P2Y11, a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

T. D. Nguyen, S. Meichle, U. S. Kim, T. Wong, and M. W. Moody

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y2 receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+]i) and stimulate Ca2+-activated Cl- and K+ channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl- channels. ATP, 2-methylthio-ATP, and ATP-gamma -S stimulated a 4- to 10-fold cAMP increase with EC50 of 10-100 µM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y2 or P1 receptors. Although UTP stimulated an 125I- efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl- conductances through P2Y2-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current (Isc) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+]i. Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type Isc increase, whereas serosal ATP stimulated a large cAMP-type Isc response. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl- conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y11 receptor mRNA, the only P2Y receptor acting via cAMP.

iodide efflux; adenosine 5'-triphosphate; Ussing chamber


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

THE EXTRACELLULAR FUNCTION of ATP and other nucleotides as messenger agents in regulating cellular metabolism and ion transport is now well established. The effects of these agents are mediated through specific P2 receptors composed of two major subclasses, the P2X receptors possessing two transmembrane domains and intrinsic ion channel activity and the P2Y receptors possessing seven transmembrane domains (8, 23, 24). The P2Y family includes selective purinoceptors (P2Y1 receptor activated preferentially by ADP and ATP), nucleotide receptors responsive to adenine and uracil nucleotides (P2Y2 receptor activated equipotently by ATP and UTP and P2Y8 receptor activated equally by all triphosphate nucleotides), and pyrimidinoceptors (P2Y3 and P2Y6 receptors activated preferentially by UDP and P2Y4 receptor activated preferentially by UTP). Although these P2Y receptor subtypes couple to phospholipase C to stimulate an increase in intracellular free Ca2+ concentration [Ca2+]i (1, 9, 23), the recently cloned P2Y11 receptor has the unique property of activating adenylyl cyclase in addition to phospholipase C (4). This receptor exhibits 33% amino acid identity with the P2Y1 receptor, its closest homolog. When this receptor was expressed in 1321N1 astrocytoma cells and in CHO-K1 cells, the rank order of agonist potency was ATP-gamma -S = 2' and 3'-O-(4-benzoylbenzoyl)-ATP > ATP > 2-methylthio-ATP > ADP; UTP and UDP were inactive (4, 5). Northern blot probing showed the P2Y11 receptor to be present in HL-60 leukemia cells and, weakly, in the small intestine. Madin-Darby canine kidney (MDCK) cells are the only other known cell type that expresses these receptors (22). Although P2Y11 receptors may mediate cAMP-dependent differentiation in HL-60 cells, the expression and function of these receptors in different epithelial cells remain to be further defined (6, 11, 27). In this report, we define a secretory role for P2Y11 receptors on pancreatic duct epithelial cells (PDEC).

PDEC mediate secretion of fluid and electrolytes and, together with acinar cells, which mediate secretion of digestive proenzymes, constitute pancreatic exocrine secretion. The secretory function of PDEC results from many ion transport pathways, including, on the apical surface of the cell, the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, a distinct Ca2+-activated Cl- channel, and a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Cl- exchanger, and, on the basolateral surface of the cell, K+ channels, a Na+-(HCO3)n cotransporter, and a Na+/H+ antiporter. We previously reported (16) that ATP and UTP interact with specific P2Y2 receptors expressed on both apical and basolateral surfaces of cultured dog PDEC to promote an increase in [Ca2+]i, stimulate mucin secretion, and activate both Cl- and K+ conductances. We now report that ATP also interacts with a P2Y receptor (most likely the P2Y11 receptor) on the basolateral surface of these cells to activate cAMP-dependent Cl- channels. To our knowledge, this is the first description of a secretory function for these receptors in epithelial cells.


    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Chemicals and reagents. ATP, UTP, ATP-gamma -S, 2-methylthio-ATP, alpha ,beta -methylene ATP, adenosine, suramin, reactive blue, and tissue culture medium and supplements were from Sigma (St. Louis, MO), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was from Calbiochem (La Jolla, CA). Na125I (16 mCi/mg iodide) was purchased from Amersham (Arlington Heights, IL).

Cell culture. Canine PDEC isolated from the accessory pancreatic duct were cultured on Transwell inserts (Costar, Cambridge, MA) coated with Vitrogen (Collagen Corporation, Palo Alto, CA) and suspended over a feeder layer of normal human gallbladder myofibroblasts. This system exposes the PDEC to growth factors from myofibroblasts that are necessary for their propagation and differentiation (20). We previously demonstrated (15, 17) that these PDEC have many of the characteristics expected of pancreatic duct cells, such as mucin secretion and expression of Ca2+-activated K+ channels, Ca2+-activated Cl- channels, and cAMP-activated CFTR Cl- channels. These cells were also used successfully to examine the secretory effects of agents acting via cAMP, such as vasoactive intestinal polypeptide (15, 20), and increased [Ca2+]i, such as histamine and trypsin (18, 19). The cells used in this report were between passages 9 and 30. Unless specified otherwise, each experiment and corresponding comparisons used monolayers seeded, cultured, and studied at the same time.

