Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPK alpha 1 and alpha 2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPK alpha(-/-)) or endothelial-specific deletion (AMPK alpha(Delta EC)) of the AMPK alpha subunits. In control and AMPK alpha 1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPK alpha 1 or alpha 2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPK alpha 1 subunit attenuated phenylephrine-mediated contraction in an eNOS-and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPK alpha 1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPK alpha 1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.