Department of Medicine, University of Washington, and Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108
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ABSTRACT |
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Extracellular
triphosphate nucleotides, such as ATP, may regulate various cellular
functions through specific cell surface receptors. We examine in this
report the different secretory effects of ATP and analogs on
nontransformed dog pancreatic duct epithelial cells (PDEC). We observed
that 1) ATP, UTP, adenosine
5'-O-(3-thiotriphosphate), and,
to a lesser extent, ,
-methylene-ATP, but not adenosine, stimulated
125I
efflux from PDEC, suggesting a primary role for
P2Y2 receptors, 2) ATP-stimulated
125I
efflux was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid,
diphenylamine-2-carboxylate, and DIDS, suggesting mediation through
Ca2+-activated
Cl
channels,
3) ATP stimulated an
86Rb+
efflux sensitive to BaCl2 and
charybdotoxin, thus likely occurring through
Ca2+-activated
K+ channels,
4) serosal or luminal addition of
UTP activated apical Cl
conductance and basolateral K+
conductance when nystatin-permeabilized PDEC were studied in an Ussing
chamber, suggesting the expression of
P2Y2 receptors on both sides of
the cell, 5) ATP stimulated mucin
secretion, and 6) ATP increases
intracellular Ca2+ concentration
([Ca2+]i).
In conclusion, ATP and UTP interact with
P2Y2 receptors on nontransformed
PDEC to increase
[Ca2+]i,
stimulate mucin secretion, and activate ion conductances; these
findings have implications for pancreatic exocrine function in both
health and disease, such as cystic fibrosis.
chloride channels; potassium channels; mucin; short-circuit current; cystic fibrosis; adenosine 5'-triphosphate
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INTRODUCTION |
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EXTRACELLULAR TRIPHOSPHATE nucleotides, such as ATP and UTP, may regulate various cellular functions. Indeed, the ability of these compounds to stimulate secretion by airway epithelial cells is the basis for the proposed use of UTP in the treatment of cystic fibrosis (CF; see Ref. 12). In the digestive system, ATP has many secretory effects on cells from hepatic (9), biliary (16), and colonic (8, 17) origin. In the pancreas, ATP has recently been shown to stimulate the secretion of anions by CFPAC-1 cells, derived from a pancreatic adenocarcinoma of a patient with CF (5). In a separate study, ATP also stimulated the secretion of mucin by Capan-1 cells, derived from a different pancreatic adenocarcinoma (18). These effects of ATP are mostly mediated by receptors that recognize both ATP and UTP, currently designated as P2Y2 receptors (or P2U receptors in the older nomenclature; see Ref. 11).
We recently established a method for long-term culture of
nontransformed epithelial cells from the main pancreatic duct of a dog
(22). These cells proved to be well differentiated and polarized and to
possess many of the characteristic functions of pancreatic duct
epithelial cells (PDEC), such as mucin secretion (22), cAMP and
Ca2+-activated
Cl channels (19), and
Ca2+-activated
K+ channel (19a). We now describe
the effect of ATP and analogs on these cells. Specifically, we
determined 1) the activation of
Cl
and
K+ conductances by ATP,
2) the stimulation of mucin
secretion by ATP, 3) the type and
localization of the responsible receptors, and
4) the signal-transduction pathway
involved.
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METHODS |
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Chemicals and reagents. ATP, UTP,
adenosine
5'-O-(3-thiotriphosphate)
(ATPS),
,
-methylene-ATP, adenosine,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),
charybdotoxin, and tissue culture medium and supplements were from
Sigma (St. Louis, MO). Diphenylamine-2-carboxylate (DPC) was from Fluka
(Ronkonkoma, NY); 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)
was from Research Biochemicals International (Natick, MA); and
thapsigargin, ionomycin, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were from Calbiochem (La Jolla, CA). Na125I (16 mCi/mg iodide) was
purchased from Amersham (Arlington Heights, IL), and
86RbCl (4.66 mCi/mg rubidium) was
from NEN (Boston, MA).
N-acetyl-D-[3H]glucosamine
(4.5 Ci/mmol) was from ICN (Costa Mesa, CA).
