Calcium-dependent regulation of the membrane potential and extrajunctional acetylcholine receptors of rat skeletal muscle

The resting membrane potential and the development of extrajunctional acetylcholine receptors were measured in strips of rat diaphragm muscle that had been cultured for 48 h. The resting membrane potential fell and the density of extrajunctional acetylcholine receptors increased during this period. The addition of the divalent ionophore A23187 to the culture medium inhibited these changes. The maximal effective concentration of A23187 was 1.0 μ m. At this concentration the ionophore inhibited the fall in membrane potential by 12.0 mV and the increase in extrajunctional acetylcholine receptor development by 61%. The ionophore was also effective in partially restoring these properties in muscles that had been denervated for 3 days prior to culturing. The actions of A23187 required the presence of calcium. The effects of A23187 were similar to inhibitors of protein and ribonucleic acid synthesis, although its effect on acetylcholine receptor development was greater than its effect on overall protein synthesis (23–36% inhibition). Caffeine (5 m m) was also effective in inhibiting the changes in membrane properties of cultured denervated muscle: the membrane potential was raised 6.2 mV and extrajunctional acetylcholine receptor development was inhibited by 40%. Dibutyryl guanosine cyclic 3',5'-monophosphate at concentrations of 0.2 and 1.0 m m had no significant effect on the membrane potential or extrajunctional acetylcholine receptor development of cultured denervated muscle. Similar concentrations of dibutyryl adenosine cyclic 3',5'-monophosphate raised the membrane potential but had little effect on the development of extrajunctional acetylcholine receptors. Of the agents tested, only the divalent ionophore A23187 or caffeine inhibited the fall in membrane potential and the increase in extrajunctional acetylchonine receptors that occurred in cultured muscles. Both A23187 annd caffeine are known to elevate cytoplasmic levels of calcium ions. Since contractile activity prevents denervation-induced changes in muscle and is associated with a rise in the free intracellular calcium ion concentration, it is suggested that its effects on the regulation of the resting membrane potential and the development of extrajunctional acetylcholine receptors may be exerted through cytoplasmic calcium ions.