372 Silencing of Gene Expression for Corticotropin-Releasing Factor (CRF) in the Colon Attenuates Stress-Induced Acceleration of Colonic Transit in Rats Sumei Liu, Mei-Hu Qu, Wei Ren, Aditi Bhargava, Jen Chang, Julia J. Hoy, Guo-Du Wang, Fie Zou, Xiyu Wang, Xiaohong Sun, Yun Xia, Jackie D. Wood We tested a hypothesis that silencing of CRF expression in the enteric nervous system (ENS) attenuates stress-evoked alterations of colonic motor function. Laser capture microdissection (LCM), real time RT-PCR, and immunofluorescence were used to study expression of CRF in the ENS of the rat, mouse and guinea pig colon. Injection of double-stranded RNA (dsRNA) for CRF (dsCRF, 20 μg/rat) into the rat colon was used to achieve RNA interference (RNAi)-mediated inhibition of CRF gene expression. DsRNA for β-globulin was a control (dsControl). Four days after dsRNA administration, rats were exposed to restraint stress for 2 h. Fecal output was monitored at 15 min intervals for 2 h. LCM and RT-PCR revealed CRF mRNA expression in myenteric neurons in rat proximal and distal colon. CRF-like immunoreactivity (IR) was expressed in nerve cell bodies and fibers in the myenteric and submucosal plexuses of the rat, mouse, and guinea pig colon. CRF-IR neurons were 5.4±0.3% of the myenteric population and 21.9±4.5% of the submucosal population in rat colon. In rat colonic myenteric plexus, 45.0±10.5% of CRF-IR neurons expressed choline acetyltrans- ferase-IR and 97.1±1.4% expressed nitric oxide synthase-IR. In the rat colonic submucosal plexus, all CRF-IR neurons expressed vasoactive intestinal peptide-IR. Stress induced wide- spread immunostaining for c-fos in colonic myenteric ganglia. Double labeling showed that most of the c-fos-positive neurons in stressed animals expressed either CRF-IR or CRF1receptor-IR. Intramural injection of dsRNA for CRF (dsCRF) “knocked-down” basal CRF expression in rat colon, while CRF expression in dsControl animals was unaffected. CRF mRNA was decreased by 60% after four days. CRF peptide-IR was undetectable or very low in myenteric and submucosal neurons of the rat colon. DsControl and dsCRF- treated rats had similar daily food intake, body weight gain, and cumulative fecal output during the 4 days post injection. In dsControl-treated rats, restraint stress induced a significant increase in fecal output when compared to non-stressed controls (stress: 9.7 ± 1.3 vs. non- stress: 2.7 ± 0.8; P < 0.01). Restraint stress did not induce a significant increase in fecal output in dsCRF-treated rats as compared to non-stressed rats (stress: 3.2 ± 1.4 vs. non- stress: 2.7 ± 1.4; P > 0.05). Stress changes CRF expression in the colonic ENS and is a factor in stress-evoked colon dysmotility. (Support: NIH R01 DK37238 and R01 DK57075 (JD Wood), PhRMA Research Starter Award (S Liu), UNC Center for Functional GI & Motility Disorders seed grant (S Liu), and Hellman Grant (A Bhargava). 373 Regulation of Small Intestinal EC Cell Serotonin Release - the Role of Taste and Mechanical Forces Mark Kidd, Bjorn I. Gustafsson, Ignat Drozdov, Oyvind Hauso, Irvin M. Modlin Introduction: The enterochromaffin (EC) cells of the diffuse neuroendocrine cell system occur as single cells throughout the gastrointestinal mucosa. The apical processes of these cells have been postulated to act as luminal- and mechano-sensors that on stimulation activates EC cell secretion of bioactive agents including serotonin. There is evidence that altered secretion of serotonin from EC cells is a key factor in hypersecretion and the dysmotility-associated with IBS and IBD. In the absence of pure isolated normal EC cells or immortalized EC cell lines, it has not previously been possible to investigate the mechanistic basis of EC cell function. Aim: Using pure (98-99%) human EC cells that we have isolated with FACS sorting, and the immortal