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At least 109 records · Page 6Linked to original sources
PubMed1994

Evidence that glutamate is released from capsaicin-sensitive primary afferent fibers in rats: study with on-line continuous monitoring of glutamate.

The aim of this study is to elucidate whether the excitatory amino acid glutamate is released from capsaicin-sensitive primary afferent fibers, and to compare the releasing effect of capsaicin on glutamate with that on substance P. The release of glutamate was measured using a fluorometric on-line continuous monitoring system, in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, 2-min application of capsaicin produced an increased outflow of glutamate, as well as an increase in the release of immunoreactive substance P from dorsal horn slices of the rat. The release of glutamate was concentration-dependently increased by capsaicin at concentrations in the range of 0.1-3 microM, and the release evoked by 10 microM capsaicin was not higher than that evoked by 3 microM. On the other hand, capsaicin at concentrations of 1-10 microM produced a concentration-dependent increase in the release of immunoreactive substance P, without effect at 0.1 microM. The amount of glutamate release evoked by 3 microM capsaicin was about 42.8 pmol.mg-1 protein, and 290 times that of immunoreactive substance P. The release of glutamate by 3 microM capsaicin was suppressed by the depletion of calcium from the superfusate. Capsaicin at 3 microM failed to increase the release of glutamate from the dorsal horn slices of the rats made an L4-L6 dorsal rhizotomy. These results suggest that capsaicin evoked the release of glutamate from primary afferent fibers in the dorsal horn and that glutamate may play an important role in pain transmission between primary afferent fibers and dorsal horn neurons.

Animals↗
PubMed2005

Altered glutamate and GABA release within thalamocortical circuitry in metabotropic glutamate receptor 4 knockout mice.

The metabotropic glutamate receptor 4 is highly expressed presynaptically on thalamocortical neurons that are involved in the pathogenesis of generalized absence seizures. Mutant mice devoid of metabotropic glutamate receptor 4 are completely resistant to absence seizures induced by low doses of GABA type A receptor antagonists. The purpose of this study was to test the hypothesis that there is altered glutamate and GABA release within thalamocortical circuitry in mice devoid of metabotropic glutamate receptor 4. Extracellular GABA and glutamate release were determined in ventrobasal thalamus, the nucleus reticularis thalami and laminae I-III, and IV-VI of cerebral cortex (laminae I-III of cerebral cortex, and laminae IV-VI of cerebral cortex) using in vivo microdialysis techniques on awake, free moving mice. A significant increase of both basal and K(+)-evoked glutamate release was detected in the ventrobasal thalamus, the nucleus reticularis thalami and laminae IV-VI of cerebral cortex of mice devoid of metabotropic glutamate receptor 4 mice. There also was a significant increase in both basal and K(+)-evoked GABA release in the mice devoid of metabotropic glutamate receptor 4, but a significant decrease of GABA release in laminae IV-VI of cerebral cortex. However, there was no alteration of either GABA or glutamate release in laminae I-III of cerebral cortex, cortical laminae that are not involved in absence seizures. These data indicate that deletion of the metabotropic glutamate receptor 4 gene results in a selective perturbation of glutamate and GABA release within the thalamocortical circuitry involved in the pathogenesis of absence seizures.

Animals↗
PubMed2003

Group I metabotropic glutamate receptors modulate glutamate and gamma-aminobutyric acid release in the periaqueductal grey of rats.

In this study, we investigated the effects of group I metabotropic glutamate (mglu) receptor ligands on glutamate and gamma-aminobutyric acid (GABA) extracellular concentrations at the periaqueductal grey level by using in vivo microdialysis. An agonist of group I mglu receptors, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG, 1 and 2 mM], as well as a selective agonist of mglu(5) receptors, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 2 and 4 mM), both increased dialysate glutamate and GABA concentrations. 7-(Hydroxyimino)cyclopropa-[b]-chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 1 mM), a selective mglu(1) receptor antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP, 0.5 mM), a selective mglu(5) receptor antagonist, perfused in combination with DHPG, antagonized the effect induced by DHPG on the extracellular glutamate and GABA concentrations. MPEP (0.5 mM), perfused in combination with CHPG, antagonized the increased glutamate and GABA extracellular levels induced by CHPG. MPEP (1 mM) decreased the extracellular concentrations of glutamate but did not modify the dialysate GABA concentrations. Moreover, as the intra-periaqueductal grey perfusion of (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, 100 microM], a selective N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, did not change the extracellular concentrations of glutamate, this suggests that the MPEP-induced decrease in glutamate is not a consequence of NMDA receptor blockade. These data show that group I mglu receptors in the periaqueductal grey may modulate the release of glutamate and GABA in awake, freely moving rats. In particular, mglu(5), but not mglu(1), receptors seem to be functionally active on glutamate terminals.

