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NEUROPHARMACOLOGY
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois
Received for publication
December 23, 2003
Accepted
March 15, 2004.
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Abstract |
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,
-methylene adenosine triphosphate also potently inhibited specific [3H]A-317491 binding to both P2X2/3 and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X2/3 and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X2/3 receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.
; Lewis et al., 1995; Vulchanova et al., 1997). The selective localization of P2X3 receptors on sensory neurons has generated considerable interest in the role of this receptor in the propagation of pain signaling (Burnstock, 2000; Jarvis and Kowaluk, 2001). The P2X3 receptor is natively expressed as a functional homomer and as a heteromultimeric combination (P2X2/3 receptors) with the slowly desensitizing P2X2 receptor (Chen et al., 1995; Lewis et al., 1995; Lynch et al., 1999). These P2X3-containing channels share highly similar pharmacological profiles (Bianchi et al., 1999) but differ in their acute desensitization kinetics (Collo et al., 1996; Burgard et al., 1999). As a consequence, activation of the heteromeric P2X2/3 receptors can be pharmacologically distinguished from P2X2 receptor-mediated responses since P2X2/3 receptors are sensitive to low concentrations of the P2X3 agonist ,-meATP, whereas the P2X2 receptor is not (Bianchi et al., 1999; Lynch et al., 1999).
Characterization of P2 receptor pharmacology has been hindered by the general lack of high-affinity ligands that selectively interact with specific P2 receptor subtypes (Jacobson et al., 2002). Currently, seven homomeric P2X receptors and eight G-protein-coupled P2Y receptors have been cloned and characterized in recombinant expression systems (see Jacobson et al., 2002). Historically, ATP-derived analogs generally function as nonselective agonists for these receptors, and antagonist pharmacophores have been based on weakly active and nonselective agents like suramin and PPADS (Jacobson et al., 2002). More recently, nucleotide analogs like TNP-ATP, a high-affinity antagonist of P2X1, P2X3, and P2X2/3 receptors and diinosine pentaphosphate, a nanomolar P2X1 antagonist, have been described (Lewis et al., 1998; King et al., 1999).
The utility of radioligand binding methodologies to characterize P2 receptors has also suffered from the lack of pharmacological selectivity. Although the binding of a number of radiolabeled ATP analogs has been described (Jacobson et al., 2002), these radioligands have been shown to bind with high nanomolar affinity for recognition sites that are not functional P2 receptors (Motte et al., 1996; Schachter and Harden, 1997; Schachter et al., 1997; Yu et al., 1999). A-317491 is a high-affinity non-nucleotide antagonist of P2X3 and P2X2/3 receptors that has high selectivity for blocking P2X3-containing channels as compared with its ability to interact with other P2 receptors and many other receptors, enzymes, and ion channels (Jarvis et al., 2002). The present studies were undertaken to characterize the ability of [3H] A-317491 to specifically label recombinant human P2X3 and P2X2/3 expressed in 1321N1 cells, a cell line that has previously been shown to be devoid of endogenous functional P2 receptors (Schachter et al., 1997; Yu et al., 1999; McDonald et al., 2002).
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Materials and Methods |
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; Lynch et al., 1999). In brief, the homomultimeric cell lines were constructed by transfecting individual human P2X (P2X2 and P2X3) receptor cDNAs into 1321N1 cells using standard lipid-mediated transfection methods. Similarly, the heteromultimeric cell lines were constructed by transfecting human P2X2a cDNA into stably transfected human P2X3-expressing cells, designated here as P2X2/3 (Lynch et al., 1999). Cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics as follows: P2X3, 300 µg/ml G418; P2X2a, 100 µg/ml hygromycin; and P2X2/3, 150 µg/ml G418 and 75 µg/ml hygromycin.
Cell Membrane Preparation. Cell membranes were prepared from confluent cells in 225-cm2 plates. Cells were rinsed, scraped in phosphate-buffered saline (PBS), and pelleted by centrifugation at 1000 rpm for 10 min and stored at -80°C until use. For each assay, cells were thawed and resuspended in 50 mM Tris-HCl, pH 7.4 and homogenized using a polytron (setting 6 for 20 s). Cell membranes were centrifuged (48,000g for 20 min), and the resulting pellet was resuspended in buffer. Cell membranes were washed using this centrifugation and resuspension procedure three times. Total protein concentrations were determined by the method of Bradford (1976) and adjusted to 100 µg/ml.
