The binding of a 2-chloroethylamine derivative of oxotremorine (BM 123) to muscarinic receptors in the rat cerebral cortex.

The interaction of a mustard analogue of oxotremorine, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), with muscarinic receptors in the rat cerebral cortex was investigated using 3H-ligand-binding methods. When cortical homogenates were preincubated with BM 123 (1.0 mM), washed extensively, and then assayed for the binding of the specific muscarinic antagonist, [3H](-)-N-methylscopolamine, a decrease in binding capacity was noted without an accompanying change in affinity. The rate at which BM 123 alkylated muscarinic receptors was sensitive to temperature, with little or no receptor alkylation occurring at 0 degree. Thus, it was possible to estimate the affinity of BM 123 and its transformation products for muscarinic receptors by measuring their ability to competitively inhibit 3H-ligand binding to cortical homogenates at 0 degree. When measured by competitive inhibition of [3H]oxotremorine-M and [3H](-)-N-methylscopolamine binding, the concentrations of the aziridinium ion of BM 123 required to displace 50% of specific 3H-ligand binding were 3.5 nM and 4.5 microM, respectively. In contrast, the parent 2-chloroethylamine and its alcoholic hydrolysis product were much less active. The kinetics of the alkylation of muscarinic receptors by BM 123 were consistent with a model in which the aziridinium ion rapidly forms reversible complexes with superhigh high and low affinity sites which slowly convert to covalent complexes. The rate of alkylation of the superhigh affinity site was slowest whereas the converse was true for the low affinity site. It was possible to alkylate the high and low affinity sites selectively with BM 123 by taking advantage of kinetic differences in the rates of alkylation of these two sites. Atropine, oxotremorine, and oxotremorine-M antagonized the rate of alkylation of muscarinic receptors in a manner that was consistent with competitive inhibition.