Event: 932

Key Event Title


KE4 : Uncoupling, eNOS

Short name


Uncoupling, eNOS

Biological Context


Level of Biological Organization

Cell term


Cell term
endothelial cell

Organ term


Key Event Components


Process Object Action
nitric oxide synthase, endothelial functional change

Key Event Overview

AOPs Including This Key Event


AOP Name Role of event in AOP
Hypertension KeyEvent



Taxonomic Applicability


Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI
Bos taurus Bos taurus High NCBI
Mus musculus Mus musculus High NCBI
Rattus norvegicus Rattus norvegicus High NCBI

Life Stages


Life stage Evidence
All life stages Not Specified

Sex Applicability


Term Evidence
Unspecific Not Specified

Key Event Description


Endothelial nitric oxide synthase (eNOS) is responsible for the generation of vascular nitric oxide (NO), a protective molecule that is involved in the regulation of endothelium-dependent vasodilation, vascular tone, and blood pressure (Förstermann and Münzel, 2006). To generate NO, eNOS hydroxylates L-arginine to N-hydroxy-L-arginine and then oxidizes N-hydroxy-L-arginine to L-citrulline and NO. This enzymatic process requires NADPH, Ca2+/calmondulin, flavin mononucleotide, flavin adenine dinucleotide and its cofactor tetrahydrobiopterin (BH4). Limiting BH4 levels or S-glutathionylation of eNOS can lead to eNOS uncoupling in which eNOS produces superoxide (or other reactive oxygen species) and less NO. The uncoupling of eNOS has been demonstrated to cause endothelial dysfunction, and is implicated in a number of cardiovascular diseases such as hypertension, atherosclerosis, hypercholesterolemia, and diabetes mellitus (Dumitrescu et al., 2007).

How It Is Measured or Detected


The activity of eNOS can be measured indirectly through superoxide and NO production. Superoxides can be detected using several standard methods including lucigenin-enhanced chemiluminescence (Münzel et al., 2002; Tarpey et al., 1999), electron paramagnetic resonance (EPR) spin-trapping (Roubaud et al., 1997), and HPLC/fluorescence detector-based assay using dihydroethidium (Fink et al., 2004; Zhao et al., 2003). NO production can be measured through the conversion of L-arginine to L-citrulline (de Bono et al., 2007) , in situ fluorescent signal detection with fluorescent indicator DAF-2 DA (Itoh et al., 2000; Nagata et al., 1999; Qiu et al., 2001), EPR spin-trapping (Xia et al., 2000), and the determination of total nitrate and nitrite concentration (Crabtree et al., 2009; Du et al., 2013).

Domain of Applicability


eNOS uncoupling has been demonstrated in humans, cows, mice and rats (Chen et al., 2010; Crabtree et al., 2009; De Pascali et al., 2014; Du et al., 2013; Jayaram et al., 2015).



de Bono, J.P., Warrick, N., Bendall, J.K., Channon, K.M., and Alp, N.J. (2007). Radiochemical HPLC detection of arginine metabolism: Measurement of nitric oxide synthesis and arginase activity in vascular tissue. Nitric Oxide 16, 1–9.

Chen, X., Xu, J., Feng, Z., Fan, M., Han, J., and Yang, Z. (2010). Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation. Acta Pharmacol. Sin. 31, 813–820.

Crabtree, M.J., Tatham, A.L., Al-Wakeel, Y., Warrick, N., Hale, A.B., Cai, S., Channon, K.M., and Alp, N.J. (2009). Quantitative regulation of intracellular endothelial nitric-oxide synthase (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I expression. J. Biol. Chem. 284, 1136–1144.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem. Biophys. Res. Commun. 436, 462–466.

Dumitrescu, C., Biondi, R., Xia, Y., Cardounel, A.J., Druhan, L.J., Ambrosio, G., and Zweier, J.L. (2007). Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc. Natl. Acad. Sci. U. S. A. 104, 15081–15086.

Fink, B., Laude, K., McCann, L., Doughan, A., Harrison, D.G., and Dikalov, S. (2004). Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am. J. Physiol. Cell Physiol. 287, C895–C902.

Förstermann, U., and Münzel, T. (2006). Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113, 1708–1714.

Itoh, Y., Ma, F.H., Hoshi, H., Oka, M., Noda, K., Ukai, Y., Kojima, H., Nagano, T., and Toda, N. (2000). Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. 287, 203–209.

Jayaram, R., Goodfellow, N., Zhang, M.H., Reilly, S., Crabtree, M., De Silva, R., Sayeed, R., and Casadei, B. (2015). Molecular mechanisms of myocardial nitroso-redox imbalance during on-pump cardiac surgery. Lancet Lond. Engl. 385 Suppl 1, S49.

Münzel, T., Afanas’ev, I.B., Kleschyov, A.L., and Harrison, D.G. (2002). Detection of Superoxide in Vascular Tissue. Arterioscler. Thromb. Vasc. Biol. 22, 1761–1768.

Nagata, N., Momose, K., and Ishida, Y. (1999). Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide. J. Biochem. (Tokyo) 125, 658–661.

Qiu, W., Kass, D.A., Hu, Q., and Ziegelstein, R.C. (2001). Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence. Biochem. Biophys. Res. Commun. 286, 328–335.

Roubaud, V., Sankarapandi, S., Kuppusamy, P., Tordo, P., and Zweier, J.L. (1997). Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal. Biochem. 247, 404–411.

Tarpey, M.M., White, C.R., Suarez, E., Richardson, G., Radi, R., and Freeman, B.A. (1999). Chemiluminescent Detection of Oxidants in Vascular Tissue Lucigenin But Not Coelenterazine Enhances Superoxide Formation. Circ. Res. 84, 1203–1211.

Xia, Y., Cardounel, A.J., Vanin, A.F., and Zweier, J.L. (2000). Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. Free Radic. Biol. Med. 29, 793–797.

Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vásquez-Vivar, J., and Kalyanaraman, B. (2003). Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368.