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PubMed1977

The pathway of glutamate metabolism in rat brain mitochondria.

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.

Aminooxyacetic Acid↗
PubMed1996

A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity.

The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.

Animals↗
PubMed2000

Elevated cortical extracellular fluid glutamate in transgenic mice expressing human mutant (G93A) Cu/Zn superoxide dismutase.

Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 microM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2002

Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death.

Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, L-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 microM, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 microM, NMDA receptor) or MCPG (500 microM, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2((5)H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2.

Animals↗
PubMed2004

Neurotensin enhances endogenous extracellular glutamate levels in primary cultures of rat cortical neurons: involvement of neurotensin receptor in NMDA induced excitotoxicity.

Primary cultures of cortical neurons were employed to investigate the modulatory effects of neurotensin on glutamate excitotoxicity and the possible neuroprotective actions of the neurotensin receptor antagonist SR48692. NT(1-13) and its biologically active fragment NT(8-13) at 10 nM (30 min) increased endogenous glutamate levels. The inactive fragment NT(1-7) (10-100 nM; 30 min) was ineffective. SR48692, applied 20 min before NT and maintained in contact with cells during NT exposure as well as a low calcium medium (from the onset of the experiment) prevented the NT(1-13)-induced increase in extracellular glutamate levels. The addition of NMDA (0.01-10 micro M; 10 min) to the medium concentration-dependently increased extracellular glutamate levels. When 0.1 nM NT(1-13) was added in combination with 0.01 micro M NMDA, in concentrations by themselves ineffective, a significant increase in glutamate levels was observed. SR48692 at 100 nM counteracted the increase in glutamate levels induced by 0.1 nM NT(1-13) plus 0.01 micro M NMDA. The inhibitor of the protein kinase C (PKC) calphostin C (0.1 micro M; 10 min before NT) prevented the increase in glutamate levels induced by the combined treatments. The morphological analysis indicated that 10 nM NT(1-13) enhanced the glutamate (10 min)-induced apoptosis. The peptide was added 30 min prior to glutamate and maintained in contact with cells during the glutamate exposure. The presence of 100 nM SR48692 (20 min before NT) antagonized this effect of NT(1-13). These findings support the view of a pathophysiological role for NT in the cerebral cortex. Thus, under pathological conditions NT by enhancing glutamate outflow and by amplifying the NMDA-mediated glutamate signaling may be involved in increasing the degeneration of cortical neurons.

Animals↗
PubMed2006

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana.

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.

Arabidopsis↗
PubMed2003

Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters.

BACKGROUND: During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures. METHODS: Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively. RESULTS: At clinically relevant concentrations (0.05-10 microm), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol. CONCLUSION: Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.

Amino Acid Transport System X-AG↗
PubMed1999

Glutamate-like immunoreactivity during hair cell recovery after gentamicin exposure in the chinchilla vestibular sensory periphery.

OBJECTIVE: Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista ampullaris after gentamicin ototoxicity. STUDY DESIGN: In five groups of three animals each, ototoxicity was produced by placing gentamicin (50 microg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1, 2, 4, 8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed. METHODS: For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immunoreaction was detected using the avidinbiotinylated-complex technique and diaminobenzidine was the chromogen. RESULTS: Normal sensory epithelia demonstrated type I and type II hair cells with moderate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-like immunoreactivity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type II hair cells surviving ototoxic treatment (15%-18%) contained moderate glutamate-like immunoreactivity, supporting cells showed no immunoreactivity, and nerve terminals and fibers displayed strong immunoreactivity. At 4 and 8 weeks after treatment, recovered hair cells (80%) had greater glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level. CONCLUSION: Glutamate may be used as an indicator of hair cell differentiation and as an index of the molecular recovery of hair cells after ototoxicity.

Animals↗
PubMed1998

Modulation by zinc of the glutamate transporters in glial cells and cones isolated from the tiger salamander retina.

1. Zinc may be released from some presynaptic glutamatergic neurons, including hippocampal mossy fibres and retinal photoreceptors. We whole-cell-clamped glial (Müller) cells isolated from the salamander retina to investigate the effect of zinc on glutamate transporters in these cells. Glutamate-evoked currents in these cells are generated largely by carriers homologous to the mammalian GLAST/EAAT1 transporter. 2. Zinc inhibited both glutamate uptake into the cells, and glutamate release by reversal of the uptake process. The IC50 for inhibition of uptake (< 1 microM) was similar to or below the values for zinc modulating NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and GABA receptors, and 100-fold less than the calculated value for the rise in extracellular zinc concentration evoked by depolarization with potassium in area CA3 of the hippocampus. 3. Although zinc altered the apparent affinity of the transporter for glutamate and Na+, it did not act simply by binding competitively to the glutamate-, Na(+)-, K(+)- or H(+)-binding sites on the transporter. Zinc inhibited both forward and reversed glutamate transport from the outside of the cell membrane, but not from the inside. The inhibitory action of zinc on uptake was voltage independent, indicating a zinc-binding site outside the membrane field. 4. As well as inhibiting glutamate transport, zinc potentiated activation of the anion conductance in the Müller cell glutamate transporter. However, zinc reduced the current mediated by the anion conductance in the cone synaptic terminal glutamate transporter (homologous to the mammalian EAAT5), indicating that zinc has different actions on different glutamate transporter subtypes. 5. By acting on glutamate transporters, zinc may have a neuromodulatory role during synaptic transmission and a neuroprotective role during transient ischaemia.

