A700 AGA ABSTRACTS

3832

ROLE OF CGMP SIGNAL PATHWAY IN OXIDANT-INDUCEDDISRUPTION OF ACTIN CYTOSKELETON AND OF INTESTI·NAL BARRIER PERMEABILITY IN HUMAN EPITHELIALCELLS.Sri Komanduri, Ali Banan, Yang Zhang, Ali Keshavarzian, Rush Univ,Chicago, IL.

Nitric Oxide (NO) overproduction has been identified as a mediator ofincreased intestinal epithelial permeability. Excess NO is known to activatesoluble guanylyl cyclase (GC), subsequently forming cGMP & ultimatelyactivating protein kinase G (PKG) in various cellular models. Aims: Weinvestigated the role of cGMP & PKG pathways in oxidant-induced actincytoskeletal disruption & colonic epithelial hyperpermeability. Methods:Human colonic cell (Caco-2) monolayers were incubated with a range ofconcentrations of oxidant (H20 2; 0.025 - 5.0 mM for 30 min) in thepresence or absence of agents (15 min preincubation) that mimic I activateor inhibit the GC, cGMP & PKG pathway. These included agents knownto increase cGMP levels: dibutryl-cGMP (a membrane permeable cGMPanalouge, 0.005 - 1.0 mM). Agents known to decrease cGMP levels: ODQ(a selective GC inhibitor, IH-[1,2,4]-oxadiazolo-[4,3,-a]-quinoxalin-I-one,I - 10 JLM) & KT5823 (a selective PKG inhibitor, I - 50 JLM). Effects oncell viability, monolayer permeability & actin cytoskeleton were thendetermined (n = 6 per group, *P<0.05). Monolayer barrier permeabilitywas determined by fluorescein sulfonic acid clearance & viability wasassessed by a combination of calcein-AM I ethidium homodimer-I. Cyto­chemical staining of the actin by fluorescein isothiocyanate (FITC)-conju­gated phalloidin was also performed followed by detailed examination byhigh-resolution laser confocal (LSCM). Results: Oxidants in a dose depen­dent manner (0.1 - 5 mM) & significantly increased monolayer permeabil­ity (without any significant loss of cell viability) &, concomitantly, dis­rupted the actin cytoskeleton (as described by the beading and fragmenta­tion of F-actin in the peripheral apical ring of actin). Incubation with 0.025mM dibutryl-cGMP alone had no adverse effect on either the cytoskeletonor monolayer permeability. However, preincubation with this dose ofdibutryl-cGMP & a non-damaging dose of H20 2 (e.g., 0.01 & 0.05 mM)caused both significant actin disruption & monolayer hyperpermeability (potentiation ). Moreover, pretreatment with either an inhibitor of GC or ofPKG (ODQ & KT5823. respectively) prevented both oxidant-induced actincytoskeletal disruption & monolayer hyperpermeability. Conclusion: Datasuggest that oxidant-induced loss of intestinal epithelial monolayer barrierfunction and of actin stability are mediated, at least in part, by a cGMP­dependent pathway.

3833

INHIBITION OF TNF MRNA TRANSLATION BY AN IMMUNO­MODULATORY PEPTIDE.Suhasini Iyer, Dimitris Kontoyiannis, Jacky Woo, Nancy Mori, GeorgeKollias, Roland Buelow, SangStat Med Corp, Fremont, CA; HellenicPasteur Institute, Athens, Greece.

RDP58 is a lO-amino acid peptide consisting of D-amino acids with thefollowing sequence: NH2-r-nle-nle-nle-r-nle-nle-nle-g-y-CONH2, wherenle indicates D-norleucine. RDP58 was developed by computer aidedrational design based on HLA-derived peptides which were shown tomodulate immune responses in vitro and in vivo. Studies on the mechanismof action revealed effective inhibition of TNF production by the peptide. Invitro, RDP58 was tested with monocytel macrophage cell lines. Unstimu­lated RAW264.7 and THP-I cells produced undetectable amounts ofTNFin culture. On stimulation with LPS, a significant increase in TNF concen­trations was observed with levels of 2.46 ng/mI and 3.04 ng/ml inRAW264.7 and THP-l cells respectively. RDP58 inhibited LPS-inducedTNF production in mouse (RAW264.7) and human (THP-I) cells, in adose-dependent manner with half-maximal inhibition seen at 20 uM. Adetailed analysis of the mechanism of peptide-mediated TNF inhibitiondemonstrated no effect of RDP58 on TNF gene transcription and mRNAsteady state levels. However, analysis by immunoprecipitation of 35S­methionine labeled TNF from cells stimulated with LPS in the presence orabsence of RDP58, showed specific inhibition of TNF protein synthesis.RDP58 did not affect general protein synthesis which was evidenced byimmunoprecipitation of HLA class I from identically treated cell extracts.The expression of TNF is regulated on a transcriptional and a translationallevel. The regulation of TNF translation is partially dependent on the 3'untranslated region (UTR) of the mRNA molecule. To investigate themechanism of RDP58 mediated inhibition of TNF mRNA translation,

