transgenic mouse. We show that constitutive expression of IL-13 in the small bowel inducesmodification of intestinal epithelial architecture including villus blunting (WT: 627.03 ±53.21 vs IL13Tg: 462.57 ± 47.66 μm, p < 0.0001, n=34) , goblet cell hyperplasia (WT:0.063 ± 0.024 vs IL13Tg: 0.123 ± 0.029, p<0.0001, n=22), increased epithelial proliferationand migration (villus / crypt, Wt: 4.977 ± 0.873 vs IL-13Tg: 3.382 ± 0.680, p<0.0001, n=25). Modification of epithelial architecture was associated with increased Cl- secretion (TER:Wt: 153.333 ± 16.449 vs IL13Tg: 92.004 ± 12.671Ω/cm2, p=0.0014, n=3). Pharmacologicalanalyses In Vitro and In Vivo determined IL-13 stimulated Cl- secretion to be driven byCFTR Cl- channel activity. IL-13 mediated dysregulation of epithelial architecture and barrierfunction was dependent on IL-13Rα1 and not IL-13Rα2. These observations demonstratea central role for IL-13 in the regulation of intestinal epithelial cell function and suggestthat IL-13 may have an important role in food allergy.

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Lymphotoxin Alpha-Expressing Lymphoid-Tissue Inducer Cells are Essentialfor the Development of Intestinal TH17 CellsTakanori Kanai, Yasumasa Kanai, Yuuichi Ono, Yohei Mikami, Akira Okazawa, AtsushiHayashi, Sujino Tomohisa, Katsuyoshi Matsuoka, Tadakazu Hisamatsu, SusumuOkamoto, Nagamu Inoue, Akihiko Yoshimura, Toshifumi Hibi

Background: Naturally-occurring IL-17-producing CD4(+) T (Th17) cells reside in the intest-inal mucosa of healthy mice under SPF conditions. It has been reported that mice lackingRORγt defect intestinal Th17 cells and lymphoid-tissue inducer (LTi) cells that are essentialfor the formation of lymphoid organs, such as Peyer's patches and cryptopatches. Thus, itremains unknown whether the function of RORγt, the presence of LTi cells, or the formationof lymphoid organs is crucial for the development of Th17 cells. Methods: To determinethis, we analyzed lymphotoxin alpha-deficient (LTα(-/-)) mice, which lack lymphoid organs,but retain normal RORγt gene. Results: The ratio of intestinal Th17 cells in LTα(-/-) micewas significantly reduced as compared to that in age-matched wild type (WT) mice. Surpris-ingly, the ratio of intestinal IL-17-producing CD3(-)CD4(+)NK1.1(-)CD11c(-)IL-7Rα(high)CCR6+ LTi-like cells was conversely increased in LTα(-/-) mice, but not inRORγt(-/-) mice. To assess a reciprocal regulation of Th17 cells and LTi cells, we comparedLTα (-/-) x RAG-2(-/-) mice to RAG-2(-/-) mice. We confirmed that both mice lack intestinalTh17 cells, but the absolute number and the ratio of intestinal LTi cells in LTα(-/-) x RAG-2(-/-) mice were significantly greater than those in RAG-2(-/-) mice, suggesting that intestinalLTi cells are negatively regulated by LTα signaling pathway, but are produced independentlyof intestinal Th17 cells. To clarify a necessity of lymphoid tissues for the development ofintestinal Th17 and LTi cells, we next conducted a parabiosis system between Ly5.1(+) WTmice and Ly5.2(+) LTα(-/-) mice. Four wks after parabiosis surgery, substantial number ofTh17 cells irrespective host or donor cells emerged in intestinal mucosa of LTα(-/-) mice,whereas substantial number of host-derived LTi cells persisted in intestinal mucosa of LTα(-/-) mice. Finally, to reveal whether LTα signaling pathway affects the development of intestinalTh17 cells in colitic condition, we transferred LTα(-/-) or WT CD4(+)CD45RB(high) T cellsinto LTα(-/-) x RAG(-/-) mice. Six wks after transfer, both groups of mice developed colitiswith comparable induction of colonic lamina propria IL-17-producing CD4(+) T cells aswell as IFN-γ-producing cells. Conclusions: Collectively, all the present results suggest thatthe development of intestinal Th17 cells is regulated by LTα-LTβR signaling pathway viaLTα-expressing LTi cells in physiological, but not colitogenic, conditions.

