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PubMed2006

Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-D-aspartate (NMDA) receptor activation.

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO(-)). Recent studies have implicated ONOO(-) in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO(-). The present investigation examined glutamate-induced ONOO(-) formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO(-) production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO(-) levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO(-)-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO(-) production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO(-) generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.

Animals↗
PubMed2006

Does glutamate influence myocardial and peripheral tissue metabolism after aortic valve replacement for aortic stenosis?

BACKGROUND & AIMS: Glutamate plays an important role for myocardial metabolism in association with ischaemia. Patients with coronary artery disease characteristically demonstrate increased uptake of glutamate. Improved recovery of myocardial metabolism and haemodynamic state after coronary surgery has been reported in patients treated with glutamate infusion. However, the effect of glutamate has not been studied after other cardiac surgical procedures. In addition, the effects of glutamate on peripheral tissue metabolism remain to be described. METHODS: Twenty patients undergoing surgery for aortic stenosis were studied after randomisation to blinded infusion of glutamate or saline during 1h immediately after skin closure. Myocardial and leg tissue metabolism were assessed with organ balance techniques. RESULTS: Postoperative glutamate infusion induced a marked increase in myocardial and leg tissue uptake of glutamate. This was associated with a significant uptake of lactate in the heart. The negative arterial-venous differences of amino acids and free fatty acids across the leg were significantly smaller in the glutamate group. Haemodynamic state remained stable and did not differ between groups. CONCLUSION: The heart and peripheral tissues consumed the exogenously administered glutamate after surgery for aortic stenosis. Potentially favourable effects of glutamate on myocardial and peripheral tissue metabolism are suggested.

Aged↗
PubMed2005

Glutamate level in optic nerve head is increased by artificial elevation of intraocular pressure in rabbits.

Neurons can be damaged by the activation of glutamate receptors, but whether glutamate is related to the development of glaucomatous optic neuropathy is still controversial. The purpose of this study was to measure the acute changes in the glutamate levels in the optic nerve head (ONH) of rabbits induced by an artificial elevation of the intraocular pressure (IOP). A concentric microdialysis probe was inserted into the ONH of rabbits via the pars plana. The probe was perfused with Ringer's solution, and the levels of glutamate in 10-min dialysate samples were measured repeatedly using high-performance liquid chromatography. After the glutamate level was stabilized for at least 60 min, the IOP was adjusted to three levels; 120 mm Hg (n=11), 60 mm Hg (n=12), and 15 mm Hg (control group; n=11). The IOP was altered by changing the height of a bottle of Ringer's solution, which was connected to the anterior chamber by a 23-gauge needle. The IOP levels were maintained for 60 min, and the glutamate levels were determined every 10 min during the 60 min. The mean basal levels of glutamate in the 10-min dialysate were not significantly different among the three groups. The glutamate levels remained unchanged and stable in the controls, but elevation of the IOP significantly increased the level of the glutamate in the dialysate (IOP60, P=0.012; and IOP120, P=0.005: repeated measures ANOVA). Elevation of the IOP causes an increase in the glutamate levels in the ONH of rabbits. This suggests a possible interaction between glutamate metabolism and the IOP in the ONH.

Animals↗
PubMed2006

Brain neuroprotection by scavenging blood glutamate.

Excess glutamate in brain fluids characterizes acute brain insults such as traumatic brain injury and stroke. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As blood glutamate scavenging has been demonstrated to increase the efflux of excess glutamate from brain into blood, we tested the prediction that oxaloacetate-mediated blood glutamate scavenging causes neuroprotection in a pathological situation such as closed head injury (CHI), in which there is a well established deleterious increase of glutamate in brain fluids. We observed highly significant improvements of the neurological status of rats submitted to CHI following an intravenous treatment with 1 mmol oxaloacetate/100 g rat weight which decreases blood glutamate levels by 40%. No detectable therapeutic effect was obtained when rats were treated IV with 1 mmol oxaloacetate together with 1 mmol glutamate/100 g rat. The treatment with 0.005 mmol/100 g rat oxaloacetate was no more effective than saline but when it was combined with the intravenous administration of 0.14 nmol/100 g of recombinant glutamate-oxaloacetate transaminase, recovery was almost complete. Oxaloacetate provided neuroprotection when administered before CHI or at 60 min post CHI but not at 120 min post CHI. Since neurological recovery from CHI was highly correlated with the decrease of blood glutamate levels (r=0.89, P=0.001), we conclude that blood glutamate scavenging affords brain neuroprotection Blood glutamate scavenging may open now new therapeutic options.

