Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Abstract

Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca²⁺/calmodulin, eNOS has several regulatory sites. Ser¹¹⁷⁹ phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser¹¹⁷⁹ in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser¹¹⁷⁹ was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser⁴⁷³ at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca²⁺ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca²⁺ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC)α and PKCδ or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKCδ is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser¹¹⁷⁹ phosphorylation-dependent eNOS activation through a Ca²⁺-dependent, PKCδ-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.