esophageal epithelium. Design Gene expression microarray was used to survey gene expres-sion in the esophagus at three critical stages, specification, metaplasia and maturation. Theesophagi were isolated from wild-type, Nrf2-/-, Keap1-/-, or Nrf2-/-;Keap1-/- embryos oryoung adults. Array data were statistically analyzed for differentially expressed genes andpathways. Histochemical and immunohistochemical staining were used to verify potentialinvolvement of the Wnt pathway, PPARβ/δ and the PI3K/Akt pathway in the developmentof esophageal epithelium. Results Dynamic gene expression pattern accompanied the mor-phological changes of the developing esophagus at the critical stages. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated inthe maturation phase. The Wnt pathway was active early and became inactive later in themetaplasia stage. In addition, global transcript profiling of the esophagus isolated fromKeap1-/- mice showed increased expression of Nrf2 downstream targets and genes involvedin keratinization. Microarray data also suggested that esophageal hyperkeratosis in theKeap1-/- mice was due to activation of PPARβ/δ and the PI3K/Akt pathway. ConclusionsMorphological changes of the esophageal epithelium are associated with dynamic changesin gene expression. The activity of Nrf2/Keap1 pathway is required for the maturation ofthe esophagus.

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Mucin Side Chain Sugar Residues Expression in Helicobacter pylori, NSAID,and Idiopathic Peptic UlcersYaron Niv, Doron Boltin, Marisa Halpern, Miriam Cohen, Zohar Levi, Alex Vilkin, SaraMorgenstern, Samuel B. Ho

Introduction: Idiopathic peptic ulcer is rising in the Western world, and may have a differentetiology and pathogenesis than H, pylori or NSAID. The main protective factor of the gastricmucosa is the mucus unstirred layer. Change inmucin expressionmay lead to ulcer formation.Aim: To determine the expression pattern of mucin's type 2 backbone structure and sialicacid residues in H. pylori, NSAID, and idiopathic-gastric ulcers. Methods: We randomlyselected 92 patients with H. pylori (Group 1, n=30), NSAID (Group 2, n=18), combined H.pylori and NSAID associated gastric ulcers (Group 3, n=24), and patients with idiopathicgastric ulcers (Group 4, n=20). ECA (specific for Neu5Acα2-6gal/GalNAc) and SNA (specificfor Galβ1-4GlcNAc, type 2 backbone structure exposed after removal of sialic acid), lectinstaining performed on sections of the mucosa from the ulcer margins. Inflammation scorewas assessed according to the Sidney system. Results: Bleeding and mortality rates weresignificantly higher in patients with idiopathic ulcer. The expression of sialic acid residuesstained by SNA lectin was higher in these patients, Group 4, than in Group 1, reachingsignificance in the foveola (P < 0.0001, and P = 0.004, respectively). EMA staining intensitywas significantly higher in the foveola than in the glands, 13.49±3.08 versus 5.71±4.72 (P< 0.0001), with no difference between Group 1 and Group 4. Conclusion: This observationmight be important, since increased number of sialic acid residues may decrease the protectionefficiency against acid, pepsin, toxic material or bacterial adhesion.

Su1741

Klotho, an Aging Suppressor, and Fibroblast Growth Factor 23 is Involved inGastric Ulcer HealingTetsuya Tanigawa, Toshio Watanabe, Yuji Nadatani, Koji Otani, Hirohisa Machida,Hirotoshi Okazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, YasuhiroFujiwara, Tetsuo Arakawa

