P2Y receptors mediate Ca²⁺ signaling in duodenocytes and contribute to duodenal mucosal bicarbonate secretion

Abstract

Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y₂ receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y₂-receptor agonist, but not ADP (10 μM), significantly induced murine duodenal short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 μM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 μM), a store-operated channel blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice, but markedly impaired response occurred in P2Y₂ knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 μM). Both ATP and UTP, but not ADP (1 μM), raised cytoplasmic-free Ca²⁺ concentrations ([Ca²⁺]_cyt) with similar potencies in SCBN cells. ATP-induced [Ca²⁺]_cyt was attenuated by U-73122 (10 μM), La³⁺ (30 μM), or 2-APB (10 μM), but was not significantly affected by nifedipine (10 μM). UTP (1 μM) induced a [Ca²⁺]_cyt transient in Ca²⁺-free solutions, and restoration of external Ca²⁺ (2 mM) raised [Ca²⁺]_cyt due to capacitative Ca²⁺ entry. La³⁺ (30 μM), SK&F96365 (30 μM), and 2-APB (10 μM) inhibited UTP-induced Ca²⁺ entry by 92, 87, and 94%, respectively. Taken together, our results imply that activation of P2Y₂ receptors enhances DMBS via elevation of [Ca²⁺]_cyt that likely results from an initial increase in intracellular Ca²⁺ release followed by extracellular Ca²⁺ entry via store-operated channel.

purinergic receptors (purinoceptors) are a family of widely expressed transmembrane receptors and are composed of two major classes based on their relative responses to adenosine and nucleotides (i.e., ATP, ADP, UTP, and UDP) (1). P1 receptors are activated by adenosine, while nucleotides activate P2 receptors. P2 receptors are subdivided into ionotropic P2X receptors, which are ligand-gated channels, and metabotropic (G protein-coupled) P2Y receptors (39). Activation of P2X receptors (P2X_1-7 and P2XM) increases the plasma membrane permeability to Na⁺ or Ca²⁺. P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) classically signal mainly through G protein-dependent pathways, most commonly via G_q-dependent pathways, activating phospholipase C (PLC) and mobilizing intracellular Ca²⁺ (1, 39). Purinergic receptors have been well characterized in many epithelial tissues, including the gastrointestinal tract, airway, and kidney (6, 10, 39).

Both ATP and UTP are known to modulate electrolyte transport in the intestinal tract, pancreatic duct, and gallbladder (14, 24, 32, 40, 55). The effects of ATP or UTP on epithelial Cl⁻, bicarbonate (HCO₃⁻), K⁺ secretion, and Na⁺ reabsorption have been studied extensively in mouse, rat, or guinea pig tissues and in cultured human cells (Caco-2, T84), mostly by the use of Ussing chambers (24, 28, 37, 39–42, 54). It has been demonstrated that P2Y receptors play an important role in regulating Cl⁻ secretion in colon (24, 54) and HCO₃⁻ secretion in pancreatic duct and gallbladder epithelia (14, 32). However, little is known about the expression and function of purinergic receptors in duodenum, in particular, their role in duodenal mucosal bicarbonate secretion (DMBS). Recently, duodenal brush-border intestinal alkaline phosphatase activity was reported to affect HCO₃⁻ secretion in rats (2), presumably by hydrolysis of endogenous luminal ATP. This finding implies that purinergic receptors are involved in the regulation of DMBS. Moreover, an increase in cytoplasmic-free Ca²⁺ concentrations ([Ca²⁺]_cyt) by P2 receptors is associated with intestinal epithelial ion and fluid secretion (39, 40). An increase in [Ca²⁺]_cyt in duodenal epithelial cells elicits DMBS, which is a predominant physiological mechanism to defend gastric acid-induced duodenal injury (17, 20, 62). Surprisingly, although [Ca²⁺]_cyt is a critical second messenger that regulates duodenal epithelial ion transport (17, 18, 62), the regulatory mechanisms of [Ca²⁺]_cyt homeostasis in duodenal epithelial cells and P2Y receptor-mediated [Ca²⁺]_cyt in these cells are largely unknown.

Therefore, in the present study, we used duodenal epithelial cells, murine duodenal tissues, and intact mice to ask the following. 1) Are P2Y receptors expressed in duodenal epithelial cells, and, if so, which subtypes are expressed? 2) What mechanisms are involved in P2Y receptor-mediated [Ca²⁺]_cyt homeostasis in duodenal epithelial cells? Also, 3) do P2Y receptors regulate DMBS via Ca²⁺ signaling pathways? Our findings reveal that the P2Y₂-receptor subtype is expressed in duodenal epithelial cells, and that this receptor subtype appears to play an important role in Ca²⁺-mediated DMBS.

MATERIALS AND METHODS

Animals.

This study was approved by the University of California, San Diego Animal Subjects Committee. Female adult Harlan C-57 black mice, wild-type C57BL/6, and P2Y₂ receptor knockout (backcrossed and inbred onto a C57BL/6 background) mice were housed in an animal care room with a 12:12-h light-dark cycle and were allowed free access to food and water. Our laboratory's use of P2Y₂-receptor knockout mice has been described previously (53). Before each experiment, mice were deprived of food and water for at least 1 h, but for no longer than 90 min.

