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Regulation of P2Y₁ Receptor-Mediated Signaling by the Ectonucleoside Triphosphate Diphosphohydrolase Isozymes NTPDase1 and NTPDase2

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (C.A.-C., T.K.H. J.L.B.); and Inspire Pharmaceuticals Inc., Durham, North Carolina (J.L.B.)

Received for publication September 2, 2004.

Accepted for publication October 19, 2004.

	Abstract

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Ectonucleoside triphosphate diphosphohydrolases (NTPDases) control the concentration of released extracellular nucleotides, but the precise physiological roles played by these isozymes in modulation of P2 receptor signaling remain unclear. Activation of the human P2Y₁ receptor was studied in the presence of NTPDase1 or NTPDase2 expressed either in the same cell as the receptor or in P2Y₁ receptor-expressing cells cocultured with NTPDaseexpressing cells. Coexpression of NTPDase1 with the P2Y₁ receptor resulted in increases in the EC₅₀ for 2'-methylthioadenosine 5'-diphosphate (2MeSADP; 12-fold), ADP (50-fold), and ATP (10-fold) for activation of phospholipase C. Similar effects were observed when the P2Y₁ receptor and NTPDase1 were expressed on different cells. These results are explained by the capacity of NTPDase1 to hydrolyze both nucleoside triphosphates and diphosphates. NTPDase2 preferentially hydrolyzes nucleoside triphosphates, and the presence of NTPDase2 under either coexpression or coculture conditions did not change the EC₅₀ of 2MeSADP, ADP, or adenosine 5'-O-(2-thiodiphosphate) for activation of the P2Y₁ receptor. However, the EC₅₀ for ATP was 15-fold lower in the presence of NTPDase2 than in cells expressing the P2Y₁ receptor alone. Whereas expression of NTPDase1 decreased basal activity of the P2Y₁ receptor, the presence of the NTPDase2 resulted in P2Y₁ receptor-dependent increases in basal activity. These results suggest that basal activity of the P2Y₁ receptor is maintained by paracrine or autocrine release of receptor agonists and that the biological and/or pharmacological response mediated by P2Y receptors in target tissues is highly dependent on the types of ectonucleotidases expressed in the vicinity of the receptor.

The actions of extracellular nucleotides are mediated by two distinct families of cell surface receptors. P2X receptors are ligand-gated ion channels that conduct extracellular cations in response to ATP. Seven receptors (P2X₁ through P2X₇) comprise this family found largely, but not exclusively, on excitatory tissues (Khakh et al., 2000). P2Y receptors are a group of eight molecularly defined G protein-coupled receptors that exist both in the central and autonomic nervous systems as well as on most nonexcitatory cells (Abbracchio et al., 2003). Like P2X receptors, which are activated by ATP (but not ADP), the sparsely expressed P2Y₁₁ receptor is the only P2Y receptor that is activated selectively by ATP (Ralevic and Burnstock, 1998). The P2Y₁, P2Y₁₂, and P2Y₁₃ receptors are selectively activated by ADP, the P2Y₂ receptor is activated equipotently by UTP and ATP, the P2Y₄ receptor is selectively activated by UTP, the P2Y₆ receptor is selectively activated by UDP, and the P2Y₁₄ receptor is activated by nucleotide sugars, (e.g., UDP-glucose and UDP-galactose) (Ralevic and Burnstock, 1998; Abbracchio et al., 2003).

The concentration of extracellular nucleotides is regulated by a variety of surface-located enzymes known as ectonucleotidases (Zimmermann, 2000). In addition to their potential role in termination of purinergic signaling, the ectonucleotidases may prevent P2 receptor desensitization (Enjyoji et al., 1999) and control the availability of ligands for either nucleotide or adenosine receptors (Bonan et al., 2001; Sévigny et al., 2002). The most prominent of these ectoenzymes are members of the ectonucleoside 5'-triphosphate diphosphohydrolase (NTPDase) family (Zimmermann, 2000). Although seven NTPDases (1 through 6 and 8) (Zimmermann, 2000, 2001; Bigonnesse et al., 2004) have been extensively studied at the biochemical and molecular levels, their cellular actions remain to be defined. The most widely expressed NTPDases, NTPDase1 and NTPDase2, exhibit tissue distributions (e.g., in neural, vascular, and secretory tissues) that roughly correspond with reported distributions of several P2X and P2Y receptors (Vlajkovic et al., 2002a,b). The substrate selectivities of these isozymes suggest that they may play markedly different roles in regulation of P2Y receptor-mediated signaling because NTPDase1 (also known as ecto-ATPDase, CD39, apyrase) hydrolyzes both nucleoside tri- and diphosphates to their corresponding monophosphates (Kaczmarek et al., 1996; Heine et al., 1999), whereas NTPDase2 (ecto-ATPase, CD39L1) selectively hydrolyzes nucleoside triphosphates to their corresponding diphosphates (Kegel et al., 1997; Kirley, 1997; Mateo et al., 1999).