Radioligand Binding. Radioligand binding methodology was generally adapted from procedures described by Yu et al. (1999) and modified to enhance specific binding to cell membranes expressing human P2X2/3 and P2X3 receptors. All radioligand binding assays were run in triplicate in a final volume of 0.25 ml. Based on preliminary ligand association binding studies using cell membranes containing human P2X2/3 receptors, binding reactions were conducted at 4°C for 90 min. Binding incubations conducted at 23°C or 37°C did not alter the level of total radioligand bound but increased nonspecific binding (B. Bianchi, unpublished data). Ligand competition studies were carried out using 3 to 4 nM [3H]A-317491 and 7 to 12 different concentrations of test compounds in 50 mM Tris-HCl, pH 7.4 containing 10 mM CaCl2. Nonspecific binding was defined in the presence of 100 µM A-317491 or 100 µM BzATP, which yielded similar levels of nonspecific binding. Binding reactions were initiated by the addition of 10 to 20 µg of protein. Ligand saturation studies were conducted using 12 to 16 concentrations (0.01–150 nM) of [3H]A-317491. All binding reactions were terminated by rapid filtration through a glass fiber filter mat (Whatman GF/B; Brandel, Inc., Gaithersburg, MD) using a 48-well Brandel cell harvester (Model M-48; Brandel). Membranes were washed twice with 5 ml of ice-cold 50 mM Tris-HCl, pH 7.4. Filter circles were incubated at room temperature in 5 ml of Ecolume (MP Biomedicals, Irvine, CA) for 12 h and counted in a Beckman LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). IC50 values were determined using a four-parameter logistic Hill equation (Graphpad Prism, San Diego, CA). Protein determinations were carried out as described by Bradford (1976).
Radioligand binding procedures using cell membranes expressing human P2X3 receptors required modification to minimize receptor desensitization during cell harvesting. Apyrase (1 U/ml, Sigma-Aldrich, St. Louis, MO) was added to confluent cells 30 min before harvesting in an effort to eliminate extracellular ATP. P2X3-expressing cell membrane preparations and radioligand methods were conducted as described for P2X2/3 receptors with the exception that the protein concentration was increased to 20 µg per assay tube.
Measurement of Intracellular Ca2+ Levels. P2X3 and P2X2/3 receptor function was determined on the basis of agonist-mediated increases in cytosolic calcium concentration as previously described (Bianchi et al., 1999). A fluorescent calcium chelating dye (Fluo-4; Molecular Probes, Eugene, OR) was used as an indicator of the relative levels of intracellular calcium in a 96-well format using the Fluoresence Imaging Plate Reader (Molecular Devices, Sunnyvale, CA). Cells were grown to confluence and loaded with the acetoxymethylester form of Fluo-4 (2 µM) in Dulbecco's PBS for 1 to 2 h at room temperature. Prior to the assay, each 96-well tissue culture plate was washed three times with Dulbecco's PBS to remove extracellular Fluo-4 AM. Fluorescence data were collected at 1-s intervals. Agonists were added 10 s after the start of the experimental run. Data shown are relative fluorescence units, based on cell density and the efficiency of Fluo-4 loading. IC50 values were determined using a four-parameter logistic Hill equation (GraphPad Prism).
Materials
Purine and pyrimidine nucleotide and nucleoside analogs, suramin PPADS, and cibacron blue were obtained from Sigma/RBI (Natick, MA). TNP-ATP and Fluo-4 AM were obtained from Molecular Probes. A-317491 and its enantiomer A-317344 were synthesized at Abbott Laboratories (Abbott Park, IL). Tissue culture media was from Hyclone Laboratories (Logan, Utah), and all other reagents were from Invitrogen (Carlsbad, CA).
