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INTRODUCTION |
It is well established that pancreatic
HCO
secretion performs a critical role in digestion.
HCO
protects duodenal epithelia by neutralizing
gastric acid and maintains an optimum intraluminal pH for digestive
enzymes (1). Accumulating evidence suggests that
HCO
-rich pancreatic fluid secretion is also
important for maintaining the patency of intrapancreatic ductal trees
(2-4). Transepithelial HCO
transport is the
principal driving force for fluid secretion by pancreatic duct cells,
and reduced HCO
concentrations result in
acidification of the luminal environment. The rheologic properties of
mucus are critically affected by mucin concentrations and the pH of
solvent. In general, mucin precipitation and viscosity are
progressively increased as the pH and the volume of secreted fluid
decrease (5, 6). Indeed, the most prominent changes observed in the
pancreatic juice of obstructive ductal diseases, such as cystic
fibrosis (CF)1 or chronic
pancreatitis, are reductions in secreted volume and HCO
concentration (2, 7).
Although there is some disagreement on the precise mechanism of ductal
HCO
secretion, it is widely accepted that the bulk
of HCO
secretion is mediated by
Cl
/HCO
exchange on the luminal
membrane (8). Previously, we showed that cystic fibrosis transmembrane conductance regulator (CFTR) activates
Cl
/HCO
exchange activity in
heterologous expression systems and in the luminal membrane of
pancreatic duct (9, 10). Notably, the CFTR-dependent
HCO
/Cl
transport ratio in each
CF-causing CFTR mutation appears to correlate well with the reported
disease severity, especially with the pancreatic status of patients
(11).
An increase in intracellular cAMP by secretin is one of the major
signals of pancreatic HCO
secretion. Activation of
the CFTR Cl
channel and the CFTR-dependent
Cl
/HCO
exchange activities was shown to be responsible for cAMP-induced HCO
secretion (1,
8). It was also shown that cAMP increases electrogenic
HCO
permeability in the luminal membrane of guinea
pig pancreas through a mechanism that has not been identified (12). In
addition, intracellular calcium signaling evoked by various stimuli,
such as activations of cholinergic, purinergic (P2R), and
protease-activated receptors (PAR), is also known to increase fluid and
HCO
secretion either directly or by potentiating
cAMP-mediated mechanisms (1, 13-16). Cholinergic activation through
vagal efferent fibers is the major physiologic stimulus for the
cephalic phase of pancreatic secretion. Recent studies have
demonstrated that P2Rs control fluid and the HCO
secretion of many epithelial cells including pancreatic duct cells, in
an auto- or paracrine manner (13, 16). Moreover, PARs, which can be activated by interstitially released trypsin during pancreatic inflammation, have been suggested to increase pancreatic secretion (14,
17). However, little is known about the precise mechanism of
calcium-induced HCO
and fluid secretion in
pancreatic duct cells.
In the present study, we report that calcium signals activate CFTR- and
Cl
-dependent HCO
transport
in the luminal membrane of pancreatic duct-derived cells. ATP and
trypsin were found to increase the luminal
Cl
/HCO
exchange activity of CAPAN-1 cells expressing wild type (WT)-CFTR, and this was inhibited by buffering [Ca2+]i with
1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA). The effects of ATP on luminal
Cl
/HCO
exchange were not prominent in CFPAC-1 cells bearing a defective form (
508) of CFTR, although ATP
evoked significant calcium signaling in the cells. Notably, adenoviral
transfection of WT-CFTR in CFPAC-1 cells increased ATP-activated
luminal Cl
/HCO
exchange. These results
provide the molecular basis for calcium-induced bicarbonate secretion by pancreatic duct cells and possibly by other epithelial cells.
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MATERIALS AND METHODS |
Chemicals and Solutions--
Fura-2-AM and
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) were
purchased from Molecular Probes (Eugene, OR). PAR2-activating
peptide (SLIGRL-NH2) and PAR4-activating peptide (AYPGKF-NH2) were synthesized at the Korea Basic
Science Institute (Seoul, Korea). All other chemicals including ATP,
trypsin, and thrombin were purchased from Sigma.
The standard perfusate was termed solution A and contained (in
mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, and 10 HEPES (pH 7.4 with
NaOH). In the measurements of Cl
/NO
exchange (see Fig. 3) and
[Cl
]o/[HCO
]i
exchange (see Fig. 8, B and C), Cl
was replaced with equimolar NO
or gluconate in
solution A. The HCO
-buffered NaCl solution B
contained (in mM) 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4 with NaOH). The
HCO
-buffered Cl
-free solution C
contained (in mM) 120 Na+-gluconate, 5 K+-gluconate, 1 MgSO4, 9.3 hemicalcium
cyclamate, 10 D-glucose, 5 HEPES, and 25 NaHCO3
(pH 7.4 with NaOH). To prepare HCO
-buffered high
K+ (100 mM K+) solution D, 95 mM NaCl in solution B was replaced with 95 mM KCl. To prepare HCO
-buffered, high K+
(100 mM K+), Cl
-free solution E,
95 mM Na+ gluconate in solution C was replaced
with 95 mM K+ gluconate. All
HCO
-buffered solutions were continuously gassed with
95% O2 and 5% CO2 to maintain the pH of
solutions. The osmolarity of all solutions was adjusted to 310 mosM
with the major salt prior to use.
