AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor-oxaloacetate (OA) or alpha-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

In Vitro Release of Endogenous β- Alanine, GABA, and Glutamate, and Electrophysiological Effect of β-Alanine in Pigeon Optic Tectum

The efflux of 20 amino acids, induced by either high K+ concentration or veratrine, was determined in pigeon tectal slices. Ca2+-dependent, K+-induced release of beta-alanine, gamma-aminobutyric acid (GABA), and glutamate was observed. Veratrine caused release of the same amino acids plus glycine in a tetrodotoxin-sensitive manner. beta-Alanine had a strong inhibitory effect on the activity of tectal neurons which was blocked by strychnine but not by bicuculline. The results indicated a transmitter function for beta-alanine in the optic tectum, and were consistent with the previously proposed transmitter role of GABA and glutamate in this structure.     

Inhibitory glutamate receptor channels

Inhibitory glutamate receptors (IGluRs) are a family of ion channel proteins closely related to ionotropic glycine and γ-aminobutyric acid (GABA) receptors; They are gated directly by glutamate; the open channel is permeable to chloride and sometimes potassium. Physiologically and pharmacologically, IGluRs most closely resemble GABA receptors; they are picrotoxin-sensitive and sometimes crossdesensitized by GABA. However, the amino acid sequences of cloned IGluRs are most similar to those of glycine receptors. Ibotenic acid, a conformationally restricted glutamate analog closely related to muscimol, activates all IGluRs. Quisqualate is not an IGluR agonist except among pulmonate molluscs and for a unique multiagonist receptor in the crayfishAustropotamobius torrentium. Other excitatory amino acid agonists are generally ineffective. Avermectins have several effects on IGluRs, depending on concentration: potentiation, direct gating, and blockade, both reversible and irreversible. Since IGluRs have only been clearly described in protostomes and pseudocoelomates, these effects may mediate the powerful antihelminthic and insecticidal action of avermectins, while explaining their low toxicity to mammals. IGluRs mediate synaptic inhibition in neurons and are expressed extrajunctionally in striated muscles. The presence of IGluRs in a neuron or muscle is independent of the presence or absence of excitatory glutamate receptors or GABA receptors in the cell. Generally, extrajunctional IGluRs in muscle have a higher sensitivity to glutamate than do neuronal synaptic receptors. Some extrajunctional receptors are sensitive in the range of circulating plasma glutamate levels, suggesting a role for IGluRs in regulating muscle excitability. The divergence of the IGlu/GABA/Gly/ACh receptor superfamily in protostomes could become a powerful model system for adaptive molecular evolution. Physiologically and pharmacologically, protostome receptors are considerably more diverse than their vertebrate counterparts. Antagonist profiles are only loosely correlated with agonist profiles (e.g., curare-sensitive GABA receptors, bicuculline-sensitive AChRs), and pharmacologically identical receptors may be either excitatory or inhibitory, and permeable to different ions. The assumption that agonist sensitivity reliably connotes discrete, homologous receptor families is contraindicated. Protostome ionotropic receptors are highly diverse and straightforward to assay; they provide an excellent system in which to study and integrate fundamental questions in molecular evolution and adaptation.     

The components required for amino acid neurotransmitter signaling are present in adipose tissues

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABA(A)Ralpha2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

Glutamate in plants: metabolism, regulation, and signalling

Glutamate occupies a central position in amino acid metabolism in plants. The acidic amino acid is formed by the action of glutamate synthase, utilizing glutamine and 2-oxoglutarate. However, glutamate is also the substrate for the synthesis of glutamine from ammonia, catalysed by glutamine synthetase. The alpha-amino group of glutamate may be transferred to other amino acids by the action of a wide range of multispecific aminotransferases. In addition, both the carbon skeleton and alpha-amino group of glutamate form the basis for the synthesis of gamma-aminobutyric acid, arginine, and proline. Finally, glutamate may be deaminated by glutamate dehydrogenase to form ammonia and 2-oxoglutarate. The possibility that the cellular concentrations of glutamate within the plant are homeostatically regulated by the combined action of these pathways is examined. Evidence that the well-known signalling properties of glutamate in animals may also extend to the plant kingdom is reviewed. The existence in plants of glutamate-activated ion channels and their possible relationship to the GLR gene family that is homologous to ionotropic glutamate receptors (iGluRs) in animals are discussed. Glutamate signalling is examined from an evolutionary perspective, and the roles it might play in plants, both in endogenous signalling pathways and in determining the capacity of the root to respond to sources of organic N in the soil, are considered.     

The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish.

