Activation of colonic
proteinase-activated receptor-2 (PAR(2)) caused
inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR(2)-activating
peptide,
SLIGRL, on colonic paracellular permeability and
inflammation at two different doses, 5 and 100 microg per mouse, in an attempt to discriminate between both PAR(2)-mediated effects. We further investigated the possible involvement of
interferon gamma (IFN-gamma) and
calmodulin-dependent activation of
myosin light chain kinase (MLCK), and alterations of zonula occludens-1 (ZO-1) localization in PAR(2)-induced responses. Thus, at the lower dose,
SLIGRL increased colonic permeability without causing
inflammation. Western blotting showed phosphorylation of mucosal
myosin light chain (MLC) expression after both doses of
SLIGRL. Moreover, while the MLCK inhibitor,
ML-7, abolished the permeability effects of the low dose of
SLIGRL, it only partially inhibited that of the high dose. In IFN-gamma-deficient mice (B6 ifng(-/-)), the increases in permeability were similar for both doses of
SLIGRL and prevented by
ML-7. In addition, MLCK immunoprecipitation revealed an increase of
calmodulin binding to MLCK in the mucosa of mice treated with either dose of
SLIGRL. Finally, we have shown that direct activation of PAR(2) on enterocytes is responsible for increased permeability and ZO-1 disruption. Moreover,
SLIGRL at a dose that does not produce
inflammation increases permeability via
calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN-gamma secretion, while the increased permeability in response to the high dose of PAR(2) agonist involves IFN-gamma secretion.