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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 732-738, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4768

Effects of Extracellular Triphosphate Nucleotides and Nucleosides on Airway Smooth Muscle Cell Proliferation

Marie-Claire Michoud, Giuseppe Napolitano, Karim Maghni, Vasanthi Govindaraju, Annalisa Cogo and James G. Martin

Meakins-Christie Laboratories and Montreal Chest Institute Research Center, McGill University, Montreal, Quebec, Canada

Address correspondence to: M. C. Michoud, Meakins Christie Laboratories, McGill University, 3626 St Urbain Street, Montreal, PQ, H2X 2P2 Canada. E-mail: Marie-Claire.Michoud{at}McGill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular ATP and uridine triphosphate (UTP) have a range of effects on a wide variety of cells through the activation of P2 receptors. The aim of this work was to establish if stimulation with ATP and UTP enhances airway smooth muscle (ASM) cell proliferation and to determine the type of receptor mediating this effect. Proliferation of rat ASM cells was assessed through bromodeoxyuridine (BrdU) uptake and by cell counting. At concentrations of 10-6 and 10-5 M, ATP and UTP induced significant increases in BrdU incorporation. ATP analogs specific for the P2X and P2Y1 receptor subtypes had no effect. UDP (a P2Y6 receptor agonist) produced significant decreases in BrdU incorporation and cell counts. Adenosine, the metabolite of ATP, produced an increase in cell proliferation through stimulation of the A1 receptor. A2 and A3 receptor stimulation had no effect. Reverse transcription and polymerase chain reaction analysis showed that mRNA transcripts for the P2Y2, P2Y4, P2Y6, A1, A2, and A3 receptor subtypes were present in cultured ASM cells. These data show that extracellular UTP, ATP, and their metabolites may affect airway remodeling by increasing or by reducing (P2Y6 receptor) ASM cell proliferation.

Abbreviations: {alpha}-ß-methyleneadenosine 5'-triphosphate, {alpha}-ß-MeATP • ß,{gamma}-methyleadenosine 5'triphosphate, ß,{gamma}-MeATP • 2-methylthioadenosine triphosphate, 2MeS-ATP • adenosine, ADO • airway smooth muscle, ASM • adenosine 5'-0 (3-thiotriphosphate), ATP-{gamma}-S • bromodeoxyuridine, BrdU • N6-cyclopentyladenosine, CPA • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • Hanks' balanced salt solution, HBSS • 1-deoxy-1-(6-{[(3-Iodophenyl)methyl]amino}-9H-purin-9-yl)-N-methyl-ß-D-ribofuranuronamide, IB-MECA • 5'-N-ethyl-carboxamidoadenosine, NECA • phorbol 12-myristate-13-acetate, PMA • tracheal smooth muscle, TSM • uridine triphosphate, UTP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since the publication of the report by Drury and Szent-Györgyi in 1929 that extracellular adenine nucleosides and nucleotides have pronounced effects on the circulatory system, mediating vasodilatation, heart block, and decrease in blood pressure, the role of these compounds in the cardiovascular system has been extensively investigated (1–3). Numerous reports have shown that extracellular ATP has positive inotropic and chronotropic effects on the myocardium and induces both vascular smooth muscle cell contraction and relaxation. It also regulates vascular smooth muscle cell proliferation and apoptosis. These effects of extracellular ATP are mediated by the activation of specific P2 receptors present on the cell membrane. The P2 receptors (previously known as purinergic receptors) consist of two families: the ligand gated ion receptors or P2X receptors (seven subtypes) and the G-protein–coupled receptors or P2Y receptors (seven subtypes) (4–6).

In the respiratory system, it has been shown that extracellular ATP and UTP produce an increase in surfactant synthesis by alveolar type II cells (7) and enhance chloride ion and airway surface fluid secretions. Again, these effects are mediated by the stimulation of specific P2 receptor subtypes on epithelial and goblet cells (8–10). These properties of the triphosphate nucleotides have led to the investigation of the possible use of aerosolized uridine triphosphate (UTP) as a treatment to enhance airway fluid transport in patients with cystic fibrosis (11–13), a disease believed to be caused by a defect in chloride transport.