EC cell line, KRJ-I, we investigated whether candidate luminal factors (tastants/olfactants) and mechanical strain regulated serotonin release. Methods and Results: Using RT-PCR, we identified that normal and neoplastic EC cells express transcripts for tastant, olfactory and mechano-responsive G-protein coupled receptors: T1R3 (sweet), T2R1 (bitter), OR1G1 (class II olfactory receptor), the stretch (ADORA) receptors (1, 2a/b, 3) as well as transporters for glutamine (SNAT1/2), glucose (GLUT1/3) and bile salts (ABST). In EC cell culture, tastants including caffeine, luminal amino acids like tyramine and octopamine, and the olfactants, thymol and eugenol, stimulated 5HT secretion (EC50= 0.05-0.8μM; 2-8-fold release, 60 mins). Glutamine and the bile salt, sodium deoxycholate, also stimulated 5-HT release (EC50=0.002-0.2μM; 2-fold release) but glucose inhibited this (IC50=0.003μM). Serotonin release was associated with ERK phosphorylation (1.5-fold, p<0.02) and calcium influx. Application of cyclic strain in a computer controlled flexible- bottom well vacuum system (10cpm, 10% radial strain) resulted in a time-dependent (2-6 hrs) increase in serotonin release (2.5-fold, p<0.03) and tryptophan hydroxylase transcription (2-fold, p<0.04), effects which could be reversed by the ADORA2a antagonist, alloxazine A-49 AGA Abstracts (1μM). Conclusions: We demonstrate that EC cells are gut luminal- and mechanical-sensors that respond to luminal factors including tastants, olfactants and bile salts as well as mechan- ical forces and regulate serotonin secretion through mechanisms that include Ca2+ influx, ERK phosphorylation and tryptophan hydroxylase transcription. Elucidation of factors that regulate EC cell secretion is necessary to understand the role of the EC cell in disease and to facilitate the development of agents that can selectively target EC cell function. 374 Effect of a Novel Trans-Galactooligosaccharide Prebiotic On Faecal Microbiota and Symptoms in Patients with Irritable Bowel Syndrome David B. Silk, Amanda J. Davis, Jelena Vulevic, Glenn Gibson, George Tzortzis INTRODUCTION: Gut flora-mucosal interactions may be involved in the pathogenesis of irritable bowel syndrome (IBS), and the probiotic bifidobacterium infantis 35624 has been shown to alleviate symptoms in IBS patients. Prebiotics are non digestible food supplements that are fermented by host bacteria thereby altering the microbiota of the host often by stimulating healthy bacteria. We have synthesized a novel trans-galactooligosaccharide prebi- otic and report here the first ever study undertaken to investigate the efficacy of prebiotic therapy in IBS. AIMS & METHODS: 44 patients with Rome II positive IBS completed a 12 week single centre parallel cross over controlled clinical trial. (2 weeks baseline, 4 weeks placebo (PL), 2 weeks washout, 4 weeks prebiotic (PB). Patients were randomized to receive either 3.5 g/d PL, PB, 7 g/d PL PB or PL PL. The primary efficacy variable was the effect of PB on bifidobacterium enhancement of stools. The secondary efficacy variables included IBS symptoms (abdominal pain/discomfort, bloating/distension, bowel movement difficulty) monitored weekly and scored according to a 6 point Likert scale. Stool frequency and form (Bristol stool scale) SGA, anxiety and depression and QOL scores were also monitored. RESULTS: PB but not PL significantly enhanced the bifidobacterium proportion of faecal microflora (3.5 g/d p< 0.005; 7g/d p<0.001). PL PL was without effect on any of the clinical parameters monitored. PB 3.5 g/d significantly improved stool consistency (p<0.05), flatulence (p<0.05), composite score of symptoms (p<0.005) and SGA (p<0.001). PB 7g/d significantly improved SGA (p<0.05) and anxiety scores (p<0.05). CONCLUSON: The trans- galactooligosaccharide prebiotic is significantly bifido bacterium enhancing in IBS patients and is effective in alleviating symptoms. These findings suggest that the prebiotic has potential as a therapeutic agent in IBS and should prompt the undertaking of large randomized controlled trials of suitably formulated prebiotics in IBS patients. 