Immediate and Delayed Consequences of Xanomeline Wash-Resistant Binding at the M3 Muscarinic Receptor

Abstract

Xanomeline is thought to be a M1/M4 functionally selective agonist at muscarinic receptors. We have previously demonstrated that it binds in a unique manner at the M1 receptor. In the current study, we examined the ability of xanomeline to bind to the M3 receptor and determined the long-term consequences of this mode of binding in Chinese hamster ovary cells expressing M3 receptors. Xanomeline binds in a reversible and wash-resistant manner at the M3 receptor and elicits a functional response under both conditions. Long-term exposure to xanomeline resulted in changes in the binding profile of [3H]NMS and a decrease in cell-surface receptor density. Additionally, pretreatment with xanomeline was associated with antagonism of the functional response to subsequent stimulation by conventional agonists. Our results indicate that xanomeline binds to and activates the M3 muscarinic receptor in a wash-resistant manner, and that this type of binding results in time-dependent receptor regulation.

Involvement of a Subpopulation of Neuronal M4Muscarinic Acetylcholine Receptors in the Antipsychotic-like Effects of the M1/M4Preferring Muscarinic Receptor Agonist Xanomeline

“…Since xanomeline is not entirely M 1 /M 4 -specific (Jakubík et al, 2006;Noetzel et al, 2009), classical cholinomimetic side effects, e.g., gastrointestinal side effects, were observed in humans (Bodick et al, 1997;Shekhar et al, 2008). Previous efforts to develop highly selective orthosteric agonists of the individual muscarinic subtypes have not been successful.…”

Mechanisms of M3 Muscarinic Receptor Regulation by Wash-Resistant Xanomeline Binding

“…1 a and b, respectively). Once again, this is in contrast to the effects of xanomeline under similar conditions whereby inhibition of [ 3 H]NMS binding results in a biphasic curve and reaches a maximum of nearly 100% [13] . Previous research demonstrated a decrease in protein content following xanomeline pretreatments at the M1 receptor [14] .…”

“…Binding occurs in a reversible manner at the orthosteric (primary) binding site and in a wash-resistant manner at an allosteric (secondary) binding site [12] . We have demonstrated that this unique binding profile also occurs at the M3 receptor [13] . At both the M1 and M3 receptors, wash-resistant xanomeline binding leads to a long-term decrease in cell-surface receptor expression and attenuation of receptor function [13,14] .…”

“…We have demonstrated that this unique binding profile also occurs at the M3 receptor [13] . At both the M1 and M3 receptors, wash-resistant xanomeline binding leads to a long-term decrease in cell-surface receptor expression and attenuation of receptor function [13,14] . This is similar to the well-documented changes in receptor expression and sensitivity induced by conventional reversible muscarinic agonists [15][16][17][18] .…”

“…Previous research demonstrated that brief treatment of cells expressing the M3 receptor with xanomeline resulted in a monophasic inhibition curve of [ 3 H]NMS binding whereas long-term treatment produced a biphasic binding inhibition curve [13] . Experiments were designed to confirm that the observed effects of xanomeline are due to its unique interactions with the M3 receptor rather than a general property of agonists.…”