Secretory effects of ATP on nontransformed dog pancreatic duct epithelial cells

Toan D. Nguyen, Mark W. Moody, Christopher E. Savard, and Sum P. Lee

Department of Medicine, University of Washington, and Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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, beta ,gamma -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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Chemicals and reagents. ATP, UTP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), beta ,gamma -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
<IT>r</IT> = [ln(R<SUB>1</SUB>) − ln(R<SUB>2</SUB>)]/(<IT>t</IT><SUB>1</SUB> − <IT>t</IT><SUB>2</SUB>)
where R1 and R2 are the percentage of counts initially loaded remaining in the cells at times t1 and t2.

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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Abstract
Introduction
Methods
Results
Discussion
References

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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Fig. 1.   Effect of ATP and analogs on 125I- efflux from pancreatic duct epithelial cells (PDEC). 125I- efflux from PDEC was determined as detailed in METHODS, and the efflux rate coefficient (per min) was calculated. Each data point is the mean and SE from 3 experiments. A: after 1 min of baseline determination, ATP was added to the different final concentrations shown. B: after 1 min of baseline determination, 100 µM of either ATP or UTP was added. C: after 1 min of baseline determination, 100 µM of adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) or beta ,gamma -methylene-ATP was added. B and C: open circle , vehicle.

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 ATPgamma 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 beta ,gamma -methylene-ATP stimulated a smaller response. This potency profile, ATP = UTP = ATPgamma 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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Fig. 2.   Effect of inhibitors of Cl- channels on ATP-stimulated 125I- efflux from PDEC. Effect of 100 µM of ATP in the presence (bullet ) or absence (open circle ) of inhibitor was studied. 125I- efflux from PDEC was determined as detailed in METHODS, and the efflux rate coefficient (per min) was calculated. Each data point is the mean and SE from 3 experiments. A: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was added to a final concentration of 500 µM at the beginning of the experiment, and, after 1 min of baseline determination, 100 µM ATP was added. DMSO and ethanol were used to dissolve NPPB; its final concentration was 0.1% (vol/vol) and 0.9% (vol/vol), respectively, in both treatment and control buffers. B: diphenylamine-2-carboxylate (DPC) was added to a final concentration of 2.5 mM at the beginning of the experiment, and, after 1 min of baseline determination, 100 µM ATP was added. Ethanol was used to dissolve DPC; its final concentration was 1% (vol/vol) in both treatment and control buffers. C: DIDS was added to a final concentration of 500 µM at the beginning of the experiment, and, after 1 min of baseline determination, 100 µM ATP was added. DMSO was used to dissolve DIDS; its final concentration was 0.1% (vol/vol).

                              
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Table 1.   Inhibition of ATP-mediated activation of Cl- and K+ channels

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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Fig. 3.   Effect of ATP on 86Rb+ efflux from PDEC. 86Rb+ efflux from PDEC was determined as detailed in METHODS, and the efflux rate coefficient (per min) was calculated. Each data point is the mean and SE from 3 experiments. A: after 1 min of baseline determination, 100 µM of either ATP or UTP was added. open circle , Vehicle. B: BaCl2 (bullet ; 6 mM) or vehicle (open circle ) was added at the beginning of the experiment, and, after 1 min of baseline determination, 100 µM ATP was added. NaOH was used to dissolve BaCl2; its final concentration was 0.1 mM in both treatment and control buffers. C: charybdotoxin (bullet ; 100 nM) or vehicle (open circle ) was added at the beginning of the experiment, and, after 1 min of baseline determination, 100 µM ATP was added.

Ussing chamber studies. Because these PDEC exhibit normal tight junctions (22), a cell monolayer will form a molecular and electrical barrier between the luminal compartment, in contact with the apical surface, and the serosal compartment, in contact with the basolateral surface. This property was exploited to localize the P2Y2 receptors and to study apical Cl- and basolateral K+ conductances.