Determination of intracellular cAMP. Confluent monolayers of PDEC grown on Transwell inserts were washed with (in mM) 140 NaCl, 4.7 KCl, 1.2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, and treated with ATP or analogs for 20 min. The cells were then scraped and transferred (using 1 ml of water) to a test tube containing 1 ml of 5% perchloric acid. After 15 min at 4°C, the samples were centrifuged at 1,500 g for 10 min and 0.4 ml of 30% potassium bicarbonate was added to the supernatant to precipitate potassium perchlorate. After 45 min at 4°C the samples were again centrifuged at 1,500 g for 10 min, and the supernatant was lyophilized overnight. The dried sample was resuspended in 1.2 ml of buffer, and the cAMP concentration was determined by radioimmunoassay using a kit from Diagnostic Products (Los Angeles, CA).

Efflux studies. The use of cellular 125I- effluxes to study the activation of Cl- conductances has been validated (28) and was effective for characterizing Cl- conductances on canine PDEC (15, 16, 18, 19).

Transwell membranes supporting confluent monolayers of PDEC were excised from the insert and washed twice 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 PDEC were next loaded with the radioactive tracer through a 45-min incubation at 37°C with 1.5 ml of efflux buffer containing ~2 µCi/ml Na125I and washed four times with 2 ml of isotope-free buffer. Efflux of 125I- 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), except for BAPTA-AM, which was also included in the 45-min loading period. The radioactivity of these sequential samples and the radioactivity remaining in the cells at the end of the experiment were measured using a gamma counter (Isodata 120; ICN, Huntsville, AL).

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 r = [ln (R1) - ln (R2)]/(t1 - t2), where R1 and R2 are the percentage of counts initially loaded remaining in the cells at times t1 and t2, respectively.

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 and 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 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 to the serosal compartment and a buffer consisting of (in mM) 135 Na-gluconic acid, 1.2 CaCl2, 1.2 MgCl2, 2.4 K2HPO4, 0.6 KH2PO4, 10 HEPES, and 10 glucose to the luminal compartment. The buffers in both compartments were warmed with a water jacket at 37°C.

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/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.

Identification of P2Y11 mRNA by RT-PCR. Confluent PDEC were harvested from Transwell inserts by trypsinization for 30 min at 37°C followed by centrifugation at 500 g for 10 min at 4°C. The cells were washed once with PBS, and total cellular RNA was extracted and purified according to the guanidinium-isothiocyanate method (3) with reagents supplied by Qiagen (Valencia, CA). Typically, 3 mg of total RNA was isolated from 12 Transwell inserts. RNA was further purified by DNaseI treatment for 30 min at 37°C with an absorbance ratio at 260 nm/280 nm of 2.0. A sample of 0.15 mg of RNA was reverse-transcribed in (mM) 10 Tris · HCl, 50 KCl, and 1.5 MgCl2, pH 8.3, supplemented with 0.0125 U/ml of placental RNase inhibitor, 1.25 mM random decamer, 50 mM dNTP, and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion, TX). Reverse transcription was allowed to occur for 1 h at 42°C, followed by inactivation for 10 min at 92°C. The cDNA was next PCR-amplified using 1 U of SuperTaq DNA polymerase (Ambion) in 50 µl of reaction mixture containing 125 µM dNTP and 0.5 µM reverse/forward primer mix, using the following thermocycling profile: one 10-min cycle at 95°C, followed by 40 cycles of 30 s at 95°C, 90 s at 60°C, and 90 s at 72°C, and a final holding at 72°C for 10 min. The primers, derived from the human DNA sequence of the P2Y11 receptor and synthesized by IDT (Coralville, IO), were forward 5'-CTGGTGGTTGAGTTCCTGGT-3' and reverse 5'-GTTGCAGGTGAAGAGGAAGC-3' (expected human product size: 234 bp) (22). The PCR product was then visualized by ultraviolet lighting after 8% polyacrylamide gel electrophoresis at 100 V for 30 min. The DNA in the dominant band at ~220 bp was eluted from the gel and sequenced using an ABI 373 sequencer (Applied Biosystems, Foster City, CA) and an ABI prism dye terminator sequencing protocol. Sequence homology was determined using the advanced BLAST program from the National Center for Biotechnology Information.

Statistics. Unless specified otherwise, results are 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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INTRODUCTION
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Determination of intracellular cAMP. As shown in Fig. 1A, compared with a control incubation, a 5.5-fold increase in intracellular cAMP, from 0.178 ± 0.039 to 1.002 ± 0.246 (relative units; n = 6 for each), was observed in PDEC treated with 100 µM of ATP for 20 min (P < 0.01, unpaired 2-tailed t-test with 10 df). When 14 experiments (each with n >=  3) were combined, the increase in cAMP produced by 100 µM ATP was 5.0 ±1.5-fold greater than that in the control incubation. A similar increase was also observed with the ATP analog 2-methylthio-ATP (1.083 ± 0.181, increased over control with P < 0.001). In contrast, treatment with UTP showed no significant increase in cAMP (0.213 ± 0.020, P = 0.441 compared with control).


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Fig. 1.   Effect of ATP and analogs on intracellular cAMP. A: confluent monolayers of pancreatic duct epithelial cells (PDEC), cultured in parallel, were exposed to 100 µM ATP, UTP, or 2-methylthio-ATP (2-MeS-ATP) or vehicle (water) for 20 min, and intracellular cAMP was determined as described in EXPERIMENTAL PROCEDURES. The means ± SE of the cAMP concentrations (in relative units) from 6 monolayers for each treatment are shown. Each unit is equivalent to 12 pmol cAMP/filter of confluent PDEC. **Statistically significant increases in cAMP with ATP and MeS-ATP compared with vehicle [P < 0.01, unpaired 2-tailed t-test, 10 degrees of freedom (df)]. B: confluent monolayers of PDEC were exposed to 100 µM ATP, ADP, or ATP-gamma -S or vehicle (water) for 20 min, and intracellular cAMP was determined. The means ± SE of the cAMP concentrations (in relative units) from 6 monolayers for each treatment are shown. **Statistically significant increases (P < 0.001, unpaired 2-tailed t-test, 10 df) in cAMP obtained with ATP, ADP, or ATP-gamma -S compared with vehicle; dagger statistical difference (P < 0.05) between the increases in cAMP elicited with ADP and ATP; #statistical difference (P < 0.01) between the increases in cAMP elicited with ATP-gamma -S and ATP. C: confluent monolayers of PDEC were exposed to 100 µM ATP or adenosine or vehicle (water) for 20 min, and intracellular cAMP was determined. The means ± SE of the cAMP concentration (in relative units) from 7 monolayers for each treatment are shown. **Statistically significant increase in cAMP observed only with ATP compared with control treatment (P < 0.01, unpaired 2-tailed t-test, 12 df). D: confluent PDEC monolayers preincubated with 1 µM indomethacin (Indo) or vehicle (water) were subsequently exposed to 100 µM ATP, and the cAMP concentration was determined. The means ± SE of the cAMP concentration (in relative units) from 6 monolayers for each treatment are shown. **Statistically significant increases in cAMP obtained with ATP in either control untreated cells (ATP) or cells pretreated with indomethacin vs. vehicle in control untreated cells (P < 0.001, unpaired 2-tailed t-test, 10 df).

As shown in Fig. 1B, ADP also produced an increase in cAMP (ADP 0.683 ± 0.041 vs. control 0.374 ± 0.039; n = 6, P < 0.001, unpaired 2-tailed t-test with 10 df). This increase was significantly smaller than the increase obtained with ATP [ATP: 0.981 ± 0.092; P < 0.05, unpaired 2-tailed t-test with 10 degrees of freedom (df)]. ATP-gamma -S, the nondegradable analog of ATP, also produced an increase in cAMP, which was larger than that produced by ATP (3.173 ± 0.475; P < 0.001 compared with control, P < 0.01 compared with ATP). Because ATP may also interact with P1 adenosine receptors to activate adenylate cyclase, the response to adenosine was examined to assess mediation of the ATP effect through adenosine receptors. As shown in Fig. 1C, in the presence of a positive control response to ATP, adenosine did not produce an increase in cAMP (adenosine 0.418 ± 0.042, control 0.555 ± 50; n = 7).

In MDCK cells, UTP and ATP indirectly increase intracellular cAMP through P2Y2 receptors by stimulating the cellular release of arachidonic acid, which, on conversion to prostaglandins, interacts with prostaglandin receptors on the same cells to activate adenylate cyclase. This effect was abolished with addition of the cyclooxygenase inhibitor indomethacin. To evaluate whether this pathway was also operative in PDEC, we examined the effect of 1 µM indomethacin on the cAMP increase stimulated by ATP. As shown in Fig. 1D, preincubation with indomethacin did not inhibit the cAMP increase stimulated by ATP; there may even be an enhanced response, which did not reach statistical significance (control 0.107 ± 0.011, ATP 0.248 ± 0.028, ATP + indomethacin 0.373 ± 0.051; n = 6; P < 0.001 for cAMP increase with either ATP or ATP + indomethacin vs. control; P = 0.059 for cAMP increase with ATP in presence vs. absence of indomethacin, 2-tailed t-test, 10 df).

In Fig. 2, the dose responses of cAMP stimulation to ATP, ATP-gamma -S, 2-methylthio-ATP, and alpha ,beta -methylene ATP were compared. The effects of all of these agents were concentration dependent, and for ATP, ATP-gamma -S, and 2-methylthio-ATP, EC50 were between 10 and 100 µM. alpha ,beta -Methylene-ATP was less potent and produced an effect only at 1 mM, not 100 µM.


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Fig. 2.   Concentration dependence of stimulation of cAMP increase by ATP, ATP-gamma -S, 2-MeS-ATP, and alpha ,beta -methylene ATP. Confluent monolayers of PDEC were exposed to different concentrations of ATP (triangle ), ATP-gamma -S (open circle ), 2-MeS-ATP (), and beta ,gamma -methylene-ATP () for 20 min, and the means ± SE of cAMP concentrations were measured (n = 6 for ATP, n = 4 for all others). Each unit is equivalent to 12 pmol cAMP/filter of confluent PDEC. For each agonist, the monolayers used were cultured in parallel and studied on the same day; the corresponding results are thus comparable. Between different agonists, however, the magnitude of the cAMP increase may not be directly comparable because the series of experiments were performed on different days. To facilitate comparison between the different agonists, the scale for ATP is magnified and values are shown in italics.

Iodide efflux studies. To correlate the ATP-stimulated increase in cAMP with a biological event, activation of Cl- conductances was assessed through studies of 125I- efflux. We previously established (15) that activation of either the cAMP-activated CFTR or the Ca2+-activated Cl- channel is reflected by an increase in the 125I- efflux rate coefficient. As shown in Fig. 3, 100 µM of either UTP (Fig. 3A) or ATP (Fig. 3B) stimulated an increase in 125I- efflux. In Fig. 3A, the 125I- efflux increase stimulated by UTP was totally abolished by preincubation with the cell-permeant Ca2+ chelator BAPTA-AM (50 µM), suggesting that this efflux was totally mediated through an increase in [Ca2+]i. Indeed, in the control experiments shown in Fig. 3, C and D, the 125I- effluxes resulting from either Ca2+ influx (treatment with 1 µM of the calcium ionophore A-23187) or mobilization of intracellular calcium (treatment with 2 µM thapsigargin) were also completely inhibited by BAPTA-AM. In contrast, the efflux rate increase stimulated by ATP was only partially inhibited by the same calcium chelator (manifested by a delayed and smaller peak response; Ref. 15), suggesting that this efflux was only partially mediated by increased [Ca2+]i (Fig. 3B). As previously reported (16), 100 µM adenosine did not stimulate an increase in 125I- efflux (Fig. 3E); this finding is consistent with the earlier absence of adenosine effect on cAMP production.


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Fig. 3.   Effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) on the increased 125I- efflux stimulated by ATP and UTP. 125I- efflux from PDEC was determined as detailed in EXPERIMENTAL PROCEDURES, and the efflux rate coefficient was calculated. For each panel, the means ± SE from 3 experiments are shown. A: after 1 min of baseline determination, 100 µM UTP was added to PDEC previously treated with 50 µM BAPTA-AM () or untreated controls (open circle ). B: after 1 min, 100 µM ATP was added to PDEC previously treated with 50 µM BAPTA-AM () or untreated controls (open circle ). C: after 1 min of baseline determination, 1 µM A-23187 was added to PDEC previously treated with 50 µM BAPTA-AM () or untreated controls (open circle ). D: after 1 min of baseline determination, 2 µM thapsigargin was added to PDEC previously treated with 50 µM BAPTA-AM () or untreated controls (open circle ). E: after 1 min of baseline determination, 100 µM adenosine was added to PDEC (); 125I- efflux from untreated controls is also shown (open circle ).

Ussing chamber studies. To examine further the ATP activation of Cl- conductances on PDEC, confluent monolayers of PDEC were mounted in Ussing chambers and their basolateral membranes were permeabilized to small monovalent ions with 0.36 mg/ml nystatin. This procedure allowed examination of ion transport through the apical membranes of PDEC. A serosal right-arrow luminal Cl- concentration gradient of 135 mM was also maintained using appropriate buffers to drive Cl- through activated Cl- conductances, an event reflected by an increase in Isc. Under these conditions, both forskolin (100 µM), acting through cAMP, and the calcium ionophore A-23187 (1 µM), acting through increased [Ca2+]i, stimulated an increase in Isc. However, the Isc increase stimulated through the cAMP pathway (Fig. 4B) was much larger than the Isc stimulated by increased [Ca2+]i (Fig. 4A). Indeed, the average maximal Isc increase after forskolin was 67.5 ± 3.3 µA/cm2 (n = 6) vs. 8.1 ± 1.9 (n = 7) µA/cm2 after A-23187.


