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In vivo effects of phenytoin and phenobarbital on GABA receptors in rat cerebral cortex and cerebellum
Neuroscience Letters, 46 (1984) 255-260
255
Elsevier Scientific Publishers Ireland Ltd. NSL 02692
IN VIVO EFFECTS OF P H E N Y T O I N A N D P H E N O B A R B I T A L ON G A B A R E C E P T O R S IN R A T C E R E B R A L C O R T E X A N D C E R E B E L L U M
YOSHIHISA TATSUOKA, YUZURU KATO, KAZUHIDE YOSHIDA and HIROO IMURA
The Second Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606 (Japan) (Received November 21st, 1983; Revised version received February 20th, 1984; Accepted February 23rd, 1984)
Key words: GABA receptor - phenytoin - phenobarbital - cerebral cortex - cerebellum
The in vivo effects of anticonvulsants on specific binding of [3H]GABA in the rat brain were examined in male Wistar rats. Acute treatment with phenobarbital increased specific [3H]GABA binding in the cerebral cortex, whereas repeated treatment with phenobarbital failed to change [3H]GABA binding. [3H]GABA binding in the cerebellum was not influenced by phenobarbital administration. Acute treatment with phenytoin produced no change in [3H]GABA binding, whereas repeated treatment with phenytoin caused a significant increase in [3H]GABA binding in the cerebellum, but not in the cerebral cortex. The effects of these anticonvulsants may be due, at least in part, to GABA receptor-mediated mechanisms.
It is well known that 3~-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system and plays an important role in the mechanism by which convulsions are controlled [8]. The administration o f anticonvulsants such as phenytoin and phenobarbital is known to alter the brain GABA content in vivo [11, 13]. Phenobarbital affects GABA receptors in vivo [9] and in vitro [1], and is considered to act through the GABA-benzodiazepine-barbiturate receptor complex [10]. However, the results reported are still controversial. A possible interaction between phenytoin and benzodiazepines has been suggested [17]. However the effect of these anticonvulsants on GABA receptors in vivo remains to be elucidated. We studied in the present experiments the changes of GABA receptors in rat cerebral cortex and cerebellum after single and repeated treatment with phenytoin or phenobarbital, by means of radioreceptor assay. Male Wistar rats weighing 170-200 g were housed in an air-conditioned room (23 + 1°C) under artificial light (lights on at 06.00 h, off at 18.00 h). Animals were fed laboratory chow (Oriental Yeast Co., Tokyo) and water ad libitum. For acute treatment, the rats were divided into 3 groups at random and each group of animals was injected intraperitoneally with phenytoin sodium salt (50 mg/kg), phenobarbital sodium salt (30 mg/kg) or saline solution (I ml/kg) at 08.00 h. One hour after the injection, rats were sacrificed by decapitation. For repeated treatment the same 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
256
doses of phenytoin, phenobarbital or saline as in the acute experiment were administered intraperitoneally twice a day (08.00 and 18.00 h) for 10 days. On the morning of the eleventh day (08.00 h), the final injection was given in the same dose. One hour later, the rats were sacrificed by decapitation. Immediately after decapitation the cerebral cortex and the cerebellum were dissected rapidly. Subcellular fractionation was performed with the method described previously [16]. Briefly, synaptosomal fractions were prepared from the cerebral cortex and the cerebellum of each group using the method described by Gray and Whittaker [3]. Synaptosomal fractions were suspended in distilled water and centrifuged. To remove endogenous GABA and its binding inhibitors [4], these pellets were frozen and stored at - 2 0 ° C for at least 18 h. Then they were thawed and sonicated in 50 mM Tris-HC1 (pH 7.4) buffer with a Polytron PT-10, and the homogenates were centrifuged twice and frozen again to store at - 2 0 ° C until assayed. On the day of the assay, the synaptic membranes were thawed, repelleted and resuspended with the same buffer to a protein concentration of 0.2-0.4 mg/ml. Protein