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Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS‐elicited Ca 2+ stimulates ERK, CREB, early genes
Author(s) -
Espinosa Alejandra,
Leiva Aida,
Peña Marisol,
Müller Mariolly,
Debandi Anibal,
Hidalgo Cecilia,
Angélica Carrasco M.,
Jaimovich Enrique
Publication year - 2006
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.20745
Subject(s) - apocynin , nadph oxidase , reactive oxygen species , ryanodine receptor , nad+ kinase , depolarization , chemistry , nad(p)h oxidase , microbiology and biotechnology , mitochondrial ros , intracellular , biochemistry , biology , biophysics , enzyme
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 p47 phox and gp91 phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H 2 O 2 ) at concentrations (100–200 µM) that did not alter cell viability; H 2 O 2 induced a transient intracellular Ca 2+ rise, measured as fluo‐3 fluorescence. Minutes after Ca 2+ 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 H 2 O 2 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. J. Cell. Physiol. 209: 379–388, 2006. © 2006 Wiley‐Liss, Inc.