Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide

Abstract

We have examined the role of the glutathione redox cycle as an antioxidant defense mechanism in cultured bovine and human endothelial cells by disrupting the glutathione redox cycle at several points. Endothelial glutathione reductase was selectively inhibited with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU). Cellular stores of reduced glutathione were depleted by reaction with diethylmaleate (DEM) or 1-chloro-2,4-dinitrobenzene (CDNB) or by inhibition of glutathione synthesis with buthionine sulfoximine (BSO). Whereas several strains of untreated bovine and human endothelial cells were resistant to lysis by enzymatically generated hydrogen peroxide, BCNU-treated cells were readily lysed in a time- and dose-dependent manner. Glucose-glucose oxidase-mediated lysis of BCNU-treated bovine endothelial cells was catalase-inhibitable and directly related to BCNU concentration and endogenous glutathione reductase activity. Pretreatment of bovine endothelial cells with BCNU did not potentiate lysis by distilled water, calcium ionophore, lipopolysaccharide, or hypochlorous acid. Depletion of cellular reduced glutathione by reaction with DEM or CDNB or by inhibition of glutathione synthesis by BSO also potentiated endothelial lysis by enzymatically generated hydrogen peroxide. Inhibition of endothelial glutathione reductase by BCNU or depletion of reduced glutathione by BSO increased endothelial susceptibility to lysis by hydrogen peroxide generated by phorbol myristate acetate-activated neutrophils. We conclude that the glutathione redox cycle plays an important role as an endogenous antioxidant defense mechanism in cultured endothelial cells.