Cell culture. Dog PDEC were isolated
from the accessory pancreatic duct of a dog. These cells were
subsequently cultured in Eagle's MEM containing 10% fetal bovine
serum, 2 mM L-glutamine, 20 mM HEPES, 100 IU/ml penicillin,
100 µg/ml streptomycin, 5 µg/ml bovine insulin, 5 µg/ml human
transferrin, and 5 ng/ml sodium selenite and were grown on
24-mm-diameter Transwell inserts (Costar, Cambridge, MA), coated with
0.5 ml of a 1:1 solution of Eagle's MEM/Vitrogen (Collagen, Palo Alto,
CA). The Transwell inserts allow the PDEC to share a common medium with
a feeder layer of myofibroblasts cultured on the bottom of the well in
which the insert is suspended. The myofibroblast cells were isolated
from the serosal surface of a normal human gallbladder using trypsin (21); these cells produce growth factors necessary for maintaining and
propagating well-differentiated PDEC. We previously demonstrated that
these PDEC have many of the characteristics expected of pancreatic duct
cells, such as mucin secretion and expression of
Ca2+-activated
K+ channels (19a),
Ca2+-activated
Cl channels, and
cAMP-activated CF transmembrane conductance regulator (CFTR)
Cl
channels (19, 22). The
cells used in this report were between passages
9 and 30.
Efflux studies. The use of cellular
125I
and
86Rb+
effluxes to study the activation of
Cl
and
K+ conductances has been validated
(29) and was previously effective for characterizing the
Cl
and
K+ conductances on these PDEC
(19a; see Ref. 19).
PDEC were grown to confluence on Transwell inserts as described above.
The membranes containing the cells were then excised from the insert
and washed two times with 1 ml of efflux buffer consisting of (in mM)
140 NaCl, 4.7 KCl, 1.2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES,
pH 7.4. The cells on these membranes were then loaded with the
radioactive tracer through a 45-min incubation at 37°C with 1.5 ml
of efflux buffer containing either ~2 µCi/ml Na125I or ~1 µCi/ml
86RbCl. The cells were next washed
four times with 2 ml of isotope-free buffer. The isotope efflux was
measured by sequential addition and removal of 1 ml of isotope-free
buffer at 15-s intervals for a 5-min period. To establish baseline
efflux, no secretagogue was added for the first minute; in the
remaining 4 min, the secretagogue tested was included in the buffer.
When inhibitors were tested, they were added at the beginning of the
experiment (including the baseline monitoring period). The
radioactivity of these sequential samples and the radioactivity
remaining in the cells at the end of the experiment were measured for
125I
using a gamma counter (Isodata 120; ICN, Huntsville, AL) and for
86Rb+
using a liquid scintillation counter (Tri-Carb model 1600TR; Packard,
Meriden, CT).
The radioactivity contained in the cells at a particular time point was calculated as the sum of the radioactivities released in subsequent efflux samples and remaining in the cells at the end of the experiment. The efflux rate coefficient (r) for a certain time interval was also calculated using the formula
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In certain experiments, peak stimulated efflux rate coefficients were compared. These peak rates were calculated by subtracting the baseline rate coefficient, the lowest efflux rate before the addition of ATP, from the peak stimulated efflux rate coefficient, the highest efflux rate after the addition of ATP.
Ussing chamber studies. Confluent monolayers of PDEC and their supporting membrane were excised from the Transwell system and mounted in modified Ussing chambers with an aperture area of 0.95 cm2. In this system, the luminal compartment is in contact with the apical surface of the PDEC, whereas the serosal compartment is in contact with the basolateral surface of the cell.
