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J Biol Chem, Vol. 274, Issue 45, 31784-31791, November 5, 1999

Purinoceptors Evoke Different Electrophysiological Responses in Pancreatic Ducts
P2Y INHIBITS K⁺ CONDUCTANCE, AND P2X STIMULATES CATION CONDUCTANCE^*

Susanne E. Hede, Jan Amstrup, Bettina C. Christoffersen, and Ivana Novak

From the August Krogh Institute, Copenhagen University, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca²⁺ and pH measurements, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. The data show two types of purinoceptors and cellular responses. UTP and ATP produced large Ca²⁺ transients, a decrease in intracellular pH, 8-10-mV depolarization of the membrane voltage, and a decrease in the whole-cell conductance. The membrane effects were due to closure of K⁺ channels, as confirmed by dependence on extracellular K⁺. UTP/ATP effects could be associated with P2Y₂ purinoceptors, and RT-PCR revealed mRNAs for P2Y₂ and P2Y₄ receptors. On the other hand, 2',3'-O-4-benzoylbenzoyl-ATP induced Ca²⁺ influx and ~20-mV depolarization of the membrane voltage with a concomitant increase in the whole-cell conductance. These effects were dependent on extracellular Na⁺, not Cl, indicating opening of cation channels associated with P2X₇ purinoceptors. RT-PCR showed mRNAs for P2X₇ and P2X₄ receptors. In microperfused ducts, luminal (but not basolateral) ATP caused large depolarizations of membrane voltages recorded with microelectrodes, consistent with luminal localization of P2X₇ receptors. Thus, P2Y₂ (and possibly P2Y₄) purinoceptors inhibit K⁺ channels and may not support secretion in native ducts. P2X₇ (and possibly P2X₄) receptors are associated with cation channels and may contribute to regulation of secretion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Extracellular nucleotides are regulators of a wide range of cellular functions in various cells, including regulators of epithelial transport (1). They act through specific P2 receptors, which, according to their molecular structure and signal transduction, have been divided into two distinct receptor families: G protein-coupled receptors (P2Y receptors) and ligand-gated ion channels (P2X receptors) (1). In epithelia cultured from upper respiratory airways, intestine, and exocrine glands, including pancreas, UTP and ATP stimulate Cl transport through P2Y receptors, which were described pharmacologically as P2U receptors and, in many cases, may correspond to the cloned P2Y₂ receptors (2-11). However, similar action of pyrimidines on Cl transport could also be exerted through P2Y₄ or P2Y₆ (12, 13). Due to their effects on Cl transport, nucleotides were proposed as therapeutic agents for treatment of cystic fibrosis, as they could bypass the defective function of the cystic fibrosis transmembrane regulator Cl conductance and restore secretion by activation of a Ca²⁺-dependent Cl conductance (8). Interestingly, recent studies on exocrine glands indicate that the P2Y receptors and associated signal transduction pathways are not static, but dynamic. For example, the P2Y₂ receptor expression increases dramatically in tissue culture of salivary gland cells (14), and the P2Y₁ receptor activity changes during the gland development (15). Thus, it is important to consider which P2 receptors are present in freshly prepared cells from exocrine glands. Although there is evidence for some P2Y-type receptors (4, 11, 16, 17), more commonly accounted purinoceptors are those linked directly to cation influx (18-23). Occupation of these purinoceptors leads to stimulation of Cl transport, volume changes, and secretion of enzymes and peptides (19, 21, 24), but in some cases, interference with the Ca²⁺ signaling utilized by other receptor pathways has been reported (25, 26). These receptors have been described pharmacologically as P2Z receptors, but it is unclear if these correspond to the pore-forming P2X₇ receptors originally found in immunoreactive cells (1). Interestingly, in situ hybridization analysis shows P2X₄ receptors on salivary gland acini, but not on ducts (27). Thus, from pharmacological studies, it seems that exocrine glands can express receptors belonging to P2X and P2Y families, but their identity, localization, and function are unclear.

