AGA Abstracts 177 The Roles of TNFR Signaling and NOX1 in Epithelial β-Catenin Activation During Colitis Daniela Posca, Hyunji Ryu, Elizabeth Managlia, Goo Lee, Preetika Sinh, Rebecca B. Katzman, Patrizia Doldo, Terrence A. Barrett TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that P- β-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p<0.05). These findings suggested TNF signaling regulates β-catenin activation independent of its effects on inflammation. Studies in wild type (WT) mice showed that T cell activation (anti- CD3mAb treatment) In Vivo induces both TFNR1 and TNFR2 mRNA in IEC. IHC of bone marrow chimera (BMC) mice generated by reconstituting WT or TNFR1/2 knockout (KO) mice with WT BM (as BM->host) revealed that T cell activation increased numbers of P-β- cat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genome- wide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNF- induced epithelial β-catenin activation in UC and CD. 178 Targeting the Actin Cytoskeletal Protein Fascin as a Novel Modulator of Intestinal Epithelial Cell Proliferation and Colon Cancer Richard P. Stevenson, Kevin Myant, Thomas Jamieson, Yafeng Ma, Ang Li, Ee Hong Tan, Shigeko Yamashiro, Massimo Pignatelli, Owen Sansom, Laura M. Machesky Background: Recent evidence suggests that stem cells are important for cancer metastasis and that the epithelial-to-mesenchymal transition also involves transition toward stemness. Current thinking suggests that Lgr5, a 7-transmembrane spanning G-protein, also marks a certain population of stem cells capable of regenerating a crypt and that the specialized immune secretory cell, the paneth cell, is important for maintenance of the stem cell niche. Fascin is an actin bundling protein, absent from normal epithelium, but upregulated in a subpopulation of regenerating cells following damage to the intestinal epithelium. Evidence suggests that fascin is a target of β-catenin-TCF, a central player in the Wnt signaling pathway and implicated in the differentiation and regeneration of intestinal epithelium. Fascin is also highly expressed in more than 50 epithelial cancers including colorectal with expression levels correlating with disease severity. Aims: To study the role of fascin in both intestinal regeneration and the neoplastic transformation of intestinal epithelial cells subjected to chronic inflammation. Results: We initially compared fascin knockout "KO" and normal wild type "WT" mice for their response to 3.5% Dextran sodium sulphate ("DSS" - a non- genotoxic agent and mimic of inflammatory bowel disease) over 5 days to induce acute colitis. Fascin KO mice lost more weight and showed more severe clinical signs than WT. Immunohistochemical analysis also demonstrated an increase in the number of regenerating intestinal crypts in the WT mice compared with the fascin KO. Using recently established methods for crypt culture In Vitro, we found that the diameter of the fascin KO crypts increased at a slower rate and they also underwent fewer crypt fission events compared with the WT. Preliminary analysis demonstrates a reduction in stem cell markers (e.g. Lgr5) in the cultured crypts from the fascin KO mouse compared with WT. We further sought to determine the role of fascin in tumourigenesis using a colitis associated tumour model - a single intra-peritoneal injection of Azoxymethane (causes the activation of β-catenin muta- tions) followed by 3 courses of 2% DSS. After 70 days the colons are harvested and the polyp number and tumour burden determined. Both the number of polyps and tumour burden were greater in the WT than the fascin KO. Conclusion: Fascin plays a key role in intestinal regeneration and tumourigenesis, potentially through enhanced stem cell signalling following Wnt activation. Further knowledge of the interaction between fascin and β-catenin- TCF is needed to understand the role of fascin in intestinal regeneration and tumourigenesis more clearly. 