Cysteine, Glutathione, and Thiol Redox Balance in Astrocytes.

This review discusses the current understanding of cysteine and glutathione redox balance in astrocytes. Particular emphasis is placed on the impact of oxidative stress and astrocyte activation on pathways that provide cysteine as a precursor for glutathione. The effect of the disruption of thiol-containing amino acid metabolism on the antioxidant capacity of astrocytes is also discussed.

Thiol redox homeostasis in neurodegenerative disease.

This review provides an overview of the biochemistry of thiol redox couples and the significance of thiol redox homeostasis in neurodegenerative disease. The discussion is centred on cysteine/cystine redox balance, the significance of the xc(-) cystine-glutamate exchanger and the association between protein thiol redox balance and neurodegeneration, with particular reference to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and glaucoma. The role of thiol disulphide oxidoreductases in providing neuroprotection is also discussed.

The transsulfuration pathway: a source of cysteine for glutathione in astrocytes.

Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (γ-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the γ-glutamylcycle leading to formation of glutathione (L: -γ-glutamyl-L: -cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the x (c) (-) cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from the indispensable amino acid, methionine, has recently been identified as a significant contributor to GSH synthesis in astrocytes. The purpose of this review is to evaluate the importance of the transsulfuration pathway in these cells, particularly in the context of a reserve pathway that channels methionine towards cysteine when the demand for glutathione is high, or under conditions in which the supply of cystine by the x (c) (-) exchanger may be compromised.

A flow-cytometric method for continuous measurement of intracellular Ca(2+) concentration.

Alterations in intracellular Ca(2+) concentration are amongst the most rapid responses to a variety of stimuli in mammalian cells. In the nervous system in particular, responses occur within nanoseconds. A major challenge in intracellular Ca(2+) analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca(2+) concentration has been monitored by confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry, although there are issues with the number of cells analyzed or gaps in recording due to the addition of compounds, with significant loss of detail of a rapid Ca(2+) response. The new generation of flow cytometers (such as Accuri C6) resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic Ca(2+) measurements. This system was tested with commonly used Ca(2+) modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca(2+) uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca(2+) entry via store-operated Ca(2+) channels (SOCC). This well-established pathway can be partially inhibited by 2-aminoethoxydiphenyl borate (2-APB), a blocker of SOCC. Both the increase with TG alone and the partial increase when coincubated with 2-APB were observed with continuous recording along with calibration curves using an Accuri C6 flow cytometer. With these new cytometers, dynamic Ca(2+) concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness.

Glutathione depletion causes a JNK and p38MAPK-mediated increase in expression of cystathionine-gamma-lyase and upregulation of the transsulfuration pathway in C6 glioma cells.

Cancer cells have a high demand for cysteine as precursor of the antioxidant, glutathione, that is required to promote cell growth and division. Uptake of cystine by the x(c)(-) cystine-glutamate exchanger provides the majority of cysteine, but a significant percentage may be derived from methionine, via a transsulfuration pathway. Our aim was to evaluate the relative contribution of the exchanger and the transsulfuration pathway to glutathione synthesis in astrocytoma/glioblastoma cells, using the C6 glioma cell line as a model system. Blockade of the x(c)(-) exchanger with the gliotoxins l-alphaaminoadipate or l-beta-N-oxalylamino-l-alanine (400 microM) caused a loss of cellular cysteine and depletion in glutathione to 51% and 54% of control, respectively, after 24 h. Inhibition of the transsulfuration pathway with propargylglycine (1 mM, 24 h) depleted glutathione to 77% of control. Co-incubation of cells with gliotoxin and propargylglycine reduced glutathione to 39% of control at 24 h and to 20% at 48 h. Expression of cystathionine-gamma-lyase, the rate-limiting enzyme of the transsulfuration pathway, was significantly increased following incubation of the cells with gliotoxins. Incubation of C6 cells with diethylmaleate for 3 h led to a significant reduction in glutathione (63%), whereas expression of cystathionine-gamma-lyase was increased by 1.5-fold. Re-feeding methionine to diethylmaleate-treated cells incubated in the absence of cystine or methionine resulted in a significant recovery in glutathione that was blocked by propargylglycine. Co-incubation of C6 cells with diethylmaleate and the JNK-inhibitor, SP600125, abolished the increase in expression of cystathionine-gamma-lyase that had been observed in the presence of diethylmaleate alone. Similar results were obtained with the p38(MAPK) inhibitor, SB203580. It is concluded that glutathione depletion causes a JNK- and p38(MAPK)-mediated increase in expression of cystathionine-gamma-lyase that promotes flux through the transsulfuration pathway to compensate for loss of glutathione in C6 glioma cells.