399

CA-Sensing Receptor Regulates Ca2+-Mediated Duodenal BicarbonateSecretion via SOC and IKCa ChannelsXiao Dong, Chase Wong, Christine L. Estrema, Biguang Tuo, Hui Dong

Background and Aims: Ca-sensing receptor (CaSR), a G protein-coupled receptor, is expre-ssed in intestinal epithelium and mediates fluid secretion; however, the involvement of CaSRin the regulation of duodenal bicarbonate secretion (DBS) is largely unknown to date.Therefore, we sought to investigate the role of CaSR in Ca2+-mediated DBS and the underlyingcellular mechanisms. Methods: Murine duodenal mucosae were mounted in Ussing cham-bers. Both short-circuit current (Isc) and HCO3- secretion were measured In Vitro by pHstat. Spermine (1-10 mM), extracellular Ca2+ (4 mM) and Gd3+ (0.5 mM) were used asCaSR activators, and 2-APB (100 μM) and SK&F69365 (10 μM) were used to selectivelyblock store-operated channels (SOC). Clotrimazole (30 μM) or chromanol 293B (10 μM)was used to selectively inhibit intermediate Ca2+-activated K+ channels (IKCa) or KCNQ1channels, respectively. Cytosolic free Ca2+ concentration ([Ca2+]cyt) or intracellaular pH(pHi) in single SCBN cell, a duodenal crypt cell line, was measured with a digital cell imagingsystem. Results: Superfusion of spermine (1-10 mM) raised [Ca2+]cyt in duodenal epithelialcells in a dose-dependent manner (p<0.01, n=50 cells). Spermine (3-5 mM)-induced anincrease in [Ca2+]cyt was almost completely abolished by selective blockers of SOC, 2-APB(100 μM) or SK&F96365 (10 μM) (p<0.01, n=40-50 cells). Moreover, extracellular Ca2+(4 mM) or Gd3+ (0.5 mM) also raised [Ca2+]cyt in duodenal epithelial cells (p<0.01, n=30-40 cells). Spermine (1 mM) or Gd3+ (0.5 mM) markedly stimulated DBS (p<0.01, n=5) without altering duodenal Isc (p>0.05, n=5) in CFTR wild type mice, but did not affectbasal DBS and duodenal Isc (p>0.05, n=6) in CFTR knock out mice. Spermine- or Gd3+-stimulated DBS in CFTR wild type mice was significantly inhibited by U73122 (10 μM), aselective PLC inhibitor (p<0.01, n=5). Clotrimazole markedly inhibited spermine-inducednet peak DBS (p<0.01, n=6). However, chromanol 293B did not significantly affect spermine-induced net peak DBS (p>0.05, n=6), which was similar in KCNQ1 wild type and knockout mice (p>0.05, n=6). Again, either spermine (1 mM) or Gd3+ (0.5 mM) significantlyinduced HCO3- flux through CFTR channels on the membrane of SCBN cells (p<0.01, n=40-50 cells). Conclusions: CaSR is functionally expressed in duodenal epithelial cells andinvolved in Ca2+-mediated DBS. SOC and IKCa (but not KCNQ1) may serve as downstreammolecular targets for the CaSR-mediated DBS. Our findings not only propose a novel rolefor CaSR in regulating DBS but also suggest this receptor could be a potential therapeutictarget in disease states characterized by reduced DBS, such as peptic ulcer disease.

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400

Capsaicin Causes Mitochondrial Proton Buffering in Parietal CellsSascha Kopic, Michael K. Murek, Danielle Collins, Mohammad Reza Boroumand, John P.Geibel

Capsaicin is the active and pungent ingredient in various types of chilli peppers and is anagonist of the Ca2+ channel TRPV1. The role of capsaicin in the genesis of gastric refluxand gastric ulcer disease has been subject of controversial discussion in the past. Thisstudy investigated the effects of capsaicin on gastric acid secretion. Immunogold electronmicroscopy (EM) was used to identify the subcellular localization of TRPV1 in rat parietalcells. Hand dissected single rat gastric glands were loaded with the pH sensitive fluorescentdye BCECF, the mitochondrial Ca2+ indicator dye X-Rhod, and the acid indicator Lysosensorgreen. Following loading, changes in intracellualar pH, mitochondrial Ca2+ and spacialmovement of acid was monitored with the use of a digital imaging system in response tocapsaicin exposure. The NH4Cl prepulse technique was used for pH measurements inorder to acidify the cytosol and to thereby provide enough substrate for proton transportmechanisms to be observed. Immunogold EM revealed localization of TRPV1 on the innermitochondrial membrane. No plasma membrane staining was identified. Capsaicin exposurecauses robust cytosolic alkalinization that can be inhibited by the TRPV1 inhibitor AMG9810or mimicked by the capsaicin analogue resiniferatoxin. Alkalinization is Na+ independentand cannot be inhibited by omeprazole, suggesting non NHE or H+,K+-ATPase mediatedproton movement. Cytosolic alkalinization is only inhibitable by blocking mitochondrialCa2+,H+-Exchanger with eosinB, suggesting capsaicin mediated buffering of protons intothe mitochondria. Results were subsequently confirmed in isolated human gastric glands.Mitochondrial Ca2+ measurements revealed that capsaicin increases mitochondrial Ca2+ whenCa2+,H+-Exchanger is blocked with eosinB. Monitoring of acid movement with Lysosensorgreen showed no capsaicin induced acid movement into the lumen of the gastric gland.Exposure to carbachol, a classic secretagogue and stimulant of gastric acid secretion, resultedin acid movement into the lumen. This study for the first time identifies an organellarlocalization of TRPV1. Our results indicate that capsaicin causes mitochondrial buffering ofcytosolic protons via a mitochondrial Ca2+,H+-Exchanger. In addition we have demonstratedthat capsaicin is not a direct stimulant of gastric acid secretion. Opening of mitochondrialTRPV1 may result in Ca2+ influx into mitochondria which provides substrate for Ca2+,H+-Exchange. We thus speculate that capsaicin has a beneficial effect by reducing acid secretiondue to buffering of excessive cytosolic protons into the mitochondria

