proliferation in disease models including the dextran sodium sulfate (DSS) and azoxyme-thane(AOM)/DSS colitis induced carcinogenesis models. In colon myofibroblasts, epi deletionresults in a marked decrease in Hhip mRNA, suggesting that epimorphin modulates Hhsignaling. Aims: 1. Determine the effect of epi deletion on hedgehog signaling in colonmyofibroblasts 2. Evaluate Ihh expression in AOM/DSS-treated dysplastic WT and epi-/-

colons. Methods: Colon myofibroblasts were incubated with Sonic hedgehog (Shh, whichis functionally equivalent to Ihh; 0.9 μg/ml) for 24 h and Hh end target (Hhip, Gli1, Patch1)mRNA expression was evaluated by qRT-PCR. Ihh expression in dysplastic and adjacentnon-dysplastic descending colons of AOM/DSS treated mice was evaluated by immunohisto-chemistry . Results: Expression of Hh end-targets [Hhip (3.5 fold, p<0.05), Gli1 (6.13,p<.005), and Patch1 (2.8, p=0.07)] increased in WT myofibroblasts treated with Shh. Epi-/-

colon myofibroblasts treated with Shh had increased expression of Gli1 (2.3 fold, p<0.01);however, absolute levels of Gli1 remained 3.7 fold less than WT (p<0.005). Treatment withShh did not induce Hhip and Patch1 expression in epi-/- myofibroblasts. Ihh expression inWT non-dysplastic tissue (4 out of 4 samples) was limited to the epithelium; cytoplasmicexpression was strongest in the epithelial cuff. The intensity of Ihh staining in non-dysplasticepi-/- tissue (2 out of 4 samples) was stronger than in WT samples, although the expressionpattern was similar. Ihh expression in WT (n=4) dysplastic tissue was heterogeneous, charac-terized by less intensely stained as well as unstained epithelial cells. In contrast, Ihh immuno-reactivity in epi-/- dysplastic tissue was highly variable. Conclusions: Epi deletion bluntsthe normal colon myofibroblast response to Hh signals. The effects of epi deletion on Hhsignaling likely contribute to the enhanced epithelial proliferative phenotype in DSS andAOM/DSS treated epi-/- mice.

962

Cortistatin Inhibits Intestinal Secretion in the Guinea Pig Ileum In VitroSumei Liu, Wei Ren, Jackie D. Wood

Background and Aims: Cortistatin (CST) is a novel neuropeptide with high structural homo-logy with somatostatin. CST binds with high affinity to all somatostatin receptors and alsohas actions not shared by somatostatin. We previously found that CST is expressed byenteric neurons and acts as an inhibitory neurotransmitter in the submucosal plexus. Theaim of the present study was to evaluate the effect of CST on neurogenic intestinal secretion.Methods: Submucosal/mucosal flat-sheet preparations from guinea pig ileum were mountedin Ussing flux chambers for measurement of short-circuit current (Isc) as an indicator ofmucosal secretion. Transmural electrical field stimulation (EFS) evoked characteristic biphasicincreases in Isc, with an early rapidly rising phase followed by a sustained phase. Results:Application of CST-14 (rat), CST-17 (human), CST-29 (rat), or CST-29 (human) to thesubmucosal side of the preparation decreased baseline Isc in a concentration-dependentmanner with an IC50 of 90.5 nM, 52.1 nM, 102.9 nM, or 71.6 nM, respectively. Thesomatostatin-2 receptor (SSTR-2) antagonist CYN 154806 (1 μM) significantly suppressedCST-14r (100 nM), CST-17h (100 nM), CST-29r (100 nM), or CST-29h (100 nM)-evokedΔIsc by 36.0 ± 8.2% (n=5; P < 0.05), 47.2 ± 11.2% (n=5; P < 0.01), 51.7.0 ± 8.4% (n=5;P < 0.01), or 46.9 ± 7.5% (n=5; P < 0.01), respectively. The neuronal blocker tetrodotoxin(300 nM) significantly reduced CST-14r (100 nM), CST-17h (100 nM), CST-29r (100 nM),or CST-29h (100 nM)-evoked ΔIsc by 36.5 ± 2.7% (n=5; P < 0.01), 48.9 ± 11.2% (n=5;P < 0.01), 49.5 ± 3.6% (n=5; P < 0.01), or 53.7 ± 4.2% (n=5; P < 0.01), respectively. Allof the CSTs (1 nM - 1 μM) also produced a concentration-dependent reduction of the EFS-evoked biphasic secretory responses. The effect of CSTs on the EFS-evoked responses wasantagonized by CYN 154806 (1 μM). Immunohistochemical staining with a CST-17 poly-clonal antibody revealed expression of CST-17 immunoreactivity (IR) in guinea pig submuco-sal neurons. CST-17-IR was localized in cholinergic secretomotor neurons but was absentfrom non-cholinergic secretomotor neurons and calbindin-IR neurons. CST-17 was co-expressed with somatostatin in submucosal plexus neurons. Conclusions: The results suggestthat CST acts at SST-2 receptors in the submucosal plexus to suppress secretomotor neuronalexcitability and neurogenic intestinal secretion. Acknowledgement: Supported by NIH R01DK37238 (JDW) and R01 DK57075 (JDW).

