CALL FOR PAPERS Oxidant Signaling in Lung Cells NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-

Gertzberg, Nancy, Paul Neumann, Victor Rizzo, and Arnold Johnson. NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF. Am J Physiol Lung Cell Mol Physiol 286: L37–L48, 2004. First published June 13, 2003; 10.1152/ajplung. 00116.2003.—We tested the hypothesis that the NAD(P)H oxidasedependent generation of superoxide anion (O2 ) mediates tumor necrosis factor(TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47 and p22 were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2 was measured by the fluorescence of 6-carboxy-2 ,7 -dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47 translocation, 2) an increase in p22 protein, 3) increased localization of p47 with p22, 4) O2 generation, and 5) increased permeability to albumin. p22 antisense oligonucleotide prevented the TNF-induced effect on p22, p47, O2 , and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2 and permeability to albumin was also prevented by the O2 scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2 , mediates TNF-induced barrier dysfunction in PMEM.