1 Physiologisches Institut der Ludwig-Maximilians-Universität, D-80336 München, Germany
2 Max von Pettenkofer-Institut der Ludwig-Maximilians-Universität, D-80336 München, Germany
Correspondence
Simon D. Lytton
Simon.lytton{at}t-online.de
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ABSTRACT |
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Present address: SeraDiaLogistics, Hertlingstr. 1, 81545 München, Germany.
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INTRODUCTION |
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In this study, we set out to investigate if the exposure of epithelial cells to physiological concentrations of NH4Cl and H. pylori-derived NH3/ alters their tight-junction behaviour and if so, to what extent the effects are manifest at the level of tight-junction protein expression. In previous work, ammonium treatment of Madin-Darby canine kidney (MDCK) cells was found to be associated with the expression of low-molecular-weight (LMW) occludin (Vastag et al., 2005
), and the expression levels of both claudins and occludin have been shown to influence tight-junction function in human colon carcinoma cell lines (Li et al., 2004
; Bojarski et al., 2004
). The aim of the present study was to assess the time-course of the effects of ammonium on Caco-2 cell tight junctions and to determine if the ammonium derived from H. pylori metabolism of urea modifies the expression of occludins. The rationale was that the H. pylori-mediated disruption of gastric epithelial tight junctions is linked to the formation of a LMW occludin that is dependent on bacterial urease activity and the production of ammonium. This proposed mode of action provides an alternative to the vacuolating cytotoxin VacA, or the translocation of the bacterial protein CagA, encoded by the cag pathogenicity island (cag-PAI), both of which have been shown to influence TER (Amieva et al., 2003
; Guillemin et al., 2002
; Papini et al., 1998
). In this work, we show that the reduction of TER by H. pylori has an ammonium-specific component that is independent of VacA and CagA. We also demonstrate that the level of ammonium produced by H. pylori is correlated with the expression of LMW occludin. These results suggest that H. pylori-derived ammonium exerts a rapid and specific change in the production of LMW occludin that coincides with the disruption of tight-junction function.
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METHODS |
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Cell cultures.
Cells of the human colon carcinoma epithelial cell line Caco-2 (ATCC clone HTB-37) were obtained from the laboratory of Professor Gstraunthaler (Institut für Physiologie Innsbruck, Austria) and cultured in Minimum Eagle's alpha Medium (Invitrogen) supplemented with heat-inactivated fetal calf serum (20 %), L-glutamine (2 mM), non-essential amino acids (2 mM), penicillin and streptomycin (100 U ml1 each), and amphotericin B (2·5 U ml1). Caco-2 cells between passages 4 and 6 only were grown on semipermeable transwell tissue-culture inserts of uncoated polycarbonate (8 µm pore size, 10 mm diameter, Nunc) or on collagen- and fibronectin-coated cover slips (12 mm, Marienfeld GmbH, Lauda-Koenigshofen, Germany). In addition to the Caco-2 cells mentioned above, Caco-2 cells of passage 85, MDCK cells of passages 2730, and human lymphoid T cell line Jurkat (gift of Professor G. Hacker, Institute for Medical Microbiology, Technische Universität München) were grown in plastic Petri dishes (15 mm, Griener Bio-One) and flasks (75 cm2, Nunc). The confluence of epithelial cell monolayers was assessed by phase-contrast microscopy.
Bacterial strains and culture conditions.
The H. pylori wild-type strain P12 and its isogenic mutants were grown on GC agar plates (Difco) supplemented with horse serum (8 %), vancomycin (10 µg ml1), trimethoprim (5 µg ml1) and nystatin (1 µg ml1), and incubated for 2448 h in a microaerobic atmosphere (85 % N2, 10 % CO2, 5 % O2). The P12ureA mutant was constructed by allelic replacement mutagenesis with plasmid pBS1, containing a TnMax8 transposon insertion in the ureA gene (S. Odenbreit and others, unpublished results); P12
vacA
cagA has been described previously (Gebert et al., 2003
).
Preparation of cell extracts and culture supernatants.
