Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells

Hideo Suzuki1, Akinori Yanaka1, Takeshi Shibahara1, Hirofumi Matsui1, Akira Nakahara1, Naomi Tanaka1, Hiroshi Muto2, Takashi Momoi3, and Yasuo Uchiyama4

1 Departments of Gastroenterology and Endoscopy, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575; 2 Toride Ishikai Hospital, Toride 300-0032; 3 Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421; and 4 Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH4Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH4Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH4Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.

Helicobacter pylori; gastric epithelial cells


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROLONGED INFECTION with Helicobacter pylori causes chronic gastritis and gastric atrophy, which are closely associated with the development of gastric carcinoma (2, 9, 30). A number of studies have shown that H. pylori induces apoptosis of surface mucous cells and cells in gastric glands (7, 16, 17, 20, 22, 31, 36, 50, 52), a process that may be associated with the progression of gastric atrophy (4, 32, 45). Although several factors have been proposed with regard to H. pylori-induced apoptosis of gastric epithelial cells, the precise mechanisms by which H. pylori causes apoptosis have not been well clarified.

One of the major factors for H. pylori-induced apoptosis is ammonia. H. pylori possesses strong urease activity and produces high concentrations of ammonia within the mucous gel layer as a result of hydrolysis of urea (10, 30). H. pylori-derived ammonia causes injury in isolated human gastric epithelial cells in vitro (40). Ammonia, at concentrations detected in gastric juice in H. pylori-infected subjects, causes gastric mucosal injury (10, 33, 44, 48), retards gastric mucosal restitution (43), and induces apoptosis of gastric epithelial cells (13, 40). The mechanisms by which ammonia induces apoptosis of gastric epithelial cells remain unclear, however.

Ammonia exists as two different forms, NH3 and NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. NH3, a small lipophylic molecule (mol wt 17.0), is readily permeable across the phospholipid bilayer of cell membranes (18). In contrast, NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, a monovalent cation, does not passively diffuse cell membrane but passes through only cation channels located mainly on the basolateral cell membrane of gastric epithelial cells (3). Because ammonia has a pKa of 9.0 (18), most of the ammonia exists as NH<UP><SUB>4</SUB><SUP>+</SUP></UP> within acidic gastric lumen, under physiological conditions. However, treatment of the gastric mucosa with acid inhibitors increases luminal pH and thereby enhances generation of NH3 (18). Therefore, it seems reasonable to assume that gastric luminal ammonia diffuses across gastric mucosa readily at high luminal pH and thereby causes severe damage at high luminal pH (53). These considerations suggest that an increase in luminal pH by treatment with acid inhibitors may play an important role in the progression of ammonia-induced gastric injury.

A number of clinical studies suggest that acid suppression therapy by proton pump inhibitors (PPIs) exaggerates H. pylori-induced corpus gastritis (5, 24, 28, 49) and that long-term treatment with PPIs accelerates progression of gastric atrophy in patients with H. pylori infection (23, 25). We have also shown that prolonged acid inhibition by long-term use of PPI exaggerates corpus gastritis and enhances apoptosis of gastric corpus mucosa in H. pylori-infected mice in vivo (54). However, the issue of how or why PPIs exaggerate corpus gastritis in H. pylori-infected gastric mucosa has not been studied in detail.

On the basis of these background studies, we hypothesized that high luminal pH caused as a result of acid suppression therapy may enhance conversion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> to NH3 at the apical surface of gastric epithelial cells, thereby accelerating diffusion of luminal ammonia into the mucosa and accelerating the apoptotic pathway of the gastric epithelial cells.

In the present study, we examined the effects of ammonia on the viability of gastric surface mucous cells at different luminal pH. In addition, we also analyzed mechanisms by which ammonia induces death of gastric epithelial cells using morphological and biochemical methods.


    MATERIALS AND METHODS
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Cell Culture

GSM06 cells, a gastric surface mucous cell line derived from transgenic mice harboring the simian virus 40 (SV40) large T-antigen gene (41), were cultured in DMEM (Dulbecco) supplemented with 10% fetal bovine serum, 1% insulin-transferrin-selenium-X (GIBCO), and 10 ng/ml epidermal growth factor (Wako Pure Chemical Industries, Osaka, Japan) until the cells established confluency. The incubation medium was replaced with a fresh solution at 3-day intervals. GSM06 cells were cultured in Earle's buffered medium, containing (in mM) 1.8 CaCl2, 5.4 KCl, 0.8 MgSO4, 116 NaCl, 26.2 NaHCO3, 1 NaH2PO4 · H2O, and 5.55 glucose as the standard medium. pH in the incubation medium was adjusted to 5.0, 6.0, or 7.0. The cells were incubated in the presence or absence of 10 or 30 mM NH4Cl at different ambient pH.

Electrophysiological Analysis

Functional characteristics of GSM06 monolayers were evaluated by electrophysiological analysis. GSM06 cells were cultured on permeable supports (cell culture inserts 3092) (Falcon) for 7 days until confluency was established. The GSM06 monolayers were then incubated in lucite Ussing chambers in vitro. The serosal and the luminal sides were bathed with HEPES-Ringer-100% O2 (pH 7.4) and with 150 mM NaCl (pH 7.4), respectively. Transmucosal electrical resistance was measured as an index of the gastric mucosal barrier function. Changes in electrical resistance during exposure to graded doses of HCl (pH 7.4-2.5) or NH4Cl (1-100 mM) were examined. HCl or NH4Cl was added to either the luminal or the serosal solution.

Morphological Analysis by Electron Microscopy

Some cells incubated under the experimental conditions were fixed for electron microscopy with the buffer containing 1.25% glutaraldehyde, 2.5% paraformaldehyde, 0.06% picric acid, and 0.06% CaCl2 with 0.1 M cacodylate buffer (pH 7.2) for 5 min on ice. They were then postfixed with 1% OsO4 buffered with 0.1 M cacodylate buffer (pH 7.2) for 5 min on ice. After dehydration with a graded series of ethanol, the samples were removed from the dishes by propylene oxide and were embedded in Epon 812 (TAAB). Thin sections, cut with an ultramicrotome (Ultracut-N, Reichert-Nissei) and stained with 1% uranyl acetate and lead citrate were observed with a Hitachi H-7100 electron microscope.

Cell Death Assay

To examine viability of GSM06 cells, lactate dehydrogenase (LDH) release from the cells into the medium was determined. The LDH content in the culture medium was measured by using a LDH assay kit (Kyokuto Pharmaceutical, Tokyo, Japan). Total cellular LDH content was measured after lysis with 0.1% Tween 20. The cell death rate was estimated by calculating ratios of released LDH to total (cellular and released) LDH. In some experiments, LDH release was assessed in RGM-1 cells, a cell line derived from normal rat gastric mucosal cells (19).

Morphological Analysis by Laser Scanning Microscopy

The cells cultured in chambered slides (Nunc) were fixed for laser scanning microscopy, with 4% paraformaldehyde buffered with phosphate buffer, containing 4% sucrose, for 15 min at room temperature (15). In addition, cells were cultured in the presence of NH4Cl with or without 100 µM acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), an inhibitor of caspase-3-like proteinases. These fixed cells were thoroughly washed with PBS and treated with 0.3% H2O2 in methanol for 30 min. To examine caspase-3 activity in dying cells, the cells were doubly stained for activated caspase-3 and terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) as reported previously (15, 34). Briefly, the cells were stained with a site-specific antibody against caspase-3 for 24 h at 4°C, which recognizes the carboxy terminus of the cleaved site of p20/17 but not the proform (8, 15, 21), and then incubated with FITC-conjugated goat anti-rabbit IgG for 1 h at room temperature. To detect nuclear DNA fragmentation, the cells were incubated with 100 U/ml TdT and 10 nmol/ml biotinylated 16-2'-dUTP (Boehringer-Manheim-Yamanouchi, Osaka, Japan) in TdT buffer (100 mM sodium cacodylate, pH 7.0, 1 mM cobalt chloride, 50 µg/ml gelatin) in a humid atmosphere at 37°C for 1 h. Further incubation with Texas red-conjugated avidin (Nichirei) was carried out for 30 min at room temperature. They were then viewed with a confocal laser scanning microscope (LSM-GB 200, Olympus, Tokyo, Japan).

Biochemical Analysis

To examine the molecular mechanism for ammonia-induced death of GSM06 cells, the cells cultured under various experimental conditions were biochemically analyzed. Fragmentation of DNA into oligonucleosomes in the treated cells was examined by electrophoresis of genomic DNA. Genomic DNA obtained from the cells was prepared for electrophoresis by a modification of the method described originally by Sambrook et al. (37). Each sample was subjected to electrophoresis on a 2% agarose gel and was visualized under ultraviolet light after staining with 250 ng/ml ethidium bromide.

To assess the potential involvement of the caspase family of proteinases, the activation of caspases-3 and -9 was determined by detecting their cleaved products by immunoblotting. The cells were lysed with a buffer containing 150 mM NaCl, 50 mM Tris, and 1% Triton X-100, including a proteinase inhibitor cocktail (Boehringer Mannheim). After being centrifuged twice at 15,000 g for 10 min at 4°C, the supernatants were measured for protein concentrations using the bicinchoninic acid protein assay system (Pierce, IL), and immunoblotting was performed. Each sample was separated by tricine SDS-PAGE (38) in 15% (wt/vol) acrylamide. Electrophoretic transfer of proteins from polyacrylamide gels to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Tokyo, Japan) was performed according to the method described by Towbin et al. (46). The sheets were soaked in PBS containing 5% bovine serum albumin (Sigma Chemical) to block nonspecific binding and then incubated with anti-caspase-9 (MBL, Nagoya, Japan) or anti-caspase-3 (anti-p20/17). Immunodetection was carried out with a chemiluminescent enhanced chemiluminescence kit (Amersham, Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's recommended protocol.

Proteolytic activity of caspase-3-like proteinases was also examined in cells cultured under various experimental conditions, using the method described elsewhere (6). Briefly, cytosolic extracts of the cells were prepared by repeated freezing and thawing of cells in 100 µl of extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 µM cytochalasin B, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain, and 10 µg/ml chymopain) as described. Cell lysates were then diluted with 0.5 ml IL-1beta -converting enzyme standard buffer {100 mM HEPES-KOH buffer, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfo- nate, 10 mM DTT, and 0.1 mg/ml ovalbumin} and were incubated at 30°C for 30 min with 1 µM of the fluorescent substrate (7-methoxycoumarin-4-yl)acetyl-Asp-Glu-Val-Asp-Ala-Pro-Lys(Dnp)-NH2 (MOCAc-DEVDAPK) (The Peptide Institute, Osaka, Japan). Specific caspase-3-like activity was determined by subtracting the values obtained in the presence of 1 µM Ac-DEVD-CHO and expressed as optical density (OD) (× 103) per milligram protein.

The involvement of cytochrome c and bax in the cell death cascade was also examined in GSM06 cells under various experimental conditions. The cells were rinsed with PBS after removal of medium, scraped from dishes, and centrifuged at 1,200 g for 5 min at 4°C. Pelleted cells were homogenized in 0.25 M sucrose by passing them six times through a 27-gauge needle at 4°C and then centrifuged at 1,200 g for 5 min at 4°C. The supernatant was referred to as postnuclear supernatant. After the postnuclear supernatants were centrifuged at 20,000 g for 30 min, the supernatants were further centrifuged at 100,000 g for 1 h, and their supernatants were used as the cytosolic fractions. This fraction was then incubated with the lysis buffer and subjected to SDS-PAGE and immunoblotting. Immunostaining was performed using anti-bax and anti-cytochrome c antibody (PharMingen, San Diego, CA) and detected using the method described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Electrophysiological Features of GSM06 Cell Monolayers

Effects of luminal or serosal acidification on electrical resistance. In this series of experiments, serosal or luminal acidification was induced by a stepwise decrease in the serosal or the luminal pH, respectively. During the serosal or the luminal acidification, the luminal or the serosal pH was kept constant at 7.4 throughout the experiment, respectively. Electrical resistance at each pH value was measured after the cells were exposed to the pH for 10 min.

During serosal acidification, electrical resistance significantly decreased in parallel with the decrease in the serosal pH from 7.4 to 4.0. (Fig. 1A). In contrast, during luminal acidification, electrical resistance did not change during the decrease in the luminal pH from 7.4 to 4.0. Further reduction in the luminal pH to <3.5 caused a significant decrease in electrical resistance. These results suggest the GSM06 cell monolayers have excellent polarity as gastric epithelial cells, in terms of the relatively impermeable nature of the apical membrane to H+.


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Fig. 1.   Effects of HCl or NH4Cl on electrophysiology of GSM06 cells. Dose-response studies on the effects of luminal or serosal acid (HCl) (A) and NH4Cl (B) on resistance (R) in GSM06 cells. Transmucosal electrical resistance across the cell monolayers was measured. Graded doses of H+ or NH4Cl were added to either the serosal or the luminal side. The value of resistance is shown as a percentage of basal. Data are expressed as means ± SD. A: a P < 0.05, b P < 0.01, significant difference from the corresponding values at pH 7.4. B: a P < 0.05, b P < 0.01, significant difference from the corresponding values in the absence of NH4Cl. x P < 0.05, y P < 0.01, significant difference from the corresponding values in the presence of luminal NH4Cl.

Effects of luminal or serosal NH4Cl on electrical resistance. In this series of experiments, serosal or luminal NH4Cl was added by a stepwise increase in the serosal or the luminal NH4Cl concentration ([NH4Cl]) from 0 to 100 mM, respectively. During exposure to the serosal or the luminal NH4Cl, the luminal or the serosal side was bathed with NH4Cl-free HEPES-buffered Ringer solution throughout the experiment, respectively. Electrical resistance at each [NH4Cl] value was measured after the cells were exposed to the [NH4Cl] for 10 min.

Both the luminal and the serosal NH4Cl, at concentrations between 3 and 100 mM, induced dose-dependent decreases in electrical resistance. The magnitude of the decrease in electrical resistance induced by the serosal NH4Cl was significantly greater than that induced by the equivalent concentrations of the luminal NH4Cl, indicating that serosal membrane of the GSM06 cells is relatively more permeable to ammonia than the apical membrane. These results are in concert with our previous results in intact sheets of guinea pig gastric mucosa in vitro (43).

Morphological Features of GSM06 Cells by Electron Microscopy

The cells cultured at pH 7.0 in the absence of NH4Cl showed cuboidal shape with a few cytoplasmic processes. The cells possessed large irregularly shaped nuclei with one or two distinct nucleoli (Fig. 2A). The cells had some vacuolar structures and microvilli on the apical membrane. Tight junctions were observed between the cells, indicating that the GSM06 cell monolayers were polarized, as do normal gastric epithelial cells (Fig. 2A). The same cellular features were observed in the cells incubated at pH 5.0 and pH 6.0 in the absence of NH4Cl (data not shown).


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Fig. 2.   Effects of NH4Cl on ultrastructure of GSM06 cells. A: typical GSM06 cell monolayer, cultured at pH 7.0 in the absence of NH4Cl. The cells possessed some microvilli on the apical membrane, a few vacuolar structures and lysosomes within the cytoplasm, and a large irregularly shaped nucleus with a distinct nucleolus. Original magnification, ×5,000; scale bar, 2 µm. A, inset: in paneled photograph located at top right, tight junction structure was clearly observed between the cells (original magnification, ×15,000). B: typical GSM06 cells cultured in the presence of 30 mM NH4Cl at pH 7.0 for 3 h. The cells contain numerous vacuoles in the cytoplasm. Tight junctions were not observed, whereas some microvilli were observed. Original magnification, ×5,000; scale bar, 2 µm. C: typical GSM06 cells cultured in the presence of 30 mM NH4Cl at pH 7.0 for 6 h. The cells contained numerous vacuoles and autophagosome/lysosome-like structures and a nucleus with condensed chromatin. Original magnification, ×7,000; scale bar, 2 µm. N, nucleus; TJ, tight junction; MV, microvillus; Ly, lysosome; MG, mucous granule; V, vacuole; A, autophagosome.

Because 3-100 mM NH4Cl caused significant decrease in electrical resistance at pH 7.4 (Fig. 1B), we examined the effects of 10 and 30 mM NH4Cl on morphology of GSM06 cells. Incubation of the cells with 10 mM NH4Cl for up to 6 h did not cause dramatic changes in morphology at any pH between 5.0 and 7.0 (data not shown), suggesting that electrical resistance may be a more sensitive index than morphological alteration in response to exposure to NH4Cl. In contrast, the cells incubated with 30 mM NH4Cl for 3 h showed numerous vacuolar structures containing part of the cytoplasm. Tight junctions were not observed between the cells (Fig. 2B). Further incubation of the cells with NH4Cl for 6 h induced not only cell shrinkage but also electron-dense materials and myelin-like figures in the cytoplasm. More than 30% of the cells demonstrated typical morphological configuration of apoptotic cells, characterized by pyknosis with margination and condensation of nuclear chromatin and shrinkage of cytoplasm especially when the cells were incubated at pH 7.0 (Fig. 2C), suggesting that GSM06 cells undergo apoptosis in the presence of NH4Cl at neutral ambient pH. These features were rarely observed in the cells incubated for 6 h at lower pH even in the presence of NH4Cl (data not shown).

Effects of NH4Cl on Cell Viability

Incubation of the GSM06 cells in the presence of 30 mM NH4Cl for 6 h increased LDH release significantly, in a dose-dependent (10-30 mM) and pH-dependent (pH 5.0-7.0) manner (Fig. 3A). In contrast, in the absence of NH4Cl, incubation of the cells for 6 h did not increase LDH release at any pH (Fig. 3A).


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Fig. 3.   Effects of NH4Cl on viability of GSM06 cells and RGM-1 cells at different ambient pH. A: effects of NH4Cl on GSM06 cells. B: effects of NH4Cl on RGM-1 cells. The GSM06 and the RGM-1 cells were cultured in the absence or in the presence of NH4Cl (10 or 30 mM) at pH 5, 6, and 7 for 6 h, respectively. The cell death rate was expressed by calculating ratios of released lactate dehydrogenase (LDH) activity to total (cellular and released) LDH activity. Vertical bar indicates ±SD. a P < 0.05, significant difference from the corresponding values in the absence of NH4Cl. b P < 0.01, significant difference from the corresponding values at pH 5.0. c P < 0.01, significant difference from the corresponding values at pH 6.0.

Incubation of the RGM-1 cells with 30 mM NH4Cl for 6 h also caused a significant increase in LDH release, in a dose-dependent (10-30 mM) and pH-dependent (pH 5.0-7.0) manner (Fig. 3B).

Morphological Features of GSM06 Cells by Laser Scanning Microscopy

To examine the ultrastructural features of the dying cells, the GSM06 cells cultured in the presence of 30 mM NH4Cl for 6 h were doubly stained with TUNEL and an anti-activated form of caspase-3, which recognizes the carboxy terminus of cleaved caspase-3 (p20/17) (15). The GSM06 cells cultured in the absence of NH4Cl did not show any cells stained positively with TUNEL and activated caspase-3 at all pHs examined (Fig. 4, A-C). In contrast, positive staining for both TUNEL (Texas red labeling) and activated caspase-3 (FITC labeling) were detected in the cells cultured in the presence of NH4Cl at pH of 5.0, 6.0, and 7.0 (Fig. 4, D-F). The number of the doubly stained cells increased significantly in parallel with the elevation of ambient pH (Fig. 4, D-F). The double staining of the GSM06 cells with TUNEL and activated casapase-3 induced by NH4Cl was completely abolished in the presence of a specific inhibitor of caspase-3, Ac-DEVD-CHO (Fig. 4, G-I), indicating that activation of caspase-3 is involved in this dying process.


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Fig. 4.   Effects of NH4Cl on caspase-3 activity and apoptosis of GSM06 cells. The GSM06 cells were cultured in the absence (A-C) or presence (D-F) of 30 mM NH4Cl. The pH in the medium was kept constant at 5.0 (A, D, G), 6.0 (B, E, H), and 7.0 (C, F, I) for 6 h. Some cells were incubated with a specific caspase-3 inhibitor, Ac-DEVD-CHO (G-I) for 6 h. The cells doubly stained with activated caspase-3 (FITC labeling) and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL; Texas red labeling) increased at higher pH, effects completely abolished in the presence of the caspase-3 inhibitor. Scale bar, 20 µm.

Biochemical Analysis of GSM06 Cells Cultured in the Presence of NH4Cl

Effects of NH4Cl on electrophoresis of genomic DNA. To further confirm nuclear alterations in GSM06 cells during incubation with NH4Cl, DNA fragmentation into oligonucleosomes was analyzed by electrophoresis. Genomic DNAs obtained from the cells at 6 h after addition of NH4Cl clearly showed clear formation of a ladder at pH 7.0, less so at pH 6.0, and very faintly at pH 5.0 (Fig. 5). No fragmentation was observed in genomic DNAs obtained from the cells cultured in the absence of NH4Cl (Fig. 5). These results suggest that death of GSM06 cells induced by NH4Cl was apoptotic and that ammonia-induced apoptosis occurs more favorably at higher ambient pH.


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Fig. 5.   Effects of NH4Cl on electrophoresis of genomic DNA. Electrophoresis of DNA from GSM06 cells cultured in the absence of NH4Cl at pH 5, 6, and 7 (lanes 1, 2, and 3) showed no fragmentation. In contrast, DNA fragmentation into oligonucleosomes was shown intensely in the cells cultured in the presence of 30 mM NH4Cl at pH 7.0 (lane 6), less so in those at pH 6.0 (lane 5), and not at all in those at pH 5.0 (lane 4). Lane M shows 100-bp DNA ladder markers.

Effects of NH4Cl on immunoblotting of caspase-3 and caspase-9. Because the active form of caspase-3 was immunopositive in GSM06 cells incubated in the presence of NH4Cl, the caspase cascade was further examined by immunoblotting. Activated forms of caspases-9 and -3 were detected in the extracts of GSM06 cells, which had been incubated for 6 h in the presence of NH4Cl at any pHs (Fig. 6). The amounts of proteins of caspase-9 and caspase-3 increased significantly as the elevation of ambient pH (Fig. 6). In contrast, the activated forms of caspases-9 and -3 were not detected at any pHs, when the cells were cultured in the absence of NH4Cl for 6 h.


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Fig. 6.   Effects of NH4Cl on expression of caspases-3 and -9 in GSM06 cells. A: caspase-3. Caspase-3 was activated only when the cells were cultured in the presence of NH4Cl. The intensity of the protein bands detected at 17 kDa (p17) increased in a pH-dependent manner. The antibody used recognizes the carboxy terminus of p17 only. B: caspase-9. Procaspase-9 [46 kDa (p46)] was detected in both cells with or without NH4Cl. The intensity decreased only in the NH4Cl-treated group incubated at high ambient pH. Corresponding to the decrease in the intensity of procaspase-9 bands, the intensity of its active form detected at 35 kDa markedly increased at high ambient pH.

Effects of NH4Cl on DEVDase activity. DEVDase (caspase-3-like proteinase) activity was measured in the cells cultured with or without 30 mM NH4Cl. The DEVDase activity was low in the cells cultured in the absence of NH4Cl, whereas it showed a pH-dependent increase when 30 mM NH4Cl was present in the culture media (Fig. 7).


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Fig. 7.   Effects of NH4Cl on caspase-3-like activity. The casapase-3-like activity was measured using a fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-Asp-Glu-Val-Asp-Ala-Pro-Lys(Dnp)-NH2 (MOCAc-DEVDAPK). NH4Cl increased DEVDase (caspase-3-like) activity in GSM06 cells in a concentration-dependent and pH-dependent manner. In particular, the activity was highest in cells cultured in the presence of either 10 or 30 mM NH4Cl at ambient pH 7.0. In contrast, no activity was shown in the cells cultured without NH4Cl. Data are expressed as means ± SD. a P < 0.05 and b P < 0.05, significant difference from the corresponding values at pH 5.0 and at pH 6.0, respectively. c P < 0.001, significant difference from the corresponding values in the presence of 10 mM NH4Cl.

Effects of NH4Cl on cytochrome c release and expression of bax protein. Because the activation of caspase-9 was involved in the ammonia-dependent apoptosis of GSM06 cells, the next series of experiments was conducted to examine if ammonia induces cytochrome c release from mitochondria into the cytosol and to determine if bax protein is involved in the ammonia-induced release of mitochondrial cytochrome c.

Incubation of the GSM06 cells with 30 mM NH4Cl at pH 7.0 induced release of cytochrome c from mitochondria to cytosol (Fig. 8A). In addition, the NH4Cl-induced release of cytochrome c increased over time for 6 h after addition of NH4Cl, without altering expression of bax protein (Fig. 8B). These results support the contention that ammonia-dependent apoptosis of GSM06 cells is mediated by the release of cytochrome c from mitochondria, followed by the activation of caspases-9 and-3, and further suggest that bax is not involved in the upstream initiation of the ammonia-induced cytochrome c release.


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Fig. 8.   Effects of NH4Cl on cytochrome c release and bax expression. A: effects of NH4Cl on cytochrome c release from mitochondria (Mt) to cytosol. B: changes in bax expression and cytochrome c release after addition of NH4Cl. Postnuclear supernatant (PNS) prepared from the GSM06 cells was separated into cytosolic fractions by centrifugation at 100,000 g for 1 h. The immunoblot demonstrated that cytochrome c was released from mitochondria to cytosol at 6 h after addition of NH4Cl at pH 7.0 (A). Incubation of the cells with 30 mM NH4Cl at pH 7.0 increased release of cytochrome c over time for 6 h but did not affect bax expression throughout the experiment (B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that NH4Cl decreases viability of GSM06 cells, especially at high ambient pH. Morphological examination shows that many of the cells incubated with NH4Cl underwent shrinkage and condensation of nuclear chromatin. Biochemical analysis revealed that NH4Cl induces release of cytochrome c from mitochondria into the cytosol, activation of caspase cascade, caspases-9 and -3, and fragmentation of genomic DNA into oligonucleosomes. Dual staining of TUNEL and activated caspase-3 using a cleavage site-directed antibody against caspase-3 (8, 15, 21) revealed that DNA fragmentation in the cells was associated with the activation of caspase-3. In addition to TUNEL staining, DNA fragmentation into oligonucleosomes was also confirmed by electrophoresis. These data strongly suggest that NH4Cl-induced apoptosis of GSM06 cells is mediated by cytochrome c and caspases-9 and -3.

It has been well known that release of cytochrome c triggers activation of caspase 9, leading to activation of caspase-3, and eventually causes apoptosis in a variety of conditions (12, 26, 39). Cytochrome c released from mitochondria, together with dATP, has been shown to bind Apaf-1, which resembles CED-4 in Caenorhabditis elegans and recruits procaspase-9, which is autoactivated (26). The activation of caspase-3 activates caspase-activated DNase (CAD) by cleaving an inhibitor of CAD, ICAD, resulting in DNA fragmentation into oligonucleosomes in nuclei (27).

Recent studies in human gastric mucosa have shown that bax protein is involved in H. pylori-induced apoptosis of gastric epithelial cells (20). Because bax protein has been shown to increase mitochondrial membrane permeability and to enhance release of cytochrome c (12), one would assume that bax may be involved in the ammonia-induced apoptosis of GSM06 cells. Our results, however, strongly suggest that bax is not involved in the upstream initiation of ammonia-induced cytochrome c release in the GSM06 cells (Fig. 8B), because ammonia enhanced release of cytochrome c cells over time for 6 h but did not affect bax expression throughout the experiment (Fig. 8B). Because ammonia has been shown to open mitochondrial permeability transition pore (1), which plays an important role in cytochrome c release from mitochondria (56), we assume that ammonia induces cytochrome c release presumably through enhancement of mitochondrial membrane permeability without altering bax expression. Such a case has also been reported in apoptosis induced by selenium (56) or by free radicals (11).

It has been suggested that H. pylori causes mutation of p53 gene, which affects apoptosis of gastric epithelial cells (52, 55). Because p53 gene in GSM06 cells is inactivated by induction of SV40 large T gene, our results obtained from GSM06 cells may not be directly comparable to those of other native gastric epithelial cells, in which p53 gene is normally operative. We believe, however, that our postulated pathway for the ammonia-induced apoptosis of gastric epithelial cells is not influenced by inactivation of p53 gene in GSM06 cells, because a recent study in primary cultures of guinea pig chief cells, which are considered to have no mutation in p53 gene, has also shown that ammonia induces apoptosis of chief cells by enhancing cytochrome c release, followed by activation of caspase-9 and caspase-3 (51), which is exactly the same pathway as we proposed in the present study.

It is well known that H. pylori has strong urease activity and produces high concentrations of ammonia, which diffuses into the gastric lumen (47). Concentrations of ammonia in the gastric juice of H. pylori-infected subjects have been shown to reach 30 mM (47). Because H. pylori resides largely beneath the surface mucous layer adjacent to the gastric epithelial cells, the luminal surface of gastric epithelial cells is exposed to high concentrations of ammonia. Because the present study suggests that the GSM06 cell monolayers possess excellent functional polarity, as do normal gastric epithelial cells (Fig. 1, A and B), it seems reasonable to assume that in the present study we have added NH4Cl on the luminal surface of the GSM06 cell monolayers. Thus we believe that the concentrations of ammonia (10-30 mM) used in the present study are clinically relevant with those in the gastric lumen in H. pylori-infected subjects.

It has been reported that numerous apoptotic cells are detected in the gastric surface mucous epithelium and gastric glands of H. pylori-infected subjects (31, 33). However, the mechanisms by which H. pylori induces apoptosis of gastric epithelial cells have remained unclear. A number of studies have proposed several different mechanisms by which H. pylori causes apoptosis of gastric epithelial cells. First, H. pylori produces a variety of toxic substances, which may cause apoptosis of gastric epithelial cells. For example, ammonia, known to dissipate mitochondrial membrane potential and to inhibit mitochondrial energy consumption (48), causes apoptosis of gastric mucosal cells (13, 40). Monochloramine (NH2Cl), a highly toxic substance generated as a result of the reaction of ammonia with neutrophil-derived free radicals, induces DNA fragmentation in gastric epithelial cells (42). H. pylori urease itself has been shown to induce apoptosis of gastric epithelial cells, which depend on the expression of class II major histocompatibility complex molecules by the target cells (7). H. pylori also produces other substances such as vacuolating cytotoxin (vac A) and lipopolysaccharide, which cause apoptosis of gastric epithelial cells (17, 22). In addition to these H. pylori-derived substances, H. pylori induces inflammatory responses in host gastric mucosa, which also modulate apoptosis in gastric epithelial cells. For instance, H. pylori infection upregulates expression of inducible nitric oxide synthase (29), IL-1beta (35), and TNF-alpha (35), which enhances Fas-mediated apoptosis in host gastric mucosa (14, 36), all of which have been thought to play important roles in H. pylori-induced apoptosis of gastric epithelial cells in vivo.

The present study demonstrates that high ambient pH accelerates ammonia-induced death of both GSM06 cells and RGM-1 cells. Because the elevation of luminal pH enhances conversion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> to NH3 (18), it seems reasonable to assume that at higher ambient pH relatively high concentrations of NH3 diffuse the cell membrane more readily and enhance cytochrome c release from mitochondria, thereby accelerating the apoptotic pathway.

In addition to the present findings, our preliminary results in human gastric mucosal biopsies suggest that ammonia-induced apoptosis of human gastric mucosal cells is also enhanced at high ambient pH (data not shown). Furthermore, our previous studies in intact sheets of bullfrog gastric mucosa in vitro have shown that gastric luminal ammonia decreases gastric mucosal potential difference and resistance only at high luminal pH (53). The present results are in agreement with the findings by others. For example, ammonia accelerates cell death in rabbit isolated gastric glands in vitro at high ambient pH (13). Oral administration of ammonia causes severe damage in rat gastric mucosa only when the luminal pH was maintained at high values (48). Taken together, we believe that the pH dependency of the effects of ammonia on cell death is not a specific finding of GSM06 cells but rather is a common nature of various cell types.

We believe that the present results are relevant with the clinical observations that long-term use of PPI exaggerates corpus gastritis and accelerates gastric mucosal atrophy in patients with H. pylori infection (5, 23-25, 28, 49). Although no studies have directly shown that acid suppression treatment enhances apoptosis of H. pylori-infected human gastric mucosa, we have previously shown that prolonged acid inhibition by long-term use of PPI enhances apoptosis of gastric corpus mucosa and accelerates progression of corpus atrophy in H. pylori-infected mice in vivo (54), suggesting that acid suppression therapy causes gastric epithelial cell apoptosis within H. pylori-infected mucosa. Because exaggeration of gastritis is accompanied by the increase in epithelial cell apoptosis in H. pylori-infected gastric mucosa (52), we believe that these clinical findings could be attributable at least in part to the enhancement of ammonia-induced apoptosis of gastric epithelial cells at high luminal pH.

In summary, the present results suggest that 1) ammonia at concentrations that are detectable in the H. pylori-infected gastric lumen causes apoptosis of GSM06 cells through release of cytochrome c from mitochondria, thereby activating the caspase cascade followed by DNA fragmentation, and 2) high ambient pH enhances ammonia-induced apoptosis of the cells, an effect that may be associated with the clinical findings that prolonged acid suppression therapy exaggerates corpus gastritis and accelerates gastric atrophy in patients with H. pylori infection.


    ACKNOWLEDGEMENTS

We gratefully thank Drs. W. Silen and S. Ito (Harvard Medical School) for critical review of the manuscript. We also gratefully thank Drs. N. Sugiyama and Y. Tabuchi (Molecular Biology Research Laboratory and Exploratory Research Laboratories, Daiichi Pharmaceutical) for the kind gift of GSM06 cells and for technical advice.


    FOOTNOTES

This work was supported by Grant-in-Aid for Scientific Research 10670450 and by priority areas from the Ministry of Education, Science, Sports, and Culture of Japan.

Address for reprint requests and other correspondence: A. Yanaka, Depts. of Gastroenterology and Endoscopy, Institute of Clinical Medicine, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan (E-mail: ynk-aki{at}md.tsukuba.ac.jp).

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. Section 1734 solely to indicate this fact.

June 20, 2002;10.1152/ajpgi.00482.2001

Received 12 November 2001; accepted in final form 14 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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