Localization of aquaporins in the proximal and distal colon

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Tumor Necrosis Factor ot (TNFa) Potentiates the Ion Secretion in Mouse Distal Colon Judith Cj Oprins, Claudia Van der Burg, Jack A. Groot, Swammerdam Institute for Life Science, Amsterdam Netherlands

Background:We have previously shown that TNF~ potentiates the ion secretion induced by muscarinic and histaminic receptor activation in HT29cI.19Acells. We obtained evidencefrom phospholipid analysesthat this was due to a stronger activation of protein kinase C (PKC) by upregulation of the phospholipaseD (PLD) route. In T84 cells, TNFa also upregulatedthe PLD pathway. However, in these cells the carbachol-induced ion secretion was not affected due to a lower expression of PKCa. The aim of this study was to investigate whether TNF~ had a potentiating effect on the ion secretion in nativetissue, and if so, via which mechanism TNF,~ exerts its effect. Methods:Short circuit current (1~) across mouse distal colon was measured in Ussing chambers. Tissue was exposed during 3 hours to 50 ng/ml TNF~ and compared with tissue without TNF~ incubation. Pmstaglandinsynthesis and neuronal activation were inhibited by 1/LM indomethacin and 0.3/~M TIX respectively.Amiloride (100/~U) was added to block mucosal Na+-intlux. After 3 hours, activation of the muscarinic receptor with carbachol resulted in an increase in I,o with 88 _+ 26 #A.cm 2. After 3 hours of exposure to TNFathe changein current was increased2.4 fold 1214-+ 25 mA.cm-2). A similar activation of the responseto histamine was observed (31 _+ 6 pA.cm2 histamine vs. 80 -+ 12 pA.cm2 TNFc~ + histamine). To investigatethe role of the PLD pathway propranolol was used to block the conversion of phosphatidic acid (the primary product of PLD activation) to DAG. Application of 100 ~.M propranolol to the mucosal side of the cells 15 minutes prior to application of carbachol, resulted in a diminished response (25 _+ 10/.~A.cm-2). Conclusion: TNF~xpotentiatesthe carbachol-induced ion secretion in mouse distal colon, resembling the effect in the cell line HT29cI.19A. The data suggest that the PLD pathway is also involved in carbachol-induced ion secretion in native tissue. We hypothesizethat also in native tissue TNFa exerts its potentiating effect via this pathway. An upregulation of the PLD pathway by TNFe could be involved in the disturbed pathophysiology occurring in patients suffering chronic inflammatory diseases,which are characterizedby elevatedlevels of TNFa and mast cell mediators. 2700 Expression of K+-Activated ATPase in Apical Membranes of the Human Distal Colon. Ravinder K. Gill, Univ of Illinois, VA Chicago: Westside, Chicago, IL; Biju P. Kunhiraman, VA Chicago: Westside, Chicago, IL; Seema Saksena, SangeetaTyagi, PradaepK. Dudeja, Univ of Illinois, VA Chicago: Westside, Chicago, IL Active K+ absorption in mammalian distal colon is energizedby an apical H+-K*-ATPase, a member of the gene family of P-type ATPases, but distinct from the gastric H+-K+-ATPase. The presenceof H+-K÷-ATPasehas previously been established in distal colon of rat, rabbit and guinea pig. However, the molecular identity of the transporter involved in active K+ absorption in the human colon is not known. The present study was, therefore, undertaken to establish the presenceof K+-activated ATPaseactivity in the human colonic apical membranes. Apical membranevesicles (AMVs) were isolated and purified from mucosal scrapings of organ donor colonic tissue using a differential centrifugation and divalent cation (Mg2+) precipitation technique. H+-K+-ATPaseactivity was measured in membrane preparations as a difference in the ATPaseactivity in the presenceof Mg2+ alone or in the presenceof Mg2+ plus K÷. Immunoblotting studies utilizing antibodies raised to a fusion protein derived from rat colonic H+-K+-ATPasecDNA detected a protein band with expected relative size of -110 Kda. The protein was only expressedin the apical human colonic membranesand almost no expressionwas detected in the basolateralmembranes. In the apical membranes,the expression of H*-K+-ATPase protein was substantially higher in the distal as compared to the proximal colon. In order to assess,the relativeabundanceof H+-K*-ATPasein various colonic regions, a semi-quantitative RT-PCR (using gone specific primers) was performed on total RNA extracted from pinch biopsy samplestaken from different regions of human colon. The relative abundance of human H÷-K+-ATPasemRNA was -1.5-fold higher in the descending colon compared to ascending and transverse colon. Further characterization of the human colonic H+-K+-ATPaserevealedthat the enzymatic activity associated with this protein was: i) significantly higher in distal human colonic AMV compared to proximal colonic region; it) saturable with increasing K* concentrations with a I~ for K÷ of 1.97 mM; iii) saturable with increasing concentrations of ATP; iv) inhibited by ouabain at concentrations of 1-3 mM; v) inhibitable by vanadate (100 ~M); vi) fully activated by Rb÷ and NH4÷ but not by Na÷ and Cs÷. Conclusion: Our data demonstratethe presenceof an H+-K+-ATPasein the human distal colonic AMVs and suggests that it may be involved in active K+ absorption in this region of the intestine. (Supported by Department of Veteran Affairs, NIDDK: DK 54016, DK 33349) 2701 A New Model of C02 Regulation of Colonic Na* Absorption Alan N. Cbarney, Richard W. Egnor, VA Medical Ctr, New York, NY; Valentin Zaharia, New York Univ Sch of Medicine, New York, NY; RamanashreeV. Gummakonda, VA Medical Ctr, New York, NY Background: C02 affects colonic Na÷ absorption by providing H* for apical Na÷/H+ exchange via a carbonic anhydrase(CA)-dependentprocess. We recently proposedthat CO2also affects the movementof NHE3-containingvesiclesto and from the apical membrane.This suggestion was based on an inverse relationship between ambient PCO2and the number of subapical vesicles observed in rat colonic epithelial cells by transmission EM. Methods: We tested for the presence of vesicle NHE3 by permeablizing colonic cells and exposing them to rabbit anti-NHE3 antibody and then goat anti-rabbit IgG conjugated to peroxidase. We tested for endocytosis by labeling the apical membrane of colonic cells with FITC-labeledphytohemagglutinnin and Cy-3-1abeledanti-NHE3 antibody and observing fluorescence as PC02 was lowered. We then examinedwhether wortmannin, an inhibitor of PI-3 kinase and exocytosis affected CO2-induced stimulation of colonic Na+ absorption. Ion transport across colonic

