Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux.
An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.
Hunter
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Nathanson
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D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D007473:Ion Channels / Q000187:drug effects; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D011868:Radioisotopes; D011976:Receptors, Muscarinic / Q000032:analysis*; D012413:Rubidium
D007473:Ion Channels; D011868:Radioisotopes; D011976:Receptors, Muscarinic; D001285:Atropine; D002217:Carbachol; D012413:Rubidium; D011188:Potassium