Divisions of 1 Nephrology and Hypertension and 3 Digestive Diseases, Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0585; and 2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Pancreatic dysfunction in patients with cystic fibrosis (CF) is
felt to result primarily from impairment of ductal
HCO3 secretion. We provide molecular
evidence for the expression of NBC-1, an electrogenic
Na+-HCO
3
cotransporter (NBC) in cultured human pancreatic duct
cells exhibiting physiological features prototypical of CF duct
fragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cells
and CFPAC-1 cells transfected with wild-type CF transmembrane
conductance regulator (CFTR)]. We further demonstrate that
1)
HCO
3 uptake across the basolateral
membranes of pancreatic duct cells is mediated via NBC and
2) cAMP potentiates NBC activity
through activation of CFTR-mediated
Cl
secretion. We propose
that the defect in agonist-stimulated ductal HCO
3 secretion in patients with CF is
predominantly due to decreased NBC-driven
HCO
3 entry at the basolateral
membrane, secondary to the lack of sufficient electrogenic driving
force in the absence of functional CFTR.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; electrogenic sodium-bicarbonate cotransporter; pancreatic dysfunction
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) remains a major healthcare problem
worldwide (29). An autosomal recessive disease, CF results from mutational inactivation of a cAMP-sensitive
Cl channel [CF
transmembrane conductance regulator (CFTR)] with resultant
impairments in the respiratory, pancreatic, hepatobiliary, and
genitourinary systems (29). With regard to the pancreas, pancreatic
dysfunction is felt to result primarily from impairment of
secretin-stimulated ductal
Cl
and
HCO
3 secretion (17, 22,
23). Experimental and histopathological evidence suggests
that the reduction in secretin-stimulated
HCO
3 secretion from pancreatic duct
epithelial cells alters intraductal pH sufficiently to precipitate proteins secreted from acinar cells, resulting in protein plugs, and to
disrupt vesicular trafficking in the apical domain of the acinar cell
(16, 31). Together, these alterations lead to pancreatic fibrosis and
insufficiency in 80% of cases (31).
Although gene therapy has provided the scientific and medical
communities with an appealing potential therapeutic option in the
treatment of the CF defect, important obstacles have yet to be overcome
(2, 21, 42). An alternative hope is that a better understanding of the
physiological role(s) of CFTR in various organs will lead to novel
strategies capable of correcting the functional defect in the absence
of functional CFTR. The currently accepted model of pancreatic ductal
HCO3 secretion suggests that
1) intracellular
HCO
3 accumulates due to basolateral
diffusion of CO2 and subsequent
action of carbonic anhydrase, 2) G
protein-coupled receptors (e.g., secretin and vasoactive intestinal
peptide) activate cAMP-sensitive CFTR, and 3) resultant increases in luminal
Cl
drive a
Cl
/HCO
3
exchanger (reviewed in Refs. 11, 18, 34). On the basis of the belief
that HCO
3 accumulates intracellularly
in a passive, essentially unregulated fashion, recent physiological
studies have focused on alternate channels capable of conducting
Cl
into the pancreatic duct
lumen (43, 44).
However, a recent report indicated that
HCO3 uptake at the basolateral
membrane of pancreatic duct cells in guinea pig is
Na+ dependent (19). We have
recently reported the molecular cloning, cellular distribution, and
functional expression of NBC-1, an electrogenically driven
Na+-HCO
3
cotransporter (NBC) found in human renal epithelial cells (9). Northern
blot hybridization utilizing an NBC-1 cDNA probe detected a similarly
sized transcript in normal human kidney and pancreas (9). Taken
together, these findings raise the possibility that either NBC-1 or a
highly homologous electrogenically driven
Na+-nHCO
3
cotransporter may mediate HCO
3 uptake
in human pancreatic duct cells (where
n represents an unknown number
2).
If this is in fact the case, an important testable hypothesis is that
cAMP-mediated activation of CFTR creates an electrogenic potential that
drives HCO
3 entry into pancreatic duct
epithelium via a
Na+-nHCO
3 cotransporter.
In an attempt to identify a model system(s) that would allow us to gain
initial insights into the functional role of
Na+-HCO3
cotransport in both normal and CF pancreatic duct epithelium, duct
cells prototypical of normal or CF duct fragments (CAPAN-1 or CFPAC-1
cells, respectively) were utilized. Interestingly, CAPAN-1 cells,
derived from a well-differentiated human ductal pancreatic
adenocarcinoma with ductal morphology, represent the only human
tumor-derived cell line known to express the cAMP-sensitive CFTR in a
confluence-dependent manner and exhibit other functional
characteristics prototypical of normal pancreatic ducts (7, 12, 24).
CFPAC-1 cells were derived from a well-differentiated human ductal
pancreatic cancer resected from a patient with CF due to the F508
mutation in CFTR (32). These cells have been extensively studied and
documented to have normal pancreatic ductal physiology, excepting the
lack of cAMP-sensitive Cl
secretion (15, 32). Furthermore, a recent report from our group
indicated the development of stably transfected CFPAC-1 cell lines
bearing functional wild-type CFTR (in this paper referred to as
CFPAC-WT), and confirmed the recovery of cAMP-sensitive Cl
secretion in those cells
(15).
