April 1995 Intestinal Disorders A321

• CI" TRANSPORT CHARACTERISTICS OF AN IMMORTALIZED HUMAN EPITHELIAL CELL LINE (N3) DERIVED FROM THE NORMAL TRANSVERSE COLON. J. Sahi, J.S. Stauffer, T.J. Layden, M.P. Moyer and M.C. Ran. Departments of Physiology and Biophysics & Medicine, University of Illinois at Chicago, Chicago, IL and Department of Surgery, University of Texas, San Antonio, TX.

N3 is a normal, non-transformed epithelial cell line derived from the human transverse colonic mucosa (Stauffer et aI, Gastro.106:A273, '94). We characterized these cells further and studied their" CI- transport properties. In culture, the cells have a doubling time of 2-3 days, and growth plateaus at day 8. On non-porous substrates such as plastic or plastic coated with collagen type IV, the cells reach =90% confluence in 7 days with no further growth. On porous substrates such as filters coated with collagen IV or complex fibrillar collagen, the cells are 100% confluent by day 7; they develop a transepithelial resistance (R) of 206 and 252 ohm.cm 2 respectively. R (ohm.cm 2) was lower when cells were grown on transwells coated with collagen I (158), laminin (121) and MatrigeI (151). Second messenger-mediated CI- transport in N3 cells was studied using the Cl'-sensitive fluoroprobe 6-methoxy-quinolyl acetoethyl ester (MQAE). Cells grown on coverslips were MQAE-loaded (n=5 experiments). C1- influx was dependent on [Cl']o, and basal CI influx was 0 .21+0.02 raM/second. The cyclic AMP-dependent secretagogues forskolin (I#M), 8Br-cAMP (10#M), and PGE~ (1/zM) increased CI- permeability to 0 .39+0.06, 0 .53+0 .09 and 0 .49+0.03 respectively. The cGMP-dependent regulators, heat stable enterntoxin (i/zM) increased CI influx by 2.2 fold to 0 .47+0 .08 whereas 8Br-cGMP (10/zM) increased it by 1.3 fold to 0 .28+0.06. The protein kinase C activator phorbol dibutyrate (1/~M) increased CI permeability to 0 .45+0.05. The CI channel blocker diphenylamine 2-carboxylic acid (50#M) inhibited basal and second messenger Stimulated CI-permeabilities by 87% and =50% respectively. The Na-K-2CI cotransport inhibitor, furosemide (I#M) inhibited both basal and second messenger stimulated CI" permeabilities by =35%. Thus normal cell lines derived from the human transverse colon exhibit cAMP, cGMP and PKC sensitive CI permeabilities that involve the Na-K-2CI cotransporter and CL channels. This resembles the CI transport and its regulation in primary cultures of human distal colonic crypts (Sahi et al, AJP 266:G846, '94). The R values are similar to those reported for intact human colon (124 ohm.cm2; Gastro. 93: 441, '87). These features make N3 cells a promising model to study normal transverse colonic function (Supported by DK08449-03 & DK38510).

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• BACTERO1DES FRAGILIS TOXIN DEMONSTR,~,TES POLAR ACTION ON THE CYTOSKELETON AND CHLORIDE SECRETION OF T84 MONOLAYERS. Roxan F. Saidi, Frances G. Chambers, Irene G. Eromor, Sherin S. Koshy, Roger L. Van Tassell, Cynthia L. Sears. Divs. of Gastroenterology and Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD and Virginia Polytechnic Institute and State University, Blacksburg, VA.

