First Department of Medicine, Eberhard-Karls University Tübingen, D-72076 Tübingen, Germany
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ABSTRACT |
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Several Na+/H+ exchanger (NHE) isoforms are expressed in the stomach, and NHE1 and NHE2 knockout mice display gastric mucosal atrophy. This study investigated the cellular distribution of the NHE isoforms NHE1, NHE2, NHE3, and NHE4 in rabbit gastric epithelial cells and their regulation by intracellular pH (pHi), hyperosmolarity, and an increase in cAMP. Semiquantitative RT-PCR and Northern blot experiments showed high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells. Fluorescence optical measurements in cultured rabbit parietal and mucous cells using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and NHE isoform-specific inhibitors demonstrated that in both cell types, intracellular acidification activates NHE1 and NHE2, whereas hyperosmolarity activates NHE1 and NHE4. The relative contribution of the different isoforms to pHi- and hyperosmolarity-activated Na+/H+ exchange in the different cell types paralleled their relative expression levels. cAMP elevation also stimulated NHE4, whereas an increase in osmolarity above a certain threshold further increased NHE1 and not NHE4 activity. We conclude that in rabbit gastric epithelium, NHE1 and NHE4 regulate cell volume and NHE1 and NHE2 regulate pHi. The high NHE1 and NHE2 expression levels in mucous cells may reflect their special need for pHi regulation during high gastric acidity. NHE4 is likely involved in volume regulation during acid secretion.
Na+/H+ exchanger isoform 1; Na+/H+ exchanger isoform 2; Na+/H+ exchanger isoform 4; stomach; intracellular pH regulation; intracellular pH; volume regulation
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INTRODUCTION |
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NA+/h+ exchangers (NHE) belong to a gene family of ion-transport proteins whose basic function is the exchange of extracellular Na+ for intracellular H+, thus causing either a rise in intracellular pH (pHi) or, if coupled to the action of other transporters, an increase in cell volume. These principal functions enable NHE to play a key role in a number of cellular processes, including proliferation, migration, transepithelial ion transport, gene regulation, and cell metabolism (38, 39).
pHi regulation in gastric epithelial cells has met with special interest because of the periodic presence of a very low luminal pH. Therefore, the capability of these cells to regulate their pHi after an intracellular acid load was investigated soon after the development of fluorescent dyes that allowed pHi measurements in cells too small for microelectrode puncture (22). Studies (28) demonstrated the existence of a NHE on all three major cell types in rabbit stomach capable of normalizing pHi after an acid load. However, data on the physiological relevance of Na+/H+ exchange in the stomach are controversial, and conflicting data have been presented concerning the role of Na+/H+ exchange in the initiation of acid secretion (19, 21, 29) and the maintenance of a near-neutral pHi during a luminal acid load (18, 33, 41). Recent data from our laboratory and others (16, 18, 30, 33) suggest a role for Na+/H+ exchange in gastric mucosal pHi homeostasis during luminal acidification, stimulation-associated volume regulation (1, 31), and gastric epithelial wound healing (15).
Up to this time, six Na+/H+ isoforms have been cloned from mammalian tissue. Rat gastric mucosa expresses NHE1, NHE2, NHE3, and NHE4 (20). A basolateral location for NHE1 and NHE4 has been demonstrated in rat stomach (23, 32), whereas an apical location is assumed for NHE3 and NHE2 on the basis of their apical location in rat kidney and intestine (3, 4, 13), where they have a proven (NHE3) or potential role (NHE2) in Na+ reabsorption and proton secretion. NHE1 knockout mice display gastric histopathology (2), and NHE2 knockout mice have as their most striking feature a severe gastric atrophy with an almost complete disappearance of parietal and chief cells (26). NHE4 is expressed in the stomach with particularly high expression levels (20). All of these findings suggest that the different NHE isoforms have an important, and likely distinct, physiological function in the gastric mucosa. To learn more about the physiological functions of the different NHEs with a special emphasis on their role in gastric physiology, we investigated 1) the relative mRNA expression levels of NHE1, NHE2, NHE3, and NHE4 in the different cell types of the rabbit gastric mucosa and 2) the activation of the different NHE isoforms by low pHi, hyperosmolarity, and cAMP in cultured rabbit parietal and mucous cells.
