Expression and distribution of the Na+-HCOminus 3 cotransporter in human pancreas

Christopher R. Marino1,2,3, Virginia Jeanes2, Walter F. Boron4, and Bernhard M. Schmitt4

1 Veterans Affairs Medical Center, Departments of 2 Medicine and of 3 Physiology and Biophysics, University of Tennessee, Memphis, Tennessee 68163; and 4 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The cellular mechanisms of HCO-3 secretion in the human pancreas are unclear. Expression of a Na+-HCO-3 cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single ~130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na+-K+ pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of HCO-3 by human pancreatic duct cells involves the basolateral uptake of Na+ and HCO-3 via NBC, an electrogenic Na+-HCO-3 cotransporter.

ion transport; immunofluorescence; membrane proteins; cell physiology; pancreatic ducts


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE MAJOR physiological functions of the exocrine pancreas is the secretion of a HCO-3-rich, alkaline fluid. Stimulated primarily by secretin, the pancreas secretes HCO-3 that combines with HCO-3 secreted by the intestinal mucosa and the biliary tree to neutralize gastric acid entering the duodenum. It is well established that the duct epithelial cell is the principal site of HCO-3 secretion in the pancreas (see Ref. 4 for review).

Micropuncture techniques have demonstrated that the ductular tree exchanges HCO-3 for Cl- (9, 27), indicating a role for apical anion exchange in HCO-3 secretion. Recently, a cAMP-activated Cl- channel has been detected on the apical membrane of pancreatic duct cells. Patch-clamp (16, 17), in situ hybridization (43), and immunolocalization studies (12, 29) have identified this Cl- channel in rat and human pancreas as the cystic fibrosis transmembrane conductance regulator (CFTR). These observations have been combined into a model of pancreatic HCO-3 secretion (4, 41) in which the apical transport of HCO-3 from cytoplasm into the duct lumen occurs by functionally coupling Cl-/HCO-3 exchange to secretin-stimulated, cAMP-activated Cl- secretion via CFTR. Apical HCO-3 secretion by this mechanism requires a relatively high intracellular HCO-3 concentration ([HCO-3]). Based primarily on studies in the rat, it appears that the intracellular HCO-3 arises from the reactions CO2 + H2O right-arrow H2CO3 right-arrow HCO-3 + H+. Carbonic anhydrase catalyzes the first of these reactions. According to this model (4, 41), the H+ is extruded across the basolateral membrane by a Na+/H+ exchanger, which is energized by the Na+ gradient generated by the Na+-K+ pump. The K+ entering the cell via the Na+-K+ pump exits via a basolateral K+ channel.

However, the described model does not account for all physiological observations. For example, mathematical modeling suggests that the scheme cannot explain the high luminal [HCO-3] seen after stimulation with secretin in species such as the guinea pig, pig, and humans (4, 41). In the pig, a vacuolar-type H+ pump is the principal route of basolateral H+ extrusion (44). Moreover, the carbonic anhydrase inhibitor acetazolamide inhibits HCO-3 secretion by no more than 50% in all species studied, including humans (3, 14, 15), rabbit (25, 42), cat (9, 10), dog (5, 31), and pig (33), suggesting that duct cells directly import HCO-3 across the basolateral membrane. Additional evidence for a direct HCO-3 uptake mechanism comes from studies in which the effect of altering [HCO-3] or PCO2 on HCO-3 secretion was examined. These studies indicate that pancreatic HCO-3 secretion is strongly controlled by basolateral [HCO-3], whereas PCO2 has no major effect (2, 35, 42). In contrast, luminal [HCO-3] in the parotid does vary directly with arterial PCO2 (7). Finally, intracellular pH (pHi) studies on rat and guinea pig ducts have directly demonstrated the presence of a basolateral HCO-3 uptake mechanism that is dependent on Na+ and inhibited by DIDS, consistent with the presence of either a Na+-dependent Cl-/HCO-3 exchanger or a Na+-HCO-3 cotransporter (NBC) (23, 30a, 44, 45).

