Differentiation-dependent alterations in the extracellular ATP-evoked calcium fluxes of cultured skeletal muscle cells from mice

Although extracellular adenosine triphosphate (ATP) has been generally accepted as the regulator of cellular differentiation, the relative contribution of the various purinoreceptor subtypes to purinergic signalling at distinct stages of skeletal muscle differentiation is still poorly understood. Here we measured extracellular ATP-evoked changes in intracellular calcium concentration and surface membrane ionic currents (IATP), calculated the calcium flux (FL) entering the myoplasmic space and compared these parameters at different stages of differentiation on cultured mouse myotubes. The ATP-evoked FL displayed an early peak and then declined to a steady level. With differentiation, the early peak became separated from the maintained component and was absent on mature myotubes. Repeated ATP applications caused desensitization of the response in both immature and differentiated myotubes, owing mainly to the reduction of the early peak of FL in the former and to a decline of both components in the latter group of cells. Depolarization of the cell or removal of external calcium suppressed the early peak. IATP showed no inactivation, and its voltage dependence displayed strong inward rectification. The concentration dependence of IATP can be fitted using a Hill equation, yielding an EC50 of 56 μM. Results are consistent with the parallel activation of both P2X and P2Y receptors.