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PubMed1999

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes).

Ionotropic glutamate receptor agonists, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release. We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.

4-Aminopyridine↗
PubMed1996

Phosphocreatine-dependent glutamate uptake by synaptic vesicles. A comparison with atp-dependent glutamate uptake.

ATP-dependent uptake of glutamate into synaptic vesicles has been well documented. Stimulation of glutamate uptake into synaptic vesicles by other high-energy phosphates has not been described. In this paper, we examine the stimulation of phosphocreatine (PCr)-induced glutamate uptake and determine whether this stimulation is secondary to conversion of PCr to ATP. We found the following. 1) PCr stimulates glutamate uptake into synaptic vesicles in the absence of added ATP. 2) At a glutamate concentration of 50 microM, no concentration of added ATP could produce the degree of stimulation seen in the presence of PCr. 3) 0.5 mM iodoacetamide completely inhibits synaptic vesicle creatine kinase activity but does not inhibit PCr-stimulated glutamate uptake. 4) PCr-dependent glutamate uptake, unlike ATP-dependent uptake, is not magnesium- or chloride-dependent. 5) 0.5 mM N-ethylmaleimide, a selective H+-ATPase inhibitor, completely inhibits ATP-dependent glutamate uptake but only slightly inhibits PCr-dependent glutamate uptake. 6) PCr-dependent glutamate uptake is sensitive to valinomycin, a K+/H+ translocator, whereas the ATP-dependent uptake is not. Therefore, it appears that in addition to the well-known ATP-dependent glutamate uptake system, there is a previously unreported PCr-dependent glutamate uptake system in synaptic vesicles. The total glutamate uptake by synaptic vesicles is likely the sum of both ATP- and PCr-dependent glutamate uptake.

Adenosine Triphosphate↗
PubMed1976

Effect of growth conditions on glutamate transport in the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

The effects of growth conditions on the glutamate transport activity of intact cells and membrane vesicles and on the levels of glutamate-binding protein in wild-type Escherichia coli K-12 CS101 and in two glutamate-utilizing mutants, CS7 and CS2TC, were studied. Growth of CS101 on aspartate as the sole source of carbon or nitrogen resulted in a severalfold increase in glutamate transport activity of intact cells and membrane preparations to levels characteristic of the operator-constitutive mutant CS7. The high glutamate transport activity of mutant CS7 was not depressed further by growth on aspartate. Synthesis of glutamate-binding protein was not enhanced by aspartate in either strain. Mutant CS2TC produces a heat-labile repressor of glutamate permease synthesis and is therefore able to grow on glutamate at 42 C but not at 30 C. CS2TC cells grown in a glycerol-minimal medium at the restrictive temperature (30 C) exhibit low glutamate transport activity. Growth on aspartate at 30 C results in derepressed synthesis of glutamate permease. Cells grown on glycerol at 42 C have high glutamate transport activity. No further derepression is obtained upon growth on aspartate. Growth of CS101 and CS7 in "rich broth" greatly reduces the levels of glutamate-binding protein but does not appreciably affect glutamate transport by whole cells or membrane preparations. The identity of the carrier and the role of the binding protein in glutamate transport are discussed in the light of these findings.

Aspartic Acid↗
PubMed1998

Anion currents and predicted glutamate flux through a neuronal glutamate transporter.

Kinetic properties of a native, neuronal glutamate transporter were studied by using rapid applications of glutamate to outside-out patches excised from Purkinje neurons. Pulses of glutamate activated anion currents associated with the transporter that were weakly antagonized by the transporter antagonist kainate. In addition, kainate blocked a resting anion conductance observed in the absence of glutamate. Transporter currents in response to glutamate concentration jumps under a variety of conditions were used to construct a cyclic kinetic model of the transporter. The model simulates both the anion conductance and the glutamate flux through the transporter, thereby permitting several predictions regarding the dynamics of glutamate transport at the synapse. For example, the concentration-dependent binding rate of glutamate to the transporter is high, similar to binding rates suggested for ligand-gated glutamate receptors. At saturating glutamate concentrations, transporters cycle at a steady-state rate of 13/sec. Transporters are predicted to have a high efficiency; once bound, a glutamate molecule is more likely to be transported than to unbind. Physiological concentrations of internal sodium and glutamate significantly slow net transport. Finally, a fixed proportion of anion and glutamate flux is expected over a wide range of circumstances, providing theoretical support for using net charge flux to estimate the amount and time course of glutamate transport.

