Studies on the GABAergic system in astrocytoma and neuroblastoma cells in culture

The GABAergic system was investigated in C-6 astrocytoma cells and C-1300 neuroblastoma cells in culture and compared to that in mouse brain. The activities of glutamate decarboxylase, GABA-transaminase, succinic semialdehyde dehydrogenase and glutamate dehydrogenase were measured. In the cultured cells, only glutamate dehydrogenase activity was equal or greater than that of mouse cerebral cortex. Glutamate decarboxylase in both cell lines was 2%, while GABA-transaminase and succinic semialdehyde dehydrogenase activities were less than 20% of those found in brain. In spite of the disparate enzyme activities, GABA, glutamate, and -ketoglutarate concentrations were similar in the cell lines and cerebral cortex. The anticonvulsant drugs sodium valproate and aminooxyacetic acid increased cortical GABA concentrations but either had no effect or decreased GABA in the cells in a complete medium. The convulsant isoniazid decreased GABA in mouse brain but had no effect in either cell line. In the absence of pyridoxal in the medium, some drug effects could be induced in the cultured cells. It is concluded that the differing responses of the GABAergic system in the mouse brain and cell lines may be attributed in part to the fact that the cells do not represent an integrated system and are of tumor origin.     

Succinic Semialdehyde as a Substrate for the Formation of γ-Aminobutyric Acid

The conversion of succinic semialdehyde into gamma-aminobutyric acid (GABA) by GABA-transaminase was measured in rat brain homogenate in the presence of different concentrations of the cosubstrate glutamate. The calculated kinetic parameters of succinic semialdehyde for GABA-transaminase were a limiting Km value of 168 microM and a limiting Vmax value of 38 mumol g-1 h-1. Combination with previously obtained data for the conversion of GABA into succinic semialdehyde revealed a kEq value of 0.04, indicating that equilibrium of GABA-transaminase is biased toward the formation of GABA. The increased formation of GABA in the presence of succinic semialdehyde was not due to an increased conversion of glutamate into GABA by glutamic acid decarboxylase. Therefore these results indicate that succinic semialdehyde can act as a precursor for GABA synthesis.     

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Kinetic characterization of GABA-transaminase from cultured neurons and astrocytes

The enzymatic mechanism and the kinetic parameters of GABA-transaminase extracted from cultured mouse cerebral cortex neurons and astrocytes were studied. Neuronal as well as astrocytic GABA-transaminase obeyed a bi bi ping-pong reaction mechanism. The estimated K_m-values for -ketoglutarate and GABA were significantly lower for astroglial GABA-transaminase compared to the neuronal enzyme suggesting a possible existence of cell specific isozymes of GABA-transaminase. The observed enzymatic mechanism and the magnitude of the estimated kinetic parameters imply that GABA-transaminase synthesized in the two types of cultured neural cells is mechanistically and kinetically equivalent to the enzyme synthesized in the brainin vivo.     

Purification of rat brain succinate-semialdehyde dehydrogenase and study of its inhibition by branched chain fatty acids]

Rat brain succinic semialdehyde deshydrogenase has been purified 1300 fold. This enzyme is inhibited non competitively by the same branched chain fatty acids which inhibit GABA-transaminase competitively with respect to GABA. The respective activities of GABA-T and SSADH found in rat brain indicate that at anticonvulsant doses, the acids dipropylacetic and 2-methyl 2-ethyl caproic preferentially inhibit GABA-transaminase thus inducing a rise in cerebral GABA level. This increase is therefore not due to metabolism of the succinic semialdehyde by GABA-T.     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

Neurochemistry of GABAergic activities in the central nervous system ofLocusta migratoria

