Protection of cultured rat gastric cells against oxidant-induced damage by exogenous glutathione

Background/Aims: Reduced glutathione (GSH) is an intracellular protectant against oxidants. The present study determined whether extracellular GSH protects against oxidant damage or whether an uptake system of GSH is present in cultured gastric cells. Methods: Hydrogen peroxide was generated by glucose oxidase and glucose. Cytotoxicity was assessed by 51Cr release. Intracellular GSH was assayed by the method of Tietze. Results: Pretreatment with extracellular GSH decreased H2O2-induced 51Cr release. Treatment with GSH enhanced cellular GSH content. Protection by pretreatment with GSH was prevented by buthionine sulfoximine (an inhibitor of γ-glutamylcysteine synthetase). Enhancement of intracellular GSH was also prevented by buthionine sulfoximine. Acivicin (an inhibitor of γ-glutamyl transpeptidase) prevented intracellular accumulation of GSH from extracellular GSH. Cysteine was effective in preventing damage and enhancing intracellular GSH content, whereas both glutamine and glycine were not. Conclusions: Extracellular GSH protects cultured gastric cells from H2O2 damage by accelerating intracellular GSH synthesis; this is mediated by membrane-bound γ-glutamyl transpeptidase acting on extracellular GSH (which supplies these cells with cysteine) and then by intracellular γ-glutamylcysteine synthetase.