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MATERIALS AND METHODS |
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Materials. Unless otherwise specified, all reagents were obtained from Sigma-Aldrich and Fluka (Deisenhofen, Germany) or Merck (Darmstadt, Germany) at tissue culture grade, molecular biology grade, or the highest grade available.
Rabbit gastric cell purification for molecular biology studies. Parietal, chief, and mucous cells were purified from rabbit gastric mucosa, and the homogeneity of the three cell fractions was assessed by light microscopy after staining cytospin preparations as described previously (27, 28). The mucous cell fraction consists of 90-95% periodic acid-Schiff granule-positive cells, whereas the parietal cell fraction shows a purity of 95-98% and the chief cell population contains <2% contaminating cells. These findings were confirmed by the expression level of the H+-K+-ATPase in the different cell fractions as determined by Northern blot analysis (see Ref. 24 for data).
RNA isolation and Northern blot analysis.
Isolation of total and purified poly(A)+ RNA and Northern
blot analysis were carried out as described previously (11, 12, 24). Membranes were probed (see Fig. 1) with rabbit NHE1
(5'-untranslated region and nt 1-1524 of coding sequence), rabbit
NHE2 (nt 1628-2954 of coding sequence and 3'-untranslated region),
rabbit NHE3 (nt 1183-2496 of coding sequence), rabbit NHE4
[~1.7-kb PCR fragment, containing 680-bp coding sequence, 970-bp
3'-untranslated region, and ~50-bp poly(A) signal], rabbit
H+-K+-ATPase -subunit (nt 2646-3050 of
GenBank accession no. X64694), rabbit glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (nt 239-592 of GenBank accession no.
L23961), rat NHE2 (nt 174-2032 of coding sequence), and rat NHE4
(nt 272-2151 of coding sequence and 3'-untranslated region).
Rabbit NHE1, NHE2, and NHE3 cDNA fragments were kindly provided by
C. M. Tse, C. Yun, and M. Donowitz, rat NHE2, NHE4, and pepsinogen
by J. Orlowski and G. Shull, and rabbit NHE4 by Z. Wang and G. Shull.
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Semiquantitative RT-PCR.
Semiquantitative PCR was carried out as described previously (9,
24). Homologous primers for rabbit NHE1, NHE2, NHE3, NHE4,
GAPDH, and histone 3.3a were deduced from published sequence information or after sequencing an appropriate cDNA fragment (Table 1). The identity of the NHE1, NHE2, NHE3,
and NHE4 amplimers was confirmed by restriction analysis (see Fig.
1C). For semiquantitative PCR, the products were separated
on an agarose gel, and the optical density of the ethidium
bromide-stained bands was measured using the ImageMaster VDS system and
software (Amersham Pharmacia, Freiburg, Germany). The amplification
efficiency of the gene of interest and histone 3.3a was determined by
calculating the slope after semilogarithmic plotting of the values
against the cycle number (see Fig. 2A). The virtual
relationship integrated optical density (ODI) of the studied gene vs.
ODI of histone 3.3a was calculated (see Fig. 2B). To compare
NHE1, NHE2, NHE3, and NHE4 expression levels, the OD values of
the different PCR products were corrected according to their length.
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Purification and culture of rabbit parietal and mucous cells. Cell culture was adapted from the method of Chew et al. (10) as we (1, 24) have described in detail previously. To assess the functional integrity, the ability of the parietal cells to respond to secretory stimuli was periodically determined by measurement of [14C]aminopyrine (AP; Amersham Pharmacia) uptake into the cells as described by Chew et al. (10). Mucous cells were evaluated optically only. For fluorescence measurements, mucous cells were selected that had settled in groups of three or more and in which large mucous granules could be visualized under the microscope.
