Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GLUTAMATES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,369 records · Page 76Linked to original sources
PubMed1992

A novel prodrug of salicylic acid, salicylic acid-glutamic acid conjugate utilizing hydrolysis in rabbit intestinal microorganisms.

The fate of salicylic acid-glutamic acid conjugate (salicyl-glutamic acid) following oral, intravenous, intracecal and rectal administration (60, 10, 5 and 5 mg/kg, respectively: salicylic acid equivalent) was examined in rabbits. Salicylic acid was detected in the blood 2 h after oral administration of salicyl-glutamic acid and it reached the maximum level (69.4 micrograms/ml) at 18 h after the dose. A high blood concentration of salicylic acid (24.8 micrograms/ml) was observed up to 36 h. But only a small amount of salicyl-glutamic acid was detected in the blood (less than 2.5 micrograms/ml, as salicylic acid). In contrast, unchanged salicyl-glutamic acid was found mainly in the blood following intravenous administration of salicyl-glutamic acid, suggesting that presystemic de-conjugation of salicyl-glutamic acid predominantly occurred. The intestinal mucosal de-conjugation of salicyl-glutamic acid was negligible in the in situ intestinal sac preparation with complete mesenteric venous blood collection. Immediate and very extensive salicylic acid formation in the cecum was found following intracecal administration of salicyl-glutamic acid. After oral pretreatment of rabbits with kanamycin sulfate (6 x 400 mg), a significant inhibition of salicylic acid formation following intracecal administration of salicyl-glutamic acid was observed, indicating that the intestinal microorganisms were responsible for the biotransformation of salicyl-glutamic acid. Also, in vitro incubation of salicyl-glutamic acid with gut contents showed that the primary location of hydrolysis was the hind gut.

Animals↗
PubMed2002

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9.

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.

Bacteria↗
PubMed2005

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells.

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na+-dependent EAA transporters glutamate/ aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. MeHg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-3H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p < 0.001) increased following treatments with 5 or 10 microM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p < 0.001, both at 5 and 10 microM MeHg) increased and GLT-1 transporter protein levels significantly (p < 0.001) decreased following MeHg exposure (5 microM). (3) MeHg exposure led to significant inhibition (p < 0.05) of glutamate uptake by GLAST (both 5 and 10 microM MeHg), whereas GLT-1 transporter activity was significantly (p < 0.01) increased following exposure to 5 and 10 microM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.

Animals↗
PubMed2004

Coregulation of glutamate uptake and long-term sensitization in Aplysia.

In Aplysia, long-term facilitation (LTF) at sensorimotor synapses of the pleural-pedal ganglia is mediated by an increase in the release of a neurotransmitter, which appears to be glutamate. Glutamate uptake also is increased in sensory neurons 24 hr after the induction of long-term sensitization (Levenson et al., 2000b). The present study investigated whether the same signaling pathways were involved in the long-term increase in glutamate uptake as in the induction of LTF. Thus, roles for cAMP, PKA (cAMP-dependent protein kinase), MAPK (mitogen-activated protein kinase), and tyrosine kinase in the regulation of glutamate uptake were tested. We found that 5-HT increased cAMP and activated PKA in sensory neurons. Exposure of pleural-pedal ganglia to analogs of cAMP or forskolin increased glutamate uptake 24 hr after treatments. Inhibitors of PKA (KT5720), MAPK (U0126 and PD98059), and tyrosine kinase (genistein) blocked the long-term increase in glutamate uptake produced by 5-HT. In addition, bpV, a tyrosine phosphatase inhibitor, facilitated the ability of subthreshold levels of 5-HT to increase glutamate uptake. Inhibition of PKC, which is not involved in LTF, had no effect on the long-term increase in glutamate uptake produced by 5-HT. Furthermore, activation of PKC by phorbol-12,13-dibutyrate did not produce long-term changes in glutamate uptake. The results demonstrate that the same constellation of second messengers and kinases is involved in the long-term regulation of both glutamate release and glutamate uptake. These similarities in signaling pathways suggest that regulation of glutamate release and uptake during formation of long-term memory are coordinated through coregulation of these two processes.

