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

We examined the spatiotemporal pattern of intracellular Ca2+ liberatio n 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 equilibra ted in Ca2+-free medium and voltage clamped at -50 mV evoked localized fluorescence transients of variable amplitude with less than 0.5 s de lay, Under the same experimental conditions fluorescence transients we re elicited by ACh also in mouse primary myotubes, Ca2+ transients wer e 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) t hrough the whole-cell patch-clamp recording pipette or by extracellula r application in permeabilized cells, InsP(3) diffusion into the myopl asm caused Ca2+ spikes with 5 +/- 1 s (mean +/- SEM) delay from the ru pture of the membrane patch. Spikes were followed by sustained increas es In fluorescence or by damped oscillations. In permeabilized myotube s, InsP(3) induced the release of sequestered (Ca2+Ca)-Ca-45 with a ha lf-maximally effective concentration (EC(50)) of 0.28 +/- 0.05 mu M, a nd Hill coefficient of 0.79 +/- 0.09. It is concluded that the ACh-act ivated inward current in mouse myotubes is coupled to cytosolic Ca2+ m obilization from internal InsP(3)-sensitive pools.