Pancreatic ducts modify enzyme- and Cl-rich secretion originating from acini by adding a bicarbonate-rich fluid. Bicarbonate secretion is achieved by coupling of ion transport through the luminal cystic fibrosis transmembrane regulator Cl channels and the luminal Cl/HCO₃ exchanger (28-30). The driving force for the Cl exit is kept by the K⁺ exit via the basolateral K⁺ channels (29, 31-33). The main regulators of pancreatic bicarbonate secretion are secretin, vasoactive intestinal peptide, acetylcholine, and noradrenaline (30, 34, 35). In earlier studies, we showed that isolated ducts also respond to ATP by releasing stored Ca²⁺ and initiating Ca²⁺ influx through channels different from those used by acetylcholine (36-38). Furthermore, we demonstrated that ATP inhibits secretin-evoked changes in the membrane voltage (37). We used Ca²⁺ signals and pharmacological profiles to describe two types of receptors: one responding to UTP and ATP and belonging to the P2Y superfamily (P2Y₂-like receptor) and another responding to BzATP¹ and ATP and belonging to the P2X superfamily (P2X₇-like receptor) (36). Although epithelial P2 receptors are associated with Ca²⁺ signaling in ducts, it is not clear how this is further translated into ion transport and thus secretion in native pancreatic ducts. Therefore, the aim of this study was to use the patch-clamp methods and intracellular Ca²⁺ measurements to elucidate how purinergic receptors regulate ion transport in pancreatic ducts. Furthermore, we aimed at identifying these purinoceptors using molecular biological methods, which were applied to this epithelium for the first time. For this purpose, we used pancreatic duct fragments prepared from collagenase digests of whole rat pancreas. The advantage of this preparation is that we can obtain pure duct cells free of the surrounding connective tissue, blood vessels, and nerves, all likely purinoceptor contaminants. These ducts retain receptors, Ca²⁺ signals, and electrophysiological responses comparable to the microdissected ducts (29, 30, 32, 34, 39). The disadvantage of the preparation is that ducts are too fragile for microperfusion; and thus, the polarity of the responses cannot be studied.

In this study, we show that UTP/ATP inhibit K⁺ channels and thus most likely cannot support secretion. On the other hand, BzATP stimulates cation conductance and may therefore regulate secretory processes stimulated by other secretagogues. RT-PCR analysis revealed that ducts contain mRNAs for P2Y₂ and P2X₇ receptors, likely candidates for the observed effects, as well as mRNAs for P2Y₄ and P2X₄ receptors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All standard chemicals including collagenase V, trypsin inhibitor, UTP, ATP, and BzATP were obtained from Sigma (Copenhagen, Denmark). Fura-2/AM and BCECF/AM were obtained from Molecular Probes, Inc. (Leiden, The Netherlands). Tissue culture media and primers for PCR were provided by Life Technologies, Inc.

Preparation of Ducts-- Female Wistar rats (100-200 g), kept on a standard laboratory diet, were killed by cervical dislocation. The pancreas was excised and subjected to collagenase digestion as described previously (39). Briefly, the pancreas was cut into small pieces and incubated with collagenase V (1.3 mg/ml) in Dulbecco's modified Eagle medium 1000/Ham's F-12 mixture containing trypsin inhibitor (0.17 mg/ml). Pancreatic ducts compose <5% of the total pancreatic tissue, and single intercalated and small intralobular duct fragments (outer diameter of 10-40 µm and length of 100-200 µm) were identified with the aid of a dissection microscope. Any remaining loose connective tissue and small blood vessels were stripped with sharpened forceps. Subsequently, ducts were transferred into a chamber placed on an inverted microscope (Axiovert 100TV (Carl Zeiss, Inc.) or DM IRBE (Leica Inc.)) and inspected with 40× or 63× objectives for "cleanliness." Fig. 1 shows an example of small intralobular ducts obtained using these methods. These "clean" ducts were used for patch-clamp experiments, for molecular biology, and for most fluorescence measurements. For physiological experiments, suction pipettes held ducts on a side such that both the luminal and basolateral sides of the epithelium were accessible to the bathing solutions. For one series of experiments, small intralobular and extralobular ducts (outer diameter of 20-50 µm) were microdissected from fresh rat pancreas. These microdissected ducts were held between concentric pipettes at each end, and the lumen was perfused according to methods described earlier (32). Such ducts were used for microelectrode recordings.

View larger version (72K): [in this window] [in a new window]   Fig. 1.   Pancreatic ducts. Shown is an example of two pancreatic ducts freed from the surrounding connective tissue by collagenase digestion and microdissection. Both ducts are intralobular; the duct in the right panel is closest to acinar cells. Bars indicate 20 µm. Differential contrast images were obtained with a 63× water objective in a Leica confocal laser scanning microscope.

During all physiological experiments, the chamber was continuously perfused at a rate of 10-15 ml/min to avoid mechanical disturbances, which can cause release of ATP.² Control solutions contained 145 mM Na⁺, 3.6 mM K⁺, 1.5 mM Ca²⁺, 1 mM Mg²⁺, 125 mM Cl, 25 mM HCO₃, 2 mM phosphate, and 5 mM glucose. The pH was equilibrated to 7.4 with 5% CO₂ in O₂. The temperature was kept constant at 37 °C during all experiments. In some experiments, K⁺, Na⁺, or Cl concentrations were modified as follows: K⁺ was increased from 3.6 to 20, 25, or 50 mM by equimolar substitution for Na⁺; Na⁺ was decreased from 145 to 5 mM and substituted with N-methyl-D-glucamine; and Cl was decreased from 125 to 5 mM and substituted with gluconate. The pancreatic ducts were stimulated with ATP, UTP, and BzATP in concentrations of 0.1 mM. In a few experiments, concentrations of agonists were varied from 10⁸ to 10³ M, except for BzATP, which was not used at concentrations higher than 0.1 mM due to the cost.

