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.
- P2Y2 receptor
- cytoplasmic-free Ca2+
- capacitative Ca2+ entry
- store-operated channels
- duodenal ion transport
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 (P2X1-7 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 Gq-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
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 (Isc) 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 = Kd × (Sf2/Sb2) × (R − Rmin)/(Rmax − R), where Kd (225 nM) is the dissociation constant for Ca2+, R is the measured fluorescence ratio, and Rmin and Rmax are minimal and maximal ratios, respectively (25). Sf2/Sb2 is the fluorescence ratio for Ca2+-free and -bound indicators measured at 380 nm.
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.
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.
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.
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 Isc (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 Isc and HCO3− secretion. However, addition of ADP (10 μM) did not significantly induce duodenal Isc or HCO3− secretion (Fig. 2). Thus activation of the P2Y2 receptors likely increases [Ca2+]cyt via SOC and stimulates duodenal Cl− and HCO3− secretion.
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.
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.
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.
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.
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.
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 Gq/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 Isc and DMBS. UTP stimulated duodenal Isc 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 Isc than on HCO3− secretion (Fig. 2A vs. 2B), most likely because Isc 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 Isc 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.
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.
↵* X. Dong and E. J. Smoll contributed equally to this work.
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