Application of competition kinetics to investigate rat brain muscarinic receptors.

We have recently developed a method for obtaining kinetic parameters of unlabeled ligands that is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. We employed this method to investigate the kinetics of muscarinic agonist binding to rat brain medulla-pons homogenates. The agonists studied were acetylcholine, carbamylcholine and oxotremorine, with N-methyl-4-[3H]-piperidyl benzilate employed as the radiolabeled ligand. Our results suggest that the binding of muscarinic agonists to high-affinity sites is characterized by dissociation rate constants higher by two orders of magnitude than those of antagonists and by similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low-affinity agonist binding sites are in the association rate constants, which were 2 to 5 orders of magnitude lower for agonists. The data demonstrate that isomerization of the muscarinic receptors following ligand binding is insignificant in the case of agonists, unlike that of antagonists. Moreover, as is shown in the medulla-pons preparation, agonist-induced interconversion between high- and low-affinity binding sites does not occur to an appreciable extent.