Ischemia-reperfusion injury is accompanied by endothelial
hypoxia and reoxygenation that trigger oxidative stress with enhanced
superoxide generation and diminished
nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial
NO synthase (eNOS) cofactor
tetrahydrobiopterin can trigger eNOS uncoupling, in which the
enzyme generates
superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical
cysteine residues of the
reductase site that serves as a redox switch to control eNOS coupling. While
superoxide can deplete
tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following
hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to
hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process.
Hypoxia and reoxygenation of aortic endothelial cells triggered
xanthine oxidase-mediated
superoxide generation, causing both
tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with
tetrahydrobiopterin along with increasing intracellular levels of
glutathione greatly preserved eNOS activity after
hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to
hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to
reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the
enzyme from NO to
superoxide production. Thus, in endothelial reoxygenation injury, normalization of both
tetrahydrobiopterin levels and the
glutathione pool are needed for maximal restoration of eNOS function and NO generation.