In humans, the difficult access to viable tissue has greatly limited physiological studies of pancreatic HCO-3 secretion. Some components of the above models may be applicable to human pancreas, as suggested by the presence of CFTR on the apical membrane of human duct cells (17, 29). Furthermore, the detection of carbonic anhydrase II by immunocytochemistry (26) and RT-PCR (38) in human pancreatic duct cells is compatible with a role of this enzyme in the production of intracellular HCO-3. However, the other major elements of HCO-3 secretion by human pancreatic duct cells remain unknown.

Recently, interest has emerged in the potential role of NBC as a mechanism for increasing intracellular [HCO-3] in pancreatic duct cells (22-25, 30a, 44, 45). Fueled by the cloning of NBCs from salamander kidney (37), rat kidney (36), and human kidney (8), as well as from human pancreas (1) and human heart (11), molecular approaches are now becoming available to test this hypothesis. Northern blots have shown abundant expression of NBC mRNA in whole human pancreas (1, 8, 11). In mouse, an in situ hybridization study has demonstrated the presence of NBC mRNA in both acinar and duct cells (1). In humans, however, it is not known whether NBC is present in the pancreatic duct cells and, if so, whether NBC is present at the apical and/or the basolateral membrane.

In the present study, we have used antisera directed against rat kidney NBC to immunolocalize the NBC protein in human pancreas. Our results establish for the first time that NBC is strongly expressed on the basolateral membrane of human duct epithelial cells. This finding points to an important role of the NBC in HCO-3 secretion by the human pancreatic duct cell.


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INTRODUCTION
METHODS
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Materials

Unless otherwise indicated, all materials were obtained from Sigma Chemical (St. Louis, MO).

Northern Blotting

A commercially available blot of human pancreatic poly(A)+ RNA (Clontech) was probed with nonisotopically labeled riboprobes complementary to nucleotides 1014-1173 and 2784-3105 of rat kidney NBC (36). These sequences encode the same polypeptides contained in the fusion proteins used to raise the anti-NBC sera (see Antibodies). To obtain these riboprobes, we amplified the above sequences by PCR (PCR purification kit; Qiagen, Chatsworth, CA) and cloned them into the pSV-SPORT1 vector. Positive clones were propagated in Escherichia coli (DH5alpha ; GIBCO, Gaithersburg, MD) and plasmid DNA was prepared. Linearized plasmid DNA was then used to generate antisense RNA probes labeled with digoxigenin, termed NBC3-AS and NBC5-AS, by in vitro transcription (T7 MEGAscript kit, Ambion, Austin, TX).

The poly(A)+ RNA blot was prehybridized for 30 min at 65°C ("Northern MAX," Ambion), followed by hybridization for 90 min at 65°C with a mix of riboprobes NBC3-AS and NBC5-AS (~1 nM each, in "ZipHyb," Ambion). Blots were washed stringently at 65°C with 2× SSC (1× SSC: 150 mM NaCl and 15 mM trisodium citrate), 0.1% SDS (2 × 15 min), 0.5× SSC, 0.1% SDS (2 × 15 min), and 0.2× SSC, 0.1% SDS (1 × 15 min). The ensuing immunodetection protocol of bound digoxigenin label was carried out at room temperature. Membranes were blocked for 30 min with Blotto (3% Carnation nonfat dry milk in PBS, treated with 0.1% diethyl pyrocarbonate for 1 h, followed by brief boiling in a microwave oven). Afterward, they were reacted with sheep anti-digoxigenin Fab fragments, coupled to alkaline phosphatase (Boehringer Mannheim). Subsequently, the membrane was washed in Blotto, equilibrated in detection buffer (0.1 M diethanolamine in 0.13 M NaCl, pH 9.5), and reacted with "CDP-Star" substrate (Boehringer Mannheim; 0.25 mM in detection buffer). The chemiluminescent signal was recorded on X-OMAT-AR film (Kodak, Rochester, NY).

