A334 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• ULTRASTRUCTURAL LOCALIZATION~OF A NOVEL COLON EPITHELIAL PROTEIN ASSOCIATED WITH ULCERATIVE COLITIS. R. Umashanker*, N. Gujral*, K.M~ Das*, P.S. Amenta+. Departments of Pathology+ and Medicine*,Robert Wood Johnson Medical School, New Brunswick, NJ.

Several studies demonstrated that the monoclonal antibody, 7Et2H n (IgM isotype), reacts specifically to colon epithelium and the reactive protein colocalizes with IgGl autoantibody, specifically found in ulcerative colitis (J Immunol 139:77, 1987; Gut 34:650, 1993). These data suggest that 7Ej2HI2 reactive protein is a putative autoantigen in ulcerative colitis~ Immunocytochemical studies using immunoperoxidase and immunofluorescence methods and 7EnHl2 moAb suggested localization of the protein at the peripheral areas of colonic enterocytes, probably along the cell membrane. However, its precise cellular localization is unclear. In this study, we examined localization of the reactive protein at the ultrastructural level using immunoelectron microscopy with paraformaldehyde fixed, fresh frozen tissue. Biopsy specimens were taken from the distal colon of six subjects with normal colonoscopic examinations. Fourteen specimens were studied by electron microsoopy immunohistochemistry. The reactivity of 7EnHl2 moAb was exclusively localized ~o the microvilli at the apical domain of colonic epithelial Cells and along lateral membrane. Anti-laminin antibody was used as a control~and it was restricted to the basement membrane.

This study shows the presence of the 7EI2HI2- reactive protein at the apical surface along the microvilli, as well as along the lateral membranes of absorptive colonic epithelial cells. Further characterization of this novel protein may provide an important clue regarding its possible role in ulcerative colitis.

• NITRIC OXIDE MEDIATES IFN-T-INDUCED HYPERPERMEABILITY IN CULTURED CACO-2BBe MONOLAYERS N. Unno, M.J. Menconi, M. Smith, M.P. Fink. Dept. of Surgery, Beth Israel Hospital & Harvard Medical School, Boston~ MA

IFN- T increases permeability of T84 intestinal epithelial cell monolayers (Madara and Stafford, J.Clin.Invest.83:724,1989). We recently demonstrated that exogenous nitric oxide (NO) increases permeability in Caco,2BBe intestinal epithelial monolayers (Saltzman et. al., Am.J.Physio L, i n press). Accordingly, we hypothesized that IFN-T-induced hyperpermeahility in intestinal epithelia is mediated via up-regulation of endogenous NO biosynthesis. Using monnlayers of Caco-2BBe cells grown 0n'permeable supports, We assessed permeability by measuring the apical to basolateral clearance (expressed as nL/cm2/h) of the. hydrophilic probe, fluorescein sulphonic acid (FS; mw 478 D). Incubation of monolayers with IFN- T increased permeability to FS in a time-dependent fashion. Using IFN-3, at 1000 U/ml, permeability was 12,4 + 1.1 on day 3, 24.3 + 0.6* on day 5, and 100.6 + 6,9* on day 7 (* indicates p< .01 versus monolayers incubated under identical conditions without IFN--~). The effect of seven days of exposure to IFN-,), was dose-dependent (control, 10~9 + 2.1; IFN-T 250 U/mi, 25.0 + 3.1"; 500 U/ml, 53.2 + 12.3"; 1000 U/ml, 113.6 + 14.2") Basolateral exposure ofmonolayers to IFN-T increased permeability but apical exposure did not (control, 16.0 + 1.5; apical only, 14.1 + 0.9; basolateral only, 115.2 + 15.7"; apical plus basolateral, 109.6 + 15.2" ). IFN- v-induced hyperpermeability was ameliorated by incubation with the NO synthase inhibitors, L-NAME (5 mM), L-NMMA (5 mM), and L-NA (1 mM) (see Table below). The concentrations of NO metabolites, nitrate/nitrite (NO3" /NO2"), in the media were increased by IFN- T (Table), NO synthase inhibitors a t t e n u a t e d I F N - T - i n d u c e d p r o d u c t i o n o f NO3-/NO2".

Group Permeability (nL/cm2/h) NO3-/NO2"(nmols/well) Control 17.9 4- 2.1' 0.45 4- 0.29* IFN-T 133.6 4- 13.9 14.1 4- 1.4 IFN-T +L-NAME 47.9 4. 13.0 t 8.9 4. 1.3' IFN-T +L-NMMA 27.3 4- 2.4* 4.2 4- 0.9 ~ IFN-T +L-NA 40.5 4- 7.0 t 7.8 4- 2.0 t t indicates p < .01 in comparison with IFN- T group (ANOVA).

We conclude that IFN-T causes hyperpermeability in Caco-2BBe monolayers via NO-dependent mechanism.

• INHIBITION OF CALCIUM-DEPENDENT CHLORIDE SECRETION BY CARBACHOL AND EGF: POSSIBLE INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE. J.M. Uribe. F. Sanchez de Medina, A.E. Traynor-Kaplan, and K.E. Barrett. Dept. of Medicine, Univ. of California, San Diego, School of Medicine, San Diego, CA 92103.