Monolayer Culture and Adenoviral Transfection--
CAPAN-1 (ATCC
HTB-79) and CFPAC-1 cells (ATCC CRL-1918) were purchased from the
American Type Culture Collection, Manassas, VA. CAPAN-1, a metastatic
pancreatic cancer cell line expressing WT-CFTR, was maintained in RPMI
1640 medium (Invotrogen) supplemented with 10% fetal bovine
serum and penicillin (50 units/ml)/streptomycin (50 µg/ml). CFPAC-1,
a pancreatic duct cell line derived from a patient with CF bearing a
F508-CFTR mutation, was maintained in Iscove's modified Dulbecco's
medium supplemented with 10% fetal bovine serum and penicillin (50 units/ml)/streptomycin (50 µg/ml). CAPAN-1 and CFPAC-1 cells were
removed from the culture flasks using trypsin/EDTA washed by
centrifugation/resuspension in fresh medium. Aliquots of this
suspension were plated (3 × 105
cells/cm2) on permeable supports fabricated from
Transwell-Clear Polyester membrane (0.4 µm pore diameter; Costar,
Cambridge, MA), which was coated with collagen (30 µg/ml) for 30 min.
Membranes bearing cultured cells (culture area 0.3 cm2)
were floated on the culture medium in Petri dishes and incubated for
4-5 days until the cells formed a functionally polarized monolayer.
Adenoviral vector expressing WT-CFTR (Ad-CFTR) was purchased from the
Institute of Human Gene Therapy, Philadelphia, PA. The E1-E3 deleted
viruses were amplified in HEK 293 cells and purified using a
CsCl2-based method (18). Ad-CFTR and Ad-
-Gal (mock vector) viruses were titrated by limiting dilutions or plaque assays
and stored in small aliquots to avoid repeated freeze-thaw cycles.
Filter-grown CFPAC-1 cells were washed twice with phosphate-buffered saline, and 150 µl of the virus-containing medium was applied to the
luminal side of the cells for 5 h. The cells were then rinsed with
fresh media and incubated in the culture medium and were used for
pHi measurements 48-72 h after transfection.
RT-PCR and Immunohistochemistry--
The expression of CFTR in
transfected cells was verified by RT-PCR, immunostaining, and
immunoblotting using a procedure described previously (19). The
mRNA transcripts of human WT-CFTR and
F508-CFTR were analyzed by
RT-PCR in pancreatic duct-derived cells. Total RNA was prepared from
CAPAN-1 and CFPAC-1 cells using Trizol solution (Invitrogen) and
reverse-transcribed using random hexa-primers and an RNase
H
reverse transcriptase (Invitrogen). The cDNA was
amplified using specific primers and a Taq polymerase
(Promega, Madison, WI), and the products were separated on a 2%
agarose gel containing 0.1 µg/ml ethidium bromide. The primer
sequences used were as follows: WT-CFTR, sense
(5'-GGCACCATTAAAGAAAATATCATCTT-3') and antisense
(5'-TAATTTGGGTAGTGTGAAGGGTTC-3'), size of PCR product 145 base pairs;
F508-CFTR, sense (5'-GGCACCATTAAAGAAAATATCATTGG-3') and antisense
(5'-TAATTTGGGTAGTGTGAAGGGTTC-3'), size of PCR product 142 base pairs.
Immunostaining of monolayers was performed using monoclonal antibodies
against the C terminus of CFTR (Clone 24-1; R&D Systems, Minneapolis,
MN). Briefly, membrane-cultured cells were fixed and permeabilized by
incubation in cold methanol for 10 min at
20 °C and then stained
with the primary antibodies against CFTR and the fluorescently labeled
secondary antibodies. Images were collected using a Zeiss LSM510
confocal microscope with serial Z-sections. For immunoblotting, cell
lysates (100 µg of protein) were suspended in SDS sample buffer and
separated by SDS-polyacrylamide gel electrophoresis. The separated
proteins were then transferred to polyvinylidene difluoride membranes,
which were blocked by a 1-h incubation at room temperature in 5%
nonfat dry milk in a solution containing 20 mM Tris-HCl, pH
7.5, 150 mM NaCl, and 0.05% Tween 20. CFTR proteins were
detected by 1-h incubations with monoclonal antibodies against the R
domain of CFTR (Clone 13-1; R&D Systems) and the appropriate secondary
antibodies. The staining intensities of immunostaining and
immunoblotting were analyzed using an imaging software (MCID version
3.0; Brook University, St. Catharines, Ontario, Canada).
Measurement of [Ca2+]i and
[Cl
]i--
Measurements of
[Ca2+]i in the monolayers were
performed using protocols reported previously with slight modification (20). Briefly, after achieving confluency, the cells were loaded with
Fura-2 by incubating in a medium containing 3 µM
Fura-2-AM and 1.6 µM pluronic F127 (for 30 min at
37 °C). A membrane bearing Fura-2-loaded cells was mounted in a
miniature Ussing chamber (AKI Institute, University of Copenhagen,
Denmark) attached to the stage of an inverted microscope. The membrane
was located in between the two-half chambers, which separate the
chamber into a luminal (upper) and a basolateral (lower) compartment. A
transparent coverslip was placed at the bottom of the perfusion
chamber, which allowed fluorescence measurements from the dye-loaded
monolayers using objective lenses having a long working distance (more
than 2 mm). Separate luminal and basolateral perfusates were delivered to the chamber after warming (37 °C). The Fura-2 fluorescence was
recorded (Delta Ram; PTI Inc., South Brunswick, NJ) at excitation wavelengths of 350 nm and 380 nm, and the 350/380 fluorescence ratio
was calibrated by exposing the cells to solutions containing high and
low concentrations of Ca2+ and 10 µM
ionomycin (21).
