Divergent functions of apical membrane Na−H exchanges in apical membrane of surface epithelial cells of rat colon

extrusion rate(0.43_+O.04pH units/min,(n-25). To confirm that we were affecting NHE an additional series was conducted in the presence of either Amiloride(lmM) or the amiloride analogue EIPA (2/~M). In both pH,recovery was completely inhibited under all experimental conditions, recovery was O.O0_+O.OtpH units/min. We conclude that the dista~ colon has a functional divalent cation receptor that has direct interactions with basolateral NHE activity. Furthermore, activation of this receptor leadsto net proton efflux from cell to blood. Modulation of transport proteins via the divalent cation receptor should provide new insights into the maintenance of body calcium ion homeostasis. 2694 Role of Apical Anion Exchangersin ph~ RegulaUooIn Polarized Rat Distal Colonic Surface Cells Krishnan Selvi, Vazhaikkurichi M Rajendran,Yale Univ, New Haven, CT; Satish K. Singh, Boston Univ, Boston, MA Background/Objectives:In rat distal colon, CI absorption is electroneutraland believedto be due to CFanion exchangers.CI-HCO3,CI-OH, and CI-SCFAexchangeshave been identified in apical (AP) membrane vesicles. In intact polarized epithelial cells, vectorial ion transport processes that also move acids and bases can be characterizedby monitoring intracelluiar pH (pHi). We sought to determine the presence and impact, if any, of AP CI-HC03, CI-OH and/or Cl-hutyrate exchangeson pHi. Methods: We utilized an optical Useing-type chamber to image polarized surface-cells in situ while independentlysuperfusing serosol and mecosal aspects of a sheet of rat distal colon. Following alkali loads, we assessed AP W-loading recovery by analysis of digitized images of the pH-sensitive dye, BCECF,to obtain pHi vs. time records from individual surface cells. Experimentswere performed at 37°(;, pH 7.4 in a Ringer solution buffered either with 32 mM HEPES(HCO3-frea)or 5% C0-]22 mM HC03 Isotonic mannitol bathed the serosa throughout all experiments. Results: Whether in the absence or presence of HC03, removing mucosal CI rapidly alkalinized surface cells by 0.30 pHi units. Upon reintroducing CI, pHi rapidly returned to baseline, but was blocked by DIDS (500/~M), consistent with CI-OH and/or CI-HC03exchangeactivity at resting pHi. However, when we inhibited carbonic anhydrase and minimized endogenous HC03 production with ethoxyzolamide (10/~M), CI removal no longer produced an alkalinization in the absence but still did in the presence - of HCO3.We next alkalinizedcells with a propionic acid pulse under HCO~-freeconditions plus ethoxyzolamide.Recovery from this alkaline pHi required either (a) CI and HC03 together or (b) CI and Na-butyrate (5 raM) together. 80th of these acidifying recoverieswere blocked by DIDS. Conclusions: The apical membrane of rat distal colon surface cells possesses (a) pHi dependent, stilbenc-sensitive CI-HC03 exchange and (b) pHi-dependent,stilbene-sensitive,HCO3-independentCI-bstyrate exchange,both of which can participate in pHi regulation. A CI-OH exchangeprocess described in membranevesicles may be present but does not appear to function in the control of intracellular pH.

