Upstream eventS-Glutathionylation, eNOS
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Peptide Oxidation Leading to Hypertension||adjacent||High||Moderate|
|Homo sapiens||Homo sapiens||High||NCBI|
|Bos taurus||Bos taurus||High||NCBI|
|Rattus norvegicus||Rattus norvegicus||High||NCBI|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
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.
Evidence Supporting this KER
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 aortae 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 aortae 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 demonstrate a clear interaction between S-glutathionylation and eNOS uncoupling, therefore this link has strong biological plausibility.
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 arbitray units; measured by western blot), decreased NO production (100% to 34.2 ± 1.7%) and increased superoxide generation (relative intensity of 3.5 to 49.2; by fluorescence) 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 and 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 in HUVECs showed treatment with 100nM/L Ang II increased S-glutathionylated eNOS protein expression by 1.42 fold, and was associated with an increase in superoxide generation of nearly 50% at the same dose. DTT (which removes PR-SG residues) was shown to partially reverse this effect (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.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of 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.