Antibodies

Anti-(MBP-NBC3) and anti-(MBP-NBC5) are rabbit antisera raised against fusion proteins containing maltose-binding protein (MBP) and rat kidney NBC amino acid sequences 338-391 and 928-1035, respectively (36). The fusion proteins were expressed in E. coli and affinity purified on amylose resin before immunization. The generation, characterization, and specificity of the antisera have been described (39).

Mouse monoclonal antibody (MAb-6H) against the beta -subunit of rat Na+-K+ pump (40) was generously provided by Michael J. Caplan (Yale School of Medicine, New Haven, CT). Mouse MAb against the COOH terminus of human CFTR was obtained from Genzyme Diagnostics (Cambridge, MA). FITC- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit and anti-mouse F(ab')2 fragments, respectively, were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Immunoblotting

Frozen human pancreas was solubilized in boiling SDS (3%), containing 0.4 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and a 1:200 dilution of a protease inhibitor cocktail (in mg/ml) of 0.2 leupeptin, 0.2 chymostatin, 0.2 pepstatin, 0.5 soybean trypsin inhibitor, and 0.5 aprotinin. Lysate (100 µg) was prepared in Laemmli sample buffer, heated to 100°C for 5 min, and subjected to 6% SDS-PAGE. Separated proteins were transferred to a membrane (Immobilon, Millipore), which was then blocked with Blotto (5% Carnation nonfat dry milk in PBS containing 0.1% Tween 20) and immunoblotted with NBC antisera at a 1:250 dilution. Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). For specificity controls, anti-NBC sera were preabsorbed with the respective immunogenic fusion protein (0.9 mg/ml) overnight at 4°C before the immunoblotting procedure.

Immunofluorescence Microscopy

Indirect immunofluorescence microscopy was performed as previously described (29). Blocks of normal human pancreas frozen in OCT (Tissue-Tek; Miles, Elkhart, IN) were provided by Dr. Alina F. Jukkola (Pathology Service, VA Medical Center, Memphis, TN). Cryosections (5-6 µm thick) were fixed in methanol at -20°C for 20 min and then blocked with 1% BSA in PBS (100 mM NaCl, 10 mM phosphate buffer, pH 7.4) containing a 1:1,000 dilution of the protease inhibitor cocktail. Primary antibodies were applied to tissue sections for 1-2 h at room temperature in a moist chamber. NBC antisera were diluted 1:250 in BSA-PBS, and bound antibodies were detected with FITC-conjugated goat anti-rabbit F(ab')2 fragments. Anti-CFTR and anti-Na+-K+ pump labeling were both detected with TRITC-conjugated goat anti-mouse F(ab')2 fragments. For double-label experiments, rabbit and mouse primary antibodies were applied simultaneously, followed by the simultaneous application of FITC-conjugated anti-rabbit and TRITC-conjugated anti-mouse secondary antibodies. Fluorescence signals were visualized on an Axiophot fluorescent microscope (Zeiss) and digitally stored using Photoshop 4.01 software (Adobe Systems, Mountain View, CA). Photoshop was not used to modify images other than to adjust brightness or contrast for improved signal definition. The specificity of NBC primary antibody labeling was determined through preabsorption experiments performed as described for immunoblot experiments.


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INTRODUCTION
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Expression of NBC mRNA and Protein in Human Pancreas

We assessed NBC expression in the human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Northern blot analysis with rat kidney NBC riboprobes detected a single ~9.5-kb mRNA species in normal human pancreas (Fig. 1A). Previous studies have reported sizes of ~7.6 kb and ~7.7 kb (1) for the pancreatic NBC mRNA; the discrepancies may be due to poor separation of high-molecular-mass mRNAs on these blots.