ATP-Binding Cassette Transporters↗
PubMed2004

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells.

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.

Animals↗
PubMed2005

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging.

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.

Animals↗
PubMed1996

Presynaptic modulation by L-glutamate and GABA of sympathetic co-transmission in rat isolated vas deferens.

1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect, whilst kainate (Kain, 1 microM-1 mM) caused an inhibition of the EFS-induced contractions. Effects of Kain could be abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX, 10 microM). NMDA, Quis and Kain had no effect on the exogenous ATP- or noradrenaline-induced contractions. 7. It is concluded that the excitatory amino acid L-glutamate modulates the electrically evoked vas deferens contraction through conversion to the inhibitory amino acid GABA by a specific GABA transaminase. The GABA formed may then act on GABAB receptors and cause inhibition of the contraction through a presynaptic mechanism.

Animals↗
PubMed2003

Increased tonic activation of presynaptic metabotropic glutamate receptors in the rat supraoptic nucleus following chronic dehydration.

Chronic dehydration induces structural changes in the hypothalamic supraoptic nucleus (SON), including increased glutamate synapses and retraction of astroglial processes. We performed whole-cell recordings in acute hypothalamic slices to determine whether these changes increase tonic activation of presynaptic metabotropic glutamate receptors (mGluRs) by increasing ambient glutamate in the SON. Activation of presynaptic group III mGluRs caused a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in SON neurones that was significantly attenuated in slices from dehydrated rats (-27.8 %) compared with untreated rats (-41.7 %), suggesting a higher basal occupancy of mGluRs by ambient glutamate during dehydration. Blocking group III mGluRs caused an increase in the frequency of mEPSCs that was significantly higher in slices from dehydrated rats (+42.8 %) than untreated rats (+31.4 %), suggesting greater tonic activation of presynaptic mGluRs by ambient glutamate during dehydration. Increasing ambient glutamate levels by inhibiting astrocyte glutamate uptake resulted in a decrease in mEPSC frequency due to increased activation of presynaptic mGluRs. This was attenuated in slices from dehydrated rats (-35.4 %) compared with slices from untreated rats (-48.8 %), suggesting diminished astrocytic glutamate uptake during dehydration. Immunochemical analyses revealed a robust expression of the GLT-1 transporter protein in the SON, which was diminished in SON punches from dehydrated rats compared with untreated controls. Thus, dehydration leads to increased tonic activation of presynaptic mGluRs on glutamate terminals, consistent with a decrease in glutamate buffering capacity. The resulting reduction in glutamate release probability may compensate for the increase in glutamate release sites that occurs during dehydration.

Animals↗
PubMed2006

The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia.

Maintaining a low extracellular glutamate concentration in the central nervous system is important for terminating synaptic transmission and preventing excitotoxic cell death. The stoichiometry of the most abundant glutamate transporter, GLT-1, predicts that a very low glutamate concentration, approximately 2 nM, should be reached in the absence of glutamate release, yet microdialysis measurements give a value of approximately 1 microM. If other glutamate transporters had a different stoichiometry, the predicted minimum glutamate concentration could be higher, for example if those transporters were driven by the cotransport of 2 Na+ (rather than of 3 Na+ as for GLT-1). Here we investigated the ionic stoichiometry of the glutamate transporter GLAST, which is the major glutamate transporter expressed in the retina and cerebellum, is expressed in other adult brain areas at a lower level than GLT-1, and is present throughout the brain early in development when expression of GLT-1 is low. Glutamate transport by GLAST was found to be driven, as for GLT-1, by the cotransport of 3 Na+ and 1 H+ and the counter-transport of 1 K+, suggesting that the minimum extracellular glutamate concentration should be similar during development and in the adult brain. A less powerful accumulation of glutamate by GLAST than by GLT-1 cannot be used to explain the high glutamate concentration measured by microdialysis.

Amino Acid Transport System X-AG↗
PubMed2004

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione.

In cultured rat cerebellar granule cells, glutamate or N-methyl-d-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca(2+) levels ([Ca(2+)](i)), reactive oxygen species (ROS) generation, and cell death (respective EC(50) values for glutamate were 12, 30, and 38 microM) but no increase in caspase-3 activity. Removal of extracellular Ca(2+) blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca(2+)](i) increase. This indicates that glutamate-induced cell death is attributable to [Ca(2+)](i) increase and ROS generation, and the [Ca(2+)](i) increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca(2+)](i) increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca(2+)](i) increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca(2+)](i) increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca(2+)](i) increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity.

Animals↗
PubMed2001

Regulatory responses to an oral D-glutamate load: formation of D-pyrrolidone carboxylic acid in humans.

