Critical role of phospholipase Cγ1 in the generation of H ₂O₂-evoked [Ca²⁺]_i oscillations in cultured rat cortical astrocytes

Reactive oxygen species, such as the superoxide anion, H₂O ₂, 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 μM. The H₂O₂-induced [Ca ²⁺]_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. H₂O ₂ activated phospholipase C (PLC) γ1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H ₂O₂-induced [Ca²⁺]_i oscillations. In addition, RNA interference against PLCγ1 and the expression of the inositol 1,4,5-trisphosphate-sequestering "sponge" prevented the generation of [Ca²⁺]_i oscillations. H₂O ₂-induced [Ca²⁺]_i oscillations and PLCγ1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H ₂O₂ production, PLCγ1 activation, and [Ca ²⁺]_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 H ₂O₂ generate [Ca²⁺]_i oscillations by activating PLCγ1 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.