Electrophysiological Measurements-- The cell responses to nucleotides were monitored in whole-cell nystatin patch-clamp recordings adopted for pancreatic ducts (39). Patch pipettes had resistances of 3-5 megaohms, and the initial seal was at least 1 gigaohm. The success rate of obtaining seals was <10%, and stable cell membrane voltage recordings were obtained only in a fraction of these. The cell membrane under the pipette was permeabilized to make direct electrical access to duct cells. Nystatin (0.02-0.1 mM) was dissolved in a pipette solution adjusted to pH 7.2 that had the following composition: 11 mM Na⁺, 125 mM K⁺, 32 mM Cl, 96 mM gluconate, 1 mM Mg²⁺, 6 mM phosphate, and 5 mM glucose; and Ca²⁺ was adjusted to 0.1 µM with EGTA. A flowing 1 M KCl electrode was used as a reference. The membrane voltage (V_m) was continuously monitored during experiments in a current-clamp mode (zero current clamp). Periodically, the whole-cell current (I) was measured in a voltage-clamp mode, where the voltage was first clamped to the spontaneous cell voltage and then in increasing and decreasing 10-mV steps of 500-ms duration. In some experiments, V_m was clamped at the spontaneous cell voltage, and continuous whole-cell currents were measured. Where possible, the total conductance (G_t) was corrected for the series resistance to obtain the cell membrane conductance. The series resistance was estimated by the compensation circuit of the patch-clamp amplifier (EPC 9, Heka, Lambrecht, Germany), and in some cases where this was not possible, series resistance and cell membrane conductance estimations were made subsequently by fitting an exponential function to the initial part of the current trace (40). Since duct cells can be variably coupled to each other, G_t changes with the agonists are relative rather than absolute. In several experiments, ducts were microdissected and microperfused according to methods described earlier (32). Single duct cells were impaled with 100-200-megaohm Ling-Gerard microelectrodes, and V_m was recorded with a WPI electrometer.

Measurements of Intracellular Ca²⁺ and Intracellular pH-- The fura-2 method was used to estimate [Ca²⁺]_i in pancreatic ducts. Briefly, as we have described earlier (37), ducts were loaded with 0.5-1.0 µM fura-2/AM, and the fluorescence intensity of 10-20 cells was measured with a photomultipler at 510 nm after excitation at 340, 360, and 380 nm. The fluorescence ratio (340/380 nm) was an estimate of [Ca²⁺]_i. Signals were calibrated in situ with ionomycin (1.0-5.0 µM). For pH_i measurements, ducts were loaded with BCECF/AM as described earlier (41). Emission intensity of duct cells was measured at 510 nm after excitation at 490 and 440 nm. The fluorescence ratio (490/440 nm) was an estimate of pH_i. Signals were calibrated in situ with the protonophore carbonyl cyanide p-chlorophenylhydrazone or nigericin (1 or 10 µM).

Statistics-- Physiological data are presented as original recordings, summaries, and means ± S.E. In most experiments, control and test measurements were made within one duct cell or one duct, and n refers to measurements on different ducts. The paired Student's t test was applied, and p < 0.05 was accepted as significant. The F-test was used to test for the difference between two regression coefficients obtained for the current-voltage relations.

