. Cristae removed from both ears of 30 animals (0.6 mg wet weight approximately per organ) were osmotically shocked by thorough homogenization in 100 vol. of triple distilled H20 in a glass-teflon tissue grinder and dispersed in an Ultrasonic Power Unit sonicator for 5 s. The homogenates were frozen and stored overnight at 0 °C, then they were thawed and sonicated for 10 min with cycles of 15 s sonication and 5 s of rest. This preparation was kept in the freezer for 24 h, being then thawed and centrifuged at 65,000 g for 20 min. The final pellet was stored frozen and used after 6-7 weeks time at which maximum specific binding was observed. For the binding assay, the membrane pellets were resuspended before use and homogenized in 100 vol. based on the original weight of 50 mM Tris-citrate buffer pH 7.4 and incubated with 0.05% Triton X-100 (Sigma Co) for 30 min at 37 °C; the membrane suspension was then centrifuged at 65,000 g for 15 min. The supernatant was discarded and the pellet was washed twice by resuspension in the same buffer and repeated sedimentation at 65,000 g for 15 min. The resulting pellet was resuspended in suitable volumes of 50 mM Tris-citrate buffer to yield a final protein concentration of 0.014-0.017 mg/ml. The binding assay was carried out according to the centrifugation method of Enna and Snyder s except that centrifugation took place at 65,000 g instead of at 48,000 g and incubation was performed at room temperature; [3H]GABA (38 Ci/mmol) at a concentration of 8 nM was used as a ligand. To measure bound [3H]GABA, the pellet resulting from the binding assay was digested with 0.2 ml of tissue solubilizer (NCS, Amersham) and counted after 24 h of addition of Triton-based scintillation fluid 13 in a Packard TriCarb Scintillation Spectrometer. Specifically bound ligand was calculated as the difference between the
amount of radioactive ligand associated to the membrane fraction of parallel binding experiments performed in the presence and in the absence of 1 mM unlabeled GABA. The percentage of unspecific binding under these conditions was less than 30%. Sodium dependence of [3H]GABA interactions was explored in parallel experiments in which incubation took place in either 50 mM Tris-citrate buffer pH 7.4 containing 118 mM NaCI or in 50 mM Tris Citrate buffer pH 7.4 with 118 mM choline chloride. In competition studies, 4-6 different concentrations (from 0.01 to 200/~M) of unlabeled GABA (Sigma Co), muscimol (Sigma Co), bicuculline methiodide (Sigma Co), or (_+)-baclofen (a generous gift of Dr. S. J. Enna) were tested for their ability to displace specific binding in the presence of 8 nM [3H]GABA. Incubation was performed for 5 min, when experimentally determined equilibrium was reached. For the saturation experiments [3H]GABA was present in the samples in 14 different concentrations in a range of 0.25-188 nM. Each experiment was performed in triplicate. The protein content was measured by the method of Lowry et al. 21. When [3H]GABA interaction with chick inner ear membranes was assessed at 8 nM GABA in the absence of sodium (substituting NaC1 for choline chloride) a specific [3H]GABA binding of 0.34 pmol/mg protein was obtained. Whereas when assayed in the presence of sodium, a value of 0.35 pmol/mg protein of specifically bound [3H] was obtained. (These are the results of 3 independent triplicate experiments run in parallel with less than 10% variation). Thus essentially the specific binding of [3H]GABA to vestibular membranes of the chick inner ear was not dependent on sodium ions. Therefore, the competition as well as the saturation experiments could be safely performed in the absence of sodium. Results obtained in the competition experiments when unlabeled GABA, muscimol, bicuculline methiodide or (+)-baclofen were used as displacers of bound [3H]GABA to membranes of the chick inner ear are shown in Table I. Unlabeled G A B A was able to displace bound [3H]GABA with an IC50 of 0.033 ~M whereas muscimol and bicuculline methiodide performed the same IC50 for displacement at 0.010 /zM and 105 /zM respectively. In contrast, (+)-baclofen did not have any effect even at concentrations as high as 200/zM. These compounds are tra-
181 TABLE I Inhibition of specific [3H]GABA binding to membranes of the chick inner ear by various model compounds Specific [3H]GABA-bound displacement by each compound used was determined by using 8 nM of [3H]GABA in the presence of 4-6 different concentrations (from 0.01 to 200/~M) of each compound. The concentrations of the compounds inhibiting 50% of specific binding (IC50 values) were calculated by log-probit analysis. Results are mean values of 4-5 experiments run in triplicate. Compound GABA Muscimol Bicuculline methiodide (+)-Baclofen
IC5o value (I~M) 0.033 0.010 105 >200
