Inhibition of human gastric H+-K+-ATPase alpha -subunit gene expression by Helicobacter pylori

Monika Göõz, Charles E. Hammond, Kellie Larsen, Yurii V. Mukhin, and Adam J. Smolka

Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H+-K+-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H+-K+-ATPase alpha -subunit (HKalpha ) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10-4 M) increases in cAMP, free intracellular Ca2+, and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10-7 M) and staurosporine- (10-7 M) and calphostin C-sensitive (5 × 10-8 M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10-8 M), genistein-sensitive (5 × 10-5 M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H2 and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.

gastric adenocarcinoma cells; luciferase; promoter regulation; acid secretion; H2 receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THIS STUDY TESTS THE HYPOTHESIS that Helicobacter pylori infection of human gastric epithelial cells inhibits gene expression regulated by the 5'-flanking region of the H+-K+-ATPase alpha -subunit (HKalpha ) gene. H. pylori is a spiral, microaerophilic, Gram-negative bacterium that colonizes the gastric mucosa in 25-50% of the population in developed countries and 70-90% in developing countries (52). H. pylori is a causative agent of peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma (18). In addition, disturbances of normal acid secretory mechanisms accompany H. pylori gastric infection, resulting in either hypo- or hyperchlorhydria, depending on the clinical setting. Thus in human studies, acute H. pylori infection has been associated with hypochlorhydria (25, 37-39, 41); in animal studies, dogs (23) and ferrets (34) were rendered achlorhydric after infection with H. felis and H. mustelae, respectively. Chronic H. pylori infection either stimulates acid secretion, decreasing gastric pH and predisposing to duodenal ulcer (20), or causes impaired acid secretion with increased risk for gastric cancer (19, 33, 43).

Studies (8, 32) of gastric mucosal cells infected in vitro with H. pylori have demonstrated inhibition of acid secretory function. Thus in rabbit (8) and guinea pig gastric cell isolates (32), [14C]aminopyrine accumulation (measuring parietal cell intracellular acidification) was reduced after H. pylori infection. A nonhuman-infecting Helicobacter species (H. felis) also inhibited acid secretion in rabbit parietal cells (54). In human parietal cells, H. pylori infection inhibited histamine-, carbachol-, and dibutyryl cAMP-stimulated acid secretion (28, 29). Sonicates of H. pylori also reduced acid secretion in rabbit parietal cells (8), and vacuolating toxin in supernatants of H. pylori had similar inhibitory effects on guinea pig parietal cells (32). Other putative acid-inhibitory factors associated with H. pylori are acid inhibitory factor 2, present in an organic solvent extract of H. pylori (7), and a 92-kDa protein (27). In contrast, an H. pylori fatty acid (cis-9,10-methylene-octadecanoic acid) has been reported to stimulate acid secretion in isolated guinea pig parietal cells (4).

Gastric acid secretion is mediated by an Mg2+-dependent, K+-stimulated, H3O+-transporting, P-type ATPase (H+-K+-ATPase, EC 3.6.1.36) (21, 44). The alpha -subunit of the enzyme (HKalpha , Mr, ~94,000) is a polytopic integral protein of tubulovesicular and secretory canalicular membranes in acid-secreting gastric parietal cells. Close interaction of HKalpha with an integral monotopic glycosylated beta -subunit (HKbeta , Mr, ~60,000-80,000) is required for functional electroneutral exchange of luminal K+ for cytoplasmic protons (9). The acid secretagogues histamine, gastrin, and carbachol induce HKalpha gene transcription in enriched canine parietal cell preparations (5). Pretreatment of the cells with omeprazole, a specific, irreversible, covalently bound inhibitor of H+-K+-ATPase, also induced HKalpha gene transcription (6). Several HKalpha promoter response elements have been associated with induction of HKalpha gene transcription. A 5'-flanking sequence motif (GATA) of rat HKalpha and HKbeta genes transfected into HeLa cells binds the parietal cell-specific transcription factors GATA-GT1, 2, and 3, with concomitant transcription of both genes (42, 51). Deletional analysis of canine HKalpha 5'-flanking sequences transfected into canine parietal cells showed that binding of the transcription factor Sp1 to a site between bases -54 and -45 activated constitutive HKalpha transcription (40). A similar approach showed that epidermal growth factor (EGF)-induced transcriptional activation of HKalpha gene was accompanied by protein binding to a 5'-flanking sequence between bases -162 and -156 (30). Most recently, a preliminary study (41) showed HKalpha promoter activation when p53, a transcription factor involved in cell cycle regulation and apoptosis, interacts with an HKalpha 5'-flanking segment between bases -162 and -144. The repercussions of H. pylori gastric infection on HKalpha gene transcription have not been reported.

To study the mechanisms whereby H. pylori gastric infection may affect expression levels of the H+-K+-ATPase gene, we cloned human and rat HKalpha 5'-flanking sequences into a mammalian expression vector upstream of firefly luciferase coding sequence. Transfection of human gastric adenocarcinoma cells (AGS cells) with these vectors allowed luminometric measurement of transient luciferase expression as a measure of HKalpha 5'-flanking sequence activity in response to acid secretagogues and H. pylori. Transfection studies in this AGS cell model revealed specific regulation of HKalpha promoter activity by histamine, EGF, and H. pylori and implicated protein kinases A (PKA) and C (PKC) and protein tyrosine kinase (PTK) as mediators of such regulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Ham's F-12, HEPES, antibiotic-antimycotic solution (10,000 U/ml penicillin G, 25 g/ml amphotericin B, and 10,000 mg/ml streptomycin), and Hanks' balanced salt solution (HBSS) without phenol red were acquired from Cellgro Mediatech (Herndon, VA). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA). Calphostin C, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and phorbol-12-myristate-13-acetate (PMA) were purchased from Calbiochem-Novabiochem (La Jolla, CA). Histamine (free base), cimetidine, genistein, staurosporine, EGF, IBMX, and EGTA were purchased from Sigma Chemical (St. Louis, MO). Restriction enzymes were obtained from Promega (Madison, WI), and Cytodex-3 microcarrier beads were from Amersham Pharmacia Biotech (Piscataway, NJ). All other reagents were of molecular biology grade or the highest grade of purity available.

Cells and bacteria. Human AGS cells (ATCC CRL 1739) were maintained in AGS medium (Ham's F-12, 10% fetal bovine serum, 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, and 100 µg/ml streptomycin) at 37°C in a 5% CO2-95% air incubator and used between passages 42 and 56. A strain of H. pylori positive for vacuolating cytotoxin (VacA+), cytotoxin-associated protein (CagA+), and urease was obtained from the American Type Culture Collection (Rockville, MD) (ATCC no. 49603). H. pylori were cultured on 5% horse blood agar plates (Remel, Lenexa, KS), which were incubated in a BBL Campy Pouch microaerophilic system (Becton Dickinson, Cockeysville, MD) at 37°C. Cultures were routinely screened for urease activity and discarded unless positive. For AGS cell infections, H. pylori were harvested between 48 and 72 h after inoculation of agar plates, resuspended in sterile PBS, and enumerated by absorbance at 600 nm (1 OD600nm = 2.4 × 108 colony-forming units/ml) (31). For experiments using heat-killed H. pylori, bacteria were suspended in sterile PBS and heated at 80°C for 30 min. Esherichia coli (strain DH5alpha , Life Technologies, Grand Island, NY) were seeded onto agar plates and incubated at 37°C overnight. A single colony was inoculated into 10 ml LB broth without antibiotics and cultured overnight in a shaking incubator (225 rpm) at 37°C. The bacterial suspension was centrifuged at 4°C at 1,000 g, and sedimented bacteria were washed twice with sterile PBS. E. coli were resuspended in PBS for infection of AGS cells.

HKalpha promoter-reporter gene constructs. Genomic DNAs encompassing the 5'-flanking regions of both human and rat gastric H+-K+-ATPase genes were kindly provided by Dr. Gary Shull, University of Cincinnati. The human DNA was precipitated and dissolved in TE buffer (Tris · HCl and EDTA). A 2.2-kbp segment of the promoter sequence (ending at the translation start site) was amplified by PCR (Gene-Amp XL-PCR kit, Perkin-Elmer, Norwalk, CT) using forward (5'-AATATGGTACCTCGACTCGA-TCCGTCCACCTCA-3') and reverse (5'-AATATAAGCTTGCCTGTGCTCCCACCCA-ACA-3') oligonucleotide primers that add Kpn I and Hind III restriction sites to the 5' and 3' ends, respectively, of the 2.2-kbp promoter sequence. Approximately 1 ng of DNA was used as a template, and the reaction proceeded at 94°C for 1 min and 61°C for 8 min, for 30 cycles. After digestion with Kpn I and Hind III, the PCR product was ligated into the Kpn I and Hind III cloning sites of luciferase expression vector pGL2-Basic (Promega). The rat HKalpha 5'-flanking region was excised from the donor plasmid with Nhe I and Bgl II, yielding a 2.2-kbp segment that was ligated into the corresponding cloning sites of pGL2-Basic. All plasmid constructs were purified by CsCl double banding. At least three separate preparations of each plasmid construct were used to acquire experimental data from AGS cell transfections.

Transient transfection and luciferase assay. AGS cells were plated into six-well plates (300,000 cells/well) and incubated for 18 h at 37°C in 5% CO2-95% air. The cells were washed once with 2 ml Opti-MEM I serum-reduced medium (Life Technologies). An aliquot (2 µg) of either rat or human HKalpha promoter-luciferase plasmid DNA was mixed with 100 µl Opti-MEM I (solution A). Lipofectamine reagent (10 µl, Life Technologies) was mixed with 90 µl Opti-MEM I (solution B). Solutions A and B were mixed, incubated at room temperature for 30 min, and layered onto the cells. The pGL2-Control plasmid, containing SV40 promoter and enhancer, was used as a positive control; a negative control was provided by transfection with pGL2-Basic plasmid, containing neither promoter nor enhancer. Cells were then incubated for 5 h at 37°C in 5% CO2-95% air. After cell treatments as specified below, the cells were lysed using passive lysis buffer (Promega) according to the manufacurer's suggested protocol. The luciferase-catalysed cellular light emission (relative light units; RLU) reflecting HKalpha promoter activity was measured for 30 s (AutoLumat LB 953, Wallac, Gaithersburg, MD), and luciferase activity was expressed as a percentage of unstimulated control. As a control for interassay variability, control wells in each experiment were cotransfected with an aliquot (0.04 µg) of Renilla pRL-TK plasmid DNA (Promega). The ratio of basal HKalpha promoter-driven light emission to that driven by the Renilla pRL-TK plasmid DNA was relatively constant in the control wells from experiment to experiment. Calculated interassay variability was minimal (CV% = 6.3%), reflecting the high reproducibility of the lipofectamine transfection protocol.

AGS cell treatments. Transfected AGS cells were placed in fresh serum-free Ham's F-12 medium without antibiotic and incubated for 30 min at 37°C with cimetidine (10-6 to 10-4 M) or vehicle, followed by a further 5 h incubation at 37°C with histamine or vehicle. Transfected AGS cells were incubated for 24 h with H. pylori (0.6-24 × 107 bacteria/ml) at multiplicities of infection (MOI) ranging from 20 to 800 or with E. coli (0.6-6 × 107 bacteria/ml) at MOI ranging from 20 to 200. Viability of cells after H. pylori infection was 97%, measured by trypan blue exclusion and the LIVE/DEAD viability kit using the manufacturer's suggested protocol (Molecular Probes). Probes of intracellular signaling pathways, such as EGF, PMA, staurosporine, calphostin C, OAG, and genistein, were incubated with transfected cells at concentrations and times as specified in RESULTS.

cAMP measurement. AGS cells were grown to confluence in six-well culture plates. AGS medium was replaced with fresh, serum-free AGS medium containing 0.4 mM IBMX without or with histamine (10-6 to 3 × 10-4 M) or with histamine (10-4 M) and cimetidine (10-4 M) for 30 min at 37°C. Medium was then aspirated from the wells and replaced with 2 ml ice-cold ethanol (66% vol/vol). Cell suspensions recovered from wells were centrifuged for 10 min at 2,000 g, and the supernatants were lyophilized and stored at -70°C until measurement. Cellular cAMP content was determined using a competitive cAMP enzyme immunoassay kit (Signal Transduction Products, San Clemente, CA).

Free intracellular Ca2+ measurement. AGS cell free intracellular Ca2+ concentration ([Ca2+]i) was measured by a high-throughput optical screening system for cell-based fluorometric assays (47) [fluorometric imaging plate reader system (FLIPR), Molecular Devices, Sunnyvale, CA]. The instrument simultaneously reads all 96 wells of a test microplate with kinetic updates in the subsecond range and includes a 6-W argon ion laser (Coherent, Santa Clara, CA), an optical scanning system, an integrated 96-tip pipettor to transfer reagents from two 96-well addition trays to the test microplate, temperature control (37 ± 0.1°C), and a charge-coupled device camera. AGS cells were seeded (~50,000 cells/well) into a 96-well clear-bottom black test microplate (Corning Costar, Cambridge, MA) and placed overnight in a 5% CO2-95% air incubator at 37°C. Cells were dye loaded with 4 µM fluo 3-AM ester (excitation at 488 nm, emission at 540 nm; Molecular Probes) in a loading buffer (1 × HBSS, pH 7.4, with 20 mM HEPES and 2.5 mM probenecid) for 1 h at 37°C. Probenecid avoids measurement of artifactually elevated Ca2+ signals by blocking anion transporter-mediated accumulation of fluo 3 free acid in intracellular Ca2+ storage compartments. The test microplate was washed four times with loading buffer and loaded into the FLIPR instrument. Cimetidine, vehicle, or EGTA was then added from the first addition tray to specified wells of the test microplate. Emission intensities in each well of the test microplate were measured simultaneously at 6-s intervals for 10 min. Histamine and EGTA, or vehicle, was then added from the second addition tray to specified wells of the test microplate. Emission intensities in each well of the test microplate were then measured simultaneously at 1-s intervals for 6 min. Differences in fluo 3 emission intensities between vehicle-treated wells and cimetidine-, histamine-, or EGTA-treated wells, respectively, were calculated by FLIPR software and displayed as time courses of emission intensity for each condition, expressed as the percentage of maximal emission intensity elicited by 10-3 M histamine.