Confluent Caco-2 cell monolayers were infected by inoculation of bacterial suspensions into MEM medium supplemented with urea (2·515 mM) at a m.o.i. of 50. After 1624 h incubation at 37 °C in humidified CO2 (5 %), the Caco-2-infected monolayers were washed twice in 5 ml ice-cold PBS and immediately frozen at 70 °C in lysis buffer [1 % Triton X-100, 20 mM HEPES, 150 mM NaCl, 1·5 mM MgCl2, containing protease inhibitors leupeptin (2 µg ml1), sodium vanadate (2 mM), PMSF (4 mM), pepstatin (1 mM), aprotinin (10 µg ml1) and benzamidine (5 mM)]. The thawed lysates were sonicated (5x15 s) on ice. Protein from the pellet and supernatant fractions after centrifugation (13 000 g for 45 min) was measured by Bio-Rad Assay Dye Reagent and analysed on SDS-PAGE immunoblots. The medium from H. pylori/Caco-2 cell co-cultures was centrifuged (4000 g for 30 min), the pH was recorded, and the filtered (0·2 µm, Millipore) supernatants, referred to as co-culture supernatants (ccs), were kept frozen at 20 °C.
Ammonium determination.
Ammonium concentrations in the ccs were measured on an automated clinical chemistry analyser (ARCHITECT c8000, Abbott Laboratories) using the ammonia assay (Roche Diagnostics), an enzymic method based on glutamate dehydrogenase and the NADPH-dependent conversion of 2-oxoglutarate to L-glutamate (Kirsten et al., 1963).
Patient tissue sampling.
Endoscopic biopsies from patients with stomach carcinoma (n=9) or patients without gastric carcinoma (n=3) were obtained from defined locations in the antral or corpus mucosa or the tumour site (Rieder et al., 2001) in accordance with the Ethics Committee approval of Ludwig-Maximilians-Universität, München, project no. 240/01. Specimens were immediately frozen in liquid nitrogen and stored at 70 °C. Freshly thawed specimens (approx. 1 mmx1 mm) were transferred on ice to 2 ml lysis buffer and homogenized four times with a Dounce teflon homogenizer. The Triton X-100-soluble extracts were analysed on SDS-PAGE immunoblots. The H. pylori status of each patient was determined by assay of urease activity in gastric biopsies and by testing of patient sera for H. pylori-positive IgG and IgM antibodies.
SDS-PAGE and immunoblotting.
SDS-PAGE was performed by the method of Laemmli (1970). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (Amersham Biosciences) in a tank mini-blot apparatus (Scie-Plas Biochrom Ltd, Southam, UK) at constant current (45 mA for 16 h at 4 °C). Unreacted sites of the nitrocellulose membrane were blocked (2 h at room temperature) in non-fat milk (5 %) in PBS/Tween 20 (0·1 %), pH 7·4. The nitrocellulose membrane was then incubated with the anti-occludin or anti-ZO-1 antibodies at 1 : 2000 dilution for 3 h at room temperature and washed three times with PBS/Tween 20 (0·1 %), pH 7·4. The horseradish peroxidase (HRP)-conjugated secondary antibodies goat anti-mouse IgG or goat anti-rabbit IgG were diluted 1 : 12 000 in blocking buffer and incubated with nitrocellulose membranes (1 h at room temperature). After three washings, the nitrocellulose membranes were developed using enhanced chemiluminescent reagent (Pierce) on photographic film (Hyperfilm ECL, Amersham Biosciences). The images of the scanned nitrocellulose blots were processed with Adobe Photoshop version 6.0 software and the densitometry (arbitrary units, a.u.) of the immunoreactive occludin proteins was calculated by Image J software (NIH).
Occludin cDNA analysis.
Occludin cDNA was amplified by PCR of reverse-transcribed RNA from Caco-2 cells using Invitrogen Superscript II. Oligonucleotides were obtained from Metabion (Martinsried, Germany). The following primer sequences were used: FOCLN218 (5'-CAAACCGAATCATTATGCAC-3'), FOCLN431 (5'-CTGGGACAGAGGCTATGGAA-3') and ROCLN1726 (5'-GCATCAGCCTTCTATGTTTTC-3'). PCR products were analysed on 1 % agarose gels.
Electrophysiological and paracellular flux measurements.
