Critical role of phospholipase Cγ1 in the generation of H2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes

Reactive oxygen species, such as the superoxide anion, H2O2, and the hydroxyl radical, have been considered as cytotoxic by-products of cellular metabolism. However, recent studies have provided evidence that H2O2 serves as a signaling molecule modulating various physiological functions. Here we investigated the effect of H2O2 on the regulation of intracellular Ca2+ signaling in rat cortical astrocytes. H2O2 triggered the generation of oscillations of intracellular Ca2+ concentration ([Ca2+](i)) in a concentration-dependent manner over the range 10-100 mu M. The H2O2-induced [Ca2+](i) oscillations persisted in the absence of extracellular Ca2+ and were prevented by depletion of intracellular Ca2+ stores with thapsigargin. The H2O2-induced [Ca2+](i) oscillations were not inhibited by pretreatment with ryanodine but were prevented by 2-aminoethoxydiphenyl borate and caffeine, known antagonists of inositol 1,4,5-trisphosphate receptors. H2O2 activated phospholipase C (PLC) gamma 1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-induced [Ca2+](i) oscillations. In addition, RNA interference against PLC gamma 1 and the expression of the inositol 1,4,5-trisphosphate-sequestering "sponge" prevented the generation of [Ca2+](i) oscillations. H2O2-induced [Ca2+](i) oscillations and PLC gamma 1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLC gamma 1 activation, and [Ca2+](i) increases, which were attenuated by N-acetylcysteine and diphenyleneiodonium and by the overexpression of peroxiredoxin type II. Therefore, we conclude that low concentrations of exogenously applied H2O2 generate [Ca2+](i) oscillations by activating PLC gamma 1 through sulfhydryl oxidation-dependent mechanisms. Furthermore, we show that this mechanism underlies the modulatory effect of endogenously produced H2O2 on epidermal growth factor-induced Ca2+ signaling in rat cortical astrocytes.