Jun-ichi Sagara

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.

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine‐free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na+‐independent, glutamate‐sensitive route previously found in various types of cells and designated as system x−c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.

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.

Characterization of cytosolic glutathione S-transferase in cultured astrocytes.

To elucidate the contribution of glutathione S-transferase (GST) and glutathione peroxidase (GPx) to the protection against oxidative stress in rat brain, we prepared GST and GPx from newborn rat liver, brain and cultured astrocytes, and investigated the characteristics and kinetics of the enzymes. The activity of cytosolic GST of the cultured astrocytes toward 1-chloro-2,4-dinitrobenzene (CDNB) was much higher than that of GPx toward peroxides. The GST activity toward 4-hydroxy-2-nonenal (4HNE) was almost the same as the GPx activity. GST isozymes were purified from the cytosolic fraction of the liver and astrocytes. In the case of the astrocytes, a major GST isozyme with an isoelectric point (pI) of 9.02 accounted for approximately 40% of total GST activity toward CDNB, while hepatic GST isozymes showed seven peaks in the basic region. Each of astrocytes and liver showed a single GST peak with high activity toward 4HNE, namely AVIII and LVIII, respectively, and both of them had a similar pI value of about 6.7. The kinetic parameters of AVIII and LVIII were found to be similar to each other. These data suggest that the same types of GST isozymes are expressed in the astrocytes and liver, and take part mainly in the detoxification of 4HNE.

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.

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.

Beneficial effect of antioxidants in purified neurons derived from rat cortical culture.

Brain cell suspensions obtained from cerebrum of fetal rats were cultured and after 5 days neurons were separated from the residual cells. These purified neurons, which were replated on the dish, started to die within 24 h in culture. Glutathione content of these neurons decreased rapidly to less than one-tenth of the initial level after 24 h. In the presence of alpha-tocopherol, a well-known antioxidant, the neurons survived for at least 3 days, though glutathione content remained very low. Butylated hydroxyanisol had similar effect, but ascorbic acid and uric acid had no or very little effect. Serotonin, which is assumed to have an antioxidant activity, kept the neurons alive for 3 days. These results suggest that neurons separated from the other types of cells cannot survive due to the oxidative stress, which may otherwise be neutralized by a mechanism involving glutathione, and that antioxidants including serotonin has a beneficial effect on these purified neurons.

Stimulation of 125I-3-iodo-α-methyl-l-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

INTRODUCTION Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. METHODS Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 degrees C or under ice-cold conditions. RESULTS Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. CONCLUSIONS The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.

Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells).

INTRODUCTION High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of (14)C(U)-L-tyrosine ((14)C-Tyr), (125)I-4-iodo-L-meta-tyrosine (4-(125)I-mTyr), (125)I-6-iodo-L-meta-tyrosine (6-(125)I-mTyr), (125)I-3-iodo-α-methyl-L-tyrosine ((125)I-IMT) and (125)I-3-iodo-L-tyrosine (3-(125)I-Tyr) using Chinese hamster ovary cells (CHO-K1). METHODS Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na(+) at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na(+)-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na(+)-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN(3) and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. RESULTS (14)C-Tyr exhibited affinity for systems L, A and ASC. 4-(125)I-mTyr and 3-(125)I-Tyr exhibited high specificity for system L, whereas 6-(125)I-mTyr and (125)I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-(125)I-mTyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN(3), DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-(125)I-mTyr. CONCLUSIONS 4-(125)I-mTyr exhibited the greatest system L specificity (93.46 ± 0.13%) of all of the tested amino acids.