Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5–10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2′3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X4, P2X5, P2X7, P2Y1 and P2Y4 receptors. siRNA-reduced protein expression of P2X5, P2X7 and P2Y1 receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X7 siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X7 siRNA caused significant reduction in the agonist-elicited ionic currents IATP and IBzATP, with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X4, P2X5 and P2X7 and metabotropic P2Y1 and P2Y4 purinoreceptors participates in forming the calcium transients of multinucleated myotubes.