membranes (AJP276:G359, 1999). Previousstudies in which cAMP inhibited HCO3-dependent Na absorption, hut not butyrate-dependentNa absorption (the tatter was a surprising and unexplainedobservation) raisedthe possibility that distinct (and different) NHE isoforms were required for HCO3-dependentand butyrate-dependentNa absorpion. Since prior studies have linked NHE-3 isoform to HCO3-dependentNa absorption, we proposed that NHE-2 and NHE3 isoforms were selectively linked to butyrate- and HCO3-dependentNa absorption, respectively, and in these present studies this possibility was tasted by using HOE694,an amiloride analogue that selectively inhibits NHE-2, but not NHE-3 isoforms. Unidirectional ~Na fluxes were performed under voltage clamp conditions in HCO3-free,HCO3-Ringerand butyate-Rioger solutions. One mM amiloride inhibited net Na absorption in both HC03- and butyrate-Ringer solutions, while 10 pM EIPA,an amilorideanaloguethat inhibits electroneutralNa-CIabsorption (but does not alter electrogenic Na absorption) preventedthe increase in net Na absorption induced by the additon of HC03. In contrast, 50 p.M HOE694, a concentration that inhibits NHE-2, but not NHE-3 isoforms, did not prevent HCO3-stimulationof net Na absorption (4.2 -+ 2.0 vs 3.6 -+ 1.3 pEq/h.cm2). However, 50 pM HOE694 completely inhibited butyratestimulation of net Na absorption (0.8 ~- 0.8 vs 5.0 -+ 0.8/~Eq/h.cm2). These results indicate that butyrate-dependentNa absorption is linked to NHE-2 isoform, while NHE-3 isoform is responsible for HCO3-dependentNa absorption. The failure of cAMP to inhibit butyratedependentNa absorption is consistentwith cAMP inhibition of NHE-3,but not NHE-2isoforms. 2697 SDmolation of NHE2-modiated H*-extrusioe by cAMP in Rat Distal Colon Selvi Krishnan, Vazhaikkurichi M. Rajendran, Henry J. Binder, Yale Univ, New Haven, CT; Satish K. Singh, Boston Univ, Boston, MA Background: In rat distal colon, electroneutralNa absorption and intracellular pH (pHi) regulation are mediated by Na-H exchange(NHE); molecular studies have identified NHE2and NHE3 isoforms in apical (AP) membranes in rat distal colon. Recently, we found that (a) HCO3dependentetectroneutralNa absorption is mediated by NHE3 and blocked by cAMP while (b) butyrate-dependentelectroneutral Na absorption is mediated by NHE2 and not inhibited by cAMP, providing a possible mechanismfor the higher efficacy of SCFA-generatingoral rehydration solutions in cholera. Objective:To determine whether cAMP affects the roles of NHE2 and/or NHE3 in pHi homeostasis in rat distal colon. Methods: We utilized an optical Ussingtype chamberto image polarizedsurface-cells in situ while independentlysuperfusing serosel and raocosal aspects of a sheet of rat distal colon. Following NH~CI-inducedacidification, we H'-extrusion by analysis of digitized images of the pH-sensitive dye, BCECF,to obtain pHi vs. time records from individual surface cells. We monitored the effect on AP Nadependent alkalinization of (a) specifically inhibiting NHE2 with HOE-694 (50 ~M) and (b) functionally inhibiting NHE3 but not NHE2with DBcAMP(1 raM). Experimentswere performed at 37°C, pH 7.4 in a HCO~-freeHEPES-bufferedRinger. isotonic mannitol bathed the serosa in all experiments. Results: Recoveryfrom H*-Ioads required mucosal Na and was blocked by amiloride (1raM), consistent with Na-Hexchange.Specificallyinhibiting NHE2with HOE694 (50 pM) decreasedthe rate of AP Na-dependentH+-extrusion by 60%. In contrast, inhibiting NHE3 (but not NHE2) with DBcAMP decreasedthe rate of Na-dependentH*-extrusion by only 18%. Thecombination of cAMP and HOE-694blockedH* -extrusion completely,consistent with a 36% increase in NHE2 activity over baseline NHE2 activity when NHE3 is inhibited with cAMP. Conclusions: (a) Under basal conditions, both NHE2 and NHE3 can contribute to pHi homeostasis,with NHE2 dominating. (b) When NHE3 is inhibited by cAMP, a substantial increase in NHE2 activity mediates compensatory H--extrusion. We speculate that SCFAassociatedhomeostaticH--extrusion stimulates compensatoryNHE2activity that, unlike NHE3 activity, is not inhibited by e~evatedintracellular rAMP levels.