ATP-Binding Cassette Transporters↗
PubMed1992

Differential in vivo and in vitro effect of gentamicin on glutamate synthesis and glutamate deamination in rabbit kidney-cortex tubules and mitochondria.

The effect of gentamicin on both glutamate synthesis and glutamate deamination was studied in kidney-cortex mitochondria and tubules isolated from both control and gentamicin-treated animals. In kidney-cortex mitochondria which were permeabilized in order to make a free access of substrates and antibiotic to the glutamate dehydrogenase, gentamicin appeared to be a very potent inhibitor of glutamate synthesis, resulting in about 60% decrease of the enzyme activity at 5 mM concentration. Other aminoglycoside antibiotics decreased the enzymatic activity, in the following order: gentamicin > neomycin = tobramycin = kanamycin > biodacyna > amikacin > streptomycin. This, in principle, corresponds to their known nephrotoxic potential observed in vivo. The inhibitory action of antibiotics was abolished by neither ADP nor leucine, allosteric activators of glutamate dehydrogenase. Surprisingly, gentamicin did not decrease the rate of ammonia formation from glutamate when added to both renal tubules and mitochondria isolated from control rabbits. This indicates that the antibiotic exerts its inhibitory effect on glutamate dehydrogenase activity in the direction of glutamate synthesis only. In contrast, the rate of both glutamate deamination and glutamate synthesis was about 40% lower in renal tubules and mitochondria isolated from kidney-cortex of animals which were given antibiotics for 10 days. In view of these results it seems that (i) the depression of ammoniagenesis in gentamicin-treated animals may be due to a decrease of glutamate dehydrogenase content and (ii) under conditions in vitro the aminoglycoside inhibits the enzyme activity in the direction of glutamate synthesis while it does not affect the glutamate deamination.

Adenosine Diphosphate↗
PubMed1983

Effect of carbohydrate on plasma and erythrocyte glutamate levels in humans ingesting large doses of monosodium L-glutamate in water.

In previous studies, plasma glutamate concentration was lower when equivalent doses of monosodium L-glutamate (MSG) were given with a ready-to-feed liquid formula meal (Sustagen; 0.4 g protein, 1.1 g carbohydrate, 0.06 g fat, 6.6 kcal energy/kg body weight) rather than in water. This difference was suggested to reflect a carbohydrate effect on mucosal cell glutamate metabolism. To test this hypothesis, a large dose of monosodium L-glutamate (150 mg/kg body weight) dissolved in water, with or without added carbohydrate, was administered to eight healthy adult subjects. Carbohydrate was administered at 1.1 g/kg body weight in the form of partially hydrolyzed corn starch (Polycose). In the absence of carbohydrate, the mean (+/- SD) peak plasma glutamate concentration was 59.4 +/- 46.5 mumol/dl, and the incremental area under the plasma glutamate concentration time curve was 3391 +/- 2360 mumol/(dl x min). The addition of carbohydrate to the glutamate solution significantly decreased (p = 0.001) both the mean peak plasma glutamate concentration (7.18 +/- 3.48 mumol/dl) and the incremental area under the plasma glutamate concentration-time-curve (451 +/- 20.8 mumol/(dl x min). Erythrocyte glutamate and aspartate concentrations were not affected by glutamate loading in either test. Delayed gastric emptying did not account for the carbohydrate effect. Carbohydrate is postulated to serve as a pyruvate source for mucosal cells, facilitating the transamination of glutamate and its subsequent metabolism. This process would reduce the release of glutamate to the peripheral circulation.

Adult↗
PubMed1978

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.

Amino Acids↗
PubMed2003

Inhibition of non-vesicular glutamate release by group III metabotropic glutamate receptors in the nucleus accumbens.

