Critical role of phospholipase Cgamma1 in the generation of H2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes.
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 10-100 microM. 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) gamma1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-induced [Ca2+]i oscillations. In addition, RNA interference against PLCgamma1 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 PLC1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLCgamma1 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 PLCgamma1 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.
Hong
Jeong Hee
JH
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Moon
Seok Jun
SJ
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Byun
Hae Mi
HM
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Kim
Min Seuk
MS
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Jo
Hae
H
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Bae
Yun Soo
YS
Division of Molecular Life Science, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea.
Lee
Syng-Ill
SI
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Bootman
Martin D
MD
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, CB2 4AT Cambridge, United Kingdom.
Roderick
H Llewelyn
HL
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, CB2 4AT Cambridge, United Kingdom; Department of Pharmacology, University of Cambridge, CB2 1PD Cambridge, United Kingdom.
Shin
Dong Min
DM
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea. Electronic address: dmshin@yumc.yonsei.ac.kr.
Seo
Jeong Taeg
JT
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea. Electronic address: jeong@yumc.yonsei.ac.kr.
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004815:Epidermal Growth Factor; D004963:Estrenes; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011760:Pyrrolidinones; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin
D004963:Estrenes; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D006861:Hydrogen Peroxide; D051966:Phospholipase C gamma; D002118:Calcium