In this work, we report the results of studies assessing
1) the molecular identity and
functional expression of this pancreatic Na+-nHCO3
cotransporter in CAPAN-1, CFPAC-1, and CFPAC-WT cells,
2) the effect of membrane
depolarization on Na+-dependent,
HCO
3 cotransport following acid loading of these cells, and 3) the
ability of cAMP to influence intracellular pH
(pHi) recovery in the same cells
either bearing or lacking functional CFTR. Our findings suggest that
HCO
3 uptake in these prototypical
pancreatic duct cell models is electrogenically regulated by
cAMP-sensitive CFTR. These findings have important ramifications
regarding our understanding of the physiological role of CFTR in these
cells and provide important novel insights into the pancreatic
pathophysiology of CF.
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MATERIALS AND METHODS |
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Cell lines. CAPAN-1 and CFPAC-1 cells were obtained from the American Type Culture Collection and cultured as previously described by our group (15, 32). Stably transfected CFPAC-1 cells bearing functional CFTR (CFPAC-WT) were cultured in a similar fashion, excepting the addition of G418 (1 mg/ml) to the medium (15).
RNA isolation and Northern blot hybridization.
Total cellular RNA was extracted from CAPAN-1 and CFPAC-1 cells
according to the established methods (13), quantitated
spectrophotometrically, and stored at 80°C. Total RNA
samples (30 µg/lane) were fractionated on a 1.2%
agarose-formaldehyde gel, transferred to Magna NT nylon membranes,
cross-linked by ultraviolet light, and baked. Hybridization was
performed according to Church and Gilbert (14). The membranes were
washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). A
32P-labeled cDNA
fragment corresponding to nucleotides 509-3237 of the mRNA
encoding human NBC-1 was used as a specific probe.
Functional localization of
Na+-HCO3
cotransport.
CFPACWT cells were grown in collagen-coated 30-mm Millicell HA
culture dish inserts (porosity 0.45 µm) for 10 days and
assayed for the presence of
HCO
3-dependent
22Na+
uptake from the luminal or basolateral surface. The integrity of
confluent cell monolayers was assessed by a lack of significant transport of
22Na+
from the upper compartment to the lower compartment under control conditions (>1% of the
22Na+
appeared in the lower compartment after 5 min, which is a good indicator of the integrity of the monolayer grown on a filter). Acid
loading was accomplished by NH4Cl
pulse at each side of the filter (38). The
NH4Cl-containing solution
consisted of (in mM) 75 N-methyl-D-glucamine
(NMDG) chloride, 25 KHCO3, and 40 NH4Cl. The uptake medium consisted
of (in mM) 10 22NaCl, 105 NMDG
chloride, and 25 KHCO3; 0.5 mM
ouabain was added to the basolateral uptake medium (to inhibit
Na+-K+-ATPase),
and 2 mM amiloride was added to the apical or basolateral uptake medium
(to inhibit
Na+/H+
exchange). A Na+-free solution
(Na+ was replaced by equimolar
NMDG as chloride salt) that in addition contained 2 mM amiloride was
used to bathe the side opposite the uptake medium. All solutions
contained (in mM) 4 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH
7.4).
Cell pH measurement.
Changes in pHi were monitored
using the pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM as described (3, 38, 39). Cells were grown to confluence on
glass coverslips and incubated in the presence of 5 µM BCECF in a
solution consisting of (in mM) 115 tetramethylammonium (TMA) chloride,
25 KHCO3, and 10 HEPES (pH 7.4)
and gassed with 5% CO2-95% O2.1
The monolayer was then perfused with the appropriate solutions in a
thermostatically controlled holding chamber (37°C) in a Delta Scan
dual-excitation spectrofluorometer (PTI, South Brunswick, NJ). The
fluorescence ratio at excitation wavelengths of 500 and 450 nm was
utilized to determine pHi values.
Calibration curves were established daily by the KCl-nigericin
technique. HCO3-free or
HCO
3-containing solutions were used to
determine the HCO
3 dependence of the
transporter. The Na+ and
HCO
3 dependence of the transporter was examined by monitoring pHi
recovery (dpHi/dt, in pH unit/min) from an acid load following
NH3/NH+4
withdrawal, as employed for Refs. 4 and 9. The
NH+4-containing solution consisted of (in mM)
75 TMA (or NMDG) chloride, 40 NH4Cl, and 25 KHCO3. The
Na+-containing solution consisted
of 115 mM NaCl and 25 mM KHCO3. For voltage-clamp studies of cAMP effect, the
Na+-containing solution consisted
of (in mM) 45 NaCl, 25 NaHCO3, and
either 70 TMA chloride or 70 KCl. All solutions contained (in mM) 0.8 K2HPO4,
0.2 KH2PO4,
1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4).