Ussing chamber experiments.

Ussing chamber experiments were performed as described previously (19). Briefly, after mice were anesthetized with halothane and euthanized by cervical dislocation, the abdomen was opened by a midline incision. The proximal duodenum was removed and immediately placed in ice-cold isoosmolar mannitol and indomethacin (10 μM) solution to suppress trauma-induced release of prostaglandins. The duodenal tissue from each animal was stripped of seromuscular layers, divided, and examined in three chambers (window area, 0.1 cm²). Experiments were performed under continuous short-circuited conditions (Voltage-Current Clamp, VCC 600; Physiological Instruments, San Diego, CA) with luminal pH maintained at 7.40 by continuous infusion of 5 mM HCl under the automatic control of a pH-stat system (ETS 822; Radiometer America, Westlake, OH). The volume of titrant infused per unit time was used to quantitate HCO₃⁻ secretion. Measurements were recorded at 5-min intervals, and mean values for consecutive 5- or 10-min periods were averaged. The rate of luminal HCO₃⁻ secretion was expressed as micromoles per centimeters squared per hour. The short-circuit current (I_sc) was measured in microamperes and converted into microequivalents per centimeter squared per hour. After a 30-min period during which basal parameters were measured, inhibitors were added to both sides of the tissues for another 10 min, followed by addition of UTP or ADP to both sides of the tissues. Electrophysiological parameters and HCO₃⁻ secretion were then recorded for an additional 60 min.

Acid-stimulated HCO₃⁻ secretion in vivo.

In vivo experiments were performed using a well-validated technique, as described previously (27, 62), with the HCO₃⁻ concentration of samples measured via a CO₂-sensitive electrode. Mice were anesthetized by intraperitoneal injection of a Hypnorm/Midazolam cocktail (25% Hypnorm plus 25% Midazolam, 10 mg/kg). Respiratory rate and response to toe-pinch of the animals were carefully monitored. After initiation of anesthesia, the abdomen was opened, and the duodenum accessed through two small incisions: one just below the ribcage on the left side, and the other just below the sternum. The stomach was located through the first incision, and a small incision was made just proximal to the pyloric sphincter. A soft polyethylene catheter was inserted into the stomach through this incision, gently pushed through the pyloric sphincter, and tied firmly into position with silk suture thread around the outside of the pyloric sphincter, thus isolating the proximal duodenum (5–10 mm) from the stomach. The junction of the pancreatic duct and the duodenum was located through the incision below the sternum. A small incision was made in the duodenum, and a second polyethylene catheter was inserted and tied into place just proximal to the junction with the pancreatic duct without interrupting the duodenal blood supply. Thus pancreatic secretions were excluded from the test duodenal segment while the blood supply remained intact. Throughout the duration of the experiment, the duodenum maintained a healthy pink color and was kept moist within the abdominal cavity.

After surgery, the proximal duodenum was perfused (0.15 cc/min) with isotonic saline for 20 min. After this initial washout and recovery period, basal HCO₃⁻ secretion was measured for 20 min. The mouse was then given an intraperitoneal injection of drugs, as indicated by the experimental design or control vehicle (DMSO), and HCO₃⁻ secretion was measured for 6 min. The duodenal segment was then perfused with 10 mM HCl in isotonic saline for 5 min. After acidification, the segment was gently flushed to remove any residual acid and allowed a 5-min washout period. HCO₃⁻ secretion was then measured for an additional 42 min. After each experiment, the length of the duodenal test segment was measured in situ to the nearest millimeter. Animals could be sustained under these experimental conditions for >2 h. Sample volumes were measured by weight to the nearest 0.01 mg. The amount of HCO₃⁻ in the effluents was quantitated by use of a CO₂-sensitive electrode (Thermo Orion, Beverly, MA). The electrode was calibrated before each day's use by constructing a semilogarithmic standard curve using known HCO₃⁻ concentrations. A 1-ml aliquot from each 6-min perfusion period was individually sampled, yielding a reading in millivolts. This reading was then converted back to an HCO₃⁻ concentration, as dictated by the previously generated standard curve. In this way, HCO₃⁻ outputs were determined for each 6-min period and expressed as micromoles per centimeter per hour. Stimulated HCO₃⁻ outputs are presented as HCO₃⁻ output over time and as net HCO₃⁻ output (peak minus average basal output).

SCBN cell culture.

SCBN, a canine duodenal epithelial cell line, was grown according to published methods (11, 12). It grows as polarized confluent monolayers and expresses Ca²⁺-dependent Cl⁻ secretion. Cells of passages 23–33 were grown to confluence (∼5 days) in 75-cm² flasks. Cells were fed with fresh Dulbecco's modified Eagle medium supplemented with 10% FBS, l-glutamine, and streptomycin every 2–3 days. After the cells had grown to confluence, they were replated onto 12-mm round coverslips (Warner Instruments, Hamden, CT) and incubated for at least 24 h before use.

[Ca²⁺]_cyt measurement by digital Ca²⁺ imaging.

