Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain.

Acute and chronic ammonia toxicity was produced in the mice by intraperitoneal injection of ammonium chloride (200 mg/kg) and by exposure of mice to ammonia vapours (5% v/v) continuously for 2 days and 5 days respectively. The ammonia content was elevated in the cerebellum, cerebral cortex and brain stem and in liver. In acute ammonia intoxication there was a decrease in the monoamine oxidase (MAO) activity in all the three regions of brain. In chronic ammonia toxicity (2 days of exposure) a significant increase in the activity of MAO was observed in the cerebral cortex while in cerebellum and brain stem there was a significant decrease. In cerebral cortex and cerebellum there was a rise in the activity of MAO as a result of exposure to ammonia vapours for 5 days. A significant decrease was observed in the activity of glutamate decarboxylase (GAD) in all the three regions of the brain both in acute and chronic ammonia toxicity (2 days). There was a decrease in the activity of this enzyme only in the cerebral cortex in the animals exposed to ammonia for 5 days. The activity of GABA-aminotransferase (GABA-T) showed a significant rise in cerebellum and a fall in the brain stem in acute ammonia toxicity. In chronic ammonia toxicity GABA-T showed a rise in all the three regions of brain. Chronic ammonia toxicity produced a significant decrease in the content of glutamate in all the three regions without a significant change in the content of aspartate. GABA and glutamine. The content of alanine increased in all the three regions of brain under these experimental conditions. The ratio of glutamate + aspartate/GABA and glutamate/glutamine showed a decrease in all the three regions as a result of ammonia toxicity.     

Glutamate Decarboxylase and GABA Aminotransferase Levels in Different Regions of Rat Brain on the Onset of Leptazol Induced Convulsions

The activities of Glutamate decarboxylase (GAD) and Gamma aminobutyric acid (GABA) were studied in three regions of rat brain in heightened neuronal activity resulting in convulsions by Leptazol. These enzymes were studied in preconvulsive, convulsive and post convulsive phases. The activity of GAD decreases significantly in the preconvulsive phase in all the three regions of brain followed by a significant increase during the convulsive and post convulsive phase in cerebral cortex and cerebellum. The activity of GABA-T decreases maximal during the preconvulsive phase followed by convulsive phase. The activity of this enzyme tended to increase to control values when the postconvulsive phase was reached. Therefore, it is suggested that the concomitant decrease of GAD activity and GABA concentration, is probably an important factor in the onset of convulsions.     

Influence of short-lasting bilateral clamping of carotid arteries (BCCA) on GABA turnover in rat brain structures

We have previously shown that short-lasting reduction of cerebral blood flow by bilateral clamping of carotid arteries (BCCA) results in long-lasting increase in regional GABA concentration and decrease in seizure susceptibility in rats. In the present experiments, the effect of BCCA on GABA turnover and the enzymes involved in GABA synthesis and degradation were studied in rats. Regional GABA turnover was measured by means of GABA accumulation induced by the GABA-transaminase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Fourteen days after BCCA, GABA turnover was significantly increased in hippocampus, substantia nigra and cortex, but not different from sham-operated controls in several other brain regions, including striatum, hypothalamus and cerebellum. The activity of glutamate decarboxylase (GAD) measured ex vivo did not show any changes in investigated structures, while the activity of GABA-T was slightly increased in hippocampus. The increased GABA turnover in some brain regions may explain our previous findings of increased GABA content in these brain regions and decreased sensitivity of BCCA treated animals to the GABA_A-receptor antagonist bicuculline.     

Effects of some anticonvulsant drugs on brain GABA level and GAD and GABA-T activities

The effect of anticonvulsant drugs was examined on brain GABA levels and GAD and GABA-T activities. The level of GABA was increased by the treatment with diphenylhydantoin. The drug had no effect on GABA-T activity, whereas GAD activity was inhibited. Carbamazepine increased the GABA level but did not effect GAD and GABA-T activities. Diazepam had no effect on GABA level and GAD activity, whereas it caused a slight inhibition of GABA-T activity. Phenobarbital administration decreased GABA level only at the higher concentration. Clonazepam effected only GAD activity. Some anticonvulsant drugs generally increase brain GABA level; however the lack of correlation with an effect on the GAD and GABA-T activities indicate that other factors than metabolism, such as membrane transport processes, are involved in the mechanism of action of anticonvulsant drugs.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

EFFECTS OF AMINOOXYACETIC ACID AND l-GLUTAMIC ACID-γ-HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ-aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2-oxoglutarate aminotransferase (GABA-T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA-T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l -glutamic acid-hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.     