Statistical analyses. Values are expressed as means ± SE. The significance of differences between mean values was examined using ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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Molecular expression of NBC-1 in pancreatic duct cells. On the basis of the high level of expression of NBC-1 mRNA in whole normal human pancreas (9), we first examined whether this transporter is expressed in cultured human duct cells, CAPAN-1 and CFPAC-1 cells. Northern hybridizations utilizing a 32P-labeled probe corresponding to nucleotides 509-3237 of human NBC-1 identified an 8.1-kb transcript in both CAPAN-1 cells and CFPAC-1 cells (Fig. 1). The expression of this pancreatic NBC was compared with rat kidney cotransporter (Fig. 1). NBC-1 mRNA levels in the duct cells (Fig. 1) were lower than in the whole pancreas (9).
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Functional expression of NBC in pancreatic duct cells.
Cells were grown to confluence on glass coverslips and incubated with
BCECF. In the presence of 1 mM amiloride (an inhibitor of
Na+/H+
exchange), switching to a
Na+-containing solution resulted
in rapid pHi recovery from
acidosis in the presence of HCO3 (Fig.
2, A and
B). The recovery from cell acidosis
was inhibited in both cell lines by 200 µM DIDS (Fig. 2). The results
of four separate experiments showed DIDS-sensitive,
Na+-dependent
HCO
3-mediated
pHi recovery rates of 0.11 ± 0.03 and 0.16 ± 0.03 pH unit/min in CAPAN-1 and CFPAC-1 cells,
respectively (Fig. 2, C and
D). NBC activity in CFPAC-WT cells
showed rates very similar to CFPAC-1 cells (0.15 ± 0.03 pH
unit/min, n = 4). In the absence of
HCO
3 but in the presence of
Na+ and amiloride, no
pHi recovery from acidosis was
detected, confirming the HCO
3
dependence of pHi recovery in Fig. 2. In the absence of Na+ and
HCO
3, there was no
pHi recovery from acidosis, indicating functional absence of
H+-ATPase in both cell lines (Fig.
2, E and
F).
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Basolateral localization of
Na+-HCO3
cotransport in pancreatic duct cells.
To examine the localization of the NBC in pancreatic duct cells,
CFPAC-WT were grown to confluence in 30-mm Millicell HA culture dish
inserts (porosity 0.45 µm) for 8-10 days and
assayed for the presence of acid-stimulated,
HCO
3-dependent 22Na+
uptake from the upper compartment (luminal surface) or lower compartment (basolateral surface) (see Functional
localization of
Na+-HCO
3
cotransport). As illustrated in Fig.
3A,
HCO
3-dependent, acid-stimulated 22Na+
influx was observed predominantly at the basolateral surface (there is
a minimal degree of
22Na+
influx at the luminal surface that could be due to overgrowth of cells
on the filters, which can disturb complete polarization of the cells).
Taken together, these experiments indicate that the electrogenic NBC
mediates HCO
3 influx into the duct
cells across the basolateral membrane.
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Electrogenicity of
Na+-HCO3
cotransport in pancreatic duct cells.
The kidney NBC is highly electrogenic and transports three
HCO
3 for each
Na+ (35, 37, 45), indicating that
it carries a net negative charge. As such, this transporter is very
sensitive to alterations in the membrane potential (6, 8, 28, 36).
Indeed, in the kidney, the
Na+-3HCO
3
cotransporter that is located on the basolateral membrane of the
proximal tubule and carries HCO
3 from
the cell to the blood is only driven by inside-negative membrane potential (6, 8, 28, 36). To determine whether alterations in the
membrane potential can affect the rate of
Na+-HCO
3
cotransport in pancreatic duct cells, CFPAC-1 cells were acid loaded by
the NH+4 pulse method and switched to a
Na+-containing solution (70 mM
NaCl) that had either low (1.8 mM) or high (72 mM)
K+. As indicated in Fig.
3B, in the presence of valinomycin
(which increases permeability of the membrane to
K+ and causes the flow of
K+ down its gradient), the rate of
Na+-dependent
HCO
3 influx decreased significantly in
low-K+ solution (summary of 4 separate experiments showed that NBC activity decreased by 54% in
low-K+ solution vs.
high-K+ solution,
P < 0.02, n = 4). Taken together, these results
indicate that changing the membrane potential regulates
Na+-HCO
3
cotransport activity.
Effects of cAMP on
Na+-HCO3
cotransport in pancreatic duct cells.
It has been shown that stimulation of CFTR by secretin, which works via
cAMP, increases HCO
3 secretion (5, 11,
34). In view of the fact that
Na+-HCO
3
cotransport is highly electrogenic and is regulated by membrane
potential (Fig. 3B), we entertained
the possibility that stimulation of ductal
HCO
3 secretion by CFTR activation
could be due to enhanced HCO
3 entry at
the basolateral membrane via stimulation of the NBC (resulting from
depolarization of the membrane potential secondary to
Cl
secretion by CFTR). We
therefore examined the effect of 8-bromoadenosine 3'5'-cyclic monophosphate (8-BrcAMP) on
Na+-HCO
3
cotransport in cells with functional or mutant CFTR. Cells were exposed
to cAMP during the NH+4 withdrawal step. As
indicated, addition of 500 µM 8-BrcAMP significantly enhanced the NBC
activity in CAPAN-1 cells, increasing it by ~2.8-fold (Fig.