[Ca²⁺]_cyt levels in SCBN cells were measured by fura 2 fluorescence ratio digital imaging, as described previously (62). Briefly, SCBN cells, grown on coverslips, were loaded with 5 μM fura 2-acetoxymethyl ester (AM) (dissolved in 0.01% Pluronic F-127 plus 0.1% DMSO in physiological salt solution described below) at room temperature for 50 min and then washed in normal physiological salt solution for at least 20 min. Thereafter, the coverslips with SCBN cells were mounted in a perfusion chamber on a Nikon microscope stage. Cells were initially superfused with physiological salt solution for 5 min and then switched to Ca²⁺-free or Ca²⁺ solutions containing different drugs. The ratio of fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illuminations) from the cells, as well as background fluorescence, was collected at room temperature (22°C) with the use of a ×40 Nikon UV-Fluor objective and an intensified CCD camera (ICCD200). The fluorescence signals emitted from the cells were monitored continuously using a MetaFluor Imaging System (Universal Imaging, Downingtown, PA) and were recorded in an IBM-compatible computer for later analysis. [Ca²⁺]_cyt was calculated from fura 2 fluorescent emission excited at 340 and 380 nm using the ratio method based on the equation [Ca²⁺]_cyt = K_d × (Sf₂/Sb₂) × (R − R_min)/(R_max − R), where K_d (225 nM) is the dissociation constant for Ca²⁺, R is the measured fluorescence ratio, and R_min and R_max are minimal and maximal ratios, respectively (25). Sf₂/Sb₂ is the fluorescence ratio for Ca²⁺-free and -bound indicators measured at 380 nm.

RT-PCR analysis.

Total RNA from SCBN cells or mouse duodenal mucosa was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Five micrograms of total RNA were converted into cDNA with reverse transcriptase. After inactivation at 70°C for 10 min, 1 μl of the reaction mixture was incubated in buffer containing 0.2 mM dATP, dCTP, dGTP, and dTTP, 0.2 μM oligonucleotide primers, as shown below, 3 mM MgCl₂, 500 mM KCl, and a 10× buffer consisting of 200 mM Tris·HCl (pH 8.0), together with 1 unit of Taq polymerase (Invitrogen). Primers were synthesized by Integrated DNA Technologies (Coralville, IA). Mouse P2Y₂-specific sense and antisense primers (GenBank accession number is NM_008773) were 5′-AGCCCATTACGTGACTGTCC-3′ and 5′-CTGAGGCAGGAAACAGGAAG-3′, respectively. Mouse P2Y₄-specific sense and antisense primers (GenBank accession number is NM_020621) were 5′-AACCAGGAAGCTGGGGTACT-3′ and 5′-GGAGGTTCCTTAGGGTCAGC-3′, respectively. Canine P2Y₂-specific sense and antisense primers (GenBank accession number is XM_542321) were 5′-CGTCAACGTGGCTTACAAGA-3′ and 5′-AATCCTCACTGCTGGTGGAC-3′, respectively. Canine P2Y₄-specific sense and antisense primers (GenBank accession number is XM_845543) were 5′-GTGCTACTCGCTGATGGTGA-3′ and 5′-AAGCGGAGCATGAGGTAGAA-3′, respectively. GAPDH sense and antisense primers, as described by Ijichi et al. (29), were 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′, respectively. The samples were amplified in an automated thermal cycler (GeneAmp 2400; Applied Biosystems). DNA amplification conditions included an initial 3-min denaturation step at 94°C, 35 cycles of 30 s at 94°C, 30 s at 57°C, 40 s at 72°C, and a final elongation step of 10 min at 72°C. The products were electrophoresed on a 1.5% agarose gel, stained with ethidium bromide (0.5 μg/ml), and then photographed under UV light.

Chemicals and solutions.

UTP, ATP, ADP, SK&F96365, U-73122, nifedipine, suramin, and pyridoxal-phosphate-6-azo(benzene-2-4-disulphonic acid) tetrasodium salt (PPADS) were purchased from Sigma. 2-Aminoethoxydiphenyl borate (2-APB) was purchased from Tocris Bioscience (Ellisville, MO). Fura 2-AM was from Molecular Probes (Eugene, OR). The other chemicals were obtained from Fisher Scientific (Santa Clara, CA). The mucosal solution used in Ussing chamber experiments contained the following (in mM) and was bubbled with 100% O₂: 140 Na⁺, 5.4 K⁺, 1.2 Ca²⁺, 1.2 Mg²⁺, 120 Cl⁻, 25 gluconate, and 10 mannitol. The serosal solution contained the following (in mM) and was bubbled with 95% O₂ + 5% CO₂: 140 Na⁺, 5.4 K⁺, 1.2 Ca²⁺, 1.2 Mg²⁺, 120 Cl⁻, 25 HCO₃⁻, 2.4 HPO₄²⁻, 2.4 H₂PO₄⁻, 10 glucose, and 0.01 indomethacin.

The physiological salt solution used in digital Ca²⁺ measurement contained the following (in mM): 140 Na⁺, 5.0 K⁺, 2 Ca²⁺, 147 Cl⁻, 10 HEPES, and 10 glucose. For the Ca²⁺-free solution, Ca²⁺ was omitted, and 0.5 mM EGTA was added to prevent possible Ca²⁺ contamination. The osmolalities for all solutions were ∼284 mosmol/kgH₂O.

Statistical analysis.

