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Am J Physiol Gastrointest Liver Physiol 291: G1171-G1179, 2006. First published July 6, 2006; doi:10.1152/ajpgi.00099.2006
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HORMONES AND SIGNALING

A vH+-ATPase is present in cultured sheep ruminal epithelial cells

Benjamin Etschmann,1 Katrin Sophie Heipertz,1 Annabelle von der Schulenburg,1 and Monika Schweigel2

1Department of Veterinary Physiology, Free University of Berlin, Berlin; and 2Research Institute for the Biology of Farm Animals (FBN), Department of Nutritional Physiology "Oskar Kellner," Dummsterstorf, Germany

Submitted 2 March 2006 ; accepted in final form 30 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, the existence and functional activity of a vacuolar-type H+-ATPase (vH+-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH+-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH+-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH+-ATPase and Na+/H+ exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pHi) was determined in nominally HCO3–-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl– concentration ([Cl–]e) from 136 to 36 mM. The initial pHi of REC was 7.4 ± 0.1 in nominally HCO3–-free, HEPES-buffered NaCl medium and 7.0 ± 0.1 after acid loading with butyrate. Selective inhibition of the vH+-ATPase with foliomycin decreased pHi by 0.19 ± 0.03 pH units. On the basis of the observed decreases in pHi resulting from inhibition of vH+-ATPase as well as of subtypes 1 and 3 of NHE, vH+-ATPase activity appears to account for ~30% of H+ extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H+ extrusion, respectively. Lowering of [Cl–]e induced a pHi decrease (–0.51 ± 0.03 pH units) and impaired pHi recovery from butyrate-induced acid load. Moreover, reduction of [Cl–]e abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pHi, indicating a role of Cl– in the function of these H+ extrusion mechanisms. We conclude that a vH+-ATPase is expressed in ovine REC and plays a considerable role in the pHi regulation of these cells.

active transport; pHi regulation; Na+/H+ exchanger; BCECF

IN RUMINATING ANIMALS, the forestomach, particularly the rumen, plays a considerable role in the absorption of nutrients, mainly of short-chain fatty acids and of electrolytes. For years, the transport of these substrates has been known to be energized by a Na+-motive force generated by the Na+,K+-ATPase, which is expressed at high levels (19, 27) and accounts for ~22–25% of the rumen O2 and, consequently, ATP utilization (26). Recently, we found indirect functional evidence that a second active transport mechanism, namely a vacuolar-type H+-adenosine triphosphatase (vH+-ATPase), is present in the cell membrane of sheep rumen epithelial cells (REC) (41). In this study, Mg2+ influx and thereby the free intracellular Mg2+ concentration was reduced after application of the specific vH+-ATPase inhibitors bafilomycin A1 and foliomycin. We interpreted these results to reflect an important role of the putative ruminal vH+-ATPase for the establishment and maintenance of the inside negative membrane potential in REC, which is the main driving force for the Mg2+ influx in these cells. A link between electrogenic H+ secretion by vH+-ATPases and ion transport and/or regulation of cytosolic pH has been found in various epithelia, e.g., frog skin, mammalian renal collecting duct, and epididymis (8, 12, 17).

The objectives of the present study were to explore the presence of vH+-ATPase transcription and translation in ovine REC by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Moreover, it was investigated whether the enzyme contributes to the regulation of the intracellular pH (pHi) as well as pHi recovery from an acid load under different conditions. This was assayed by determining the REC pHi with the aid of the membrane-permeable fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and by making use of two known vH+-ATPase characteristics: 1) inhibition by foliomycin (6, 11) and 2) modulation of H+ transport by extracellular Cl– (13, 25, 37, 44). Transport inhibitors of subtype 1 (HOE 694) and 3 (S3226) Na+/H+ exchangers (NHE) were used to differentiate between NHE-mediated H+ efflux and H+ extrusion by vH+-ATPase and to examine a possible interaction between these transport mechanisms. To eliminate contributions from HCO3–-transporting systems, all experiments were performed in media nominally lacking this anion.

We found direct molecular evidence for the expression of vH+-ATPase in ovine REC and established that the transport protein is functionally active in these cells. Although our data confirm that NHE activity plays the main role (~70%) in REC pHi regulation in the absence of HCO3–, vH+-ATPase is a considerable complementary mechanism responsible for ~30% of total H+ release under our experimental conditions. For both, NHE and vH+-ATPase activity, sensitivity for extracellular Cl– was established.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, kanamycin) and FCS were purchased from Sigma (St. Louis, MO). Dulbecco's PBS and collagen were obtained from Biochrom (Berlin, Germany). BCECF-AM and pluronic acid were from Molecular Probes (Eugene, OR). Foliomycin was purchased from ICN (Aurora, OH). RT-PCR and quantitative RT-PCR (qRT-PCR) reagents were purchased from Bio-Rad (Hercules, CA). All other chemicals were obtained from Sigma.

Cell culture. The tissues used for the isolation of REC originated from sheep aged at least 6 mo and were obtained at a local slaughterhouse. Primary cultures of REC were prepared as described by Galfi et al. (16). Briefly, REC were isolated by fractional trypsination and grown in medium 199 containing 10% FCS, 1.36 mM glutamine, 20 mM HEPES, and antibiotics (gentamycin 50 mg/l, kanamycin 100 mg/l) in an atmosphere of humidified air-5% CO2 at 38°C. Experiments were performed between 6 and 12 days after seeding.

Solutions for measuring the pHi. pHi was measured in HEPES-buffered high-Na+ solution containing (in mM) 100 NaCl, 45 N-methyl-D-glucamine-Cl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4 or in HEPES-buffered high-Na+ solution with butyrate (in mM: 125 NaCl, 20 Na-butyrate, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4). To investigate the effects of lowering the extracellular Cl– concentration ([Cl–]e), 100 mM NaCl was replaced by Na-gluconate. In one series of experiments, the effect of foliomycin on pHi recovery was investigated in HCO3–-containing solution (in mM: 125 NaCl, 20 NaHCO3, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, pH 7.4) preequilibrated with 5% CO2 and 95% air.

