AMPK phosphorylation of KCa2.3 alleviates angiotensin II-induced endothelial dysfunction.

Abstract

The endothelial small-conductance calcium-activated potassium channels (K_Ca2.3) are indispensable for endothelium-dependent hyperpolarization (EDH) response, mainly in resistance arteries. We recently demonstrated in diet-induced obese mice that adenosine monophosphate-activated protein kinase (AMPK) upregulates endothelial K_Ca2.3 expression and improves endothelial function. However, the molecular mechanism of regulation of K_Ca2.3 by AMPK remains less explored. Using techniques of bioinformatics, molecular biology and wire myograph system, we examined K_Ca2.3 phosphorylation by AMPK in human umbilical vein endothelial cells (HUVECs), human embryonic kidney 293 (HEK-293T) cells and second-order mesenteric resistance arteries from angiotensin II-induced hypertensive mice. In HUVECs, treatment with activators of AMPK (AICAR, metformin, and MK-8722) significantly increased phosphorylation of K_Ca2.3 Thr106 (human), which was antagonized by AMPK inhibitor compound C. In HEK-293T cells, K_Ca2.3 current was enhanced by AMPK activation or phosphomimetic mutant K_Ca2.3 (T106D), which was abolished after de-phosphomimetic mutant (T106A) or deletion of K_Ca2.3 of Thr106 site (T106Del). In mice with angiotensin II infusion, 2-week treatment with AICAR or overexpressing phosphomimetic mutant K_Ca2.3 Thr107D (mouse) restored K_Ca2.3-mediated EDH-dependent relaxation in mesenteric resistance arteries together with reversal of early phase hypertension. Our study demonstrates for the first time that AMPK activation mediates K_Ca2.3 phosphorylation in endothelial cells with enhanced channel activity. This effect ameliorates endothelial dysfunction of mesenteric resistance arteries and alleviates angiotensin II-induced early phase hypertension in mice.