Although the effects of extracellular ATP and UTP on airway epithelial cells have been extensively investigated, the effects of these compounds on airway smooth muscle (ASM) cells are not well characterized. Flezar et al (14) have reported that intratracheal instillation of ATP produces an increase in airway resistance in vivo in rats. In vitro, the published data on airway tone are conflicting; both relaxant and contractile effects on airway smooth muscle have been observed (15–18). These discrepancies are likely due to the type of preparation studied and the wide variety of cell types present. The presence or absence of epithelium is of particular pertinence, because this tissue is an important source of bronchoactive mediators such as nitric oxide and prostaglandin E2 (19, 20). Indeed, Fedan and coworkers (16) report that the effects of ATP and other purine nucleotides depend on whether the agonist is applied to either the luminal or serosal surface (luminal versus serosal) of the trachea. Aksoy and Kelsen (18) have confirmed that the ATP-induced airway relaxation is dependent on the presence of the epithelium.

Pharmacologic studies from our laboratory have shown that exposure of ASM cells to extracellular triphosphate nucleotides (ATP and UTP) produces an increase in intracellular Ca2+ and that this increase is mediated by the P2Y2 receptor. Furthermore, the ATP-induced increase in cytosolic Ca2+ was enhanced by preincubation of the cells with adenosine, a metabolite of ATP, although adenosine itself had no measurable effect on the intracellular free Ca2+ concentrations ([Ca2+]i) (21, 22). These data thus showed that extracellular ATP modulates ASM contractility directly and through the effects of its metabolite adenosine.

Exposure of ASM to extracellular adenine and uridine nucleotides may not have effects that are limited to the regulation of contraction. Indeed, these nucleotides induce the proliferation of various cell types, such as renal mesangial cells, breast cancer cell lines, mammalian cell lines, and vascular smooth muscle cells (23–25). Considering that airway smooth muscle cells are exposed to extracellular ATP secreted by airway epithelial cells and/or mast cells (26, 27), and considering that P2Y2 receptors (the receptors for both adenine and uridine nucleotides) are present on airway smooth muscle cells, we hypothesized that exposure of ASM cells to endogenous or exogenous triphosphate nucleotides could induce smooth muscle cell proliferation. If such an action were to occur, it could lead to an increase in ASM mass and perhaps also contribute to other aspects of the airway remodeling commonly observed in inflammatory airway diseases. Thus, the aim of this study was to measure the effects of adenine and uridine nucleotides on ASM cell proliferation and to determine which type of receptor mediates this response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Primary cultures of tracheal smooth muscle (TSM) cells were prepared as previously described (22). Briefly, 7- to 9-wk-old male Fisher rats (Harlan Sprague Dawley, Walkerville, MD) were injected intraperitoneally with a lethal dose of pentobarbital, and the tracheas were removed and cut longitudinally through the cartilage and placed in Hanks' balanced salt solution (HBSS). Tissue digestion was obtained by incubating the tracheas for 45 min at 37°C in HBSS containing 0.2% collagenase type IV and 0.05% elastase type V. The dissociated cells were then collected by centrifugation, resuspended in culture medium containing 1:1 Dulbecco's modified Eagle's medium: Ham's F12 medium (DMEM:F12) supplemented with 10% fetal bovine serum (FBS), 0.244% NaHCO3, penicillin (100 U/ml) and streptomycin (100 µg/ml) and plated in 25 cm2 culture flasks. Confluent cells were detached with a 0.25% trypsin-0.02% EDTA solution and grown on 24-well plates for cell counting and 96-well plates for 5-bromo-2-deoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). Cells from the 1st to 5th passage were used. They were identified as smooth muscle cells by positive immunohistochemical staining for smooth muscle–specific {alpha}-actin and the absence of cytokeratin (28).

Growth Assay
Cell counting. Cells were plated onto 24-well plates at a density of 104 per well in DMEM:F12 containing 10% FBS. Forty-eight to seventy-two hours later, the cells were growth-arrested by switching the culture medium to DMEM:F12 containing 0.5% FBS for 48 h. The cells were then exposed (in duplicate) to various mitogenic agents diluted in DMEM–0.5% FBS, or to the same amount of diluent (0.1 ml DMEM–0.5% FBS) for a period of 48 h. Then, they were detached with 0.5 ml of HBSS containing 0.25% trypsin and 0.02% EDTA and counted on a standard hemacytometer (American Optical, Buffalo, NY).