Scanning electron microscopy. AGS cells (2 × 106) and Cytodex-3 microcarrier beads (8 × 104, cell-to-bead ratio = 25) were suspended in 10 ml AGS medium in a rotating high-aspect-ratio culture vessel (Synthecon, Houston, TX), and the cells were grown to confluency (3 days) at 37°C in a 5% CO2-95% air incubator. Aliquots of the bead/cell suspension were placed on Thermonox glass coverslips in six-well plates, and H. pylori were added to the cells at an estimated MOI of 10. After 24 h, the growth medium was removed, and beads with adherent cells were fixed for 30 min in 2% glutaraldehyde/cacodylate buffer at room temperature, rinsed in 0.1 M cacodylate buffer with 7% sucrose, and postfixed in 2% aqueous osmium tetroxide for 2 h. Gold-coated samples were examined in the JEOL LV5410 scanning electron microscope.

Interleukin-8 assay. Samples of medium from transfected, H. pylori-infected AGS cells were removed from the wells after 24 h and stored at -70°C. Interleukin-8 (IL-8) concentrations of the samples were determined by ELISA (human IL-8 Duo-Set ELISA development system, R&D Systems, Minneapolis, MN).

Statistical analysis. Comparisons between treatment groups were made by using unpaired Student's t-tests and ANOVA. Findings of P < 0.05 were taken to indicate statistical significance. The Statistica software package (Statsoft, Tulsa, OK) was used for this purpose.


    RESULTS
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Analysis of modulation of HKalpha promoter activity in gastric epithelial cells was achieved by transfecting human AGS cells with a plasmid (pGL2-Basic) containing 2.2 kbp of the 5'-flanking region of the human or rat HKalpha gene fused to a reporter (firefly luciferase) gene plasmid. The activation status of the HKalpha promoter in response to gastric secretory agonists, antagonists, and H. pylori was then measured by exposing transfected cells to luciferin and expressing the resulting light emission intensity in RLU, using the light emission of transfected but untreated cells as the basal level.

As a first step in validating transfected AGS cells as an appropriate experimental system for studies of alteration of HKalpha gene expression by H. pylori infection, we sought to establish the responsiveness of the cells to the acid secretagogue histamine, in terms of increases in cAMP levels (22). AGS cells were incubated with histamine alone or with histamine and cimetidine (an H2-receptor antagonist), and then cAMP concentrations in a cytoplasmic extract of the cells were measured using a competitive enzyme-linked immunoassay. As shown in Fig. 1, histamine elicited dose-dependent increases in AGS cell cAMP concentration, with the greatest increase (39% over basal cAMP concentration) occurring at 10-4 M histamine. Coincubation of the cells with 10-4 M cimetidine completely suppressed elevation of AGS cell cAMP by histamine.


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Fig. 1.   Histamine stimulates cAMP formation in gastric adenocarcinoma (AGS) cells, and the stimulation is inhibited by cimetidine. AGS cells in serum-free Ham's F-12 medium containing 0.4 mM IBMX were treated for 30 min at 37°C with different concentrations of histamine (10-6-3 × 10-4 M) alone or with histamine (10-4 M) and cimetidine (10-4 M). cAMP concentrations in extracts of the AGS cells were measured as described in MATERIALS AND METHODS. Bars depict %unstimulated control and show results from 3 individual experiments. * P < 0.05, ** P < 0.01 vs. unstimulated control; ## P < 0.05 vs. 10-4 M histamine alone. The cAMP content of untreated AGS cells was 3.75 ± 0.75 pmol/106 cells (mean ± SE, n = 3).

To further substantiate the functionality of the putative H2 receptor on AGS cells, we measured [Ca2+]i in AGS cells treated with histamine. Gastric parietal cells have been shown to respond to histamine stimulation with transient increases in [Ca2+]i (36). AGS cells were loaded with the nonratiometric fluorescent Ca2+ probe fluo 3, and relative changes in [Ca2+]i in AGS cells in response to histamine, EGTA, and cimetidine were measured as changes in fluorescence emission intensities (540 nm) of intracellular fluo 3. As shown in Fig. 2, histamine elicited dose-dependent increases in AGS cell [Ca2+]i. In the presence of extracellular EGTA (1.5 mM), the increase in [Ca2+]i caused by 1 mM and 300 µM histamine was ~20% of the EGTA-free response (Fig. 2, A and B), and no response was observed with 100 µM histamine (Fig. 2C). In the presence of cimetidine (10-3 M), the increase in [Ca2+]i caused by 1 mM and 200 µM histamine was ~20% of the cimetidine-free response (Fig. 2, D and E). Persistence of some histamine-stimulated [Ca2+]i elevation in the presence of 10-3 M cimetidine suggests that AGS cells may express H2 receptor subpopulations with differing cimetidine sensitivity. Nevertheless, the data indicate that histamine-stimulated transient increases in AGS cell [Ca2+]i are mediated at least in part by endogenous histamine H2 receptors coupled to a plasma membrane Ca2+ influx pathway. The mechanism of such coupling in AGS cells remains to be clarified.


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Fig. 2.   Histamine induces elevation of intracellular free Ca2+ concentration ([Ca2+]i) in AGS cells. Cells were loaded with fluo 3 for 1 h and stimulated with histamine for 6 min with or without prior pretreatment (10 min) with either 1.5 mM EGTA (A, B, C) or 1 mM cimetidine (D, E). Changes in [Ca2+]i were measured as fluorescence changes at 540 nm every 6 s during pretreatment and every second after histamine stimulation. Histamine addition to AGS cells (at the time indicated by arrows) caused dose-dependent transient elevations of [Ca2+]i that were inhibited by EGTA (A, B, and C) and cimetidine (D and E). Data are expressed as %fluo 3 emission intensity at 540 nm, where 100% is the fluo 3 emission intensity obtained with 1 mM histamine. Fluorescence traces represent the average of concurrent measurements of 8-12 identical sample wells. Each pair of traces is a representative result from 1 of 3 experiments.

Because histamine is known to induce HKalpha gene transcription by a receptor-mediated signaling pathway in isolated canine gastric parietal cells (5), we measured the activation status of HKalpha promoter in AGS cells transfected with the pGL2 HKalpha -reporter plasmid. Figure 3 shows the dose-dependent stimulation of HKalpha promoter activity by histamine treatment of transfected AGS cells. At a concentration of 10-4 M, histamine stimulated the human HKalpha promoter sequence activity by 103 ± 8% (n = 7). Preincubation of transfected AGS cells with 10-4 M cimetidine, an H2 receptor antagonist, restricted histamine-dependent activation of HKalpha promoter sequence to 38 ± 2.2% (n = 7); cimetidine alone (10-4 M) exerted no significant activation of HKalpha promoter sequence.


