Cytosolic Ca 2+ movements of endothelial cells exposed to reactive oxygen intermediates: Role of hydroxyl radical‐mediated redox alteration of cell‐membrane Ca 2+ channels

The mode of action of reactive oxygen intermediates in cysosolic Ca 2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. Cytosolic Ca 2+ movements provoked by X/XO consisted of an initial Ca 2+ release from thapsigargin‐sensitive intracellular Ca 2+ stores and a sustained Ca 2+ influx through cell‐membrane Ca 2+ channels. The Ca 2+ 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 • O 2 − influx through anion channels was responsible for the Ca 2+ movements, in which • OH generation catalyzed by intracellular transition metals (i.e., Haber‐Weiss cycle) was involved. After an initial Ca 2+ elevation provoked by X/XO, cytosolic Ca 2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca 2+ decrease. Extracellular addition of sulphydryl (SH)‐reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca 2+ channel blocker, Ni 2+ , abolished the sustained increase in Ca 2+ , suggesting that Ca 2+ influx through cell‐membrane Ca 2+ channels was extracellularly regulated by the redox state of SH‐groups. The X/XO‐provoked change in cellular respiration was inhibited by Ni 2+ or dithiothreitol as well as inhibitors of Haber‐Weiss cycle, suggesting that Ca 2+ influx was responsible for • OH‐mediated cytotoxicity. We concluded that intracellular • OH generation was involved in the Ca 2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca 2+ elevation was partly responsible for the oxidants‐mediated cytotoxicity.British Journal of Pharmacology (1999) 126 , 1462–1470; doi: 10.1038/sj.bjp.0702438