To measure [Cl
]i, CAPAN-1 and
CFPAC-1 cells were grown on coverslips, which form the bottom of a
different type of chamber suitable for single perfusion.
[Cl
]i was measured with a
Cl
-sensitive dye MQAE using the procedure
described before with minor modifications (10). Cells were suspended in
solution A containing 10 mM of MQAE and incubated for 30 min at room temperature and for 40 min at 0 °C. After chamber
assembly, external MQAE was washed out by perfusing the cells with
solution A. MQAE fluorescence was measured at an excitation wavelength
of 350 nm and emission wavelength of 450 nm. At the end of each
experiment, a calibration procedure was performed using standard
Cl
-containing solutions with 150 mM
K+, 5 µM nigericin, and 10 µM
tributyltin cyanide.
Measurement of pHi--
Measurements of
pHi in the monolayers were performed using a
pH-sensitive fluorescent probe BCECF. Cells were loaded with BCECF by
being incubated for 10 min at room temperature in solution A containing
2.5 µM BCECF-AM and mounted in the miniature Ussing
chamber. BCECF fluorescence was recorded and calibrated using a
protocol previously described (9). Briefly, the fluorescence at
excitation wavelengths of 490 and 440 nm was recorded using the
recording setup (Delta Ram; PTI Inc.) and the 490/440 ratios were
calibrated intracellularly by perfusing the cells with solutions containing 145 mM KCl, 10 mM HEPES, and 5 µM nigericin with the pH adjusted to 6.2-7.6.
[Cl
]i/[HCO
]o
exchange activities were estimated from the initial rate of
pHi increase as a result of
[Cl
]o removal. Initial rates of
pHi changes were obtained from the first
derivative of the traces using a single exponential fit.
Buffer capacity was calculated by measuring
pHi in response to 5-40 mM
NH4Cl pulses (22). Intrinsic buffer capacity (
i) of CAPAN-1 cells (17.6 ± 0.8 mM/pH unit at pHi 7.2) was
significantly lower than that of CFPAC-1 cells (21.6 ± 1.4 mM/pH unit). Total buffer capacity
(
t) was calculated from
t =
i + 2.3[HCO
]i (22). At
pHi 7.2 with 5% CO2 gassing,
[HCO
]i was estimated to be
14.4 mM. Thus, in HCO
-buffered solutions
the
t of CAPAN-1 (50.7 mM/pH
unit) was only 7% different from that of CFPAC-1 (54.7 mM/pH unit). In addition, all the transporter activity
comparisons were made within the same cell types. Therefore, the
results of Cl
/HCO
exchange activity
were expressed as pH unit/min, and this value was directly analyzed
without compensating for
t.
Statistical Analysis--
The results of multiple experiments
are presented as means ± S.E., and statistical analysis was
carried out by using analysis of variance or Student's t
test as appropriate. p < 0.05 was considered statistically significant.
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RESULTS |
P2Rs and PARs in Pancreatic Duct-derived Cells--
To investigate
the role of calcium signaling on luminal
Cl
-dependent HCO
transport
mechanisms, we first characterized the calcium-evoking receptors in
CAPAN-1 and CFPAC-1 cells (Fig. 1).
Applications of ATP on either the luminal or the basolateral membrane
increased [Ca2+]i in both CAPAN-1
and CFPAC-1 cells, and the activation of P2Rs caused biphasic
[Ca2+]i increases, thus a rapid
[Ca2+]i peak which lasted for 30 s-1 min followed by a small sustained
[Ca2+]i increase (Fig.
1A). Repetitive short ATP applications at 3- to 5-min
intervals evoked corresponding calcium signals, and during the 30 min
of observation, the amplitudes of the
[Ca2+]i peaks did not decrease.
P2Rs on the luminal membrane showed a higher affinity for ATP than
those on the basolateral membrane (Fig. 1B). The
EC50 values of luminal and basolateral ATP for
[Ca2+]i increase in CAPAN-1 cells
were 3.1 µM and 20.6 µM, respectively.
CFPAC-1 cells showed similar ATP responses. Thus, CFPAC-1 cells
responded to ATP applications with higher affinity for luminal side
(EC50 = 6.1 µM) than basolateral
(EC50 = 19.9 µM). These results are in good
agreement with those of our previous reports that native submandibular
and pancreatic ducts express multiple P2Rs in both luminal and
basolateral membranes (13, 21).

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Fig. 1.
Effect of P2R and PAR agonists on
[Ca2+]i.
