Differential expression and regulation of Na+/H+ exchanger isoforms in rabbit parietal and mucous cells

Heidi Rossmann, Thorsten Sonnentag, Alexander Heinzmann, Barbara Seidler, Oliver Bachmann, Dorothee Vieillard-Baron, Michael Gregor, and Ursula Seidler

First Department of Medicine, Eberhard-Karls University Tübingen, D-72076 Tübingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 alpha -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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Fig. 1.   A: high-stringency Northern blot analysis of ~2 µg twice-purified poly(A)+ RNA from rabbit kidney cortex, colonic mucosa, gastric mucosa, parietal cell fraction, and mucous cell fraction, probed with 32P-labeled homologous Na+/H+ exchanger (NHE) isoforms NHE1 and NHE3 cDNA fragments, as described in MATERIALS AND METHODS. Hybridization of the filter with a cDNA fragment of the H+-K+-ATPase alpha -subunit ensures the quality of the cell separation. Rehybridization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe confirms loading of intact RNA in all lanes. Exposure times: 16 h for NHE1 and NHE3 (short exposure), 3 days for NHE3 (long exposure), 3 h for H+-K+-ATPase, and 6 h for GAPDH. B: high-stringency Northern blot analysis of ~20 µg once-purified poly(A)+ RNA from rabbit parietal and mucous cell fractions, probed with 32P-labeled homologous NHE1, NHE2, and NHE4 cDNA fragments, as described in MATERIALS AND METHODS. Exposure times: 12 h for NHE1, 40 h for NHE2, and 17 h for NHE4. C: amplification and restriction analysis of NHE1, NHE2, and NHE4 cDNA fragments. As expected, the NHE1 fragment (left) is cut by Ava I (lane 1: 232 and 124 bp) but not by Sma I (lane 2). The NHE2 (right) fragment is cut by Hinc II (lane 6: 113 and 67 bp), but not by Sau96 I (lane 5), which should cut a nonspecifically amplified NHE4 PCR product. Conversely, NHE4 (right) is cut by Hae III (lane 2), but not by Hind II (lane 1), which should cut a nonspecifically amplified NHE2 PCR fragment. Lanes 3 and 7 (right): undigested PCR products. Lanes 3 (left) and 4 (right): molecular weight standards. Even after 40 cycles, no NHE3 band was amplified from rabbit gastric mucosa.

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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Table 1.   Primers used for probe generation and semiquantitative RT-PCR experiments



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Fig. 2.   Semiquantitative RT-PCR analysis of rabbit NHE1, NHE2, and NHE4 expressed in gastric epithelial cell types. A: parallel curves for NHE1 (356 bp) and histone 3.3a (523 bp), NHE2 (180 bp) and histone 3.3a, and NHE4 (270 bp) and histone 3.3a amplified from rabbit gastric mucosa are shown. The ratio of the gene of interest to histone 3.3a was determined during the exponential phase of both reactions as described previously (see Ref. 24). The ethidium bromide-stained bands, which were analyzed, are shown below each diagram. ODI, integrated optical density; R, amplification efficiency; n cycles indicates the number of PCR cycles, as do the numbers given for the stained bands. NHE3 could not be amplified within 40 cycles. B: NHE1 vs. histone 3.3a, NHE2 vs. histone 3.3a, and NHE4 vs. histone 3.3a fragments were amplified from rabbit gastric mucosa, parietal, mucous, and chief cells. The ratio of the gene of interest to histone 3.3a, representing the relative expression level of the studied gene, was plotted as a bar graph for the studied tissues (n = 3).

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 and beta -actin are known to be expressed quite differently among the gastric epithelial cell types, the Northern blots (Fig. 1) do not provide quantitative information. Although histone 3.3a was found to be evenly expressed in the different gastric cell types, the short poly(A) tail prevents its enrichment and use as a control for poly(A)+ Northern blot analysis (24). Therefore, a semiquantitative PCR technique, as described previously (24), was used to compare the NHE expression levels in the different epithelial cell types (Fig. 2). NHE1 mRNA expression levels were highest in mucous cells, lower in parietal cells, and lowest in chief cells. NHE2 mRNA expression levels were lower than NHE1 levels, but the distribution between the different cell types was similar to that of NHE1. In contrast, NHE4 was expressed to a similar extent in parietal and chief cells and significantly lower in mucous cells (Fig. 2).

These data demonstrate NHE1, 2, and 4 expression in rabbit gastric epithelial cells. The findings show a differential NHE isoform expression in the different gastric cell types, with high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells.

Role 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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Fig. 3.   Intracellular pH (pHi) trace (A and C) and initial proton efflux rates (B and D) after acidification of cultured rabbit parietal (A and B) and mucous cells (C and D) by the ammonium prepulse technique in the presence and absence of HOE-642 or dimethylamiloride (DMA). HOE-642 at 1 µM (this concentration is selective for NHE1) inhibits 80% of the Na+/H+ exchange rate during pH recovery in parietal cells and 62% in mucous cells. HOE-642 at 50 µM (at this concentration NHE1 and NHE2 are inhibited) abolished the remaining proton flux in parietal and mucous cells. TMA, tetramethylammonium; n.s., not significant.

In mucous cells, initial proton efflux rates after pHi 6.4 were 65 ± 16 mM/min and therefore significantly higher than in parietal cells (Fig. 3, C and D). HOE-642 at 1 µM inhibited only about two-thirds of this proton efflux, whereas 25 and 50 µM HOE-642 inhibited 87% and 91%, which were not significantly different from that inhibited by 500 µM DMA (92%). These results demonstrate that 1) a comparable acid load stimulates a significantly higher Na+/H+ exchange-mediated proton efflux rate in mucous cells than in parietal cells and 2) this proton efflux is predominantly mediated by NHE1 in both cell types, with the rest being mediated by NHE2.

Kapus et al. (17) observed different pHi/pHo relationships in NHE1-, NHE2-, and NHE3-transfected activator protein-1 (AP-1) cells and a relative shift of the pHi dependence of acid-induced proton efflux to more alkaline values compared with those of NHE1- and NHE3-transfected cells. Therefore, we investigated if the relationship of NHE1- to NHE2-mediated proton efflux changes with an increase in external and internal pH. Figure 4 shows the pHi curves (Fig. 4A) and proton efflux rates (Fig. 4B) during a change in the perfusate of cultured parietal cells from pH 7.4 (pHi 7.2) to pH 8. Interestingly, 41% of the ensuing intracellular alkalinization was inhibited by 500 µM DMA and therefore mediated by Na+/H+ exchange, whereas the other 59% was likely due to proton or base conductances. Under these experimental conditions, NHE1 and NHE2 contributed similarly to the Na+/H+ exchange-mediated alkalinization, and again NHE4 did not contribute. This is comparable to an 80% NHE1-20% NHE2 distribution when parietal cells were acidified to pHi 6.4 and pHo 7.4. Thus the percentage of proton flux mediated by NHE1 and NHE2 in a cell type expressing both isoforms is indeed dependent on pHi and pHo. Of note, total Na+/H+ exchange rates were 1.6 mM/min in the former and 20 mM/min in the latter condition.


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Fig. 4.   pHi trace (A) and proton flux rates (B) in cultured parietal cells after changing from a pH 7.4 perfusate to a pH 8 perfusate. Under these circumstances, the pHi slowly increased (A) and 41% of the base influx rate was inhibited by 500 µM DMA and therefore due to Na+/H+ exchange (B). In this situation, 1 µM HOE-642 inhibited 41% of DMA-sensitive base flux and 50 µM HOE-642 inhibited an additional 57%. Therefore, in this condition of high pHi and extracellular pH (pHo), total parietal cell Na+/H+ exchange rate was ~1.6 mM/min (compared with 20 mM/min at pHi 6.4) but the % of NHE1 to NHE2 activity was 1:1.4, compared with 4:1 at pHi 6.4 and pHo 7.4.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 5C). Thus DMA-sensitive proton efflux, calculated on the basis of this underlying proton influx, is 1.65 ± 0.03 mM/min. HOE-642 at 1 µM inhibited 55% of the hyperosmolarity-induced proton efflux, and 25 and 50 µM HOE-642 had only a marginal further inhibitory effect, whereas 500 µM DMA, which also inhibits NHE4, completely prevented alkalinization (Fig. 5, B and D). In mucous cells, hyperosmolarity also caused a DMA-sensitive cellular alkalinization by 0.25 ± 0.06 pH units, and the proton efflux rate during the initial linear phase of alkalinization was 1.35 ± 0.12 mM/min. Of this proton efflux rate, 65% was inhibited by 1 µM HOE-642, 9% by 50 µM HOE-642, and the residual flux by 500 µM DMA (Fig. 5, E-H).