Measurement of pHi by spectrofluorometry. For the determination of pHi, cells were loaded with 1 µM BCECF-AM and subsequently washed twice in Dulbecco's PBS. REC were incubated for a further 30 min to allow for complete deesterification and washed twice before measurement of fluorescence. Intracellular pH was determined by measuring the fluorescence of the probe-loaded REC in a spectrofluorometer (LS-50 B, Perkin-Elmer) using the fast-filter accessory, which allowed fluorescence to be measured at 20-ms intervals with excitation for BCECF at 440 and 480 nm and emission at 515 nm. All measurements were made at 37°C in a 3-ml cuvette containing 2 ml of cell suspension (10% cytocrit) under stirring.

BCECF signals were calibrated to pH by using the ionophore nigericin (10 µM) to equilibrate intra- and extracellular H+ concentration. The procedure was repeated for various pH values between 6.0 and 8.0.

For data evaluation, 10-s data sets each were averaged at the beginning of the measurement and then after 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, and 550 s. The final pHi was determined as the mean pHi of the last 10 s of the measurement. Thus, for the calculation of any given pHi value, 500 data points were used.

Western blot analysis. The monoclonal antibody (13D11-B2, Molecular Probes) used in this study is directed against the yeast vH+-ATPase 60-kDa subunit. For Western blots, washed REC were solubilized in ice-cold Frackelton buffer [in mM: 10 Tris buffer pH 7.5, 50 NaCl, 30 Na-pyrophosphate, 50 NaF, 0.1 Na3O4V, 1 phenylmethylsulfonyl fluoride, 1 DTT, complemented with 1% Triton X-100 and with a protease inhibitor mixture (Roche)]. The protein concentration was determined by the Bradford assay (Bio-Rad). Protein samples (40–80 µg) were separated by SDS(12.5%)-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose filters. After transfer, filters were blocked with 3% nonfat dry milk in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20 (TBS-T) for 1 h. Following blocking, filters were incubated at 4°C with the primary antibody (1:5,000 dilution) overnight, washed three times for 10 min in TBS-T, and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution). After incubation with the secondary antibody, filters were washed three times for 10 min in TBS-T and developed photographically by chemiluminescence (Super Signal, Pierce).

RT-PCR. Using bovine cDNA consensus sequence sets (GeneBank accession nos. J03244 and NM_176671), specific primers for vH+-ATPase subunits B and E were generated and subsequently used in RT-PCR experiments to detect vH+-ATPase mRNA molecules in ovine ruminal epithelium and in ovine isolated REC. The primer sequences for subunits E and B of vH+-ATPase were 5'-gccaatgagaaagcaga-3'/5'-ggtccagccgactttcg-3' and 5'-gaggagatgattcagactgg-3'/5'-ttcatggcttgtacatcctt-3', respectively. PCR products were cloned into bacterial vectors (pDrive, Qiagen) and subjected to cycle sequencing. The resulting sequence data were compared with the homologous cDNA sequences from Bos taurus.

qRT-PCR. To assay the relative expression levels of NHE subtypes 1, 2, and 3 (NHE1, -2, and -3, respectively) in REC, the corresponding mRNAs were detected using qRT-PCR with gene-specific primer pairs (NHE1: 5'-cctctacagctacatggcctac-3' and 5'-gggagatgttggcttcca-3'; NHE2: 5'-gaggcgtgtgtgaacgag-3' and 5'-gggtgaagcgggtgataaa-3'; NHE3: 5'- agctacgtggccgaggg-3' and 5'-agacagaggcctccacggt-3'). Reverse transcriptions were performed using a commercial kit (iScript cDNA synthesis kit, Bio-Rad) according to the manufacturer's instructions. qRT-PCR experiments were conducted using 35 cycles of a two-step PCR protocol with universal annealing conditions (2 min at 58°C and 30 s at 95°C) and a melt curve (55°C to 95°C with 10-s holds at each half degree) in a thermocycler instrument (iQCycler, Bio-Rad) using a commercial SYBR-Green kit (SYBR Green Supermix, Bio-Rad) according to the manufacturer's instructions. Baseline definition and cycle threshold (CT) positioning were automatically performed with the qRT-PCR signal detection software (iQ software, v3.0) using default settings. The sensitivity, specificity, and efficiency of all qRT-PCR assays were determined for all target genes in pilot experiments by using 10-fold cDNA dilution series and subsequent sequence analysis of the resulting PCR products. All qRT-PCR assays were specific and had efficiencies greater than 90% (data not shown). CT values were normalized against beta-actin CT values detected using qRT-PCR in the same samples and used to calculate relative expression levels as previously described (9, 30). Results were determined as relative expression levels of NHE3 and NHE2 compared with the relative expression level of NHE1 in REC.

Statistical analysis. If not otherwise stated, data are presented as means ± SE. Significance was determined by Student's t-test, paired t-test, or Tukey's analysis of variance as appropriate. P < 0.05 was considered to be significant. All statistical calculations were performed by using SigmaStat (Jandel Scientific).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
pHi of REC. BCECF-loaded REC in suspension were equilibrated for 5 min in either HEPES-buffered NaCl medium, HEPES-buffered NaCl medium with butyrate, or HEPES-buffered NaCl medium containing butyrate in which the Cl– concentration was reduced from 136 to 36 mM (high-Na+/low-Cl– medium). The extracellular pH was 7.4 in all solutions. After equilibration, the pHi was measured over a 10-min period. The time course of the REC pHi for all three conditions is shown in Fig. 1. The initial and final pHi of REC incubated in HEPES-buffered NaCl medium were 7.46 ± 0.09 and 7.42 ± 0.06, respectively. In the media containing butyrate, the presence of this highly lipophilic short-chain fatty acid led to a reduction of pHi. In REC exposed to HEPES-buffered NaCl medium with 20 mM butyrate and Cl– concentrations of either 136 or 36 mM, the pHi measured at the beginning of the measuring period was reduced to 7.00 ± 0.07 and 6.48 ± 0.03, respectively, indicating that the effect of butyrate was more pronounced in the medium containing a low-Cl– concentration. In both media, the acidification induced by butyrate was followed by a pHi recovery that amounted to 0.57 ± 0.05 and 0.38 ± 0.05 pH units in media with a Cl– concentration of 136 mM or 36 mM, respectively. At the end of the 10-min measurement period, pHi had recovered to 7.58 ± 0.05 in medium with a [Cl–]e of 136 mM, but only to 6.90 ± 0.07 in the Cl–-reduced (36 mM) medium.


