Upstream eventDecrease, AKT/eNOS activity
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|
|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
AKT can phosphorylate eNOS which leads to increased eNOS enzymatic activity and subsequent NO production (Dimmeler et al., 1999; Fulton et al., 1999). Inhibition of AKT attenuates eNOS phosphorylation and its activity, resulting in decreased NO bioavailability and endothelial dysfunction.
Evidence Supporting this KER
Two studies demonstrated that AKT can directly phosphorylate eNOS at Ser1177/Ser1179, leading to increased eNOS enzymatic activity and subsequent NO production in COS cells (Dimmeler et al., 1999; Fulton et al., 1999). Expressing active AKT in bovine microvascular endothelial cells also induced NO release (Fulton et al., 1999). Inhibition of Akt or a mutation of eNOS at AKT-sensitive sites attenuated eNOS phosphorylation and its activity, resulting in decreased NO bioavailability and endothelial dysfunction (Dimmeler et al., 1999; Fulton et al., 1999; Uruno et al., 2005). Treatment of BAECs with SIN-1 resulted in a reduction in both eNOS activity and NO production (Das et al., 2014). Cigarette smoke extract (CSE) treatment inhibited AKT and eNOS and NO release in VEGF-stimulated HUVECs (Michaud et al., 2006), and methylglyoxal and high glucose reduced eNOS bradykinin-stimulated eNOS activity and NO production in HUVECs (Dhar et al., 2010). Overall, the biological plausibility for decreased AKT/eNOS activity leading to NO depletion is strong.
Include consideration of temporal concordance here
Empirical support for this linkage is strong since various stressors (ischemia, peroxynitrite, SIN-1, insulin+orotic acidura, etc.) can cause a decrease in AKT or eNOS activity which then leads to increased eNOS uncoupling and decreased NO levels.
Treatment of HUVECs with 30 μM methylglyoxal (MG) and 25 mM high glucose (HG) concentrations for 24 hours caused a decrease in eNOS activity (control: 100%, MG: 55%, HG: 66%), while a shorter treatment for 3 hours caused a decrease in NO production (control: 20 μM, MG: 12 μM, HG: 16 μM) (Dhar et al., 2010).
Treatment of BAECs with SIN-1 for 2 hours decreased eNOS activity from 100% to 70% and NO production from 100% to 53% (Das et al., 2014).
In HUVECs, exposure to 5 μmol/L peroxynitrite for 5 minutes reduced AKT phosphorylation by 30% (Song et al., 2007) while exposure to 50 μmol/L peroxynitrite for 3 hours reduced NO production from 90% to 6.5% (Zou et al., 2002), demonstrating that AKT phosphorylation occurs before NO production at a lower dose.
Prolonged myocardial ischemia (>30 min) in isolated rat hearts caused decreased eNOS activity (58% reduction) and increased superoxide generation (from relative fluorescence unit of <0.01 to 0.3), suggesting a depletion in NO production (Dumitrescu et al., 2007).
Treatment of HUVECs with 50 μM insulin for 30 minutes increased phosphorylation of AKT (control: 1, insulin: 5.7) and eNOS (control: 1, insulin: 1.9) as well as NO production (control: 100%, insulin: 157%), but these effects were reversed by additional treatment with 100 μM orotic aciduria (OA) for one hour (Akt: 1.8; eNOS: 0.28; NO: 106%) (Choi et al., 2015). Similar results were observed with addition of uric acid to insulin-treated HUVECs (Choi et al., 2014).
Treatment with all-trans retinoic acid (ATRA) and retinoic acid receptor agonist Am580 at 1 μmol/L for 48 hours also increased AKT (control: 100%, ATRA: 304%, Am580: 212%) and eNOS (control: 100%, ATRA: 276%, Am580: 209%) phosphorylation and NO production (control: 100%, ATRA: 170%, Am580: 169%) in human endothelial cells (Uruno et al., 2005).