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Fig. 1.   Expression of the electrogenic Na+-HCO-3 cotransporter (NBC) in human pancreas. A: detection of NBC mRNA by nonisotopic Northern hybridization. Human pancreatic poly(A)+-RNA was probed simultaneously with 2 cRNAs complementary to exactly the sequences of NBC cDNA that encode the amino acid epitopes recognized by the antisera used in this study. B: detection of NBC protein by immunoblot. Lysates of normal human pancreas (100 µg/lane) were probed with the following: lane A, no primary antibody; lane B, polyclonal anti-(MBP-NBC3) serum preincubated with MBP-NBC3 fusion protein; lane C, anti-(MBP-NBC3) serum preincubated with MBP-NBC5 fusion protein; lane D, anti-(MBP-NBC3) serum without preincubation; lane E, polyclonal anti-(MBP-NBC5) serum preincubated with MBP-NBC5 fusion protein; lane F, anti-(MBP-NBC5) serum preincubated with MBP-NBC3 fusion protein. Both antisera recognize a single ~130-kDa protein. This signal was abolished by preabsorption with the immunogenic MBP-NBC fusion protein (lanes B and E) but not by preabsorption with the other MBP-NBC fusion protein (lanes C and F). All antisera were used at 1:250 dilution.

Immunoblot analysis of normal human pancreas with both anti-(MBP-NBC3) and anti-(MBP-NBC5) antisera demonstrated the presence of a single 130- to 135-kDa immunoreactive protein (Fig. 1B). The signal was broad, consistent with the supposition that NBC is a glycoprotein, based on the identification of three consensus sites for N-linked glycosylation in the human pancreatic NBC protein (1, 11). The apparent molecular mass of the pancreatic NBC protein is higher than the 116-kDa mass predicted from its cDNA (1, 11), consistent with posttranslational modification such as glycosylation.

The specificity of the immunoblot signals for each NBC antiserum was demonstrated by signal loss after preabsorption with the respective MBP-fusion protein immunogen (Fig. 1B). In contrast, preincubation of each antiserum with the opposite MBP-fusion protein did not extinguish the 130- to 135-kDa signal, indicating that the signal was specific for the NBC epitopes and not due to antibodies directed against the MBP component of the fusion proteins.

These experiments confirm the expression of NBC mRNA in the human pancreas and directly demonstrate the presence of the NBC protein.

Localization of NBC Protein in Pancreas

We next examined the cellular and membrane distribution of NBC in human pancreas by indirect immunofluorescence microscopy. The fluorescence signals generated by anti-(MBP-NBC3) serum were NBC epitope specific, as shown by antibody preabsorption experiments (Fig. 2, A and B). We made similar observations with anti-(MBP-NBC5) serum (not shown). Furthermore, these two different NBC antisera produced similar labeling patterns by immunofluorescence microscopy on cryosections of normal human pancreas (Fig. 2, C and D), further supporting the notion that the signal faithfully reflects NBC expression in this tissue.


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Fig. 2.   Immunodetection of the electrogenic NBC in human pancreas: specificity and reproducibility with 2 different antisera. Indirect immunofluorescence microscopy on methanol-fixed, ~5-µm- thick cryosections of normal human pancreas. A: polyclonal anti-(MBP-NBC3) serum, parenchymal labeling pattern. B: anti-(MBP-NBC3) serum preabsorbed with immunogenic MBP-NBC3 fusion protein; lack of immunolabeling demonstrates specificity of labeling seen in A and C. C: anti-(MBP-NBC3) serum, ductular and parenchymal labeling pattern. D: polyclonal anti-(MBP-NBC5) serum, ductular and parenchymal labeling pattern; note the similarity between labeling patterns obtained with anti-(MBP-NBC3) serum and anti-(MBP-NBC5) serum. Bars, 50 µm.

In the large interlobular ducts, which can be identified morphologically, both antisera labeled the periphery of the epithelial cells lining the ducts (Fig. 2, C and D). Higher magnification shows that this labeling is confined to the basolateral membranes, with no detectable NBC immunoreactivity at the apical side (Fig. 3A). The antisera also labeled the small intercalated ducts that drain the acini but not the basolateral membranes of acinar cells (Fig. 3B). We did not detect labeling of pancreatic islet cells (not shown).