concentrations were determined by the method of Lowry et al. [7], with bovine serum albumin as standard. [3H]GABA binding assay was performed by the method described previously [16]. A 0.5 ml aliquot of synaptic membrane suspension was incubated with 0.1 ml of a solution containing 1.5-2.0 nM of [3H]GABA (specific activity 57 Ci/mmol, Radiochemical Center, Amersham) and 0.4 ml of Tris-HCl buffer (pH 7.4) with various concentrations of unlabeled GABA at 4°C for 30 min. Unbound [3H]GABA was removed by a glass filter paper (Whatman G F / B ) under vacuum, and radioactivity retained on the filter paper was counted with a Packard Tricarb Scintillation Spectrometer. The difference between the amount of radioactivity bound in the presence and absence of 0.1 mM unlabeled GABA was termed specific [3H]GABA binding. All binding determinations were conducted in triplicate. Statistical evaluation was conducted by one-way analysis of variance in combination with Duncan's new multiple range test [2]. TABLE I EFFECTS OF ACUTE TREATMENT W I T H ANTICONVULSANT ON SPECIFIC [3H]GABA BINDING IN THE CEREBRAL CORTEX AND THE CEREBELLUM One hour after the injection of phenytoin or phenobarbital, cerebral cortical or cerebellar synaptic membranes were prepared and GABA binding was studied as described in the text. Specific [3H]GABA binding was measured as the difference in binding obtained with incubation in the presence or absence of 0.1 mM unlabeled GABA. The means ± S.E. are shown. *P<0.01 vs controls. Treatment
Saline Phenytoin Phenobarbital
(n)
(6) (6) (6)
Specific binding (fmol/mg protein) Cerebral cortex
Cerebellum
179 + 17 176 + 9.4 260 +_ 14"
408 ± 53 438 ± 65 379 ± 39
257
T A B L E II E F F E C T OF A C U T E T R E A T M E N T W I T H B I N D I N G IN T H E C E R E B R A L C O R T E X
PHENOBARBITAL
ON
SPECIFIC
[3H]GABA
Synaptic m e m b r a n e o f the cerebral cortex was incubated with [3H]GABA and various concentrations of unlabeled G A B A as described in the next. Binding parameters were obtained by Scatchard analysis. The means ± S.E. are shown. * P < 0 . 0 2 vs controls; * * P < 0 . 0 1 vs controls. Treatment
Saline Phenobarbital
Kd (nM)
(n)
(4) (4)
Bmax ( p m o l / m g protein)
gdl
gd2
Bmaxl
5.2 _+ 0.3 5.3 + 0.6
123 ± 15 126 ± 12
0.40 ± 0.03 2.7 ± 0.07 0.65 ± 0 . 0 1 " ' 3 . 9 + 0.20*
Bmax2
Table I shows the effects of a single dose injection of an anticonvulsant on specific [3H]GABA binding in the cerebral cortex and the cerebellum. Mean (+ S.E.) specific [3H]GABA binding in the cerebral cortex was significantly increased by phenobarbital injection compared with that in the control group (260 + 14 fmol/mg protein vs control 179 + 17 fmol/mg protein, P<0.01). Specific [3H]GABA binding in the cerebral cortex was not changed by phenytoin. A single injection of either phenytoin or phenobarbital caused no significant change in [3H]GABA binding to cerebellar synaptic membranes. To further characterize the stimulating effect of phenobarbital on [3H]GABA binding in the cerebral cortex, we analyzed binding data by Scatchard plots [14], which suggested two binding sites. Binding parameters determined by the Rosenthal method [12] are shown in Table II. A high affinity site (Kat 5.2 + 0.3 nM; Bmaxl 0.40 + 0.03 pmol/mg protein) and a low affinity site (Kd2 123 + 15 nM; Bmax22.7 + 0.07 pmol/mg protein) were obtained in the cerebral cortex. A single injection of phenobarbital increased the capacity of both high (Bmax1 0.65 _+ 0.01 pmol/mg protein) and low (Bin=2 3.93 _+ 0.2 pmol/mg protein) affinity binding sites in the cerebral cortex, but had no effect on the Ka of either high (Kdl 5.3 _+ 0.6 nM) or low (Ka2 126 _+ 12 nM) affinity binding sites. T A B L E II1 EFFECTS OF R E P E A T E D T R E A T M E N T W I T H A N T I C O N V U L S A N T S ON SPECIFIC [3H]GABA B I N D I N G IN T H E C E R E B R A L C O R T E X A N D C E R E B E L L U M After 10 days of treatment with phenytoin or phenobarbital, cerebral cortical or cerebellar synaptic membranes were prepared and G A B A binding was studied as described in the text. The means + S.E. are shown. * P < 0 . 0 1 vs controls. Treatment
(n)
Saline Phenytoin Phenobarbital