Monolayers of CAPAN-1 or CFPAC-1 cells were loaded with Fura-2, and
[Ca2+]i was measured in separate
luminal and basolateral perfusates. A, ATP evoked a biphasic
[Ca2+]i increase, a rapid peak
followed by a small sustained
[Ca2+]i increase. B,
both basolateral and luminal ATP increased
[Ca2+]i, although luminal ATP
showed higher affinity. Note that 10-fold lower concentration of ATP
was used on the luminal side. C, trypsin-evoked calcium
signaling only when applied to the basolateral side. D,
among the several PAR-activating peptides (AP), only
PAR-2-activating peptide-evoked
[Ca2+]i increase. CAPAN-1 and
CFPAC-1 cells showed similar
[Ca2+]i responses to P2R or PAR
agonists. Traces A and D were taken
from experiments on CAPAN-1 cells, and B and C
from experiments on CFPAC-1 cells. LM, luminal membrane;
BLM, basolateral membrane.
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Increasing evidence suggests that pancreatic duct cells express PARs
and that this plays significant roles in both physiologic and
pathological states (14, 17, 23). Trypsin (1 µM) evoked a
rapid Ca2+ peak when applied to the basolateral side of
CAPAN-1 and CFPAC-1 cells, but not to the luminal side (Fig.
1C). Basolateral applications of isoform-specific activating
peptide (AP) showed that pancreatic duct cells express functional PAR-2
receptors (Fig. 1D). Unlike purinergic activation,
repetitive applications of PAR agonists did not cause any calcium
signaling due to receptor internalization and degradation, which is a
characteristic of all known PARs (23). After a resting period of 30 min, the cells began to respond to PAR agonists, possibly because of
new receptor expression on the plasma membrane (data not shown).
P2R and PAR Activate Electroneutral
Cl
/HCO
Exchange on the
Luminal Membrane--
Initially, we examined the effects of purinergic
and protease-activated receptors on the
Cl
-dependent HCO
transport
mechanisms in physiologic buffers, which contained 5 mM
K+,129 mM Cl
, and 25 mM HCO
(gassing with 5%
CO2). The basal pHi of CFPAC-1 cells
(7.23 ± 0.05) seemed to be higher than that of CAPAN-1 cells
(7.11 ± 0.06). To better observe lumen-specific
Cl
/HCO
exchange activity, the
basolateral side of the monolayer was first applied to
Cl
-free solutions. In a previous paper, we showed that
this maneuver prevents the dissipation of luminally accumulated
HCO
through the forward mode of basolateral
Cl
/HCO
exchange (outward
HCO
movement) (10). Although basolateral
Cl
-free solution may activate the reverse mode of
basolateral Cl
/HCO
exchange (inward
HCO
movement), intracellular alkalinization was very
small;
pHi increases in CAPAN-1 and CFPAC-1
cells were 0.05 ± 0.02 and 0.04 ± 0.03, respectively. This
was attributed to the higher luminal to basolateral antiporter
distribution ratio of the pancreatic duct cells (8, 24).
Luminal Cl
-dependent HCO
transport was estimated by measuring the initial
pHi increase due to
[Cl
]i/[HCO
]o
exchange. In physiologic buffers, the activation of P2R or PAR
decreased the observed luminal Cl
/HCO
exchange in both CAPAN-1 and CFPAC-1 cells (Fig.
2). A similar phenomenon was observed
previously in isolated submandibular duct cells and microperfused
pancreatic duct cells, when they were activated by cAMP (10). We reason that this is because of the simultaneous activation of Cl
exit pathways, such as Cl
channels or
K+-Cl
cotransport. Because
[Cl
]i decreases very rapidly due
to the activation of Cl
exit pathways, it attenuates the
driving force for
[Cl
]i/[HCO
]o
exchanges. Actually, it has been shown that both CAPAN-1 and CFPAC-1
cells express Ca2+-activated Cl
channels on their luminal membranes (25).

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Fig. 2.
Measurements of luminal
Cl /HCO exchange
in physiologic buffers. A and B, monolayers of CAPAN-1 and
CFPAC-1 cells were loaded with BCECF, and pHi
was measured in a double perfusion chamber. In physiologic buffers
containing 5 mM K+, activation of either P2R or
PAR decreased the observed luminal
Cl /HCO exchange. C, results
shown are the means ± S.E. of five experiments.
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Therefore, in the next set of experiments we measured electroneutral
luminal Cl
/HCO
exchange in high
[K+]o conditions. When
countercurrent K+ movement is blocked by high
K+ (100 mM) buffers, electrogenic
Cl
pathways are impaired, and thus most of the
Cl
movement is the result of electroneutral transport
(see Fig. 4A). In addition, high external K+
also blocks outward Cl
movement through
K+-Cl
cotransport. This was recently
identified in the basolateral membrane of rat pancreatic duct cells
(26). Moreover, it was found that bathing the cells in high
K+-containing buffers reduces
Cl
/NO
exchange in pancreatic
duct-derived cells (Fig. 3). It has
previously been shown that the measurement of
Cl
/NO
exchange in
HCO
-free buffer (nominally Hepes-buffered)
accurately reports Cl
channel activity in intact cells
(11). Changes in [Cl
]i were
measured by applying Cl
-free,
NO
-containing perfusates in coverslip grown cells.
