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

Abstract

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 P2X(4), P2X(5), P2X(7), P2Y(1) and P2Y(4) receptors. siRNA-reduced protein expression of P2X(5), P2X(7) and P2Y(1) receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X(7) 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-P2X(7) siRNA caused significant reduction in the agonist-elicited ionic currents I (ATP) and I (BzATP), with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X(4), P2X(5) and P2X(7) and metabotropic P2Y(1) and P2Y(4) purinoreceptors participates in forming the calcium transients of multinucleated myotubes.

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+signals and an IL-6 autocrine loop

“…For the same reason, to allow ATP diffusion to the T-tubules and surpass ectonucleotidases metabolization, we need to use higher ATP concentrations and time exposures for whole muscle experiments (500 M, 4 h) than for newborn-derived myotubes (10 -100 M for 15-60 min). All the studies relating P2Y receptor activity with skeletal muscle physiology, using varying experimental models, have used high M ATP concentrations (50 -180 M) (18,20,21,56,57,80). It is relevant to note that, considering that ATP release is a local event, only Յ1% intracellular ATP pool needs to be released to maximally activate all receptors.…”

The calcium channel α2/δ1 subunit interacts with ATP5b in the plasma membrane of developing muscle cells

“…The role of ATP in regulating skeletal muscle function has been extensively examined. It has been shown that ATP activates satellite cells for the formation and regeneration of skeletal muscle (1,4,35), regulates intracellular calcium concentration and contractility (7,21,29), and can influence metabolism (27). Moreover, extracellular ATP is necessary for the repair of skeletal muscle in mdx mice, a model for Duchenne muscular dystrophy (28,42).…”

The role of purinergic signalling in the musculoskeletal system

“…Many studies have demonstrated the expression of multiple P2 receptor subtypes by skeletal muscle (Banachewicz et al, 2005;Cheung et al, 2003;Deli et al, 2007;Janssens et al, 1996); however, the pattern of purinergic receptor expression is strongly influenced by developmental stage and species (see (Burnstock et al, 2013)). …”