[3H]-317491 (9.9 Ci/mmol) was synthesized as shown in Fig. 1 at Abbott Laboratories. 3-(3-phenoxyphenyl)benzaldehyde (2) (0.782 mmol; purchased from Acros Organics, Fairlawn, NJ) in 30 ml of absolute ethanol was treated with (1S)-1,2,3,4-tetrahydro-1-naphthalenylamine (1) (0.112 ml, 0.782 mmol; purchased from Lancaster). After stirring at ambient temperature for 4 h, the mixture was treated with NaBT4 (0.033 g, 0.860 mmol) in one portion. After stirring an additional 18 h, the reaction mixture was concentrated under reduced pressure and the residue dissolved in diethyl ether and quenched by addition of a solution of 1 N NaOH. The phases were allowed to separate, and the aqueous phase was extracted with diethyl ether. The organic phases were combined, washed with water, brine, dried (Na2SO4), filtered, and the filtrate concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 15% ethyl acetate/hexanes) to provide [3H]-N-(3-phenoxybenzyl)-N-[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amine (3) as a colorless oil (0.226 g, 79%). 1H NMR (300 MHz, CDCl3) 
6.8 to 7.53 (13H), 3.77 to 4.1 (m, 3H), 2.6 to 2.95 (m, 2H), 1.4 to 2.2 (m, 5H); MS (ESI+) 330 (M+H)+.
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1,2,4,5-Benzenetetracarboxylic dianhydride (0.270 g, 1.24 mmol) and triethylamine (0.215 ml, 1.55 mmol) in tetrahydrofuran (60 ml) were treated drop wise with N-(3-phenoxybenzyl)-N-[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amine (3) (0.618 mmol) in tetrahydrofuran (10 ml) at -78°C. The reaction mixture was allowed to stir for 16 h, gradually warming to ambient temperature. The mixture was treated with saturated aqueous Na2CO3 solution, stirred vigorously for 30 min, and then carefully acidified using 12 M HCl. The acidified solution was extracted with ethyl acetate. The organic extracts were combined, washed with 1 N HCl, brine, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 100:1:1 ethyl acetate/HCO2H/H2O) to provide [3H]-5-({(3-phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino}carbonyl)-1,2,4-benzenetricarboxylic acid (4) as a white solid (0.292 g). 1H NMR (300 MHz, DMSO-d6) 6.5 to 8.3 (m, 15H), 3.8 to 5.6 (m, 3H), 2.5 to 2.7 (m, 2H), 1.6 to 2.1 (m, 4H); MS (ESI+) 566 (M+H)+.
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Results |
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Ligand association experiments indicated that 3 nM [3H]A-317491 binding to P2X2/3 receptors reached equilibrium within 90 min at 4°C (Fig. 3). Ligand dissociation studies demonstrated that [3H]A-317491 binding was reversible, and distinct high- and low-affinity binding components were detected (Fig. 3). A high-affinity dissociation t1/2 value of 23 ± 1 min with corresponding K1 (0.0816 ± 0.009 min-1 nM-1) and K-1 (0.0387 ± 0.013 min-1) values was estimated from these experiments, resulting in a derived affinity constant value (Kd) of 0.5 nM. Ligand saturation studies indicated that [3H]A-317491 labeled two distinct classes of sites on P2X2/3 cell membranes, a high-affinity site (KdH = 0.93 ± 0.19 nM, apparent Bmax = 10.24 ± 1.35 pmol/mg protein) and a lower affinity site (KdL = 86.80 ± 22.0 nM, apparent Bmax = 23.8 ± 3.79 pmol/mg protein) (Fig. 4). Analysis of [3H]A-317491 binding over a lower range of ligand concentrations (0.01–7 nM) indicated that specific [3H]A-317491 binding was saturable and labeled a single class of high-affinity recognition sites (Kd = 1.07 ± 0.11 nM and apparent Bmax = 11.76 ± 0.41 pmol/mg protein) that was similar to the estimate of high-affinity [3H]A-317491 binding obtained in the two-site analysis (Fig. 5).