RNA Isolation, cDNA Synthesis, and Polymerase Chain Reaction Amplification-- Total RNA was isolated from small pancreatic ducts (prepared from collagenase digests of rat pancreas) (Fig. 1) using total RNA isolation reagent (Advanced Biotechnologies Ltd.) according to the manufacturer's instructions. First strand cDNA was synthesized utilizing an anchored oligo(dT) primer using Reverse-iT (Advanced Biotechnologies Ltd.). PCR conditions were identical for all primers (Table I). 2 µl of reverse-transcribed RNA was added to a solution of 10 µM each forward and reverse primers, 1.5 mM MgCl₂, PCR buffer (7.5 mM Tris-HCl, pH 8.8, 20 mM (NH₄)₂SO₄, and 0.01% (v/v) Tween 20), 0.5 mM dNTPs, 1.25 units of Red Hot DNA polymerase (all from Advanced Biotechnologies Ltd.), and distilled H₂O in a total volume of 50 µl. Temperature cycling proceeded as follows: 1 cycle at 94 °C for 5 min and 35 cycles at 94 °C for 30 s, 55 °C for 60 s, and 72 °C for 90 s, followed by 72 °C for 10 min. PCR products were then subjected to gel electrophoresis on a 1% agarose gel containing ethidium bromide. Possible contamination of blood vessels and acinar cells was ruled out by RT-PCR amplification of thrombin and amylase using two specific primers (Table I). Thrombin is present in endothelial cells, but not duct cells; and similarly, amylase is present in acinar cells, but not duct cells. As a positive control for duct cells, carbonic anhydrase II primers were used. As tissue controls, we used blood vessels obtained from collagenase digests of pancreas as well as the whole pancreas and testis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of Nucleotides on Cell Calcium-- Fig. 2A shows the effect of extracellular ATP and BzATP (0.1 mM) on [Ca²⁺]_i in pancreatic ducts. ATP stimulation, similar to that of acetylcholine, evokes a characteristic biphasic Ca²⁺ response consisting of a Ca²⁺ peak and a Ca²⁺ plateau. The peak response is largely due to release of Ca²⁺ from intracellular stores, whereas the plateau reflects largely influx of Ca²⁺ from extracellular medium (36, 38). UTP evoked a similar response, but a more pronounced Ca²⁺ influx component. On the other hand, stimulation with BzATP led to a monophasic Ca²⁺ response, which was almost fully dependent on extracellular Ca²⁺ and thus due to Ca²⁺ influx. On the basis of these and other observations, we postulated that there are at least two types of receptors belonging to both P2Y and P2X families (36). Fig. 2B summarizes the maximal Ca²⁺ responses for the three purinergic analogues used in this study. The EC₅₀ values for UTP, ATP, and BzATP were estimated at 5, 4, and ~20 µM, respectively. ATP, UTP, and BzATP were the best agonists regarding the intracellular Ca²⁺ signals compared with ITP, 2-methylthio-ATP, ,-methylene-ATP, and adenosine, as shown earlier (36). Since native pancreatic ducts are not suitable for extensive electrophysiological experiments (see "Experimental Procedures"), we focused on using ATP, UTP, and BzATP as pharmacological markers for further patch-clamp experiments aimed at elucidating how purinoceptors couple to ion transport.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of UTP, ATP, and BzATP on intracellular Ca2+. A, effect of agonists (0.1 mM) on Ca2+ transients in a single duct; B, concentration-response curves for ATP, UTP, and BzATP. Responses are given as changes in the fluorescence ratio 340/380 nm. Each point is a mean ± S.E. for 3-11 experiments.

Effect of UTP and ATP on Cell Membrane Voltage and Conductance-- In contrast to the Ca²⁺ responses evoked by UTP and ATP, the V_m responses measured in whole-cell patch-clamp experiments were relatively small, although there was some variability between cells. On average, UTP and ATP evoked ~10-mV changes in V_m, and in several cells, the effects were very small, as shown in an original recording in Fig. 3A. In contrast, BzATP depolarized V_m reversibly by ~20 mV in the same cell. Fig. 3B shows a whole-cell current measurement in another duct cell, where again the ATP and UTP responses were small, whereas BzATP stimulated a sustained inward current of ~100 pA at a holding potential of 60 mV. In this experiment, carried out in a current-clamp mode, ATP depolarized V_m by 9 mV, and UTP by 4 mV (not shown). Similar paired experiments were performed on 14 ducts, and Fig. 4A shows the summary of the V_m data for the three agonists. The summary shows that UTP and ATP caused small but significant V_m depolarizations of 8 ± 2 and 10 ± 2 mV, respectively. BzATP depolarized V_m by 22 ± 3 mV in the same experiments, and this effect was significantly larger compared with the UTP and ATP responses (p < 0.005).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of ATP, UTP, and BzATP on membrane voltage and current in single duct cells. A, original recording of Vm in a duct cell stimulated with 0.1 mM nucleotides. For most of the time, a spontaneous Vm was recorded (zero current clamp), except for short intervals when Vm was clamped in 10-mV steps above and below the spontaneous Vm for the whole-cell current measurements. B, recording of the whole-cell current in another duct cell where Vm was clamped at 60 mV.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Nucleotides depolarize membrane voltage but have different effects on the total conductance. The effects of nucleotides on Vm and Gt are summarized for 13-14 experiments. Protocols are the same as described in the legend of Fig. 3; and in most cases, all three nucleotides were tested on each duct. A, individual experiments are depicted in the upper panel showing control values for Vm just prior to and after the application of nucleotides and the Vm values with UTP, ATP, and BzATP (0.1 mM). The mean Vm changes ± S.E. for each nucleotide are summarized in the lower panel. B, shown is a summary of the mean Gt changes ± S.E. obtained from the I/V relations in these experiments. Asterisks indicate that the test values for nucleotides were significantly different from their corresponding controls (*, p < 0.05; **, p < 0.005; ***, p < 0.0005). nS, nanosiemens.

