Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca2+-activated K+ channels (KCa) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of KCa with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with Nω-nitro-l-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance KCa (SKCa) channels with apamin and intermediate-conductance KCa (IKCa) channels with triarylmethane-34 (TRAM-34). Apamin–TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca2+ buffering with BAPTA elicited a similar increment in superoxide. Apamin–TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOSThr495). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOSThr495 observed during apamin and TRAM-34 application. Our results indicate that blockade of SKCa and IKCa activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOSThr495. These findings disclose a novel mechanism involved in the control of NO production.

► SKCa and IKCa channels interact with NO production in endothelial cells. ► ACh-induced vasodilation and NO production depend on SKCa and IKCa channel opening. ► Inhibition of SKCa and IKCa increases O2•− formation by NAD(P)H oxidase activation. ► NAD(P)H oxidase-derived O2•− inhibits eNOS by its phosphorylation at threonine 495. ► eNOS activity is controlled by SKCa and IKCa through NAD(P)H oxidase/O2•− signaling.