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As noted above, similar binding studies using cell membranes from 1321N1 cells expressing homomeric P2X3 receptors failed to yield reliable specific [3H]A-317491 binding (Fig. 2). However, incubation of the intact P2X3-expressing cells with apyrase (1 U/ml) prior to and during cell harvesting and preparation of the membranes produced markedly enhanced specific [3H]A-317491 binding (Fig. 6A). Apyrase treatment did not significantly alter 3 nM [3H]A-317491 binding to P2X2/3 receptors and was only effective in enhancing [3H]A-317491 binding to P2X3 cell membranes when added prior to cell harvesting (data not shown). Apyrase treatment also did not enhance specific [3H]A-317491 binding to cell membranes from wild-type 1321N1 cells or cells expressing P2X2a receptors. As was found for [3H]A-317491 binding to P2X2/3 receptors, specific [3H]A-317491 binding to P2X3 receptors was enhanced in the presence of 10 mM CaCl2 such that specific binding represented approximately 70 ± 5% of total binding. Using these same conditions, specific [3H]A-317491 binding to P2X1 receptors was not observed regardless of the absence or presence of 1 U/ml apyrase (Fig. 6B). Ligand association experiments indicated that specific 3 nM [3H]A-317491 binding to P2X3 receptors reached equilibrium within 30 min at 4°C (Fig. 7). Ligand dissociation studies indicated that specific [3H]A-317491 binding to P2X3 receptors was reversible, and distinct high- and low-affinity binding components were detected (Fig. 7). A high-affinity dissociation t1/2 value of 33 ± 1 min with corresponding K1 (0.0155 ± 0.004 min-1 nM-1) and K-1 (0.0210 ± 0.003 min-1) values were estimated from these experiments, resulting in a derived affinity constant value (Kd) of 1.4 nM. Ligand saturation studies indicated that [3H]A-317491 also labeled two distinct classes of sites on P2X3 cell membranes, a high-affinity site (KdH = 8.37 ± 1.40 nM, apparent Bmax = 4.11 ± 0.54 pmol/mg protein) and a lower affinity site (KdL = 788.4 ± 62.19 nM, apparent Bmax = 140.3 ± 17.15 pmol/mg protein) (Fig. 8). A similar one-site analysis of high-affinity binding to P2X3 receptors, as was done for P2X2/3 receptors (Fig. 5), was not possible due to lack of clearly saturable binding at these lower radioligand concentrations (Fig. 9).
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In ligand competition studies, the P2X receptor agonists, 2-meSATP and BzATP, potently inhibited the specific binding of [3H]A-317491 to P2X2/3 receptors (Fig. 10; Table 1). Also shown in Table 1 are the corresponding agonist pEC50 and relative efficacy values for P2X receptor agonists to activate the P2X2/3 receptor. In addition to 2-meSATP and BzATP, several other ATP analogs showed moderate nanomolar affinity to inhibit [3H]A-317491 binding. These agonists were approximately 10-fold less potent in stimulating P2X2/3 receptor-mediated calcium flux. UTP and ,,meADP inhibited [3H]A-317491 binding only at micromolar concentrations (Table 1). The diadenosine polyphosphates Ap4A, Ap5A, and Ap6A also potently competed for [3H]A-317491 binding and activated P2X2/3 receptors, whereas Ap2A and Ap3A were significantly less active in inhibiting [3H]A-317491 binding and did not activate P2X2/3 receptors (Table 1).
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A-317491 was the most potent compound to compete for [3H]A-317491 binding to P2X2/3 receptors (Fig. 11; Table 1), whereas the enantiomer of A-317491, A-317334, was approximately 150-fold less active. TNP-ATP also potently inhibited [3H]A-317491 binding, whereas the prototypic P2X antagonists suramin and PPADS were significantly less active. Both A-317491 and TNP-ATP were also potent antagonists of P2X2/3 receptor-mediated calcium influx.
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The pharmacology of [3H]A-317491 binding to P2X3 receptors was very similar to that observed for the P2X2/3 receptor. Table 2 shows the derived Ki values for both P2X receptor agonists and antagonists to compete for [3H]A-317491 binding at both P2X2/3 and P2X3 receptors. Like their activity at P2X2/3 receptors, both BzATP and 2-meSATP potently inhibited [3H]A-317491 binding (Table 2; Fig. 10). Unlabeled A-317491 also potently competed for [3H]A-317491 binding and was approximately 100-fold more active than its enantiomer, A-317344 (Fig. 11). The rank order of potency for P2X receptor agonists to compete for [3H]A-317491 binding to P2X3 and P2X2/3 receptors showed a high positive correlation (r = 0.98, P < 0.001). In addition, there was good correspondence (r = 0.86, P < 0.01) in the activity of these ligands to compete for high-affinity [3H]A-317491 binding and to alter P2X2/3 receptor activation (Fig. 12). The derived Ki values for P2X receptor ligands to interact with P2X3 receptors were generally less than those for P2X2/3 receptors (Table 2), consistent with the data generated in the ligand saturation and kinetic experiments. For P2X2/3 receptors, there was a general trend for steeper competition curves (nH values) as compared with P2X3 receptors (Table 2). There was also a trend for weaker compounds (Ki values >1 µM) to show nH values that were less than unity.