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Fig. 4.   Effect of forskolin, A-23187, and ATP on basolaterally permeabilized PDEC monolayers studied in Ussing chambers. Confluent monolayers of PDEC were mounted in Ussing chambers, and their basolateral membrane was permeabilized to monovalent ions with 0.36 mg/ml nystatin and studied in the presence of a serosal right-arrow luminal Cl- concentration gradient of 135 mM as detailed in EXPERIMENTAL PROCEDURES. A and B: PDEC monolayers cultured in parallel and studied concurrently were exposed to either 1 µM A-23187 (A) or 100 µM forskolin (B) added to the luminal compartment after 5 min of baseline recording. The recording shown is representative of 4 different experiments. C and D: PDEC monolayers cultured in parallel and studied concurrently were exposed to 100 µM ATP added to either the luminal (C) or the serosal (D) compartment. The recording shown is representative of 3 different experiments.

We previously reported (16) that UTP, interacting with P2Y2 receptors on either the apical or basolateral surfaces of PDEC, stimulated a small Isc increase typical of a Ca2+-activated response when added to either the serosal or luminal compartment of the Ussing chamber. When ATP was evaluated in the same manner, luminal addition stimulated the same small Ca2+-activated type Isc increase (Fig. 4C). In contrast, addition of ATP to the serosal compartment stimulated a large Isc increase (Fig. 4D) typical of the cAMP-activated response shown in Fig. 4B. From three different experiments, the mean maximal Isc increase elicited by 100 µM luminal ATP was 6 ± 2 µA/cm2 whereas the mean maximal Isc elicited by 100 µM serosal ATP was 101 ± 14 µA/cm2. When all experiments with measurement of the Isc response to the serosal addition of ATP were reviewed, the mean maximal Isc increase was 65.8 ± 7.5 µA/cm2 (n = 14). In further experiments not shown, a similar response was observed with either 100 µM ATP-gamma -S or 100 µM 2-methylthio-ATP. Adenosine, at a concentration of 100 µM, failed to elicit any increase in Isc when added to both compartments of the Ussing chamber.

To further verify the identity of the signaling mechanism mediating ATP action, we evaluated the effect of BAPTA-AM, which completely inhibited the Isc response resulting from increased [Ca2+]i after treatment with A-23187 (data not shown). When cells were treated with 50 µM BAPTA-AM, starting 30 min before the recording, the Isc increase resulting from the luminal addition of ATP was totally inhibited (Fig. 5A, top) whereas the large Isc increase resulting from the serosal addition of ATP was still elicited (Fig. 5A, bottom). Suramin and reactive blue, two inhibitors of P2 receptors, were also used to verify that the large Isc increase stimulated by serosal ATP was indeed mediated by P2 receptors. The Isc increase resulting from the serosal addition of ATP was totally inhibited when PDEC monolayers were treated with either 1 µM suramin (Fig. 5B) or 100 µM reactive blue (Fig. 5C).


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Fig. 5.   Effect of BAPTA-AM, suramin, and reactive blue on ATP response by PDEC in Ussing chambers. Confluent monolayers of PDEC were mounted in Ussing chambers, and their basolateral membrane was permeabilized to monovalent ions using 0.36 mg/ml nystatin and studied in the presence of a serosal right-arrow luminal Cl- concentration gradient of 135 mM as detailed in EXPERIMENTAL PROCEDURES. The tracings shown were obtained from monolayers cultured in parallel and studied on the same day. A: PDEC monolayers were preincubated with 50 µM BAPTA-AM for 30 min and, after an additional 5 min of baseline recording, were exposed to either 100 µM luminal ATP (top) or 100 µM serosal ATP (bottom). The recording shown is representative of 3 different experiments performed on different days: the average short-circuit current (Isc) elicited from serosal addition of ATP was 70 ± 5.6 µA/cm2; luminal addition of ATP elicited no discernible Isc increase in 2 experiments and a transient Isc spike of 2.5 µA/cm2 in 1 experiment. B: PDEC monolayers were treated with the P2 inhibitor suramin (1 µM) starting 15 min before the experiment. After 5 min of baseline recording, 100 µM ATP was added to the serosal side. This total inhibition is representative of 3 different experiments; the corresponding control Isc increase to ATP in the absence of suramin was 55.2 ± 20.5 µA/cm2. C: PDEC monolayers were treated with the P2 inhibitor reactive blue (100 µM) starting 15 min before the experiment. After 5 min of baseline recording, 100 µM ATP was added to the serosal side. This total inhibition is representative of 3 different experiments; the corresponding control Isc increase to ATP in the absence of reactive blue was 46.4 ± 5.1 µA/cm2.