Results are expressed as means ± SE. Differences between means were considered to be statistically significant at P < 0.05 using Student's t-test or one-way ANOVA followed by Newman-Keuls post hoc test, as appropriate.

RESULTS

mRNA expression of P2Y receptors in SCBN cells and mouse duodenal epithelia.

There are eight subtypes of P2Y receptors, but P2Y₂ and P2Y₄ are the major subtypes identified in intestinal epithelia (10, 39). We, therefore, attempted to identify these two subtypes of P2Y receptors in duodenal epithelial cells by using RT-PCR analysis. Figure 1 shows that transcripts for P2Y₂ were readily detected in both SCBN cells and mouse duodenal epithelium. P2Y₄ products were also detected in mouse duodenal epithelium at low levels. However, P2Y₄ products were undetectable in SCBN cells. Therefore, P2Y₂ is the main subtype of P2Y receptors expressed in duodenal epithelial cells.

Fig. 1.

Expression of P2Y receptors in mouse duodenal epithelium and the SCBN cell line. Total RNA was isolated from mouse duodenal epithelium and SCBN cells. P2Y-receptor transcripts were detected by RT-PCR analysis. M, molecular weight marker; B, blank control in PCR reaction that contains all ingredients except cDNA. GAPDH was used as an internal loading control. These data are representative of 3 separate experiments with identical results.

UTP-induced murine duodenal ion transport in vitro.

Although expression of P2Y receptors and their role in regulating colonic Cl⁻ secretion and HCO₃⁻ secretion from pancreatic ductal and gallbladder epithelia have been studied (14, 24, 32), little is known about P2Y receptors in duodenal epithelial ion secretion. UTP selectively stimulates P2Y receptors (13, 52), and thus we used it in Ussing chambers to test whether the P2Y receptors play a role in murine duodenal ion transport. In the first set of experiments, we studied the involvement of P2Y receptors and membrane ion channels in the duodenal epithelial ion transport by using pharmacological agents. As shown in Fig. 2, after the baseline became stable for 30 min, addition of UTP (10 μM) to the mucosal side of the chambers induced duodenal I_sc (Fig. 2A). UTP (10 μM) also stimulated duodenal HCO₃⁻ secretion, which reached a peak around 10 min later and lasted for at least 30 min. Therefore, we calculated net peak HCO₃⁻ secretion from the difference between the baseline and the peak value 10 min after addition of UTP (Fig. 2B). Treatment of the duodenal tissues with suramin (300 μM), a P2Y-receptor antagonist (1), or 2-APB (100 μM), a cell-permeable antagonist of inositol 1,4,5-trisphosphate (IP₃) receptor and store-operated channels (SOC) (9), abolished UTP-induced duodenal I_sc and HCO₃⁻ secretion. However, addition of ADP (10 μM) did not significantly induce duodenal I_sc or HCO₃⁻ secretion (Fig. 2). Thus activation of the P2Y₂ receptors likely increases [Ca²⁺]_cyt via SOC and stimulates duodenal Cl⁻ and HCO₃⁻ secretion.

Fig. 2.

Effects of nucleotides on murine duodenal epithelial ion transport in vitro. A: time course of nucleotide-induced murine duodenal short-circuit current (I_sc). After baseline was measured for 30 min, solvent or UTP (10 μM) was added to the mucosal side of Ussing chambers, but ADP (10 μM) was added to both sides, as indicated by an arrow. B: net peak duodenal bicarbonate (HCO₃⁻) secretion calculated from the difference between the baseline and the peak value at 10 min after addition of UTP (10 μM) or ADP (10 μM). In some experiments, the duodenal tissues were pretreated with suramin (300 μM) or 2-aminoethoxydiphenyl borate (2-APB; 100 μM) for 10 min before addition of UTP. Con, control. Values are mean ± SE; n = 5–6 for each group. **P < 0.01 vs. control. ##P < 0.01 vs. UTP alone.

To test the polarized function of P2Y₂ receptors, we added UTP (10 μM) mucosally or serosally and then assessed UTP-stimulated HCO₃⁻ secretion: mucosal or serosal addition of UTP induced comparable duodenal HCO₃⁻ secretion (Fig. 3A). In a second set of experiments, we assessed the role of P2Y₂ receptors in mediating duodenal HCO₃⁻ secretion using a genetic approach. As Fig. 3B illustrates, UTP-stimulated HCO₃⁻ secretion was markedly impaired in P2Y₂ knockout mice compared with that in wild-type mice, even though carbachol-stimulated HCO₃⁻ secretion was similar in P2Y₂ knockout and wild-type mice. The latter results indicate that the capacity to respond to another G protein-coupled receptor, the muscarinic cholinergic receptor, is unaltered in the duodenal epithelium of P2Y₂ knockout animals. These data thus provide further evidence for the role of P2Y₂ receptors in mediating duodenal HCO₃⁻ secretion.

Fig. 3.

Effect of UTP on duodenal HCO₃⁻ secretion in vitro from wild-type and P2Y₂ knockout mice. A: net peak duodenal HCO₃⁻ secretion calculated from the difference between the baseline and the peak value at 10 min after mucosal or serosal addition of UTP (10 μM) in wild-type mice. B: comparison of net peak duodenal HCO₃⁻ secretion between wild-type (P2Y₂^+/+) and P2Y₂ knockout (P2Y₂^−/−) mice after addition of UTP (10 μM) to both sides or serosal addition of carbachol (CCh; 100 μM) in Ussing chambers. Values are means ± SE; n = 8–9 for each group. **P < 0.01 vs. P2Y₂^+/+.

Involvement of P2Y receptors in acid-stimulated DMBS in vivo.

Our experiments conducted with Ussing chambers showed that the P2Y receptors are functionally expressed in murine duodenal epithelia and are involved in duodenal epithelial ion transport. To further investigate whether the P2Y receptors in duodenal epithelia have physiological roles, we measured acid-stimulated duodenal HCO₃⁻ secretion in whole animals. Figure 4A shows a time course study of HCl-stimulated murine DMBS in vivo. Duodenal luminal perfusion of HCl (10 mM) resulted in a robust increase in DMBS in control mice, which reached a maximal level at 30 min after acid stimulation and declined thereafter to the baseline level. Net peak HCO₃⁻ secretion, calculated from the difference between the baseline and the peak value at 30 min, was used to assess acid-stimulated HCO₃⁻ secretion (Fig. 4B). Luminal perfusion of suramin (1 mM) significantly attenuated acid-stimulated DMBS; net peak HCO₃⁻ secretion was inhibited by 67% (Fig. 4). PPADS (30 μM), another P2Y-receptor antagonist with a chemical structure different from that of suramin (1), also attenuated acid-stimulated DMBS (Fig. 4A) and the net peak HCO₃⁻ secretion (Fig. 4B). From these results, we conclude that P2Y receptors in duodenal epithelium can regulate acid-stimulated duodenal HCO₃⁻ secretion in vivo.

Fig. 4.

Involvement of P2Y receptors in acid-stimulated duodenal mucosal HCO₃⁻ secretion in vivo. A: time course of duodenal mucosal HCO₃⁻ secretion induced by luminal perfusion of 10 mM HCl in controls of mice treated with suramin (1 mM) or pyridoxal-phosphate-6-azo(benzene-2-4-disulphonic acid) tetrasodium salt (PPADS; 30 μM) for 10 min. B: net peak HCO₃⁻ secretion calculated from the difference between the baseline and the peak value at 30 min after HCl perfusion. Values are means ± SE; n = 5–6 for each group. **P < 0.01 vs. control.

Nucleotide-induced Ca²⁺ mobilization in duodenocytes.

ATP and UTP induce Ca²⁺ mobilization via activation of the P2Y receptors in many types of mammalian cells (32), but their roles in Ca²⁺ mobilization in duodenal epithelial cells have not been fully investigated. Therefore, we measured the kinetics of [Ca²⁺]_cyt in SCBN cells by using a digital Ca²⁺ imaging system. ATP (1 μM) treatment in Ca²⁺-containing solutions markedly raised [Ca²⁺]_cyt, which consisted of a rapid rise to a peak, followed by rapid decline to a plateau phase that was relatively sustained until ATP was withdrawn (Fig. 5A). In some instances, we observed oscillation in [Ca²⁺]_cyt during ATP treatment. The ATP-induced increase in [Ca²⁺]_cyt was reproducible (Fig. 5A) and inhibited by pretreatment with suramin (100 μM, data not shown). The response to UTP (1 μM) was qualitatively and quantitatively indistinguishable from that to ATP (1 μM) (Fig. 5B). However, ADP (1 μM) failed to elicit discernible changes in [Ca²⁺]_cyt, although higher concentration of ADP (10 μM) induced a slight increase in [Ca²⁺]_cyt (Fig. 5C). We found that 1 μM UTP or ATP induced similar increases in [Ca²⁺]_cyt measured at 1 min after superfusion of nucleotides in Ca²⁺-containing solutions, but response to ADP was much smaller (Fig. 5D). These data are consistent with our RT-PCR and duodenal epithelial ion transport data described above, indicating that P2Y₂ receptor is a major subtype of P2Y receptors functionally expressed in duodenal epithelial cells.

Fig. 5.

Nucleotide-induced Ca²⁺ mobilization in duodenal epithelial cells in Ca²⁺-containing solutions. ATP- (A), UTP- (B), and ADP-induced (C) cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in SCBN cells was determined with digital Ca²⁺ imaging. D: ATP and UTP at 1 μM raised reproducible [Ca²⁺]_cyt, which consists of a transient and a plateau phase, while ADP induced a much smaller one at the same concentration. Values are mean ± SE; n = 40 cells for each tracing or bar. **P < 0.01 vs. ATP.

ATP-induced intracellular Ca²⁺ release and Ca²⁺ entry via SOC.

