Molecular and functional evidence for electrogenic and electroneutral Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporters in murine duodenum

J. Praetorius1, H. Hager3, S. Nielsen3, C. Aalkjaer2, U. G. Friis1, M. A. Ainsworth1, and T. Johansen1

1 Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark-Odense University, Winsloewparken 21, DK-5000 Odense C; 2 Department of Physiology, University of Aarhus Ole Worms Alle 160, and 3 Department of Cell Biology, Institute of Anatomy, University of Aarhus University Park 233/234, DK-8000 Aarhus C, Denmark


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Inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pHi) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl- increased pHi, whereas an acidification was observed on removal of Na+ or both Na+ and Cl-. The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO<SUB>3</SUB><SUP>−</SUP> transport as well as basolateral electrogenic HCO<SUB>3</SUB><SUP>−</SUP> transport in proximal duodenal villus cells.

intracellular pH; ion transport; fluorescence spectroscopy; sodium-bicarbonate cotransporter proteins


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BICARBONATE SECRETION FROM proximal duodenal epithelial cells is regarded as an important protective factor against gastric acid (5) and has been shown to include electrogenic as well as electroneutral transport processes (34, 35, 40). These processes are dependent on basolateral Na+ and are sensitive to inhibition by the stilbene derivative DIDS, an inhibitor of various Cl- channels as well as most HCO<SUB>3</SUB><SUP>−</SUP> transporters (12). A model for proximal duodenal acid/base transport was presented by Isenberg and coworkers (19): luminal excretion of HCO<SUB>3</SUB><SUP>−</SUP> by Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange and an anion channel was accompanied by basolateral Na+/H+ exchange and Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. The basolateral transporters could be involved in both maintaining intracellular pH (pHi) and in providing HCO<SUB>3</SUB><SUP>−</SUP> for apical extrusion (also generated from CO2 and H2O by action of the carbonic anhydrase). Whether duodenal Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport is electrogenic or electroneutral remains unknown, and the basolateral localization of the transporter has not been verified. The focus on this transporter has increased since the molecular cloning, sequencing, and electrogenic characterization of the electrogenic renal and pancreatic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporters (NBC) (2, 30) and the electroneutral NBC (13, 27). Hence, the present study was undertaken to investigate the molecular and functional expression of NBCs in duodenal epithelial cells. We aimed to discriminate between electrogenic and electroneutral NBC forms by molecular biological tools, to demonstrate the cellular localization of the transporters, and to determine the direction of the Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport near steady-state pHi in isolated cells.


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Materials. Amiloride hydrochloride, antibiotic-antimycotic solution (A9909, containing penicillin, streptomycin, and amphotericin), and RPMI 1640 medium (R7388) were obtained from Sigma Chemical (St. Louis, MO). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Primers for RT-PCR were synthesized by DNA-Technology. All other chemicals were of analytical grade. Drugs were dissolved in DMSO or ethanol, and final concentrations of DMSO or ethanol were below 0.1% (vol/vol), except for the RPMI medium at 0.4% (vol/vol). Antibodies directed against 15 amino acids of the COOH terminal of the electroneutral NBC from rat aorta (raNBCn1) or the electrogenic NBC1 from rat kidney (rkNBC1) were raised in rabbits (24, 39).

Cell preparation. C57BL/6 mice weighing 16.7 ± 5.4 g were killed at 34 ± 7 days of age (mean ± SD; n = 28) by cervical dislocation. After laparotomy, a 15-mm segment of proximal duodenum was excised starting from 1 mm distal to the pylorus. The segment involved the entire horizontal part of the murine duodenum (proximal to hepatic and pancreatic outlets). Identical segments were used for functional experiments and mRNA extraction. The lumen was rinsed and exposed to a citrate solution for 5 min to remove the mucous layer. Final concentrations were (in mM): 134.2 Na+, 9.5 K+, 97.5 Cl-, 5.6 HPO<SUB>4</SUB><SUP>2−</SUP>, 8.0 H2PO<SUB>4</SUB><SUP>−</SUP>, 27.0 citrate, and 10 glucose. pH was adjusted to 7.4, and 5.6 µM indomethacin was added. The luminal surface was then exposed to the EDTA-containing solution for 20 min at 37°C. Final concentrations (in mM) were: 153.3 Na+, 4.7 K+, 140.2 Cl-, 8.2 HPO<SUB>4</SUB><SUP>2−</SUP>, 1.5 H2PO<SUB>4</SUB><SUP>−</SUP>, 1.0 EDTA, and 10 glucose. pH was adjusted to 7.4, and 5.6 µM indomethacin was added. Indomethacin was present during the cell isolation to prevent stimulation of HCO<SUB>3</SUB><SUP>−</SUP> secretion secondary to release of prostaglandin caused by the manipulation of the duodenum. Epithelial cells were separated from the structural components of the duodenum by gentle manipulation as described earlier (26).

Design of PCR primers. To identify highly conserved regions of the Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporters, we aligned sequences of known electrogenic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporters from salamander kidney NBC (GenBank accession no. AF001958) (30), rat renal NBC (AF004017) (29), mouse pancreatic NBC (AF020195), and human kidney NBC (AF007216) (11). Primers were constructed to recognize either conserved regions of the electrogenic NBC types or regions that were specific to the renal or pancreatic type, respectively. For detection of an electroneutral NBC, primers were constructed on similarity between rat electroneutral NBCn1 (AF070475) (13) and human electroneutral NBC3 (AF047033) (27). Table 1 shows the various primer sequences. Start base numbers correspond to those of rat renal NBC1, mouse pancreatic NBC1, or rat electroneutral NBCn1.

                              
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Table 1.   Primers for RT-PCR

RT-PCR and sequence analysis. Isolated duodenal epithelial cells were lysed, and total RNA was extracted using the RNeasy mini kit (Qiagen). RNA (125 µg/ml) was denatured in the presence of 62.5 µg/ml oligo(dT) primer (GIBCO BRL) and 187.5 µg/ml yeast tRNA at 95°C for 3 min in a volume of 8 µl. Denatured RNA was reverse transcribed by incubation with (final concentrations): 0.5 mM (of each base) deoxyribonucleotides (Pharmacia), 10 mM dithiothreitol, 1 IU/µl RNasin (Promega), 0.5 µg/µl BSA, 10 U/µl RT (GIBCO BRL), and buffer in a total volume of 20 µl for 60 min at 37°C. The reaction was terminated by heating at 95°C for 4 min.