Permeabilization of the basolateral membrane of PDEC to small monovalent ions after serosal addition of nystatin was used to exclusively study the conductances located on the apical membrane. A serosal-to-luminal 135 mM Cl- gradient was generated to drive Cl- across activated apical Cl- conductances; this electrogenic flow was reflected as an increased Isc. In such a system, UTP stimulated an increased Isc when added to either the serosal or the luminal side of PDEC monolayers (Fig. 4A). The response after addition to either side was sustained; the mean peak Isc increase stimulated by serosal UTP was 6.84 ± 1.33 µA/cm2, whereas the peak Isc increase stimulated by luminal UTP was 7.65 ± 1.85 µA/cm2 (n = 3). The ability of UTP to stimulate secretion from either the serosal or the luminal compartment suggests that the corresponding P2Y2 receptors are located on both the apical and basolateral sides of these PDEC.


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Fig. 4.   Effect of UTP on apical Cl- conductance using permeabilized PDEC monolayers mounted in Ussing chambers. As described in METHODS, monolayers of confluent PDEC were mounted in Ussing chambers, and nystatin was added to the serosal compartment to permeabilize the basolateral membrane and target the apical membrane for studies. Short-circuit current (Isc; µA/filter of 0.95 cm2) measured in the presence of a serosal-to-luminal 135 mM Cl- gradient was monitored. In A-D, both tracings were derived from monolayers cultured and studied at the same time. Each experiment was repeated at least 3 times. A: after 5 min of baseline determination, UTP was added to a final concentration of 100 µM to either the serosal compartment, in contact with the basolateral membrane, or the luminal compartment, in contact with the apical membrane. UTP stimulated an increased Isc from either the luminal or serosal compartment in all 3 experiments; the average Isc increase was 6.84 ± 1.33 µA/cm2 for the serosal addition and 7.65 ± 1.85 µA/cm2 for the luminal addition. B: cells were preincubated with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (+BAPTA) or vehicle (control) for >= 10 min. After 5 min of baseline monitoring, 100 µM UTP was added to the serosal and luminal compartments of both the control and BAPTA-treated cells. Suppression of UTP effect by BAPTA was observed in all 3 experiments. DMSO was used to dissolve BAPTA-AM; its final concentration was 0.1% (vol/vol) for both treated and control monolayers. C: after 10 min of baseline observation, 500 µM NPPB (+NPPB) or vehicle (control) was added to the luminal compartment, and, after an additional 5 min, 100 µM UTP was added to the serosal and luminal compartments of both the control and NPPB-treated monolayers. DMSO and ethanol were used to dissolve NPPB; its final concentration was 0.1% (vol/vol) and 0.9% (vol/vol), respectively, in both treatment and control buffers. Suppression of UTP effect by NPPB was observed in all 3 experiments. D: after 5 min for baseline observation, 500 µM DIDS (+DIDS) or vehicle (control) was added to the luminal compartment and, after an additional 5 min, 100 µM UTP was added to the serosal and luminal compartments of both control and DIDS-treated monolayers. DMSO was used to dissolve DIDS; its final concentration was 0.1% (vol/vol). DIDS-treated monolayers showed 58 ± 16% (peak Isc increase) or 55 ± 15% (area under curve, 10 min after addition of UTP) of the control response (determined from 4 experiments).

Correlation between Isc increases and Cl- flow through activated Cl- channels was also verified. Indeed, when the Cl- gradient was reversed (luminal-to-serosal instead of serosal-to-luminal), the polarity of the Isc change was also reversed, confirming the dependence of these Isc changes on Cl- flow (data not shown). The Isc increase stimulated by UTP was abolished by 500 µM NPPB, added into the luminal compartment to inhibit the apical membrane Cl- conductances (Fig. 4C). It was partially inhibited by 500 µM DIDS, also added into the luminal compartment (Fig. 4D). When the peak Isc increases stimulated by UTP were compared, the mean Isc increase in the presence of DIDS was 58 ± 16% of control; when the areas under the curve (10-min period after addition of UTP) were compared, the mean area in the presence of DIDS was 55 ± 15% of control (n = 4). The complete inhibition by NPPB and the partial inhibition by DIDS in Ussing chambers are comparable to the effects observed with 125I- efflux studies.