Applications of 0 mM Cl
, 145 mM
NO
solution to CAPAN-1 cells rapidly decreased
[Cl
]i, and ATP stimulation
further enhanced this [Cl
]i
decrease by 43% (Fig. 3A). As shown in Fig. 3B,
perfusing the cells with a 100 mM K+-containing
solution inhibited Cl
/NO
exchange
activity in control and ATP-stimulated states by 71 and 73%,
respectively. Measurements in CFPAC-1 cells showed similar results and
summarized results are presented in Fig. 3C.

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Fig. 3.
Measurements of
Cl /NO
exchange. Cl /NO exchange
was measured in HCO -free buffer (nominally
Hepes-buffered), which reports Cl channel activity in
intact cells. [Cl ]i was measured
with the aid of the Cl -sensitive dye MQAE in
coverslip-grown CAPAN-1 and CFPAC-1 cells. A, applications
of NO solution to CAPAN-1 cells rapidly decreased
[Cl ]i, and ATP further enhanced
the [Cl ]i decrease.
B, perfusing the cells with a high K+-containing
solution inhibited Cl /NO exchange
activities. C, the results shown are the means ± S.E.
of four to six experiments.
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Surprisingly, when measured in high-K+ containing buffers,
the activation of P2R or PAR highly increased luminal
Cl
-dependent HCO
transport
in CAPAN-1 cells (Fig. 4, C and
D). An average of 5-6 experiments are summarized in Fig.
4B. The basal activity of luminal
Cl
/HCO
exchange was 0.145 ± 0.010 pH unit/min, which increased to 0.383 ± 0.044 and
0.481 ± 0.052 by ATP and trypsin treatment, respectively. On the
other hand, the effect of calcium agonists on luminal
Cl
/HCO
exchange was very small in
CFPAC-1 cells. As shown in Fig. 4, E and F,
CFPAC-1 cells showed only 27% increase in
Cl
/HCO
exchange activity when
treated with ATP versus the corresponding 164%
activity increase in CAPAN-1 cells (Fig. 4B).

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Fig. 4.
Measurements of luminal
Cl /HCO exchange
in high K+ buffers. The activities of
luminal Cl /HCO were measured in
perfusate containing a high concentration of K+ (100 mM) to block Cl movement through electrogenic
or K+-coupled pathways. A, schematic diagram of
Cl and HCO movement in high
K+ buffer. B, results shown are the means ± S.E. of five to six experiments. Activation of P2R- (C)
or PAR-stimulated (D) luminal
Cl /HCO exchange in CAPAN-1 cells, but
not in CFPAC-1 cells (E and F).
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Expression of WT-CFTR Augments the Effect of Calcium Agonists on
Luminal Cl
/HCO
Exchange in
CFTR-impaired Cells--
The finding that the effect of calcium
agonists is nearly abolished in CFTR-impaired cells prompted us to
investigate the role of CFTR. Accordingly, we measured luminal
Cl
/HCO
exchange activity in
membrane-cultured CFPAC-1 cells 48-72 h after transfection with
variable doses of Ad-CFTR. The expression of WT-CFTR in CFPAC-1
monolayer was verified by several molecular methods (Fig.
5).

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Fig. 5.
Exogenous expression of WT-CFTR in CFPAC-1
cells by Ad-CFTR transfection. A, RT-PCR. mRNA
transcripts of WT- and F508-CFTR were analyzed in pancreatic
duct-derived cells. Both WT- and F508-CFTR transcripts were detected
in Ad-CFTR transfected CFPAC-1 cells. B, immunoblotting of
CFTR protein was performed 48 h after Ad-CFTR transfection using
monoclonal antibodies against the R-domain of CFTR. C,
semi-quantitative confocal microscopy on luminal membrane CFTR
expression. Monolayers of pancreatic duct-derived cells were labeled
with antibodies against the C terminus of CFTR and with fluorescently
tagged secondary antibodies. Under the same image-acquisition setup for
each sample, luminal surface images were collected using a Zeiss LSM510
microscope after serial Z-axis sections. Summarized results are the
averages of three separate experiments.
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The presence of the mRNA transcripts of WT- and
F508-CFTR in
RT-PCR of CAPAN-1 and CFPAC-1 cells, respectively, confirmed the
cell-line characteristics. As expected, both WT- and
F508-CFTR transcripts were found in CFPAC-1 cells transfected with Ad-CFTR (Fig.
5A). By immunostaining, when the cells were transfected with
30 and 200 m.o.i. (multiplicity of infection) of Ad-CFTR, 31 ± 7% and 83 ± 6% of the cells, respectively, were found to be
CFTR-positive. When the amount of CFTR protein was compared by
immunoblotting, the CFTR expression in CFPAC-1 cells transfected with
200 m.o.i. of Ad-CFTR was 2.7-fold that of CAPAN-1 cells (Fig.
5B). On the other hand, when the same comparison was made by
the luminal surface immunostaining using semi-quantitative confocal
microscopy, the luminal membrane CFTR expression in 200 m.o.i.
transfected CFPAC-1 cells was 1.4-fold that of CAPAN-1 cells (Fig.
5C). Several possible mechanisms can explain this discrepancy between the results obtained by immunoblotting and surface
confocal microscopy, including the possibility of higher protein
maturation in the native expression of CAPAN-1 cells. However, this
mechanism was not further investigated in the present study.