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Fig. 3.   Histamine activates human H+-K+-ATPase 5'-flanking sequence in AGS cells transfected with HKalpha -reporter gene plasmid. AGS cells were incubated with different concentrations of histamine (8 × 10-7-10-4 M) to establish the maximal stimulatory concentration as described in MATERIALS AND METHODS. The stimulatory effect of histamine (10-4 M) was inhibited by cimetidine (10-4 M). HKalpha promoter activities in treated transfected cells are shown as %change in luciferase activity expressed in relative light units (RLU) compared with untreated transfected control cells. Bars represent means ± SE, n = 7. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. untreated control; # P < 0.05 vs. 10-4 M histamine-treated cells.

Having demonstrated histamine-dependent activation of the HKalpha promoter in transfected AGS cells, we sought to identify components of intracellular signaling pathways that might be involved in this response. In view of several inconsistent reports of PKC involvement in acid secretory signal transduction in parietal cells (10, 11, 40), we first studied the effect on the human HKalpha promoter of activation of PKC by PMA. Transfected AGS cells were preincubated for 1 h with either calphostin C (5 × 10-8 M), a highly specific PKC inhibitor, or staurosporine (10-7 M), a relatively nonspecific PKC inhibitor, and then incubated for 5 h with or without PMA. Figure 4A shows that PMA stimulated the HKalpha promoter sequence in a dose-responsive manner, with maximal (2.7-fold) stimulation elicited at 10-7 M PMA. Concurrent incubation of the cells with PMA and calphostin C restricted HKalpha promoter activation to 1.7-fold of basal, whereas staurosporine eliminated the PMA response completely. Because PMA specificity is not restricted to activation of PKC, we measured the effect of the diacylglycerol analog OAG on PKC-mediated HKalpha promoter activation. Figure 4B shows that OAG exerted a dose-responsive stimulation of human HKalpha promoter transfected into AGS cells and that this stimulation was preempted by treatment of the cells with calphostin C (5 × 10-8 M). These data indicate a role for PKC in regulation of HKalpha gene transcription initiation in gastric epithelial cells.



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Fig. 4.   Human HKalpha promoter transfected into AGS cells is activated by treatment of the cells with phorbol-12-myristate 13-acetate (PMA) or with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Transfected AGS cells were incubated with different concentrations of PMA or OAG with or without preincubation with either staurosporine or calphostin C as described in MATERIALS AND METHODS. HKalpha promoter activities in treated transfected cells are shown as %change in luciferase activity expressed in RLU compared with untreated transfected control cells. A: effect of PMA (10-7 M) was partially antagonized by the specific protein kinase C (PKC) inhibitor calphostin C (5 × 10-8 M), and complete inhibition occurred with the less specific inhibitor staurosporine (10-7 M). B: effect of OAG (2.5 × 10-4 M) was antagonized by the specific PKC inhibitor calphostin C (5 × 10-8 M). Values are means ± SE; n = 3. * P < 0.05, ** P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. maximal PMA or OAG concentration.

Because PTK have also been implicated in regulation of parietal cell acid secretion (3, 12, 35) and indeed may also activate PKC with acid secretory sequelae (55), we next studied the effect on the human HKalpha promoter of activation of PTK by EGF. Figure 5 shows that EGF stimulated the human HKalpha promoter in a dose-responsive manner, with maximal stimulation elicited at 10-8 M EGF. Concurrent incubation of the cells with EGF and the PTK inhibitor genistein (5 × 10-5 M) eliminated the EGF response; genistein alone had no significant effect on HKalpha promoter activity.


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Fig. 5.   Human HKalpha promoter is activated by epidermal growth factor (EGF), and this activation is overcome by genistein. Transfected cells were incubated with different concentrations of EGF with or without prior pretreatment (30 min) with 5 × 10-5 M genistein in serum-free medium. HKalpha promoter activities in treated transfected cells are shown as %change in luciferase activity expressed in RLU compared with untreated transfected control cells. Values are means ± SE; n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. unstimulated control; ### P < 0.001 vs. 10-8 M EGF-treated cells. NS, not significant.

Having shown responsiveness of an exogenous HKalpha promoter sequence in AGS cells to physiologically relevant stimuli, including histamine, PMA, OAG, and EGF, we then asked whether H. pylori infection of the cells would impact on promoter activation by these stimuli, thereby providing potential molecular mechanisms at the level of H+-K+-ATPase gene expression for the observed clinical pathophysiology of H. pylori infection. To exclude the possibility that any observed changes in HKalpha promoter activity after H. pylori infection were simply artifacts arising from reduced AGS cell viability, we measured infected cell viability by both trypan blue exclusion and a fluorescence-based assay. Over a range of H. pylori MOI (20-400), AGS cells showed >95% viability 24 h after H. pylori infection (data not shown). Although plasma membrane integrity was clearly unaffected by H. pylori, at least by these criteria, scanning electron microscopy revealed significant differences between uninfected and infected cells. Normal, uninfected AGS cells growing on the surface of Cytodex 3 microcarrier beads displayed dense surface arrays of microvilli and microplicae (Fig. 6A). In contrast, after 24 h incubation with H. pylori, AGS cell surface microvilli and microplicae were greatly reduced in number, and numerous plasma membrane blebs were in evidence (Fig. 6B). In this photomicrograph, three H. pylori organisms are seen attached to cell plasma membranes at the interface between adjacent AGS cells. To verify that AGS cells, having undergone reporter plasmid transfection and H. pylori infection, nonetheless retained functional physiological responses in spite of these morphological changes, we tested the ability of the cells to mount an IL-8 secretory response to H. pylori infection. Figure 7 shows that at H. pylori MOI of up to 100, transfected AGS cells exhibited a robust secretion of IL-8, raising the medium IL-8 concentration from the lower limit of ELISA detection (~30 pg/ml) to over 800 pg/ml. The specificity of this response for viable H. pylori was shown by the significantly attenuated IL-8 response to infection with heat-killed H. pylori.


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Fig. 6.   Helicobacter pylori attachment to AGS cells induces changes in surface membrane morphology. Scanning electron microscopy of normal (A) and H. pylori-infected (B) AGS cells was carried out as described in MATERIALS AND METHODS. Cells were incubated with H. pylori for 24 h. Adherent H. pylori organisms are indicated by an arrowhead.



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Fig. 7.   Helicobacter pylori (Hp) induces interleukin-8 (IL-8) secretion in AGS cells. Transfected cells were incubated for 24 h with different concentrations of live or heat-killed H. pylori. Multiplicity of infection (MOI) ranged from 20 to 100. IL-8 concentrations of supernatants were measured as described in MATERIALS AND METHODS and expressed as pg/ml. Values are means ± SE; n = 3. * P < 0.05, *** P < 0.001 vs. uninfected controls; # P < 0.05, ## P < 0.01 vs. heat-killed H. pylori administered at the same MOI.

