Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high-affinity sodium-dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l -trans-pyrrolidine-2,4-dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl -threo-β-hydroxyaspartate and l -aspartate-β-hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate or by intrastriatal coinjection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.     

Cerebellar Granule Neurons are More Vulnerable to Transient Transport-Mediated Glutamate Release than to Glutamate Uptake Blockade. Correlation with Excitatory Amino Acids Levels

The extracellular concentration of glutamate is highly regulated due to its excitotoxic nature. Failure of glutamate uptake or reversed activation of its transporters contributes to neurodegeneration related to some pathological conditions. We have compared the neurotoxicity of the substrate glutamate uptake inhibitor, l-trans-pyrrolidine-2,4-dicarboxylate (PDC), which promotes glutamate release by heteroexchange, with that of DL-threo-beta-benzyloxyaspartate (DL-TBOA), a non-substrate inhibitor, in cerebellar granule cell cultures. PDC substantially increases the extracellular concentration of glutamate during 30 min exposure and causes neuronal death at high concentrations, while DL-TBOA neurotoxicity is only observed after long-term exposure (8–24 h). During mitochondrial inhibition by 3-nitropropionic acid (3-NP), PDC-induced neuronal death is facilitated, but not that of DL-TBOA. In cultures containing a higher population of astrocytes DL-TBOA-induced increase in glutamate levels is more pronounced, but neuronal death is only triggered in the presence of 3-NP. Results suggest that cerebellar granule neurons are more vulnerable to acute transport-mediated glutamate release than to uptake blockade, which correlates with the extracellular excitatory amino acids levels.     

l-Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l-Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l -glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l -Serine-O-sulfate was an agonist at mGluR1a (EC₅₀ = 70 µM), mGluR2 (EC₅₀ = 25 µM), and mGluR4 (EC₅₀ = 324 µM). l -Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, l -cysteine, and dl -threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC₅₀ values of 43, 64, 463, and 488 µM, respectively, and displaced l -[³H]glutamate binding from membranes prepared from these cells with respective IC₅₀ values of 48, 44, 79, and 139 µM. However, d -aspartate,l -trans-pyrrolidine-2,4-dicarboxylate, l -threo-3-hydroxyaspartate, and l -aspartate-β-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC₅₀ values of 73, 54, 57, and 430 µM) but did not displace l -[³H]glutamate binding. These compounds inhibited Na⁺-dependent l -glutamate uptake into baby hamster kidney cells with IC₅₀ values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l -glutamate transporter, was significantly attenuated in the presence of l -glutamate decarboxylase (EC 4.1.1.15) or l -alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl -trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l -glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.     

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

Neurotransmitter l-glutamate released at central synapses is taken up and “recycled” by astrocytes using glutamate transporter molecules such as GLAST and GLT. Glutamate transport is essential for prevention of glutamate neurotoxicity, it is a key regulator of neurotransmitter metabolism and may contribute to mechanisms through which neurons and glia communicate with each other. Using immunocytochemistry and image analysis we have found that extracellular d-aspartate (a typical substrate for glutamate transport) can cause redistribution of GLAST from cytoplasm to the cell membrane. The process appears to involve phosphorylation/dephosphorylation and requires intact cytoskeleton. Glutamate transport ligands l -trans-pyrrolidine-2,4-dicarboxylate and dl-threo-3-benzyloxyaspartate but not anti,endo-3,4-methanopyrrolidine dicarboxylate have produced similar redistribution of GLAST. Several representative ligands for glutamate receptors whether of ionotropic or metabotropic type, were found to have no effect. In addition, extracellular ATP induced formation of GLAST clusters in the cell membranes by a process apparently mediated by P2 receptors. The present data suggest that GLAST can rapidly and specifically respond to changes in the cellular environment thus potentially helping to fine-tune the functions of astrocytes. The authors J.-W. Shin and K. T. D. Nguyen have contributed equally.     

Glutamate Uptake Impairment and Neuronal Damage in Young and Aged Rats In Vivo

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l - trans -pyrrolidine-2,4-dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3-month-old and 22–24-month-old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.     

Epidermal Growth Factor and Basic Fibroblast Growth Factor Protect Dopaminergic Neurons from Glutamate Toxicity in Culture

Abstract: In this report we characterize the toxicity of the excitatory amino acid l -glutamate with respect to dopaminergic neurons cultured from embryonic rat mesencephalon. We also demonstrate that two growth factors, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), can protect these neurons from damage. Micromolar concentrations of l -glutamate, as well as agonists that specifically activate N -methyl- d -aspartate (NMDA) and non-NMDA receptors, are all toxic to dopamine neurons in a concentration-dependent manner, as reflected by decreases in high-affinity dopamine uptake and confirmed by decreases in numbers of tyrosine hydroxylase-immunoreactive neurons. Although the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione could attenuate the effects of quisqualate, treatment with this antagonist could not eliminate the effects of glutamate itself. Similarly, (±)-2-amino-5-phosphonopentanoic acid was effective against NMDA toxicity but could not protect cells from quisqualate toxicity. Thus, each type of receptor could mediate neurotoxicity independently of the other. The presence of EGF or bFGF in the culture medium conferred a relative resistance of dopaminergic neurons to glutamate and quisqualate neurotoxicity by increased glutamate transport. However, treatment of the cultures with l - trans -pyrrolidine-2,4-dicarboxylic acid, an inhibitor of glutamate transport, attenuated but did not eliminate the protective effects of both growth factors against glutamate toxicity. When cultures were incubated with conditioned medium from growth factor-treated cultures, neuroprotection was also achieved. These results suggest that both EGF and bFGF can protect neurons from neurotoxicity in culture by increasing the capacity of the culture for glutamate uptake as well as by the secretion of soluble factors into the medium.     

Evaluation of Drugs Acting at Glutamate Transporters in Organotypic Hippocampal Cultures: New Evidence on Substrates and Blockers in Excitotoxicity

Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (±)-threo-3-methylglutamate and DL-threo--benzyloxyaspartate), in the presence of 300 M added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity.     

Evidence Disputing the Link Between Seizure Activity and High Extracellular Glutamate

Abstract: As seizures in experimental models can be induced by the activation and suppressed by the inhibition of glutamate receptors, it is often proposed that a high extracellular glutamate level subsequent to excessive presynaptic release and/or altered glutamate uptake is epileptogenic. The purpose of this study was to ascertain the link between seizure activity and high extracellular glutamate. To assist the detection of any putative rise in extracellular glutamate during seizures, microdialysis was coupled to enzyme-amperometric detection of glutamate, which provides maximal sensitivity and time resolution. Electrical activity and field potential were also recorded through the dialysis membrane to confirm that epileptic activity was present at the sampling site. No increase in dialysate glutamate content was detected during picrotoxin-induced seizures, even when the K⁺ concentration in the perfusion medium was raised to 50% above that measured previously during paroxysmal activity. In addition, sustained inhibition of glutamate uptake by l - trans -pyrrolidine-2,4-dicarboxylate increased the extracellular glutamate level >20-fold but did not produce electrophysiological changes indicative of excessive excitation. These findings indicate that seizures are not necessarily accompanied by an increased extracellular glutamate level and that increased glutamatergic excitation in epilepsy may result from other abnormalities such as increased density of glutamate receptors, enhanced activation subsequent to reduced modulation, or sprouting of glutamatergic synapses.     

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [³H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing ³H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [³H]acetate plus [U-¹⁴C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [³H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [³H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, ¹⁴C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of ¹⁴C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na⁺-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [³H]glutamate, constancy of the specific activity of extracellular [³H]glutamate, decrease in the specific activity of extracellular [¹⁴C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.     

Evidence for Neuronal Origin and Metabotropic Receptor-Mediated Regulation of Extracellular Glutamate and Aspartate in Rat Striatum In Vivo Following Electrical Stimulation of the Prefrontal Cortex

Abstract: Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200–300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 µ M tetrodotoxin or depletion of extracellular Ca²⁺, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor l - trans -pyrrolidine-2,4-dicarboxylic acid (200 µ M ) increased resting Glu and Asp levels by 300–500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1 S ,3 R )-1-aminocyclopentane- trans -1,3-dicarboxylate (200 µ M ). This effect was blocked by the metabotropic Glu receptor antagonist ( RS )-α-methylcarboxyphenylglycine (MCPG; 200 µ M ). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of l - trans -pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.     

