AGIO AGAABSTRACTS

3140MOUSE GUANYLIN AND UROGUANYLIN ARE PARADOXI­CALLY INCREASED IN TISSUE AND SERUM IN RESPONSE TOOSMOTIC DIARRHEA.Kris A. Steinbrecher, James T. Knapmeyer, Mitchell B. Cohen, Acad HospMed Ctr, Cincinnati, OH.

Guanylin (Gn) and uroguanylin (Ugn) are endogenous peptide hormonesthat are highly homologous to the diarrhea-causing E. coli enterotoxins.These putative secretagogues are released from intestinal epithelia into theintestinal lumen and systemic circulation. Once secreted, Gn and Ugn bindtheir transmembrane receptor Guanylate Cyclase C (GC-C), initiatingsignal transduction pathways that lead to fluid secretion in the intestine.However, there is little evidence to suggest that this pathway plays asignificant role in intestinal or systemic fluid homeostasis in the wholeanimal. In an effort to determine whether Gn or Ugn respond to changes inintestinal fluid balance, we hypothesized that a diet rich in lactose wouldosmotically draw water into the intestinal lumen of mice and cause acompensatory downregulation of GnlUgn. Mice were fed either a standardcontrol diet or a 75% lactose/25% control diet for either 2 days or 4 days.Mice were sacrificed and gut and carcass weights were obtained at eachtime point. Gn and Ugn levels were determined by Northern and Westernanalysis in intestine, kidney and serum. Animals fed the lactose diet for 2days developed overt diarrhea. weight loss and significantly increasedgut-to-carcass ratios. GC-C RNA levels were unchanged. Surprisingly. 2days on the lactose diet resulted in significantly increased Gn and UgnRNA and protein levels throughout the intestine and increased Ugn RNAand protein levels in the kidney. In addition, circulating levels of Ugn werealso increased at 2 days. These increases were specific to the lactose diet.No change was seen in GnlUgn RNA after 2 days of fasting or 2 days offluid deprivation. By 4 days, when the diarrhea had resolved, RNA levelsof both GnIUgn had returned to baseline. Protein levels remained elevatedonly in cecum and kidney. We conclude that this model of osmotic diarrhearesults in paradoxically increased levels of these peptide ligands in epithe­lia of the intestine and kidney as well as an increased release of Ugn intothe blood. These data are contradictory to current thinking concerning thephysiological role of these ligands in fluid homeostasis. We thereforespeculate that Gn and Ugn, or their prohormones, have a major functiondistinct from their potential roles as intestinal secretagogues.

3141THE OXIDANT MONOCHLORAMINE POTENTIATES AGO­NIST·STIMULATED CL SECRETION BY DIRECT ACTIVATIONOF BASOLATERAL MEMBRANE K CONDUCTANCE IN HUMANINTESTINAL T84 CELLS.Kazunori Sugi, Mark W. Musch, Anca Di, Deborah J. Nelson, Eugene B.Chang, Univ of Chicago, Chicago, IL.

Background: Monochloramine (MC), a physiologically important and cellpermeant oxidant, potentiates the secretory effect of both calcium- andcAMP-mediated secretagogues in T84 cells. This effect would significantlyamplify the effects of mucosal inflammation in causing net secretion andwatery diarrhea. Through indirect measures, we determined that the oxi­dant was affecting a Rb efflux pathway and hypothesized that was a resultof direct activation andlor alteration of the calcium-sensitivity of this Kchannel. In this study. we tested this hypothesis by performing whole cellelectrophysiological measurements in T84 cells under Ca-clamped condi­tions. Methods: Whole cell recordings of T84 cells were performed atroom temperature in a nominally Ca++-free NMDG-aspartate bath solu­tion. Current/voltage (IIV) relationships were determined in the cells priorto and following exposure to O.lmM MC in solutions in which K+ was theonly permeant species and its theoretical reversal potential being 81 mV.Internal solutions were buffered to either 0,40, or 100 nM Ca++ usingBAPTA or EGTA. Results: The addition of MC stimulated a currentpotential which reached a plateau after 2-5 min under Ca-free conditionswhere the mean current at 180 mV increased 28 fold. At 40 nM Ca++, anincrease of 31 fold was observed. Interestingly, the TEA-sensitivity of theK conductance was lost following addition of MC, whereas it continued tobe sensitive to the inhibitor quinine (lmM), possibly due to covalentmodification of the transporter. The electrophysiological finding thus con­firm previous observations made with measurement of Rb efflux. [Conclu­sions] The oxidant monochloramine potentiates the secretory effects ofboth Ca++ - and cAMP-stimulated secretion by a direct effect on calcium­activated basolateral K channel conductance. lowering its Ca-activationthreshold. This resulting increase in K recycling combined with agonist­stimulated increases in apical membrane anion conductance would mark­edly increase the magnitude of active secretion. This effect may play animportant role in amplifying and prolonging the secretory response ofinflamed intestinal mucosa and enhancing the severity of diarrhea.