The dependence of this secretory effect on an increased [Ca2+]i was also confirmed using BAPTA-AM (Fig. 4B). Preincubation of PDEC with 50 µM BAPTA-AM for >= 10 min to deplete [Ca2+]i also abolished the subsequent response to UTP.

The effect of UTP on the basolateral Ca2+-activated K+ channel was examined in a similar manner, except that the apical membrane was permeabilized and a luminal-to-serosal gradient of 114 mM K+ was generated. Ouabain was also added to inhibit the Na+-K+-ATPase pump. Under these conditions, UTP also stimulated an increased Isc of 24.4 ± 3.2 µA/cm2 (n = 7). When the K+ gradient was reversed from luminal-to-serosal to serosal-to-luminal, the polarity of the Isc change was also reversed, suggesting that the Isc change reflected K+ flow (Fig. 5A). The smaller amplitude of the Isc change exhibited with the serosal-to-luminal gradient may reflect a relative directionality for the K+ conductance; it was previously observed when Ca2+ ionophore A-23187 was used (19a).


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Fig. 5.   Effect of UTP on basolateral K+ conductance using permeabilized PDEC monolayers mounted in Ussing chambers. As described in METHODS, monolayers of confluent PDEC were mounted in Ussing chambers, and nystatin was added to the luminal compartment to permeabilize the apical membrane and target the basolateral membrane for studies. Isc (µA/filter of 0.95 cm2) measured in the presence of a luminal-to-serosal 114 mM K+ gradient (unless specified otherwise) was monitored. In A and B, both tracings were obtained from monolayers cultured and studied at the same time. Each experiment was repeated at least 3 times. A: after 5 min of baseline monitoring, UTP was added to both compartments of permeabilized monolayers, subject to either a luminal-to-serosal or serosal-to-luminal K+ gradient. Isc increase stimulated by UTP and its reversal upon reversal of the K+ gradient were observed in 3 experiments. B: after 5 min of baseline monitoring, 100 nM charybdotoxin (+charybdotoxin) or vehicle (control) was added to the serosal compartment and, after an additional 5 min, 100 µM UTP was added to the serosal and luminal compartments of both the control and charybdotoxin-treated monolayers. Inhibition of UTP effect by charybdotoxin was observed in all 3 experiments.

The efffect of UTP was also abolished by charybdotoxin, added onto the serosal compartment to inhibit basolateral K+ conductance (Fig. 5B). This inhibition was observed with 86Rb+ efflux studies and verifies that the Isc stimulated under these conditions reflects K+ flow through the Ca2+-activated K+ channel.

Studies of mucin secretion. As shown in Table 2, ATP stimulated an increase in mucin secretion in a dose-dependent manner at concentrations >= 10 µM. Consistent with the 125I- efflux studies, an equivalent response was also obtained with UTP and ATPgamma S. Agents that stimulated an increase in [Ca2+]i, such as thapsigargin (10 µM) or A-23187 (1 µM), also stimulated mucin secretion, although no response was obtained with adenosine.

                              
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Table 2.   Effect of ATP on mucin secretion by PDEC

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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Fig. 6.   Effect of ATP on PDEC intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in PDEC was determined using fura 2 as described in METHODS, and the ratio of fluorescence emitted at 510 nm when the cells were excited at 340 and 380 nm is shown. A: after 5 min of baseline determination, cells were exposed to 100 µM ATP. After an additional 10 min, cells were exposed to 14 µM ionomycin to permeate the cells to the 1 mM CaCl2 in the buffer. Each tracing is derived from an individual cell, and data are representative of 3 experiments. B: after 10 min of baseline determination, cells were exposed to 10 µM thapsigargin. After an additional 15 min, cells were exposed to 100 µM ATP and, after an additional 5 min, exposed to 14 µM ionomycin. Each tracing is derived from an individual cell, and data are representative of 4 experiments. DMSO was used to dissolve thapsigargin and ionomycin; its final concentration was 0.2% (vol/vol).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 = ATPgamma S > beta ,gamma -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, ATPgamma 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 beta ,gamma -methylene-ATP, which shows a smaller, delayed response. Alternatively, it is possible that beta ,gamma -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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