Glutamate Induces Glutathione Efflux Mediated by Glutamate/Aspartate Transporter in Retinal Cell Cultures

This study was undertaken in order to characterize the role of the glutamate/aspartate transporter (GLAST) in the glutathione (GSH) efflux induced by glutamate. Our results demonstrated that retinal cell cultures exhibit two mechanisms of GSH release, one Na⁺-independent and other Na⁺-dependent. Glutamate and aspartate induced GSH efflux only in presence of Na⁺. Treatment with PCD (L-trans-Pyrrolidine-2,4-dicarboxylate), a transportable glutamate uptake blocker, increased GSH release indicating that GSH can be carried by glutamate transporters in retinal cell cultures. Added to this, treatment with zinc ion cultures, a recognized inhibitor of GLAST blocked GSH efflux evoked by glutamate. Treatment with NMDA antagonist (MK-801) did not have any effect on the GSH release induced by glutamate. These results suggest that glutamate induces GLAST-mediated release of GSH from retinal cell cultures and this could represent an important mechanism of cellular protection against glutamate toxicity in the CNS.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

κ Receptor Activation Attenuates l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Glutamate Levels in the Striatum

Abstract: The effects of local κ receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of l - trans -pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) were investigated. l - trans -PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective κ-opioid receptor agonist U-69593 (1-100 n M ) significantly attenuated l - trans -PDC-stimulated glutamate levels in a concentration-dependent manner. The selective κ receptor antagonist nor -binaltorphimine (1-100 n M ) reversed the U-69593-induced decrease in l - trans -PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by κ receptor activation. In addition, nor -binaltorphimine significantly elevated basal extracellular glutamate levels, implying that κ receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that l - trans -PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of l - trans -PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, κ receptors regulate the calcium-dependent component of l - trans -PDC-evoked extracellular glutamate levels in the striatum.     

The Glutamate Uptake System in Presynaptic Vesicles: Further Characterization of Structural Requirements for Inhibitors and Substrates

Noncyclic fluorine-substituted and cyclic analogs of glutamic acid were tested for their ability to inhibit glutamate uptake in isolated bovine presynaptic vesicles, in order to assess the specific structural requirements of the glutamate translocation system in the vesicle membrane. Cyclic analogs that permitted close interaction between the positive and negative charges of the glutamate molecule were effective inhibitors; maximum inhibitory potency was observed with L-trans-1-aminocyclopentane-1,3-dicarboxylic acid (l-t-ACPD), while d-t-ACPD was less active. Analogs with a larger or smaller ring (as in trans-1-aminocyclohexane-1,3-dicarboxylic acid or trans-1-aminocyclobutane-1,3-dicarboxylic acid) were also inhibitory, but somewhat less so. trans-ACPD was also taken up by the vesicles with a time course and ATP dependence similar to uptake of glutamate, and this uptake was inhibited by glutamate. The K _m value for t-ACPD uptake was similar to its K _i for inhibition of glutamate uptake, while its rate of uptake was lower than that of glutamate. Fluorine-substituted noncyclic analogs with substitutions at the 4-carbon were less effective than glutamic acid itself, although 4,4-difluoroglutamic acid was equal in activity to the unsubstituted compound. Inhibition by these derivatives appeared to be competitive in nature, and they probably were also transported by the vesicle uptake system. Special issue article in honor of Dr. Frode Fonnum.     

