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Article

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

by
Nina Zippel
1,
Annemarieke E. Loot
1,
Heike Stingl
1,2,
Voahanginirina Randriamboavonjy
1,2,
Ingrid Fleming
1,2 and
Beate Fisslthaler
1,2,*
1
Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany
2
DZHK (German Centre for Cardiovascular Research) partner site RhineMain, Theodor Stern Kai 7, 60590 Frankfurt, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(9), 2753; https://doi.org/10.3390/ijms19092753
Submission received: 2 August 2018 / Revised: 7 September 2018 / Accepted: 11 September 2018 / Published: 13 September 2018
(This article belongs to the Special Issue AMP-Activated Protein Kinase Signalling)

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 AMPKα1 and α2 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 (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-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 AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 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 AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.
Keywords: endothelial nitric-oxide synthase; vasodilation; phenylephrine; vasoconstriction; endothelial cells; ionomycin endothelial nitric-oxide synthase; vasodilation; phenylephrine; vasoconstriction; endothelial cells; ionomycin
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MDPI and ACS Style

Zippel, N.; Loot, A.E.; Stingl, H.; Randriamboavonjy, V.; Fleming, I.; Fisslthaler, B. Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation. Int. J. Mol. Sci. 2018, 19, 2753. https://doi.org/10.3390/ijms19092753

AMA Style

Zippel N, Loot AE, Stingl H, Randriamboavonjy V, Fleming I, Fisslthaler B. Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation. International Journal of Molecular Sciences. 2018; 19(9):2753. https://doi.org/10.3390/ijms19092753

Chicago/Turabian Style

Zippel, Nina, Annemarieke E. Loot, Heike Stingl, Voahanginirina Randriamboavonjy, Ingrid Fleming, and Beate Fisslthaler. 2018. "Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation" International Journal of Molecular Sciences 19, no. 9: 2753. https://doi.org/10.3390/ijms19092753

APA Style

Zippel, N., Loot, A. E., Stingl, H., Randriamboavonjy, V., Fleming, I., & Fisslthaler, B. (2018). Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation. International Journal of Molecular Sciences, 19(9), 2753. https://doi.org/10.3390/ijms19092753

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