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Fig. 5.   pHi trace (A-C: parietal cells; E-G: mucous cells) and initial proton efflux rates (D: parietal cells; H: mucous cells) after addition of hyperosmolar solution to cultured parietal and mucous cells. A and D: exposure of cultured parietal cells to 400 mosmol/kgH2O caused a rapid pHi increase. B and D: HOE-642 at 1 µM (this concentration is selective for NHE1) inhibits 55% of the Na+/H+ exchange rate; 25 or 50 µM HOE-642 (concentrations selective for NHE1 and NHE2) inhibits an additional 10%. The remaining flux rate is inhibited by 500 µM DMA (C and D) and represents NHE4 activity. E and H: the same type of experiments performed in cultured mucous cells yielded qualitatively similar results. HOE-642 at 1 µM inhibits 65% of the Na+/H+ exchange rate (F and H); 50 µM HOE-642 inhibits an additional 9%, and 500 µM DMA (G and H) inhibits the remaining flux rate (26%).

These results demonstrate that the hyperosmolarity-induced Na+/H+ exchange activation in both parietal and mucous cells is predominantly due to NHE1 and NHE4. In the two cell types, the relative contribution of NHE1 and NHE4 to hyperosmolarity-induced Na+/H+ exchange paralleled their relative expression levels, i.e., a higher NHE4 contribution in parietal cells, which also express more NHE4 than mucous cells. NHE2 contribution to hyperosmolarity-activated Na+/H+ exchange was present but minimal both in parietal and mucous cells. These findings demonstrate that hyperosmolarity is able to activate NHE2, consistent with previous results (17) in NHE2-transfected cell lines, but that hyperosmolarity-induced NHE2 activation is marginal in gastric epithelial cells.

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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Fig. 6.   Initial proton efflux rates induced by hyperosmolarity at different osmolar strengths. It is obvious that a fairly modest increase in osmolarity stimulated a Na+/H+ exchange rate that was inhibited by 32% by 1 µM HOE-642 (representing inhibition of NHE1 activity), an additional 8% by 50 µM HOE-642 (representing inhibition of NHE2 activity), and a further 59% by 500 µM DMA (representing inhibition of NHE4 activity). With increasing osmolar strength, Na+/H+ exchange rate was strongly increased, and this was largely due to an increase in NHE1 activity; n = 5 at each osmolarity and for each inhibitor concentration.

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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Fig. 7.   pHi trace (A-C) and hyperosmolarity-induced initial proton efflux rates (D-F) under control conditions (A and D), after inhibition of NHE1 (1 µM HOE-642, B and E), and after inhibition of NHE1 and NHE2 (25 µM HOE-642, C and F) in the presence and absence of forskolin (Forsk) in cultured parietal cells. In the presence of forskolin, hyperosmolarity caused a higher proton efflux rate in the presence and absence of 1 and 25 µM HOE-642; n = 3-7. Because the aim of these experiments was to test whether a difference existed between forskolin and hyperosmolarity vs. hyperosmolarity alone, independent of the presence of HOE-642, all flux rates were compared with Wilcoxon's rank test for paired samples and P was found to be <0.01.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> generation. Thus the exposure of gastric mucous cells to the high luminal proton concentration, their secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, their exposure to highly anisotonic luminal fluids during meals, and their migration throughout most of their short life span may all necessitate high NHE1 activity. We found an extraordinarily high Na+/H+ exchange rate in cultured mucous cells in response to an intracellular acid load, and two-thirds of this rate was due to NHE1 activity. On the other hand, hyperosmolarity elicited no stronger NHE1 activation in mucous than in parietal cells, despite the vastly different NHE1 expression levels. Therefore, we speculate that the very high NHE1 expression in gastric mucous cells may be related primarily to their necessity to extrude protons into the interstitium during HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and to prevent intracellular acidosis during periods of high luminal acid concentration. The fact that the NHE1 knockout mouse displays gastric but not intestinal histopathology (2) also suggests that this defect may not so much reduce their proliferative capacity (because this should have similarly severe effects in the rapidly proliferating small intestinal cells) but may weaken the gastric barrier to acid.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, which occurs during acid secretion. Indeed, we found in this study that, whereas overall Na+/H+-mediated acid extrusion rates decrease with increasing pHi and pHo, the percentage of NHE1- to NHE2-mediated acid extrusion decreases with increasing pHi and pHo. It is therefore feasible that NHE2 performs a substantial part of acid extrusion when the intracellular pH of gastric epithelial cells is near neutral and the interstitial pH high.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (31). In contrast to many other cell types, acid-secreting parietal cells do not regulate volume via Na+-K+-2Cl- 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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