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induced endothelial barrier dysfunction and nitration of actin
1 Research Service, Veterans Affairs Medical Center, Albany, NY, USA
2 Albany Medical College, Albany, NY, USA
3 SUNY Albany, Albany, NY, USA
* To whom correspondence should be addressed. E-mail: arnold.johnson{at}med.va.gov.
We tested the hypothesis that tumor necrosis factor-
(TNF) induces a peroxynitrite (ONOO-) dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated 
-actin (NO2--actin). The permeability of PMEM were assessed by the clearance rate of Evans Blue labeled albumin. PMEM lysate -actin was extracted with a DNase-sepharose column. The extracted -actin was quantified in terms of its nitrotyrosine/-actin ratio by using anti-nitrotyrosine and anti--actin antibodies, sequentially, in a dot blot assay. The cellular compartmentalization of NO2--actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with -actin-immunofluorescence or F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 hr and 4.0 hr resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/-actin ratio at 0.5 hr with minimal association of the nitrotyrosine with F-actin polymers. The TNF-induced increase in the nitrotyrosine/-actin ratio and permeability were prevented by the anti-ONOO- agent urate. The data indicate that TNF-induces an ONOO-- dependent barrier dysfunction which is associated with the generation of NO2--actin.
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