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

We tested the hypothesis that endothelial cell nitric oxide synthase (ecNOS ) mediates the tumor necrosis factor (TNF)-alpha -induced increase in nitri c 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 (NO2-, the oxidation product of NO) , and barrier function (albumin clearance rate). PEM were incubated with TN F (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 prot racted decrease (4-24 h) in ecNOS protein levels. The other NOS isotypes, i nducible and brain NOS, could not be detected in the PEM using RT-PCR and W estern blot assay. ecNOS antisense oligonucleotide decreased ecNOS protein, which prevented the increase in NO and albumin permeability at TNF-4 h. Sp ermine-NONOATE, the NO agonist, ablated the protective effect of ecNOS anti sense oligonucleotide on albumin permeability in response to TNF-4 h. Howev er, ecNOS antisense oligonucleotide had no effect on the TNF-induced increa se 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 th e acute (i.e., 4 h) barrier dysfunction; however, the prolonged (i.e., 24 h ) increase in the TNF-induced increase in endothelial permeability is indep endent of NO.