1. Muscarinic M(2) receptors normally inhibit the production of cyclic AMP via 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) 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 differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in their 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 proportionate to the receptors' low affinity for the G(s) proteins. It tends to be abolished by receptor internalization.

Institute of Physiology Academy of Sciences of the Czech Republic Vídenská 1083 14220 Prague Czech Republic

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BONHAUS D.W., CHANG L.K., KWAN J., MARTIN G.R. Dual activation and inhibition of adenylyl cyclase by cannabinoid receptor agonists: evidence for agonist-specific trafficking of intracellular responses. J. Pharmacol. Exp. Ther. 1998;287:884–888. PubMed

BUCKLEY N.J., BONNER T.I., BUCKLEY C.M., BRANN M.R. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol. 1989;35:469–476. PubMed

BURFORD N.T., NAHORSKI S.R. Muscarinic m1 receptor-stimulated adenylate cyclase activity in Chinese hamster ovary cells is mediated by Gs alpha and is not a consequence of phosphoinositidase C activation. Biochem. J. 1996;315:883–888. PubMed PMC

CARRUTHERS A.M., WARNER A.J., MICHEL A.D., FENIUK W., HUMPHREY P.P.A. Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO-K1 cells and involvement of Gαs proteins. Br. J. Pharmacol. 1999;126:1221–1229. PubMed PMC

CAULFIELD M.P. Muscarinic receptors – characterization, coupling and function. Pharmacol. Ther. 1993;58:319–379. PubMed

CHENG Y.-C., PRUSOFF W.H. Relation between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973;22:3099–3108. PubMed

DITTMAN A.H., WEBER J.P., HINDS T.R., CHOI E.-J., MIGEON J.C., NATHANSON N.M., STORM D.R. A novel mechanism for coupling of m4 muscarinic acetylcholine receptors to calmodulin-sensitive adenylyl cyclases: crossover from G protein-coupled inhibition to stimulation. Biochemistry. 1994;33:943–951. PubMed

DOLEŽAL V., LISÁ V., TUČEK S. Differential effects of the M1–M5 muscarinic acetylcholine receptor subtypes on intracellular calcium and on the incorporation of choline into membrane lipids in genetically modified Chinese hamster ovary cell lines. Brain Res. Bull. 1997;42:71–78. PubMed

EASON M.G., JACINTO M.T., LIGGETT S.B. Contribution of ligand structure to activation of alpha2-adrenergic receptor subtype coupling to Gs. Mol. Pharmacol. 1994;45:696–702. PubMed

EASON M.G., KUROSE H., HOLT B.D., RAYMOND J.R., LIGGETT S.B. Simultaneous coupling of alpha2-adrenergic receptors to two G-proteins with opposing effects. J. Biol. Chem. 1992;267:15795–15801. PubMed

EASON M.G., LIGGETT S.B. Identification of a Gs coupling domain in the amino terminus of the third intracellular loop of the alpha2A-adrenergic receptor. J. Biol. Chem. 1995;270:24753–24760. PubMed

EGLEN R.M., MONTGOMERY W.W., WHITING R.L. Negative and positive inotropic responses to muscarinic agonists in guinea pig atria in vitro. J. Pharmacol. Exp. Ther. 1988;247:911–917. PubMed

FRASER C.M., ARAKAWA S., MCCOMBIE W.R., VENTER J.C. Cloning, sequence analysis, and permanent expression of a human alpha2-adrenergic receptor in Chinese hamster ovary cells. J. Biol. Chem. 1989;264:11754–11761. PubMed

GURWITZ D., HARING R., HELDMAN E., FRASER C.M., MANOR D., FISCHER A. Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands. Eur. J. Pharmacol. 1994;267:21–31. PubMed

IMAI S., OHTA H. Positive inotropic effects induced by carbachol in rat atria treated with islet-activating protein (IAP) – association with phosphatidylinositol breakdown. Br. J. Pharmacol. 1982;94:347–354. PubMed PMC

