AGA Abstracts current studies were designed to define the signaling pathways involved in NHE3 promoter activation by PMA (100 nM, 16h). We examined the effect of various signal transduction pathway inhibitors on the PMA-induced NHE3 expression in C2BBe1 cells transfected with NHE3 promoter-luciferase constructs. Inhibition of ERK1/2 (PD98059, 50 μM and U0126, 10 μM) and P38 (SB203580, 25 μM) led to a 40- and 50% decrease in PMA-induced NHE3 promoter activity, respectively. AG490, a JAK-2 inhibitor, showed no effect. Examining PMA effects on ERK1/2 phosphorylation by Western blot analysis showed a time-dependent activation of ERK1/2. Additionally, our previous studies showed minimal inhibition of PMA- induced NHE3 promoter activity in response to the PKC inhibitor, Chelerythrine chloride (2 μM). To further assess the role of PKC on the stimulation of NHE3 promoter activity in response to PMA, transfected cells were treated with or without various PKC inhibitors in the presence and absence of PMA and luciferase activity was measured. For these studies, the general PKC inhibitor chelerythrine chloride (2 μM), specific conventional PKC (cPKC) inhibitor GÖ6976 (0.05-5 μM), and novel PKC (nPKC) inhibitor GÖ6983 (5 μM) were used. Bryostatin-1 (100 nM), a non-phorbol ester PKC activator was utilized as a control. Chelerythrine chloride and GÖ6976 exhibited minimal effect on PMA-induced promoter activity, while GÖ6983 abolished the increase in NHE3 promoter activity in response to PMA entirely. Exposure to Bryostatin-1 showed ~20% increase in promoter activity, an effect blocked by GÖ6976. In conclusion, our data suggest that PMA-induced stimulation of NHE3 expression may be independent of cPKCs; however, nPKC isozyme(s) may regulate an intermediate component of the PMA-induced signaling pathway, such as MAPKs, leading to NHE3 stimulation. W1650 Interdigestive Secretion from the Small Intestine Is Selectively Enhanced in Idiopatic Bile Acid Malabsorption Antal Bajor, Kjell-Arne Ung, Lena Ohman, Evan Thomas, Joel C. Bornstein, Magnus Simren, Henrik Sjovall The exact mechanism behind the severe diarrhoea in idiopathic bile acid malabsorption (BAM) is unknown. The aim of the study was to assess the role of small intestinal secretomotor function. Intestinal secretomotor neurons are mechanosensitive, i.e. the degree of activity will depend on the current motor pattern. Methods: We measured motor activity by pressure recording and transmural potential difference (PD), a marker for electrogenic chloride secre- tion, in the proximal jejunum in 18 healthy volunteers and 11 patients with BAM. PD was measured between calomel half-cells using a saline infusion as flowing electrode and a subcutaneous infusion as reference. The BAM diagnosis was based on a pathological bile acid retention (75SeHCAT) test (<10% retention day 7). The amount of phase III associated secretion (secretion occurring in association with distally migrating maximal motor activity) was estimated from mean PD during phase III, measured at one point, multiplied with the length of the secreting segment. If several phase III periods occurred, their contributions were added. Secretomotor activity was measured during three hours in the interdigestive state and one hour after a 500 kcal test meal. Results: No significant differences were seen in fasted contraction frequency (1.8±0.2 contractions*min-1 in both groups), number of phase III periods, phase III duration or fed contraction frequency (3.4±0.2 vs 3.0±0.2) between controls and BAM patients. There was a non-significant tendency to a more rapid propagation velocity of phase III in BAM patients (controls: 6.6±0.9; BAM: 9.2±1.3 cm*min- 1, p=0.095). The phase III segment length was significantly longer in BAM patients (42±6 vs 60±5 cm, p=0.033). PD during phase I (motor quiescence) was not significantly different from zero in either group. During phase II (intermittent motor activity) and phase III (maximal motor activity), mean PD was significantly higher in BAM patients (phase II: 0.7+- 0.2 vs 1.5+-0.3, p<0.05, phase III: 4.5+-0.3 vs 6.4+-0.3, p<0.01). The PD response to the test meal was virtually identical in the two groups. Estimated phase III-associated active chloride secretion (= mean phase III PD at one recording