Gamma-aminobutyric Acid (GABA)

Vernadakis and Woodbury, Inhibitions of the Nervous System and Gamma-aminobutyric Acid (1960),532 demonstrated that PHT enhanced the conversion of free glutamic acid to glutamine and GABA in rat brain. This was in contrast to the effect of cortisol, which shunted free glutamic acid to the Krebs cycle and away from glutamine and GABA.

532. Vernadakis, A. and Woodbury, D. M., Effects of diphenylhydantoin and adrenocortical steroids on free glutamic acid, glutamine, and gamma-aminobutyric acid concentrations of rat cerebral cortex, Inhibitions of the nervous system and gamma-aminobutyric acid, 242-248, Pergamon Press, Oxford, 1960.

Bhattacharya, Kishor, Saxena and Bhargava, Archives Internationales de Pharmacodynamie et de Therapie (1964),522 demonstrated that PHT increased brain GABA in mice.

522. Bhattacharya, S. S., Kishor, K., Saxena, P. N., and Bhargava, K. P., A neuropharmacological study of gamma-aminobutyric acid (GABA), Arch. Int. Pharmacodyn., 150: 295-305, 1964.

Verster, Garoutte, Ichinosa and Guerrero-Figueroa, Federation Proceedings (1965),454 observed that the uptake of [14C]-GABA by mouse brain was increased by 35% with PHT.

454. Verster, F. de B., Garoutte, J., Ichinosa, H., and Guerrero-Figueroa, R., Mode of action of diphenylhydantoin, Fed. Proc., 24: 390, 1965.

Saad, El Masry and Scott, Communications in Behavioral Biology (1972),1495 studied the effect of PHT on the GABA content of normal mouse cerebral hemispheres and of cerebral hemispheres depleted of GABA. PHT (25-50 mg/kg) increased normal cerebral hemisphere GABA, and also increased cerebral hemisphere concentrations of GABA which had been previously reduced by isoniazid.

1495. Saad, S. F., El Masry, A. M., and Scott, P. M., Influence of certain anticonvulsants on the concentration of 8-aminobutyric acid in the cerebral hemispheres of mice, Communications in Behav. Biol., 9: February, 1972.

Weinberger, Nicklas and Berl, Neurology (1976),2115 demonstrated in rat brain synaptosomes that PHT and pentobarbital facilitated glutamate uptake. However, PHT (10-100 µM) facilitated GABA uptake to a lesser extent than pentobarbital. The authors suggest that these differences of action may relate to PHT's lack of hypnotic effect.

2115. Weinberger, J., Nicklas, W. J. and Berl, S., Mechanism of action of anticonvulsants, Neurology 26: 162-166, 1976.

Ayala, Johnston, Lin and Dichter, Brain Research (1977),1732 observed that PHT (25-200 µM) increases and prolongs GABA-mediated postsynaptic conductance in the crayfish stretch receptor.

1732. Ayala, G. F., Johnson, D., Lin, S., and Dichter, H. N., The mechanism of action of diphenylhydantoin on invertebrate neurons: II. Effects on synaptic mechanisms, Brain Res., 121: 259-70,1977.

Deisz and Lux, Neuroscience Letters (1977),1794 and Aickin, Deisz and Lux, Journal of Physiology (1978),1716 found that PHT (as low as .001 µM) increased inhibitory synaptic conductance and prolonged inhibitory postsynaptic potentials of crayfish stretch receptor neurons. The authors conclude that PHT decreases the rate of closing of the GABA-activated chloride channel, thereby enhancing inhibition.

1716. Aickin, C. C., Deisz, R. A. and Lux, H. D., The effect of diphenylhydantoin and picrotoxin on post-synaptic inhibition, J. Physiol., 284: 125-6, 1978.

Tunnicliff, Smith and Ngo, Biochemical and Biophysical Research Communications (1979),3031 found that PHT (10 µM) competitively inhibited the binding of [3H]-diazepam to rat cerebral cortex synaptic membranes. GABA increased the binding affinity of both drugs three-to four-fold. From their data, the authors suggest that PHT and diazepam bind at the same GABA-related site and that PHT's ability to reduce neuronal hyperexcitability may be related to this effect.

3031. Tunnicliff, G., Smith, J. A., Ngo, T. T., Competition for diazepam receptor binding by diphenylhydantoin and its enhancement by gamma-aminobutyric acid, Biochem. Biophy. Res. Commun., 91: 1018-24, 1979.