Activation of the G protein-coupled receptors in many types of mammalian epithelial cells can induce Ca²⁺ release from intracellular stores, which, in turn, promotes opening of SOC in the plasma membrane that leads to further Ca²⁺ entry (47, 48, 50). However, the regulatory mechanisms of [Ca²⁺]_cyt in duodenal epithelial cells are largely unknown. Therefore, we tested whether ATP induced an intracellular Ca²⁺ release and Ca²⁺ entry via SOC in duodenal epithelial cells. We found that ATP (1 μM) in Ca²⁺-containing solutions reproducibly increased [Ca²⁺]_cyt (Fig. 6A). Nifedipine (10 μM), a voltage-gated Ca²⁺ channel (VGCC) blocker, did not significantly affect ATP-induced [Ca²⁺]_cyt (Fig. 6B). However, U-73122 (10 μM), a PLC inhibitor, partially inhibited ATP-induced [Ca²⁺]_cyt (Fig. 6C). La³⁺ (30 μM) and 2-APB (10 μM), two SOC blockers (9, 65), abolished ATP-induced [Ca²⁺]_cyt (Fig. 6, D and E). Figure 6F summarizes the effects of ATP on [Ca²⁺]_cyt measured at 1 min after the cells were exposed to ATP (1 μM) in the absence or the presence of different inhibitors in Ca²⁺-containing solutions. In the case of Ca²⁺-free solutions, although ATP (1 μM) also increased [Ca²⁺]_cyt, the response was transient and followed by rapid decline to the baseline level before withdrawal of ATP (n = 50 cells, data for ATP not shown here, but similar data for UTP are shown in Fig. 7). Unlike in Ca²⁺-containing solutions, ATP could not reproducibly induced Ca²⁺ signaling events in Ca²⁺-free solutions. These results suggest that stimulation of P2Y receptors in duodenal epithelial cells induces Ca²⁺ release from the intracellular stores and extracellular Ca²⁺ entry, likely via SOC rather than VGCC.

Fig. 6.

ATP-induced Ca²⁺ mobilization via phospholipase C/inositol 1,4,5-trisphosphate pathway and store-operated channels (SOCs) in duodenal epithelial cells in Ca²⁺-containing solutions. A: ATP (1 μM, indicated by a horizontal bar) increased [Ca²⁺]_cyt in a control study. The ATP-induced [Ca²⁺]_cyt was partially inhibited by U-73122 (10 μM; C), abolished by LaCl₃ (30 μM; D) and 2-APB (10 μM; E), but not altered by nifedipine (10 μM; B). F: effects of ATP on [Ca²⁺]_cyt measured at 1 min after the cells were exposed to ATP (1 μM) in the absence or the presence of different inhibitors in Ca²⁺-containing solutions. Values are means ± SE; n = 40 cells for each tracing. **P < 0.01 vs. control.

Fig. 7.

UTP-induced capacitative Ca²⁺ entry (CCE) via SOCs in SCBN cells. In the absence of extracellular Ca²⁺ and presence of nifedipine (10 μM), extracellular application of UTP (1 μM) induced a transient increase in [Ca²⁺]_cyt. A: approximately 5 min later, when the [Ca²⁺]_cyt transients declined back to the basal level, restoration of 2 mM external Ca²⁺ induced a large increase in [Ca²⁺]_cyt due to Ca²⁺ entry. Pretreatment of cells with 10 μM 2-APB (B), 30 μM La³⁺ (C), or 30 μM SK&F96365 (D) abolished CCE via SOCs. Effects of the compounds on UTP-induced intracellular Ca²⁺ release (E) and their effects on CCE (F) are summarized. Values are mean ± SE; n = 50 cells for each tracing. **P < 0.01 vs. control.

UTP-induced capacitative Ca²⁺ entry through SOC in duodenocytes.

Induction of intracellular Ca²⁺ release followed by SOC opening leads to further Ca²⁺ entry after activation of the G protein-coupled receptors, known as capacitative Ca²⁺ entry (CCE), and is a well-documented response in mammalian cells (47, 48). Most nonexcitable cells do not express VGCC. Therefore, CCE through SOC is considered an important mechanism to control [Ca²⁺]_cyt homeostasis and regulation of Ca²⁺-dependent biological processes in epithelial cells (7, 21, 48). We thus examined whether SOC is functionally expressed and whether CCE is involved in P2Y-mediated [Ca²⁺]_cyt in duodenal epithelial cells by assessing response to UTP.

UTP (1 μM) induced a marked [Ca²⁺]_cyt transient in Ca²⁺-free solutions (Fig. 7A). Restoration of external Ca²⁺ (2 mM) ∼5 min after [Ca²⁺]_cyt transients declined back to the basal level (i.e., when the store was depleted) increased [Ca²⁺]_cyt due to Ca²⁺ entry (Fig. 7A). Nifedipine (10 μM) was applied throughout the experiments to prevent the involvement of VGCC. Pretreatment of cells with 2-APB (10 μM), a cell-permeable antagonist of IP₃ receptor and SOC blocker, partially inhibited UTP-induced intracellular Ca²⁺ release and abolished CCE through SOC (Fig. 7B). La³⁺ (30 μM), a nonselective SOC blocker that does not alter UTP-induced intracellular Ca²⁺ release, abolished UTP-induced CCE (Fig. 7C). Similarly, SK&F96365 (30 μM), a selective SOC blocker, abolished UTP-induced CCE in duodenocytes (Fig. 7D). Figure 7E summarizes the effects of La³⁺, SK&F96365, and 2-APB on UTP-induced intracellular Ca²⁺ release, which was significantly inhibited only by 2-APB (59%). However, La³⁺, SK&F96365, and 2-APB significantly inhibited CCE by 92, 87, and 94%, respectively (Fig. 7F). Thus SOC likely plays an important role in P2Y₂-receptor-mediated [Ca²⁺]_cyt homeostasis in duodenal epithelial cells.

