Nobuo Makino

Redox Imbalance in Cystine/Glutamate Transporter-deficient Mice

Cystine/glutamate transporter, designated as system x–c, mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT–/– mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT–/– mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT–/– mice was lower than that in the xCT–/– mice. The embryonic fibroblasts derived from xCT–/– mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT–/–-derived cells and permitted growth. These results demonstrate that system x–c contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.

Glutathione Efflux from Cultured Astrocytes

Abstract: The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25°C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis‐Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p‐Chloromercuribenzenesulfonic acid, a thiol‐reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.

Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture

H2O2 is a key substance in the oxidative stress. To evaluate the antioxidant activity of intact human umbilical vein endothelial cells (HUVEC), we measured the H2O2 removal rate by the cell in monolayer culture at various H2O2 concentrations (1-300 microM). It was shown that the removal reaction can be divided into two kinetically different reactions: reaction 1 apparently following the Michaelis-Menten kinetics and reaction 2 following the first-order kinetics. Reaction 1, which was diminished by treatment with diethyl maleate, could be attributed to GPx. Reaction 2, which was inhibited by aminotriazole, was principally attributable to catalase, though non-enzymatic reactions may contribute to it partially. Furthermore, we have constructed a mathematical model for the H2O2 elimination including the pentose phosphate pathway enzymes, GSSG reductase and GSH peroxidase. On the basis of the known kinetics and observed activities of the enzymes, the model could reproduce well the observed concentration dependence of the H2O2 removal rate. It was suggested from the simulation study that GSSG reductase is more important than G6PD in determining the rate of the NADPH-dependent H2O2 elimination.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS‐mediated oxidant injury. Cystine/glutamate transporter, designated as system xc– and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian‐cultured cells. In the present paper, we have investigated system xc– in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc– nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc– activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT‐deficient (xCT−/−) mice had lower cysteine content than those from the wild‐type mice. GSH levels in the xCT−/−cells decreased rapidly when they were cultured, whereas those in the wild‐type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT−/−cells compared with the wild‐type cells. These results suggest that system xc– plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase.

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and brain ischemia, and hydrogen peroxide (H(2)O(2)) plays a central role in the stress. In this study, we have examined the kinetics of H(2)O(2) elimination by PC12 cells as a neuronal model in connection with the enzyme activities supporting the reaction. Similarly to other cell lines previously studied, H(2)O(2) removal kinetics could be divided into two reactions: one apparently following the Michaelis-Menten kinetics (GSH-dependent reaction) and the other following the first-order kinetics (mainly catalyzed by catalase). Based on the enzyme activities in the cell homogenate, it was inferred that glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the GSH- and NADPH-dependent H(2)O(2) elimination by PC12 cells. This is in contrast with fibroblasts and endothelial cells previously examined, in which glutathione reductase (GR) is rate-limiting in the reaction sequence. Treatment of PC12 cells with nerve growth factor increased G6PD activity in the cell homogenate and H(2)O(2) removal activity of the whole cells, with a concomitant increase in the resistance against H(2)O(2) toxicity. These results suggest the importance of G6PD in the antioxidant function of brain and pathogenesis of the oxidative stress-related diseases.

Kinetics of hydrogen peroxide elimination by astrocytes and C6 glioma cells: Analysis based on a mathematical model

Oxidative stress is implicated in a variety of disorders including neurodegenerative diseases, and H(2)O(2) is important in the generation of reactive oxygen and oxidative stress. In this study, we have examined the rate of extracellular H(2)O(2) elimination and relevant enzyme activities in cultured astrocytes and C6 glioma cells and have analyzed the results based on a mathematical model. As compared with other types of cultured cells, astrocytes showed higher activity of glutathione peroxidase (GPx) but lower activities for GSH recycling. C6 cells showed relatively low GPx activity, and treatment of C6 cells with dibutyryl-cAMP, which induces astrocytic differentiation, increased catalase activity and H(2)O(2) permeation rate but exerted little effect on other enzyme activities. A mathematical model [N. Makino, K. Sasaki, N. Hashida, Y. Sakakura, A metabolic model describing the H(2)O(2) elimination by mammalian cells including H(2)O(2) permeation through cytoplasmic and peroxisomal membranes: comparison with experimental data, Biochim. Biophys. Acta 1673 (2004) 149-159.], which includes relevant enzymes and H(2)O(2) permeation through membranes, was found to be fitted well to the H(2)O(2) concentration dependences of removal reaction with the permeation rate constants as variable parameters. As compared with PC12 cells as a culture model for neuron, H(2)O(2) removal activity of astrocytes was considerably higher at physiological H(2)O(2) concentrations. The details of the mathematical model are presented in Appendix.

Kinetic studies on the removal of extracellular tert-butyl hydroperoxide by cultured fibroblasts

In fibroblasts toxic hydroperoxides are removed mainly by GSH peroxidase. The reaction depends on NADPH, since GSSG must be reduced by GSSG reductase for recycling. In this work we have studied the kinetics of tert-butyl hydroperoxide (tBH) removal by cultured fibroblasts in relation to the GSSG reduction. The rate of the reaction showed biphasic dependence on tBH concentration. About a third of the reaction was saturated below 10 microM tBH, while the rest of the reaction showed less steep dependence, reaching a plateau at 200 microM tBH. The latter reaction is thought to be due to GSH peroxidase, and the concentration dependence could be explained on the basis of reaction kinetics of GSH peroxidase and GSSG reductase. The maximum rate of tBH removal was estimated as 40-50 nmol tBH/min/mg of protein, while the glutathione reductase activity is the solubilized cell was 33.0 +/- 3.5 nmol GSSG/min/mg of protein. It was concluded that, under the oxidative stress as in the present experiments, the step catalyzed by GSSG reductase is rate-limiting in the reaction sequence.

Interactions of Nitric Oxide and Oxygen in Cytotoxicity: Proliferation and Antioxidant Enzyme Activities of Endothelial Cells in Culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O2 in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-1-ium-1,2-diolate) (DETA-NONOate) (100–500 μM), under normoxia (air), hypoxia (< 0.04% O2) or hyperoxia (88–94% O2). It was found that the dose dependency on NONOate was little affected by the ambient O2 concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H2O2 elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 μM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24 h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H2O2 removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.

Conflicting effects of N-acetylcysteine on purified neurons derived from rat cortical culture

We examined the protective effects of N-acetylcysteine (NAC) on the death of glia-free neurons in culture. Under normoxic conditions, the protection by NAC was observed only in cystine-free but not complete medium. When the cells were cultured under hypoxic conditions, NAC much elongated their survival even in the presence of cystine. H2O2 was found to be generated to considerable concentration in the presence of both NAC and cystine, and the administration of catalase prevented the cell death. These results suggest that the harmful effect of NAC is because of H2O2 generated by autoxidation of cysteine, which derives from the reaction between NAC and cystine. The present results raise the possibility that NAC can act as either antioxidant or prooxidant depending on the milieu.

The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model. The model consists of seven reactions and three transports, which are grouped into four categories: autoxidation of thiols into disulphides, thiol-disulphide exchange and reactions of thiols with medium components, as well as the cellular influx and efflux of thiols and disulphides. The obtained results are interpreted that cystine (CSSC) after entering astrocyte is reduced to CSH, most of which is released to medium and autoxidized to CSSC. The efflux of GSH was estimated to be considerably slower than that of CSH, and most of the excreted GSH is converted to cysteine-glutathione disulphide principally through the thiol-disulphide exchange. The results seem to indicate that astrocytes provide neurons mainly with CSH, rather than GSH, as the antioxidant material for neuroprotection.