Animals↗
PubMed2003

Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at controlling intracellular levels of glutamate and ultimately the releasable pool of glutamate in neurons.

Amino Acid Transport System X-AG↗
PubMed1993

Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a 'caged' L-glutamate.

1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged' L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8. The rate constant was proportional to [H+] and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1980

Salmonella typhimurium mutants with altered glutamate dehydrogenase and glutamate synthase activities.

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH(4))(2)SO(4) as the nitrogen source (Asm(-)) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.

Chromosome Mapping↗
PubMed2001

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance.

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.

Aminooxyacetic Acid↗
PubMed1988

A re-examination of the interaction of N-acetyl-L-aspartyl-L-glutamate with a subpopulation of rat brain membrane L-[3H]glutamate binding sites.

The neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG) has been reported to act at a subpopulation of putative quisqualate receptors on the basis of its competitive inhibition of specific binding of L-[3H]glutamate and on the basis of quisqualate-sensitive binding of [3H]NAAG radiolabeled on the glutamate moiety. Recently, a membrane-bound metallopeptidase, N-acetylated alpha-linked acidic dipeptidase (NAALADase), which cleaves NAAG to N-acetyl-aspartate (NAA) and glutamate, has been characterized and has been shown to exhibit optimal activity under incubation conditions used to measure NAAG binding sites. Accordingly, we have examined whether NAALADase mediated release of glutamate from NAAG might account for the receptor binding results. Insertion of empirically derived kinetic constants for NAALADase hydrolysis of NAAG into a theoretical model for peptide-derived glutamate inhibition of [3H]glutamate binding reveals that NAAG can appear to displace, with high affinity, a subpopulation of [3H]glutamate labeled sites, and yet have little or no intrinsic activity for these sites. Furthermore, empirical data relating time and protein concentration to NAAG displacement of [3H]glutamate binding are more consistent with a proteolytic mechanism rather than an equilibrium interaction of the peptide with membrane receptors. Coupled with recent findings attributing the Cl- -dependent glutamate binding to a sequestration phenomenon, these results demonstrate that the inferred action of NAAG at glutamate synaptic receptors through previous radioligand binding studies is probably incorrect. Furthermore, these studies offer a general caution regarding the conclusions about subpopulations of receptors drawn from receptor binding assays conducted with ligands, which may be structurally modified by enzymatic processes.

Animals↗
PubMed2002

Feeding-induced decrease in extracellular glutamate level in the rat nucleus accumbens: dependence on glutamate uptake.

In vivo microdialysis combined with high-performance liquid chromatography and electrochemical detection was used to monitor extracellular glutamate levels in the medial nucleus accumbens of Sprague-Dawley rats during their feeding behaviour. Consumption of a palatable new diet or a diet to which rats were previously exposed caused a decrease in extracellular level of glutamate in the nucleus accumbens during and after feeding. The presentation of an inedible object (a piece of rubber) instead of the expected food caused a marked increase in extracellular glutamate levels. In contrast, if the piece of rubber was presented to rats that did not expect food delivery, the extracellular level of glutamate remained unchanged during the rubber presentation. The feeding-induced decrease in the extracellular glutamate level did not depend on food deprivation and was completely prevented by intraaccumbal infusions through the dialysis probe of 10 mM D,L-threo-beta-hydroxyaspartate (a glutamate uptake inhibitor). Intraaccumbal infusions of 10 microM S-(-)-raclopride L-tartrate (a D2/D3 dopamine receptor antagonist) or 1 microM tetrodotoxin (a voltage-dependent Na(+) channel blocker) also completely reversed the decrease in extracellular glutamate level in response to food intake. The D1/D5 dopamine receptor antagonist SCH-23390 (10 microM) administered into the nucleus accumbens had no significant effect on the feeding-induced decrease in extracellular glutamate level. From the data obtained we suggest that the decrease in the extracellular level of glutamate in the medial nucleus accumbens in response to feeding appears to arise from a temporal increase in glutamate uptake that is probably operated by dopamine inputs to the nucleus accumbens via D2/D3 receptors. Our findings also suggest that the dissociation between the expected biological value of a presented object and the reality might be an important determinant for regulation of glutamate release in this brain area during feeding behaviour.

Analysis of Variance↗
PubMed1996

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

ATP-Binding Cassette Transporters↗
PubMed1995

Glutamate 404 is involved in the substrate discrimination of GLT-1, a (Na+ + K+)-coupled glutamate transporter from rat brain.