GASTROENTEROLOGY Vol. 118, No.4

3142DIURNAL VARIATION IN SGLTI INDUCTION AND FUNCTION.Ali Tavakkolizadeh, Urs V. Berger, David B. Rhoads, Robert Shen, LynneL. Levitsky, Michael J. Zinner, Stanley W. Ashley, Edward E. Whang,Brigham & Women's Hosp, Harvard Med Sch, Boston, MA; Harvard MedSch, Boston, MA; MA Gen Hosp, Harvard Med Sch, Boston, MA.

Background: Diurnal variation in intestinal sugar absorption has beendescribed, but its underlying mechanisms are unknown. We hypothesizedthat this phenomenon maybe due to periodicity in SGLTI transcription andactivity. Materials and Methods: Rats in a 12 hour light-dark cycle (07:00-19:00) were sacrificed at 10:00, 16:00, 22:00, or 04:00. Proximaljejunum was mounted in Ussing chambers and ~I sc in response toaddition of 30mM 3-0-Methylglucose (3-0-MG) was recorded. 3H_Ia_belled 3-0-MG fluxes were calculated before and after addition of lOOIJ.Mphloridzin. SGLTl mRNA expression was evaluated using in-situ hybrid­ization. Results: 3-0-MG induced ~I sc and absorptive fluxes were sig­nificantly greater at 16:00 and 22:00 (Table I). These increases werephloridzin inhibitable. There was increased mRNA expression along theentire crypt-villus axes, including the upper third of the villi, at the abovetime points. At 04:00 and 10:00, SGLTl mRNA was confined to the cryptand lower villi. Conclusions: The diurnal variation in sugar absorption maybe due to periodicity in SGLTl transcription and function.

Table 1

TIme M..l!iAlcm2) Jm-. J~m J...

10:00 844±7.2 2.83±O.16 1.3?±O.30 1,4?±O,4116:00 1374±13.8· 4.22±O.25' 1.3?±O.15 2.86±O.33·22:00 136,4±12.8· 3.36±O.28. 1.10±O.15 2.26±O2204:00 1167±8.5 3.01±0.26 0.8?±0.0? 2.14±O22

• p<0.05 compared to10:00 using ANOVA and post-hoc Tukey testJm-', J.-m, Jne. measured in!lmole.cm'.hr'

3143EVIDENCE FOR TWO DISTINCT TRANSPORTERS INVOLVEDIN CHLORIDE ABSORPTION IN THE HUMAN COLONIC API­CAL MEMBRANE VESICLES.Sangeeta Tyagi, Reena J. Kavilaveettil, Shadwan Alsafwah, Krishnamur­thy Ramaswamy, Pradeep K. Dudeja, Univ of Illinois at Chicago, Chicago,IL.

Recent studies have indicated that mutations in human DRA (DownRegulated in Adenoma) gene might be related to Congenital ChlorideDiarrhea and that DRA may be responsible for intestinal apical Cl'-HC0:iexchange. The cDNA sequence for DRA is homologous to S04' transport­ers but not to anion exchangers such as AEs. Also, in-vitro functionalstudies of DRA have shown it to be capable of 504'. oxalate and CI'transport. Our earlier studies have established the presence of a distinctCI'-OH' (HC0:i) exchanger with minimal affinity for S04' . However, todate, the mechanism(s) of S04' transport across the human colonic apicalmembrane and the possible role of this transporter in oxalate and crtransport were not elucidated. The aim of our current studies was toelucidate the mechanism of S04' uptake in human proximal colonic apicalmembrane vesicles (AMV) and to define its interactions with oxalate andcr transport. Our results demonstrate the presence of a pH-gradient drivencarrier-mediated S04'-OH' exchange process in these membranes, basedon the following: i) a marked increase in S04' uptake in the presence of anoutwardly directed OR gradient, ii) significant inhibition of S04' uptakeby the anion exchange inhibitor DIDS, and iii) demonstration of saturationkinetics (Km for S04' : 0.80 ± 0.17 mM and Vmax: 649 ± 74 pmoVmgproteinll 0 sec). To elucidate the role of S04'-OH' exchanger in oxalate andCI transport, the effects of oxalate and CI- on S04' uptake were examined.The OH'- gradient driven S04' uptake was significantly inhibited byincreasing concentrations of Cl' (1-10 mM) in the incubation media with aK; for Cl of9.3 ± 1.4 mM, indicating lower affinity for chloride. Also, theS04'-OH- exchange was competitively inhibited by oxalate with a Kilower than for Cl. In contrast, the pH and HC0:i gradient-stimulated Cluptake was not inhibited by the presence of increasing concentrations ofS04' (5-50 mM). Also, S04'-OH- exchange activity was highly sensitiveto inhibition by DIDS compared to Cl'-OH' (HC0:i) exchange with a lowsensitivity. These data strongly suggest that the S04'-OH'and CI'-HC0:iexchangers are distinct transporters. Based on inhibitor profile and sub­strate affinities for various anions, we speculate that two distinct transport­ers may be involved in Cl' transport in the human intestinal luminalmembranes: i) an anion exchanger with high affinity for S04' and oxalatebut low affinity for Cl; ii) a distinct Cl'-OH' (HCO;) exchanger with lowaffinity for S04'.