Combination of Ca2+-activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

Background and purpose: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. Experimental approach: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+](i)) were measured. Key results. A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ activated K channels abolished acetylcholine (10 mu M)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+](i). Cyclopiazonic acid (CPA, 30 mu M), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+](i), but inhibited the sustained NO release induced by CPA. Conclusions and Implications: Acetylcholine increases endothelial cell [Ca2+](i) by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.