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A variety of agonists and antagonist ligands including adenosine, cycolpentyladenosine, CGS 21680, CGS 15943, theophylline, cyclopentylxanthine, and dipyridamole that interact with P1 adenosine receptors (A1, A2A, A2B, and A3) or adenosine uptake sites did not significantly inhibit [3H]A-317491 binding (<10% inhibition) at concentrations up to 100 µM.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Direct study of ligand recognition sites for the individual P2 receptor subtypes has been complicated by the paucity of available P2 receptor-selective radioligands (Jacobson et al., 2002). P2X receptor binding studies have been reported using [3H]ATP, [3H],MeATP, or [35S]ATP
S (Bo and Burnstock, 1989; Michel and Humphrey, 1996; Michel et al., 1996, 1997). Several of these ligands, notably [35S]dATPS and [35S]ADPS, have also been used to study P2Y receptor subtypes (Cooper et al., 1989; Van Rhee et al., 1993; Wilkinson and Boarder, 1995). However, the validity of P2 receptor binding assays using these nucleotide-derived radioligands has to be carefully considered since these ligands typically lack appropriate selectivity for individual P2 receptor subtypes (Michel et al., 1997; Jacobson et al., 2002; North, 2002), and a number of studies have demonstrated that high-affinity binding of these radioligands can be obtained in cellular and membrane preparations that do not contain functional P2 receptors (Motte et al., 1996; Schachter and Harden, 1997; Yu et al., 1999). More recently, progress has been made in the development of pharmacologically specific radioligand binding assays for some P2Y receptors. Examples include the respective use of [3H]2MeSATP and [3H]MRS2279 as specific agonist and antagonist labels for recombinant human P2Y1 receptors (Takasaki et al., 2001; Waldo et al., 2002). In the latter case, the non-nucleotide radioligand [3H]MRS2279 was also shown to specifically label P2Y1 receptors in native tissues (Waldo et al., 2002).
The study of ligand recognition at P2X receptors may also be complicated by the fact that functional P2X ligand-gated ion channels exist as oligomeric combinations with specific subunit arrangements. Both structural and biophysical modeling studies suggest that three agonist molecules interacting with a minimal trimeric conformation of individual P2X receptor subunits is necessary for receptor function (Nicke et al., 1998; Ding and Sachs, 1999; Stoop et al., 1999; North, 2002). This model has received further support by the recent demonstration that the likely conformation of the heteromeric P2X2/3 receptor is comprised of two adjacent P2X3 subunits and one P2X2 subunit (Jiang et al., 2003). However, the effect of subunit arrangement or interaction on antagonist binding is incompletely understood.
The present data demonstrate that specific [3H]A-317491 binding can be reliably detected using a straightforward cell membrane preparation from 1321N1 cells expressing human heteromeric P2X2/3 receptors (Lynch et al., 1999). Since P2X3 receptors can rapidly desensitize following brief (Chen et al., 1995; Lewis et al., 1995) or even functionally undetectable exposure to low concentrations of agonist (McDonald et al., 2002), the fast desensitizing properties of the homomeric P2X3 receptor necessarily confound the analysis of antagonist activity (Burgard et al., 2000; Spelta et al., 2002; Neelands et al., 2003). In the present studies, reliable [3H]A-317491-specific binding to cell membranes from P2X3-expressing cells could only be detected when apyrase (1 U/ml) was included in the cell medium both before and during cell membrane preparation. Similar to the results obtained for P2X2/3 receptors, both high- and low-affinity [3H]A-317491 binding components were detected in the ligand saturation and kinetic binding studies of the P2X3 receptor following apyrase treatment. Thus, unlike the slowly desensitizing P2X2/3 receptor, elimination of free ATP, both before and during membrane preparation, is necessary to reliably detect specific [3H]A-317491 binding to the fast homomeric P2X3 receptor. Taken together, these data indicate that agonists may show increased affinity for desensitized P2X3 receptors (McDonald et al., 2002), but antagonist affinity is greatly reduced at desensitized receptors.
