ACETYLCHOLINE-ACTIVATED INWARD CURRENT INDUCES CYTOSOLIC CA2+ MOBILIZATION IN MOUSE C2C12 MYOTUBES

We examined the spatiotemporal pattern of intracellular Ca2+ liberation in mouse myotubes by means of fluorescence imaging of cytosolic free Ca2+ together with the simultaneous recording of membrane whole-cell currents, Acetylcholine (ACh) applications to C2C12 myotubes equilibrated in Ca2+-free medium and voltage clamped at -50 mV evoked localized fluorescence transients of variable amplitude with less than 0.5 s delay, Under the same experimental conditions fluorescence transients were elicited by ACh also in mouse primary myotubes, Ca2+ transients were inhibited in myotubes clamped at depolarized potentials (-10 mV to +50 mV), or equilibrated in a Na+,Ca2+-free medium, as well as in cells loaded with heparin, or with inositol (1,4,5) trisphosphate (InsP(3)). To investigate whether InsP(3) could induce Ca2+ mobilization, [Ca2+](i) determinations were carried out in myotubes loaded with InsP(3) through the whole-cell patch-clamp recording pipette or by extracellular application in permeabilized cells, InsP(3) diffusion into the myoplasm caused Ca2+ spikes with 5 +/- 1 s (mean +/- SEM) delay from the rupture of the membrane patch. Spikes were followed by sustained increases In fluorescence or by damped oscillations. In permeabilized myotubes, InsP(3) induced the release of sequestered (Ca2+Ca)-Ca-45 with a half-maximally effective concentration (EC(50)) of 0.28 +/- 0.05 mu M, and Hill coefficient of 0.79 +/- 0.09. It is concluded that the ACh-activated inward current in mouse myotubes is coupled to cytosolic Ca2+ mobilization from internal InsP(3)-sensitive pools.