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

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

L-trans-PDC enhances hippocampal neuronal activity by stimulating glial glutamate release independently of blocking transporters

The glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) reversibly enhanced hippocampal neuronal activity in the rat and mouse dentate gyrus. The PDC action was still found in mice lacking the glial glutamate transporter GLT-1. PDC did not influence the rate of spontaneous miniature excitatory postsynaptic currents and spontaneous inhibitory postsynaptic currents, ionotropic glutamate receptor currents, or GABA-evoked currents in cultured rat hippocampal neurons. PDC increased glutamate released from cultured hippocampal astrocytes from normal rats, normal mice, and GLT-1 knock-out mice, that is not inhibited by deleting extracellular Na(+), while the drug had no effect on the release from cultured rat hippocampal neurons. The results of the present study thus suggest that PDC stimulates glial glutamate release by a mechanism independent of inhibiting glutamate transporters, which perhaps causes an increase in synaptic glutamate concentrations, in part responsible for the enhancement in hippocampal neuronal activity.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N-acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N-acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon-glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58-83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLUR(II). No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.     

Effects of endogenous glutamate on extracellular concentrations of taurine in striatum and nucleus accumbens of the awake rat: Involvement of NMDA and AMPA/kainate receptors

Summary. Using microdialysis, the effects of endogenous glutamate on extracellular concentrations of taurine in striatum and nucleus accumbens of the awake rat were investigated. The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was used to increase the extracellular concentration of glutamate. PDC (1, 2 and 4 mM) produced a dose-related increase of extracellular concentrations of glutamate and taurine in striatum and nucleus accumbens. Increases of extracellular taurine were significantly correlated with increases of extracellular glutamate, but not with PDC doses, which suggests that endogenous glutamate produced the observed increases of extracellular taurine in striatum and nucleus accumbens. The role of ionotropic glutamate receptors on the increases of taurine was also studied. In striatum, perfusion of the antagonists of NMDA and AMPA/kainate glutamate receptors attenuated the increases of extracellular taurine. AMPA/kainate, but not NMDA receptors, also reduced the increases of extracellular taurine in nucleus accumbens. These results suggest that glutamate-taurine interactions exist in striatum and nucleus accumbens of the awake rat. Received March 5, 1999/Accepted September 22, 1999     

Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.     

Ionotropic glutamate receptors are involved in malondialdehyde production in anesthetized rat brain cortex: a microdialysis study

Elevated extracellular glutamate levels can increase malondialdehyde production in the brains of anesthetized rats. Thus, we investigated whether ionotropic glutamate receptors are involved in glutamate-induced malondialdehyde production. A microdialysis probe was implanted in the brain cortex of anesthetized rats. The malondialdehyde level in microdialysates was analyzed using an HPLC system. Three different ionotropic glutamate receptor agonists were used. At a concentration of 1.5 mM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA, a selective AMPA receptor agonist) induced a dramatic increase in extracellular malondialdehyde production (as much as 14-fold relative to the basal value). N-Methyl-D-aspartic acid (NMDA, a selective NMDA receptor agonist) also induced an increase in extracellular malondialdehyde production; however, the increase was not as much as that observed in the perfusion of AMPA receptor agonist. Kainic acid (a selective kainate receptor agonist) did not significantly increase malondialdehyde production. When co-perfused with L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 31.4 mM), a glutamate uptake transport inhibitor that can increase the extracellular glutamate levels, AMPA receptor antagonist [1-(4-aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride, 1.0 mM] can significantly reduce PDC-induced malondialdehyde production. Although NMDA receptor antagonist [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate, MK801] also can decrease the PDC-induced malondialdehyde production, it was not as effective as the AMPA receptor antagonist. These results suggest that ionotropic receptors are involved in the glutamate-induced increase in malondialdehdye production. Specifically, AMPA receptor seems to be predominant in the glutamate-induced malondialdehdye production in anesthetized rat brain cortex.     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

Adenosine enhances glial glutamate efflux via A2a adenosine receptors

The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 microM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)+ mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions.     

Transient increase in the high affinity [3H]-L-glutamate uptake activity during in vitro development of hippocampal neurons in culture

The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587-593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 microM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 microM [3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta-benzyloxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.     

