Abstract 1665: Angiotensin II Inactivates eNOS Through Tyrosine Phosphorylation
We recently reported that in endothelial cells, the proline-rich tyrosine kinase PYK2 is activated by fluid shear stress and phosphorylates eNOS on tyrosine 657. Tyrosine phosphorylation of eNOS in vitro completely inhibits enzyme activity and limits NO production in response to shear stress. Overexpression of PYK2 in isolated arteries abrogated NO-mediated vasodilatation. In atherosclerotic lesions, eNOS is partially inactive even in the presence of sufficient co-factors. Since PYK2 is also known to be activated by angiotensin (Ang) II and reactive oxygen species, we speculated that this mechanism plays a role in the loss of NO bioavailability in pathologies associated with increased Ang II and H2O2 levels. In native porcine aortic endothelial cells and in freshly isolated murine aortae, Ang II and H2O2 both enhanced the phosphorylation of eNOS on Tyr657. This effect was correlated with increased PYK2 phosphorylation and the association of PYK2 with eNOS as shown by co-immunoprecipitation. Furthermore, in vivo oxidative stress induced by endotoxins also increased p-Tyr657eNOS levels in mouse aortae. In cultured endothelial cells, H2O2 had a dual effect on eNOS phosphorylation and activity. At low concentrations (1 − 100 μmol/L) H2O2 stimulated the phosphorylation of Tyr657 without affecting that of either Ser1177 or Thr495. At these concentrations, H2O2 also limited basal NO production and markedly impaired NO production in response to bradykinin and ionomycin. Furthermore, low (30 μmol/L) concentrations of H2O2 impaired acetylcholine-induced vasodilatation in murine aorta. At higher concentrations (300–500 μmol/L) of H2O2, increased intracellular Ca2+ elicited the phosphorylation of Ser1177 and increased NO production. Taken together, PYK2 activation under pathological conditions, i.e. increased Ang II and H2O2 causes the phosphorylation of eNOS on Tyr657 impeding NO production and endothelium dependent vasodilatation. This mechanism, in addition to eNOS uncoupling, may underlie the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin-angiotensin system and elevated redox stress.