The ability of [3H]A-317491 to label low-affinity binding components in membrane preparations from cells expressing P2X2/3 and P2X3 receptors was unexpected. Although the high-affinity binding components for both the P2X2/3 and P2X3 receptor assays are pharmacologically consistent with a specific labeling of functional P2X3-containing channels and highly correlated with each other, the nature of the lower affinity binding components detected in both the saturation and kinetic binding studies remains ambiguous. Detection of low-affinity [3H]A-317491 binding cannot be readily attributable to errors in defining nonspecific binding because high concentrations of both nucleotide and non-nucleotide agonist and antagonist ligands inhibited 3 nM [3H]A-317491-specific binding to a similar degree. As shown in Figs. 2 and 6, reliable specific binding of 3 nM [3H]A-317491 was not detected in membranes prepared from null cells or cells expressing P2X2, or P2X3 receptors in the absence of apyrase treatment. In addition, specific 3 nM [3H]A-317491 binding was not detected in cell membranes from P2X1-expressing cells in the absence or presence of apyrase treatment. These results indicate that high-affinity [3H]A-317491 binding to functional P2X3-containing channels can be clearly distinguished from other lower affinity [3H]A-317491 recognition sites.
The 1321N1 cell line expressing the heteromeric P2X2/3 receptor also likely expresses functional homomeric P2X3 and P2X2a receptors (Lynch et al., 1999; Jarvis and Burgard, 2002; Neelands et al., 2003). The present data show that [3H]A-317491 did not bind with high affinity to P2X2a receptors or to P2X3 receptors in the absence of apyrase treatment. Based on the ligand saturation data, it is likely that [3H]A-317491 does recognize desensitized or otherwise nonfunctional P2X3 receptor subunits, albeit at approximately 85-fold lower potency as compared with its high-affinity recognition sites. It also cannot be ruled out that [3H]A-317491 binds with low affinity to non-P2 receptor recognition sites that have high affinity for previously characterized nucleotide-derived agonist radioligands (Yu et al., 1999). It is interesting to note that the ligand saturation studies indicated that the low-affinity binding component found in the P2X3 cell membrane preparation was larger than that found for P2X2/3 receptors. However, precise characterization of these low-affinity binding sites was complicated by their relatively poor saturatability and the higher amount of nonspecific binding observed at [3H]A-317491 concentrations above 20 nM. This fact likely contributes to the extremely high apparent Bmax values obtained for these low-affinity sites. Additional binding studies using higher ligand concentrations, intact null cells, or cells expressing either P2X3 or P2X2/3 or receptors, and centrifugation techniques also did not provide reliable methods for characterizing these low-affinity [3H]A-317491 binding sites in further detail (B. Bianchi, unpublished data).
As was found for the P2X2/3 receptor, high-affinity [3H]A-317491 binding to P2X3 receptors that had been treated with apyrase readily reached equilibrium, was reversible, and was pharmacologically consistent with the labeling of functional P2X3 receptors. P2X receptor agonists were generally 10- to 100-fold more potent in competing for high-affinity binding to both P2X3 and P2X2/3 receptors as compared with their activities to evoke receptor mediated calcium influx (Table 2). In addition, there is some evidence to indicate that negative cooperativity exists for P2X antagonist block of the putative trimeric conformation of the P2X2/3 receptor (Spelta et al., 2002; Jiang et al., 2003). Thus, the subunit conformation and interaction of functional P2X receptors likely contributes to the observed differences in receptor binding and activation potencies. It is interesting to note that the agonist potencies to compete for high-affinity [3H]A-317491 binding (at both P2X2/3 and P2X3 receptors) are in close agreement with the agonist concentrations necessary to effectively desensitize P2X3 receptors and that these concentrations evoke minimal or no detectable receptor activation (McDonald et al., 2002). Taken together, these observations suggest that initial agonist binding to P2X3 and P2X2/3 receptors can occur with low nanomolar affinity but that channel activation requires additional agonist binding interactions that may occur with reduced affinity relative to the initial binding interaction.
The continued emergence of potent non-nucleotide-based P2 receptor-selective antagonist radioligands, like [3H]A-317491, will be useful for the further characterization of individual P2 subunits and the development of novel receptor-selective ligands. A limitation of the present studies is the relatively low specific activity of the radioligand. Based on the ligand saturation data, [3H]A-317491 labeled approximately 150,000 to 300,000 receptors per cell in the recombinant 1321N1 cell lines. An increase in specific activity of [3H]A-317491 may increase the signal to noise ratio of the radioligand and facilitate the use of lower radioligand concentrations. An increase in specific activity would also facilitate the characterization of P2X3-containing channels in native tissues. These studies also highlight the difficulty in discriminating functional receptor interactions from nonfunctional ligand recognition sites using radioligand binding techniques. Within the P2X receptor family, high-affinity antagonist binding using [3H]A-317491 appears to provide the needed receptor/nonreceptor discrimination. The development of other radioligands with increased selectivity for functional P2X receptor subunits relative to nonfunctional or nonreceptor recognition sites will further clarify the pharmacological properties of these ATP-sensitive ligand-gated ion channels.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ,-meATP, ,-methylene adenosine triphosphate; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium; TNP, 2',3'-O-(2,4,6, trinitrophenyl); PBS, phosphate-buffered saline; BzATP, 2,3-O-(4-benzoylbenzoyl)-ATP; 2-meSATP, 2-methylthio-ATP; nH, Hill slope.