Previously published studies have shown D-glutamate to be the most potent natural inhibitor of glutathione synthesis known, yet how D-glutamate is handled in humans is unknown. Therefore, we administered an oral D-glutamate load to four healthy volunteers and monitored the plasma D-glutamate concentration and excretion over a 3-h postload period. Compared with time controls, the plasma D-glutamate concentration increased 10-fold in the 1st h and then reached a plateau over the remaining time course. In contrast, plasma D-pyrrolidone carboxylic acid increased progressively throughout the 3-h time course to a level 10-fold higher than the D-glutamate plasma concentration. Excretion of D-glutamate progressively increased despite a constant filtered D-glutamate load rising from only 5 to 95% of the filtered amount. Excretion of D-pyrrolidone carboxylic acid increased with the rise in filtered load without significant reabsorption. The amount of D-pyrrolidone carboxylic acid excreted over the 3-h time course was 10 times the amount excreted as D-glutamate and accounted for almost 20% of the administered D-glutamate. These findings indicate that plasma D-glutamate concentration is tightly regulated through two mechanisms: 1) the transport into cells and metabolic conversion to D-pyrrolidone carboxylic acid and excretion, and 2) the enhancement of D-glutamate clearance by the kidneys.

Absorption↗
PubMed1998

Postsynaptic response kinetics are controlled by a glutamate transporter at cone photoreceptors.

We evaluated the role of the sodium/glutamate transporter at the synaptic terminals of cone photoreceptors in controlling postsynaptic response kinetics. The strategy was to measure the changes in horizontal cell response rate induced by blocking transporter uptake in cones with dihydrokainate (DHK). DHK was chosen as the uptake blocker because, as we show through autoradiographic uptake measurements, DHK specifically blocked uptake in cones without affecting uptake in Mueller cells. Horizontal cells depolarized from about -70 to -20 mV as the exogenous glutamate concentration was increased from approximately 1 to 40 microM, so horizontal cells can serve as "glutamate electrodes" during the light response. DHK slowed the rate of hyperpolarization of the horizontal cells in a dose-dependent way, but didn't affect the kinetics of the cone responses. At 300 microM DHK, the rate of the horizontal cell hyperpolarization was slowed to only 17 +/- 8.5% (mean +/- SD) of control. Translating this to changes in glutamate concentration using the slice dose response curve as calibration in Fig. 2, DHK reduced the rate of removal of glutamate from approximately 0.12 to 0.031 microM/s. The voltage dependence of uptake rate in the transporter alone was capable of modulating glutamate concentration: we blocked vesicular released glutamate with bathed 20 mM Mg2+ and then added 30 microM glutamate to the bath to reestablish a physiological glutamate concentration level at the synapse and thereby depolarize the horizontal cells. Under these conditions, a light flash elicited a 17-mV hyperpolarization in the horizontal cells. When we substituted kainate, which is not transported, for glutamate, horizontal cells were depolarized but light did not elicit any response, indicating that the transporter alone was responsible for the removal of glutamate under these conditions. This suggests that the transporter was both voltage dependent and robust enough to modulate glutamate concentration. The transporter must be at least as effective as diffusion in removing glutamate from the synapse because there is only a very small light response once the transporter is blocked. The transporter, via its voltage dependence on cone membrane potential, appears to contribute significantly to the control of postsynaptic response kinetics.

ATP-Binding Cassette Transporters↗
PubMed2001

Characteristics of glutamate-evoked temporomandibular joint afferent activity in the rat.

Injection of glutamate into the rat temporomandibular joint (TMJ) capsule can reflexly induce a prolonged increase in the electromyographic (EMG) activity of the jaw muscles, however, the characteristics of TMJ afferents activated by glutamate have not been investigated. In the present study, we examined the effect of glutamate injection into the TMJ capsule on jaw muscle EMG activity and the extracellularly recorded activity of single trigeminal afferents that had receptive fields in the TMJ tissue and antidromically identified projections to the brain stem subnucleus caudalis (Vc) in rats of both sexes. Glutamate (0.05--1.0 M, 10 microl) injection into the TMJ capsule evoked EMG activity in a dose-related manner; however, at concentrations of 0.5 and 1.0 M, glutamate-evoked digastric muscle responses were greater in female than in male rats. In experiments where jaw muscle EMG and afferent activity were recorded simultaneously, glutamate (0.5 M, 10 microl) injection into the TMJ capsule evoked activity in the jaw muscles as well as in 27 (26 A delta and 1 C-fiber afferent) of 34 trigeminal afferents that could be activated by blunt mechanical stimulation of the TMJ tissue. In these experiments, glutamate-evoked jaw muscle activity was significantly increased for 6 min after the glutamate injection, whereas afferent activity was significantly increased only during the first minute after the glutamate injection. The glutamate-evoked afferent activity was inversely related to conduction velocity and, in afferents with conduction velocities <10 m/s, was significantly greater in female (n = 6) than in male (n = 10) rats. These results suggest that glutamate excites putative nociceptive afferents within the TMJ to a greater degree in female than in male rats. This sex-related difference in afferent discharge may, in part, underlie sex-related differences in glutamate-evoked jaw muscle EMG activity.

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