Multiple functional P2X and P2Y receptors in the luminal and basolateral membranes of pancreatic duct cells

Xiang Luo1, Weizhong Zheng1, Ming Yan1, Min Goo Lee2, and Shmuel Muallem1

1 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and 2 Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea


    ABSTRACT
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Purinergic receptors in the basolateral and luminal membranes of the pancreatic duct can act by a feedback mechanism to coordinate transport activity in the two membranes during ductal secretion. The goal of the present work was to identify and localize the functional P2 receptors (P2R) in the rat pancreatic duct. The lack of selective agonists and/or antagonists for any of the cloned P2R dictated the use of molecular and functional approaches to the characterization of ductal P2R. For the molecular studies, RNA was prepared from microdissected pancreatic intralobular ducts and was shown to be free of mRNA for amylase and endothelial nitric oxide synthase (markers for acinar and endothelial cells, respectively). A new procedure is described to obtain an enriched preparation of single duct cells suitable for electrophysiological studies. Localization of P2R was achieved by testing the effect of various P2R agonists on intracellular Ca2+ concentration ([Ca2+]i) of microperfused intralobular ducts. RT-PCR analysis suggested the expression of six subtypes of P2R in the pancreatic duct: three P2YR and three P2XR. Activation of Cl- current by various nucleotides and coupling of the receptors activated by these nucleotides to G proteins confirmed the expression of multiple P2R in duct cells. Measurement of [Ca2+]i in microperfused intralobular ducts suggested the expression of P2X1R, P2X4R, probably P2X7R, and as yet unidentified P2YR, possibly P2Y1R, in the basolateral membrane. Expression of P2Y2R, P2Y4R, and P2X7R was found in the luminal membrane. The unprecedented expression of such a variety of P2R in one cell type, many capable of activating Cl- channels, suggests that these receptors may have an important role in pancreatic duct cell function.

rat pancreatic duct; purinergic receptors; intracellular calcium; chloride channels


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THE PANCREATIC DUCT SECRETES most of the fluid and determines the final electrolyte composition of the pancreatic juice (3, 7). Acinar cells secrete digestive enzymes and a small volume of a plasmalike isotonic fluid into the common acinar lumen, which then flows into intercalated ducts. As the fluid passes through the ductal system, duct cells absorb the Cl- and secrete HCO-3 and fluid to the lumen. Transcellular Cl- transport by the duct is the major function controlling ductal secretion. This activity is of particular interest because it is intimately regulated by cystic fibrosis transmembrane conductance regulator (CFTR) (3). In cystic fibrosis, Cl- absorption and HCO-3 secretion are impaired, which results in hyperconcentration of digestive enzymes and mucines and leads to obstruction of intralobular ducts (34).

Several hormones and neurotransmitters regulate pancreatic ductal secretion. The best studied so far are regulation by secretin and cholinergic stimulation. Secretin stimulates the generation of cAMP (12) to activate CFTR in the luminal membrane (LM) (13, 14). Cholinergic stimulation of the M3 muscarinic receptors (16) increases ductal intracellular Ca2+ concentration ([Ca2+]i) (4, 17, 31, 40), strongly depolarizes the basolateral membrane (BLM) potential (17), and stimulates fluid secretion (4).

Another agonist acting on pancreatic duct (8, 17) and other CFTR-expressing epithelial cells (20, 25, 30, 32, 33, 37) is ATP, which interacts with P2 receptors (P2R). P2R are unique in epithelial physiology because they are expressed in both the BLM and LM. This provides the cells with a receptor system to coordinate transepithelial function through central and paracrine/autocrine inputs. Thus the basolateral receptors are stimulated by ATP secreted by purinergic neurons. ATP secreted by acinar cells stored in secretory granules and by ductal ATP-binding cassette transporters such as CFTR to the duct lumen are likely the substrate for the luminal P2R. This form of regulation is attracting considerable attention, as it may be of clinical relevance (34, 35). Characterization of P2R expressed in epithelial cells is partial and not conclusive. Most published studies relied on a pharmacological approach of determining sequence of potency to several P2R agonists. On the basis of such studies, it has been suggested that airway epithelial cells express P2Y2R in the LM and P2Y3R in the BLM (20). Nasal epithelial cells were suggested to express P2Y2R in the BLM and P2Y6R in the LM (24). P2Y1R and/or P2Y2R were found in the BLM, and P2zR was found in the LM of submandibular duct cells (2, 25, 37). The response of isolated ducts to ATP (17) and other nucleotides led Christofferson et al. (8) to suggest the expression of P2uR and P2zR in pancreatic duct cells. Membrane localization of P2R was not determined. The only conclusive work was reported on submandibular acinar cells, in which molecular and functional studies reported the expression of P2X4R in these cells (5).

