Mo1817

Lactobacillus Acidophilus Attenuates Dysregulation of DRA Function andExpression in Inflammatory ModelsVarsha Singh, Geetu Raheja, Seema Saksena, Ravinder K. Gill, Anas Alakkam, AlipBorthakur, Gail A. Hecht, Waddah A. Alrefai, Pradeep K. Dudeja

SLC26A3 [DRA (down-regulated in adenoma)] plays an important role in mediating theluminal Cl-/HCO3- exchange process in the intestine. A decrease in DRA function andexpression has been implicated in various diarrheal disorders and mutations in its gene are thebasis for the congenital chloride diarrhea disease. Also DRA mRNA expression is significantlydecreased by the proinflammatory cytokines IL-1β and IFN-γ as well as in patients withulcerative colitis. Therefore, agents that can protect DRA function and expression can beexploited as anti-diarrheal therapies. Probiotics e.g. Lactobacilli, are important commensalbacteria in the gut and have been used for the treatment of diarrheal disorders. In thisregard, previous studies from our laboratory have shown that Lactobacillus acidophilus (LA)stimulated Cl-/HCO3- exchange activity via a dual mechanism including an increase in thesurface levels of DRA in response to short-term treatment (3h) and by a transcriptionalmechanism in response to long-term treatment (8-24h). However, whether probiotics canincrease DRA expression under inflammatory conditions is not known. Two model systemswere selected for these studies. For In Vitro studies, post-confluent Caco2 cells were exposedto IFN-γ (30ng/ml) basolaterally in the presence or absence of L. acidophilus (LA) or itsconditioned media (CM) for 24h and Cl-/HCO3- exchange activity was measured by DIDSor niflumic acid-sensitive 125I uptake. DRA promoter activity was measured by luciferaseassays in Caco-2 cells transiently transfected with the DRA promoter (-1183/+144). For InVivo studies, the effect of LA (oral gavage of 3x109 CFU, single dose on day 1) was investigatedin dextran sodium sulfate-induced (3% DSS in drinking water x 7 days) mild experimentalcolitis in C57BL/6J mice. Results demonstrated that pretreatment of Caco-2 cells with LAor LA-CM for one hour followed by cytokine treatment significantly alleviated the inhibitoryeffects of IFN-γ on Cl-/HCO3- exchange activity. Also, the decrease in DRA promoter activitywas blocked by pretreatment of cells with LA-CM for 24 h. Oral gavage of live LA into miceattenuated DSS colitis, reflected by maintenance of body weight, colonic length and colonicweight. Also, the decrease in DRA mRNA and protein levels associated with DSS colitis wasblocked by LA administration. Confocal microscopic analysis of apical DRA staining in thedistal colon showed a significant reduction in DSS-treated mice which was blocked by LAco-administered withDSS. In conclusion, the positive effects of the probiotic LA in attenuatinginflammation-induced down-regulation of DRA function, expression and promoter activitysupport the therapeutic potential of this probiotic in inflammatory diarrheal disorders.(Supported by NIDDK and Dept. of Veterans Affairs)

Mo1818

A New Animal Model of Intestinal FibrosisMartin Hausmann, Katharina Leucht, Thomas Rechsteiner, Michaela Krebs, ChristianBenden, Silvia Kellermeier, Michael Fried, Annette Boehler, Gerhard Rogler

BACKGROUND: Fibrosis in inflammatory bowel disease (IBD) is a consequence of chronicinflammation and can occur in Crohn's disease (CD) and ulcerative colitis (UC). Fibrosisand subsequent strictures are a frequent cause for surgery in patients with CD. So far thereis no satisfying conservative treatment of intestinal fibrosis which is partially due to the lackof reliable animal models of intestinal fibrosis. We therefore established a simple procedurefor a heterotopic intestinal transplant model of intestinal fibrosis in rats. METHODS: MaleBrown-Norway (donor) and Lewis (recipient) rats weighing 200 to 250 g were obtainedfrom Harlan Sprague Dawley Inc., the Netherlands, and kept under specific pathogen-freeconditions. Heterotopic transplantation of the terminal ileum was performed. Donor terminalileum (1 cm) was extracted and transplanted subcutaneously into the neck of recipientanimals. Grafts were harvested at day 7, 14 and 21 after transplantation. The experimentalprotocol was approved by the local Animal Care Committee of the University Zurich. Crosssections of paraffin-embedded tissues were stained with (Mayer's) hematoxylin-eosin (H&E)and Elastica van Gieson (EvG). Immunohistochemistry for Keratin (DAKO, Z0622) andTGF-β (Santa Cruz, sc-146) was performed. RESULTS: Heterotopic allografts and isograftsunderwent a sequence of epithelial loss during the first week after transplantation. After 14days the intestinal lumen was partially or completely obliterated by fibrous tissue. Two tothree weeks after transplantation several keratin positive cells were found scattered in thefibrotic tissue again. In freshly isolated small bowel immunohistochemistry showed TGF-βexpression in intestinal epithelial cells and in the lamina propria along the crypt-villus axis.Three weeks after transplantation TGF-β expression could be found in the complete fibroustissue. EvG staining showed a significantly increased size of the collagen deposit below theepithelial layer three weeks after transplantation compared to freshly isolated small bowel(113.7 ± 85.7 μm, n = 36 and 19.3 ± 6.2 μm, n = 72, p < 0.001). A multitude of collagendeposits could be found in the fibrotic tissue. CONCLUSION: The heterotopic intestinaltransplant model of intestinal fibrosis in rats showed corner stones of fibrosis development:epithelial loss, fibrous tissue, TGF-β expression and collagen deposit. This model will behelpful for the development of preventive therapies for patients with IBD suffering fromstrictures and fibrosis.

