Critical role of phospholipase C_ϒ1 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 C_ϒ1 in the generation of H₂O₂-evoked [Ca²⁺]_i oscillations in cultured rat cortical astrocytes. Journal of Biological Chemistry, 281(19) pp. 13057–13067.

DOI: https://doi.org/10.1074/jbc.M601726200

URL: http://www.jbc.org/content/281/19/13057

Abstract

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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