1 Department of Internal
Medicine I, Rabbit parietal
cells express three
Na+/H+
exchanger isoforms (NHE1, NHE2, and NHE4). We investigated the effects
of carbachol, histamine, and forskolin on
Na+/H+
exchange activity and acid formation in cultured rabbit parietal cells
and tested the effect of NHE isoform-specific inhibition on
agonist-induced
Na+/H+
exchange. Carbachol (10
NHE1; NHE2; NHE4; stomach; acid secretion; histamine; forskolin; carbachol; intracellular pH; volume regulation
THE INVESTIGATION OF ION homeostasis and intracellular
pH (pHi) control in the gastric
parietal cells during acid secretion has yielded controversial results
(7, 14, 15, 27, 30). Investigators have all found an increase in the
anion flux rate via the basolateral anion exchanger, although the mode
of activation has remained obscure (7, 15, 18, 27), and many have also observed a slight increase in the
pHi during secretagogue
stimulation of the parietal cells (14, 15, 18). It has remained unclear whether this increase was due to the action of the
H+-K+-ATPase
per se or activation of an
Na+/H+
exchanger (15, 18, 27). Also, the functional significance, if any, of
this pHi increase is controversial
(7, 15, 18, 27). Thus it is still largely unclear how the parietal cell accomplishes ion, pHi, and volume
homeostasis during acid formation.
Several years ago, a method was developed by Chew and co-workers (6)
that allowed the maintenance of isolated rabbit parietal cells in
culture for a period of up to 2 wk with preserved
H+-K+-ATPase
expression and a better response to secretagogues than seen in freshly
isolated cells. Saccomani et al. (21) demonstrated that cultured
parietal cells display a polarized membrane arrangement. We have
therefore speculated that parietal cells maintained in culture may be
an interesting model to study possible changes in
pHi and ion transport processes in
response to hormonal stimulation. In this study, we examine the effects
of secretagogues and hyperosmolarity on parietal cell
Na+/H+
exchange activity and pHi and
investigate which of the three different
Na+/H+
exchanger isoforms expressed in rabbit parietal cells (namely, NHE1,
NHE2, and NHE4) are activated by the respective secretagogues.
Materials.
4-Isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate
(HOE-642) was prepared by Hoechst (Frankfurt, Germany).
3-(Cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo-[1,2a]-pyridine (Sch-28080) was from Schering (Berlin, Germany). Collagenase was from
Worthington (Freehold, NJ). Pronase E was from Merck (Darmstadt, Germany). BSA (cell culture grade) was from Paesel und Lorei
(Frankfurt, Germany).
[14C]aminopyrine was
from Amersham (Braunschweig, Germany). A 1:1 mixture of Ham's F-12 and
DMEM (with HEPES and L-glutamine
and without HCO Isolation and culture of rabbit parietal cells.
Cells were isolated as described previously (25-27) with minor
differences as described below. Cell culture was adapted from the
method published by Chew et al. (6). Rabbit gastric cells were
enzymatically dispersed after high-pressure perfusion of the stomach in
situ as previously described (25, 27). Elutriation was performed using
a Beckman JM 6-C centrifuge with a JE-5.0 quick-assembly rotor. The
elutriation buffer was composed of (in mM) 140 NaCl, 14 HEPES, 7 Tris,
3 KH2PO4,
2 K2HPO4,
1.2 CaCl2, 1.2 MgSO4, and 20 glucose and 1 g/l
BSA, 0.5 mM dithiothreitol (DTT), and 10 mg/l gentamicin, pH 7.4. The
cell suspension was loaded into the 5-ml small chamber, and cells were
elutriated at a constant rotor speed of 1,750 rpm in four fractions
with flow rates of 15, 30, 35, and 65 ml/min. Fraction
4, primarily consisting of parietal and chief cells,
was then loaded on top of a Nycodenz step gradient (2:1, 1:1, and 1:2
dilution of Nycodenz-elutriation buffer) and centrifuged at 1,000 g for 8 min without brake. The second
band (thick yellow) was aspirated and diluted to 20 ml in culture
medium (DMEM-Ham's F-12 medium containing 2 g/l albumin, 800 nM
insulin, 5 mg/l transferrin, 5 µg/l sodium selenite, 10 nM
hydrocortisone, 8 nM EGF, 5 mg/l geneticin, 50 mg/l novobiocin, and 100 mg/l gentamicin). The suspension was then washed, and 250,000 cells/dish were placed onto 22-mm coverslips (for fluorescence studies)
or directly into 35-mm culture dishes (for aminopyrine uptake studies)
that were coated with 50 µl of Matrigel (diluted 1:7 in ice-cold
H2O). Cells were cultured in a
humidified incubator at 37°C in air. The medium was changed every
day.