A potential problem with the pharmacological approach, which is highlighted in several recent reviews and other articles (5, 6, 11, 19, 22, 23, 26, 29), is the uncertainty with which P2R can be identified when more than one P2R is expressed in the same cell type. All available agonists and antagonists show cross-reactivity and similar affinity for at least two P2R types (1, 5, 11, 23). Furthermore, the same P2R expressed in different cells can display a different profile of responsiveness to P2 agonists (11, 23), and two P2R can assemble into one functional unit (26). Molecular characterization together with functional studies can circumvent, in part, some of these difficulties (5, 23, 26). In the present work we prepared RNA from microdissected pancreatic intralobular ducts, which was free of mRNA for amylase and endothelial nitric oxide synthase (eNOS; markers for acinar cells and endothelial cells in blood capillaries, respectively) to analyze by RT-PCR the P2R expressed in pancreatic duct cells. Surprisingly, the pancreatic duct appears to express six subtypes of P2R: three P2YR and three P2XR. Measurement of Cl- current activation by various nucleotides and coupling of the receptors activated by these nucleotides to G proteins confirmed the expression of multiple P2R in duct cells. Finally, measurement of [Ca2+]i in microperfused intralobular ducts suggested the expression of several P2XR and P2YR in the BLM and LM of the pancreatic duct. The expression of such a variety of P2R in the duct suggests that these receptors have an important role in pancreatic duct cell function.


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Chemicals and solutions. All nucleotides were purchased from Sigma, except 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) which was from RBI (Natick, MA). Fura 2-AM was from Molecular Probes (Eugene, OR). The standard perfusion solution (bath and lumen) contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). This solution was supplemented with 10 mM sodium pyruvate, 0.02% soybean trypsin inhibitor, and 1 mg/ml BSA to form pancreatic solution A (PSA). The standard solution was the bath solution during current recording. The pipette solution contained (in mM) 140 KCl, 10 HEPES (pH 7.3 with NaOH), 1 MgCl2, 1 ATP, and 0.1 EGTA. These conditions were found as optimal to record the inward current in duct cells, which was carried mostly by Cl-.

Microdissection of pancreatic duct and fluorescence recording. Intralobular ducts were microdissected from the rat pancreas and microperfused exactly as described before (40). Rats (male or female, Sprague-Dawley, 75-100 g) were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium and killed by exposure to air saturated with methoxyflurane. The pancreas was removed and placed in PSA for microdissection. For measurement of [Ca2+]i, two segments of intralobular duct were microdissected. One was kept on ice until use and the other was transferred to a perfusion chamber. After insertion of the luminal perfusion micropipette, the duct was loaded with fura 2 by luminal perfusion for 10-15 min with a solution containing 2.5 µM fura 2-AM. Bath perfusion started before application of the dye to the lumen and, except during application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) to the bath, the bath perfusions did not stop until the end of each experiment. In three experiments BzATP was applied to the bath, as were all other agonists, by continuous perfusion. Because of limited agonist availability and cost, in four experiments bath perfusion was stopped, and BzATP was applied to the bath by infusion of 1 ml solution (~13 chamber volumes) containing 1 mM BzATP. Bath stimulation was terminated by resumption of perfusion. In all experiments luminal BzATP was applied by perfusion. Fura 2 fluorescence was recorded at excitation wavelengths of 355 and 380 nm as detailed in Ref. 36. Results are expressed as the 355/380 fluorescence ratios because contamination with extracellular dye, which tended to remain in the connective tissue, prevented meaningful calibration (see also Ref. 40).

Preparation of RNA. To prepare RNA, most of the pancreatic ductal tree was microdissected and carefully cleaned from adherent cells and connective tissue as much as possible (see Fig. 1 in Ref. 40). Then five to seven segments of the intralobular duct were cut, inspected for absence of acinar cells, blood capillaries, and cell debris (see Fig. 1, A and B), and transferred to a clean Eppendorf tube. The ducts were washed twice with PSA and packed and treated with Trizol for extraction of RNA. The ductal origin of the RNA was verified by PCR analysis of marker mRNAs. RNA extracted from submandibular gland tissue (all cells) was used as a source for positive controls. As shown in Fig. 1C, pancreatic ductal RNA contained mRNA for CFTR and was free from mRNA for amylase (marker for acinar cells) and eNOS (marker for pancreatic blood capillaries; see Ref. 37).