Essman and Essman, Brain Research Bulletin (1980),2482 studied the effects of PHT, pentobarbital and diazepam (all at 100 µM) on GABA uptake by synaptosomes isolated from different rat brain regions in resting and post-convulsion states. In the resting state, all three agents significantly increased GABA uptake by cerebral cortical nerve endings (74%), but decreased uptake in hippocampal synaptosomes. Only pentobarbital increased cerebellar synaptosome GABA uptake (28%). In the post-convulsion state, GABA up-take was decreased in the cerebral synaptosomes and increased in the hippocampal synaptosomes by all three agents.

2482. Essman, W. B., Essman, E. J., Anticonvulsant effects upon regional synaptosomal GABA uptake: differences in convulsed rats, Brain Res. Bull., 5(2): 821-4, 1980.

Gallagher, Mallorga and Tallman, Brain Research (1980),2518 using extracellular unit recording and microionotophoretic techniques in rat brain, found that PHT enhanced benzodiazepine inhibition of neuronal firing. This increased effect correlated with enhanced specific binding of benzodiazepines due to an increase in the total number of binding sites in animals pre-treated with intraperitoneal PHT (100 mg/ kg) sixty minutes before testing. The effects of PHT on benzodiazepine binding were different from, and independent of, those of GABA. (See also Refs. 2519, 2520.)

2518. Gallager, D. W., Mallorga, P., Tallman, J. F., Interaction of diphenylhydantoin and benzodiazepines in the CNS, Brain Res., 189: 209-20, 1980.

2519. Gallagher, J. P., lnokuchi, H., Nakamura, J., Shinnick-Gallagher, P., Effects of anticonvulsants on excitability and GABA sensitivity of cat dorsal root ganglion cells, Neuropharmacology, 20: 427-33, 1981.
2520. Gallagher, J. P., Inokuchi, H., Shinnick-Gallagher, P., Actions of anti-convulsants on GABA-depolarizations and action potentials recorded from a mammalian sensory neuron, Soc. Neurosci. Abstr., 5: 588, 1970.

Adams, Constanti and Banks, Federation Proceedings (1981),2274 studying the effects of GABA and inhibitory nerve cells on crayfish abdominal stretch receptor neurons, found that inhibitory postsynaptic current decay was prolonged by PHT (50 µM). They suggest that PHT slows the closing of channels opened by GABA, thus augmenting GABA's effect.

2274. Adams, P. R., Constanti, A., Banks, F. W., Voltage clamp analysis of inhibitory synaptic action in crayfish stretch receptor neurons, Fed. Proc., 40(11): 2637-41, 1981.

Patsalos and Lascelles, Journal of Neurochemistry (1981),2851 studied the effects of ten-day PHT treatment (50 mg/kg) on rat regional brain concentrations of GABA, glutamate, aspartate, and taurine in rats. PHT reduced cerebellar GABA, taurine and aspartate, and hypothalamic GABA and aspartate. Of the other agents tested, sodium valproate raised GABA and taurine, and phenobarbital raised GABA, in most brain regions.

2851. Patsalos, P. N., 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(2); 688-95, 1981.

Bowling and De Lorenzo, Science (1982),2351 reported PHT binding to micromolar affinity benzodiazepine receptors present in rat brain membrane. PHT, in therapeutic concentrations, exhibited specific, saturable membrane binding, which could be displaced by diazepam. This binding affinity was not significantly influenced by GABA or muscimol, a GABA agonist. The authors suggest that this receptor is important to the actions of both PHT and diazepam in reducing abnormal neuronal hyperexcitability. (See also Ref 2350.)

2351. Bowling, A. C., De Lorenzo, R. J., Micromolar affinity benzodiazepine receptors: identification and characterization in central nervous system, Science, 216: 1247-50, 1982.
2350. Bowling, A. C., De Lorenzo, R. J., Anticonvulsant receptors; identification and characterization in brain, Neurology, 32 (2): A224, 1982.

Czuczwar, Turski and Kleinrok, Neuropharmacology (1982),2421 reported that PHT (8 mg/kg), intraperitoneally, potentiated the antipentylenetetrazol activity of clonazepam and nitrazepam in mice. The authors suggest that a PHT-induced increase in the number of benzodiazepine receptors accounts for this effect.

2421. Czuczwar, S. J., Turski, L., Kleinrok, Z., Effects of combined treatment with diphenylhydantoin and different benzodiazepines on pentylenetetrazol- and bicuculline-induced seizures in mice, Neuropharmacology, 21(6): 563-7, 1982.

