TEA inhibits ACh-induced EDRF release: endothelial Ca2+-dependent K+ channels contribute to vascular tone
The effects of K+-channel blockers on the acetylcholine (ACh)-induced relaxation of vascular smooth muscle, intracellular free Ca2+ concentration ([Ca2+](i)) elevation, and ACh-evoked outward K+ current of endothelial cells of rabbit aorta were studied using bioassay, spectrofluorimetry, and patch-clamp techniques, respectively. In bioassay experiments, ACh caused relaxation of endothelium-denuded aortic rings in a concentration-dependent manner when perfused through an endothelium-intact donor segment of aorta but not when perfused directly onto the recipient aortic ring. ACh-induced relaxation was inhibited by perfusion of tetraethylammonium ions (TEA; 5 mM) through the donor but not by perfusion directly onto the recipient segment. Glibenclamide had no effect on ACh-induced relaxation of the bioassay ring in either situation. ACh increased [Ca2+](i) at the endothelial surface of aortic strips but not at the adventitial surface. TEA inhibited ACh-induced [Ca2+](i) elevation, whereas glibenclamide had no effect. In patch-clamp experiments with freshly isolated endothelial cells, ACh evoked a biphasic outward current which was completely abolished by TEA (3 mM). It is concluded that Ca2+-dependent K+ channels are important for increasing [Ca2+](i) during agonist stimulation and consequently for the synthesis/release of endothelium-derived relaxing factors (EDRFs). Furthermore, endothelial ATP- sensitive K+ channels do not contribute to ACh-induced relaxation or evoke an increase in endothelial [Ca2+](i) of rabbit thoracic aorta.