subtypes CRF1 and CRF2 and in vitro studies utilizing the HMC1 mast cell line demonstrated that CRF directly stimulates the selective release of vascular endothelial growth factor (VEGF); however, tryptase and TNF-a release was not induced by CRF which in contrast to the present studies in native porcine ileal tissues [41]. The lack of effect of CRF on tryptase or TNFa observed in HMC1 cells by Cao et al. [41] is likely due to the simplified cell culture conditions and therefore lack of potentially critical co-factors that are present in native tissues. In support of this hypothesis, Asadi et al. [45] demonstrated that pre-treatment of the human mast cell line LAD2 with substance P induced CRF1 expression and enhanced CRF-mediated TNF-a and IL-8 release. This suggests that neural mediators may play a crucial role in priming the mast cell for CRF signaling; however, demonstration of these signaling pathways in vivo have yet to be demonstrated. Overall, we show here that in porcine ileum, CRF induces the release of mast cell proteases and TNF-a which contribute significantly to CRF-induced disturbances in intestinal epithelial barrier function; furthermore these pathways are regulated by the enteric nervous system. Given the clinical significance of these pathways in stress-related intestinal disorders such as IBS and IBD, a greater understanding of how these signaling pathways are integrated during health and disease are needed. Materials and Methods Ethics Statement The North Carolina State University Institutional Animal Care and Use Committee approved all studies. Animals and Intestinal Tissue Collection Yorkshire x Hampshire cross-bred pigs of either sex and between 628 weeks of age were used in this study. Prior to experiments and tissue harvest, pigs were sedated with a combination of xylazine (1.5 mg im) and ketamine (11 mg/kg im) followed by euthanasia with an intravenous overdose of pentobarbital via a catheterized ear vein. Initial sedation was used to minimize stress before we obtained intestine for subsequent intestinal studies. Segments of ileum were harvested immediately after euthanasia and prepared for Ussing chamber studies. Chemicals and Reagents Rat/human CRF (0.01–0.5 mM) and Astressin B (1 mM) were obtained from The Salk Institute, La Jolla, CA. CRF doses were based on comparable doses of CRF used in several experiments utilizing cell culture models [41,45,46] and physiological ranges found in peripheral tissues including primary rat adrenals [47] and human endometria [48]. Cromolyn sodium, porcine anti-TNF-a neutralizing antibody, protease inhibitor cocktail, c48/80, and tetrodotoxin (TTX) were obtained from Sigma Chemical. The PI cocktail contained the chymase inhibitor Soybean Trypsin Inhibitor (100 mg/mL) and the tryptase inhibitor Aprotinin (Sigma, 10 mM). Ussing Chamber Experiment Ileal mucosa was stripped from the seromuscular layer in oxygenated (95% O2–5% CO2) Ringer solution (in mmol/l: 154 Na+, 6.3 K+, 137 Cl2, 0.3 H2PO4-, 1.2 Ca2+, 0.7 Mg2+, 24 HCO32; pH 7.4). Tissues were then mounted in 1.13 cm2 aperture Ussing chambers, as described in previous study [5,49]. Tissues were bathed on the serosal and mucosal sides with 10 mL of Ringer solution. The serosal bathing solution contained 10 mM glucose, which was osmotically balanced on the mucosal side with 10 mM mannitol. Bathing solutions were oxygenated (95% O2– Mechanisms of CRF-Induced Intestinal Dysfunction 5% CO2) and circulated in water-jacketed reservoirs maintained at 37uC. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. Tissues were maintained in the short-circuited state, except for brief intervals to record the open-circuit PD. Transepithelial electrical resistance (TER; V?cm2) was calculated from the spontaneous PD and short- circuit current (Isc), as previously described [50]. After a 30-min equilibration period on Ussing chambers, TER was recorded at 15-min intervals over a 4-hr period and then averaged to derive the basal TER values for each experimental treatment. Mucosal-to-serosal Fluxes of FITC-labeled Dextran Mucosal-to-serosal fluxes of (FITC)-dextran (4 kDa; Sigma- Aldrich, St. Louis, MO) were performed at the same time as TER was measured. After a 15-min period on Ussing chambers, FITC Dextran (0.25 mM) was added to the mucosal side of Ussing chamber-mounted tissues. The FD4 was allowed to equilibrate for 15 min after which 100 mL samples (in triplicate) were taken from the serosal side of tissues at 30-minute intervals and transferred into a 96 well assay plate. The presence of FD4 fluorescence intensity of each sample was measured by fMax Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA) and concentrations were determined from standard curves generated by serial dilution of FD4. FD4 flux was measured for 180 minutes post-CRF exposure and presented as the rate of FD4 flux (mg/min). Experimental Protocol After mounting on Ussing chambers, ileal tissues were allowed to equilibrate for a 15–30 minute period until tissue TER and Isc values stabilized. Thirty minutes prior to CRF exposure, Pharmacologic antagonists (Astressin B, cromolyn, PI, anti-TNFa, and TTX) were added to the serosal side of ileal mucosa, where the actions of these compounds are expected to exert direct effects on submucosal target cells. CRF was added to the serosal chamber and TER and FD4 flux were measured over a 180-min period. Histological Examination At the end of the Ussing chamber experiments, ileal tissues fixed in 10% neutral buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin for histological analysis. Tissues were visualized for changes in intestinal pathology as a result of experimental agonist treatments (Meiji Microscope Solutions, Model OMFL400). Mast Cell Histological Analysis Counts Frozen sections of ileum (10 mm thick) were prepared and then fixed for 1 h in Carnoy’s fixative (60% ethanol-30% chloroform- 10% glacial acetic acid). Sections were then stained for 45 min at room temperature with 0.5% toluidine blue in 0.5 N HCl in PBS. Mast cell counts were conducted on five different fields per slide from n = 4 pigs per treatment group. Data is presented as percentage of mast cells exhibiting degranulation. All histological analyses were performed by at least 2 reviewers who were blinded to experimental treatments. Measurement of TNF-a Supernatant from the serosal bathing solution of Ussing Chambers was collected at time 240 min (end of experiment) for analysis of TNF-a using a commercial ELISA kit (R&D Systems) according to manufacturer’s instructions. Samples were frozen PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39935