Dual effects of muscarinic M-2 acetylcholine receptors on the synthesis ofcyclic AMP in CHO cells: dependence on time, receptor density and receptoragonists

1 Muscarinic M-2 receptors normally inhibit the production of cyclic AMP vi a G(i) proteins, but a stimulatory component occurs in their effect at high agonist concentrations, believed to be based on the activation of G(s) pro teins. We investigated the conditions which determine the occurrence and ex tent of the stimulatory component in CHO cells stably expressing muscarinic M-2 receptors. 2 Biphasic concentration-response curves (decline followed by return toward s control values) were obtained after 10 min incubation with carbachol, oxo tremorine-M, acetylcholine, arecoline and arecaidine propargyl ester, but t he upward phase was missing with oxotremorine, methylfurmethide, furmethide and pentylthio-TZTP, Shortening the incubation favoured the occurrence of the stimulatory component. Carbachol (1 mM) and oxotremorine-M (1 mM) broug ht about net stimulation (above 100% of control) of cyclic AMP synthesis du ring 2 min incubations. The stimulatory components disappeared after the de nsity of receptors had been lowered with oxyphenonium mustard. 3 All agonists stimulated the synthesis of cyclic AMP in cells pretreated w ith pertussis toxin. 4 Most differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in t heir efficacy and the induced receptor internalization. 5 We propose that the G(s)-mediated stimulatory component of the effect of muscarinic M-2 receptors on cyclic AMP synthesis only occurs if the density of activated receptors is high enough to saturate the G(i) proteins and pr oportionate to the receptors' low affinity for the G(s) proteins. It tends to be abolished by receptor internalization.