Acute and short term effects of ethanol on the metabolism of glutamic acid and GABA in rat brain

Although alcoholic intoxication is attributed to its pharmacological effects on the cell membranes in brain, the rapid metabolic utilisation of the same alters the metabolism of brain affecting the metabolism of glutamate and GABA which have varied metabolic roles besides serving a major proportion of synaptic activity. A study on the effects of ethanol, both acute and short-term, on glutamate (glu) and GABA metabolism in various regions of rat brain was carried out. Increased activities of glutamic acid decarboxylase (GAD) and aspartic acid aminotransferase (AST) in all brain regions, but decreased activity of glutamic acid dehydrogenase (GDH) in cerebral cortex (CC) and cerebellum (CB) following ethanol administration in brain was observed. Differential effects of ethanol were also obtained on the contents of glu and aspartate (asp), which were increased in CC, CB, and brain stem (BS) regions, as opposed to GABA content, which, although found to increase in acute toxicity, showed a decrease in all of the above brain regions in short-term toxicity. It is concluded that the above changes in glu, asp and GABA represent the consequences of metabolic utilization of alcohol in the brain, probably more a state of cerebral excitation than depression, and the changes may be a compensatory phenomenon.     

Response of the cockroach brain gamma-aminobutyric acid system to isonicotinic acid hydrazide and mercaptopropionic acid

The presence of gamma-aminobutyric acid (GABA) as well as glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) enzymes was demonstrated in the cockroach (Periplaneta americana) brain. Isonicotinic acid hydrazide (INH) in vivo (2.19 mumol/g) inhibited brain GAD activity, the inhibition lasted for about 2 hours and the normal activity levels reappeared at 4 h after INH administration. Brain GABA levels increased initially but then declined and were restored to normal levels at 4 h after INH administration. GABA-T activity was strongly inhibited by INH and a total 100% inhibition was observed at 2-3 h following INH treatment. The GABA-T activity, however, began to recover after 3 h but only 37% of the total enzyme activity was released from inhibition. Mercaptopropionic acid (MPA) in vivo (32 micrograms/g) inhibited brain GAD activity and depleted GABA level also. Results indicate that INH response of the cockroach brain GABA system is similar to that reported for the chick brain but differs from that of the mammalian brain.     

A Regional Study of 4-Aminobutyrate Metabolism and Amino Acid Levels in Rat Brain Following Chronic Oral Administration of Ethanolamine O-Sulphate

Abstract: Ethanolamine O-sulphate (EOS) dissolved in the drinking water (5mg-ml⁻¹) was administered ad libitum to rats for 26 days. At the end of this period, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) activities, 4-aminobutyrate (GABA) concentration, and the levels of six other amino acids were measured in various brain regions. Significant inhibition of GABA-T accompanied by significant increases in GABA content were observed throughout the brain, although the magnitudes of these effects varied according to region. GAD activity was significantly reduced in most brain regions, although this effect was apparently not related to cofactor availability or the direct actions of EOS or increased GABA concentration. Glutamine levels were significantly reduced to approximately 72% of control values in all brain regions. Aspartate levels were significantly reduced to approximately 84% of control values in all regions except the striatum and cerebellum. Minor changes in other amino acid levels were also detected. These neurochemical changes which accompanied the primary effect of EOS on GABA-T are discussed in terms of indirect secondary metabolic changes rather than nonspecific enzyme inhibition by EOS.     

GABA-ergic system in brain regions of glutamate-lesioned rats

Subcutaneous administration of high doses of glutamate to rats during their first 10 days after birth produced a great reduction of GABA content and GAD activity in the adult mediobasal hypothalamus, both in male and female. In addition GABA content and GAD activity showed a slight significant decrease in female cerebellum and male striatum. Glutamate treatment was also followed by a significant increase in GABA content and GAD activity of male substantia nigra, cerebellum, hippocampus and of female olfactory bulb. No reduction in GABA-T activity was observed in different brain areas studied except in mediobasal hypothalamus. The results support the view that glutamate treatment had a direct toxic effect on GABA-ergic neurons in mediobasal hypothalamus. The changes in GAD activity observed in all areas studied may reflect the neuroendocrine changes determined by nucleus arcuate lesions.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

