Glutamate stimulates ascorbate transport by astrocytes

Cell Biochemistry and Biophysics, 2013

Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbaterelease was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na ? -dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated.

Brain Research, 2000

Expression of the Na -ascorbate cotransporter, SVCT2, was detected in rat brain and in primary cultures of cerebral astrocytes by Northern blot analysis. SVCT2 expression in cultured astrocytes increased in response to the cyclic AMP analog, dibutyryl cyclic AMP. A 1 mathematical model of ascorbic acid transport was developed to evaluate the hypothesis that Na -ascorbate cotransport across the plasma membrane regulates the steady state intracellular concentration of ascorbic acid in these cells. The outcomes predicted by this model were compared to experimental observations obtained with primary cultures of rat cerebral astrocytes exposed to normal and pathologic conditions. Both cotransport activity and intracellular ascorbic acid concentration increased in astrocytes activated by dibutyryl cyclic 1 AMP. Conversely transport activity and ascorbic acid concentration were decreased by hyposmotic cell swelling, low extracellular Na 1 concentration, and depolarizing levels of extracellular K . In cells incubated for up to 3 h in medium having an ascorbic acid concentration typical of brain extracellular fluid, the changes in intracellular ascorbic acid concentration actually measured were not 1 significantly different from those predicted by modeling changes in Na -ascorbate cotransport activity. Thus, it was not necessary to specify alterations in vitamin C metabolism or efflux pathways in order to predict the steady state intracellular ascorbic acid concentration. These results establish that SVCT2 regulates intracellular ascorbic acid concentration in primary astrocyte cultures. They further indicate 1 that the intracellular-to-extracellular ratio of ascorbic acid concentration at steady state depends on the electrochemical gradients of Na and ascorbate across the plasma membrane.

2000

The excitatory transmitter glutamate (Glu), and its analogs kainate (K A), and D-aspartate (D-Asp) produce significant pH changes in glial cells. Transmitter-induced pH changes in glial cells, generating changes in extracellular pH, may represent a special form of neuronal-glial interaction. We investigated the mechanisms underlying these changes in intracellular H ϩ concentration ([H ϩ ] i ) in cultured rat hippocampal astrocytes and studied their correlation with increases in intracellular Na ϩ concentration ([Na ϩ ] i ), using fluorescence ratio imaging with 2Ј,7Ј-bis(carboxyethyl)-5,6carboxyfluorescein (BCECF) or sodium-binding benzofuran isophthalate (SBFI). Glu, K A, or D-Asp evoked increases in [Na ϩ ] i ; Glu or D-Asp produced parallel acidifications. K A, in contrast, evoked biphasic changes in [H ϩ ] i , alkaline followed by acid shifts, which were unaltered after Ca 2ϩ removal and persisted in 0 Cl Ϫ -saline, but were greatly reduced in CO 2 /HCO 3 Ϫ -free or Na ϩfree saline, or during 4,4Ј-diisothiocyanato-stilbene-2,2Јdisulphonic acid (DIDS) application. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked K A-evoked changes in [H ϩ ] i and [Na ϩ ] i , indicating that they were receptor-ionophore mediated. In contrast, CNQX increased the [H ϩ ] i change and decreased the [Na ϩ ] i change induced by Glu. D-Asp, which is transported but does not act at Glu receptors, induced [H ϩ ] i and [Na ϩ ] i changes that were virtually unaltered by CNQX. Our study indicates that [Na ϩ ] i increases are not primarily responsible for Glu-or K A-induced acidifications in astrocytes. Instead, intracellular acidifications evoked by Glu or D-Asp are mainly caused by transmembrane movement of acid equivalents associated with Glu/Asp-uptake into astrocytes. K A-evoked biphasic [H ϩ ] i changes, in contrast, are probably attributable to transmembrane ion movements mediated by inward, followed by outward, electrogenic Na ϩ /HCO 3 Ϫ cotransport, reflecting K Ainduced biphasic membrane potential changes.

Neurochemical Research, 2003

The regulation of the Na ϩ -dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with L-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca 2ϩ / diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [ 3 H]-D-aspartate uptake. This reduction was dose-and time-dependent and sensitive to PKC inhibitors. Furthermore, the L-glutamate-dependent [ 3 H]-D-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the L-glutamate effect. In sharp contrast, in cortical astrocytes, both L-glutamate and TPA pre-exposure result in an augmentation of the [ 3 H]-D-aspartate uptake. These findings suggest that the Na ϩ -dependent glutamate uptake GLAST undergoes a region-specific regulation.

Annals of the New York Academy of Sciences, 1991

Neuroscience Letters, 2005

To determine if extracellular ascorbate, which may increase by several hundred micromolar in striatum during behavioral activation, directly alters glutamate transmission, we monitored striatal glutamate transients evoked by electrical stimulation of cerebral cortex in anesthetized rats tested with varying concentrations of ascorbate (0, 50, 200, and 500 microM) by reverse dialysis. Capillary electrophoresis coupled with laser-induced fluorescence detection was used to analyze dialysates collected at 3-s intervals. Ascorbate elevated striatal glutamate in a concentration-dependent fashion. Addition of 500 microM ascorbate not only more than doubled basal glutamate levels relative to the ascorbate-free condition, but significantly increased both the magnitude of the electrically evoked glutamate response as well as its subsequent return to baseline. In fact, the time required to return to within 10% of the pre-stimulation baseline increased by >100s. Reverse dialysis of iso-ascorbate, in contrast, had no effect on stimulation-evoked glutamate release arguing against an antioxidant effect. It appears, therefore, that the level of extracellular ascorbate plays a critical role in regulating corticostriatal glutamate transmission.

