This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 49/3/577    most recent 01.HYP.0000255954.80025.34v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Motley, E. D. Articles by Eguchi, S. Search for Related Content PubMed PubMed Citation Articles by Motley, E. D. Articles by Eguchi, S. Related Collections Cell signalling/signal transduction Peripheral vascular disease Thrombin Endothelium/vascular type/nitric oxide Related Article

(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

Related Article:

Regulation of Endothelial Nitric Oxide Synthase by Thrombin

Rhian M. Touyz
Hypertension 2007 49: 429-431. [Extract] [Full Text] [PDF]

This article has been cited by other articles:

				T. Kobayashi, K. Taguchi, S. Nemoto, T. Nogami, T. Matsumoto, and K. Kamata Activation of the PDK-1/Akt/eNOS pathway involved in aortic endothelial function differs between hyperinsulinemic and insulin-deficient diabetic rats Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1767 - H1775. [Abstract] [Full Text] [PDF]

				A. Bellis, D. Castaldo, V. Trimarco, M. G. Monti, P. Chivasso, J. Sadoshima, B. Trimarco, and C. Morisco Cross-Talk Between PKA and Akt Protects Endothelial Cells From Apoptosis in the Late Ischemic Preconditioning Arterioscler Thromb Vasc Biol, August 1, 2009; 29(8): 1207 - 1212. [Abstract] [Full Text] [PDF]

				B. Olszewska-Pazdrak, T. W. Hein, P. Olszewska, and D. H. Carney Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1162 - C1170. [Abstract] [Full Text] [PDF]

				R. M. Osipov, C. Bianchi, R. T. Clements, J. Feng, Y. Liu, S.-H. Xu, M. P. Robich, J. Wagstaff, and F. W. Sellke Thrombin Fragment (TP508) Decreases Myocardial Infarction and Apoptosis After Ischemia Reperfusion Injury. Ann. Thorac. Surg., March 1, 2009; 87(3): 786 - 793. [Abstract] [Full Text] [PDF]

				C. G. da Silva, A. Specht, B. Wegiel, C. Ferran, and E. Kaczmarek Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells Circulation, February 17, 2009; 119(6): 871 - 879. [Abstract] [Full Text] [PDF]

				R. Martinelli, M. Gegg, R. Longbottom, P. Adamson, P. Turowski, and J. Greenwood ICAM-1-mediated Endothelial Nitric Oxide Synthase Activation via Calcium and AMP-activated Protein Kinase Is Required for Transendothelial Lymphocyte Migration Mol. Biol. Cell, February 1, 2009; 20(3): 995 - 1005. [Abstract] [Full Text] [PDF]

				H. Suzuki, E. D. Motley, K. Eguchi, A. Hinoki, H. Shirai, V. Watts, L. N. Stemmle, T. A. Fields, and S. Eguchi Distinct Roles of Protease-Activated Receptors in Signal Transduction Regulation of Endothelial Nitric Oxide Synthase Hypertension, February 1, 2009; 53(2): 182 - 188. [Abstract] [Full Text] [PDF]

				A. M. Bair, P. B. Thippegowda, M. Freichel, N. Cheng, R. D. Ye, S. M. Vogel, Y. Yu, V. Flockerzi, A. B. Malik, and C. Tiruppathi Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-{kappa}B Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase C{delta} J. Biol. Chem., January 2, 2009; 284(1): 563 - 574. [Abstract] [Full Text] [PDF]

				C. Chabot, K. Spring, J.-P. Gratton, M. Elchebly, and I. Royal New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival Mol. Cell. Biol., January 1, 2009; 29(1): 241 - 253. [Abstract] [Full Text] [PDF]

				H. Kinoshita, N. Matsuda, H. Kaba, N. Hatakeyama, T. Azma, K. Nakahata, Y. Kuroda, K. Tange, H. Iranami, and Y. Hatano Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5'-Triphosphate-Sensitive K+ Channel Function Impaired by High Glucose in the Human Artery Hypertension, September 1, 2008; 52(3): 507 - 513. [Abstract] [Full Text] [PDF]

				H. Nakashima, G. D. Frank, H. Shirai, A. Hinoki, S. Higuchi, H. Ohtsu, K. Eguchi, A. Sanjay, M. E. Reyland, P. J. Dempsey, et al. Novel Role of Protein Kinase C-{delta} Tyr311 Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II Hypertension, February 1, 2008; 51(2): 232 - 238. [Abstract] [Full Text] [PDF]

				R. M. Touyz Regulation of Endothelial Nitric Oxide Synthase by Thrombin Hypertension, March 1, 2007; 49(3): 429 - 431. [Full Text] [PDF]