Pharmacological evaluation of the long-term effects of xanomeline on the M₁ muscarinic acetylcholine receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M₁ and M₄ receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M₁ muscarinic receptor. Pretreatment of CHO cells that stably express the M₁ receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomelineinduced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.