To begin to address the physiological roles of NTPDase isozymes in P2Y receptor-mediated signaling, we engineered 1321N1 human astrocytoma cells to stably coexpress the P2Y₁ receptor with either NTPDase1 or NTPDase2. Given the selectivity of activation of the human P2Y₁ receptor by ADP, we addressed whether NTPDase2 and NTPDase1 exhibit different functional relationships in P2Y₁ receptor-mediated signaling; the former produces the cognate P2Y₁ receptor agonist from ATP, and the latter ectoenzyme reduces the agonist action of both ATP and ADP at the P2Y₁ receptor. The functional consequence of proximity of NTPDases to the P2Y receptor also was examined in experiments in which the P2Y₁ receptor was coexpressed on the same cell as the ectoenzymes versus studies in which the P2Y₁ receptor and ectoenzymes were present on different cells. Finally, we addressed the underlying contributions of nucleotide release and ectoenzyme activity to basal P2Y₁ receptor signaling in cocultures of cells expressing the P2Y₁ receptor with cells expressing NTPDases.

	Materials and Methods

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Materials. ADP, 2MeSADP, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). ATP was from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK) and ADPS was from Calbiochem-Novabiochem (La Jolla, CA). The selective P2Y₁ receptor antagonist MRS2179 (tetra-ammonium salt) was obtained from Tocris (Ballwin, MO). Hygromycin B was from Roche Diagnostics Corporation (Indianapolis, IN). G-418 was obtained from Invitrogen (Carlsbad, CA). Plasmid purification kits were purchased from QIAGEN (Valencia, CA). [myo-³H]inositol (20 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). [³H]MRS2279 (89 Ci/mmol) was synthesized as described by Waldo et al. (2002). All tissue culture reagents were from the Lineberger Comprehensive Cancer Center tissue culture facility at the University of North Carolina (Chapel Hill, NC). We are grateful to Dr. John Olsen (Department of Medicine, University of North Carolina) for his kind gifts of PA317 cells and pLXSN and pLXPIH vectors.

Cell Culture. The murine packaging cell lines PA317 and 1321N1 human astrocytoma cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air/5% CO₂. 1321N1 cells stably expressing NTPDase1 or NTPDase2 and pLXPIH vector control cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and 600 µg/ml hygromycin B, whereas 1321N1 cells stably expressing the human P2Y₁ receptor (Schachter et al., 1996) were grown in 600 µg/ml G-418-containing medium. In some experiments, cells were cocultured such that human P2Y₁ receptor expressing cells were grown in the presence of an equivalent number of cells expressing either NTPDase1 or NTPDase2.

Stable Expression of the Human NTPDase1, NTPDase2, and P2Y₁ receptor in 1321N1 Human Astrocytoma Cells. Retrovirus harboring the human NTPDase1 or NTPDase2 coding sequences or the control retrovirus were produced as described previously (Comstock et al., 1997). In brief, complementary DNAs encoding the human NTPDase1 or NTPDase2 were cloned into the retroviral expression vector pLXPIH (Mateo et al., 1999); then, recombinant retroviral particles were produced by calcium phosphate-mediated transfection of PA317 murine packaging cells (Comstock et al., 1997). Transfected cells were incubated for 48 h at 32°C in the presence of 5 µM sodium butyrate, and the cell supernatant containing packaged retroviruses was collected, filtered, and used to infect wild-type 1321N1 human astrocytoma cells. Infection was carried out for 2 h in the presence of 8 µg/ml Polybrene. After 48 h of culture, positive clones were selected in culture medium containing 600 µg/ml hygromycin B for NTPDase-expressing cells. To examine the functional consequences of proximity of NTPDases to the human P2Y₁ receptor, both proteins were expressed in the same cell by reinfection of clonal human P2Y₁ receptor expressing cells with either NTPDase1 or NTPDase2 recombinant retroviruses.

Assay of Ectonucleotidase Activity. The pLXPIH vector control cells and 1321N1 cells expressing human P2Y₁ receptor or the NTDPases were seeded into 48-well plates at 4 x 10⁴ cells per well and assayed after cells reached confluence. In brief, the cells were washed twice with 500 µl of phosphate-free saline solution consisting of 125 mM NaCl, 5.2 mM KCl, 20 mM HEPES, pH 7.4, 2 mM CaCl₂, 1.2 mM MgCl₂, and 5 mM D-glucose, and incubated at 37°C in a water bath. Fifty µl of ATP and ADP were added to each well (final volume, 200 µl), and cells were incubated for 5 min. The incubation was terminated by transferring 170 µl of the cell-free supernatants to a new plate containing 170 µl of 20 mM EDTA at 4°C. Ectonucleotidase activity was measured as the release of inorganic phosphate from the nucleotides. Inorganic phosphate was determined colorimetrically using a modification of the malachite green-based assay (Lanzetta et al., 1979). In general, 30 µl of cell supernatants were combined with 100 µl of malachite green reagent and mixed, and absorbance was determined at 590 nm in a plate reader.