375 Role of Ghrelin in the Relation Between Hyperphagia and Accelerated Gastric Emptying in Mice with Streptozotocin-Induced Diabetes Pieter-Jan Verhulst, Betty De Smet, Inge Y. Saels, Theo Thijs, Dieder Moechars, Luc Ver Donck, Theo L. Peeters, Inge Depoortere Background and aim: Ghrelin is an important orexigenic peptide with gastroprokinetic properties. Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying and increased plasma ghrelin. This study aimed to investigate the causal relationships between these three events and the mechanisms involved, by comparing changes in ghrelin receptor knockout (GHS-R -/- ) mice and wild-type (GHS-R +/+ ) mice. Methods: STZ-diabetes was induced by bolus injection of STZ (160mg/kg). Food intake, body weight and blood glucose were monitored daily. Gastric emptying was measured with the 13 C- octanoic acid breath test at day 6, 10, 16 and 22 after the induction of diabetes. The mRNA expression of NPY, AgRP and POMC in the hypothalamus was quantified by real-time PCR at day 27. Contractility elicited by electrical field stimulation (EFS) was measured isometrically in fundic smooth muscle strips before and at day 27 after the induction of STZ-diabetes. Results: Plasma ghrelin levels were 5 fold higher in diabetic (GHS-R +/+ :1888±393 pg/ml; GHS-R -/- :2237±752 pg/ml) than in non-diabetic mice (GHS-R +/+ :528±149 pg/ml; GHS-R -/- : 435±73 pg/ml). The start of the hyperphagia was delayed from day 5 till day 9 in the GHS-R -/- mice and was more pronounced (P < 0.01) in GHS-R +/+ than in GHS-R -/- mice between day 12 and day 21. In the hypothalamus of GHS-R +/+ mice NPY mRNA increased 9-fold, AgRP mRNA 35-fold, while POMC mRNA decreased 7-fold. In diabetic GHS-R -/- mice the increase of NPY and AgRP was less pronounced, while POMC mRNA was unaffected. No difference in gastric emptying was observed between non-diabetic GHS-R +/+ (T 1/2 : 109±5 min) and GHS-R -/- mice (T 1/2 : 110±7 min). After the induction of diabetes gastric emptying was equally accelerated in both genotypes from day 16 on (T 1/2 : GHS-R +/+ : 56±10 min; GHS-R -/- : 55±9 min). Alterations in food intake and gastric emptying were correlated but the hyperphagia preceded the changes in gastric emptying. In fundic smooth muscle strips of diabetic GHS-R +/+ and GHS-R -/- mice, neuronal relaxations were reduced while EFS- induced off-contractions were equally enhanced. In strips from both genotypes, the concen- tration-response curve to ACh and substance P was shifted to the left. Conclusion: Diabetic hyperphagia is predominantly regulated by central mechanisms in which the ghrelin signaling pathway plays a predominant role by affecting the expression of NPY and AgRP in the hypothalamus. The accelerated gastric emptying, which occurs independent from ghrelin, is not the cause of the increased food intake and involves local changes in neuronal and smooth muscle contractility which may impair accommodation. 376 AZD3355, a Novel GABA B Receptor Agonist, Inhibits Transient Lower Esophageal Sphincter Relaxations in the Dog Anders Lehmann, Lena Brändén, Anita Carlsson, Thomas Elebring, Jörgen Jensen, Jan P. Mattsson, Karolina A. Nilsson, Anna Uvebrant, Sverker von Unge Transient lower esophageal sphincter relaxation (TLESR) is the major cause of gastroeso- phageal reflux and plays a crucial role in the etiology of gastroesophageal reflux disease (GERD). Baclofen, a GABA B receptor agonist, potently inhibits TLESR and reflux, and ameliorates GERD symptoms. Since baclofen produces CNS side-effects, its utility in the AGA Abstracts