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Am J Physiol Lung Cell Mol Physiol 280: L914-L922, 2001;
1040-0605/01 $5.00
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Vol. 280, Issue 5, L914-L922, May 2001

Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction

Kathleen Bove1, Paul Neumann2, Nancy Gertzberg2, and Arnold Johnson1,2

1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Center for Cardiovascular Science, Albany Medical College, Albany, New York 12208

We tested the hypothesis that endothelial cell nitric oxide synthase (ecNOS) mediates the tumor necrosis factor (TNF)-alpha -induced increase in nitric oxide (NO) and albumin permeability in pulmonary microvessel endothelial monolayers (PEM). PEM lysates were analyzed for ecNOS mRNA (RT-PCR), ecNOS protein (Western immunoblot), NO levels (NO<UP><SUB>2</SUB><SUP>−</SUP></UP>, the oxidation product of NO), and barrier function (albumin clearance rate). PEM were incubated with TNF (50 ng/ml) for 0.5, 2, 4, and 24 h. TNF induced a decrease in ecNOS mRNA at 2, 4, and 24 h. TNF induced an acute (0.5 h) increase followed by a protracted decrease (4-24 h) in ecNOS protein levels. The other NOS isotypes, inducible and brain NOS, could not be detected in the PEM using RT-PCR and Western blot assay. ecNOS antisense oligonucleotide decreased ecNOS protein, which prevented the increase in NO and albumin permeability at TNF-4 h. Spermine-NONOATE, the NO agonist, ablated the protective effect of ecNOS antisense oligonucleotide on albumin permeability in response to TNF-4 h. However, ecNOS antisense oligonucleotide had no effect on the TNF-induced increase in albumin permeability at 24 h despite prevention of the increase in NO. The data indicate that the isotype ecNOS mediates generation of NO and the acute (i.e., 4 h) barrier dysfunction; however, the prolonged (i.e., 24 h) increase in the TNF-induced increase in endothelial permeability is independent of NO.

antisense; edema; messenger ribonucleic acid; permeability; transcription


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