TER was measured with a dual-voltage ohmmeter clamp (Vastag et al., 2005); readings in ohms using Ag/AgCl electrodes were determined from the voltage response to 5 µA cm2 current for 150 ms every 2 s. The TER results are expressed as the measured resistance in ohms multiplied by the area of the filter (1 cm2). All conditions were established in duplicate for each experiment. Inserts were used between days 16 and 22 after seeding, when the TER was greater than 500
cm2. Measurements of TER were recorded at the start of each experiment and at indicated time intervals, and are expressed in arbitrary units (a.u.) as the ratio of TER at each time point divided by TER at time 0. In order to provide the Caco-2 cells with sufficient stores of glutamate and growth factors, the medium was removed at 1012 h intervals and fresh medium containing either NH4Cl or ccs was added immediately following the measurement of TER. The lucifer yellow (LY) permeability assay of BD Biosciences (Chong et al., 1997
) was used to assess the permeability of the Caco-2 cells on the transwell filters. Monolayers were washed once with Hanks' balanced saline solution (HBSS), pH 7·4, and volumes of 50 µl 0·4 M LY 476 Da in HBSS were placed in the apical compartment and 270 µl of the HBSS transport buffer in the basal compartment. After 1 h incubation at 37 °C in 5 % CO2, the LY fluorescence flux was determined at 485 nm excitation and 530 nm emission using a fluorescent plate reader (TECAN, Kircheim, Germany). Permeability coefficients (Pcoeff) were calculated according to the formula Pcoeff=V/(AxCi)xCf/t, where V is the volume of the basal chamber, A the area of the membrane insert (0·804 cm2), Ci the initial concentration or fluorescence units in the apical compartment, Cf the final concentration or fluorescence units in the basal compartment, and t the assay time in seconds.
Immunofluorescence.
Confluent Caco-2 cell monolayers, 1014 days after seeding on collagen- or fibronectin-coated glass cover slips, were exposed to NH4Cl- or NH3/-containing H. pylori/Caco-2 ccs. At various time intervals, the cells were fixed in 4 % paraformaldehyde/PBS (15 min, 37 °C) and incubated (30 min, room temperature) in blocking buffer consisting of PBS containing 10 % goat serum or 10 % BSA, 0·1 % Triton X-100, 0·1 % Tween 20, pH 7·4. The primary mouse or rabbit antibodies, diluted 1 : 800 in goat serum blocking buffer, or the goat anti-N 19-terminal occludin diluted 1 : 800 in BSA blocking buffer, were added for 1·5 h at room temperature. After removal of antibodies, the cells were washed three times in PBS, 0·1 % Triton X-100, 0·1 % Tween 20, pH 7·4. Indirect detection of occludin protein was performed using the Cy3-coupled (Molecular Probes) or Alexa Fluor-coupled (Molecular Probes) goat secondary antibodies. After five washings, the cells were embedded in Mowiol 4-88 (Hoechst, Frankfurt). For visualization, an Olympus BX50F microscope equipped with an Olympus FluoView laser scanning system was used. Confocal images acquired at 0·75 nm sections were projected using Image J software (NIH) and processed by Adobe Photoshop version 6.0 software.
Data analysis.
Data are presented as the mean±SEM. The significance of difference between the means was evaluated by Student's t test for unpaired samples or by ANOVA; P<0·05 was considered significant.
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RESULTS |
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Ammonium-mediated decrease of TER is reversible
To determine if the effect of H. pylori-derived ammonium on tight-junction function is reversible, the 48 h Caco-2 cell cultures were washed and reinstated in normal control medium (Fig. 1b). The TER measurements of cultures that were exposed to the ammonium-containing
vacA
cagA-ccs or wt-ccs showed a significant upward readjustment of their TER to a range of 0·70·8 a.u. within 24 h. These results provide evidence that H. pylori-derived ammonium alters the tight-junction function of Caco-2 cells, but that the effects are reversible upon removal of ammonium.
H. pylori-derived ammonium induces the production of LMW occludin in Caco-2 cells
Previous work has shown that antibodies produced against pure forms of occludin bind to proteins in the 6066 kDa area (Mankertz et al., 2002; Bojarski et al., 2004
). However, an additional low-molecular-weight form of occludin (LMW occludin) can be induced in MDCK cells by treatment with NH4Cl (Vastag et al., 2005
). Accordingly, the dose-dependent effects of NH4Cl on LMW occludin production were assessed in Caco-2 cells after 24 h treatment with 050 mM NH4Cl (Fig. 2
). The appearance of LMW occludin of 42 kDa was most prominent after exposure to 15 mM NH4Cl or 15 mM (NH4)2SO4 (results not shown). The production of 6066 kDa occludins (Fig. 2
) or the tight-junction protein ZO-1 (results not shown) did not significantly change during treatment with ammonium salts.
