, Volume 25, Issue 6, pp 1377-1386

Myosin Light Chain Kinase Inhibition: Correction of Increased Intestinal Epithelial Permeability In Vitro

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Abstract

Purpose

To examine whether myosin light chain kinase (MLCK) inhibitors can reduce intestinal epithelial permeability increases in vitro.

Materials and Methods

Isolated rat, mouse and human colonic tissue mucosae and Caco-2 monolayers were exposed to cytochalasin D (cD) and sodium caprate (C10), in the absence and presence of the MLCK inhibitors, ML-9 and D PIK. Transepithelial electrical resistance (TEER) and Papp of [14C]-mannitol or FITC-dextran 4000 (FD-4) were measured. Western blots were used to measure MLC phosphorylation.

Results

Increases in Papp of [14C]-mannitol and decreases in TEER were induced by tight junction openers. These changes were attenuated by ML-9. D-PIK offset the FD-4 Papp increase induced by C10 in Caco-2 only, while ML-9 and PIK inhibited MLC directly. cD induced constriction of peri-junctional actin in Caco-2 monolayers, but this was prevented by ML-9. Although mannitol fluxes across colonic mucosae from dextran-sulphate (DSS)-treated mice were higher than control, they were not ameliorated by either ML-9 or PIK in vitro.

Conclusions

ML-9 inhibits paracellular permeability increases in several intestinal epithelial models. D-PIK reduced stimulated paracellular fluxes in Caco-2 monolayers, but not in tissue. Pre-established increases were not modified by two MLCK inhibitors in a mouse model of IBD.