NHE2 is the predominant pH1-regulator in murine colonic crypt cells

all three retoviral vectors and GFP+ cells isolated by flow cytometry. GFP protein was readily detected in all three pooled cell lines. Cdxl protein was observed only in the HCT116/MIGR1~ x l cells. Endogenous Cdx2 protein was weakly detected in both HCT116/MIGR1 and HCTt16/MIGR1-Cdx1 cell lines, but high Cdx2 levels were only observed in the HCT116/ MIGR1-Cdx2 cells. Transfection of a Cdx-responsivapromoter-reporter construct into these cell lines demonstratedactivation of the reporter only in the Cdx-expressinglines. Preliminary studies of DNAcontent and DNA synthetic rates suggestsCdx2 is superior to Cdxl at inducing a GO/G1 accumulation and reducing DNA synthesis in HCT116 cells. CONCLUSIONS:The MIGR1, MIGR1-Cdxl, and MIGR1-Cdx2 retroviral vectors infect a wide number of human colon cancer cell lines efficiently and yield high levels of gene expression. They provide a useful means of rapidly generating cell lines with high levels of Cdxl and Cdx2 protein expression. The effects of Cdxl and Cdx2 expression on the differentiation and proliferation of human colon cancer ceils can now be explored more comp~eteJy.

3775 NHE2 Is the Predominant pHi-regulator in Murine Colonic Crypt Cells. Oliver Bachmann, Heidi Rossmann, Katrin Wuechner, Med Dept I, Tuebingen Germany; Juergen Strobel, Roche Diagnostics, PenzbergGermany; Michael Gregor, Ursula Seidler, Med Dept I, Tuebingen Germany Background&Aim: The Na÷/H÷ exchangerisoforms NHE1, NHE2 and NHE3 are expressedin murine colon, but their role in the regulation of colonocyte intracellular pH has not been clearly defined. In particular, the functional significance of NHE2 has remained obscure. The aim of this study was to quantify the contribution of the different NHE isoforms to pH, regulation in murine colonic crypts and surface cells. Methods: Epithelial crypts and surface cells were isolatedfrom murine proximal colon, and intraceilularpH was monitored floorometrioally with the pH-sensitiva dye BCECF.Na*/H * exchange was activated by an intracellular acid load using the NH4÷ prepulse technique, and by applying a hyperosmolar solution. The contribution of the different NHE isoforms was assessed using increasing concentrations of the NHE-specifiocompound HOE642 (1/~M inhibits NHE1, 50/~M inhibits NHE1 and NHE2) and 700/~M di-methyl-amiloride (DMA, inhibits all NHE isoforms in murine colon). NHE 13 expression levels were determined by semiquantitativePCR using 18s rRNA as an internal control. Results: In crypts, the total Na+-sensitivaacid extrusion rate after acidification in Oz/ HEPES buffered medium was 18.7-+1.7 mM/min, while in surface cells, the flux rate was 9.31 -+0.29 mM/min. Sequential inhibition of the different isoforms showed that in crypts, NHE1 is responsible for 40% of acid-induced Na÷/H*-exchange rate, NHE2 for 50%, and NHE3 for 10%, whereas in surface cells, NHE1 and NHE2 mediate 20% each and NHE3 mediates 60%. Hyperosmolar solution (400 mosm) caused a proton flux rate of 0.97-+0.07 mM/min, which was reduced by 40% by NHE1 inhibition. The rest was inhibited by NHE2 inhibition. Expression studies showed that NHE1 is almost equally expressed in crypts and surface cells, whereas NHE2 is more abundant in crypts than in surface cells and NHE3 expression levels are higher in surface cells than in crypts. Thus the expressionpattern goes in parallel with the functional results. Conclusion: In murine colon, NHE1, NHF_2and NHE3 participate in pH regulation. Taken together, our results indicate that NHE3 is of particular importancefor pH, regulation in routine colonic surfacecells, whereasNHE2is the predominant pH, regulator in crypts.

