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.
Highlights
During exercise, several pathways are activated in skeletal muscle in order to maintain cellular homeostasis [1]
We have previously reported that ATP release from muscle cells is frequency-dependent, ATP is released from muscle fibers at low frequency but not at high frequency [26]; in agreement with those results, we observed an increase of reactive oxygen species (ROS) production at 20Hz but not at 90Hz, suggesting that ROS increase during muscle activity is dependent on release of extracellular ATP (Fig 1D)
We describe a novel mechanism for ROS production via Protein kinase C (PKC)-NADPH oxidase 2 (NOX2) activation induced by extracellular ATP released from adult cultured muscle cells after electrical stimulation which mimics the physiological activation of skeletal muscle during exercise (Fig 6)
Summary
Several pathways are activated in skeletal muscle in order to maintain cellular homeostasis [1]. NADPH oxidases are proteins that transfer electrons across biological membranes. The movement of cytoplasmic subunits, p67phox, p47phox, p40phox and Rac GTPase from the cytoplasm to the membrane form the active NOX2 enzyme complex [4]. Skeletal muscle cells express NOX2 [6] and several authors suggest that this is one of the main sources of ROS during muscle contraction or electrical stimuli [7, 8], in addition, NOX protein subunits were detected in transverse tubules and triads isolated from rabbit skeletal muscle but not in sarcoplasmic reticulum vesicles [9], electrical stimulation induces NOX2 activation in skeletal muscle cells [7]; the mechanism of activation, is not fully understood
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