Pharmacological analysis of the novel mode of interaction between xanomeline and the M₁ muscarinic acetylcholine receptor

Previous findings in our laboratory suggested that the M₁ muscarinic acetylcholine receptor (mAChR) agonist xanomeline exhibits a novel mode of interaction that involves persistent binding to and activation of the M₁ mAChR, subsequent to extensive washout, as well as a possible insurmountable element. In the present study, we examined this interaction in greater detail, using Chinese hamster ovary cells transfected with the genes for the M₁ mAChR and neuronal nitric oxide synthase. Pretreatment of cells with xanomeline, followed by extensive washout, resulted in elevated basal levels of neuronal nitric oxide synthase activity that were suppressed by the antagonists atropine or pirenzepine in a concentration-dependent manner. Analysis of the data yielded estimates of Schild slope factors and pK(B) values for the antagonists that were consistent with a model of simple competition between these latter agents and the persistently bound form of xanomeline. The ability of the antagonists to produce parallel dextral shifts of the concentration-response curves to carbachol and xanomeline was also investigated. The interaction between xanomeline and pirenzepine appeared to be insurmountable, but this may have been due to an equilibrium artifact. In contrast, the interaction between xanomeline and atropine conformed to a model of competition, indicating that the mode of interaction of free xanomeline at the M₁ mAChR is pharmacologically identical with that of the persistently bound form. Radioligand binding studies also showed that the presence of various concentrations of xanomeline had no significant effect on the calculated affinity of atropine or pirenzepine in inhibiting the binding of [³H]N-methylscopolamine. Overall, these findings suggest that the persistent attachment of xanomeline to the M₁ mAChR does not prevent this agonist from interacting with the classic binding site in a competitive fashion.