ditionally used to discriminate a G A B A A (muscimol and bicuculline) from a G A B A B type of receptors ((+)-baclofen). The order of potency displayed (muscimol > G A B A >> bicuculline) indicates the possible presence of a G A B A A receptor although bicuculline was much less potent in displacing [3H]G A B A than has been shown in nervous system membranest0,30, 31. As potency of bicuculline to displace bound G A B A in rat brain and retinal membranes has been augmented by incubating in the presence of chaotropic agents3,aA0,26, experiments with thiocyanate and perchloric ions are needed and will be performed later in our research. O u r result however is comparable with results in chick retinal membranes in which an IC50 of displacement of bound [3H]GABA for bicuculline of around 100/tM was obtained20. Further, an IC50 of 100/~M for bicuculline determined in a physiological preparation was obtained at crustacean neuromuscular junction 27. In contrast, (+)-baclofen, a selective agonist of G A B A B receptors1,2, 29 was ineffective in displacing bound [3H]GABA. This is not surprising, since under the conditions used, namely Tris-citrate buffer in the absence of Ca 2+ or Mg 2÷, G A B A B binding sites cannot be detectedL Therefore, even if G A B A B receptors are present in the chick inner ear membranes, they would have not been demonstrated with the incubation medium used. Additional pharmacological experimentations, using other G A B A agonists such as T H I P , imidazole acetic acid, 3-aminopropane sulfonic acid, etc. are now in progress in our laboratory to completely discard this latter possibility. Fig. 1 shows the results of specific [ 3 H ] G A B A
c
v=
-
il
60-
e 40-
,.,
0.1
0.2
0,3
Bound E'H'] GABA (pmoles/mg
0,4
0,5
0,6
protein)
Fig. 1. Scatchard plot of saturation of specific [3H]GABA binding to a crude membrane preparation of chick inner ear. An equilibrium dissociation constant of 19 nM and a maximum binding capacity of 0.58 pmol/mg protein were calculated from the plot with a PDP-11/34 computer by linear regression and drawn with a Hewlett-Packard 7225B plotter. A linear correlation coefficient of-0.988 was also calculated. The saturation curve is shown in the inset. Data in its ordinates represent specifically bound [3H]GABA expressed as pmol/mg protein plotted against GABA concentrations. Each point in the graph is the mean of 3 independent observations each in triplicate. Data of greater GABA concentrations are not shown but were taken into account for the Scatchard plot calculations. The methodology followed and the calculation of the data are described in the text. binding as measured at increasing concentrations of the ligand. As can be observed, the specific binding of [ 3 H ] G A B A to chick inner ear membranes is saturable (Fig. 1, inset) and the binding data yielded a linear Scatchard plot that is consistent with a single class of binding sites. The values for an apparent equilibrium constant (Kd) of 19.4 nM and a maximum binding capacity (Bmax) of 0.59 pmol/mg protein were calculated by linear regression analysis of the data of the Scatchard plot. The finding, in a Triton-treated, extensively washed, frozen and thawed membrane preparation of the chick inner ear, of a single population of binding sites for [ 3 H ] G A B A as indicated by the linear Scatchard plot (Fig. 1) differs from the two populations of binding sites for G A B A consistently found in nervous tissue 3,9,10,15,17. However, it resembles the encountering of only one high-affinity site in human anterior pituitary 14. Notwithstanding, the K d values and number of binding sites found in the chick inner ear membranes for [ 3 H ] G A B A are very similar to what has been shown for the high-affinity G A B A binding site
182 TABLE II
high-affinity, saturable, s o d i u m - i n d e p e n d e n t G A B A
Comparison of [3H]GABA binding data found in several nervous tissue membrane preparations with [3H]GABA binding values obtained in chick vestibular membranes
binding system of the same magnitude, kinetic con-
Preparation
Ka (nM)
Bmax (pmol/mg protein)
Reference
vous system m e m b r a n e fractions, strongly suggests the possible presence of a G A B A A receptor in our
Chick inner ear Human anterior pituitarya Rat retina b Rat brainb Rat brainb Rat cerebellumb Rat brainb,c
19.4"
0.59
Our work
40 18 16 32 32 20
0.65 0.60 0.60 1.17 1.4 0.48
Grandison et a1.14 Enna and Snyder9 Enna and Snyder1° Greenlee et a1.15 Browner et al. 3 Jordan et al. 17
stants and properties in front of agonists and antagonists obtained for G A B A receptors in various ner-
preparation and may well represent a physiological
Only one high-affinity site was encountered. b Although two sites were present, data refer only to the highaffinity one. ¢ Highly purified synaptic plasma membrane fraction. a
one. Studies in which the coincidence of the presence of a G A B A postsynaptic site and of glutamate decarboxylase together with a high-affinity G A B A transport system in discrete regions of m a m m a l i a n brain have been used in support for the participation of G A B A as n e u r o t r a n s m i t t e r in those regions 7. As all these parameters have been d e m o n s t r a t e d in isolated chick vestibular cristae, a n e u r o t r a n s m i t t e r role for GABA
may be postulated in these organs.
Our thanks to Dr. S. J. E n n a for a generous gift of in the formerly m e n t i o n e d nervous tissue preparations3,9,10,15A7. Table II compares the results ob-
(___)-baclofen; to Ing. Federico Fern~indez Cancino for help with the computer-based calculations and to
tained in the chick i n n e r ear m e m b r a n e s with those
Mrs. Virginia G o d i n e z and Miss Yoland Diaz de Castro for secretarial assistance. Supported in part by
data. In conclusion: the presence in a crude m e m b r a n e preparation of isolated chick vestibular cristae, of a
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Grant P C C B B N A 020897 from Consejo Nacional de Ciencia y Tecnologia ( C O N A C Y T ) , M6xico.
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