Immunofluorescence.
Immunofluorescence studies were carried out as described by Soroka et
al. (28) with minor differences. Briefly, cultured cells were incubated
for 45 min in buffer B
(114.4 mM NaCl, 5.4 mM KCl, 5 mM Na2HPO4, 1 mM
NaH2PO4, 1.2 mM MgSO4, 2 mM
CaCl2, 2 g/l BSA, 10 mM glucose, 0.5 mM DTT, 1 mM pyruvate,
and 10 mM HEPES, pH 7.4) ± 1 mM histamine, washed with
buffer C (60 mM PIPES, 5 mM EGTA, and
2 mM MgCl2, pH 6.8, prewarmed to
37°C), and fixed for 15 min at room temperature with 3% freshly
prepared paraformaldehyde in buffer C.
After cells were washed twice with PBS, they were permeabilized with
0.2% Triton X-100 in PBS for 10 min and washed twice again with PBS.
Antibody HK-12.18 was applied at 1:25 dilution in 0.1% BSA-PBS (pH
7.15) for 3 h. Cells were incubated for 30 min with FITC-labeled goat
anti-mouse IgG as second antibody and then photographed using an AGFA
RS-1000 film and 3-min exposure.
Fluorescence microscopy for determination of
pHi.
Cultured cells were incubated with 5 µM BCECF-AM (Molecular Probes)
in buffer (in mM: 140 NaCl, 14 HEPES, 7 Tris, 3 KH2PO4, 2 K2HPO4,
1.2 CaCl2, 1.2 MgSO4, and 20 glucose) for 30 min
at 37°C. Cells were then washed free of BCECF-AM and incubated for 30 min at 37°C in buffer and washed again, and the coverslips were
mounted in a custom-made perfusion chamber that was placed on the
heated stage of an inverted fluorescence microscope (Nikon Diaphot-TMD). Preheated and continuously gassed solutions were connected by a manifold to the chamber, allowing rapid fluid changes without interruption of flow. Cells were alternately excited at 440 ± 10 nm and 490 ± 10 nm at a rate of 100/s. Emitted light from
an individual group of cells was collected through a 510-nm dicroic
mirror, a 530-nm long-pass filter, and an adjustable diaphragm and
recorded by a photomultiplier. Data acquisition (1 ratio value/s) and
processing were performed using the software provided by the manufacturer (Photon Technologies, Wedel, Germany) except for the
excitation shutter, which was controlled by a custom-made program that
allowed changes between continuous and intermittent (to reduce
photobleaching) illumination during the experiment. At the end of each
experiment, the 440 nm-to-490 nm ratio was calibrated to
pHi after clamping
pHi to extracellular pH using the
high K+-nigericin method (100 mM
potassium gluconate, 40 mM KCl, 14 mM HEPES, 7 mM Tris, 3 mM
KH2PO4,
2 mM
K2HPO4,
1.2 mM CaCl2, 1.2 mM
MgSO4, 20 mM glucose, and 10 µM
nigericin, pH 6.6 or pH 7.4). Background fluorescence was found to be
negligible and was not corrected for.
Determination of the intrinsic buffering capacity.
Intrinsic buffering capacity ( [14C]aminopyrine uptake.
[14C]aminopyrine
uptake was studied as described by Chew and Hersey (5), with minor
differences. After cells were washed twice and preincubated for 60 min
in buffer B, cells were incubated with
1 ml of buffer B plus 0.05 µCi
[14C]aminopyrine for
30 min. Agonists were then added for 45 min. Next, the supernatant was
discharged, and cells were washed twice with buffer
B and then lysed with 1 ml 3% Triton X-100, 500 µl of which were counted in a beta counter.
Statistics.
Results are given as means ± SE.
H+ fluxes were calculated by
performing linear regression analysis on individual
pHi traces during the first
1-2 min of stimulation (linear phase). ANOVA was used for multiple
comparisons.
Validation of the parietal cell culture.
Before the start of the experiments in this study, a number of
experiments were performed to validate the parietal cell culture. Immunocytochemical staining of
H+-K+-ATPase
revealed that almost all cells in the culture express this protein even
after 7 days of culture (Fig.
1). Cell number was determined
over the length of the culture, and cell aliquots were stimulated daily
over the length of the culture. We observed a dramatic increase in
basal [14C]aminopyrine
uptake between day zero and
day one, whereas agonist-stimulated [14C]aminopyrine
uptake remained similar; therefore, the ratio of 14C uptake between stimulated and
unstimulated cells declined sharply (Fig.
