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 necrosis factor-alpha (TNF-alpha)-induced alterations in permeability and actin of pulmonary artery endothelial monolayers (PAEM). The permeability 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 antibodies to the COOH terminus of actin and dinitrophenylhydrazone (DNP), respectively. PAEM were incubated with TNF-alpha (100 U/ml) for 4 h. Incubation 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) permeability to Evans blue dye-albumin. The .NO synthase inhibitor aminoguanidine (100 microM) prevented the TNF-alpha-induced increase in NO2-, nitrotyrosine immunofluorescence, oxidized p42, and permeability. Coincubation with L-arginine (200 microM) or the .NO mimic spermine-NO (1 microM) prevented the ablation of the response to TNF-alpha by aminoguanidine. The data indicate that TNF-alpha-induced increases in endothelial permeability and oxidized protein are mediated by .NO in PAEM.
Ferro
T J
TJ
Department of Medicine, Albany Medical College, New York, USA.
Gertzberg
N
N
Selden
L
L
Neumann
P
P
Johnson
A
A
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000032:analysis; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000503:physiopathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006835:Hydrazones / Q000276:immunology; D008970:Molecular Weight; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009573:Nitrites / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D012709:Serum Albumin / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
C001072:2,4-dinitrophenylhydrazone; D000199:Actins; D000906:Antibodies; D004791:Enzyme Inhibitors; D006146:Guanidines; D006835:Hydrazones; D009573:Nitrites; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; D013096:Spermine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; C004479:pimagedine