Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes.
Controlled generation of reactive oxygen species (ROS) may contribute to physiological intracellular signaling events. We determined ROS generation in primary cultures of rat skeletal muscle after field stimulation (400 1-ms pulses at a frequency of 45 Hz) or after depolarization with 65 mM K+ for 1 min. Both protocols induced a long lasting increase in dichlorofluorescein fluorescence used as ROS indicator. Addition of diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase, PEG-catalase, a ROS scavenger, or nifedipine, an inhibitor of the skeletal muscle voltage sensor, significantly reduced this increase. Myotubes contained both the p47phox and gp91phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H2O2) at concentrations (100-200 microM) that did not alter cell viability; H2O2 induced a transient intracellular Ca2+ rise, measured as fluo-3 fluorescence. Minutes after Ca2+ signal initiation, an increase in ERK1/2 and CREB phosphorylation and of mRNA for the early genes c-fos and c-jun was detected. Inhibition of ryanodine receptor (RyR) decreased all effects induced by H2O2 and NAD(P)H oxidase inhibitors DPI and apocynin decreased ryanodine-sensitive calcium signals. Activity-dependent ROS generation is likely to be involved in regulation of calcium-controlled intracellular signaling pathways in muscle cells.
Espinosa
Alejandra
A
Escuela de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Leiva
Aida
A
Peña
Marisol
M
Müller
Mariolly
M
Debandi
Anibal
A
Hidalgo
Cecilia
C
Carrasco
M Angélica
MA
Jaimovich
Enrique
E
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004558:Electric Stimulation; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000235:genetics; D016762:Genes, fos / Q000235:genetics; D016758:Genes, jun / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000187:drug effects; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D017362:Cyclic AMP Response Element-Binding Protein; D008562:Membrane Glycoproteins; D021122:Protein Subunits; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium