Induction of Na+/K(+)-ATPase activity by long-term stimulation of nicotinic acetylcholine receptors in C2C12 myotubes

1 To investigate the role of long-term stimulation of nicotinic acety]choline receptors (AChRs) on the regulation of membrane potential, non-contracting C2C12 myotubes were stimulated for 1-4 days with carbachol (10 mu M) and membrane potentials were measured by the intracellular microelectrode technique after washing out of the drug. 2 The membrane potential (-45.7mV) gradually increased by 10.1 mV to -55.8mV during 4 days treatment, which was caused by enhanced electrogenic Na+/K+-pumping. 3 The concentration-dependent enhancement of Na+/K+-ATPase activity in long-term carbachol-treated myotubes (4 days, EC(50) = 5.3 mu M) was prevented by co-treatment with the competitive nicotinic AChR antagonist, pancuronium but not by the muscarinic antagonist, atropine. 4 Enhanced Na+/K+-ATPase activity still developed in carbachol-stimulated myotubes during cotreatment (4 days) with the nicotinic AChR-channel blocker, chlorpromazine (1 mu M). Membrane depolarization as such, obtained by incubation in high K+ medium (40mM, 4 days) did not enhance Na+/K+-ATPase activity. 5 Non-treated myotubes possessed a high-affinity ouabain binding site (K-d = 119nM) in association with the low Na+/K+-pumping activity. Long-term stimulation of myotubes (4 days) with carbachol or with a combination of carbachol and chlorpromazine was accompanied by the development of an additional low-affinity ouabain binding site (K-d = 13 mu M). 6 Binding of monoclonal antibodies directed against either alpha(1)- or alpha(2)-subunit of Na+/K+-ATPase were both increased in myotubes treated with carbachol (4 days). 7 These results support the concept that nicotinic AChRs regulate Na+/K+-ATPase activity, independent of the functionality of the receptor-operated ion-channel.