Uncertainties and Inconsistencies
In the context of this AOP, decreased activity of AKT is likely due to proteasomal degradation following exposure to a (oxidative) stressor (Abdehghany et al. 2017). However, decreased eNOS activity could be due to multiple causes, highlighted in this AOP. Firstly, if AKT expression levels are reduced, it follows that eNOS phosphorylation will be reduced. Secondly, similar to AKT, eNOS itself has been shown to be susceptible to proteasomal degradation (Abdehghany et al. 2017). Thirdly, depletion of BH4 and/or S-glutathionylation has been shown to uncouple eNOS, leading to reduced NO levels and increased superoxide levels. The relative contribution of each of these eNOS perturbation routes to NO depletion is currently unknown.
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?
In two studies (Song et al, 2007; Das et al., 2014), it appears that a minimum of 30% reduction in eNOS activity or AKT phosphorylation caused a change in NO production in as little as five minutes. Other studies showed that 50-60% reduction in AKT phosphorylation/eNOS activity will lead to decreased NO bioavailability (Dhar et al., 2010; Dumitrescu et al., 2007). The studies above demonstrated that there are several known modulators for these two key events including peroxynitrite, high glucose, methylglyoxal, insulin and ATRA.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The relationship between decreased AKT/eNOS activity and NO depletion is supported by studies performed in humans, cows, and rats.
AbdelGhany, T., Ismail, R., Elmahdy, M., Mansoor F, Zweier J, Lowe, F., and Zweier, JL. (2017). Cigarette Smoke Constituents Cause Endothelial Nitric Oxide Synthase Dysfunction and Uncoupling due to Depletion of Tetrahydrobiopterin with Degradation of GTP Cyclohydrolase. Nitric Oxide (Under review).
Choi, Y.-J., Yoon, Y., Lee, K.-Y., Hien, T.T., Kang, K.W., Kim, K.-C., Lee, J., Lee, M.-Y., Lee, S.M., Kang, D.-H., et al. (2014). Uric acid induces endothelial dysfunction by vascular insulin resistance associated with the impairment of nitric oxide synthesis. FASEB J. 28, 3197–3204.
Choi, Y.-J., Yoon, Y., Lee, K.-Y., Kang, Y.-P., Lim, D.K., Kwon, S.W., Kang, K.-W., Lee, S.-M., and Lee, B.-H. (2015). Orotic Acid Induces Hypertension Associated with Impaired Endothelial Nitric Oxide Synthesis. Toxicol. Sci. 144, 307–317.
Das, A., Gopalakrishnan, B., Druhan, L.J., Wang, T.-Y., De Pascali, F., Rockenbauer, A., Racoma, I., Varadharaj, S., Zweier, J.L., Cardounel, A.J., et al. (2014). Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation. Br. J. Pharmacol. 171, 2321–2334.
Dhar, A., Dhar, I., Desai, K.M., and Wu, L. (2010). Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose. Br. J. Pharmacol. 161, 1843–1856.
Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A.M. (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605.
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.
Fulton, D., Gratton, J.P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F., Papapetropoulos, A., and Sessa, W.C. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597–601.
Michaud, S.E., Dussault, S., Groleau, J., Haddad, P., and Rivard, A. (2006). Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: role of NO and reactive oxygen species. J. Mol. Cell. Cardiol. 41, 275–284.
Song, P., Wu, Y., Xu, J., Xie, Z., Dong, Y., Zhang, M., and Zou, M.-H. (2007). Reactive nitrogen species induced by hyperglycemia suppresses Akt signaling and triggers apoptosis by upregulating phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in an LKB1-dependent manner. Circulation 116, 1585–1595.
Song, P., Xie, Z., Wu, Y., Xu, J., Dong, Y., and Zou, M.-H. (2008). Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells. J. Biol. Chem. 283, 12446–12455.
Uruno, A., Sugawara, A., Kanatsuka, H., Kagechika, H., Saito, A., Sato, K., Kudo, M., Takeuchi, K., and Ito, S. (2005). Upregulation of nitric oxide production in vascular endothelial cells by all-trans retinoic acid through the phosphoinositide 3-kinase/Akt pathway. Circulation 112, 727–736.
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.