Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli , chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides , chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn 5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H⁺-linked glutamate carrier of E. coli , was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides .     

GABA synthesis by cultured fibroblasts obtained from persons with Huntington's disease.

Abstract— A consistent observation in particular regions of brains of persons having died with Huntington's disease (HD) is a reduction in the concentration of γ-aminobutyric acid (GABA) and a decrease in the activity of its synthetic enzyme, glutamate decarboxylase (EC 4.1.4.15). GABA levels are also reduced in HD cerebrospinal fluids. This study suggests that skin fibroblasts obtained from persons with HD can be used to study their GABA system. A rapid and specific assay for [¹⁴C]glutamate– [¹⁴C]GABA based on Aminex A-7 chromatography has been developed. Cell monolayers and homogenates of HD cells convert [¹⁴C]glutamate to [¹⁴C]GABA. GABA synthesis by HD cell homogenates is pyridoxal dependent and is inhibited by 1 mm -aminooxyacetic acid. GABA synthesis by HD and control cell homogenates also show the same thermal sensitivity as rat brain GAD. When compared to non-HD human cells the HD cells reveal disturbances in the non-neuronal GABA metabolic pathway. Concentrated HD cell homogenates synthesize approx 3 times the amount of GABA as control cells. When diluted both extracts made similar amounts of GABA. Synthesis of GABA by HD cell homogenates is not inhibited by cysteine sulfinate. Decarboxylation of glutamate in these cells is therefore most likely due to glutamate decarboxylase and not cysteine sulfinate decarboxylase. HD cells in monolayer also synthesize 3 times the amount of GABA as compared to control cells. In addition, glutamate upake is altered in HD cells. This report indicates there may be a different pattern of enzyme regulation between HD and control cells.     

Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme

Branched-chain amino acid aminotransferase (BCAT), which has pyridoxal 5'-phosphate as a cofactor, is a key enzyme in the biosynthetic pathway of hydrophobic amino acids (leucine, isoleucine, and valine). The enzyme reversibly catalyzes the transfer of the amino group of a hydrophobic amino acid to 2-oxoglutarate to form a 2-oxo acid and glutamate. Therefore, the active site of BCAT should have a mechanism to enable recognition of an acidic amino acid as well as a hydrophobic amino acid (double substrate recognition). The three-dimensional structures of Escherichia coli BCAT (eBCAT) in complex with the acidic substrate (glutamate) and the acidic substrate analogue (glutarate) have been determined by X-ray diffraction at 1.82 and 2.15 A resolution, respectively. The enzyme is a homo hexamer, with the polypeptide chain of the subunit folded into small and large domains, and an interdomain loop. The eBCAT in complex with the natural substrate, glutamate, was assigned as a ketimine as the most probable form based upon absorption spectra of the crystal complex and the shape of the residual electron density corresponding to the cofactor-glutamate bond structure. Upon binding of an acidic substrate, the interdomain loop approaches the substrate to shield it from the solvent region, as observed in the complex with a hydrophobic substrate. Both the acidic and the hydrophobic side chains of the substrates are bound to almost the same position in the pocket of the enzyme and are identical in structure. The inner side of the pocket is mostly hydrophobic to accommodate the hydrophobic side chain but has four sites to coordinate with the gamma-carboxylate of glutamate. The mechanism for the double substrate recognition observed in eBCAT is in contrast to those in aromatic amino acid and histidinol-phosphate aminotransferases. In an aromatic amino acid aminotransferase, the acidic side chain is located at the same position as that for the aromatic side chain because of large-scale rearrangements of the hydrogen bond network. In the histidinol-phosphate aminotransferase, the acidic and basic side chains are located at different sites and interact with different residues of the disordered loop.     

Kynurenine Pathway Measurements in Huntington''s Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.     