Background and Aim: The klotho gene was originally identified as an aging suppressorgene, which extends lifespan when overexpressed and accelerates the development of aging-like phenotypes when disrupted in mice. Klotho protein is involved in modulation of mineralmetabolism and aging, and recent researches revealed that Klotho functions as a humoralfactor with pleiotropic activities. Klotho protein forms a complex with fibroblast growthfactor (FGF) receptors and functions as an obligate co-receptor for FGF23. In this study,we investigated whether Klotho and FGF23 is involved in the mechanism of gastric ulcerhealing. Materials and Methods: Experimental gastric ulcers were induced in C57BL/6Jmice by focal serosal application of 60% acetic acid for 30 sec. Mouse recombinant Klotho(10 and 20 μg/kg body weight/day) or vehicle (PBS) was administered intraperitoneally tothe mice with ulcers from day 0 (3 days after inducing ulceration) to day 7. On day 4 and7, the stomachs were removed and the ulcer size was measured; the ulcer size was expressedin the form of an ulcer index (the product of maximum length and minimum length of anulcer). Expression of mRNA for FGF23 and Klotho in normal gastric tissue and ulcer tissuewas determined by real-time quantitative RT-PCR. Results: Expression of mRNA for FGF23was almost undetectable in normal gastric mucosa and it was markedly induced in ulcertissue. Expression of mRNA for Klotho in gastric epithelial cells at the ulcer margin wasreduced by 90% compared with normal gastric tissue. Expression of mRNA for Klotho washardly detectable in ulcer bed. Administration of recombinant Klotho accelerated gastriculcer healing [ulcer index: 8.25 ± 2.46 mm2 in vehicle-treated group vs 3.81 ± 0.66 mm2

in Klotho (10 μg/kg body weight/day)-treated group and 4.38 ± 1.09 mm2 in Klotho (20μg/kg body weight/day)-treated group]. Conclusions: Our findings suggest that Klothopromotes gastric ulcer healing.

Su1742

NSAID-Sparing Effect of Glucosamine Hydrochloride Through PreventingCOX-2 N-Glycosylation Secures Lesser GI Toxicity Under the Warranty ofNSAID ActionHua Hong, Yoon Jae Kim, Eun-Hee Kim, Young-Min Han, Ki-Baik Hahm

Though non-steroidal antiinflammatory drug (NSAID) has been prescribed for pain alleviationas well as potent anti-inflammation popularly, the notorious GI toxicity limits its generalusage, after which “Coxib” has been invented to guarantee GI safety. Though glucosamine(GS), an agent composed of an amino monosaccharide, has been widely used for thealternative regimen of arthritis, their clinical implication in this matter has been under

S-493 AGA Abstracts

debate. Since in addition to enzymatic inhibition, change in COX-2 mRNA stability throughinhibiting COX-2 N-glycosylation can also lead to COX-2 inhibition, we hypothesized thatthe addition of GS-HCl can reduce the dose of NSAID required for authentic action of NSAIDbecause previously we reported that GS-HCl led to inhibition of COX-2 N-glycosylation andCOX-2 protein turnover (Jang et al, JBC, 2007). Stimulation of IEC-6 with indomethacinresulted in significantly increased expressions of COX-2, iNOS, ICAM-1, VCAM-1, IL-8,and IL-1 as well as increased apoptosis, whereas the co-treatment of 5mM GS-HCl andindomethacin significantly decreased expressions of these inflammatory mediators as wellas attenuated apoptosis relevant to significant inhibition of COX-2 N-glycosylation. Indo-methacin-induced gastric ulcers were significantly attenuated in animal group co-treatedwith GS-HCl and lowered dose of indomethacin under the improved efficacy of collagen-induced arthritis than high doses of indomethacin alone group. Our study provided thefirst insight that GS-HCl contributed to COX-2 weakening action through N-glycosylationinhibition, leading to novel strategy that combination of GS-HCl and lowered dosing ofNSAID secured lesser GI toxicity under the similar COX inhibiting action.

Su1743

Estrogen Receptors Regulate Intestinal Epithelial Cell Proliferation and theUnderlying MechanismsHui Dong, Chang-Whan Kim, Jeong Wook Kim, Jimmy Y. Chow, Biguang Tuo

