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Am J Physiol Lung Cell Mol Physiol 290: L674-L684, 2006. First published November 11, 2005; doi:10.1152/ajplung.00391.2005
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Peroxynitrite mediates TNF-{alpha}-induced endothelial barrier dysfunction and nitration of actin

Paul Neumann,1 Nancy Gertzberg,1 Erin Vaughan,1 Joshua Weisbrot,2 Renee Woodburn,3 William Lambert,3 and Arnold Johnson1

1Research Service of the Stratton Veterans Affairs Medical Center; 2Albany Medical College; and 3University at Albany State University of New York, Albany, New York

Submitted 12 September 2005 ; accepted in final form 8 November 2005

We tested the hypothesis that tumor necrosis factor (TNF)-{alpha} induces a peroxynitrite (ONOO–)-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO2-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with antinitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO2-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO2-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO– agent Urate. The data indicate that TNF induces an ONOO–-dependent barrier dysfunction, which is associated with the generation of NO2-beta-actin.

edema; permeability


Address for reprint requests and other correspondence: A. Johnson, 151, 113 Holland Ave., Dept. of Veterans Affairs Medical Center, Albany, NY 12208 (e-mail: jmurd{at}msn.com, arnold.johnson{at}va.gov)




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