In the next series of experiments, we investigated the electrophysiological events underlying the V_m depolarization caused by UTP and ATP. To illustrate how V_m changes correlate with the cell conductance changes, Fig. 5A shows an experiment in which the V_m responses to UTP and ATP were some of the largest obtained. The corresponding current-voltage relations in Fig. 5B show that during the UTP and ATP stimulation, V_m shifted away from the resting voltage; but notably, G_t decreased. A summary of the whole-cell conductance data for 14 experiments is given in Fig. 4B. UTP and ATP caused small but significant reductions in G_t of 17 and 20%, whereas BzATP increased G_t by 18%. In 10 of these experiments, where it was possible to correct G_t for series resistance, changes in the cell membrane conductance evoked by UTP, ATP, and BzATP were qualitatively and quantitatively similar.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   UTP and ATP depolarize Vm and decrease Gt. A, original recording of Vm and the corresponding current in a duct cell stimulated with 0.1 mM UTP and ATP. Except for the voltage-clamp protocols, spontaneous Vm was recorded. A decrease in the current response in voltage-clamp protocols during the agonist application indicates a decrease in Gt. B, I/V relation obtained from the voltage-clamp protocols shown above for the same cell.

The depolarization of V_m and the reduction in whole-cell conductance, triggered by UTP and ATP, could be due to a closure of K⁺ channels. This theory was tested in current-clamp experiments in which the bath K⁺ concentration was changed from 4 to 20 mM (see "Experimental Procedures"). In unstimulated ducts, this K⁺ concentration change resulted in ~20-mV depolarization of V_m, as shown in the original recordings in Fig. 6A. During the ATP stimulation, however, the same K⁺ concentration step had a smaller effect, depolarizing V_m by ~10 mV. Fig. 6B summarizes seven paired experiments with similar protocols in unstimulated and UTP/ATP-stimulated ducts. Thus, in the stimulated ducts, the K⁺ concentration step depolarized V_m by 13 ± 3 mV. In the same but unstimulated ducts, the K⁺ step depolarized V_m significantly more by 19 ± 1 mV (p < 0.05). In additional experiments, duct cells were voltage-clamped in bath solution containing 4, 25, or 50 mM K⁺ in the presence or absence of UTP or ATP. An example of such a paired experiment on one duct cell is shown in Fig. 6 (C and D). At 4 mM K⁺, UTP decreased G_t from 10.8 to 5.8 nanosiemens (p < 0.001) and shifted the reversal potential by 16 mV away from the K⁺ equilibrium potential (Fig. 6C). At 25 and 50 mM K⁺ (only 50 mM K⁺ is shown in Fig. 6D), the control G_t was higher compared with 4 mM K ⁺, as expected for K⁺ currents. UTP decreased G_t from 25.3 to 16.0 nanosiemens (p < 0.001), but the reversal potential was not affected as the cell voltage lies close to the equilibrium potentials for K⁺, Cl, and nonspecific cation conductance. Similar paired observations were obtained for an additional four cells.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   ATP and UTP inhibit K+ conductance. A, effect of a K+ concentration step (from 4 to 20 (K20) mM) on the Vm of a single duct cell unstimulated and stimulated with 0.1 mM ATP. B, summary of data shown as means ± S.E. for seven such experiments in which duct cells were stimulated with ATP or UTP and the Vm response to the 20 mM K+ step was compared with that in the same unstimulated cells. Asterisks indicate that the test values for the K+ step were significantly different from the control resting Vm (**, p < 0.005; ***, p < 0.0005), and the response to 20 mM K+ was significantly smaller in stimulated versus unstimulated duct cells (p < 0.05). C and D, I/V curves for one cell exposed to 4 (K4) or 50 (K50) mM K+ solution, respectively, in the presence or absence of UTP (0.1 mM). The whole-cell currents were dominated by the K+ conductance, which was inhibited by UTP.

Effect of ATP on Intracellular pH-- Taken together, UTP/ATP depolarize V_m due to closure of K⁺ channels. One candidate for the K⁺ channel, the renal K⁺ channel (see "Discussion"), is down-regulated by intracellular Ca²⁺ and acidic pH_i. Therefore, in a separate set of experiments, we monitored pH_i in duct cells loaded with BCECF in response to ATP. Fig. 7 shows the reversible effect of ATP (0.1 mM) on the pH_i of a pancreatic duct. For comparison, this figure also shows the pH_i of another duct stimulated with a classical duct secretagogue, secretin (1 nM). In summary, ATP decreased pH_i from 7.51 ± 0.08 to 7.40 ± 0.10 in eight ducts (p < 0.005). In contrast, secretin had no effect on pH_i in similar experiments, i.e. pH_i was 7.49 ± 0.05 in resting ducts and 7.50 ± 0.05 in stimulated ducts (n = 16).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   ATP decreases pHi. Shown is the effect of extracellular ATP (0.1 mM) on pHi in one pancreatic duct. For comparison, this original recording shows the effect of secretin (1 nM) on pHi recorded in another pancreatic duct.

