M. Angélica Carrasco and Enrique Jaimovich have contributed equally to this work.
Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes†
Article first published online: 8 AUG 2006
Copyright © 2006 Wiley-Liss, Inc.
Journal of Cellular Physiology
Volume 209, Issue 2, pages 379–388, November 2006
How to Cite
Espinosa, A., Leiva, A., Peña, M., Müller, M., Debandi, A., Hidalgo, C., Angélica Carrasco, M. and Jaimovich, E. (2006), Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes. J. Cell. Physiol., 209: 379–388. doi: 10.1002/jcp.20745
- Issue published online: 29 AUG 2006
- Article first published online: 8 AUG 2006
- Manuscript Accepted: 8 JUN 2006
- Manuscript Received: 26 JAN 2006
- Fondo Nacional de Investigación en Areas Prioritarias (FONDAP) Grant. Grant Number: 15010006
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 µM) 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. J. Cell. Physiol. 209: 379–388, 2006. © 2006 Wiley-Liss, Inc.