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Fig. 1.   Bright-field images of pancreatic duct segments and isolated cells and PCR analysis of ductal mRNA. Segments of intralobular pancreatic ducts were microdissected, cleaned, and washed as described in METHODS. A and B: magnifications of ×100 and ×400, respectively. D and E: isolated cells at a magnification of ×400. D: cells obtained after initial digestion and suspension of cells in a solution containing 10 mg/ml BSA. E: enriched preparation of duct cells. C: RT-PCR analysis of mRNA prepared from submandibular gland (SMG) tissue and from microdissected intralobular pancreatic ducts (Panc duct) similar to those in A and B. Primers used to amplify each gene product are listed in Table 1. Gene products amplified were beta -actin (beta  Act), cystic fibrosis transmembrane conductance regulator (CFTR), amylase (Amy), and endothelial isoform of nitric oxide synthase (eNOS).

Preparation and enrichment of single pancreatic duct cells. Our experience in isolation of single duct cells from the submandibular gland (40) indicated that duct cells have lower density than acinar cells. On the basis of this observation, we used the following procedure to obtain a preparation enriched in single pancreatic duct cells. Rats were killed as detailed above for microdissection of intralobular ducts. The pancreas was minced and digested with collagenase and trypsin by a standard published procedure (39). Most of the acini were removed by a 10-s centrifugation at 500 rpm using a Sorvall centrifuge model RC-3B. The supernatant was removed to a clean tube, and the cells were collected by a 3-min centrifugation at 1,500 rpm. The pelleted cells were washed twice with PSA, resuspended in 8 ml of ice-cold PSA containing 10 mg/ml BSA, and placed in a standard 15-ml conical plastic tube. The low abundance of duct cells in this preparation is shown in Fig. 1D. The tube was placed on ice, and the cells were allowed to sediment at 1 g. Every 10 min the pelleted cells were carefully collected from the bottom of the tube with a Pasteur pipette for microscopic inspection to verify removal of mostly acinar cells. After 7-10 collections, the remaining cells were harvested by a 3-min centrifugation at 1,500 rpm, resuspended in PSA, and kept on ice until use. Figure 1E shows the enrichment in duct cells relative to acinar cells in this preparation. Pancreatic duct cells were distinguished from acinar cells by their size and appearance. The secretory granules can be easily identified in the larger acinar cells, whereas the smaller duct cells had a smooth appearance. In accordance with their different sizes, during electrophysiological recordings duct and acinar cells were distinguished by their different capacitance. In a typical series of experiments, the capacitance of acinar cells averaged 13.6 ± 2.6 pF (n = 11), with a minimum and a maximum of 9.2 and 17.8 pF, respectively. In 14 duct cells from the same preparations, the average capacitance was 4.4 ± 0.8 pF, with a minimum and a maximum of 3.2 and 6.3 pF, respectively. Finally, all 11 acinar cells strongly responded to 1 mM carbachol stimulation, showed no response to 100 µM adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), and subsequently strongly responded to 1 nM cholecystokinin octapeptide (CCK-8). All 14 duct cells strongly responded to stimulation with carbachol and ATPgamma S (see RESULTS for protocol) and showed very small or no response to CCK-8. Based on these observations, we believe that all responses (summarized in Table 2 below) are from pancreatic duct cells and are not contaminated by recording from other cell types.

RT-PCR analysis. Approximately 2 ng of total RNA extracted with the RNAzol kit were used to perform the RT reaction with the GeneAMP RNA-PCR kit (Perkin-Elmer) in a 20-µl reaction volume as specified by the manufacturer. Random hexamers were used as RT primers for cDNA synthesis by incubation for 15 min at 42°C, 5 min at 95°C, and 5 min at 4°C. PCR primers were designed for mRNA of each gene product using Gene Runner software version 3.00 as well as published sequences. All primers, reaction conditions, and expected product sizes are listed in Table 1. PCRs were started by a 10-min hot start with a Perkin-Elmer AmpliTaq Gold DNA polymerase. For all primers, 35 cycles of cDNA amplifications were carried out in a PTC-100 thermal cycler (MJ Research). The PCR products were separated on a 2% agarose gel containing 0.1 µg/ml ethidium bromide. The identity of all amplified products was verified by sequencing.

                              
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Table 1.   RT-PCR primers and reaction conditions

Electrophysiology. The whole cell configuration of the patch-clamp technique (15) was used to record the inward current at a holding potential of -40 mV. All current recordings were performed at room temperature. Current was recorded with a patch-clamp set up from Axon Instruments using pCLAMP 6 and a DigiData 1,200 interface. The patch-clamp output was filtered at 10 Hz. Leak current was subtracted from all records. The amplitude of the currents stimulated by carbachol and ATPgamma S was within 15% in any given cell preparation but varied considerably between cell preparations (range 70-200 pA/pF); therefore, the results in Table 2 are given as number of responding cells.

