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PubMed1981

Correlation between glutamate deamination and glutamine deamidation in rat kidney mitochondria.

Intramitochondrial glutamate removal through deamination may regulate renal ammoniagenesis from glutamine. However, little information is available to determine if there is a strong association between glutamine deamidation and the removal within mitochondria of the glutamate subsequently formed after deamidation. Using rat renal mitochondria, we found that ammonia production, glutamate appearance, and amide nitrogen disappearance were near equal aerobically and anerobically, when no ADP-generating system was present. When an ADP-generating system was added (ATP alone, ATP+ malonate, or ATP+2,4 DNP), more ammonia was formed aerobically from glutamine. Additionally, less glutamate appeared even though more amide nitrogen disappeared. Intramitochondrial concentrations of glutamate decreased. When pyruvate and alpha-ketoglutarate were added to the system, ammoniagenesis, deamidation, and deamination decreased; while glutamate built up in both the medium and mitochondria. In our mitochondrial system, we found a significantly positive correlation between glutamate deamination and glutamine deamidation, between glutamate accumulation and intramitochondrial glutamate concentrations; and a significantly negative correlation between glutamate deamination and glutamate accumulation, between glutamine deamidation and intramitochondrial glutamate concentrations, and between glutamate deamination and glutamate accumulation. We conclude that there is a biochemical relationship between glutamine deamidation and deamination of the glutamate subsequently formed. We propose that increased deamination lowers mitochondrial concentrations of glutamate and increases deamidation. In contrast, slowing deamination increases mitochondrial concentrations of glutamate and decreases deamidation.

Adenosine Diphosphate↗
PubMed1989

Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death.

The mechanism of delayed neurotoxicity, triggered by glutamate, was studied in 7-8-day-old primary cultures of rat cerebellar granule cells. Treatment of cultures for 15 min with 50 microM glutamate in Mg2+ -free medium, followed by removal of the excitoxin, resulted in neuronal death, which started to appear 2-3 hr after the termination of glutamate treatment. The number of dead neurons increased gradually in the next few hours and 80-85% of neurons were found dead 24 hr later. Antagonists of N-methyl-D-aspartate-sensitive glutamate receptors (phencyclidine) or 1.2 mM MgCl2, but not the antagonist of N-methyl-D-asparatate-insensitive glutamate receptors (6-cyano-7-nitroquinoxaline-2,3-dione), abolished the neurotoxic effect of kainate. Development of glutamate-induced neuronal death depends strongly on Ca2+. Removal of extracellular Ca2+ (with 1mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) immediately after the termination of glutamate exposure and before the appearance of the early signs of neuronal death (post-glutamate period) dramatically reduced neuronal degeneration. Neurotoxic concentrations of glutamate induced sustained increase of 45Ca2+ uptake in the post-glutamate period. The delayed increase of 45Ca2+ uptake, as well as the delayed neurotoxicity, were not affected by post-glutamate treatment with phencyclidine, dibenzocyclohepteneimine; DL-2-amino-5-phosphonovalerate, or MgCl2 or with voltage-dependent Ca2+ channel blockers (nitrendipine, verapamil, diltiazem). Neurotoxic concentrations of glutamate also induced a delayed sustained increase of [3H]phorbol-12,13-dibutyrate binding, reflecting an increased translocation of protein kinase C (PKC) from cytosol to the cell membrane during the post-glutamate period. Pretreatment of neurons with the ganglioside GT1b (trisialosylgangliotetraglycosylceramide), followed by removal of free GT1b from the incubation medium, prevented PKC translocation, the sustained increase of 45Ca2+ uptake in the post-glutamate period, and the delayed neuronal death. We suggest that the sustained activation and translocation of PKC primed by glutamate receptor stimulation may be the triggering event causing the protracted increase of neuronal Ca2+ influx. This influx is insensitive to voltage-dependent Ca2+ channel blockers and glutamate receptor antagonists. It appears that this delayed increase of Ca2+ influx may be important in causing neuronal death.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1975

Placental transfer of glutamate and its metabolites in the primate.