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx.

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca2+ ATP pump inhibitor, thapsigargin (10 microM) produced a time-dependent increase in the Vmax for D-[3H]aspartate transport that reached a maximum at 15 min (143% of control; P<0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the Vmax of sodium-dependent D-[3H]aspartate transport (190% of control; P<0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in D-[3H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 microM) or SKF 96365 (10 microM) caused a net inhibition of D-[3H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in D-[3H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells.

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.

Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid.

At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.

Astroglial plasticity and glutamate function in a chronic mouse model of Parkinson's disease.

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases, astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1. However, in Parkinson's disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse model of Parkinson's disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8 weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the progressive nature of the pathophysiology associated with Parkinson's disease.

Gliotoxins disrupt alanine metabolism and glutathione production in C6 glioma cells: a 13C NMR spectroscopic study.

Gliotoxins are a group of amino acids that are toxic to astrocytes, and are substrates of high-affinity sodium-dependent glutamate transporters. In the present study, C6 glioma cells were preincubated for 20 h in the presence of 400 microM L-alpha-aminoadipate, L-serine-O-sulphate, D-aspartate or L-cysteate, as well as in the presence of the poorly transported L-glutamate uptake inhibitor, L-anti-endo-methanopyrrolidine dicarboxylate. In experiments following [3-13C]alanine metabolism, all toxins caused a decreased incorporation of label into glutamate. Production of labelled lactate changed only when cells were incubated in the presence of L-alpha-aminoadipate or L-serine-O-sulphate. Incubation with L-anti-endo-methanopyrrolidine dicarboxylate caused no change in the amount of label incorporated into either glutamate or lactate. When glutathione production was followed using 1 mM [2-13C]glycine, differential effects of the gliotoxins were revealed. Most notably, both L-serine-O-sulphate and L-alpha-aminoadipate caused significant increases in labelling of glutathione. Once again, L-anti-endo-methanopyrrolidine dicarboxylate was without effect. Overall, we have shown that the gliotoxins cause disruption to alanine metabolism and glutathione production in C6 glioma cells, but that there are notable differences in their mechanisms of action. In the absence of any disruption to metabolism by L-anti-endo-methanopyrrolidine dicarboxylate, it is concluded that their mode of action involves more than inhibition of glutamate transport.

An investigation into the role of calcium in the modulation of rat synaptosomal D-[3H]aspartate transport by docosahexaenoic acid.

The effect of the polyunsaturated fatty acid cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the high-affinity, sodium-dependent uptake of D-[3H]aspartate into purified rat brain synaptosomes was examined. Incubation of the synaptosomes with 20 microM DHA caused over 50% inhibition of the maximum velocity (V(max)) of D-[3H]aspartate transport. This inhibition was significantly potentiated by pre-exposure of the synaptosomes to the fatty acid for 10 min prior to the start of the transport assay. Less highly unsaturated fatty acids such as arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid), linolenic acid (cis-9,12,15-octadecatrienoic acid) and oleic acid (cis-9-octadecenoic acid) were significantly less potent than DHA. Removal of extracellular calcium, or reduction of the intracellular calcium concentration using the intracellular calcium chelator BAPTA/AM (10 microM), did not reduce the inhibition caused by DHA. On the other hand, an increase in the concentration of intracellular calcium mediated by thapsigargin (25 microM) or the calcium ionophore A23187 (10 or 100 nM) led to a reduction in the rate of D-[3H]aspartate transport in the absence of DHA. The CaM kinase II inhibitor, KN-93, reduced D-[3H]aspartate uptake independently of whether DHA was also present, but had no effect on the inhibition of D-[3H]aspartate uptake by either A23187 or thapsigargin. We conclude that whereas DHA inhibits synaptosomal D-[3H]aspartate uptake in a calcium-independent manner, a calcium-based mechanism exists that can also modulate glutamate transporter activity.