Infection of transfected AGS cells for 24 h with H. pylori was accompanied by marked inhibition of both human and rat HKalpha 5'-flanking sequence basal activation of luciferase gene transcription/translation (Fig. 8). Significantly, human HKalpha promoter was far more sensitive to H. pylori infection than rat HKalpha promoter; half-maximal inhibition of human HKalpha promoter occurred at an H. pylori MOI of ~65, while equivalent inhibition of rat HKalpha promoter required an MOI of 650. Heat-killed H. pylori at an MOI of 65 had no inhibitory effect on the human HKalpha promoter, again demonstrating the specificity of the inhibition for viable H. pylori. As a further test of specificity, the effect of another Gram-negative bacterium, E. coli DH5alpha , on human HKalpha promoter status was measured. Transfected AGS cells were incubated for 24 h at 37°C with E. coli at MOI between 20 and 200; no inhibition of human HKalpha promoter activity was detected at even the higher MOI tested (data not shown). Clearly, the differential inhibition of basal activation of human and rat HKalpha promoter by H. pylori is consistent with the specificity of the organism for human hosts.


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Fig. 8.   Helicobacter pylori inhibits basal human and rat HKalpha promoter activity. Cells were transfected with human or rat promoter and incubated for 24 h with different concentrations of live or heat-killed H. pylori. , Human promoter, live H. pylori; black-diamond , human promoter, heat-killed H. pylori; black-triangle, rat promoter, live H. pylori. MOI ranged from 20 to 800. Values are means ± SE; n = 7. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. heat-killed H. pylori administered at the same MOI.

Given the sensitivity of basal HKalpha promoter activation to H. pylori infection, at least in the setting of the AGS cell line, we then assessed the extent to which acid secretagogue-induced promoter activation was impacted by H. pylori infection of the cells. Aliquots of transfected AGS cells were incubated with histamine (10-4 M), PMA (10-7 M), or EGF (10-8 M), in the presence or absence of H. pylori (at MOI = 50 or 100), and HKalpha promoter activation was measured 24 h later. As noted before, histamine alone caused a 2.4-fold activation of HKalpha promoter; this stimulation was reduced almost to basal levels by H. pylori infection at MOI of 50 and to 80% of basal level by MOI of 100 (Fig. 9A). Incubation of HKalpha promoter-transfected AGS cells with PMA caused a 10-fold stimulation of promoter activity; when the cells were infected with H. pylori at an MOI of 50, PMA caused only 4.3-fold stimulation of HKalpha promoter activity, and infection at MOI of 100 allowed only 2.2-fold stimulation (Fig. 9B). Finally, incubation of HKalpha promoter-transfected AGS cells with EGF (10-8 M) caused a twofold stimulation of promoter activity (Fig. 9C). However, H. pylori infection of the cells at an MOI of 50 and 100 reduced HKalpha promoter activity below the basal level, to 75% and 60%, respectively. The marked differences in promoter activity measured in this set of experiments, compared with activities shown in Figs. 4A and 5, reflect the longer secretagogue incubation times here (24 h instead of 5 h). At MOI of 50 and 100, H. pylori infection alone inhibited basal HKalpha promoter activity by 42% and 56%, respectively (Fig. 8). Together, these data are consistent with downregulation of HKalpha promoter activity by H. pylori infection being mediated at least in part by modulation of PKA and PKC and PTK activities.


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Fig. 9.   H. pylori (Hp) inhibits histamine- (Hist), PMA-, and EGF-induced activation of the human HKalpha promoter. Transfected cells were treated with 10-4 M histamine (A), 10-7 M PMA (B), or 10-8 M EGF (C) alone or together with H. pylori at MOI of 50 or 100 for 24 h. HKalpha promoter activities in treated transfected cells are shown as %change in luciferase activity expressed in RLU compared with untreated transfected control cells. Values are means ± SE; n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Ordinate in B is expressed in log terms.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we tested the hypothesis that H. pylori infection of human gastric epithelial cells mobilizes intracellular signaling pathways that inhibit H+-K+-ATPase gene expression. Human and rat H+-K+-ATPase 5'-flanking sequences fused to a luciferase reporter gene were transiently transfected into human AGS, and responsiveness of the HKalpha promoter sequence to acid secretory agonists and antagonists and to H. pylori infection was measured.

The gastric epithelial origin of AGS cells makes this cell line particularly useful for in vitro studies of H. pylori pathophysiology. For example, in response to H. pylori infection, AGS cells have been shown (14) to secrete the cytokine IL-8, a neutrophil chemotactic factor increased in H. pylori-infected patients with active gastritis. This process is initiated by H. pylori attachment to AGS cells, which promotes reorganization of actin and associated cellular proteins such as vasodilator-stimulated phosphoprotein (46) and has also been shown to induce tyrosine phosphorylation of 145-kDa host protein (45). The subsequent activation of nuclear factor-kappa B and its translocation into the nucleus where interaction with IL-8 promoter response elements stimulates IL-8 gene expression were also analyzed in AGS cells (31, 48).

However, the derivation of AGS cells from transformed gastric adenocarcinoma tissue raises the possibility that AGS cell responses to H. pylori may be different from normal gastric mucosal cell responses, particularly after the distinctly nonphysiological lipofectamine-mediated transfection of the cells with HKalpha -reporter gene plasmids. In this context, our demonstration of high AGS cell viability, H. pylori-mediated effacement of surface microvilli [reported in primary human gastric epithelial cells from patient gastric biopsies (49)], robust secretion of IL-8 in response to H. pylori infection, and apparently functional histamine H2-receptor signal transduction pathways suggests that the cell line retains significant physiological parallels with normal gastric epithelial cells. IL-8, an activator of neutrophils, T cells, and basophil histamine degranulation, plays a prominent role in mediating gastric inflammation and epithelial cell degeneration and as such may have significant effects on acid output in vivo. In this in vitro study using only epithelial cells, downregulation of HKalpha promoter activity cannot be attributed to IL-8 secreted by AGS cells. We observed that E. coli DH5alpha infection of AGS cells did not inhibit HKalpha promoter activity, even though E. coli DH5alpha induces robust IL-8 secretion in AGS cells (48).

Another particularly significant parallel with gastric cells emerges from our measurements of the relative susceptibility of human and rat HKalpha promoter to inhibition by H. pylori. Because we transfected both rat and human HKalpha promoters into the same host (human AGS cells), the differential susceptibility of promoter downregulation to H. pylori infection must originate in specific sequence differences between the 2.2-kbp 5'-flanking segments of the two HKalpha genes. These sequence differences are guiding our continued study of promoter regulation by H. pylori. Clearly, the human promoter's far greater sensitivity to downregulation by H. pylori is entirely consistent with the fact that, with the exception of nonhuman primates (17), the human stomach is the only substantial reservoir of H. pylori.