The cDNA product was amplified by PCR. The following was added to 3 µl of cDNA produced by the RT reaction (final concentrations): 250 µM (of each base) deoxyribonucleotides, 50 U/ml Taq polymerase (Boehringer Mannheim), and 0.5 µM of each of the two primers, PCR buffer, and water to a final volume of 20 µl. After a denaturation at 95°C for 3 min, 30 cycles of PCR amplification were performed: denaturation at 95°C for 30 s, annealing at 52-60°C (dependent on primer specifications) for 30 s, and polymerization at 72°C for 30 s. The last cycle included 10 min at 72°C. A 15-µl sample of the PCR product and loading buffer was analyzed by electrophoresis on a 2% agarose gel. The gel was stained with ethidium bromide and photographed under ultraviolet illumination. Negative controls were yeast tRNA without duodenal RNA in the RT reaction and PCR and the omission of RT in RNA-containing samples during the RT reaction. RNAs from kidney, pancreas, or aorta extracts were used as positive controls for the renal and pancreatic NBC1 isoforms or the electroneutral NBCn1, respectively. Bands of predicted molecular weight for each PCR product were excised from the gel and purified using the QIAquick gel extraction kit (Qiagen). Both strands were sequenced using the corresponding specific primers and analyzed on a 310 genetic analyzer (ABI PRISM; PE Applied Biosystems).

Immunohistochemistry. Duodenum from normal NMR1 mice were fixed by perfusion via the right heart ventricle with 2% paraformaldehyde in 0.1 M cacodylat buffer (pH 7.4). Blocks were dehydrated and embedded in paraffin. For light- and laser-confocal microscopy, the paraffin-embedded proximal duodena were cut at 2 µm on a rotary microtome (Micron). The staining was carried out using indirect immunofluorescence or indirect immunoperoxidase. The sections were dewaxed and rehydrated. On the sections for immunoperoxidase, endogenous peroxidase were blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigen, we pretreated the sections by boiling them in 1 mM Tris (pH 9) supplemented with 0.5 mM EGTA using a microwave oven. Nonspecific binding of immunoglobulin was quenched by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with relevant antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After a wash in PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min, the sections for laser confocal microscopy were incubated in Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 for 60 min. After a wash in PBS for 3 × 10 min, the sections were mounted in glycerol supplemented with antifade reagent (n-propyl gallate). For immunoperoxidase, the sections were washed, followed by incubation in horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (DAKO P448) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The peroxidase activity was visualized by 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Kem En Tek) dissolved in PBS supplemented with 0.1% H2O2. Counterstaining was carried out using Mayer's hematoxylin. Finally, the sections were dehydrated in graded alcohol and mounted in hydrophobic mounting medium (Eukitt). The microscopy was carried out using a Leica DMRE light microscope and a Zeiss LSM510 laser confocal microscope.

Determination of pHi. The recording of pHi was performed by cuvette-based spectroscopy using the membrane-permeant form of the pH-sensitive fluorescent dye BCECF-AM as previously described (26). Aliquots (200 µl) of cell suspension in RPMI were kept at 2°C until use. The RPMI medium was adjusted to 1.0 mM Ca2+, 1 mg/ml albumin, 5.6 µM indomethacin, 5 IU/ml penicillin, 5 µg/ml streptomycin, and 12.5 ng/ml amphotericin B. Antibiotics/antimycotics were added to prevent microbial growth before and during dye loading. The cells were transferred to 37°C for 15 min, and dye was loaded in 5 µM of BCECF-AM for an additional 15 min and then washed. Cuvettes containing 1.8 ml of cell suspension were placed in a dark chamber at 37°C, provided with H2O-saturated 95% O2-5% CO2 under an air-tight cap, and continuously stirred. The emitted light at 535 nm was recorded, and a ratio was calculated for excitations at 490 and 440 nm. The fluorescence ratio (490/440) was calibrated to pHi by the high K+/nigericin technique (37). The data points were fitted to a third-order polynomial function: pHi(x) = 4.1702 + 5.1816 × x - 3.4078 × x2 + 1.002 × x3. The total buffering capacity (beta tot) was determined from the calculated contribution from the HCO<SUB>3</SUB><SUP>−</SUP>/CO2 buffer system (10), and the intrinsic buffering capacity was obtained previously (26) involving stepwise decreasing NH<SUB>4</SUB><SUP>+</SUP> concentrations. The equation was beta tot= [2.3 × 9.6 × 10-7/10-pHi] + [186.1 - 23.5 × pHi]. The data from the experiments are presented as pHi vs. time traces. Mean values of pHi were determined from the inverse exponential fit of the individual traces before and after the medium was changed. At predefined levels of pHi, the slope of the fitted recovery curve was determined (Delta pHi/dt). The net H+ efflux at the given pHi was calculated by multiplication of the Delta pHi/dt with the buffering capacity for the pHi value. The light emission was quenched in recordings from samples containing amiloride or DIDS. Therefore, these samples were calibrated in the presence of the inhibitors. Data are presented as means ± SD. The Mann-Whitney rank-sum test was used for statistics, and values of P < 0.05 were considered significant.