Figure 1
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  		Fig. 1. Time course of the intracellular pH (pHi) of ruminal epithelial cells (REC) incubated in HEPES-buffered NaCl medium, in HEPES-buffered NaCl medium with butyrate, or in HEPES-buffered high-Na+/low-Cl– medium with butyrate. Measurements were made after a 5-min preincubation in the respective solution. Values are means ± SE of 5–8 single experiments. **P < 0.01 vs. the respective control (HEPES-buffered NaCl medium or HEPES-buffered NaCl medium with butyrate).

 
Effect of foliomycin on the pHi of REC incubated in HEPES-buffered NaCl medium with butyrate. BCECF-loaded REC were suspended in foliomycin (2 µmol/l)-containing HEPES-buffered NaCl medium with butyrate (20 mM). After a 5-min preincubation period in this medium, the pHi was measured continuously over a 10-min period. In Na+-containing media, a NHE is known to be active in REC (42). Therefore, in some experiments, HOE 694 and S3226, known as specific inhibitors of NHE1 and of NHE3, respectively, were applied in addition to foliomycin. Control measurements were performed with cells handled in the same way, but without any blocker present in the solutions. Compared with the pHi of such control cells, foliomycin-treated REC showed a decreased pHi (Figs. 2 and 3A). Figure 2 gives a summary of the pHi reduction induced by foliomycin alone or by a combination of foliomycin with HOE 694 and/or S3226. At the beginning of the measurement, the pHi of foliomycin-treated REC was reduced by 0.19 ± 0.03 pH units, which corresponds to a 56 ± 12% increase of the intracellular H+ concentration. At the end of the measuring period, the pHi difference between control and foliomycin-treated cells amounted to –0.18 ± 0.07 pH units. To investigate the contributions of NHE1 and NHE3 to the adjustment of pHi, the specific inhibitors HOE 694 (100 µmol/l) and S3226 (10 µmol/l) were applied in addition to foliomycin. Compared with foliomycin alone, this induced a further reduction of the pHi measured at the beginning or end of the experiment. The observed pHi decrease amounted to 0.23 ± 0.10 and 0.32 ± 0.08 pH units in the presence of foliomycin and S3226, and to 0.45 ± 0.13 and 0.63 ± 0.20 pH units in the presence of all three blockers, respectively (Fig. 2).


Figure 2
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  		Fig. 2. Effect of the vacuolar-type H+-ATPase (vH+-ATPase) inhibitor foliomycin alone or in combination with the Na+/H+ exchanger (NHE) inhibitors HOE 694 and/or S3226 on the pHi of REC. Ten-minute measurements were made after a 5-min preincubation in either HEPES-buffered NaCl medium with 20 mM butyrate (control) or the same media with blockers as indicated. The mean pHi reduction from pHi measured in control medium without inhibitors (see Fig. 1) was calculated for the given time points and is shown for each condition. Blocker concentrations are 2, 10, and 100 µmol/l for foliomycin, S3226, and HOE 694, respectively. Values are means ± SE of 4–5 single experiments. *P < 0.05 and **P < 0.01 vs. control.

 

Figure 3
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  		Fig. 3. Effect of vH+-ATPase and of NHE inhibitors on pHi recovery after an acid load with butyrate. A: records from 2 experiments showing the effects of foliomycin (2 µmol/l) on pHi and on the pHi recovery. The latter is given by the numbers on the right-hand side of each trace. Foliomycin reduced the pHi in both experiments. In contrast, pHi recovery is inhibited in experiment 1 only. B: magnitude of the mean change of the pHi recovery induced by single or combined application of vH+-ATPase and of NHE inhibitors. Blocker concentrations are 2, 10, and 100 µmol/l for foliomycin, S3226, and HOE 694, respectively. Values are means ± SE of 4–6 single experiments. *P < 0.05 vs. control (HEPES-buffered NaCl medium with 20 mM butyrate).

 
Influence of foliomycin on the butyrate-induced pHi recovery of REC. As shown in Fig. 1, the butyrate-induced intracellular acidification is followed by a pHi recovery. On average, this amounted to 0.57 ± 0.05 units within 10 min. An inhibitory effect of foliomycin on the pHi recovery was seen in part of the experiments performed but was masked by a strong NHE activation in other experiments (Fig. 3). Characteristic original traces showing such different results are shown in Fig. 3A. Although a combined application of foliomycin and S3226 led to more consistent inhibitory effects, a significant inhibition of the butyrate-induced pHi recovery (–0.19 ± 0.08 pH units) was seen only when HOE 694 was applied in addition to these inhibitors (Fig. 3B). These results point to a predominant role of NHE in butyrate induced pHi recovery under our experimental conditions. Therefore, the specific role of the NHE3 and NHE1 was investigated by single or combined application of S3226 and HOE 694 (Fig. 3B). As shown in Fig. 3B, the pHi recovery was reduced by 0.08 ± 0.04 and 0.07 ± 0.02 pH units in medium containing S3226 (10 µmol/l) or HOE 694 (100 µmol/l), whereas the presence of both inhibitors caused a decrease by 0.15 ± 0.05 pH units.