Background and Aims: We recently reported that estrogen and estrogen receptors (ERs)play an important role in human intestinal mucosal protection and epithelial cell homeostasis;however, the underlyingmechanisms are largely unknown.We therefore sought to investigatethe cellular and molecular mechanisms by which estrogen and ERs protect human intestineand regulate epithelial cell proliferation. Methods: SCBN cell, an intestinal epithelial cryptcell line, and CHO cells stably expressing Na+/Ca2+ exchanger (NCX1-CHO) were used.17b-estradiol (E2, 1-10 nM) was used as a ERs agonist. Gramicidin (1 ug/ml) or ouabain(1 uM) was used to raise intracellular Na+ concentrations and stimulate the Ca2+ entrymode of NCX1. RT-PCR and Western blot analysis were used to detect expression of ERs,NCX1 and CFTR. Cytosolic free Ca2+ concentration ([Ca2+]cyt) in single cells was measuredwith a digital cell imaging system and XTT assay was applied to study cell proliferation.Results: The expression of ER subtypes (ERa and ERb) was detected by RT-PCR and Westernblot analysis in SCBN cells. E2 at 1-10 nM significantly suppressed cell proliferation for 24and 48 h (p<0.001, n=10); however, progesterone at 1-10 nM did not significantly affectcell proliferation. E2-induced suppression of cell proliferation was reversed by CFTRinh-172 (10 uM). The expression of CFTR and NCX1 were confirmed in SCBN cells. To furtherelucidate the mechanisms underlying E2-induced suppression of cell proliferation, NCX1-CHO cells were used. After intracellular Na+ concentrations were raised by gramicidin (1ug/ml) or ouabain (1 uM) to stimulate the Ca2+ entry mode of NCX1, [Ca2+]cyt wasincreased in NCX1-CHO cells but not in control CHO cells (n=50 cells for each group,p<0.001). Similarly, either gramicidin or ouabain increased proliferation of NCX1-CHOcells (n=5, p<0.05), but not control CHO cells (n=5, p>0.05). Interestingly, gramicidin-induced proliferation of NCX1-CHO cells was significantly suppressed by E2 (n=5, p<0.05).Conclusions: ERs are functionally expressed in intestinal epithelial cells to regulate cellproliferation. Our findings not only reveal the novel roles for CFTR and NCX1 in regulatingintestinal epithelial cell proliferation but also suggest these molecules may be involved inERs-suppressed epithelial cell proliferation that is associated with intestinal inflammationand carcinogenesis.

Su1744

Some Water-Soluble Vitamins are Likely to Be Deficient at the Time of PepticUlcer Development: Time-Course Changes in Blood Concentrations of Water-Soluble VitaminsWataru Sato, Kazumasa Miyake, Hiroyuki Nagoya, Yasuhiro Kodaka, Tomotaka Shindo,Nobue Ueki, Masafumi Kusunoki, Tetsuro Kawagoe, Seiji Futagami, Katya Gudis, ChoitsuSakamoto

[Background and aim] Unlike fat-soluble vitamins (V), water-soluble V have a metabolicfeature. Water-soluble V are relatively easy to become depleted because they are unstable,excreted readily in urine, and difficult to be stored in the body. Lack of water-soluble Vwhich have an antioxidant action can be one of the causative factors of the onset of pepticulcer (PU), though the relationship between PU development and such V is not clear yet.Of the water-soluble V B6 and V B12 and folic acid are involved in the metabolism ofhomocysteine which can cause arteriosclerosis and phlebothrombosis. In other words, homo-cysteine concentrations in blood may be an indicator of such vitamin deficiency and arterio-sclerosis. Therefore, in this study, lack of water-soluble V in PU development was evaluatedby measuring blood concentrations of water-soluble V and homocysteine in patients withhemorrhagic PU. [Methods] This prospective study included the patients who needed hospit-alization for the treatment of PU. Infusion using a vitamin preparation was not performedduring hospitalization. Fasting blood concentrations of water-soluble V (V B1,V B2, V B6,V B12, V C, and folic acid) and homocysteine were measured at 3 time points (at the timeof admission and hospital discharge and 3 months after discharge). Of the 20 consecutivepatients who met the inclusion criteria, 10 patients who completed measurements at 3 timepoints were analyzed. All of these 10 patients were male. [Results] Patients were 3 withgastric ulcer and 7 with duodenal ulcer, with an average age of 58.4±15.1 years, and withan average hospitalization period of 13.7±6.1 days. Scarring of ulcers and biopsy resultsconfirmed no malignant findings in all 10 patients at 3 months after hospital discharge.Mean blood concentrations of V B1, V B2, V B6, and V C at the time of admission were atthe lower limit of normal. Time-specific effects had been confirmed by one-way repeated-measures analysis of variance in mean concentrations of V B1, V B2 and V B6 (P< 0.05),but not V B12, V C, folic acid and homocysteine, Mean V B2 concentration at the time ofhospital discharge was significantly higher compared with at the time of admission (P<0.05).Furthermore, mean concentrations of V B1, V B2, and V B6 significantly increased 3months after discharge compared with at the time of admission (P<0.05). Moreover, meanconcentrations of V B1 and V B6 significantly increased 3 months after discharge comparedwith at the time of discharge (P<0.05). [Conclusion] Since V B1, V B2, and V B6 appear