Animals↗
PubMed2004

Evaluation of glutamate loss from damaged retinal cells of dogs with primary glaucoma.

OBJECTIVE: To determine whether retinal damage in dogs with primary glaucoma (PG) is consistent with ischemia-induced glutamate toxicosis. SAMPLE POPULATION: Retinal tissue sections from 25 dogs with PG and 12 normotensive control dogs. PROCEDURE: Retinal sections from control and glaucomatous dogs were stained for morphometric and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses to determine whether retinal damage was consistent with glutamate toxicosis. Immunohistochemical analysis was performed to detect ischemia-like loss of glutamate from neurons in damaged areas. RESULTS: In severely damaged glaucomatous retinas, all neurosensory layers had focal regions that were thin or disrupted. There was less thinning of the outer nuclear layer (ONL) and inner nuclear layer (INL) in moderately damaged retinas than in severely damaged retinas. Acute signs of damage in the INL included cells with dark, condensed chromatin and lightly stained cytoplasm interspersed with a few TUNEL-positive cells, which was consistent with glutamate toxicosis. Glutamate immunoreactivity was reduced in thin areas and in damaged cells of the INL and ONL, which was consistent with glutamate release in damaged areas. Glutamate immunoreactivity was increased in putative Müller cells in damaged areas, which also was consistent with glutamate release. CONCLUSIONS AND CLINICAL RELEVANCE: Retinal damage in dogs with PG differs in intensity in focal areas. Damage in affected regions resembles damage induced by glutamate. Glutamate is lost from damaged neurons and accumulates in Müller cells, which is consistent with increased glutamate release contributing to the damage. Glutamate antagonists may protect INL cells in dogs with glaucoma.

Analysis of Variance↗
PubMed2000

SNARE protein-dependent glutamate release from astrocytes.

We investigated the cellular mechanisms underlying the Ca(2+)-dependent release of glutamate from cultured astrocytes isolated from rat hippocampus. Using Ca(2+) imaging and electrophysiological techniques, we analyzed the effects of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i.e., a slow inward current (SIC) and/or an increase in the frequency of miniature synaptic currents. We found that the Ca(2+)-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transporters, because the astrocyte-induced glutamate-mediated responses in neurons were affected neither by inhibitors of glutamate transporters (beta-threo-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxylate) nor by replacement of extracellular sodium with lithium. We show that Ca(2+)-dependent glutamate release from astrocytes requires an electrochemical gradient necessary for glutamate uptake in vesicles, because bafilomycin A(1), a vacuolar-type H(+)-ATPase inhibitor, reduced glutamate release from astrocytes. Injection of astrocytes with the light chain of the neurotoxin Botulinum B that selectively cleaves the vesicle-associated SNARE protein synaptobrevin inhibited the astrocyte-induced glutamate response in neurons. Therefore, the Ca(2+)-dependent glutamate release from astrocytes is a SNARE protein-dependent process that requires the presence of functional vesicle-associated proteins, suggesting that astrocytes store glutamate in vesicles and that it is released through an exocytotic pathway.

ATP-Binding Cassette Transporters↗
PubMed1977

Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases.

In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.

Alanine Transaminase↗
PubMed1991

Phylogenetic studies on the synaptic vesicle glutamate transport system.

The ATP-dependent uptake of glutamate into synaptic vesicles isolated form mammalian brains is well characterized. Glutamate uptake requires an electrochemical proton gradient, is specific for glutamate over other amino acids, and is stimulated by chloride. To determine whether these characteristics are fundamental to the vesicular uptake system, vesicles were isolated from the brain and central nervous ganglia of several vertebrate and invertebrate species, which included goldfish, frogs, turtles, pigeons, rats, Drosophila, and crayfish, and these vesicles were assayed for glutamate uptake activity. ATP-dependent glutamate was found in all of the vertebrate species tested, but was not detected in Drosophila or crayfish vesicles. The nature of the vesicular uptake of glutamate was similar among all the vertebrates: the specificity for glutamate remained high, transport was energized by a vacuolar (V)-type ATPase, 2-4 mM chloride stimulated uptake three- to sixfold, and Km for glutamate was between 0.5 and 2 mM. While these major characteristics of the uptake system remained conserved among the vertebrates tested, minor differences were seen in glutamate specificity, the steady-state level of glutamate obtained in the vesicles, and Vmax of the glutamate uptake systems. These results indicate that the synaptic vesicle glutamate uptake system is present throughout the vertebrate class, and that while minor changes in the transport system have occurred, its major functional characteristics, such as stimulation by chloride and strict substrate specificity, have been conserved for over 350-400 million years.

