Cardiac β-adrenergic receptor: Evaluation of FM 24 as a new and very slowly dissociable blocker

Abstract

The compound 1-(2-exo-bicyclo[2, 2, 1]hept-2-ylphenoxy)-3-[(1-methyl-ethyl) amino]-2-propanol, hydrochloride (FM 24) was found to inhibit isoproterenol-dependent adenylate cyclase from rat cardiac particulate fraction with an apparent dissociation constant of 0.2 μM as compared to 0.01 μM for propranolol. The inhibition brought about by FM 24 was apparently non-dissociable since it could not be reversed by extensive washing, unlike that of propranolol. Preincubation of the particulate fraction for 2 min with either antagonist led to a 2 to 3-fold increase in the apparent affinity of FM 24 for the system, with no change in the apparent affinity of propranolol. Kinetic experiments revealed that the effect of propranolol could be reversed by addition of an excess of isoproterenol while that of FM 24 could not. This drug had no effect on the basal and fluoride-dependent activities of the enzyme. Assays performed with [U- 14C]FM 24 indicated that the long lasting inhibition was not related to conversion of FM 24 into an active metabolite. FM 24 blocked also the specific binding of (−)-[ 3H]dihydroalprenolol to rat heart membranes. Isoproterenol and propranolol protected the β-receptor binding sites against inactivation by FM 24. Scatchard analysis of (−)-[ 3H]dihydroalprenolol binding in the presence and absence of β-anatgonists revealed that exposure of membranes to FM 24 resulted in a reduction in the number of receptor binding sites, while the affinity of the unreacted sites for (−)-[ 3H]dihydroalprenolol remained unchanged. By contrast, exposure of membranes to propranolol led to a reduction in the apparent affinity for (−)-[ 3H]dihydroalprenolol without significant reduction in the number of binding sites. These findings strongly suggest that FM 24 is a slowly dissociable β-receptor antagonist, since no covalent binding to the β-receptor sites is likely to occur.