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 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. 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-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.
Az-ma
T
T
Department of Anaesthesiology and Critical Care Medicine, Hiroshima University, School of Medicine, Japan. azm@ma3.seikyou.ne.jp
Saeki
N
N
Yuge
O
O
D000818:Animals; D001011:Aorta; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010618:Phenanthrolines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000502:physiology*; D013552:Swine; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D007502:Iron Chelating Agents; D016877:Oxidants; D010618:Phenanthrolines; D017382:Reactive Oxygen Species; D019820:Xanthine; D017665:Hydroxyl Radical; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D009532:Nickel; D006861:Hydrogen Peroxide; D014969:Xanthine Oxidase; D002118:Calcium; D004229:Dithiothreitol; C025205:1,10-phenanthroline