Mo1819

SRC Dynamic Increase in Direct Binding to the PLC-γ SH2 Domain(S), butNot NHE3, is Necessary to Form Signaling Complexes to Allow CarbacholMediated Inhibition of NHE3 Activity in CACO-2/Bbe CellsNicholas C. Zachos, Luke J. Lee, Olga Kovbasnjuk, Xuhang Li, Mark Donowitz

BACKGROUND: Intestinal electroneutral Na absorption involves the BB Na/H exchanger 3(NHE3), which when inhibited contributes to diarrhea. Elevated levels of intracellular Ca2+

([Ca2+]i) inhibit NHE3 activity in the intact intestine. Apical PLC-γ is necessary for [Ca2+]iinhibition of NHE3: 1) PLC-γ directly binds NHE3 which is necessary for [Ca2+]i to inhibitNHE3; 2) PLC-γ exerts lipase independent functions through its SH2 domains, which whenblocked, prevent Ca2+ inhibition of NHE3. [Ca2+]i inhibition of NHE3 also involves Src;

S-657 AGA Abstracts

since the Src inhibitor, PP2, prevents carbachol (CCH) inhibition of NHE3 in the intactintestine. However, the mechanism responsible for this effect is unknown, although Srcdirectly binds PLC-γ. In this study, we tested the hypothesis that the SH2 domain of PLC-γ links Src to NHE3 containing complexes to mediate Ca2+ regulation of NHE3 activity.METHODS: Polarized Caco-2/BBe cells grown on filters were transiently infected with a3HA-NHE3 adenovirus construct and studied 48 h later. NHE3 activity was measured byBCECF/fluorimetry in cells treated +/- PP2 (10μM), in the presence or absence of 10μMCCH. Total cell lysates were obtained (with 0.1% Triton X-100) for tyrosine phosphorylationof Src (Y416; Y527) and PLC-γ (Y783), and co-immunoprecipitation (Co-IP). Direct bindingof Src and PLC-γ was determined by Far Western blots. RESULTS: 1) CCH rapidly decreasedNHE3 activity by ~40%, which was abolished with PP2 treatment. 2) CCH treatment rapidlyinduced tyrosine phosphorylation of Src but not PLC-γ. Phosphorylation of Src at Y416(activation site) increased from 1-10 min post-CCH treatment, while the Y527 (inactivationsite) decreased over the same time course. 3) Co-IP studies demonstrated that Src associatedwith PLC-γ, but not NHE3, under basal conditions, an interaction that increased after CCH(<5min), which was prior to dissociation of PLC-γ and NHE3 (>5 min post CCH). 4) Directbinding to Src only occurs through the SH2 domain(s) of PLC-γ, since blocking the SH2domain of PLC-γ with a specific tyrosine phosphorylated peptide inhibitor, but not anunphosphorylated negative control peptide, prevented direct binding of Src to PLC-γ. CON-CLUSIONS: This study demonstrated that Src 1) Activity is necessary for [Ca2+]i inhibitionof NHE3 activity, 2) Activation occurs rapidly, as early as 1 minute, after CCH treatment,3) Binds directly to PLC-γ SH2 domain but not NHE3, 4) The Src/PLC-γ interaction isdynamic, initially increasing and then decreasing under elevated [Ca2+]i conditions with theincreased effect occurring over the time of NHE3 inhibition, and 5) PLC-γ exerts lipaseindependent effects in its involvement in elevated [Ca2+]i inhibition of NHE3 activity bybringing Src into NHE3 containing multi-protein complexes prior to dissociation of PLC-γfrom NHE3 and subsequent NHE3 endocytosis.

