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 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, 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.
Bove
K
K
Research Service, Stratton Veterans Affairs Medical Center, Albany, New York 12208, USA.
Neumann
P
P
Gertzberg
N
N
Johnson
A
A
D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005070:Evans Blue / Q000737:chemistry; D008833:Microcirculation; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011652:Pulmonary Circulation; D012333:RNA, Messenger / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000493:pharmacokinetics; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D005070:Evans Blue; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III