Endothelial AMP-Activated Kinase ?1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation.

ABSTRACT: 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?1 and ?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?^-/-) or endothelial-specific deletion (AMPK?^?EC) of the AMPK? subunits. In control and AMPK?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?1 or ?2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPK?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?1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPK?1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

SUBMITTER: Zippel N 

PROVIDER: S-EPMC6165563 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature