title: Induction of Na+/K(+)-ATPase activity by long-term stimulation of nicotinic acetylcholine receptors in C2C12 myotubes.
abstract: 1. To investigate the role of long-term stimulation of nicotinic acetylcholine receptors (AChRs) on the regulation of membrane potential, non-contracting C2C12 myotubes were stimulated for 1-4 days with carbachol (10 microM) and membrane potentials were measured by the intracellular microelectrode technique after washing out of the drug. 2. The membrane potential (-45.7 mV) gradually increased by 10.1 mV to -55.8 mV 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, EC50 = 5.3 microM) 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 co-treatment (4 days) with the nicotinic AChR-channel blocker, chlorpromazine (1 microM). Membrane depolarization as such, obtained by incubation in high K+ medium (40 mM, 4 days) did not enhance Na+/K(+)-ATPase activity. 5. Non-treated myotubes possessed a high-affinity ouabain binding site (Kd = 119 nM) 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 (Kd = 13 microM). 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.
authors.affiliation: Department of Pharmacology/Clinical Pharmacology, University of Groningen, The Netherlands.
authors.forename: R H
authors.initials: RH
authors.lastname: Henning
authors.forename: S A
authors.initials: SA
authors.lastname: Nelemans
authors.forename: J
authors.initials: J
authors.lastname: van den Akker
authors.forename: A
authors.initials: A
authors.lastname: den Hertog
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018680:Cholinergic Antagonists; D004594:Electrophysiology; D004790:Enzyme Induction / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology*; D009132:Muscles / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010042:Ouabain / Q000493:pharmacokinetics; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000276:immunology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D018680:Cholinergic Antagonists; D011950:Receptors, Cholinergic; D010042:Ouabain; D002217:Carbachol; D000254:Sodium-Potassium-Exchanging ATPase