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.
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Jeong Hee
JH
Hong
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Seok Jun
SJ
Moon
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Hae Mi
HM
Byun
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Min Seuk
MS
Kim
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Hae
H
Jo
Division of Molecular Life Science, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea.
Yun Soo
YS
Bae
Department of Oral Biology, Brain Korea 21 Project for Medical Science, Oral Science Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea.
Syng-Ill
SI
Lee
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, CB2 4AT Cambridge, United Kingdom.
Martin D
MD
Bootman
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, CB2 4AT Cambridge, United Kingdom; Department of Pharmacology, University of Cambridge, CB2 1PD Cambridge, United Kingdom.
H Llewelyn
HL
Roderick
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.
Dong Min
DM
Shin
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.
Jeong Taeg
JT
Seo
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