Determination of dissociation constants and relative efficacies of oxotremorine analogs at muscarinic receptors in the guinea-pig ileum by pharmacological procedures.

The muscarinic activities in the isolated guinea-pig ileum of oxotremorine, its acetamide analog, I, and of carbachol were resolved into affinity and efficacy components. The method used involved irreversible blockade of spare receptors with propylbenzilylcholine mustard (PrBCM). This method also was employed to determine dissociation constants (KA) and relative efficacies of the enantiomers of two oxotremorine analogs (II and III) with partial agonist properties. The KA values thus obtained for the enantiomers of Compounds II and III were almost identical to those estimated pharmacologically by two independent methods, one of which did not make use of an irreversible antagonist. The dissociation constants of the enantiomers of the competitive antagonist IV, determined against carbachol, were the same before and after inactivation of about 90% of the receptors with PrBCM. These results appear to justify the use of PrBCM for the determination of dissociation constants and relative efficacies of muscarinic agonists, despite claims in the recent literature to the contrary. The KA values of oxotremorine (6.79 X 10(-7) M) and carbachol (1.64 X 10(-5) M) were in good agreement with those determined pharmacologically and biochemically in other laboratories. The efficacy of carbachol was 7.2-fold higher than that of oxotremorine which was only slightly less efficacious than Compound I. A survey of structure-activity relationships among the eight oxotremorine analogs studied suggested that the structural requirements for achieving high affinity are independent of those leading to high efficacy.