RT-PCR. Because the only P2 receptor to date known to be coupled to adenylate cyclase is the P2Y11 receptor, expression of this receptor in canine PDEC was studied through RT-PCR of the RNA from these cells, using unique primers from the known sequence of the human P2Y11 receptor. As shown in Fig. 6A, a single band, with a size approximating the one expected for the fragment deduced from the human P2Y11 receptor (234 bp), was observed (lane b). Without reverse transcriptase, no signal was seen (Fig. 6A, lane c), excluding possible genomic DNA contamination.


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Fig. 6.   RT-PCR of P2Y11 receptors in PDEC. A: total cellular RNA was purified from PDEC, and the presence of P2Y11 mRNA was probed by RT-PCR as described in EXPERIMENTAL PROCEDURES. The DNA fragment deduced from the human DNA sequence of the P2Y11 receptor is expected to be 234 bp. Lane a shows the DNA ladder (size standards on left); lane b shows the PCR product (position marked by arrow on right); in lane c, reverse transcriptase was omitted to exclude genomic DNA contamination. B: DNA fragment shown in lane b of A was eluted from the acrylamide gel and sequenced. Of the 221-bp fragment, 3 segments were compared with the human P2Y11 sequence: segments A and C (underlined) exhibited 88 and 100% homology, respectively, with the P2Y11 human sequence, whereas segment B (in italics) showed no match.

This band was eluted from the gel and analyzed. It consisted of 221 bp, of which 167 bp were clearly identified. This sequence consisted of a 116-bp segment and a 37-bp segment (segments A and C in Fig. 6B) that exhibited 88% and 100% identity, respectively, with the human sequence and an intervening 14-bp segment (segment B in Fig. 6B) with no match, for an average identity of 84%.


    DISCUSSION
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REFERENCES

The exocrine function of the pancreas is mediated by acinar cells, which secrete digestive enzymes, and PDEC, which secrete electrolytes. For PDEC, activation of Cl- channels is a key event in electrolyte secretion; in cystic fibrosis, for example, impaired expression or function of CFTR, a cAMP-activated Cl- channel, results in decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, inspissation of pancreatic juice in pancreatic ducts, ductal obstruction, and pancreatic insufficiency (12, 13). We previously demonstrated (16) that, in cultured nontransformed PDEC, ATP and UTP can activate Ca2+-activated Cl- channels through P2Y2 receptors. We now demonstrate that ATP, but not UTP, can also activate the cAMP-stimulated CFTR Cl- channel via increased cAMP and the P2Y11 receptor.

We first observed that cAMP in PDEC was increased after treatment with ATP. In other epithelial cells, ATP can stimulate an increase in cAMP indirectly. Indeed, in T84 cells, ATP partly stimulates secretion through breakdown to AMP and adenosine and subsequent interaction with the adenosine P1 receptor (7), whereas, in MDCK cells, ATP, acting via P2Y2 receptors, stimulates the release of arachidonic acid release, which in turn activates prostaglandin receptors on conversion to prostaglandins (21). Mediation of the ATP effect through either of these pathways was excluded for PDEC. Response to ATP-gamma -S, a nonhydrolyzable analog of ATP, suggests that ATP, and not a degradation product, is the bioactive agent. Furthermore, adenosine itself did not stimulate an increase in cAMP, excluding a role for the P1 receptor. With regard to the P2Y2-arachidonic acid-prostaglandin pathway, the inability of UTP to increase cAMP suggests that, unlike the case of MDCK cells, P2Y2 receptors on PDEC do not indirectly mediate this effect. In addition, blockade of arachidonic acid conversion to prostaglandin by the cyclooxygenase inhibitor indomethacin did not inhibit ATP-stimulated cAMP increase. By exclusion, this effect appears to be mediated by a direct interaction between ATP and the PDEC, most likely the P2Y11 receptor, the only P2Y receptor coupled to adenylate cyclase.

Although the average fivefold increase in cAMP is modest (compared with the >= 10-fold increase observed with forskolin; data not shown), it was sufficient to produce a biological effect. Indeed, this increase in cAMP caused an activation of the CFTR Cl- channel, a crucial component for the secretory function of PDEC. This activation was established using two different modalities, measurements of 125I- efflux and of Isc in Ussing chambers. Monitoring 125I- efflux to assess activation of Cl- conductances, first described by Venglarik et al. (28), has been validated for the Ca2+-activated Cl- channel and cAMP-activated CFTR channel in these PDEC (15). As previously shown, UTP, interacting with the P2Y2 receptor, stimulated 125I- efflux through the Ca2+-activated Cl- channel (16); as expected, this effect was completely inhibited by Ca2+ chelation with BAPTA-AM. ATP also stimulated an increase in 125I- efflux; however, only a portion of this increase was inhibited by BAPTA-AM. These findings are consistent with the hypothesis that although ATP, like UTP, may interact with the P2Y2 receptor to increase [Ca2+]i and stimulate 125I- efflux through the Ca2+-activated Cl- channel, it also interacts with the P2Y11 receptor to stimulate a BAPTA-resistant 125I- efflux through the cAMP-dependent CFTR Cl- channel.