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sto become easily deficient at the onset of PU, the deficiency of water-soluble V may beone of the causative factors of PU development. In patients who have developed PU,supplementation of water-soluble V is desirable for ulcer prevention.Time-course changes in blood concentrations of water-soluble vitamins and homocysteine

Su1745

Differential Expression of Short-Chain Fatty Acid Receptor FFA2 and FFA3 inForegutIzumi Kaji, Yasutada Akiba, Jonathan D. Kaunitz, Shin-ichiro Karaki, Atsukazu Kuwahara

Background and aim: Short-chain fatty acids (SCFAs), such as acetate, propionate, andbutyrate have a variety of bioactivity both in physiological and in pathological conditionsin the gastrointestinal (GI) tract. Therefore, SCFA receptors FFA2 (GPR43) and FFA3 (GPR41)may be the therapeutic target molecules for mucosal injury. We have reported that FFA2and FFA3 are expressed in L-cells in terminal ileum and colon, and long-term ingestion ofdietary fiber increases the number of FFA2-positive cells. However, the expression of FFA2and FFA3 has not been studied in the upper GI tract. The aim of the present study is toclarify the distribution of FFA2 and FFA3 in the upper GI tract. Methods: We examinedthe expression of FFA2 and FFA3 in rat upper GI tract, dorsal root ganglion (DRG) andnodose ganglion (NG) using RT-PCR and immunohistochemistry with specific antibodies.Results: Stable mRNA expression of FFA2 and FFA3 was detected in whole wall of glandularstomach, antrum, proximal and distal duodenum, and jejunum, but not in esophagus andforestomach. FFA2 and FFA3 were expressed both in the mucosa and in the submucosa/muscle layer in the duodenum, while only in the mucosa in the colon. Immunoreactivity(IR) for FFA2 was localized at a part of chromogranin A (CgA)-positive enteroendocrinecells in the glandular stomach, distal duodenum, and jejunum. In contrast to the colonicFFA2-positive cells with glucagon-like peptide-1 (GLP-1) expression, most FFA2-IR cellsin the upper GI tract contained 5-HT, indicating enterochromaffin cells. Only in pyloric-duodenum junction, FFA2 was expressed in CgA-negative enterocytes. On the other hand,FFA3-IR cells were CgA and 5-HT-positive, partially in glandular stomach and throughoutduodenum and jejunum. In the ileum, FFA3-IR was found in GLP-1-positive L cells. FFA3but not FFA2 was expressed in the DRG neurons, while both FFA2 and FFA3 were expressedin NG neurons. Conclusion: The segmental and cell-type differences in the expression ofFFA2 and FFA3 in the upper GI tract suggest that FFA2 and FFA3 have distinct roles inthe nutrient sensing and mucosal physiological responses in the upper GI tract.

Su1746

Pathogenesis of Steroid-Induced Gastric Ulceration in Rats: Involvement ofProstaglandin Deficiency, Pepsin Secretion, and Healing Impairment ActionAya Yamaguchi, Shusaku Hayashi, Yuka Ishikawa, Shino Kunimi, Kikuko Amagase, KojiTakeuchi

