Phosphorylation and activation of the endothelial nitric oxide synthase byfluid shear stress

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eN OS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+](i)), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here w e demonstrate that fluid shear stress elicits the phosphorylation of eNOS o n tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream t arget, Akt (protein kinase B), prevented the maintained serine phosphorylat ion and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting se rine/threonine phosphatases, increased eNOS activity by approximately twofo ld, as assessed by the accumulation of intracellular cyclic GMP, without in creasing the intracellular concentration of free Ca2+. These data suggest t hat shear stress activates a pathway involving PI3K and the serine/threonin e kinase Akt, which phosphorylates eNOS. This phosphorylation directly incr eases eNOS activity at resting [Ca2+](i), thus rendering the shear stress-i nduced activation of eNOS apparently Ca2+-independent.