P2Y₁ Receptor-Stimulated Phospholipase C Activity. Agonist-induced inositol phosphate production was measured in 1321N1 cells grown to confluence on 48-well plates. Twelve hours before the assay, the inositol lipid pool of the cells was radiolabeled by incubation in 200 µl of serum-free, inositol-free Dulbecco's modified Eagle's medium, containing 0.4 µCi of [myo-³H]inositol. No changes of medium were made subsequent to the addition of [³H]inositol. On the day of the assay, cells were challenged with 50 µl of the 5-fold concentrated solution of receptor agonists (ATP, ADP, 2MeSADP, or ADPS) in 200 mM HEPES, pH 7.3, containing 50 mM LiCl for 20 min at 37°C. Incubations were terminated by aspiration of the drug-containing medium and addition of 450 µl of ice-cold 50 mM formic acid. After 15 min at 4°C, samples were neutralized with 150 µl of 150 mM NH₄OH. [³H]Inositol phosphates were isolated by ion exchange chromatography on Dowex AG 1-X8 columns as described previously (Filtz et al., 1994)

Radioligand Binding Assay. Control cells and 1321N1 cells expressing human P2Y₁ receptor alone or coexpressed with either NTPDase1 or NTPDase2 were seeded into 12-well plates at 4 x 10⁵ cells per well and assayed after cells reached confluence. On the day of assay, the cells were incubated at 4°C with 9 nM [³H]MRS2279 (approximately 30,000 cpm) in 20 mM Tris, pH 7.5 at 4°C, 145 mM NaCl, and 5 mM MgCl₂ in a volume of 400 µl. Specific binding was usually defined as total [³H]MRS2279 binding minus binding occurring in the presence of a 10 µM concentration of the P2Y₁ receptor-specific antagonist MRS2179. Incubations were for 30 min in an ice-water bath. Binding reactions were terminated by aspiration of the radioligand-containing medium followed by a quick single wash of 1 ml of binding buffer and addition of 1 ml of 0.1 M NaOH. After neutralizing each sample with 0.1 M HCl, the radioactivity was quantified by liquid scintillation spectrometry.

Data Analysis. Agonist potencies from concentration-response curves were obtained by nonlinear regression analysis using Prism software (GraphPad Software, San Diego, CA). All experiments were performed in triplicate assays and repeated at least three times.

	Results

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The human P2Y₁ receptor is selectively activated by ADP (Schachter et al., 1996; Leon et al., 1997; Palmer et al., 1998). For example, the purified human P2Y₁ receptor binds ADP with 20-fold higher affinity than ATP, and ATP is a relatively weak partial agonist compared with ADP at the purified receptor (Waldo and Harden, 2004). Thus, ATP is very unlikely to be a physiological agonist of the P2Y₁ receptor. However, given the large basal and mechanically induced release of ATP that occurs from most if not all cells, extracellular ATP potentially serves as a major source for P2Y₁ receptor activating ADP.

With the goal of studying the influence of ecto-NTPDases on P2Y receptor-mediated signaling we generated six different stable lines of 1321N1 human astrocytoma cells. A control 1321N1 cell line was isolated from cells infected with empty pLXPIH retrovirus. Stable cell lines expressing either the human P2Y₁ receptor, NTPDase1, or NTPDase2 alone were selected after infection of 1321N1 cells with retroviruses harboring the corresponding gene, and stable lines coexpressing the P2Y₁ receptor with either NTPDase1 or NTPDase2 also were selected.

