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Key Event Relationship Overview

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Description of Relationship

Upstream Event Downstream Event/Outcome
eNOS, S-Glutathionylation eNOS, Uncoupling

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding
Oxidative Stress Leading to Hypertension Directly Leads to Strong Moderate

Taxonomic Applicability

Name Scientific Name Evidence Links
Homo sapiens Homo sapiens NCBI
Bos taurus Bos taurus NCBI
Rattus norvegicus Rattus norvegicus NCBI

How Does This Key Event Relationship Work

Oxidative stress can trigger S-glutathionylation of eNOS at cysteine residues Cys689 and Cys908, which are known to be critical for normal eNOS function (Zweier et al., 2011). S-glutathionylation directly causes eNOS uncoupling, a state in which eNOS switches from producing NO to generating superoxide, thus impairing endothelium-dependent vasodilation and contributing to endothelial dysfunction. Uncoupling of eNOS via S-glutathionylation is different from BH4-mediated eNOS uncoupling in that superoxide is produced in the reductase domain rather than the oxygenase domain and superoxide generation cannot be inhibited by L-NG-Nitroarginine methyl ester (L-NAME), suggesting that S-glutathionylation occurs independent of calcium/calmodulin and heme.

Weight of Evidence

Biological Plausibility

In vitro experiments showed that S-glutathionylation of eNOS significantly decreased NO activity in dose-dependent manner and greatly increased superoxide generation (Chen et al., 2010). NOS inhibitor L-NAME partially blocked superoxide generation. These results were observed in bovine aortic endothelial cells (BAECs) and in vessels of spontaneously hypertensive (SHR) rats. Treatment of BAECs with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, induced eNOS S-glutathionylation, resulting in decreased NO and increased superoxide generation. Gene silencing of eNOS in BAECs also induced superoxide production. In vessels of SHR rats, S-glutathionylation as determined by immunohistology was associated with a decrease in endothelium-dependent vasodilation as a result of decreased NO. Exposure of human aortic endothelial cells (HAECs) to ultrafine particles (UFP) caused S-glutathionylation of eNOS and a dose-dependent decrease in NO production (Du et al., 2013). Decreased NO production was found to be mediated by S-glutathionylation since overexpression of glutaredoxin, an inhibitor of S-glutathionylation, significantly reduced UPF-mediated decrease in NO production. Cardiac reperfusion patients exhibited decreased eNOS activity which was identified to be a result of eNOS S-glutathionylation (Jayaram et al., 2015). Additional evidence was observed in BAECs undergoing hypoxia and reoxygenation in which eNOS S-glutathionylation increased by three-fold compared to control cells and NO production was decreased (De Pascali et al., 2014). These effects were reversed with treatment of N-acetyl-l-cysteine, which increased cellular concentration of GSH. These results provide extensive understanding between S-glutathionylation and eNOS uncoupling, therefore this link has strong biological plausibility.

Empirical Support for Linkage

Include consideration of temporal concordance here

Treatment with BCNU (25 μM, 80 μM) resulted in increased eNOS S-glutathionylation, increased superoxide generation and decreased NO production in a dose-dependent manner in BAECs (Chen et al., 2010).

Exposure to hypoxia/reoxygenation and treatment with angiotensin II (Ang II) demonstrated a response-response relationship between eNOS S-glutathionylation and superoxide generation in human and bovine endothelial cells (De Pascali et al., 2014; Galougahi et al., 2014). Following hypoxia and reoxygenation in BAECs, increased S-glutathionylation of eNOS (0.3 to 1), decreased NO production (100% to 34.2 ± 1.7%) and increased superoxide generation (relative intensity of 3.5 to 49.2) were observed (De Pascali et al., 2014). Treatment with 100 nmol/L Ang II for 1 hour caused an increase in both eNOS S-glutathionylation (from 1 to 1.42 fold) and superoxide generation (from 81.1 to 113 fluorescence signal unit) (Galougahi et al., 2014).

Overall, there is moderate empirical support for eNOS S-glutathionylation leading to eNOS uncoupling.

Uncertainties or Inconsistencies

No uncertainties or inconsistencies were found for this KER.

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?

No study explored how much change in S-glutathionylation of eNOS is required to lead to eNOS uncoupling. One study showed that a fold change of 1.42 after Ang II treatment was associated with an increased in superoxide generation (Galougahi et al., 2014). Ang II and hypoxia/reoxygenation were demonstrated to be modulators of the response-response relationship between S-glutathionylation of eNOS and eNOS uncoupling as these two stressors caused a change in both key events (De Pascali et al., 2014; Galougahi et al., 2014). More experiments using other stressors or oxidants are needed to further understand of this relationship.

Evidence Supporting Taxonomic Applicability

The evidence supporting this key event relationship are from human subjects, HAECs, BAECs, and SHR rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013; Jayaram et al., 2015).


Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

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.

Galougahi, K.K., Liu, C.-C., Gentile, C., Kok, C., Nunez, A., Garcia, A., Fry, N.A.S., Davies, M.J., Hawkins, C.L., Rasmussen, H.H., et al. (2014). Glutathionylation Mediates Angiotensin II–Induced eNOS Uncoupling, Amplifying NADPH Oxidase-Dependent Endothelial Dysfunction. J. Am. Heart Assoc. 3, e000731.

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.

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.