Activation of the CFTR Cl- channel is further verified with the use of confluent PDEC monolayers mounted in Ussing chambers. The protocol used in this report (basolateral permeabilization and serosal-to-luminal Cl- gradient) allows Cl- flow through activated Cl- channels to be reflected as an increased Isc. In addition, Cl- flow through the Ca2+-activated Cl- channel can also be differentiated from flow through the cAMP-stimulated CFTR Cl- channel on the basis of magnitude of the maximal Isc increase elicited. Indeed, additional experiments confirmed that the large Isc increase stimulated by forskolin through increased cAMP is DIDS insensitive, and thus mediated by the CFTR Cl- channel, whereas the smaller Isc increase stimulated by A-23187 through increased Ca2+ is DIDS sensitive, and thus mediated through the Ca2+-activated Cl- channel (Nguyen et al., manuscript in preparation).

With this system, we previously demonstrated (16) the presence of P2Y2 receptors on both apical and basolateral membranes of PDEC. Indeed, either apical or serosal UTP stimulated a small Isc increase compatible with activation of the Ca2+-activated Cl- channel. As shown in Fig. 4C, apical P2Y2 receptors interacted with luminal ATP to activate Ca2+-activated Cl- channels, resulting in a small Isc increase that was inhibited by BAPTA (Fig. 5A, top). In contrast, as shown in Fig. 4D, serosal ATP elicited a large Isc increase, as seen with activation of the cAMP-activated Cl- channel. Mediation of this large Isc increase through the cAMP pathway is further supported by its resistance to BAPTA (Fig. 5A), a Ca2+ chelator that previously abolished the Ca2+-type Isc increase elicited by UTP acting via the P2Y2 receptor (16). The basolateral receptor responsible for the large Isc increase elicited by serosal ATP is a P2 receptor, because it was inhibited by both suramin and reactive blue. Of the P2 receptors, we previously observed (16) that stimulation of the basolateral P2Y2 receptor by serosal UTP resulted in only a small Isc increase that was inhibited by BAPTA, compatible with activation of the Ca2+-activated Cl- channel. Thus, although the P2Y2 receptor may account for a small component of the Isc response to serosal ATP, it cannot account for the large Isc increase resistant to BAPTA. Serosal ATP-gamma -S and 2-methylthio-ATP both elicited the same type of large Isc response, consistent with the agonist profile for the stimulation of cAMP production. Therefore, the large Isc response from serosal ATP is most likely mediated by the P2Y11 receptor.

Many P2Y and P2X receptors have been demonstrated on PDEC (10, 14). P2Y receptors are classically coupled to phospholipase C, the only exception being the recently cloned P2Y11 receptor, which is also coupled to adenylate cyclase (4). Pharmacologically, the P2Y11 receptor is the likely candidate to mediate the stimulation of cAMP increases in PDEC by ATP. Indeed, ATP, ATP-gamma -S, 2-methylthio-ATP, and ADP, but not UTP, stimulated an increase in cAMP and a large cAMP-type Isc increase in Ussing chambers consistent with the agonist profile of P2Y11 receptors expressed in transfected CHO cells (5). For the putative P2Y11 receptor on HL-60 leukemia cells and for the P2Y11 receptor expressed on transfected CHO cells, the relative potency of the different nucleotides are ATP-gamma -S > ATP > 2-methylthio-ATP > ADP (4-6). As shown in Fig. 1, testing at 100 µM concurrently, we obtained an efficiency profile of ATP-gamma -S > ATP = 2-methylthio-ATP > ADP, similar to that observed with the human P2Y11 receptor except for the equivalence between ATP and 2-methylthio-ATP. As shown in Fig. 2, the EC50 for ATP, ATP-gamma -S, and 2-methylthio-ATP were all between 10 and 100 µM, values intermediate between those observed for the HL-60 P2Y11 receptor (EC50 of 30, 100, and 300 µM for ATP-gamma -S, ATP, and 2-methylthio-ATP, respectively) and the transfected human P2Y11 receptors (EC50 of ~3, 10, and 30 µM for ATP-gamma -S, ATP, and 2-methylthio-ATP, respectively). It is possible that these differences reflect variance between human and canine P2Y11 receptors, the cell model used (HL-60 vs. PDEC vs. transfected cells), or the partial degradation of ATP or analogs by nucleotidases present on the surfaces of these cells. The latter possibility is further supported by the strongest response to ATP-gamma -S. Through RT-PCR followed by sequencing, we verified the presence of mRNA for P2Y11 receptors on these PDEC. Of note, the canine cDNA fragment sequenced showed 84% homology with its human counterpart.

