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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

The Effects of Extracellular Purines and Pyrimidines on Human Airway Smooth Muscle Cells

Seymour Heisler Laboratory of the Montreal Chest Institute Research Center and Meakins Christie Laboratories, McGill University (V.G., J.G.M., K.M., M.-C.M.), University of Montreal Hospital Center (P.F.), Montreal, Quebec, Canada

Received May 17, 2005; accepted August 10, 2005.

	Abstract

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Extracellular ATP and UTP modulate the function of many cell types through the stimulation of specific P2 receptors, and the inhalation of UTP has been proposed as a therapeutic means of increasing mucociliary clearance in cystic fibrosis patients. The aim of this study was to determine whether P2 receptors are present and functional in human airway smooth muscle (HASM) cells. Experiments were conducted on primary cultures of HASM cells. Reverse transcription-polymerase chain reaction and Western blot analysis showed that P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptor subtypes are expressed. Exposure to extracellular ATP, UTP, ADP, and UDP at concentrations ranging from 10^-6 to 10^-4 M, produced significant increases in intracellular Ca²⁺ that peaked to 491 ± 51 nM (p < 0.001) with ATP 10^-5 M and to 321 ± 30 nM with UTP 10^-4 M. ATP and UTP also induced HASM cell contraction, decreasing cell length by 9.9 ± 4.3 and 5.6 ± 2.0%, respectively. Pretreatment of the cells with UTP for short periods of time (10 and 30 min) enhanced the peak Ca²⁺ release to UTP, whereas repeated and prolonged pretreatment with UTP decreased it. These results indicate that several subtypes of P2Y receptors are present and functional in HASM cells. They also show that the response of the receptors is increased after short periods of exposure to UTP and decreased after prolonged and repeated exposure. Considering that ATP and UTP are endogenous mediators and that analogs of UTP could be used as a therapeutic modality, the role of extracellular triphosphate nucleotides in physiological and pathophysiological processes in the airways warrants further investigation.

Although the effects of extracellular purine nucleotides have been extensively investigated in the cardiovascular, urogenital, gastrointestinal, and nervous systems, research on the effects of these compounds in the respiratory system is more recent. In the respiratory system, ATP and UTP act on various cell types: ATP is one of the most powerful known agonists for surfactant secretion by alveolar type II cells, and it also increases Cl^- secretion and arachidonic acid metabolism when applied to the apical surface of cultured airway epithelial cells (Knowles et al., 1991; Hwang et al., 1996). Mucin secretion by submucosal glands and goblet cells is also increased in the presence of ATP (Kim and Lee, 1991). These effects are mediated by specific receptors, the P2 receptors, present on the cell plasma membrane. It has also been reported that endogenous sources of ATP and UTP are present in the airways, indicating that these compounds are physiological mediators. Indeed, airway epithelial cells are a significant source of ATP and UTP, which can be released after simple mechanical stimuli (Lazarowski et al., 1997a) and cellular swelling (Hazama et al., 1999). In addition, Lazarowski et al. (2000) have reported that the concentration of ATP in the extracellular fluid overlying cultured human bronchial epithelial cells remains stable, despite continuous ATP hydrolysis, indicating the presence of constant sources of ATP. The powerful effects of extracellular triphosphate nucleotides on mucociliary clearance have led some investigators to investigate the therapeutic potential of P2Y receptor stimulation for the improvement of mucociliary clearance in subjects with cystic fibrosis (Knowles et al., 1995; Bennett et al., 1996). Kreda et al. (2000) have also described another therapeutically oriented use of the P2Y2/4 receptor and its ligand UTP as a gene transfer vector.

The effects of ATP on the airway smooth muscle of various animal species have produced conflicting results. In vivo, Flezar et al. (1992) have reported that intratracheal instillation of ATP produces an increase in airway resistance in rats. In vitro, the published data on airway tone are conflicting; both relaxant and contractile effects of ATP on airway smooth muscle have been observed (Fedan et al., 1993a,b, 1994). These discrepancies are likely due to the type of preparation studied and the presence or absence of epithelium. In rat airway smooth muscle cells, extracellular ATP and UTP induce cell proliferation and trigger a substantial increase in intracellular Ca²⁺ ([Ca²⁺]_i), one of the main determinants of smooth muscle contraction (Michoud et al., 1997, 1999). Also, a recent publication by Bergner and Sanderson (2002) shows that ATP and UTP induce airway smooth muscle cell contraction in mouse lung slices.

