Evidence for muscarinic receptors in endothelial cells from combined functional and binding studies

Abstract

The aim of this study was to characterize muscarinic receptors of the bovine coronary artery by means of a combination of mechanical relaxation and contraction responses and radioligand binding data. Fresh helical strips of bovine coronary artery with intact endothelium relaxed in response to low concentrations (0.03−1 μM) of acetylcholine (ACh) and contracted at higher concentrations while endothelium-denuded strips only contracted. The ED 50 for relaxation was 0.13 μM and that for contraction 1.8 μM (without endothelium); in the presence of endothelium, contraction dose-response curves were shifted to the right and the maximum contraction was reduced. In order to determine the location of the receptors mediating vasorelaxation, apparent affinity constants (K A) of ACh for relaxant and contractile effects were determined by irreversible blockade of receptors with propyl benzilycholine mustard (PBCM). The affinity constants (K A) were 0.22 μM for relaxation and 13 μM (with endothelium) and 20 μM (without endothelium) for contraction. In competition binding experiments against the muscarinic antagonist, [ 3H]N-methylscopolamine ([ 3H]NMS), the apparent affinity (K 1) of ACh for binding sites in homogenates of endothelium-free coronary artery was 16 μM which was not different from the affinity constant determined in functional contraction experiments. Thus, the affinity constant of ACh determined for relaxation responses with endothelium-preserved vessels had no correlate in the binding affinity as determined with endothelium-free arteries. These findings indicate that bovine coronary arteries are relaxed by ACh through muscarinic receptors located on the endothelium whereas contractions are mediated by receptors on smooth muscle cells.