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Fig. 3.   Immunolocalization of the electrogenic NBC in human pancreas; subcellular localization and double-labeling with antibodies directed against Na+-K+ pump or cystic fibrosis transmembrane conductance regulator (CFTR). Immunofluorescence labeling as in Fig. 2. A: interlobular duct in cross-section, labeled with anti-(MBP-NBC3) serum. NBC immunoreactivity is found along basolateral membranes (arrowheads), whereas apical membranes do not show any labeling. L, duct lumen. Bar, 10 µm. B: 3 acini of human pancreas drained by small intercalated ducts, labeled with anti-(MBP-NBC3) serum; high magnification micrograph. Brightness enhancement was used to delineate the 3 acinar clusters. Significant expression of NBC is evident in small intercalated ducts but not in basolateral membranes of acinar cells (arrowheads). Bar, 10 µm. C and D: double-labeling of single tissue section with anti-(MBP-NBC3) serum (C) and monoclonal antibody (MAb)-6H against the Na+-K+ pump beta -subunit (D), a marker for basolateral membrane of pancreatic duct cells. Note superimposition of NBC and Na+-K+ pump immunoreactivities. Bars, 20 µm. E and F: double-labeling of single tissue section with anti-(MBP-NBC3) serum (E) and MAb against CFTR (F), a marker for apical membrane of pancreatic duct cells. Same anatomic structures stain positive for NBC and CFTR, but respective subcellular expression patterns differ. Bars, 10 µm.

NBC labeling was also present throughout the parenchyma of the gland (Fig. 3, C and E) and appeared to be even more intense than that of the large interlobular ducts (Fig. 3A). The tubular appearance and branching pattern of some of these labeled structures were suggestive of intercalated and intralobular ducts. Because the pattern of NBC immunoreactivity by itself is not sufficient for the unequivocal identification of these structures, we performed double-label immunofluorescence microscopy experiments with antibodies against established duct-cell marker proteins. One of these antibodies (mouse MAb-6H) is directed against the beta -subunit of the rat Na+-K+ pump, which is highly expressed on the basolateral membrane of pancreatic duct cells (6, 40). As illustrated in Fig. 3, C and D, NBC labeling by anti-(MBP-NBC3) serum and Na+-K+ pump labeling by MAb-6H were completely superimposable. The same result was obtained in double-label experiments with anti-(MBP-NBC5) serum and MAb-6H (not shown). These colocalization experiments confirm that 1) the labeled cells in the parenchyma are of ductular origin and 2) NBC is expressed on the basolateral membrane of these cells, i.e., epithelial cells of the intralobular and intercalated ducts. This basolateral localization parallels our findings from the larger interlobular ducts (Fig. 3A), whose conspicuous morphology allowed assignment of the NBC labeling to the basolateral membrane in single-label experiments.

We also studied NBC localization in the parenchyma with a second duct-cell marker, a CFTR antibody that labels the apical membrane (20, 29). In double-label experiments, the anti-CFTR monoclonal antibody labeled the same structures as anti-(MBP-NBC3) serum (Fig. 3, E and F), further confirming the ductular nature of these structures. The subcellular localization of the labeling within these structures, however, was clearly different for anti-(MBP-NBC3) serum and anti-CFTR. These results confirm the basolateral localization of NBC in human pancreatic duct cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Recently, the cloning of NBCs (1, 8, 11, 36, 37) has made it possible to examine these transporters at the molecular level in human and other tissues. Previous work has shown that poly(A)+ RNA extracted from whole human pancreas contains high levels of NBC mRNA (1, 8). It is unclear, however, whether this NBC is present at the main sites of HCO-3 secretion, the pancreatic ducts, which account for only ~14% of the mass of the human pancreas.

Localization of NBC in Human Pancreas

Pancreatic ducts. We now demonstrate that the human pancreas expresses both NBC mRNA and NBC protein, and show the histological and subcellular localization of the NBC protein in human pancreas. In larger ducts, NBC is present in the basolateral membrane of the duct epithelial cells. Similarly, the strong NBC immunoreactivity within the parenchyma localizes exclusively to basolateral membranes of small and intermediate ducts, as demonstrated by double-label experiments with NBC and either a basolateral marker (i.e., the Na+-K+ pump) or an apical marker (i.e., CFTR). Our observation that NBC and CFTR are present, respectively, at the basolateral and apical membranes of the same duct cells is consistent with the notion that both NBC and CFTR are important elements of the cellular HCO-3-secretory machinery.