De Belleroche, Dick and Wyrley-Birch, Life Sciences (1982),2435 found that PHT, diazepam, clonazepam and phenobarbital inhibited potassium-stimulated, but not resting, [14C]-GABA release from tissue slices of rat cerebral cortex. Carbamaze-pine, at concentrations up to 100 M, had no effect.

2435. De Belleroche, J., Dick, A., Wyrley-Birch, A., Anticonvulsants and trifluoperazine inhibit the evoked release of GABA from cerebral cortex of rat at different sites, Life Sci., 31: 2875-82,1982.

File and Lister, Neuroscience Letters (1983),2493 found that, in rats, PHT (10 mg/ kg) reversed the anxiogenic and convulsant effects of Ro 5-4864, a selective ligand for benzodiazepine micromolar receptors. The authors suggest that PHT has actions at central nervous system micromolar receptors.

2493. File, S. E., Lister, R. G., The anxiogenic action of Ro 5-4864 is reversed by phenytoin, Neurosci. Lett., 35: 93-6,1983.

Pozdeev, Mediator Processes in Epilepsy (1983),2878 studied the effects of PHT (37.5-125 mg/kg), twice a day for three to seven days, on brain neurotransmitter systems. Whole brain analysis of rats treated with 37.5 mg/kg intraperitoneally for seven days showed increased levels of taurine (48%), glutamate (18%), glycine (63%), and aspartate (21%). GABA levels decreased by 15% and glutamic acid decarboxylase and GABA-transferase activities increased by 27% and 32% respectively. The author notes that animals treated with PHT at this dosage appeared well and their motor activity and appetite increased. At higher dosage levels (for example, 75 mg/kg twice a day for three days), where there was evidence of toxicity, taurine levels decreased 50%; GABA levels increased by 11% with evidence of inhibition of GABAergic systems; glutamate levels increased 20%; and the metabolic inactivation of glutamate was decreased. Serotonergic and glycinergic systems showed increased activity. In some animals the neurotransmitter systems returned to normal with continued treatment. The author concludes that PHT's effects cannot be explained by an influence on a single neurotransmitter, but rather by alterations in the balance of excitatory and inhibitory neurotransmitter systems.

2878. Pozdeev, V. K., The effect of diphenylhydantoin on the function of the brain transmitter systems, Mediator Processes in Epilepsy, Nauka, Leningrad, 85-92, 1983.

Skerritt and Johnston, Clinical and Experimental Pharmacology and Physiology (1983),2961 evaluated the in vitro effects of phenobarbital, carbamazepine and PHT on potassium-stimulated release of [14C]-GABA and D-aspartate from slices of rat cerebral cortex. PHT (25-200 µM) and phenobarbital, but not carbamazepine, selectively inhibited the release of the excitatory amino acid D-aspartate. There was also some inhibition of GABA release, but it was less than that for D-aspartate. The authors note that interactions with both sodium and calcium appear necessary for PHT's effects.

2961. Skerritt, J. H., Johnston, G. A., Inhibition of amino acid transmitter release from rat slices by phenytoin and related anticonvulsants, Clin. Exp. Pharmacol. Physiol., 10: 52733, 1983.

Macdonald, McLean and Skerritt, Federation Proceedings (1985),2747reported that, at concentrations achieved in human cerebrospinal fluid, PHT, carbamazepine and valproic acid limited sustained repetitive firing, but did not alter postsynaptic GABA responses in primary dissociated cultures of mouse neurons. The barbiturates and benzodiazepines, on the other hand, increased postsynaptic GABA responses. The authors conclude that PHT's primary effect in mouse spinal cord neurons is on repetitive firing, rather than postsynaptic GABA response.

See also Refs. 1780, 2273, 2307, 2389, 2399, 2494, 2645, 2670, 2686, 2745, 2748, 2797; 2798, 2864, 2921, 2954, 3004, 3046.