Backgrounds & Aims: Glucocorticoids are important hormones released from adrenal glandsand show diverse actions via the activation of the nuclear receptors. These hormones arefrequently used for treatment of various diseases such as bronchial asthma, rheumatoidarthritis or ulcerative colitis and so on. It is known that these steroids cause lesions or ulcersin the stomach as an inevitable side effect, yet the mechanisms underlying these adverseeffects remain to be unexplored. In the present study, we demonstrated using prednisolone thedevelopment of ulcers in rat stomachs and investigated the mechanism of gastric ulcerogenicresponse to this steroid by examining the influence on gastric secretion, prostaglandin (PG)production and wound healing as well as the effects of various agents on these ulcers.Methods: Male SD rats were used. Prednisolone (12.5~50 mg/kg) was given SC once dailyfor 4 days, and the animals were killed 24 h after the final administration. Various drugssuch as mifepristone (a glucocorticoid receptor antagonist), omeprazole, cimetidine, pepstatin(a specific pepsin inhibitor) and indomethacin were given PO twice daily (30 min beforeand 8 h after each administration of prednisolone) for 4 days. Acid secretion was examinedin pylorus-ligated rats, and pepsin secretion was examined in fistula rats. PGE2 content wasmeasured by EIA, while COX-1/COX-2 expressions were determined by RT-PCR. The healingimpairment effect was assessed in a wound healing model using RGM1 cells. Results:Prednisolone induced liner or punctate hemorrhagic lesions in the stomach; the severity ofthese lesions was increased in dose- and time-dependent manners, the lesion score at 50mg/kg after 4 day-treatment being 60.1±7.8 mm2. These lesions were dose-dependently andsignificantly attenuated by mifepristone, a glucocorticoid receptor antagonist, and preventedsignificantly by omeprazole, cimetidine and pepstatin, while indomethacin had no effect.Prednisolone did not affect acid secretion but significantly increased pepsin secretion. Themucosal PGE2 content was significantly decreased by 4 days treatment of prednisolone,without much change in COX-1/COX-2 expression. On the other hand, prednisolone dose-dependently and significantly impaired the process of wound healing of RGM1 cells, and thiseffect was also a mifepristone-inhibitable. Conclusion: These results suggest that prednisolonecauses severe lesions in the stomach via the activation of the nuclear receptors, and thepathogenic mechanism of these lesionsmay involve both reduced PGE2 content and increasedcorrosive action of acid/pepsin as well as the healing impairment action. This ulcer model

S-494AGA Abstracts

is useful for screening the drugs that may effectively prevent the adverse reaction in thestomach during steroid therapy.

Su1747

Apical Dopamine D2-Like Receptor Mediated Dopamine-Induced DuodenalBicarbonate Secretion in RatXiaoyan Feng, Lifei Zheng, Xiaofeng Li, Jin Song, Yue Zhang, Jinxia Zhu

Background and Aims: Gastric epithelial cells can synthesize dopamine (DA), which hasbeen proved to have the protective effect on the gastrointestinal (GI) mucosa. However, themechanism remains largely unknown. The present study aims to investigate the mechanismunderlying the effect of DA on the duodenal bicarbonate secretion (DBS) in rat. Methods:Short-circuit current (Isc) and real-time pH titration were performed to investigate theduodenal epithelial ion transport. Immunofluorescence was used to examine the distributionof DA receptors in the duodenal epithelium. Stomach perfusion In Vivo and radioimmuno-assay (RAI) were used to detect DA content in rat gastric perfusate. Results: ExogenousDA (0.01-100μM), when added to the basolateral side of duodenal mucosa, induced aconcentration-dependent Isc decrease with an apparent IC50 of 5.3μmol/l (n=7). The Iscdownward deflection was partly (1μM) and completely (10μM) inhibited by D1-like receptorantagonist, SCH-23390, from -12.4±2.0 to -5.8±1.3 μA/cm2 (n=5, P<0.05) and to -2.6±0.4μA/cm2 (n=5, P<0.01), respectively. Furthermore, the result of real-time pH titration indic-ated that apical pH was not significantly affected by basolateral addition of DA (10μM).However, apical application of DA significantly increased apical pH although no alterationwas observed in Isc. The apical addition of DA (10μM) increased DBS from 1.9±0.1 to2.7±0.2μmol/cm2*h (n=7, P<0.001), which was completely inhibited by D2-like receptorantagonist, sulpiride at 1.0 and10μM (n=5). Immunofluorescence results indicated that theimmunoreactivities of all five kinds of DA receptors were observed in the duodenal epithelia.Intravenous infusion of histamine (2mg/kg/h, n=5, P<0.05) or decreasing pH in gastricperfusate (pH=2.5, n=5, P<0.01) significantly increased DA content in gastric perfusate from99.0±8.6 and 121.1±8.9 to 123.6±6.7 and 155.8±13.8nM, by 25.8% and 28.7%, respectively.The secreted DA from gastric epithelia might enter duodenum with gastric juice throughpylorus and have an active effect on DBS. Conclusions: Luminal, not serosal DA may increaseDBS through apical D2-like receptors, while serosal DA may involve the duodenal iontransport through D1-like receptors. This study reveals an unreported DA-regulated DBS,which may provide a new insight on the modulation and protection of DA on the rat duo-denum.

