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.     

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.     

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.     

Effect of L-cycloserine on brain GABA metabolism

The administration of L-cycloserine to mice resulted in a dramatic decrease in the activities of 4-aminobutyrate:2-oxoglutarate aminotransferase (GABA-T) and L-alanine:2-oxoglutarate aminotransferase (ALA-T) in both brain and liver. L-Aspartate:2-oxoglutarate aminotransferase was inhibited only slightly, and brain glutamic acid decarboxylase not at all. Liver ALA-T activity returned to near normal levels within 24 h of L-cycloserine administration whereas liver GABA-T and brain ALA-T activities had returned only halfway to normal levels in the same time period. The recovery in the activity of brain GABA-T was even slower. A consequence of the inhibition of brain GABA-T activity was an elevation in the GABA content of the tissue which was maximal 3 h after L-cycloserine administration and which was still noticeable 8 h after the drug treatment. L-Cycloserine was also a potent in vitro inhibitor of brain GABA-T activity. The inhibition was competitive with respect to GABA, the Ki value being 3.1 X 10(-5) M. The prior administration of L-cycloserine to mice significantly delayed the onset of isonicotinic acid hydrazide induced convulsions.     

THE EFFECT ON GABA METABOLISM IN BRAIN OF ISONICOTINIC ACID HYDRAZIDE AND PYRIDOXINE AS A FUNCTION OF TIME AFTER ADMINISTRATION

—The effect of intramuscularly administered INH on brain levels of GABA in chicks was dependent on the amount injected. A subconvulsant dose of INH (1·1 mmol/kg) produced a slow steady decline in the level of GABA, whereas a convulsant dose (2·19 mmol/kg) brought about a sequential fall and rise in GABA level. This sequence of events reflected changes in the relative activities of GAD and GABA-T brought about by the hydrazide. The administration of pyridoxine together with the INH (2·19 mmol/kg) prevented the onset of seizures and lessened the effect of the INH on GABA levels and GAD activity but not on GABA-T activity. The possibility that a deranged GABA metabolism is responsible for hydrazide-induced seizures is discussed.     

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.     

Use of Inhibitors of γ-Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid

The technique of estimating gamma-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats, but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/kg i.p., 5-30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation.     

Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system

In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.     

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.     

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.     

EFFECT OF DIAZEPAM AND PENTOBARBITAL ON AMINOOXYACETIC ACID-INDUCED ACCUMULATION OF GABA

Abstract— The effect of diazepam and pentobarbital on γ-aminobutyric acid (GABA) levels, the aminooxyacetic acid (AOAA)-induced accumulation of GABA, and the in vitro activity of l -glutamate 1-carboxyl-lyase (EC 4.1.1.15) [GAD] were studied in various regions of rat brain. Diazepam increased GABA levels in the substantia nigra, diminished the AOAA-induced accumulation of GABA in the caudate nucleus, cingulate, parietal and entorhinal cortex and had no effect on GABA accumulation in the pyriform and cerebellar cortex. After pentobarbital, GABA levels were elevated in the caudate nucleus but decreased in the parietal and pyriform cortex; the AOAA-induced accumulation of GABA also diminished in all cortical regions studied. No correlation was found between the apparent changes in GABA synthesis, as estimated by accumulation after inhibition of 4-aminobutyrate-2-oxoglu-tarate (EC 2.6.1.19) [GABA-T] with AOAA, and the changes in GABA levels induced by these drugs. The reduction in AOAA-induced GABA accumulation after diazepam and pentobarbital treatment was most pronounced in regions which showed the greatest accumulation of GABA after AOAA administration. Neither diazepam nor pentobarbital administration affected the activity of GAD in homogenates of cingulate cortex. Chlorpromazine, at a dose which decreased spontaneous activity, enhanced the AOAA-induced GABA accumulation in the cingulate cortex, suggesting that drug-induced sedation is not necessarily associated with decreased GABA synthesis. While regional differences were observed in the effects of diazepam and pentobarbital on GABA synthesis, both agents appear to inhibit GABA synthesis in vivo and both do so, in at least some brain areas, at subsedative doses.     

Molecular structure--activity relationship of hydrazides inhibiting glutamic acid decarboxylase, GABA-alpha-oxoglutarate aminotransferase, and monoamine oxidase activities in chick brain.

Several aryl and heteroaryl hydrazides were synthesized and evaluated for their inhibitory effects on glutamic acid decarboxylase (GAD), GABA-alpha-oxoglutarate aminotransferase (GABA-T), and monoamine oxidase (MAO) enzyme systems in chick brain 24 h after their intramuscular administration (0.75 mmol/kg). All compounds produced a reduction in GAD, GABA-T, and MAO activity. Structure-activity relationships indicated that the ring structure had a greater influence on the degree of GAD and GABA-T inhibition than did the N'-terminal group. In contrast, structural requirements for MAO inhibition were much more restrictive. The intramuscular administration of benzoic acid hydrazide to chicks 24 h prior to their being exposed to oxygen at high pressure provided significant protection against the onset of the hyperbaric oxygen-induced seizures.     

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.     

THE ROLE OF GABA METABOLISM IN THE CONVULSANT AND ANTICONVULSANT ACTIONS OF AMINOOXYACETIC ACID

Abstract— At high dosage levels AOAA acted as a convulsant agent in mice and rats but in lower amounts it was an effective anticonvulsant agent against INH-induced seizures, by tripling the time to the onset of the convulsions. AOAA elevated brain GABA levels as a result of a preferential inhibition of the GABA-T enzyme system but, contrary to previous reports, the activity of the GAD enzyme system was also inhibited, even by relatively low dosage levels of AOAA. The state of excitability of the brain following the administration of AOAA was related, within the limits of the present study, to changes in GAD activity and GABA levels, but additional data are required before the relationship can be properly evaluated.     

SUSTAINED DRUG-INDUCED ELEVATION OF BRAIN GABA IN THE RAT1

Abstract— The contents of GABA, homocarnosine, and β-alanine can be raised in rat brain for long periods of time by the continued administration of phenelzine, aminooxyacetic acid (AOAA), or isonicotinic acid hydrazide (INH). These 3 compounds apparently act by preferential inhibition of the enzyme GABA aminotransferase (GABA-T). Oral administration of phenelzine (20 mg/kg per day) caused a 25–50 per cent increase in GABA levels in rat brain, but produced appreciable toxic side effects. A similar increase in GABA levels in brain resulted from oral administration to rats of INH in a dosage of 60 mg/kg per day, without production of any obvious toxic effects. Simultaneous administration of large doses of pyridoxine did not abolish the GABA-elevating effect of INH. Brain GABA levels in the rat were increased by approx. 50 per cent by daily injections of AOAA (2.5 mg/kg per day). At this low dosage, AOAA injections in rats could be continued for at least 6 weeks without producing evident toxic effects. Oral administration of large amounts of GABA, on the other hand, failed to increase the content of GABA in the brains of rats not treated with GABA-T inhibitors, and failed to produce any further increase of brain GABA levels in rats treated with AOAA.     

Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

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.     

Inhibitors of crayfish glutamic acid decarboxylase

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN^– and Cl^–), and some cations (e.g. Zn⁺²). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.