2747. Macdonald, R. L., McLean, M. J., Skerritt, J. H., Anticonvulsant drug mechanisms of action, Fed. Proc., 44: 2634-9, 1985.
1780. Connors, B. W., Pentobarbital and diphenylhydantoin effects on the excitability and GABA sensitivity of rat dorsal root ganglion cells, Society for Neuroscience, 9th Annual Meeting, Nov. 2-6,1979.
2273. Abdul-Ghani, A. S., Goutinho-Neito, J., Druce, D., Bradford, H. F., Effects of anticonvulsants on the in vivo and in vitro release of GABA, Biochem. Pharmacol., 30(4): 363-8, 1981.
2307. Banerjee, A., Turner, A. J., Guha, S. R., GABA dehydrogenase activity in rat brain, Biochem. Pharmacol., 31(20): 3219-23,1982.
2389. Cheng, S. C., Brunner, E. A., Effects of anesthetic agents on synaptosomal GABA disposal, Anesthesiology, 55: 34-40,1981.
2399. Connors, B. W., A comparison of the effects of pentobarbital and diphenylhydantoin on the GABA sensitivity and excitability of adult sensory ganglion cells, Brain Res., 207: 357-69,1981.
2494. File, S. E., Pellow, S., RO 5-4864, a ligand for benzodiazepine micromolar and peripheral binding sites: antagonism and enhancement of behavioral effects., Psychopharinacology, 80(2): 166-70,1983.
2645. Kaneko, S., Sato, T., Kirahashi, K., Hill, R. G., Taberner, P. V., Effect of carbamazepine on primary afferent depolarization and gamma-aminobutyric acid metabolism comparison with phenytoin, Igaku No. Ayumi, 121(12): 1030-2, 1982.
2670. Krnjevic, K., GABA-mediated inhibitory mechanisms in relation to epileptic discharges, Basic Mechanisms of Neuronal Hyperexcitability, Jasper, H. H. and Van Gelder, N. M., Eds., Alan R. Liss, Inc, New York, 249-80, 1983.
2686. Lalonde, R., Botez, M. I., Subsensitivity to muscimolinduced catalepsy after long-term administration of phenytoin in rats, Psychopharmacology, 86: 77-80, 1985.
2745. Macdonald, R. L., Barbiturate and hydantoin anticonvulsant mechanisms of action, Basic Mechanisms of Neuronal Hyperexcitability, Jasper, H. H. and Van Gelder, N. M., Eds., Alan R. Liss, Inc., New York, 361-87, 1983.
2748. Macdonald, R. L., Skerritt, J. H., McLean, M. J., Anticonvulsant drug cations on GABA responses and sustained repetitive firing neurons in cell culture, Neuropharmacology, 23(7): 843-4, 1984.
2797. Mimaki, T., Deshmukh, P. P., Yamamura, H. I., Effect of phenytoin on benzodiazepine receptors in rat brain, Advances in Epileptology: 12th Epilepsy International Symposium, Dam, M., et al., Eds., Raven Press, New York, 73-9, 1981.
2798. Minchin, M. C., Iversen, L. L., Release of [3H] gamma-aminobutyric acid from glial cells in rat dorsal root ganglia, J. Neurochem., 23: 533-40,1974.
2864. Petersen, E. N., DMCM: a potent convulsive benzodiazepine receptor ligand, Eur. J. Pharmacol., 94: 117-24, 1983.
2921. Saladini, M., Gabana, M. A., Bracco, F., Effect of antiepileptic drugs on the cerebral amino acid uptake in vitro, Ital. J. Neurol. Sci., 2(4): 351-9, 1981.
2954. Simmonds, M. S., Distinction between the effects of barbiturates, benzodiazepines and phenytoin on responses to gamma-aminobutyric acid receptor activation and antagonism by bicuculline and picrotoxin. Br. J. Pharmacol., 73(3): 739-47,1981.
3004. Tappaz, M., Pacheco, H., Effects of convulsant and anticonvulsant drugs on the spontaneous and induced release of GABA (14C) from slices of rat cerebral cortex, J. Pharmacol., 4:433-52,1973.
3046. Varotto, M., Roman, G., Battistin, L., Pharmacological influences on the cerebral level and transport of GABA. Effect of some antiepileptic drugs on the cerebral level of GABA, Boll. Soc. Ital. Biol. Sper., 57(8): 904-8, 1981.

Wong and Teo, Neurochemical Research (1986),3499 observed PHT's effect on synaptic functions, especially those of the excitatory glutamic acid (Glu) and the inhibitory ?-aminobutyric acid (GABA) systems, which are important in seizure activity. It was suggested that PHT might limit the propagation of seizures through the balance of Glu and GABA pathways. PHT was observed to inhibit competitively the sodium-dependent high affinity synaptosomal transport of both GABA and Glu.

3499. Wong, P.T. and Teo, W.L., The effect of phenytoin on glutamate and GABA transport, Neurochem. Res., 11(9): 1379-82, 1986.

Kaneko, Hirano, Kondo, Otani, Fukushima, Hishida and Matsunaga, Japanese Journal of Psychiatry and Neurology (1988),3500 conduced a study to try to clarify the actions of PHT on the GABAergic system of the rat brain. They note that although PHT has not been shown to have a significant effect on GABA metabolism in the mouse brain or on GABA-mediated presynaptic inhibition in the rat brain, it does interact with benzodiazepine receptors, which are, in turn, coupled to GABA receptors.