Enzymes of Cerebral GABA Metabolism and Synaptosomal GABA Uptake in Acute Liver Failure in the Rabbit: Evidence for Decreased Cerebral GABA-Transaminase Activity

Abstract: Measurements of the activities of the two key enzymes in cerebral GABA metabolism—glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T)—were performed in normal rabbits and in rabbits with hepatic encephalopathy due to galactosamine-induced liver failure. Furthermore the uptake of GABA by synaptosomes was studied. Hepatic encephalopathy was associated with a marked decrease in the activity of GAB A-T. This decrease in activity was already apparent in galactosamine-treated rabbits before the onset of hepatic encephalopathy. Sera and serum ultrafiltrates of rabbits with hepatic encephalopathy but not of normal rabbits or of rabbits with uremic encephalopathy were shown to inhibit GABA-T activity in vitro . Cerebral GAD activity and synaptosomal GABA uptake in rabbits with hepatic encephalopathy and in untreated animals were not different. These later findings indicate that hepatic encephalopathy is not associated with alterations of presynaptic GABA nerve terminals in the central nervous system. The demonstration of a decrease in cortical GABA-T activity provides indirect evidence for decreased GABA turnover in the brains of rabbits with hepatic encephalopathy and thus is compatible with augmented GABA-ergic inhibitory neurotransmission contributing to the neural inhibition of hepatic encephalopathy.     

Comparative studies on the GABA-transaminase immunoreactivity in rat and gerbil brains.

Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system (CNS). Degradation of GABA in the CNS is catalyzed by the action of GABA transaminase (GABA-T). However, the neuroanatomical characteristics of GABA-T in the gerbil, which is a useful experimental animal in neuroscience, are still unknown. Therefore, we performed a comparative analysis of the distribution of GABA-T in rat and gerbil brains using immunohistochemistry. GABA-T immunoreactive neurons were observed in the regions which contained GABAergic neurons of both animals: corpus striatum; substantia nigra, pars reticulata; septal nucleus; and accumbens nucleus. GABA-T + neurons were restricted to layers III and V in the rat. Unlike the rat GABA-T + neurons were observed in layers II, III, and V of the gerbil cerebral cortex. These results suggest that the expression of GABA-T in the gerbil brain may be similar to that in the rat brain, except in the cerebral cortex.     

Correlation between alterations in brain GABA metabolism and seizure excitability following administration of GABA aminotransferase inhibitors and valproic acid—a re-evaluation

The time course of the effects of aminooxyacetic acid, γ-vinyl GABA, γ-acetylenic GABA, gabaculine, ethanolamine-O-sulphate (EOS) and valproic acid (VPA) on brain GABA content and the activities of glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T), the enzymes involved in biosynthesis and degradation of GABA, was re-determined and compared with the action on the electroconvulsive threshold in mice. All drugs caused significant increases in the seizure threshold, and the temporal pattern of this effect correlated rather well with the induced elevation of brain GABA. However, no clear relationship was found between the extent of GABA increase and the relative increase of seizure threshold. Except for VPA, the time course of the increment in brain GABA followed closely the inhibition of GABA-T. The activity of GAD was gradually decreased by γ-acetylenic GABA and a slow decline of GAD activity was also observed after γ-vinyl GABA. EOS and gabaculine suggesting a feedback repression of GAD synthesis by highly elevated GABA concentrations. Concomitant with significant reduction of GAD activity, a decrease in seizure threshold occurred though brain GABA levels remained markedly elevated. On the other hand, following administration of VPA the effect of GABA levels was paralleled by an increase in GAD activity indicating that the GABA-elevating action of this drug can be attributed at least in part to an activation of GABA synthesis. The data suggest that reduction of GAD activity may be an inevitable consequence of increasing brain GABA concentrations over a certain extent and this effect seems to limit the anticonvulsant efficacy of GABA-T inhibitors.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

γ-Aminobutyric Acid Metabolism and Behavioral Effects After Intraventricular Injection of Spermine in Chicks

Effects of intraventricularly injected spermine on behavior and electrocortical activity and gamma-aminobutyric acid (GABA) metabolism after a single dose of 1.13 mumol/animal were studied. Decrease in locomotor activity, sedation or sleep, and electrocortical synchronization that lasted approximately 2 h were observed. In addition spermine caused a significant increase in GABA content in diencephalon and brainstem, 30 min after administration. Concomitantly a significant increase of glutamate decarboxylase (GAD) activity was observed in cerebral hemispheres, diencephalon, and brainstem. Reduction in gamma-aminobutyrate: alpha-oxoglutarate amino-transferase (GABA-T) levels occurred in the diencephalon along with a significant increase of GABA-T in the brainstem. The present results demonstrate that spermine has the capacity to affect GABA metabolism and are in favor of the suggestion that endogenous polyamines may modulate GABAergic mechanisms.     