The expression of CFTR significantly enhanced the effect of ATP on
luminal Cl
/HCO
exchange in a
dose-dependent manner. In non-transfected and Ad-
-Gal
(mock vector)-transfected cells, ATP increased luminal
Cl
/HCO
exchange activity by 27 and
24%, respectively. However, in the exogenously CFTR-expressed cells by
transfecting with 30 and 200 m.o.i. of Ad-CFTR, ATP increased luminal Cl
/HCO
exchange activity by 60 and 246%, respectively (Fig. 6).

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Fig. 6.
WT-CFTR expression augments the effect of
calcium agonists on luminal
Cl /HCO exchange
in CFPAC-1 cells. CFPAC-1 cells were transfected with various
doses of Ad-CFTR, and luminal Cl /HCO
exchange was measured using the methods described in Fig. 3.
Transfection of Ad-CFTR increased the stimulatory effect of ATP on
luminal Cl /HCO exchange
(A). The averages of five experiments are shown in
panel B.
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Calcium Is Required for the P2R- or the PAR-induced Activation of
Luminal Cl
/HCO
Exchange--
As depicted in Fig. 1, stimulation by P2R or PAR caused
a rapid [Ca2+]i rise and a
short-lived [Ca2+]i peak in
pancreatic duct-derived cells. Interestingly, treatment with P2R or PAR
agonists 2-3 min before the application of Cl
-free
solution did not increase the luminal
Cl
/HCO
exchange (Fig.
7A), although treatment with
agonists simultaneously with or 15 s after Cl
-free
solution strongly activated CFTR-dependent luminal
Cl
/HCO
exchange (Fig. 4). These
results suggest that the Cl
-dependent
HCO
transport activity is highly correlated with
[Ca2+]i. To investigate the
Ca2+-dependence of the P2R- or the PAR-induced effects,
CAPAN-1 cells were preloaded with BAPTA-AM (50 µM) for 30 min and luminal Cl
/HCO
exchange
activities were measured after ATP or trypsin stimulation. Chelating
intracellular calcium with BAPTA completely abolished the P2R- or
PAR-induced activation of luminal
Cl
/HCO
exchange (Fig. 7, B
and D).

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Fig. 7.
Calcium is required for the P2R- or the
PAR-induced activation of luminal
Cl /HCO exchange.
A, application of ATP 2 min before Cl -free
solution did not increase luminal
Cl /HCO exchange. B and
C, effect of ATP on luminal
Cl /HCO exchange was measured in
CAPAN-1 cells after pretreatment with the calcium chelator
BAPTA-AM (B, 50 µM for 30 min) or the
PKA inhibitor H89 (C, 30 µM for 1 h).
BAPTA pretreatment completely abolished the stimulatory effect of ATP
on luminal Cl /HCO exchange, whereas
H89 pretreatment did not. D, summary data of calcium
chelator (B) and PKA inhibitor (C) pretreatments.
Means ± S.E. of five to six individual traces of P2R or PAR
activation are shown. **, p < 0.01
|
|
It has been suggested that Gq-coupled agonists may activate CFTR
Cl
channel function by increasing responsiveness to
endogenous cAMP/PKA pathways (27). To determine whether P2R or PAR
activates CFTR-dependent Cl
/HCO
exchange via the PKA pathway, cells were pretreated with a high concentration (30 µM)
of the PKA inhibitor H89 for 1 h, and the activity of
Cl
/HCO
exchange was measured with
buffers containing 50 nM H89. As shown in Fig. 7,
C and D, the inhibition of the PKA pathway did
not decrease P2R- or PAR-induced activation of luminal
Cl
/HCO
exchange. The PKA-inhibitory effect of H89 was confirmed by the finding that the same H89 treatment inhibited the forskolin-induced activation of luminal
Cl
/HCO
exchange by 93% in CAPAN-1 cells.
Measurements of Cl
/OH
Exchange and
[Cl
]o/[HCO
]i
Exchange--
Finally, we demonstrated that the increases in
pHi by
[Cl
]o removal in the present
study were caused by a Cl
/HCO
exchanger, although other mechanisms, for example,
Cl
/OH
exchange or the exchange of
Cl
and HCO
via Cl
channels may also have a similar effect. First, the activity of
Cl
/OH
exchange was measured in CAPAN-1
cells using HCO
- and CO2-free solutions
(Hepes-buffered). As shown in Fig.
8A, Cl
/OH
exchange activity was not observed in
the luminal membrane of CAPAN-1 cells. Applications of
Cl
-free solution caused a slight decrease or no changes
in pHi, and ATP treatment did not evoke a
pHi increase in Hepes-buffered solutions. Hence,
the activity of Cl
/OH
cannot explain the
increases in pHi by
[Cl
]o removal in
HCO
- and CO2-containing solutions (Fig.
4).

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|
Fig. 8.
Measurements of
Cl /OH exchange and
[Cl ]o/[HCO ]i
exchange. A, Cl /OH
exchange was measured in CAPAN-1 cells using HCO -
and CO2-free solutions (Hepes-buffered).
