Shear stress regulates endothelial NO synthase (eNOS) by the protein kinase A (PKA)-dependent mechanisms

Exposure of endothelial cells to shear stress stimulates NO production by phosphorylating eNOS at Ser/sup 1179/ in the phosphoinositide-3-kinase (PI3K)- and PKA-dependent mechanisms. The eNOS contains additional potential phosphorylation sites including Ser/sup 116/, Thr/sup 497/ and Ser/sup 635/. Here, we studied phosphorylation of these potential sites in response to shear stress, vascular endothelial growth factor (VEGF) and 8-Br-cAMP in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser/sup 635/ that was consistently slower than that at Ser/sup 1179/. The Thr/sup 497/ was rapidly dephosphorylated by 8-Br-cAMP, but not by shear and VEGF. None of the stimuli phosphorylated the Ser/sup 116/. While shear-dependent phosphorylation of Ser/sup 635/ was not affected by PI3K inhibitors wortmannin and LY294002, it was blocked by either treating cells with a PKA inhibitor H89 or infecting with an adenoviral vector expressing PKA inhibitor (PKI). These results suggest that shear stimulates at least two independent signaling pathways regulating eNOS: one that activates a PI3K-dependent PKA pathway and the other that activates a PI3K-independent PKA pathway. In summary, these studies suggest an essential role of PKA in regulation of eNOS and endothelial cell biology in response to shear stress.