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Involvement of Parasympathetic Pelvic Efferent Pathway in Acceleration ofDistal Colonic Transit and Defecation in Response to Psychological Stress inRatsKazunori Suda, Hiromi Setoyama, Mitsuhisa Kawai, Satoshi Matsumoto, MasanobuNanno

Background: Stress is a major contributing factor to the pathophysiology of IBS and acceler-ates colonic transit and increases in defecation. The parasympathetic vagal and pelvic nervesregulate colonic motility and defecation in physiological state. It was shown that physicalstress altered colonic functions via vagal efferent in rats. However, it remains still unclearwhether the pelvic efferent pathway is involved in accelerating colonic and rectal functionsinduced by stress. The aim of this study was to investigate whether distal colonic transitand defecation are accelerated via pelvic efferent nerve in psychological stress-loaded rats.We also evaluated the effect of HPA-axis activation on stress -induced defecation. Method:The rats were subjected to water avoidance stress (WAS) or sham stress for 1-hr or 2-hr.The number of fecal pellets was counted for 1-hr in WAS- or sham stress-rats. Adrenalectomy(Adx), subdiaphragmatic vagotomy (Vag), pelvic nerve rectal branches (Rectal nerves) transec-tion (RNT), or each sham operation was performed at 1 week before WAS. Colonic transitwas assessed by the distribution of phenol red injected from catheter located in proximalcolon. The distal part of colonic and rectal transit was measured over a 2-hr WAS periodfrom the evacuation rate of a bead inserted into distal colon. c-Fos immunoreactivity (IR),used as a marker of neuronal activation, was monitored in longitudinal muscle/myentericplexus (LMMP) whole mount preparations of proximal and distal colon after WAS and shamstress. Results: WAS significantly accelerated colorectal transit and defecation with a markedelevation of plasma corticosterone (CORT) levels. Increased defecation induced by WASwas significantly reduced by Vag and RNT, even though CORT levels were elevated. WAS-induced acceleration of transit in distal colon and rectum was significantly reduced by RNT,compared with sham-operation. The number of c-Fos-IR cells were significantly increasedby WAS in LMMP of proximal and distal colon. In RNT- rats, an increase in c-Fos IR cellsafter WAS was decreased in distal colon, but not in proximal colon, whereas Vag did notaffect an increase of c-Fos IR cells in distal colon. In contrast, in Adx-rats WAS increasedthe number of defecation without an elevation of plasma CORT. Conclusion: It is concludedthat psychological stress accelerated distal colonic transit and rectal transit via a parasympath-etic efferent pelvic pathway. It seems that the stimulation of pelvic nerve in response tostress activates myenteric neurons in distal colon, followed by an increase of colonic motility.On the other hand, a vagal efferent pathway affects proximal colonic transit. However, anincrease in defecation induced by acute psychological stress might not involve the activationof the HPA axis.

Tu1980

Altered Eating Behavior by Subdiaphragmatic Truncal Vagus NerveStimulation in RatsHelene Johannessen, David Revesz, Yosuke Kodama, Chun-Mei Zhao, Gjermund Johnsen,Ronald Mårvik, Baard Kulseng, Georg Kuhn, Thorleif Thorlin, Elinor Ben-Menachem,Duan Chen