Fluorescence microscopy for determination of pHi. pHi measurements are described elsewhere in detail (1). Cultured cells were loaded with 5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Leiden, The Netherlands) and incubated for 30 min in buffer A (120 mM NaCl, 14 mM HEPES, 7 mM Tris, 3 mM KH2PO4, 1.2 mM CaCl2, 1.2 mM MgSO4, and 20 mM glucose, pH 7.4 gassed with O2), then alternately excited at 440 ± 10 and 490 ± 10 nm at a rate of 100/s. Emission wavelength was 530 nm. At the end of each experiment, the 440 nm-to-490 nm ratio was calibrated to pHi after clamping pHi to extracellular pH (pHo) using the high K+-nigericin method as described previously (1). Cellular acidification was achieved by an ammonium prepulse [2-15 min with 40 mM NH4Cl or (NH4)2SO4].
Determination of the intrinsic buffering capacity. Intrinsic buffering capacity was determined as previously described by Boyarsky et al. (Ref. 7; see Ref. 24 for values).
Statistics. Results are given as means ± SE. Proton fluxes were calculated by performing linear regression analysis on individual pHi traces during the first 1 to 2 min of stimulation (linear phase). Unless otherwise indicated, Student's t-test was used for paired samples and ANOVA was used for multiple comparisons.
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RESULTS |
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Molecular characterization of NHE isoforms expressed in rabbit gastric mucosa and their distribution in different epithelial cell types. As detected by Northern blot analysis and RT-PCR (Fig. 1) NHE1, NHE2, and NHE4 are expressed in rabbit gastric mucosa, whereas NHE3 is not. A 4.8- and 4.4-kb mRNA could be easily detected in rabbit kidney and colon by hybridization with a homologous NHE3 cDNA fragment (Fig. 1A) and a PCR product of the expected size and restriction pattern was amplified from colonic mucosa. In contrast, a faint band appeared only in the mucous cell lane after a 3-day exposure of the X-ray film, and RT-PCR amplified an NHE3 cDNA from rabbit stomach inconstantly and only at very high cycle numbers. In contrast to the situation in rat stomach (Ref. 20; B. Seidler, unpublished data), NHE3 expression is very low in rabbit gastric epithelial cells.
Incubation of a Northern blot with a homologous NHE2 fragment revealed several transcript sizes (Fig. 1B): a broad band at 5.2-6.5 kb, whose broadness is due to residual rRNA, a band at 4.5 kb, and a faint band at 4.9 kb. The rabbit NHE2 mRNA sizes correspond to those described by Tse et al. (35, 43). Because NHE2 and NHE4 show stretches of high homology, the cloning of a rabbit NHE4 cDNA fragment was done in the 3'-region [corresponding to nt 1979 of the rat sequence to the poly(A) signal] of the presumed NHE4 nucleotide sequence, where the homology (as deduced from the comparison of the rat NHE2 and NHE4 sequences) was expected to be extremely low, and this was confirmed by sequencing. Three mRNAs were detected by a homologous NHE4 probe, but with distinct transcript sizes compared with NHE2: one broad band at 4.9-6.2 kb and two more bands at 4.1 and 3.75 kb (Fig. 1B). The different sizes of the hybridization products seen with NHE2 and NHE4 cDNA prove that, under high-stringency conditions, the probes do not hybridize with mRNA of the other isoform. Because rabbit NHE4 Northern blot analysis has not been described yet, we cannot compare our transcript sizes with those of others. To quantify gene expression, a suitable internal control is necessary. Because GAPDH andRole of NHE1, NHE2, and NHE4 in parietal and mucous cell
pHi recovery.
To evaluate the physiological significance of the different NHE
isoforms in parietal and mucous cells, we evaluated their contribution
to the Na+/H+ exchange-mediated recovery from
an intracellular acid load. Cultured parietal and mucous cells were
acidified by an ammonium prepulse to approximately pH 6.4, and
pHi recovery was measured fluorometrically without and with
inhibitors in concentrations that selectively inhibit NHE1, NHE1 and
NHE2, or NHE1, NHE2, NHE3, and NHE4 (5, 25). Parietal cell
resting pHi in a HEPES-O2 buffer was 7.24 ± 0.03, which corresponds to previous measurements (19,
21), whereas mucous cell resting pHi was
significantly higher than parietal cell pHi (7.43 ± 0.04). When acidified to pHi of ~6.4, parietal cells
recovered with an initial proton efflux rate of 21 ± 1.1 mM/min
(Fig. 3, A and B).
Of this proton efflux, 80% was inhibited by 1 µM HOE-642
(very specific for NHE1 inhibition at this low concentration), and
almost all of the residual flux was inhibited by 25 or 50 µM HOE-642,
which also inhibits NHE2 (25). There was no significant
difference between 25 or 50 µM HOE-642 (data not shown), indicating
that either concentration is suitable for full NHE2 inhibition but not
NHE4 inhibition. The residual proton efflux under 500 µM
dimethylamiloride (DMA) (which also inhibits NHE4; Ref. 5)
was not significantly different from that under 25 µM HOE-642,
indicating that NHE1 and NHE2, but not NHE4, mediate pHi
recovery from an intracellular acid load.
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Activation of parietal and mucous cell NHE1, NHE2, and NHE4 by
hyperosmolarity.
The two basic and distinct cellular functions of
Na+/H+ exchange are pHi and volume
regulation. Therefore, we next investigated the activation of the
different NHE isoforms in parietal and mucous cells by hyperosmolarity.
Exposure of cultured parietal cells to 400 mosmol/kgH2O caused a rapid pHi increase of
0.21 ± 0.03 pH units (Fig.
5A). DMA (500 µM) completely
inhibited proton efflux and unmasked a slow acidification after
exposure to 400 mosmol/kgH2O, possibly due to anion
exchange activation, which is expected to have only a relatively small
effect on pHi at the given low pHi and in the
absence of CO2-HCO
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Effect of increasing osmolar strength on NHE1, NHE2, NHE3, and NHE4
activity.
In hepatocytes, increasing osmolar strength sequentially recruits
additional volume-regulatory mechanisms (40). We therefore wondered if a similar situation existed for NHE1 and NHE4 activation in
parietal cells. The perfusate of BCECF-loaded cultured parietal cells
was changed from 300 to 350, 400, or 500 mosmol/kgH2O, and the resulting pHi increase was measured. Figure
6 demonstrates the parietal cell proton
efflux rates due to Na+/H+ exchange stimulation
on exposure to 350, 400, and 500 mosmol/kgH2O. We
were surprised by the results: total DMA-sensitive proton efflux stimulated by 350 mosmol/kgH2O was 1.4 mM/min, and 0.82 mM/min or 60% was due to NHE4 activation. Higher osmolar strengths
increased overall Na+/H+ exchange rates
dramatically to 3.42 mM/min at 500 mosmol/kgH2O. Surprisingly, the increase in Na+/H+ exchange
rates was largely due to an increase in NHE1 activity, which increased
from 0.46 mM/min at 350 mosmol/kgH2O to 2.31 mM/min at 500 mosmol/kgH2O, whereas NHE4 activity increased from 0.82 mM/min at 350 mosmol/kgH2O to 1.13 mM/min at 500 mosmol/kgH2O. Hyperosmolarity-induced NHE2 activity was
minimal at all osmolar strengths. Thus the ratio of NHE1 to NHE4
activity changed from 0.56 at 350 mosmol/kgH2O to 1.5 at
400 mosmol/kgH2O and to 2.24 at 500 mosmol/kgH2O. These results suggest that both NHE isoforms are already activated in parietal cells at fairly modest increases in
osmolar strength, where NHE4 is the predominant active isoform, but
that NHE1 activity can increase far more dramatically than NHE4
activity at higher osmolarity.
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Activation of NHE4 by cAMP. We (1) have previously reported that acid secretagogues differentially activate the NHE isoforms in cultured rabbit parietal cells, with forskolin- or histamine-induced Na+/H+ exchange being mediated predominantly (55 and 65%) by NHE4. In this study, we found that parietal cell Na+/H+ exchange rates stimulated by 400 mosmol/kgH2O were somewhat higher than those previously measured under forskolin or histamine stimulation (1) but that the percentage of NHE4-mediated Na+/H+ exchange was lower (~40%). Volume measurements demonstrated that 400 mosmol/kgH2O causes only a slightly larger cell shrinkage than forskolin (31) but that forskolin and hyperosmolarity do not cause a stronger cell shrinkage than hyperosmolarity alone (Sonnentag, unpublished results). We therefore wondered if forskolin might activate NHE4 independently of cell shrinkage.
To further explore this possibility, we measured the hyperosmolarity-induced proton efflux rate in the absence and after sequential NHE1, and NHE1 and NHE2, inhibition in the presence and absence of forskolin (Fig. 7). We found that, in the presence of forskolin, hyperosmolarity consistently stimulated a higher proton efflux rate in both the absence and the presence of 1 and 25 µM HOE-642 than did hyperosmolarity alone. This suggests that an intracellular increase in cAMP levels has a distinct stimulatory effect on NHE4 that is not explained by cAMP-mediated cell shrinkage.
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DISCUSSION |
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The present study was undertaken to explore the cellular distribution of the NHE isoforms expressed in rabbit gastric mucosa and to investigate whether the different isoforms may have different modes of regulation and potential physiological significance in the stomach.
A previous study (14) demonstrated that NHE1 is strongly
expressed in the gastric mucosa compared with other segments of the
gastrointestinal tract and the kidney. This study demonstrates particularly high NHE1 expression levels in gastric mucous cells. NHE1
is thought to be involved in pHi and volume homeostasis and to be activated during cellular proliferation and migration
(38). Several studies suggest that protons enter the
surface cells during periods of strong luminal acidification, and both
Kiviluoto et al. (18) and we (30, 33) have
demonstrated that Na+/H+ exchange is one of the
homeostatic mechanisms involved in the maintenance of a near-neutral
pHi in the amphibian gastric mucosa. Also, evidence
(14) from the duodenum demonstrates that NHE1 is involved
in HCO
The acid-activated Na+/H+ exchange rate not inhibited by 1 µM HOE-642 (and therefore not mediated by NHE1) was inhibited by 25 or 50 µM HOE-642 and therefore was most likely due to NHE2. Both the percentage of and the absolute value for acid-activated Na+/H+ exchange rate sensitive to 50 µM but not 1 µM HOE-642 was far higher in mucous than in parietal cells. These findings correspond very well with the relative expression levels for NHE2 in mucous and parietal cells and make it highly likely that the acid-activated Na+/H+ exchange activity inhibited by 50 µM but not 1 µM HOE-642 is due to NHE2.
