Muscarinic M 2 receptors preferentially couple with the G i/o class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M 2 receptors (CHO-M 2 cells) whether direct interaction of M 2 receptors with G s and G q/11 G-proteins is responsible for the latter effects. Suppression of the G s ␣ subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M 2 cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC 50 of 79 M. Removal of the G q , G 11 , or both ␣ subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G s ␣ subunit had no effect. There are five subtypes of muscarinic receptors designated as M 1 to M 5 receptors (Bonner, 1989;Bonner et al., 1987Bonner et al., , 1988. Although individual subtypes preferentially interact with particular classes of G-proteins (Jones et al., 1991;Caulfield et al., 1994;Felder, 1995), specificity of their functional outcome is not absolute (Kostenis et al., 1997a(Kostenis et al., ,b, 2005Milligan and Kostenis, 2006). We previously observed in Chinese hamster ovary cells (CHO), which specifically express individual subtypes of muscarinic receptors (Buckley et al., 1989), that stimulation of muscarinic M 2 receptors preferentially inhibits adenylyl cyclase via G i/o G-proteins . However, higher agonist concentrations lead to anomalous concentration-dependent reversal of this effect . Obliteration of M 2 muscarinic receptormediated inhibition of adenylyl cyclase by pertussis toxin treatment results in revealing of strong stimulation of cAMP production. Furthermore, it was demonstrated that stimulation of porcine M 2 receptors increases IP production in a concentration-dependent manner , Vogel et al., 1995

1 Muscarinic M 2 receptors normally inhibit the production of cyclic AMP via G i proteins, but a stimulatory component occurs in their eect at high agonist concentrations, believed to be based on the activation of G s proteins. We investigated the conditions which determine the occurrence and extent of the stimulatory component in CHO cells stably expressing muscarinic M 2 receptors. 2 Biphasic concentration-response curves (decline followed by return towards control values) were obtained after 10 min incubation with carbachol, oxotremorine-M, acetylcholine, arecoline and arecaidine propargyl ester, but the 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) brought about net stimulation (above 100% of control) of cyclic AMP synthesis during 2 min incubations. The stimulatory components disappeared after the density of receptors had been lowered with oxyphenonium mustard. 3 All agonists stimulated the synthesis of cyclic AMP in cells pretreated with pertussis toxin. 4 Most dierences between agonists regarding the stimulatory component of their eect on cyclic AMP synthesis could be explained by dierences in their ecacy and the induced receptor internalization. 5 We propose that the G s -mediated stimulatory component of the eect 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 proportionate to the receptors' low anity for the G s proteins. It tends to be abolished by receptor internalization. British Journal of Pharmacology (2001)

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