GASTROENTEROLOGY Vol. 118, No.4

experiments were performed with macrophages from transgenic animalsexpressing modified human TNF genes. Replacement of the TNF mRNA3'UTR region with that from the human b-globin mRNA, resulted incomplete reversal of the inhibitory effect of RDP58 on TNF production.Interestingly, elimination of the AU-rich element from TNF mRNA hadonly minor effects on peptide-mediated inhibition of TNF synthesis, indi­cating that yet unidentified elements in the 3'UTR of TNF mRNA arenecessary for the peptide's effect. Given the role ofTNF in the etiology ofinflammatory diseases such as ulcerative colitis and Crohn's disease. theuse of a small molecular weight TNF translation inhibitor in the treatmentof these patients would be of great benefit.

3834

IFN-r INDUCED DOWNREGULATION OF EXPRESSION ANDFUNCTION OF APICAL MEMBRANE NA+IH+ EXCHANGERS,NHE2 AND NHE3, IN RAT INTESTINE AND HUMAN INTESTI­NAL CAC02lBBE (C2) CELLS.Flavio Rocha, Leo Lishansky, Mark W. Musch, Crescence M. Bookstein,Kazunori Sugi, Yue Xie, Michael Field, Eugene B. Chang, Univ ofChicago, Chicago, IL; Columbia Univ, New York, NY.

Background: Interferon-v (y-IFN) chronically inhibits intestinal epithelialNa absorption and may contribute to diarrhea in mucosal inflammation. Wedemonstrated that y-IFN causes a dose- and time-dependent functionaldownregulation of the apical membrane Na transporter NHE2 and NHE3 inintestinal Cacoz/bbe (C2) cells and rat intestine. However, the mechanismsunderlying the y-IFN effects were not known. Therefore, we determined ifchronic stimulation by y-IFN specifically downregulates Na absorption bydecreasing the protein and mRNA expression of NHE2 and NHE3 at thetranscriptional and/or post-transcriptional level. Methods: Serum-stimu­lated (30%) C2 cells were grown on collagen-coated transwells for 14 daysto induce apical NHE activity, They were treated with 3 or 30 ng/ml -y-IFNfor 24, 48, and 72 hrs. Adult rats were treated with 25K units y-IFN for 48hrs via IP injection. Brush border membrane vesicles from the homoge­nized cells and intestinal samples were harvested for Western blot analysisof NHE2 and NHE3 protein. Rat intestine was harvested to determinemRNA content of these isoforms by Northern blot. Results: -y-IFN treat­ment of C2 cells significantly decreased both NHE2 and NHE3 proteinexpression. Beginning at 48 hrs, NHE2 protein expression was reduced to89% and 45% of control with 3 and 30 ng/ml of -y-IFN, respectively.Greater effects on NHE3 protein expression were observed where abun­dance was reduced to 54% and 19% of control with 3 and 30 ng/ml of-y-IFN,respectively. The effect was maximal for both doses at 72 hrs whereprotein expression of both isoforms was reduced to 10% of control. In therat, NHE2 and NHE3 protein expression was also significantly reduced to30% of control at 48 hrs in both the ileum and colon. Ileal NHE2 mRNAwas significantly reduced to 20% of control, whereas in colon, it was onlydecreased to 65% of control. NHE3 mRNA was reduced to 28% and 44%of control in the ileum and colon, respectively. These effects were specific,as no changes in villin or brush border hydrolases expression or functionwere observed. [Conclusions] -y-IFNcauses a significant and specific, time­and dose-dependent downregulation of apical NHE2 and NHE3 proteinand mRNA level, suggesting decreased gene transcription. However. post­transcriptional events may also be involved in this effect, especially withNHE2. Thus, y-IFN regulation of intestinal epithelial NHE2 and NHE3expression and function appears to be quite distinct.

3835

HYPOXIA REGULATES EPITHELIAL TRANSCRIPTION FAC­TOR EXPRESSION THROUGH ACTIVATION OF THE UBIQ­UITIN-PROTEASOME DEGRADATION PATHWAY.Cormac T. Taylor, Sean P. Colgan, Brigham & Women's Hosp, Boston,MA.

Introduction: Epithelial hypoxia occurs during inflammation in a number ofintestinal diseases. Previously, we demonstrated an epithelial inflammatoryresponse to hypoxia which includes induction of tumor necrosis factoralpha (TNFa). This response appears to be orchestrated by the transcriptionfactors CREB and NFI<B. Here, we have investigated a potential pathwayby which hypoxia may alter expression of these transcription factors in thecolonic epithelial cell line, T84. Methods: Confluent T84 monolayers wereexposed to hypoxia (p02 20 torr, 0-48 hours). Differential mRNA displayanalysis was performed to determine altered gene arrays by hypoxia.