Effect of a reduced extracellular Cl– concentration on the inhibitor effects. The activity of vH+-ATPase has been shown to require Cl– ions (25, 37). Therefore, we tested the effect of a reduced extracellular Cl– concentration ([Cl–]e = 36 mM) on the aspect of pHi that is sensitive to foliomycin. In contrast to the media with normal [Cl–]e, the starting pHi of REC incubated in the Cl–-reduced solution was not significantly decreased by treatment with foliomycin or by application of foliomycin in combination with S3226 (Figs. 4 and 5). However, pHi fell significantly (–0.14 ± 0.01 pH units) when HOE 694 was present in addition to foliomycin and S3226 (Fig. 5). At the end of the measuring period, the pHi of foliomycin-treated cells was reduced by 0.07 ± 0.03 pH units compared with controls, but the effect was not significant (Figs. 4 and 5). As shown in Fig. 5, a further depression of pHi (–0.20 ± 0.06 pH units) was obtained by an additional application of S3226, which is not increased by applying a combination of foliomycin, S3226 and HOE 694 (–0.16 ± 0.01 pH units).


Figure 4
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  		Fig. 4. Effect of a reduced extracellular Cl– concentration on foliomycin effects. REC were incubated in either control solution [HEPES-buffered high-Na+/low-Cl– medium with butyrate, with extracellular Cl– concentration ([Cl–]e) = 36 mM], or in the same medium with foliomycin (2 µmol/l). Values are means ± SE of results from 5 different experiments; pHi is shown plotted against time. Inset: representative original recordings of pHi.

 

Figure 5
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  		Fig. 5. Effect of the vH+-ATPase inhibitor foliomycin alone or in combination with the NHE inhibitors HOE 694 and/or S3226 on the pHi of REC. Ten-minute measurements were made after a 5-min preincubation in either HEPES-buffered high-Na+/low-Cl– (36 mM) medium with 20 mM butyrate (control) or the same media with blockers as indicated. The mean pHi change from pHi measured in control medium without inhibitors (see Fig. 1) was calculated for the given time points and is shown for each condition. Blocker concentrations are 2, 10, and 100 µmol/l for foliomycin, S3226, and HOE 694, respectively. Values are means ± SE of 5 single experiments. *P < 0.05 vs. control.

 
Figure 6 shows the mean values of pHi recovery changes upon application of certain inhibitors to the Cl–-reduced medium. With foliomycin present in the solution, only a slight decrease (–0.07 ± 0.04 pH units/10 min) occurred. Interestingly, a combined application of all three blockers (S3226, HOE 694, and foliomycin) has a similar, negligible effect (–0.05 ± 0.03 pH units/10 min) on pHi recovery. A stronger, significant reduction was observed with S3226 plus foliomycin (–0.14 ± 0.04 pH units/10 min).


Figure 6
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  		Fig. 6. Effect of vH+-ATPase and of NHE inhibitors on butyrate-induced pHi recovery in Cl–-reduced solution. Results are expressed as change from the pHi recovery measured under control conditions (HEPES-buffered high-Na+/low-Cl– medium with 20 mM butyrate, [Cl–]e = 36 mM). Blocker concentrations are 2, 10, and 100 µmol/l for foliomycin, S3226, and HOE 694, respectively. Bars represent means ± SE of 5 single experiments. *P < 0.05 vs. control.

 
Immunoblot of the REC vH+-ATPase. The results of the Western blot analyses are shown in Fig. 7. As can be seen, in immunoblots of protein lysates from REC, the anti-vH+-ATPase antibody labels a 60-kDa protein representing subunit B of vH+-ATPase (Fig. 6, lanes A and B). A 60-kDa immunoreactive band was also detected in protein lysates of yeast cells that were used as positive control (Fig. 7, lane D).


Figure 7
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  		Fig. 7. Immunoblot of the 60-kDa vH+-ATPase subunit in REC. Using an antibody directed against the 60-kDa subunit of vH+-ATPase (clone 13D11), specific bands were detected at the expected size of 60 kDa (A and B). Protein-free supernatant (SN) was used as negative control (C), and yeast cells were used as positive control (D). MW, molecular weight.

 
RT-PCR. RT-PCR experiments to detect the subunits E and B of vH+-ATPase in ovine ruminal epithelium and isolated REC showed specific bands of the expected size. The primers for subunit E were designed to amplify base pairs 67 to 604 in the coding sequence of vH+-ATPase subunit E. The corresponding band detected after agarose gel electrophoresis of RT-PCR products from cDNA using these primers was ~550 bp in size. Messenger RNA from vH+-ATPase subunit B was detected by using primers designed to amplify base pairs 499 to 1307 and showed an electrophoretic band at ~800 bp (Fig. 8). RT-PCR products (550 and 800 bp) from the ruminal epithelium were cloned into bacterial vectors and subsequently sequenced in part. The attained sequences were compared with the bovine sequences of vH+-ATPase subunits E and B that had been used in primer design. The sequences of vH+-ATPase subunits E and B showed a 99% (277/278) and 96% (433/448) homology between bovine and ovine sequences, respectively (Figs. 9 and 10). The sequence data were published in GeneBank (accession nos. AJ829476 and AJ829758). RT-PCR products from isolated REC were treated as described for RT-PCR products from ruminal epithelium. The attained sequence data from isolated ovine REC showed 100% homology to the sequences from ovine ruminal epithelium (data not shown).


Figure 8
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  		Fig. 8. Products from RT-PCR experiments to detect vH+-ATPase subunits B and E were resolved by gel electrophoresis, and specific bands of the expected sizes (550 and 800 bp) were detected. These results point to the presence of vH+-ATPase mRNA in the rumen epithelium. Neg., negative.

 

Figure 9
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  		Fig. 9. Sequence analysis of the cloned RT-PCR product of vH+-ATPase subunit E (vHE). The sequence of the cloned RT-PCR product has a 99% homology to the corresponding bovine sequence.

