Extracellular ATP signaling affects contractility of skeletal muscle.

Muscle contraction consumes more than 50% of the ATP produced by the striated cells, moreover, the working muscle also releases some of its precious nucleotide into the extracellular milieu. However, growing evidence shows that this release is not a pure waste. It appears in fact that skeletal muscle cells utilize extracellular ATP to stimulate relevant autocrine/paracrine signaling pathways, related, for example to the differentiative programm. Present work was aimed at investigating the purinergic signaling of adult skeletal muscle. First, we confirmed, at the single fiber level, the release of ATP during contractile activity. The electrically stimulation (five 30-mV pulses of 2 ms duration with 500 ms interval) of cultured rat flexor digitorum brevis muscle fibers, isolated by the collagenase method, caused a substantial, rapid (seconds) release of ATP into the extracellular fluid, whose level returned to baseline with an estimated half-decay time of about 20 min. The decrease of the ATP concentration in the extracellular milieu represents an indirect observation of the presence of an ATP-hydrolyzing activity at the muscle fibers surface, that we measured as high as 41 ± 4 nmol Pi/mg protein (n = 3). Consistently, RT-PCR and WB analyses show the presence of NTPDase-1 and NTPDase-2, NPP1, and -sarcoglycan in muscle fibers. Different purinergic receptors have been reported to be expressed by striated cells. In particular we detected the presence of P2 receptor transcripts and proteins, such as P2X1, P2X4, P2X5, P2X6 and P2X7 as well as P2Y1, P2Y2 and P2Y6 in adult muscle fibers. Moreover, we demonstrated that P2X4 is localized mainly in the T-tubule membranes, i.e., the critical site of excitation-contraction coupling of skeletal muscle. Because all the elements of extracellular ATP signaling are present in skeletal muscle, we speculated that both the enhanced Ca2+ entry as well as the subsequent activation of Ca2+-dependent intracellular processes could modulate muscle contraction, especially during sustained contractile activity. Therefore, we examined different in vitro stimulation protocols on a typical slow- twitch muscle (soleus) in order to mimic sustained contractile activity. We found that stimulation of soleus muscle at low frequencies (between 0.016 and 0.05 pulses/s) produced a slow progressive rise of twitch tension (potentiation) which, after 40 twitches, was about 20% higher than the initial value. We then applied different protocols devoted either to prevent or stimulate any possible effect of extracellular ATP in tension potentiation. The removal of extracellular ATP, by specific enzymes (hexokinase/apyrase), the inhibition of P2X receptors by a selected cocktail of P2 blockers (PPADS, suramin RB-2), or Ca2+-free conditions, all abolished tension potentiation. The addition of Zn+ or ivermectin, at levels known to stimulate P2X4, were without effects, suggesting that the receptor is already fully activated. On the contrary, elevated doses of Zn+, ivermectin or 2meSATP reduced twitch tension potentiation. Taking together, these data reveal that ATP-mediated Ca2+ entry plays, by a still unknown mechanism/s, an important role in modulating the contractile activity of skeletal muscle and add new hints regarding the extracellular ATP signaling of this tissue