1 Present Address: Cephalon Inc., West Chester, PA. 
Address correspondence to: Dr. Michael F. Jarvis, R4PM AP9A/2, 100 Abbott Park Rd., Abbott Park, IL 60064-6123. E-mail: michael.jarvis{at}abbott.com
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Bianchi BR, Alexander K, Park H, Metzger R, Touma E, Lynch KJ, Jarvis MF, Kowaluk EA, and Van Biesen T (1999) Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376: 127-138.[CrossRef][Medline]
Bo XN and Burnstock G (1989) [3H]-alpha, beta-methylene ATP, a radioligand labelling P2-purinoceptors. J Auton Nerv Syst 28: 85-88.[CrossRef][Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Burgard EC, Niforatos W, Van Biesen T, Lynch KJ, Kage KL, Touma E, Kowaluk EA, and Jarvis MF (2000) Competitive antagonism of recombinant P2X(2/3) receptors by 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Mol Pharmacol 58: 1502-1510.
Burgard EC, Niforatos W, Van Biesen T, Lynch KJ, Touma E, Metzger RE, Kowaluk EA, and Jarvis MF (1999) P2X receptor-mediated ionic currents in dorsal root ganglion neurons. J Neurophysiol 82: 1590-1598.
Burnstock G (2000) P2X receptors in sensory neurons. Br J Anaesth 84: 476-488.
Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, and Wood JN (1995) A P2X purinoceptor expressed by a subset of sensory neurons. Nature (Lond) 377: 428-431.[CrossRef][Medline]
Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099-3108.[CrossRef][Medline]
Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A, and Buell G (1996) Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16: 2495-2507.
Cooper CL, Morris AJ, and Harden TK (1989) Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2Y-purinergic receptor. J Biol Chem 264: 6202-6206.
Ding S and Sachs F (1999) Single channel properties of P2X2 purinoceptors. J Gen Physiol 113: 695-719.
Jacobson KA, Jarvis MF, and Williams M (2002) Perspective: purine and pyrimidine (P2) receptors as drug targets. J Med Chem 45: 4057-4093.[CrossRef][Medline]
Jarvis MF and Burgard EC (2002) Antagonism of P2X3-containing channels: commentary on Spelta et al. Br J Pharmacol 135: 1343-1344.[CrossRef][Medline]
Jarvis MF, Burgard EC, Mcgaraughty S, Honore P, Lynch K, Brennan TJ, Subieta A, van Biesen T, Cartmell J, Bianchi B, et al. (2002) A-317491, A novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc Natl Acad Sci USA 99: 17179-17184.
Jarvis MF and Kowaluk EA (2001) Pharmacological characterization of P2X3 homomeric and heteromeric channels in receptors nociceptive signaling and behavior. Drug Dev Res 52: 220-231.[CrossRef]
Jiang L-H, Kim M, Spelta V, Bo X, Surprenant A, and North RA (2003) Subunit arrangement in P2X receptors. J Neurosci 23: 8903-8910.
King BF, Liu M, Pintor J, Gualix J, Miras-Portugal MT, and Burnstock G (1999) Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors. Br J Pharmacol 128: 981-988.[CrossRef][Medline]
Lewis C, Neidhart S, Holy C, North RA, Buell G, and Surprenant A (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature (Lond) 377: 432-435.[CrossRef][Medline]
Lewis CJ, Surprenant A, and Evans RJ (1998) 2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)-A nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels. Br J Pharmacol 124: 1463-1466.[CrossRef][Medline]
Lynch KJ, Touma E, Niforatos W, Kage KL, Burgard EC, Van Biesen T, Kowaluk EA, and Jarvis MF (1999) Molecular and functional characterization of human P2X(2) receptors. Mol Pharmacol 56: 1171-1181.