To study apical Cl
conductance, the basolateral surface of the PDEC was permeabilized by
adding 0.36 mg/ml nystatin to the serosal compartment for 20 min before
the addition of the agonist or inhibitor. A serosal-to-luminal
Cl
gradient was generated
by adding, to the serosal compartment, a buffer consisting of (in mM)
135 NaCl, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4,
0.6 KH2PO4,
10 HEPES, and 10 glucose and, to the luminal compartment, a buffer
consisting of (in mM) 135 sodium gluconic acid, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4,
0.6 KH2PO4,
10 HEPES, and 10 glucose.
To study basolateral K+ conductance, the apical surface of PDEC was permeabilized with nystatin. A luminal-to-serosal K+ gradient was generated by adding, to the luminal compartment, a buffer consisting of (in mM) 10 NaCl, 1.25 CaCl2, 1 MgCl2, 118 potassium gluconate, 10 HEPES, and 25 glucose, titrated to pH 7.4 with NaOH, and, to the serosal compartment, a buffer consisting of (in mM) 10 NaCl, 1.25 CaCl2, 1 MgCl2, 4 potassium gluconate, 114 N-methyl-D-glucamine, 10 HEPES, and 25 glucose, titrated to 7.4 with acetic acid. The buffer in both compartments was warmed with a water jacket at 37°C. Ouabain (100 µM) was added to inhibit the Na+-K+-ATPase pump and maintain intracellular ATP.
Spontaneous potential differences were short-circuited using an automatic voltage clamp (model DVC-1000; WPI, Sarasota, FL) with an Ag-AgCl2 electrode, and the current necessary to maintain this short-circuit current (Isc) was continuously recorded using a MP100 analog-to-digital converter and the Acknowledge 2.0 software program (BioPak Systems, Goleta, CA). For studies of resistance, a current of 100 µA was maintained across the monolayer, the resulting voltage was recorded, and the corresponding resistance was calculated using Ohm's law (voltage = current × resistance). Instrument calibration was performed before each experiment using a membrane without cells.
Mucin secretion studies. Mucins synthesized by PDEC grown to confluence were labeled for 24 h using acetyl-D-[3H]glucosamine (~2 µCi/Transwell insert), a mucin precursor. The cell monolayers were then washed with sterile 100 mM NaCl and 20 mM Na2HPO4, pH 7.4, reincubated in a serum-free medium, and treated for 2 h with ATP or analogs added to the apical compartment. After this incubation, the medium of the apical compartment was collected and centrifuged at 1,000 g for 5 min to discard cell debris, and a 0.5-ml aliquot of the supernatant was then sampled for further studies. The labeled glycoproteins released in this sample were next precipitated using 10% tricholoracetic acid-1% phosphotungstic acid, and the associated radioactivity was determined in a scintillation counter. This radioactivity was then extrapolated to the total volume of the overlying medium and expressed as a percentage of the control values obtained with untreated cells. Cell integrity was verified by monitoring the release of lactate dehydrogenase. This assay technique has been previously validated (13).
Measurement of intracellular cytosolic Ca2+. PDEC were seeded at low density on glass coverslips and were studied the next day. These cells were loaded with 4 µM fura 2-AM for 60 min at 37°C in culture medium, washed, mounted onto a LU-CB1 Leiden microincubator (Medical System, Greenvale, NY), and perfused with Ringer solution at 37°C. Fluorescence in single cells was detected using a Diaphot 200 inverted microscope (Nikon, Tokyo, Japan), fitted with a Xenon short arc lamp (Ushio, Tokyo, Japan) and a TE-CCD detector (Princeton Instrument, Trenton, NJ), and programmed with the Image-1 Metamorph sofware (Universal Imaging, Westchester, PA). In this system, intracellular free Ca2+ ([Ca2+]i) is reflected by the ratio of the fluorescence emitted at 510 nm when the cells are alternately excited at 340 and 380 nm.
Statistics. Unless specified otherwise, results were expressed as means ± SE. When appropriate, statistical significance was determined with Student's t-test using the Statview 512+ software program (Brainpower, Calabasas, CA). ![]() |
RESULTS |
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Studies of iodide efflux. The effect
of ATP on
125I
efflux by PDEC was first assessed. As shown in Fig.
1A,
ATP stimulated
125I
efflux in a concentration-dependent manner: a small response was
detected at 10 µM, and the maximal response was observed at 1 mM,
with peak efflux rate coefficients of 0.36 ± 0.15/min and 1.35 ± 0.16/min, respectively (mean ± SE,
n = 3). This response was
rapid, as the peak efflux rates occurred 15-30 s after the addition of ATP.
