© 1996 American Heart Association, Inc.
Articles |
Binding of the Novel Nonxanthine A2A Adenosine Receptor Antagonist [3H]SCH58261 to Coronary Artery Membranes
the Departments of Medicine (L.B., J.C.S., J.R.), Pharmacology (L.B., D.M.D., S.P.B.), and Anesthesiology (D.M.D.), College of Medicine, University of Florida, Gainesville, and Schering-Plough Research Institute (A.M., S.D., E.O.), San Raffaele Science Park, Milan, Italy.
Correspondence to Luiz Belardinelli, MD, Professor of Medicine, University of Florida, PO Box 100277, Gainesville, FL 32610-0277. E-mail ramsey.med@shands.ufl.edu.
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Abstract |
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This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-
]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900±61, 892±35, and 959±76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261>HE-NECA=CSC=CGS 21680=XAC>NECA=R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radioligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.
Key Words: A2A adenosine receptor • coronary artery • radioligand • A2A receptor antagonist • SCH58261
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Introduction |
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Adenosine is an endogenous coronary vasodilator that has been proposed to play a major role in the metabolic regulation of coronary blood flow.1 The coronary vasodilatation caused by adenosine and adenosine analogues is mediated primarily by the A2AAdoR subtype present in the vascular wall.2 3 Much is known about the physiology and pharmacology of adenosine receptor–mediated coronary vasodilatation.2 3 However, it has not been possible to demonstrate the presence of A2AAdoRs in coronary arterial tissue, vascular smooth muscle, or endothelial cells by radioligand binding. Despite numerous attempts to label A2AAdoRs of coronary arteries with radioisotopes such as [14C]aden-osine,4 [3H]NECA,2 3 4 [3H]CGS 21680,2 3 4 and, more recently, 125IAPE,5 very few specific radioligand binding sites have been detected.
Three high-affinity selective A2AAdoR antagonists have been developed recently and either tritiated or iodinated6 7 8 : the [3H]8-styrylxanthine derivative KF 17837S6 and the nonxanthine heterocyclic compounds [125I]ZM 2413857 and [3H]SCH58261.8 All three radioligands have been shown to bind with high affinity (0.7 to 7.0 nmol/L) to A2AAdoRs in membranes prepared from either brain striatum or Chinese hamster ovary cells expressing a recombinant canine A2A receptor.6 7 8 However, it has yet to be determined whether these new radioligands bind to coronary artery A2AAdoRs with high affinity and in a saturable manner.
In the present study, we report that [3H]SCH58261 binds to a saturable high-affinity site in membranes prepared from porcine coronary arteries. The [3H]SCH58261 binding site has pharmacological properties consistent with its identification as an A2AAdoR. For comparison, we have also characterized the binding of [3H]SCH58261 to A2AAdoRs of membranes obtained from porcine striatum and from the rat pheochromocytoma cell line, PC12. The potency of SCH58261 to antagonize the A2AAdoR-mediated accumulation of cAMP in PC12 cells was also determined.
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Materials and Methods |
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Chemicals
SCH58261 and [3H]SCH58261 (specific activity, 68.6 Ci·mmol-1; radiochemical purity, 99%) were synthesized as previously described.9 XAC, R-PIA, CSC, NECA, and CGS 21680 were purchased from Research Biochemicals, Inc. The selective A2AAdoR agonist HE-NECA was a gift from Schering-Plough Research Institute. Rolipram was a gift from Berlex Labs. Stock solutions (50 mmol/L for R-PIA, 20 mmol/L for other drugs) of these chemicals were prepared in DMSO. The final concentration of DMSO in the incubation buffer of the radioligand binding assays was
0.1% (vol/vol). Concentrations of DMSO up to 1% did not reduce binding of [3H]SCH58261 to porcine striatal membranes (not shown). Rabbit anti-cAMP antibody was purchased from Accurate Chemical and Scientific Corp. Adenosine deaminase and ScAMP-TME were purchased from Sigma Chemical Co.
Membrane Preparations
Membranes were prepared from porcine coronary arteries, porcine brain striatum, and PC12 cells for use in quantification of specific [3H]SCH58261 binding. The protein content of membrane suspensions was determined using the Bradford method (Bio-Rad) with bovine serum albumin as standard.