Background/aim: The brain-gut axis has been considered as an autonomic neurohumoralpathway regulating eating behavior, intestinal motility and energy homeostasis. Eating dis-orders, irritable bowel syndrome (IBS) and obesity could be resulted from dysautonomia.The aim of the present study was to establish an animal model of subdiaphragmatic truncalvagus nerve stimulation (stVNS) in order to control eating behavior. Methods: Male Sprague-Dawley adult rats and the standard maintenance rat diet were used. Electrodes were surgicallyattached to both anterior and posterior subdiaphragmatic vagal trunks through an abdominalmidline incision. The electrodes were connected by a lead wire to a pulse generator thatwas implanted subcutaneously on the back. The pulse generator was operated by an externalwireless controller that can be programmed individually and manually. Measurements ofeating behavior, food intake and metabolic parameters were carried out for 48 hours by thecomprehensive laboratory animal monitoring system (CLAMS). The high resolution CLAMSdata were generated by monitoring all parameters every five minutes, and the data from thelast 24 hours were used for the statistical analysis. Results: In response to short-term stVNSat 0.5 mA, 30 Hz, 500 μ seconds pulse width, cycle of ON 30 seconds/OFF 5 minutes for24 hours, number of meals and meal size (expressed as grams/meal) were reduced, whilemeal duration (minutes/meal) and intermeal interval (minutes) were increased during bothdaytime and nighttime. Food intake (grams/100 grams body weight) was reduced duringboth daytime and nighttime. In response to long-term stVNS over a time period of 5-10weeks, starting at 0.5 mA and increasing to 1.0 and 2.0 mA with 30 Hz, 500 μ secondspulse width, cycle of ON 30 seconds/OFF 5 minutes for 24 hours, satiety ratio (intermealinterval minutes/gram meal size) was increased, while food intake was reduced duringnighttime but not daytime in comparison with sham-operated and age-matched controls.Sleep percentage, water intake, urine production, and energy expenditure (kcal/hour/100grams body weight) were unchanged in response to either short- or long-term stVNS.Respiratory exchange ratio (VCO2/VO2) was unchanged in response to short-term stVNS,but reduced to long-term stVNS during both daytime and nighttime; the values remainingabove 1.0. Conclusions: The eating behavior could be altered in either short- or long-termby stVNS. This rat model of stVNS might be useful to explore how to control the eatingbehavior, and hopefully lead to the development of a new, reversible minimal invasivetherapy for eating disorders, IBS and obesity. Acknowledgment: The research leading tothese results has received funding from the European Union Seventh Framework Programme(FP7/2007-2013) under grant agreement n°266408.

S-892AGA Abstracts

Tu1981

The Selective 5-HT4 Receptor Agonist, Td-8954, is Efficacious in a Guinea PigModel of Post-Operative IleusScott Armstrong, Christina B. Campbell, Meagan Chan, Henry Liu, David T. Beattie

Post-operative ileus (POI) is a common disorder which occurs following abdominal surgeryand results in a temporary impairment of gastrointestinal (GI) motility. Currently, the onlyFDA-approved therapy is alvimopan (Entereg®), a peripheral μ-opioid receptor antagonist,indicated to accelerate the time to GI recovery following partial large or small bowel surgerywith primary anastomosis. The efficacy of alvimopan is presumably due to its preventingendogenous and exogenous opioid agonist-induced reductions in GI motility. An agentwhich has inherent GI prokinetic activity may have a greater effect on a multi-factorialdisorder like POI. 5-HT4 receptor agonists have been shown to be efficacious in various GIdisorders of slow transit (e.g., chronic idiopathic constipation and constipation-predominantirritable bowel syndrome; Emmanuel et al., 2002; Manini et al., 2010). <br /> To investigatethe potential of a 5-HT4 agonist in POI, an In Vivo model of surgically-induced delayed GItransit was developed in the guinea pig. The surgical procedure involved a laparotomyfollowed by a temporary (5 minute) removal of the GI tract (duodenum to rectum), andwas performed in the absence of exogenous opioid administration. To measure whole GItransit, a non-absorbable carmine red marker was dosed orally 30 minutes after completionof the surgery and the time for appearance of the dye in the fecal material was recordedhourly for up to 12 hours. The median whole GI transit time for a non-surgical sham animalwas 7 hours. The surgical procedure produced a delay in GI transit of approximately 3hours. Subcutaneous administration (30minutes post-surgery) of the selective 5-HT4 receptoragonists, prucalopride (0.3, 3, and 10 mg/kg) or TD-8954 (0.003, 0.03, 0.3, and 3 mg/kg),dose-dependently reversed the surgically-induced delay in transit. Pre-treatment with theselective 5-HT4 receptor antagonist, piboserod (3 mg/kg subcutaneously, 15 minutes post-surgery) blocked the effects of both prucalopride and TD-8954 in this POI model confirmingthat the prokinetic effects of both compounds were 5-HT4 receptor mediated. Alvimopan(0.003, 0.03, 0.3, and 3 mg/kg subcutaneously) failed to have any effect on the surgically-induced ileus suggesting a minimal role of endogenous opioids in this model. These datasupport the further evaluation of TD-8954 as a treatment for POI in humans. <br />References: <br /> Emmanuel AV, Roy AJ, Nicholls TJ, Kamm MA (2002) Aliment PharmacolTher 16:1347-1356 <br /> Manini ML, Camilleri M, Goldberg M, Sweetser S, McKinzie S,Burton D, Wong S, Kitt MM, Li Y-P, Zinmeister AR (2010) Neurogastroenterol Motil 22: 42-e8