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The receptors mediating this ATP effect were characterized next. We
first demonstrated that 100 µM adenosine did not stimulate an
increase in
125I
efflux (data not shown), suggesting that adenosine receptors are absent
in these cells. Thus it is unlikely that ATP produces an effect after
conversion to adenosine and through an adenosine receptor. Furthermore,
as shown in Fig. 1C, an increased
125I
efflux was also observed with 100 µM ATP
S, a nonhydrolyzable analog of ATP, suggesting that degradation of ATP was not necessary for
stimulation of
125I
efflux. To further characterize the responsible receptor subtype, the
effects of different ATP analogs were studied. As shown in Fig. 1,
B and
C, both ATP and UTP, at 100 µM,
stimulated a similar response in these cells, whereas 100 µM of
,
-methylene-ATP stimulated a smaller response. This potency
profile, ATP = UTP = ATP
S, is characteristic for the
P2Y2 receptor (8, 9, 14, 15, 20,
30, 31).
To verify that the ATP-stimulated
125I
efflux occurred through activated
Cl
conductances, different
inhibitors of Cl
channels
were tested. As shown in Fig.
2 and Table
1, 500 µM NPPB, 2.5 mM DPC, and 500 µM
DIDS inhibited ATP-stimulated
125I
efflux by 96, 87, and 51%, respectively. When this inhibitory profile
was compared with the ones previously observed for the Ca2+-activated
Cl
channel (93%, 80%, and
37% inhibition, respectively, by NPPB, DPC, and DIDS) and the
cAMP-activated CFTR Cl
channel (53%, 100%, and no inhibition, respectively, by NPPB, DPC,
and DIDS; see Ref. 19), this pattern most closely matched that of the
Ca2+-activated
Cl
channel. Thus the
125I
efflux stimulated by ATP was mainly mediated through
Ca2+-activated
Cl
channels on PDEC.
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Studies of 86Rb+ efflux. Through studies of 86Rb+ efflux, we recently demonstrated that PDEC also express functional basolateral Ca2+-activated K+ channels (19a). The effect of ATP on the efflux of 86Rb+ from PDEC was therefore evaluated. In Fig. 3A, 100 µM of either ATP or UTP stimulated an increase in 86Rb+ efflux. As shown in Fig. 3, B and C, and in Table 1, this efflux was inhibited by 44% with 6 mM BaCl2 and by 64% with 100 nM charybdotoxin, agents that classically inhibit K+ channels. These values are similar to the inhibitions of 36 and 63% produced, respectively, by BaCl2 and charybdotoxin, when the Ca2+ ionophore A-23187 was used to stimulate 86Rb+ in PDEC (19a). These findings suggest that ATP and UTP also stimulate Ca2+-activated K+ channels in PDEC.
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Studies of cytosolic Ca2+. Because the signal-transduction pathway mediated by P2Y2 receptors usually involves an increased [Ca2+]i, we determined the effect of ATP on [Ca2+]i, using the fluorescent chelator fura 2. As shown in Fig. 6A, 100 µM ATP stimulated a transient increase in [Ca2+]i. When the experiment was repeated with 10 µM thapsigargin, which increases cytosolic Ca2+ and depletes intracellular stores by inhibiting the reuptake of Ca2+ into endoplasmic reticulum, an increase in [Ca2+]i was also noted (Fig. 6B). Of note, prior treatment with thapsigargin preempted the subsequent response to ATP, suggesting that the ATP-stimulated increase in [Ca2+]i required intact intracellular stores of Ca2+.
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DISCUSSION |
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Although the secretory function of PDEC is an important component of
the exocrine pancreas, functional studies in this field have been
hampered by the difficulty in developing a practical and representative
model for the secretory function of these cells. The method developed
by Oda et al. (22) for successfully culturing nontransformed dog PDEC
could provide a solution to this issue. Indeed, we have shown that
these cells secrete mucin (22) and functionally express two distinct
types of Cl channels, the
cAMP-activated CFTR Cl
channel and a Ca2+-activated
Cl
channel (19), as well as
a Ca2+-activated basolateral
K+ channel (19a). These cultured
nontransformed PDEC would therefore constitute an ideal model to study
the effects of ATP on pancreatic ductal secretion.
