Acute Mechanical Stretch Promotes eNOS Activation in Venous Endothelial Cells Mainly via PKA and Akt Pathways

Abstract

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser1177, Ser633 and Ser615 and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.

Periodic Mechanical Stress Activates PKCδ-Dependent EGFR Mitogenic Signals in Rat Chondrocytes via PI3K-Akt and ERK1/2

“…PI3K-Akt signals could respond to mechanical stimuli and modulate proliferation in several nonchondrocytic cell types [28,29]. This study investigated and considered PI3K-Akt signals to be candidate signals for inducing chondrocyte proliferation by periodic [30,31].…”

Protein kinase D activity controls endothelial nitric oxide synthesis

“…Upon the application of fluid shear stress (Dimmeler et al, 1999;Gallis et al, 1999), acute mechanical stretch (Hu et al, 2013) or UV light (Park et al, 2011), eNOS becomes rapidly phosphorylated on Ser1179. Likewise, treatment of endothelial monolayers with VEGF (Fulton et al, 1999;Michell et al, 1999), insulin (Salt et al, 2003), estrogens (Haynes et al, 2000), endothelin-1 (Liu et al, 2003) or bradykinin (Fleming et al, 2001;Schneider et al, 2003) leads to eNOS Ser1179 phosphorylation.…”

Modulation of low shear stress-induced eNOS multi-site phosphorylation and nitric oxide production via protein kinase and ERK1/2 signaling

“…This result illustrated that eNOS-Ser633 as a protective amino acid residue is dephosphorylated via the dephosphorylation of PKA by LSS. Numerous experiments under physiological stimuli, including shear stress, have suggested that PKA increased the level of phosphorylation at the site of eNOS-Ser633 or other sites, and NO production (16)(17)(18)(19). These results suggested that the effects of shear stress on the phosphorylation of eNOS-Ser633 and PKA depend on shear stress value.…”