Ca2+ is mobilized by hydroxyl radical but not by superoxide in RTH-149 cells: the oxidative switching-on of Ca2+ signaling.
Differential effects of superoxide and hydroxyl radical on intracellular calcium were investigated in trout hepatoma cells (RTH-149). [Ca2+]i variations were recorded using confocal imaging, fluo-3 loading, and exposure to various mixtures consisting of hypoxanthine/xanthine oxidase (HX/XO), and of sub-stimulatory concentrations of H2O2 and Cu2+ . No [Ca2+]i variation was found with HX/XO, a slight [Ca2+]i rise with a mixture of Cu2+ and HX/XO, a sustained rise with Cu2+ and H2O2, and the highest rise with Cu2+, H2O2 and HX/XO. Fluorimetric assay using dihydrorhodamine 123 revealed a correlation between the oxidizing power of a mixture and its effect on [Ca2+]i. The [Ca2+]i rise induced by Cu2+, H2O2 and HX/XO, was partially reduced in Ca2+ free medium or in the presence of SOD, converted into Ca2+ transient by verapamil, and almost abolished by the PLC inhibitor U73122 or in the presence of the hydroxyl radical quencher TEMPOL. Data indicate that Ca2+ is mobilized by hydroxyl radical but not by superoxide. The mechanism consists of PLC activation causing intracellular Ca2+ release, while Ca2+ entry potentiates Ca2+ release thus leading to sustained [Ca2+]i rise. A role of hydroxyl radicals in the oxidative switching-on of Ca2+ signaling is discussed.
Burlando
Bruno
B
DISAV, Università del Piemonte Orientale, Via Bellini 25 G, 15100 Alessandria, Italy. burlando@unipmn.it
Viarengo
Aldo
A
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004963:Estrenes / Q000494:pharmacology; D005456:Fluorescent Dyes; D017665:Hydroxyl Radical / Q000494:pharmacology*; D019271:Hypoxanthine / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011760:Pyrrolidinones / Q000494:pharmacology; D012235:Rhodamines / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology*; D014337:Trout; D014700:Verapamil / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism
D004963:Estrenes; D005456:Fluorescent Dyes; D016877:Oxidants; D011760:Pyrrolidinones; D012235:Rhodamines; C058319:dihydrorhodamine 123; D013481:Superoxides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019271:Hypoxanthine; D017665:Hydroxyl Radical; D014700:Verapamil; D014969:Xanthine Oxidase