Previous in vitro studies have shown that group III metabotropic glutamate receptors (mGluRs) regulate synaptic glutamate release. The present study used microdialysis to characterize this regulation in vivo in rat nucleus accumbens. Reverse dialysis of the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) decreased, whereas the antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP) increased the extracellular level of glutamate. The decrease by L-AP4 or the increase by MSOP was antagonized by co-administration of MSOP or L-AP4, respectively. Activation of mGluR4a by (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid or mGluR6 by 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid had no effect on extracellular glutamate. (R,S)-4-Phosphonophenylglycine (PPG), another group III agonist with high affinity for mGluR4/6/8, reduced extracellular glutamate only at high concentrations capable of binding to mGluR7. The increase in extracellular glutamate by MSOP was tetrodotoxin-independent, and resistant to both the L-type and N-type Ca2+ channel blockers. L-AP4 failed to block 30 mm K+-induced vesicular glutamate release. Blockade of glutamate uptake by d,l-threo-beta-benzyloxyaspartate caused a Ca2+-independent elevation in extracellular glutamate that was reversed by L-AP4. Finally, (S)-4-carboxyphenylglycine, an inhibitor of cystine-glutamate antiporters, attenuated the L-AP4-induced reduction in extracellular glutamate. Together, these data indicate that group III mGluRs regulate in vivo extracellular glutamate in the nucleus accumbens by inhibiting non-vesicular glutamate release.

Aminobutyrates↗
PubMed1996

Effects of dexmedetomidine on hypoxia-evoked glutamate release and glutamate receptor activity in hippocampal slices.

BACKGROUND: The selective alpha(2) agonist dexmedetomidine may improve neurologic outcome after incomplete ischemia in animals when it is administered either before or after the start of the ischemic insult. To clarify further the mechanisms by which alpha(2) agonists may provide neuroprotective effects, the authors tested the hypotheses that dexmedetomidine decreases synaptic and extrasynaptic glutamate release stimulated by potassium chloride or hypoxia, and decreases postsynaptic glutamate receptor activity during aerobic or hypoxic conditions. METHODS: Glutamate released from brain slices (300-microns thick) from rat hippocampus was measured in a cuvette during two experimental stresses: (1) potassium chloride-evoked depolarization (30 mM) with and without 10 nM, 100 nM, or 1,000 nM dexmedetomidine; and (2) hypoxia (95% N2 - 5% CO2) with and without 100 nM dexmedetomidine. Glutamate release was quantified by fluorescence assay using 1 mM nicotinamide adenine dinucleotide, and 5 international units per ml glutamate dehydrogenase. The formation of nicotinamide dinucleotide reduced from nicotinamide adenine dinucleotide by glutamate dehydrogenase was measured fluorometrically (excitation light 340 nm, emission intensity 460 nm) in the solution above the slice. Glutamate receptor activity was determined by the change in cytosolic calcium concentration in CA1 neurons in the presence and absence of 100 nM dexmedetomidine during administration of N-methyl-D-aspartate (100 microM) and during simulated ischemic penumbra conditions (PO2 = 20 mmHg, glutamate 3 mM). Calcium concentration was measured using a microscope fluorometer in fura 2-loaded rat hippocampal brain slices. RESULTS: Dexmedetomidine attenuated potassium chloride-evoked glutamate release by 37%, 51%, (P = 0.03) and 27%, respectively, for the 10 nM, 100 nM, and 1,000 nM concentrations, and decreased (at 100 nM) the increase in glutamate release in response to hypoxia by 61% (P < 0.0001). Dexmedetomidine (100 nM) had no effect on N-methyl-D-aspartate or hypoxia plus 3 mM L-glutamate-mediated calcium changes. CONCLUSIONS: The selective alpha(2)-adrenergic agonist dexmedetomidine decreases evoked glutamate release from hippocampal rat brain slices during depolarization or hypoxic stress, but does not alter calcium changes mediated by the stimulation of glutamate receptors during aerobic or hypoxic conditions.

Adrenergic alpha-Agonists↗
PubMed1981

Glutamate as a putative transmitter in the cerebellum: stimulation by GABA of glutamic acid release from specific pools.

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.

Animals↗
PubMed1991

Glutamate dehydrogenase reaction as a source of glutamic acid in synaptosomes.

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.

Amino Acids↗
PubMed2005

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons.

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.

