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 Ser¹¹⁷⁹ in the phosphoinositide-3-kinase (PI3K)- and PKA-dependent mechanisms. The eNOS contains additional potential phosphorylation sites including Ser¹¹⁶, Thr⁴⁹⁷ and Ser⁶³⁵. 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 Ser⁶³⁵ that was consistently slower than that at Ser¹¹⁷⁹. The Thr⁴⁹⁷ was rapidly dephosphorylated by 8-Br-cAMP, but not by shear and VEGF. None of the stimuli phosphorylated the Ser¹¹⁶. While shear-dependent phosphorylation of Ser⁶³⁵ 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.