P2X4, P2X7, and P2X1 are also expressed on the basolateral (P2X1 and P2X4) and/or apical (P2X7) membranes of rat PDEC (10, 14). Although characterization of the secretory effects of P2X receptors is not within the scope of this report, the possibility that these receptors may mediate some of the findings in this report should be addressed.

Because P2X receptors function as ATP-gated channels and are not coupled to G protein, they will not mediate the observed increases in cAMP produced by ATP and analogs. Furthermore, P2X receptors are cation conductances (Ca2+ >> Na+ > K+) and should not directly mediate 125I- efflux. On the other hand, they may mediate 125I- through increased [Ca2+]i; this effect, however, should be inhibited by BAPTA-AM. Indeed, the same BAPTA-AM pretreatment totally inhibited the efflux stimulated by increased [Ca2+]i obtained with either the Ca2+ ionophore A-23187 (influx of extracellular Ca2+) or thapsigargin (mobilization of Ca2+ from intracellular stores). Thus the BAPTA-resistant effect exhibited in Fig. 3D is unlikely to be mediated through P2X receptors. With regard to the Ussing chamber experiments, the basolateral membranes of the PDEC are already permeabilized to small monovalent ions with nystatin. Therefore, the possible activation of the Na+ and K+ conductances intrinsic to P2X receptors on the basolateral surfaces of these cells by serosal ATP should not affect Isc. Alternatively, activation of P2X receptors may indirectly stimulate Ca2+-activated Cl- channels through cellular influx of Ca2+. Again, the corresponding Isc increase should be inhibited by BAPTA-AM and exhibit the characteristics of a current mediated by Ca2+-activated Cl- channels. In the aggregate, although the role of functional P2X receptors in these canine PDEC remains to be examined, it is unlikely that these receptors account for all the findings attributed to P2Y11 receptors in this report.

ATP mediates both autocrine and paracrine functions (8, 24-26). On PDEC, the differential polarity of ATP receptor subtypes coupled to distinct signaling pathways may allow ATP to produce different effects as it interacts with either the serosal or apical surface of these cells. Through P2Y2 receptors expressed on both surfaces of the PDEC, ATP present in either the pancreatic ductal lumen or serosal compartment will be able to stimulate phospholipase C to activate both the Ca2+ and protein kinase C signaling pathways. On the other hand, because functional P2Y11 receptors are only expressed on the basolateral surface of the PDEC, only serosal ATP will be able to stimulate adenylate cyclase and activate the cAMP signaling pathway. This distinction may be relevant because the sources of luminal and serosal ATP may differ. In the biliary system, ATP may be released by bile duct epithelial cells into the luminal compartment for a paracrine effect on biliary secretion or an autocrine effect on cell volume regulation (2, 24). The same mechanism may be operative for ATP release into the pancreatic juice. Of note, should ATP efflux from epithelial cells be mainly mediated through CFTR (26), the apical expression of this channel would favor efflux into the luminal compartment. In addition, because the common bile duct and main pancreatic duct share an outflow into the duodenal lumen, bile may be refluxed into the pancreatic duct lumen. The ATP contained in that bile may interact with the apical surface of PDEC.

Serosal ATP, on the other hand, may be derived from interstitial components such as nerve endings. Alternatively, during inflammation, ATP may be released from injured cells or from inflammatory cells. The restriction of P2Y11 receptors to the basolateral surface of PDEC will ensure that the serosal ATP from these sources effects a different response.

To our knowledge, demonstration of a secretory effect mediated by the P2Y11 receptor would be the first description of a function for this receptor in epithelial cells. Indeed, P2Y11 receptors have only been described in HL-60 leukemia (4) and MDCK (22) cells. In HL-60 cells P2Y11 receptors may mediate cAMP-stimulated differentiation (6, 11, 27), and their function in MDCK cells, beyond cAMP stimulation, is still undefined. The sparseness of information on P2Y11 receptors may relate to their recent identification; our report further supports their importance in epithelial cell biology.

In summary, we have ascribed a secretory function to the P2Y11 receptors in epithelial cells of pancreatic ductal origin. These findings support further studies to define a role for these receptors and for ATP in the (patho)physiology of epithelial cells.


    ACKNOWLEDGEMENTS

The authors thank Dr. Sum Lee and Chris Savard for advice in the culture of PDEC and the cAMP assays.


    FOOTNOTES

This research was partially funded by the Department of Veterans Affairs, Cystic Fibrosis Foundation (RDP R565), and the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-55885).

Address for reprint requests and other correspondence: T. D. Nguyen, GI Section (S-111-Gastro), Puget Sound VA Health Care System, 1660 S. Columbian Way, Seattle, WA 98108 (E-mail: t1nguyen{at}u.washington.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 May 2000; accepted in final form 8 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(5):G795-G804