 

Figure 10
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  		Fig. 10. Sequence analysis of the cloned RT-PCR product of vH+-ATPase subunit B (vHB). The sequence of the cloned RT-PCR product has a 96% homology to the corresponding bovine sequence.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
pHi under control conditions. The resting pHi of REC suspended in HEPES-buffered, HCO3–-free, high-Na+ medium was found to be 7.4 ± 0.1, which is in the range (7.2 to 7.5) reported in one study with REC (36) and in studies with other cell systems (13, 34) under HCO3–-free conditions. As expected, exposure of REC to solutions containing butyrate leads to intracellular acidification (–0.45 ± 0.07 pH units), resulting mainly from permeation of the lipid-soluble protonated form of the fatty acid across the cell membrane. Entry of ionized butyrate, which is mediated via exchange against intracellular HCO3– is of minor importance under our experimental conditions. After uptake into the cells, the nonionized butyrate readily dissociates, thereby delivering H+ to the cell interior. This substantial (185%) butyrate-induced elevation of intracellular H+ concentration ([H+]i) rapidly activates cell-alkalinizing mechanisms and pHi recovers to values not significantly different from the control values observed in butyrate-free media. As shown by Müller et al. (36), in HCO3–-buffered solutions a Na+-HCO3– symporter operates as the dominant mechanism for recovery from an acid load. However, HEPES-buffered, nominally HCO3–-free medium was used in the present study. Under these conditions, the pHi recovery induced by butyrate is mainly due to stimulation of NHE activity in these cells (36, 40, 42), but there is only sparse information on the contribution of specific NHE subtypes to this process. Recently, our group showed the expression of the NHE subtypes 1 and 3 in REC (40). In this study, selective inhibition of NHE1 (by use of HOE 694, 100 µmol/l) and of NHE3 (by use of S3226, 10 µmol/l) led to a 24 and 27% reduction of the pHi recovery, respectively. In the present experiments, the pHi recovery induced by butyrate was reduced by 33% in HOE 694- and by 20% in S3226-treated REC. With the NHE1 inhibitor HOE 694 present, Müller et al. reported a 69% inhibition of the butyrate-induced pHi recovery. However, because of the relatively high dosage of 200 µmol/l used in this study, effects on other NHE subtypes seem likely. HOE 694 is known to inhibit NHE2 and in concentrations higher than 100 µmol/l it has been shown to inhibit the NHE3 to a considerable extent (10, 20). Using qRT-PCR in primary cultured REC, we found that the relative abundance of NHE1-mRNA is 400- and 7,000-fold of those of NHE3 and NHE2, respectively (data not shown). These data suggest that NHE2-mRNA is present only in traces in these cells and that this NHE subtype contributes only marginally to pHi recovery in REC. In conclusion, our results show that, beyond NHE1, NHE3 mediates a significant proportion of the butyrate-induced pHi recovery and that both mechanisms are active not only at acidic but also at physiological pHi values. Nevertheless, recovery from intracellular acidification was reduced by ~50% following NHE1 and NHE3 inhibition and, consequently, additional processes must contribute to the counterregulation of pHi and pHi homeostasis.

In our previous work, a Na+-independent H+ extrusion and an inhibition of electrodiffusive Mg2+ uptake by vH+-ATPase inhibitors have been observed in REC (41). Since these preliminary data point to a possible role of vH+-ATPases in REC pHi regulation and solute transport, functional and molecular experiments were performed in this study to find direct evidence for the existence of such transport proteins in these epithelial cells. To this end, we first investigated whether selective inhibition of vH+-ATPase by foliomycin (5, 6, 11) is sufficient to change pHi responses in cultured ovine REC incubated in HEPES-buffered NaCl medium with 20 mM butyrate.

Influence of foliomycin and of NHE inhibitors on the pHi of REC. Five minutes after application of foliomycin, the REC pHi was effectively reduced by 0.19 ± 0.03 pH units. This pHi change is in the order (–0.08 to –0.26 pH units) of results from other authors who studied nonpigmented ciliary epithelium, mouse bladder epithelium, and macrophages by use of specific vH+-ATPase inhibitors (21, 23, 34, 43). In part of the experiments, a time-dependent attenuation of the foliomycin-induced pHi decrease occurred, leading to a stronger variability of the foliomycin effect (–0.18 ± 0.07 pH units) observed at the end of the measuring period. This result is indicative of secondary adjustments of the pHi by other H+ extruding mechanisms, such as NHE. Most NHE subtypes show a steep elevation of exchange activity with increasing [H+]i resulting from increased affinity of the allosteric modifier side of the exchanger to intracellular H+ (3). After foliomycin application, there is a substantial, 60% elevation of [H+]i coupled with an increased driving force for H+ secretion via NHE (ENHE = ENa+-EH+) that amounted to +107 mV compared with +95 mV under control conditions. These considerations are supported by our results showing that the addition of specific NHE1 and NHE3 inhibitors (HOE 694 and/or S3226) to foliomycin-containing REC suspensions induced a further depression of pHi. The data are consistent with additive effects of vH+-ATPase and NHE inhibition on pHi. On the basis of decreases in pHi resulting from selective inhibition of vH+-ATPase and of NHE1 and NHE3, vH+-ATPase activity accounts for ~30%, NHE3 activity for 20%, and NHE1 for 50% of the H+ extrusion under our experimental conditions. Similar results have been found by Wu et al. (46) in bovine corneal epithelial cells.