401

Lubiprostone Stimulates HCO3- Secretion in Isolated Mouse Stomachs In VitroThrough Activation of Prostaglandin EP1 ReceptorsMasafumi Koyama, Shusaku Hayashi, Kento Takahashi, Koji Takeuchi

Background/Aim: Lubiprostone, a bicyclic fatty acid derived from prostaglandin (PG) E1,is considered a possible chloride channel type-2 (ClC-2) opener and is used for the treatmentof chronic constipation. A recent study showed that lubiprostone stimulated CFTR-dependentduodenal HCO3- secretion in rats, and this action was mediated by the activation of EP4receptors. However, because the characteristics of HCO3- secretion differ in the stomach andthe duodenum, including EP receptor subtypes, it remains unknown whether lubiprostoneproduces similar effects on HCO3- secretion in the stomach. In the present study, we thusexamined the effect of lubiprostone on HCO3- secretion in the mouse stomach In Vitro,focusing on the possible involvement of EP receptor activation. Methods: Male C57BL/6Jmice were used after 18 h fasting. The gastric mucosa was stripped of the muscular layers,mounted between two halves of a lucite chamber and bathed in unbuffered saline gassedwith 100% O2 on the mucosal side and HCO3- Ringer's gassed with 95% O2-5% CO2 onthe serosal side, and these solutions were continuously circulated by a gas-lift system. TheHCO3- secretion was measured at pH 7.0 by a pH-stat method using 2 mM HCl. Lubiprostone(1x10-4~3x10-4 M) or PGE2 (10-4 M) was applied to the serosal side. ONO-8711 (EP1antagonist: 10-6~10-5M) and AE3-208 (EP4 antagonist: 10-6 M) were added serosally 30min before lubiprostone or PGE2. The mRNAs expression of ClC-2 and EP1~EP4 wereexamined by RT-PCR. Results: The isolated mouse stomach consistently secreted HCO3- atrates of 0.12~0.18 μEq/h as basal secretion, in the absence or presence of 0.1% DMSO, asolvent for the agents used in the present study. The application of lubiprostone stimulatedgastric HCO3- secretion in a concentration-dependent manner, and this action was attenuatedby the EP1 antagonist ONO-8711 but not the EP4 antagonist AE3-208. Likewise, the HCO3-secretion was increased by addition of PGE2, and this response was inhibited by theEP1 but not EP4 antagonist. Lubiprostone had no effect on the mucosal PGE2 content.Indomethacin did not affect the increased HCO3- response to lubiprostone. The expressionof ClC-2 channel as well as EP1-EP4 mRNAs was observed in the mouse gastric mucosa.Conclusion: These results suggest that lubiprostone stimulates HCO3- secretion in thestomach, and this action is mediated by the activation of EP1 receptors but not dependenton endogenous PG production. The present study also shows the different characteristicsof HCO3- secretion in the stomach and duodenum.

402

Luminal ATP Release is Impaired in Intestinal Alkaline Phosphatase Akp3Knockout MiceChikako Watanabe, Yasutada Akiba, Takanari Nakano, Sonoko Narisawa, Jose L. Millan,Jonathan D. Kaunitz

Brush border intestinal alkaline phosphatase (IAP) pH-dependently regulates the concentra-tion of luminally-released ATP, which stimulates duodenal bicarbonate secretion (DBS) viaP2Y receptors. Luminal ATP is degraded by IAP and ecto-nucleoside triphosphate diphos-phohydrolase (ENTPDase). We hypothesized that lack of IAP increases luminal ATP concen-tration, enhancing DBS. Since ATP-binding cassette (ABC) transporters (ABCB or ABCCfamily) or pannexin (Panx) hemichannels may mediate ATP release from epithelial cells, wealso hypothesized the alteration of expression of these ATP release associated channels. Wemeasured DBS with pH and CO2 electrodes and ATP output with luciferin-luciferase in a

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