Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors

PLoS One. 2014 Feb 18;9(2):e88910. doi: 10.1371/journal.pone.0088910. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / drug effects
  • CHO Cells
  • Calcium / metabolism
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cricetinae
  • Cricetulus
  • Cyclic AMP / metabolism
  • Extracellular Space / drug effects
  • Extracellular Space / metabolism
  • Humans
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism
  • Kinetics
  • N-Methylscopolamine / pharmacology
  • Pyridines / pharmacology*
  • Receptor, Muscarinic M1 / agonists*
  • Receptor, Muscarinic M1 / antagonists & inhibitors
  • Receptor, Muscarinic M1 / metabolism*
  • Receptor, Muscarinic M4 / agonists*
  • Receptor, Muscarinic M4 / antagonists & inhibitors
  • Receptor, Muscarinic M4 / metabolism*
  • Thiadiazoles / pharmacology*
  • Time Factors


  • Pyridines
  • Receptor, Muscarinic M1
  • Receptor, Muscarinic M4
  • Thiadiazoles
  • xanomeline
  • Cyclic AMP
  • Calcium
  • N-Methylscopolamine

Grants and funding

This work was supported by the Academy of Sciences of the Czech Republic project [AV0Z 50110509] and support [RVO:67985823], the Grant Agency of the Czech Republic grants [305/09/0681] and [P304/12/G069]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.