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Detection of NBC forms by RT-PCR and sequence analysis. RT-PCR was performed with mRNA from duodenal epithelial cells to study the molecular expression of NBCs. Initially, RT-PCR was performed to detect a nucleotide fragment that was conserved in the renal and the pancreatic type electrogenic NBC1. As illustrated in Fig. 1A, we detected a band of the expected molecular size of 760 bp in both duodenum and the control tissue (kidney extract). The nucleotide sequence of the duodenal PCR product shared 99.3% identity with mouse pancreatic NBC (AF020195) corresponding to 100% homology of the predicted amino acid sequences. However, a similar homology of the predicted amino acid sequence was also found to rat renal NBC (AF004017) based on 96.3% nucleotide identity. The band was detected only when reverse transcription was performed (Fig. 1A). It should be noted that, besides the band of expected molecular size, a weaker band appears (lane 6). This is also true for several lanes of the following RT-PCR figures. The nucleotide sequence analysis was performed only on bands of predicted length. To discriminate between the renal and pancreatic type NBCs, primers were constructed to recognize each of the two NH2 terminal sequences. The complete coding region of each of the NH2 terminals was covered by one of the specific sense primer and a common antisense primer located in the conserved region of the sequence. As illustrated, bands were detected for the renal type NBC (209 bp; Fig. 1B) as well as for the pancreatic type NBC (341 bp; Fig. 1C) using duodenal cDNA. Similar bands were obtained with the kidney and pancreas control lanes (Fig. 1, B and C). The sequences of both duodenal PCR products were then determined. The renal type NBC NH2 terminal fragment obtained from duodenal cDNA shared 98.9% nucleotide identity with rat renal electrogenic NBC, yielding 100% homology of the predicted amino acid sequences. The pancreatic type NBC NH2 terminal from duodenal cDNA shared 98.0% nucleotide identity with mouse pancreatic electrogenic NBC, amounting to 100% homology of the predicted amino acid sequences. In Fig. 1C, beta -actin bands were detected of a molecular size of ~206 bp. The use of a beta -actin primer pair as positive control for the presence of cDNA also served as a marker for the purity of the mRNA used for reverse transcription. The primers are located on each side of an intron in the genomic sequence. PCR products of ~270 bp are obtained if the reverse-transcribed mRNA (cDNA) is contaminated by chromosomal DNA. The code for the transmembrane domain and COOH terminal of mouse duodenal NBC1 was then sequenced by multiple RT-PCR reactions using primer pairs designed on the basis of homology between mouse and rat electrogenic NBC isoforms (Table 1). By use of the sequence for the renal type NH2 terminal, the complete coding region of the murine renal type NBC1 was a 3,035-bp sequence sharing 94.5% nucleotide identity with rat renal electrogenic NBC, amounting to 98.6% homology of the predicted amino acid sequences. The complete mouse renal type NBC was given GenBank accession no. AF141934. By use of the sequence for the pancreatic type NH2 terminal, the complete coding region shared 94.9% nucleotide identity with mouse pancreatic NBC, amounting to 98.4% homology of the predicted amino acid sequences. The recent cloning and sequencing of the electroneutral NBCn1 provided the opportunity to study the expression of mRNA encoding this protein. As shown in Fig. 1D, a band of the predicted molecular size of 362 bp was detected with duodenal epithelial cell cDNA. A similar band was detected using rat aorta cDNA as positive control. beta -Actin bands were detected of a molecular size of ~206 bp. The duodenal fragment of the electroneutral NBCn1 shared 96.0% nucleotide identity with rat NBCn1 and 100% amino acid homology. The mouse NBCn1 fragment was given the Genbank accession no. AF218295.


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Fig. 1.   Demonstration of mRNA encoding Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter (NBC) forms in duodenal epithelial cells. Specific primers for various NBCs were used for RT-PCR. Template for the reverse transcription was either mRNA from isolated duodenal epithelial cells (Duo) or extract from kidney (Kid), pancreas (Pan), or aorta (Aor). In negative control lanes, H2O was used instead of template (H2O), or mRNA from isolated duodenal epithelial cells was used without reverse transcription (Duo-). Arrows indicate expected molecular sizes. A: detection of mRNA encoding a highly conserved 760-bp segment of the electrogenic NBC1 using primer pair NBCd(+)/NBCe(-) in lanes 2, 3, 4, 6, 7, and 8. Lanes 1 and 5 are the standard markers. B: detection of mRNA encoding a 209-bp NH2 terminal specific to the renal type NBC1 using the primer pair NBCa(+)/NBCc(-) in lanes 2, 3, and 4. Lane 1 is the standard marker. C: detection of mRNA encoding a 341-bp NH2 terminal specific to the pancreatic type NBC1 using the primer pair NBCb(+)/NBCc(-) in lanes 2, 4, and 6. A beta -actin primer pair provided positive control with duodenal mRNA in lanes 3, 5, and 7. Lane 1 is the standard marker. D: detection of mRNA encoding a 362-bp segment of the electroneutral NBCn1 using the primer pair NBCo(+)/NBCp(-) in lanes 2, 4, and 6. A beta -actin primer pair provided positive control with duodenal mRNA in lanes 3, 5, and 7. Lane 1 is the standard marker.

Immunolocalization of NBC isoforms. Staining of the basolateral membranes of proximal duodenal villus cells was obtained with the rabbit anti-NBCn1 antibody as shown in Fig. 2, A and C. The rabbit anti-rkNBC1 antibody also stained the basolateral membranes of villus cells from mouse proximal duodenum (Fig. 2, B and D). The crypt cells were not labeled with the anti-NBCn1 antibody (Fig. 2E) or by the anti-rkNBC1 antibody (Fig. 2F). The basolateral labeling found with both antibodies was confirmed by laser confocal images using immunofluorescence (Fig. 2, G and H). For both antibodies, the labeling was completely prevented by omission of primary antibody and by preadsorption with 0.2 mg/ml of the relevant peptide (performed with both peroxydase and fluorescence labeling; not shown). It is noted that the anti-rkNBC1 antibody labeled the apical and not the basolateral membrane in initial experiments performed without perfusion fixation of the tissue or microwave antigen retrieval (not shown).


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Fig. 2.   Immunocytochemical analyses of the cellular and subcellular localization of the electroneutral NBCn1 and the electrogenic NBC1 in mouse duodenum using immunoperoxidase and immunofluorescence labeling in the paraffin-embedded tissue sections. A and B: low magnification reveals that NBCn1 (A) and NBC1 (B) are expressed in enterocytes situated on the villi. No expression is seen in the enterocytes of the crypts, in the duodenal glands, or in other components of the duodenal wall. C and D: high magnification of villi shows expression of NBCn1 (C) and NBC1 (D) and reveals that both transporters are localized in the basolateral membranes of the enterocytes, whereas the apical membrane shows no labeling. E and F: high magnification of cross-sections of crypts shows no labeling of NBCn1 (E) or NBC1 (F). G and H: laser confocal images of immunofluorescence-labeled sections. Optical sections of 1-µm thickness superimposed on a Normarski DIC image show that labeling of NBCn1 (G) and NBC1 (H) is associated with basolateral domains of the cells in the columnar epithelium, whereas no labeling is seen at the apical domains. Bars, 10 µm.

