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(Hypertension. 2007;49:577.)
© 2007 American Heart Association, Inc.

Original Articles

Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Evangeline D. Motley; Kunie Eguchi; Myla M. Patterson; Phillip D. Palmer; Hiroyuki Suzuki; Satoru Eguchi

From the Department of Biomedical Sciences (E.D.M., K.E., M.M.P., P.D.P.), Division of Cardiovascular Biology, Meharry Medical College, Nashville, Tenn; and the Cardiovascular Research Center and the Department of Physiology (K.E., H.S., S.E.), Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Evangeline D. Motley, Department of Biomedical Sciences, Division of Cardiovascular Biology, Meharry Medical College, 1005 D. B. Todd Blvd, Nashville, TN 37208. E-mail emotley{at}mmc.edu

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) and PKC or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC 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, PKC-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.

Key Words: thrombin • NO • endothelial NO synthase • protein kinase C

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Regulation of Endothelial Nitric Oxide Synthase by Thrombin

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