Effect of BzATP on Cell Membrane Voltage and Conductance-- In another series of experiments, we investigated the ionic basis for BzATP-evoked depolarization in V_m and increased whole-cell conductance. Fig. 8 shows examples of two protocols carried out in four experiments. Fig. 8A is the original recording of whole-cell currents in a duct stimulated with BzATP in a control 145 mM Na⁺ solution and in a test 5 mM Na⁺ solution. Fig. 8B shows the results of a voltage-clamp protocol carried out on another cell stimulated with BzATP in control and low Na⁺ solutions. The current-voltage relation indicates that BzATP increases the whole-cell conductance, but only in the presence of control Na⁺ concentrations. Hence, these experiments show that BzATP opens cation channels that allow Na⁺ and also Ca²⁺ influx, as shown earlier (36). In another set of experiments (n = 4), we tested whether BzATP stimulation affects the Cl conductance. Fig. 9 presents an example of one of four experiments in which the bath Cl concentration was decreased from 125 to 5 mM. This maneuver seemed to have no effect on BzATP-evoked V_m changes, which indicates that the Cl conductance was unaffected. For comparison, in ducts stimulated with secretin or isoproterenol, a similar Cl concentration step causes 15-20-mV depolarization, which is due to an increase in the cystic fibrosis transmembrane regulator Cl conductance (30, 34).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   BzATP increases inward current that is dependent on extracellular Na+. A, continuous recording of a whole-cell current in a duct cell stimulated with 0.1 mM BzATP with 145 and 5 (5Na) mM Na+ solutions as indicated by the bars; B, I/V relation taken from recordings made in another cell stimulated with BzATP with control (145 mM Na+) and 5 mM Na+ (5Na) in the extracellular medium. Increases in the whole-cell conductance with BzATP were apparent only with high extracellular Na+.

View larger version (8K): [in this window] [in a new window]   Fig. 9.   BzATP effect on Vm is independent of extracellular Cl. Shown is a continuous recording of Vm in a duct cell stimulated with 0.1 mM BzATP as indicated by the bar. Some minutes later, the same cell was stimulated again, and extracellular Cl was decreased from 125 to 5 mM (5Cl).

Microelectrode Recordings on Perfused Ducts-- In one series of experiments, the V_m of single duct cells was monitored with microelectrodes on microperfused ducts obtained by microdissection, and one such experiment is shown in Fig. 10A. ATP application from the basolateral side led to a small depolarization of 2 ± 1 mV (n = 8), sometimes preceded by a few millivolts of hyperpolarization (also see Ref. 37). These V_m changes are very similar to UTP/ATP responses observed using the whole-cell patch-clamp recordings in duct fragments (Figs. 3-6). In contrast, luminal application of ATP resulted in a significantly larger and reversible depolarization of 17 ± 3 mV (n = 10). These V_m changes were similar to the BzATP responses observed in duct fragments (Figs. 3 and 4).

View larger version (9K): [in this window] [in a new window]   Fig. 10.   Luminal ATP has a bigger effect than basolateral ATP on Vm. Shown is an original recording of Vm taken with a microelectrode in a microperfused duct. ATP (0.1 mM) was applied from the basolateral side (ATP bl), or it was infused luminally (ATP lum) as indicated by the bars. The recording also shows the effect of basolateral 20 mM K+ (K20).

P2 Purinoceptor Subtype Expression in Pancreatic Duct Cells Analyzed by RT-PCR-- Since the pharmacological data presented above indicated the existence of both P2X and P2Y receptors, we used RT-PCR to investigate which subtypes of the purinergic receptor were expressed in ducts. As shown in Fig. 11A, by use of primers for the P2X family (P2X₁ to P2X₇), we could detect transcripts of both P2X₄ and P2X₇. There were two bands for P2X₇; the upper band was seen only when total RNA from pancreatic ducts was used, and not when RT-PCR was performed on total RNA from other tissues. Whether the upper band is due to mispriming or to a yet unknown P2X receptor requires further investigations. Regarding the P2Y family of purinergic receptors, we did not include primers for P2Y₃, as the sequence available from GenBank^TM is non-mammalian, or primers for P2Y₅ and P2Y₇, as their identity as purinoceptors is doubtful (1). In the case of the P2Y receptors, we were able to detect transcripts of P2Y₂ and P2Y₄, but not of P2Y₆. As a control for duct epithelium, we show expression of mRNA for carbonic anhydrase II. The samples were free of RNA contaminants from acini and blood vessels since no products for amylase and thrombin were detected. The primers that gave no product in the ducts (P2X₁, P2X₂, P2X₃, P2X₅, P2X₆, P2Y₁, and P2Y₆) were tested on total RNA from testis, blood vessels, and whole pancreas to verify the ability of these primers to give products (Fig. 11B).