Characterization of Separated Cell Types from the Developing Rat Cerebellum: Transport of Glutamate and Aspartate by Preparations Enriched in Purkinje Cells, Granule Neurones, and Astrocytes

Preparations of structurally preserved cerebellar perikarya (cells) were found to express high-affinity transport systems for glutamate but not for certain putative transmitter substances (including monoamines, glycine and taurine) and non-transmitter amino acids. The characteristics of the high-affinity glutamate transport system were similar to those of other preparations of brain tissue: [³H]glutamate uptake by the cells was Na⁺-dependent and was inhibited competetively by other acidic amino acids. The rank order of apparent affinities of the carrier for acidic amino acids was L-aspartate > L-glutamate > D-aspartate ? D-glutamate (the affinity for D-glutamate being over two orders of magnitude lower than for the other three amino acids). Comparison of high-affinity [³H]glutamate uptake in preparations enriched in different cell types showed that although the affinities are similar (2-4 fiM), the rate is outstandingly high in astrocytes (V_max 18 nmol/min per mg protein). Significantly, uptake into the putatively glutamatergic granule cells was very low. These observations were supported by autoradiographic findings which showed that the predominant sites of [3H]glutamate uptake in cerebellar cultures enriched in interneurones are the astrocytes. Furthermore, the V_max in cultures enriched in astrocytes was as high as that in separated astrocytes. Thus, it seems that the principal cell type involved in acidic amino acid uptake in the cerebellum is the astrocyte, and this must be taken into consideration when high-affinity uptake is used as a marker for glutamatergic transmitter systems. Furthermore, the selective cellular distribution of glutamate transport sites, together with the uneven distribution of enzymes related to glutamate metabolism observed previously, indicates that a metabolic interaction takes place between the different cell types, supporting the current hypothesis on metabolic compartmentation in the brain.     

Effects of p-chlorophenylalanine and alpha-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells.

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.     

Elimination of KATP channels in mouse islets results in elevated [U-13C]glucose metabolism, glutaminolysis, and pyruvate cycling but a decreased gamma-aminobutyric acid shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Huntington disease and Tourette syndrome. II. Uptake of glutamic acid and other amino acids by fibroblasts

Injection of kainic acid, a rigid analog of the excitatory neurotransmitter glutamic acid (glu), into the neostriatum of rats produces a condition that mimics Huntington disease (HD) in at least 12 different morphological and biochemical parameters. These results suggested that one of the possible basic mechanisms in HD is a defect in the presynaptic of glial uptake of glu, resulting in chronic hyperstimulation and death of a specific set of neurons. To test this hypothesis, the uptake of glu was studied in 12 carefully matched sets of control-HD pairs and two lines of Tourette syndrome fibroblasts. Although the first six sets suggested a glutamate transport defect in HD cells, examination of 12 sets indicated that there were no significant differences between control and HD cells. The fibroblasts showed both a high and low affinity uptake of glutamic acid. Sodium dependent uptake of L-glutamate (L-glu) minus D-glutamate (D-glu) at 100, 1,000, and 10,000 Micrometers glutamate was normal in HD and Tourette syndrome cells.     

Starvation and chemoreception in antarctic benthic invertebrates

Sensitivity (chemoreception) to different amino acids was studied in six invertebrate species: Serolis polita, Glyptonotus antarcticus, Abyssochromene plebs, Waldeckia obesa, Odontaster validus, and Sterechinus neumayeri. The sensitivity was estimated by the changes in basic metabolism (respiration rate). Starvation increased the sensitivity in all the species. The metabolism rates increased in the presence of L-glutamic acid in G. antarcticus, A. plebs, O. validus, and St. neumayeri. The serine and arginine amino acids had a significant impact on the metabolism of the necrophagous species S. polita and W. obesa. The chemical information may be mediated by means of L-glutamic acid via glutamate receptors, which can be blocked by kynurenic acid, as occurs in the experiments with G. antarcticus and A. plebs.     

Plasma and Cerebrospinal Fluid Amino Acids in Epileptic Patients

Altered plasma and cerebrospinal fluid amino acid levels may be associated with human epilepsy. We studied three groups of patients, those with a generalized epileptic syndrome, juvenile myoclonic epilepsy, patients with refractory localization-related epilepsies, and patients with acute seizures (within 24 h). Plasma levels of amino acids were studied in all patient groups, as were those in the cerebrospinal fluid (CSF) of patients with acute seizures. After acute seizures, the amino acid changes in the CSF were limited to a reduction in the level of taurine, whereas the levels of most amino acids in plasma were decreased. On the other hand, levels of the excitatory amino acids glutamate and aspartate were increased. The most notable finding in the juvenile myoclonic epilepsy patients was an increase in glutamate level in the plasma. Our study supports the conception of an altered metabolism of glutamate in generalized epilepsies.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

(1)H NMR based metabolomics of CSF and blood serum: a metabolic profile for a transgenic rat model of Huntington disease