Role of vH+-ATPase in butyrate-induced pHi recovery. Our results show that NHE1 and NHE3 are responsible for ~50% of the butyrate-induced pHi recovery with NHE1 being most important and that the activity of the latter is stimulated in the presence of foliomycin. Therefore, in about half of the experiments, an inhibitory effect of foliomycin on the pHi recovery was masked, mainly by strong activity of NHE1. This hypothesis is supported by an additional series of experiments performed with REC incubated in CO2(5%)/HCO3– (20 mM)-containing solutions. Under such conditions, the Na+-HCO3– cotransporter is mainly (88%) responsible for pHi recovery and NHE activity is only of minor importance (36). Hence, we found a consistent inhibitory foliomycin effect on the pHi recovery that was reduced to 0.17 ± 0.01 pH units/10 min (P < 0.05) compared with that (0.22 ± 0.02 pH units/10 min) observed in nontreated cells (results not shown). However, it cannot be excluded that NHE3 and, possibly, other undefined mechanisms may compensate in part for cytoplasmic pH recovery changes that otherwise would occur when vH+-ATPase is inhibited in the HEPES-buffered medium containing butyrate. For instance, a cooperative interaction between vH+-ATPase and NHE3 has been proposed to exist in rabbit nonpigmented ciliary epithelium as well as in cardiomyocytes (23, 24). In the case of cardiomyocytes, it was suggested that the vH+-ATPase-mediated H+ efflux may serve to hinder cytoplasmatic Na+ overload due to Na+ entry via Na+/H+ exchange. In REC, activation of the cAMP-protein kinase A pathway leads to NHE3 inhibition and to activation of unspecific cation channels, which are known to mediate Na+ influx (28, 29). It remains to be determined whether vH+-ATPase in REC is activated and plays a specific functional role in the mechanisms of solute transport under such conditions.

Role of [Cl–]e for vH+-ATPase and NHE activity. In the present study lowering of [Cl–]e led to a pHi reduction (–0.5 pH units) that was much stronger than that (–0.2 pH units) observed in an earlier study (41) when we used HCO3–-buffered, butyrate-containing medium. Such data fit well to a Cl– sensitivity of the NHE and the vH+-ATPase that have been identified to operate as the dominant pHi-regulating mechanisms in the HEPES-buffered medium used in the present study. In accordance with this assumption, the inhibitory effect of foliomycin on the starting pHi seen in high-Cl– medium was abolished. Moreover, the parts of pHi sensitive to S3226 and HOE 694 were significantly smaller in the solution with reduced [Cl–]. The reduction of the blocker effect determined at the end of the measuring period amounted to 52 ± 24% (foliomycin), 37 ± 20% (foliomycin + S3226), and 73 ± 1% (foliomycin + S3226 + HOE 694). For other cell systems a role of Cl– in vH+-ATPase function has been demonstrated both in subcellular organelles (13, 14, 44) and in H+ extrusion across the cell membrane (13, 25, 37). The mechanism(s) underlying the effect of low-Cl– medium on REC vH+-ATPase activity remains to be determined, but one of these may be reduced electrical shunting. A number of studies have shown that the electrogenic pump needs a parallel anion current to shunt the generated potential and that a Cl– current accomplished by a distinct Cl– channel is related with vH+-ATPase activity (14, 25, 32, 33). Moreover, regulation of the channel conductance, e.g., by cAMP-dependent protein kinase, seems to be an important mechanism by which the vH+-ATPase activity is modulated (45). Alternatively, decreased vH+-ATPase-mediated H+ extrusion may be attributed to insufficient saturation of a specialized anion binding site in the intramembranal sector of the vH+-ATPase with Cl– (25) and/or a decrease in the number of pumps exposed on the REC surface (31). Endo- and exocytic translocation of vH+-ATPases preexisting in intracellular organelles is a general mechanism operating in many cell types as a means of adjusting vH+-ATPase activity in response to various signals or stimuli (1, 4, 7). A negative effect of Cl– channel blocking on vesicle trafficking has been demonstrated, but the mechanism of this action is not known (31).

Our results show for the first time that Cl– is required for normal NHE activity in REC. In other tissues Cl– ions have been reported to modulate basal NHE activity as well as NHE activity induced by hyperosmolality (2, 22, 35, 38), and a distinct Cl–-dependent Na+/H+ exchange has been identified in the apial membrane of colonic crypt cells (38, 39). Similarly to the regulation of vH+-APase activity by Cl–, it has been suggested that the Cl–-dependent stimulation of NHE activity involves brush border insertion of vesicles containing NHE (31, 35) and/or interaction of intracellular Cl– with the COOH terminus or with an associated protein, e.g., a Cl– channel (2, 39). Intracellular chelation of Ca2+ and thus a decreased Ca2+ sensitivity of NHE activation and a Cl– requirement of various intracellular signaling pathways have also been discussed (18).

In CO2/HCO3–-containing media a reduction of [Cl–]e has no effect on the butyrate-induced pHi recovery (41). In contrast, in the HEPES-buffered medium used in this study lowering of [Cl–]e impairs cell pH recovery from an acid load by ~33% and, in special, its HOE 694-sensitive part. Hence, a strong inhibition of the NHE1 activity must be responsible for the main part of the reduced H+ extrusion rate. As indicated by the foliomycin and S3226 effects, the vH+-ATPase- and NHE3-mediated components seem to be more important under such conditions. Surprisingly, simultaneous application of foliomycin, S3226, and HOE 694 does not cause a stronger inhibition of the pHi recovery than foliomycin alone or in combination with S3226. This requires an explanation, and it seems possible that proton sequestration in intracellular organelles leads to the observed effect.

Molecular evidence for the expression of a vH+-ATPase in REC. Partial sequencing of the 600- and 800-bp RT-PCR products generated from ovine ruminal epithelium and from isolated ovine REC cDNA with vH+-ATPase E- and B-subunit specific primers demonstrated a 99 and 96% identity to respective bovine vH+-ATPase subunits (GeneBank accession nos. J03244, NM_176671, AJ829476, and AJ829758). Our results show that the mRNA of the vH+-ATPase E- and B-subunit exists in both cultivated REC and in ruminal tissue. Furthermore, we show Western blot evidence for the existence of the vH+-ATPase B-subunit in REC. The monoclonal antibody used, which is directed against the yeast 60-kDa B-subunit of the vH+-ATPase, detected a protein with a corresponding molecular weight of ~60 kDa in REC as well as in the yeast cell vacuole membranes used as positive control.