View larger version (63K): [in this window] [in a new window]   Fig. 11.   P2 purinoceptor subtype expression in pancreatic duct cells analyzed by RT-PCR. A, total RNA from pancreatic duct cells was reverse-transcribed, and the resulting cDNA was PCR-amplified using the indicated primers (see Table I). Lane Mw contains molecular weight markers. B, PCR primers that gave no signal in pancreatic ducts are shown here as positive controls obtained from testis, blood vessels, and whole pancreas. The gels shown are representative of at least three independent experiments. CAII, carbonic anhydrase II.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The key observations are that UTP/ATP evoked large Ca²⁺ signals, a small depolarization of the membrane voltage, and a decrease in the whole-cell conductance, effects due to an inhibition of K⁺ channels. BzATP/ATP increased cell Ca²⁺ and caused a large depolarization of the membrane voltage and an increase in the whole-cell membrane conductance, effects due to opening of cation channels and influx of Na⁺ and Ca²⁺. Thus, the two types of electrophysiological and Ca²⁺ responses are due to at least two types of purinoceptors in pancreatic ducts. RT-PCR analysis showed that native pancreatic ducts possess mRNAs for P2Y₂, P2Y₄, P2X₄, and P2X₇ purinoceptors. These are the first molecular biological data on native pancreatic duct epithelium.

P2Y Receptors-- Recently, it became evident that pyrimidines can exert their action via several P2Y purinoceptors (1). In expression systems, the P2Y₂ receptor is stimulated equally well with UTP and ATP; the human P2Y₄ receptor has a greater selectivity for UTP over ATP; and the P2Y₆ receptor is stimulated preferentially with UDP (42, 44, 45). In pancreatic ducts, we did not find expression of mRNA for P2Y₆, which is the luminal receptor on respiratory and intestinal epithelia responsible for Ca²⁺-mediated Cl secretion (12, 13). However, we detected transcripts for both P2Y₂ and P2Y₄ receptors (Fig. 11). Unfortunately, rat P2Y₂ and P2Y₄ receptors show similar pharmacology regarding agonists and accept ATP and UTP equally well (46). Both cell Ca²⁺ and electrophysiological responses are slightly more pronounced with UTP compared with ATP. However, this may simply be due to the ability of ATP to cross-react with the second type of receptors (P2X) discussed below. Thus, we cannot on this basis conclude which receptor is associated with the physiological response we observed in rat ducts. Nevertheless, stimulation of P2Y₂/P2Y₄ leads to a large Ca²⁺ transient and inhibition of K⁺ channels (Figs. 4-6). The inhibition of K⁺ channels is an unexpected finding, as in many similar epithelia, including cultured human and dog pancreatic duct epithelia (see the Introduction), P2Y₂-like receptors stimulate K⁺ fluxes as well as Cl fluxes (2, 3, 7, 10, 47). Some differences between our results and these data may be accounted for by different experimental conditions, as mentioned in the Introduction, or by different transport properties of epithelia derived from small intralobular ducts compared with main/large pancreatic ducts used for the tissue culture (48). Nonetheless, there are a few recent reports indicating that P2Y₂ receptors can inhibit M-type K⁺ currents in neurons (49), IsK/KvLQT1 K⁺ channels in vestibular dark cells (50), and Ca²⁺-activated K⁺ channels in spermatogenic cells (51). It is not yet clear what cellular mechanisms cause, on one hand, an increase in cell Ca²⁺ and, on the other hand, an inhibition of K⁺ channels. Inhibitory mechanisms could be indirect, involving calmodulin II, protein kinase C, or membrane-bound phosphatases; or they could be directly exerted by intracellular Ca²⁺, e.g. the eag K⁺ channel and renal K⁺ channels, or by acidic pH_i (52-56). Our earlier studies on ducts show that the major cell K⁺ conductance resides on the basolateral as opposed to the luminal membrane (32, 33); but except for a few patch-clamp studies on pancreatic ducts (31, 57), the identity of K⁺ channels is unknown. However, from our earlier studies, we know that the basolateral K⁺ channels are inhibited by acidic pH_i; and consequently, ducts cannot secrete (33). Interestingly, in this study, we have shown that ATP actually decreases pH_i (Fig. 7). Thus, a fall in pH_i or changes in cell Ca²⁺ and associated messengers could close the K⁺ channels in pancreatic ducts. Given that P2Y₂/P2Y₄ receptors inhibit K⁺ channels in pancreatic duct cells, they cannot support secretion, as Cl efflux cannot be followed by K⁺ efflux (see the Introduction). Perhaps then P2Y₂/Y₄ receptors could be involved in a down-regulation of secretion evoked by the true secretagogues.