point*phase III segment length*n- umber of phase III:s*3 hours-1) was significantly higher in the BAM patients (273+-38 mV vs 738+-214, p<0.01). The median 75SeHCAT retention value in the BAM patients was 2.5% (0.85-4.67) at day seven. There was a significant inverse monoexponential correlation between the value of the 75SeHCAT test and estimated chloride secretion (p<0.05). Conclu- sion: Interdigestive jejunal secretion seems to be selectively enhanced in BAM. W1651 Ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT) Is Upregulated in Rat Model of Diabetes Mellitus Fadi Annaba, Pradeep Kumar, Amika Singla, Ke Ma, Redouane Boumendjel, Seema Saksena, Rhonda D. Kineman, Ravinder K. Gill, Waddah A. Alrefai Diabetes mellitus is associated with high levels of plasma cholesterol due to an increase in intestinal bile acid absorption and expansion of their pool in enterohepatic circulation. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. In this regard, ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT) is responsible for active reabsorption of majority of bile acids. However, alterations in ASBT function and expression in diabetes mellitus have not been examined. Current studies were designed to investigate the modulation of ASBT in streptozotocin (Stz)-induced diabetes mellitus in rats. Diabetes mellitus was induced in Sprague-Dawley rats by low doses of Stz (20 mg/kg body weight) administered IP on five consecutive days. Rats were sacrificed 2 weeks after the last Stz injection and blood and small intestinal tissues were collected. Plasma insulin levels were significantly decreased in diabetic rats compared to control confirming hypoinsulinemia (0.5±0.04 vs 2.4±0.3 ng/ml). Insulin (6U/day) was given to a group of diabetic rats via s.c osmotic pumps for 3 days before sacrifice. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time QRT-PCR and Western blotting. Functional studies were performed by mounting ileal tissues on Ussing chambers for measurement of Na+-dependent 3H- taurocholic acid (TC) transepithelial flux. Our data showed that ASBT mRNA and protein expression were significantly elevated by ~7 fold and ~5 fold, respectively, (P<0.05) in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Similarly, mRNA expression of oraginc solute transporters alpha and beta (expressed on the basolateral membrane) were significantly increased ~ 4 fold (P<0.05) in A-710 AGA Abstracts diabetic rats. These data indicated that the bile acid transepithelial transport processes are upregulated in diabetes mellitus. Consistently, ileal Na+-dependent transepithelial 3H-TC net flux (relative to DNA content for normalizing epithelial hyperplasia) was significantly increased in diabetic rats (~ 4 fold; P<0.05) that returned to normal with insulin. These studies demonstrated that ASBT function and expression are upregulated in rats with Stz- induced diabetes mellitus. The increase in ASBT expression may underlie the mechanisms by which bile acid pool is increased contributing to disturbances in cholesterol homeostasis associated with diabetes mellitus [supported by NIH/NIDDK]. W1652 Calcium Inhibition of the Intestinal Anion Exchanger Down Regulated in Adenoma (DRA) Requires the Interaction with PDZK1 Georg Lamprecht, Andreas Heil, Vera Gaco, Julia Schäfer, Jerrold R. Turner, Michael Gregor Introduction: NHE3 and DRA work in parallel to mediate electroneutral NaCl absorption in the intestine. Elevated Ca i inhibits NHE3 activity and NaCl absorption in transfected cells and in the intact intestine through mechanisms involving the PDZ proteins E3KARP or PDZK1. Like NHE3, DRA possesses a PDZ-binding domain, but the functional consequences of this PDZ interaction for DRA regulation are unknown. In the upper gastrointestinal tract DRA interacts with CFTR and is stimulated by cAMP together with CFTR. On the other hand, DRA is expected to be inhibited by cAMP together with NHE3 in the lower gastrointesti- nal tract. This has raised the question whether DRA is at all regulated individually or only together with its partner transport proteins. Methods: Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in HEK cells and inducibly in Caco-2/BBE cells. DRA mediated Cl/HCO 3 exchange was measured as pH i changes. Ca i was assessed