As part of our validation of the AGS cell line as an appropriate model for studies of HKalpha promoter regulation, we sought to establish that functional histamine H2 receptors were present on the cell surface. Our measurements of histamine-stimulated, cimetidine-sensitive elevations of [Ca2+]i and cAMP in AGS cells provide strong evidence for the presence of H2 receptors on these cells functionally coupled to an adenylate cyclase pathway (cAMP generation) and to another pathway promoting transient elevation of free intracellar Ca2+. Induction of [Ca2+]i pulses in AGS cells by 100-300 µM histamine (Fig. 2) is consistent with [Ca2+]i oscillations induced by 10-4 M histamine in ~50% of rabbit parietal cells (36), suggesting that H2 receptor coupling to intracellular signaling pathways may be similar in AGS and parietal cells. Although 10-5 and 10-6 M histamine also elicited [Ca2+]i pulses in rabbit parietal cells (36), those measurements derived from observations of single cells and show considerable heterogeneity in [Ca2+]i responsiveness to histamine among cells in the same preparation. In contrast, 10-4 M histamine induced only minimal elevations of [Ca2+]i in <10% of canine parietal cells (15). Paradoxically, histamine stimulation (10-4 M) of rat hepatoma cells transfected with canine histamine H2 receptor caused concurrent transient elevations of [Ca2+]i and cAMP (16). Our finding that maximal elevation of AGS cell cAMP was elicited by 10-4 M histamine is consistent both with the H2 receptor-transfected hepatoma cell study (16) and with the original observations in isolated rabbit gastric glands of maximal cAMP elevation by 10-4 M histamine (10). In the latter study, 10-4 M histamine stimulated a 200% increase in cAMP concentration over basal levels, whereas in the present study, AGS cell stimulation at the same histamine concentration yielded cAMP elevation of 50%. The difference in responsiveness may reflect fewer H2 receptors on AGS cells or subclasses of H2 receptors with differing pharmacological profiles. Together, our cAMP and Ca2+ data clearly point to expression of functional histamine H2 receptors on AGS cells, a property of this cell line that has not previously been described.

Our demonstration of HKalpha promoter activation in response to histamine treatment of AGS cells recapitulates histamine-induced HKalpha gene transcription in isolated canine parietal cells (5) and is yet another line of evidence that functional histamine receptors are present on AGS cells. Interestingly, histamine H2 receptors have been shown to be expressed on MKN-45 gastric carcinoma cells (2). Further studies of histamine-dependent signal transduction pathways in AGS cells should facilitate the dissection of molecular mechanisms of HKalpha gene regulation.

The inhibitory effects of H. pylori infection on basal as well as histamine, PMA, OAG, and EGF-induced activation of the human HKalpha promoter transfected into AGS cells are possibly reflective of the transient hypochlorhydria accompanying acute H. pylori infection in humans (25, 37-39, 43). The in vitro data acquired in the present study are consistent with clinical studies showing that normal acid secretion is restored in patients with hypochlorhydria when their H. pylori infection is eradicated (19) and that H+-K+-ATPase mRNA levels in such patients are increased after H. pylori eradication (24). The data can thus be interpreted in terms of H. pylori attachment to gastric epithelial cells causing downregulation of HKalpha gene transcription, resulting in fewer functional proton pumps being synthesized, and a consequent increase in gastric pH that would favor mucosal colonization by the organism.

The molecular mechanisms by which H. pylori shuts down H+-K+-ATPase gene expression appear to require the participation of at least two intracellular signaling pathways. Inhibition of OAG-induced activation of HKalpha promoter by calphostin C and of PMA-induced HKalpha activation by staurosporine and H. pylori implicate PKC as a common intermediary in both up- and downregulation of H+-K+-ATPase gene expression. The physiological role of PKC in cytoplasmic rather than nuclear regulation of acid secretion is somewhat controversial, phorbol esters having been shown to both inhibit and activate acid secretion (53), depending on the state of activation of the adenylate cyclase pathway. In addition, several different PKC isoforms have been identified in parietal cells, one or more of which are involved in cytoplasmic tubulovesicular transformations leading to secretory canalicular activation of H+-K+-ATPase (12). At the nuclear level, by activating PKC, phorbol esters such as PMA have been shown to induce the protooncogenes c-fos and c-jun, whose expression leads to formation of the heterodimeric transcription factor activator protein-1 (1). More in-depth studies of mobilization of AGS cell transcription factors in the presence and absence of H. pylori will clarify the coupling of intracellular kinases to HKalpha promoter activation.

Our data showing stimulation of human HKalpha promoter activity by EGF, and inhibition of that stimulation both by genistein and by H. pylori, may point to a role for extracellular signal-regulated protein kinases (ERKs) as mediators of H. pylori effects on HKalpha gene expression. Genistein is a broad-spectrum inhibitor of protein kinases; however, the concentration we used in this study (50 µM) is well below the genistein IC50 for PKC and PKA (350 µM). EGF exerts its properties of growth promotion, regulation of endocrine and exocrine secretion, and intestinal electrolyte transport by receptor binding and subsequent induction of ERKs and the early response gene c-fos (26). In isolated parietal cells, acute administration of EGF inhibits acid secretion (11), whereas chronic EGF increases acid secretion (11, 30). The reversal of these effects by PTK inhibitors (30), which decrease phosphorylation of at least one ERK isoform (11), and the induction of acid secretion by prolonged activation of ERKs (50), have been interpreted in terms of HKalpha promoter response elements receiving input from ERK- and c-fos-dependent pathways (50). As we have shown, human HKalpha 5'-flanking sequence transfected into AGS cells is responsive to EGF receptor stimulation; whether the response is mediated by mobilization of an endogenous ERK pathway remains to be clarified, as does the mechanism by which H. pylori overcomes EGF-stimulated HKalpha promoter activation.

The significant finding of this study was that H. pylori inhibits basal and agonist-stimulated activation of human H+-K+-ATPase 5'-flanking sequence transfected into AGS cells. In addition, histamine, EGF, PMA, and OAG activated the promoter in an antagonist-sensitive manner, indicating that AGS cells possess functional histamine and EGF receptors and that both PTK and PKC and PKA signaling pathways play a role in promoter regulation. Also significant was the localization of H. pylori species specificity with respect to proton pump regulation to the 5'-flanking sequence of the HKalpha gene. The downregulation of human HKalpha promoter by H. pylori, measured in this study in the transfected AGS cell model, may represent the in vitro correlate of the hypochlorhydria reported in clinical studies of acute H. pylori infection. Further studies are required to establish whether H+-K+-ATPase mRNA levels and H+-K+-ATPase expression itself are reduced as a consequence of acute H. pylori infection. In addition, identification of the H. pylori-induced factor(s) responsible for downregulation of HKalpha promoter activity is clearly a high priority.


    ACKNOWLEDGEMENTS

We thank Dr. Gary Shull for generous provision of plasmids containing rat and human H+-K+-ATPase 5'-flanking DNA, Drs. John Raymond and Spencer Shorte for discussions and assistance with FLIPR (Public Health Service shared equipment Grant S10-RR-13005), Drs. Steve Frawley and Scott Willard for discussions and assistance with luminometry, and Dr. Tom Gettys for discussion of cAMP assays. We acknowledge the expert assistance of the Oligonucleotide Synthesis Facility, the Biomolecular Computing Resource Facility, and the Electron Microscopy Facility at the Medical University of South Carolina.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43138 and National Aeronautics and Space Administration Grant NAG8-1385 (A. J. Smolka).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. J. Smolka, Dept. of Medicine, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: smolkaaj{at}musc.edu).