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segments was measured in Ussing chambers in HC03 Ringer under short-circuit conditions. Results: Immunoperoxidaselabeling revealed reaction product along microvilli and vesicle membranes indicating the presence of NHE3. We also found evidence of apical membrane endocytesis: apical membrane fluorescence was internalized when PC02 was reduced. This endocytosis was not prevented by increasing cell pH by other means or by inhibition of CA. In Ussing chambers, when bathing solution PC02 was increased from 21 to 70 mmHg in control tissues, J%s and j , a increased and I,, and PD decreased. In the presence of wortmannin, the C02-stimulated increment in J%= was markedly reduced: 5.3 _+ 0.8 (9) vs. 1.3 -+ 0.9 ~q.cm2.h -~ (8), PO.05, n = 6 per group). In contrast, administration of the adenosinetype A2b receptoragonist 5'-N-ethylcarboxamidoadenosine(NECA)evokeda chloride and concentration-dependentIsc-increase,which peakedafter 5 rain (L~Isc 16.4_+2.5, 10.2-+1.6, 6.5_+1.0, 2.8~0.7, 0.9-+0.2/~/cm2) following administration of 10-4, 10-5, 106,10-7,10-8 M of NECA, respectively, n=6 per group), and returned to baseline 10 min after NECAadministration. Semsal adenosine (lO-4M)-induced Isc-increasewas reduced by 62%, 40%, and 7% in the semsel presenceof 10-6, 10-7, and 10-8 M, respectively,of the neurotensin type 1 receptor antagonist SR 48692 (n=6 per group). In contrast, 10-6 M of the substance P-(NK-1)-receptorantagonist CP-96,345 did not have an effect on adenosineinduced Isc-increase.CONCLUSION:Our data show that an adenosinetype 2b receptoragonist stimulates chloride secretion in human colon in vitro. Our results showing that the high affinity neurotensin receptor is involved in the mediation of adenosine responses, indicate a "cross talk" betweenadenosineand neurotensin in the human colon leadingto a diarrheal response. 2703 Localization 01 Aquaporins in The Proximal And Distal Colon. Laurie E. Wallace, Carolyn O. Mahoney, Univ of Calgary, Calgary Canada;Edward V. O'Lcughlin, New Children's Hosp, New South Wales Australia; Donald G. Gall, James A. Hardin, Univ of Calgary, Calgary Canada The aquaporins are a family of membrane proteins that are thought to form water channels in lipid bilayers. Aquaporins have been identified throughout the gastrointestinal tract. Due to its prominent role in water reabsorption aquapofin expression was examined in colonic tissue. Aim: To examine aquaporin protein expression and localization in proximal and distal colon. Methods: NZW rabbits (1Kg) were fasted. Proximal and distal colonic segments were fixed in 10% formalin, cryosectioned and then probed with aquaporin (AQP) 1-9 antibodies using immunocytochemistry.Labelingspecificity was determinedby immunostainingsections in the absence of primary antibodies. Results: AQP1 staining was cytoplasmic and was associatedwith epithelial ceils in the upper crypt of the distal colon. AOP2 immunoreactivity was observed on the apical aspect of the surface epithelium of the proximal and distal colon. AQP3 staining was localizedto the basolateral aspect of the surface epithelium in the distal colon. AQP4 was expressedalong the basolateralaspect of the crypt and surface epithelium in both the proximal and distal colon. AQP7 staining was cytoplasmic and associated with scattered epithelialcells throughout the proximal and distal colon. AQP8 staining was basolateral on both crypt and surface epithelium of proximal and distal colon. No staining was observedfor AQP5,6 and 9 in colonic epithelium. Conclusions:Aquaporinsare localizedto the apicaland basolateralaspectsof the colonic epithelium.Thefindings suggestthat aquaporinsin the proximal and distal colon may play a role in transepithelial water transport. 2704 pH Stimulation of Ileal fie÷ Absorption Does Not Involve Membrane Trafficking Alan N. Chamey, Richard W. Egnor, RamanashreeV. Gummakonda, VA Medical Ctr, New York, NY Background: Na+ absorption in the rat ileum is specifically responsive to extracellular pH. Reductions in pH caused by increases in PCO2or decreasesin [HC03] in HC03 Ringer, and reductions in pH in HEPESRinger are equally effective in stimulating Na+ absorption. The mechanism of action of pH on ileal transport does not involve changes in bulk cytoplasmic