Strains of Bacteroides fragilis associated with diarrhea in children produce a heat-labile 20 kDa protein toxin (BFF). BFT stimulates secretion in lamb ligated intestinal loops and alters intestinal epithelial cell morphology in vivo and in vitro. The purpose of this study was to examine the activity of BFF on polarized T84 monolayers. In Ussing Chambers, BFT had two effects. First, BET diminished monolayer resistance (R). The time course, magnitude and concentration-dependency differed when BFT was applied to the apical vs. basolateral membranes. A maximal decrease in R of 50% occurred at 6 hrs after incubation with apical BFT (I00 ug/ml). In contrast, 30 ng/ml of basolateral BFF yielded an 80% decrease in R by 90 rain of incubation whereas 500 ng/ml of basolateral BET diminished R by 80% by 60 rain of incubation. Second, only basolateml BET increased short circuit current (lsc; indicative of C1- secretion): The effect on lsc was concentration-dependent (threshold and maximal concentrations of 30 ng/ml and 500 ng/ml, respectively) and short-lived. Time course experiments indicated that Isc returned to baseline as R continued to decrease suggesting different mechanisms for these two activities. No release of lactic dehydmgenase was detectable and synthesis of proteins by the mon01ayers was intact after treatment of T84 monolayers with 500 ng/ml of basolateral BET for 2.5 hrs indicating lack of.injury to the monolayers. By light microscopy, monolayers treated with apical BET(500 ng/ml) revealed ballooning of the apical surface of cells and rounding detectable by 6 hrs and maximal after 18 hrs. Monolayers treated with basolateral BET (500 ng/ml, 90 rain) and stained with rhodamine-phalloidin to identify F actin revealed diminished and flocculated staining at the tight junctional ring and thickening of linear F actin strands (resembling stress fibers) at the basolateral monolayer surface. Transmission electron microscopy (EM) after basolateral BFF revealed loss of microvilli and complete dissolution of some tight junctions (zonula occludens and zonula adherens) without loss of desmosomes. In addition, the cells appeared swollen compared to controls and contained new supranuclear inclusions of uncertain identity. Scanning EM suggested unravelling of the microvilli of some, but not all, cells in the monolayer. We conclude that BET is a non- lethal toxin acting in a polar manner on T84 monolayers to: 1) alter monolayer resistance through an effect on the cell cytoskeleton and 2) stimulate C1- secretion. The effect on R may result from BET altering the F actin structure of the intestinal epithelial cell. BET may contribute to diarrheal disease by altering the epithelial barrier and stimulating C1- secretion.

EFFECT OF MUCUS ON INTESTINAL MUCOSAL PERMEABILITY : STUDIES IN CACO-2 CELLS. O. Saitoh, K. Nakagawa, H, Matsumoto, K. Sugi, K. Maemura, M. Yoshizumi, K. Takada, L Hirata, K. Katsu. 2nd Dept. of Int. Med., Osaka Medical College, Osaka, Japan.

The mucus gel layer has been considered to play an important role in protecting the intestinal mucosa from chemical or physical injury. The aim of this study was to examine the role of mucin, the major constituent of the mucus gel layer, in maintaining the intestinal mucosal barrier. METHODS: Caco-2 cells were cultured as monolayers for 20 days on microporous membranes(0.6cm 2) in culture inserts. To confirm differentiation, transepithelial resistance and alkaline phosphatase(ALP) activities of the cell homogenate were determined. Paracellular permeability was measured by 3H- mannitol flux(apical-to-basolateral transport for 3 hours). To examine the effect of intratuminal stimulatory factors on the permeability, sodium taurocholate(TC), oleic acid(OA) or OA+TC were added to the apical side prior to the permeability experi- ments. For inhibition experiments, mucin (porcine gastric mucin, Sigma; 5, 10, 20, 40mg/ml) was layered over the apical surface of the monolayers prior to addition of 2mM OA in 4 mM TC. Trypan blue and propidium iodide were used as cell viability markers. RES U LTS: transport of ~H-mannitol (% per hour) control 1.2440.1 % TC(4,8,!6,32mM) 1.2440.3, 1.0-t-0.2, 1.5440.3, 15.9-4-1.4 ~ OA(10,20,40mM) 1.5_+0.2, 2.4+_0.1 b , 4.7-t-0.3 b OA(10,20,40mM)/4mM TC 2.2_+.0.6 a, 6.9-----0.8 b , 9.2i__0.7 b mucin(5,10,20,40mg/ml) 5.2441.0, 3.3+_1.1 °, 2.7-----0.5 d, 1.4___0.6'

+ 20raM OA/4mM TC mean+_SEM (n= 4-6). a: p<0.05, b: p<0.01 vs control; c: p<0.05, d: p<0.01 vs 20mM OA/4mM TC (Wilcoxon test).

CONCLUSIONS: OA and TC, both postprandial intestinal contents, could increase the intestinal permeability in a dose dependent manner. The mucus gel layer inhibited the increased permeability induced by intraluminal factors, and this action may contribute to the intestinal mucosal barrier function.