On the basis of high colonic expression levels and evidence for an
apical location, the physiological role of NHE2 has been discussed as
an alternative Na+ absorption mechanism in intestine and
kidney (8, 34, 36, 37, 44). Surprisingly, NHE2-deficient
mice show no intestinal or renal abnormalities and no diarrhea or
electrolyte imbalance but do display severe gastric mucosal atrophy
with total reduction of mucosal thickness and a particularly severe
reduction of parietal and chief cells, corresponding to a complete loss
of the acid secretory capacity by the age of 2-3 mo
(26). Speculations as to the underlying mechanism for
these gastric changes focused on NHE2 as an important player in
parietal cell volume regulation and on a potential role of NHE2 in the
mucosal protection mediated by the surface cells (26). Our
data show that NHE2 expression is fairly low and NHE2 activation by
hyperosmolarity is minimal in parietal cells, making the first of the
two hypotheses very unlikely. NHE2 is highly expressed in mucous cells
and is activated in these cells by low pH but only minimally by
hyperosmolarity, thus strengthening the hypothesis that NHE2 may be
involved in mucosal protection by the surface cells. When
Na+/H+ exchange was studied in NHE2-transfected
AP-1 cells, a remarkable feature was that NHE2 activity was strongly
increased by raising the pHo (42). In isolated
frog gastric mucosa, we (30 and unpublished observations) have
previously observed that raising interstitial buffering capacity and
thus interstitial pH caused an enhanced Na+/H+
exchange-mediated basolateral proton extrusion, increasing epithelial pHi. On the basis of these findings, Schultheis et al.
(26) speculated that NHE2 may be the NHE isoform that
mediates Na+/H+ exchange activation by an
increase in interstitial HCO
This hypothesis would be based on the assumption that gastric NHE2 is located in the basolateral membrane. Our attempts to localize NHE2 in rabbit stomach have failed so far, and no data are available on NHE2 localization in the stomach of other species. If we assume an apical location, as has been described in kidney (8) and intestine (13), its activity would result in proton secretion into the lumen. Possibly, the high carbonic anhydrase II expression levels in parietal cells require an alternative acid secretion mechanism in situations when the proton pump is not activated, and this explains the preferential degeneration of parietal cells in NHE2-knockout mice. However, we have never observed acid secretion in omeprazole-inhibited stripped mouse stomach in the Ussing chamber, a preparation that exhibits high agonist-induced acid secretory rates (I. Blumenstein and U. Seidler, unpublished observations).
Within the gastrointestinal tract, NHE4 is exclusively expressed in the
stomach, and our results demonstrate that parietal cells have markedly
higher NHE4 expression levels than surface cells. Interestingly,
despite high expression levels, NHE4 did not contribute to the
Na+/H+ exchange-mediated pHi
recovery from an intracellular acid load, demonstrating that NHE4 is
not activated by low pHi. On the other hand, the
hyperosmolarity-induced Na+/H+ exchange
activity was in a significant way due to NHE4, and the relative
contribution of NHE4 to hyperosmolarity-induced
Na+/H+ exchange activity paralleled the
relative NHE4 expression levels in both parietal and mucous cells.
These data demonstrate for the first time that hyperosmolarity
activates NHE4 in a cell type with endogenous NHE4 expression. These
data explain why Bookstein et al. (5, 6) only observed
Na+/H+ exchange activity in NHE4 transfected
Na+/H+ exchange-deficient fibroblasts during
hyperosmolar culture conditions and demonstrate that hyperosmolar
conditions are not a prerequisite for NHE4 expression, just for
activity. We then wondered whether parietal cells sequentially recruit
NHE1 and NHE4 with increasing degrees of hyperosmolarity, as has been
shown (40) to occur in hepatocytes with
Na+/H+ exchange,
Na+-K+-2Cl cotransport, and
Na+ conductance. However, we found that NHE4 becomes
activated at very moderate osmolar strength, where it is the
predominant isoform, and that it is NHE1, not NHE4, that can
dramatically increase its flux rate with increasing osmolar strength.
Thus the percentage of NHE4- to NHE1-mediated Na+ uptake
shifts dramatically with increasing osmolar strength from a 2:1
relationship at 350 mosmol/kgH2O to a 1:2 relationship at 500 mosmol/kgH2O.