179 Enteric Glia Mediate Nicotinic-Stimulated Epithelial Ion Transport in Mouse Colon Revealed by Acute Glial Metabolic Inhibition Sarah MacEachern, Bhavik Patel, Wallace K. MacNaughton, Keith A. Sharkey Background & Aims: Enteric glia contribute to a variety of gastrointestinal functions. Recent reports have suggested that enteric glia are involved in the modulation of epithelial ion transport via the release of nitric oxide (NO), but direct evidence is lacking. Using a model of glial metabolic inhibition, we aimed to test the hypothesis that enteric glia are directly involved in the regulation of epithelial ion transport. Methods: Experiments were conducted S-44 AGA Abstracts in distal colon from wild-type CD1 mice. Full-thickness colonic segments were mounted in Ussing chambers and held under voltage-clamp conditions. The net electrogenic movement of ions across the epithelium was recorded as short-circuit current (ISC). Whole-mount longitudinal muscle-myenteric plexus preparations were utilized for electrochemical detec- tion of NO. Diamond microelectrodes were positioned near the ganglionic surface to provide real-time measurements of NO release. Tissues were treated with the glial metabolic poison fluoroacetate (FA; 5 mM; 30-120min) and then stimulated with the nicotinic agonist DMPP (10 μM), electrical field stimulation (EFS, 50V, 10Hz, 5s) and forskolin (10 μM) in Ussing experiments or veratridine (10 μM) in electrochemical detection experiments, in the presence or absence of nitric oxide synthase (NOS) I & II inhibitors. Results: Nicotinic stimulation with DMPP resulted in a biphasic change in ISC characterized by an increase in chloride secretion (Phase I; 47±9 μA/cm2, n=6) followed by a decrease below baseline (Phase II; -42±6 μA/cm2) that returned to baseline in 290±49 s. Pre-treatment with FA eliminated Phase II of the response, and the remaining response was sensitive to NOS I inhibition. In contrast, EFS resulted in a monophasic ΔISC (37±9 μA/cm2, n=8) that was sensitive to both NOS I and NOS II inhibition. FA treatment had no significant effect on the EFS response (25±5 μA/cm2, n=8) or on the epithelial ISC response to forskolin. DMPP causes the release of NO detected electrochemically from the myenteric plexus (953±18 pA, n=6). This response was reduced by both NOS I and II inhibition, and together these inhibitors completely blocked the response. FA treatment reduced the NO released following DMPP treatment (503±8 pA, n=5) and the remaining response was sensitive only to NOS I inhibition. Veratridine resulted in NO release (581±11 pA, n=5), which was sensitive only to NOS I inhibition, and was not altered following FA treatment (546±16 pA, n=5). Conclusions: Through the use of glial metabolic inhibition, we demonstrate that nicotinic stimulation of the myenteric plexus causes the release of NO from enteric neurons and enteric glia to regulate ion transport. In contrast, activation of enteric neurons using EFS or veratridine causes the release of NO from enteric neurons and regulates ion transport in a glial- independent manner. 