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

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. 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 scavenger (5,5‐dimethyl‐1‐pyrroline‐N‐oxide), or an anion channel blocker (disodium 4, 4′‐diisothiocyano‐2, 2′‐stilbenedisulphonic acid), suggesting that •O2− influx through anion channels was responsible for the Ca2+ movements, in which •OH generation catalyzed by intracellular transition metals (i.e., Haber‐Weiss cycle) was involved. 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. 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‐mediated cytotoxicity. We concluded that intracellular •OH 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. British Journal of Pharmacology (1999) 126, 1462–1470; doi:10.1038/sj.bjp.0702438