Activation of Glutamate Receptors Stimulates the Formation of Nitrite in Synaptosomes from Rat Cerebellum

Abstract: Synaptosomes from rat cerebellum were used to investigate the involvement of different glutamate receptor subtypes in the control of the synthesis of nitric oxide (NO), measured as its breakdown product nitrite (NO₂^-). Synaptosomes incubated in the presence of NAD|PH and l -arginine produced measurable levels of NO₂^-, which were reduced by addition of Nω-nitro-l -arginine methyl ester, an inhibitor of nitric oxide synthase. The selective ionotropic glutamate receptor agonist N-methyl-d -aspartate (NMDA) induced a pronounced increase in NO₂^-formation, which was prevented by Nω-nitro-l -arginine methyl ester and by the specific NMDA receptor antagonist Dl -2-amino-5-phosphonovaleric acid (AP-5). The NMDA-induced increase in NO₂^-formation was blocked by chelation of extracellular Ca²⁺ with EGTA. Both l -glutamate and the selective agonist for the metabotropic glutamate receptors (β)-1-aminocyclopentane-trans-1,3-dicarboxylic acid raised NO₂^-production, which retumed to control levels after addition of Nω-nitro-l -arginine methyl ester. The selective glutamate ionotropic receptor agonist (R,S)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid did not cause any change in NO₂ formation. The stimulatory effect of l -glutamate was blocked by the metabotropic glutamate receptor antagonist Dl -2-amino-4-phosphonobutyric acid but was unaffected by the selective NMDA receptor blocker AP-5. Removal of extracellular Ca²⁺ by EGTA did not affect the action of l -glutamate; whereas W-7, an inhibitor of calmodulin, and dantrolene, a compound that blocks the mobilization of Ca²⁺ from intracellular stores, abolished the effect of l -glutamate on NO₂^-formation. It is suggested that stimulation of ionotropic NMDA receptors activates NO metabolism by causing an influx of Ca²⁺ from the extracellular space, whereas activation of metabotropic receptors by l -glutamate provokes a mobilization of Ca²⁺ from intracellular stores, which stimulates nitric oxide synthase activity by forning Ca²⁺/calmodulin complexes.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport-Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or dl -2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the V _max of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10⁻⁵ M ) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l - trans -pyrrolidine-2,4-dicarboxylic acid (1 m M ). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.     

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.     

Extracellular Glutamate Levels Are Chronically Elevated in the Brains of LP-BM5-Infected Mice: A Mechanism of Retrovirus-Induced Encephalopathy

Abstract: Mice infected with the LP-BM5 leukemia retrovirus mixture develop a progressive immunodeficiency with associated behavioral, histological, and neurochemical alterations consistent with glutamatergic hyperactivation. To gain insight into the contribution of excitatory amino acids to the neurodegeneration observed in these mice, their concentrations were measured in the CSF and striatal microdialysates. Glutamate concentrations were significantly elevated in CSF but not plasma as early as 4 weeks postinoculation. Steady-state glutamate levels in striatal microdialysates were increased threefold and could be reduced 40% by application of l -α-aminoadipate, an inhibitor of microglial glutamate transport. Stimulation of infected mice with KCl/ l - trans -2,4-pyrrolidine dicarboxylate further increased glutamate levels 170–270% above those evoked in control mice. Tetrodotoxin suppressed the depolarization-evoked increase in glutamate by 88% in control mice, but it had only negligible effects in 40% of infected mice. Analysis of glutamate transport and catabolism suggests that abnormal astrocytic function does not contribute to the increase in basal extracellular glutamate levels. These findings are the first direct evidence that infection with an immunodeficiency-inducing retrovirus leads to a chronic elevation of extracellular free glutamate levels in the brain, which contributes to the neurodegenerative and cognitive deficits observed in these mice.     

Involvement of Metabotropic Glutamate Receptors in Ischemia-Induced Taurine Release in the Developing and Adult Hippocampus

Metabotropic glutamate receptors have recently been envisaged as involved in both potentiation and prevention of ischemic and excitotoxic neuronal damage. The release of the inhibitory amino acid taurine is markedly enhanced in ischemia in both the immature and mature mouse hippocampus. The modulation of [³H]taurine release by metabotropic receptor agonists and antagonists was studied in hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. Agonists of group I, II and III metabotropic glutamate receptors generally reduced the ischemia-induced release in adult animals. In the immature hippocampus the group I agonists (S)-3,5-dihydroxyphenylglycine and (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate, which mainly enhance neuronal excitation, potentiated initial taurine release in ischemia. Ionotropic glutamate receptor agonists also enhance the ischemia-induced taurine release in developing mice. This glutamate-activated taurine release may thus constitute an important protective mechanism against excitotoxicity in the immature hippocampus.