Sodium-coupled glutamate transporters, located in the plasma membrane of nerve terminals and glial processes, serve to keep its extracellular glutamate concentration below extracellular levels. Moreover, they help in conjunction with diffusion to terminate the transmitter's action in synaptic transmission. We have investigated the role of negatively charged amino acid residues of GLT-1, a cloned (Na+ + K+)-coupled glutamate transporter from rat brain. Using site-directed mutagenesis we modified these negative residues, which are located in hydrophobic surroundings and are highly conserved within the glutamate transporter family. Out of five residues meeting these criteria, three, aspartate 398, glutamate 404, and aspartate 470, are critical for heterologously expressed glutamate transport. This defective transport cannot be attributed to the mere requirement of a negative charge at these positions. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to that of wild type. No cryptic activity was revealed by reconstitution experiments aimed to monitor the activity of transporter molecules not located in the plasma membrane. Significantly, whereas all of the mutants at the glutamate 404 position exhibit impaired transport of glutamate, they possess considerable transport of D- and L-aspartate, up to 80% of wild type values. Binding of glutamate is not impaired in these mutants. Our observations indicate that the glutamate 404 residue may be located in the vicinity of the glutamate-aspartate permeation pathway.

ATP-Binding Cassette Transporters↗
PubMed2006

Microglial glutamate uptake is coupled to glutathione synthesis and glutamate release.

The physiological function of microglial glutamate uptake has been debated as it is about 10% of that measured for astrocytes. This study addresses how glutamate, taken up from the extracellular space, is utilized by microglia. It was found that purified rat microglia incubated for 60 min with (3)H-glutamate had an increased intracellular accumulation of (3)H-glutamate after 12 h incubation with tumour necrosis factor alpha (TNF-alpha) but not after incubation with lipopolysaccharide (LPS). Furthermore, LPS- but not TNF-alpha-treated cells showed an increased efflux of (3)H-labelled compounds, presumably glutamate through the X(C) (-) system and treatment with LPS or TNF-alpha increased the microglial glutathione concentrations and led to an increased incorporation of (3)H-glutamate into glutathione. Depending on the stimuli, 3-6% of the total labelled contents were found in the form of glutathione and 25-35% in the form of glutamate. These results show that microglial glutamate uptake is directly coupled to glutathione synthesis and release of glutamate and/or glutamate metabolites. Additionally, the increased glutathione contents after LPS or TNF-alpha treatment were able to reduce microglial cell death after H(2)O(2) challenge, showing a potential (self)-protective function for microglial glutamate transporter expression and glutathione synthesis.

Animals↗
PubMed2004

Adenosine A 2A receptor antagonists prevent the increase in striatal glutamate levels induced by glutamate uptake inhibitors.

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Activation of adenosine A(2A) receptors increases extracellular glutamate levels, while A(2A) receptor antagonists reduce stimulated glutamate outflow. Whether a modulation of the glutamate uptake system is involved in the effects elicited by A(2A) receptor blockers has never been investigated. This study examined the ability of adenosine A(2A) receptor antagonists to prevent the increase in glutamate levels induced by blockade of the glutamate uptake. In rats implanted with a microdialysis probe in the dorsal striatum, perfusion with 4 mm l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), or 10 mm dihydrokainic acid (DHK, a non-transportable competitive inhibitor that mainly blocks the glial glutamate transporter GLT-1), significantly increased extracellular glutamate levels. The effects of PDC and DHK were completely prevented by the adenosine A(2A) receptor antagonists SCH 58261 (0.01 mg/kg i.p.) and/or ZM 241385 (5 nm via probe). Since an impairment in glutamate transporter function is thought to play a major role in neurodegenerative disorders, the regulation of glutamate uptake may be one of the mechanisms of the neuroprotective effects of A(2A) receptor antagonists.

Adenosine A2 Receptor Antagonists↗
PubMed1998

Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake.

Glutamate transport across the plasma membrane of neurons and glia is powered by the transmembrane electrochemical gradients for sodium, potassium, and pH, but there is controversy over the number of Na+ cotransported with glutamate. The stoichiometry of glutamate transporters is important because it determines a lower limit to the extracellular glutamate concentration, [glu]o, in both normal and pathological conditions. We used whole-cell clamping to study the stoichiometry of the glial transporter GLT-1, the most abundant glutamate transporter in the brain, expressed under control of the Tet-On system in a Chinese hamster ovary (CHO) cell line selected for low endogenous glutamate transport. After the induction of GLT-1 expression with doxycycline, glutamate evoked a Na+-dependent inward current with the voltage dependence and pharmacology of GLT-1 and acidified the cell cytoplasm. Raising [K+]o around cells clamped with electrodes containing sodium and glutamate evoked an outward reversed uptake current. These responses were reduced by the specific GLT-1 blocker dihydrokainate (DHK). DHK evoked an outward current with NO3-, but not with Cl-, as the main intracellular anion, suggesting that the anion conductance of the transporter is active even without external glutamate but generates little current in the absence of highly permeable anions like NO3-. Measuring the reversal potential of the transporter current in various ionic conditions suggested that the transport of one glutamate anion is coupled to the cotransport of three Na+ and one H+ and to the countertransport of one K+. This suggests that in ischemia, when [K+]o rises to 60 mM, the reversal of glutamate transporters will raise [glu]o to >50 microM.

