Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption.
Oxidants such as monochloramine (NH(2)Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH(2)Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH(2)Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH(2)Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH(2)Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH(2)Cl. The actin-stabilizing agent phalloidin did not prevent NH(2)Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH(2)Cl, observed as a redistribution of the alpha-subunit of basolateral Na(+)-K(+)-ATPase to the apical membrane, an effect not found with the apical membrane protein Na(+)/H(+) exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.
Musch
Mark W
MW
The University of Chicago Hospitals, 5841 S. Maryland Ave., MC 6084, Chicago, IL 60637, USA.
Walsh-Reitz
Margaret Mary
MM
Chang
Eugene B
EB
D000199:Actins / Q000096:biosynthesis / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects; D002700:Chloramines / Q000633:toxicity; D017097:Electric Impedance; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects; D008565:Membrane Proteins / Q000096:biosynthesis / Q000502:physiology*; D018613:Microscopy, Confocal; D062793:Occludin; D016877:Oxidants / Q000633:toxicity*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D062826:Zonula Occludens-1 Protein
D000199:Actins; D002700:Chloramines; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D016877:Oxidants; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase; C030816:chloramine