2). We then used acridine orange to stain
the acidic membrane compartments in the parietal cells and observed
that, in contrast to freshly isolated cells, in which the secretory
membranes are collapsed, quite a few parietal cells display red vesicle
structures that shift their color to green under omeprazole treatment
(data not shown). Thus the reason for the high basal
[14C]aminopyrine
uptake is spontaneous acid formation in many of the cultured cells.
Histamine treatment resulted in the appearance of more or enlargement
of preexisting acidic compartments.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
4 M)
was the weakest acid secretagogue but caused the strongest Na+/H+
exchange activation, which was completely blocked by 1 µM HOE-642 (selective for NHE1); histamine
(10
4 M) and forskolin
(10
5 M) were stronger
stimulants of
[14C]aminopyrine
accumulation but weaker stimulants of
Na+/H+
exchange activity. HOE-642 (1 µM) reduced forskolin-stimulated Na+/H+
exchange activity by 35%, and 25 µM HOE-642 (inhibits NHE1 and -2)
inhibited an additional 13%, but 500 µM dimethyl amiloride (inhibits
NHE1, -2, and -4) caused complete inhibition. The presence of 5%
CO2-HCO
3
markedly reduced agonist-stimulated H+ efflux rates, suggesting that
the anion exchanger is also activated. Hyperosmolarity also activated
Na+/H+
exchange. Our data suggest that, in rabbit parietal cells,
Ca2+-dependent stimulation causes
a selective activation of NHE1, whereas cAMP-dependent stimulation
activates NHE1, NHE2, and more strongly NHE4. Because intracellular pH
(pHi) did not change in the
presence of
CO2-HCO
3
and concomitant activation of
Na+/H+
and anion exchange is one of the volume regulatory mechanisms, we
speculate that the physiological significance of secretagogue-induced Na+/H+
exchange activation may not be related to
pHi but to volume regulation during acid secretion.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
3), HEPES, Tris,
epidermal growth factor (EGF) (murine; cell culture grade), histamine,
carbachol, insulin, transferrin, sodium selenite, hydrocortisone,
gentamicin, novobiocin, and geneticin were from Sigma (Deisenhofen,
Germany); ranitidine was from Glaxo Wellcome (Bad Oldesloe, Germany),
and Nycodenz was from Nycomed (Oslo, Norway). Matrigel was from Becton Dickinson (Bedford, MA).
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
was from Molecular Probes (Leiden, The Netherlands). Antibody HK12.18
(against the cytoplasmatic domain of hog
H+-K+-ATPase)
was a generous gift from Dr. Adam Smolka (University of South Carolina,
Charleston, SC). All other chemicals were either from Sigma or from
Merck at tissue culture grade or the highest grade available.
i) was determined as
previously described by Boyarsky et al. (2, 3). Typical
pHi-dependent intrinsic buffering
curves with a
i
value of 70 mM/pH unit at pHi of
6.6, 38 mM/pH unit at 7.0, and 21 mM/pH unit at 7.4 were obtained and
used to calculate H+ efflux rates
for each given pHi. The shape of
the curve resembles those of many other cell types and is not shown.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Immunohistochemical staining of
H+-K+-ATPase
in cultured parietal cells at culture day
7. A: diffuse staining
of the cytoplasm in ranitidine-treated cells.
B: during histamine stimulation, large
vacuoles appear in the parietal cells and
H+-K+-ATPase
staining is seen preferentially in the membrane of these vacuoles.
Vacuoles are acidic as determined by 9-aminoacridine accumulation, and
acidification is reversed by omeprazole treatment (not shown).
View larger version (36K):
[in a new window]
Fig. 2.
Open bars indicate
[14C]aminopyrine
uptake values in ranitidine-treated cells, and hatched bars indicate
uptake in forskolin-stimulated parietal cells over the first 7 days of
culture. Top line ( ): decrease in
cell number/dish during that time period. Bottom
line (
): stimulation factor (stimulated
[14C]aminopyrine
uptake/basal
[14C]aminopyrine
uptake values). A sharp increase in basal
[14C]aminopyrine
uptake values occurs between day 0 and
day 1, whereas forskolin-stimulated
[14C]aminopyrine
uptake values do not show this phenomenon. This results in a sharp
decrease in the ratio between stimulated and control
[14C]aminopyrine
uptake values (
). To find a reason for this increase in basal
[14C]aminopyrine
uptake values, we studied the cell microfluorometrically using acridine
orange dye. In freshly isolated cells (day
0), red vacuoles were rarely observed, indicating
collapsed secretory canaliculi. At day
1, quite a few cells displayed red vacuoles that
increased in size and number during forskolin stimulation. Thus the
increase in basal
[14C]aminopyrine
uptake values is due to spontaneous acid formation in unstimulated
controls. cpm, Counts/min.