                              
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Table 2.   Characterization of agonists that activate ionotropic and metabotropic P2R in pancreatic duct cells


    RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION
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Pancreatic duct cells express multiple P2R. The first indication for the expression of multiple P2R in pancreatic duct cells was obtained by measuring the properties of the ATP-activated inward current. The inward current was carried mostly by Cl-. Hence ATP and carbachol activated a nonselective cation current and a Cl- current. Accordingly, the reversal potential under the ionic composition of the solutions used in the present work was close to 0 mV. Substitution of external Na+ with K+ and internal K+ for Na+ had minimal effect on the current recorded at -40 mV. On the other hand, reducing external Cl- concentration to 33 mM (at internal Cl- of 150 mM) resulted in a current with a reversal potential of -38 ± 3 mV, which is close to the Nernst reversal potential for Cl-. Stimulation of duct cells by bath perfusion with a solution containing supramaximal concentration of carbachol (1 mM) or application of ATP by puffing a solution containing 5 mM ATP activated the Cl- current to a similar extent (Fig. 2A). ATP also activated the Cl- current when it was applied by bath perfusion (Fig. 2B). Removal and readdition of Ca2+ to the perfusion medium showed that ATP activated both Ca2+-dependent and Ca2+-independent Cl- currents (n = 9). These findings are similar to those reported in several cell types (20, 24, 30, 32, 33, 37), in which ATP binds to multiple P2R to activate at least two different Cl- channels.


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Fig. 2.   ATP activates multiple P2 receptors in pancreatic duct cells. Whole cell configuration of patch-clamp technique was used to record inward current. A: cell was stimulated by bath application of 1 mM carbachol (Car), inhibited with 10 µM atropine (Atr), and then stimulated by puffing a solution containing 5 mM ATP. B: cell was stimulated by bath application of 1 mM ATP. Where indicated, Ca2+ was removed and readded to bath by perfusion with a Ca2+-free solution. C: pipette solution contained 2 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), and the cell was dialyzed with the pipette solution for 5 min before the first stimulation. As indicated, the cell was stimulated with 1 mM carbachol, inhibited with 10 µM atropine, and stimulated by puffing a solution containing 5 mM ATP.

P2R are classified into two classes, metabotropic (P2Y) and ionotropic (P2X) (1, 23, 29). Because P2YR, but not P2XR, are coupled to G proteins (6, 22, 23, 29), a convenient test for the expression of P2XR is to measure the effect of inhibition of G proteins with guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) on agonist-dependent activation of Cl- current. Figure 2C shows that including 2 mM GDPbeta S in the pipette solution abolished the response of the cells to stimulation with carbachol, a Gq-coupled receptor (39). The same cells responded normally to stimulation with ATP. In fact, the magnitude of the currents activated by ATP in control cells and cells infused with GDPbeta S was similar in all experiments.

Activation of Ca2+-dependent and Ca2+-independent Cl- currents by ATP shown in Fig. 2 suggested expression of more than one type of P2R in these cells. This excluded the ability to identify the type of P2R expressed in pancreatic duct cells by binding or activity assays based on determining the potency sequence of P2R agonists. Indeed, in preliminary studies we measured the apparent affinity for the nucleotides listed in Table 2 to activate Cl- current in the presence or absence of GDPbeta S and could not obtain definitive results. Perfusion of several nucleotides such as 2-MeS-ATP and alpha beta -methylene-ATP (alpha beta -MeATP) resulted in variable stimulating signals and relatively rapid desensitization of the response to other P2 agonists, including ATP. Therefore, in most experiments nucleotides were applied by a puffer pipette. We noticed that in this setup ~5- to 10-fold higher concentrations of ATP or ATPgamma S in the puffer pipette were needed to obtain the same intensity of stimulation observed when the agonists were applied by perfusion. In addition, the need to change the puffer pipette for each change in agonist concentration precluded obtaining accurate dose-response curves for the agonists used. Nonetheless, half-maximal responses for the agonists listed in Table 2 were obtained at concentrations between 25 µM (alpha beta -MeATP) and 100 µM (BzATP). This profile did not conform to any known P2R type. Thus we concluded that pharmacological characterization is unreliable in identifying the P2R expressed in pancreatic duct cells.