The effects of ATP and UTP on human airway smooth muscle (HASM) cells have not, to our knowledge, been determined. Thus, the aim of this work was to determine whether P2 receptors are present in HASM cells and to characterize the direct and indirect effects of ATP, UTP, and their metabolites on these cells. In an attempt to mimic the in vivo conditions that would prevail if cystic fibrosis patients were treated with inhaled P2Y receptor ligands, the modifying effects of short and prolonged periods of exposure to UTP on smooth muscle cell responses were also measured. Indeed, because cystic fibrosis patients frequently have inflamed and ulcerated airways, therapies based on the inhalation of P2Y receptor ligands to stimulate airway clearance could also affect the underlying airway smooth muscle cells.

	Materials and Methods

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Cell Cultures
Cultures of HASM cells were prepared from surgical and lung transplant specimens as described by Panettieri et al. (1989). Segments of lobar or main bronchus measuring 5 x 2 mm were incubated for 90 min at 37°C in 10 ml of Hanks' balanced salt solution buffer (5 mM KCl, 0.3 mM KH₂PO₄, 138 mM NaCl, 4 mM NaHCO₃, and 5.6 mM Na₂HPO₄) to which 640 U of collagenase type IV, 10 mg of soybean trypsin inhibitor, and 100 U elastase (type IV) had been added. The digested tissue was then filtered through a 125-µm Nytex mesh, and the resulting cell suspension was centrifuged. The pellet was reconstituted in culture medium containing 5% fetal bovine serum, insulin, human fibroblast growth factor, gentamicin sulfate, amphotericin-B, human epidermal growth factor (Cambrex Bio Science Walkersville, Walkersville, MD) and plated in a 25-cm² culture flask. Confluent cells were detached with a 0.025% trypsin and 0.02% EDTA solution and grown on 25-mm-diameter glass coverslips for Ca²⁺ imaging and on 150 x 25-mm culture dishes for protein and total RNA extraction. Confluent cells from first to fourth passage were used. They were identified as smooth muscle cells by positive immunohistochemical staining for smooth muscle specific -actin and positive identification of myosin light chain kinase and calponin by Western blotting. The project was approved by the local Institutional Review Boards.

Measurement of Intracellular Ca²⁺
Cells grown on 25-mm-diameter coverslips in 35-mm-diameter wells were used 10 to 14 days postplating. As described previously, cells were incubated for 20 to 30 min at 37°C with Hanks' buffer (137 mM NaCl, 4.2 mM NaHCO₃, 10 mM glucose, 3 mM Na₂HPO₄, 5.4 mM KCl, 0.4 mM KH₂PO₄, 1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.8 mM MgSO₄, and 5 mM HEPES) containing 5 µM Fura 2-acetoxymethyl ester (Fura-2AM) and 0.02% polyoxyethylene/polyoxypropylene co-polymer (Pluronic F-127) (Michoud et al., 1997). The loaded cells were then washed and the coverslips placed in a Leiden chamber (Medical Systems Corp., Greenville, NY) containing 450 µl of Hanks' buffer on the stage of an inverted microscope equipped for cell imaging with 40x oil objective (Nikon, Montreal, QC, Canada). The cells were imaged using an intensified camera (Videoscope IC 200) and PTI software (Photon Technology International, Lawrenceville, NJ) at a single emission wavelength (510 nm) with double excitatory wavelengths (340 and 380 nm). The fluorescence ratio (340:380) was measured in individual cells (n = 8 per slide), and [Ca²⁺]_i was calculated using a dissociation constant of Ca²⁺ to Fura-2 of 224 nM (Tsien et al., 1985). Maximum ratio was determined in cells exposed to 10^-5 M ionomycin in the presence of 1.3 mM CaCl₂ and minimum ratio in Ca²⁺ free Hanks' buffer to which EGTA (10^-3 M) and ionomycin (10^-5 M) had been added. Background fluorescence and autofluorescence were automatically subtracted.