We observed that P2Y2 (previously
known as P2U) receptors mediated
the effects of ATP. This conclusion is based on the relative secretory
potency of ATP and its analogs: ATP = UTP = ATPS >
,
-methyleneATP >> adenosine (8, 10, 30, 31). In contrast to T84 colonic and airway epithelial cells, in which adenosine receptors have been shown to partially mediate the effects of ATP (8,
27, 28), in PDEC, the lack of response to a high concentration of
adenosine suggests that none of the adenosine receptors is involved.
The absence of these receptors suggests that, for PDEC, the active
agent is ATP and not its degradation product, adenosine. This
conclusion is also consistent with the potent effect of the
nonhydrolyzable ATP analog, ATP
S.
It should be recognized, however, that although the current data
suggest a major role for P2Y2
receptors in mediating the ATP effect, contribution from other
P2 receptor subtypes cannot be
excluded. Such receptors may mediate the effect of
,
-methylene-ATP, which shows a smaller, delayed response.
Alternatively, it is possible that
,
-methylene-ATP stimulates a
response through a degradation product that can interact with
P2Y2 receptors; the delayed
diminished response may just reflect such degradation. In any case, the
P2Y2 receptor appears to serve a
major role in mediating the effect of ATP and UTP on PDEC.
P2Y2 receptors have been shown to mediate the effects of ATP in many epithelial cell types, including nasal (12), SPOC1 airway goblet (1), colonic adenocarcinoma T84 (8), pancreatic adenocarcinoma CFPAC (5), bile duct (16), and HTC hepatoma (10) cells. In the pancreas, P2Y2 purinoceptors also appear to stimulate the anion secretion from CFPAC pancreatic adenocarcinoma cells (5). However, because only ATP, and not UTP, stimulated mucin secretion from CFTR-corrected CFPAC cells and Capan-1 cells, it is possible that P2Y2 purinoceptors may not mediate the effect of ATP on mucin secretion from these pancreatic adenocarcinoma cells (18). These latter findings may be reflective of the malignant origin of these cells and highlight the desirability of studies performed with nontransformed, normal PDEC.
The ability of the PDEC to express tight junctions so that cell
monolayers form a physical barrier between the apical and basolateral
surfaces allowed us to functionally determine the polar distribution of
the P2Y2 receptor in Ussing
chambers. The ability of UTP to activate
Cl conductances when added
to either the serosal or luminal compartment suggests that the
P2Y2 receptors are located on both
the apical and basolateral plasma membranes of PDEC. This localization
is different from the restricted apical localization of
P2Y2 receptors in
Necturus gallbladder cells (7) and in
CFPAC cells (5) and the basolateral localization in T84 cells (8).
Using basolaterally or apically permeabilized cells to study the
conductance, respectively, of the apical or basolateral membrane, and
using a Cl or
K+ gradient to examine the
Cl
or
K+ conductances on these
membranes, we were able to correlate
125I
efflux with the activation of apical
Cl
conductances and to
correlate
86Rb+
efflux with the activation of basolateral
K+ conductances. Indeed, UTP
stimulated an increased
Isc from
basolaterally permeabilized cells that was dependent on the direction
of the Cl
gradient,
suppressed by NPPB, and partially inhibited by DIDS (Ca2+-activated
Cl
conductance). UTP also
stimulated an increased
Isc from apically permeabilized cells that was dependent on the direction of the K+ gradient and inhibited by
charybdotoxin (Ca2+-activated
K+ conductance).
In many systems, the P2Y2 receptor
is coupled to increased
[Ca2+]i.
In PDEC, stimulation of this pathway is consistent with the activation
by UTP of Ca2+-activated
Cl and
K+ conductances. The primary role
for increased
[Ca2+]i
was further supported by the ability of the intracellular
Ca2+ chelator BAPTA-AM to inhibit
the activation by UTP of Cl
conductance in the Ussing chamber. Finally, activation of this pathway
was also confirmed by direct measurement of
[Ca2+]i
in PDEC. Because this increase was mimicked and subsequently preempted
by thapsigargin, it was primarily derived from
Ca2+ released from intracellular
stores.
