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We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M2 and M3 muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M2 and M3 muscarinic receptors (CHO M2+M3 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 M2 cells, whereas the other site exhibited properties like those of CHO M3 cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M3 and CHO M2+M3 cells, but not in CHO M2 cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M3 and CHO M2+M3 cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M2 and CHO M2+M3 cells and a less robust inhibition in CHO M3 cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M3 and CHO M2+M3 cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M2, CHO M3, and CHO M2+M3 cells. The pharmacological antagonism of the cAMP response in CHO M2+M3 cells resembled that expected for a response mediated independently by both M2 and M3 receptors. [Copyright &y& Elsevier]

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3-1.0 microM) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M(3) muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum.