Biphasic release of endothelium-derived relaxing factor(s) by acetylcholine from perfused canine femoral arteries. Characterization of muscarinic receptors

Abstract

Experiments were designed to compare the release of endothelium-derived relaxing factor(s) in response to various muscarinic receptor agonists from canine femoral arteries mounted in organ chambers or perfused in a bioassay system. In rings of femoral arteries, suspended for isometric tension recording in organ chambers, acetylcholine induced endothelium-dependent relaxations during contractions evoked by prostaglandin F(2a). Atropine and pirenzepine antagonized these relaxations in a competitive manner, atropine with a higher affinity (K(B) = 1.9 x 10-9 M) than pirenzepine (K(B) = 5.4 x 10-7 M). Carbachol and McN-A-323 also evoked endothelium-dependent relaxations, and pirenzepine inhibited these responses with a similar low potency. The order of relative potency of the agonists in organ chamber studies was acetylcholine = carbachol >> McN-A-343. Isolated segments of femoral arteries with endothelium were perfused (2 ml/min) with modified Krebs-Ringer-bicarbonate solution containing indomethacin; the perfusate was bioassayed for endothelium-derived relaxing factor(s) by means of a ring of coronary artery without endothelium. When infused above but not below the femoral artery, low concentrations (10-8-10-7 M) of acetylcholine caused transient relaxations of the bioassay ring contracted with prostaglandin F(2a); higher concentrations of acetylcholine caused sustained decreases in tension. Atropine inhibited the two phases of the concentration-relaxation curve with similar potencies. Pirenzepine inhibited both phases in a competitive manner but exhibited significantly higher potency against the first- (ED50, 1.9 x 10-9 M) than against the second-phase responses (ED50, 2.1 x 10-7 M). Compound McN-A-343 induced only transient decreases in tension, whereas carbachol caused sustained relaxations. The order of relative potency of the agonists in bioassay studies was McN-A-343 = acetylcholine >> carbachol (for transient responses) and acetylcholine = carbachol >> McN-A-343 (for sustained responses). These experiments indicate that, in isolated femoral arterial rings studied in the organ chamber, muscarinic receptors with low affinity for pirenzepine appear to be involved in the endothelium-dependent relaxations evoked by various muscarinic receptor agonists. In perfused segments of the same artery, acetylcholine stimulates the intraluminal release of endothelium-derived relaxing factor(s) in a manner that produces a biphasic concentration-relaxation curve in a superfused bioassay preparation. Although this system does not allow rigorous receptor analysis, it is tentatively postulated that muscarinic receptors with different properties mediate the two phases of the response.