Effect of Inhibitors of GABA Aminotransferase on the Metabolism of GABA in Brain Tissue and Synaptosomal Fractions

Abstract: Five inhibitors of the GABA degrading enzyme GABA-aminotransferase (GABA-T), viz., gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate, and aminooxyacetic acid, as well as GABA itself and the antiepileptic sodium vdproate were administered to mice in doses equieffective to raise the electroconvulsive threshold by 30 V. The animals were killed at the time of maximal anticonvulsant effect of the respective drugs and GABA, GABA-T and glutamate decarboxylase (GAD) were determined in whole brain and synaptosomes, respectively. The synaptosomal fraction was prepared from brain by conventional ultracentrifugation procedures. All drugs studied brought about significant increases in both whole brain and synaptosomal GABA concentrations, and, except GABA itself, inhibited the activity of GABA-T. Furthermore, all drugs, except GABA and γ-acetylenic GABA, activated GAD in the synaptosomal fraction. This was most pronounced with ethanolamine O -sulphate, which induced a twofold activation of this enzyme but exerted only a weak inhibitory effect on GABA-T. The results suggest that activation of GAD is an important factor in the mechanism by which several inhibitors of GABA-T and also valproate increase GABA concentrations in nerve terminals, at least in the relatively non-toxic doses as used in this study.     

SOME EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE METABOLISM OF γ-AMINOBUTYRIC ACID IN RAT BRAIN

(1) The inhibitor of γ-aminobutyrate transaminase (GABA-T), amino-oxyacetic acid (AOAA), drastically reduced the activity of GABA-T to 30 per cent of the control value, with a corresponding increase of brain GABA, but had no effect on the activity of glutamate decarboxylase (GAD). (2) The monoamine oxidase (MAO) inhibitors phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine, lowered GABA-T activity to 58, 49 and 48 per cent, respectively; this was associated with a marked elevation of brain GABA. (3) The action of phenelzine and phenylpropylhydrazine in vivo and in vitro could be abolished by pre-treatment of the tissue with the structurally related MAO inhibitors phenylisopropylhydrazine and trans-2-phenylcyclopropylamine. These had no action on the GABA system in vivo, either on the GABA content or on the GABA-T activity. These latter drugs, however, were unable to influence the effects of AOAA either on GABA or on GABA-T. (4) The possible mechanism of action on GABA and the enzyme activities of the GABA system is discussed.     

Elevation of Brain GABA Content by Chronic Low-Dosage Administration of Hydrazine, a Metabolite of Isoniazid

Abstract: When γ-aminobutyric acid aminotransferase (GABA-T) activity was measured in vitro in rat brain, neither isoniazid (INH) nor four of its known metabolites (isonicotinic acid, acetylisoniazid, acetylhydrazine, diacetylhydrazine) inhibited the enzyme in concentrations (5 mM) far higher than those likely to be achieved when INH is administered to man. In contrast, hydrazine (5 μM) caused a 50% inhibition of GABA-T without inhibiting glutamic acid decarboxylase (GAD). Rats were injected daily for 109 days with hydrazine (0.08 or 0.16 mmol/kg/day), after which amino acid contents and enzyme activities were measured in their brains. Both hydrazine doses caused significant elevations of whole brain GABA content and reductions of GABA-T activity, but did not affect GAD activity. Chronic administration of hydrazine at thee doses did not reduce weight gain or alter rat behavior, nor did it produce any irreversible pathologic changes in liver or alterations in hepatic aryl hydrocarbon hydroxylase activity. However, hydrazine treatment caused changes in the contents of many brain amino acids besides GABA, and markedly increased concentrations of ornithine, tyrosine, and α-aminoadipic acid in rat plasma. Inhibition of GABA-T activity and the other biochemical alterations observed in patients given high doses of INH probably result from hydrazine formed in the metabolic degradation of INH. Thus administration of hydrazine might be a more direct means of elevating brain GABA content in patients where this seems indicated, and might not entail a greater risk of adverse effects.