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
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ABSTRACT |
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The cellular
mechanisms of HCO3 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
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INTRODUCTION |
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ONE OF THE MAJOR physiological functions of the
exocrine pancreas is the secretion of a
HCO3-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 HCO3 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
H2CO3
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
[HCO3] 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
HCO3 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
[HCO3] 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 HCO3 secretion by the human pancreatic
duct cell.
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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 (DH5The 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 -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 ![]() |
RESULTS |
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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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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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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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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 -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.
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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
HCO3 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 HCO3-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+-HCO3 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 HCORuling 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+-HCO3 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 HCO3 Secretion by
Human Pancreas
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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
HCO3 uptake into human pancreatic duct
cells. Thus NBC is likely to play an important role in
HCO
3 secretion in the human pancreas.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abuladze, N.,
I. Lee,
D. Newman,
J. Hwang,
K. Boorer,
A. Pushkin,
and
I. Kurtz.
Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter.
J. Biol. Chem.
273:
17689-17695,
1998
2.
Ammar, E. M.,
D. Hutson,
and
T. Scratcherd.
Absence of a relationship between arterial pH and pancreatic bicarbonate secretion in the isolated perfused cat pancreas.
J. Physiol. (Lond.)
388:
495-504,
1987[Abstract].
3.
Anand, B. S.,
R. Goodgame,
and
D. Y. Graham.
Pancreatic secretion in man: effect of fasting, drugs, pancreatic enzymes, and somatostatin.
Am. J. Gastroenterol.
89:
267-270,
1994[Medline].
4.
Argent, B. E.,
and
R. M. Case.
Pancreatic ducts: cellular mechanism and control of bicarbonate secretion.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1994, p. 1473-1498.
5.
Banks, P. A.,
and
P. T. Sum.
Mode of action of acetazolamide on pancreatic exocrine secretion.
Arch. Surg.
102:
505-508,
1971[Medline].
6.
Bundgaard, M.,
M. Moeller,
and
J. H. Poulsen.
Localization of sodium pump sites in cat pancreas.
J. Physiol. (Lond.)
313:
405-414,
1981[Abstract].
7.
Burgen, A. S. V.,
and
N. G. Emmelin.
Physiology of the Salivary Glands. London: Arnold, 1961.
8.
Burnham, C. E.,
H. Amlal,
Z. Wang,
G. E. Shull,
and
M. Soleimani.
Cloning and functional expression of a human kidney Na+: HCO3 cotransporter.
J. Biol. Chem.
272:
19111-19114,
1997
9.
Case, R. M.,
A. A. Harper,
and
T. Scratcherd.
The secretion of electrolytes and enzymes by the pancreas of the anaesthetized cat.
J. Physiol. (Lond.)
201:
335-348,
1969[Medline].
10.
Case, R. M.,
T. Scratcherd,
and
R. D. Wynne.
The origin and secretion of pancreatic juice bicarbonate.
J. Physiol. (Lond.)
210:
1-15,
1970[Medline].
11.
Choi, I.,
M. F. Romero,
N. Khandoudi,
A. Bril,
and
W. F. Boron.
Cloning and characterization of a human electrogenic Na+-HCO3 cotransporter isoform (hhNBC).
Am. J. Physiol.
276 (Cell Physiol. 45):
C576-C584,
1999
12.
Crawford, I.,
P. C. Maloney,
P. L. Zeitlin,
W. B. Guggino,
S. C. Hyde,
H. Turley,
K. C. Gatter,
A. Harris,
and
C. F. Higgins.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Proc. Natl. Acad. Sci. USA
88:
9262-9266,
1991[Abstract].
14.
Dreiling, D. A.,
H. D. Janowitz,
and
M. Halpern.
The effect of a carbonic anhydrase inhibitor, Diamox, on human pancreatic secretion. Implications on the mechanism of pancreatic secretion.
Gastroenterology Suppl.
54:
765-767,
1968.
15.
Dyck, W. P.,
N. C. Hightower,
and
H. D. Janowitz.
Effect of acetazolamide on human pancreatic secretion.
Gastroenterology
62:
547-552,
1972[Medline].
16.
Gray, M. A.,
J. R. Greenwell,
and
B. E. Argent.
Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.
J. Membr. Biol.
105:
131-142,
1988[Medline].
17.
Gray, M. A.,
A. Harris,
L. Coleman,
J. R. Greenwell,
and
B. E. Argent.
Two types of chloride channel on duct cells cultured from human fetal pancreas.
Am. J. Physiol.
257 (Cell Physiol. 26):
C240-C251,
1989
18.
Gray, M. A.,
S. Plant,
and
B. E. Argent.
cAMP-regulated whole cell chloride currents in pancreatic duct cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C591-C602,
1993
19.
Haws, C. M.,
and
P. M. Quinton.
No measurable bicarbonate conductance through CFTR in Calu-3 cells (Abstract).
Pediatr. Pulmonol.
15:
A108,
1998.
20.
Hyde, K.,
C. J. Reid,
S. J. Tebbutt,
L. Weide,
M. A. Hollingsworth,
and
A. Harris.
The cystic fibrosis transmembrane conductance regulator as a marker of human pancreatic duct development.
Gastroenterology
113:
914-919,
1997[Medline].
21.
Illek, B.,
J. R. Yankaskas,
and
T. E. Machen.
cAMP and genistein stimulate HCO3 conductance through CFTR in human airway epithelia.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L752-L761,
1997
22.
Ishiguro, H.,
S. Naruse,
M. C. Steward,
M. Kitagawa,
S. B. Ko,
T. Hayakawa,
and
R. M. Case.
Fluid secretion in interlobular ducts isolated from guinea-pig pancreas.
