EFFECTS OF GLUTAMATE AND OTHER AMINO ACIDS ON THE RETINA1

The application of unlabelled glutamate to the isolated chicken retina charged with [¹⁴C]glutamate caused an increase in the tissue transparency and a release of the label into the superfusion fluid. The processes causing the change in transparency were‘desensitized’by a prolonged application of unlabelled glutamate, whereas the release of the labelled amino acid was relatively unaffected. Mg²⁺ tended to depress the change in transparency caused by stimulation with unlabelled glutamate but had little effect on the release of labelled glutamate from the retina. The effect of a Ca²⁺-free superfusion fluid on the transparency and release of glutamate varied from retina to retina. Aspartate (in higher concentrations) elicited a change in transparency and release of the label in a manner similar to that of glutamate. Glutamine caused a change in transparency accompanied by a release of labelled glutamine and in some experiments the release of a small amount of labelled glutamate. Homocysteic acid elicited marked changes in transparency but no release of labelled glutamate. Pyroglutamate depressed both the change in transparency and the release of labelled glutamate caused by the unlabelled amino acid. Gamma-aminobutyric acid and glycine had no effect on the transparency of the tissue or on the release of amino acids. We have discussed the possibility that a release of glutamate from the intracellular compartment into the extracellular space is involved in the mechanism of spreading depression.     

Decline in ribonucleic acid and protein synthesis with loss of viability during the early hours of imbibition of rye (Secale cereale L.) embryos.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.     

Studies on the protonation constants and solvation of alpha-amino acids in dioxan-water mixtures

To gain more information about the effect of solvent on alpha-amino acids, the stoichiometric protonation constants of 10 alpha-amino acids (glycine, DL-alanine, DL-valine, L-leucine, L-isoleucine, DL-phenylalanine, L-serine, L-threonine, L-asparagine, and L-glutamine) in different dioxan-water mixtures have been determined potentiometrically using a combined pH electrode system calibrated in concentration units of hydrogen ion at 25 degrees C with an ionic strength of 0.10 M. For all amino acids studied, it was observed that the log K(1) values relating to the protonation equilibria of the anionic form are almost unaltered by the change in solvent composition. However, the log K(2) values corresponding to the formation equilibria of cationic form increase with the increase in dioxan content. The variation of these constants is discussed on the basis of specific solute-solvent interactions and structural changes of amino acids from water to dioxan-water media. The zwitterionic to neutral form ratio of these acids in dioxan-water mixtures is also discussed.     

Effects of metabolic inhibitors on the release of glutamate from the retina

—The superfused, isolated retina of the chicken was used to investigate the mechanisms responsible for the increase in retinal transparency and the release of glutamate associated with stimuli known to elicit spreading depression (SD). We sought to distinguish between (1) mechanisms involving glutamate-induced increase in Na⁺ permeability and consequent uptake of extracellular material into the intracellular compartment and (2) mechanisms involving interference with operation of the Na⁺ pump that would result in a similar uptake of extracellular materials. Tetrodotoxin (which inhibits inward movements of Na⁺) depressed the transparency increase caused by stimulation with glutamate but not that elicited by application of KCl. Ouabain (which inhibits the Na⁺ pump) caused a marked increase in tissue transparency. The application of inhibitors of the aerobic metabolism, such as DNP or cyanide, or deprivation of O₂ had no effect on the retinal transparency; results suggesting that the energy for the Na⁺ pump could be supplied by glycolysis. Indeed iodoacetate (which inhibits glycolysis) caused a marked change in transparency. Furthermore we found evidence for a compound in the superfusion fluid supplemented with iodoacetate that may be a reaction product of glutamate and iodoacetate. In some preparations superfusion with glucose-free solutions caused a slowly developing increase in transparency and release of glutamate; in others the increase in transparency was more sudden and there was a larger release of glutamate. Seemingly, interference with the tissue metabolism can cause an uptake of extracellular material either by arrest of the Na⁺ pump or by the release of glutamate, depending on the conditions of the experiment.     

Chemotactic response of Escherichia coli to chemically synthesized amino acids.

In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness. The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both. Seven of the commonly occurring amino acids are repellents. This work was carried out with chemically synthesized amino acids.     

RELEASE OF GLUTAMATE AND OTHER AMINO ACIDS FROM ARTHROPOD NERVE–MUSCLE PREPARATIONS

—A study has been made of the release of glutamate and other amino acids from perfused leg preparations from Carcinus and Schistocerca, at rest and during stimulation of motor nerves to the leg muscles. Stimulation caused an increase in release af many amino acids. No evidence was obtained to link glutamate release from such preparations with synaptic function. If release of transmitter glutamate is to be demonstrated in arthropods, techniques must be devised to avoid contamination with large quantities of non-transmitter glutamate.     

Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis)

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.     

Pyridoxal 5'-phosphate dependent enzymes in the nematode Nippostrongylus brasiliensis.

Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.     

