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Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes

Authors

  • Alejandra Espinosa,

    1. Escuela de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Aida Leiva,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Marisol Peña,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Mariolly Müller,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Anibal Debandi,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Cecilia Hidalgo,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • M. Angélica Carrasco,

    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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  • Enrique Jaimovich

    Corresponding author
    1. Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
    • ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile.
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  • M. Angélica Carrasco and Enrique Jaimovich have contributed equally to this work.

Abstract

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.

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