Influence of acid/base transport inhibitors and removal of extracellular Cl- or Na+ on pHi. The function of NBCs was initially investigated by pHi recordings near steady-state pHi. In Fig. 3 A-E, the recording was initiated as the medium was switched from HEPES-buffered salt solution (solution 1, Table 2) to a CO2/HCO<SUB>3</SUB><SUP>−</SUP>-HEPES-buffered salt solution (solution 2). After 5 min, the composition of the medium was changed either by addition of inhibitors (3.6-7.2 µl of inhibitor in DMSO to a 1.8-ml sample followed by gentle mixing) or by centrifugation and resuspension in Na+- and/or Cl--depleted media. The pHi was determined after 5 min and after 10 min of measurement, i.e., after 5 min exposure to CO2/HCO<SUB>3</SUB><SUP>−</SUP> and 5 min after the change of medium. The net H+ efflux was calculated at the last second before and at the first second after the change of medium. Initially, the effects of inhibitors of HCO<SUB>3</SUB><SUP>−</SUP> transporters (200 µM DIDS) or Na+/H+ exchange (1 mM amiloride) were studied. As illustrated in Fig. 3, A and F, addition of DIDS effectively increased both pHi and the net H+ efflux (both P < 0.05; n = 5). In contrast, amiloride had only a minor effect on both pHi [not significant (ns)] and net H+ efflux (P < 0.05; n = 5). Next, the dependence on extracellular Na+ and Cl- was studied (Fig. 3, B and F). Removal of extracellular Cl- caused an increase of the net H+ efflux (P < 0.05; n = 5), resulting in a slight elevation of pHi (statistically significant only if values before and after manipulation were day matched). Na+ removal or removal of both Na+ and Cl- caused a decrease of pHi and negative net H+ efflux values (all P < 0.05; n = 5). Accordingly, acid extrusion and/or alkaline uptake were Na+ dependent, and a component of alkaline extrusion was Cl- dependent. The pHi decrease in Na+-free medium is largely independent of extracellular Cl-.


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Fig. 3.   Effect of H+ and HCO<SUB>3</SUB><SUP>−</SUP> transport inhibitors on intracellular pH (pHi) and dependence of pHi on extracellular Cl- and Na+. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded duodenal epithelial cells were exposed to 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP> (solution 2) as fluorescence recording was initiated. After 5 min, the medium was changed with respect to the presence of inhibitors and/or the ionic composition in the continued presence of 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP>. Traces were performed on the same day and are representative of 5 experiments. Mean pHi values ± SD determined after 5 and 10 min are indicated on traces. DIDS and amiloride were used in final concentrations of 200 µM and 1 mM, respectively. A: effects of DIDS and amiloride on pHi. After 5 min, DIDS (open circle ) or amiloride () was added to the cell suspension as indicated on the trace. B: effect of removal of Cl- and/or Na+ from the medium. The cells were washed and resuspended as indicated on the traces in Cl--free (open circle ), Na+-free (), or NaCl-free (triangle ) solutions (solution 3, solution 4-, or solution 5, respectively). C: effect of Na+ removal and simultaneous addition of DIDS or amiloride on pHi. The cells were washed and resuspended in Na+-free solution (solution 4) in the presence of either DIDS (open circle ) or amiloride (). D: effect of Cl- removal and simultaneous addition of DIDS or amiloride on pHi. The cells were washed and resuspended in Cl--free solution (solution 3) in the presence of DIDS (open circle ) or amiloride (). E: effect of DIDS on pHi in relatively Cl--depleted cells. Samples were pretreated for 30 min in Cl--free HEPES-buffered solution supplied with 100% O2 (solution 7), and DIDS was added after 5-min exposure to the Cl--free and CO2/HCO<SUB>3</SUB><SUP>−</SUP>-containing solution 3 (open circle ). F: mean net H+ efflux before and after changing the composition of the solution. The traces from 5 experiments were fitted to inverse exponential functions in the intervals of 0-5 min and 5-10 min. Means ± SD of the net H+ efflux were calculated from the total buffering capacity, and the slope of the fitted recovery curve was derived from the inverse exponential fits immediately before (cross-hatched) and after (open) the manipulation took place.


                              
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Table 2.   Solutions

The inhibitors amiloride and DIDS were then used simultaneously with removal of either Na+ or Cl- to study the pHi changes further. As shown in Fig. 3, C and F, amiloride had no effect on the pHi decrease induced by Na+ removal (ns; n = 5), whereas the negative net H+ efflux induced by Na+ removal was reduced in the presence of amiloride (P < 0.05). Removal of Na+ and simultaneous addition of DIDS changed the course of the pHi recording compared with removal of Na+ alone: an initial pHi decrease was followed by slow pHi increase (ns after 5 min). The negative net H+ efflux after Na+ removal was less pronounced in the presence of DIDS than in its absence (P < 0.05; n = 5). Hence, both amiloride- and DIDS-sensitive processes are involved the pHi decrease on Na+ removal. The removal of Cl- in the presence of DIDS increased pHi and the net H+ efflux (both P < 0.05; n = 5), as shown in Fig. 3, D and F. The effects of DIDS on the pHi and net H+ efflux were actually more pronounced in the absence of extracellular Cl- (P < 0.05); the DIDS-sensitive alkaline extrusion seems to be independent of extracellular Cl-. The pHi was unaffected by Cl- removal in the presence of 1 mM amiloride (ns; n = 5), resulting in only a slight effect on the net H+ efflux (ns). It is noted that the net H+ efflux on Cl- removal in the presence of amiloride seems of intermediate size compared with amiloride or Cl- removal alone (both ns). Also, the net H+ efflux before changing the medium was very modest, and only minimal differences were observed between the samples (ns).

Duodenal epithelial cells were incubated for 30 min in a Cl--free HEPES buffer to reduce intracellular Cl-. DIDS was applied after 5 min of incubation in Cl--free solution with CO2/HCO<SUB>3</SUB><SUP>−</SUP>. DIDS elevated pHi of duodenal cells pretreated without extracellular Cl-, as exemplified in Fig. 3E (P < 0.05; n = 5). The net H+ efflux was likewise increased after addition of DIDS (P < 0.05). These increases were of similar magnitude to that induced by DIDS in control solution (ns) and smaller than the net H+ efflux on Cl- removal without prior intracellular Cl- reduction (P < 0.05).

