Mechanisms of CRF-Induced Intestinal Dysfunction Figure 6. The role of mast cell activation in CRF-mediated changes in FD4 flux. Treatment of porcine ileal tissues mounted on Ussing chambers with CRF increased mast cell degranulation indicated by the increased percentage of degranulated mast cells (C) and the increased release of mast cell tryptase (A) and TNF-a (B). Treatment of porcine ileal tissues with cromolyn sodium 30 minutes prior to exposure to 0.5 mM CRF blocked mast cell degranulation (C) and TNF-a release (B) and prevented CRF-mediated increases in FD4 permeability (D). c48/80 (5 mg/mL) induced marked degranulation of mast cells (C) and increases in FD4 flux (D) that were inhibited with sodium cromolyn. Data for each FD4 flux experiments are expressed as means 6 SE for n = 628 pigs. Symbols (*,{) differ from other treatments by p,0.05; ANOVA. doi:10.1371/journal.pone.0039935.g006 could explain the above data presenting large changes in paracellular permeability to uncharged ions but little changes in TER. Alternatively, disparate effects of CRF on FD4 flux and TER may also be due to time-dependent changes in TER. Chronic administration of CRF increases conductance and thus decreases in TER in rodents [11] whereas our ex vivo model would represent acute CRF effects. Nerve-mast cell interactions have been an increasing subject of interest due to their role in several intestinal disorders [35]. The interplay between CRF, mast cells, and enteric nerves has been documented in rodents [36,37] and humans [38]; however, the nature of these interactions remains poorly understood. In the present study, the barrier-disrupting effects of CRF were prevented by the nerve blocker, TTX demonstrating that enteric neural signaling is a critical element in CRF-mediated intestinal barrier dysfunction. This important role of the enteric nervous system in stress-induced intestinal barrier disruption has been demonstrated previously in rodent models [6,21]; however, it remains unclear how enteric neuronal signaling modulates this important pathway. In our studies, we showed that enteric neural inhibition blocked both baseline and CRF-mediated mast cell degranulation. This suggests that enteric neural signaling may modulate intestinal barrier function under baseline and stressed conditions via the control of mast cell activation. CRF receptors are expressed on enteric neurons [39,40] and mast cells [41,42] and therefore it is difficult to delineate the precise signaling pathways with the pharmacologic approach used in the present study. CRF could be mediating its effect on mast cell PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39935