BrdU ELISA. Cell proliferation was also determined using a colorimetric assay based on the measurement of BrdU incorporation during DNA synthesis (Boehringer Mannheim, Montreal, PQ, Canada). The BrdU ELISA was performed according to the manufacturer's instructions. Briefly, confluent cells were detached by trypsinization, counted, and plated onto 96-well microtiter-plates at a density of 1,000 cells/well in DMEM:F12 supplemented with 10% FBS for 48 h. The growth medium was then switched to DMEM:F12 containing 0.5% FBS only, and the cells exposed to this medium for a period of 48 h to induce quiescence and synchronize the cell cycle. The vehicle (DMEM:F12 containing 0.5% FBS) and test substances were then added in triplicate or sextuplicate wells (10 µl/well). For the concentration curves to UDP, 100 U/ml hexokinase were added to the vehicle (DMEM:F12 containing 1.0% FBS and 25 mM glucose) to prevent the possible conversion of UDP into UTP (29). Six hours later, the thymidine analog BrdU was added (10 µl/well) and the cells incubated for a further 18 h, during which the BrdU was incorporated into the newly synthesized DNA of proliferating cells. Total exposure time of the cells to test substances was thus 24 h. The culture medium was then removed, and the cells denaturated with FixDenat solution and incubated for 90 min with 1:100 diluted mouse anti BrdU mAbs conjugated to peroxidase. After removing the antibody conjugate, substrate solution was added for 20 min and the reaction stopped by adding 1 M H2SO4 solution. The absorbance (optical density; OD) was measured within 5 min at 450 nm with a reference wavelength at 690 nm using an ELISA plate reader (400 ATC; SLT Lab instruments, Salzburg, Austria.) (30).

PKC downregulation. Cells were plated on 96 microtiter plate and allowed to grow as described above. Twenty-four and forty-eight hours after switching the medium to DMEM–0.5% FBS, phorbol myristate acetate (PMA 0.8 and 1.6 x 10-6 M) was added to selected wells. BrdU incorporation was then measured as described above.

Detection of P1 and P2 Receptor Subtypes mRNA Expression in Cultured TSM Cells
Total cellular RNA was extracted from confluent cultured TSM cells (passages 2–5) cells with TRIZOL (Gibco BRL, Burlington, ON, Canada) and the gene of interest was amplified by reverse transcription–polymerase chain reaction (RT-PCR) (31). Briefly, RNA pellets were dissolved in RNAse- and DNase-free-tested water (Ambion Inc., Austin, TX). Strand cDNA was made in a 20-µl reaction, using 2 µg of total RNA as template, oligo(dT)12–18 primer and Superscript II enzyme, in presence of acetylated BSA (Gibco BRL) and RNAguard RNase (Pharmacia Biotech, Baie d'Urfe, PQ, Canada) as enzyme inhibitors. The PCR mixture consisted of (final concentration) 1.5 mM MgCl2, 1x PCR buffer, 0.2 mM dNTP mixture, 2.5 U Platinum Taq polymerase (Gibco BRL), 20 pmol of the upstream and downstream primers, and the synthesized cDNA strand. The samples were amplified in a Programmable Thermal Controller (PTC-100; MJ Research Inc., Watertown, MA). The general PCR conditions were: 1 min denaturation at 92°C, 2 min for the annealing, and 3 min of extension at 72°C. The annealing temperature, the number of cycles and the sequence of PCR primer set used to amplify each gene of interest are indicated in Table 1. Most of the PCR primer sequences were selected on different exons of the respective genes to exclude genomic amplification. PCR products were visualized by ethidium bromide staining after gel-agarose (2% for all genes) electrophoresis and the correct size of the bands was determined by comparison with the DNA molecular weight markers (Roche Molecular Biochemicals, Montreal, PQ, Canada). The calculation of the size of the amplicons on agarose gels was performed using the Fluoro 800 Advanced Fluorescence, Chemiluminescence and Visible Light Imager (Alpha Innotech Corporation, Montreal, PQ, Canada). In all PCR experiments, a negative control was included which corresponded to a negative control for the RT reaction (no RNA) followed by PCR amplification of a RT aliquot. All RT-PCR experiments were performed in parallel on total RNA isolated from the TSM cells of four animals. PCR primers were synthesized and purified by FLPC at the Sheldon Biotechnology Centre (Montreal, PQ, Canada).