When radioactive glutamate was infused into pregnant rhesus monkeys, 69 to 88 per cent of radioactivity in the maternal plasma remained in association with glutamate while 10 to 22 per cent was converted to glucose. In the fetal plasma, glucose and lactate accounted for more than 80 per cent of radioactivity, with less than 2 per cent of the label found in glutamate. Maternal glutamate infusions resulting in a ten- to twenty-fold increase in maternal plasma glutamate levels (60 to 100 mumoles per 100 ml.) had no effect upon fetal glutamate levels. Infusions producing maternal glutamate levels 70 times normal (280 mumoles per 100 ml.) did result in some transfer of glutamate to the fetal circulation. Labeled glutamate administered to the fetus at 1.5 to 2.4 Gm. per kilogram of fetal weight did not result in glutamate transfer to the maternal circulation. Infusion of glutamate to the fetus at 5 Gm. per kilogram of fetal weight increased fetal plasma glutamate levels to 2, 000 mumoles per 100 ml. and resulted in some transfer of glutamate to maternal circulation. Glutamate metabolites (lactate and glucose) were readily transferred across the placenta in either direction. These studies indicate that the primate placenta is virtually impermeable to glutamate unless extreme elevations of plasma glutamate are induced.

Amino Acids↗
PubMed1994

Glutamate-induced swelling of single astroglial cells in primary culture.

Glutamate induced an increase in cell volume within one minute and evoked cytosolic Ca2+ transients in type 1 astroglial cells in primary culture obtained from the cerebral cortex of newborn rat. Even the metabotropic glutamate receptor agonists (1S,3R)-1-aminocyclopentane- 1,3-dicarboxylic acid (1S-3R-ACPD) and L(+)-2-amino-4 phosphonobutyric acid (L-AP4) induced a cell swelling with ACPD inducing a parallel Ca2+ transient while L-AP4 did not. A new method was used where rapid changes in relative cell volume could be followed at the single cell level. Relative volume changes in cultured single astroglial cells were examined by microspectrofluorimetry after loading the cells with the highly fluorescent intracellular probe fura-2/AM. At its isosbestic point, 358 nm, fura-2 is ion-insensitive and the fluorescent signals emitted are related only to the intracellular dye concentration. By varying the excitation wavelengths, changes in intracellular Ca2+ transients could be recorded simultaneously with the relative volume variations of the individual cells. Thus, as rapid changes in cell volume were followed, the results from this method could be of physiological significance. Glutamate-induced cell swelling was blocked by BaCl2 and by tetraethylammonium, suggesting that K+ channels are operative in glutamate-induced cell swelling. Furthermore, the glutamate-induced swelling was blocked by the Na+; K+, and 2Cl- co-transport inhibitor furosemide. The glutamate-induced swelling was partially blocked by pertussis toxin and partially blocked also by the glutamate carrier-blocker dihydroaspartate. When the ionotropic glutamate receptor alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid was blocked with the antagonist 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo(F)quinoxaline, glutamate still induced a swelling, suggesting that this receptor was not directly involved in the glutamate-induced volume increase. Even in situations of blocked or partially blocked swelling, intracellular Ca2+ transients could be obtained. Furthermore, the glutamate-induced swelling was evoked even in low extracellular Ca2+ concentrations. Our data suggest that glutamate-induced rapid swelling is a complex process at the molecular level. One hypothetical mechanism might be that glutamate interacts with metabotropic glutamate receptors and induces a release of Ca2+ from internal stores. Furthermore glutamate interacts with K+ channels, and probably at least one co-transporter and the sodium-dependent high-affinity uptake glutamate carrier, resulting in cell swelling.

Aminobutyrates↗
PubMed1991

Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes.

L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. The affinity batch chromatography procedure described previously [Chen et al. (1988) J. Biol. Chem. 263, 417-427] was used to obtain a fraction enriched in glutamate-binding proteins. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. [14C]Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of [14C]methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. The increase in methylamine influx was dependent on the concentration of L-glutamic acid with an estimated Kact for L-glutamate equal to 0.2 microM for synaptic membrane proteins and 0.32 microM for purified proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. Glutamate-activated methylamine flux was completely inhibited by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of Na+ led to a transient increase in the influx of the lipid-permeable anion probe S14CN-. Electrophoretic analysis of partially purified proteins reconstituted in liposomes indicated enrichment of several bands, the most prominent being those of molecular size equal to approximately 69, 60, 35, and 25 kDa. Antibodies raised against the purified 71- and 63-kDa glutamate-binding proteins reacted strongly with the approximately 69-kDa band of reconstituted proteins and markedly decreased the initial rate of glutamate-activated cation flux. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the approximately 69-kDa protein in the function of these ion channels.