Hydrolysis of ATP and ADP was quantified in the extracellular medium as a measure of the ectoenzyme activity present on the 1321N1 cell lines expressing NTPDase1, NTPDase2, NTPDase1-P2Y₁ receptor, or NTPDase2-P2Y₁ receptor. As illustrated in Fig. 1, the capacity of NTPDase1- and NTPDase2-expressing cells to hydrolyze adenine nucleotides was markedly increased relative to the very low hydrolysis observed under these conditions in vector-infected cells. The initial rate of ATP and ADP hydrolysis was quantified in all six cell lines (Table 1). Whereas hydrolysis was near background in vector and P2Y₁ receptor-expressing cells, robust and similar rates of hydrolysis of ATP were observed between NTPDase1- and P2Y₁ receptor-NTPDase1-expressing cells and between NTPDase2- and P2Y₁ receptor-NTPDase2-expressing cells. Thus, stable cell lines expressing NTPDases alone and NTPDases coexpressed with the P2Y₁ receptor were produced such that similar amounts of functional ectoenzyme activities were observed. As has been previously established for these molecularly defined enzymes, NTPDase1 hydrolyzed ATP and ADP similarly, whereas NTPDase2 showed selectivity for ATP over ADP (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Ectonucleotidase activities of NTPDase1- and NTPDase2-expressing 1321N1 cells. NTPDase1-expressing cells (left) or NTPDase2-expressing cells (right) were prepared as described under Materials and Methods. Cells were incubated with increasing concentrations of ATP, ADP, or AMP for 3 min at 37°C, and nucleotide hydrolysis was measured as the release of inorganic phosphate. Data shown are mean ± S.E.M. of triplicate assays from a representative experiment repeated at least three times.

P2Y receptors are not natively expressed by 1321N1 human astrocytoma cells, but stable expression of the human P2Y₁ receptor conferred [³H]inositol phosphate responses to ADP and to adenine diphosphate analogs. Concentration effect curves to ADPS were generated to determine whether the apparent potency of this nonhydrolyzable ADP analog was altered when the P2Y₁ receptor was coexpressed (in the same cell) with NTPDase1 versus NTPDase2 or when P2Y₁ receptor-expressing cells were cocultured with 1321N1 cells stably expressing either NTPDase1 or NTPDase2. As illustrated in Fig. 2, the EC₅₀ value for ADPS was approximately 1 µM in cells expressing P2Y₁ receptor alone or in P2Y₁-expressing cells cocultured with NTPDase1 or NTP-Dase2. It is interesting that the EC₅₀ of ADPS was approximately 20-fold lower in cells coexpressing the P2Y₁ receptor with NTPDase1 but was only marginally lower in cells coexpressing the P2Y₁ receptor with NTPDase2, suggesting different functional interactions between the P2Y₁ receptor and NTPDase1 versus NTPDase2. Furthermore, the molecular proximity between the receptor and the ectoenzyme is apparently critical, because, in contrast to the result obtained under coexpression conditions, the EC₅₀ of ADPS did not change when P2Y₁ receptor-expressing cells were cocultured with NTPDase1-expressing cells.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of ectoenzymes on the pharmacological activity of a nonhydrolyzable nucleotide analog. P2Y1 receptor-promoted [3H]inositol phosphate accumulation was quantified after incubation of cells with the indicated concentrations of ADPS for 20 min at 37°C. Activity of the P2Y1 receptor was studied in the presence of NTPDase1 or NTPDase2 coexpressed in the same cell (right) or after coculture of P2Y1 receptor-expressing cells with either NTPDase1- or NTPDase2-expressing cells (left). The accumulation of [3H]inositol phosphates was normalized to the maximal response produced by ADPS. Data are mean ± S.E.M. of triplicate determinations, and results shown are representative of at least three independent experiments.

NTPDase1 catalyzes conversion of both tri- and diphosphates to their corresponding nucleoside monophosphates. Therefore, functional apposition of this ectoenzyme with the P2Y₁ receptor might be expected to reduce the capacity of both nucleoside triphosphates and nucleoside diphosphates to activate their cognate P2Y receptors. This was observed to be the case for both ADP (Fig. 3) and ATP (Fig. 4) for activation of the P2Y₁ receptor. It is noteworthy that this inhibitory action of NTPDase1 was observed irrespective of whether the ectoenzyme was coexpressed on the same cell as the P2Y₁ receptor or was expressed at similar levels on an independent cell that was cocultured at 1:1 ratio with the P2Y₁ receptor-expressing cells. Thus, the capacity of adenine nucleotides to promote P2Y₁ receptor-mediated signaling in response to adenine nucleotides added to the bulk medium is similarly attenuated by NTPDase1 expressed in close apposition with the P2Y₁ receptor or expressed at a distance in another cell.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Expression of NTPDase1 but not NTPDase2 markedly decreases the apparent potency of ADP. P2Y1 receptor-promoted [3H]inositol phosphate accumulation was studied in the presence of NTPPase1 or NTPDase2 either coexpressed with the receptor (right) or in P2Y1 receptor-expressing cells cocultured with NTPDase1 or NTPDase2-expressing cells (left). The accumulation of [3H]inositol phosphates was normalized to the maximal response produced by ADP. Data are mean ± S.E.M. of triplicate determinations, and results shown are representative of at least three independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of expression of NTPDase isozymes on P2Y1 receptor-mediated responses to ATP. P2Y1 receptor-promoted [3H]inositol phosphate accumulation was studied in the presence of NTPDase1 or NTPDase2 either coexpressed with the receptor (right) or in P2Y1 receptor-expressing cells cocultured with NTPDase1- or NTPDase2-expressing cells (left). The accumulation of [3H]inositol phosphate was normalized to the maximal response produced by ATP. Data are the mean ± S.E.M. of triplicate determinations, and are representative of at least three independent experiments.