4, A and
C). The rate of
pHi recovery increased from 0.10 ± 0.02 pH unit/min in the absence of cAMP to 0.28 ± 0.04 pH
unit/min in the presence of cAMP (P < 0.001, n = 4). These experiments were performed in the presence of an outward
Cl
gradient (extracellular
Cl
was replaced with
gluconate). However, potentiation of
Na+-HCO
3
cotransport activity by cAMP was significantly attenuated when external
Cl
was present (apparently
due to the lack of a favorable chemical gradient for the outward
movement of Cl
), strongly
suggesting that the stimulatory effect of cAMP on the NBC is indirect
and is mediated via activation of CFTR (data not shown).
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Potentiation of
Na+-HCO3
cotransport by cAMP is only observed in duct cells expressing
functional CFTR.
To determine whether the presence of a functional CFTR is indeed
necessary for enhancement of the NBC by cAMP (Fig. 4), CFPAC-1 cells
that express the defective CFTR were examined. As shown in Fig. 4,
B and
D, in the presence of an outward
Cl
gradient, addition of
cAMP had no significant effect on the NBC activity. To determine
whether stimulation of
Na+-HCO
3
cotransport by cAMP is indeed due to functional CFTR and is not a
cell-specific property, CFPAC-1 cells stably transfected with wild-type
CFTR (CFPAC-WT) were examined. Addition of cAMP resulted in an
approximately twofold increase in the rate of
Na+-HCO
3
cotransport activity in CFPAC-WT cells (Fig.
5, A
and C). The rate of
pHi recovery increased from 0.15 ± 0.02 pH unit/min in the absence of cAMP to 0.29 ± 0.04 pH
unit/min in the presence of cAMP (P < 0.005, n = 4). The stimulatory
effect of cAMP on
Na+-HCO
3
cotransport was prevented when extracellular Cl
was present (data not
shown).
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Potentiation of
Na+-HCO3
cotransport by cAMP in pancreatic duct cells is via membrane
depolarization.
The above results indicate that cAMP stimulates the NBC in cells
expressing only functional CFTR. They further indicate that stimulation
of the NBC by cAMP is due to activation of CFTR. Activation of CFTR by
cAMP in vitro or in vivo leads to
Cl
secretion and subsequent
cell membrane depolarization (reviewed in Refs. 11, 18, 34). To
determine whether alteration in cell membrane potential was responsible
for stimulation of
Na+-HCO
3
cotransport, Na+-dependent
HCO
3 transport in CFPAC-WT cells was
examined in response to cAMP under voltage-clamped conditions. Cells
were acidified and exposed to cAMP in the presence of the K+ ionophore valinomycin and high
external K+ to clamp the membrane
potential (35, 37). As shown in Fig. 5,
B and
D, the stimulatory effect of cAMP on
the NBC was blocked by voltage clamping in
high-K+ solution.
CFTR does not carry HCO3 in
pancreatic duct cells.
It has been shown that stimulation of CFTR by secretin, which works via
cAMP, increases HCO
3 secretion
(5, 11). One possibility with respect to the underlying
stimulatory mechanism of ductal HCO
3
secretion by cAMP was that CFTR had the capability to carry
HCO
3, analogous to findings in
tracheal epithelial cells (27, 33). To determine whether CFTR mediates
HCO
3 efflux, duct cells were loaded
with HCO
3 and monitored for
pHi in the presence of
HCO
3 but the absence of
Na+ in the media (to keep the NBC
inactive). As indicated in Fig. 5E,
addition of cAMP had no significant effect on
pHi in the presence of an outward
Cl
gradient (0 mM external
Cl
). Similarly, cAMP had
no effect on pHi in the absence of
an outward Cl
gradient.
These results indicate that CFTR has minimal affinity for
HCO
3 in the pancreatic duct cells.
RT-PCR analysis of pancreatic NBC-1 cDNA. To determine whether the pancreas NBC-1 expresses the protein kinase A (PKA) binding site, a region around the PKA phosphorylation consensus site of the kidney NBC-1 was amplified using RT-PCR, cloned, and sequenced. Accordingly, total RNA from human pancreas was subjected to reverse transcription using an oligo(dT) primer. After the reverse transcription and digestion with RNase H, the pancreatic cDNA was purified on a Microcon-30 ultrafilter (Amicon) to remove the oligo(dT) primers and digested RNA. PCR was then performed using the primers 5'-CAA AGA GTC ACT GGA ACC CTT G and 5'-AAG AGA GAA GCA GAG AGA GCG. Analysis by gel electrophoresis revealed a single band, indicating a single amplification product. The band was excised from the agarose gel, purified, and ligated into a pGEM-T plasmid (Promega). After transformation by electroporation and antibiotic selection, two independent clones were selected. The two clones were grown up, and plasmids were isolated from each for sequencing. Because of the central location of the PKA consensus site within each of the cDNA inserts, it was possible to get high-quality sequence of this region by sequencing from each end of the two plasmids. Thus the sequence of the PKA consensus phosphorylation site was determined by sequencing in both directions from two independent clones. Because bidirectional sequencing was not performed on the flanking regions, some sequence ambiguities remain in those areas. However, all four overlapping sequences showed an intact PKA consensus sequence.
