ABSTRACT
Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Bacterial Translocation/drug effects , Citrobacter rodentium/pathogenicity , Colitis/prevention & control , Colon/drug effects , Enterobacteriaceae Infections/drug therapy , Intestinal Mucosa/drug effects , Vasoactive Intestinal Peptide/administration & dosage , Animals , Azepines/pharmacology , Bacterial Adhesion , Caco-2 Cells , Claudin-3 , Colitis/metabolism , Colitis/microbiology , Colitis/pathology , Colon/metabolism , Colon/microbiology , Colon/pathology , Disease Models, Animal , Enterobacteriaceae Infections/complications , Enterobacteriaceae Infections/metabolism , Enterobacteriaceae Infections/microbiology , Humans , Injections, Intraperitoneal , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Mannitol/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Myosin Light Chains/metabolism , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosin-Light-Chain Kinase/metabolism , Naphthalenes/pharmacology , Occludin , Permeability , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Tight Junctions/drug effects , Tight Junctions/metabolism , Tight Junctions/microbiology , Time Factors , Zonula Occludens-1 ProteinABSTRACT
Cytosolic phospholipase A(2)(cPLA(2)), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA(2) distribution in pancreatic beta-cells is different from that of most other mammalian cells: it is evenly distributed throughout the beta-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca(2+) in the MIN6 beta-cell line also stimulated a redistribution of cPLA(2) immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca(2+) being responsible for translocation of cPLA(2). These observations suggest that cPLA(2) may be compartmentalised in unstimulated beta-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca(2+).