Determination of acid secretion by
[14C]aminopyrine uptake after
stimulation with different agonists.
To demonstrate appropriate acid secretory responsiveness and to
determine differences in potency of the agonists of acid formation used
in this study,
[14C]aminopyrine
uptake was measured on stimulation and compared with a control that was
pretreated with ranitidine to prevent effects from potential endogenous
histamine release. Carbachol (104 M) stimulated
[14C]aminopyrine
uptake 2.6 ± 0.6-fold (n = 5),
10
5 M histamine stimulated
uptake 4.0 ± 0.7-fold, and
10
5 M forskolin stimulated
uptake 3.9 ± 0.5-fold compared with control. The difference between
[14C]aminopyrine
uptake values in control incubated in the presence and absence of
10
5 M ranitidine was not
significant, indicating that contamination with histamine-releasing
cells is small.
Effects of agonists of acid secretion on
pHi and
Na+/H+
exchange activity.
The resting pHi in clusters of
four to six cultured parietal cells, measured microfluorometrically
with the fluorescent dye BCECF, was found to be 7.16 ± 0.02 pH
units over the whole time course of the experiments. Carbachol, which
was the weakest agonist for
[14C]aminopyrine
uptake, caused a consistent increase in parietal cell resting
pHi of 0.2 ± 0.03 pH units
(Fig.
3A),
which was inhibited by the concomitant addition of
106 M atropine or
intracellular Ca2+ chelation by
preincubation with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (data not shown). Forskolin and histamine, which were both
stronger agonists for
[14C]aminopyrine
uptake, also caused increases of parietal cell
pHi of 0.15 ± 0.02 and 0.13 ± 0.03 pH units (P < 0.001, n = 5-7) (Fig. 3A). Omeprazole pretreatment caused
rapid cellular acidification after stimulation, but the
pHi increase was unchanged (Fig.
4A). Sch-28080 also did not influence the
pHi increase. On the other hand,
Na+ removal completely abolished
the response to all secretagogues, and 100 µM dimethyl amiloride
(DMA) completely inhibited the response to carbachol but
not to histamine and forskolin (data not shown), whereas 500 µM DMA
completely inhibited the pHi
increase seen with any secretagogue (data not shown). This suggests
that
Na+/H+
exchanger activation was the underlying mechanism for the
pHi rise with all tested
secretagogues. To quantify
Na+/H+
exchange activation, we determined the
H+ efflux rate during the initial
1-2 min after secretagogue stimulation (linear
pHi increase) (Figs.
3C and
4B and Table
1).
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Effects of NHE-specific inhibition on the agonist-mediated pH rise. The differential sensitivities of the carbachol-, histamine-, and forskolin-induced Na+/H+ exchange activation to DMA suggested to us that different isoforms are likely stimulated by the different agonists. We therefore tested the effect of the NHE1-specific inhibitor HOE-642 on secretagogue-induced Na+/H+ exchange activation. HOE-642 has been shown to block the different NHE subtypes in a strictly concentration-dependent manner, which made it a very useful compound for us to identify the NHE isoforms involved in secretagogue-induced Na+/H+ exchanger activation. Preincubation with 1 µM HOE-642, a concentration that will fully inhibit NHE1 in transfected fibroblasts (22), completely inhibited the carbachol-induced H+ efflux but only 28% and 35% of the histamine- and forskolin-induced flux rates, respectively (Fig. 3, B and C). Increasing the concentration of HOE-642 to 25 µM, a concentration that will fully inhibit NHE2 activity, caused an additional 13% inhibition of the initial H+ flux rate, but 500 µM DMA, which has been shown to inhibit NHE4 activity in transfected fibroblasts (1), caused complete inhibition (Fig. 5, B and C). Because only these three isoforms are expressed in rabbit parietal cells, the results suggest that Ca2+-mediated stimulation causes a selective stimulation of the rabbit parietal cell NHE1, whereas cAMP-dependent stimulation activates predominantly NHE4 and to a lesser extent NHE1 and NHE2.
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Effect of hyperosmolarity on parietal cell
Na+/H+
exchange.