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DISCUSSION |
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Cultured pancreatic duct cells (CAPAN-1 and CFPAC-1) express
the NBC-1 mRNA (Fig. 1) and NBC activity (Fig. 2). The
NBC is located on the basolateral membrane domain and dictates
HCO3 uptakes in duct cells (Fig.
3A). This transporter is highly
electrogenic and is regulated by alterations in membrane potential
(Fig. 3B). cAMP potentiates
HCO
3 uptake via NBC only in duct cells
expressing functional CFTR (Figs. 4 and
5A), by depolarizing the cell
membrane (Fig. 5B).
According to the currently accepted model of
HCO3 secretion in the pancreatic duct
cells, basolateral HCO
3 transport is
via diffusion of CO2 and
subsequent action of cytosolic carbonic anhydrase (5, 11, 34). The
resultant carbonic acid dissociates into
H+ and
HCO
3, with
H+ transported back to the blood
via a basolateral H+-ATPase
and/or a
Na+/H+
exchanger (5, 11, 34). Our data indicate that intracellular accumulation of HCO
3 occurs as a
result of direct transport of HCO
3 via
a basolateral electrogenic NBC rather than by diffusion of
CO2. This is consistent with the expression of NBC-1 in the pancreatic duct cells (Fig. 1) and is in
agreement with recent reports indicating functional expression of NBC
in basolateral membranes of pancreatic duct cells in guinea pig and rat
(19).
NBC-1 is expressed in both renal proximal tubule cells (10) and
pancreatic duct cells (Figs. 1-4). The pancreatic NBC-1 is apparently a spliced variant of kidney NBC-1 (1). In the kidney proximal tubule cells, the
Na+-3HCO3
cotransporter mediates the transport of
Na+ and
HCO
3 from cell to blood against
chemical gradients for both ions, as cellular concentrations of
Na+ and
HCO
3 are less than those of blood (6, 8, 28, 36). As such, the only driving force for outward movement of
Na+-3HCO
3
cotransport is the favorable membrane potential (inside-negative
potential). Indeed, with basolateral membrane potentials at
65
to
70 mV in the kidney proximal tubule cells (8, 28, 36, 45),
the stoichiometry of three HCO
3 per
Na+ allows for the efflux of the
Na+-3HCO
3
cotransporter from cell to blood against chemical gradients for both
Na+ and
HCO
3. Membrane potentials of
60
mV or lower (i.e.,
50 or
40 mV) will not be able to
overcome the uphill chemical gradients for
Na+ and
HCO
3 (45). In the pancreatic duct
cells, basolateral membrane potentials of
35 mV or lower (which
has been shown to be the case in the stimulated state) (26) guarantee that the direction of the NBC is from the blood to the cell. In other
words, this transporter mediates the influx of
HCO
3 to duct cells, a phenomenon
completely opposite to that in the kidney proximal tubule cells. This
change in the direction of the transport results from the activity of
the apical CFTR that is absent in the kidney proximal tubule cells.
We have examined the effect of cAMP on NBC-1 in kidney HEK-293 cells
(transiently transfected with the NBC-1 cDNA) and found that its
activity is decreased in response to cAMP
(dpHi/dt decreased from 0.24 to
0.16 pH unit/min in response to cAMP,
P < 0.04, n = 4 for each group). Inhibition of
Na+-3HCO3
cotransport in the kidney by cAMP is consistent with published reports
on regulation of this transporter in kidney proximal tubule cells (30).
Kidney NBC-1 has only a single PKA phosphorylation site
(at the 3' end of the coding region at amino acid 979), which
likely mediates the inhibitory effect of cAMP (9). One possibility with
respect to the opposite effects of cAMP on
Na+-3HCO
3
cotransport activity in the kidney and pancreas (inhibition vs.
stimulation, respectively) was that pancreatic NBC lacked the PKA
binding site (resulting from either deletion or alternate splicing),
leaving the stimulatory effect of membrane depolarization unopposed.
Sequence analysis of the 3' end of the pancreatic NBC-1
cDNA, however, showed an intact PKA consensus sequence in pancreatic NBC-1 (Fig.
6).2
The stimulatory effect of cAMP on the pancreas cotransporter strongly
suggests that the inhibitory phosphorylation effect would have to be
superseded by the depolarizing effect of CFTR activation. Alternatively, it is plausible that the inhibitory effect of PKA in the
kidney proximal tubule cells is mediated via an intermediary protein
(41), and that protein could be absent in the pancreatic duct cells,
leaving the stimulatory effect of membrane depolarization unopposed.