Huntington disease (HD) is a hereditary brain disease. Although the causative gene has been found, the exact mechanisms of the pathogenesis are still unknown. Recent investigations point to metabolic and energetic dysfunctions in HD neurons. Both univariate and multivariate analyses were used to compare proton nuclear magnetic resonance spectra of serum and cerebrospinal fluid (CSF) taken from presymptomatic HD transgenic rats and their wild-type littermates. N-acetylaspartate (NAA), was found to be significantly decreased in the serum of HD rats compared to wild-type littermates. Moreover, in the serum their levels of glutamine, succinic acid, glucose and lactate are significantly increased as well. An increased concentration of lactate and glucose is also found in CSF. There is a 1:1 stoichiometry coupling glucose utilization and glutamate cycling. The observed increase in the glutamine concentration, which indicates a shutdown in the neuronal-glial glutamate-glutamine cycling, results therefore in an increased glucose concentration. The elevated succinic acid concentration might be due to an inhibition of succinate dehydrogenase, an enzyme linked to the mitochondrial respiratory chain and TCA cycle. Moreover, reduced levels of NAA may reflect an impairment of mitochondrial energy production. In addition, the observed difference in lactate supports a deficiency of oxidative energy metabolism in rats transgenic for HD as well. The observed metabolic alterations seem to be more profound in serum than in CSF in presymptomatic rats. All findings suggest that even in presymptomatic rats, a defect in energy metabolism is already apparent. These results support the hypothesis of mitochondrial energy dysfunction in HD.     

Effect of cold-temperature exposure and acclimation on amino acid pool changes and enzyme activities of rat brown adipose tissue

The amino acid pool composition and its concentration ratios with respect to blood and plasma, as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamine synthetase, adenylate deaminase and glutamate dehydrogenase have been studied in the interscapular brown adipose tissue of control, 12-h cold-exposed and 15-day cold-acclimated rats. Cold temperature affected the amino acid metabolism and pool composition more intensely after 15 days than after 12-h cold-exposure, even though the patterns of change were very similar in both groups. Cold temperatures induced a decrease in glutamine and an increase in glutamate concentration in the tissue. This probably increased the metabolism of branched chain amino acids and caused a decrease in adenylate deaminase activity. It also seemed to increase alanine utilization. We concluded that amino acid metabolism in brown adipose tissue is enhanced by cold temperature acclimation.     

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.     

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.     

DL-7-azatryptophan and citrulline metabolism in the cyanobacterium Anabaena sp. strain 1F.

An alternative route for the primary assimilation of ammonia proceeds via glutamine synthetase-carbamyl phosphate synthetase and its inherent glutaminase activity in Anabaena sp. strain 1F, a marine filamentous, heterocystous cyanobacterium. Evidence for the presence of this possible alternative route to glutamate was provided by the use of amino acid analogs as specific enzyme inhibitors, enzymological studies, and radioistopic labeling experiments. The amino acid pool patterns of continuous cultures of Anabaena sp. strain 1F were markedly influenced by the nitrogen source. A relatively high concentration of glutamate was maintained in the amino acid pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamate in nitrogen metabolism. The addition of 1.0 microM azaserine increased the intracellular pools of glutamate and glutamine. All attempts to detect any enzymatic activity for glutamate synthase by measuring the formation of L-[14C]glutamate from 2-keto-[1-14C]glutarate and glutamine failed. The addition of 10 microM DL-7-azatryptophan caused a transient accumulation of intracellular citrulline and alanine which was not affected by the presence of chloramphenicol. The in vitro activity of carbamyl phosphate synthetase and glutaminase increased severalfold in the presence of azatryptophan. Results from radioisotopic labeling experiments with [14C]bicarbonate and L-[1-14C]ornithine also indicated that citrulline was formed via carbamyl phosphate synthetase and ornithine transcarbamylase. In addition to its effects on nitrogen metabolism, azatryptophan also affected carbon metabolism by inhibiting photosynthetic carbon assimilation and photosynthetic oxygen evolution.     

Excitatory Amino Acid-Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.     

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.     

Effect of cysteamine on amino acid pools of cultured fibroblasts and the uptake of L-proline

Cysteamine when incubated with human normal and cystinotic fibroblasts produces significant alterations in cellular amino acid pools with a decrease in proline, glycine, histidine, and methionine as well as branch chain and dibasic amino acids. Serine content was increased while aspartate, glutamate and glutamine were unchanged. The uptake of L-proline by cysteamine treated cells was impaired suggesting that altered uptake of amino acids may be a factor in the cysteamine effect.