PT - JOURNAL ARTICLE AU - Lauren T. May AU - Yvonne Lin AU - Patrick M. Sexton AU - Arthur Christopoulos TI - Regulation of M<sub>2</sub> Muscarinic Acetylcholine Receptor Expression and Signaling by Prolonged Exposure to Allosteric Modulators AID - 10.1124/jpet.104.073767 DP - 2005 Jan 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 382--390 VI - 312 IP - 1 4099 - http://jpet.aspetjournals.org/content/312/1/382.short 4100 - http://jpet.aspetjournals.org/content/312/1/382.full SO - J Pharmacol Exp Ther2005 Jan 01; 312 AB - The effects of prolonged exposure of M2 muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3′-phthalimidopropyl)-ammonium bromide (C7/3′-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [3H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 μM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 μM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M2 mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation. The American Society for Pharmacology and Experimental Therapeutics