PATHWAYS OF GLUTATHIONE METABOLISM AND TRANSPORT IN ISOLATED PROXIMALTUBULAR CELLS FROM RAT-KIDNEY

Cellular uptake and metabolism of exogenous glutathione (GSH) in fresh ly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L -buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell populatio n. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivic in/BSO produced 3- to 4-fold increases in intracellular GSH within 10- 15 min. These significantly higher intracellular concentrations were m aintained for up to 60 min. At lower concentrations of extracellular G SH, an initial increase in intracellular GSH concentrations was observ ed, but this was not maintained for the 60-min time course. In the abs ence of inhibitors, intracellular concentrations of GSH increased to l evels that were 2- to 3-fold higher than initial values in the first 1 0-15 min, but these dropped below initial levels thereafter. In both t he absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (CSSG) and degradation of GSH to gluta mate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intra cellular GSH to GSSG in a concentration-dependent manner and extracell ular GSSG was transported into PT cells, but limited intracellular red uction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH un der these conditions. Hence, direct uptake of GSH, rather than reducti on of GSSG or resynthesis from precursors, may be the primary mechanis m to maintain intracellular thiol redox status under toxicological con ditions. Since PT cells are a primary target for toxicants, the abilit y of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.