In conclusion, the present results clearly demonstrate that the vH+-ATPase gene is present in the ovine rumen epithelium and in isolated REC as mRNA and protein and that the enzyme is functionally active. Reduction of baseline pHi by the selective H+-ATPase inhibitor foliomycin and sensitivity of the pHi regulation to [Cl–]e are consistent with H+ extrusion activity dependent on vH+-ATPase in the cell membrane of ovine REC. The latter was responsible for ~30% of the H+ extrusion under our experimental conditions (nominally CO2/HCO3–-free medium). Our data confirm that REC regulate their pHi mainly through NHE mechanisms in the nominal absence of HCO3–/CO2 and more important quantified the contribution of NHE1 (~50%) and NHE3 (~20%) in this process. In addition, an influence of [Cl–]e on the NHE activity in REC was first shown in this study. At the present time, we are far away from a conclusive definition of the physiological function(s) of vH+-ATPase in REC. However, taking into consideration the very different pHi sensitivities of NHE (maximal activity at acidic pHi) and vH+-ATPase (maximal activity at neutral pHi), it appears possible that vH+-ATPase may play a considerable role in REC pHi regulation, besides being involved in the establishment of transport gradients.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This study was supported by a student gratification of the Free University of Berlin to Sophie Heipertz.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the valuable assistance of Dr. Elisabeth Froschauer (Institute of Microbiology and Genetics, University of Vienna) with the Western blots. Thanks to Dr. H.-J. Lang (HMR-Germany GmbH, Frankfurt a. Main, Germany) for kindly providing us with HOE 694 and S3226.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Schweigel, Research Institute for the Biology of Farm Animals-FBN, Dept. of Nutritional Physiology "Oskar Kellner," Wilhelm-Stahl-Allee 2, 18196 Dummerstorf; Germany (e-mail: mschweigel{at}fbn-dummerstorf.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.