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  		TABLE 1 Summary of PCR primer sequences and PCR conditions used

 
Chemicals
Elastase, collagenase, ATP, UTP, ADO, PMA, and PDGF-BB were purchased from Sigma (St. Louis, MO). DMEM, FBS, penicillin, and streptomycin were from Gibco Canada (Mississauga, ON, Canada). Ham's F-12 was from ICN (Mississauga, ON, Canada). 1-Deoxy-1-(6-{[(3-Iodophenyl)methyl]amino}-9H-purin-9-yl)-N-methyl-ß-D-ribofuranuronamide (IB-MECA), {alpha}-ß-methyleneadenosine 5'-triphosphate ({alpha}-ß-MeATP), ß,{gamma}-methyleadenosine 5'-triphosphate (ß,{gamma}-MeATP), 2-methylthioadenosine triphosphate (2MeS-ATP), adenosine 5'-0 (3-thiotriphosphate) (ATP-{gamma}-S), and 5'-N-ethyl-carboxamidoadenosine (NECA) were obtained from RBI Research Biochemicals (Natick, MA). Uridine 5'-diphosphate (UDP) and hexokinase were from Boehringer Mannheim.

Statistical Analysis
The data are expressed as mean ± SEM. The Dunnett t test was used to determine statistical significance of differences between means for data obtained after the application of various test agents and control conditions. A value of P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P2 Receptor Stimulation
Figures 1 and 2 show the concentration-response curves for ATP and UTP obtained with the BrdU incorporation assay. ATP induced concentration-dependent increases in BrdU incorporation (Figure 1), reaching a maximum of 39.5 ± 14% above control at 10-5 M (n = 6, P < 0.006). At the concentration of 10-4 M, ATP stimulation had the opposite effect, and a decrease in BrdU incorporation of 25.7 ± 7% (n = 6, P < 0.002) below control was observed. A decrease in BrdU incorporation was also observed with ATP-{gamma}-S, an analog of ATP which is more slowly hydrolyzed, at 10-4 M: 54.4 ± 8.2% (P < 0.006, n = 5). Exposure to UTP also produced concentration-dependent increases in BrdU incorporation (Figure 2), reaching a maximum of 44.3 ± 10% above control at 10-5 M (n = 6, P < 0.01). As a positive control, the increase in optical density produced by the ASM mitogen PDGF (100 ng/ml) was measured and was 123.0 ± 11.1% above control (n = 29, P < 0.000).



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  		Figure 1. Effects of ATP on BrdU incorporation. Values represent the mean ± SEM and are expressed as percentage change from control. The numbers in parentheses represent the number of experiments. *P < 0.05, test agent versus control.

 


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  		Figure 2. Effects of UTP on BrdU incorporation. Values represent the mean ± SEM and are expressed as percentage of control. The numbers in parentheses represent the number of experiments. *P < 0.05, test agent versus control.

 
Cell Counts
Compared with exposure to the diluent only, exposure to UTP 10-4 M produced an increase in cell counts of 9.73 ± 4.08 x 10 (3) (122.0 ± 8.9% of baseline, n = 8, P < 0.05), ATP 10-4 M an increase of 10.63 ± 1.86 (123.2 ± 3.6%, P < 0.000, n = 32), ATP-{gamma}-S 10-4 M an increase of 10.87 ± 3.59 x 103 (129.4 ± 11.0%, n = 6, P < 0.025), and PDGF an increase of 31.0 ± 7.88 x 103 (159.0 ± 13.0%, n = 8, P < 0.005).

In a separate series of experiments, the ATP (10-5 M) induced increase in BrdU incorporation was abolished following downregulation of PKC with PMA 0.8 x 10-6 M (192.8 ± 49.3% of control versus 101.3 ± 7.3%, n = 6, P < 0.005) and PMA 1.6 x 10-6 M (76.3 ± 5.8%, n = 6, P < 0.005).

Table 2 shows the effects of four selective agonists for the P2X subtypes of receptor. In concentrations ranging from 10–9–10-5 M, neither ß{gamma}-MeATP (n = 8) nor {alpha}ß-MeATP (n = 7) or 2-meSATP, had a significant effect on BrdU incorporation. At 10-4 M, ß{gamma}-MeATP induced a significant decrease of 22.9 ± 9.1% in BrdU incorporation.