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Because occludin is postulated to play a role in the transepithelial migration of activated lymphocytes, we investigated whether or not the exposure of Jurkat T cells to ammonium would modify their occludin expression. As described for peripheral blood lymphocytes (Alexander et al., 1998), we found 60 kDa occludin in immunoblots of Jurkat T cells that were activated for 24 h with 30 ng ml1 phorbol ester (phorbol myristoyl acetate, PMA) and 125 ng ml1 of the calcium ionophore A23187, but no occludin expression in Jurkat T cells maintained in normal RPMI culture medium. LMW occludins were not detected in Jurkat T cells that were exposed to ammonium salts during their activation (results not shown). These results suggest that ammonium does not induce the formation of LMW occludins in T lymphocytes and that the effects of ammonium on occludin expression are specific for epithelial cells.
LMW occludin production induced by H. pylori ccs correlates with ammonium production
To determine if the ammonium generated in the H. pylori/Caco-2 co-cultures is sufficient for the formation of 42 kDa occludin, the ccs were assayed for their effects on occludin production in non-infected Caco-2 cells (Figs 4 and 5
). The immunoblot intensities of 42 kDa occludin, but not the intensities of 6066 kDa occludins, showed a significant correlation with the amount of ammonium production (Fig. 4
). The ammonium levels in the diluted wt-ccs, 2·7513 mM
, were in agreement with the amounts expected from the stoichiometric conversion of urea to ammonium, whereas the supernatants of
ureA-ccs had 0·1250·75 mM
. The kinetics of ammonium-dependent 42 kDa occludin production showed a first-order exponential rate with k=0·4303 and t
=1·6 h (Fig. 5
, left panel). During the course of 8 h NH4Cl exposure, the production of 42 kDa occludin increased approximately threefold compared to no major change of 6066 kDa occludin production (Fig. 5
, left panel).
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Evidence that the 42 kDa occludin is derived from N-terminal cleavage of full-length protein
To determine whether the LMW occludin is a product of ammonium-dependent post-translational events (i.e. protease cleavage) or transcription from alternative mRNA templates (as reported by Mankertz et al., 2002), we performed N-terminal-specific immunoblots of protein extracts and RT-PCR of cDNA obtained from Caco-2 cells after 24 h NH4Cl treatment (Fig. 6
). The polyclonal antibody specific for occludin N-terminal peptide detected only the 66 kDa protein in extracts of cultures treated with NH4Cl and control cultures, whereas immunoblots of the same extracts with the anti-C-terminal antibody revealed the 42 kDa occludin (Fig. 6a
). In RT-PCR experiments, the use of 5' FOCLN218 and 3' oligonucleotide primer ROCLN1726 gave a single amplification product of 1530 bp that corresponds to the full-length occludin transcript. To favour amplification of shorter-length transcripts, the cDNA concentration was reduced by 10-fold in the presence of FOCLN218 and ROCLN1726 primers (Fig. 6b
, lane 2). In addition, another 5' oligonucleotide (FOCLN431), corresponding to amino acid position 89Trp after the first transmembrane domain, was used with ROCLN1726 (results not shown). Both of these conditions gave only a single PCR product. The lack of evidence for an alternative mRNA transcript generated during ammonium treatment and the absence of 42 kDa occludin in N-terminal-specific antibody immunoblots suggest that the effect of ammonium on occludin expression is at the post-translational level and likely to involve N-terminal cleavage events.
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DISCUSSION |
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The rapid restoration of TER in polarized epithelial cells that have been treated with H. pylori NH3/ and the abolishing of the 42 kDa occludin after removal of NH3/
are consistent with the fact that occludins are not an essential structural component of tight junctions, but instead play a role in the modulation of epithelial barrier function. Our findings support the prevailing view that the breakdown of the transepithelial barrier function of Caco-2 cells is reversible (Tavelin et al., 2003
; Nusrat et al., 2000
) and that epithelial cells have the capacity to undergo dynamic remodelling of their tight-junction protein apparatus in response to external stimuli, without suffering permanent cell damage (Bojarski et al., 2004
; Tavelin et al., 2003
; Ivanov et al., 2004
; Singh et al., 2000
). The production of LMW occludin during 14 h wt-ccs exposures indicates that occludin turnover is rapidly perturbed in the presence of H. pylori. It has been shown previously that H. pylori infection of AGS epithelial cells leads to an induction of occludin expression which is dependent on CagA (Guillemin et al., 2002
). Moreover, a disturbance of epithelial tight junctions in response to translocated CagA has been found in MDCK cells (Amieva et al., 2003
). These effects of H. pylori infection are only documented for full-length occludins. The study of Amieva et al. (2003)
does not examine changes in occludin distribution in H. pylori-infected versus uninfected cells. Our detection of a 42 kDa occludin in Caco-2 cells is in accordance with the fact that the redistribution of occludins (possibly due to formation of LMW occludin complexes) occurs in epithelial cells solely in the presence of ammonium and is independent of the cag pathogenicity island of H. pylori. Here we show no difference in occludin distribution between wild-type and
vacA
cagA mutant strains, and suggest that ammonium produced by H. pylori urease activity constitutes an additional stress to epithelial tight-junction integrity that is independent of CagA or VacA. The action of inflammatory cytokines, known to disrupt epithelial barrier function by apoptosis-independent mechanisms (Bruewer et al., 2003
; Nusrat et al., 2000
), is not likely to explain the presence of LMW occludins in epithelial tissue in so far as we have found no inducible 42 kDa occludin production by Caco-2 under treatment with IFN
or TNF-
(S. D. Lytton, unpublished observations).
