Effects of xanomeline at the M3 muscarinic receptor

Objective: There is a high level of homology at the primary binding site across all five subtypes of muscarinic receptors, making it difficult to develop an agonist that selectively binds to a single subtype. Xanomeline has been touted in the literature as being a functionally selective M1/M4 muscarinic receptor agonist. These receptors are targets for the treatment of Alzheimer's disease and schizophrenia, respectively. However, side effects associated with xanomeline include changes in salivation and gastrointestinal tract problems, which are mediated by the M3 receptor. The goal of the current research was to determine the binding and functional effects of xanomeline treatment on M3 muscarinic receptors expressed in Chinese hamster ovary cells. Methods: Cells were treated with xanomeline for one hour followed by washing and either used immediately or after waiting for 23 hours. Control groups included cells that were not exposed to xanomeline and cells that were treated with xanomeline for 24 hours. Radioligand binding and functional assays were conducted to determine the effects of xanomeline treatment. GraphPad Prism 4.0 was used for data and statistical analysis. Results: Xanomeline is able to bind reversibly and in a wash-resistant manner to the M3 muscarinic receptor. It exhibits partial agonistic activity at these receptors, also with reversible and persistent components. Incubation with xanomeline for one hour followed by washing and waiting 23 hours leads to long-term changes in the binding properties and function of the receptor. Conclusions: Our results demonstrate that exposure to xanomeline for a brief period of time followed by washing and waiting leads to long-term changes in receptor binding and function. Mechanistically these changes could be due to internalization or down-regulation of the receptor or allosteric modification of the receptor conformation. Experiments are currently being undertaken to differentiate between these possibilities. Work supported by grants T32DE007288 and R01NS25743.