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  		TABLE 2 Effects of ATP analogs on BrdU incorporation

 
The effects of UDP, a specific agonist for the P2Y6-receptor subtype, are shown in Figures 3A and 3B. At the four concentrations tested, UDP produced significant decreases in BrdU uptake, reaching a peak of 22.8 ± 6.5% at 10-7 M (P = 0.03, n = 6). To confirm that a decrease in BrdU incorporation below control values indeed reflected a decrease in cell numbers, measurements were made by cell counting at concentrations of UDP 10-5 M and 10-4 M. The results confirmed that cell counts were decreased by 31.4 ± 8.4% (n = 7, P < 0.025) following stimulation with UDP 10-5 M.



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  		Figure 3. (A) Effects of UDP on BrdU incorporation. Values represent the mean ± SEM and are expressed as percentage of control (n = 10). (B). Effects of UDP on cell counts. Values represent the mean ± SEM (n = 5–6). *P < 0.05, test agent versus control.

 
P1 Receptor Stimulation
Concentration–response curves for adenosine (ADO) and CPA, an A1 selective agonist, are shown in Figure 4. At the concentrations of 10-7 M and 10-6 M, ADO significantly increased BrdU incorporation above baseline levels, reaching a peak at 20.0 ± 5.0% (n = 8). CPA had a small but consistent effect on ASM growth at concentrations ranging from 10-10–10-7 M with a peak increase of 24.6 ± 6.0% at 10-8 M (n = 8, P < 0.05). NECA, the selective agonist for the A2 receptor subtype had no effect at concentrations ranging from 10-12–10-7 M; at 10-6 M, it produced a small but significant decrease in BrdU incorporation: -9.5 ± 4.0% (n = 5, P < 0.03). IB-MECA, an A3-receptor agonist, had no significant effect on cell proliferation (n = 4).



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  		Figure 4. Effects of ADO and CPA on BrdU incorporation. The data are expressed as percentage of control. Values represent the mean ± SEM (n = 9 for ADO and 8 for CPA). *P < 0.05, **P < 0.01, test agent versus control.

 
P1 and P2 Receptor mRNA Expression
To confirm the results obtained with pharmacologic agonists, the expression of the mRNA encoding for the P2 receptor subtypes responding to ATP, UTP, and UDP: P2Y2, P2Y4, P2Y6 was looked for and detected by RT-PCR from each of four different animals for P2Y2 and P2Y6, and three animals in the case of the P2Y4. The expression of genes encoding for the P1 receptor subtypes was also detected in four animals. The A1, A2B, and A3 subtypes were found in four of the four samples. In all cases, the products of amplification were of the expected size: P2Y2: 339 bp; P2Y4: 376 bp; P2Y6: 449 bp; A1: 958 bp; A2B: 797 bp; and A3: 522 bp (Figures 5 and 6).



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  		Figure 5. mRNA expression for P2Y2, P2Y6 and P2Y4 receptors measured by RT-PCR. Lanes 1–4 each represent a different animal.

 


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  		Figure 6. mRNA expression for A1, A2B, and A3 measured by RT-PCR. Lanes 1–4 each represent a different animal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of extracellular ATP as a ubiquitous mediator of cell function has become increasingly apparent over the last few years, with the description of several specific membrane surface receptors and the realization that sources of extracellular ATP are more numerous than was previously thought (2, 32). Indeed, in addition to being secreted as a neurotransmitter in the central and peripheral nervous systems, ATP is secreted by numerous cell types, including mastocytes, epithelial cells, and smooth muscle cells (26, 27). Recent publications by Lazarowski and coworkers (27) and by Ostrom and colleagues (33) show that ATP is continuously secreted by epithelial cells, and that it acts as an autocrine and paracrine signaling molecule through the activation of specific P2 receptors. In vascular smooth muscle cells and various other cell types, including glioma cells and various cell lines such as pc12, 3T3, and 3T6, stimulation of P2Y receptors by ATP and other triphosphate nucleotides has a well-defined mitogenic effect (25, 34–37). In vascular smooth muscle cells, for example, the mitogenic effect of ATP is greater than or equal to that of recognized mitogenic agents such as epidermal growth factor and insulin (36). Whether such an effect is also exerted upon ASM cells is not known. Thus, the aim of this study was to determine the effects of extracellular ATP and UTP on ASM cell proliferation and to determine which subtypes of P1 and P2 receptors mediate this response.

