Upstream eventOxidation, Glutathione
Key Event Relationship Overview
AOPs Referencing Relationship
|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 Stage Applicability
|All life stages||High|
Key Event Relationship Description
Under oxidative stress, reduced glutathione (GSH) is oxidized to glutathione disulfide (GSSG), which then induces S-glutathionylation of eNOS at its cysteine residues, resulting in eNOS uncoupling (Chen et al., 2010).
Evidence Supporting this KER
In an in vitro system, GSSG induced S-glutathionylation of human eNOS in a dose-dependent manner, which was reversed by reducing agents 2-mercaptoethanol and dithiothreitol (Chen et al., 2010). Cysteine residues 689 and 908 were identified as the critical sites for S-glutathionylation of eNOS since their mutatation resisted glutathionylation. These results were confirmed in bovine aortic endothelial cells (BAECs) and in aortae of spontaneously hypertensive (SHR) rats. Treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, induced an increase in GSSG and eNOS S-glutathionylation as confirmed by mass spectrometry in which Cys 689 was shown to be more than 50% S-glutathionylated. Aortae of SHR rats showed S-glutathionylation via immunohistology whereas normotensive rats had little S-glutathionylation. In human aortic endothelial cells (HAECs) exposed to ultrafine particles (UFP), GSSG levels were increased in parallel with protein S-glutathionylation, which was confirmed by ELISA to be eNOS S-glutathionylation (Du et al., 2013). This was recapitulated in LDLR-null mice exposed to UFP. Additional evidence was observed in BAECs undergoing hypoxia and reoxygenation where eNOS S-glutathionylation increased by three-fold compared to control cells and GSSG levels were increased (De Pascali et al., 2014). These effects were reversed with treatment of N-acetyl-l-cysteine, which increased cellular concentration of GSH. These results support strong biological plausibility for this key event relationship.
Include consideration of temporal concordance here
Treatment with GSSG (0.5 mM, 1 mM, 2 mM) for one hour induced a dose-dependent increase in human eNOS S-glutathionylation in vitro whereas BCNU (25 μM, 80 μM) also resulted in increased eNOS S-glutathionylation in a dose-dependent manner in BAECs (Chen et al., 2010).
Exposure to UFP and hypoxia/reoxygenation demonstrated a response-response relationship between GSH oxidation and eNOS S-glutathionylation in HAECs and BAECs, respectively (Du et al., 2013; De Pascali et al., 2014). Exposure to 50 μg/mL UFP for 6 hours caused a reduction in GSH from 17.1±1.8 μM in controls to 12.0±2.4 μM in treated cells, which is indicative of increased GSSG, and increased eNOS S-glutathionylation from 0.008±0.001 OD450 (as measured by ELISA) in controls to 0.042±0.006 OD450 in treated cells (Du et al., 2013). Similarly, following hypoxia and reoxygenation in BAECs, decreased GSH levels (1.64 ± 0.03 to 0.32 ± 0.03 μmol/mg) and increased S-glutathionylation of eNOS (0.3 to 1) were observed (De Pascali et al., 2014).
Overall, there is moderate empirical support for glutathione oxidation leading to eNOS S-glutathionylation.
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?
Based on results by Chen et al.(2010), a concentration of 0.5 mM GSSG was able to induce a significant increase in eNOS S-glutathionylation; and increasing concentrations of GSSG affected eNOS S-glutathionylation in a dose-responsive manner in vitro. UFP and hypoxia/reoxygenation were demonstrated to be modulators of the response-response relationship between glutathione oxidation and S-glutathionylation of eNOS (Du et al., 2013; De Pascali et al., 2014). Additional experiments using other stressors or oxidants would be beneficial in further understanding of this relationship, specifically around the temporal aspect (i.e. does glutathione oxidation occur at an earlier time point than eNOS S-glutathionylation?).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The evidence supporting this key event relationship are from BAECs, HAECs, LDLR-null mice, and SHR rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).
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.