Both glutamate and gamma-aminobutyric acid (GABA) are involved in pituitary hormone release in fish. Glutamate serves 2 purposes, both as a neurotransmitter and as a precursor for GABA synthesis. Glutamate can be catabolized to GABA by the actions of 2 distinct but related enzymes, glutamate decarboxylase 65 (GAD65) and GAD67. They derive from 2 different genes that likely arose from an early gene duplication prior to the emergence of teleosts more than 400 million years ago. There is good evidence for the involvement of GABA in luteinizing hormone (LH) release in fish. The mechanism of GABA action to stimulate LH release appears to be a combination of effects on GnRH release, potentiation of gonadotropin hormone-releasing hormone (GnRH) action, and in some cases directly at the LH cell. These actions appear to be dependent on such factors as sex or sex steroid levels, and there may also be species differences. Nevertheless, the stimulatory effects of GABA on LH are present in at least 4 fish species. In contrast, convincing data for the inhibitory effects of GABA on LH release have only been observed in 1 fish species. The sites and mechanisms of action of amino acid neurotransmitters on LH release have yet to be fully characterized. Both 130N-methyl-D-aspartic acid (NMDA) and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors are likely to have important roles. We suggest that it is a receptor similar to the GABA(A) type which mediates the effects of GABA on LH release in fish, at least partially acting on the GnRH neuron, but likely directly acting at the gonadotroph as well. GABA may also be involved in regulating the release of other pituitary hormones in fish, namely follicle stimulating hormone (FSH = GTH-I), prolactin, and growth hormone. Based on the findings described in this review, a working model for the involvement of glutamate and GABA in the regulation of LH release in teleost fish is proposed.     

Biosynthesis of the polyoxins, nucleoside peptide antibiotics: a new metabolic role for L-isoleucine as a precursor for 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid).

The biosynthetic origin of the carbon skeleton of 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid) is described. This unique cyclic amino acid is the C terminus of the nucleoside peptide antibiotics, the polyoxins, elaborated by Streptomyces cacaoi var, asoensis. In vivo experiments show that 14-C from [1-14-C]isoleucine, [U-14-C]isoleucine, [1-14-C]methionine, [U-14-C]methionine, [U-14-C]threonine, and [1-14-C]glutamate is incorporated into polyoximic acid; however, 14-C from [5-14-C]glutamate and [methyl-14-C]methionine is not incorporated. The distribution of 14-C in polyoximic acid clearly shows that the intact carbon skeleton of L-isoleucine is utilized directly. The incorporation of 14-C from [U-14-C]methionine, [U-14-C]threonine, and [1-14-CA1glutamate into polyoximic acid occurred only after their conversion to isoleucine via 2-ketobutyrate. A scheme is presented in which either of the two beta-unsaturated amino acids isolated from Bankera fuligineoalba, L-2-amino-3-hydroxymethyl-3-pentenoic acid or L-2-amino-3-formyl-3-penetenoic acid, is regarded as a possible intermediate amino acid between isoleucine and polyoximic acid.     

Metabolism and functions of gamma-aminobutyric acid

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca²⁺ activates Ca²⁺/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H⁺ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.     

Glutamic acid and gamma-aminobutyric acid neurotransmitters in central control of breathing.

We review recent cross-disciplinary experimental and theoretical investigations on metabolism of the amino acid neurotransmitters glutamic acid and gamma-aminobutyric acid (GABA) in the brain during hypoxia and hypercapnia and their possible role in central control of breathing. The roles of classical modifiers of central chemical drive to breathing (H+ and cholinergic mechanisms) are summarized. A brief perspective on the current widespread interest in GABA and glutamate in central control is given. The basic biochemistry of these amino acids and their roles in ammonia and bicarbonate metabolism are discussed. This review further addresses recent work on central respiratory effects of inhibitory GABA and excitatory glutamate. Current understanding of the sites and mechanisms of action of these amino acids on or near the ventral surface of the medulla is reviewed. We focus particularly on tracer kinetic investigations of glutamatergic and GABAergic mechanisms in hypoxia and hypercapnia and their possible role in the ventilatory response to hypoxia. We conclude with some speculative remarks on the critical importance of these investigations and suggest specific directions of research in central mechanisms of respiratory control.     

13C Nuclear Magnetic Resonance Evidence for γ-Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.     