Our data show that a single administration of ATP or UTP at various concentrations produces an increase in BrdU incorporation, an index of increased DNA synthesis. The maximum increase in incorporation (40% above control) was observed at the concentration of 10-5 M for both ATP and UTP. This increase is comparable to that reported by Erlinge and coworkers (36) for vascular smooth muscle cells after 19 h of incubation with ATP or UTP, and by Wang and colleagues (25), who found a doubling of [3H]thymidine incorporation after 40 h of incubation with ATP. Interestingly, Erlinge and associates (36) report a considerable increase in DNA synthesis (600% of control) if the cells are incubated with ATP for longer periods of time. As ATP is degraded to ADO by ectonucleotidases present at the cell surface, the increase in DNA synthesis observed after 72 h incubation is likely due to a combined effect of ATP and ADO, and indicates that extracellular ATP is a powerful direct and indirect mitogenic agent. In this study, the mitogenic effects of ATP and UTP measured by an increase in BrdU incorporation were confirmed by an increase in cell numbers. These increases are comparable to those reported by other authors for ATP and to that of other mitogenic agents such as {alpha}-thrombin (36, 38).

Extracellular ATP effects are mediated by a number of membrane receptors subdivided into two classes: the P2X or ion-linked receptors, of which seven subtypes have been identified, and the P2Y or G-protein–coupled receptors. There are no specific antagonists for the various subtypes of receptors (4). Thus, their functional significance is assessed by their affinity for various analogs of ATP and their response to UTP (4). The data we obtained show that the rank order of the agonists was UTP = ATP and that the P2X agonists {alpha}-ß-MeATP, ß,{gamma}-MeATP, and 2-MeSATP had little effect (Figures 1 and 2 and Table 1). These data suggest that the increase in BrdU incorporation is mediated by a P2Y rather than a P2X type of receptor. The increase in proliferation mediated by ATP and UTP, together with the data showing that mRNA encoding for P2Y2 and P2Y4 receptors is present in our cells, suggests that both subtypes of P2 receptors (the P2Y2, which responds to ATP and UTP, and the P2Y4, which respond to UTP and not to ATP) are involved in the response to these triphosphate nucleotides.

Interestingly, at the concentration of 10-4 M, a decrease in BrdU incorporation was observed following exposure to both ATP and ATP-{gamma}-S while cell counts increased. This decrease in BrdU incorporation is consistent with published data showing that high doses of extracellular ATP produce a sharp decrease in [3H]thymidine incorporation (35, 39, 40). Similarly to BrdU incorporation, [3H]thymidine incorporation (which is also an index of increased DNA synthesis) is usually measured 18 h after the marker has been added, i.e., 24 h following the addition of the mitogenic agent. Cell numbers, on the other hand, are usually counted 48 h following agonist exposure to allow for a completion of a full cell cycle, which in the case of tracheal smooth muscle cells lasts ~ 26 h (28). We submit that if the entry into the S phase of the cell cycle (which lasts ~ 12 h) is delayed following exposure to a given agonist, it might not be completed by the time BrdU incorporation is measured. Such a situation would be reflected as a decrease in BrdU incorporation in the agonist-exposed cells compared with control cells, in which the addition of the vehicle produces a small increase in DNA synthesis. Cell counts, on the other hand, as they are performed 48 h after the addition of the agonist, would not be affected by a delay in the S phase entry unless it is very prolonged. Thus, it is conceivable that in conditions where the entry into S phase is delayed, techniques such as BrdU or [3H]thymidine incorporation measure a decrease in incorporation, whereas cell numbers actually increase. This hypothesis is supported by the work of Huwiler and coworkers (41), who showed that the stimulation of P2Y receptors activates the p38-stress-activated protein kinase, and by that of Page and colleagues (42), who report that p38 activation negatively regulates cyclin D1 expression, a G1 cyclin required for cell cycle traversal.

