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

Abstract

Background/Aims: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. Methods: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. Results: The above treatment protocol with xanomeline resulted in similar effects of the binding of [³H]NMS and [³H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. Conclusion: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.

Long-Term Activation upon Brief Exposure to Xanomleline Is Unique to M1 and M4 Subtypes of Muscarinic Acetylcholine Receptors

“…Only indirect evidence from in vivo and behavioral experiments supports xanomeline selectivity [7] , [10] . In contrast, xanomeline activates all subtypes of muscarinic receptors with the same potency [20] , [23] , [24] ( Fig. 1 and Table 1 ), and the affinity of xanomeline reversible as well as wash-resistant binding is the same at all receptor subtypes [25] .…”

“…The fundamental question where xanomeline selectivity in vivo comes from remains unanswered. Three possibilities may be considered, where xanomeline functional selectivity may be based on: a) pharmacodynamics (receptor level); b) differential receptor regulation (cell level) [24] , [26] ; c) pharmacokinetics (system level).…”

“…Recent data suggest that xanomeline functional preference could be based on differential regulation of muscarinic receptor subtypes [24] , [26] . It has been shown repeatedly that regulation of muscarinic receptors differs among receptor subtypes [31] – [33] and is agonist dependent [34] .…”

“…Functional subtype preference of xanomeline among muscarinic receptors is rather puzzling. Its reversible binding and receptor activation occur with the same affinity and potency at all subtypes of muscarinic receptors [20] , [23] , [24] . Also xanomeline wash-resistant binding occurs at all receptor subtypes with the same affinity [25] .…”

“…So far, the only observed qualitative exception from uniform behavior of xanomeline at muscarinic receptors is functional antagonism by wash-resistant xanomeline at M 5 receptors [22] . There are also differences in kinetics of xanomeline binding and activation between M 1 and M 2 receptors [20] and in long-term effects and receptor regulation between M 1 and M 3 receptors [24] , [26] .…”