Because we (1) previously observed that the contribution
of NHE4 to histamine- or forskolin-induced
Na+/H+ exchange activity in cultured rabbit
parietal cells was significantly higher (>50% of total
Na+/H+ exchange activity) than the contribution
to hyperosmolarity-induced Na+/H+ exchange
activity in this study (~40%) but that a medium change to 400 mosmol/kgH2O and stimulation by forskolin result in a
similar degree of cellular shrinkage (31), we wondered if
cAMP per se stimulates NHE4. After finding that forskolin plus
hyperosmolarity does not result in a stronger degree of parietal cell
shrinkage than hyperosmolarity alone (Sonnentag, unpublished
observations), we tested the effect of simultaneous application of
forskolin and hyperosmolarity on NHE4 activation. We found that the
simultaneous application of forskolin and a hyperosmolar medium
resulted in a significantly higher Na+/H+
exchange rate than hyperosmolarity alone. A similar difference was
observed in the absence of inhibitors and in the presence of 1 and 25 µM HOE-642, suggesting that the potentiating effect of forskolin was
due to enhanced NHE4 activity (although we did not perform enough
experiments to allow statistical evaluation for each inhibitor
separately). The data suggest that hyperosmolarity and cAMP are
independent and/or additive activators of NHE4. In cultured rabbit
parietal cells, rapid and pronounced cellular volume loss occurs during
cAMP-mediated stimulation of acid secretion, followed by rapid volume
recovery that is to a large part mediated by
Na+/H+ and
Cl/HCO
cotransport (31), and one reason for the strong NHE4
expression in parietal cells may be the necessity to recruit another
volume regulatory mechanism besides NHE1 during episodes of cellular shrinkage. The fact that cAMP activates NHE4 may enable parietal cells
to simultaneously activate both the apical secretory mechanisms and the
basolateral homeostatic mechanisms during cAMP-mediated stimulation of
acid secretion.
In summary, this report demonstrates that gastric epithelial cells express the NHE isoforms NHE1, NHE2, and NHE4, with particularly high expression levels for NHE1 and NHE2 in mucous cells and high NHE4 expression levels in parietal and chief cells. Because gastric mucous and parietal cells can be purified and cultured, we were for the first time able to functionally study native NHE2- and NHE4-expressing cells. Surprisingly, acid-induced Na+/H+ exchange activation was mediated by NHE1 and NHE2 in both cell types, with minimal NHE4 contribution. On the other hand, hyperosmolarity-induced Na+/H+ exchange activation was mediated by NHE1 and NHE4, and the expression levels paralleled the relative contribution of these isoforms to pHi-controled and hyperosmolarity-induced Na+/H+ exchange. NHE4 was activated both by hyperosmolarity and cAMP. We therefore conclude that in the gastric epithelium NHE1 and NHE2 regulate pHi, whereas NHE1 and NHE4 regulate cell volume.
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ACKNOWLEDGEMENTS |
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We thank Perikles Kosmidis for technical help, Wolf-Christian Siegel for help with primary cell culture and fluorometric experiments, John Orlowski, Chen Ming Tse, Chris Yun, Mark Donowitz, Zhuo Wang, Gary Shull, and Andreas Pfeifer for supplying NHE cDNA fragments, and Richard Wahl for the use of the isotope laboratory.
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FOOTNOTES |
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This work was supported in part by Deutsche Forschungsgemeinschaft Grants Se-460/9-1-9-4 and Se 460/2-5, Eberhard-Karls University Tübingen Fortüne Program Grant Nr-137 (F-1281038), Bundesministerium für Bildung und Forschung Grant Fö-01KS9602, and the Tübingen Interdisciplinary Center for Clinical Research.
This work includes experiments performed by T. Sonnentag and A. Heinzmann toward fulfillment of the requirements for their doctoral theses.
Address for reprint requests and other correspondence: U. Seidler, Abteilung Innere Medizin I, Eberhard-Karls Universität Tübingen, Otfried-Müller Str. 10, D-72076 Tübingen, Germany (E-mail: ursula.seidler{at}uni-tuebingen.de).
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.
Received 21 July 2000; accepted in final form 16 March 2001.
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