963

Influence by Glucagon-Like Peptide-2 (GLP-2) on Spontaneous MechanicalActivity and Electrically-Evoked Cholinergic Contractions in Mouse ProximalColonAlessandra Rotondo, Antonella Amato, Rosa Serio, Flavia Mule'

Background and aims: Glucagon-like peptide-2 (GLP-2) is a hormone produced by enteroen-docrine L cells of the small and large intestine and secreted in a nutrient-dependent manner.GLP-2 affects multiple facets of intestinal physiology, including growth, absorption andmotility. Recent evidence have shown that GLP-2 can act peripherically to modulate mousestomach capacity. However, the mechanism of action of this peptide in regulating theintestinal motility remains to be characterized. The purpose of the present study was toexamine, In Vitro, the influence of GLP-2 on spontaneous mechanical activity and onelectrically-evoked cholinergic contractions in mouse proximal colon.Methods: Experimentswere performed In Vitro recording changes in intraluminal pressure from mouse isolatedcolonic segments. Electrical field stimulation (EFS) (trains of stimuli: duration 5 s, supramaxi-mal voltage, 8 hz and 0.5 ms pulse duration) was applied at intervals of 70 s to induceneurally-mediated contractions. Results: In our experimental conditions, mouse proximalcolon exhibited spontaneous phasic contractions of about 10 cm H2O in amplitude. Applica-tion of GLP-2 (0.1 nM- 300 nM) produced a concentration-dependent reduction of thespontaneous contractions, which was abolished by the desensitization of GLP-2 receptor(GLP-2 10 nM for 45 min) or by TTX (1 μM). The inhibitory effect of GLP-2 was notaffected by apamin (0.1 μM), a blocker of small conductance Ca2+-dependent K+ channels,or by Nω-nitro-L-arginine methyl ester (L-NAME) (300 μM), an inhibitor of nitric oxidesynthase, but it was antagonized by atropine (1 μM), which per se reduced significantly theamplitude of spontaneous contractions. EFS evoked contractions with an amplitude of about12 cm H2O, which always followed a small relaxation (about 0.5 cm H2O). Contractileresponses to EFS were abolished by atropine (1 μM) or tetrodotoxin (TTX) (1 μM), suggestingtheir cholinergic origin. GLP-2 (0.1 nM- 300 nM) produced a concentration-dependent

S-141 AGA Abstracts

reduction of the cholinergic evoked contractions and this effect was blocked by GLP-2receptor desensitization. The peptide inhibitory effect on the amplitude of the cholinergicevoked contractions was unaffected by pre-treatment with L-NAME. GLP-2 (30 nM) failedto affect the concentration-response curves for contractile effects evoked by exogenouscarbachol (10 nM - 30 μM). Conclusions: The present results demonstrate for the first timethat GLP-2 is able to modulate negatively the spontaneous mechanical activity and theexcitatory cholinergic neurotransmission through prejunctional GLP-2 receptors. Supportedby PRIN 2007, ITALY

964

Intestinal CCK Cells Possess Dendritic-Like Pseudopods Suggesting aParacrine ActionRashmi Chandra, Leigh A. Samsa, Steven Vigna, Rodger A. Liddle