ATP-Binding Cassette Transporters↗
PubMed1994

L-carnitine increases the affinity of glutamate for quisqualate receptors and prevents glutamate neurotoxicity.

We have shown that acute ammonia toxicity is mediated by activation of the NMDA type of glutamate receptors. Although it is well known that L-carnitine prevents acute ammonia toxicity, the underlying molecular mechanism is not clear. We suspected that L-carnitine would prevent ammonia toxicity by preventing the toxic effects of glutamate. We have tested this hypothesis using primary cultures of neurons. L-carnitine prevented glutamate neurotoxicity in a dose-dependent manner similar to that required to prevent ammonia toxicity in animals. It is also shown that L-carnitine increases selectively the affinity of glutamate for the quisqualate type of glutamate receptors, while the affinity for the kainate and NMDA receptors is slightly decreased. L-carnitine prevents the increase in cytoplasmic Ca2+ induced by addition of glutamate. The Ca2+ levels rose 4.8-fold following addition of 1 mM glutamate, however, when the neurons were incubated previously with 5 mM L-carnitine, the Ca2+ levels increased only by 50%. Also, AP-3, an antagonist of the metabotropic receptor prevents the protective effect of L-carnitine against glutamate neurotoxicity. We suggest, therefore, that the protective effect of L-carnitine against glutamate toxicity is due to the increased affinity of glutamate for the metabotropic receptor. This mechanism could also explain the protection by L-carnitine against acute ammonia toxicity.

Ammonia↗
PubMed1990

Low affinity binding to glutamate receptor sites correlates with depolarizing responses induced by glutamate and quisqualate in striatal synaptoneurosomes.

In the present study, binding affinity of glutamate and quisqualate to striatal synaptoneurosome membranes in the guinea-pig was compared with concentration-dependence of depolarizing responses induced by these agents. The displacement of radioactive glutamate from receptor binding site by unlabelled glutamate and quisqualate revealed a nonhomogeneous population of binding sites. A high affinity component of binding was observed with an inhibition constant of 0.04 microM for glutamate and 0.45 microM for quisqualate, as well as a low affinity component with an inhibition constant of 10 microM for glutamate and 87 microM for quisqualate. Changes of the membrane potential in striatal synaptoneurosomes induced by glutamate and quisqualate were detected by measuring the absorbance of a potential sensitive cyanine dye. Glutamate and quisqualate induced constantly a depolarization in synaptoneurosome particles. Concentration-response curves showed that half-maximal depolarization was obtained with 10 microM glutamate and 100 microM quisqualate. The comparison of the displacement data with the changes in the membrane potential in the present investigation indicate that in vitro glutamate and quisqualate depolarize striatal synaptoneurosome particles through low affinity binding to receptor site for glutamate.

Animals↗
PubMed2003

The roles of different types of glutamate receptors involved in the mediation of nucleus submedius (Sm) glutamate-evoked antinociception in the rat.

Based on our previous findings that glutamate microinjected into the thalamic nucleus submedius (Sm) inhibits dose-dependently the rat tail-flick (TF) reflex, this study investigated which glutamate receptor subtype is involved in mediating this effect. The effects of an NMDA (N-methyl-D-aspartate), non-NMDA or metabotropic glutamate receptor (mGluR) antagonist microinjected into Sm on the TF reflex were examined in untreated or in Sm glutamate treated (microinjection into the Sm) rats. The TF latencies were measured in each of these groups of rats every 5 min. Injection of DNQX [6,7-dinitroquinoxaline-2,3(1H,4H)-dione], a non-NMDA receptor antagonist, or (+/-)-MCPG [(+/-)-alpha-methyl-4-carboxyphenylglycine], a mGluR antagonist, into the Sm blocked the inhibitory effects induced by a subsequent microinjection of glutamate into the same Sm site. The TF latency increased only by 6.6+/-1.6 or 9.0+/-1.1%, respectively, of the baseline value, which was markedly less than that (51.3+/-8.4 or 50.7+/-5.3%) obtained from injection of glutamate only (P<0.001, n=8). However, pre-microinjection of MK-801 [(+)-5-methyl-10,11-dibenzo[a,d]cyclohepten-5,10-imine], an NMDA receptor antagonist, into the Sm had no effect on the Sm glutamate-evoked inhibition of the TF reflex. The TF latency change (40.0+/-11.1%) was not significantly different (P>0.05, n=8) compared with that obtained from glutamate injection alone. These observations suggest that non-NMDA and metabotropic glutamate receptors, but not NMDA receptors, are involved in mediating Sm glutamate-evoked antinociception.

Animals↗
PubMed1998

Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate.

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.

Amino Acid Isomerases↗