Cl /OH exchange activities were not observed
in the luminal membrane of CAPAN-1 cells. Five separate experiments
showed similar results. B, to evaluate the effect of
electrogenic HCO pathways, the forward mode of
Cl /HCO exchange
([Cl ]o/[HCO ]i
exchange) was measured in Cl -containing and
Cl -free solutions. C, stimulatory effect of
ATP on the forward mode of
[Cl ]o/[HCO ]i
exchange. Summarized results are the means ± S.E. of five to six
experiments.
|
|
It has been shown that HCO
can pass through several
Cl
channels including CFTR and the
Ca2+-activated Cl
channel, although its
permeability is much lower than that of Cl
(8, 12). It is
possible that depolarization caused by Cl
exit may evoke
the inward flux of HCO
through Cl
channels during the reverse mode measurements of
Cl
/HCO
exchange
([Cl
]i/[HCO
]o
exchange, Fig. 4). However, the fact that bathing the cells in 100 mM K+ decreased Cl
channel
activity (Fig. 3) but enhanced the pHi increase by [Cl
]o removal in
ATP-stimulated states (compare Fig. 2 with Fig. 4) strongly suggests
that HCO
movement through Cl
channels
cannot explain the present finding. In addition, we reevaluated the
effect of electrogenic HCO
pathways using the
forward mode of Cl
/HCO
exchange
([Cl
]o/[HCO
]i
exchange, Fig. 8B). When we changed the perfusates from
HCO
- and CO2-containing solutions to
Hepes-buffered solutions, pHi abruptly increased
due to the rapid diffusion of CO2. Cells can regain optimum
pHi by two mechanisms, via the forward mode of
[Cl
]o/[HCO
]i
exchange and alternatively via the electrogenic HCO
exit through Cl
channels. In this state, the directions
of Cl
and HCO
movement through
Cl
channels are both outward. Therefore, the exchange of
Cl
and HCO
through anion channels
cannot occur, and the HCO
movement through Cl
channels can be isolated from the activity of
Cl
/HCO
exchange by measuring the
pHi recovery in Cl
-free solutions.
Because CO2 can nonspecifically diffuse through both
luminal and basolateral membranes, coverslip-grown cells were used
for the following experiments. When applying the
Hepes-buffered solution, cells were stimulated with ATP to fully
activate Cl
channels. The pHi
recovery rate of CAPAN-1 cells from alkali load was 0.246 ± 0.025 pH unit/min (at pHi 7.6) in
Cl
-containing solutions. However, in
Cl
-free solutions this value dropped to 0.041 ± 0.014 pH unit/min. These results imply that HCO
movement through Cl
channels mediates less than 17% of
the total HCO
transport and that this
mechanism performs only a minor role in CAPAN-1 cells (Fig.
8B).
We also verified the stimulatory effect of ATP on the forward mode of
[Cl
]o/[HCO
]i
exchange (Fig. 8C). Experiments were also performed in high
K+-containing solutions to minimize the effect of
electrogenic pathways. In 100 mM K+-containing
solution, the basal pHi recovery rate was 0.081 ± 0.009 pH unit/min (at pHi 7.6) and
ATP stimulation increased the pHi recovery rate
by 72% (0.140 ± 0.009 pH unit/min, at pHi
7.6). Comparing to 164% increase by ATP in the reverse mode of
[Cl
]o/[HCO
]i
exchange (Fig. 4B), the increase rate was smaller
in the forward mode of [Cl
]o/[HCO
]i
exchange (Fig. 8C). This was possibly due to the
progressive increase in [Cl
]i by
the blockade of Cl
exit pathways in high
K+-containing solutions.
 |
DISCUSSION |
The coordinated actions of various transporters on both the
luminal and basolateral membranes of pancreatic duct cells perform transepithelial HCO
transport. Although the precise
mechanisms of how duct cells can secrete such high concentrations of
HCO
(up to 140 mM) are unknown, the
following two processes are generally accepted as important pathways of
ductal HCO
secretion (8). First, CFTR performs a
critical role in HCO
secretion. This is supported by
the observation that HCO
secretion is severely
defective in the pancreatic juice of CF patients (2, 3). Second,
Cl
/HCO
exchange at the luminal
membrane mediates significant portions of HCO
secretion and accounts for the transport of at least 70-80
mM concentrations of HCO
in the
pancreatic juice (8). Recently, we demonstrated that the activity of
luminal Cl
/HCO
exchange depends on
CFTR expression (9, 10). A series of studies support the notion that
CFTR-dependent pancreatic HCO
secretion
is critical for maintaining the patency of ductal trees (2, 3, 11). Thus, the impairment of this process was suggested to be an important pathologic mechanism in mutated CFTR-related pancreatic damage and
possibly other organ pathologies (4).
It has long been known that calcium agonists such as
cholecystokinin or acetylcholine can increase fluid and
HCO
secretion and that these have a strong
potentiatory effect when combined with cAMP agonists, such as secretin
or vasoactive intestinal polypeptide (VIP), in in
vivo experiments (1, 15). However, the molecular mechanisms
responsible for Ca2+-induced fluid and
HCO
secretion are largely unknown. Recently, it was
suggested that the activation of the Ca2+-activated
Cl
channel by P2R activation in pancreatic duct cells can
stimulate HCO
secretion by the parallel activation of electroneutral Cl
/HCO
exchange, as
a substitute for CFTR Cl
channel (25). However, this
study focused on electrogenic Cl
conductance, not on
electroneutral Cl
/HCO
exchange, the
transporter that actually mediates HCO
transport.
Moreover, the study was undertaken in nonpolarized cultures, and thus
it is impossible to isolate the luminal membrane-specific effect.
