title: Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.
abstract: Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, United States of America.
authors.forename: Eva
authors.initials: E
authors.lastname: Šantrůčková
authors.affiliation: Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Doležal
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, United States of America.
authors.forename: Esam E
authors.initials: EE
authors.lastname: El-Fakahany
authors.affiliation: Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Jakubík
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D019832:N-Methylscopolamine / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043588:Receptor, Muscarinic M4 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013830:Thiadiazoles / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D011725:Pyridines; D043643:Receptor, Muscarinic M1; D043588:Receptor, Muscarinic M4; D013830:Thiadiazoles; C075257:xanomeline; D000242:Cyclic AMP; D002118:Calcium; D019832:N-Methylscopolamine