Carbachol (C) has two effects on Cl" secretion in T u colonocytes; an initial stimulation followed by inhibition of subsequent Ca + ÷ dependent secretion (such as that stimulated by thapsigargin (TG)). Epidermal growth factor (EGF) shares the inhibitory action. We have shown that activation of a tyrosine kinase by C and EGF is involved in the production of the 3,4,5,6 isomer of inositol tetrakisphosphate in Tu cells. This messenger likely mediates inhibition of Ca ++ dependent CI" secretion, but the pathway for its increase is unknown. Phosphatidylinusitol 3-kinase (PI3-K) and phuspholipase C-'7 (PLC-7) are tyrosine .kinase substrates and involved in the metabolism of inusitol phosphates. We thus hypothesized that C and EGF activate PI3-K, PLC-7, or both. CI" secretion was measured as changes in short circuit current (I,) in Ussing chambers. Tyrosine- phusphorylated PI3-K and PLC~ were assessed via immunoprecipitation and Western blotting. Data are means ± SEM and were analyzed by Student's t-test unless indicated. 15 mill. pretreatment of T,( monolayers with 5#M U-73122 (U), a PLC inhibitor, significantly inhibited the stimulatory effect of 104M C on I,, (AI, 8.3+2.9 vs. 23.1+4.4 #A/cm 2, n---6, p<0.05) but did not reverse the inhibitory effect of carbachol on responses to TG (2#M) (AI, U+C+TG:7.2~I.8 vs. C+TG:6.4_+2~2 vs. TG:20.5+6.5#A/cm 2, n=4). Similarly, U did not reverse the inhibitory effect of EGF (I00 ng/ml) on responses to TG (AI., U+EGF +TG:5.5+ 1.0 vs. EGF+TG: 12.2-+ 1.6 vs. TG:20.5-+6.5 pA/cm2,n=3). No increase in tyrosine-phosphorylated PLC-'7 was observed in cells treated with C or EGF. In contraSt, 15 min. pretreatment of Tu cells with the PI3-K inhibitor, LY294002 (L, 2#g/ml), potentiated the response to TG alone and partially reversed the inhibitory effect of C (AI~ L+C+TG:IS.5+0.6 vs. C+TG:5.2+1.3 vs.

TG:21.0~2.3 VS. L+TG:45.~0.0 pA/¢m 2, n=2-3, p <0.05 by Studant-Newman- Kuels) or EGF (AI~L+EGF+TG:21.9+4.1 vs, EGF+TG:8.5±3.5 vs. TG:21±2.3 vs. L+TG:45.0~--0.0 #A/cm 2, n=2-3). C or EGF both induced a large increase in tyrosine phosphorylation of the 85kDa subunit of PI 3-K after 1 rain., which fell to a lower but discemable level after 5 and 15 rain. These data suggest that 1) the stimulatory effect of C on Cl- secretion can be uncoupled from its inhibitory effect and 2) PI3-K is phosphorylated by C and EGF, and likely thereby activated. This latter effect may be part of the signal transduction cascade leading to the inhibition of Ca *+ dependent CI- secretion in T u colonocytes.

NEW [NSIGHTS ON INTESTINAL TIGHT JUNCTIONS (T J) REGULATION: A LESSON FROM V I B R I O C H O L E R A E . l~ . Uzzau, 2S.E. Goldblum, 4J.M. Anderson, 3j.B. Kaper, UA. Fasano. 1Div. of Pediatric Gastroenterology and Nutrition, 2Div. of Infectious Diseases, and 3Center for Vaccine Development, University of Maryland Baltimore, MD, and 4yale School of Medicine, New Haven, CY. When added to rabbit intestine mounted in Ussing chambers, the Zonula Occludens Toxin (ZOT) of K c h o l e r a e induces a decrease in tissue resistence,(Rt) and in tight janction (tj) complexity in a reversible fashion. We have recently suggested that ZOT mechanism of action is mediated by Protein Kinase C (PKC) ( G a s t r o e n t e r o l o g y 1994;106:A232). Aim of the present study was to gain more insights on tj regulation by using ZOT as a tj modulator. Methods: ZOT effect on tissue permeability was evaluated in rabbit small intestine mounted in Ussing chambers. Bovine pulmunary artery endothelial cells were used for studies of F- and G-aedn pools. IEC6 cells were used to study F-actin and ZO-I protein localization by fluorescence microscopy. The evaluation of PCK isoforms translocation was performed by immunoblotting. Results. Pre-treatment of IEC6 cells with phospholypase C inhibitor neomycm sulphate or with the calcium chelator Quin-2 prevented the effect of ZOT on actin reorganization. Neomycin also partially prevented the decrease in tissue resistance induced by ZOT on rabbit small intestine mounted in Ussing chambers. The actin reorganization involved reduction of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This G- to F- actin shift suggests that ZOT induces polymerization of monomefic G-actin to filamentous F-actin. This polymerizatioI, was time- and dose-dependant, was reversible, and was associated to displacement of ZO-1 protein from the tj complex. Preliminary data aimed at identifying the PKC isoform(s) involved in ZOT effect on aetin reorganization and tj modulation suggested that the Ca2÷-dependent PKC~x isoform is not involved in ZOT activity. Conclusion: ZOT seems to activate an intracellular cascade of events aimed at regulating the tj permeability. By using ZOT as a ZO modulator, it may be possible to gain more insight into the physiology oftj regulation, particularly at the cellular and molecular level.