A reliable approach to identify P2R expression is PCR analysis of P2R in a defined cell type. To achieve that, we prepared RNA from microdissected intralobular ducts. Because smooth muscle and endothelial cells in blood vessels express several P2R (6, 23), it was important to verify the absence of contaminating blood capillaries in our preparation. eNOS protein is abundantly and exclusively expressed in pancreatic capillaries (37) and is therefore a good marker for contaminating capillaries. As described in METHODS and Fig. 1, the RNA prepared from microdissected intralobular ducts contained mRNA for CFTR (ducts) but not mRNA for eNOS (blood capillaries) or amylase (acinar cells). Figure 3 shows the results of RT-PCR analysis of P2R expressed in pancreatic duct using this mRNA preparation. Pancreatic ducts were found to express at least three P2XR (P2X1, P2X4, and P2X7) and four P2YR (P2Y1, P2Y2, P2Y4, and P2Y5). The ability of the primers that gave negative results in pancreatic ducts to amplify the expected PCR product was ascertained by using mRNA preparation from a submandibular gland tissue (Fig. 3, bottom). Although no mammalian homolog of P2Y3R has been identified, Li et al. (27) provided evidence that P2Y3R is the functional avian homolog of mammalian P2Y6R. Nevertheless, primers for P2Y3R and P2Y6R were used to probe the expression of this gene in pancreatic duct. With both sets of primers no product was amplified. It is also important to note at this stage that, despite extensive efforts, no response could be found for the cloned and expressed P2Y5R when stimulated with various P2 agonists (21, 28). Thus the P2Y5R may be an orphan G protein-coupled receptor for a nonnucleotide agonist. Consequently, although P2Y5R is expressed in pancreatic duct cells, the possible function of this receptor is not considered further in the present work. The surprising number of P2R expressed in one cell type prompted several verifications of these findings (between 3 and 6 RNA preparations from different animals). Positive signals like those shown in Fig. 3 were obtained in all experiments. In addition, the identity of all RT-PCR products was verified by sequencing. The multiple P2Rs expressed in pancreatic duct cells explain well the failure of the pharmacological approach to identify the P2R expressed in these cells.


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Fig. 3.   RT-PCR analysis of P2 receptors (P2R) in pancreatic duct. PCR primers listed in Table 1 were used to analyze expression of P2R in intralobular pancreatic ducts. Positive controls for PCR primers that did not give a signal in pancreatic duct were obtained with RNA extracted from submandibular gland tissue. M denotes 100-bp ladder markers.

G protein-coupled and G protein-independent receptors. A critical question raised by the PCR analysis is whether all P2R expressed in duct cells are functional. Considering the number of P2R expressed in duct cells, the only feasible approach was to test the effect of agonists reported to be at least partially selective for defined P2R. These agonists could be divided into two groups: those that activate the ionotropic, G protein-independent P2R and those that activate the metabotropic, G protein-coupled P2R. Figure 4 shows the protocol used to identify the agonists activating the G protein-independent P2R. Duct cells were dialyzed through the patch pipette with solutions that were with or without 2 mM GDPbeta S to inhibit all G proteins. Inhibition of G protein-dependent signaling was always verified by the complete inhibition of the response to muscarinic stimulation with 1 mM carbachol. Stimulation by ATPgamma S, BzATP, and alpha beta -MeATP was not inhibited by GDPbeta S. Cells were stimulated with ATPgamma S by perfusion of a solution containing the maximal effective concentration of 100 µM. In these and all other current recording experiments, all other nucleotides were applied by puffing. A concentration of 1 mM in the puffing pipette solution was found to be maximal for all agonists. Because the response to ATPgamma S and carbachol was used as a control, their effect was tested in all cells. The response to these agonists was highly consistent where 141 out of 154 (92%) cells responded to carbachol, and 131 out of these 154 cells (85%) responded to ATPgamma S. Of the 141 cells responding to carbachol, 131 (93%) responded to ATPgamma S. Finally, 133 out of 147 cells (90%) infused with GDPbeta S responded to ATPgamma S (Table 2).


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Fig. 4.   P2R agonists that stimulate G protein-independent P2R in pancreatic duct cells. Single duct cells were dialyzed with a standard pipette solution (A, C, E) or a standard pipette solution containing 2 mM GDPbeta S (B, D, F). As indicated, cells were perfused with bath solutions containing 1 mM carbachol and 10 µM atropine. The cells were then stimulated by perfusion with a bath solution containing 100 µM adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S; A, B) or by puffing a bath solution containing 1 mM 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP; C, D) or 1 mM alpha beta -methylene-ATP (alpha beta -MeATP; E, F). Note that GDPbeta S completely inhibited the response to carbachol without affecting the response of the nucleotides. Results of all similar experiments are summarized in Table 2.

