Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase.
Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) 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 N(ω)-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 K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) 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 Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.
Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Pablo S
PS
Gaete
Mauricio A
MA
Lillo
Nicolás M
NM
Ardiles
Francisco R
FR
Pérez
Xavier F
XF
Figueroa
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001030:Apamin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D066298:In Vitro Techniques; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008638:Mesenteric Arteries / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology
D061207:Calcium Ionophores; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D011720:Pyrazoles; D051640:Small-Conductance Calcium-Activated Potassium Channels; C411671:TRAM 34; D014665:Vasodilator Agents; D013481:Superoxides; D001030:Apamin; D009569:Nitric Oxide; D015759:Ionomycin; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019255:NADPH Oxidases; D000109:Acetylcholine