Su1748

Esophageal Myofibroblasts in Neonatal and Adult Murine Esophagus Respondto Acidic Injury With an Increase in BMP4 and IL-6 SecretionJana Binkley, Isra Darwech, Elzbieta A. Swietlicki, Marc S. Levin, Deborah C. Rubin,Anisa Shaker

Epithelial-stromal interactions during gastrointestinal ulcer healing include growth factorand cytokine secretion. Prior to the development of granulation tissue, the underlying stromaat the ulcer base is exposed to injurious luminal agents. The contribution of the esophagealstroma to this injury response has not been rigorously studied in acid-induced disease. Ourhypothesis was that the esophageal stroma contains myofibroblasts that participate in acidinjury responses by secreting soluble mediators of epithelial injury and repair. Aims: 1.Establish that esophageal myofibroblasts are present in the stroma of the murine esophagus.2. Establish primary cultures of esophageal myofibroblasts and determine the secretoryresponse to hydrochloric acid (HCL). Methods: Esophagi harvested from 8 day old (n=5)and adult C57Bl/6 (n=7) mice were submitted for histology, and for α-SMA, vimentin,desmin, and cytokeratin (CK)-18 immunostaining. Myofibroblast cultures were establishedfrom neonatal esophagi by enzymatic digestion. Primary cultures were plated at equal densityin 6 well plates, grown to 80% confluence, and treated with acidified (pH 4) media for 30and 60 min. Cells for RNA and conditioned media for ELISAs were collected at 0, 3, 6,and 24 h. Bmp4, IL-6, KC (murine IL-8 homolog), and Cox-2 mRNA expression wereevaluated. IL-6 and Bmp-4 secretion were determined by ELISA. Results: Neonatal andadult esophagi demonstrated α-SMA and vimentin positive spindle-shaped cells in the laminapropria, distinct from the muscularis mucosa, suggesting they were myofibroblasts. Primarycultures demonstrated homogeneous α-SMA and vimentin immunostaining and undetectabledesmin and CK-18, consistent with the myofibroblast phenotype. Myofibroblasts grown inserum free media expressed Bmp4 mRNA and secreted Bmp4. In acid treated myofibroblaststhere was a trend toward an increase in Bmp4 mRNA after 3 and 6 h. In response to 30min of treatment with acidic media, Bmp4 secretion increased from 76.9 to 86.4 pg/ml after24 h (p<0.05). IL-6 and Cox-2 mRNA increased after 60 min of treatment with acidic media(139 fold and 8 fold respectively after 3 h vs. no treatment, p<0.05) and KC mRNA increased2 fold after 6 h (p<0.05). IL-6 protein secretion was detected 6 and 24 h after treatmentwith acidic media (12 and 34 pg/ml vs. 0 pg/ml in untreated cells, p<0.05). Conclusions:1. Cells with a myofibroblast phenotype are present in neonatal and adult murine esophagealstroma. 2. We have established and characterized the phenotype of primary cultures of α-SMA+, vimentin+ esophageal myofibroblasts. 3. Esophageal myofibroblasts constitutivelysecrete Bmp4 and increase secretion of IL-6 and Bmp4 in response to acid. 4. These findingssuggest esophageal myofibroblasts are participants in the stromal response to epithelial injuryand repair.