Enzymatic degradation protects neurons from glutamate excitotoxicity

Several enzymes with the capacity to degrade glutamate have been suggested as possible neuroprotectants. We initially evaluated the kinetic properties of glutamate pyruvate transaminase (GPT; also known as alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase under physiologic conditions to degrade neurotoxic concentrations of glutamate. Although all three enzymes initially degraded glutamate rapidly, only GPT was able to reduce toxic (500 microM) levels of glutamate into the physiologic (<20 microM) range. Primary cultures of fetal murine cortical neurons were subjected to paradigms of either exogenous or endogenous glutamate toxicity to evaluate the neuroprotective value of GPT. Neuronal survival after exposure to added glutamate ranging from 100 to 500 microM was improved significantly in the presence of GPT (> or =1 U/ml). Cultures were also exposed to the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC-dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.     

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Effects of Endogenous Glutamate on Extracellular Concentrations of GABA, Dopamine, and Dopamine Metabolites in the Prefrontal Cortex of the Freely Moving Rat: Involvement of NMDA and AMPA/KA Receptors

Using microdialysis, interactions between endogenous glutamate, dopamine, and GABA were investigated in the medial prefrontal cortex of the freely moving rat. Interactions between glutamate and other neurotransmitters in the prefrontal cortex had already been studied using pharmacological agonists or antagonists of glutamate receptors. This research investigated whether glutamate itself, through the increase of its endogenous extracellular concentration, is able to modulate the extracellular concentrations of GABA and dopamine in the prefrontal cortex. Intracortical infusions of the selective glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular glutamate. PDC (0.5, 2, 8, 16 and 32 mM) produced a dose-related increase in dialysate glutamate in a range of 1–36 M. At the dose of 16 mM, PDC increased dialysate glutamate from 1.25 to 28 M. PDC also increased extracellular GABA and taurine, but not dopamine; and decreased extracellular concentrations of the dopamine metabolites DOPAC and HVA. NMDA and AMPA/KA receptor antagonists were used to investigate whether the increases of extracellular glutamate were responsible for the changes in the release of GABA, and dopamine metabolites. The NMDA antagonist had no effect on the increase of extracellular GABA, but blocked the decreases of extracellular DOPAC and HVA, produced by PDC. In contrast, the AMPA/KA antagonist blocked the increases of extracellular GABA without affecting the decreases of extracellular DOPAC and HVA produced by PDC. These results suggest that endogenous glutamate acts preferentially through NMDA receptors to decrease dopamine metabolism, and through AMPA/KA receptors to increase GABAergic activity in the medial prefrontal cortex of the awake rat.     

Reduction in glutamate uptake is associated with extrasynaptic NMDA and metabotropic glutamate receptor activation at the hippocampal CA1 synapse of aged rats

This study aims to determine whether the regulation of extracellular glutamate is altered during aging and its possible consequences on synaptic transmission and plasticity. A decrease in the expression of the glial glutamate transporters GLAST and GLT‐1 and reduced glutamate uptake occur in the aged (24–27 months) Sprague–Dawley rat hippocampus. Glutamatergic excitatory postsynaptic potentials recorded extracellularly in ex vivo hippocampal slices from adult (3–5 months) and aged rats are depressed by DL‐TBOA, an inhibitor of glutamate transporter activity, in an N‐Methyl‐d‐ Aspartate (NMDA)‐receptor‐dependent manner. In aged but not in young rats, part of the depressing effect of DL‐TBOA also involves metabotropic glutamate receptor (mGluRs) activation as it is significantly reduced by the specific mGluR antagonist d‐methyl‐4‐carboxy‐phenylglycine (MCPG). The paired‐pulse facilitation ratio, a functional index of glutamate release, is reduced by MCPG in aged slices to a level comparable to that in young rats both under control conditions and after being enhanced by DL‐TBOA. These results suggest that the age‐associated glutamate uptake deficiency favors presynaptic mGluR activation that lowers glutamate release. In parallel, 2 Hz‐induced long‐term depression is significantly decreased in aged animals and is fully restored by MCPG. All these data indicate a facilitated activation of extrasynaptic NMDAR and mGluRs in aged rats, possibly because of an altered distribution of glutamate in the extrasynaptic space. This in turn affects synaptic transmission and plasticity within the aged hippocampal CA1 network.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.