fluoremetrically. Endogenous NHERF, E3KARP, PDZK1 and IKEPP were detected by specific antibodies. Results: 4Br-A23187 inhibited both DRA and DRA- ETKFminus in HEK cells. Stimulation of protein kinase C by 100 nM phorbol esther did not inhibit DRA. In transfected Caco-2/BBE cells the PDZ interaction motif was required for 4Br-A23187 to inhibit DRA. As a physiologically relevant agonist 100 μM UTP increased Ca i , and inhibited full-length DRA but not DRA-ETKFminus in Caco-2 cells. In DRA trans- fected HEK cells 100 μM UTP increased Ca i , but did not inhibit DRA. HEK cells endogenously express relatively little PDZK1 and E3KARP compared to Caco-2/BBE cells. Additional transfection of PDZK1 into HEK/EGFP-DRA cells was required for UTP to inhibit DRA. Conclusion: 1) DRA is individually (i.e. in HEK cells that do not express NHE3 or CFTR) inhibited by Ca i but not by dircet stimulation of protein kinase C. 2) In HEK cells the non- physiologically high Ca i triggered by 4Br-A23187 inhibits DRA independently of its PDZ interaction. But in the more differentiated cell system of Caco-2/BBE cells or following the physiological stimulation by UTP Ca i inhibition requires the PDZ interaction of DRA and the presence of PDZK1. These data suggest that the Ca signal is targeted or tailored by the binding of PDZK1 to DRA, probably involving other proteins. Together with data from PDZK1 -/- mice, these data underscore the prominent role of PDZK1 in Ca i -mediated inhibition of colonic NaCl absorption. 3) Our data suggest that in addition to stimulating chloride secretion, UTP also inhibits chloride absorption. W1653 Protein Tyrosine Phosphatase N2 (PTPN2) Knock-Down Suppresses Epithelial Ion Transport By Promoting Epidermal Growth Factor (EGF) -Induced EGF- Receptor (EGFR) and Phosphatidylinositol 3'-Kinase (PI3-K) Activation Michael Scharl, Ivan Rudenko, Kim E. Barrett, Declan F. McCole Background: We have recently shown that the proinflammatory cytokine interferon gamma (IFNγ) activates the Crohn's disease (CD) candidate gene, PTPN2, in intestinal epithelial cells (IECs). The EGFr regulates a variety of cellular signalling events and IFNγ transactivates EGFr by increasing its tyrosine phosphorylation. PTPN2 has been shown to regulate EGF- induced PI3-K activation in fibroblasts. EGFr activation and recruitment of PI3-K play a key role in regulating Ca 2+ -dependent chloride secretion in IECs. Here we study a possible role for PTPN2 in modifying EGFr regulation of Ca 2+ -dependent chloride secretion in IECs. Methods: T 84 human colonic epithelial cells were used for all studies. Protein analysis was performed by Western blotting. PTPN2 knock-down was induced by siRNA. Electrophysiolo- gical studies were conducted in Ussing chambers. Results: EGF (100 ng/ml; 5 min) treatment of T 84 cells caused EGFr tyrosine phosphorylation (n=3; p<0.05), that was further enhanced by PTPN2 knock-down (16.3±1.2 fold vs. 45.6±7.4 fold of respective control; n=3; p<0.05). Analysis of specific EGFr phosphotyrosine residues revealed that PTPN2 knock-down pro- moted EGF-induced phosphorylation of Y 992 (n=3; p<0.05) and Y 1068 (n=5; p<0.05), but not of Y 1148 or Y 1173 . Loss of PTPN2 led to increased association of the PI3-K catalytic subunit, p110, with EGFr (n=3; p<0.05) and elevated phosphorylation of the downstream marker, Akt, in response to EGF (n=3; p<0.05). As a functional consequence, PTPN2 knock- down potentiated EGF (20 min; 100 ng/ml) inhibition of carbachol (100 μM) stimulated chloride secretion (n=8; p<0.05). Interestingly, loss of PTPN2 did not affect IFNγ-induced (24 h; 1000 U/ml) EGFr transactivation, nor EGF- or IFNγ-induced phosphorylation of extracellular signal-regulated kinase 1/2. Conclusion: Our data establish a role for PTPN2 in regulating EGFr signalling in IECs in response to EGF but not IFNγ. Knock-down of PTPN2 amplifies PI3-K activation and promotes the suppression of epithelial chloride secret- ory responses, a finding that corresponds to the known hyposecretory state seen in chronic intestinal inflammation. Our findings highlight the role of PTPN2 in regulating EGFr signal- ling events in IECs and suggest that PTPN2 dysfunction could contribute to ion transport suppression associated with CD. Supported by CCFA and NIH.