(6) (6) (6)
Specific binding ( f m o l / m g protein) Cerebral cortex
Cerebellum
187 + 29 169 ± 11 173 ± 12
392 ± 16 580 ± 24* 419 _+ 13
258
I'ABI.E IV EFFECT OF REPEATED TREATMENT WITH P H E N Y T O I N ON SPECIFIC [SH]GABA BINDING IN THE CEREBELLUM Synaptic membrane of the cerebellum was incubated with [3H]GABA and various concentrations of unlabeled GABA as described in the text. Binding parameters were obtained by Scatchard analysis. The means _+ S.E. are shown. **P<0.01 vs controls; *P<0.05 vs controls. Treatment
(n)
Saline Phenytoin
(4) (4)
Ka (nM)
Bmax ( p m o l / m g protein)
Kdl
Ka2
Bmaxl
5.7 ± 0.7 6.3 ± 0.8
124 _+ 28 112 _+ 31
1.0 _+ 0.14 6.6 ± 0.8 1.9 _+ 0.12"* 9.9 ± 1.3"
Bmax2
Table III shows the effects of repeated treatment with phenytoin and phenobarbital on specific [SH]GABA binding to synaptic membranes in the cerebral cortex and the cerebellum. Phenobarbital had no significant effect on [3H]GABA binding to cerebrocortical and cerebellar synaptic membranes. Thus, the stimulating effect of phenobarbital on [3H]GABA binding to the cerebral cortex observed in acute treatment was rather blunted by repeated treatment. On the other hand, repeated treatment with phenytoin significantly (P<0.01) increased the specific binding of [3H]GABA to cerebellar synaptic membranes (580 _+ 24 fmol/mg protein vs control 392 _+ 16 fmol/mg protein). As indicated in Table IV, binding analysis showed that there were two binding sites of [3H]GABA, a high affinity site (Ka~ 5.7 +_ 0.7 nM; Bmax~ 1.0 + 0.14 pmol/mg protein) and a low affinity site (Ka2 124 + 28 nM; Bmaxz 6.6 +_ 0.8 pmol/mg protein), in the cerebellar synaptic membranes. Repeated treatment with phenytoin increased the binding capacity of both high (Bmaxt 1.9 _+ 0.12 pmol/mg protein) and low (Bmax2 9.9 + 1.3 pmol/mg protein) affinity binding sites, without any significant effect on the Ka of either high (Kat 6.3 + 0.86 nM) or low (Ka2 112 +_ 31 nM) affinity binding site. As reported previously [16], specific [3H]GABA binding sites in our radioreceptor assay system are considered to be GABA receptors. Acute treatment with phenobarbital increased the number of receptors in the cerebral cortex. The present findings are in agreement with reports of in vitro effects of phenobarbital [1, 18]. However, synaptic membranes were sonicated in the sufficient amount of buffer and centrifuged. This washing procedure was repeated 5 times before the binding assay to minimize the effects of residual anticonvulsant. Therefore, these results reflect in vivo effects of phenobarbital on GABA receptors, suggesting that GABA receptors are involved, at least in part, in the anticonvulsant effect of phenobarbital in vivo. In contrast, after repeated treatment, phenobarbital no longer stimulated GABA receptors in the cerebral cortex. This might be explained by a down regulation of GABA receptors since increased cortical GABA content following phenobarbital treatment was reported previously [11]. These findings suggest that the anticon-
259
vulsive effect of repeated treatment with phenobarbital is not due to a simple direct action on GABA receptors, but is related to a still unidentified mechanism. Although acute and repeated treatment with phenytoin had no effect on G A B A receptors in the rat cerebral cortex, phenytoin increased the number of GABA receptors in the cerebellum when given repeatedly. This may be explained by supersensitivity to GABA since decreased cerebellar GABA at this stage was reported previously [11], suggesting a possible relationship to the cerebellar ataxia seen in phenytoin intoxication. Recently, it has been reported that there exist bicuculline-insensitive GABA receptors (GABAB sites) in addition to bicuculline-sensitive GABA receptors (GABAA sites) in the central nervous system [5]. Kato et al. indicated that the affinity of GABAB sites are regulated by divalent cations in the cerebellum [6]. As specific [3H]GABA binding sites in our radioreceptor assay system are bicuculline-sensitive [16], they are considered to be GABAA sites. The effects of phenytoin and phenobarbital on GABAB receptor should be further evaluated with [3H]baclofen as a ligand.