A plasma membrane depolarization was sought, induced by application of 50 mM K+, to study the possible involvement of electrogenic transporters. Substitution of K+ by N-methyl-D-glucamine (NMDG) was used as control to ensure identical Na+ concentrations in the samples. As shown in Fig. 4A, the high concentration of K+ increased pHi slightly compared with NMDG (statistically significant only if values were day matched; n = 5). The day-matched net H+ effluxes at pHi 7.06 were similar in the two solutions (Fig. 4B; ns). The day-matched net H+ efflux at pH 7.25 with 50 mM K+ was higher than the control flux in 50 mM NMDG (Fig. 4B; P < 0.05). Figure 4C shows the actual values of the control net H+ efflux at the two levels of pHi used for the calculations.


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Fig. 4.   Effect of membrane potential change on pHi and net H+ efflux. BCECF-loaded cells were exposed to 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP>-buffered solutions containing either 50 mM of K+ (mixture of solutions 2 and 6) or 50 mM N-methyl-D-glucamine (NMDG) (control, mixture of solutions 2 and 4) as pHi measurements were initiated (both 95 mM Na+). A: influence of 50 mM K+ on pHi. The traces from a typical experiment are shown. Mean pHi values ± SD from 5 experiments are also indicated with 50 mM K+ (open circle ) or 50 mM NMDG (). The values were calculated at the initiation of the measurement and after 5 min. B: influence of 50 mM K+ on the net H+ efflux. Net H+ efflux was determined at pHi 7.06 and 7.25 in 50 mM K+ solution and in control solution (50 mM NMDG). Columns show the mean values ± SD of the net H+ efflux with 50 mM K+ in %control (n = 5). C: control values for net H+ efflux (50 mM NMDG). Columns show the mean values ± SD of the net H+ efflux at pHi 7.06 and 7.25 (n = 5).

Recovery of pHi after acidification or alkalization. The pHi regulation was then studied in a broader pH range by inducing an intracellular acidification by a prepulse of 30 mM NH<SUB>4</SUB><SUP>+</SUP>. CO2/HCO<SUB>3</SUB><SUP>−</SUP> were present only during pHi recovery to increase the probability of an inward HCO<SUB>3</SUB><SUP>−</SUP> gradient and to obtain a more pronounced acidification. Figure 5, A and B, shows the pHi dependence of the net acid extrusion of acidified duodenal epithelial cells. The traces of the recovery period were fitted to inverse exponential functions, and the mean values for H+ efflux were obtained from the curve fit at different values of pHi. For all levels of pHi, the net H+ efflux was reduced by removal of Na+ and by addition of 1 mM amiloride or a high concentration of DIDS (500 µM). The reduction of the H+ efflux was greater when Na+ was omitted than when amiloride or DIDS was added for all values of pHi (P < 0.05 in the pHi range from 6.7 to 6.95). The amiloride-sensitive component of the H+ efflux (difference between control and amiloride curves) was larger at low pHi, whereas the DIDS-sensitive component (difference between control and DIDS curves) was rather uniform within the pHi interval studied. The Na+-dependent H+ efflux amounted to 85.4 ± 1.0% of control at pHi 6.80 (P < 0.05; n = 4), whereas the amiloride-sensitive and the DIDS-sensitive H+ efflux at pHi 6.80 comprised 68.5 ± 2.3 and 25.6 ± 10.1% of control H+ efflux, respectively (for both P < 0.05; n = 4). Hence, the recovery from acid pHi contains both DIDS- and amiloride-sensitive components. The difference between the amiloride-insensitive flux and the Na+-independent flux is larger than that observed in the absence of CO2/HCO<SUB>3</SUB><SUP>−</SUP> (not shown). This indicates the participation of Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport.


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Fig. 5.   Recovery of pHi after acidification by NH<SUB>4</SUB><SUP>+</SUP> prepulse. A: BCECF-loaded duodenal epithelial cells were exposed to 30 mM of NH<SUB>4</SUB><SUP>+</SUP> for 3 min in HEPES buffer. After removal of NH<SUB>4</SUB><SUP>+</SUP>, the cells were resuspended in various solutions containing 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP>. These were control solution (solution 2), control solution with DIDS (500 µM) or amiloride (1 mM), or a solution depleted of extracellular Na+ (solution 4). The traces of 1 of 4 experiments are shown. B: net H+ efflux during pHi recovery in the same experiments. The net H+ efflux was calculated for each trace at pHi values within the range 6.6-7.2, and means ± SD from 4 experiments were calculated. C: recovery of pHi in relatively Cl--depleted cells. Duodenal epithelial cells were incubated after dye loading for 30 min in a Cl--free HEPES-buffered solution (solution 7) to reduce intracellular Cl- concentration. Control samples were incubated for 30 min in standard HEPES buffer (solution 1). A prepulse of 30 mM NH4 (SO<SUB>4</SUB><SUP>2−</SUP> salt) was applied, and after wash, the cells were resuspended in the Cl--free solution (solution 3) with 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP>.

The dependence of the pHi recovery on intracellular Cl- was then studied. Duodenal epithelial cells were incubated after dye loading for 30 min in a Cl--free HEPES-buffered solution to reduce intracellular Cl- (supplied with 100% O2). An ammonium prepulse was applied using (NH4)2SO<SUB>4</SUB><SUP>2−</SUP>, and cells were resuspended in the Cl--free solution with 24 mM of HCO<SUB>3</SUB><SUP>−</SUP> and 5% CO2 as illustrated in Fig. 5C. Control samples were incubated for 30 min in standard HEPES buffer (100% O2) before the ammonium sulfate prepulse. The net H+ efflux in Cl--depleted cells amounted to 97.4 ± 9.2% of control values calculated from the inverse exponential fitted curves in the pH range from 6.85 to 7.0 (ns at all levels of pHi; n = 5). Thus the pHi recovery seems independent of intracellular Cl-.

