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Articles in PresS, published online ahead of print July 26, 2002
Am J Physiol Lung Cell Mol Physiol, 10.1152/ajplung.00123.2002
Submitted on April 25, 2002
Accepted on July 19, 2002
1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA
2 Division of Infectitious Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
* To whom correspondence should be addressed. E-mail: vnataraj{at}mail.jhmi.edu.
As the exact mechanisms of generation of reactive oxygen species (ROS) during hyperoxia in vascular endothelium are not thoroughly understood, we investigated the role and mechanisms of regulation of NAD[P]H oxidase in hyperoxia-induced generation of ROS in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia (95% O2/5% CO2) for 1, 3, and 12 h increased the generation of O2-., hydroxyl radicals, H2O2 and total ROS. Diphenyleneiodonium (DPI) (100 µM) significantly inhibited where as rotenone (100 µM) and oxypurinol (100 µM) failed to inhibit hyperoxia-induced generation of ROS in HPAECs. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and SOD- and DPI-inhibitable generation of O2-. by intact cells as measured by lucigenin chemiluminescence. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox and p47 phox subcomponents of NAD[P]H oxidase in HPAECs. Hyperoxia-induced generation of ROS was completely blocked in cells transfected with p22 phox antisense plasmid. Hyperoxia also activated p38 mitogen-activated protein kinase (p38 MAPK) and extracellular-regulated kinase (ERK) in HPAECs. Inhibition of p38 MAPK and MEK1/2 significantly attenuated the hyperoxia-induced generation of ROS in HPAECs, suggesting the involvement of p38 MAPK and ERK. The attenuation of hyperoxia-induced ROS generation in HPAECs infected with p38 MAP kinase dominant negative adenoviral constructs further supports the role of p38 MAP kinase. In summary, our results demonstrate a role for MAP kinases in the regulation of hyperoxia-induced activation of NAD[P]H oxidase in HPAECs.
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