1 Asano, Y. and Ogasawara, N., Stimulation of GABA receptor binding by barbiturates, Europ. J. Pharmacol., 77 (1982) 355-357. 2 Duncan, D.B., Multiple range and multiple F tests, Biometrics, 11 (1955) 1-42. 3 Gray, E.G. and Whittaker, V.P., The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat., 96 (1962) 79-87. 4 Guidotti, A., Konkel, D.R., Ebstein, B., Corda, M.G., Wise, B.C., Krutzsh, H., Meek, J.L. and Costa, E., Isolation, characterization, and purification of a rat brain protein (GABA-modulin), Proc. nat. Acad. Sci. U.S.A., 79 (1982) 6084-6088. 5 Hill, D.R. and Bowery, N.G., 3H-Baclofen and 3H-GABA bind to bicuculline-insensitive GABAa sites in rat brain, Nature (Lond.), 283 (1981) 149-152. 6 Kato, K., Goto, M. and Fukuda, H., Regulation by divalent cations of ~H-baclofen binding to GABA~ sites in rat cerebellar membranes, Life Sci., 32 (1983) 879-887. 7 Lowry, O.H., Rosebrough, N.L., Farr and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 Meldrum, B., Convulsant drugs, anticonvulsants and GABA-mediated neuronal inhibition. In P. Krogsgaard-Lasen, J. ScheeI-Kruger and H. Kofod, (Eds.), GABA-Neurotransmitters, Academic Press, New York, 1979, pp. 390-405. 9 M6hler, H., Okada, T. and Enna, S.J., Benzodiazepine and neurotransmitter receptor binding in rat brain after chronic administration of diazepam or phenobarbital, Brain Res., 156 (1978) 391-395. 10 Olsen, R.W., GABA-benzodiazepine-barbiturate receptor interactions, J. Neurochem., 37 (1981) 1-13. 11 Patosalos, P.N. and Lascelles, P.T., Changes in regional brain levels of amino acid putative neurotransmitters after prolonged treatment with the anticonvulsant drugs diphenylhydantoin, phenobarbitone, sodium valproate, ethosuximide and sulthiame in the rat, J. Neurochem., 36 (1981) 688-695. 12 Rosenthal, H.E., A graphic method for the determination and presentation of binding parameters in a complex system, Analyt. Biochem., 20 (1967) 525-532. 13 Saad, S.F., Elmasry, S.M. and Scott, P.M., Influence of certain anticonvulsants on the concentration of 7-aminobutyric acid in the cerebral hemispheres of mice, Europ. J. Pharmacol., 17 (1972) 386-392.
260 14 Scatchard, G., The attraction of protein for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 600-672. 15 Sivum, S.P., Nabeshima, T. and Ho, I.K., Alterations of regional 3,-amino-butyric acid receptors in morphine tolerant mice. Biochem. Pharmacol., 30 (1981) 2187-2190. 16 Tatsuoka, Y., Kato, Y. and Imura, H., Inhibition by DN-1417 (a TRH derivative) of [3H]GABA binding in the rat brain, Neurosci. Lett., 42 (1983) 149-154. 17 Tunnicliff, G. and Smith, J.A. Competition for diazepam receptor binding by diphenylhydantoin and its enhancement by 3,-aminobutyric acid, Biochem. biophys. Res. Commun., 91 (1979) 1018-1024. 18 Willow, M. and Johnston, G.A.R., Enhancement by anesthetic and convulsant barbiturates of GABA binding to rat brain synaptosomal membranes, J. Neurochem., 1 (1981) 364-367.