Figure 6 shows the trace of one of five experiments using propionic acid prepulse to induce alkalization. Cells were exposed to CO2/HCO<SUB>3</SUB><SUP>−</SUP> solution (solution 2) as the measurement was initiated. After 3 min, an isotonic solution containing propionic acid was added in a 1:1 volume (30 mM final concentration of propionic acid). After an additional 5 min, the cells were washed and resuspended in either control solution or Na+- or Cl--depleted solutions. The recordings were performed in the continued presence of CO2/HCO<SUB>3</SUB><SUP>−</SUP>. The traces were fitted to inverse exponential functions starting 30 s after wash and resuspension, with the exception of Cl--free traces. The latter traces were fitted to linear functions. Mean values for OH- efflux were obtained from the fitted curves as measure for the outward HCO<SUB>3</SUB><SUP>−</SUP> transport. At pHi 7.55, removal of extracellular Na+ increased the net OH- efflux compared with the control net OH- efflux (P < 0.05; n = 5), whereas removal of extracellular Cl- reduced the efflux. The recovery of pHi was very low in the absence of extracellular Cl-, and mean values for the efflux could not be obtained at a common level of pHi. For all five experiments, the efflux was lowest in Cl--free medium.


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Fig. 6.   Recovery of pHi after alkalization by propionic acid prepulse. A: after 3-min exposure to control solution containing 5% CO2-24 mM HCO<SUB>3</SUB><SUP>−</SUP> (solution 2), BCECF-loaded duodenal epithelial cells were exposed to 30 mM of propionic acid for 5 min. After wash, the cells were resuspended in either control HCO<SUB>3</SUB><SUP>−</SUP> solution (solution 2) or solutions depleted of Na+ or Cl- as indicated (solution 4 or 3, respectively). The traces of 1 of 5 experiments are shown. B: net OH- efflux during pHi recovery after alkalization. Means ± SD of net OH- efflux were calculated from 5 experiments at pHi 7.55.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated the expression of multiple NBCs in isolated epithelial cells from proximal mouse duodenum. The molecular expression of the NBC forms was studied by RT-PCR, and the localization was studied by immunohistochemistry. Inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport was identified as the Na+-dependent, Cl--independent, and relatively DIDS-sensitive alkaline uptake in the presence of CO2/HCO<SUB>3</SUB><SUP>−</SUP>. Outward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport was suggested by finding a more DIDS-sensitive and Cl--independent alkaline extrusion in the presence of CO2/HCO<SUB>3</SUB><SUP>−</SUP>.

Although the functional presence of Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport has been demonstrated in acidified duodenal epithelial cells from rat and humans (4, 19), the molecular nature of the transporter has not yet been identified. The recent cloning and sequencing of many NBC forms made further analysis of duodenal Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport possible. The RT-PCR experiments and sequence analysis demonstrate the expression of at least two putative electrogenic NBC1 isoforms by use of mRNA from isolated duodenal epithelial cells. This is the first evidence for the presence of electrogenic NBCs in this tissue. The next step was to discriminate between the pancreatic and the renal types of the electrogenic NBC1, which differ only in the NH2 terminal. We expected to identify the pancreatic-type NBC1, because both the duodenal epithelium and the pancreatic ducts secrete a large amount of HCO<SUB>3</SUB><SUP>−</SUP> into the lumen of the respective organs. Nevertheless, mRNA encoding both the pancreatic and renal isoforms of the NBC1 was detected in the preparation.

The murine sequence of the renal-type NBC1 has not previously been determined. Given the importance of the murine model in membrane transport research, we sequenced the complete coding region of the cDNA. We did not find complete identity to the published mouse pancreas NBC1 in the transmembrane segment and COOH terminal of the coding sequence. Whether this could be explained by genetic differences between the mouse strains is unknown. It is noted that the predicted amino acid sequence encoding the consensus sites for DIDS binding of anion exchanger (AE) and NBC1 isoforms was conserved in the mouse renal-type NBC1: at predicted amino acids 559-562 a KMIK sequence is found, and at amino acid 768-771 a KLKK sequence is located.

The expression of mRNA encoding NBCn1 suggests that the duodenal epithelial cells are also capable of electroneutral Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. This novel detection of NBCn1 expression in the gastrointestinal tract adds to the growing list of epithelia expressing this NBC protein, e.g., the testes, lung, and kidney (13). However, the expression of at least three NBC forms in the preparation should give rise to major concern regarding the quality of the mRNA. Arguments against the occurrence of false-positive PCR products are described in the following. First, the actin primers were applied in RT-PCR experiments to detect contamination with chromosomal DNA. As described in RESULTS, the primers covered an intron-spanning segment of the actin gene, and on contamination the band would be detected at higher molecular weight than observed (~270 bp). Second, the brightest bands of the PCR were always of the predicted size, although additional bands occurred in several lanes. However, the sequence analysis provided strong evidence for the specificity of the bands with very high homology to known sequences from other species. Third, a control sample of mRNA without RT was included in the first RT-PCR experiment after each isolation of mRNA. This was performed to test the purity of the mRNA both from duodenal epithelial cells and for the control tissue. Chromosomal DNA, if present, would have caused the formation of PCR bands in these controls. Finally, no introns were detected during the sequencing of the RT-PCR products of mouse NBC forms, including the complete coding region of the murine renal-type NBC1. Different physiological functions of the NBC proteins may then be expected within the epithelial cells or along the crypt-villus axis. The epithelial cells of duodenum include numerous typical villus enterocytes (~80%), fewer crypt cells (<20%), and a few percent goblet, paneth, and enteroendocrine cells (25). Expression of one of the NBC forms in, for example, the goblet cells or endocrine cells could provide the observed result because of the high sensitivity of the method. Hence, immunohistochemical staining of the duodenum was conducted to confirm the expression of the NBC forms at protein levels and to determine its localization within the epithelium.

