Glutamate Transporters GLAST Research Articles

Methylphenidate administration determines enduring changes in neuroglial network in rats

Repeated exposure to psychostimulant drugs induces complex molecular and structural modifications in discrete brain regions of the meso-cortico-limbic system. This structural remodeling is thought to underlie neurobehavioral adaptive responses. Administration to adolescent rats of methylphenidate (MPH), commonly used in attention deficit and hyperactivity disorder (ADHD), triggers alterations of reward-based behavior paralleled by persistent and plastic synaptic changes of neuronal and glial markers within key areas of the reward circuits. By immunohistochemistry, we observe a marked increase of glial fibrillary acidic protein (GFAP) and neuronal nitric oxide synthase (nNOS) expression and a down-regulation of glial glutamate transporter GLAST in dorso-lateral and ventro-medial striatum. Using electron microscopy, we find in the prefrontal cortex a significant reduction of the synaptic active zone length, paralleled by an increase of dendritic spines. We demonstrate that in limbic areas the MPH-induced reactive astrocytosis affects the glial glutamatergic uptake system that in turn could determine glutamate receptor sensitization. These processes could be sustained by NO production and synaptic rearrangement and contribute to MPH neuroglial induced rewiring.

Intrathecal Etanercept Partially Restores Morphine's Antinociception in Morphine-Tolerant Rats via Attenuation of the Glutamatergic Transmission

Long-term exposure to morphine leads to analgesic tolerance. In addition to an opioid receptor conformational change, enhancing the glutamatergic signal transmission is also involved in morphine tolerance. Tumor necrosis factor-α has been demonstrated to correlate with neuronal plasticity via activation of glutamatergic transmission. We examined the effect of etanercept, a tumor necrosis factor-α inhibitor on morphine tolerance in rats. Male Wistar rats were implanted with 2 intrathecal (IT) catheters, and 1 IT catheter was connected to a mini-osmotic pump, used for either morphine infusion (15 μg/h) or saline (1 μL/h) infusion for 5 days. On day 5, either etanercept (50 μg) or saline (10 μL) was injected after discontinued morphine infusion. Three hours later, acute morphine (15 μg/10 μL, IT) treatment was given and all rats received a nociceptive tail-flick test. The results showed that acute etanercept (50 μg) treatment caused a significant antinociceptive effect of morphine in morphine-tolerant rats. Western blotting indicated that etanercept attenuated the downregulation of membrane glutamate transporters GLT-1 and GLAST in morphine-tolerant rats. Etanercept also inhibited the upregulation of surface AMPA-receptor and N-methyl-d-aspartate-receptor subunits, including GluR1/GluR2 and NR1/NR2A. These results demonstrate that etanercept partially restores the antinociceptive effect of morphine in morphine tolerance after a morphine challenge. Etanercept has potential for use in the clinical management of pain, particularly in patients who require long-term opioid treatment, and the effectiveness of which can be hampered by tolerance.

Long-term changes in expressions of spinal glutamate transporters after spinal cord injury

Glutamate is a major excitatory transmitter in the central nervous system that may produce cellular injury when its concentration is abnormally increased in the synaptic cleft. Glial glutamate transporters GLAST and GLT-1, which are responsible for clearing synaptic glutamate into glial cells, play an important role in the regulation of the glutamate concentration in the synaptic cleft. However, there has been no report on long-term changes in the levels of glutamate transporters following spinal cord injury. Spinal cord injury (SCI) was induced at T12 by a New York University (NYU) impactor. Segments of the spinal cord at T9–10, L1–2, L4–5 and at the epicenter were removed after SCI, and Western blots for GLAST, GLT-1 and EAAC1 were performed. GLAST and GLT-1 were significantly decreased in the epicenter from 1 day up to 8 weeks after SCI. GLT-1 was significantly decreased in the spinal segments rostral to the injury site, and GLAST expression was significantly increased in the L4–5 region of the spinal cord for 8 weeks. Because strategies to modulate the regulation of glutamate transporters may be applied, the present data serve as a reference for further research, although the long-term roles of glutamate transporters in pathological processes caused by SCI are not clear.