Animals↗
PubMed2004

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.

Animals↗
PubMed2005

Facilitatory effect of aspirin on glutamate release from rat hippocampal nerve terminals: involvement of protein kinase C pathway.

The effect of aspirin on glutamate release from isolated nerve terminals (synaptosomes) from rat hippocampus was examined. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4AP) was facilitated by aspirin in a concentration-dependent manner, but the 4AP-evoked Ca(2+)-independent release was not modified. Also, aspirin-mediated facilitation of glutamate release was completely inhibited by bafilomycin A1, which depletes vesicle content by inhibiting the synaptic vesicle H(+)-ATPase that drives glutamate uptake, not by l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a excitatory amino acid (EAA) transporter inhibitor, suggesting that the facilitation of glutamate release produced by aspirin originates from synaptic vesicle exocytosis rather than reversal of the plasma membrane glutamate transporter. In addition, aspirin did not alter either 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release, but significantly increased in 4AP-evoked Ca(2+) influx. A possible effect of aspirin on synaptosomal Ca(2+) channels was confirmed in experiments where synaptosomes pretreated with a combination of the N- and P/Q-type Ca(2+) channel blockers, which abolished the aspirin-mediated facilitation of glutamate release. The facilitatory action by aspirin observed in glutamate release was mimicked and occluded by arachidonic acid (AA) and eicosatetraynoic acid (ETYA), an analogue of AA that mimics the effect of AA but cannot be metabolized. Furthermore, this aspirin-mediated facilitation of glutamate release may depend on activation of protein kinase C (PKC), because PKC activator and PKC inhibitor, respectively, superseding or suppressing the facilitatory effect of aspirin. Together, these results suggest that aspirin exerts their presynaptic facilitatory effect, likely through AA directly to induce the activation of PKC, which subsequently enhances the Ca(2+) influx through voltage-dependent N- and P/Q-type Ca(2+) channels to cause an increase in evoked glutamate release from rat hippocampal nerve terminals.

Animals↗
PubMed2006

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors.

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.

Animals↗
PubMed2004

Mechanisms of oxygen glucose deprivation-induced glutamate release from cerebrocortical slice cultures.

Glutamate has been recognized to mediate ischemia-induced neuronal injury in the brain, but the source of extracellular glutamate during ischemic insults remains controversial. We investigated the mechanisms of glutamate release in organotypic cerebrocortical slice cultures prepared from rat neonates, using oxygen glucose deprivation (OGD) as an in vitro ischemia model. Slice cultures were submerged in glucose-free deoxygenated buffer for 20-60 min and glutamate released into the extracellular buffer was quantified. Cell injury was assessed by uptake of propidium iodide 24 h after OGD insult. OGD-induced time-dependent glutamate release and cell injury, both of which were potently inhibited by a sodium channel blocker tetrodotoxin (1 microM). Application of voltage-dependent Ca2+ channel blockers or of an inhibitor of vacuolar-ATPase significantly reduced OGD-induced glutamate release and cell injury. On the contrary, inhibitors of glutamate transporters exacerbated OGD-induced glutamate release and cell injury. Volume sensitive organic anion channel blockers also augmented OGD-induced glutamate release and cell injury. In addition, OGD-induced glutamate release was markedly reduced in neuron-depleted slice cultures that were pretreated with 100 microM NMDA. These results suggest that vesicular release of neuronal origin constitutes a crucial component of extracellular glutamate increase during ischemic insults, which triggers neuronal injury.

Animals↗
PubMed2004

Glutamate transporters regulate excitability in local networks in rat neocortex.

