Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

• Published in: Hypertension (Dallas, Tex. 1979) Vol. 49; no. 3; pp. 577 - 583
• Main Authors: Motley, Evangeline D, Eguchi, Kunie, Patterson, Myla M, Palmer, Phillip D, Suzuki, Hiroyuki, Eguchi, Satoru
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 01.03.2007
• Subjects: Adenovirus; Animals; Aorta; Calcium - metabolism; Cattle; Cells, Cultured; Endothelium, Vascular - drug effects; Enzyme Activation - drug effects; Humans; Muscle, Smooth, Vascular - drug effects; Nitric Oxide Synthase Type III - drug effects; Nitric Oxide Synthase Type III - metabolism; Phosphorylation; Protein Kinase C-alpha - metabolism; Rats; Serine - metabolism; Signal Transduction; Thrombin - pharmacology; Umbilical Veins - drug effects
• ISSN: 0194-911X; 1524-4563; 1524-4563
• DOI: 10.1161/01.HYP.0000255954.80025.34

Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca/calmodulin, eNOS has several regulatory sites. Ser 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 Ser in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser 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 Ca ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca 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 Ser phosphorylation-dependent eNOS activation through a Ca-dependent, PKCδ-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.