180 The Extracellular Calcium Sensing Receptor Mediates Local Inhibitory Reflexes Evoked by Aromatic L-Amino Acids in Guinea Pig Jejunum Rachel Gwynne, Kenny Ly, Joel C. Bornstein Purpose The mechanisms by which luminal nutrients modulate intestinal motility remain poorly understood. Application of the aromatic amino acids L-phenylalanine (L-Phe) or L- tryptophan (L-Tryp) to the intestinal mucosa excites a reflex that triggers inhibitory junction potentials in the circular muscle. This involves release of 5-HT and/or ATP from mucosal enteroendocrine (EE) cells, but how EE cells detect amino acids is unknown. This study tested whether the extracellular calcium sensing receptor (CaSR), which is sensitive to aromatic L-amino acids, is involved in sensing L-amino acids in guinea pig jejunal mucosa. Methods Segments of guinea pig jejunum were dissected to allow circular muscle cells to be impaled with glass microelectrodes close to intact mucosa. Inhibitory reflexes were evoked in the circular muscle by applying L- and D- amino acids (all 30 mM) mucosally. Involvement of the CaSR was examined using the CaSR antagonist NPS 2143 and agonist cinacalcet. Results L-Phe and L-Tryp evoked significantly larger amplitude IJPs than their respective D-isomers when tested at the same location (L-Phe 95% larger, N = 6, P < 0.001, L-tryp 65% larger, N = 4, P = 0.01). Further, the non-aromatic amino acids L-leucine (L-Leu) and L-lysine (L-Lys) were much less effective at producing IJPs at sites where L-Phe evoked them consistently (mean IJP amplitude L-Phe 3.7 ± 0.3 mV, L-Lys 0.5 ± 0.2 mV, L-Leu 0.3 ± 0.2 mV, both N = 8 and P < 0.001). The CaSR antagonist NPS 2143 at 10 μM and 30 μM reversibly reduced L-Phe evoked IJPs by 48 % (mean IJP amplitude L-Phe 4.2 ± 0.4 mV, NPS 10 μM 2.2 ± 0.7 mV, N = 4, P = 0.005) and 59 % (L-Phe 3.9 ± 0.2 mV, 30 μM NPS 1.6 ± 0.6 mV, N = 6, P < 0.001) respectively. NPS 2143 did not affect electrically evoked IJPs. Cinacalcet (CaSR agonist;10 μM or 30 μM) evoked IJPs indistinguishable from those evoked by the L-amino acids (amplitudes 3 - 10 mV, latencies 150 - 300 ms). These were abolished by tetrodotoxin (TTX, 1 μM, N = 3) and significantly reduced by PPADS (mean IJP amplitude cinacalcet 5.5 ± 1.2 mV, PPADS 10 μM 2.2 ± 0.7 mV, 60 % reduction, N = 4, P = 0.004) and the combined 5-HT 3 and 5-HT 4 antagonist, tropisetron, (cinacalcet 4.9 ± 0.3 mV, tropisetron 10 μM 2.5 ± 0.7 mV, 49 % reduction, N = 4, P = 0.009). Together PPADS and tropisetron reduced the IJPs by 74% (cinacalcet 4.2 ± 0.2 mV, PPADS and tropisetron 1.1 ± 0.4 mV, N = 4, P < 0.0001). Conclusions Aromatic L-amino acids evoke local inhibitory reflexes by activation of the extracellular CaSR in the mucosa leading to release of both 5-HT and ATP to act on the terminals of intrinsic sensory neurons. CaSRs probably act as sensory transducers for luminal amino acids in guinea-pig jejunum, thereby coupling nutrient levels to intestinal motility to regulate the time available for nutrient absorp- tion. 181 Enteric Neurons Undergo Significant Electrophysiological and Morphological Changes During Postnatal Development Jaime Pei Pei Foong, Trung V. Nguyen, John B. Furness, Joel C. Bornstein, Heather M. Young Purpose: Neurons in the adult enteric nervous system (ENS) are classified electrophysiolo- gically into two main groups: AH (action potentials have long afterhyperpolarizing potentials, AHPs), or S (synaptic, have fast excitatory postsynaptic potentials, EPSPs). AH and S neurons have distinct morphologies; AH neurons are multi-axonal (Dogiel type II, DII), S neurons are uniaxonal and most only have lamellar dendrites. Early stages of ENS development such as migration, proliferation and differentiation have been well studied, but little is known about the maturation of enteric neurons. We examined the electrophysiology and morphology of myenteric neurons of early postnatal (P0, P10-11) and adult mice. Methods: Intracellular recordings were taken from myenteric plexus and attached muscle preparations dissected from P0, P10-11 and adult mouse duodenum. Synaptic potentials were evoked via focal stimulation of internodal strands (1 pulse or 10 pulses, 20 Hz). Microelectrodes contained biocytin for post hoc examination of neuron morphology and axon projections. Results: AH-like/DII and S/uniaxonal neurons were seen at P0. Both classes of neurons underwent