Journal of Neurochemistry, 2007

It has been demonstrated that glutamatergic activity induces ascorbic acid (AA) depletion in astrocytes. Additionally, different data indicate that AA may inhibit glucose accumulation in primary cultures of rat hippocampal neurons. Thus, our hypothesis postulates that AA released from the astrocytes during glutamatergic synaptic activity may inhibit glucose uptake by neurons. We observed that cultured neurons express the sodium-vitamin C cotransporter 2 and the facilitative glucose transporters (GLUT) 1 and 3, however, in hippocampal brain slices GLUT3 was the main transporter detected. Functional activity of GLUTs was confirmed by means of kinetic analysis using 2-deoxy-D-glucose. Therefore, we showed that AA, once accumulated inside the cell, inhibits glucose transport in both cortical and hippocampal neurons in culture. Additionally, we showed that astrocytes are not affected by AA. Using hippocampal slices, we observed that upon blockade of monocarboxylate utilization by a-cyano-4-hydroxycinnamate and after glucose deprivation, glucose could rescue neuronal response to electrical stimulation only if AA uptake is prevented. Finally, using a transwell system of separated neuronal and astrocytic cultures, we observed that glutamate can reduce glucose transport in neurons only in presence of AA-loaded astrocytes, suggesting the essential role of astrocyte-released AA in this effect.

Neurochemistry International, 1997

The characteristics of high-K+ and electricallyevokedendogenousglutamate and [3H]D-aspartate release have been studied in multiple in oitrw preparations of the rat hippocampus(transverse slices, granulecellscultures, synaptosomesand mossyfibresynaptosomes)under similarexperimentalconditions. High external K+ concentrations evoked [3H]D-aspartateand endogenousglutamate overflowin a concentration-dependent manner in all preparations (except it was not possible to measure endogenous glutamate outflow from granule celk). This effect was tetrodotoxin-insensitivebut partially calciumdependent. In slices,field electrical stimulation evoked an overtlowof endogenousglutamate, but not of [3H]D-aspartate,in a frequency-dependentmanner. This effect was concentration-dependentlyamplified bytheglutamate uptake inhibitorL-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC).The electricallyevoked glutamate overtlowin the presenceof t-PDCwas tetrodotoxin-sensitiveandcalcium-dependent.In primary dentate gyrus cell cultures, electrical stimulation evoked an overtlowof [3H]D-aspartatein a frequencydependentmanner, whileendogenousglutamate outflowwas not detectable. This effectcould be inhibited by tetrodotoxin and by the N-type calcium channel blocker ro-conotoxinGVIA. Finally, the effect of adenosine has been studied in order to assess the pharmacologicalmodulability of [3H]D-aspartate and endogenous glutamate stimulation-inducedoverflow.Adenosine was found to inhibit 35mM K+-and 20Hz electrical stimulation-induced[3H]D-aspartateand endogenousglutamate overflow.These effects were all prevented by the Al receptor antagonist 8-cyclopentyl-l,3 dimethylxanthine (CPT). These data are in line with the hypothesisthat reuptake plays a role in regulating glutamate release, and that [3H]Daspartate represents a valid marker of endogenous glutamate under most (but not all) experimental conditions. Q 1997ElsevierScienceLtd 113

Neuroscience Letters, 1985

Key words: amino acid glutamate aspartate-ventricular cerebrospinal fluid -hydrocephalus cerebral ischemia

Journal of Neuroscience Research, 2013

Ascorbate (vitamin C) is a nonenzymatic antioxidant highly concentrated in the brain. In addition to mediating redox balance, ascorbate is linked to glutamate neurotransmission in the striatum, where it renders neuroprotection against excessive glutamate stimulation. Oxidative stress and glutamatergic overactivity are key biochemical features accompanying the loss of dopaminergic neurons in the substantia nigra that characterizes Parkinson's disease (PD). At present, it is not clear whether antiglutamate agents and ascorbate might be neuroprotective agents for PD. Thus, we tested whether ascorbate can prevent cell death from prolonged exposure to glutamate using dopaminergic neurons of human origin. To this purpose, dopamine-like neurons were obtained by differentiation of SH-SY5Y cells and then cultured for 4 days without antioxidant (antiaging) protection to evaluate glutamate toxicity and ascorbate protection as a model system of potential factors contributing to dopaminergic neuron death in PD. Glutamate dose dependently induced toxicity in dopaminergic cells largely by the stimulation of AMPA and metabotropic receptors and to a lesser extent by N-methyl-D-aspartate and kainate receptors. At relatively physiological levels of extracellular concentration, ascorbate protected cells against glutamate excitotoxicity. This neuroprotection apparently relies on the inhibition of oxidative stress, because ascorbate prevented the pro-oxidant action of the scavenging molecule quercetin, which occurred over the course of prolonged exposure, as is also seen with glutamate. Our findings show the relevance of ascorbate as a neuroprotective agent and emphasize an often underappreciated role of oxidative stress in glutamate excitotoxicity. Occurrence of a glutamate-ascorbate link in dopaminergic neurons may explain previous contradictions regarding their putative role in PD. V