KE5 : Decrease, AKT/eNOS activity

Endothelial nitric oxide synthase (eNOS) is responsible for the generation of nitric oxide (NO), which is an important regulator of vascular homeostasis. The activity of eNOS can be regulated through calmodulin-mediated dimerization, tetrahydrobiopterin-mediated conversion of L-arginine to L-citrulline, protein-protein interactions with heat shock protein 90 and caveolin, S-nitrosylation, acetylation and phosphorylation (Atochin et al., 2007; Qian and Fulton, 2013). eNOS has been shown to be phosphorylated at multiple sites including tyrosine (Y), serine (Ser) and threonine (Thr) residues. Serine-threonine protein kinase AKT, a multifunctional regulator of cellular processes like glucose metabolism and proliferation, can directly phosphorylate eNOS at Ser1177/Ser1179, leading to increased eNOS enzymatic activity and subsequent NO production (Dimmeler et al., 1999; Fulton et al., 1999). Inhibition of AKT or a mutation of the AKT phosphorylation site on eNOS attenuates eNOS phosphorylation and its activity, resulting in decreased NO bioavailability and endothelial dysfunction (Dimmler et al. 1999)

Western blot analysis can be performed to determine the expression levels of phosphorylated eNOS, phosphorylated Akt, total Akt and total eNOS proteins using the appropriate anti-phospho-eNOS, anti-phospho-Akt, anti-eNOS, and anti-Akt antibodies. Alternatively, eNOS activity can be measured using the conversion of L-arginine to L-citrulline assay.

ELISA kits for AKT/eNOS and phospho AKT/eNOS expression are commercially available.

Decreased Akt and eNOS activity was observed in humans, cows, mice and rats following exposure to stressors.

Cigarette smoke exposure was shown to inhibit the phosphorylation of AKT and eNOS in VEGF-stimulated human umbilical vein endothelial cells (HUVECs), resulting in decreased NO levels (Michaud et al. 2006).

In rat aortic rings, exposure to methylglyoxal and high concentrations of glucose decreased endothelium-dependent relaxation.  Further experiments in rat endothelial cells and HUVECs demonstrated a reduction in eNOS phosphorylation and activity, and reduced NO levels in response to the same stressors (Dhar et al. 2010).

In bovine aortic endothelial cells, AKT and eNOS phosphorylation were decreased following exposure to the peroxynitrite source; SIN-1, with an associated reduction in NO bioavailability.  These effects were ameliorated by treatment with the ROS scavenger DMPO (Das et al. 2014).

eNOS knockout mice are routinely used as models of hypertension.  Such mice display reduced bioavailability of NO and impaired vasodilation (Huang et al. 1995).

Reduced AKT/eNOS phosphorylation was reported under conditions of hyperglycaemia (in mice) and in HUVECs following treatment with high concentrations of glucose.  Aortic rings from hyperglycaemic mice demonstrated impaired vasodilation.  Resveratrol treatment was shown to improve vasodilation and eNOS phosphorylation in wild-type mice, but not AKT knockout mice.  Transfection of HUVECs with AKT siRNA abolished resveratrol-enhanced eNOS phosphorylation and NO release (Li et al. 2017),

Atochin, D.N., Wang, A., Liu, V.W.T., Critchlow, J.D., Dantas, A.P.V., Looft-Wilson, R., Murata, T., Salomone, S., Shin, H.K., Ayata, C., et al. (2007). The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo. J. Clin. Invest. 117, 1961–1967.

Das A, Gopalakrishnan B, Druhan LJ, Wang TY, De Pascali F, Rockenbauer A, Racoma I, Varadharaj S, Zweier JL, Cardounel AJ, Villamena FA.  Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelialcells via eNOS phosphorylation.  Br J Pharmacol. 2014, 171(9):2321-34. doi: 10.1111/bph.12572.

Dhar A, Dhar I, Desai KM, Wu L.  Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose.  Br J Pharmacol. 2010, 161(8):1843-56.

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.

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.

Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC.    Hypertension in mice lacking the gene for endothelial nitric oxide synthase.  Nature. 1995, 377(6546):239-42.

Li JY, Huang WQ, Tu RH, Zhong GQ, Luo BB, He Y.  Resveratrol rescues hyperglycemia-induced endothelial dysfunction via activation of Akt.  Acta Pharmacol Sin. 2017, 38(2):182-191.

Michaud SE, Dussault S, Groleau J, Haddad P, Rivard A.J. Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: role of NO and reactive oxygen species.  Mol Cell Cardiol. 2006 Aug;41(2):275-84.

Qian, J., and Fulton, D. (2013). Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium. Oxid. Physiol. 4, 347.