Pancreatic acini. We did not detect NBC protein in the acinar cells of the human pancreas. Although this finding does not exclude low NBC expression levels in acinar cells, it does show that NBC expression in duct cells is much higher than in all other cell types in human pancreas. In contrast, an in situ hybridization study on mouse pancreas (1) demonstrated similar levels of NBC mRNA in both duct and acinar cells. In addition, others have observed a transporter with some of the functional characteristics of a Na+-HCO-3 cotransporter in both duct and acinar cells of the rat pancreas (30, 45). These discrepancies may reflect differences in pancreatic biology among species.

Reliability of immunolabeling. Three lines of evidence indicate that our antisera label the NBC protein in human pancreas. First, the two antisera were raised against polypeptide sequences from rat kidney NBC (39) that have 100 and 96% sequence identity with human pancreas NBC (1). Indeed, both antisera recognize heterologously expressed human heart NBC (11), which is 100% identical to the human pancreas NBC (1, 11). Second, the two antisera, which are directed against different domains of the NBC protein, identify a single protein band (130-135 kDa) and generate similar labeling patterns within pancreas tissue. Third, and most importantly, we directly demonstrate the specificity of both antisera in the present study by antibody preabsorption experiments, both for immunoblotting and indirect immunofluorescence.

Physiology of Human Pancreatic NBC

Animal studies have provided compelling evidence for Na+-coupled, stilbene-sensitive uptake of HCO-3 across the basolateral membranes of pancreatic-duct cells (13, 22-24, 44, 45). These data are compatible with any of three HCO-3 transporters: 1) an electrogenic Na+-HCO-3 cotransporter, presumably with a Na+-HCO-3 stoichiometry of 1:2, 2) an electroneutral Na+-HCO-3 cotransporter, and 3) a Na+-driven Cl-/HCO-3 exchanger. In principle, one could distinguish among these transporters either by examining their electrogenicity or Cl- dependence. The electrogenicity of the basolateral HCO-3 transporter in pancreatic ducts has not been explicitly addressed in any species.

Ruling out a Na+-driven Cl-/HCO-3 exchanger in pancreatic duct cells would require demonstrating that Na+-dependent HCO-3 uptake does not require intracellular Cl-. Based on an experiment on guinea pig interlobular ducts, Ishiguro et al. (23) suggested that removal of extracellular Cl- has no effect on the recovery of pHi from intracellular acid loads. It was not possible, however, to compare pHi recovery rates at identical pHi values and it is not clear if the cells were in fact depleted of Cl-. Thus there is no compelling evidence against the involvement of Cl- in the basolateral HCO-3 uptake into pancreatic duct cells.

Abuladze and co-workers (1) have recently identified the cDNA of the human pancreas form of the Na+-HCO-3 cotransporter. Choi and co-workers (11) studied the functional properties of this transporter in the Xenopus oocyte expression system. The cDNA clone used in this study was isolated from human heart (11) but is 100% identical with the human pancreas NBC cDNA (1). The oocyte experiments showed that this NBC isoform is electrogenic, Na+ dependent, HCO-3 dependent, and inhibited by DIDS. They also showed that this transporter is labeled on immunoblots by the same antisera that we used in the present study, anti-(MBP-NBC3) and anti-(MBP-NBC5) serum (11). Conjointly, the molecular identification of the human pancreatic NBC (1), the demonstration that this transporter is electrogenic (11) and detected by our NBC antisera (11), and our immunolocalization data indicate the presence of an electrogenic NBC on the basolateral membrane of human pancreatic duct cells. This is the first evidence supporting the electrogenicity of HCO-3 transport at the basolateral membrane of pancreatic duct cells of any species. It is also the first identification of any acid-base transporter in the human exocrine pancreas.