ATP and UTP also stimulated mucin secretion from PDEC in this report. The ability to study different secretory functions in a single cell system attests to the versatility of these cultured nontransformed PDEC as a model for pancreatic ductal secretion. In contrast, in the adenocarcinoma CFPAC pancreatic cells, ATP and UTP, acting through P2Y2 purinoceptors, stimulated anion transport but not mucin secretion, whereas in Capan-1 and CFTR-corrected CFPAC cells, ATP, but not UTP, stimulated mucin secretion (5, 18).
In this report, the ATP effect could be elicited starting at 10 µM and was maximal at 0.1-1 mM; comparable dose dependencies have been described for Capan-1 and CFPAC cells expressing CFTR (18), T84 colonic cells (28), and tracheal SPOC-1 cells (1). When the P2Y2 receptor was cloned and expressed in astrocytoma cells, the maximal response on [Ca2+]i was obtained with 10-100 µM ATP or UTP (15, 23). The concentration of ATP or UTP used in most experiments in this report (100 µM) was equal to or lower than the concentration used in many studies of the secretory effects of ATP (e.g., see Refs. 1, 5, 7, 8, 10, 16, 18, 28, 30).
Further studies will be required to clarify the implications of these
findings for pancreatic exocrine function. To our knowledge, the
concentration of ATP has not been determined in pancreatic juice.
However, release of ATP from different cells, including cholangiocytes,
has been reported (6). This extracellular release may occur through the
CFTR Cl channel (24, 25,
26) or the multidrug resistance channel (3), even though some aspects
of these transports are still controversial (2). We have previously
demonstrated the presence of CFTR on PDEC (19), confirming the presence
of at least one potential pathway for extracellular release of ATP. In
human bile, ATP, possibly released through these mechanisms, has been
measured and averages 1.67 µM, with an upper range of 6.5 µM (6).
If similar concentrations of ATP are present in pancreatic juice, ATP
may modulate the secretory function of PDEC through the demonstrated apical receptors. Of interest, because bile refluxes into the pancreatic duct in biliary pancreatitis, a pathological contribution of
biliary ATP in this condition should be explored.
In CF, the defective expression or function of the CFTR
Cl channel can
theoretically be bypassed by stimulating the
Ca2+-activated
Cl
channel via
P2Y2 receptors. UTP is the
preferred agonist for this clinical use because, unlike ATP, it is not
degraded into products that induce bronchoconstriction in patients with
asthma. Trials using UTP for treatment of the pulmonary complications of CF have been promising (4, 12). Although pulmonary complications produce the majority of the morbidity and mortality of CF,
complications involving the digestive system, especially pancreatic
insufficiency, are gaining prominence with the longer survival of CF
patients. Pancreatic insufficiency in CF can be traced back to the
defective function of the CFTR
Cl
channel on PDEC. The
current demonstration of P2Y2
receptors on PDEC suggests that a similar strategy can be considered
for pancreatic disease in CF. Whereas delivery of UTP to the PDEC will
be more problematic than delivery to respiratory cells, the expression
of P2Y2 receptors on both sides of
the PDEC will allow a wider range of options.
In summary, we have demonstrated the expression of
P2Y2 receptors on both the
basolateral and apical surfaces of nontransformed PDEC, which can
interact with ATP or UTP to increase
[Ca2+]i,
activate Ca2+-activated
Cl and
K+ conductances, and stimulate
mucin secretion. These findings support further studies into the
potential role for extracellular nucleotides in pancreatic ductal
secretion and into the therapeutic potential of UTP in pancreatic CF.
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ACKNOWLEDGEMENTS |
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The intracellular studies were performed using the Calcium Imaging Facility of the Puget Sound Dept. of Veterans Affairs, and we thank Dr. Hans van Brederode for helping with the use of this facility.
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FOOTNOTES |
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This research was funded in part by grants from the Department of Veterans Affairs (to T. D. Nguyen and S. P. Lee), the National Institutes of Health (DK-50246 to S. P. Lee), and the Cystic Fibrosis Foundation (to T. D. Nguyen).
Address for reprint requests: T. D. Nguyen, GI Section (111 GI), VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108.
Received 11 August 1997; accepted in final form 30 March 1998.
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