Comparison of the pharmacological antagonism of M 2 and M 3 muscarinic receptors expressed in isolation and in combination

Abstract

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M 2 and M 3 muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [ 3 H ] N-methylscopolamine binding to CHO cells expressing both M 2 and M 3 muscarinic receptors (CHO M 2+M 3 cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M 2 cells, whereas the other site exhibited properties like those of CHO M 3 cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M 3 and CHO M 2+M 3 cells, but not in CHO M 2 cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M 3 and CHO M 2+M 3 cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M 2 and CHO M 2+M 3 cells and a less robust inhibition in CHO M 3 cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M 3 and CHO M 2+M 3 cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M 2, CHO M 3, and CHO M 2+M 3 cells. The pharmacological antagonism of the cAMP response in CHO M 2+M 3 cells resembled that expected for a response mediated independently by both M 2 and M 3 receptors.