Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine.

OBJECTIVES Although in tissue injury following hypoxia/reoxygenation (H/R) an increased endothelial formation of superoxide anions (O(2)(-)) plays an important role, it is still not fully understood which of the potential enzymatic sources of endothelial O(2)(-) are crucially involved. In this study, we particularly examined the activities of NAD(P)H oxidase and xanthine oxidase (XO) after 8 h of exposure to mild hypoxia. We further studied whether enzyme activities can be modified by NO and adenosine during hypoxic treatment. METHODS AND RESULTS In human umbilical vein endothelial cells O(2)(-) production was measured immediately after exposure to hypoxia ('early reoxygenation') or after 2 h of reoxygenation at normoxic conditions ('late reoxygenation'). In the early reoxygenation phase the O(2)(-) production was attenuated by 28.5% while it was enhanced by 58.2% after late reoxygenation. Using specific inhibitors of NAD(P)H oxidase and XO, gp91ds-tat and oxypurinol, respectively, we show that the constitutively active NAD(P)H oxidase was blocked following hypoxia while XO was activated. The presence of NO during hypoxia had no effect on NAD(P)H oxidase activity but it significantly inhibited the activation of XO. Inhibition of XO activation was, at least in part, caused by the release of adenosine from endothelial cells which induces an increased formation of NO by its A1 and A2 receptors. CONCLUSION Our results indicate that during exposure to mild hypoxia for 8 h, a change in the enzymatic source of endothelial O(2)(-) occurs: a prolonged inhibition of NAD(P)H oxidase was found while an enhanced activity of XO occurs in the reoxygenation phase. These results suggest that different strategies of antioxidant therapy should be taken into consideration in oxidative stress related to chronic hypoxia when compared to normoxic atherosclerotic tissues with an activated vascular NAD(P)H oxidase as the main source of O(2)(-).