Model of HCO-3 Secretion by Human Pancreas

In Fig. 4, we present a working model of HCO-3 secretion in human pancreas. This model integrates our finding of an electrogenic NBC in the basolateral membrane of duct cells with the few currently established elements of HCO-3 secretion in human pancreas (i.e., the presence of CFTR and carbonic anhydrase II), as well as with hypothetical mechanisms extrapolated from several animal species (4, 22-24). According to this model, intracellular HCO-3 has two potential sources: 1) hydration of CO2 to carbonic acid, catalyzed by carbonic anhydrase, and subsequent dissociation into H+ and HCO-3, and 2) direct electrogenic uptake of HCO-3 across the basolateral membrane via NBC. In the absence of agonist stimulation, the hydration of CO2 generates sufficient amounts of intracellular HCO-3. This reaction is sustained by basolateral H+ extrusion through Na+/H+ exchangers and/or H+ pumps (34, 44). The large voltage gradient across the basolateral membrane opposes the direct uptake of HCO-3 via the electrogenic NBC. Consequently, NBC may mediate little or no HCO-3 uptake into the duct cell or it may even extrude HCO-3. In the absence of stimulation, the secretion of intracellular HCO-3 across the apical membrane is mainly mediated by a Cl-/HCO-3 exchanger. Na+ as the major cation enters the duct lumen by electrodiffusion via a paracellular pathway.


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Fig. 4.   Cellular model of HCO-3 secretion in human pancreatic duct. Under resting conditions, HCO-3 is generated intracellularly from CO2, a process that is catalyzed by carbonic anhydrase (CA) and sustained by basolateral extrusion of protons via a Na+/H+ exchanger (NHE) and/or an electrogenic H+-pump. When stimulated with secretin, depolarization of the basolateral membrane switches on the direct uptake of HCO-3 together with Na+ via the electrogenic NBC, and elevation of cAMP concentration activates CFTR. Intracellular HCO-3 concentration and luminal Cl- concentration increase, thus promoting HCO-3 secretion via an apical Cl-/HCO-3 exchanger. In addition, an unknown Cl--independent, apical HCO-3 extrusion mechanism like the one described in guinea pig may also be present in human pancreas, and CFTR itself may conduct HCO-3. NKA, Na+-K+ pump.

Upon stimulation of the pancreatic duct cells with secretin, intracellular cAMP concentration rises and activates CFTR in the apical membrane. The increased Cl- conductance leads to depolarization of the basolateral and apical membranes. On the basolateral membrane, depolarization makes inward-directed electrogenic Na+-HCO-3 cotransporter energetically more favorable, and NBC takes up HCO-3 at a high rate. On the apical membrane, the activation of CFTR allows Cl- to recycle back into the lumen and thus stimulate the extrusion of HCO-3 via the Cl-/HCO-3 exchanger. In addition, CFTR may itself function as a HCO-3 channel (18, 21, 28, 32), although this remains controversial (19). It has been suggested that the electrochemical gradients of Cl- and HCO-3 across the apical membrane of stimulated ducts could not explain the high luminal [HCO-3] observed if Cl-/HCO-3 exchangers in parallel with Cl- channels were the only apical HCO-3 extrusion mechanism (24). In fact, an additional, Cl--independent mechanism of apical HCO-3 extrusion has been observed in guinea pig (22, 24) and may be operative in human pancreas too.

In conclusion, the present study demonstrates that NBC is expressed strongly in human pancreas, specifically in the basolateral membranes of pancreatic ducts. Our data suggest that the electrogenic NBC constitutes a major route for basolateral HCO-3 uptake into human pancreatic duct cells. Thus NBC is likely to play an important role in HCO-3 secretion in the human pancreas.


    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30344 (W. F. Boron), a Veterans Affairs Merit Award (C. R. Marino), and a Forschungsstipendium from the Deutsche Forschungsgemeinschaft (to B. M. Schmitt).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. M. Schmitt, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520 (E-mail: bernhard.schmitt{at}yale.edu).

Received 19 March 1999; accepted in final form 23 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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