Shear Stress Stimulates Phosphorylation of Endothelial Nitric-oxide Synthase at Ser1179 by Akt-independent Mechanisms

Boo, Yong Chool; Sorescu, George P.; Boyd, Nolan L.; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

doi:10.1074/jbc.m108789200

Cited by 406 publications

(352 citation statements)

References 42 publications

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“…However, several lines of evidence have shown that PKA also regulates the phosphorylation of eNOS at Ser 1179 , Ser 635 , and Ser 617 in bovine eNOS (Ser 1177 , Ser 633 , and Ser 615 in humans) [24,[26][27][28][29] . Ser 633/635 phosphorylation is critical in maintaining NO synthesis after the initial sensitization by Ca 2+ flux and Ser 1177/1179 phosphorylation [30] , and it is stimulated via the PKA pathway in response to shear stress and acute statin treatment in aortic endothelial cells [24,27] , suggesting that PKA-mediated Ser 633/635 phosphorylation may be another mechanism by which statins protect against myocardial no-reflow and necrosis after ischemia and reperfusion.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

Yang

Geng³

et al. 2012

Acta Pharmacol Sin

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Aim: The cholesterol-lowering drugs statins could enhance the activities of endothelial nitric oxide synthase (eNOS) and protect myocardium during ischemia and reperfusion. The aim of this study was to examine whether protein kinase A (PKA) was involved in statinmediated eNOS phosphorylation and cardioprotection. Methods: 6-Month-old Chinese minipigs (20-30 kg) underwent a 1.5-h occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD). In the sham group, the LAD was encircled by a suture but not occluded. Hemodynamic and cardiac function was monitored using a polygraph. Plasma activity of creatine kinase and the tissue activities of PKA and NOS were measured spectrophotometrically. p-CREB, eNOS and p-eNOS levels were detected using Western blotting. Sizes of the area at risk, the area of no-reflow and the area of necrosis were measured morphologically. Results: Pretreatment of the animals with simvastatin (SIM, 2 mg/kg, po) before reperfusion significantly decreased the plasma activity of creatine kinase, an index of myocardial necrosis, and reduced the no-reflow size (from 50.4%±2.4% to 36.1%±2.1%, P<0.01) and the infarct size (from 79.0%±2.7% to 64.1%±4.5%, P<0.01). SIM significantly increased the activities of PKA and constitutive NOS, and increased Ser 133 p-CREB protein, Ser 1179 p-eNOS, and Ser 635 p-eNOS in ischemic myocardium. Intravenous infusion of the PKA inhibitor H-89 (1 μg·kg -1 ·min -1 ) partially abrogated the SIM-induced cardioprotection and eNOS phosphorylation. In contrast, intravenous infusion of the eNOS inhibitor L-NNA (10 mg·kg -1 ) completely abrogated the SIM-induced cardioprotection and eNOS phosphorylation during ischemia and reperfusion, but did not affect the activity of PKA. Conclusion: Pretreatment with a single dose of SIM 2.5 h before reperfusion attenuates myocardial no-reflow and infarction through increasing eNOS phosphorylation at Ser 1179 and Ser 635 that was partially mediated via the PKA signaling pathway.

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Section: Discussionmentioning

confidence: 99%

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

Yang

Geng³

et al. 2012

Acta Pharmacol Sin

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“…Elevated eNOS activity observed in rodents with elevated EPO levels has been attributed to increased shear stress created by the increase in hematocrit, since shear stress can stimulate eNOS activity by a variety of mechanisms including increasing eNOS mRNA expression and/or stability, or eNOS protein phosphorylation. 42,43 However, in patients with polycythemia-who are not receiving exogenous EPO (and who may have very low EPO levels)-high hematocrit appears to inhibit endothelium-dependent vasodilatation in response to acetylcholine. This process can be reversed by hemodilution and normalization of blood hemoglobin concentration.…”

Section: Org Frommentioning

confidence: 99%

Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells

Beleslin-Čokić

Čokić²,

Yu³

et al. 2004

Blood

263

211

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Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell

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“…Bovine aorta endothelial cells (BAECs) harvested from descending thoracic aortas were maintained (37 o C, 5% CO2) in a growth medium [Dulbecco's modified Eagle's medium (DMEM) containing 1 g/l glucose (Gibco) and 20% fetal bovine serum (FBS, Atlanta Biologicals) without antibiotics] (Boo et al, 2002b). BAECs used in this study were between passages 5 and 10.…”

Section: Cell Culture and Shear Stress Treatmentsmentioning

confidence: 99%

“…The recombinant adenoviral construct, Ad-Akt-Myr, was used to express a constitutively active Akt mutants generated by fusing a myristoylation signal to its amino terminus Boo et al, 2002b). Recombinant adenovirus encodinggalactosidase (Ad--gal) was used as a control.…”

Section: Adenoviral Infectionmentioning

confidence: 99%

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Boo

2006

Exp Mol Med

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Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.

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Shear Stress Stimulates Phosphorylation of Endothelial Nitric-oxide Synthase at Ser1179 by Akt-independent Mechanisms

Cited by 406 publications

References 42 publications

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

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