DISCUSSION

Epithelial cells can release nucleotides (such as ATP/UTP) in response to physiological stimuli, including changes in cell volume, membrane stress, and receptor stimulation (8, 33, 36, 38, 43, 55). In the intestinal tract, stimuli, such as ingestion of food and water, can readily evoke ATP/UTP release from epithelial cells (23, 55). Purinergic receptors are expressed in epithelial cells and induce a variety of biological effects (1). Three rodent P2Y receptors are sensitive to UTP: P2Y₂, P2Y₄, and P2Y₆ (52); however, P2Y₂ and P2Y₄, but not P2Y₆, are sensitive to suramin (1, 52). P2Y₂ and P2Y₄ receptors can couple to G_q/11 proteins and activate PLC-β, resulting in production of IP₃ and mobilization of intracellular Ca²⁺. P2Y₂ receptors, a prominent subtype of luminal P2Y receptors in many epithelia, respond to agonists with the order of potency UTP = ATP > ADP and are inhibited by suramin and PPADS (1, 6, 10).

Purinergic signaling pathways contribute to the regulation of ion transport in intestinal, biliary, and pancreatic duct epithelium (14, 24, 32, 39, 40). However, little is known regarding the expression and function of the P2Y receptors in duodenal epithelium; only one report has implied that endogenous luminal ATP released from duodenal brush border is involved in DMBS (2). In the present study, we show that mRNAs of the P2Y receptors are expressed in duodenal epithelial cells and that P2Y₂ is a major subtype. Furthermore, we used UTP as a selective P2Y receptor agonist (13, 52) in studies with Ussing chambers to demonstrate that P2Y receptor activation stimulates duodenal I_sc and DMBS. UTP stimulated duodenal I_sc and DMBS over a similar time course, with effects lasting at least 30 min. Mucosal or serosal addition of UTP induced comparable effects on duodenal HCO₃⁻ secretion, suggesting that functional P2Y receptors are expressed on both the luminal and the basolateral membranes of duodenal epithelial cells, results akin to those found for other epithelial cells (24, 39, 46). The stimulatory effect of UTP was significantly inhibited by use of a P2Y-receptor antagonist or a SOC blocker. Responses to ATP are potentially mediated by numerous types of P2X and P2Y receptors or by its degradation to adenosine and the activation of the adenosine (P1) receptors (10), but activation by UTP can only be readily attributable to the activation of P2Y receptors.

To assess if P2Y receptors have physiological roles in the regulation of DMBS, we used HCl as a physiological stimulus in animals and found that acid-stimulated DMBS was attenuated similarly by two P2Y-receptor antagonists with different chemical structures. We assessed P2Y-receptor-mediated DMBS by using two different techniques to measure HCO₃⁻, i.e., pH-stat method in vitro (Figs. 2 and 3) and CO₂-sensitive electrodes in vivo (Fig. 4). The pH-stat method detects a loss of H⁺ or increase in HCO₃⁻, and thus its use can be confounded by epithelial H⁺ secretion. However, CO₂-sensitive electrodes measure HCO₃⁻ concentration (22). The rates of duodenal HCO₃⁻ in vitro seem lower than those observed in mice in vivo (Fig. 3 vs. Fig. 4), a difference likely due to 1) different units of HCO₃⁻ secretion used for in vitro vs. in vivo studies (μmol·cm⁻²·h⁻¹ vs. μmol·cm⁻¹·h⁻¹); and 2) assessment of effect of UTP alone in vitro vs. acid-induced release of multiple factors in vivo (e.g., PGE₂, ACh, 5-HT, VIP, etc.) (3, 30, 62) that may stimulate different rates of HCO₃⁻ secretion. In addition, UTP induced a larger effect on duodenal I_sc than on HCO₃⁻ secretion (Fig. 2A vs. 2B), most likely because I_sc represents a combination of duodenal epithelial Cl⁻ and HCO₃⁻ secretion. Importantly, we confirmed a role for P2Y₂-mediated DMBS by use of a genetic approach: UTP-stimulated HCO₃⁻ secretion was markedly impaired in P2Y₂ knockout mice (Fig. 4B). Taken together, our data not only support the idea that extracellular ATP can regulate DMBS (2), but also provide strong evidence for the expression of P2Y₂ subtype in duodenal epithelial cells and for a key role of P2Y₂ receptors in purinergic regulation of DMBS.

Both ATP and UTP can mobilize [Ca²⁺]_cyt via activation of the P2Y receptors in many types of mammalian cells (32, 39, 40, 49, 64). To assess such an activity in duodenal epithelial cells, we used the SCBN cell model because of the following. 1) This cell line is a well-characterized, nontransformed duodenal epithelial crypt cell line, which forms electrically tight monolayers (45). 2) It expresses functional CFTR channels and has been used in the study of Ca²⁺-dependent Cl⁻ secretion (11, 12). 3) Activation of protease-activated receptor can stimulate HCO₃⁻ secretion in SCBN cells (unpublished observations), results that are in agreement with the report of Buresi et al. (12) regarding a residual increase in I_sc in Cl⁻-free solution that was likely due to HCO₃⁻ transport evoked by activation of protease-activated receptor in these cells. 4) P2Y₂ receptors are expressed in both mouse duodenal epithelium and SCBN cells (Fig. 1). All of these features made SCBN a valuable cell model for the present study and potentially for future studies as well.

