Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells (original) (raw)
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Glutamate-Induced Inhibition of D-Aspartate Uptake in Müller Glia from the Retina
Neurochemical Research, 2000
Müller glial cells from the retina "in situ" and in primary culture, mainly express the high-affinity sodium-coupled glutamate/aspartate transporter GLAST-1, which dominates total retinal glutamate (Glu) uptake, suggesting a major role for these cells in the modulation of excitatory transmission. The possible involvement of ionotropic and metabotropic Glu receptors in the regulation of Glu uptake was studied in primary cultures of Müller glia. We demonstrate that exposure to 1 mM L-Glu induces a time-dependent inhibition of D-aspartate (D-Asp) uptake in a Na ϩ -dependent manner, as a result of a reduction in the number of transporters at the plasma membrane. The inhibition of D-Asp uptake by Glu was not mimicked by agonists or modified by antagonists of ionotropic and metabotropic Glu receptors. In contrast, transport was inhibited by GLAST-1 transportable substrates threo-hydroxyaspartate and aspartate--hydroxamate, but not by the nontransportable inhibitors trans-pyrrolidine dicarboxylate or DL-threo--benzyloxyaspartic acid. Under the same experimental conditions, L-Glu did not affect the sodium-dependent transport systems for glycine or GABA. The present results demonstrate that the specific downregulation of glutamate/aspartate transport by L-Glu is unrelated to Glu receptor activation, and results from the internalization of transporter proteins triggered by the transport process itself. Such negative feedback of Glu on Glu transport, could contribute to retinal toxicity under pathological conditions in which high extracellular concentrations of Glu are reached.
Glial glutamate transporters: New actors in brain signaling
IUBMB Life, 2011
Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na 1 -dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/ neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so-called tripartite synapse.
Regulation of high-affinity glutamate uptake activity in Bergmann glia cells by glutamate
Brain Research, 2000
The effect of glutamate receptor activation on the high-affinity sodium-dependent glutamate transport expressed in chick Bergmann glia 3 cells was examined. Pre-exposure to glutamate produced a time-and dose-dependent decrease in H-labeled D-aspartate uptake. This effect could not be reproduced by selective glutamate receptor agonists. Furthermore, it was insensitive to both ionotropic and metabotropic glutamate receptor antagonists. Replacement of extracellular sodium ions with choline in the preincubation media, abolished the reduction of the uptake. When the cells were pre-exposed to competitive transportable inhibitors of the transporter, such as D-aspartate, DL-threo-hydroxyaspartate (DL-THA), and aspartate-b-hydroxamate (ABH), the glutamate effect was mimicked. From saturation experiments, it was found that the reduction on the uptake, after glutamate treatment, is related to an increase in K . Interestingly, the m 21 effect is blocked by staurosporine, a Ca / diacylglycerol-dependent protein kinase (PKC) inhibitor. The present findings suggest that glutamate regulates its transport in a non-receptor fashion, a phenomena that is most probably linked to changes induced by the translocation process of the substrate through the transporter.
Glutamate transport by retinal M�ller cells in glutamate/aspartate transporter-knockout mice
Glia, 2005
Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/ aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Mü ller cells in GLAST Ϫ/Ϫ mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the K m value for glutamate uptake was similar in wild-type and GLAST Ϫ/Ϫ mouse retinas, but the V max was ϳ50% lower in the mutant. In Na ϩ-free medium, the V max was further reduced by 40%. In patch-clamp recordings of dissociated Mü ller cells from GLAST Ϫ/Ϫ mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Mü ller cells. [ 3 H]D-Aspartate uptake autoradiography, however, showed that Na ϩ-dependent, high-affinity transporters account for most of the glutamate uptake by Mü ller cells, and that Na ϩ-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Mü ller cells in the GLAST Ϫ/Ϫ mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Mü ller cells, new Na ϩ-dependent, high-affinity glutamate transporters remain to be identified.