NTPDase2 selectively hydrolyzes nucleoside triphosphates over nucleoside diphosphates (Table 1). Because ATP only weakly activates the ADP-activated P2Y₁ receptor, we hypothesized that ATP would be converted to ADP in the presence of NTPDase2 and, therefore, that the apparent agonist action of ATP might be augmented at P2Y₁ receptors expressed in the presence of NTPDase2. On the other hand, because NTPDase2 exhibits high selectivity for hydrolysis of nucleoside triphosphates over nucleoside diphosphates, we hypothesized that the effect of NTPDase2 would be negligible on the capacity of ADP to promote P2Y₁ receptor-mediated signaling. These ideas were supported by the results presented in Figs. 3 and 4. Thus, neither coexpression of NTPDase2 with the P2Y₁ receptor nor coculture of NTPDase2-expressing cells with P2Y₁ receptor-expressing cells altered the concentration effect curve of ADP for stimulation of [³H]inositol phosphate accumulation by the P2Y₁ receptor. In contrast, the concentration effect curve of ATP for stimulation of [³H]inositol phosphate accumulation was shifted to the left by approximately 15-fold by the presence of NTPDase2. This effect was similarly observed irrespective of whether the ectoenzyme was expressed on the same cell as the P2Y₁ receptor or was expressed on a neighboring cell.

2MeSADP is a hydrolyzable ADP analog that, similar to ADP, is metabolized by NTPDase1 and to a much less extent by NTPDase2 (data not shown). The EC₅₀ value of 2MeSADP for stimulation of [³H]inositol phosphate accumulation by the P2Y₁ receptor was approximately 10-fold higher in cells coexpressing the P2Y₁ receptor with NTPDase1 or in cocultures of NTPDase1-expressing cells with P2Y₁ receptor-expressing cells. As for ADP, neither coexpression of NTPDase2 with the P2Y₁ receptor nor coculture of NTPDase2-expressing cells with P2Y₁ receptor-expressing cells caused a significant change in the concentration-effect curve of 2MeSADP for P2Y₁ receptor activation (Table 2). 2MeSADP is a more potent agonist for the human P2Y₁ receptor than is ADP (Schachter et al., 1996; Waldo and Harden, 2004). Thus, the extent to which the apparent potency of 2MeSADP and ADP for P2Y₁ receptor activation was diminished in the presence of NTPDase1 could be the result of a combination between 1) differences in the K_d values of these agonists for the P2Y₁ receptor and 2) differences in the K_m values of NTPDase1 for these substrates. NTPDase1 probably hydrolyzes 2MeSADP and ADP with similar K_m values. Therefore, we speculate that the smaller shift to the right of the concentration-effect curve for 2MeSADP compared with ADP for P2Y₁ receptor activation occurs because 2MeSADP activates the P2Y₁ receptor at lower concentrations that are less affected by NTPDase1 activity than the higher concentrations of ADP necessary to activate the receptor.

Heterologous expression of P2Y receptors has been widely reported to result in elevation of "basal" [³H]inositol phosphate accumulation (Filtz et al., 1994; Parr et al., 1994; Lazarowski et al., 1995; Alvarado-Castillo et al., 2002). Whereas constitutive activity has been shown previously to account for elevated responses of some overexpressed recombinant G protein-coupled receptors (Seifert and Wenzel-Seifert, 2002), the most parsimonious interpretation of data for P2Y receptors to date is that endogenous nucleotides released either constitutively or after mechanical stimulation of cells account for most if not all of these "basal" activities of expressed P2Y receptors. This conclusion was based largely on observation of release of endogenous nucleotide into the extracellular medium at levels capable of P2Y receptor activation (Lazarowski et al., 1995, 2000; Ostrom et al., 2000; Joseph et al., 2003; Lazarowski et al., 2003) as well as on observation of lowered [³H]inositol phosphate accumulation in P2Y receptor-expressing cells after addition of soluble apyrase to the medium (Parr et al., 1994; Ostrom et al., 2000). The availability of cell lines engineered to coexpress the P2Y₁ receptor with either NTPDase1 or NTPDase2 as well as the availability of means to generate cocultures in which P2Y₁ receptor-expressing cells are in the presence of NTPDase1- or NTPDase2-expressing cells provide two different cell systems to more rigorously address the influence of ectoenzymes on the receptor-stimulating activities of cellular nucleotides released into the extracellular medium.