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Stimulation of NBC by cAMP (which is also observed at baseline
pHi, data not shown) in the
pancreatic duct cells expressing functional CFTR is intriguing and
highlights the coordinated regulation of apical and basolateral
HCO3 transporters in the duct cells.
In the currently accepted model of
HCO
3 secretion, the defect in
HCO
3 secretion presumably lies at the
apical membrane of the duct cells, where decreased secretion of
Cl
via CFTR inhibits the
apical
Cl
/HCO
3
exchange. It is worth mentioning that, although an apical
Cl
/HCO
3
exchanger is expressed in the duct cells (20, 46), its role in
mediating secretin-stimulated HCO
3 secretion remains controversial (20). Indeed, a recent report demonstrated that removal of luminal
Cl
or addition of DIDS only
partially inhibited secretin-stimulated HCO
3 secretion (<25%) in the guinea
pig pancreatic duct cells (20), strongly suggesting that the apical
Cl
/HCO
3
exchanger does not play a major role in secretin-stimulated
HCO
3 secretion. In addition, CFTR also
did not show any significant affinity for
HCO
3 in the duct cells (Fig.
7), which would have otherwise explained the ductal HCO
3 secretion defect in
CF. Taken together, these results suggest that the ductal
HCO
3 secretion defect in CF has an
alternative molecular basis than the currently accepted one.
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Our results indicating lack of stimulation of NBC in cells expressing
the mutant CFTR are consistent with a decreased basolateral HCO3 transport into the duct cells in
patients with CF. Although we acknowledge the need for confirmatory
studies in primary cultures of both normal and CF human pancreatic duct cells, our findings in these prototypical cells suggest that the limiting step in ductal HCO
3 secretion
in CF patients lies at the basolateral membrane of these cells, where decreased electrogenic driving force (resulting from lack of
depolarization of the membrane) prevents the entry of
HCO
3 via the NBC. Accordingly, we
propose the following HCO
3 transport
model for the pancreatic duct cells (Fig. 7). According to this model,
secretin increases intracellular cAMP, which then results in the
activation of CFTR and secretion of
Cl
, leading to
depolarization of both luminal and basolateral membranes. The
depolarization of the basolateral membrane increases the driving force
for NBC and, as a result, enhances entry into the duct cells of
HCO
3, which is then secreted at the
apical membrane (mostly via a transporter that is poorly defined at the present and partially via an apical
Cl
/HCO
3 exchanger).
Because the identity of the apical transporter mediating the majority
of the ductal HCO3 secretion remains poorly understood (20), and because all acid-base transporters seem to
be expressed in both normal and CF cells, it is logical to conclude
that an alternative strategy to gene therapy for correcting the
HCO
3 secretion defect in CF patients
could be via activation of NBC (either directly or indirectly).
According to this hypothesis, stimulation of NBC either directly (i.e., via protein kinase C analogs or angiotensin II) or indirectly (i.e.,
via cell membrane depolarization) should increase entry into the duct
cells of HCO
3, which can be secreted into the lumen via the apical HCO
3
transporter(s). Depolarizing agents such as basolateral
K+ channel blockers are excellent
candidates to indirectly stimulate NBC.
In summary, a basolateral electrogenic NBC dictates
HCO3 uptake in the pancreatic duct
cells. This cotransporter is stimulated by cAMP only in cells
expressing functional CFTR. The stimulation of the NBC by cAMP is
mediated via membrane depolarization. We propose that the defect in
ductal HCO
3 secretion in patients with
CF is predominantly due to decreased
HCO
3 entry at the basolateral membrane
as a result of decreased electrogenic driving force and subsequent
inhibition of NBC. Future studies should test the possibility that
stimulating the NBC (either directly or indirectly) can increase
HCO
3 entry into the duct cells and
therefore alleviate pancreatic HCO
3 secretion defect in patients with CF.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent contributions of Elizabeth Kopras, Charles Burnham, and Zhaohui Wang.
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FOOTNOTES |
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These studies were supported by National Institutes of Health Grants DK-46789, DK-52821, and DK-54430 (to M. Soleimani) and CA-74456 (to C. D. Ulrich) and a grant from Dialysis Clinic Inc. (to M. Soleimani).
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.
1
To use HCO3 in
Na+-free solutions for
NH+4 loading and withdrawal steps, we
initially attempted to avoid choline bicarbonate, as choline has been
shown to have nonspecific effects on
pHi. We therefore used
KHCO3. However, subsequent studies
indicated similar experimental results when either 25 mM choline
bicarbonate (and more physiological concentrations of
K+) or 25 mM
KHCO3 was used.
2
Pancreatic NBC-1 has two consensus PKA binding
sites (1). However, the additional phosphorylation site does not play
an important role in mediating the stimulatory effect of cAMP on NBC-1,
as addition of cAMP had no effect on
Na+-dependent
HCO3 cotransport in cells expressing the mutant CFTR.