In one series of experiments 10/10 cells responded to ATPgamma S, and in the same series 20/20 cells dialyzed with 2 mM GDPbeta S responded to stimulation with ATPgamma S (Table 2). Although ATPgamma S was proposed as a selective agonist for P2X2R and P2X4R (1), it was shown to be a partial agonist for P2X2R (11) and P2X3R (26). Pancreatic duct cells express P2X1R and P2X4R. Thus the effect of ATPgamma S indicates expression of functional P2X4R and possibly P2X1R in duct cells. BzATP is a potent agonist for P2X7R (1, 6, 22, 29). It is also a partial agonist for P2X1 and P2X2R (11). Thus the response of duct cells to BzATP suggests that P2X7R and P2X1R are functional in pancreatic duct cells. These findings are in agreement with a recent study reporting an effect of BzATP on [Ca2+]i of duct cells (8). The response of duct cells to alpha beta -MeATP that was not sensitive to GDPbeta S inhibition suggests that the response was due to an active P2X1R in duct cells. alpha beta -MeATP is a potent agonist for P2X1R and P2X3R (5, 9). Of the alpha beta -MeATP-sensitive P2R, pancreatic duct cells express only P2X1R. Furthermore, extensive studies showed that this nucleotide does not activate P2X2R, P2X4R, P2X5R, and P2X6R when the clones are expressed in HEK-293 cells (5, 9, 11, 26). However, in several tissues, P2X1R could be activated by 2-MeS-ATP as well as by alpha beta -MeATP (29, 22). In 14 of 14 experiments, duct cells did not respond to 2-MeS-ATP when GDPbeta S was included in the pipette solution (Table 2). Activation of P2X4R by alpha beta -MeATP cannot explain this discrepancy, since P2X4R is also more sensitive to 2-MeS-ATP stimulation than to alpha beta -MeATP (22, 29). Although likely, the absence of a GDPbeta S-insensitive 2-MeS-ATP response does not allow us to state with certainty whether P2X1R expressed in the pancreatic duct cell (Fig. 3) is functional.

The response of a second group of P2R agonists was completely inhibited by GDPbeta S, indicating coupling of these receptors to heterotrimeric G proteins. Figure 5 shows individual examples, and Table 2 summarizes the results of multiple experiments. These agonists consistently activated pancreatic duct cells. The response to UTP, 2-MeS-ATP, and ADP was observed in 17 of 19, 13 of 15, and 6 of 7 cells, respectively. In all experiments (13 with UTP, 14 with 2-MeS-ATP, and 11 with ADP), infusion of 2 mM GDPbeta S into the cells prevented the response to these agonists. In further controls, the same cells did not respond to muscarinic stimulation but showed a completely normal response to ATPgamma S (Table 2). In eight of eight experiments, we could not observe an effect of UDP on Cl- current whether UDP was applied by perfusion or by puffing. These observations should be contrasted with the findings below showing the effect of UDP on [Ca2+]i of microperfused pancreatic ducts. The reason for a lack of response of single duct cells to UDP is not clear at present. One possibility is that the [Ca2+]i increase evoked by activation of the UDP-sensitive P2R is spatially segregated from the Cl- channels.


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Fig. 5.   P2R agonists that stimulate G protein-coupled P2R in pancreatic duct cells. Single duct cells were dialyzed with standard pipette solution (A, C, E) containing 2 mM GDPbeta S (B, D, F). Where indicated the cells were stimulated by 1 mM carbachol, inhibited with 10 µM atropine, and stimulated by perfusing a solution containing 100 µM UTP (A, B) or by puffing solutions containing 1 mM 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP; C, D) or 1 mM ADP (E, F). In experiments B, D, and F, positive signals were obtained by stimulating cells with 100 µM ATPgamma S. Results of all similar experiments are summarized in Table 2.

Cross-reactivity of most P2R agonists with the various P2YR makes it difficult to determine with any certainty which P2YR are functional in duct cells. The very high sensitivity of P2Y1R to stimulation by 2-MeS-ATP (22, 29), the inhibition of the response to this agonist by GDPbeta S (Table 2), and the high potency for this agonist to increase [Ca2+]i in microperfused ducts (see Fig. 8) can be considered good evidence for a functional P2Y1R in pancreatic duct cells. Similar arguments can be made for the effect of UTP on P2Y2R and P2Y4R expressed in duct cells. These are the only P2R to show high affinity for UTP. We can be more certain of the expression of a functional P2Y4R in pancreatic duct cells, since the cells responded to UDP (see Fig. 8), an agonist for this receptor (22, 29).

Sidedness of agonist response. Physiologically and possibly clinically relevant information is the cellular and membrane localization of the P2R in pancreatic duct cells. Because activation of all known P2R increases [Ca2+]i (6, 22, 23, 29), an initial and partial answer to this question can be obtained by measuring the effect of P2R agonists on [Ca2+]i of the microperfused pancreatic duct. In the first set of experiments we tested the effect of luminal and basolateral ATP on [Ca2+]i. Figure 6A shows that application of 1 mM ATP to the BLM or LM side of the duct was equally effective in increasing [Ca2+]i. In all experiments tested (n = 16), cells responding to bath ATP also responded to bath carbachol, which usually increased [Ca2+]i to a level somewhat higher than that induced by ATP. Activation of P2XR increases Ca2+ influx, whereas activation of P2YR increases [Ca2+]i by activation of Ca2+ release from internal stores and Ca2+ influx (6). This distinction can be used to determine the type of P2R expressed on each side of the pancreatic duct. Figure 6, B and C, shows that removal of extracellular Ca2+ from the luminal and bath solutions reduced, but did not prevent, the effect of either basolateral or luminal ATP on [Ca2+]i. Readdition of Ca2+ to the bath following basolateral stimulation and to the lumen following luminal stimulation caused a marked increase in [Ca2+]i, indicative of maintained activation of Ca2+ influx during P2R stimulation. Because ATP can activate P2YR and P2XR present in both membranes (see Figs. 7 and 8), it is not clear which pathway mediates the influx. However, Ca2+ mobilization from internal stores by basolateral or luminal ATP clearly indicates that P2YR are expressed in both membranes.


