Perinatal changes in superoxide generation in the ovine lung: Alterations associated with increased pulmonary blood flow

Although alterations in ROS generating systems are well described in several vascular disorders, there is very limited information on the perinatal regulation of these systems in the lung both during normal development and in pulmonary hypertension. Thus, this study was undertaken to explore how the two predominant superoxide generating systems, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and xanthine oxidase (XO), are developmentally regulated in control lambs and in our established lamb model of increased pulmonary blood flow (Shunt) over the first 2months of life. We found that the levels of p47(phox), p67(phox), and Rac1 subunits of NADPH oxidase complex were altered. During the first two months of life there was no change in p47(phox) protein levels in either normal or Shunt lambs. However, both p67(phox) and Rac1 protein levels decreased over time. In addition, p47(phox) protein levels were significantly increased in shunt lambs at 2- and 4-, but not 8-weeks of age compared to age-matched controls while levels of the p67(phox) subunit were decreased at 8-weeks of age in the Shunts but unchanged at other time periods. Furthermore, Rac1 protein expression was significantly increased in the Shunts only at 4weeks of age. These data correlated with a significant increase in NADPH oxidase dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. During normal development XO levels significantly increased over time in normal lambs but significantly decreased in the Shunts. In addition, XO protein levels were significantly increased in the Shunt at 2- and 4-weeks of age but significantly decreased at 8-weeks. Again this correlated with a significant increase in XO dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. Collectively, our findings suggest that NADPH oxidase and XO are major contributors to superoxide generation both during the normal development and during the development of pulmonary hypertension.