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Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases
Journal article   Open access   Peer reviewed

Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases

Quan-Jiang Zhang, Shawna L McMillin, Jason M Tanner, Milda Palionyte, E Dale Abel and J David Symons
The Journal of physiology, Vol.587(Pt 15), pp.3911-3920
08/01/2009
DOI: 10.1113/jphysiol.2009.172916
PMCID: PMC2746618
PMID: 19505983
url
https://doi.org/10.1113/jphysiol.2009.172916View
Published (Version of record) Open Access

Abstract

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB - a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by approximately 50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition.
 Cyclic AMP-Dependent Protein Kinases - metabolism AMP-Activated Protein Kinases - metabolism Phosphorylation Mice, Inbred C57BL Male Phosphatidylinositol 3-Kinases - metabolism Physical Conditioning, Animal - physiology Animals Arteries - enzymology Androstadienes - pharmacology Mitogen-Activated Protein Kinase 3 - metabolism Cyclic AMP Response Element-Binding Protein - metabolism Signal Transduction - physiology Mice Protein Kinase Inhibitors - pharmacology Nitric Oxide Synthase Type III - metabolism Proto-Oncogene Proteins c-akt - metabolism Mitogen-Activated Protein Kinase 1 - metabolism

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