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  1. Adelsberg van J and Al-Awqati O. Regulation of cell pH by Ca2+-mediated exocytotic insertion of H+-ATPases. J Cell Biol 102: 1638–1645, 1986.[Abstract/Free Full Text]
  2. Aharonovitz O, Kapus A, Szaszi K, Coady-Osberg N, Jancelewicz T, Orlowski J, and Grinstein S. Modulation of Na+/H+ exchange activity by Cl–. Am J Physiol Cell Physiol 281: C133–C141, 2001.[Abstract/Free Full Text]
  3. Aronson PS. Kinetic properties of the plasma membrane Na-H exchanger. Annu Rev Physiol 47: 545–560, 1985.[CrossRef][Web of Science][Medline]
  4. Banerjee A, Li G, Alexander EA, and Schwartz JH. Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol 280: C775–C781, 2001.[Abstract/Free Full Text]
  5. Bowman EJ, Gustafson KR, Bowman BJ, and Boyd MR. Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases. J Biol Chem 278: 44147–44152, 2003.[Abstract/Free Full Text]
  6. Bowman EJ, Siebers A, and Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972–7976, 1988.[Abstract/Free Full Text]
  7. Breton S, Nsumu NN, Galli T, Sabolic I, and Smith PJS. Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract. Am J Physiol Renal Physiol 278: F717–F725, 2000.[Abstract/Free Full Text]
  8. Brown D and Breton S. Mitochondria-rich, proton-secreting epithelial cells. J Exp Biol 199: 2345–2358, 1996.[Abstract]
  9. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23–29, 2002.[Abstract]
  10. Choi JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and Baum M. Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule. J Clin Invest 105: 1141–1146, 2000.[Web of Science][Medline]
  11. Drose S, Bindseil KU, Bowman EJ, Siebers A, Zeeck A, and Altendorf K. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32: 3902–3906, 1993.[CrossRef][Medline]
  12. Ehrenfeld J and Klein U. The key role of the H+ V-ATPase in acid-base balance and Na+ transport processes in frog skin. J Exp Biol 200: 247–256, 1997.[Abstract]
  13. Fernandez R and Malnic G. H+ ATPase and Cl– interaction in regulation of MDCK cell pH. J Membr Biol 163: 137–145, 1998.[CrossRef][Web of Science][Medline]
  14. Forgac M. Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H+-ATPases. J Exp Biol 203: 71–80, 2000.[Abstract]
  15. Gäbel G, Vogler S, and Martens H. Short chain fatty acids and CO2 as regulators of Na+ and Cl– absorption in isolated sheep rumen mucosa. J Comp Physiol [B] 161: 419–426, 1991.[Medline]
  16. Galfi P, Neogrady S, and Kutas F. Culture of ruminal epithelial cells from bovine ruminal mucosa. Vet Res Commun 4: 295–300, 1980.[CrossRef]
  17. Gluck SL, Nelson RD, Lee BS, Wang Z, Guo X, Fu J, and Zhang K. Biochemistry of the renal V-ATPase. J Exp Biol 172: 219–229, 1992.[Abstract/Free Full Text]
  18. Goss GG, Jiang J, Vandorpe DH, Kieller D, Chernova MN, Robertson M, and Alper SL. Role of JNK in hypertonic activation of Cl–-dependent Na+/H+ exchange in Xenopus oocytes. Am J Physiol Cell Physiol 281: C1978–C1990, 2001.[Abstract/Free Full Text]
  19. Hansen O. Isoform of Na+,K+-ATPase from rumen epithelium identified and quantified by immunochemical methods. Acta Physiol Scand 163: 201–208, 1998.[CrossRef][Web of Science][Medline]
  20. Helmle-Kolb C, Di Sole F, Forgo J, Hilfiker H, Tse CM, Casavola V, Donowitz M, and Murer H. Regulation of the transfected Na+/H+-exchanger NHE3 in MDCK cells by vasotocin. Pflügers Arch 434: 123–131, 1997.[CrossRef][Medline]
  21. Heming TA and Bidani A. Plasmalemmal H+ extruders in mammalian alveolar macrophages. Comp Biochem Physiol A 133: 143–150, 2002.[CrossRef][Medline]
  22. Hogan EM, Davis BA, and Boron WF. Intracellular Cl– dependence of Na-H exchange in barnacle muscle fibers under normotonic and hypertonic conditions. J Gen Physiol 110: 629–639, 1997.[Abstract/Free Full Text]
  23. Hou Y and Delamere NA. Studies on H+-ATPase in cultured rabbit nonpigmented ciliary epithelium. J Membr Biol 173: 67–72, 2000.[CrossRef][Web of Science][Medline]
  24. Karwatowska-Prokopczuk E, Nordberg JA, Li HL, Engler RL, and Gottlieb RA. Effect of vacuolar proton ATPase on pHi, Ca2+ and apoptosis in neonatal cardiomyocytes during metabolic inhibition/recovery. Circ Res 82: 1139–1144, 1998.[Abstract/Free Full Text]
  25. Kaunitz JD, Gunther RD, and Sachs G. Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla. J Biol Chem 260: 11567–11573, 1985.[Abstract/Free Full Text]
  26. Kelly JM, McBride BW, and Milligan LP. In vitro ouabain-sensitive respiration and protein synthesis in ruminal epithelial papillae of Hereford steers fed either alfalfa or bromegrass hay once daily. J Anim Sci 71: 2799–2808, 1993.[Abstract]
  27. Kristensen NB, Hansen O, and Clausen T. Measurement of the total concentration of functional Na+,K+-pumps in the rumen epithelium. Acta Physiol Scand 155: 67–76, 1995.[Web of Science][Medline]
  28. Lang I and Martens H. Na transport in sheep rumen epithelium is modulated by voltage-dependent cation conductance in apical membrane. Am J Physiol Gastrointest Liver Physiol 277: G609–G618, 1999.[Abstract/Free Full Text]
  29. Leonhard-Marek S, Stumpff F, Brinkmann I, Breves G, and Martens H. Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+. Am J Physiol Gastrointest Liver Physiol 288: G630–G645, 2005.[Abstract/Free Full Text]
  30. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]
  31. Malnic G and Geibel JP. Cell pH and H+ secretion by S3 segment of mammalian kidney: Role of H+-ATPase and Cl–. J Membr Biol 178: 115–125, 2000.[CrossRef][Web of Science][Medline]
  32. Marshansky V and Vinay P. Proton gradient formation in early endosomes from proximal tubules. Biochim Biophys Acta 1284: 171–180, 1996.[Medline]
  33. Mattson JP, Lorentzon P, Wallmark B, and Keeling DJ. Characterization of proton transport in bone-derived membrane vesicles. Biochim Biophys Acta 1146: 106–112, 1993.[Medline]
  34. McKinney LC and Moran A. Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes. Am J Physiol Cell Physiol 268: C210–C217, 1995.[Abstract/Free Full Text]
  35. Miyata Y, Muto S, Yanagiba S, and Asano Y. Extracellular Cl– modulates shrinkage-induced activation of Na+/H+ exchanger in rat mesangial cells. Am J Physiol Cell Physiol 278: C1218–C1229, 2000.[Abstract/Free Full Text]
  36. Müller M, Aschenbach JR, and Gäbel G. Role of Na+/H+ exchange and HCO3– transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen. J Comp Physiol [B] 170: 337–343, 2000.[CrossRef][Medline]
  37. Pacheco G, de Becemberg IL, Gonzalez de Alfonzo R, and Alfonzo MJ. Biochemical characterization of a V-ATPase of tracheal smooth muscle plasma membrane fraction. Biochim Biophys Acta 1282: 182–192, 1996.[Medline]
  38. Rajendran VM, Geibel J, and Binder HJ. Chloride-dependent Na-H exchange. J Biol Chem 270: 11051–11054, 1995.[Abstract/Free Full Text]
  39. Sangan P, Rajendran VM, Geibel J, and Binder HJ. Cloning and expression of a chloride-dependent Na+-H+ exchanger. J Biol Chem 277: 9668–9675, 2002.[Abstract/Free Full Text]
  40. Schweigel M, Freyer M, Leclercq S, Etschmann B, Lodemann U, Böttcher A, and Martens H. Luminal hyperosmolarity decreases Na transport and impairs barrier function of sheep rumen epithelium. J Comp Physiol [B] 175: 575–591, 2005.[CrossRef][Medline]
  41. Schweigel M and Martens H. Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells. Am J Physiol Gastrointest Liver Physiol 285: G45–G53, 2003.[Abstract/Free Full Text]
  42. Schweigel M, Vormann J, and Martens H. Mechanisms of Mg2+ transport in cultured ruminal epithelial cells. Am J Physiol Gastrointest Liver Physiol 278: G400–G408, 2000.[Abstract/Free Full Text]
  43. Tomochika K, Shinoda S, Kumon H, Mori M, Moriyama Y, and Futai M. Vacuolar-type H+-ATPase in mouse bladder epithelium is responsible for urinary acidification. FEBS Lett 404: 61–64, 1997.[CrossRef][Web of Science][Medline]
  44. Wadsworth SJ and van Rossum GDV. Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes. J Membr Biol 142: 21–34, 1994.[Web of Science][Medline]
  45. Wang Y and Telfer WH. Cyclic-AMP-induced water uptake in a moth ovary: inhibition by bafilomycin and anthracene-9-carboxylic acid. J Exp Biol 201: 1627–1635, 1998.[Abstract]
  46. Wu X, Torres-Zamorano V, Yang H, and Reinach PS. ETA receptor mediated inhibition of intracellular pH regulation in cultured corneal epithelial cells. Exp Eye Res 66: 699–708, 1998.[CrossRef][Web of Science][Medline]




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