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

Cells underoxidative stress induced by peroxides undergo functional andmorphological changes, which often resemble those observed duringapoptosis. Peroxides, however, also cause the oxidation ofintracellular reduced glutathione (GSH). We investigated the relationbetween these peroxide-induced effects by using human umbilical veinendothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 andECV304. With HUVEC, tert-butylhydroperoxide (tBH) or hydrogenperoxide application in the presence of serum induced, in adose-dependent way, reorganization of the actin cytoskeleton, membraneblebbing, and nuclear condensation. These processes were accompanied bytransient oxidation of GSH. With ECRF24 cells, this treatment resultedin less blebbing and a shorter period of GSH oxidation. However,repeated tBH addition increased thenumber of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVECand ECRF24 cells, but not in ECV304 cells. Neither membrane blebbingnor nuclear condensation in any of these cell types was due toapoptosis, as evidenced by the absence of surface expression ofphosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although hightBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells,peroxide-induced cytoskeletal reorganization and bleb formationcorrelate with the degree of GSH oxidation but do not represent anearly stage of the apoptotic process.