Animals↗
PubMed1998

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Azospirillum brasilense↗
PubMed1996

Glutamate-mediated release of Ca2+ in mitral cells of the olfactory bulb.

1. Effects of glutamate on intracellular Ca2+ concentration ([Ca2+]i) were investigated in cultured olfactory bulb neurons of Xenopus laevis tadpoles. We imaged [Ca2+]i in these cells with the use of a confocal laser scanning microscope and the calcium indicator dyes Fluo3 and FuraRed. 2. In the standard bath solution, application of glutamate through a pipette resulted in an increase of [Ca2+]i in both mitral/ tufted (M/T) cells and interneurons. The increase occurred in all compartments of the cells, although in a nonhomogenous way. 3. In an ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-buffered bath solution (Ca(2+)-free Ringer solution), glutamate reproducibly induced an increase of [Ca2+]i in M/T cells but not in interneurons. This increase in [Ca2+]i had the following properties: 1) it was delayed with respect to the combined ionotropic and metabotropic response to glutamate, 2) in some cases it stopped before the end of the glutamate application, and 3) it was not affected by D,L-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione added to the bath. It was interpreted as a release of Ca2+ from intracellular calcium stores. 4. Of 47 M/T cells that showed a glutamate-mediated release of [Ca2+]i, 46 also showed Ca2+ influx through ionotropic glutamate receptors, so that intracellular release of [Ca2+]i appeared to be associated with the presence of glutamate-gated ion channels. However, of 110 M/T cells showing an ionotropic response to glutamate, only 46 showed a glutamate-mediated release of [Ca2+]i. 5. The glutamate-mediated release of [Ca2+]i in the dendrites was higher than that in the soma. No indications for calcium waves were found. 6. Quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) mimicked the effect of glutamate on the release of [Ca2+]i, with potencies quisqualate > glutamate > > ACPD. N-acetyl-aspartyl-glutamate did not induce release of [Ca2+]i. The release was partly blocked by (+)-alpha-methyl-4-carboxyphenylglycine. 7. In conclusion, some but not all M/T cells of the olfactory bulb of X. laevis show a glutamate-mediated and predominantly dendritic increase of [Ca2+]i that is associated with glutamategated channels. The pharmacological properties of the corresponding metabotropic glutamate receptor (mGluR) resemble those of the mGluR1/5 receptors in rat.

Animals↗
PubMed1994

Na(+)-dependent glutamate transporter in human retinal pigment epithelial cells.

PURPOSE: To characterize glutamate uptake in the human retinal pigment epithelial (HRPE) cell line 165 and to determine the feasibility of using this cell line as an experimental model for glutamate transport studies. METHODS: Confluent monolayers of the HRPE cells cultured in 35-mm petri dishes were used to study glutamate uptake with regard to its ion dependence, substrate specificity, and kinetics. The radioactivity associated with the cells was measured by a liquid scintillation counter. RESULTS: Glutamate uptake was noticeably stimulated by the presence of Na+ in uptake buffer. Acidic amino acids (100 microM; L-glutamate, L-aspartate, D-aspartate, and L-cysteate) and glutamate transporter inhibitors (100 microM; dihydrokainic acid, DL-threo-beta- hydroxyaspartic acid, and L-trans-pyrrolidine-2,4-dicarboxlic acid) interacted with uptake of radiolabeled glutamate. The activity of glutamate uptake depends on Na+ concentration. Glutamate uptake in the presence of Na+ does not have anion dependence. The uptake of glutamate was enhanced by external acidic environment and inhibited by 0.5 mM DIDS: Glutamate receptor antagonists (100 microM; [+/-]-2-amino-4-phosphonobutyric acid and 6-cyano-7- nitroquinoxaline-2,3-dione) did not inhibit glutamate uptake. Kinetic analysis shows that this transporter consists of at least two saturable systems. CONCLUSIONS: The results of the present study demonstrate that a glutamate transporter is expressed in the HRPE cells. Its characteristics are similar to those of glutamate transporters observed in the RPE of laboratory animals. The human cell line 165 will be a useful tool for characterization of glutamate transport in the RPE. This study also provides clear evidence for the presence of a glutamate transporter in the human RPE.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress.