Detection of P₂-Receptor Subtype mRNA Expression
Total RNA was extracted from confluent cells (10th day after passage) by TRIzol reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. To analyze mRNA, single-strand cDNA was synthesized in a 20-µl reaction using 2 µgof total RNA as a template, oligo(dT)_12-18 primer, and Superscript II enzyme in the presence of acetylated bovine serum albumin (Invitrogen) and RNAguard ribonuclease inhibitor (GE Healthcare, Montreal, QC, Canada). Conventional PCR was done for the detection of P2Y_1, P2Y₄, and P2Y₆ subtypes and real-time PCR for P2Y_2. The PCR mixture consisted of 1.5 mM MgCl₂, 1x PCR buffer, 0.2 mM dNTP mixture, 2.5 units of Platinum Taq polymerase (Invitrogen), and 20 pmol of the 5' and 3' primers. mRNA from P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptor and from cyclophilin (a housekeeping gene) was amplified from the resulting cDNA with the appropriate oligonucleotide primers (Table 1) by PCR. For the amplification of P2Y₄ and P2Y₆, the PCR conditions were as follows: initial denaturation for 5 min at 94°C followed by 35 cycles with each cycle consisting of denaturation at 94°C for 1 min, annealing at specific temperature for 1 min and extension at 72°C for 3 min. For P2Y₁, 25 cycles for the first reaction followed by 20 cycles for the second reaction were performed. Real-time PCR was performed using a fluorescence temperature cycler (Light Cycler; Roche Diagnostics, Montreal, QC, Canada). Twenty microliters of reaction mix contained 2 µl of cDNA and 4 µl of both sense and antisense primers for P2Y₂, MgCl₂ (3 mM), and 2 µl of fast start DNA SYBR Green with Taq polymerase and H₂O. PCR cycling parameters: initial denaturation step at 95°C for 10 min was followed by PCR (95°C for 15 s and extension at 72°C for 24 s) was performed for 40 cycles. After completion of the cycling process, samples were subjected to melting curve analysis for 1 cycle (denaturation at 95°C followed by 66°C and ended by 95°C and cooling). The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis, and the size of the bands was determined by comparison with the DNA molecular markers (Roche Diagnostics). To confirm the presence of P2Y₂ and P2Y₄ mRNA, PCR products were purified with GFX PCR and gel purification kit (GE Healthcare, Piscataway, NJ) and sequenced at the Institute de Recherche en Sciences de la Vie et de la Santé (University of Laval, Laval, QC, Canada). Samples for RNA extraction were obtained from four different subjects. In all experiments, human placenta (BD Biosciences Clontech, Palo Alto, CA) was used as positive control. PCR primers were synthesized at the Sheldon Biotechnology Centre (Montreal, QC, Canada).

Measurement of Contraction
HASM cells were plated in six-well plates onto 25-mm-diameter coverslips coated with laminin (1 µg/cm²) at an approximate density of 1500 cells/cm² (Hirst et al., 2000a). They were grown for 4 days in a culture medium containing 5% fetal bovine serum. The coverslips were then transferred into a Leiden chamber (Medical Systems Corp.) containing 450 µl of Hanks' buffer. Images were taken using an inverted microscope (Nikon) at 15x magnification using Nomarski optics. A charge-coupled device camera (Hamamatsu C 2400) and commercial software (Photon Technology International) were used to acquire and record the images. Images were taken once before and up to 10 min after the addition of the agonists or vehicle. Images were analyzed with the Scion software (National Institutes of Health, Bethesda, MD). The length of the cells was measured before and 10 min after the addition of the agonists and contraction expressed as the percentage decrease in cell length from the initial value.

Experimental Protocols
Pharmacological P2 Receptor Identification. Peaks in [Ca²⁺]_i were determined after stimulation with a range of concentrations (10^-6-10^-4 M) of ATP, ADP, adenosine UTP, UDP, and 2-methylthio-ATP (2-MeSATP). All test drugs were diluted in Hanks' buffer from frozen stock solutions. They were prewarmed to 37°C before being added in the appropriate concentrations in 50-µl volume. [Ca²⁺]_i was measured for 30 s before and for 2 min after the addition of the drugs. Concentration-response curves are noncumulative, each cell being exposed to a single concentration of agonist.

Short-Term Exposure of HASM Cells to UTP. Cells were cultured on glass coverslips in six-well plates. On the 10th day postplating, UTP (10^-4 M) or vehicle (Hanks' buffer) was added for 5, 10, 30, or 60 min to the culture medium. After exposure to UTP, the cells were washed and loaded with Fura-2AM for measurement of [Ca²⁺]_i.

