Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin.
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 (NO(2)-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 anti-nitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO(2)-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 NO(2)-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 NO(2)-beta-actin.
Neumann
Paul
P
151, 113 Holland Ave., Dept. of Veterans Affairs Medical Center, Albany, NY 12208, USA.
Gertzberg
Nancy
N
Vaughan
Erin
E
Weisbrot
Joshua
J
Woodburn
Renee
R
Lambert
William
W
Johnson
Arnold
A
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D008168:Lung / Q000098:blood supply*; D008833:Microcirculation; D009566:Nitrates / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000502:physiology*; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D014018:Tissue Distribution / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014527:Uric Acid / Q000494:pharmacology
D000199:Actins; D000418:Albumins; D009566:Nitrates; D020033:Protein Isoforms; D014409:Tumor Necrosis Factor-alpha; D030421:Peroxynitrous Acid; D014527:Uric Acid; C002744:3-nitrotyrosine; D014443:Tyrosine