Excitatory postsynaptic currents (EPSCs) in the neocortex are principally mediated by glutamate receptors. Termination of excitation requires rapid removal of glutamate from the synaptic cleft following release. Glutamate transporters are involved in EPSC termination but the effect of uptake inhibition on excitatory neurotransmission varies by brain region. Epileptiform activity is largely mediated by a synchronous synaptic activation of cells in local cortical circuits, presumably associated with a large release of glutamate. The role of glutamate transporters in regulating epileptiform activity has not been addressed. Here we examine the effect of glutamate transport inhibition on EPSCs and epileptiform events in layer II/III pyramidal cells in rat neocortex. Inhibiting glutamate transporters with DL-threo-beta-benzyloxyaspartic acid (TBOA; 30 microM) had no effect on the amplitude or decay time of evoked, presumably alpha-amino-3-hydroxyl-5-methyl-isoxazolepropionic acid-mediated, EPSCs. In contrast, the amplitude and duration of epileptiform discharges were significantly enhanced. TBOA resulted also in a decreased threshold for evoking epileptiform activity and an increased probability of occurrence of spontaneous epileptiform discharges. TBOA's effects were not inhibited by the group I and II metabotropic glutamate receptors antagonist (S)-alpha-methyl-4-carboxyphenylglycine or the kainate receptor antagonist [(3S,4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid]. D-(-)-2-amino-5-phosphonovaleric acid could both prevent excitability changes by TBOA and block already induced changes. Dihydrokainate (300 microM) had effects similar to TBOA suggesting involvement of the glial transporter GLT-1. Inhibiting glutamate transport increases local network excitability under conditions where there is an enhanced release of glutamate. Our results indicate that uptake inhibition produces an elevation of extracellular glutamate levels and activation of N-methyl-D-aspartate receptors.

2-Amino-5-phosphonovalerate↗
PubMed2006

Glutamate reduces secretion of l-serine in astrocytes isolated from stroke-prone spontaneously hypertensive rats.

In the CNS, l-serine (l-Ser) plays an essential role in neuronal survival by evoking a variety of biological responses in glial cells. Initially, we examined whether glutamate, hydrogen peroxide (H(2)O(2)), interleukin-1 (IL-1) beta, and sodium nitroprusside (SNP) induce the secretion of l-Ser in astrocytes isolated from Wistar Kyoto rats (WKY). The secretion of l-Ser was significantly induced with glutamate and SNP in cultured astrocytes. Next, to gain insight into the involvement of l-Ser in glutamate-induced neuroprotection, we compared the secretion of l-Ser in astrocytes isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive rats, WKY. We also examined the mRNA expression of the enzyme that produces l-Ser, 3-phosphoglycerate dehydrogenase (PHGDH), and a neural amino acid transporter, ASCT1, in the cultured astrocytes. A dose-dependent study of glutamate in astrocytes of SHRSP indicated differences in the secretion of l-Ser, and gene expression of PHGDH and ASCT1, compared with levels in the WKY astrocytes. We demonstrated that both the secretion and the gene expression were significantly attenuated in glutamate-treated astrocytes from SHRSP. Cerebral ischemia in SHRSP induced a massive efflux of glutamate, causing delayed neuronal death in region CA1 of the hippocampus. The results suggest that the attenuated secretion of l-Ser in astrocytes is involved in neuronal vulnerability and survival in SHRSP during the production of glutamate, as the secretion of l-Ser, which is stimulated by glutamate, is closely related to the protective effect against glutamate-mediated neurotoxicity. We conclude that glutamate and SNP up-regulate the secretion of l-Ser in primary astrocytes. Secretion of l-Ser is regulated in astrocytes in response to glutamate and nitric oxide and may correspond to the level of l-Ser needed for neuronal survival during brain insults such as ischemic stroke in SHRSP.

Analysis of Variance↗
PubMed1997

The inhibitory effects of beta-amyloid on glutamate and glucose uptakes by cultured astrocytes.

beta-Amyloid is the primary protein component of neuritic plaques, which are degenerative foci in brains of patients with Alzheimer's disease (AD). The effects of this naturally occurring beta-amyloid on the cells of the central nervous system have not been completely understood. beta-Amyloid increases the vulnerability of cultured neurons to glutamate-induced excitotoxic damage. Because astrocytes play a key role in uptake of extracellular glutamate and glutamate uptake is ATP-dependent, we studied the effect of beta25-35 on glutamate and glucose uptake in cultured hippocampal astrocytes following 7 days of exposure to beta25-35. Astrocytic glutamate uptake was studied at 1, 5, 10, 15, 20, and 60 min following the addition of [3H]glutamate (5 nM) to the culture media, and astrocytic glucose uptake was assessed at 60 min after the addition of [14C]glucose (600 and 640 nM) to the media. Glutamate uptake by control astrocytes was time-dependent. Astrocytes exposed to beta25-35, however, showed significantly lower glutamate uptake at all sampling times. Similarly, [14C]glucose uptake by astrocytes was inhibited by beta25-35. When glucose uptake was blocked by phloretin (10 mM), astrocytic [3H]glutamate uptake was also blocked, suggesting that the inhibitory effect of beta-amyloid on glutamate uptake is caused by diminished glucose uptake. Thus, our present study suggests a possible link between two proposed mechanisms of pathogenesis of the Alzheimer's disease: glutamate neurotoxicity and global defect in cerebral energy metabolism.

