April 1998 • G1553 RESISTANCE OF CROHN'S DISEASE (CD) MUCOSAL T-CELLS TO CELL DEATH IS INDEPENDENT OF TRIGGER: DECREASED ANTI- Fas AND NITRIC OXIDE (NO)-INDUCED APOPTOSIS. J. Itoh, S. Kugathasan, T. McCormick, E.G. Lapetina, A.D. Levine, C. Fiocchi. Case Western Reserve University, Cleveland, OH. We have previously reported preliminary evidence that CD mucosal T-cells are resistant to IL-2 deprivation-induced apoptosis (GE 108:A841, 1995). We investigated whether this resistance occurs only upon removal of an essential T-cell growth factor, or whether resistance to apoptosis represents an intrinsic reduction in susceptibility to a variety of stimuli. T-cell lines were derived from mucosal biopsies of control, CD and ulcerative colitis (UC) patients with IL-2 for 2 weeks. Apoptosis was induced by: 1) incubation with anti-Fas antibody (CHll; 0.5 lag/ml); 2) exposure to the potent NO donor S-nitroso-glutathione (GSNO; 1 mM); 3) culture with high concentrations of TNF-ct (2000 U/ml). After 24 hrs apoptotic index was measured by acridine orange/ethidium bromide staining and % viability by the MTS assay. Fas receptor expression was assessed by flow cytometry as mean fluorescence intensity (MFI). When apoptosis was induced with the CHI 1 antibody, CD T-cells displayed a significantly lower (*p < 0.01) apoptotic index than control cells, while no significant difference was detected between UC and control cells. This occurred in spite of comparable level of Fas receptor expression among the three groups. Diagnosis Apoptotic index (%) Fas (MH) Control(n= 10~11) 51.2_+3.1 112.5_+4.3 CD (n = 9-15) 37.6 _+ 3.0* 96.9 _+ 8.7 UC (n = 8-11) 42.4 _+ 5.1 94.8 -+ 8.0 When apoptosis was induced by NO, the residual viability of CD T-cells was significantly higher (**p < 0.01) than that of control cells, while the viability of UC and control cells was not significantly different. TNF-~t failed to induce apoptosis in control CD or UC T-cells. % viability Diagnosis GSNO (1 mM) TNF-ct (2000 U/ml) Control (n = 4~8) 37.5 -+ 2.8 100.3 _+ 4.2 CD (n = 3-5) 59.0 ± 4.6** 106.4 _+ 5.0 UC (n = 4-5) 46.6 _+ 4.0 99.3 _+ 6.7 These results show that, in addition to growth factor deprivation, CD T-cells are also resistant to apoptosis induced by ligation of Fas receptor and NO, stimuli that use totally different trigger mechanisms and independent death pathways. This global resistance of CD T-cells to apoptosis may extend their life span and explain their accumulation in the inflamed mucosa. G1554 EFI~'I~CT OF SPECIFIC PRE-INDUCTION OF A 60-KdA HEAT SHOCK PROTEIN ON ACETIC ACID-INDUCED COLONIC LESION IN RATS. A. Iwabuchi * M. Otaka,* A. Okuyama,* M. Jin,* S. Otani,* S. Itoh,* H. Sasahara,* H. Itoh,** Y. Tashima,** O. Masamune*. *Department of Internal Medicine -1 and **Department of Biochemistry, Akita University School of Medicine, AKITA, JAPAN [Background] Many recent studies have indicated the importance of heat shock proteins (HSPs) for survival of ceils under stress conditions. Some HSPs are known to be involved in cytoprotection against environmental stresses. We have reported that pre-induction of a 72- and 90-kDa heat shock proteins (HSP72, 90) prevented mucosal damage induced by acetic acid in rat colonic mncosa. We have also reported that thyrotropin releasing hormone (TRH) specifically induced HSP60 in the colonic mucosa. In this study, we investigated the influence of specific pre-induction of HSP60 on acetic acid- induced colonic mucosal damage. [Materials] 15-25-week old male Sprague-Dawley rats were used in this experiment. [Methods] Using specific antibodies, expression of HSP60 in the colonic mucosa was investigated by immunoblot before and after intraperitoneal TRH administration. The density of the immunologically stained bands was analyzed using scanning densitometer. The effects of pre-induction of HSP60 by TRH administration on acetic acid-induced colonic lesion were evaluated based on damaged area. [Results] (1) Expression of HSP60 was specifically and dose-dependently increased after TRH administration in rat colonic mucosa without any pathologic alteration. (2) Pre-induction of HSP60 by TRH administration had no preventive effect against acetic acid-induced colonic mucosal damage. [Conclusions] In the colonic mucosa, HSP60 induction correlates with increase of colonic motility caused by TRH administration. In addition, our findings might indicate that HSP60 has no cytoprotective function in the colonic mucosa against acetic acid-induced mucosal damage, and that the function of HSP60 could be different from those of HSP72 and 90 in the colonic mucosa with respect to cytoprotection. Intestinal Disorders A381 • G1555 FUNCTIONAL CHARACTERIZATION OF APICAL AND BASO- LATERAL ANION TRANSPORTERS IN RABBIT PROXIMAL DUODENUM. P. Jacob. G. Lamprecht, H. Rossmann, M. Gregor, U. Seidler. 