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1 Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2 Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA
3 Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
* To whom correspondence should be addressed. E-mail: deluder{at}ccm.upmc.edu.
A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO.) production secondary to induction of inducible NO. synthase is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium. C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle. Twelve h later, leakage of FITC-dextran (Mr 4 kDa) from blood into bronchoalveolar lavage (BAL) fluid was significantly increased in endotoxemic but not control mice. This decrease in bronchoalveolar barrier function was associated with up-regulation of iNOS protein expression and NF-
B activation in lung tissue. Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluoresence, decreased in lung following the injection of mice with LPS. Treatment of endotoxemic mice with an isoform-selective inducible NO. synthase (iNOS) inhibitor [L-N(6)-(1-iminoethyl)lysine; L-NIL] ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function. Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1000 U/ml IFN-
, 10 ng/ml TNF-
, and 1 ng/ml of IL-1
) increased permeability to FD4, but adding L-NIL prevented this effect. These results suggest that decreased expression and mistargeting of TJ proteins in lung following systematic inflammation may be NO.-dependent.
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