Intestinal CRF Receptor Subtypes Mediate Distinct Intestinal Barrier Function Responses Through Intestinal Mast Cell Activation

Abstract

Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.