ABSTRACT
Wire myograph and immunofluorescence techniques were used to investigate the nature and location of the potassium channels involved in mediating acetylcholine-mediated relaxation of contracted small mesenteric arteries from endothelial nitric oxide synthase knock-out, /, mice. Previous data demonstrated that the contribution of endothelium-derived hyperpolarizing factor (EDHF) to acetylcholine-mediated relaxation of contracted small mesenteric arteries from / mice was up-regulated compared to tissues from / mice. In the current study, relaxation to acetylcholine was unaffected by charybdotoxin or apamin alone, but was significantly inhibited by the combination of these agents, or tubocurarine. The additive effects of scyllatoxin and iberiotoxin did not mimic the effects of the apamin/charybdotoxin combination. Thus, acetylcholine-induced EDHF-mediated relaxation in small mesenteric arteries involved the activation of tubocurarine and apamin/charybdotoxin sensitive K channels. Immunofluorescence labeling with antibodies for small conductance potassium channels subtypes, SK2 and SK3, on mouse whole mesenteric arteries and freshly isolated single vascular smooth muscle and endothelial cells was also conducted with antibodies to -smooth muscle actin and von Willebrand factor to identify vascular smooth muscle and endothelial cells respectively. The data indicate that the SK2 was associated with the plasma membrane of endothelial and the nuclear region of vascular smooth muscle cells. SK3 was associated with the plasmamembrane of both vascular smooth muscle and endothelial cells. Together these data indicate that the apamin-sensitive channel involved in mediating endotheliumdependent hyperpolarization may be associated with both vascular smooth muscle and endothelial cells; however, SK2, which possesses the highest affinity for apamin, appears to be present in the greatest density and is only associated with the endothelial cell plasmamembrane.
