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.

Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy

“…In addition to their role in the potentiation of contraction and skeletal muscle plasticity, Cea et al () have proposed that Panx1 channels may also help maintain the normal oxidative state of skeletal muscle. Absence of Panx1 (KO mice) prevented the increase in ROS induced by skeletal muscle denervation, while the increase in ROS production induced by electrical stimulation could be blocked by carbenoxolone (Diaz‐Vegas et al, ). Altogether these data suggest that Panx1 channels are important to adult skeletal muscle health by regulating the potentiation of the contractile response, muscle plasticity that enables adaptation to demand, and the oxidative state during exercise or electrical stimuli.…”

“…The levels of the low molecular weight species (∼40 kDa) of Panx3 were significantly decreased in all dystrophic muscles assessed (c and d), while the levels of its ∼70 kDa immunoreactive species were drastically increased (c and e). Data are mean ± SD, *p ≤ 0.05 (n = 3) while the increase in ROS production induced by electrical stimulation could be blocked by carbenoxolone (Diaz-Vegas et al, 2015).…”

Extracellular ATP and other nucleotides—ubiquitous triggers of intercellular messenger release

“…They function as widespread signal Prostacyclin release from endothelial cells [195][196][197][198] Thromboxane A2 (TXA2) ATP, ADP/P2Y 12 TXA2 release from platelets, astrocytes [199][200][201] Thromboxane B2 (TXB2) ATP/P2X7 TXB2 release from primary human peritoneal macrophages and blood monocytes [202] Leukotriene B4 (LTP4) ATP/P2X7 LTP4 release from primary human peritoneal macrophages and blood monocytes [202] Sphingosine 1-phosphate ATP via ABCA family transporter Sphingosine 1-phosphate release from rat platelets [203] Nitric ROS release from numerous cell types [217][218][219][220] substances activating P2XRs and P2YRs [221]. In peripheral synapses, ATP can act as fast neurotransmitter through its own postsynaptic P2XRs.…”

“…Interestingly, ROSdependent PI3 kinase activation was involved in the activation of caspase-1 and processing and secretion of IL-1β and IL-18, suggesting that ROS can in turn be important mediators of P2X7R-induced pro-inflammatory cytokine release [219]. In mouse skeletal muscle, ATP released by electrical stimulation induced ROS production via activation of P2Y 1 Rs [220].…”

Regulation of NADPH oxidases in skeletal muscle

“…A cause and effect relationship involving Nox2 has been established with pharmacologic and genetic interventions. Specifically, contraction-induced increase in ROS was blocked or attenuated by pharmacologic agents (apocynin and Nox2 ds-tat ) [18, 25, 158, 164, 165] or knockout of Nox2 subunits [25, 166]. These data suggest that Nox2 is the main source of ROS during muscle contractions.…”