Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is a potent vasodilator and anti-thrombotic autocoid that is important for the maintenance of systemic blood pressure. When activated by changes in local blood flow or circulating hormones, NO is synthesized by a membrane bound, calcium-requiring enzyme, nitric oxide synthase (ECNOS) that converts L-arginine to NO and L-citrulline. Once generated, NO is released from the endothelium causing relaxation of vascular smooth muscle. In order to elucide how the endothelial production of NO is regulated we have cloned the cDNA for ECNOS and have shown that it contains an N-myristoylation consensus sequence, not found in neuronal or macrophage forms of NOS (both soluble isoforms). In addition, we have shown by site-directed mutagenesis, that mutation of glycine 2 (the putative myristic acid acceptor site) in the N-myristoylation consensus sequence converts membrane-associated ECNOS to a cytosolic form. Therefore, the purpose of this proposal is to test the hypothesis that N- myristoylation directs ECNOS towards the membrane where the enzyme is most responsive to changes in flow and to local hormones. To this end, we will: 1. Characterize the N-terminal fatty acid modification of ECNOS and examine its functional relevance by site-directed mutagenesis and the generation of chimeric NOS proteins; 2. Localize ECNOS protein (immunohistochemically) in EC and in cells lines stably expressing either wild-type (membrane) or mutant ECNOS (soluble); 3. Test the functional significance of N-myristoylation of ECNOS by examining the release of NO in response to calcium-mobilizing agonists or flow in endothelial cells stably transfected with either wild-type (membrane) or mutant (soluble) ECNOS. and 4. Examine in endothelial cells, if the rate of N- myristoylation or localization of myristoylated ECNOS is affected by calcium-mobilizing hormones or flow. If ECNOS is myristoylated and if myristoylation is necessary for ECNOS localization and activity, then understanding the importance of this protein modification will aid in the understanding how NO is regulated in vivo.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
Application #
Study Section
Pathology A Study Section (PTHA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Medicine
New Haven
United States
Zip Code
Rudic, R D; Bucci, M; Fulton, D et al. (2000) Temporal events underlying arterial remodeling after chronic flow reduction in mice: correlation of structural changes with a deficit in basal nitric oxide synthesis. Circ Res 86:1160-6
Papapetropoulos, A; Garcia-Cardena, G; Dengler, T J et al. (1999) Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest 79:213-23
Guillot, P V; Guan, J; Liu, L et al. (1999) A vascular bed-specific pathway. J Clin Invest 103:799-805
Shah, V; Wiest, R; Garcia-Cardena, G et al. (1999) Hsp90 regulation of endothelial nitric oxide synthase contributes to vascular control in portal hypertension. Am J Physiol 277:G463-8
Rudic, R D; Sessa, W C (1999) Nitric oxide in endothelial dysfunction and vascular remodeling: clinical correlates and experimental links. Am J Hum Genet 64:673-7
Shah, V; Toruner, M; Haddad, F et al. (1999) Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 117:1222-8
Sowa, G; Liu, J; Papapetropoulos, A et al. (1999) Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion. J Biol Chem 274:22524-31
Papapetropoulos, A; Piccardoni, P; Cirino, G et al. (1998) Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling. Proc Natl Acad Sci U S A 95:4738-42
Rudic, R D; Shesely, E G; Maeda, N et al. (1998) Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest 101:731-6
Papapetropoulos, A; Garcia-Cardena, G; Madri, J A et al. (1997) Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 100:3131-9

Showing the most recent 10 out of 22 publications