Ketogenic diet, brain glutamate metabolism and seizure control

We do not know the mode of action of the ketogenic diet in controlling epilepsy. One possibility is that the diet alters brain handling of glutamate, the major excitatory neurotransmitter and a probable factor in evoking and perpetuating a convulsion. We have found that brain metabolism of ketone bodies can furnish as much as 30% of glutamate and glutamine carbon. Ketone body metabolism also provides acetyl-CoA to the citrate synthetase reaction, in the process consuming oxaloacetate and thereby diminishing the transamination of glutamate to aspartate, a pathway in which oxaloacetate is a reactant. Relatively more glutamate then is available to the glutamate decarboxylase reaction, which increases brain [GABA]. Ketosis also increases brain [GABA] by increasing brain metabolism of acetate, which glia convert to glutamine. GABA-ergic neurons readily take up the latter amino acid and use it as a precursor to GABA. Ketosis also may be associated with altered amino acid transport at the blood-brain barrier. Specifically, ketosis may favor the release from brain of glutamine, which transporters at the blood-brain barrier exchange for blood leucine. Since brain glutamine is formed in astrocytes from glutamate, the overall effect will be to favor the release of glutamate from the nervous system.     

Taurine, glutamate and GABA modulate the outgrowth from goldfish retinal explants and its concentrations are affected by the crush of the optic nerve

Summary The amino acid taurine plays an important trophic role during development and regeneration of the central nervous system. Other amino acid systems, such as those for glutamate and gamma-aminobutyric acid (GABA), are modified during the same physiological and pathological processes. After crushing the optic nerve, goldfish retinal explants were plated in the absence and in the presence of different amino acids and amino acid receptor agonists. The length and the density of the neurites were measured at 5 days in culture. Taurine increased the length and the density of neurites. Glutamate and glycine increased them at low concentration, but were inhibitors at higher concentration. The combination of N-methyl-D-aspartate (NMDA) and glycine produced a greater inhibitory effect than NMDA alone. NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) added simultaneously with taurine impaired the stimulatory effect of the latter. GABA stimulated the emission of neurites in a concentration dependent manner. Hypotaurine also elevated the length of neurites, but cysteinesulfinic acid did not produce a significant effect. The concentrations of taurine, glutamate and GABA were determined by HPLC with fluorescent detection in the retina of goldfish at various days post-crushing the optic nerve. The levels of taurine were significantly increased at 48 h after the crush, and were elevated up to 20 days. Glutamate level decreased after the lesion of the optic nerve and was still low at 20 days. GABA concentration was not significantly different from the control. The interaction of these amino acids during the regenerative period, especially the balance between taurine and glutamate, may be a determinant in restoring vision after the crush.Abbreviations AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - GABA gamma-aminobutyric acid - NMDA N-methyl-D-aspartate     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

Action of taurine, 3-aminopropanesulfonic acid, and GABA on the hindgut and heart of the cockroach, Leucophaea maderae.

Taurine, glycine, glutamate, and gamma-aminobutyric acid (GABA) were all present in concentrations of greater than 1% of the total free amino acid content in the brain, thoracic, and abdominal ganglia of Leucophaea maderae. Hemolymph, subesophageal ganglia, and hindgut had substantial amounts of glutamate and glycine, but less than 0.3% taurine or GABA. Taurine, 3-aminopropanesulfonic acid (3-APS), cysteine-sulfinic acid (CSA), and GABA each had myotropic activity on the isolated cockroach hindgut, with 3-APS having the most consistent effect (ED50 = 0.63 mM), while taurine and CSA activities were similar to that of GABA on the hindgut. Both taurine and 3-APS had anti-arrhythmic effects on semi-isolated heart preparations of L. maderae, while GABA was inhibitory and induced arrhythmia. Bicuculline was antagonistic to the effects of GABA, taurine, and 3-APS on the hindgut, and induced arrhythmia in heart preparations; this arrhythmia was reversible by taurine, but not by GABA or 3-APS.     

Metabolism of gamma-aminobutyric acid during cold acclimation and freezing and its relationship to frost tolerance in barley and wheat

Amino acid homeostasis was investigated in frost-resistant barley seedlings under either cold- or freezing-stress conditions. Total free amino acid content varied only slightly, but a substantial conversion of glutamate to gamma-aminobutyric acid (GABA) was found that was proportional to the severity of the stress. Cold acclimation caused a significant increase in amino acid pools, and induced the expression of the GABA-shunt genes. As a consequence, GABA accumulated to a higher extent during the subsequent exposure to lower temperature. A different picture was obtained with a frost-sensitive genotype, in which glutamate decarboxylation occurred during the stress as well, but the activation of the GABA shunt seemed not to take place, and free glutamate was almost depleted. Analogous results were found in frost-resistant and frost-sensitive wheat cultivars. Feeding non-hardened plants with exogenous glutamate resulted in increased GABA accumulation under low temperature. The possibility that glutamate decarboxylation and GABA metabolism would play a role in frost tolerance is discussed.