Coronary Arteries
Porcine hearts were obtained from the abattoir of the University of Florida School of Veterinary Medicine and transported to the laboratory in ice-cold physiological saline solution. The hearts were rinsed, and the coronary arteries were flushed with physiological saline solution. The right coronary artery and the left anterior descending and circumflex coronary arteries were dissected from each heart. The arteries were cleaned of adventitial fat and connective tissue by careful and extensive trimming. The arteries were cut open longitudinally and treated with collagenase (Worthington type II, 70 U/mL) for 15 minutes at 37°C. The endothelium was removed by lightly brushing the intimal surface of the vessels with a cotton swab. Arteries were then quickly frozen in liquid nitrogen, pulverized using a mortar and pestle, and homogenized (Polytron, Brinkman Instruments) in 50 mmol/L Tris-HCl buffer (pH 7.4) containing (mmol/L) sucrose 250, EDTA 5, and PMSF 2. The homogenate was filtered through cotton gauze, and the filtrate was spun at 900g for 10 minutes. The supernatant was collected and centrifuged at 48 000g for 6 hours to pellet membranes. The membrane pellet was resuspended in 50 mmol/L Tris buffer containing 5 mmol/L EDTA and 2 mmol/L PMSF and frozen at -80°C. Immediately before receptor binding assays, membranes were thawed and washed once in 50 mmol/L Tris buffer by resuspension and centrifugation to remove EDTA and PMSF.
Striatum
Porcine striatum was obtained from Pel Freeze Inc. Striatum was minced and homogenized in 10 vol ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4). Homogenates were filtered through cotton gauze and spun at 48 000g for 15 minutes at 4°C. The supernatant was discarded, and the membrane pellet was resuspended in 10 vol 50 mmol/L Tris-HCl buffer (pH 7.4) and washed three times by centrifugation and resuspension in fresh buffer. The final pellet was frozen at -80°C until used for receptor binding assays.
PC12 Cells
PC12 cells were obtained at low passage from the American Type Culture Collection and grown in DMEM with fetal bovine serum (5%), horse serum (10%), L-glutamine (0.5 mmol/L), penicillin G (100 U/mL), streptomycin (0.1 mg/mL), and amphotericin B (2.5 µg/mL). Cells were cultured in an atmosphere of 5% CO2/95% air maintained at 37°C. Experiments were performed on 1-day preconfluent cells. PC12 cells were detached from the culture plates into chilled (4°C) 50 mmol/L Tris-HCl buffer (pH 7.4). The cell suspensions were homogenized (Polytron) at low speed for 30 seconds, diluted with Tris-HCl buffer to a volume of 45 mL, and spun at 48 000g for 15 minutes. The supernatants were discarded, and the pellets were washed three times by resuspension in Tris-HCl buffer and centrifugation. The final pellet was frozen at -80°C until used for receptor binding assays.
[3H]SCH58261 Binding Assays
Association and Dissociation Kinetics
The KAssoc and KDissoc values of [3H]SCH58261 to and from membranes prepared from porcine coronary artery, brain striatum, and PC12 cells were determined. For determination of KAssoc of [3H]SCH58261 to A2AAdoRs, membranes (0.03 to 0.05 mg) were incubated with 1 to 2 nmol/L [3H]SCH58261 in 50 mmol/L Tris buffer (pH 7.4) containing adenosine deaminase (2 U/mL) at 21°C for various durations of time (see Fig 1
). Separation of bound from free radioligand was carried out by vacuum filtration using a Brandel cell harvester. For determination of KDissoc of [3H]SCH58261 to A2AAdoRs, membranes (0.03 to 0.05 mg) were incubated with 1 to 2 nmol/L [3H]SCH58261 in 50 mmol/L Tris buffer (pH 7.4) containing adenosine deaminase (2 U/mL) at 21°C for 1 hour, then 50 µmol/L NECA was added, and radioligand bound to membranes was determined at selected times thereafter. Radioligand bound at each time was calculated as a percentage of radioligand bound in the absence of NECA. Triplicate determinations were performed at each time.
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Equilibrium Saturation Binding Assays
Total, specific, and nonspecific binding and the affinity (Kd) of the A2AA antagonist [3H]SCH58261 were determined by equilibrium saturation binding assays (see Fig 2). Increasing concentrations of [3H]SCH58261 (0.1 to 40 nmol/L) were incubated for 2 hours at 21°C with 100 µL aliquots of membrane suspension (0.03 to 0.05 mg protein), adenosine deaminase (2 U/mL), and 200 µL of 50 mmol/L Tris-HCl buffer (pH 7.4). To terminate an incubation, ice-cold buffer was added to each sample, and membrane-bound radioligand was collected onto Schleicher & Schuell glass fiber filters by vacuum filtration using a Brandel cell harvester. The radioactivity trapped on the filters was counted. Nonspecific binding was defined as binding not displaced by 50 µmol/L NECA. Nonspecific binding was subtracted from total binding to calculate specific binding. Triplicate determinations were made at each radioligand concentration.
