Peroxide-induced membrane blabbing in endothelial cells associated with glutathione oxidation but not apoptosis

Cells under oxidative stress induced by peroxides undergo functional and mo rphological changes, which often resemble those observed during apoptosis. Peroxides, however, also cause the oxidation of intracellular reduced gluta thione (GSH). We investigated the relation between these peroxide-induced e ffects by using human umbilical vein endothelial cells (HUVEC) and two HUVE C-derived cell lines, ECRF24 and ECV304. With HUVEC, tert-butyl hydroperoxi de (tBH) or hydrogen peroxide application in the presence of serum induced, in a dose-dependent way, reorganization of the actin cytoskeleton, membran e blebbing, and nuclear condensation. These processes were accompanied by t ransient oxidation of GSH. With ECRF24 cells, this treatment resulted in le ss blebbing and a shorter period of GSH oxidation. However, repeated tBH ad dition increased the number of blebbing cells and prolonged the period of G SH oxidation. ECV304 cells were even more resistant to peroxide-induced ble b formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC and ECRF24 cel ls, but not in ECV304 cells. Neither membrane blebbing nor nuclear condensa tion in any of these cell types was due to apoptosis, as evidenced by the a bsence of surface expression of phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH: although high tBH concentration s lead to nonapoptotic death. We conclude that, in endothelial cells, perox ide-induced cytoskeletal reorganization and bleb formation correlate with t he degree of GSH oxidation but do not represent an early stage of the apopt otic process.