Studies of occludin constructs bearing mutations or deletions of their N-terminal transmembrane regions provide evidence that the N-terminal and C-terminal cytosolic domains play a role in maintenance of the membrane-protein extracellular loop structure and in the organization and modulation of the tight-junction barrier (Nusrat et al., 2000; Tavelin et al., 2003
). The accumulation of LMW occludin and its impact on the function of the full-length protein in intact cells should be resolved in the future by transfection of epithelial cells with N-terminal-truncated occludins. Such studies need to discriminate between the C-terminal caspase cleavage site of occludin (Bojarski et al., 2004
) and processing of occludin by proteases that show ammonium and/or pH sensitivity, as indicated by the results presented here.
Our data underline the importance of ammonium production for H. pylori-mediated modulation of the epithelial cell barrier and suggest that ammonium contributes to H. pylori pathogenicity. Because the effects of ccs on Caco-2 TER and the effects of ammonium on 42 kDa occludin formation are both reversible upon removal of ammonium, we might predict that truncated occludins and bacterial soluble factors have a synergistic action on the disturbance of epithelial tight-junction function. The presence of LMW occludins may not only suggest a link to the disturbance of epithelial cell tight junctions, but may also warrant an investigation of whether or not their presence is associated with other clinical consequences, such as gastric inflammation, alteration in gastrointestinal absorption or a specific autoimmune response to truncated membrane protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., Nelson, W. J. & Falkow, S. (2003). Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 300, 14301434.
Blaser, M. J. & Atherton, J. C. (2004). Helicobacter pylori persistence: biology and disease. J Clin Invest 113, 321333.
Bojarski, C., Weiske, J., Schöneberg, T. & 7 other authors (2004). The specific fates of tight junction proteins in apoptotic epithelial cells. J Cell Sci 117, 20972107.
Bruewer, M., Luegering, A., Kucharzik, T., Parkos, C. A., Madara, J. L., Hopkins, A. & Nusrat, A. (2003). Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol 171, 61646172.
Chong, S., Dando, S. A. & Morrison, R. A. (1997). Evaluation of Biocoat intestinal epithelium differentiation environment (3-day cultured Caco-2 cells) as an absorption screening model with improved productivity. Pharm Res 12, 18351837.[CrossRef]
Eaton, K. A., Brooks, C. L., Morgan, D. R. & Krakowka, S. (1991). Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59, 24702475.[Medline]
Gebert, B., Fischer, W., Weiss, E., Hoffmann, R. & Haas, R. (2003). Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 301, 10991102.
Gillen, A. D., Wirz, A. A., Neithercut, W. D., Ardill, J. E. S. & McColl, K. E. L. (1999). Helicobacter pylori infection potentiates the inhibition of gastric acid secretion by omeprazole. Gut 44, 468475.
Guillemin, K., Salama, N. R., Tompkins, L. S. & Falkow, S. (2002). Cag pathogenicity island-specific responses of gastric epithelial cells to Helicobacter pylori infection. Proc Natl Acad Sci U S A 99, 1513615141.
Hagen, A. S., Wu, J. H. & Morrison, S. W. (2000). NH4Cl inhibition of acid secretion: possible involvement of an apical K+ channel in bullfrog oxyntic cells. Am J Physiol Gastrointest Liver Physiol 279, G400G410.
Handlogten, M. E., Hong, S.-P., Westhoff, C. M. & Weiner, D. (2004). Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol 287, F628F638.
Ivanov, A. I., Nusrat, A. & Parkos, C. (2004). Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol Biol Cell 15, 176188.