Amino Acid Transport System X-AG↗
PubMed2005

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2.

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Adenosine Diphosphate↗
PubMed2003

Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

Alanine Transaminase↗
PubMed2007

Nonvesicular release of glutamate by glial xCT transporters suppresses glutamate receptor clustering in vivo.

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named "genderblind" (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses.

Amino Acid Transport System y+↗
PubMed2000

Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.

PURPOSE: Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters (molecules that ordinarily regulate extracellular glutamate; E:xcitatory A:mino A:cid T:ransporter; EAAT). Elevated glutamate levels can also lead to alterations in glutamate receptor expression. It was hypothesized that selective blockade of glutamate transporters would be toxic to retinal ganglion cells. METHODS: Glutamate transporters were blocked either pharmacologically or with subtype-specific antisense oligonucleotides against EAAT1. Glutamate levels, transporter levels and ganglion cell survival were assayed. RESULTS: Pharmacological inhibition of glutamate transporters with either an EAAT2 specific inhibitor or a nonspecific inhibitor of all the subtypes of transporters was toxic to ganglion cells. Treatment with oligonucleotides against the glutamate transporter EAAT1 decreased the levels of expression of the transporter, increased vitreal glutamate, and was toxic to ganglion cells. CONCLUSIONS: These results demonstrate that normal function of EAAT1 and EAAT2 is necessary for retinal ganglion cell survival and plays an important role in retinal excitotoxicity. Manipulation of retinal glutamate transporter expression may become a useful tool in understanding retinal neuronal loss.

ATP-Binding Cassette Transporters↗
PubMed2006

Role of glutamate transporters in the clearance and release of glutamate during ischemia and its relation to neuronal death.

Glutamate neurotransmitter action on postsynaptic receptors is terminated by its clearance from the synaptic cleft by transporter proteins located in neurons and glial cells. Failure of glutamate removal can lead to neuronal death due to its well-known neurotoxic properties. Glutamate transporters are dependent on external Na+, and thus on the activity of Na+/K+ ATPases, which maintain the Na+ concentration gradient. When the energy brain requirements are not fulfilled by the appropriate blood supply of glucose and oxygen, the Na+ gradient collapses leading to impaired glutamate and aspartate removal, or even to the release of these amino acids through the reverse operation of their transporters. Such a scenario would be associated with brain ischemia and hypoglycemia due to the prompt decline in ATP levels. In addition, some evidence suggests that downregulation of glutamate transporters after the ischemic period, or the dysfunction induced by oxidation, contributes to the accumulation of extracellular glutamate and neuronal death. Neuronal damage is associated with excitotoxicity, a type of cell death triggered by the overactivation of glutamate receptors and the loss of calcium homeostasis. Throughout this review we will discuss recent evidence suggesting that failure of glutamate transport during ischemia contributes to the elevation of extracellular glutamate and to the induction of excitotoxicity. We will also discuss the contribution of glial vs. neuronal glutamate transporters in ischemic damage, and the involvement of the different glutamate transporter subtypes. We will focus on experimental data from rodent models, because many of the studies on glutamate transport and ischemic damage have been performed in these animal species.

Animals↗
PubMed2005

Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters.

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent glutamate concentrations from reaching neurotoxic levels. Glutamate transporters play an important role in ischemic neuronal death during stroke and have been implicated in epilepsy and amytropic lateral sclerosis. However, the molecular structure and the glutamate-uptake mechanism of these transporters are not well understood. The most recent models of glutamate transporters have three or five subunits, each with eight transmembrane domains, and one or two membrane-inserted loops. Here, using fluorescence resonance energy transfer (FRET) analysis, we have determined the relative position of the extracellular regions of these domains. Our results are consistent with a trimeric glutamate transporter with a large (>45 A) extracellular vestibule. In contrast to other transport proteins, our FRET measurements indicate that there are no large-scale motions in glutamate transporters and that glutamate uptake is accompanied by relatively small motions around the glutamate-binding sites. The large extracellular vestibule and the small-scale conformational changes could contribute to the fast kinetics predicted for glutamate transporters. Furthermore, we show that, despite the multimeric nature of glutamate transporters, the subunits function independently.

Amino Acid Transport System X-AG↗