Basal [³H]inositol phosphate accumulation was measured in cells expressing the P2Y₁ receptor alone or in coculture with cells expressing either NTPDase1 or NTPDase2. As illustrated in Fig. 5, expression of the P2Y₁ receptor alone resulted in a marked increase in [³H]inositol phosphate accumulation. This increase was largely inhibited by the presence of the P2Y₁ receptor antagonist MRS2179 during the incubation, suggesting that basally released extracellular nucleotide activates the receptor in a manner prevented by the addition of exogenous receptor antagonist. This contention is supported by the observation that coculture of NTP-Dase1-expressing cells with P2Y₁ receptor-expressing cells almost completely inhibited P2Y₁ receptor-dependent increases in [³H]inositol phosphate accumulation. In contrast, coculture of NTPDase2-expressing cells with P2Y₁ receptorexpressing cells resulted in a marked increase in [³H]inositol phosphate accumulation by a mechanism that also was inhibited by the presence of the P2Y₁ receptor antagonist MRS2179. The most parsimonious interpretation of these results is that cellular ATP is released into the extracellular medium, and the presence of NTPDase2 results in the formation of ADP; ADP in turn activates the P2Y₁ receptor more effectively than does ATP. Again, the inhibitory effect of MRS2179 supports the idea of autocrine regulation of the overexpressed receptor.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Differential influence of NTPDase1 and NTPDase2 on basal P2Y1 receptor-mediated signaling. Wild type cells infected with empty-vector (vector), P2Y1 receptor-expressing cells (P2Y1), NTPDase1-expressing (NTPDase1) or NTPDase2-expressing (NTPDase2) cells were cocultured as indicated at a 1:1 cell ratio in 48-well plates as described in Methods. Twenty-four hours after seeding, cells were labeled with [3H]inositol in inositol-free medium in the presence of 100 µM MRS2179 and 1 mM LiCl. After 48 h, the medium was removed, and the basal levels of [3H]inositol phosphates were measured as described under Materials and Methods. Data are mean ± S.E.M. of triplicate determinations and are representative of at least two independent experiments.

Basally released nucleotides also potentially induce down-regulation of the P2Y₁ receptor; therefore, the presence of ectoenzymes that metabolize nucleotides might be expected to differentially regulate receptor levels. Indeed, quantification of P2Y₁ receptors using [³H]MRS2279 revealed approximately 5-fold higher receptor levels in cells coexpressing NTPDase1 compared with cells expressing P2Y₁ receptor alone (Table 3). In contrast, P2Y₁ receptor levels in cells coexpressing NTPDase2 were similar to levels in cells expressing P2Y₁ receptor alone.

	Discussion

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The results reported here illustrate marked NTPDase-dependent alteration of P2Y receptor-promoted signaling. The molecular form of the NTPDase and, therefore, the relative selectivity for metabolism of extracellular ATP versus ADP determines whether the effect of ectoenzyme is to enhance or to decrease the apparent potency of the extracellular nucleotide (Fig. 6). Autocrine and paracrine regulation may account for much of the physiological responses mediated through the P2Y receptor family of signaling proteins, and the data illustrated here show a marked influence of NTPDases on the activation state of P2Y₁ receptors under both basal and agonist-stimulated conditions.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Formation of the cognate agonist of the P2Y1 receptor by NTP-Dase2 and inactivation of P2Y1 receptor-activating nucleotides by NTPDase1.The schematic illustrates the release of cellular ATP and its conversion by NTPDase2 into the cognate agonist ADP for the P2Y1 receptor (top) and the release of cellular ATP and the conversion of ATP (and ADP) to AMP (bottom), which is not an agonist of the P2Y1 receptor.

P2Y receptors are not natively expressed in 1321N1 human astrocytoma cells; therefore, this cell line has been widely applied for heterologous expression of molecularly identified P2Y receptors. NTPDase activity is also relatively low in 1321N1 cells (Lazarowski et al., 2000) and, as illustrated here, increases in the hydrolysis rates of extracellular nucleotides of 50-fold are conferred in 1321N1 cells stably expressing either NTPDase1 or NTPDase2. The reported high selectivity of NTPDase2 for ATP over ADP and lack of selectivity of NTPDase1 for ATP versus ADP also was recapitulated in the cells overexpressing NTPDases. Thus, these engineered 1321N1 cell lines provide a useful system for examining the pharmacological interactions of NTPDase-catalyzed nucleotide metabolism and P2Y₁ receptor signaling. Moreover, the system has allowed us to address for the first time the influences rendered by NTPDases when present on the same cell as the receptor versus when NTPDases are present on a neighboring cell.