Amino Acid Transport System X-AG↗
PubMed1995

Presynaptic glutamate levels in tonic and phasic motor axons correlate with properties of synaptic release.

Synaptic glutamate release involves the accumulation of cytoplasmic glutamate in synaptic vesicles, whereafter it is released by triggered exocytosis. As glutamatergic terminals are known to be functionally diverse it was of interest to examine whether the presynaptic glutamate supply differs between individual axon terminals with distinct release properties. The glutamatergic terminals in the crustacean neuromuscular system system comprise a "phasic" type which shows fatigue of release during repetitive stimulation, and a "tonic" type which can maintain transmission for long periods. Quantitative immunogold analysis showed that the axons in a tonic nerve innervating slow muscles in the abdomen contained two times higher levels of glutamate labeling over axoplasmic matrix and over mitochondria, as compared to the corresponding elements in a phasic nerve. Similar results were obtained when adjacent phasic and tonic axons in a mixed nerve innervating leg muscles were compared. In the terminal regions of tonic and phasic axons the glutamate labeling differed correspondingly over axoplasmic matrix and mitochondria, while the synaptic vesicles showed a similar strong accumulation of labeling in both types of terminal. The level of labeling for glutamine, a glutamate precursor, was closely similar in phasic and tonic axons. The axoplasmic glutamate concentration was estimated to be in the low millimolar range, through comparison with coprocessed conjugates with known glutamate concentration. These results show that fatigue-resistant tonic axons and terminals contain higher levels of glutamate than fatiguable phasic axons, presumably representing an adaptation to the markedly different impulse activities in the two types of neuron. The axonal glutamate concentrations are in the range of the Km value for vesicular glutamate transport. Thus in tonic axons the high glutamate level appears to promote an efficient refilling of synaptic vesicles during sustained release, while in phasic axons the refilling should be slower which is compatible with an infrequent release.

Animals↗
PubMed1993

Depolarization elicits, while hyperpolarization blocks uptake of endogenous glutamate by retinal horizontal cells of the turtle.

We have employed an immunoreaction against glutamate to qualitatively demonstrate varying levels of glutamate in retinal horizontal cells of the turtle. Glutamate-like immunoreactivity (GLI) in horizontal cells could be demonstrated after glutamate decarboxylase was inhibited by aminooxyacetic acid (AOAA) and its degradation to GABA was blocked. Depolarization of horizontal cells by kainic acid (KA) induces strong glutamate immunoreactivity in these cells, whereas hyperpolarization by 2,3-cis piperidine dicarboxylate (PDA) abolishes glutamate-like immunoreactivity in horizontal cells. When glutamate release from cones and bipolar cells is blocked in the absence of calcium, or when glutamate uptake is blocked by DL-threo beta-hydroxy aspartate, KA/AOAA treatment of the retina does not induce GLI in horizontal cells. Our data show that horizontal cells are capable of taking up glutamate from the endogenous retinal pool in an activity dependent way. Our interpretation of these findings is that retinal horizontal cells are capable of regulating glutamate levels in the extracellular space of the cone pedicle complex by an activity-dependent uptake system. We suggest that inhibition of glutamate uptake upon hyperpolarization rather than inhibition of GABA release may evoke the antagonistic surround response of retinal bipolar cells.

Aminooxyacetic Acid↗
PubMed1988

Calcium-dependent glutamate cytotoxicity in a neuronal cell line.

