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

Hee Hong Jeong, Jun Moon Seok, Mi Byun Hae, Seuk Kim Min, Hae Jo, Soo Bae Yun, Syng Ill Lee, Martin D. Bootman, H. Llewelyn Roderick, Min Shin Dong, Taeg Seo Jeong

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Abstract

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.

Original languageEnglish
Pages (from-to)13057-13067
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number19
DOIs
Publication statusPublished - 2006 May 12

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Jeong, H. H., Seok, J. M., Hae, M. B., Min, S. K., Jo, H., Yun, S. B., Lee, S. I., Bootman, M. D., Roderick, H. L., Dong, M. S., & Jeong, T. S. (2006). Critical role of phospholipase Cγ1 in the generation of H 2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes. Journal of Biological Chemistry, 281(19), 13057-13067. https://doi.org/10.1074/jbc.M601726200