ENDOTHELIAL BARRIER DYSFUNCTION AND P42 OXIDATION-INDUCED BY TNF-ALPHA ARE MEDIATED BY NITRIC-OXIDE

We tested the hypothesis that nitric oxide (. NO) mediates tumor necro sis factor-alpha (TNF-alpha)-induced alterations in permeability and a ctin of pulmonary artery endothelial monolayers (PAEM). The permeabili ty of PAEM, was assessed by the clearance rate of albumin labeled with Evans blue dye. The PAEM Triton-soluble (''cytoskeletal-nonassociated '') and -insoluble (''cytoskeletal-associated'') lysates were analyzed by Western blot for actin and oxidized protein using polyclonal antib odies to the COOH terminus of actin and dinitrophenylhydrazone (DNP), respectively. PAEM were incubated with TNF-alpha (100 U/ml) for 4 h. I ncubation of PAEM with TNF-alpha resulted in increases in 1) the . NO oxidation product nitrite (NO2-), 2) nitrotyrosine immunofluorescence, 3) the oxidation of p42 (tentatively identified as actin), and 4) per meability to Evans blue dye-albumin. The . NO synthase inhibitor amino guanidine (100 mu M) prevented the TNF-alpha-induced increase in NO2-, nitrotyrosine immunofluorescence, oxidized p42, and permeability. Coi ncubation with L-arginine (200 mu M) or the NO mimic spermine-NO (1 mu M) prevented the ablation of the response to TNF-alpha by aminoguanid ine. The data indicate that TNF-alpha-induced increases in endothelial permeability and oxidized protein are mediated by . NO in PAEM.