Microglial self-defence mediated through GLT-1 and glutathione

Glutamate is stored in synaptic vesicles in presynaptic neurons. It is released into the synaptic cleft to provide signalling to postsynaptic neurons. Normally, the astroglial glutamate transporters GLT-1 and GLAST take up glutamate to mediate a high signal-to-noise ratio in the synaptic signalling, and also to prevent excitotoxic effects by glutamate. In astrocytes, glutamate is transformed into glutamine, which is safely transported back to neurons. However, in pathological conditions, such as an ischemia or virus infection, astroglial transporters are down-regulated which could lead to excitotoxicity. Lately, it was shown that even microglia can express glutamate transporters during pathological events. Microglia have two systems for glutamate transport: GLT-1 for transport into the cells and the x (c) (-) system for transport out of the cells. We here review results from our work and others, which demonstrate that microglia in culture express GLT-1, but not GLAST, and transport glutamate from the extracellular space. We also show that TNF-α can induce increased microglial GLT-1 expression, possibly associating the expression with inflammatory systems. Furthermore, glutamate taken up through GLT-1 may be used for direct incorporation into glutathione and to fuel the intracellular glutamate pool to allow cystine uptake through the x (c) (-) system. This can lead to a defence against oxidative stress and have an antiviral function.

Activation of the VIP/VPAC2 system induces reactive astrocytosis associated with increased expression of glutamate transporters

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide that acts as a neuromodulator in the CNS. Recently, secretion of several functional molecules has been identified in VIP-stimulated astrocytes in vitro. However, the relationship between VIP and its specific receptors in neurological disorders remains unknown. To investigate the role of the VIP system under pathological conditions, we performed a cold injury on the right cerebrum of adult C57BL/6 mice and observed expression patterns for VIP and its receptor, VPAC2. Immunohistochemical studies revealed VPAC2 expression in reactive astrocytes around the core lesion by post-injury day 7, which then returned to contralateral levels at post-injury day 14. By contrast, VIP immunoreactivity was detected in activated microglial cells, suggesting that microglia–astrocyte interactions in the VIP/VPAC2 system are important for the tissue repair process. In primary cultured astrocytes stimulated with N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP) to mimic reactive astrocytosis, VPAC2 mRNA expression was highly up-regulated compared to that of the other VIP receptors, PAC1 and VPAC1. VPAC2 activation by the selective VPAC2 agonist, Ro25-1553, induced reactive morphological and biochemical changes from a polygonal shape to a stellate shape in cultured astrocytes. Further, Ro25-1553 increased cell surface expression of the glutamate transporters GLAST and GLT-1, which can limit excitotoxic neuronal cell death. In summary, the transient expression of VPAC2 in reactive astrocytes and the up-regulation of functional glutamate transporters suggest that the VIP/VPAC2 system induces reactive astrocytosis and plays a key role in neuroprotection against excitotoxicity in neurological disorders.

Pharmacological enhancement of glutamate transport reduces excitotoxicity in vitro

Glutamate transporters are responsible for removing glutamate from the extracellular space and have the potential to protect neurons from excitotoxicity. In the present study, the effects of ceftriaxone and (2R, 4R)-APDC (APDC) on the protein expression of GLAST and GLT-1, the rate of glutamate uptake, and neuroprotection were evaluated in a cell culture model of glutamate excitotoxicity. Mixed neuron/astrocyte cultures were prepared from 1 day old rat pups. Protein levels of GLAST and GLT-1 glutamate transporters were quantified using In-Cell Western techniques after acute or 5-day treatment with either ceftriaxone or APDC. Glutamate uptake was measured using Michaelis-Menten kinetics to evaluate the effects of 5-day treatment with ceftriaxone or APDC. Neuronal cell death in response to a 10-minute 1 mM glutamate challenge was measured following 5-day treatment with either ceftriaxone or APDC. Five-day treatment with 100 μM ceftriaxone significantly increased both GLAST and GLT-1 protein levels 31.3% and 47.5% above control, respectively, increased the Vmax 29.3%, increased the Km of glutamate uptake 117.9%, and reduced neuronal death 22.0% after a 1 mM glutamate challenge. Five-day treatment with 1 mM APDC significantly increased GLAST protein levels 27.6%, increased the Vmax 92.4%, increased the Km of glutamate transport 118.9%, and decreased neuronal death 36.8% after a 1 mM glutamate challenge. Chronic treatment with ceftriaxone or APDC provided neuroprotection from glutamate excitotoxicity while increasing GLAST and GLT-1 protein levels and increasing glutamate uptake. These compounds may have therapeutic potential in chronic excitotoxic neurodegenerative diseases.