The absence of a secretagogue-induced
pHi change in physiological
CO2-HCO3
concentrations and the apparent activation of both
Na+/H+
and anion exchange by all secretagogues suggested to us that the
physiological significance of agonist-induced
Na+/H+
exchanger activation in parietal cells could be a volume regulatory response after secretion-associated cell shrinkage. Hyperosmolarity activates
Na+/H+
exchange in many cell types (10, 11); therefore, the effect of
hyperosmolarity on parietal cell
Na+/H+
exchange was tested. Figure 7 shows a rapid
rise in pHi induced by an increase
in medium osmolarity of 100 mosM. This
pHi rise was absent in the absence
of Na+ (data not shown) or in the
presence of 500 µM DMA (Fig. 7B)
but was partially preserved in the presence of 1 µM HOE-642 (data not
shown), indicating that more than one isoform is involved.
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DISCUSSION |
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The present study was undertaken to investigate whether the different NHE isoforms expressed in rabbit parietal cells show a differential response to acid secretagogues. We found that carbachol caused the strongest activation of Na+/H+ exchange, and its effect was completely blocked by atropine or intracellular Ca2+ chelation, indicating a typical muscarinic receptor, Ca2+-dependent signaling pathway. Also, the addition of 100 µM DMA or 1 µM HOE-642 completely inhibited the carbachol-induced Na+/H+ exchange activation. Because Scholz et al. (22) demonstrated a full inhibition of the initial rates of amiloride-sensitive 22Na+ influx by 1 µM HOE-642 in NHE1-transfected PS120 fibroblasts, a <20% inhibition in NHE2-transfected fibroblasts, and no inhibition in NHE3-transfected fibroblasts, this substance is particularly well suited to distinguish between NHE1 activation and that mediated by the other isoforms. The exquisite sensitivity of carbachol-mediated Na+/H+ exchange activation to HOE-642 suggests that Ca2+-mediated stimulation is exclusively mediated via the NHE1 isoform.
Histamine and forskolin induced a pHi increase that was inhibited by 100 µM DMA and 1 µM HOE-642 to a minor extent only but was completely blocked by 500 µM DMA or the removal of Na+ from the medium, suggesting that other NHE isoforms are involved. Increasing the HOE-642 concentration to 25 µM completely inhibited the NHE2 isoform in transfected fibroblasts (22), which has also been reported to be stimulated by cAMP in transfected Na+/H+ exchanger-deficient Chinese hamster ovary cells (9). HOE-642 at 25 µM inhibited only an additional 13% of the forskolin-stimulated H+ flux rate, suggesting that the majority of cAMP-dependent Na+/H+ exchanger activation is mediated via the NHE4 isoform, since only these three isoforms are expressed in rabbit parietal cells (24).
Apart from its known amiloride resistance, little is known about the NHE4 isoform, since expression in transfected cells has been achieved by only two groups. In one laboratory (1) hyperosmolar medium was necessary to induce expression of NHE4, and in another (4) the application of stilbenes was necessary. DMA at 500 µM had been inhibitory for NHE4, and preincubation with cytochalasin D reduced NHE4 exchange activity markedly (1). An inhibitory effect of cytochalasin D was found for both the total and the HOE-642-insensitive part of forskolin-stimulated Na+/H+ exchange activity in rabbit parietal cells; although this finding does not allow positive identification, it is at least consistent with the hypothesis that the HOE-642-insensitive part is due to NHE4 activation.
We had observed this relatively amiloride-resistant histamine-induced pHi increase in cultured parietal cells many years ago but had been unable to explain the findings reasonably (20). When Orlowski et al. (17) published the cloning of rat NHE3 and NHE4 and the expression of both isoforms in rat stomach, we speculated that the cAMP-mediated pHi increase may be due to an amiloride-resistant NHE isoform. We obtained the cDNAs or PCR-cloned cDNA fragments for the different NHE isoforms for rat and rabbit and found that, whereas all four NHE isoforms are expressed in rat stomach, albeit with very different distributions among the different cell types, NHE3 is expressed in rabbit stomach at extremely low abundance and only in surface cells, whereas the other three isoforms are strongly expressed (24). We have now quantified NHE1, -2, and -4 expression in the different rabbit gastric cell types using histone 3.3a as an internal standard and found that mucous cells express very high NHE1 and NHE2 levels and low NHE4 levels, whereas parietal cells display a more even distribution, with NHE1 and NHE2 expression slightly higher than NHE4 expression (unpublished results). Although expression levels do not accurately reflect protein abundance, they usually give a rough estimate of the expected function if the levels are not low, and this was not the case for NHE expression in the stomach. Thus it was somewhat surprising to us that we found relatively little contribution of the NHE2 isoform to the secretagogue-induced pHi increase, especially in light of the finding that the NHE2 knockout mouse shows severe gastric histopathology (23). This may be in part because it is clearly difficult to obtain true estimates of NHE2 function, since one has to use a window between different drug concentrations to estimate the NHE2 contribution to a given Na+/H+ exchange rate, and this can clearly underestimate its true activity. In fact, a concentration of 1 µM inhibits NHE2 activity in transfected fibroblasts by ~20% (22) and 25 µM may not fully inhibit NHE2 at physiological extracellular Na+ concentrations. Therefore, a few percentages of the Na+/H+ exchange activation that we attribute to NHE1 and NHE4 may be NHE2 mediated. However, it is also possible that the conditions of our experiments are not those under which NHE2 function is strongly activated in the parietal cell.