Summary The biochemical elements of GABA-ergic synapses in the central nervous tissue were examined by a comparative neurochemical approach. The high concentration of GABA as well as the activities of glutamate decarboxylase and GABA-transaminase suppose a high content of GABAergic elements in the nervous system of the locust.Nerve endings isolated from the ganglia of locusts accumulated exogenous GABA in a carriermediated, sodium dependent process into compartments from where it could partially be released under depolarizing conditions. The transport was stimulated by extracellular chloride, was modulated by specific ionophores (enhanced by valinomycin, inhibited by CCCP) and could effectively be blocked by GABAergic ligands (DABA, muscimol). Binding studies revealed the existence of multiple binding sites for GABA which differ in number, affinity, pharmacology and ion dependency. The putative receptors for GABA (Na⁺-independent binding sites) in locust nervous tissue exceeded the concentrations found in vertebrate brain tissue and showed different binding pharmacology.Abbreviations GABA -amino butyric acid - GAD glutamate decarboxylase - GABA-T GABA-transaminase - DABA diamino butyric acid     

Mechanism of action of an anticonvulsant, sodium dipropylacetate

The hypothesis that the brain GABA level increase which is induced by a sodium dipropyl acetate treatment arises either through inhibition of succinic semialdehyde dehydrogenase (SSADH), or through inhibition of GABA transaminase by succinic semialdehyde (SSA), has been considered. It appeared that in vivo brain GABA level increase cannot be attributed to SSADH inhibition, and that SSA is not a GABA precursor. It has been shown that SSA is neither in vivo nor in vitro a GABA-transaminase inhibitor. 4-hydroxybenzaldehyde, a potent SSADH inhibitor did not increase GABA level at a dosage which induces a 99% inhibition of SSADH.     

Evidence for a role of high Km aldehyde reductase in the degradation of endogenous gamma-hydroxybutyrate from rat brain

gamma-Hydroxybutyrate (GHB) is a putative neurotransmitter in brain. We have already demonstrated that it is transformed into gamma-aminobutyrate (GABA) by rat brain slices incubated under physiological conditions. This conversion occurs via a GABA-transaminase reaction. Therefore, succinic semialdehyde, the oxidative derivative of GHB, appears to be the primary catabolite of GHB degradation. Apparently, the kinetic characteristics and pH optimum of GHB dehydrogenase (high Km aldehyde reductase) in vitro do not favor a role for this enzyme in endogenous brain GHB oxidation. However, in the presence of glucuronate, glutamate, NADP and pyridoxal phosphate, pure GHB dehydrogenase, coupled to purified GABA-transaminase does produce GABA from GHB at an optimum pH close to the physiological value and with a low Km for GHB.     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

In situ measurements of enzyme activities in the brain

Summary The present review focusses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver -ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of -ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires -ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Glutamine and alpha-ketoglutarate uptake and metabolism by nerve terminal enriched material from mouse cerebellum

In order to provide evidence relevant to the hypothesis that nonsynaptically derived -ketoglutarate serves as a metabolic precursor of the neurotransmitter pools of glutamate and GABA the uptake and metabolism of -ketoglutarate by nerve terminal enriched material was studied and compared to corresponding data for glutamine. Both -ketoglutarate and glutamine were transported across the cell membrane by high affinity and low affinity carriers. Under conditions prevailing in vivo -ketoglutarate probably is transported primarily by its high affinity carrier, whereas gluatmine should be transported primarily by one or more low affinity carriers. Based upon reciprocal uptake inhibition experiments glutamine appeared to be transported by the alanine preferring system, and to a lesser extent by the basic amino acid and large neutral amino acid carriers. A comparison of the rate of uptake by different cellular preparations enriched in either nerve terminals or cell bodies indicated that -ketoglutarate is transported selectively by nerve terminals. Both substrates were rapidly converted to glutamate; however, glutamine was more readily metabolized to GABA. The results of our study are consistent with the concept that both glutamine and -ketoglutarate derived from extra-neuronal sources are taken up by nerve terminals and utilized to replenish the neurotransmitter pools of glutamate and GABA.     