Prolonged and Repeated Exposures of HASM Cells to UTP. Cells were cultured on glass coverslips in six-well plates. On the 7th day postplating, either vehicle or UTP (10^-4 M) was added once a day for 3 days and [Ca²⁺]_i was measured in response to various agonists on the 10th day. The experiments on short- and long-term exposure to UTP were carried out on cells originating from four different patients.

Chemicals
Elastase and collagenase were purchased from Sigma-Aldrich (St. Louis, MO), and Fura-2AM and Pluronic F-127 were from Molecular Probes (Eugene, OR). ATP, ADP, UTP, and 2-MeSATP were purchased from Sigma-Aldrich. UDP was from Roche Diagnostics. EGTA was from Calbiochem (San Diego, CA).

Statistical Analysis
The data are expressed as mean ± S.E.M. (n, number of cells). In pharmacological experiments, the cells were obtained from four subjects. Comparison of means was performed using paired or unpaired Student's t tests as appropriate, and when multiple comparisons were made, the Bonferroni correction was applied. ANOVA followed by Student's t test was used for acute and chronic UTP exposure studies. p < 0.05 was considered to be statistically significant.

	Results

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Pharmacological Identification of P₂ Receptors. After exposure to 10^-5 M UTP, cells responded with a rapid rise in [Ca²⁺]_i, reflected by the change in fluorescence ratio for Fura-2 and a subsequent decline to a more sustained plateau (n = 3; Fig. 1A). Concentration-response curves for ATP, ADP, adenosine, UTP, and UDP are shown in Fig. 1B. ATP and ADP induced significant increases in [Ca²⁺]_i at all concentrations tested (10^-6-10^-4 M). After ATP stimulation at a concentration of 10^-5 M, the release of peak [Ca²⁺]_i was 491 ± 51 nM (p < 0.001; n = 27), whereas a concentration of 10^-4 M elicited a smaller release of Ca²⁺ with peak [Ca²⁺]_i reaching 389 ± 34 nM, indicating that a plateau had been reached. ADP also induced a significant Ca²⁺ release that peaked at 10^-5 M with a [Ca²⁺]_i of 349 ± 27 nM (p < 0.001; n = 32). Exposure to adenosine, the metabolite of ATP produced very small, albeit statistically significant increases in [Ca²⁺]_i with a peak at 150 ± 7 nM (n = 54) at 10^-5 M, indicating the presence of functional adenosine receptors. The cells also responded to pyrimidine nucleotides: at a concentration of 10^-4 M, peak Ca²⁺ release in response to UTP was 321 ± 30 nM (p < 0.005; n = 58) and 255 ± 41 nM (p < 0.005; n = 57) in response to UDP.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effects of ATP, UTP, and metabolites on [Ca2+]i. A, typical response of three individual cells to UTP 10-5 M. Noncumulative concentration-response curves to ATP, ADP, adenosine, UTP, and UDP are shown on B. The ordinate represents peak [Ca2+]i. The data presented are means ± S.E.M. *, p < 0.01; **, p < 0.001.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Peak Ca2+ release in response to 2-MeSATP and ATP is independent of extracellular Ca2+ concentration. A, increases in [Ca2+]i after increasing doses of 2-MeSATP in Ca2+ rich (white column) and in Ca2+-free medium (hatched columns). B, increase in [Ca2+]i produced by ATP. The data presented are means ± S.E.M.

Expression of mRNA for P2Y Receptor Subtypes. Conventional and real-time PCR analysis using different sets of primers for P2Y receptors (Fig. 3) showed the expression of mRNA coding for P2Y₁, P2Y₂, P2Y₄, and P2Y₆ in HASM cells. mRNA expression for P2Y_1, P2Y₄, and P2Y₆ receptors was detected with conventional PCR. P2Y₂ receptor mRNA was detected with real-time PCR. Because we had difficulty in obtaining the signals from positive controls (human placenta) with P2Y₂ receptor primers, the PCR products from P2Y₂ receptor reactions were sent for sequencing. The sequences were matched with the reported gene sequences for the P2Y₂ gene (GenBank no. NM_002564 [GenBank] ), confirming the expression of the P2Y₂ receptor. In P2Y₄, two bands were detected. Hence, the PCR products from the most prominent band were purified and sequenced. The sequenced products matched the published P2Y₄ gene sequence (GenBank no. NM_002565 [GenBank] ). We did not explore the other weakly expressed band, which might be a splice variant.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Expression of mRNA for P2Y receptor subtypes. mRNA expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptors was detected by PCR. Lanes 1-4 represent mRNA from four different individuals. M, DNA molecular marker; bp, base pair(s). The positive control is human placenta, and cyclophilin was used as the housekeeping gene.