What could be the physiological role of NHE activation during acid
secretion? In 1988, Muallem et al. (15) demonstrated Na+/H+
exchange activation by forskolin in freshly isolated rabbit parietal cells and speculated that the resultant alkalinization may be important
for activation of the highly
pHi-sensitive
Cl/HCO
3
exchanger. Thus
Na+/H+
exchange activation and the resultant
pHi increase have been discussed
as a signaling mechanism during acid secretion. We were not able to
reproduce these findings in our study (27), and we contribute our
failure to see forskolin-induced alkalinization to our use of freshly
isolated cells and because NHE4 may be particularly sensitive to the
cytoskeletal derangement that is certain to occur during cell isolation
[we did see a carbachol-induced
Na+/H+
exchange activation in freshly isolated cells, although to a lesser
extent than was seen in cultured cells (20)]. Nevertheless, we
and others had found secretagogue-induced
Cl
/HCO
3
activation in freshly isolated cells and gastric glands in the absence
of significant
Na+/H+
activation, demonstrating that a
Na+/H+
exchanger-mediated pHi increase
cannot be the only, or the most important, signal for
Cl
/HCO
3
exchange activation (7, 27).
Parietal cells in culture have been shown to display a polarized
membrane arrangement (21), indicating that, although they do not fully
resemble native parietal cells due to the lack of the appropriate basal
lamina and neighboring cells, they have reformed a part of their
cytoskeletal structure. In cultured parietal cells, each of the
secretagogues tested in the study caused a significant increase in
parietal cell pHi in a
HEPES-buffered medium but none caused a significant increase in a
medium buffered with 5%
CO2-HCO3.
This cannot be solely due to the higher buffer capacity in the presence
of
CO2-HCO
3 but suggests that an acid-loading mechanism, most likely the
Cl
/HCO
3
exchanger, is concomitantly activated. This suggests that the
physiological role of Na+/H+ exchanger
activation during acid secretion does not lie in its capability to
raise pHi.
A dual activation of
Na+/H+
and
Cl/HCO
3
exchange is one of the mechanisms for regulatory volume increase (10, 11, 13). A volume increase has been shown to have a signaling character
for metabolic and possibly transcriptional cellular events (8, 10), and
ion secretion is classically associated with cell shrinkage followed by
a regulatory volume increase (16, 32). We therefore speculate that the
secretagogue-induced
Na+/H+
exchange activation is a component of a cell volume regulatory phenomenon. Indeed, hyperosmolarity also induced
Na+/H+
exchange activation and a pHi
increase in the absence but not in the presence of 5%
CO2-HCO
3.
Detailed studies on volume regulation during stimulation of acid
secretion in the parietal cells are lacking, and this is clearly an
interesting field for further investigation.
Interestingly,
Na+/H+
exchange activation was not proportional to the intensity of
secretagogue stimulation of acid formation. If cell shrinkage were
simply a result of activation of the ion transport processes directly
involved in HCl transport across the canalicular membrane, i.e., apical
K+ and
Cl channels and the
H+-K+-ATPase,
this would be expected. However, different basolateral K+ channels are activated by
Ca2+- and cAMP-dependent agonists
(31), and this activation could result in cell shrinkage (12, 19, 29),
irrespective of the effect on acid secretion. However, if cell
shrinkage were the primary stimulus for
Na+/H+
exchange activation, then one would expect that all three tested agonists result in an NHE isoform activation that is proportional to
the agonist-induced volume loss, which, surprisingly to us, was not the
case. The same holds true for any other
Na+/H+
activation, which is secondary to the activation of another ion transporter during acid secretion, i.e.,
Na+-K+-ATPase
activation secondary to K+ channel
opening with resultant demand for intracellular
Na+. This is not compatible with
the very different effect of HOE-642 on
Na+/H+
exchange activation by the tested secretagogues and suggests a primary,
and differentially regulated, activation of the different NHE isoforms
expressed in rabbit parietal cells by the different secretagogues. The
data suggest that
Na+/H+
exchange activation during stimulation of acid secretion is directly activated by the respective agonist and that different signal transduction pathways result in the activation of a different set of
NHE isoforms. Thus it would appear that secretagogue stimulation of
parietal cells results in a primary activation of the different ion
transport pathways that are required both for the initiation of the
secretory process and for the homeostatic processes necessary to
maintain it. The observation that muscarinergic and histaminergic stimulation results in a differential activation of
Na+/H+
exchanger isoforms suggests that, in regard to
Na+/H+
exchange activation, a potentiated cellular response may not only be
the result of a positive cooperation at the level of the intracellular
signaling cascade but may also be due to the recruitment of several
different transport proteins.
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ACKNOWLEDGEMENTS |
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We thank Conny Stettner, Susanne Römer, Hildegard Gaillinger, and Michael Albert for their continuous improvement of the parietal cell culture method during the last years, which made the present work possible. We also thank Dorothee Vieillard-Baron, Heidi Rossmann, Petra Jacob, Alexandra Kretz, Irina Blumenstein, Katrin Seiler, Markus Guba, and Michael Walter for their organizational help and critical discussions.
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FOOTNOTES |
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This work was supported by Grants Se 460/9-1 and Se 460/2-5 from the Deutsche Forschungsgemeinschaft and by Grant F. 1281038 from the Fortüne program of the Eberhard-Karls University Tübingen.
This report includes experimental work performed by O. Bachmann and T. Sonnentag in fulfillment of the requirements for their doctoral theses.
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: U. Seidler, Abteilung Innere Medizin I, Universitätsklinikum Schnarrenberg, Eberhard-Karls Universität Tübingen, Otfried-Müller Str. 10, D-72076 Tübingen, Germany.
Received 28 May 1998; accepted in final form 30 July 1998.
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REFERENCES |
---|
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---|
1.
Bookstein, C.,
M. W. Musch,
A. DePaoli,
Y. Xie,
K. Rabenau,
M. Villereal,
M. C. Rao,
and
E. B. Chang.
Characterization of the rat Na+/H+ exchanger isoform NHE4 and localization in rat hippocampus.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1629-C1638,
1996
2.
Boyarsky, G.,
M. B. Ganz,
R. B. Sterzel,
and
W. F. Boron.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3.
Am. J. Physiol.
255 (Cell Physiol. 24):
C844-C856,
1988
3.
Boyarsky, G.,
M. B. Ganz,
R. B. Sterzel,
and
W. F. Boron.
pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl-HCO
3 exchangers.
Am. J. Physiol.
255 (Cell Physiol. 24):
C857-C869,
1988
4.
Chambrey, R.,
J. M. Achard,
and
D. G. Warnock.
Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform.
Am. J. Physiol.
272 (Cell Physiol. 41):
C90-C98,
1997
5.
Chew, C. S.,
and
S. J. Hersey.
Gastrin stimulation of isolated gastric glands.
Am. J. Physiol.
242 (Gastrointest. Liver Physiol. 5):
G504-G512,
1982
6.
Chew, C. S.,
M. Ljungstrom,
A. Smolka,
and
M. R. Brown.
Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G254-G263,
1989
7.
Debellis, L.,
S. Curci,
and
E. Fromter.
Microelectrode determination of oxyntic cell pH in intact frog gastric mucosa. Effect of histamine.
Pflügers Arch.
422:
253-259,
1992[Medline].
8.
Graf, J.,
and
D. Haussinger.
Ion transport in hepatocytes: mechanisms and correlations to cell volume, hormone actions and metabolism.
J. Hepatol.
24:
53-77,
1996[Medline].
9.
Kandasamy, R. A.,
F. H. Yu,
R. Harris,
A. Boucher,
J. W. Hanrahan,
and
J. Orlowski.
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
J. Biol. Chem.
270:
29209-29216,
1995
10.
Lang, F.,
G. L. Busch,
M. Ritter,
H. Volkl,
S. Waldegger,
E. Gulbins,
and
D. Haussinger.
Functional significance of cell volume regulatory mechanisms.
Physiol. Rev.
78:
247-306,
1998
11.
Lang, F.,
and
S. Waldegger.
Regulating cell volume.
Am. Sci.
85:
456-463,
1997.
12.
Li, Q.,
V. Jungmann,
A. Kiyatkin,
and
P. S. Low.
Prostaglandin E2 stimulates a Ca2+-dependent K+ channel in human erythrocytes and alters cell volume and filterability.
J. Biol. Chem.
271:
18651-18656,
1996
13.
McLeod, R. J.
How an epithelial cell swell is a determinant of the signaling pathways that activate regulatory volume decrease.
In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Boca Raton, FL: CRC, 1993, p. 191-200.
14.
Michelangeli, F.
Acid secretion and intracellular pH in isolated oxyntic cells.