Kirsten, E., Gerez, C. & Kirsten, R. (1963). An enzymatic microdetermination method for ammonia, specifically for extracts of animal tissues and fluids. Determination of NH4 ions in blood. Biochem Z 337, 312319.[Medline]
Kleiner, D. (1981). The transport of NH3 and NH4+ across biological membranes. Biochim Biophys Acta 639, 4152.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Li, N., Lewis, P., Samuelson, D., Liboni, K. & Neu, J. (2004). Glutamine regulates CACO-2 cell tight junction proteins. Am J Physiol Gastrointest Liver Physiol 287, G726G733.
Lichtenberger, L. M. & Romero, J. J. (1994). Effect of ammonium ion on the hydrophobic and barrier properties of the gastric mucus gel layer: implications on the role of ammonium in H. pylori-induced gastritis. J Gastroenterol Hepatol 9, S13S19.[Medline]
Mankertz, J., Waller, J. S., Hillenbrand, B., Tavalali, S., Florian, P., Schöneberg, T., Fromm, M. & Schulzke, J. D. (2002). Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage. Biochem Biophys Res Comm 298, 657666.[CrossRef][Medline]
Nusrat, A., Turner, J. R. & Madara, J. L. (2000a). Molecular physiology and pathophysiology of tight junctions IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 279, G851G857.
Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C. & Mrsny, R. J. (2000b). The coiled-coil domain of occludin can act to organise structural and functional elements of the epithelial tight junction. J Biol Chem 275, 2981629822.
Papini, E., Satin, B., Norais, N., de Bernard, M., Telford, J. L., Rappuoli, R. & Montecucco, C. (1998). Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin. J Clin Invest 102, 813820.
Rieder, G., Einsiedl, W., Hatz, R. A., Stolte, M., Enders, G. A. & Walz, A. (2001). Comparison of CXC chemokines ENA-78 and interleukin-8 expression in Helicobacter pylori-associated gastritis. Infect Immun 69, 8188.
Sidebotham, R. L., Worku, M., Karim, Q., Najma, Q., Dhir, N., Baron, K. & Hugh, J. (2003). How Helicobacter pylori urease may affect external pH and influence growth and mobility in the mucus environment: evidence from in vitro studies. Eur J Gastroenterol Hepatol 15, 395401.[CrossRef][Medline]
Singh, U., Van Itallie, C. M., Mitie, L. L., Anderson, J. M. & McClane, B. A. (2000). CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junctional protein occludin. J Biol Chem 275, 1840718417.
Smoot, D. T., Mobley, H. L. T., Chippendale, G. R., Lewison, J. F. & Resau, J. H. (1990). Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun 58, 19921994.[Medline]
Stingl, K., Altendorf, K. & Bakker, E. P. (2002). Acid survival of Helicobacter pylori: how does urease activity trigger cytoplasmic pH homeostasis? Trends Microbiol 10, 7074.[CrossRef][Medline]
Suzuki, H., Yanaka, A. & Muto, H. (2000). Luminal ammonia retards restitution of guinea pig injured gastric mucosa in vitro. Am J Physiol Gastrointest Liver Physiol 279, G107G117.
Suzuki, H., Yanaka, A., Shibahara, T., Matsui, H., Nakahara, A., Tanaka, N., Muto, H., Momoi, T. & Uchiyama, Y. (2002). Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells. Am J Physiol Gastrointest Liver Physiol 283, 986995.
Tavelin, S., Hashimoto, K., Malkinson, J., Lazorova, L., Toth, I. & Artursson, P. (2003). A new principle for tight junction modulation based on occludin peptides. Mol Pharmacol 64, 15301540.
Terres, A. M., Windle, H. J., Ardini, E. & Kelleher, D. P. (2003). Soluble extracts from Helicobacter pylori induce dome formation in polarized intestinal epithelial monolayers in a laminin-dependent manner. Infect Immun 71, 40674078.
Triebling, A. T., Korsten, M. A., Dlugosz, J. W., Paronetto, F. & Lieber, C. S. (1991). Severity of Helicobacter-induced gastric injury correlates with gastric juice ammonia. Dig Dis Sci 36, 10891096.[CrossRef][Medline]
Vastag, M., Neuhofer, W., Nagel, W. & Beck, F. X. (2005). Ammonium affects tight junctions and the cytoskeleton in MDCK cells. Pflügers Archiv Eur J Physiol 449, 384391.[CrossRef][Medline]
Received 17 March 2005;
revised 10 June 2005;
accepted 4 July 2005.
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