255
Elsevier Scientific Publishers Ireland Ltd. NSL 02692
IN VIVO EFFECTS OF P H E N Y T O I N A N D P H E N O B A R B I T A L ON G A B A R E C E P T O R S IN R A T C E R E B R A L C O R T E X A N D C E R E B E L L U M
YOSHIHISA TATSUOKA, YUZURU KATO, KAZUHIDE YOSHIDA and HIROO IMURA
The Second Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606 (Japan) (Received November 21st, 1983; Revised version received February 20th, 1984; Accepted February 23rd, 1984)
Key words: GABA receptor - phenytoin - phenobarbital - cerebral cortex - cerebellum
The in vivo effects of anticonvulsants on specific binding of [3H]GABA in the rat brain were examined in male Wistar rats. Acute treatment with phenobarbital increased specific [3H]GABA binding in the cerebral cortex, whereas repeated treatment with phenobarbital failed to change [3H]GABA binding. [3H]GABA binding in the cerebellum was not influenced by phenobarbital administration. Acute treatment with phenytoin produced no change in [3H]GABA binding, whereas repeated treatment with phenytoin caused a significant increase in [3H]GABA binding in the cerebellum, but not in the cerebral cortex. The effects of these anticonvulsants may be due, at least in part, to GABA receptor-mediated mechanisms.
It is well known that 3~-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system and plays an important role in the mechanism by which convulsions are controlled [8]. The administration o f anticonvulsants such as phenytoin and phenobarbital is known to alter the brain GABA content in vivo [11, 13]. Phenobarbital affects GABA receptors in vivo [9] and in vitro [1], and is considered to act through the GABA-benzodiazepine-barbiturate receptor complex [10]. However, the results reported are still controversial. A possible interaction between phenytoin and benzodiazepines has been suggested [17]. However the effect of these anticonvulsants on GABA receptors in vivo remains to be elucidated. We studied in the present experiments the changes of GABA receptors in rat cerebral cortex and cerebellum after single and repeated treatment with phenytoin or phenobarbital, by means of radioreceptor assay. Male Wistar rats weighing 170-200 g were housed in an air-conditioned room (23 + 1°C) under artificial light (lights on at 06.00 h, off at 18.00 h). Animals were fed laboratory chow (Oriental Yeast Co., Tokyo) and water ad libitum. For acute treatment, the rats were divided into 3 groups at random and each group of animals was injected intraperitoneally with phenytoin sodium salt (50 mg/kg), phenobarbital sodium salt (30 mg/kg) or saline solution (I ml/kg) at 08.00 h. One hour after the injection, rats were sacrificed by decapitation. For repeated treatment the same 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
256
doses of phenytoin, phenobarbital or saline as in the acute experiment were administered intraperitoneally twice a day (08.00 and 18.00 h) for 10 days. On the morning of the eleventh day (08.00 h), the final injection was given in the same dose. One hour later, the rats were sacrificed by decapitation. Immediately after decapitation the cerebral cortex and the cerebellum were dissected rapidly. Subcellular fractionation was performed with the method described previously [16]. Briefly, synaptosomal fractions were prepared from the cerebral cortex and the cerebellum of each group using the method described by Gray and Whittaker [3]. Synaptosomal fractions were suspended in distilled water and centrifuged. To remove endogenous GABA and its binding inhibitors [4], these pellets were frozen and stored at - 2 0 ° C for at least 18 h. Then they were thawed and sonicated in 50 mM Tris-HC1 (pH 7.4) buffer with a Polytron PT-10, and the homogenates were centrifuged twice and frozen again to store at - 2 0 ° C until assayed. On the day of the assay, the synaptic membranes were thawed, repelleted and resuspended with the same buffer to a protein concentration of 0.2-0.4 mg/ml. Protein concentrations were determined by the method of Lowry et al. [7], with bovine serum albumin as standard. [3H]GABA binding assay was performed