Ca 2+ is mobilized by hydroxyl radical but not by superoxide in RTH-149 cells: The oxidative switching-on of Ca 2+ signaling

Abstract

Differential effects of superoxide and hydroxyl radical on intracellular calcium were investigated in trout hepatoma cells (RTH-149). [Ca 2+] 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 H 2O 2 and Cu 2+. No [Ca 2+] i variation was found with HX/XO, a slight [Ca 2+] i rise with a mixture of Cu 2+ and HX/XO, a sustained rise with Cu 2+ and H 2O 2, and the highest rise with Cu 2+, H 2O 2 and HX/XO. Fluorimetric assay using dihydrorhodamine 123 revealed a correlation between the oxidizing power of a mixture and its effect on [Ca 2+] i. The [Ca 2+] i rise induced by Cu 2+, H 2O 2 and HX/XO, was partially reduced in Ca 2+ free medium or in the presence of SOD, converted into Ca 2+ transient by verapamil, and almost abolished by the PLC inhibitor U73122 or in the presence of the hydroxyl radical quencher TEMPOL. Data indicate that Ca 2+ is mobilized by hydroxyl radical but not by superoxide. The mechanism consists of PLC activation causing intracellular Ca 2+ release, while Ca 2+ entry potentiates Ca 2+ release thus leading to sustained [Ca 2+] i rise. A role of hydroxyl radicals in the oxidative switching-on of Ca 2+ signaling is discussed.