P2Y1 receptor-promoted activation of phospholipase C was quantified during 20-min incubations with nucleotide added to the bulk solution. Therefore, the observed EC₅₀ values should be considered "averages" of the relative concentration-dependence of agonists over time and under conditions in which NTPDase-catalyzed breakdown of nucleotide occurred at an indeterminate rate at the cell surface. Quantification of rapid Ca²⁺ responses to nucleotides potentially would provide acute measure of relative EC₅₀ values, but the physiological significance of such measurements after addition of nucleotides to the bulk solution is unclear. Indeed, the results as presented here with bulk addition of agonists in assays quantified over longer periods of time are more likely to approximate physiological responses that may occur with sustained release of nucleotides. Given the architectural boundaries of small pericellular spaces relative to cell surface receptors and NTPDases, measurement of short-term Ca²⁺ responses might provide relevant insight when the influence of NTPDases is studied under conditions in which the release of small boluses of cellular nucleotide is effected by, for example, mechanical stimulation of cells expressing both P2Y1 receptors and NTPDases.

The presence of overexpressed NTPDase2 did not modify the signaling response of the P2Y₁ receptor to the nonhydrolyzable agonist ADPS. Whereas coculture of cells overexpressing NTPDase1 with 1321N1 cells overexpressing the P2Y₁ receptor also did not modify the concentration dependence of ADPS for activation of phospholipase C, a reproducible 20-fold increase in the potency of ADPS was observed in cells coexpressing the P2Y₁ receptor with NTPDase1. The molecular significance of NTPDase1-promoted increase in effectiveness of ADPS is not known. However, we illustrated previously that basal release of ATP occurs in resting 1321N1 cells (Lazarowski et al., 2000), and the elegant studies of Joseph et al. (2003) using cell surface-attached luciferase indicate that the concentration of ATP quantified in the medium of 1321N1 cells underestimates that at the cell surface by many fold. Because extracellular hydrolysis of ADPS did not occur under the conditions of these experiments, one interpretation of the current data is that the presence of NTPDase1 on the same cell as the P2Y₁ receptor serves to metabolize released ATP (and ADP), preventing basal down-regulation of the P2Y₁ receptor and/or its downstream signaling cohorts. Indeed, results from preliminary studies quantifying cell surface P2Y₁ receptor expression on intact cells were consistent with the idea that the presence of ectoenzymes modifies P2Y₁ receptor levels. Thus, P2Y₁ receptor density was nearly 5-fold higher in cells that coexpress the P2Y₁ receptor with NTPDase1 than in cells that express either the P2Y₁ receptor alone or with NTPDase2.

NTPDase1 exhibits similar catalytic activities against nucleoside triphosphates and diphosphates as substrates for conversion to AMP. Therefore, cellular expression of this ectoenzyme can be expected to terminate the action of the cognate agonist for nucleoside triphosphate-activated P2Y receptors (e.g., the P2Y₂, P2Y₄, and P2Y₁₁ receptors) as well as nucleoside diphosphate-activated P2Y receptors (e.g., the P2Y₁, P2Y₆, P2Y₁₂, and P2Y₁₃ receptors). This was clearly observed for the P2Y₁ receptor, with 10- to 30-fold shifts to the right for activation curves for both ATP and ADP. The inhibitory action of NTPDase1 was similar irrespective of whether the ectoenzyme was coexpressed on the same cell as the P2Y₁ receptor or was expressed on a different cell in cocultures with P2Y₁ receptor-expressing cells. Similar results were observed previously with a fusion protein of the human P2Y₁ receptor and NTPDase1 (Alvarado-Castillo et al., 2002). Addition of ATP or ADP to the bulk medium in these experiments may approximate the physiological situation in which activating nucleotide is released from a distant cell.

In contrast to the action of NTPDase1, NTPDase2 exhibits high selectivity for nucleoside triphosphates over nucleoside diphosphates. Therefore, whereas NTPDase2 would be expected to have no effect on ADP levels in the medium, this ectoenzyme readily converts ATP to ADP. Indeed, NTPDase2 potentially could provide a major source for extracellular ADP (or UDP), because the release of cellular ADP (or UDP) has not been broadly described. Therefore, whereas the concentration effect curve of ADP for activation of the P2Y₁ receptor was unchanged by the presence of NTPDase2, the concentration effect curve of ATP was remarkably shifted to the left in the presence of this ectoenzyme. This follows from the fact that ATP is a very weak agonist at the P2Y₁ receptor (Waldo and Harden, 2004), and NTPDase2-catalyzed conversion of ATP to ADP would produce the active cognate agonist of the receptor. Again, the fact that the influence of NTPDase2 on the response to added ATP was essentially the same irrespective of whether NTPDase2 was on the same cell as the P2Y₁ receptor or on a neighboring cell cocultured with P2Y₁ receptor-expressing cells suggests that the concentrations of nucleotide in the bulk phase may largely approximate that at the level of the receptor when the P2Y₁ receptor agonist arises from a distal source.