Membranes from the neuroblastoma x embryonic retina cell hybrid cell line, N18-RE-105, bind L-[3H]glutamate with a pharmacologic profile consistent with a 'quisqualate-type' brain L-glutamate receptor. We describe here the cytotoxic effect of L-glutamate receptor agonists on intact N18-RE-105 cells. Cytotoxicity was quantitated by measurement of the release of the cytosolic enzyme, lactate dehydrogenase, into the culture medium after addition of L-glutamate and its analogs to the cell culture medium. L-Glutamate (10 mM) and its confirmationally restricted analogs, quisqualate (1 mM) and ibotenate (10 mM), caused cell lysis. In contrast, similar analogs which do not bind to N18-RE-105 cell membranes (kainic acid, N-methyl-D,L-aspartic acid and gamma-aminobutyric acid) were not cytotoxic. L-Glutamate-induced cytotoxicity was eliminated when calcium-free medium was used. Addition of inorganic or organic calcium channel antagonists also reduced the cytotoxicity of L-glutamate, even when 1.8 mM calcium was present in the medium. Cadmium chloride (10 microM) completely blocked L-glutamate toxicity, whereas manganese chloride (150 microM) and lanthanum chloride (25 microM) reduced toxicity by greater than 50%. Dihydropyridine voltage-sensitive calcium channel agonists or antagonists, had little or no significant effect on L-glutamate-induced toxicity. In contrast, the verapamil derivatives, D600 and D888, and the diltiazem derivative, MDL 12,330A reduced L-glutamate toxicity by greater than 50%. These results suggest that a subtype of voltage-sensitive calcium channels is involved in the mechanism of L-glutamate receptor mediated cytotoxicity in this cell line.

Calcium↗
PubMed1988

Pharmacology of the glutamate receptor.

Glutamate is a potent candidate of the excitatory transmitter at the invertebrate NMJ and the synapse of the vertebrate CNS. But pharmacological studies have not been enough to prove that glutamate functions as an excitatory neurotransmitter. During the past 10 years, we have been studying the effects of various compounds which demonstrate the glutamate blocking action, but the glutamate responses are more effectively blocked by the drugs than the nerve-evoked synaptic response. A marked difference was revealed by TI-233, the minimum concentration of TI-233 on EJP being about a hundred times greater than the minimum threshold concentration on the glutamate-induced responses. The subsequent studies demonstrated that the action of TI-233 was able to be explained by the open channel block of the glutamate-activated ion-channel. The difference does not confute the hypothesis that glutamate is the natural transmitter substance at the crayfish NMJ, notwithstanding the fact that the action of the transmitter candidate on the postsynaptic membrane must be identical in every respect with that of the transmitter. Once something potentially useful has been found it is necessary to know not only what a substance does but how well it does it, so that comparisons can be made and better drugs discovered. Our first task therefore was to find a powerful glutamate blocker. Recently, as a result of synthesizing a series of compounds on the base of the structure-activity relationship in drug design, a series of compounds was found to reduce markedly glutamate responses at the crayfish NMJ and the mammalian central neurones at extremely low concentrations. In addition, a novel potent excitatory amino acid, acromelic acid, was found. This compound markedly excites the crayfish opener muscle and the mammalian central neurones. Agonists and antagonists have provided a very useful tool for neuroscience research, and findings of these new pharmacological tools will lead to progress in pharmacological studies to elucidate the function of glutamate in the body, in addition to other established compounds. The recent advances in our limited understanding of pharmacology of the glutamate receptor are discussed here.

2-Amino-5-phosphonovalerate↗
PubMed2001

Role of glutamate receptors in the nucleus accumbens on behavioural responses to novel conflictive and non-conflictive environments in the rat.