by the method described previously [16]. A 0.5 ml aliquot of synaptic membrane suspension was incubated with 0.1 ml of a solution containing 1.5-2.0 nM of [3H]GABA (specific activity 57 Ci/mmol, Radiochemical Center, Amersham) and 0.4 ml of Tris-HCl buffer (pH 7.4) with various concentrations of unlabeled GABA at 4°C for 30 min. Unbound [3H]GABA was removed by a glass filter paper (Whatman G F / B ) under vacuum, and radioactivity retained on the filter paper was counted with a Packard Tricarb Scintillation Spectrometer. The difference between the amount of radioactivity bound in the presence and absence of 0.1 mM unlabeled GABA was termed specific [3H]GABA binding. All binding determinations were conducted in triplicate. Statistical evaluation was conducted by one-way analysis of variance in combination with Duncan's new multiple range test [2]. TABLE I EFFECTS OF ACUTE TREATMENT W I T H ANTICONVULSANT ON SPECIFIC [3H]GABA BINDING IN THE CEREBRAL CORTEX AND THE CEREBELLUM One hour after the injection of phenytoin or phenobarbital, cerebral cortical or cerebellar synaptic membranes were prepared and GABA binding was studied as described in the text. Specific [3H]GABA binding was measured as the difference in binding obtained with incubation in the presence or absence of 0.1 mM unlabeled GABA. The means ± S.E. are shown. *P<0.01 vs controls. Treatment
Saline Phenytoin Phenobarbital
(n)
(6) (6) (6)
Specific binding (fmol/mg protein) Cerebral cortex
Cerebellum
179 + 17 176 + 9.4 260 +_ 14"
408 ± 53 438 ± 65 379 ± 39
257
T A B L E II E F F E C T OF A C U T E T R E A T M E N T W I T H B I N D I N G IN T H E C E R E B R A L C O R T E X
PHENOBARBITAL
ON
SPECIFIC
[3H]GABA
Synaptic m e m b r a n e o f the cerebral cortex was incubated with [3H]GABA and various concentrations of unlabeled G A B A as described in the next. Binding parameters were obtained by Scatchard analysis. The means ± S.E. are shown. * P < 0 . 0 2 vs controls; * * P < 0 . 0 1 vs controls. Treatment
Saline Phenobarbital
Kd (nM)
(n)
(4) (4)
Bmax ( p m o l / m g protein)
gdl
gd2
Bmaxl
5.2 _+ 0.3 5.3 + 0.6
123 ± 15 126 ± 12
0.40 ± 0.03 2.7 ± 0.07 0.65 ± 0 . 0 1 " ' 3 . 9 + 0.20*
Bmax2
Table I shows the effects of a single dose injection of an anticonvulsant on specific [3H]GABA binding in the cerebral cortex and the cerebellum. Mean (+ S.E.) specific [3H]GABA binding in the cerebral cortex was significantly increased by phenobarbital injection compared with that in the control group (260 + 14 fmol/mg protein vs control 179 + 17 fmol/mg protein, P<0.01). Specific [3H]GABA binding in the cerebral cortex was not changed by phenytoin. A single injection of either phenytoin or phenobarbital caused no significant change in [3H]GABA binding to cerebellar synaptic membranes. To further characterize the stimulating effect of phenobarbital on [3H]GABA binding in the cerebral cortex, we analyzed binding data by Scatchard plots [14], which suggested two binding sites. Binding parameters determined by the Rosenthal method [12] are shown in Table II. A high affinity site (Kat 5.2 + 0.3 nM; Bmaxl 0.40 + 0.03 pmol/mg protein) and a low affinity site (Kd2 123 + 15 nM; Bmax22.7 + 0.07 pmol/mg protein) were obtained in the cerebral cortex. A single injection of phenobarbital increased the capacity of both high (Bmax1 0.65 _+ 0.01 pmol/mg protein) and low (Bin=2 3.93 _+ 0.2 pmol/mg protein) affinity binding sites in the cerebral cortex, but had no effect on the Ka of either high (Kdl 5.3 _+ 0.6 nM) or low (Ka2 126 _+ 12 nM) affinity binding sites. T A B L E II1 EFFECTS OF R E P E A T E D T R E A T M E N T W I T H A N T I C O N V U L S A N T S ON SPECIFIC [3H]GABA B I N D I N G IN T H E C E R E B R A L C O R T E X A N D C E R E B E L L U M After 10 days of treatment with phenytoin or phenobarbital, cerebral cortical or cerebellar synaptic membranes were prepared and G A B A binding was studied as described in the text. The means + S.E. are shown. * P < 0 . 0 1 vs controls. Treatment
(n)
Saline Phenytoin Phenobarbital
(6) (6) (6)
Specific binding ( f m o l / m g protein) Cerebral cortex
Cerebellum
187 + 29 169 ± 11 173 ± 12