Differential molecular regulation of glutamate in kindling resistant rats

Using pentylenetetrazol (PTZ) kindling, we collected hippocampal tissue from standard response and kindling resistant animals, measuring hippocampal mRNA with real-time PCR of glutamate transporters GLAST, GLT-1, and EAAC1 and the sodium-coupled neutral amino acid transporter (SNAT) 1, SNAT2, and SNAT3. In addition, we measured mRNA of glutamine synthetase (GS), phosphate-activated glutaminase (PAG), glutamic acid decarboxylase (GAD) 1, GAD2, and vesicular inhibitory amino acid transporter (VIAAT). Fully kindled animals had decreased expression of mRNA in the hippocampus for GLAST and GAD2 compared with saline injected control. mRNA for SNAT1, SNAT2, SNAT3, GS, and VIAAT was increased. After induction of generalized tonic-clonic seizures by PTZ there were no differences in mRNA at 24 h after seizures, equaling baseline quantities except for GAD1, which was decreased. When levels were measured at 30 days after a PTZ induced convulsive seizure, we found increased levels of GLT-1, SNAT1 and GS, but decreased levels of GAD1. When these animals, serving as control for the 30 day interval between the last convulsive seizure in the kindled experimental group, were analyzed, we found that GLT-1, SNAT3, GAD1 and VIAAT differed in that GLT-1 was decreased and the others increased. Animals found resistant to kindling had strikingly different mRNA patterns, with markedly up-regulated mRNA of proteins that transport glutamate into neurons and glia; SNAT1 was up regulated as well. Up-regulation of genes in kindling resistant animals supports the hypothesis that clearance of glutamate, conversion to glutamine and transport of glutamine into neurons, has the effect of raising the threshold for convulsive seizures and attenuating kindling.

Cardiac Glycosides Ouabain and Digoxin Interfere with the Regulation of Glutamate Transporter GLAST in Astrocytes Cultured from Neonatal Rat Brain

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na+, K+)-dependent ATPase (Na+/K+-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na+/K+-ATPase. We now report that Na+/K+-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na+/K+-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic.

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex

Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2(+) glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.

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.

Evidence for a role of glutamate as an efferent transmitter in taste buds

BackgroundGlutamate has been proposed as a transmitter in the peripheral taste system in addition to its well-documented role as an umami taste stimulus. Evidence for a role as a transmitter includes the presence of ionotropic glutamate receptors in nerve fibers and taste cells, as well as the expression of the glutamate transporter GLAST in Type I taste cells. However, the source and targets of glutamate in lingual tissue are unclear. In the present study, we used molecular, physiological and immunohistochemical methods to investigate the origin of glutamate as well as the targeted receptors in taste buds.ResultsUsing molecular and immunohistochemical techniques, we show that the vesicular transporters for glutamate, VGLUT 1 and 2, but not VGLUT3, are expressed in the nerve fibers surrounding taste buds but likely not in taste cells themselves. Further, we show that P2X2, a specific marker for gustatory but not trigeminal fibers, co-localizes with VGLUT2, suggesting the VGLUT-expressing nerve fibers are of gustatory origin. Calcium imaging indicates that GAD67-GFP Type III taste cells, but not T1R3-GFP Type II cells, respond to glutamate at concentrations expected for a glutamate transmitter, and further, that these responses are partially blocked by NBQX, a specific AMPA/Kainate receptor antagonist. RT-PCR and immunohistochemistry confirm the presence of the Kainate receptor GluR7 in Type III taste cells, suggesting it may be a target of glutamate released from gustatory nerve fibers.ConclusionsTaken together, the results suggest that glutamate may be released from gustatory nerve fibers using a vesicular mechanism to modulate Type III taste cells via GluR7.

Neural precursor-derived astrocytes of wobbler mice induce apoptotic death of motor neurons through reduced glutamate uptake

In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway. NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.

Co-administration of ultra-low dose naloxone attenuates morphine tolerance in rats via attenuation of NMDA receptor neurotransmission and suppression of neuroinflammation in the spinal cords

Although mechanisms underlying ultra-low dose naloxone-induced analgesia have been proposed, possible interactions with glutamatergic transmission and glial cell activation have not been addressed. In the present study, we examined the effect of ultra-low dose naloxone on spinal glutamatergic transmission and glial cell activity in rats chronically infused with morphine. In male Wistar rats, intrathecal morphine infusion (15 μg/h) for 5 days induced (1) antinociceptive tolerance, (2) downregulation of glutamate transporters (GTs) GLT-1, GLAST, and EAAC1, (3) increasing of NMDA receptor (NMDAR) NR1 subunit expression and phosphorylation, (4) upregulation of protein kinase C gamma (PKCγ) expression, and (5) glial cell activation. On day 5, morphine challenge (15 μg/10 μl) caused a significant increase in the concentration of the excitatory amino acids (EAAs) aspartate and glutamate in the spinal CSF dialysates of morphine-tolerant rats. Intrathecal co-infusion of ultra-low dose naloxone (15 pg/h) with morphine attenuated tolerance development, reversed GTs expression, inhibited the NMDAR NR1 subunit expression and phosphorylation, and PKCγ expression, inhibited glial cell activation, and suppressed the morphine-evoked EAAs release. These effects may result in preservation of the antinociceptive effect of acute morphine challenge in chronic morphine-infused rats. Ultra-low dose naloxone infusion alone did not produce an antinociceptive effect. These findings demonstrated that attenuation of glutamatergic transmission and neuroinflammation by ultra-low dose naloxone co-infusion preserves the lasting antinociceptive effect of morphine in rats chronically infused with morphine.