P2X Receptors-- In our recent study, we identified the BzATP-responsive receptor pharmacologically as the P2Z (P2X₇-like) receptor (36). BzATP or high ATP levels evoked Ca²⁺ transients resulting from a Ca²⁺ influx, which was sensitive to DIDS, Mg²⁺, La³⁺, and extracellular pH (36, 38). This study shows that BzATP activates an inward Na⁺ current, supporting the idea that this is a ligand-gated receptor. The P2Z receptor would in many cases correspond to the cloned P2X₇ receptor (58). Since we also found transcripts for the P2X₇ receptor in pancreatic ducts, the simplest interpretation is that the P2Z receptors we have described earlier are also of this type. However, the receptor does not form a pore either in our preparation or in other exocrine glands (4, 11, 17, 19-23, 26), as it does in immunoreactive cells. One possibility is that the P2X₇ receptor expressed in glands does not contain the carboxyl-terminal domain necessary for the pore formation (58). Another possibility is that our cells do not express ancillary factors necessary for the P2X₇ pore formation. This possibility is not remote, as the P2X₇-associated pore formation depends on the expression system (59). Finally, P2X receptors are two transmembrane-spanning proteins that can form heteromers, e.g. P2X₂ and P2X₃ (60); and given the expression of P2X₄ in ducts, it is possible to speculate that the P2X₄ and P2X₇ proteins could form heteromers responsible for the physiological properties we observed in glands. Nevertheless, we cannot exclude that the P2X₄ homomer could also be responsible for some of the characteristics we observed. This receptor displays a higher affinity for ATP than P2X₇ (43, 61, 62), consistent with the observed effects on Ca²⁺ transients (Fig. 2). However, since ATP also cross-reacts with the P2Y receptors described above, one cannot use the EC₅₀ values conclusively. Nevertheless, the lower sensitivity of P2X₄ to DIDS and ,-methylene-ATP (43, 62) compared with the sensitivity we observed (36) indicates that the P2X₇ response may be dominant.

The present finding that the stimulation of the P2X₇ receptor is associated with increased Na⁺/Ca²⁺ conductance is in agreement with earlier findings on salivary gland and lacrimal gland acini, which express the P2X₇-like receptors (17, 19-23, 26). Moreover, in these glands, ATP also increases protein secretion, cell volume changes, and ion fluxes, all indicative of secretory activity. With respect to pancreatic ducts, if the P2X₇ receptors increase only the Na⁺/Ca²⁺ conductance, it is difficult to envisage how this could support secretion. Clearly, there must be an opening of the luminal Cl channels and the basolateral K⁺ channels as well as activation of exchangers, as observed with true secretagogues (30, 33). Therefore, on the basis of the present data, this receptor on its own could probably not evoke secretion; rather, by virtue of its effect on Ca²⁺, it could regulate secretion evoked by other secretagogues. Since, in the perfused duct, ATP had the strongest effects on the lumen, reminiscent of those BzATP had on the duct fragments, the most likely localization of the P2X₇ receptor is on the luminal membrane. In another type of ductal epithelium, namely ducts from submandibular glands, the luminal P2X₇-like receptor seems to have the unusual effect of opening Cl channels, without reported effects on the cation conductances (4, 11). One interpretation is that salivary gland ducts do not secrete fluid as do pancreatic ducts; and thus, coupling of receptors and transporters may be different.

In conclusion, our studies demonstrate that native pancreatic ducts express mRNAs for P2Y₂ and P2Y₄ receptors as well as for P2X₄ and P2X₇ receptors. Stimulation of P2Y₂ (and possibly P2Y₄) receptors leads to an increase in cell Ca²⁺, but a decrease in cell K⁺ conductance, a process that is not compatible with secretion. Thus, UTP/ATP acting on these receptors would down-regulate secretion evoked by other secretagogues. In contrast, stimulation of P2X₇ (and possibly P2X₄) receptors is associated with increased cation conductance, a process that may modulate action of other secretagogues.

	Note Added in Proof

After acceptance of our paper, Luo and co-workers published an article (Luo, X., Zheng, W., Yan, M., Lee, M. G., and Muallem, S. (1999) Am. J. Physiol. 277, C205-C215) showing a greater variety of P2X and P2Y receptors in pancreatic tissue. Differences between these and our results may be due to purity of duct tissue used for RT-PCR.

	FOOTNOTES

^* This work was supported by the Danish Natural Science Research Council, the Novo-Nordisk Research Foundation, and the Carlsberg Foundation.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.

To whom correspondence and reprint requests should be addressed. Tel.: 45-3532-1645; Fax: 45-3532-1567; E-mail: inovak@aki.ku.dk.

² C. E. Neumann and I. Novak, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: BzATP, 2',3'-O-4-benzoylbenzoyl-ATP; RT-PCR, reverse transcription-polymerase chain reaction; BCECF, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; V_m, membrane voltage; G_t, total whole-cell conductance; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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