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

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 microM) of acetylcholine (ACh) and contracted at higher concentrations while endothelium-denuded strips only contracted. The ED50 for relaxation was 0.13 microM and that for contraction 1.8 microM (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 (KA) of ACh for relaxant and contractile effects were determined by irreversible blockade of a fraction of receptors with propyl benzilylcholine mustard (PBCM). The affinity constants (KA) were 0.22 microM for relaxation and 13 microM (with endothelium) and 20 microM (without endothelium) for contraction. In competition binding experiments against the muscarinic antagonist, [3H]N-methylscopolamine ([3H]NMS), the apparent affinity (KI) of ACh for binding sites in homogenates of endothelium-free coronary artery was 16 microM 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.