Salmonella typhimurium accumulates glutamate in response to osmotic stress. Cells in aerobic exponential growth have an intracellular pool of approximately 125 nmol of glutamate mg of protein-1. When cells were grown in minimal medium with 500 mM NaCl, KCl, or sucrose, 290 to 430 nmol of glutamate was found to accumulate. Values were lower when cells were harvested in stationary phase. Cells were grown in conventional medium, harvested, washed, resuspended in the control medium or in medium with osmolytes, and aerated for 1 h. With aeration, glutamate was found to accumulate at levels comparable to those observed in exponential cultures. Antibiotics inhibiting protein synthesis did not affect glutamate accumulation when cells were aerated. Strains with mutations in glutamate synthase (glt) or in glutamate dehydrogenase (gdh) accumulated nearly normal levels of glutamate under these conditions. A double (gdh glt) mutant accumulated much less glutamate (63.9 nmol mg of protein-1), but a 1.9-fold excess accumulated when cells were aerated with osmotic stress. Methionine sulfone, an inhibitor of glutamate synthase, did not prevent accumulation of glutamate in cells aerated with osmotic stress. Glutamate dehydrogenase is thought to have minimum activity when ammonium is limiting. Resuspending cells with limiting ammonium reduced glutamate production but did not eliminate accumulation of excess glutamate when cells were osmotically stressed. Amino oxyacetic acid, an inhibitor of transamination reactions, did not prevent accumulation of excess glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1981

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

Genes↗
PubMed2002

Glutamate transporters and retinal excitotoxicity.

Glutamate appears to play a major role in several degenerative retinal disorders. However, exogenous glutamate is only weakly toxic to the retina when glutamate transporters on Müller glial cells are operational. In an ex vivo rat retinal preparation, we previously found that exogenous glutamate causes Müller cell swelling but does not trigger excitotoxic neurodegeneration unless very high concentrations that overwhelm the capacity of glutamate transporters are administered. To determine the role of glutamate transporters in Müller cell swelling and glutamate-mediated retinal degeneration, we examined the effects of DL-threo-beta-benzyloxyaspartate (TBOA), an agent that blocks glutamate transport but that unlike most available transport inhibitors is neither a substrate for transport nor a glutamate receptor agonist. We found that TBOA triggered severe retinal neurodegeneration attenuated by ionotropic glutamate receptor antagonists. TBOA-induced neuronal damage was also diminished by riluzole, an agent that inhibits endogenous glutamate release. In the presence of riluzole, to inhibit glutamate release plus TBOA to block glutamate uptake, the addition of low concentrations of exogenous glutamate triggered severe excitotoxic neuronal damage without inducing Müller cell swelling. We conclude that TBOA-sensitive glutamate transporters play an important role in regulating the neurodegenerative effects of glutamate in the rat retina.

Amino Acid Transport System X-AG↗
PubMed2001

Glutamate-loading stimulates metabolic flux and improves cell recovery following chemical hypoxia in isolated cardiomyocytes.

