Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates:: Role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels

1 The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2 Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a (OH)-O-. scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that O-.(2)- influx through anion channels was responsible for the Ca2+ movements, in which (OH)-O-. generation catalyzed by intracellular transition metals (i.e.,Haber-Weiss cycle) was involved. 3 After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4 The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for (OH)-O-.-mediated cytotoxicity. We concluded that intracellular (OH)-O-. generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity.