Constitutive activity of the M₁–M₄ subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

Abstract

We investigated whether muscarinic receptors of the MrM 4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N-methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositul phosphates was inhibited by atropine and NVIS in CHO cells stably transfected with human ml and m3 and with rat ml muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF-DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M~-M 4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.antagonists permitted the demonstration of the constitutive activity of ~ opioid receptors [7], c~2-adrenoceptors [9], fl2-adrenoceptors [10][11][12], 5-hydroxytryptaminezc receptors [13] and bradykinin receptors [14].The present report describes the effects of four muscarinic antagonists on two processes which are controlled by muscarinic receptors, namely the synthesis of cyclic AMP (normally inhibited by the M 2 and M 4 subtypes of muscarinic receptors) and the hydrolysis of phosphoinositides (normally activated by the Ml, M3 and M5 subtypes of muscarinic receptors -review [15]). No reports were available on the action of muscarinic antagonists applied alone on these processes by the time our experiments were started. Recently, however, Burstein et al.[16] described an inhibitory effect of atropine and pirenzepine on the hydrolysis of phosphoinositides in cells equipped with M3 muscarinic receptors and excess Gq protein.

Mixed Agonist–Antagonist Properties of Clozapine at Different Human Cloned Muscarinic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells

“…Moreover, the response to clozapine was opposite to that elicited by atropine, which significantly increased cyclic AMP formation. This observation agrees with previous studies showing that the m2 receptor expressed in CHO and heart cells displays constitutive activity and that atropine and other muscarinic antagonists increase cyclic AMP accumulation likely by stabilizing the inactive conformation of the receptor (Jakubik et al 1995;Vogel et al 1995). As observed in CHO/m1 and m3 cells, the agonist efficacy of clozapine at the m2 receptor was lower than that of the full agonist carbachol, and, when the two drugs were combined, clozapine reduced the cyclic AMP inhibition elicited by carbachol to the level determined by its maximal effect.…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…K P Ͻ K P.G ). The same constraint was required in previous analyses with the ternary complex model (33), and it is consistent with reports that N- [ 3 H]-methylscopolamine is an inverse agonist (45). All other parameters were defined by a minimum in the sum of squares.…”

“…For assays with purified receptor in solution, buffer G was supplemented with 0.1% digitonin and 0.02% cholate. An aliquot of the ligand-containing solution (49 l for receptor in solution and vesicles, 43 l for receptor in nanodiscs) was added to the receptor (4 l, solution and vesicles; 10 l, nanodiscs) contained in a polypropylene microcentrifuge tube, and the reaction mixture was incubated at 30°C for 45 3 H]methylscopolamine, assays with and without GMP-PNP were performed in parallel; in studies at graded concentrations of oxotremorine-M, assays at 2-4 different concentrations of GMP-PNP were performed in parallel. Nonspecific binding was measured in the presence of unlabeled N-methylscopolamine (1 mM), which was premixed with the radioligand prior to the addition of the receptor.…”

Constitutive Activity and Inverse Agonism at the M2Muscarinic Acetylcholine Receptor

“…Thus, the ability of atropine, and a subset of other mACh receptor antagonists, to suppress constitutive activity has been reported in membrane (Hilf and Jakobs, 1992) and intact (Jakubík et al, 1995) cell preparations endogenously or recombinantly expressing M 2 mACh receptors. Any constitutive activity exhibited by the wild-type receptor can often be enhanced by mutagenesis of key domains within the GPCR (Parnot et al, 2002;Seifert and Wenzel-Seifert, 2002).…”