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Fig. 6.   Effect of bath and luminal ATP on intracellular Ca2+ concentration. Fura 2-loaded microperfused intralobular pancreatic ducts were sequentially exposed to bath solutions containing 1 mM ATP or 0.1 mM carbachol and then to a luminal solution containing 1 mM ATP (A). Separate ducts were perfused with Ca2+-free bath and luminal solutions before stimulation with bath (B) or luminal (C) ATP. Arrows, 1 mM CaCl2 added back to bath and then lumen (B) or added to lumen and then bath (C). Number of responding ducts is given in text.

The agonists characterized in Table 2 in terms of their sensitivity to GDPbeta S inhibition were used to determine the side from which they stimulate the pancreatic duct. Figure 7 shows that ATPgamma S and alpha beta -MeATP were effective only from the basolateral side. ATPgamma S was the most effective and potent agonist acting from the basolateral side of pancreatic duct (n = 13/13 experiments alone and n = 6/6 experiments when compared with ATP in the same duct). The response of the duct to alpha beta -MeATP was less consistent. The effect of 1 mM basolateral alpha beta -MeATP was observed in three of five ducts that responded to basolateral ATP. The same five ducts did not respond to luminal alpha beta -MeATP whether the lumen was exposed to alpha beta -MeATP before (not shown) or after (Fig. 7B) stimulation of the ducts with basolateral nucleotides. This analysis suggests that P2X4R and P2R activated by alpha beta -MeATP (probably P2X1) are expressed only in the BLM of pancreatic duct cells.


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Fig. 7.   P2R agonists interacting with P2R in basolateral membrane. Experimental protocol was as described in legend to Fig. 6, except that in A the duct was stimulated with increasing concentrations of bath ATPgamma S (1, 10, 100 and 1,000 µM) and then with 1 mM luminal ATPgamma S. B: duct was sequentially stimulated with 1 mM bath alpha beta -MeATP, 1 mM ATP, and then 1 mM luminal alpha beta -MeATP. Number of responding ducts is given in text.

The response of the pancreatic duct to agonists acting only from the luminal side is illustrated in Fig. 8. In all experiments (n = 7/7 in the presence of external Ca2+ and 3/3 in the absence of external Ca2+), the perfused duct responded to luminal but not basolateral UTP. Figure 8A shows the effect of 100 µM UTP. However, luminal UTP at a concentration as low as 1 µM increased [Ca2+]i, whereas up to 1 mM basolateral UTP had no effect. Thus UTP acts exclusively from the luminal side of the duct.


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Fig. 8.   P2R agonists interacting with luminal P2R. Experimental protocol was as in Fig. 7, except that ducts were stimulated with indicated nucleotides added to bath or luminal solutions, as specified by bars. In all experiments accessibility of nucleotides to basolateral membrane receptors through connective tissue was verified by demonstrating positive response to application of bath ATP.

The effect of UDP was tested only at a single concentration of 1 mM. Basolateral UDP had no effect on [Ca2+]i in four of four ducts that responded to bath ATP. In three of four of these ducts luminal UDP increased [Ca2+]i, but the effect was variable (0, 0.3, 0.6, and 0.75 ratio units). Another agonist acting exclusively from the luminal side is 2-MeS-ATP (Fig. 8C). The response of the duct to this agonist was consistent (5/6 of the ducts that responded to basolateral and/or luminal ATP), although, unlike UTP (Fig. 8A), it tended to increase [Ca2+]i less than ATP. The maximal effective concentration of 2-MeS-ATP was between 10 and 100 µM. Because of the uncertainty as to the exact P2R subtype activated by each of the luminal agonists, it is not clear which P2YR are expressed in the luminal membrane of the pancreatic duct. However, this membrane expresses most of the P2YR types since 1) the effect of all luminal agonists was inhibited by GDPbeta S (Table 2) and 2) they had no effect on [Ca2+]i when applied to the basolateral side at concentrations 10- to 100-fold higher than those effective from the luminal side.