The immunohistochemical localization of the NBCn1 protein on the basolateral membrane of villus cells suggests a role of this transporter in cellular uptake of HCO<SUB>3</SUB><SUP>−</SUP> from the interstitial fluid. The electroneutral properties of NBCn1 (1, 13) or NBC3 (27), along with a presumed inward Na+ gradient, predict a net influx of Na+ and HCO<SUB>3</SUB><SUP>−</SUP> by the protein. This influx could either serve as a basolateral donor of HCO<SUB>3</SUB><SUP>−</SUP> for apical secretion of the ion or simply function as a regulatory system to maintain a suitable pHi. The basolateral localization of the electrogenic NBC1 protein was expected, since Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport is believed to occur only at the basolateral membrane of duodenal epithelial cells (19). The renal NBC1 is localized to the basolateral membrane of the proximal tubule cells from the kidney (31) and extrudes Na+ and HCO<SUB>3</SUB><SUP>−</SUP> (9). On the other hand, the pancreatic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport appears to mediate net influx of the ions (20, 21) but is also localized to the basolateral membrane (23). In any case, the expression of an electrogenic NBC1 protein in the basolateral membrane introduces the possibility that this NBC contributes to the electrogenic alkaline transport across the epithelium of proximal duodenum. Interestingly, it seems that both electrogenic and electroneutral NBC forms are expressed in the majority of villus cells rather than being confined to different cell types within the epithelium. It should be noted that the rkNBC1 antibody was directed against amino acids common to both the pancreatic and renal NBC1. The similar binding pattern of the rkNBC1 and the raNBCn1 antibodies was not caused by cross-reactivity of the antibodies, since they were directed against the very dissimilar NH2 terminals of the proteins. Furthermore, the antibodies have been used to show differential localization of the electroneutral and the electrogenic NBCs in the kidney (24, 39).

The function of NBCs in duodenal epithelial cells was then assessed by measurements of pHi. The demonstration of different modes of Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport in duodenal epithelial cells seems very complicated, since multiple other acid/base transporters are expressed in the cells. Nevertheless, the contributions of the different transporters to near-steady-state pHi were investigated on the basis of their respective sensitivity toward inhibitors and their ionic requirement. In the model for duodenal acid/base transport presented by Isenberg and coworkers (36), basolateral inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport and Na+/H+ exchange are counteracted by apical Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange and, presumably, an anion channel. Both the Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport and Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange seem to be relatively DIDS insensitive, as judged from the previously applied high concentrations of stilbene derivative (4, 19).

The detection of alkaline transport by various HCO<SUB>3</SUB><SUP>−</SUP> transporters depends on minimal acid extrusion by the Na+/H+ exchangers near steady state in the presence of CO2/HCO<SUB>3</SUB><SUP>−</SUP>. In the present study, duodenal Na+/H+ exchange was minimal above pH 7.3, since the inhibitor amiloride had little effect on pHi near steady state in the present study. This is consistent with previous observations performed in the absence of CO2/HCO<SUB>3</SUB><SUP>−</SUP> (26). The removal of Na+ provided the pHi decrease observed earlier (4, 19). This acidification is most likely to be caused by outward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport and reversed by Na+/H+ exchange, since it was independent of extracellular Cl-, DIDS sensitive, and partly inhibited by amiloride.

It was surprising that DIDS greatly increased pHi by inhibition of alkaline extrusion at a relatively low concentration (200 µM). It should be emphasized that the pHi increase induced by DIDS was much larger than that observed with Cl- removal. This suggests the involvement of a DIDS-sensitive HCO<SUB>3</SUB><SUP>−</SUP> extruder different from the AE, since Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange by the AE2 isoform, which is present in duodenum, is about five times less DIDS sensitive than AE1 (16). Given the expression of several NBC forms in the duodenum, we hypothesized that the increase of pHi might be due to inhibition of alkaline extrusion through a more DIDS-sensitive NBC. The induced pHi increase then arises from unopposed acid extrusion and/or alkaline uptake. Under the present conditions, this is likely to be inward, relatively DIDS-insensitive Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport given the low rate of Na+/H+ exchange at the actual pHi. Interestingly, DIDS has been used in concentrations of 50-400 µM to block electrogenic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport (2, 10, 14, 30), whereas concentrations of 500 µM-1 mM of DIDS, or its analogs, induced incomplete inhibition of Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport by electroneutral NBC (13, 27) or Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport in acidified duodenal epithelial cells (3, 4). The apparent differences in sensitivity among the NBC forms toward DIDS are mirrored by the observation that the electroneutral NBC lacks one of the two putative DIDS-binding motifs that characterize the electrogenic NBC1 (13, 27, 30).

The net alkaline uptake induced by DIDS was present both when applied in normal medium and when Cl- was removed. This would not be the case if the Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchanger was the only DIDS-sensitive alkaline extruder, since this transporter would already be inhibited or reversed by Cl- removal. Furthermore, the effect of DIDS was actually greatest in Cl--free medium, suggesting additive effects of Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchanger inhibition/reversal and the inhibition of an additional DIDS-sensitive alkaline extruder. Interestingly, the alkalizing effect of DIDS was conserved in relatively Cl--depleted cells in the continued absence of extracellular Cl-. Under these conditions, Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange in both directions should be greatly minimized. This finding also excludes the participation of Na+-dependent Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange and makes the participation of an anion channel less likely.

Theoretically, the observed alkaline extrusion could be mediated by other HCO<SUB>3</SUB><SUP>−</SUP> transporters than outward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. The cystic fibrosis transmembrane conductance regulator (CFTR) has recently been proposed to mediate HCO<SUB>3</SUB><SUP>−</SUP> extrusion in duodenum (15, 17, 32). In the current study, this anion channel is unlikely to contribute significantly to pHi regulation, since CFTR was found to be expressed predominantly in duodenal crypt cells (6, 17, 38) and a subpopulation of villus cells that comprises a minor fraction of the total epithelial cell population (6, 7). Furthermore, CFTR is inhibited by DIDS only by intracellular application (18, 22). The subject of the present study was restricted to acid/base transport in unstimulated duodenal epithelial cells. Accordingly, the involvement of CFTR in cAMP-stimulated duodenal HCO<SUB>3</SUB><SUP>−</SUP> extrusion was beyond the scope of this study. If significant alkaline extrusion occurred through another relatively nonselective anion channel, the transport of HCO<SUB>3</SUB><SUP>−</SUP> through this channel should be increased under Cl--free conditions (on both sides of the plasma membrane). Such an increase of alkaline extrusion was not observed in the relative Cl--depleted cells. Until this point, the findings are consistent with the presence of outward-directed Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport in addition to the previously described acid/base transporters: Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange, Na+/H+ exchange, and inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. It should be noted that the degree of Cl- depletion is uncertain, because the intracellular Cl- concentration was not determined. Pretreatment in Cl--free solutions has been reported to induce significant reductions of intracellular Cl- levels in several cells types (8, 33). It can be hypothesized that membrane potential-insensitive alkaline uptake (presumably by NBCn1) is favored at low pHi, only to be counteracted by alkaline extrusion near steady-state pHi by a membrane potential-sensitive process (presumably NBC1). Indeed, the net H+ efflux was not affected by 50 mM K+ in acidified cells, whereas the net H+ efflux was increased at pHi 7.25 and the pHi after 5 min was slightly elevated by 50 mM K+. This supports to some extent the involvement of a membrane potential-sensitive component of duodenal HCO<SUB>3</SUB><SUP>−</SUP> transport and is thus consistent with the molecular biological demonstration and the immunohistochemistry. Nevertheless, more direct measurements are required to confirm this finding.