The amino acid glutamate is used in cardioplegic solutions, yet evidence is conflicting as to whether or not exogenous glutamate is indeed cardioprotective. This controversy may be because increasing extracellular glutamate does not necessarily lead to an increase in intracellular glutamate. In this study we aimed to determine whether isolation of myocytes in the presence of glutamate resulted in glutamate-loading of the cells, and, if so, whether such loading protected myocytes from simulated (chemical) hypoxia. Single ventricular myocytes were isolated from rat hearts in the presence and absence of 6.4 mM glutamate. Levels of glutamate and ATP were determined using HPLC, and NADH/NAD(+) was determined from cell autofluorescence. Chemical hypoxia was induced by superfusion with a solution containing 2.5 mM cyanide and no glucose. Intracellular [Ca(2+)] was measured by loading cells with indo-1, and cell length was measured using an edge-tracking device. Isolation of myocytes in the presence of glutamate resulted in increased intracellular glutamate levels compared with cells isolated in the absence of glutamate, 1324+/-108 v 948+/-124 pmol/mg protein, respectively (P<0.05). Cells loaded with glutamate showed increased NADH/NAD(+), (0.384+/-0.032 v 0.281+/-0.029, P<0.05) and greater ATP levels (36.031+/-1.633 nmol/mg protein v 19.279+/-3.327 nmol/mg protein, P<0.005) compared to control cells. When subjected to chemical hypoxia, cells underwent rigor-contracture at various timepoints, and were then reperfused following 5 min in rigor. Cells loaded with glutamate showed better recovery of diastolic [Ca(2+)], Ca(2+) transient amplitude, and improved contractile function compared with cells isolated in absence of glutamate. This study demonstrates an efficient method for loading myocytes with glutamate during cell isolation, and myocytes loaded with glutamate showed increased metabolic flux, as indexed by a higher NADH/NAD(+) and ATP content. Myocytes also exhibited better recovery from chemical hypoxia in terms of both Ca(2+) handling and cell contraction.

Adenosine Triphosphate↗
PubMed2000

Modulation of glutamate transporters (GLAST, GLT-1 and EAAC1) in the rat cerebellum following portocaval anastomosis.

Glutamate transporters have the important function of removing glutamate released from synapses and keeping extracellular glutamate concentrations below excitotoxic levels. Extracellular glutamate increases in portocaval anastomosis (PCA), so we used a portacaval anastomosis model in rats to analyze the expression of glutamate transporters (GLAST, GLT-1 and EAAC1) in rat cerebellum, 1 and 6 months after PCA, using immunohistochemical methods. In controls, EAAC1 immunoreactivity in Purkinje cells and glial GLAST and GLT-1 immunoreactivities in the molecular layer (ML) increased from young to old rats. One month after PCA, Purkinje cell bodies were not immunostained for neuronal EAAC1 glutamate transporter, whereas glial glutamate transporter expressions (GLAST and GLT-1) were decreased when compared to young controls. In rats with long-term PCA (6 months post-PCA), neuronal and glial glutamate transporter expressions were increased. The expression of the neuronal glutamate transporter EAAC1 was less intense than old controls, whereas glial glutamate transporters (GLAST and GLT-1) increased more than their controls. Since the level of the neuronal glutamate transporter (EAAC1) in long-term PCA did not reach that of the controls, GLAST and GLT-1 glutamate transporters seemed to be required to ensure the glutamate uptake in this type of encephalopathy. EAAC1 immunoreactivity also was expressed by Bergmann glial processes in long-term PCA, but this increase did not suffice to reverse the alterations caused at the early stage. The present findings provide evidence that transitory alteration of glutamate transporter expressions could be a significant factor in the accumulation of excess glutamate in the extracellular space in PCA, which probably makes Purkinje cells more vulnerable to glutamate effect.

ATP-Binding Cassette Transporters↗
PubMed2003

Glutamate-dependent glutamine, aspartate and serine release from rat cortical glial cell cultures.

Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate-glutamine cycle. In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial cultures. The glial glutamate transporters rapidly cleared exogenously applied glutamate and this was accompanied by rapid increases in aspartate and glutamine, and a delayed increase in serine levels in the glial-conditioned medium. While glutamate-induced increases in glutamine and serine were sustained for up to 24 h, increases in aspartate lasted only for up to 6 h. The glutamate-induced increases in aspartate and glutamine were dependent both on the concentration and the duration of glutamate stimulus, but were largely insensitive to the inhibition of non-N-methyl-D-aspartate receptors or the metabotropic glutamate receptor 5. Inhibition of the glutamate transporter function by L-trans-pyrrolidine 2,4-dicarboxylate decreased the rate of glutamate uptake but not completely abrogated the uptake process, and this resulted in the attenuation of rate of glutamate induced glutamine synthesis. Dexamethasone treatment increased serine and glutamine levels in conditioned medium and increased glutamate induced glutamine release suggesting an upregulation of glutamine synthase activity. These results further substantiate coupling between glutamate and glutamine, and shed light on glutamate-dependent release of serine and aspartate, which may further contribute to excitatory neurotransmission.