3778 Na÷-Glosoon Cofronspofl Coosos Oocfoaosd Traosmucosal Resistance and l i ~ Pkosldim~ofJon of Perljonctional Myosin II Regulatory Light Chain in H u m Jeimm. Jessica J. Berolund, Wayne State Univ, Detroit, MI; Martin Riegler, EtienneWenzl, Vienna Gen Ho~o, Vienna Austria; Jerrold R. Turner, Wayne State Univ, Detroit, MI Na*-nutrient cotransport-dapendant regulation of intestinal permeability occurs in intact rodents, isolated rodent mucosae, and human intestinal epithelial cell lines. This regulation requires phosphorylation of myosin II regulatory light chain (MLC). However, Na+-nutrient cotransport-depondantregulation of human intestinal permeability remains controversial.The goal of these studies is to evaluatethe effects Na+-nutrient cotransport on intestinal permeabiF ity and MLC phosphorylationin human jejunal enterocytes.METHODS:Normal jejunal mucosa isolatedduring surgery for unrelatedintestinal diseasewas mounted in Ossing chambersand equilibrated with physiological buffer containing 3mM glucose and 22mM mannitol. After 20rain stabilization,the apical buffer was exchangedfor heifer with 25mM glucose or 3mM glucose, 22raM mannitol and 2mM phloridzin (an inhibitor of Na+-glucose cotransport). Potential difference and short circuit current were measured. Sections (5p.m) of snap frozen mucosa ware postfixed in 1% pamformaldehydeand immunostainedusing polyclonal affindypurified goat anti-total MLC antibody and a separate polyclonal affindy-puritied rabbit antiphosphorylated MLC (pMLC) antisera. The latter reacts with ser-19 pMLC quantitativelyand does not recognizenonphosphorylatndMLC. The relative pMLC content was determinedusing pixel-byopixelnormalizationof the anti-pMLC signal to the anti-total MLC signal using ImegePro 4.1 software. RESULTS:During the first 15rain after activation or inhibition of Na÷.glucose cotransport, transmucosal resistanceschanged progressively until the resistance of mucosae without active Na°-glucose cotransportwas 37%+_9% greaterthan that of mucosaewith active Na*-glucose cotranspart (p
3776 Specificity of Receptor-Mediated Signal Traosduclion by E. ¢oli Type Ila HeM-Labile Toxin Susan Wimer-Mackin, Children's Hasp, Boston, MA; Randy K. Holmes, Univ Colorado Health Science, Denver, CO; Anne A. Wolf, Wayne I. Lencer, Children's Hasp, Boston, MA; Michael Jobling, Univ Colorado Health Science, Denver, CO Heat-labileenterotoxinLTIla and choleratoxin (CT) are structurally relatedAB5 toxins produced by Escherichia coil and Vibrio cholerae, respectively.In polarized human intestinal T84 cells, CT binds specifically to gangliosida GM1 and traffics retrograde into the ER to induce a toxic response. Binding of the related E. coil LTIIb toxin or a chimeric CT to ganglioside GDla does not elicit signal transduction in T84 cells (JCB 141:917, 1998). We found that toxin binding to GM1 (but not GDla) associated CT with detergent-insolublemembrane microdomains (DIGs), and this correlatedwith toxin function. Basedon these data, we proposed that toxin associationwith lipid rafts was necessaryfor its trafficking efficiently into Gotgi cistemae and ER of target epithelial cells. AIM: In the present studies, we characterizedthe role of specific gangliosidesas functional receptors for LTIla in T84 cells. METHODS:Toxin binding and function were assessed in T84 cells using cell surface ELISA and standard methods of electrophysiology. RESULTS:LTlla bound T84 apical membraneswith high affinity. After a lag phase, LTlla induced a CI- secretory responsesimilar to that induced by CT but attenuated in magnitude.When purified gangliosideswere immobilizedon plastic, CT bound preferentially to GM1, but LTlla bound both to GDlb and to GMI. Excess CTB subunit inhibited the CI secretory response induced by CT but not by LTIla. Furthermore, an LTIla-T141 variant that binds GM1 but not GDlb in vitro was unable to bind to T84 cells. Thus, GM1 cannot function as a functional receptor for LTlla. We found that LTIla bound to a cell surface receptor that was sensitive to neuraminidase and to competition by tetanus toxin C*fragment. These data are consistent with the idea that GDlb is a functional receptor for LTlla. Extraction in 1% triton X-IO0 and fractionation at 4°C showed that LTlla bound to human intestinal T84 cells or mouse Y1 adrenal cells was associatedwithlipid rafts and, as for CT, that this association correlated with toxin function. Thus, the LTIla receptor (presumably GDlb) in T84 cells, like the GM1 receptorfor CT, can drive toxin associationwith lipid rafts and mediatethe retrograde trafficking into the secretory pathway that is required for a functional responseto the toxin.