Our data show that UTP and ATP, but not ADP, were similarly effective in increasing Ca²⁺ signaling in SCBN cells, consistent with the expression of P2Y₂ receptors in these cells (1, 6, 10). Although differences in gene expression may exist between SCBN cells and primary duodenal enterocytes, ATP can raise [Ca²⁺]_cyt via activation of the P2Y receptors in primary duodenal enterocytes from mice (60) and rats (59), indicating the similarity of P2Y gene functional activity in the SCBN cell line and such enterocytes.

Two sources of Ca²⁺ are involved in [Ca²⁺]_cyt homeostasis in mammalian cells: intracellular Ca²⁺ release from sarcoplasmic reticulum/endoplasmic reticulum (ER) and extracellular Ca²⁺ entry (47, 48). In epithelial cells, P2X receptors can activate plasma membrane ion channels, permitting influx of extracellular Ca²⁺ and Na⁺ by passive transport; however, activation of P2Y receptors triggers intracellular Ca²⁺ release from the ER into the cytosol by the PLC/IP₃ pathway (10, 47, 48). Depletion of Ca²⁺ from the ER may induce CCE through SOC (6, 47, 48). Our results showed that ATP or UTP increased Ca²⁺ signaling in SCBN cells in the absence or presence of extracellular Ca²⁺. Such results, together with the inhibition by U-73122, 2-APB, or La³⁺, indicate that activation of P2Y receptors mediates intracellular Ca²⁺ release and extracellular Ca²⁺ entry via SOC (47, 48). Using a standard protocol (66, 67), we substantiated a crucial role of CCE through SOC in the control of P2Y₂-receptor-mediated [Ca²⁺]_cyt in duodenal epithelial cells, results consistent with findings for other epithelial cells (34, 35, 40, 58).

Although the molecular mechanism of CCE is not fully understood, two proteins (Stim1 and Orai proteins) appear to mediate the signaling and permeation mechanisms of SOC (57, 68). Stim1, an ER Ca²⁺ sensor, and Orai are plasma membrane proteins that constitute pore-forming subunits of SOC. Depletion of ER Ca²⁺ store induced by activation of plasma membrane receptors results in Stim1 without Ca²⁺ bound, which causes Stim1 to redistribute from the ER to plasma membrane regions near Orai. Stim1 then activates Ca²⁺-selective Orai channels (51, 57, 68).

Our findings strongly suggest that [Ca²⁺]_cyt is important in the signaling events in response to activation of the membrane P2Y receptors of duodenocytes and plays a critical role in regulating duodenal ion transport and DMBS in particular. How does activation of the P2Y receptors stimulate DMBS via Ca²⁺ signaling? An increase in [Ca²⁺]_cyt can activate apical CFTR channels and Cl⁻/HCO₃⁻ exchanger in human pancreatic duct cells (44) and murine duodenal epithelium (61, 62) and inhibit the ileal brush-border Na⁺/H⁺ exchanger (15, 16, 56). [Ca²⁺]_cyt can also increase basolateral Na⁺-HCO₃⁻ cotransport activity in murine colonic crypts (5) and activate basolateral Ca²⁺-activated K⁺ channels in murine duodenal epithelium to provide a driving force for HCO₃⁻ secretion (19). All of these actions of [Ca²⁺]_cyt may contribute to the molecular mechanisms underlying P2Y₂-Ca²⁺-mediated DMBS observed in the present study. However, further investigation is required to reveal which mechanism plays a major role in P2Y₂-Ca²⁺-mediated DMBS.

In conclusion, the present data show that activation of the P2Y₂ receptors in duodenal epithelium increases [Ca²⁺]_cyt by inducing intracellular Ca²⁺ release and extracellular Ca²⁺ entry via SOC (47, 48), thereby stimulating duodenal epithelial Cl⁻ and HCO₃⁻ secretion. Luminal acid or swelling can induce ATP release, and released ATP can function as an autocrine/paracrine regulator of human intestinal epithelial cells (26, 63). Although defining the precise role of purinergic pathways in regulating intestinal epithelial ion transport will require further investigation, the present findings imply that studies of P2Y, in particular P2Y₂ receptor, pathways are a potentially promising target for the treatment of pathological conditions in the intestinal tract, including perhaps protection from acid-induced duodenal injury (3, 4, 31). Increased understanding of the cellular and molecular mechanisms of Ca²⁺-mediated DMBS via activation of P2Y (e.g., P2Y₂) receptors thus has physiological and potential clinical significance.

GRANTS

This work was supported by American Heart Association Beginning Grant-in-Aid Award (0565025Y) and Cystic Fibrosis Foundation (DONG0610) to H. Dong, and by the University of California San Diego Digestive Diseases Research Development Center Grant (DK-080506), in which H. Dong serves as the director of cell imaging core. It was also partially supported by grants from the National Institutes of Health (NIH) (DK073090) and the University of California San Diego Academic Senate Research Grant (RH154H) to J. Y. Chow and a NIH Grant (GM 66232) to P. A. Insel.