title: ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.
abstract: During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.
authors.affiliation: Departamento de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Alexis
authors.initials: A
authors.lastname: Díaz-Vegas
authors.affiliation: Departamento de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Cristian A
authors.initials: CA
authors.lastname: Campos
authors.affiliation: Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Ariel
authors.initials: A
authors.lastname: Contreras-Ferrat
authors.affiliation: Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Mariana
authors.initials: M
authors.lastname: Casas
authors.affiliation: Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile.
authors.forename: Sonja
authors.initials: S
authors.lastname: Buvinic
authors.affiliation: Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Enrique
authors.initials: E
authors.lastname: Jaimovich
authors.affiliation: Departamento de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Centro de Estudios Moleculares de la Célula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Espinosa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004558:Electric Stimulation; D005110:Extracellular Space / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D058472:Receptors, Purinergic P2Y1; D000255:Adenosine Triphosphate; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D011493:Protein Kinase C