3779 Calctum-DopmdeM Stimulation Of The p44/42 Mitogen-Activated Protein Kinase Pathway By Acetyichoflne in isolated Human Colonic Crypts Susanne M. Lindqvist, Univ of East Anglia, Norwich United Kingdom; James M. Hereon, Norfolk and Norwich Hasp, Norwich United Kingdom; Paul A. Sharp, Univ of Surrey, Guilford United Kingdom; lan T. Johnson, Institute of Food Research, Norwich United Kingdom; Jean Mackay, Richard Tighe, Michael Rhodes, Norfolk and Norwich Hasp, Norwich United Kingdom; Mark R. Williams, Univ of East Anglia, Norwich United Kingdom BACKGROUND:The constant rapid renewal of the colonic epithelium is critical to the maintenance of normal colonic function. There is a striking spatial correlation betweenacatycholine (ACh)-induced colonic crypt calcium signals and the differentiation pathway representedby the colonic crypt-axis (Lindqvist et al., 1998). ACh initiates a calcium signal in the stem cell region located at the crypt-base that spreads along the crypt-axis, through the zone of proliferation and into the differentiated surface epithelial cells. Activation of the mitogenactivated protein (MAP) kinase pathwayis thought to be important in regulating colonic crypt cell growth, differentation and secretion. The aim of the present study was to investigate ACh-induced activation of the i)44/42 MAP kinase isoforms along the isolated human colonic crypt-axis. METHODS:Biopsy samples were obtained from the distal colon at colonoscopy (Ethics Approval) and placed in calcium-free, hepes-huffered saline (HBS) for one hour, Vigorous shaking liberated isolated crypts that were affixed to collagen-coatedcoverslips. Crypts were loaded with the calcium-sensitive dye Fura2 (1 /~ M) and placed on the stage of an inverted microscope coupled to a fluorescence imaging system (Photon Technology International). Followingcalcium imagingexperiments,crypts werefixed in 4% (w/v) paraformaldehyde in phosphate-bufferedsaline and labelled wit/] a phospho-p44/42 MAP kinase antibody in combinationwith a FITC-conjugatedsecondaryantibody. Indirect immunofluorescence was viewed by laser scanning confocal microscopy (Biorad 250 DVC). RESULTS:Concentrations of ACh (>100 nM) elicited a dose-dependantrelease of intracellular calcium (EC50 = 10/~ M) that was inhibited by atropine (1 /~ M). Brief stimulation with ACh (10 ~ M, 5 min.) was sufficient to activate p44/42 MAP kinase along the human colonic crypt-axis. Second messenger-independentmobilisation of intracellular calcium by thapsirgargin (300 nM) also activatedp44/42 MAP kinase.The MAP kinaseIdnaseinhibitor PD 98059 (50 p. M) attenuated the effects of both ACh and thapsigargin on MAP kinase phosphorylation. CONCLUSIONS: Stimulation of acetylcholine muscarinic receptors activates p44/42 MAP kinase in human colonic crypts via a calcium-dependentmechanism.Thesedata implicateacetylcholine-indocad colonic crypt calcium waves in the co-ordination and maintenanceof large intestinal epithelial cell growth and differentiation.

3777 Retroviral Mediated Cdxl or Cdx2 Expression in Human Colon Cancer COIl Lines and the Effects on Cell Growth, Matthew S. Keller, Peter G. Traber, John P. Lynch, Univ of Pennsylvania,Philadelphia, PA INTRODUCTION:Cdxl and Cdx2 are homeodoraaintranscription factors that are necessary for the correct expressionof intestine-specificgenes.They may also act to modulate proliferation. The antiproliterative effect of Cdx2 has not been well studied, and the growth effects of Cdxl have been studied, but the literature is currently conflicted. One limitation has beenthe difficulty in inducing Cdxl or Cdx2 expression in cell lines. Thus we sought to develop a method of reliably inducing Cdxl or Cdx2 expression in colon cancer cell lines that permits rapid identificationof and testing expressingcells. METHODS:We utilizedthe retroviral plssmid MSCV MIGR1 vector to deliver Cdxl or Cdx2 expression. This vector contains an internal ribosomal entry site (IRES)from the encephalomyocarditisvirus (ECMV).Thegreenflourescent reporter protein (GFP) is 3'to the IRES. We subcloned Cdxl or Cdx2 in this retovirel vector and then determined biochemical and protein expressionlevel in multiple cell lines, with the empty vector serving as a control. RESULTS:DLD1, HCT116, Cola DM, Cola 205, LS174T, LaVa, and SW480 colon cancer cells were infected easily with the MIGR1 retroviral vectors and GFP expressionwas high. HT29 and T84 cells infected well, although less efficiently than the other cell lines, and GFP expression was only modest. HCT116 cells were infected with

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