Substrate-induced up-regulation of Na+-dependent glutamate transport activity
Neurochemistry International, 2000
Sodium-dependent transporters regulate extracellular glutamate in the CNS. Recent studies suggest that the activity of several dierent neurotransmitter transporters can be rapidly regulated by a variety of mechanisms. In the present study, we report that pre-incubation of primary`astrocyte-poor' neuronal cultures with glutamate (100 mM) for 30 min nearly doubled the V max for Na + -dependent accumulation of L-[ 3 H]-glutamate, but had no eect on Na + -dependent [ 3 H]-glycine transport. Pre-incubation with glutamate also increased the net uptake of non-radioactive glutamate, providing evidence that the increase in accumulation of L-[ 3 H]-glutamate was not related to an increase in intracellular glutamate and a subsequent increase in exchange of intracellular non-radioactive glutamate for extracellular radioactive glutamate. The glutamate receptor agonists, a-amino-3hydroxy-5-methylisoxazole-4-propionate, quisqualate, and (1 S, 3R )-1-aminocyclopentane-1,3-dicarboxylic acid did not mimic the eect of pre-incubation with glutamate and the glutamate-induced increase was not blocked by receptor antagonists. However, compounds known to interact with the transporters, including L-aspartate, D-aspartate, L-(-)-threo-3-hydroxyaspartate (L-THA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), caused variable increases in transport activity and attenuated the increase induced by glutamate, suggesting that the increase is related to the interaction of glutamate with the transporters. Several studies were attempted to de®ne the mechanism of this regulation. We found no evidence for increases in transporter synthesis or cell surface expression. Inhibitors of signaling molecules known to regulate other neurotransmitter transporters had no eect on this stimulation. Using a variety of cultures, evidence is provided to suggest that this substrate-induced upregulation of glutamate transport is speci®c for the GLT-1 and GLAST subtypes and does not in¯uence transport mediated by EAAC1. These studies suggest that the interaction of glutamate with some of the subtypes of glutamate transporters causes an increase in transport activity. Conceivably, this phenomenon provides an endogenous mechanism to increase the clearance of glutamate during periods of prolonged elevations in extracellular glutamate. 7
Functional glutamate transport in rodent optic nerve axons and glia
Glia, 2008
Glutamate uptake and potential release via Na + -dependent glutamate transporters is crucial to CNS function and to various forms of injury. Evidence for glutamatemediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter is of particular clinical importance. The expression of glutamate transporters was examined in developing mouse and mature mouse and rat white matter using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both neonatal and mature optic nerve while GLT1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes but there was little membrane expression of either in the neonate. GLAST, EAAC1 and GLT1 were expressed in neonatal axons with significant amount of GLT1 present in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in developing mouse optic nerve revealing Na + -dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a significant potential sink and source for extracellular glutamate in white matter.
Are neuronal transporters relevant in retinal glutamate homeostasis?
Neurochemistry international
Exposure of isolated retinas to 30 microM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Müller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological media, in the absence of D-aspartate, resulted in the slow release of accumulated D-aspartate from the Müller cells and its accumulation into populations of photoreceptors and bipolar cells. This result indicates that after initial transport into Müller cells, reversal of direction of transport of D-aspartate, and thus by inference glutamate, by GLAST, readily occurs. D-aspartate released by Müller cells was strongly accumulated into cone photoreceptors which are known to express GLT-1, and into rod photoreceptors which we demonstrate here to express the retina specific glutamate transporter EAAT5 (excitatory amino transporter 5). Populations...
Glutamate transporters bring competition to the synapse
Current Opinion in Neurobiology, 2004
Glutamate transporters (GluTs) prevent the accumulation of glutamate and influence the occupancy of receptors at synapses. The ability of extrasynaptic NMDA receptors and metabotropic glutamate receptors to participate in signaling is tightly regulated by GluT activity. Astrocytes express the highest density of GluTs and dominate clearance away from these receptors; synapses that are not associated with astrocyte processes experience greater mGluR activation and can be exposed to glutamate released at adjacent synapses. Although less abundant, neuronal transporters residing in the postsynaptic membrane can also shield receptors from the glutamate that is released. The diversity in synaptic morphology suggests a correspondingly rich diversity of GluT function in excitatory transmission.
Journal of Neurochemistry, 2003
The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [ 3 H]D-aspartate or [ 3 H]L-glutamate into these preparations revealed sodium-de-pendent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, L-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the Ôglial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 lM dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.
Glutamate transporters: Gene expression regulation and signaling properties
Neuropharmacology, 2019
Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. During synaptic activity, glutamate is released and binds to specific membrane receptors and transporters activating, in the one hand, a wide variety of signal transduction cascades, while in the other hand, its removal from the synaptic cleft. Extracellular glutamate concentrations are maintained within physiological levels mainly by glia glutamate transporters. Inefficient clearance of this amino acid is neurotoxic due to a prolonged hyperactivation of its postsynaptic receptors, exacerbating a wide array of intracellular events linked to an ionic imbalance, that results in neuronal cell death. This process is known as excitotoxicity and is the underlying mechanisms of an important number of neurodegenerative diseases. Therefore, it is important to understand the regulation of glutamate transporters function. The transporter activity can be regulated at different levels: gene expression, transporter protein targeting and trafficking, and post-translational modifications of the transporter protein. The identification of these mechanisms has paved the way to our current understanding the role of glutamate transporters in brain physiology and will certainly provide the needed biochemical information for the development of therapeutic strategies towards the establishment of novel therapeutic approaches for the treatment and/or prevention of pathologies associated with excitotoxicity insults.