Tu1982

Restorative Effect of Ghrelin Signaling Activation on Postprandial UpperGastrointestinal Tract Dysfunction in an Experimental Stress ModelShoki Ro, Koji Yakabi, Mitsuko Ochiai, Shino Ohno, Yumi Harada, Masamichi Noguchi,Tomohisa Hattori

Background/Aims: Excessive stress in modern society is related to the onset and developmentof functional dyspepsia, and related symptoms such as abdominal fullness in these variousforms of stress are caused by central neural peptides. Urocortin 1 (UCN), a member of thecorticotropin-releasing factor (CRF) family, acts as an endogenous ligand for CRF receptors.On the other hand, ghrelin, the endogenous ligand for the growth hormone secretagoguereceptor, stimulates food intake, gastric emptying, and gastric acid secretion. We alreadydemonstrated that intraventricular (ICV) injection of UCN reduces plasma ghrelin levels,decreasing activity in feeding behavior. However, there has been little evidence to suggestthat the enhancement of ghrelin signaling improves the decrease in postprandial gastrointesti-nal motor activity under conditions of stress. In this study, we first investigated the influenceof ICV of UCN on gastrointestinal motility, and examined the effects of exogenous ghrelinor rikkunshito (RKT), a ghrelin signal enhancer, on the decreased motor activity of UCN-treated rats. Methods: ICV injection of UCN was administered to 24-h fasted and fed 8-week-old Sprague-Dawley rats to clarify the typical motility of the gastrointestinal tract instressed rats. In addition, exogenous rat acyl ghrelin (3 nmol/kg i.v.), rikkunshito (1000 mg/kg, p.o.) alone or in combination with (D-Lys3)-GHRP-6 (4 nmol/kg i.v.) was administered toUCN-treated fed rats. The motor activities in the antrum and duodenum of fasted and fedrats were measured using a strain gauge force transducer under free-moving conditions.The solid gastric emptying rate following ICV injection of UCN was evaluated using a testmeal containing glass beads. Plasma acyl-ghrelin and desacyl-ghrelin levels were measuredby enzyme-linked immunosorbent assay. Results: Contractions in the antrum were markedlydecreased after UCN injection, but the amplitudes of duodenum contractions were increasedin both the fed and fasted rats. An obvious delay in gastric emptying was seen in UCN-treated rats. Although acyl- ghrelin and desacyl-ghrelin levels in plasma of fasted rats weredecreased after UCN injection, they remained unchanged in fed rats. Administration ofexogenous acyl -ghrelin inhibited the abnormality in postprandial motor activity and delayedgastric emptying rates. The ghrelin signal enhancer, RKT, improved abnormal postprandialcontractions and inhibited delayed gastric emptying in UCN-treated rats. This effect wasinhibited by the administration of a ghrelin receptor antagonist. Conclusion: Supplementa-tion with exogenous ghrelin or enhancement of endogenous ghrelin signaling may suppressfunctional dyspepsia-related symptoms such as abdominal fullness during conditions ofstress.

