Differences in purinergic and voltage-dependent signalling during protein kinase Cα overexpression- and culturing-induced differentiation of C2C12 myoblasts

摘要

Differentiation of skeletal muscle cells both in vivo and in vitro is accompanied by the development of voltage-dependent processes and alterations in purinergic signalling. To date at least two independent methods have been used to induce differentiation in primary cultures, namely, appropriate modification of culturing conditions and overexpression of specific protein kinase C (PKC) isoenzymes. Here we characterize and compare the development of purinergic and depolarization-dependent alterations using these two methods to induce differentiation in C2C12 cells. We demonstrate that depolarization- and ATP-evoked Ca2+ responses underwent functional development during differentiation, and the characteristics of this progress were dependent on the actual differentiation-promoting stimulus. Overexpression of PKCα anticipated the appearance of robust increases in the intracellular calcium concentration upon ATP administration but failed to do so after depolarizing stimuli. Moreover, the first phase of the biphasic ATP-induced response observed in differentiated myotubes induced by culturing was not present in differentiated PKCα-overexpressing cells, suggesting that although purinergic signalling developed very early, purinergic stimuli failed to activate the voltage-dependent mechanisms of these cells even at subsequent stages of differentiation. Disruption of the coupling of purinergic signalling to depolarization-activated mechanisms may be explained by our observations that PKCα-overexpression changed the purinergic receptor pattern of immature myoblasts differently from what was seen in the course of culturing-induced differentiation. PKCα-specific alterations were characterized by the lack of increase in the expression of P2X7 receptors and the failure of P2Y4 receptors to appear and P2Y2 receptors to disappear. The effects of PKCα-overexpression were proven to be specific since the overexpression of the hyperproliferative isoenzyme PKCδ failed to induce any of the changes promoted by PKCα. Our data suggest that the method of inducing differentiation in skeletal muscle cells modifies not only the course of development but also the interaction of depolarization-dependent and purinergic pathways.