Mechanically induced release of ATP occurs by many if not most nonexcitatory cells (Lazarowski et al., 2003), and constitutive release of nucleotide under resting conditions is increasingly appreciated to occur in many cell types (Lazarowski et al., 2000; Ostrom et al., 2000; Joseph et al., 2003). The experiments carried out here measuring P2Y₁ receptor-mediated signaling in 1321N1 cells maintained under resting conditions may approximate that occurring physiologically during autocrine/paracrine regulation of cell signaling. Thus, as we and others have observed with heterologous expression of most members of the P2Y receptor family (Lazarowski et al., 2003), expression of the human P2Y₁ receptor enhanced inositol lipid signaling. That this effect occurs as a consequence of constitutive nucleotide release was supported by the opposing action of NTPDase2 versus NTPDase1 on basal signaling. The substrate selectivity of NTPDase2 would be expected to result in conversion of released ATP into the activating agonist ADP (Fig. 6), and as was illustrated in Fig. 5, basal P2Y₁ receptor signaling was doubled by NTPDase2 expression. On the other hand, NTPDase1 metabolized nucleoside tri-and diphosphates to the corresponding monophosphates (Fig. 6), and NTPDase expression almost completely inhibited basal P2Y₁ receptor-dependent inositol lipid signaling. That these opposing NTPDase-promoted changes follow from metabolism of ATP also is supported by the observation that the P2Y₁ receptor selective antagonist MRS2179 inhibited by up to 80% both basal and NTPDase2-promoted elevation of [³H]inositol phosphates.

The tissue distributions of NTPDase1 and NTPDase2 markedly differ in mammals. For example, NTPDase1 localized with microglia in brain, with the vasculature of brain and other tissues, and with activated lymphocytes (Enjyoji et al., 1999; Braun et al., 2000; Zimmermann, 2000), whereas NTPDase2 was expressed by astrocyte-like cells in the germinal zones of the adult rat brain (Braun et al., 2003), found in portal fibroblasts within the liver (Dranoff et al., 2002), associated with microvascular pericytes of the cardiac vasculature (Sévigny et al., 2002), and found in various compartments of the cochlea (Vlajkovic et al., 2002a,b). Recent studies also illustrated that whereas NTPDase1 was restricted to blood vessel walls, NTPDase2 was selectively associated with nonmyelinating glia and fibroblasts of the peripheral nervous system, where presumably NTPDase2 plays an important role in the control of nucleotide-mediated activation of peripheral neurons or glia and in the dialogue between these two cell types (Braun et al., 2004). It is interesting that a subpopulation of myenteric neurons in the murine colon expresses the ADP-activated P2Y₁ receptor (Giaroni et al., 2002), further implicating a role for NTPDase2 in ligand production. Functional and pharmacological studies indicate that P2Y receptors are widely and differentially distributed in mammalian tissues (Ralevic and Burnstock, 1998). However, quantification of the precise localization of these receptors has largely not been possible because of the lack of reliable receptor-selective antibodies or radioligands. Thus, although it is clear that the P2Y₁ receptor is found in tissues that also contain NTPDase1 and/or NTPDase2, colocalization of the receptor with these or other nucleotide-metabolizing enzymes has yet to be established unambiguously.

The work described here provides a first step in understanding the relationship of NTPDase1 and NTPDase2 expression to regulation of P2Y₁ receptor-mediated signaling. It will be important to carry these studies to native tissues with the advent of improved molecular reagents for quantifying both the enzymes and P2Y receptors.

	Acknowledgements

 
We are indebted to Eduardo Lazarowski for discussions, to Misty White and Savitri Maddileti for technical assistance, and to David Rinker for help with the manuscript.

	Footnotes

 
This work was supported by United States Public Health Service grants HL54889 and GM38213. C.A.-C. was supported with a scholarship from Fondo Nacional de Ciencia, Tecnología e Innovación, Venezuela.

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

doi: 10.1124/mol.104.006908.

ABBREVIATIONS: NTPDase, ectonucleoside 5'-triphosphate diphosphohydrolase; 2MeSADP, 2'-methylthioadenosine 5'-diphosphate; ADPS, adenosine 5'-O-(2-thiodiphosphate); MRS2179, 2'-deoxy-N⁶-methyl-adenosine 3',5'-bisphosphate; MRS2279, 2'-deoxy-N⁶-methyl-adenosine 3',5'-bisphosphate diammonium; CD39, lymphoid cell activation antigen 39 (ecto-apyrase, NTPDase1).

Address correspondence to: T. Kendall Harden, Department of Pharmacology, University of North Carolina School of Medicine, CB#7365, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

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