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.     

Regional distributions of γ-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and γ-aminobutyrate transaminase (GABA-T) in the central nervous brains of C57/BR,C3H/He,and F1 hybrid mice

The distributions of -aminobutyric acid (GABA), glutamate decarboxylase (GAD), and -aminobutyrate transaminase (GABA-T) have been studied in various brain areas of mice. These neurochemical markers, which are related to inhibitory neurotransmission, were investigated in different inbred strains of mice (C3H/He, C57/BR, and their F₁ hybrids). The regional distributions of GABA, GAD activity, and GABA-T activity in adult mice of these three strains were quite similar. No significant differences were found in any brain area for GAD or GABA-T activity. However, significant differences in GABA level were found in several brain areas among these strains of mice, especially in hypothalamus, hippcampus, olfactory bulb, and occipital cortex. These results provide further information to the possible influence of the GABAergic system in these brain areas.     

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.     

Distribution of ω-Amino Acid: Pyruvate Transaminase and Aminobutyrate: α-Ketoglutarate Transaminase in Microorganisms

The distribution of ω-amino acid transaminases in microorganisms was investigated, ω-Amino acid: pyruvate transaminase (ω-APT) was found in bacteria and yeasts, but not in actinomycetes and fungi. On the contrary, aminobutyrate: α-ketoglutarate transaminase (GABA-T) was shown in most of the microorganisms from bacteria to fungi. β-Alanine is a preferred amino donor for the co-APT reaction. Although bacterial and yeast GABA-T are inactive for β-alanine, fungal and actinomycete enzymes react with this compound and γ-aminobutyrate. In comparing these results with those of plant and mammalian enzymes, two different pathways of co-amino acid metabolism are suggested for bacteria, yeast and plants, i.e. one for β-alanine and the other for γ-aminobutyrate, catalyzed by ω-APT and GABA-T, respectively. In actinomycetes, fungi, and mammals GABA-T may be involved in the metabolism of both ω-amino acids. In addition, evolutionary changes of ω-amino acid transaminases are discussed.     

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.     

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.     

Properties of L-glutamate decarboxylase from brains of adult and newborn mice.

Abstract— l -Glutamate 1-carboxy-lyase (EC 4.1.1.15) (GAD) and 4-aminobutyrate-2-oxo-glutarate aminotransferase (EC 2.6.1.19) (GABA-T) have been purified from mouse brain (Wu et al. 1973; Schousboe et al., 1973) and their properties have been extensively studied (Wu & Roberts , 1974; Schousboe et al., 1974). The above enzymes were prepared from a water lysate of crude mitochondrial fraction, which accounted for only 25–30% of total GAD or GABA-T activities in brain. A procedure has been developed which liberates approx 85% of total GAD and GABA-T activities into supernatant. Two distinct, well-separated peaks with GAD activity and a single peak with GABA-T activity were observed when a concentrated extract from brain of adult or newborn mice was chromatographed on Sephadex G-200 or Bio-Gel A–1.5 m. The first peak appeared in the void volume and is. therefore. an entity of high molecular weight. The second peak gave elution characteristics which were identical to those of the enzyme that had been purified previously (mol wt = 85,000). These two GAD peaks were also clearly separated on polyacrylamide gel electrophoresis. The GAD activities in the two peaks showed similar pH profiles (optimum, 6.5). K_m values (1–2 mM), immunodiffusion patterns and inhibitions by anti-GAD IgG prepared against GAD purified from synaptosome-containing crude mitochondrial fraction (60–80%). The physiological implications of high molecular weight and low molecular weight forms of GAD are discussed.     