Analysis of Variance↗
PubMed2002

Glutamate-induced sensitization of rat masseter muscle fibers.

In rats, intradermal or intraarticular injection of glutamate or selective excitatory amino acid receptor agonists acting at peripheral excitatory amino acid receptors can decrease the intensity of mechanical stimulation required to evoke nocifensive behaviors, an indication of hyperalgesia. Since excitatory amino acid receptors have been found on the terminal ends of cutaneous primary afferent fibers, it has been suggested that increased tissue glutamate levels may have a direct sensitizing effect on primary afferent fibers, in particular skin nociceptors. However, less is known about the effects of glutamate on deep tissue afferent fibers. In the present study, a series of experiments were undertaken to investigate the effect of intramuscular injection of glutamate on the excitability and mechanical threshold of masseter muscle afferent fibers in anesthetized rats of both sexes. Injection of 1.0 M, but not 0.1 M glutamate evoked masseter muscle afferent activity that was significantly greater than that evoked by isotonic saline. The mechanical threshold of masseter muscle afferent fibers, which was assessed with a Von Frey hair, was reduced by approximately 50% for a period of 30 min after injection of 1.0 M glutamate, but was unaffected by injections of 0.1 M glutamate or isotonic saline. Injection of 25% dextrose, which has the same osmotic strength as 1.0 M glutamate, did not evoke significant activity in or decrease the mechanical threshold of masseter muscle afferent fibers. Magnetic resonance imaging experiments confirmed that injection of 25% dextrose and 1.0 M glutamate produced similar edema volumes in the masseter muscle tissue. Co-injection of 0.1 M kynurenate, an excitatory amino acid receptor antagonist, and 1.0 M glutamate attenuated glutamate-evoked afferent activity and prevented glutamate-induced mechanical sensitization. When male and female rats were compared, no difference in the baseline mechanical threshold or in the magnitude of glutamate-induced mechanical sensitization of masseter muscle afferent fibers was observed; however, the afferent fiber activity evoked by injection of 1.0 M glutamate into the masseter muscle was greater in female rats. The results of the present experiments show that intramuscular injection of 1.0 M glutamate excites and sensitizes rat masseter muscle afferent fibers through activation of peripheral excitatory amino acid receptors and that glutamate-evoked afferent fiber activity, but not sensitization, is greater in female than male rats.

Action Potentials↗
PubMed1997

Effects of increased extracellular glutamate levels on the local field potential in the brain of anaesthetized rats.

1. It is generally considered that glutamate-mediated transmission can be altered from a physiological to neurotoxic action when extracellular glutamate levels become excessive subsequent to impaired uptake and/or excessive release. However, high extracellular glutamate does not consistently correlate with neuronal dysfunction and death in vivo. The purpose of this study was to examine in situ the local depolarizations, as indicated by negative shifts of the extracellular field (d.c.) potential, produced by local inhibition of high-affinity glutamate uptake, with or without co-application of exogenous glutamate, in three brain regions of anaesthetized rats. 2. Microdialysis probes incorporating an electrode were used to apply exogenous glutamate and/or its uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), and to monitor the resulting changes in extracellular glutamate and d.c. potential at the sites of application within the cortex, striatum and hippocampus. 3. Perfusion of 1 to 10 mM L-trans-PDC markedly and concentration-dependently increased extracellular glutamate levels (by up to 1700% of basal level in the parietal cortex). Despite their large magnitude, glutamate changes were associated with minor negative shifts of the d.c. potential (< 2 mV), which were not suppressed by the N-methyl-D-aspartate (NMDA)-channel blocker, dizocilpine (MK-801, 2 mg kg-1, i.v.), or the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/ kainate-receptor antagonist, 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX, 30 mg kg-1, i.p.). L-trans-PDC had virtually identical concentration-dependent effects on dialysate glutamate in the hippocampus and striatum, but those induced in the cortex were around 40% larger (P < 0.002). In contrast, the associated depolarizations were around twice as large in the striatum and cortex as in the hippocampus (P < 0.002). Finally, co-application of L-trans-PDC did not enhance the d.c. potential changes evoked by perfusion of 5 or 20 mM glutamate. 4. As the neurotoxic potency of glutamate agonists is considered to be linked to excessive opening of glutamate-operated ion channels, these results challenge the notion that high extracellular glutamate levels may be the key to excitotoxicity in neurological disorders. In particular, they do not support the hypothesis that high extracellular glutamate causes the sudden negative shifts of the d.c. potential associated with ischaemia (i.e. anoxic depolarization), traumatic brain injury and spreading depression. Impaired uptake and excessive release of glutamate may well lead to excitotoxicity, but only at the synaptic level, not by spreading through the interstitial fluid.