Contraction of HASM by UTP and ATP. Figure 4 shows that after a 10-min exposure to UTP (10^-5 M), HASM cell length decreased by 9.9 ± 4.3% (p = 0.004; n = 14). ATP (10^-5 M) also produced a decrease in cell length but of lesser magnitude: 5.6 ± 2.0% (p < 0.01; n = 9).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Exposure to ATP and UTP induces contraction of HASM cells. After exposure to UTP (10-5 M) (open circle) and to ATP (10-5 M) (open diamond), there was a small but significant decrease in cell length compared with vehicle (black circle). *, p < 0.009; **, p = 0.004.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Short-term incubation with UTP enhances the response to UTP. A, representative tracings of the response to UTP 10-4 M of two cells exposed for 10 min to vehicle (dash lines) and of two cells exposed to UTP (solid lines). B, peak Ca2+ release in response to UTP (10-4 M) after cell incubation in UTP 10-4 M (hatched columns) for 10 and 30 min was significantly enhanced compared with incubation in Hanks' buffer (white columns). R represents the resting Ca2+ levels from all experiments done at different time periods. The data presented are means ± S.E.M. *, p < 0.05.

Effects of Prolonged Periods of Pretreatment of HASM Cells with UTP on Responses to P2Y Receptor Stimulation. After prolonged and repeated exposures of HASM cells to 10^-4 M UTP (72 h, three doses, each at the intervals of 24 h), [Ca²⁺]_i was measured in response to 10^-4 M UTP, 10^-4 M ATP, and 10^-4 M histamine, and the results are shown in Fig. 6. Resting [Ca²⁺]_i was comparable in vehicle- and UTP-treated groups: 105 ± 2.3 nM (n = 105) in the vehicle-treated groups and 99 ± 2.5 nM (n = 107) in the UTP-treated groups (ANOVA; N.S.). Peak [Ca²⁺]_i levels were significantly decreased in response to 10^-4 M UTP: 152 ± 12 nM (n = 35) in the cells pretreated with UTP versus 349 ± 24 nM (n = 36; p < 0.001) in the cells exposed to vehicle alone. Peak Ca²⁺ release in response to 10^-4 M ATP was also decreased in cells exposed to UTP for prolonged periods compared with those exposed to vehicle alone: 148 ± 7 nM (n = 38) versus 383 ± 17 nM (n = 38; p < 0.001). In contrast, the peak [Ca²⁺]_i measured in response to 10^-4 M histamine was comparable in both vehicle (413 ± 45 nM; n = 31) and UTP-pretreated cells (368 ± 36 nM; n = 34).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Long-term incubation with UTP decreases the response to ATP and UTP but not histamine. Peak Ca2+ release in response to UTP, ATP, and histamine (10-4 M) after prolonged and repeated exposure of HASM cells to 10-4 M UTP. Cells were treated with 10-4 UTP (hatched columns) or vehicle (black columns) once a day for 3 days. Values presented are means ± S.E.M. *, p < 0.001.

	Discussion

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Although the effects of extracellular ATP and other triphosphate nucleotides have been studied in various lung cell types, including alveolar macrophages, mast cells, alveolar type II cells, and pulmonary vascular smooth muscle cells, the effects of these compounds on airway smooth muscle cells have not been studied as extensively. Previous work on tracheal preparations have yielded conflicting results, some authors reporting that ATP induces contraction of guinea pig tracheal preparations, whereas others observed an inhibitory effect (Fedan et al., 1993a,b, 1994). Recent work by Bergner and Sanderson (2002) on mouse lung slices shows that both ATP and UTP produce airway contraction and that the contraction is due to the activation of P2Y₂ and/or P2Y₄ receptors as it requires the activation of phospholipase C for the release of intracellular Ca²⁺. Previous work from our laboratory has shown that in rat airway smooth muscle cells, extracellular ATP and UTP produce an increase in [Ca²⁺]_i, the main determinant of smooth muscle cell contraction, and promote airway smooth muscle cell proliferation (Michoud et al., 1997, 2002).