McDonald HA, Bianchi BR, McKenna DG, Briggs CA, Burgard EA, Lynch KJ, Faltynek C, Cartmell J, and Jarvis MF (2002) Potent desensitization of human P2X3 receptors by diadenosine polyphosphates, ,-meATP and ATP. Eur J Pharmacol 435: 134-142.
Michel AD and Humphrey PP (1996) High affinity P2X-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes. Br J Pharmacol 117: 63-70.[Medline]
Michel AD, Lundstrom K, Buell GN, Surprenant A, Valera S, and Humphrey PP (1996) The binding characteristics of a human bladder recombinant P2X purinoceptor, labelled with [3H]-alpha,beta-meATP, [35S]-ATPGammaS or [33P]-ATP. Br J Pharmacol 117: 1254-1260.[Medline]
Michel AD, Miller KJ, Lundstrom K, Buell GN, and Humphrey PP (1997) Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors. Mol Pharmacol 51: 524-532.
Motte S, Swillens S, and Boeynaems JM (1996) Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P2 receptors. Eur J Pharmacol 307: 201-209, 1996.[CrossRef][Medline]
Neelands TR, Burgard EC, Uchic ME, McDonald H, Niforatos W, Faltynek CR, Lynch KJ, and Jarvis MF (2003) Electrophysiological and pharmacological properties of a slowly desensitizing chimeric human P2x3 receptor. Br J Pharmacol 140: 202-210.[CrossRef][Medline]
Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, and Schmalzing G (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO (Eur Mol Biol Organ) J 17: 3016-3028.[CrossRef][Medline]
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
Schachter JB and Harden TK (1997) An examination of deoxyadenosine 5'(alphathio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor. Br J Pharmacol 121: 338-344.[CrossRef][Medline]
Schachter JB, Sromek SM, Nicholas RA, and Harden TK (1997) HEK293 human embryonic kidney cells endogenously express the P2Y1 And P2Y2 receptors. Neuropharmacology 36: 1181-1187.[CrossRef][Medline]
Spelta V, Jiang LH, Surprenant A, and North RA (2002) Kinetics of antagonist actions at rat P2X2/3 heteromeric receptors. Br J Pharmacol 135: 1524-1530.[CrossRef][Medline]
Stoop R, Thomas S, Rassendren F, Kawashima E, Buell G, Surprenant A, and North RA (1999) Contribution of individual subunits to the multimeric P2X(2) receptor: estimates based on methanethiosulfonate block at T336C. Mol Pharmacol 56: 973-981.
Takasaki J, Kamohara M, Saito T, Matsumoto M, Matsumoto S, Ohishi T, Soga T, Matsushime H, and Furuichi K (2001) Molecular cloning of the platelet P2TAC ADP receptor: pharmacological comparison with another ADP receptor, the P2Y1 receptor. Mol Pharmacol 60: 432-439.
Van Galen PJ, Melman N, Ijzerman A, and Jacobson KA (1992) Characterization of [35S]ADPS binding to bovine brain membranes. Pharmacol Commun 1: 279-290.
Van Rhee AM, Van Winden EC, Nagelkerke JF, De Bont HJ, Ijzerman AP, and Soudijn W (1993) Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells. Biochem Pharmacol 45: 801-807.[CrossRef][Medline]
Vulchanova L, Riedl MS, Shuster SJ, Buell G, Suprenant A, North RA, and Elde R (1997) Immunohistochemical study of the P2X2 and P2X3 sensory receptor subunits in rat and monkey neurons and their central terminals. Neuropharmacology 36: 1229-1242.[CrossRef][Medline]
Waldo GL, Corbitt J, Boyer JL, Ravi G, Kim HS, Ji X-D, Lacy J, Jacobson KA, and Harden KT (2002) Quantitation of the P2Y1 receptor with a high affinity radiolabeled antagonist. Mol Pharmacol 62: 1249-1257.
Wilkinson GF and Boarder MR (1995) Binding of [35S]adenosine 5'-O-(2-thiodiphosphate) to endothelial cells in culture. Biochem Pharmacol 49: 1411-1418.[CrossRef][Medline]
Yu H, Bianchi B, Metzger R, Lynch KJ, Kowaluk EA, Jarvis MF, and van Biesen T (1999) Lack of specificity of [35S]ATPS and [35S]ADPS as radioligands for ionotropic and metabotropic P2 receptors. Drug Dev Res 48: 84-93.[CrossRef]
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