The second approach to the study of the duodenal acid/base transporters was to remove the pHi from steady-state values and study the recovery of pHi. The pHi recovery of acidified cells was strongly inhibited by the absence of extracellular Na+, consistent with previous demonstration of Na+/H+ exchange and inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport (19). The fact that only a minor part of the pHi recovery is Na+ independent largely excludes the presence of other major acid extruders of alkaline uptake systems in the cells. The net H+ efflux was sensitive to a high concentration of DIDS (500 µM) in the entire pHi range. However, DIDS-sensitive H+ efflux seems to be constant from pHi 6.7 to 7.0, implying that the inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport is relatively insensitive to pHi in this range. Higher concentrations of DIDS were avoided because of massive and varying quenching of the signal by the compound. In contrast, the amiloride-sensitive component was strongly pHi sensitive, as reported previously (26), and seemed to account for the largest fraction of the pHi recovery at low pHi values. This Na+/H+exchange-mediated H+ efflux might be slightly underestimated, since the relatively amiloride-resistant Na+/H+ exchange isoform NHE3 is present in duodenal epithelial cells (26). The similar net H+ efflux from acidified control cells and relatively Cl--depleted epithelial cells supports the Cl--independent nature of the alkaline uptake observed without cellular acidification.

The outward HCO<SUB>3</SUB><SUP>−</SUP> transport was studied in cells alkalized by propionic acid prepulse. The pHi recovery of alkalized cells was strongly dependent on extracellular Cl- and was probably mediated by Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange. The minimal net OH- efflux in the absence of Cl- could, however, arise from another outward HCO<SUB>3</SUB><SUP>−</SUP> transport, slightly exceeding an ongoing inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport. We observed the highest rate of pHi recovery in the absence of extracellular Na+. In support of this view, full recovery of pHi was observed only in the absence of extracellular Na+. This seems consistent with a facilitation of outward HCO<SUB>3</SUB><SUP>−</SUP> transport because inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport was prevented by Na+ removal. Whether such outward HCO<SUB>3</SUB><SUP>−</SUP> transport is mediated by Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport or by a HCO<SUB>3</SUB><SUP>−</SUP> channel could not be determined.

Taking the molecular biological and the functional findings together, it seems likely that basolateral localized electroneutral NBCn1 mediates the relatively DIDS-insensitive inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport in villus cells, as illustrated in the modified model of duodenal acid/base transport (Fig. 7). This transport should be driven by an inward-directed chemical Na+ gradient and be independent of the electric gradient of the ions across the plasma membrane. It is suggested that basolateral electrogenic NBC1 forms are either involved in inward Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport or mediate the more DIDS-sensitive outward transport of HCO<SUB>3</SUB><SUP>−</SUP> presently demonstrated in isolated proximal duodenal epithelial cells. The electrogenic NBC isoforms are believed to transport two or three molecules of HCO<SUB>3</SUB><SUP>−</SUP> per Na+, as recently reviewed (28). Outward HCO<SUB>3</SUB><SUP>−</SUP> transport would be possible despite the inward-directed electrochemical gradient for Na+, assuming an apical transmembrane potential around -70 mV, a 3:1 stoichiometry, and a typical ionic distribution across the plasma membrane. Such outward electrogenic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransport could, however, result from the nonpolarized state of the cells during measurements. The actual ionic distribution and the membrane potential of the isolated cells are not known, and the direction of transporter in situ could be either inward or outward. In conclusion, evidence is provided for the expression of both electrogenic and electroneutral NBCs in the villus cell type of the duodenal epithelium. Apparently, NBCs are not expressed by the crypt cells of the murine duodenum. Further studies are required to verify the proposed function and direction of transport of the basolateral NBCs in a villus cell monolayer preparation or in the intact epithelium.


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Fig. 7.   A model for acid/base transport in villus cells from proximal duodenum. The model is modified from Isenberg and co-workers (19) according to the present findings. AE, Cl-/HCO<SUB>3</SUB><SUP>−</SUP> anion exchanger; NHE, Na+/H+ exchanger; NBCn1, electroneutral NBC; NBC1, electrogenic NBC (nHCO<SUB>3</SUB><SUP>−</SUP>, 2 or 3 molecules of HCO<SUB>3</SUB><SUP>−</SUP> are transported per Na+ molecule); Ch?, previously proposed anion channel. NHE2 and NHE3 are suggested to be localized to the apical membrane as in other segments of the small intestine (18).


    ACKNOWLEDGEMENTS

We are grateful to Ole Madsen, Mette Fredenslund, and Annette K. Rasmussen for skilled technical assistance.


    FOOTNOTES

This work was supported by the following foundations: Lily B. Lunds Fond; Overlægerådet Odense Universitetshospital; Direktør Ib Henriksens Fond; Nygård Fonden, Katrine and Vigo Skovgaards Fond, Hørslev-Fonden, and Danish Medical Research Council (9601778).

Address for reprint requests and other correspondence: J. Praetorius, 10 Center Drive, Bldg. 10, Rm. 6N323, Bethesda, MD 20892 (E-mail: praetorj{at}nhlbi.nih.gov).

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 27 January 2000; accepted in final form 9 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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4.   Ainsworth, MA, Hogan DL, Rapier RC, Amelsberg M, Dreilinger AD, and Isenberg JI. Acid/base transporters in human duodenal enterocytes. Scand J Gastroenterol 33: 1039-1046, 1998[ISI][Medline].

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7.   Ameen, NA, Martensson B, Bourguinon L, Marino C, Isenberg J, and McLaughlin GE. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J Cell Sci 112: 887-894, 1999[Abstract/Free Full Text].

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14.   Choi, I, Romero MF, Khandoudi N, Bril A, and Boron WF. Cloning and characterization of a human electrogenic Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter isoform (hhNBC). Am J Physiol Cell Physiol 276: C576-C584, 1999[Abstract/Free Full Text].

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