J. Membr. Biol.
38:
31-50,
1978[Medline].
15.
Muallem, S.,
D. Blissard,
E. J. Cragoe, Jr.,
and
G. Sachs.
Activation of the Na+/H+ and Cl/HCO
3 exchange by stimulation of acid secretion in the parietal cell.
J. Biol. Chem.
263:
14703-14711,
1988
16.
Nauntofte, B.
Regulation of electrolyte and fluid secretion in salivary acinar cells.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G823-G837,
1992
17.
Orlowski, J.,
R. A. Kandasamy,
and
G. E. Shull.
Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.
J. Biol. Chem.
267:
9331-9339,
1992
18.
Paradiso, A. M.,
M. C. Townsley,
E. Wenzl,
and
T. E. Machen.
Regulation of intracellular pH in resting and in stimulated parietal cells.
Am. J. Physiol.
257 (Cell Physiol. 26):
C554-C561,
1989
19.
Ritter, M.,
E. Woll,
D. Haussinger,
and
F. Lang.
Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene.
FEBS Lett.
307:
367-370,
1992[Medline].
20.
Römer, S.,
U. Seidler,
and
M. Classen.
Effect of histaminergic and cholinergic stimulation on pHi in cultured parietal cells (Abstract).
Gastroenterology
106:
836,
1994.
21.
Saccomani, G.,
C. G. Psarras,
P. R. Smith,
K. L. Kirk,
and
R. L. Shoemaker.
Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells.
Am. J. Physiol.
260 (Cell Physiol. 29):
C1000-C1011,
1991
22.
Scholz, W.,
U. Albus,
L. Counillon,
H. Gogelein,
H. J. Lang,
W. Linz,
A. Weichert,
and
B. A. Scholkens.
Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion.
Cardiovasc. Res.
29:
260-268,
1995[Medline].
23.
Schultheis, P. J.,
L. L. Clarke,
P. Meneton,
M. Harline,
G. P. Boivin,
G. Stemmermann,
J. J. Duffy,
T. Doetschman,
M. L. Miller,
and
G. E. Shull.
Targeted disruption of the murine NHE2 Na+/H+ exchanger gene causes reduced viability of gastric parietal cells and loss of net acid secretion.
J. Clin. Invest.
101:
1243-1253,
1998
24.
Seidler, B.,
H. Rossmann,
A. Murray,
J. Orlowski,
C. M. Tsen,
M. Donowitz,
G. Shull,
M. Gregor,
and
U. Seidler.
Expression of the Na+/H+ exchanger isoform NHE1-4 mRNA in the different epithelial cell types of rat and rabbit gastric mucosa (Abstract).
Gastroenterology
112:
290,
1997.
25.
Seidler, U.,
M. Beinborn,
and
K. F. Sewing.
Inhibition of acid formation in rabbit parietal cells by prostaglandins is mediated by the prostaglandin E2 receptor.
Gastroenterology
96:
314-320,
1989[Medline].
26.
Seidler, U.,
K. Carter,
S. Ito,
and
W. Silen.
Effect of CO2 on pHi in rabbit parietal, chief, and surface cells.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G466-G475,
1989
27.
Seidler, U.,
S. Roithmaier,
M. Classen,
and
W. Silen.
Influence of acid secretory state on Cl-base and Na+-H+ exchange and pHi in isolated rabbit parietal cells.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G81-G91,
1992
28.
Soroka, C. J.,
C. S. Chew,
D. K. Hanzel,
A. Smolka,
I. M. Modlin,
and
J. R. Goldenring.
Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.
Eur. J. Cell Biol.
60:
76-87,
1993[Medline].
29.
Suzuki, Y.,
M. Ohtsuyama,
G. Samman,
F. Sata,
and
K. Sato.
Ionic basis of methacholine-induced shrinkage of dissociated eccrine clear cells.
J. Membr. Biol.
123:
33-41,
1991[Medline].
30.
Thomas, H. A.,
and
T. E. Machen.
Regulation of Cl/HCO3 exchange in gastric parietal cells.
Cell Regul.
2:
727-737,
1991[Medline].
31.
Ueda, S.,
D. D. Loo,
and
G. Sachs.
Regulation of K+ channels in the basolateral membrane of Necturus oxyntic cells.
J. Membr. Biol.
97:
31-41,
1987[Medline].
32.
Valverde, M. A.,
J. A. O'Brien,
F. V. Sepulveda,
R. Ratcliff,
M. J. Evans,
and
W. H. Colledge.
Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-mediated secretagogue-induced volume decrease in small-intestinal crypts.
Pflügers Arch.
425:
434-438,
1993[Medline].