Figure 1 : The motor activities in the antrum and duodenum

Figure 2 : The solid gastric emptying rate

Tu1983

Activation of TRPA1, but Not of TRPV1, Improves Gut Motility in a MurineModel of Postoperative IleusKazuaki Tsuchiya, Nagisa Ohno, Kunitsugu Kubota, Atsushi Kaneko, MasahiroYamamoto, Toru Kono

Background and Aim: Agonists of transient receptor potential vanilloid (TRPV1) and transientreceptor potential ankyrin 1 (TRPA1) have been reported to induce contraction of intestinalsmooth muscle In Vitro, but it is unclear whether activation of TRPV1 or TRPA1 affectsgastrointestinal (GI) transit In Vivo. In this study, we investigated the effects of capsaicin(TRPV1 agonist), allyl isothiocyanate (AITC, TRPA1 agonist), cinnamaldehyde (CA, TRPA1agonist), daikenchuto (TU-100, TRPA1/TRPV1 agonist), and 6-shogaol (6SG, TRPA1/TRPV1agonist), the main constituent of TU-100, on murine postoperative ileus (POI) induced bysurgical manipulation of mouse intestines. TU-100, a traditional Japanese medicine that hasbeen prescribed to improve paralytic POI in Japan, is currently undergoing clinical trialson various intestinal diseases such as postoperative ileus, functional constipation, and Crohn'sdisease in the USA (http://clinicaltrials.gov/ct2/results?term=TU-100). Methods: Male C57Bl/6 mice underwent laparotomy and large bowel manipulation. Fluorescein isothiocyanate-dextran (FD70) was administered orally at 23.5 h postoperatively, and the mice were killedat 30 min post gavage. GI transit was estimated by calculating the geometric center (GC)value. Serotonin release from RIN-14B cell, an enterochromaffin-like cell line, was measuredby EIA. Results: Colonic manipulation decreased small intestinal transit to 70.2±2.7% ofcontrol. TRPA1 agonists (AITC, CA, TU-100, and 6SG) significantly improved the delayedGI transit induced by intestinal manipulation by 68.3±12.9%, 83.6±9.8%, 65.7±13.8%, and26.3±6.7%, respectively. On the other hand, the TRPV1 agonist capsaicin had no effect.Freshly isolated intestinal epithelial cells and RIN-14B cells expressed abundant TRPA1mRNA but no TRPV1 mRNA. POI was induced in TRPV1-deficient mice to a similar degreeas in wild-type mice, and TU-100 was effective in POI of TRPV1-deficient mice. AITC, CA,TU-100, and 6SG stimulated RIN-14B cell to release serotonin. Conclusions: These resultssuggest that TRPA1 is essential to induction and alleviation of POI while TRPV1 stimulationmay play little role. Stimulation of TRPA1 may be a novel therapeutic approach for thetreatment of POI.

S-893 AGA Abstracts

Tu1984

Hydroxy α-Sanshool, a Major Ingredient of TU-100 (Daikenchuto), AugmentsMigrating Motor Complex of Proximal Colon Triggered by Other ProkineticStimuliKunitsugu Kubota, Katsuya Ohbuchi, Nobuhiro Ohtake, Masahiro Yamamoto, Yuka Sudo,Yasuhito Uezono

