Critical role of phospholipase Cϒ1 in the generation of H2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes

Hong, Jeong Hee; Moon, Seok Jun; Byun, Hae Mi; Kim, Min Seuk; Jo, Hae; Bae, Yun Soo; Lee, Syng-Ill; Bootman, Martin D.; Roderick, H. Llewelyn; Shin, Dong Min and Seo, Jeong Taeg (2006). Critical role of phospholipase Cϒ1 in the generation of H2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes. Journal of Biological Chemistry, 281(19) pp. 13057–13067.




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 10–100μm. 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) γ1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-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. H2O2-induced [Ca2+]i oscillations and PLCγ1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLCγ1 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 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.

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