Amyloid beta-Peptides↗
PubMed1999

7-Nitroindazole, a selective inhibitor of nNOS, increases hippocampal extracellular glutamate concentration in status epilepticus induced by kainic acid in rats.

The glutamate extracellular concentration is controlled by metabolic and neuronal pathways via release and uptake mechanisms. Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. In this study, the influence of neuronally derived NO on hippocampal glutamate extracellular concentration was investigated in conditions of intense metabolic activation, i.e., during status epilepticus induced by systemic kainic acid (KA). Glutamate, arginine and citrulline concentrations were measured by microdialysis coupled to HPLC. Experiments were performed in conscious rats implanted with a microdialysis probe within the hippocampal CA3 area. Three groups were used: (1) rats treated with KA i.p. (12 mg/kg) and vehicle locally, via the microdialysis probe (n = 9); (2) rats given KA i.p. and a selective inhibitor of neuronal NO synthase, 7-nitroindazole (7-NI, 1.25 mM) locally (n = 13); (3) rats treated with saline i.p. and 7-NI locally (n = 7). Infusion of 7-NI or vehicle was performed throughout the second hour of status epilepticus. In groups 1 and 3, no significant modifications of extracellular glutamate, arginine and citrulline concentrations were measured. In group 2, the local application of 7-NI in the hippocampus during status epilepticus significantly increased extracellular glutamate and arginine concentrations, whereas citrulline concentration remained constant. The concomitant increases of extracellular glutamate and arginine concentrations under local 7-NI perfusion in seizure conditions, suggest that glutamate and arginine are linked in a common metabolic pathway and/or that glutamate is involved in the cross-talk between glia and neurons. A cerebrovascular effect of 7-NI which triggers glutamate release may also occur.

Animals↗
PubMed2000

Reciprocal regulation of nitric oxide and glutamate in the nucleus tractus solitarii of rats.

Nitric oxide (NO) and glutamate are both important mediators of the central cardiovascular regulation in the nucleus tractus solitarii. Our previous studies revealed that the central cardiovascular effects of NO in the nucleus tractus solitarii could be inhibited by glutamate receptor blockade. On the other hand, nitric oxide synthase (NOS) inhibitor attenuated the cardiovascular effects of glutamate. Thus, NO and glutamatergic systems appear to interact in central cardiovascular regulation. The present study examined whether NO and glutamate may affect each other's release/production in the nucleus tractus solitarii. A microdialysis probe was implanted into the nucleus tractus solitarii of male Sprague-Dawley rats, and the changes in the extracellular levels of glutamate and NO were determined by high performance liquid chromatography coupled with electrochemical detection and an NO analyzer, respectively. The results showed that NO solution elicited >10 fold increases in the extracellular level of glutamate, which returned to normal 60 min after the end of NO perfusion. The NO donor N-acetyl-penicillamine (SNAP) had an effect similar to NO solution. Furthermore, the glutamate level was reduced to 61% of basal value by perfusion with the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). When glutamate receptor agonist N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylixoxazole-4-propionic acid (AMPA) was administered into the nucleus tractus solitarii, the extracellular NO level was increased by 70-100%, whereas glutamate receptor antagonists (MK-801 hydrogen maleate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) did not alter the basal levels of NO. These results suggest that NO and glutamate may enhance each other's release/production in the nucleus tractus solitarii. This reciprocal regulation of NO and glutamate may be important in central cardiovascular control in the nucleus tractus solitarii.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2003

Glutamate loading protects freshly isolated and perfused adult cardiomyocytes against intracellular ROS generation.