Received 17 August 1999; accepted in final form 4 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angel, P, and Karin M. The role of Jun, Fos and the AP1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072: 129-157, 1991[ISI][Medline].

2.   Arima, N, Yamashita Y, Nakata H, Nakamura A, Kinoshita Y, and Chiba T. Presence of histamine H2-receptors on human gastric carcinoma cell line MKN-45 and their increase by retinoic acid treatment. Biochem Biophys Res Commun 176: 1027-1032, 1991[ISI][Medline].

3.   Atwell, MM, and Hanson PJ. Effect of pertussis toxin on the inhibition of secretory activity by prostaglandin E2, somatostatin, epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate in parietal cells from rat stomach. Biochim Biophys Acta 971: 282-288, 1988[ISI][Medline].

4.   Beil, W, Birkholz C, Wagner S, and Sewing KF. Helicobacter pylori fatty acid cis 9,10-methyleneoctadecanoic acid increases [Ca2+]i, activates protein kinase C and stimulates acid secretion in parietal cells. Prostaglandins Leukot Essent Fatty Acids 59: 119-125, 1998[ISI][Medline].

5.   Campbell, VW, and Yamada T. Acid secretagogue-induced stimulation of gastric parietal cell gene expression. J Biol Chem 264: 11381-11386, 1989[Abstract/Free Full Text].

6.   Campbell, VW, and Yamada T. Effect of omeprazole on gene expression in canine gastric parietal cells. Am J Physiol Gastrointest Liver Physiol 260: G434-G439, 1991[Abstract/Free Full Text].

7.   Cave, DR, King WW, and Hoffman JS. Production of two chemically distinct acid inhibitory factors by Helicobacter pylori. Eur J Gastroenterol Hepatol 5 Suppl: S23-S27, 1995.

8.   Cave, DR, and Vargas M. Effect of Campylobacter pylori protein on acid secretion by parietal cells. Lancet 2: 187-189, 1989[ISI][Medline].

9.   Chen, P-X, Mathews PM, Good PJ, Rossier BC, and Geering K. Unusual degradation of alpha -beta complexes in Xenopus oocytes by beta -subunits of Xenopus gastric H+-K+-ATPase. Am J Physiol Cell Physiol 275: C139-C145, 1998[Abstract/Free Full Text].

10.   Chew, CS, Hersey SJ, Sachs G, and Berglindh T. Histamine responsiveness of isolated gastric glands. Am J Physiol Gastrointest Liver Physiol 238: G312-G320, 1980[Abstract/Free Full Text].

11.   Chew, CS, Nakamura K, and Petropoulos AC. Multiple actions of epidermal growth factor and TGF-alpha on rabbit gastric parietal cell function. Am J Physiol Gastrointest Liver Physiol 267: G818-G826, 1994[Abstract/Free Full Text].

12.   Chew, CS, Zhou C-J, and Parente J. Calcium-independent protein kinase C isoforms may modulate parietal cell HCl secretion. Am J Physiol Gastrointest Liver Physiol 272: G246-G256, 1997[Abstract/Free Full Text].

13.   Chiba, T, Fisher SK, Agranov BW, and Yamada T. Autoregulation of muscarinic and gastrin receptors on gastric parietal cells. Am J Physiol Gastrointest Liver Physiol 256: G356-G363, 1989[Abstract/Free Full Text].

14.   Crabtree, JE, Peichl P, Wyatt JI, Stachl U, and Lindley IJ. Gastric interleukin-8 and IgA IL-8 autoantibodies in Helicobacter pylori infection. Scand J Immunol 37: 65-70, 1993[ISI][Medline].

15.   DelValle, J, Tsunoda Y, Williams JA, and Yamada T. Regulation of [Ca2+]i by secretagogue stimulation of gastric parietal cells. Am J Physiol Gastrointest Liver Physiol 262: G420-G426, 1992[Abstract/Free Full Text].

16.   DelValle, J, Wang L, Gantz I, and Yamada T. Characterization of H2 histamine receptor: linkage to both adenylate cyclase and [Ca2+]i signaling systems. Am J Physiol Gastrointest Liver Physiol 263: G967-G972, 1992[Abstract/Free Full Text].

17.   Dubois, A, Fiala N, Heman-Ackah LM, Drazek ES, Tarnawski A, Fishbein WN, Perez-perez GI, and Blaser MJ. Natural gastric infection with Helicobacter pylori in monkeys: a model for spiral bacteria infection in humans. Gastroenterology 106: 1405-1417, 1994[ISI][Medline].

18.   Dunn, BE, Cohen H, and Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 10: 720-741, 1997[Abstract].

19.   El-Omar, EM, Oien K, El-Nujuni A, Gillen D, Wirz A, Dahill S, Williams C, Ardill JE, and McColl KE. Helicobacter pylori infection and chronic gastric acid hyposecretion. Gastroenterology 113: 15-24, 1997[ISI][Medline].

20.   El-Omar, EM, Penman ID, Ardill JE, Chittajallu RS, Howie C, and McColl KE. Helicobacter pylori infection and abnormalities of acid secretion in patients with duodenal ulcer disease. Gastroenterology 109: 681-691, 1995[ISI][Medline].

21.   Forte, JG, Hanzel DK, Urushidani T, and Wolosin JM. Pumps and pathways for gastric HCl secretion. Ann NY Acad Sci 574: 145-158, 1989[Abstract].

22.   Forte, JG, and Soll AH. Cell biology of hydrochloric acid secretion. In: Handbook of Physiology. The Gastrointestinal System. Salivary, Gastric, Pancreatic, and Hepatobiliary Secretion. Bethesda, MD: Am. Physiol. Soc, 1989, sect. 6, vol. III, chapt. 11, p. 207-228.

23.   Fox, JG, Blanco MC, Yan L, Shames B, Polidoro D, Dewhirst FE, and Paster BJ. Role of gastric pH in isolation of Helicobacter mustelae from the feces of ferrets. Gastroenterology 104: 86-92, 1993[ISI][Medline].

24.   Furuta, T, Baba S, Takashima M, Shirai N, Xiao F, Futami H, Arai H, Nanai H, and Kaneko E. H+/K+-adenosine triphosphatase mRNA in gastric fundic gland mucosa in patients infected with Helicobacter pylori. Scand J Gastroenterol 34: 384-390, 1999[ISI][Medline].

25.   Graham, DY, Alpert LC, Lacey-Smith J, and Yoshimura HH. Campylobacter pylori infection is a cause of epidemic achlorhydria. Am J Gastroenterol 83: 974-980, 1995.

26.   Hill, CS, and Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80: 199-211, 1995[ISI][Medline].

27.   Huang, LL, Cave DR, and Kane AV. Purification and characterization of an acid inhibitory protein from Helicobacter pylori (Abstract). Gastroenterology 108: 839, 1995.

