Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability.
BACKGROUND
Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood.


METHODS
In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration.


RESULTS
Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeable TJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and beta-catenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice.


CONCLUSIONS
In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.
Cuzzocrea
S
S
Institute of Pharmacology, School of Medicine, University of Messina, Italy. salvator@www.unime.it
Mazzon
E
E
De Sarro
A
A
Caputi
A P
AP
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005609:Free Radicals / Q000494:pharmacology*; D005614:Freeze Fracturing; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007408:Intestinal Absorption; D007421:Intestine, Small / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D062793:Occludin; D018384:Oxidative Stress; D010750:Phosphoproteins / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism*; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D015054:Zymosan / Q000494:pharmacology; D051176:beta Catenin
D001549:Benzamides; C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D005609:Free Radicals; D008565:Membrane Proteins; D009566:Nitrates; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C569043:Tjp1 protein, mouse; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; C025160:3-aminobenzamide; D015054:Zymosan; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011065:Poly(ADP-ribose) Polymerases