The possible role of glutamic acid locally applied into the nucleus accumbens on exploratory behaviours measured in 'conflictive' and 'non-conflictive' environments was studied in adult male rats. As a model of conflictive environment, the elevated asymmetric-plus maze (APM) was used. As a model of a non-conflictive environment, a modified holeboard enriched with an object (OVM) was used. In order to characterize the possible glutamic acid receptors involved, the following antagonists were also used: AP3 (antagonist of the metabotropic glutamic acid receptor), AP7 (antagonist of NMDA glutamic acid receptor, and CNQX (antagonists of kainate/AMPA glutamic acid receptor). Results showed that injection of glutamic acid into the nucleus accumbens induced in the APM a decrease of exploration and an increase of the permanency score (non-exploratory behaviours) of the 'High and Low wall' arm. However, in the 'Two High Walls' arm, glutamic acid decreased permanency. In the OVM, no major changes in the motor activity were observed with glutamic acid. Nevertheless, the vertical activity (an index of rearing) and head-dipping were inhibited by the amino-acid treatment. In the APM, the decrease of exploration induced by glutamic acid was blocked by all three receptor antagonists. In the non-exploratory behaviours, the facilitatory effect observed in the 'High and Low walls' arm was blocked only by AP7 and CNQX. The inhibitory action of glutamic acid on the permanency score in the 'Two High Walls' arm was not blocked by the receptors antagonists. In the OVM, AP7 and CNQX were effective in blocking the inhibition of glutamic acid on the vertical activity, but in head-dipping, only AP3 and CNQX were able to block the effect of the amino acid on this behaviour. In conclusion, the present results are compatible with the concept that glutamatergic input fibres to the nucleus accumbens modulate the expression of exploratory behaviour induced by novelty in conflictive and non-conflictive conditions.

Animals↗
PubMedDate not supplied

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells.

Glutamate is taken up avidly by glial cells in the central nervous system. Glutamate uptake may terminate the transmitter action of glutamate released from neurons, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

Ambystoma↗
PubMed1977

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.

Adenosine Triphosphate↗
PubMed1988

The actions of L-glutamate at the postsynaptic membrane of the squid giant synapse.

The actions of L-glutamate on the postsynaptic membrane of the squid giant synapse were investigated using two methods of application: ionophoresis and bath perfusion. Bath perfusion of 10 mmoll-1 sodium glutamate did not produce an appreciable depolarization of the postsynaptic membrane but reversibly blocked the neurally evoked postsynaptic potential (PSP). The postsynaptic membrane depolarized when L-glutamate was applied ionophoretically. The sensitivity to glutamate application was not uniform, but sharply localized to sites which may correspond to synaptic contacts made by branching colaterals from the postsynaptic axon. The relationship between membrane potential and amplitude of the glutamate-activated postsynaptic potential (PSP) examined under current-clamp conditions was linear over the voltage range studied (-110 to -60 mV) with an extrapolated reversal potential of -36 mV. The amplitude of the glutamate-activated PSP was reduced either by replacing Na+ in the external solution with Tris+ (Na+-free) or by raising the extracellular K+ concentration to 20 mmoll-1 and was abolished by removing both Na+ and Ca2+ from the bath solution. The PSP amplitude was insensitive to changes in the extracellular Mg2+ concentration. The extrapolated reversal potential of the glutamate PSP was shifted to more positive potentials in both Na+-free and raised-K+ bathing solutions and was unchanged by anion substitution. The depolarization induced by L-glutamate increased with increasing ionophoretic current and reached a maximum with large pulses. Double logarithmic plots of the coulomb dose-response relationship gave a limiting slope in the range 1.7-2.2, suggesting that two glutamate molecules are required for receptor activation. The time course of desensitization of the glutamate response was studied using a double-pulse method. The initial decrease in the ratio, PSP2/PSP1, is followed by a slower time-dependent recovery of the postsynaptic response with a time constant of 8.5 s. Prolonged perfusion of the squid giant synapse with concanavalin A failed to abolish desensitization of the glutamate-evoked PSP.

Animals↗
PubMed2001

Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death.

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as "scavengers" of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.

Journal Article↗
PubMed1996

The glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures.

Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC50 in these cultures of 320 +/- 157 microM. In astrocyte-poor cultures, an EC50 for PDC of 50 +/- 5 microM was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [3H]-D-aspartate, first we demonstrated that PDC caused significant efflux of [3H]-D-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC50 of 18 +/- 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [3H]-D-aspartate release. PDC-evoked release of [3H]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.

Animals↗