A γ-aminobutyrate pathway in mammalian kidney cortex

Rat kidney cortex converts l-glutamate to γ-aminobutyrate by a decarboxylation reaction which differs from the corresponding reaction in brain. Renal l-glutamate decarboxylase has two apparent K_m values for glutamate in homogenates (0.4 and 2.5 mM). γ-Aminobutyrate is converted by a transaminase whose capacity appears to exceed the decarboxylase. γ-Aminobutyrate is converted ultimately to succinate and CO₂.γ-Aminobutyrate stimulates respiration of kidney cortex slices in vitro and the compound crosses cell membranes in kidney by a respiration-linked, mediated process.Chronic acidosis lowers renal γ-aminobutyrate in the rat; brain γ-aminobutyrate is unaffected by acidosis. Glutamic acid decarboxylase and γ-aminobutyrate transaminase activities are unchanged in acidosis. α-Methylglutamate, an inhibitor of renal glutaminase, lowers the γ-aminobutyrate and glutamate content of rat kidney in normal and acidotic states. Aminooxyacetic acid in vivo, an inhibitor of γ-aminobutyrate transaminase, causes a striking increase in renal γ-aminobutyrate during chronic acidosis.At concentrations of glutamate in vitro, which are similar to the tissue glutamate content in vivo, the γ-aminobutyrate pathway accounts for approximately one-fourth of glutamate disposal in rat kidney cortex slices.     

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.     

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.     

Free amino acid production during tomato fruit ripening: a focus on l-glutamate

In tomato, free amino acids increase dramatically during fruit ripening and their abundance changed differentially. More evident is l-glutamate which gives the characteristic “umami” flavor. Glutamate is the principal free amino acid of ripe fruits of cultivated varieties. In this paper, we examined the capacity of tomato fruits to process endogenous as well as exogenous polypeptides during the ripening transition, in order to analyze their contribution to the free amino acid pool. In addition, the activity of some enzymes involved in glutamate metabolism such as γ-glutamyl transpeptidase (γ-GTase), glutamate dehydrogenase (GDH), α-ketoglutarate-dependent γ-aminobutyrate transaminase (GABA-T), alanine and aspartate aminotransferases was evaluated. Results showed that peptidases were very active in ripening fruits, and they were able to release free amino acids from endogenous proteins and glutamate from exogenously added glutamate-containing peptides. In addition, red fruit contained enough γ-GTase activity to sustain glutamate liberation from endogenous substrates such as glutathione. From all the glutamate metabolizing enzymes, GDH and GABA-T showed the higher increase in activities when the ripening process starts. In summary, tomato fruits increase free amino acid content during ripening, most probably due to the raise of different peptidase activities. However, glutamate level of ripe fruit seems to be mostly related to GDH and GABA-T activities that could contribute to increase l-glutamate level during the ripening transition.     

Cerebral myelinogenesis in theSnell dwarf mouse: Stimulatory effects of GH and T4 restricted to the first 20 days of postnatal life

We attempted to define the critical time period during early postnatal life when GH and T₄ are essential for myelination. We administered bGH and T₄ toSnell dwarf mice during the first and second 20 days after birth. Positive results were obtained only when hormones were given during the first 20 days of postnatal life. We observed a distinct increase in brain weight, DNA content, CNPase activity and a remarkably increased level of spontaneous locomotion activity with a diurnal periodicity. Morphological observations of brain sections stained for myelin basic protein (MBP) correlated the biochemical findings. The later administration of hormones was ineffective. Our interpretation is that the administration of exogenous hormones led to increased myelinogenesis through their stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

GABA-transaminase and glutamic acid decarboxylase changes in the brain of rats treated with pyrithiamine

Pyrithiamine, a thiamine phosphokinase inhibitor, was fed to rats on a thiamine-deficient diet, producing weight loss, ataxia and loss of righting reflex in 10 days. Some rats were then sacrificed; others were returned to a normal diet, to be sacrificed only when their weight had returned to pre-experimental levels. Rats were sacrificed for assay of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) activities in homogenates of eight brain regions or were perfused for -aminobutyric acid transaminase (GABA-T) histochemistry. GAD activity was significantly reduced in symptomatic rats in the thalamus > cerebellum > midbrain > pons/medulla. GABA-T staining was similarly reduced, with greatest losses in the thalamus > inferior colliculus > pons > medulla. ChAT activity was not significantly altered in any brain area. Following return to a normal diet, GAD activity was significantly recovered in all areas except the thalamus. GABA-T staining recovered, at least partially, in all areas affected.     

GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES

Abstract— Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated K _m values were 0.31 m m for γ-aminobutyrate, 0.16 m m for α-ketoglutarate, and 3.8 μ m for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors.
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors.     

EFFECTS OF DI-n-PROPYLACETATE,AN ANTICONVULSIVE COMPOUND,ON GABA METABOLISM1

The effects on GABA metabolism of an anticonvulsant drug, di-n-propylacetate (DPA), were studied. Given intraperitoneally DPA increases the brain GABA content and does not change its biosynthesis from glutamic acid. However, it inhibits in vitro both glutamate decarboxylase and aminobutyrate transaminase (GABA-T) activities. The inhibition is more pronounced on the GABA-T and this observation might explain the increase of GABA level.     

Discontinuation effects of oxazepam and diazepam treatment on brain GABA metabolism in rats

The effects of oxazepam and diazepam (both at 10 mg/kg, i.p.) during continuous treatment for 15 days and following discontinuation after 5 days onwards on cerebral glutamic acid decarboxylase (GAD) and GABA-aminotransferase (GABA-T) have been studied. It has been found that during continuous treatment as well as following discontinuation after 5 days, a significant increase in GAD activity is observed in case of diazepam but not in case of oxazepam. On the other hand, a marked decrease in GABA-T activity is observed during continuous treatment up to 15 days with both diazepam and oxazepam but during discontinuation phase, the decreased GABA-T activity tends to increase and attain normal value much earlier in case of oxazepam than diazepam. This differential effect of oxazepam and diazepam on γ-aminobutyric acid (GABA) metabolism, following discontinuation of treatment, may possibly contribute to the difference in withdrawal effects associated with the two benzodiazepines.     

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 ± 2.5 μmol/g protein per h (mean ± S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar α-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammoniagenesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.     

BRAIN 4-AMINOBUTYRATE

A systematic study was made of aminobutyrate transaminase (4-aminobutyrate: 2-oxoglutarate aminotransferase, I.U.B.2.6.1.19) in the developing rat brain. Assays of enzymic activity were made in whole brain of seven to twenty-one animals at each day of age from birth to day 30. The numerous analyses allowed statistical treatment and recognition of an abrupt change in the rate of synthesis of aminobutyrate transaminase, as judged by activity/g wet wt., beginning at day 12. From birth through day 11 there was a linear increase of low slope in enzymic activity, from day 12 through day 26 there was a linear increase of markedly increased slope. During the first period γ-aminobutyrate increased from 14 to 25 m-moles/kg fresh wt./hr; during the second the increase was from 25 to 113. The change of slope of linear increase in rat brain aminobutyrate transaminase began abruptly midway in the ‘critical period’ of rat brain development which extends roughly from day 10 to day 14. The onset of peak increase of rat brain succinate semialdehyde dehydrogenase (I.U.B.1.2.1.16) by contrast, occurred at day 6, heralded the ‘critical period’, and preceded by 6 days the onset of peak increase of the transaminase. These time-locked changes in rate of increase of two enzymes of a biochemical pathway uniquely functional in neural tissue may be of significance in determining the development of the nervous system in the young animal.     

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.     

ω-Fluoromethyl Analogues of ω-Amino Acids as Irreversible Inhibitors of 4-Aminobutyrate: 2- Oxoglutarate Aminotransferase

Abstract— ω -Monofluoromethyl and ω-difluoromethyl analogues of the known substrates of GABA-T, β -alanine, γ -aminobutyric acid, and 5-aminopentanoic acid, are time dependent inhibitors of purified 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T). The inhibitory activity decreases with increasing chain length. In vitro , inhibitory activity decreases with increasing fluorine substitution of the methyl group. In vivo , β -difluoromethyl- β -alanine and 2,4-difluoro-3-aminobutyric acid are the most potent GABA-T inhibitors ever reported. Trifluoromethyl derivatives are devoid of GABA-T inhibitory activity in vitro or in vivo.