Rottlerin Inhibits (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)-ATPase Activity in Brain Tissue and Alters <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na⁺, K⁺)-dependent ATPase (Na⁺, K⁺-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na⁺, K⁺-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na⁺, K⁺-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na⁺, K⁺-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.     

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.     

Neuronal-induced and glutamate-dependent activation of glial glutamate transporter function

The activity of high-affinity glutamate transporters is essential for the normal function of the mammalian central nervous system. Using a combined pharmacological, confocal immunocytochemical, enzyme-based microsensor and fluorescence imaging approach, we examined glutamate uptake and transporter protein localization in single astrocytes of neuron-containing and neuron-free microislands prior to pre-synaptic transmitter secretion and during functional neuronal activity. Here, we report that the presence or absence of neurons strikingly affects the uptake capacity of the astroglial glutamate transporters GLT1 and GLAST1. Induction of transporter function is activated by neurons and this effect is mimicked by pre-incubation of astrocytes with micromolar concentrations of glutamate. Moreover, increased glutamate transporter activation is reproduced by endogenous release of glutamate via activation of neuronal nicotinic receptors. The increase in transport activity is dependent on neuronal release of glutamate, is associated with the local redistribution (clustering) of GLT1 and GLAST1 but is independent of transporter synthesis and of glutamate receptor activation. Together, these results suggest an activity-dependent neuronal feedback system for rapid astroglial glutamate transporter regulation where neuron-derived glutamate is the physiological signal that triggers transporter function.     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

Glutamatergic hypothesis of schizophrenia: involvement of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-dependent glutamate transport

Summary Hypothetical model based on deficient glutamatergic neurotransmission caused by hyperactive glutamate transport in astrocytes surrounding excitatory synapses in the prefrontal cortex is examined in relation to the aetiology of schizophrenia. The model is consistent with actions of neuroleptics, such as clozapine, in animal experiments and it is strongly supported by recent findings of increased expression of glutamate transporter GLT in prefrontal cortex of patients with schizophrenia. It is proposed that mechanisms regulating glutamate transport be investigated as potential targets for novel classes of neuroactive compounds with neuroleptic characteristics. Development of new efficient techniques designed specifically for the purpose of studying rapid activity-dependent translocation of glutamate transporters and associated molecules such as Na⁺, K⁺-ATPase is essential and should be encouraged.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes

Neutral amino acid transporter ASCT2, together with high-affinity glutamate transporters, belongs to the SLC1 gene family of Na(+)-dependent solute carriers and is one of the major transporters of glutamine in cultured astrocytes. Besides glutamine and other high-affinity substrates--alanine, serine, cysteine or threonine, ASCT2 can also translocate protonated glutamate. The present study elucidated substrate-dependent trafficking of ASCT2 in differentiated primary cultures of human fetal astrocytes. The differentiation induced by 8-bromo-cAMP caused dramatic up-regulation of two co-localized and functionally linked astroglial proteins--glutamate transporter GLAST, that is the only high-affinity router of glutamate into cultured astrocytes, and glutamine synthetase (GS), a cytosolic enzyme that converts at least a part of the arriving glutamate into glutamine. In order to distinguish individual intracellular effects of these two substrates on ASCT2, in some cultures glutamine synthetase was effectively knocked down using siRNA silencing technique. In control conditions, regardless of GS levels, almost the entire ASCT2 immunoreactivity was restricted to the cytosol. Both glutamine and alanine, though to different extents, induced partial redistribution of ASCT2 from the cytosolic compartment to the plasma membrane. However, in cultures with high GS expression, micromolar concentrations of glutamate exhibited more pronounced effect on ASCT2 trafficking than the preferred substrates of this carrier. In contrast, glutamate had no effect on ASCT2 distribution in cultures devoid of GS. D-Aspartate, a metabolically inert substrate effectively transported by GLAST, had no effect in any cell culture utilized. It seems that intracellular glutamine produced by GS from glutamate that, in turn, is supplied by GLAST, is a more potent inducer of ASCT2 trafficking to the cell surface than the ASCT2-mediated translocation of extracellular substrates. At lower pH values (6.2-6.7), the cell surface pool of ASCT2 was significantly larger than at physiological pH. In addition, high concentrations of glutamate, independently from GLAST or glutamate receptor activation, induced further arrival of ASCT2 to the plasma membrane. The pH-dependent functional activation of ASCT2 and the ASCT2-mediated glutamate uptake may play important roles during ischemic acidosis or synaptic activity-induced local acidification.     

Localization and expression of the glutamate transporter,excitatory amino acid transporter 4, within astrocytes of the rat retina

Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.This work was supported by grants from the National Health and Medical Research Council (Grant #208950) and Retina Australia.     

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Müller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min. Following euthanasia, the retina was processed for D-aspartate, GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Müller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal, its ability to transport D-aspartate into Müller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease.     

P2X7 receptors mediate NADH transport across the plasma membranes of astrocytes

NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels.     

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.     

Estrogen and tamoxifen reverse manganese-induced glutamate transporter impairment in astrocytes

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17β-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor β1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor β1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.     

Interactions between valproate,glutamate, aspartate,and GABA with respect to uptake in astroglial primary cultures

Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na⁺-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [³H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [³H]l-glutamate and [³H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [³H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.     

Rottlerin,an inhibitor of protein kinase Cdelta (PKCdelta), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCdelta-independent

Protein kinase C (PKC) regulates the activity and/or cell surface expression of several different neurotransmitter transporters, including subtypes of glutamate transporters. In the present study, the effects of pharmacological inhibitors of PKC were studied in primary astrocyte cultures that express the glutamate aspartate transporter (GLAST) subtype of glutamate transporter. We found that general inhibitors of PKC, bisindolylmaleimide I (Bis I), bisindolylmaleimide II (Bis II), staurosporine and an inhibitor of classical PKCs, Gö6976, had no effect on Na⁺‐dependent glutamate transport activity. However, rottlerin, a putative specific inhibitor of PKCδ, decreased transport activity with an IC₅₀ value (less than 10 µm ) that is comparable to that reported for inhibition of PKCδ. The effect of rottlerin was very rapid (maximal effect within 5 min) and was due to a decrease in the capacity (V_max) for transport. Rottlerin also caused a drastic loss of GLAST immunoreactivity within 5 min, suggesting that rottlerin accelerates GLAST degradation/proteolysis. Rottlerin had no effect on cell surface or total expression of the transferrin receptor, providing evidence that the effect on GLAST cannot be attributed to a non‐specific internalization/degradation of plasma membrane proteins. Down‐regulation of PKCδ with chronic phorbol ester treatment did not block rottlerin‐mediated inhibition of transport activity. These results suggest a novel mechanism for regulation of the GLAST subtype of glutamate transporter and indicate that there is a rottlerin target that is capable of controlling the levels of GLAST by controlling the rate of degradation or limited proteolysis. It appears that the target for rottlerin may not be PKCδ.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

In vitro uptake of glutamate in GLAST-and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal

Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/ aspartats transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [³H]-D-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST-transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.     

The GLT‐1 (EAAT2; slc1a2) glutamate transporter is essential for glutamate homeostasis in the neocortex of the mouse

Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with [1‐¹³C]glucose and [1,2‐¹³C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and ¹³C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and ¹³C labeling of metabolites, glutamate level was decreased but labeling from [1‐¹³C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2‐¹³C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.

     

Gliotoxic action of glutamate on cultured astrocytes

Because of the well-documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L-glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L-glutamate resulted in a concentration- and time-dependent increase in the release of LDH. L-Glutamate-induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L-glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L-glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L-glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.