In HASM cells, our results show that exposure to extracellular ATP, UTP, and their metabolites produced a significant increase in [Ca²⁺]_i. The peak Ca²⁺ release occurred within 30 s of exposure to the agonists and was followed by a lower but sustained plateau period. The data also show that adenosine had very little effect on [Ca²⁺]_i, confirming that the increase in [Ca²⁺]_i observed after ATP exposure was due to a direct effect of ATP rather than to an effect of its metabolite adenosine.

The effects of adenine and uridine nucleotides are mediated by a number of specific P2 receptors divided into two families. The P2X-receptors are ligand-gated ion channels and seven subtypes (P2 x 1-7) have been identified on the basis of their molecular structure (Harden et al., 1998). The P2Y receptors are G protein-coupled receptors and eight subtypes are widely recognized (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) (Abbracchio et al., 2003). The missing subtypes in the P2Y_1-N sequence represent the receptors cloned from nonmammalian vertebrates or receptors that are currently under functional characterization. The P2Y₁ receptor responds to ADP > ATP but not to UTP, whereas the P2Y₂ receptor responds to both ATP and UTP, and the P2Y₄ responds to UTP > ATP in humans (Kennedy et al., 2000). The P2Y₆ subtype responds preferentially to UDP and has little affinity for ATP, and the P2Y₁₁ responds to ATP but not to UTP. The results we obtained showing that ATP, ADP, UTP, and UDP produce an increase in [Ca²⁺]_i suggest that the P2Y₁,₂,₄,₆ receptor subtypes are present on HASM cells. The response to ADP being comparable in amplitude to that of ATP suggests that the P2Y₁ subtype is present. To confirm this latter observation, a dose-response curve to 2-MeSATP, a selective agonist of the P2Y₁, P2X₁, and P2X₃ receptors, was obtained. Exposure to this agonist produced an increase in [Ca²⁺]_i that was independent of the presence of Ca²⁺ in the extracellular medium, indicating that the peak [Ca²⁺]_i was due to Ca²⁺ release from intracellular stores rather than from extracellular sources, which is consistent with the response being mediated by the P2Y₁ (G-protein coupled) rather than by a P2X (ion gated channel) receptor subtype. UDP, a specific agonist of the P2Y₆ receptor (Communi et al., 1996), also produced an increase in [Ca²⁺]_i, indicating that this receptor must be present on HASM cells. This increase in [Ca²⁺]_i occurred within 30 s after the addition of the agonist and thus is attributable to UDP itself and not to a possible transphosphorylation of UDP into UTP as this reaction is slow to occur (Lazarowski et al., 1997b).

Although selective antagonists are available for P2Y₁ and P2Y₁₂, highly selective antagonists are not available for other subtypes of P2Y receptors. This makes further pharmacological characterization of the receptors difficult. Hence, we used RT-PCR analysis to confirm the presence of P2Y receptor mRNA transcripts. Because our pharmacological data indicated the presence of G protein-coupled P2Y subtypes of the receptors rather than P2X receptors, identification of mRNA transcripts of P2X receptors was not explored. These results obtained in cultured cells do not preclude the possibility of a more significant contribution of the P2X receptors in vivo. Indeed, the P2X₁ receptor, which is present and functional in contractile vascular smooth muscle cells, is down-regulated in cultured vascular smooth muscle cells (Erlinge, 2004). Thus, it is possible that the same phenomenon occurs in airway smooth muscle cells. The RT-PCR analysis of mRNA expression confirmed that HASM cells express P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptors.

