Critical Role of Phospholipase Cγ1 in the Generation of H2O2-evoked [Ca2+]i Oscillations in Cultured Rat Cortical Astrocytes

• Published in: The Journal of biological chemistry Vol. 281; no. 19; p. 13057
• Main Authors: Jeong Hee Hong, Seok Jun Moon, Hae Mi Byun, Min Seuk Kim, Hae Jo, Yun Soo Bae, Syng-Ill Lee, Martin D. Bootman, H. Llewelyn Roderick, Dong Min Shin, Jeong Taeg Seo
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 12.05.2006
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M601726200

Reactive oxygen species, such as the superoxide anion, H 2 O 2 , and the hydroxyl radical, have been considered as cytotoxic by-products of cellular metabolism. However, recent studies have provided evidence that H 2 O 2 serves as a signaling molecule modulating various physiological functions. Here we investigated the effect of H 2 O 2 on the regulation of intracellular Ca 2+ signaling in rat cortical astrocytes. H 2 O 2 triggered the generation of oscillations of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in a concentration-dependent manner over the range 10–100μ m . The H 2 O 2 -induced [Ca 2+ ] i oscillations persisted in the absence of extracellular Ca 2+ and were prevented by depletion of intracellular Ca 2+ stores with thapsigargin. The H 2 O 2 -induced [Ca 2+ ] 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. H 2 O 2 activated phospholipase C (PLC) γ1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H 2 O 2 -induced [Ca 2+ ] i oscillations. In addition, RNA interference against PLCγ1 and the expression of the inositol 1,4,5-trisphosphate-sequestering “sponge” prevented the generation of [Ca 2+ ] i oscillations. H 2 O 2 -induced [Ca 2+ ] i oscillations and PLCγ1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H 2 O 2 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 2 O 2 generate [Ca 2+ ] i oscillations by activating PLCγ1 through sulfhydryl oxidation-dependent mechanisms. Furthermore, we show that this mechanism underlies the modulatory effect of endogenously produced H 2 O 2 on epidermal growth factor-induced Ca 2+ signaling in rat cortical astrocytes.