The last group of P2R agonists are those effective from both sides. Figure 9 shows that these are ADP and BzATP. The effect of ADP was observed in two of three ducts responding to ATP, and in the two responding ducts ADP had a similar effect on [Ca2+]i when applied to the bath or the luminal sides. Note that the effect of ADP was completely sensitive to GDPbeta S (Table 2), indicating that ADP activated only P2YR. In fact, ADP is the only agonist activating P2YR found to be effective from the basolateral side. Whether ADP activates the same P2YR on both sides is not known at present, since at least two of the P2YR expressed in pancreatic duct cells (Fig. 3) can be activated by ADP (22, 29).


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Fig. 9.   P2R agonists interacting with P2R in both the luminal and basolateral membranes. As indicted, the duct was sequentially stimulated with bath and luminal BzATP (0.1 or 1 mM) or bath and luminal ADP (1 mM). Number of responding ducts is given in text.

The effect of basolateral and luminal BzATP was observed in all five of the seven ducts that responded to bath and/or luminal ATP. However, as illustrated in Fig. 9A, luminal BzATP was more effective than bath BzATP in increasing ductal [Ca2+]i. Although we were unsuccessful in demonstrating a response to bath BzATP in ducts continuously stimulated with 1 mM luminal BzATP, we do not believe that the effect of bath BzATP on [Ca2+]i is due to leakage into and activation of luminal receptors, since the same was not noted with any of the nucleotides acting only from the luminal side (Fig. 8). Hence it seems likely that the P2X7R identified in the present work is expressed in both the LM and BLM of the pancreatic duct. Expression of P2X7R was also found in the LM of submandibular duct cells (37, 38).

The myriad P2R expressed in each side of the pancreatic duct raises two questions. 1) Does each pancreatic duct cell express all P2R? 2) Why does the pancreatic duct express so many P2R? At present, we can only partially address the first question and only speculate with regard to the second question. The only approach currently available to address the number of P2R types expressed in each cell is the functional approach. This approach is limited to measurements from single dissociated cells, like those summarized in Table 2. Although the pancreatic duct contains at least two major cell types and probably several subtypes (10), we are quite confident that all duct cells express the P2R activated by ATPgamma S. Of the 141 single duct cells that responded to carbachol, 131 responded to ATPgamma S, and all cells responding to any of the agonists listed in Table 2 also responded to ATPgamma S. Because the sample is not as large with the other agonists, it is not clear how many P2R types each cell expresses. However, we note that with all agonists the percentage of responding cells was very high, approaching 100%. This was also the case for single cells stimulated with UTP (17/19 responding). This finding is somewhat different from a recent study, which reported that approximately one-third of the ducts that showed a normal response to ATP did not respond to UTP (8). Considering the different experimental systems and cell preparation used in the two studies, the different observations can be explained in many ways.

The high percentage of cells responding to all P2R agonists tested suggests that each duct cell expresses most, if not all, the P2R identified and the expression of multiple P2R in each membrane. This is not without precedent. Molecular analysis showed expression of multiple R2R in all regions of the brain (5). Measurement of short-circuit current in primary cultures of nasal and airway epithelial cells in monolayers suggested expression of at least three P2R in these cells (20, 24, 30, 32, 33). Several studies showed that submandibular duct cells express at least three P2R (2, 37, 38). A combined molecular and functional analysis, similar to that reported here for the pancreatic duct, is likely to show that the cells mentioned above and other epithelial cells express more than the three suggested P2R. Preliminary studies with isolated submandibular gland ducts suggest that this is indeed the case (not shown). Expression of such a high number and diverse P2R types in secretory epithelia suggests that P2R plays an important role in controlling epithelial cell function. This is further supported by the finding that P2R is expressed in both membranes. The P2R may specialize in activating particular ion channels to control cell activity. So far there are two examples of such a specialization. In airway epithelia luminal ATP activates the outward rectifier Cl- channel (20, 30), and in submandibular gland cells P2X7R activate a CFTR-like Cl- channel (37). Additional specialization may be achieved by expression of selective P2R in microdomains of the LM and BLM to allow a localized secondary regulation of transepithelial transport. Further refinement of cell preparation and recording techniques is needed to probe the physiological significance of the multitude of P2R in pancreatic duct cells and other epithelia.


    ACKNOWLEDGEMENTS

We thank Roshi Mehdibeigi for technical support and Karen Miler for administrative assistance.


    FOOTNOTES

This work was funded by National Institutes of Health Grants DE-12309 and DK-38938.

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

Address for reprint requests and other correspondence: S. Muallem, The Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9040 (E-mail: smuall{at}mednet.swmed.edu).

Received 4 January 1999; accepted in final form 28 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Am J Physiol Cell Physiol 277(2):C205-C215
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