Increased iNOS activity is essential for hepatic epithelial tight junction dysfunction in endotoxemic mice.
We tested the hypothesis that increased production of nitric oxide (NO*) by inducible NO* synthase (iNOS) is a key factor responsible for alterations in the expression, localization, and function of key tight junction (TJ) proteins in mice challenged with lipopolysaccharide (LPS, endotoxin). Endotoxemia was associated with hepatobiliary epithelial barrier dysfunction, as evidenced by increased plasma-to-bile leakage of FITC-labeled dextran (relative molecular mass 40 kDa) and increased circulating levels of bile acids and conjugated bilirubin. Immunoblotting revealed decreased expression of zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin in liver after injection of C57Bl/6J mice with 2 mg/kg Escherichia coli 0111:B4 LPS. Nonidet P-40-insoluble (i.e., TJ-associated) occludin and ZO-1 were virtually undetectable 12 and 18 h after injecting LPS. Immunofluorescence microscopy also revealed deranged subcellular localization of ZO-1 and occludin in endotoxemic mice. Pharmacological inhibition of iNOS activity using l-N6-(1-iminoethyl)lysine (5 mg/kg) or genetic ablation of iNOS ameliorated LPS-induced changes in hepatobiliary barrier function, and these strategies partially preserved TJ protein expression and localization. Steady-state levels of occludin and ZO-3 transcripts decreased transiently after injecting LPS but returned toward normal by 12 and 24 h after induction of endotoxemia, respectively. These results support the view that iNOS-dependent NO* production is an important factor contributing to hepatobiliary epithelial barrier dysfunction resulting from systemic inflammation and suggest that iNOS induction may play a role in the development of cholestatic jaundice in patients with severe sepsis.
Han
Xiaonan
X
Deptartment of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Fink
Mitchell P
MP
Uchiyama
Takashi
T
Yang
Runkuan
R
Delude
Russell L
RL
D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity; D001659:Biliary Tract / Q000473:pathology; D015153:Blotting, Western; D004731:Endotoxins / Q000097:blood / Q000633:toxicity*; D004768:Enterotoxins / Q000633:toxicity; D004790:Enzyme Induction / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology / Q000502:physiology; D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D008070:Lipopolysaccharides / Q000633:toxicity; D008099:Liver / Q000201:enzymology / Q000473:pathology*; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D062793:Occludin; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000201:enzymology* / Q000473:pathology / Q000502:physiology*
D001427:Bacterial Toxins; D004731:Endotoxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D009569:Nitric Oxide; C027538:heat-labile enterotoxin, E coli; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D000410:Alanine Transaminase