Glutamate loading has been shown to protect single isolated perfused cardiomyocytes against metabolic inhibition and wash-off. The mechanism underpinning this protection is unknown. This study aimed to investigate whether reactive oxygen species (ROS) are generated by single isolated perfused cardiomyocytes and whether the protective effect of glutamate loading on cell metabolism is linked to ROS. Single rat cardiomyocytes were isolated with or without glutamate to stimulate glutamate loading. ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate in various stressful conditions including metabolic inhibition and wash-off with/without antimycin A or myxothiazol; simulated ischaemia (without cyanide) and glucose reintroduction; and H(2)O(2) perfusion. Reduced glutathione (GSH) levels were measured in control and glutamate-loaded cells with/without exposure to H(2)O(2). Finally, the effect of glutamate on glutathione reductase and glutathione peroxidase activity was measured. In every stressful condition studied, ROS production was significantly lower in glutamate-loaded cells compared to controls. This occurred regardless of whether ROS were produced intracellularly (e.g. from the respiratory chain inhibited with antimycin A) or via the extracellular precursor H(2)O(2). Glutamate-loaded cells also maintained their morphological integrity at higher H(2)O(2) concentrations than control cells. Furthermore, during H(2)O(2) exposure GSH levels decreased in glutamate-loaded cells but stayed constant in control cells. Glutamate stimulated the activity of glutathione peroxidase in a concentration-dependent fashion. These results provide new evidence to show that the cardioprotective effect of glutamate loading may be mediated through an enhanced ability to destroy ROS in the cell.

Animals↗
PubMed1999

Retinal ganglion cell dysfunction induced by hypoxia and glutamate: potential neuroprotective effects of beta-blockers.

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2-50 microM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 microM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 microM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

Action Potentials↗
PubMed2001

Glutamate regulates the viability of retinal cells in culture.

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.

Adenosine Triphosphate↗
PubMed2001

Endogenous glutamate-taurine interaction in striatum and nucleus accumbens of the freely moving rat: studies during the normal process of aging.

Using microdialysis, the effects of endogenous glutamate on extracellular concentrations of taurine, in striatum and nucleus accumbens of the awake rat during the normal process of aging were investigated. The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was perfused through the microdialysis probe to increase the endogenous concentration of glutamate. Young (2-4 months), middle aged (12-14 months), aged (27-32 months) and very aged (37 months) male Wistar rats were used. PDC (1, 2 and 4 mM) produced a dose-related increase of extracellular concentrations of glutamate and taurine in striatum and nucleus accumbens in all groups of age. Increases of glutamate, but not of taurine, decreased during aging in striatum. In nucleus accumbens there were no age-related changes in the increases of glutamate and taurine induced by PDC. In all age groups, increases of extracellular taurine were significantly correlated with increases of extracellular glutamate. However, when these taurine-glutamate correlations for all groups of age were compared, no statistical differences were found. These results show first that a decrease in the increases of glutamate produced by the glutamate reuptake inhibitor in striatum, but not in nucleus accumbens, is produced by age; second that glutamate-taurine interaction in striatum and nucleus accumbens does not change during the normal process of aging.

Aging↗
PubMed1998

Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection.

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by glutamate. Neither PC12 nor PC12/bcl-2 cells showed a significant incidence of apoptosis in response to glutamate. Conventional phospholipid analysis by high-performance TLC and phosphorous determination showed no significant changes in the phospholipid composition of either cell line incubated with </=15 mM glutamate. Phospholipid peroxidation was quantified in the cells using our newly developed method based on fluorescence-HPLC analysis of metabolically incorporated oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid. Unlike previous studies that measured total phospholipid oxidation, this novel technology permitted quantitation of oxidative stress in different classes of labeled phospholipids (the amount of labeled phospholipids in the cells did not exceed 1% of total phospholipids). Significant peroxidation of phosphatidylcholine and phosphatidylethanolamine occurred in PC12 cells treated with >5 mM glutamate. The peroxyl radical initiator 2,2'-azobis(2,4-dimethylvaleronitrile) caused a pronounced loss of all major phospholipid classes in PC12 cells, but no loss of cell viability. No phospholipid peroxidation was detected in PC12/bcl-2 cells incubated with </=15 mM glutamate or with 2, 2'-azobis(2,4-dimethylvaleronitrile). These results directly demonstrate that peroxidation of membrane phospholipids is not responsible for the cytotoxicity of glutamate in PC12 cells. Total cellular thiol, protein thiol and GSH reserves were quantified by a previously described electron paramagnetic resonance spectrometric method. Total thiols were ca. 1.5-fold greater in PC12/bcl-2 than in PC12 cells. Glutamate (</=5 mM) caused a progressive and equally significant decrease in total thiols and GSH in both PC12 and PC12/bcl-2 cells. High glutamate concentrations caused oxidation of protein sulfhydryls in PC12 cells, but not in PC12/bcl-2 cells. The results suggest that the changes in cellular milieu caused by bcl-2 gene transfection protect PC12 cells from the toxic effects of glutamate in a manner consistent with prevention of protein sulfhydryl oxidation.

Adrenal Gland Neoplasms↗