GLAST stability and activity are enhanced by interaction with the PDZ scaffold NHERF-2

The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na +/H + exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced half-life of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.

Ethanol and phencyclidine interact with respect to nucleus accumbens dopamine release: differential effects of administration order and pretreatment protocol.

Executive dysfunction is a common symptom among alcohol-dependent individuals. Phencyclidine (PCP) injection induces dysfunction in the prefrontal cortex of animals but little is known about how PCP affects the response to ethanol. Using the in vivo microdialysis technique in male Wistar rats, we investigated how systemic injection of 5 mg/kg PCP would affect the dopamine release induced by local infusion of 300 mM ethanol into the nucleus accumbens. PCP given 60 min before ethanol entirely blocked ethanol-induced dopamine release. However, when ethanol was administered 60 min before PCP, both drugs induced dopamine release and PCP's effect was potentiated by ethanol (180% increase vs 150%). To test the role of prefrontal cortex dysfunction in ethanol reinforcement, animals were pretreated for 5 days with 2.58 mg/kg PCP according to previously used ‘PFC hypofunction protocols’. This, however, did not change the relative response to PCP or ethanol compared to saline-treated controls. qPCR illustrated that this low PCP dose did not significantly change expression of glucose transporters Glut1 (SLC2A1) or Glut3 (SLC2A3), monocarboxylate transporter MCT2 (SLC16A7), glutamate transporters GLT-1 (SLC1A2) or GLAST (SLC1A3), the immediate early gene Arc (Arg3.1) or GABAergic neuron markers GAT-1 (SLC6A1) and parvalbumin. Therefore, we concluded that PCP at a dose of 2.58 mg/kg for 5 days did not induce hypofunction in Wistar rats. However, PCP and ethanol do have overlapping mechanisms of action and these drugs differentially affect mesolimbic dopaminergic transmission depending on the order of administration.

Discriminative behavioral assessment unveils remarkable reactive astrocytosis and early molecular correlates in basal ganglia of 3-nitropropionic acid subchronic treated rats

Reactive astrocytosis seems to be strongly implicated in the development and maintenance of inflammatory and neurodegenerative disorders. We design a new toxic model treatment with 3-nitropropionic acid (3-NP), a mitochondrial complex II irreversible inhibitor, to induce in rats Huntington's disease (HD) like syndrome, characterized by hindlimb dystonia, involuntary choreiform movements and reduced global activity. In an attempt to find out whether molecular and morphological changes in the neuro-glial network could be involved in the pathogenesis of this disease, we developed a protocol of subchronic intra-peritoneal 3-NP intoxication. Moreover we set up specific, highly discriminative, behavioral tests to detect very early mild motor disabilities in 3-NP treated rats. This treatment did not cause severe cell death. However, in the Caudate-Putamen (CPu) of all 3-NP treated animals we found a massive astrogliosis, revealed by increased GFAP levels, paralleled by changes of the glial glutamate transporter GLAST distribution. To these glial changes we detected a transcriptional upregulation of c-fos and Sub-P in the striatal medium spiny neurons (MSN). We propose that this model of 3-NP intoxication along with the designed set of behavioral analyses allow to unmask in a very early phase the motor deficits and the underlying morpho-molecular changes associated to the onset of motor disabilities in the HD-like syndrome. Therefore this model unveil the key role played by the different components of the tripartite synapse in the pathogenesis of the HD, a putative non-cell-autonomous disease.

Glutamate Transporter Coupling to Na,K-ATPase

Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The alpha1-alpha 3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [(3)H]D-aspartate, [(3)H]L-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 microm ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [(3)H]D-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.

Rottlerin Inhibits (Na+, K+)-ATPase Activity in Brain Tissue and Alters d-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-delta) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-delta 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-delta-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.

Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc − in mouse retinal Müller glial cells

Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system x(c)(-) (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na(+)-dependent and -independent uptake of [(3)H] glutamate was assessed as a measure of GLAST and system x(c)(-) function, respectively. Hyperglycemia did not alter the uptake of [(3)H] glutamate by GLAST or system x(c)(-); neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 microM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by approximately 10% but increased system x(c)(-) activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in V(max), but not K(m). Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system x(c)(-). Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system x(c)(-) activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione.