Gastroenterology Section, Palo Alto Veterans Affairs Health Care System, Palo Alto 94304; and Gastroenterology Division, Stanford University School of Medicine, Stanford, California 94305-5487
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ABSTRACT |
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Acid produces a dynamic effect on the cell phenotype of Barrett's esophagus (BE) ex vivo. An acid pulse induces hyperproliferation, whereas continuous acid exposure promotes differentiation. To examine the mechanism for acid pulse-induced hyperproliferation, we studied the Na+/H+ exchanger (NHE), which plays a role in the control of intracellular pH and cell proliferation. NHE was inhibited pharmacologically in endoscopic BE biopsies using amiloride analogs. Cell proliferation was assessed after pulsed or continuous acid exposure using tritiated thymidine incorporation assays and immunohistochemical analysis of proliferating cell nuclear antigen expression. The NHE-dependent intracellular pH response to an acid pulse was examined by pH-sensitive microfluorimetry using a Barrett's adenocarcinoma cell line TE7. NHE inhibition significantly reduced the hyperproliferative acid-pulse effect. Furthermore, the acid-pulse activation of NHE occurred via increased transporter activity (22Na uptake) without any change in NHE-1 protein levels. Inhibition of protein kinase C (PKC), an NHE activator, also reduced the hyperproliferative response. The response of TE7 cells to an acid pulse was similar to that of BE biopsies in terms of cell proliferation and NHE and PKC dependence. Acid-pulse exposure of TE7 cells resulted in intracellular acidification followed by reneutralization to an intracellular pH greater than preacidosis values. We conclude that NHE may mediate the hyperproliferative response of BE to an acid pulse via changes in intracellular pH.
sodium-hydrogen exchanger; TE7 cells; cell proliferation; cell differentiation; gastroesophageal reflux disease; esophageal adenocarcinoma
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INTRODUCTION |
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BARRETT'S ESOPHAGUS (BE) is a common endoscopic finding, in which normal squamous esophageal epithelium is replaced by specialized intestinal epithelium containing goblet cells (2, 20, 32, 43, 44). This metaplastic Barrett's epithelium usually occurs in the context of chronic and severe gastroesophageal reflux with a high acid content (5, 45, 46) and is associated with an ~30-fold increased risk for the development of adenocarcinoma of the esophagus and gastric cardia (3, 36).
We previously showed, using an ex vivo system, that the effect of acid on the cell phenotype of BE is dynamic, depending on the pattern of acid exposure (14). The effect of prolonged acid exposure was compared with a short acid pulse to mimic the variable pattern of reflux disease in which individual reflux events may be rapid or prolonged and are influenced by posture and circadian rhythms (9, 38). Whereas continuous acid exposure promoted BE epithelial cell differentiation, a 1-h acid pulse induced cell proliferation. Hence, the variable patterns of acid exposure in vivo may contribute to the complex, heterogeneous nature of BE with its consequent variable risk of neoplastic progression (13). Specifically, BE cells pulsed with acid would proliferate preferentially and may in turn have a higher risk of developing dysplasia (10, 14, 55).
The mechanism underlying the hyperproliferative response of BE to an acid pulse may relate to the Na+/H+ exchanger (NHE) in view of its role in the control of intracellular pH (pHi), as well as the initiation of cell growth and proliferation in some cell types (17, 27, 33). Furthermore, NHE is increasingly recognized as playing an important role in several disease states, such as acid-base disorders, reperfusion stunning in cardiac ischemia, and carcinogenesis, which have NHE-mediated alterations in pHi secondary to changes in their extracellular pH as a common feature (21, 41).
NHE is a ubiquitous transporter, present in the cell membranes of most mammalian cells. It exists as five known isoforms of NHE (NHE-1 to NHE-5), which are characterized by their differential responses to an acid load and their different sensitivities to the NHE inhibitory drug amiloride (19, 31). Upregulation of NHE in response to an acute acid load is mediated via phosphorylation by protein kinase C (PKC) and/or tyrosine kinases, depending on the specific isoform, whereas chronic upregulation occurs as a result of increased transcription/translation (40). The most amiloride-sensitive isoform known is NHE-1, and this isoform has been shown to be upregulated in response to acute acid exposure as a result of PKC-induced phosphorylation (35). Whereas the role of NHE in the control of pHi is well established, the mechanism by which NHE may lead to a cell proliferative response has been the subject of much debate (17). However, in view of the pH sensitivity of the cell cycle, it is possible that the role of NHE in cell proliferation relates to its control of pHi (27).
The ion transporters responsible for the control of pHi in BE have not yet been characterized; however, NHE has been shown to be an important acid extruder in squamous esophageal cells and in the Barrett's adenocarcinoma cell line TE7 (11). In addition, mRNA levels of NHE are significantly elevated in BE compared with the levels in biopsies from adjacent squamous esophageal epithelium and esophagitis (6). This suggests an important functional role for this exchanger in Barrett's epithelium.
In this study, we used human endoscopic biopsies ex vivo to investigate the role of NHE in the hyperproliferative response of BE to an acid pulse. Cell proliferation was examined using tritiated thymidine incorporation and proliferating cell nuclear antigen (PCNA) expression in the presence or absence of pharmacological inhibitors of NHE. To investigate the mechanism for NHE upregulation in response to acute acid exposure, we assessed NHE protein levels and exchanger activity and the effect of PKC inhibition (51) and activation on BE cell proliferation. We then investigated the effect of an acid pulse on pHi, using the Barrett's adenocarcinoma cell line TE7 (29).
Our data demonstrate that the mechanism for the hyperproliferative response of BE to an acid pulse is dependent on NHE. In response to a 1-h acid pulse the activity of NHE is upregulated with no change in NHE-1 protein levels. Furthermore, NHE activation appears to be mediated, at least in part, by PKC-induced phosphorylation of the exchanger or other regulatory molecules. In the pHi studies in TE7 cells, a dynamic response to an acid pulse is demonstrated that is dependent on NHE and PKC. Based on our findings, we postulate that the increased activity of NHE in BE, in response to an acid pulse, may induce a proliferative response via alterations in pHi.
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MATERIALS AND METHODS |
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Patients, tissue collection, and antibodies. Endoscopic mucosal samples of BE were obtained from patients with previously documented BE undergoing endoscopy for cancer surveillance at the Palo Alto Veterans Affairs Health Care System and Stanford University Hospital. Biopsies were collected under a protocol approved by the Administrative Panel on Human Subjects in Medical Research. All mucosal samples were divided into two parts using a razor blade. One part was Formalin fixed and subsequently analyzed microscopically by an independent histopathologist, and the second part was placed immediately in supplemented medium (see below) and used for the experiments described.
Rabbit anti-NHE-1 antibody was kindly provided by M. C. Rao (Physiology and Biophysics, University of Illinois in Chicago and the Westside Veterans Affairs Medical Center, Chicago, IL). Mouse anti-PCNA as well as peroxidase-labeled anti-rabbit and anti-mouse IgG were purchased from Sigma Chemical (St. Louis, MO) and used as recommended by the manufacturer.Organ culture. Organ culture was performed essentially as described previously (52). Briefly, mucosal biopsies were randomly assigned to acid or control groups to avoid sampling bias. The biopsies were placed on a sterilized stainless steel grid within a 35-mm petri dish, so that culture medium (3 ml) just covered the surface of the biopsy. The petri dishes were placed on racks in a sterile sealed jar (Torsion Balance, Clifton, NJ) and perfused with 95% O2-5% CO2 and then cultured at 37°C. The jar was gassed before incubation and regassed each time it was opened. The medium used was medium 199, which contained bicarbonate, supplemented with 10% heat-inactivated FCS, 1 µg/ml of insulin (26), streptomycin (500 U/ml), and penicillin (250 U/ml). For acidic culture conditions, the medium was acidified with 0.1 M HCl (~20% vol/vol) to achieve the desired pH, then filtered using a 0.45-µm Millipore filter. In all experiments, a volume of distilled water (~20% vol/vol) was added to the control nonacidified medium to achieve an osmolality equal to that of the acid-treated medium (300 mosmol/kg). To confirm tissue viability after organ culture, lactate dehydrogenase (LDH) assays were performed (8) using an aliquot of tissue culture medium taken at the end of the culture period and an LDH assay kit (Sigma Chemical). LDH had to be <10% of a positive control (Triton X-100-treated biopsy) for the tissue to be considered viable.
Protein extraction and immunoblot analysis. Tissues were homogenized (4°C) in 1% deoxycholate, 1% Nonidet P-40, and 0.1% SDS in PBS, pH 7.4, containing 5 mM EDTA, 15 µg/ml aprotinin, 10 µM leupeptin, 10 µM pepstatin, and 0.1 mM phenylmethylsulfonyl fluoride. The protease inhibitors were added just before solubilization, and homogenization was done using Kontes glass tissue grinders. Lysates were centrifuged at 16,000 g (20 min at 4°C), and protein concentrations were measured by the bicinchoninic acid protein assay as recommended by the manufacturer (Pierce, Rockford, IL). Proteins were separated using 10% SDS-PAGE and then transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) in transfer buffer containing 12.5 mM Tris · HCl (pH 8.3), 100 mM glycine, and 20% methanol (40 V for 12 h at 4°C). After transfer, membranes were blocked for 2 h in 5% dry nonfat milk in PBS containing 0.2% Tween 20 (MT buffer), then for 1 h (22°C) in 1:1,000 dilution of the anti-NHE-1 primary antibody. Membranes were washed in MT buffer and then incubated with peroxidase-conjugated secondary antibody (1:1,000 dilution) for 1 h at 22°C. After washing in MT buffer for 1 h, bands were visualized using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).
Cell proliferation assays.
All endoscopic biopsies were weighed and then processed immediately for
organ culture. Using an organ culture system as described above, we
added 1 µCi/ml
[3H]thymidine
(Dupont-New England Nuclear, Wilmington, DE) to the culture medium and
incubated it for up to 24 h. For the acid-pulse experiments,
[3H]thymidine was
added to the culture medium with or without acid for the 1-h pulse.
Fresh medium (pH 7.4) and
[3H]thymidine were
then added for the additional culture period of up to 24 h in both
acid-pulse and control groups. At the end of the incubation, the
explants were lifted from the grid and rinsed thoroughly with unlabeled
culture medium to remove unincorporated radioactivity. The tissue
sections were then homogenized, passed through a 19-gauge needle, and
processed using a cell harvester (Skatron, Tranby, Norway). The
incorporated radioactivity was measured and expressed as counts per
minute per milligram of tissue (35). To inhibit NHE,
5-(N,N-dimethyl)-amiloride
(DMA) and
5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which has a higher specificity for the NHE-1 isoform, were used
(41). DMA or EIPA were added to the culture medium at final
concentrations of 0.2 mM and 50 µM, respectively. The doses of
amiloride analogs used were determined by dose-dependency studies with
HT-29 cells (12) and are compatible with the doses used in similar
studies (17, 25, 32, 48). For the PKC-related studies, 1 µM
bisindolylmaleimide (BIM; Boehringer Mannheim), a specific PKC
inhibitor, and 0.1 µM phorbol 12-myristate 13-acetate (PMA; Sigma
Chemical) were used (22) as recommended by the
manufacturer. BIM and PMA were prepared as stock solutions
in DMSO (5 and 0.1 mM, respectively) and stored at 20°C.
Control samples from the same patient (in the absence of inhibitor)
were run in parallel for each experiment.
Immunohistochemistry. After 24-h organ culture experiments at pH 7.4 and in acid-pulse conditions with or without DMA, the Barrett's mucosal samples were placed in formaldehyde and subsequently paraffin embedded and sectioned according to standard techniques. For immunohistochemical analysis of PCNA, tissue sections were deparaffinized, rehydrated with PBS (pH 7.2), and then incubated with 1:100 dilution of anti-PCNA for 1 h (22°C). After washing, peroxidase-conjugated goat anti-mouse IgG (1:100) was added and visualization was performed using 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, CA). Sections were counterstained lightly with hematoxylin. Photographs were taken using Ektachrome Kodak Elite 400 color slide film, which was then scanned to generate prints. To score the PCNA labeling, we assessed two zones, the luminal surface and the gland cells, as described previously (37). At least 50 surface cells were counted from each of three representative areas for each patient; and similarly, at least six glands in each biopsy section were assessed. Only distinct nuclear staining was counted as positive, and a mean score of positive cells was generated from the patients studied.
Measurement of 22Na uptake activity. DMA-sensitive 22Na uptake was measured as a direct indicator of NHE activity. Endoscopic biopsies were cultured in 2 ml Na+-free medium, either at pH 7.4 or pH 3.5, with or without 0.2 mM DMA, for 45 min in an organ culture system. The Na+-free medium was composed of 5 mM MgCl2, 100 mM KCl, 5.5 mM glucose (alpha-D), 10 mM CaCl2, 1.6 mM K2HPO4 0.4 mM KH2PO4, 0.5 mM aspartic acid, and 20 mM HEPES-Tris, (pH 7.4). After incubating in Na+-free medium, 1.5 µCi/ml 22Na (Amersham, Oakville, Ontario, Canada) was added to the medium, together with 1 mM ouabain (Sigma Chemical) to prevent Na+ efflux (33). After 15 min incubation, biopsies were washed three times in Na+-free medium, and uptake of 22Na was determined using a gamma counter. 22Na uptake through NHE was calculated as the difference between Na uptake in the presence and absence of DMA.
Culture of TE7 cells. TE7 cells (29) were kindly provided by Dr. T. Nishihira (The Second Department of Surgery, Tohoku University School of Medicine, Japan). The cells were seeded at 4 × 104/cm2 and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine and grown as monolayers in 10-cm tissue culture dishes. To test for the effect of acid, the culture medium was titrated with 0.1 M HCl to the desired pH (pH 5-6.5), as described for the organ culture studies. Cell proliferation experiments were then performed with or without DMA as described for the organ culture system, except that TE7 cells were trypsinized before processing with the cell harvester. For pHi studies, TE7 cells were subcultured onto Lab-Tek coverglass chamber slides (Applied Scientific, San Francisco, CA).
pHi experiments.
Because NHE is the major acid extruder in TE7 cells (11), these
experiments were conducted using
HCO3-free, HEPES-buffered saline
solution (HBS) to focus specifically on the role of NHE after an acid
pulse (49). For calibration experiments isosmolar
Na+-free HBS was prepared by
replacing Na+ with
K+. To mimic physiological
esophageal acid exposure and to maintain consistency with the
proliferation experiments, we used acidified HBS (0.1 M HCl) rather
than the NH4Cl prepulse technique
typically used for pHi experiments
(4). Furthermore, the acid pulse used in these experiments was modified
(3 min at pH 6.5) compared with the proliferation experiments, to
minimize photobleaching and remain in the linear range of the
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM pH-sensitive dye (Molecular Probes, Eugene, OR). After an
acid pulse, TE7 cells were then exposed to HBS at pH 7.4 in the absence
and presence of the pharmacological inhibitors DMA and PKC as described
for the proliferation experiments. TE7 cells on Lab-Tek coverglass
chamber slides were loaded with BCECF by exposure to 1 µM BCECF-AM
for 20 min in serum
/amino
acid
HBS at (37°C).
This allows uncharged BCECF-AM to cross the cell membrane, where it is
then cleaved by intracellular esterases to the charged,
membrane-impermeant form, BCECF. After loading, cells were washed twice
with fresh medium and then examined using a Nikon Diaphot-300 inverted
microscope with a ×40 DIC-fluor objective lens. Light from a
xenon bulb, alternatively excited BCECF-loaded cells at wavelengths of
490 nm (pH sensitive) and 440 nm (pH insensitive) and emission
fluorescence was collected at 535 nm. Use of the 490/440 ratio enables
accurate measurement of pHi
irrespective of variations in intracellular dye concentration.
Intensities at the two wavelengths were generated every 5 s, and the
data were collected by an intensified charge-coupled device camera (Dage MTI, Michigan City, IN). A C-imaging 1280 system equipped with
SIMCA (simple calcium) ratio dual-imaging software (Compix, Mars, PA)
was used to image and process the signal, which was subsequently
analyzed using a Microsoft Excel software package on a Macintosh
computer. Ratios of BCECF fluorescence at 490/440 were
converted to pHi by exposing
dye-loaded cells to high K+
medium, containing 15 µmol/l nigericin for a range of pH values (47).
These extracellular pH values were varied over a range of 6-8, by
titration with 1 M HCl or 1 M NaOH, followed by generation of a
calibration curve for each experiment.
Statistical analysis. Statistical evaluation was performed on a Macintosh computer using Student's paired t-test and two-tailed t-test (7) and a Microsoft Excel software package. All results were expressed as means ± SE and were representative of pooled data from at least three experimental groups.
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RESULTS |
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Inhibition of NHE attenuates hyperproliferative effect of acid pulse. To investigate whether NHE is responsible for the enhanced cell proliferation observed in BE after an acid pulse, we blocked NHE, of which NHE-1 is the most amiloride sensitive (30), using DMA. After NHE inhibition, cell proliferation was assessed using [3H]thymidine incorporation and PCNA expression. As shown in Fig. 1, A and B, an acid pulse significantly increased the rate of cell proliferation by 24 h as determined by [3H]thymidine incorporation (P < 0.05), similar to previous results (14). Of note, however, DMA attenuated tritiated thymidine uptake after an acid pulse (P < 0.05; Fig. 1B), while the control group (pH 7.4; Fig. 1A) was unaffected. Because of the effect of DMA after an acid pulse, there was no significant difference in [3H]thymidine incorporation compared with the non-acid-treated group at 12 (P = 0.18) and 24 h (P = 0.19). It is therefore unlikely that DMA has a nonspecific, toxic effect on acid-treated cells. Similar results were obtained using the amiloride analog EIPA (Fig. 1E). These results suggest that NHE activity plays an important role in the observed hyperproliferative effect of an acid pulse.
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Acid increases NHE activity. For an acid pulse to cause NHE-mediated Barrett's cell hyperproliferation, one potential consequence is upregulation of Na+/H+ exchange by either increased transcription/translation of the protein or an effect on NHE activity that is posttranslational (e.g., phosphorylation or regulation by an associated protein). Because NHE-1 is the most amiloride-sensitive isoform and because NHE-1 mRNA is increased threefold in BE compared with normal squamous esophagus (6), immunoblotting was performed with an anti-NHE-1 antibody. Immunoblotting demonstrated that expression of the NHE-1 exchanger was not increased after an acid pulse compared with pH 7.4 (Fig. 2A). In contrast, NHE activity increased significantly after short-term exposure to acid (Fig. 2B), and this increase of 22Na uptake was abrogated in the presence of DMA. Taken together, these data indicate that an acid pulse activates NHE, which in turn is associated with increased cell proliferation of glandular BE cells ex vivo.
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Inhibition of PKC attenuates hyperproliferative response of BE to acid pulse. Upregulation of NHE activity in response to acute pH changes is thought to result from alterations in NHE phosphorylation that are mediated, at least in part, by PKC (40). To study the role of PKC in the acid pulse-induced hyperproliferative response of BE, we carried out the proliferation assays in the presence of either a specific inhibitor or activator of PKC. As shown in Fig. 3A, PKC inhibition using BIM markedly attenuated the hyperproliferative effect of an acid pulse, while proliferation in the control (no acid) group was unaffected. In contrast, the presence of PMA, a PKC activator, significantly increased cell proliferation in the control group (Fig. 3B). In the acid-pulse group, PMA did not increase cell proliferation further, suggesting that cell proliferation was maximally stimulated under these conditions (Fig. 3B). Therefore, PKC may be a mechanism for the acid-pulse activation of NHE in BE.
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TE7 cells provide a model for studying pHi response to acid pulse. We investigated the effect of an acid pulse on pHi in BE, because the primary cellular role of NHE is to control pHi, usually by acting as an acid extruder (27). Furthermore, acid pulse-induced changes in pHi might play a part in the NHE-dependent hyperproliferative response of BE that we have described, in view of the pH dependence of the cell cycle (27). In the absence of an immortalized premalignant Barrett's epithelial cell line, these single-cell experiments were conducted using TE7 cells derived from Barrett's adenocarcinoma (29). First, we examined and demonstrated that TE7 cells behave similarly to ex vivo BE biopsies, in that they manifest a hyperproliferative response to an acid pulse (Fig. 4). We have previously conducted preliminary studies demonstrating that NHE is an important acid extruder in TE7 cells (11), similar to many other cell types, including rabbit squamous esophageal cells (49, 50). Therefore, in these studies we have investigated the effect of an acid pulse on the pHi of TE7 cells specifically with regard to the role of NHE. The results showed that superfusion with acidic solution initially decreased pHi by 0.34 ± 0.05 pH units during the 3-min period of acid exposure. In 40-45% of the cells analyzed in a representative experiment (n = 5), the initial intracellular acidification was rapidly followed by realkalinization that caused the pH to increase by 0.75 ± 0.12 pH units over the next 10 min, thereby reaching a peak that was greater than preacidosis values (an increase over baseline of 0.35 ± 0.05 pH units, Fig. 5A). This overshoot in pHi gradually returned to pretreatment values by 20 min (not shown). In contrast, cells that did not demonstrate an alkaline overshoot effect returned to their baseline pHi. To examine whether NHE was necessary for this alkaline overshoot effect, DMA was added to the cell perfusion medium, which resulted in progressive intracellular acidosis and abolition of the alkaline overshoot effect (Fig. 5B). In view of the attenuation of acid pulse-induced hyperproliferation of BE on PKC inhibition (Fig. 3A), we also examined the effect of BIM on pHi after an acid pulse. Addition of BIM prevented realkalinization in a similar fashion to DMA (Fig. 5C). After the removal of both DMA and BIM from the medium, the pHi gradually returned to baseline over a period of 15-20 min (similar to the recovery after the alkaline overshoot effect), suggesting that no irreversible cell damage had occurred. These results suggest that after an acid pulse there is an alkaline overshoot effect in a proportion of TE7 cells, which is NHE dependent. It is our hypothesis that this alkaline overshoot may be responsible for the observed acid pulse-induced hyperproliferation response.
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DISCUSSION |
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The ex vivo experiments described in this study demonstrate that NHE plays an important role in the increased cell proliferation seen in BE after short-term exposure to acid. Specifically, pharmacological blockade of NHE obliterates the hyperproliferative effect of an acid pulse. Furthermore, the upregulation of NHE in response to short-term acid exposure occurs as a result of increased exchanger activity, with no increase in NHE-1 levels. PKC activity appears to be necessary for the observed proliferative response to an acid pulse and raises the hypothesis that PKC is acting downstream of NHE activation. pHi studies, using TE7 cells, demonstrated that after an acid pulse there is an alkaline-overshoot effect that is dependent on NHE and PKC. The mechanism underlying the hyperproliferative response of BE to an acid pulse may therefore be a consequence of changes in pHi.
Role of NHE in cell proliferation. NHE is involved in a number of cellular processes, including the regulation of pHi and, under some circumstances, the control of cell growth and proliferation (17). Whether NHE is essential or merely permissive for cell proliferation has been the subject of much debate (17). Evidence suggesting a role for NHE in cell proliferation includes the following: 1) NHE is activated by mitogenic stimuli (17), 2) in the absence of mitogens, cell proliferation is induced by cytoplasmic alkalinization but arrested by cytoplasmic acidification (57), 3) cell proliferation is dependent on extracellular Na+ (48), 4) cell proliferation can be inhibited by amiloride and its analogs (34), and 5) mutant cell lines in which NHE operates in the reverse mode (Na+ outward, H+ inward) do not proliferate at their expected rate (1). Furthermore, NHE has been shown to be necessary for cell proliferation and hence tumorigenesis in a nude mouse model of human bladder carcinoma (33). We therefore examined the relationship between cell proliferation and NHE in BE by pharmacological inhibition of this transporter ex vivo, and we demonstrated a statistically significant reduction in acid-induced cell proliferation in the presence of the amiloride-like drugs, DMA and EIPA (Fig. 1). However, caution is needed when interpreting the [3H]thymidine incorporation data, because of the complex nature of the mucosal explants containing multiple cell types (Fig. 1C). It was for this reason that PCNA immunohistochemistry was performed that duplicated the [3H]thymidine findings. These experiments also confirmed that the glands are the main proliferative compartment in BE and that other cell types, such as lymphocytes, are not a significant proliferative population, similar to previous studies (16). Taken together, these data suggest a role for NHE in acid-induced cell proliferation in BE, although it remains unclear whether activation of NHE alone is sufficient to induce cell proliferation in this epithelium and which NHE isoform is responsible.
Mechanism for NHE upregulation in BE. For short-term acid exposure in BE to result in an increase in cell proliferation via NHE, acid must directly upregulate Na+/H+ exchange. This could result from either an increase in NHE transcription/translation or in transporter activity. Although the five known isoforms of NHE share 40-60% homology in their amino acid sequence (31), they have differences in their tissue specificity, subcellular localization, and acid and amiloride sensitivity (41). We focused on NHE-1 expression, because NHE-1 is the most amiloride-sensitive isoform and NHE-1 mRNA is significantly increased in Barrett's esophagus. Although NHE-2 mRNA is present in Barrett's esophagus, protein expression has not been demonstrated and squamous esophageal epithelium does not express this isoform (6). Furthermore, in studies using cultured renal inner-medullary collecting duct cells, activity of NHE-2, the other amiloride-sensitive isoform, has been shown to be reduced in response to acidosis. This contrasts with the increased NHE-1 activity observed in response to acidosis in these cells (42).
For short-term acid exposure in BE to result in an increase in cell proliferation via NHE, acid must directly upregulate Na+/H+. This could result from either an increase in NHE transcription-translation or increased transporter activity. Our results show an increase in amiloride-sensitive NHE activity (Fig. 2B), with no increase in NHE-1 protein levels up to 24 h after an acid pulse (Fig. 2A), which in turn is the time course over which significant cell proliferation occurs (14). This is supported by previous studies showing that upregulation of NHE in response to short-term acid-exposure results from increased activity of the exchanger (39, 40), while chronic acid exposure (hours or days) is dependent on protein synthesis to upregulate NHE (22, 24). Protein levels of other NHE isoforms were not examined given the marked increase in NHE activity. Current evidence suggests that acute activation of the antiporter is mediated via phosphorylation by PKC and/or tyrosine kinases (18, 28, 33). The kinase(s) responsible for phosphorylation of NHE appears to be specific for particular NHE isoforms. For example, while all exchanger isoforms have potential PKC phosphorylation sites, direct phosphorylation of NHE by PKC has only been shown for NHE-1 (35). In contrast, phosphorylation of NHE-3 has been shown indirectly to occur by tyrosine kinases (56). Here we demonstrate that pharmacological inhibition of PKC obliterated the acid-induced hyperproliferative response of BE to a level below that of the control group (pH 7.4) (Fig. 3A), suggesting that PKC may be necessary to maintain baseline cell proliferation after acid exposure. In contrast, cell proliferation was induced at pH 7.4 by PMA, a PKC activator (Fig. 3B). It remains to be determined whether PKC is exerting its mitogenic effect in BE via the NHE exchanger or independently. However, our observation supports previous experiments in rat renal proximal tubule and vascular smooth muscle cells, in which PKC was shown to be an important mechanism for NHE activation, which in turn resulted in an increase in cell proliferation (40, 53).A postulated mechanism for acid-induced cell proliferation via increased NHE activity in BE. Although the mechanism by which activation of NHE results in cell proliferation is unclear (17), several studies implicate NHE-induced changes in pHi (17, 33). For example, activation of NHE has been shown to alkalinize the cytoplasmic pH by 0.2-0.3 pH units, and this alkalinization is thought to be sufficient and important for the initiation of cell proliferation (33). In addition, cell cycle studies have shown that a rapid increase in pHi may be important to bring cells from G0/G1 and into S phase (27). Using TE7 cells, we demonstrated an alkaline overshoot effect in a significant proportion of cells, which increased the pHi above preacidosis values by an average of 0.35 ± 0.05 pH units and this effect was dependent on NHE and PKC activities (Fig. 5). The reason for the heterogeneous pHi response of TE7 cells to an acid pulse is unclear; however, it does correlate with the number of cells in G0/G1 and hence it may be related to the cell-cycle stage. Extrapolating these results to BE, an acid-induced alkaline overshoot may be responsible for the hyperproliferative response observed ex vivo. For example, low pH activates the NHE-1 exchanger in BE and as long as the pH remains low, cells are arrested in the cell cycle and remain unable to proliferate. However, as soon as the external pH is normalized, as would occur at the end of an acid-reflux event, a temporary cytoplasmic alkalinization by the activated NHE may be sufficient to stimulate cells to move from G0/G1 to S phase and hence result in a proliferative burst (Fig. 6). This hypothesis is in accordance with the temporary alkaline overshoot seen in cardiac myocytes in response to acute acidosis and in a model of reperfusion stunning of ischemic myocardium (54). Further support for this hypothesis is provided by the observation that some cell types can be induced to proliferate by an exogenously imposed cytoplasmic alkalization (57) and by experiments in which we alkalinized the medium of TE7 cells in culture and examined their proliferative response. In these experiments, TE7 cells were pulsed for 1 h with pH of 7.4 (control), 3.5 (acid control), 7.7, 8.0, 8.5, and 9.0 and then cultured at pH 7.4 for up to 24 h. We noted an increase in the proliferation of TE7 cells with an alkaline pulse that was similar but more rapid than that observed with 1-h acid pulse (data not shown).
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ACKNOWLEDGEMENTS |
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We are very grateful to Paul Matsudaira and Mrinalini Rao for providing the necessary antibodies. We thank Kris Morrow for preparing the figures.
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FOOTNOTES |
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This study was supported by TAP Pharmaceuticals (G. Triadafilopoulos), Veterans Administration Career Development and Merit Awards (M. B. Omary), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38707. R. C. Fitzgerald is a Fellow of the Association for International Cancer Research.
Address for reprint requests: G. Triadafilopoulos, Gastroenterology Section (111-GI), Veterans Affairs Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304.
Received 17 November 1997; accepted in final form 19 March 1998.
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