An NMR study of alterations in [1-13C]glucose metabolism in C6 glioma cells by gliotoxic amino acids.

A series of glutamate analogues, known as gliotoxins, are toxic to astrocytes in culture, and are inhibitors or substrates of high affinity sodium-dependent glutamate transporters. The mechanisms by which these gliotoxins cause toxicity are not fully understood. The effects of a series of gliotoxic amino acids (L-alpha-aminoadipate, L-serine-O-sulphate, D-aspartate and L-cysteate) on metabolism of [1-13C]glucose were examined in C6 glioma cells using 13C nuclear magnetic resonance (NMR) spectroscopy. The cells were preincubated in the presence of sub toxic concentrations of each gliotoxin (400 micromol/l) for 20 h. This was followed by incubation (4 h) with [1-13C]glucose (5.5 mmol/l) in the presence and absence of each gliotoxin. The incorporation of 13C label into the observed metabolites was analysed. Following preincubation with L-alpha-aminoadipate, D-aspartate, and L-serine-O-sulphate there was a significant decrease in the incorporation of 13C label into glutamate, alanine and lactate from [1-13C]glucose. In the presence of L-cysteate production of labelled glutamate was decreased, while there was no significant effect on the concentrations of labelled lactate and alanine. There was no change in the quantity of LDH released into the medium after incubation of the cells with any of the gliotoxins. Overall these results indicate that the presence of gliotoxins profoundly alters the flux of glucose to lactate, alanine, aspartate and glutamate.

In vitro neuronal and vascular responses to 5-hydroxytryptamine: modulation by 4-methylthioamphetamine, 4-methylthiomethamphetamine and 3,4-methylenedioxymethamphetamine.

4-Methylthioamphetamine and 4-methylthiomethamphetamine are thioarylethylamines structurally related to 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy'). This study compared effects of these agents and MDMA on 5-hydroxytryptamine (5-HT) signalling systems in the brain and vasculature in vitro. Both 4-methylthioamphetamine and 4-methylthiomethamphetamine (100 micro M) reduced the rate of specific high affinity [3H]5-HT reuptake in isolated rat brain synaptosomes to 14% and 10% of control, respectively. The concentration required for half-maximal inhibition (IC(50)) of [3H]5-HT reuptake by 4-methylthioamphetamine (0.27 micro M) was significantly lower (P<0.005) than that for inhibition by MDMA (1.28 micro M) and that for inhibition by 4-methylthiomethamphetamine (0.89 micro M). Both 4-MTA and 4-MTMA caused a significant release of preloaded [3H]5-HT from synaptosomes, but were significantly less effective than MDMA at the concentrations tested (1-100 micro M). In isolated rat aorta, a 15-min preincubation with 4-methylthioamphetamine or 4-methylthiomethamphetamine significantly reduced the maximal contraction (E(max)) induced by 5-HT to 71% or 91% of control (P<0.05 in each case), respectively. In addition, 4-methylthiomethamphetamine (100 micro M), but not 4-methylthioamphetamine, significantly increased the concentration of 5-HT required for half-maximal contraction (EC(50)) from 4.13 to 20.08 micro M (P<0.0001). In contrast, MDMA did not significantly alter the E(max) or the EC(50) of 5-HT-induced aortic contraction. It is concluded that both 4-methylthioamphetamine and 4-methylthiomethamphetamine are potent inhibitors of [3H]5-HT reuptake in the brain. Furthermore, unlike MDMA, they both directly inhibit 5-HT-mediated vascular contraction. These results suggest that these compounds may be potentially more harmful than MDMA in the context of human misuse.

Cerebral cystine uptake: a tale of two transporters.

Transport of cystine across the cell membrane is essential for synthesis of the major cellular antioxidant glutathione. Cystine uptake in the brain occurs by both the Na(+)-independent x(c)(-) cystine-glutamate exchanger and the X(AG)(-) family of high-affinity, Na(+)-dependent glutamate transporters. New evidence concerning the role of cystine transport in the defence against oxidative stress is described.