Microiontophoresis (MIP) was performed on the 32 neurons in the cuneate nuclei of 7 male Wistar rats. The effects were complete and partly dependent on whether PHT-Na or PHT in propylene glycol (PHT-PG) was used. Phenobarbital and midazolam were also used for comparison. The results were as follows: PHT was applied at 40 M. Potentiation of GABA by PHT-PG occurred in 8 neurons. The overall potentiation frequency was 60.9% (14 of 23 neurons) (31.1 8.9, which was equivalent to those of other antiepileptic drugs). PHT-PG decreased neuronal firing (NF) in 30.4% of the neurons, and an increase in NF was observed in 30.4% of the neurons. PHT-Na potentiated GABA in 78% of the tested neurons, with a magnitude of potentiation equal to that of PHT-PG. There was also an increase in NF in 11.1% of the neurons tested (and a decrease in NF in 11.1%).

The effects of PHT (10-5 to 10-3M) on GABA uptake were also determined in 6 brain regions (hypothalamus, striatum, midbrain, hippocampus, cortex, and cerebellum) in 3 Wistar rats. PHT-Na increased GABA uptake in a dose-dependent manner. The authors suggest that PHT increases GABA uptake by glia and has an indirect potentiating effect on the postsynaptic GABA receptor.

3500. Kaneko, S., Hirano, T., Kondo, T., Otani, K., Fukushima, Y., Hishida, R., Matsunaga, M., Antiepileptic drug phenytoin potentiates GABA in the rat cuneate nucleus, Jpn. J. Psych. Neurol., 42(3): 643-45, 1988.

Ruiz, Hamon and Verge, European Journal of Pharmacology (1989),3501 studied the central mechanism of action of PHT by investigating adaptive changes in GABA and -adrenoceptors in the rat cerebellum. PHT was administered to pregnant Wistar rats once a day for 17 days.

Chronic PHT treatment during development resulted in a marked reduction in binding [3H]muscimol, whereas the binding of [3H]flunitrazepan was less affected. The results indicated that long-term facilitation of GABAergic transmission by chronic 30-day PHT treatment, caused a decreased binding capacity for the GABAA agonist [3H]muscimol, and for the benzodiazepine, [3H]flunitrazepan.

3501. Ruiz, G., Hamon, M., and Verge, D., Chronic phenytoin treatment decreases GABA, but not betaadrenoceptors in the cerebellum of young rats, Eur. J. Pharmacol.,168: 251-5, 1989.

Swaiman and Machen, Brain Development (1991),3502 studied the effects of 7-day Phenobarbital and phenytoin exposure on 14-day-old glial cell cultures of fetal murine cortex. Biochemical markers monitored were Ro5-4684-displaceable 3H-flunitrazepam binding, 3H--alanine uptake, glutamine synthetase activity, and protein content. Phenobarbital concentrations were 30, 60, and 120 g/ml and phenytoin concentrations 15, 30, 60 g/ml. There were no discernible phase microscopic changes at any concentration of either drug.

Phenobarbital produced no significant changes in the biochemical measures monitored. Exposure to phenytoin produced no biochemical changes at 15 g/ml, but did produce significant changes at 30 and 60 g/ml. There was an increase in Ro5-4684-displaceable 3H-flunitrazepam binding signifying or an increase in the number of binding sites and perhaps an increased population of glial cells although, the unchanged protein content suggest that the number of glial cells was not increased. There was a decrease with 30 and 60 g/ml phenytoin of 3H--alanine uptake suggesting interference with normal membrane transport of this compound. The latter effect may mirror changes in GABA uptake in glial cells in the presence of phenytoin.

Ed. note: The effects of PHT recited were seen only at levels well above the therapeutic range of plasma PHT levels, i.e., 10-20 g/ml.

3502. Swaiman, K.F. and Machen, V.L., Effects of Phenobarbital and phenytoin on cortical glial cells in culture, Brain Dev., 13: 242-6, 1991.

See also Refs.

3503. Kapetanovic, .I.M, Yonekawa, W.D., Kupferberg, H.J., The effects of phenytoin (PHT) on 4-aminopyridine (4AP)-induced changes in neurotransmitter amino acids (AA) in rat hippocampus in vitro, Soc. Neurosci. Abstr., 18: 379, 1992.

3504. Zhang, H., Hu, Y., He, S., In vitro study: the mechanisms of antiepileptic drugs on experimental epilepsy, Epilepsia, 42(S7): 231-232, 2001.

Advisory