28.   Jablonowski, H, Hengels KJ, Kraemer N, Geis G, Opferkuch W, and Strohmeyer G. Effects of Helicobacter pylori on histamine and carbachol stimulated acid secretion by human parietal cells. Gut 35: 755-757, 1994[Abstract].

29.   Jablonowski, H, Hengels KJ, Kraemer N, Geis G, Opferkuch W, and Strohmeyer G. Effect of Helicobacter pylori on dbc-AMP stimulated acid secretion by human parietal cells. Hepatogastroenterology 41: 546-548, 1994[ISI][Medline].

30.   Kaise, M, Muraoka A, Yamada J, and Yamada T. Epidermal growth factor induces H+-K+-ATPase alpha -subunit gene expression through an element homologous to the 3' half-site of the c-fos serum response element. J Biol Chem 270: 18637-18642, 1995[Abstract/Free Full Text].

31.   Keates, S, Hitti YS, Upton M, and Kelly CP. Helicobacter pylori infection activates NF-kappa B in gastric epithelial cells. Gastroenterology 113: 1099-1109, 1997[ISI][Medline].

32.   Kobayashi, H, Kamiya S, Suzuki T, Kohda K, Muramatsu S, Kuramada T, Ohta U, Miyazawa M, Kimura N, Mutoh N, Shirai T, Takagi A, Harasawa S, Tani N, and Miwa T. The effect of Helicobacter pylori on gastric acid secretion by isolated parietal cells from a guinea pig. Scand J Gastroenterol 31: 428-433, 1996[ISI][Medline].

33.   Lee, A, Dixon MF, Danon SJ, Kuipers E, Megraud F, Larsson H, and Mellgard B. Local acid production and Helicobacter pylori: a unifying hypothesis of gastroduodenal disease. Eur J Gastroenterol Hepatol 7: 461-465, 1995[ISI][Medline].

34.   Lee, A, Krakowa S, Fox JG, Otto G, Eaton KA, and Murphy JC. Role of Helicobacter felis in chronic canine gastritis. Vet Pathol 29: 487-494, 1992[Abstract].

35.   Lewis, JJ, Goldenring JR, Asher VA, and Modlin IM. Effects of epidermal growth factor on signal transduction in rabbit parietal cells. Am J Physiol Gastrointest Liver Physiol 258: G476-G483, 1990[Abstract/Free Full Text].

36.   Ljungstrom, M, and Chew CS. Calcium oscillations and morphological transformations in single cultured gastric parietal cells. Am J Physiol Cell Physiol 260: C67-C78, 1991[Abstract/Free Full Text].

37.   Marshall, BJ, Armstrong JA, McGechie DB, and Glancy RJ. Attempt to fulfill Koch's postulates for pyloric Campylobacter. Med J Aust 142: 436-439, 1985[ISI][Medline].

38.   McColl, KE, el-Omar EM, and Gillen D. Alterations in gastric physiology in Helicobacter pylori infection: causes of different diseases or all epiphenomena? Ital J Gastroenterol 29: 459-464, 1997[ISI].

39.   Morris, A, and Nicholson G. Experimental and accidental C. pylori infection in humans In: Campylobacter pylori in Gastritis and Peptic Ulcer Disease, edited by Blaser MJ.. New York: Igaku-Shoin, 1989, p. 61-72.

40.   Muraoka, A, Kaise M, Guo Y-J, Yamada J, Song I, DelValle J, Todisco A, and Yamada T. Canine H+-K+-ATPase alpha  subunit gene promoter: studies with canine parietal cells in primary culture. Am J Physiol Gastrointest Liver Physiol 271: G1104-G1113, 1996[Abstract/Free Full Text].

41.   Muraoka, A, Kaise M, Saito K, Matsueda K, Shoda R, Yamato S, Umeda N, Todisco A, and Yamada T. Tumor suppressor gene P-53 wild-type regulates stomach-specific H+-K+-ATPase alpha  subunit gene promoter (Abstract). Gastroenterology 116: 471, 1999.

42.   Nishi, T, Kubo K, Hasebe M, Maeda M, and Futai M. Transcriptional activation of H+-K+-ATPase genes by gastric GATA binding proteins. J Biochem (Tokyo) 121: 922-929, 1997[Abstract].

43.   Rademaker, JW, and Hunt RH. Helicobacter pylori and gastric acid secretion: the ulcer link? Scand J Gastroenterol Suppl 187: 71-77, 1991[Medline].

44.   Sachs, G. The gastric proton pump: the H+-K+-ATPase. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, 2nd ed., 865-881.

45.   Segal, ED, Falkow S, and Tomkins LS. Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host proteins. Proc Natl Acad Sci USA 93: 1259-1264, 1996[Abstract/Free Full Text].

46.   Segal, ED, Lange CA, Covacci Tomkins LS, and Falkow S. Induction of host signal transduction pathways by Helicobacter pylori. Proc Natl Acad Sci USA 94: 7595-7599, 1997[Abstract/Free Full Text].

47.   Schroeder, KS, and Neagle BD. FLIPR: A new instrument for accurate, high throughput optical screening. J Biomol Screening 1: 75-80, 1996.

48.   Sharma, SA, Tummuru MK, Blaser MJ, and Kerr LD. Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells. J Immunol 160: 2401-2407, 1998[Abstract/Free Full Text].

49.   Smoot, DT, Resau JH, Naab T, Desbordes BC, Gilliam T, Bull-Henry K, Curry SB, Nidiry J, Sewchand J, Mills-Robertson K, Frontin K, Abebe E, Dillon M, Chippendale GR, Phelps PC, Scott VF, and Mobley HLT Adherence of Helicobacter pylori to cultured human gastric epithelial cells. Infect Immun 61: 350-355, 1993[Abstract].

50.   Takeuchi, Y, Yamada J, Yamada T, and Todisco A. Functional role of extracellular signal-regulated protein kinases in gastric acid secretion. Am J Physiol Gastrointest Liver Physiol 273: G1263-G1272, 1997[Abstract/Free Full Text].

51.   Tamura, S, Wang X-H, Maeda M, and Futai M. Gastric DNA-binding proteins recognize upstream sequence motifs of parietal cell-specific genes. Proc Natl Acad Sci USA 90: 10876-10880, 1993[Abstract].

52.   Taylor, DN, and Parsonnet J. Epidemiology and natural history of H. pylori infections. In: Infections of the Gastrointestinal Tract, edited by Blaser MJ, Smith PF, Ravdin J, Greenberg H, and Guerrant RL.. New York: Raven, 1995, p. 551-564.

53.   Urushidani, T, and Forte JG. Signal transduction and activation of acid secretion in the parietal cell. J Membr Biol 159: 99-111, 1997[ISI][Medline].

54.   Vargas, M, Lee A, Fox JG, and Cave DR. Inhibition of acid secretion from parietal cells by non-human-infecting Helicobacter species: a factor in colonization of gastric mucosa? Infect Immun 59: 3694-3699, 1991[ISI][Medline].

55.   Wang, L, Wilson EJ, Osburn J, and DelValle J. Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C. Gastroenterology 110: 469-477, 1996[ISI][Medline].


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