Our results demonstrate that calcium agonists activate CFTR- and
Cl
-dependent HCO
transport
in the luminal membrane of pancreatic duct cells. 1) Activation of P2R
or PAR stimulated luminal Cl
/HCO
exchange only in CAPAN-1 cells expressing WT-CFTR, but not in CFPAC-1
cells bearing an impaired CFTR, although the calcium agonists evoked
similar [Ca2+]i elevation in both
cells (Figs. 1 and 4). 2) The stimulatory effects of calcium agonists
on luminal Cl
/HCO
exchange were well
correlated with the duration of high
[Ca2+]i peak, and these effects
were completely abolished by BAPTA pretreatment (Fig. 7).
Although other Gq-coupled events such as the activation of PKC (27) or
the direct stimulation by heterotrimeric G proteins (28) cannot be
completely excluded as a possible stimulatory mechanism, the above
findings suggest that [Ca2+]i
increase is a prerequisite for the P2R- or PAR-mediated stimulation of
luminal Cl
/HCO
exchange. 3) Notably,
exogenous CFTR expression by Ad-CFTR transfection substantially
increased the stimulatory effect of calcium agonists on luminal
Cl
/HCO
exchange in CFPAC-1 cells (Fig. 6).
Although cAMP is regarded as the major intracellular signal for
HCO
secretion, agonists that evoke calcium signals
also stimulate HCO
secretion in many CFTR-expressing
epithelia. Therefore, it is not surprising that not only cAMP-activated
but also Ca2+-activated HCO
secretion
was found to be impaired in the intestinal epithelia of CFTR(
/
)
mice (29, 30). In the present study, purinergic stimulation increased luminal Cl
/HCO
exchange only by 27%
in CFTR-impaired CFPAC-1 cells. However, this value jumped to 164 and
246% in endogenous (CAPAN-1, Fig. 4B) and exogenous
CFTR-expressing cells (CFPAC-1 transfected with 200 m.o.i. of
Ad-CFTR, Fig. 6), respectively. These findings are comparable with the
data obtained from mouse duodenum in which cholinergic stimulation
increased HCO
secretion by 168% in CFTR(+/+) mice
but only by 67% in CFTR(
/
) mice (30). When combined, these results
suggest that CFTR plays an important role in the epithelial
HCO
secretion mediated by various intracellular
signals. Therefore, we expect that without the restoration of CFTR
expression in the luminal membrane, the application of calcium agonists
such as ATP may regain only a limited amount of HCO
secretion in CF epithelia, if any.
The present findings may be applicable to other CFTR-expressing
epithelia. For example, the primary sweat from CF patients is acidic
and hypertonic, suggesting that there is some defect in
HCO
-driven fluid secretion (31, 32). Interestingly,
the physiologic stimuli for sweat secretion, both sympathetic and
parasympathetic, are mediated by calcium-coupled cholinergic receptors.
Although it was originally hinted from studies upon sweat glands that
the disease-related protein, which we call now CFTR, is a
cAMP-activated Cl
channel (33, 34), the cAMP signal is
not the major physiologic signal in human sweat glands (35). According
to the present study, a defect in Ca2+-activated
HCO
transport, which depends on CFTR expression, can
be used to explain the puzzling abnormalities of CF sweat.
In addition to the classical cholinergic and cholecystokinin pathways,
recent studies have revealed the importance of other calcium-related
receptors, such as P2R and PAR, in the regulation of pancreatic fluid
and HCO
secretion. An interesting suggestion is that
cholinergic stimulation releases ATP from zymogen granules in acinar
cells, and that this in turn activates P2Rs in the luminal membrane of
duct cells (16). Combining the present data of P2R-induced
HCO
secretion with the above idea provides a
possible molecular basis for the strong potentiation of pancreatic
fluid secretion by cholinergic stimulations in vivo.
The activation of PARs, which may be prominent during pancreatic
inflammation due to proteolytic cleavage by tryptase released from mast
cells or autoactivated trypsin from acinar cells (23), evokes a
complicated pattern of pancreatic juice secretion, characterized by a
prompt increase followed by a transient suppression and a subsequent
protracted increase (14). Observations made in the present study may
provide explanations for this complicated pattern of secretion. Initial
PAR activation caused a single calcium peak, but subsequent application
of agonists did not evoke calcium signaling due to receptor
internalization and degradation. Cells began to regain their response
to PAR agonists 30 min after initial stimulation caused by new receptor
expression on the membrane (see Fig. 1 and the relevant result
section). Therefore, the secretory activity by PAR stimulation closely
follows [Ca2+]i increase and
subsequent Cl
/HCO
exchange activity.
In conclusion, the regulation of HCO
transport by
calcium signaling demonstrated here may be of particular significance
for the understanding of the physiologic and pathologic roles of
calcium-mediated bicarbonate secretion. Activation of CFTR- and
Cl
-dependent HCO
transport
provide the molecular basis for pancreatic fluid and bicarbonate
secretion by calcium agonists. In addition, impairments in the above
mechanism can explain the puzzling defect of calcium-induced
HCO
secretion in several CF epithelia including
intestinal mucosa and sweat glands, which, in turn, highlights the
general importance of CFTR in epithelial bicarbonate secretion induced
by various stimuli.