1. Medical Dept. of the Eberhard-Kads University Ttibingen, F.R.G. Background: The current ion transport model for rabbit duodenal bicarbonate secretion involves an apical anion exchanger and a basolateral Na +HCO~- cotransporter whose transport characteristics are unknown. Aim of the study: To investigate the presence and characteristics of rabbit duodenal apical and basolateral anion transporters and compare them with those in the jejunum, gastric mucosa (AE2 anion exchanger) and kidney cortex (recently cloned Na +HCO~- cotransporter). Methods: Using valinomycin-treated apical (BBMv) and basolateral membrane (BLMv) vesicles from rabbit proximal duodenal, jejunal and gastric mucosa and kidney cortex, we studied 1. CI-, HCO3- or gluconate (100 raM) driven 36C1--uptake into duodenal and jejunal BBMv and BLMv and gastric BLMv, 2. pH-and HCO3- gradient driven 22Na+-uptake into duodenal and renal BLMv, and compared transport rates, stilbene sensitivity and effect of Na+. Results: Enrichment factors for alkaline phosphatase were 24-+ 2.9 in duodenal and 30.4 _+ 2.6 in jejunal BBMv, for Na+/K+ATPase 14.9_+ 1.3 in duodenal BLMv, 18.1 _+3.1 in gastric BLMv and 7.5_+ 1.4 in renal BLMv. 1. Anion exchange: We found a rapid DIDS-sensitive 36Cl- uptake with a maximum of 80 ± 13 and of 84 _+ 19 nmol Cl'/mg protein into duodenal and jejunal BBMv at 20 sec and a large overshoot. In duodenal BLMv, maximal DIDS-sensitive 36C1 was 24 nmol Cl-/mg protein with a smaller and delayed overshoot. The DIDS inhibition curve was S-shaped with an IC~oof 580 _+ 29 laM (n=9) in duodenal BBMv which was not significantly different from that previously observed in ileal and jejunal BBMv, 380 -+48 laM (n=4) in duodenal BLMv, but only 30_+ 9 IAM in gastric BLMv (n=5, p < 0.005). Furthermore, the maximal DIDS-sensitive 36C1- uptake into duodenal BBMv loaded with 100 mM CI- was threefold of that into BBMv loaded with 100 mM HCO3-, whereas it was similar into C1- and HCO3- loaded gastric BLMv. The presence of equal transmembrane concentrations of Na+ enhanced transport rates only in duodenal and jejunal BLMv. 2. Na+HCO~ contransport: We found a significantly higher 22Na+ uptake into duodenal BLMv in the presence of a pH- and HCO3- gradient compared to a pH- gradient alone. 0.5 mM dimethyl-amiloride (DMA) completely inhibited the pH-gradient driven 22Na+ uptake in the absence but not in the presence of HCO3". 3 mM DIDS had no effect in the absence of HCO3- but inhibited a part of the 22Na+ uptake in the presence of HCO~-. Similar findings were obtained in renal BLMv. The percentage of 22Na+ uptake mediated by Na+/HCO3 - cotransport (in relation to Na+/H + exchange) decreased with increasing [Na+], and was relatively small (max. 30% at 0.1 mM [Na+],,) in duodenal BLMv, whereas it remained large in renal BLMv (70% at 4 mM [Na+].). Conclusions: A highly active anion exchanger exists in rabbit duodenal BBM with low DIDS sensitivity and a threefold higher transport rate for CI- than for HCO3". A less active anion exchange process exists in duodenal BLM. Both exchangers are functionally distinct from the gastric AE2 anion exchanger. A Na+/HCO3 - cotransport process was identified in the duodenal BLM. • G1556 TRANSCRIPTIONAL REGULATION OF THE DRA GENE: A MODEL FOR THE ALTERATION OF DIFFERENTIATED GENE EXPRES- SION IN INTESTINAL PATHOBIOLOGY. W. Jiang and G. D. Wu. Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA The differentiated columnar epithelium is the interface between the contents of the intestinal lumen and the internal environment of the animal and, therefore, expresses genes important for the functional activity at this interface. We have been studying the transcriptional regulation of the DRA gene (_D_own Regulated in Adenoma) as a model to elucidate the mechanisms by which differentiated intestinal gene expression is modulated in disease states such as intestinal neoplasia, inflammation, and metaplasia. The expression of DRA, a chloride/sulfate anion transporter that is expressed at high levels in the differentiated surface epithelium of the colon, is lost in adenomas and adenocarcinomas. Furthermore, DRA has been identified as the gene which is mutated in patients with congenital chloride diarrhea. We have previously shown that DRA expression is dramatically reduced in animal models of inflammatory bowel disease and in patients with ulcerative colitis. IL-113, reduces DRA mRNA expression in vitro by inhibiting gene transcription. Reporter gene analysis has shown that there are important elements within 700 bp of the start of transcription that direct intestinal cell line specific gene transcription. DNAse 1 footprinting and EMSAs have identified three elements within this region that interact with specific DNA binding proteins. In order to study DRA transcription in vivo, transgenic mice were developed using the human growth hormone (HGH) gene as a reporter. Preliminary analysis of these animals demonstrates that 3.6 kb of the DRA 5'-flank directs gene expression specifically to the differentiated epithelium throughout the entire gastrointestinal tract including, unexpectedly, the squamous epithelium in mouse foregut. This result suggests that the squamous and enterocytic intestinal epithelium share transcriptional mechanisms that direct intestine-specific gene transcription. Furthermore, regulatory elements within the DRA gene responsible for inhibiting aberrant expression in the squamous epithelium are not located in our initial transgene construct. These mechanisms may be operative in the development of Barrett's (intestinal) metaplasia. Alteration of differentiated gene expression is a reflection of fundamental changes in the phenotypic characteristics of the intestinal epithelium in pathobiology. Elucidation of the mechanisms responsible for these alterations will help to provide insights into the pathogenesis of these disease states.