Relationship: 956



Uncoupling, eNOS leads to Depletion, Nitric Oxide

Upstream event


Uncoupling, eNOS

Downstream event


Depletion, Nitric Oxide

Key Event Relationship Overview


AOPs Referencing Relationship


AOP Name Adjacency Weight of Evidence Quantitative Understanding
Peptide Oxidation Leading to Hypertension adjacent High High

Taxonomic Applicability


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

Sex Applicability


Sex Evidence
Unspecific High

Life Stage Applicability


Term Evidence
All life stages High

Key Event Relationship Description


The uncoupling of eNOS occurs through a number of mechanisms like BH4 depletion or S-glutathionylation of eNOS and leads to a reduction in NO bioavailability and an elevation in superoxide production, contributing to endothelial dysfunction (Zweier et al., 2011).

Evidence Supporting this KER


Biological Plausibility


It is well-established that uncoupling of eNOS causes eNOS to switch from producing NO to generating superoxides (Förstermann and Münzel, 2006). Many studies that reported BH4 depletion leading to eNOS uncoupling or S-glutathionylation leading to eNOS uncoupling measured levels of NO and superoxide which are indicative of eNOS uncoupling.

Empirical Evidence


Include consideration of temporal concordance here

Multiple experiments demonstrated that eNOS uncoupling results in increased superoxide formation and decreased NO production, which provide strong empirical support for this KER.

Prolonged myocardial ischemia (>30 min) in isolated rat hearts decreased eNOS activity (58% reduction) and increased superoxide generation from <0.01 relative fluorescence unit/mg to 0.3 as measured using a fluorescence detector-based assay (Dumitrescu et al., 2007). Similarly, cardiac reperfusion patients had decreased eNOS activity (40% reduction) and increased superoxide production from 37.83 to 65.02 light unit/s/mg as detected using lucigenin-enhanced chemiluminescence (Jayaram et al., 2015).

In BAECs, treatment with 10 mmol/L DAHP or 25 μM 4-HNE for 24 hours led to a reduction in NO (control: 1678 pmol/mg, DAHP: 1274 pmol/mg, 4-HNE: 1106 pmol/mg) and increased superoxide formation (control: 59 pmol/mg, DAHP: 97 pmol/mg, 4-HNE: 122 pmol/mg) (Whitsett et al., 2007). Similar results were observed in another study using DAHP-treated BAECs; superoxide was increased (control: 100%, DAHP: 257%) and NO was decreased (control: 96%, DAHP: 60%) (Wang et al., 2008).

In BAECs undergoing hypoxia and reoxygenation (H/R), NO decreased from 34.2±1.7% to 63.7±3.0% and superoxide increased from relative intensity of 3.5 to 49.2, demonstrating that these key events are modulated together (De Pascali et al., 2014).

BCNU (25 μM, 80 μM) resulted in increased superoxide (25 μM: 432 fluorescence intensity, 80 μM:759 fluorescence intensity) and decreased NO (25 μM: 61%, 80 μM:36%) in a dose-dependent manner in BAECs (Chen et al., 2010).

Exposure to 50 μmol/L peroxynitrite for 3 hours led to an increase in superoxide from 2 nmol/min/well to 8.6 nmol/min/well and a decrease in NO from 90% to 6.5% in BAECs (Zou et al., 2002).

Uncertainties and Inconsistencies


There are no uncertainties or inconsistencies.

Quantitative Understanding of the Linkage


Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

The uncoupling of eNOS automatically results in NO depletion and increased superoxide production. The experimental studies above included a number of modulators of the response-response relationship, such as peroxynitrite, BCNU, and DAHP.

Response-response Relationship




Known modulating factors


Known Feedforward/Feedback loops influencing this KER


Domain of Applicability


The relationship between NO depletion and eNOS uncoupling was observed in humans, cows, and rats as demonstrated by the above studies.



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.

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.

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.

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

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.

Wang, S., Xu, J., Song, P., Wu, Y., Zhang, J., Chul Choi, H., and Zou, M.-H. (2008). Acute inhibition of guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and elevates blood pressure. Hypertension 52, 484–490.

Whitsett, J., Picklo, M.J., and Vasquez-Vivar, J. (2007). 4-Hydroxy-2-nonenal increases superoxide anion radical in endothelial cells via stimulated GTP cyclohydrolase proteasomal degradation. Arterioscler. Thromb. Vasc. Biol. 27, 2340–2347.

Zou, M.-H., Hou, X.-Y., Shi, C.-M., Nagata, D., Walsh, K., and Cohen, R.A. (2002). Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J. Biol. Chem. 277, 32552–32557.

Zweier, J.L., Chen, C.-A., and Druhan, L.J. (2011). S-glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling. Antioxid. Redox Signal. 14, 1769–1775.