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 Ca(2+)-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+,Ca(2+)-free medium as well as in cells loaded with heparin, or with inositol (1,4,5) trisphosphate (InsP3). To investigate whether InsP3 could induce Ca2+ mobilization, [Ca2+]i determinations were carried out in myotubes loaded with InsP3 through the whole-cell patch-clamp recording pipette or by extracellular application in permeabilized cells. InsP3 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, InsP3 induced the release of sequestered 45Ca2+ with a half-maximally effective concentration (EC50) of 0.28 +/- 0.05 microM, 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 InsP3-sensitive pools.
Giovannelli
A
A
Dipartimento Medicina Sperimentale Universitá dell' Aquila, Roma, Italy.
Grassi
F
F
Limatola
C
C
Mattei
E
E
Ragozzino
D
D
Eusebi
F
F
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D004963:Estrenes / Q000378:metabolism / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D009132:Muscles / Q000187:drug effects* / Q000378:metabolism*; D010539:Permeability; D011760:Pyrrolidinones / Q000378:metabolism / Q000494:pharmacology
D003470:Culture Media; D004963:Estrenes; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D000109:Acetylcholine; D002118:Calcium