P2 receptors can be stimulated by extracellular ATP and/or UTP through endogenous as well as exogenous sources. The airway epithelium is one endogenous source of extracellular ATP and UTP. Indeed, airway epithelial cells constitutively secrete ATP and UTP and can also release ATP and/or UTP after various biological and mechanical stimuli (Lazarowski et al., 1997a, 2000). Considering that airway epithelium and smooth muscle cells are in proximity, it is likely that the ATP and/or UTP released by epithelial cells can have a direct effect on HASM cells, particularly in pathological conditions such as asthma and cystic fibrosis, in which the airway epithelium is disrupted. Also, aerosolized UTP has been proposed as a therapeutic agent to enhance mucociliary clearance in cystic fibrosis patients as the stimulation of P2Y₂ receptors and to a lesser extent of P2Y₄ and of P2Y₆ (via UDP, the metabolic product of UTP) (Lazarowski and Boucher, 2001) on human epithelial cells produces an increase in chloride ion secretion and ciliary beat frequency. The potential limitation of this therapy is that G protein-coupled receptors, including P2Y₂ receptors, undergo desensitization upon repeated agonist exposure (Wilkinson et al., 1994). Although the mechanism of homologous desensitization has been well established for adrenergic receptors (Hausdorff et al., 1990), the pathways for the desensitization of P2Y receptors are not well characterized. Brown et al. (1991) have shown that incubation of airway epithelial cells with UTP for 5 min or longer decreases the total inositol phosphate turnover, suggesting that agonist induced desensitization is a characteristic of its receptor. In the present study, we have determined the effects of short and prolonged, repeated exposures to UTP on the increase in intracellular Ca²⁺ in response to de novo UTP stimulation. The results show that UTP mediated increases in [Ca²⁺]_i were enhanced when the cells were exposed to UTP for 10 and 30 min. Although we did not specifically investigate the mechanisms underlying the increased response, possible mechanisms include an up-regulation of the receptor binding affinity or the activation of a tyrosine kinase pathway and of the smooth reticulum-ATPase Ca²⁺ pump activity (Kito et al., 2001; Carbajal et al., 2005). The former seems less likely since we have used concentrations of UTP that are close to maximal.

The data also show that Ca²⁺ release was decreased in response to both UTP and ATP after longer and repeated exposure to UTP (once a day for 3 days). The response to histamine was not affected by preexposure to UTP, suggesting that desensitization was confined to the P2 receptors. These data are consistent with studies by Clarke et al. (1999), who showed that in epithelial cells, the desensitization of P2Y₂ receptors depends on the duration of exposure to UTP. Although we have not addressed the mechanism of desensitization of the receptors, work from other authors suggests that uncoupling and sequestration of the P2Y₂ receptors, caused by phosphorylation of the carboxy terminus of the receptor by a G protein-coupled receptor kinase (Garrad et al., 1998), and agonist-promoted internalization are likely mechanisms (Sromek and Harden, 1998).

It has been reported that P2Y receptors are up-regulated in proliferating cells and that cell culture conditions can modulate the smooth muscle cell phenotype from contractile to synthetic (for review, see Hirst et al., 2000b). Although we cannot rule out this possibility in our experimental conditions, the results we obtained show that the presence of the P2Y receptors on HASM is not restricted to cultured cells of the synthetic/proliferative phenotype because the cells also contracted in the presence of ATP and UTP. In addition, phenotypic heterogeneity of smooth muscle cells has been described in blood vessels and in airways. Indeed, using flow cytometric analysis, Halayko et al. (1997, 2005) described subtypes of smooth muscle cell populations in freshly dissociated trachealis muscle. Based on DNA content analysis, 87% of the cells were diploid and 13% tetraploid. In addition, groups of cells with high or low content in myosin and -actin could be found. In vascular smooth muscle, extracellular nucleotides have different effects, depending on the phenotype of the cell. Stimulation of P2Y receptors produces a contraction of the contractile phenotype and mitogenesis of the synthetic phenotype and is implicated in vessel wall remodeling (Erlinge, 2004). Analogous mechanisms are probable in airway tissue.

In conclusion, these data show that several subtypes of P2Y receptors are present in HASM cells. Their stimulation produces an increase in [Ca²⁺]_i, and cellular contraction. In vivo stimulation of the P2Y receptors present on HASM cells, whether of the synthetic or of the contractile phenotype, could contribute to airway function and also to airway remodeling. Considering that the P2Y receptor ligands ATP and UTP are endogenous mediators, their role in physiological and pathophysiological processes in the airways warrants further investigation.

	Footnotes

 
This work was supported by a grant from the Canadian Cystic Fibrosis Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.089698.

ABBREVIATIONS: HASM, human airway smooth muscle; AM, acetoxymethyl ester; PCR, polymerase chain reaction; 2-MeSATP, 2-methylthio-ATP; ANOVA, analysis of variance; RT, reverse transcription.

Address correspondence to: Dr. Marie-Claire Michoud, Meakins Christie Laboratories, Department of Medicine, McGill University, 3626 St Urbain, Montreal H2X 2P2, QC, Canada. E-mail: marie-claire.michoud{at}mcgill.ca

	References

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