BACKGROUND: Cholecystokinin (CCK) is secreted by neuroendocrine (I) cells that comprise0.1 to 0.5% of the mucosal cells in the villi and crypts of the upper small intestine. Ingestedfats and proteins stimulate release of CCK. Using CCK promoter- driven GFP expression intransgenic mice, we analyzed the morphology of CCK cells present in the mouse duodenum.METHODS: CCK-GFP transgenic mice were fasted overnight and perfusion fixed withparaformaldehyde. The duodenum was harvested and 5-10 micron cryosections were col-lected. Sections were stained using a combination of antibodies against GFP, CCK (19-36),chromogranin A, trefoil factor 3 and sucrase-isomaltase. Images were collected using confocalmicroscopy and analyzed by three-dimensional reconstruction. RESULTS: GFP and CCKcolocalized in the neuroendocrine cells of transgenic mice with little aberrant GFP expression.CCK-containing cells were either flask- or spindle-shaped cells and were more abundant inthe crypts than the villi. We observed dendritic-like cellular processes (similar to pseudopodsdemonstrated for gut somatostatin-containing D cells) in some CCK cells. Most of thepseudopods were short; the longest process visualized (about 15 microns) extending acrossthree cells. In villi, the processes were sometimes curved and wove between the neighboringcells. In crypts, pseudopods usually extended to the adjacent cell. Dual processes were seenin a small percentage of the cells; often a single extension bifurcated into two. Threedimensional reconstructions suggested that these processes were not unidirectional and itis unlikely that they are used in migration of CCK cells from the crypt up the longitudinalaxis of the villus. Abundant CCK immunostaining was present in the pseudopods, suggestingthat they could release CCK onto the target cell. In order to identify the type of cells beingtargeted by the processes, sections were co-stained with antibodies to chromogranin A(neuroendocrine cell marker), trefoil factor 3 (goblet cell marker) and sucrase-isomaltase(enterocyte cell marker). Pseudopods were never targeted to chromogranin A positive cellslocated near CCK cells. In the villus, CCK cells were occasionally present adjacent to gobletcells, but often the plane of the two types of cells was different. Staining of enterocyteswith sucrase-isomaltase antibody demonstrated that most CCK cell processes extended toenterocytes. CONCLUSIONS: CCK cells have cellular processes similar to those observedfor somatostatin cells. Since CCK is present in these processes, it is likely that they are usedin paracrine secretion. We are currently elucidating the function of these processes.

965

The Brain-Gut-Microbiome Axis: Gender and Region-Specific Effects of theMicrobiome on Central Neurotransmitter and Neurotrophin LevelsPaul Scully, Gerard Clarke, Sue Grenham, Patrick Fitzgerald, Cliona M. O'Mahony, DeclanP. McKernan, Monica Tramullas, Fergus Shanahan, Timothy G. Dinan, John F. Cryan

Background: Bacterial colonisation of the intestine plays a major role in the post-nataldevelopment and maturation of the immune and endocrine systems. These processes arekey factors underpinning central nervous system (CNS) signalling. Dysregulation of thebrain-gut-enteric microbiota axis is implicated in functional disorders including irritablebowel syndrome (IBS). However, few studies have directly investigated the impact of microbi-ota on CNS functionality. Aim: To compare germ-free animals with conventional animalsof both genders for neurotransmitter and brain-derived neurotrophic factor (BDNF) concen-trations in distinct brain regions. BDNF has been implicated in brain development andstress-sensitivity. Methods: Germ-free Swiss Webster (GFSW) and conventionally colonisedSwiss Webster mice (CCSW) were compared. Mice were sacrificed at 6 weeks of age. Highperformance liquid chromatography coupled to electrochemical detection (HPLC-ECD) wasused to assess the concentrations of neurotransmitters (Serotonin (5-HT), noradrenaline,dopamine) and theirmetabolites (5-HIAA) in the frontal cortex, hippocampus, hypothalamus,and brainstem. Quantitative real-time PCR was used to evaluate BDNF expression patternsResults: There was a significant elevation in the concentration of both serotonin (5-HT)(420 ± 27.7 vs 555 ± 24ng/g tissue, p < 0.05) and its main metabolite 5-HIAA (365 ± 13.7vs 544 ± 9 ng/g tissue, p< 0.001) in the hippocampus of the male GFSW mice vs theirmale CCSW equivalents. There was a concurrent 1.4 fold decrease in BDNF expression inthe GFSW male mice vs the male CCSW animals (p<0.05). No alterations in indices of 5-HT function were found in the amygdala of the male groupings and the perturbationsdescribed in the hippocampus of the males were not apparent when the female GFSW vsCCSW were compared. Conclusion: The results demonstrate that CNS neurotransmissioncan be profoundly disrupted by the absence of normal gut microbiota. Furthermore, theeffects were both gender and region-specific, an important observation which should informfuture studies. Together, our findings identify specific higher brain centres that are underthe influence of the gastrointestinal microenvironment.

AG

AA

bst

ract

s