Background and Aim: TU-100, a traditional Japanese medicine prescribed to amelioratepost-operative ileus (POI), is currently undergoing clinical trials on various intestinal diseasessuch as POI, functional constipation, and Crohn's disease in the USA (http://clinicaltrials.gov/ct2/results?term=TU-100). TU-100 has been reported to significantly accelerate ascendingcolon emptying in healthy volunteers. In this study, we evaluated the effect of TU-100 oncolonic transit using chronically colon-catheterized rats. Further, the effect of hydroxy α-sanshool (HAS), a major ingredient of TU-100 that is readily absorbed into the systemicblood, on migrating motor complex (MMC) of isolated segments of rat proximal colon wascompared with those of a TRPV1 agonist, capsaicin, and an acetylcholine analog, bethanechol.Further, because HAS has been reported to be an agonist to TRPV1 and TRPA1 as well asa blocker of certain two-pore domain potassium channels (KCNKs), we characterized thepotential of HAS as TRPV1/TRPA1/KCNKs ligands. Methods: In Vivo: TU-100 was adminis-tered orally, and 30 min after barium sulfate was injected into the colon by a cannulapermanently placed into the proximal colon in rats. X-ray radiography was used to quantifythe colonic transit. In Vitro: Isolated segments of proximal rat colons were placed in anorgan bath containing aerated Kreb's solution at 32-33°C. MMC was evaluated by monitoringthe intestinal tract pressure and videography (DDW 2011, Sa2046). Whole cell currentswere measured by two-electrode voltage clamp with the Xenopus oocyte expression systemin the cells transfected with KCNKs cRNA. The expression of KCNKs in rat colons wasevaluated by immunohistochemistry and RT-PCR. Results: TU-100 accelerated colonic transitsignificantly. In isolated rat colon segments, capsaicin evoked a sharp peak of high pressureonly once. Bethanechol (2 μM) evoked repetitive strong MMC. TU-100 and the high dose(6 μM) of HAS evoked strong MMC with a peculiar squeezing action. On the other hand,the low dose (3 μM) of HAS alone did not evoke MMC; however, it profoundly modulatedthe effects of both capsaicin and bethanechol qualitatively and quantitatively (Fig. 1). HASwas considerably weak as a TRPV1/TRPA1 agonist (i.e., binding to TRPV1 and TRPA1, andinflux of Ca2+ and Na+ in TRPV1- or TRPA1-expressing cells). Detailed analysis of two-electrode voltage clamp studies with Xenopus oocytes expressing KCNK3 suggested thatHAS may prolong and strengthen the action potential triggered by endogenous prokineticstimuli. The existence of KCNK3 in rat colon smooth muscle layer was confirmed byimmunostaining and RT-PCR analysis. Conclusions: HAS may augment MMC triggered byother endogenous and exogenous prokinetic stimuli. Inhibition of KCNK3 channel couldbe a mechanism for this augmentation.

Tu1985

Effects of DA-9701, a Novel Prokinetic Agent on Gastric Motor Function inGuinea PigHyun Chul Lim, Hyojin Park, Sang In Lee, Jaehoon Jahng, Yong Joo Lee

Introduction: DA-9701 has been newly formulated with Pharbitis Semen and CorydalisTuber for treatment of functional dyspepsia. Pharbitidis Semen has been used for analgesiceffects on the abdomen and Corydalis Tuber has been used for analgesics and anti-ulcerantsin the oriental medicine. The gastroprokinetic effect of DA-9701 has been shown to accelerategastric emptying and increase gastrointestinal transit in rats. The purpose of this study wasto clarify the pharmacological mechanisms of DA-9701 on gastrointestinal motility via InVitro study. Methods: To investigate the effect of DA-9701 on gastric contraction, theamplitudes of contraction were checked through tissue bath study with electrical fieldstimulation (1-5 Hz) on antral circular muscle. To evaluate the pharmacological mechanism,amplitudes of contraction were checked after pre-treatment with atropine, dopamine, andGR113808 (5-HT4 receptor antagonist). To investigate the gastrointestinal transit of DA-9701, the distance of charcoal migration from pylorus to distal intestine was measured afteradministration of DA-9701, and additional experiments with infusion of atropine, dopamine,and GR113808 were performed. Results: DA-9701 improved contraction amplitude of gastricmuscle in dose-dependent manner (Figure 1), and atropine, dopamine, and GR113808suppressed DA-9701 induced contraction. DA-9701 shortened the gastrointestinal transittime in dose-dependent manner(Figure 2) and atropine, dopamine, and GR113808 signific-antly suppressed DA-9701 induced shortening of gastrointestinal transit time. Conclusion:DA-9701 increased the gastric contraction through anti-dopaminergic, serotonergic andcholinergic effects.

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