2695 Primary Role of Na Deficit and Secondary Hyperatdosterenlsm in Altered Epithelial Function After Total Prectocolestomy(TPC) in Rats Kouhei Fukushima, Shun Sato, Taku Kitayama, Hitoshi Yonezawa,Kaori Koyama, Hiroo Naito, Yuji Funayama,Chikashi Shibata, Kanichi Takahashi, Tatsuya Ueno, Aldhiko Hashimoto, Iwao Sasaki, Tohoku Univ Graduate Sch of Medicine, Sendal Japan Background & Aims: We demonstrated that TPC resulted in remarkable increase of plasma aldosterone (Aid), Aid-mediated Na absorption and mRNA induction of 11~hydroxysteroid dehydrogenasetype 2 (11~HSD2), which determines the specificity of Aid to its receptor (AJP:276:G975:00, GE:116:A1349:99). The aims of the present study were to investigate whether or not hyperaldosteronismaccompanied with Na deficit rather than other factors associated with surgical manipulation, TPC, has a primary role for enhanced Aid-mediated Na absorption and induction of 11~HSD2 mRNA in the small intestine. Methods: Male Sprague-Dawleyrats were treated with Na depletion or control diet for 4 weeks, sacrificed and plasma was separated for measurement of Aid concentration. Terminal ileal mucosae were mounted in Ussing chamber and short circuit current (Isc) were measured under the stimulation with Aid (10-7M) for 8 hrs. At the end of experiments, amiloride was added and decrease of Isc was recorded. Alternatively, RNA was extracted from kidney and epithelial cells (EC) isolated from upper, middle and lower small intestine. Expression of 11p-HSD2 mRNAwas investigatedby northern blotting. Results:Sodium depletedrats exhibitedcomparable level of plasma Aid to that of TPC rats. When control mucose was examined,Aid had a minimal effect on Isc. In contrast, elevationof Isc of Na depleted rats was remarkably higher than that of control. P significant increase of amilofide-sensitive Isc was also observed, suggesting that Aid enhanced amiloride-sensitive Isc in the terminal ileum of Na depleted rats. Those changes were mediated by AId-mineralocorticoid receptor binding since the elevation of Isc were completely inhibited by the pre-incubation with spironolactone. 11,8HSD2 mRNA was not detected in EC from upper and middle small intestine of both control and Na depleted rats. On the other hand, 11p-HSD2 mRNA was clearly induced only in the lower small intestine of Na depleted rats but not in the kidney. Conclusion: Secondary hyperaldosteronism developed by Na depletion diet resulted in enhanced reactivity to Aid, associatedwith 11,8-HSD2mRNAinduction in the terminal ileum as well as by TPC.In contrast, in the kidney, 11~HSD2 mRNA was induced only in TPC rats as previously demonstratedbut not in rats treated with Na depletiondiet, suggestingthat other factors associatedwith surgical manipulation possibly affect induction of 11~HSD2 mRNA in the kidney of TPC rats.

2698 The Tachyidnins Substance P (SP) and Neurokinin A (NK-A) Induce intestinal Secretion in Guinea Pig Tissue, Which Can Be 8locked by GR-203040, a NK-1 Specific Antagonist Silke Schwengberg, Detlef Wermelskirchan, Janssen ResearchFdn, Neuss Germany Background: In the gastrointestinal (GI) tract, tachykinins are important neurotransmittersof the enteric neurons that are intrinsic to the gut and control various aspects of digestive function, e.g. motor activity and secretion. Their effects in the GI tract are mediated by 3 different receptor subtypes named NK-1, NK-2, and NK-3, which belong to the group of Gprotein coupled receptors. SP binds preferentiallyto NK-1, NK-A to NK-2, and NK-B to NK3, but each of the tachykinins is a full agonist of all 3 receptor subtypes. Aim: To investigate whether SP and NK-A can induce secretion in the guinea pig colon, and whether this secretion can be blocked by receptor-specific antagonists. Methods: Guinea pig colonic muoose was mounted in Ussing chambers. Secretion is expressed as changes in short circuit current (Alsc; pNcm2). After equilibration for 30 min, a dose responsecurve of the tachykinins was performed. To enhance receptor-specific binding, (AlaS-/~AlaB)NK-Awas used instead of the natural peptide. In further experiments,the tissue was pretreatedwith different concentrations of specific antagonists 30 min before the tachykinins are added. Results: Secretion was induced dose-dependentlyby SP and NK-A. SP was more potent (maximal L~lsc = 179 pN cm2 at 1E-8M) than NK-A (maximal .~ Isc = 148p.A/cm2 at 1E-5M). The action of SP was strongly blocked by the NK-1 antagonist GR-203040 (80% inhibition at 1E-9M). The effect of NK-A was blocked by the same antagonist with a similar potency, although GR-203040 was shown to be NK-1 specific in receptor binding assays. In contrast, the NK-2 specific antagonist SR-48968 inhibited NK-A induced secretion signiticantiy only at 1E-6M, whereas in human receptor binding studies a plCSOof 8.4 was obtained. SR-48968 has no effect on SP inducedsecretion (not significant at 1E-6M). Conclusion: Using a functional assay,intestinal secretion from guinea pig mucosecan be induceddose dependentlyby SP and NK-A.Although the NK-A peptide is claimedto be NK-2 specific, the secretion induced by NK-A can be blocked effectively by the NK-1 receptor antagonist GR-203040. This indicates that in guinea pigs, NK-A induced secretion may be mediated by the NK-1 receptor.

2696 Divergent Funstions of Apical Membrane Na-H Exchanges in Apical Membrane of Surface Epithelial Cells of Rat Colon Krishnan Selvi, Vazhaikkurichi M. Rajendran,Henry J. Binder, Yale Univ, New Haven, CT Absorption of both HCO3-dependentand botyrate (a model short chain fatty acid)- dependent electroneutral Na-CIin the rat distal colon requiresan apical membraneNa-H exchange(NHE). Recent studies have identifiedtwo distinct NHE (NHE-2, NHE-3) isoforms in rat colonic apical

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