NAD+-dependent formation ofγ-aminobutyrate (GABA) from glutamate

Mitochondria and nuclei of various tissues, including brain and liver, are capable of producing-aminobutyrate (GABA) fromL-glutamate, but poorly, if at all, fromD-glutamate. The amino nitrogen of glutamate is found in the reaction product. The enzymes responsible for GABA formation were solubilized from crude liver cell nuclei by Triton X-100. The reaction is NAD⁺ dependent Oxygen, FMN, Mg²⁺, and pyridoxalphosphate enhanced GABA formation. NADP⁺, coenzyme A, ornithine, 2-oxoglutarate, and aminooxyacetic acid, among others, inhibited the formation of GABA. On the basis of the available information the reaction sequence, is formulated tentatively as follows:     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Evidence for an incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophicum

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (=-ketoglutarate), aspartate (=oxaloacetate) and alanine (=pyruvate) was studied. The organism was grown on H₂ plus CO₂ at pH 6.5 in the presence of 1 mM [U-¹⁴C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO₂ and C-2-C-5 from succinate indicating that in M. thermoautotrophicum -ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from -ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO₂ fixation.     

Effect of thiamine deprivation and thiamine antagonists on the level of gamma-aminobutyric acid and on 2-oxoglutarate metabolism in rat brain

Brain levels of y-aminobutyric acid (GABA), glutamate and 2-oxoglutarate, activities of glutamate decarboxylase GABA-transaminase plus succinic semiaidehyde dehydrogenase and blood levels of glutamate and 2-oxoglutarate were determined in normal, thiamine-deprived, oxythiamine-treated and pyrithiamine-treated rats. Brain GABA levels were significantly reduced in thiamine-deprived and pyrithiamine-treated rats, but the activities of the enzymes of the GABA shunt pathway were not affected. Brain levels of glutamate were decreased and of 2-oxoglutarate increased in all three types of deficiency. This was associated with similar decreases in glutamate and increases in 2-oxoglutarate in the blood in all three deficient groups. Intraventricular injections of 2-[U-¹⁴C] oxoglutarate into the brain in these four groups of rats resulted in some significant differences in distribution of ¹⁴C in various TCA-pathway intermediates and satellite compounds in the brain. Increases in ¹⁴C-label were observed for glutamine and 2-oxoglutarate in all three deficient groups as compared to controls. The ¹⁴C content of succinate, fumarate and aspartate was decreased in the thiamine deprived and PTh-treated groups and [¹⁴C]glutamate was decreased in all three deficient groups. The ¹⁴C content of GABA was not significantly affected.     

Anaerobic metabolism of goldfish,Carassius auratus (L.). Influence of anoxia on mass-action ratios of transaminase reactions and levels of ammonia and succinate

Summary Changes in the concentrations of ammonia, glutamate, alanine, aspartate, -ketoglutarate, oxaloacetate and succinate were measured in freeze-clamped lateralred muscle, dorsal white muscle and liver, and in rapidly cooled blood of goldfish after 12 h of anoxia. Alanine accumulation, succinate accumulation and aspartate depletion are observed in all tissues examined; in the liver the concentrations of glutamate increase and those of ammonia decrease. The mass-action ratio of the glutamate-pyruvate transaminase-catalyzed reaction stays within one order of magnitude from thermodynamic equilibrium in the direction of alanine formation. The mass-action ratio of the glutamate-oxaloacetate transaminase reaction is far from equilibrium when measured oxaloacetate concentrations are used. When levels of free oxaloacetate are calculated from LDH and MDH equilibrium constants, the mass-action ratio of glutamate-oxaloacetate transamination is close to equilibrium in the direction of aspartate formation. Since neither alanine nor glutamate decreases, and since ammonia gradients suggest a continuous ammonia production in all tissues examined, anaerobic proteolysis is assumed. A possible coupling between amino acid catabolism and ethanol production is discussed.Abbreviations ALA alanine - ASP aspartate - EDTA ethylene diamine tetraacetate - FP _ox oxidated flavoprotein - FP _red reduced flavoprotein - FUM fumarate - GLU glutamate - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - IMP inosine monophosphate - KG -ketoglutarate - LDH lactate dehydrogenase - MAL malate - MAR mass action ratio - MDH malate dehydrogenase - OAA oxaloacetate - PYR pyruvate - sAMP adenylosuccinate - SDH succinate dehydrogenase - SUCC succinate     

Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene

We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transribed unidirectionally and their orientation within the cluster is 5-gabD-gabT-gabP-3. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD⁺-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP⁺-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-d-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The K _m of GABA uptake was found to be 11.8 M and the V_max 0.33 nmol/min · mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.