Mo1820

NHE3 Contains Two Cytoskeleton Associated C-Terminal, Ezrin ContainingSignaling Complexes, One for Acute Stimulation and One for Inhibition,Which Together Account for Acute NHE3 RegulationBoyoung Cha, Jianbo Yang, Cheng Zhang, Rafiquel Sarker, Molee Chakraborty, NicholasC. Zachos, Sachin Mohan, Mark Donowitz

Background: The epithelial BB Na/H exchanger NHE3 associates with the actin cytoskeletonvia binding with its C-terminus to the cytoskeleton linking protein ezrin in two locations,one binding ezrin directly and the other indirectly, the latter via the NHERF, multi-PDZdomain scaffolding proteins. The direct ezrin binding domain is juxtamembrane, and involvesthree positively charged, clustered aa, K516, R520 and R527, and is necessary for NHE3trafficking. The indirect ezrin binding domain involves binding to NHERF1 and NHERF2,occurs between aa 586-605 and is necessary for NHE3 complex formation and dynamicassociation with the cytoskeleton. The hypothesis tested in this study was that these twodomains were involved in different aspects of regulation of NHE3. Methods: Point mutantsof NHE3 (NHE3 R520F and R527F, called NHE3 F1D; and NHE3 L596E and M598K,called NHE3 F2EK) were made and stably expressed in PS120, NHE null fibroblasts someof which also stably expressed NHERF1 and/or NHERF2. NHE3 activity was determined byfluorometry/BCECF under basal, serum stimulated and Ca2+ ionophore inhibited conditions.Results: 1) NHE3 failed to bind to NHERF1 and NHERF2 based on co-precipitations andoverlays when the NHE3 mutant L596E,M598K was studied as an intact protein and as aH6-fusion of aa 590-686. This part of NHE3 contains a class II PDZ binding domain (x-Φ-x-Φ). 2) Both NHE3F1D and NHE3F2EK had basal activity reduced to ~50% of wildtype. 3) The NHE3FID but not the NHE3F2EK mutant had reduced to abolished serumstimulation, absent inhibition induced by the Akt inhibitor (Akti,20 uM), and the Rho kinaseinhibitor (Y27632, 2ug/ml). 4) TheNHE3F2EK but not NHE3 FID exhibited absent inhibitionby Ca2+ (A23187, 0.5 uM) and lack of effect of the PI-3K inhibitors (LY294002, 50 uM;wortmannin, 100 nM). Conclusions: 1) Separate domains of the NHE3 C-terminus havebeen identified as being involved in regulation of basal, acutely stimulated and inhibitedNHE3 activity. These domains are defined based on their association with the actin cytoskel-eton via ezrin binding to the NHE3 C-terminus. 2) The direct ezrin binding domain isnecessary for trafficking of NHE3 and controls the non-PI-3K aspect of basal as well asacutely stimulated NHE3 activity. 3) The indirect ezrin binding domain includes a class IIPDZ binding domain and is necessary for the PI-3K dependent aspect of basal NHE3 transportand for the acute inhibition by elevated Ca2+. 4) The interactions of these two regulatorydomains account for all recognized aspects of acute regulation of NHE3 activity. 5) Cytoskel-eton association of signaling complexes should be considered as a general mechanism ofregulation of transport proteins.

Mo1821

Butyrate Decreases Intestinal Serotonin Transporter Expression via EpigeneticMechanismsRavinder K. Gill, Waddah A. Alrefai, Daniel Maher, Anoop Kumar, Pradeep K. Dudeja,Varsha Singh, Sangeeta Tyagi, Seema Saksena

The serotonin transporter (SERT) terminates signaling of serotonin by its rapid uptake andinternalization. Our previous studies demonstrated differential expression of SERT withhigher expression in the small intestine compared to colon in both mice and humans; albeitthe mechanisms underlying are not understood. In this regard, the short chain fatty acid(SCFA), butyrate (produced from undigested dietary fiber) is present in much higher concen-trations in the colon compared to small intestine. Butyrate is also a prototype of histonedeacetylase inhibitors (HDAC) that is known to activate or silence genes by epigeneticmodifications. We hypothesized that butyrate exposure modulates SERT expression thatmay underlie its low expression in the colon. Current studies therefore, investigated theeffects of butyrate on SERT function and expression and delineated the underlying mechan-isms. Caco-2 monolayers were used as In Vitro model. In Vivo effect of butyrate was assessedby pectin feeding to mice for 7 days, which results in high colonic SCFA levels by anaerobicfermentation. Results demonstrated that sodium butyrate treatment of Caco-2 cell decreased

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