J. Physiol. (Lond.)
511,pt.2:
407-422,
1998
23.
Ishiguro, H.,
M. C. Steward,
A. R. Lindsay,
and
R. M. Case.
Accumulation of intracellular HCO3 by Na+-HCO
3 cotransport in interlobular ducts from guinea-pig pancreas.
J. Physiol. (Lond.)
495,pt.1:
169-178,
1996[Abstract].
24.
Ishiguro, H.,
M. C. Steward,
R. W. Wilson,
and
R. M. Case.
Bicarbonate secretion in interlobular ducts from guinea-pig pancreas.
J. Physiol. (Lond.)
495,pt1:
179-191,
1996[Abstract].
25.
Kuijpers, G. A.,
I. G. Van Nooy,
J. J. de Pont,
and
S. L. Bonting.
The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors.
Biochim. Biophys. Acta
778:
324-331,
1984[Medline].
26.
Kumpulainen, T.,
and
P. Jalovaara.
Immunohistochemical localization of carbonic anhydrase isoenzymes in the human pancreas.
Gastroenterology
80:
796-799,
1981[Medline].
27.
Lightwood, R.,
and
H. A. Reber.
Micropuncture study of pancreatic secretion in the cat.
Gastroenterology
72:
61-66,
1977[Medline].
28.
Linsdell, P.,
J. A. Tabcharani,
J. M. Rommens,
Y. X. Hou,
X. B. Chang,
L. C. Tsui,
J. R. Riordan,
and
J. W. Hanrahan.
Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions.
J. Gen. Physiol.
110:
355-364,
1997
29.
Marino, C. R.,
L. M. Matovcik,
F. S. Gorelick,
and
J. A. Cohn.
Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.
J. Clin. Invest.
88:
712-716,
1991[Medline].
30.
Muallem, S.,
and
P. A. Loessberg.
Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO3 transporters.
J. Biol. Chem.
265:
12806-12812,
1990
30a.
Ondarza, J., de,
and
S. R. Hootman.
Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G124-G134,
1997
31.
Pak, B. H.,
S. S. Hong,
H. K. Pak,
and
S. K. Hong.
Effects of acetazolamide and acid-base changes on biliary and pancreatic secretion.
Am. J. Physiol.
210:
624-628,
1966[Medline].
32.
Poulsen, J. H.,
H. Fischer,
B. Illek,
and
T. E. Machen.
Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
Proc. Natl. Acad. Sci. USA
91:
5340-5344,
1994[Abstract].
33.
Raeder, M.,
and
O. Mathisen.
Abolished relationship between pancreatic HCO3 secretion and arterial pH during carbonic anhydrase inhibition.
Acta Physiol. Scand.
114:
97-102,
1982[Medline].
34.
Raeder, M. G.
The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion.
Gastroenterology
103:
1674-1684,
1992[Medline].
35.
Rawls, J. A.,
P. J. Wistrand,
and
T. H. Maren.
Effects of acid/base changes on pancreatic secretion.
Am. J. Physiol.
205:
651-657,
1963.
36.
Romero, M. F.,
P. Fong,
U. V. Berger,
M. A. Hediger,
and
W. F. Boron.
Cloning and functional expression of rNBC, an electrogenic Na+-HCO3 cotransporter from rat kidney.
Am. J. Physiol.
274 (Renal Physiol. 43):
F425-F432,
1998
37.
Romero, M. F.,
M. A. Hediger,
E. L. Boulpaep,
and
W. F. Boron.
Expression cloning and characterization of a renal electrogenic Na+/HCO3 cotransporter.
Nature
387:
409-413,
1997[Medline].
38.
Rosewicz, S.,
E. O. Riecken,
and
U. Stier.
Transcriptional regulation of carbonic anhydrase II by retinoic acid in the human pancreatic tumor cell line DANG.
FEBS Lett.
368:
45-48,
1995[Medline].
39.
Schmitt, B. M.,
D. Biemesderfer,
M. F. Romero,
E. L. Boulpaep,
and
W. F. Boron.
Immunolocalization of the electrogenic Na+-HCO3 cotransporter in mammalian and amphibian kidney.
Am. J. Physiol.
276 (Renal Physiol. 45):
F27-F38,
1999
40.
Smith, Z. D.,
M. J. Caplan,
B. Forbush,
and
J. D. Jamieson.
Monoclonal antibody localization of Na+-K+-ATPase in the exocrine pancreas and parotid of the dog.
Am. J. Physiol.
253 (Gastrointest. Liver Physiol. 16):
G99-G109,
1987
41.
Sohma, Y.,
M. A. Gray,
Y. Imai,
and
B. E. Argent.
A mathematical model of the pancreatic ductal epithelium.
J. Membr. Biol.
154:
53-67,
1996[Medline].
42.
Swanson, C. H.,
and
A. K. Solomon.
Micropuncture analysis of the cellular mechanisms of electrolyte secretion by the in vitro rabbit pancreas.
J. Gen. Physiol.
65:
22-45,
1975[Abstract].
43.
Trezise, A. E.,
and
M. Buchwald.
In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.
Nature
353:
434-437,
1991[Medline].
44.
Villanger, O.,
T. Veel,
and
M. G. Raeder.
Secretin causes H+/HCO3 secretion from pig pancreatic ductules by vacuolar-type H+-adenosine triphosphatase.
Gastroenterology
108:
850-859,
1995[Medline].
45.
Zhao, H.,
R. A. Star,
and
S. Muallem.
Membrane localization of H+ and HCO3 transporters in the rat pancreatic duct.
J. Gen. Physiol.
104:
57-85,
1994[Abstract].