BIOSYNTHESIS AND MAINTENANCE OF GSH IN PRIMARY ASTROCYTE CULTURES - ROLE OF L-CYSTINE AND ASCORBATE

We have studied the optimal conditions to maintain the astrocyte GSH l evels under normal and oxidative stress conditions. The rate of GSH sy nthesis from L-methionine was statistically lower than from L-cystine or N-acetyl-cysteine in astrocytes treated with diethyl-maleate, which is a substrate of GSH S-transferases. This is in accordance with the fact that cystathionase activity was not detectable. The transport of L-cystine mediated by the Na+-independent system Xc(-) is the limiting step in GSH synthesis in astrocytes. Incubation with tert-butyl hydro peroxide (t-booH) reduced GSH concentration in astrocytes. Thus reduct ion was ameliorated in part by the addition of ascorbate or dehydroasc orbate. When L-cystine and ascorbate were added together to the t-booH -treated astrocytes, the GSH concentration was indistinguishable from controls. Electron micrographs of astrocytes treated with t-booH showe d an increased number of vacuoles and mitochondrial swelling. This was prevented by ascorbate and dehydroascorbate. The physiological implic ations of the availability of GSH precursors and ascorbate in the main tenance of GSH in astrocytes are discussed.