Exposure to UDP, the metabolite of UTP, and a specific agonist for the P2Y6 receptor subtype, produced a decrease in BrdU incorporation and a decrease in cell numbers compared with the control samples, indicating an antimitotic effect. Surprisingly, this antimitotic effect of UDP diminished as UDP concentration increased. A possible explanation for this unusual observation is that, in spite of hexokinase and glucose being present in the extracellular medium to prevent the reconversion of UDP into UTP by an ecto-nucleoside disphosphokinase (29), some UDP is being converted into UTP (which would be more likely as the agonist concentration increases). In that case, the proliferative effects of UTP would counteract the effects of UDP and cause an apparent decrease in the inhibitory effect of UDP. Although the P2Y6 receptor has been cloned since 1995 and mRNA transcripts have been found abundantly in rat and human tissues (4), the physiologic effects of P2Y6 receptor activation are not well characterized. It has been reported that the P2Y6 receptor is coupled to a Gq/11 protein and that its stimulation produces a sustained increase in the formation of IP3, lasting more than an hour (4). Lazarowski and coworkers (29) have measured an increase in chloride secretion following the exposure of nasal airway epithelial cells to UDP. To our knowledge, the data reported here are the first ones to show that stimulation of the P2Y6 receptors on smooth muscle cells has an antimitotic effect. Very recently, it has been reported that P2Y6 receptor stimulation induces vascular smooth muscle cell proliferation and that this effect is inhibited by the presence of the adenylyl cyclase activator forskolin (43). This observation underlines the fact that the effects of P2Y receptor stimulation frequently have opposite effects on second messenger generation, depending on whether they are coupled to more than one type of G protein and on the type of cells studied (4). Indeed, in macrophages, stimulation of the P2Y6 receptor is coupled to both pertussis-sensitive and -insensitive G proteins which activate cPLA2. The subsequent release of prostaglandin E2 in turn produces an increase in cAMP through the activation of a specific receptor (4). Further work will be needed to determine the mechanisms by which P2Y6 stimulation has an antimitotic effect in ASM cells. The possible intracellular signaling pathways include the activation of p38 and/or an increase in cAMP (4).

The effect of adenosine, the metabolite of ATP, on smooth muscle cell proliferation was also assessed. Adenosine increased BrdU incorporation in a dose-dependent manner, and this effect appears to be mediated by the A1 receptor subtype as CPA; a selective A1 receptor agonist had the same effect. Stimulation of the A2B and A3 receptor subtypes with the selective agonists NECA and IB-MECA had little or no effect on cell proliferation, although mRNA expression for these receptors was present. In vascular smooth muscle cells and some other cell types, exposure to ADO has an antimitotic effect mediated by the stimulation of A2B receptors coupled to an inhibition of MAP kinase activity (44). In this work, we do not observe this effect, although A2B receptors are present as demonstrated by RT-PCR. In addition, no effect of A3 stimulation on cell proliferation was measured, although we have shown in previous work that this receptor is not only expressed but also functional (21). Indeed, stimulation of rat ASM cells with IB-MECA, an A3 receptor agonist, activates phopholipase A2 and causes the subsequent formation of arachidonic acid, thereby promoting the entry of extracellular calcium and enhancing the effect of contractile agonists such as serotonin. The data reported here on the effects of A1-, A2-, and A3-specific agonists on BrdU incorporation suggest that the effect of ADO on smooth muscle cell proliferation could be dependent on the relative concentration of A1 versus A2b receptors. In this respect, it is interesting to note that our measurements have been performed on Fisher rat tracheal smooth muscle cells, a strain of animal with airway hyperresponsiveness when compared with other rat strains such as Lewis or Sprague-Dawley (28). Whether the distribution of adenosine receptors and thus the response to adenosine would be the same in other rat strains, or whether it would be more comparable to the predominantly A2b receptor effect seen in vascular smooth muscle cells, remain to be determined.

In conclusion, we have shown that extracellular triphosphate nucleotides modulate ASM cell proliferation through the activation of specific P2Y receptors present on the cell surface. Interestingly, stimulation of these receptors (P2Y2 and P2Y6) has opposite effects on cell proliferation, suggesting that adenine and uridine nucleotides and their metabolites may play a role in maintaining a balance in the process of airway remodeling. In addition, these data show that adenosine also stimulates ASM cell mitogenesis through the activation of A1 receptor subtype, and confirm that the recently discovered adenosine receptor subtype A3 receptor is present on rat airway smooth muscle cells.


   Acknowledgments
 
This work was supported by an operating grant from the Canadian Cystic Fibrosis Foundation.

Received in original form November 6, 2001

Received in final form July 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dubyak, G. R., and J. S. Fedan. 1990. The biologic actions of extracellular adenosine triphosphate. Compr. Ther. 16:57–61.[Medline]
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