392 ± 16 580 ± 24* 419 _+ 13
258
I'ABI.E IV EFFECT OF REPEATED TREATMENT WITH P H E N Y T O I N ON SPECIFIC [SH]GABA BINDING IN THE CEREBELLUM Synaptic membrane of the cerebellum was incubated with [3H]GABA and various concentrations of unlabeled GABA as described in the text. Binding parameters were obtained by Scatchard analysis. The means _+ S.E. are shown. **P<0.01 vs controls; *P<0.05 vs controls. Treatment
(n)
Saline Phenytoin
(4) (4)
Ka (nM)
Bmax ( p m o l / m g protein)
Kdl
Ka2
Bmaxl
5.7 ± 0.7 6.3 ± 0.8
124 _+ 28 112 _+ 31
1.0 _+ 0.14 6.6 ± 0.8 1.9 _+ 0.12"* 9.9 ± 1.3"
Bmax2
Table III shows the effects of repeated treatment with phenytoin and phenobarbital on specific [SH]GABA binding to synaptic membranes in the cerebral cortex and the cerebellum. Phenobarbital had no significant effect on [3H]GABA binding to cerebrocortical and cerebellar synaptic membranes. Thus, the stimulating effect of phenobarbital on [3H]GABA binding to the cerebral cortex observed in acute treatment was rather blunted by repeated treatment. On the other hand, repeated treatment with phenytoin significantly (P<0.01) increased the specific binding of [3H]GABA to cerebellar synaptic membranes (580 _+ 24 fmol/mg protein vs control 392 _+ 16 fmol/mg protein). As indicated in Table IV, binding analysis showed that there were two binding sites of [3H]GABA, a high affinity site (Ka~ 5.7 +_ 0.7 nM; Bmax~ 1.0 + 0.14 pmol/mg protein) and a low affinity site (Ka2 124 + 28 nM; Bmaxz 6.6 +_ 0.8 pmol/mg protein), in the cerebellar synaptic membranes. Repeated treatment with phenytoin increased the binding capacity of both high (Bmaxt 1.9 _+ 0.12 pmol/mg protein) and low (Bmax2 9.9 + 1.3 pmol/mg protein) affinity binding sites, without any significant effect on the Ka of either high (Kat 6.3 + 0.86 nM) or low (Ka2 112 +_ 31 nM) affinity binding site. As reported previously [16], specific [3H]GABA binding sites in our radioreceptor assay system are considered to be GABA receptors. Acute treatment with phenobarbital increased the number of receptors in the cerebral cortex. The present findings are in agreement with reports of in vitro effects of phenobarbital [1, 18]. However, synaptic membranes were sonicated in the sufficient amount of buffer and centrifuged. This washing procedure was repeated 5 times before the binding assay to minimize the effects of residual anticonvulsant. Therefore, these results reflect in vivo effects of phenobarbital on GABA receptors, suggesting that GABA receptors are involved, at least in part, in the anticonvulsant effect of phenobarbital in vivo. In contrast, after repeated treatment, phenobarbital no longer stimulated GABA receptors in the cerebral cortex. This might be explained by a down regulation of GABA receptors since increased cortical GABA content following phenobarbital treatment was reported previously [11]. These findings suggest that the anticon-
259
vulsive effect of repeated treatment with phenobarbital is not due to a simple direct action on GABA receptors, but is related to a still unidentified mechanism. Although acute and repeated treatment with phenytoin had no effect on G A B A receptors in the rat cerebral cortex, phenytoin increased the number of GABA receptors in the cerebellum when given repeatedly. This may be explained by supersensitivity to GABA since decreased cerebellar GABA at this stage was reported previously [11], suggesting a possible relationship to the cerebellar ataxia seen in phenytoin intoxication. Recently, it has been reported that there exist bicuculline-insensitive GABA receptors (GABAB sites) in addition to bicuculline-sensitive GABA receptors (GABAA sites) in the central nervous system [5]. Kato et al. indicated that the affinity of GABAB sites are regulated by divalent cations in the cerebellum [6]. As specific [3H]GABA binding sites in our radioreceptor assay system are bicuculline-sensitive [16], they are considered to be GABAA sites. The effects of phenytoin and phenobarbital on GABAB receptor should be further evaluated with [3H]baclofen as a ligand.
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