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.

Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha.

We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/beta, 100 microM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-induced increases in endothelial permeability in PEM.

Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation.

We tested the hypothesis that protein kinase (PK)G activation in response to nitric oxide ((*)NO) mediates tumor necrosis factor (TNF)-alpha-induced activation of the transcription factor activating protein-1 (AP-1) in pulmonary microvessel endothelial monolayers (PEM). The DNA-binding activity of AP-1 was assessed using the electrophoretic mobility shift assay. TNF treatment (1,000 U/ml) for 4 h induced a significant increase in DNA binding of AP-1. The effects of TNF were prevented by the superoxide radical scavenger superoxide dismutase (SOD) (100 U/ml), the (*)NO synthase inhibitor aminoguanidine (100 microM), the guanylate cyclase inhibitor ODQ (100 microM), and the PKG inhibitors KT5823 (1 microM) and 8-bromo-cyclic guanosine monophosphate (cGMP)-thioate (100 microM). Spermine-NO (1 microM) and L-arginine (400 microM) prevented the aminoguanidine-induced ablation of AP-1 activation in response to TNF. Phosphorylation of H-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activity of cGMP-dependent protein kinase (PKG). TNF for 0.5 h induced an increase in PKG activity that was prevented by aminoguanidine, ODQ, KT5823, and 8-bromo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 microM), when given alone, increased PKG activity but induced significant DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN-1 (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-1. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2, which contains a regulation sequence responsive to AP-1. The pharmacologic profile of TNF-induced CAT activity was identical to TNF-induced DNA binding by AP-1. Thus, TNF-induced AP-1-dependent gene transcription is modulated by (*)NO-dependent mediated activation of PKG.

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.

Effects of pulmonary edema on regional pulmonary perfusion in the intact dog lung.

Regional pulmonary blood flow was determined in dogs during varying degrees of pulmonary edema induced by infusing 179.2-659.4 ml/kg normal saline over 2-3 h. Pulmonary hemodynamics and regional blood flows were measured during the base-line period and at 30 min postinfusion. The degree of pulmonary edema was determined by the final extravascular lung water-to-bloodless dry lung weight ratio (W/D). In dogs developing gross alveolar edema (W/D of 10.70 +/- 0.88 vs. 3.10 +/- 0.30 in controls), the blood flow was shifted to either upper or dependent lung regions. The shift to the upper regions was associated with an increased (P < 0.05) pulmonary arterial pressure (Ppa), whereas the shift to the dependent lung was not associated with a significant change in Ppa. Breathing 100% O2 did not prevent these shifts, suggesting that they were not due to localized hypoxic pulmonary vasoconstriction. The flow distribution patterns were also not related to regional differences in edema. In contrast to the changes during fulminant edema, blood flow distribution did not change after moderate levels of pulmonary edema (W/D of 6.03 0.69), suggesting that gross alveolar flooding is required for a redistribution of pulmonary blood flow. Flow redistribution to the upper lung during airway flooding may be due to increase in Ppa, whereas the increased flow in the dependent lung during the same degree of edema may be due to "bulging" of alveolar vessels into the air spaces, secondary to a decrease in surface activity.