Address for reprint requests: M. Soleimani, Division of Nephrology and Hypertension, University of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, Ohio 45267-0585.
Received 11 August 1998; accepted in final form 18 September 1998.
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REFERENCES |
---|
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---|
1.
Abdulazade, 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.
Alton, E. W.,
D. M. Geddes,
D. R. Gill,
C. F. Higgins,
S. C. Hyde,
J. A. Innes,
and
D. J. Porteous.
Towards gene therapy for cystic fibrosis: a clinical progress report.
Gene Ther.
5:
291-292,
1998[Medline].
3.
Amlal, H.,
and
M. Soleimani.
Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1194-C1205,
1997
4.
Amlal, H.,
Z. Wang,
C. E. Burnham,
and
M. Soleimani.
Functional characterization of a cloned Na+:HCO3 cotransporter.
J. Biol. Chem.
273:
16810-16815,
1998
5.
Argent, B. E.,
and
R. M. Case.
Pancreatic ducts: cellular mechanism and control of HCO3 secretion.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 1473-1497.
6.
Aronson, P. S.,
M. Soleimani,
and
S. M. Grassl.
Properties of the renal Na+-HCO3 cotransporter.
Semin. Nephrol.
11:
28-36,
1991[Medline].
7.
Becq, F.,
M. Fanjul,
I. Mahieu,
Z. Berger,
M. Gola,
and
E. Hollande.
Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin.
Pflügers Arch.
420:
46-53,
1992[Medline].
8.
Boron, W. F.,
and
E. L. Boulpaep.
The electrogenic Na/HCO3 cotransporter.
Kidney Int.
36:
392-402,
1989[Medline].
9.
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
10.
Burnham, C. E.,
Z. Wang,
H. Amlal,
G. E. Shull,
and
M. Soleimani.
Cloning, renal distribution, and regulation of the rat Na+-HCO3 cotransporter.
Am. J. Physiol.
274 (Renal Physiol. 43):
F1119-F1126,
1998
11.
Case, R. M.,
and
B. E. Argent.
Pancreatic duct cell secretion. Control and mechanisms of transport.
In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by V. L. W. Go,
and E. P. DiMagno. New York: Raven, 1993, p. 301-350.
12.
Chambers, J. A.,
and
A. Harris.
Expression of the cystic fibrosis gene and the major pancreatic mucin gene, MUC1, in human ductal epithelial cells.
J. Cell Sci.
105:
417-422,
1993
13.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
14.
Church, G. M.,
and
W. Gilbert.
Genomic sequencing.
Proc. Natl. Acad. Sci. USA
81:
1991-1995,
1984[Abstract].
15.
Drumm, M. L.,
H. A. Pope,
W. H. Cliff,
J. M. Rommens,
S. A. Marvin,
L. Tsui,
F. S. Collins,
R. A. Frizzell,
and
J. M. Wilson.
Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer.
Cell
62:
1227-1233,
1990[Medline].
16.
Freedman, S. D.,
H. F. Kern,
and
G. A. Scheele.
Acinar lumen pH regulates endocytosis, but not exocytosis, at the apical plasma membrane of pancreatic acinar cells.
Eur. J. Cell Biol.
75:
153-162,
1998[Medline].
17.
Gaskin, K. J.,
P. R. Durie,
M. Corey,
P. Wei,
and
G. G. Forstner.
Evidence for a primary defect of pancreatic HCO3 secretion in cystic fibrosis.
Pediatr. Res.
16:
554-557,
1982[Medline].
18.
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].
19.
Ishiguro, H.,
C. Steward,
A. G. 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:
169-178,
1996[Abstract].
20.
Ishiguro, H.,
C. Steward,
R. W. Wilson,
and
R. M. Case.
Bicarbonate secretion in interlobular ducts from guinea pig pancreas.
J. Physiol. (Lond.)
495:
179-191,
1996[Abstract].
21.
Knowles, M. R.,
K. W. Hohneker,
Z. Zhou,
J. C. Olsen,
T. L. Noah,
P. Hu,
M. W. Leigh,
J. F. Engelhardt,
L. J. Edwards,
K. R. Jones,
M. Grossman,
J. M. Wilson,
L. G. Johnson,
and
R. C. Boucher.
A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis.
N. Engl. J. Med.
333:
823-831,
1995
22.
Kopelman, H.,
M. Corey,
K. Gaskin,
P. Durie,
Z. Weizman,
and
G. Forstner.
Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid and secretory defect in the cystic fibrosis pancreas.
Gastroenterology
95:
349-355,
1988[Medline].
23.
Lebenthal, E.,
A. Lerner,
and
D. K. Rolston.
The pancreas in cystic fibrosis.
In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by V. L. W. Go,
and E. P. DiMagno. New York: Raven, 1993, p. 1041-1081.
24.
Levrat, J. H.,
C. Palevody,
M. Daumas,
G. Ratovo,
and
E. Hollande.
Differentiation of the human pancreatic adenocarcinoma cell line (Capan-1) in culture and co-culture with fibroblasts dome formation.