JAKUBÍK J., BAČÁKOVÁ L., EL-FAKAHANY E.E., TUČEK S. Subtype selectivity of the positive allosteric action of alcuronium at cloned M1-M5 muscarinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 1995;274:1077–1083. PubMed

JAKUBÍK J., BAČÁKOVÁ L., EL-FAKAHANY E.E., TUČEK S. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol. Pharmacol. 1997;52:172–179. PubMed

JAKUBÍK J., BAČÁKOVÁ L., LISÁ V., EL-FAKAHANY E.E., TUČEK S. Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc. Natl Acad. Sci. U.S.A. 1996;93:8705–8709. PubMed PMC

JAKUBÍK J., EL-FAKAHANY E.E., TUČEK S. Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors. J. Biol. Chem. 2000;275:18836–18844. PubMed

JOHNSON R.A., ALVAREZ R., SALOMON Y. Determination of adenylyl cyclase catalytic activity using single and double column procedures. Methods Enzymol. 1994;238:31–56. PubMed

JONES S.V.P., HEILMAN C.J., BRANN M.R. Functional responses of cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol. 1991;40:242–247. PubMed

KENAKIN T., BOSELLI C. Promiscuous or heterogeneous muscarinic receptors in rat atria? I. Schild analysis with simple competitive antagonists. Eur. J. Pharmacol. 1990;191:39–48. PubMed

KRUMINS A.M., BARBER R. The stability of the agonist β2-adrenergic receptor-Gs complex: evidence for agonist-specific states. Mol. Pharmacol. 1997;52:144–154. PubMed

MICHAL P., EL-FAKAHANY E.E., TUČEK S. Agonist-specific effects of the activation of muscarinic acetylcholine receptors detected in experiments with the synthesis of cyclic AMP. J. Neurochem. 1999a;73:S134.

MICHAL P., LYSÍKOVÁ M., EL-FAKAHANY E.E., TUČEK S. Clozapine interaction with the M2 and M4 subtypes of muscarinic receptors. Eur. J. Pharmacol. 1999b;376:119–125. PubMed

MIGEON J.C., NATHANSON N.M. Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors. Preferential coupling of m4 receptors to Gi alpha-2. J. Biol. Chem. 1994;269:9767–9773. PubMed

MILLIGAN G.Altering the relative stoichiometry of receptors, G proteins and effectors: effects on agonist function General Pharmacology: The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems 2000Berlin: Springer Verlag; 363–389.ed. Kenakin, T. & Angus, J.A. pp

NASMAN J., JANSSON C.C., AKERMAN K.E.O. The second intracellular loop of the alpha(2)-adrenergic receptors determines subtypes-specific coupling to cAMP production. J. Biol. Chem. 1997;272:9703–9708. PubMed

NEUBIG R.R. Membrane organization in G-protein mechanisms. FASEB J. 1994;8:939–946. PubMed

PEPPERL D.J., REGAN J.W. Selective coupling of alpha2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells. Mol. Pharmacol. 1993;44:802–809. PubMed

PEREZ D.M., HWA J., GAIVIN R., MATHUR M., BROWN F., GRAHAM R.M. Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor. Mol. Pharmacol. 1996;49:112–122. PubMed

SAUTEL M., MILLIGAN G. Loss of activation of Gs but not Gi following expression of an α2A-adrenoceptor-Gi1α fusion protein. FEBS Lett. 1998;436:46–50. PubMed

STRANGE P.G. G-protein coupled receptors – conformations and states. Biochem. Pharmacol. 1999;58:1081–1088. PubMed

VOGEL W.K., MOSSER V.A., BULSECO D.A., SCHIMERLIK M.I. Porcine m2 muscarinic acetylcholine receptor-effector coupling in Chinese hamster ovary cells. J. Biol. Chem. 1995;270:15485–15493. PubMed

WEBB W.W., PAPPANO A.J. Muscarinic agonist-induced positive inotropic response in chick atria. Life Sci. 1995;57:2365–2376. PubMed

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