微生物发酵法生产L-丝氨酸及L-半胱氨酸研究进展

L-丝氨酸及L-半胱氨酸在食品、医药和化妆品等行业有着广泛的应用,在植物和微生物中两者从头合成的前体物均为3-磷酸甘油酸。微生物发酵生产这两种氨基酸以其社会、经济及环境效益展现出良好的前景。针对近年来微生物发酵法生产L-丝氨酸及L-半胱氨酸的研究成果,本文综述了当前国内外学者在该领域研究的热点,即代谢途径及其调节、相关氨基酸的转运及运输、菌种及菌株改造、新菌种的开发等。最后结合当前生物技术的新发展,对今后的研究方向进行了展望。     

Fluorometric study on the interaction of amino acids and ATP with valyl-tRNA synthetase from Bacillus stearothermophilus

Interactions of several amino acids and nucleotides with valyl-tRNA synthetase [EC 6.1.1.9] (VRS) from Bacillus stearothermophilus were investigated using as a probe the ligand-induced quenching of protein fluorescence (lambda ex = 295 nm, lambda em = 340 nm) of VRS. L-Valine, L-threonine, L-isoleucine, L-glutamic acid, L-leucine, and D-valine caused fluorescence quenching. Among them, L-threonine had a Kd value comparable to that for the cognate substrate, L-valine, but the other amino acids were bound more weakly as estimated by the fluorescence titration method. L-Alanine, L-histidine, and L-serine did not cause any fluorescence change. Among the nucleotides tested (ATP, ADP, AMP, GTP, ITP, CTP, and UTP), only ATP caused the fluorescence change. In the presence of an excess amount of ATP, only L-valine and L-threonine, among the tested amino acids, induced the fluorescence quenching, and the binding of L-valine was greatly favored under this condition. This is consistent with the results of the ATP-PP1 exchange reaction by VRS, in which only L-valine and L-threonine, of these 9 amino acids tested, could serve as substrates, and the Km value for L-valine was much smaller than that for L-threonine. Thus the binding of ATP to VRS enhances the substrate specificity of VRS towards amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemotaxis toward amino acids by Bdellovibrio bacteriovorus.

Chemotaxis toward amino acids by Bdellovibrio bacteriovorous strain UKi2 was studied by the capillary technique of Adler (J. Gen. Microbiol. 74:77-91, 1973). Chemotaxis was shown to be optimal when the capillaries were incubated at between 15 and 40 degrees C for 30 min; the optimal pH was between 7.0 and 8.2. The chemotactic response was proportional to the density of the suspension of bdellovibrios up to a density of 10(8) cells/ml. B. bacteriovorus was attracted to L-asparagine, L-cysteine, L-glutamine, glycine, L-histidine, L-lysine, and L-threonine. The possible roles of chemotaxis in the life of B. bacteriovorus are discussed.     

Effect of Peroxides on [3H]d-Aspartate Release from Bovine Isolated Retinae

In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-asparagine and L-leucine in pancreatic islets

Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. L-Asparagine augmented the oxidation of L-leucine, an effect possibly attributable to activation of 2-ketoisocaproate dehydrogenase. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

Effect of α-Amino-4-Phosphonobutyrate on the Release of Endogenous Glutamate and Aspartate from Cortical Synaptosomes of Epileptic Rats

The effect of the glutamate antagonist alpha-amino-4-phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from non-epileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and gamma-aminobutyric acid (GABA) from synaptosomal preparations of cobalt-induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5-1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.     

Selective increase in the extracellular D-serine contents by D-cycloserine in the rat medial frontal cortex

A partial agonist of the N-methyl-D-aspartate (NMDA) receptor, D-cycloserine, acting at its glycine modulatory site, ameliorates the neuropsychiatric symptoms that are mimicked by NMDA antagonists and include cognitive disturbances, antipsychotic-resistant schizophrenic symptoms and cerebellar ataxia. To obtain a further insight into the mechanisms of the therapeutic efficacies of D-cycloserine, we investigated the effects of the systemic administration of D-cycloserine on the extracellular contents of an endogenous NMDA co-agonist, D-serine, in the medial frontal cortex of the rat using an in vivo dialysis technique. An acute intraperitoneal injection of D-cycloserine (50 and 100 mg/kg) caused an increase in extracellular concentrations of D-serine without significant effects on those of L-serine, glycine, L-glutamate, L-aspartate, L-glutamine, L-asparagine, L-alanine, L-threonine and taurine in the medial frontal cortex. The selective increase in the extracellular D-serine contents may, at least partially, be associated with the facilitating effects of D-cycloserine on the NMDA receptor functions in addition to its direct stimulation of the NMDA receptor glycine site.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction L-asparagine and L-leucine in pancreatic islets

1. Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. 2. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. 3. L-Asparagine augmented the oxidation of L-leucine, and effect possibly attributable to activaion of 2-ketoisocaproate dehydrogenase. 4. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. 5. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.     

Biochemical Studies of Olfaction: Binding Specificity of Odorants to a Cilia Preparation from Rainbow Trout Olfactory Rosettes

Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L-threonine, L-serine, and L-alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H-labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of L-[3H]lysine to site L was unaffected by addition of L-threonine, L-serine, or L-alanine, establishing its independence from site TSA. L-Arginine inhibited binding of L-[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.     

The Synthesis of Neuroactive Amino Acids from Radioactive Glucose and Glutamine in the Rat Retina: Effects of Light Stimulation

The effect of light stimulation in vitro on the labelling of neuroactive amino acids derived from [14C]glucose or [14C]glutamine in the rat retina has been studied. [14C]Glutamine, at 700 microM, provided about 50% of the tissue pools of glutamate, aspartate, and GABA; and the labelling of these decreased on light stimulation, both in the photoreceptor cells (glu and asp) and in the inner retina (glu, asp, and GABA). In contrast, there were no significant changes in the entry of label derived from [14C]glucose, although similar trends were apparent in the data obtained for the photoreceptor cell layer. The pools may, therefore, be separate. Other results support the contention that glucose is the principal energy source for the retina, its entry into non-amino acid derivates being decreased on light stimulation.