Int. J. Cancer
42:
615-621,
1998.
25.
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].
26.
Novak, I.,
and
R. Greger.
Effect of bicarbonate on potassium conductance of isolated perfused rat pancreatic ducts.
Pflügers Arch.
419:
76-83,
1991[Medline].
27.
Poulsen, J. H.,
and
T. E. Machen.
HCO3-dependent pHi regulation in tracheal epithelial cells.
Pflügers Arch.
432:
546-554,
1996[Medline].
28.
Preisig, P. A.,
and
R. J. Alpern.
Basolateral membrane H-OH-HCO3 transport in the proximal tubule.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F751-F756,
1989
29.
Rosenstein, B. J.,
and
P. L. Zeitlin.
Cystic fibrosis.
Lancet
351:
277-282,
1998[Medline].
30.
Ruiz, O. S.,
and
J. A. Arruda.
Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F560-F565,
1992
31.
Scheele, G. A.,
S. Fukuoka,
H. F. Kern,
and
S. D. Freedman.
Pancreatic dysfunction in cystic fibrosis occurs as a result of impairments in luminal pH, apical trafficking of zymogen granule membranes, and solubilization of secretory enzymes.
Pancreas
12:
1-9,
1996[Medline].
32.
Schoumacher, R.,
J. Ram,
M. C. Iannuzzi,
N. A. Bradbury,
R. W. Wallace,
C. Tom Hon,
D. R. Kelly,
S. M. Schmid,
F. B. Gelder,
R. A. Rado,
and
R. A. Frizzell.
A cystic fibrosis pancreatic adenocarcinoma cell line.
Proc. Natl. Acad. Sci. USA
87:
4012-4016,
1990[Abstract].
33.
Smith, J. J.,
and
M. J. Welsh.
cAMP stimulates bicarbonate secretion across normal but not cystic fibrosis epithelia.
J. Clin. Invest.
89:
1148-1153,
1992[Medline].
34.
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].
35.
Soleimani, M.,
and
P. S. Aronson.
Ionic mechanism of Na-HCO3 cotransport in rabbit renal basolateral membrane vesicles.
J. Biol. Chem.
264:
18302-18308,
1989
36.
Soleimani, M.,
G. Bizal,
P. S. Aronson,
and
J. Bergman.
Acute regulation of Na+:HCO3 cotransport system in kidney proximal tubules.
In: Molecular and Cellular Mechanisms of H+ Transport, edited by B. H. Hirst. Berlin: Springer-Verlag, 1994, p. 309-317.
37.
Soleimani, M.,
S. M. Grassl,
and
P. S. Aronson.
Stoichiometry of the Na+-HCO3 co-transporter in basolateral membrane vesicles isolated from rabbit renal cortex.
J. Clin. Invest.
79:
1276-1280,
1987[Medline].
38.
Soleimani, M.,
G. Singh,
G. L. Bizal,
S. R. Gullans,
and
J. A. McAteer.
Na+/H+ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells: expression, functional localization, and differential regulation.
J. Biol. Chem.
269:
27973-27978,
1994
39.
Soleimani, M.,
B. W. Watts,
G. Singh,
and
D. W. Good.
Effect of long-term hyperosmolality on Na/H exchanger isoform NHE-3 in LLC-PK1 cells.
Kidney Int.
53:
423-431,
1998[Medline].
40.
Wang, Z.,
P. J. Schultheis,
and
G. E. Shull.
Three terminal variants of the AE2 Cl/HCO
3 exchanger are encoded by mRNAs transcribed from alternative promoters.
J. Biol. Chem.
271:
7835-7843,
1996
41.
Weinman, E. J.,
W. Dubinsky,
and
S. Shenolikar.
Regulation of the renal Na+-H+ exchanger by protein phosphorylation.
Kidney Int.
36:
519-525,
1989[Medline].
42.
Wilson, J.
Gene therapy for cystic fibrosis: challenges and future directions.
J. Clin. Invest.
96:
2547-2554,
1995[Medline].
43.
Winpenny, J. P.,
A. Harris,
M. A. Hollinsworth,
B. E. Argent,
and
M. A. Gray.
Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells.
Pflügers Arch.
435:
796-803,
1998[Medline].
44.
Winpenny, J. P.,
B. Verdon,
H. L. McAlroy,
W. H. Colledge,
R. Ratcliff,
M. J. Evans,
M. A. Gray,
and
B. E. Argent.
Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice.
Pflügers Arch.
430:
26-33,
1995[Medline].
45.
Yoshitomi, K.,
B. C. Burckhardt,
and
E. Fromter.
Rheogenic sodium-bicarbonate co-transport in the peritubular cell membrane of rat renal proximal tubule.
Pflügers Arch.
405:
360-366,
1985[Medline].
46.
Zhao, H.,
R. A. Star,
and
S. Muallem.
Membrane localization of H+ and HCO3 transporters in the rat pancreatic duct.
J. Gen. Physiol.
194:
57-85,
1994.