Animals↗
PubMed1996

Transport and metabolism of L-glutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes.

The intracellular glutamate concentration of oxygenated, isolated adult rat heart cells incubated with 0.15 mM glutamate amounts to 2.89 +/- 0.6 mM. Under these conditions the velocity of glutamate transport was 24.3 +/- 1.6 pmol.min-1.mg protein-1 and occurs via a high-affinity carrier characterized by an apparent affinity (K(m)) value of 0.18 +/- 0.03 mM. At high glutamate concentrations ( > 1mM) this high-affinity transport system is superimposed by additional uptake processes of a low affinity but a high capacity for glutamate. The 1.6-fold increased uptake of glutamate observed during 30 min of anoxic incubation of cardiomyocytes does not prevent an intracellular decrease in this amino acid to a concentration of 0.49 mM. After 15 min reoxygenation of cardiomyocytes the intracellular glutamate content increases to the control values of oxygenated cells. Only 2.4% of the glutamate increase after reoxygenation is due to the transport o glutamate from the incubation medium. The competitive inhibitor of transaminases, aminooxyacetate, prevents both the observed intracellular decrease in glutamate during anoxia and the increase in intracellular glutamate after reoxygenation of cardiomyocytes. Half of the amino groups needed for the synthesis of glutamate originate from intracellular alanine, which increases during anoxia and is metabolized during reoxygenation of cardiomyocytes. The velocity of the glutamate uptake of cardiomyocytes incubated in a medium containing 10 mM L-glutamate amounted to 728 +/- 140 pmol.min-1.mg protein-1. During anoxic incubation of cardiomyocytes at this high extracellular glutamate concentration, the intracellular glutamate breakdown may be compensated by a simultaneous uptake of this amino acid via the transport processes characterized by a high capacity

Amino Acids↗
PubMed1984

Effect of pH on glutamate efflux from rat kidney mitochondria.

To provide further insight into the relationship between glutamate transport and metabolism in rat kidney mitochondria, the effect of pH on [U-14C]-glutamate efflux from [U-14C]glutamate-loaded mitochondria was tested. Glutamate efflux, measured in rotenone-inhibited energized mitochondria at 28 degrees C, was first order with respect to matrix glutamate. Glutamate efflux was tested at different medium pH values ranging from 6.4 to 7.9. Efflux was fastest at pH 7.9 with a rate constant of 0.988 min-1 and decreased progressively at lower pH values such that the rate constant at pH 6.4 was 0.359 min-1. A highly significant correlation between pH and the rate constant of glutamate efflux was observed. When medium pH was held constant at 7.0, matrix pH (range pH 7.2-8.4) did not affect the rate of glutamate efflux. The reduction in glutamate efflux at low pH provides an explanation for the increased concentration of glutamate observed in the mitochondrial matrix space of isolated rat kidney mitochondria incubated at acid medium pH. The elevated glutamate concentration in turn contributes to the accelerated oxidative deamination of glutamate by glutamate dehydrogenase at acid pH. Moreover, these findings suggest that the reduction in renal cortex glutamate levels observed in acute acidosis in vivo may be explained by a decreased rate of glutamate transport from mitochondria to cytosol coupled with an augmented rate of glutamate oxidation in the matrix.

Animals↗