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Competition Binding Assays
Competition assays to determine the affinities of unlabeled SCH58261, HE-NECA, CSC, NECA, XAC, CGS 21680, and R-PIA for the [3H]SCH58261 binding site were performed. Membrane suspensions (0.2 to 0.25 mg protein) were incubated for 1 hour at room temperature in 50 mmol/L Tris-HCl buffer (pH 7.4) containing adenosine deaminase (2 U/mL), [3H]SCH58261 (0.2 to 1.0 nmol/L), and progressively higher concentrations of the competing agent. Triplicate determinations were performed at each concentration of the competing agent.
Determination of cAMP Accumulation
PC12 cells were rinsed three times with HBSS, detached using a cell lifter, and pelleted by centrifugation at 500g for 5 minutes. Aliquots of the cell suspension (0.1 to 0.2 mg protein) were placed in microfuge tubes with 250 µL of HBSS containing rolipram (50 µmol/L) and warmed to 37°C. Drugs were added to the cell suspensions, and incubations were continued for 10 minutes. To terminate incubations, tubes were placed in a boiling water bath for 5 minutes. The samples were then cooled to room temperature, diluted by the addition of 1 mL of 10 mmol/L Tris-HCl buffer at pH 7.4, and then centrifuged for 2 minutes at 13 000g. The cAMP content of the supernatant was determined by modification of a radioimmunoassay described by Harper and Brooker.10 Briefly, an aliquot of the supernatant (0.01 mL) was mixed with 0.04 mL of HBSS, 0.05 mL of 50 mmol/L sodium acetate buffer (pH 6.2) containing 10 mmol/L CaCl2, 12 500 dpm of [125I]ScAMP-TME, and 0.05 mL of anti-cAMP antibody (1:2000 dilution with 0.1% bovine serum albumin in distilled water). The samples were then incubated at 4°C for 16 hours. At the end of the incubation, 70 µL of a 50% (wt/vol) hydroxyapatite suspension was added to each tube. The suspensions were gently agitated and then incubated for 10 minutes at 4°C. Antibody-bound radioactivity adsorbed to hydroxyapatite was collected onto glass fiber filters by vacuum filtration using a Brandel cell harvester. Radioactivity retained by the filter was counted in a gamma counter. Nonspecific binding of [125I]ScAMP was defined as radioactivity bound in the presence of 3 µmol/L unlabeled cAMP and was subtracted from total binding. The amount of cAMP present in samples was calculated from a standard curve using known amounts of cAMP.
Data Analysis
Data are presented as mean±SEM, and the range of values (minimum and maximum) or 95% confidence limits for the number (n) of experiments is indicated. Significance of differences among group means was determined by ANOVA followed by Tukey's testing. A value of P<.05 was considered to indicate a significant difference.
Binding parameters describing results of saturation (ie, Bmax, Kd, and the Hill coefficient) and competitive displacement experiments (Ki and the Hill coefficient) were determined using the radioligand binding analysis programs RADLIG version 4.0 (Elsevier-Biosoft) and GraphPad version 2.0 (GraphPad Software Inc). Kinetic parameters (KAssoc and KDissoc) were determined using nonlinear regression analysis. The value of Ki (and pKi) for the displacement of [3H]SCH58261 binding by each agonist and antagonist was calculated using the Cheng-Prusoff transformation.11 The significance of differences among mean pKi values (Table 3, Fig 4) was determined using two-way (for comparison across seven drug and three membrane preparation groups) ANOVA followed by Tukey's test (Sigma Stat 2.0, Jandel). In potency series, drugs are listed in order of affinity (from higher to lower values of pKi). The symbols ">" and "=" between two adjacent drugs indicate that the difference in their potencies was and was not statistically significant, respectively. For the significance of differences between the values of pKi for any two nonadjacent drugs in a potency series given in this study, refer to Table 4.
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P<.05 vs corresponding values for striatum and PC12 cells. 
P<.05 vs corresponding values for striatum and PC12 cells. P=NS vs striatum and PC12 cells.
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The potency of SCH58261 to antagonize CGS 21680 stimulation of cAMP formation in PC12 cells was estimated using Schild analysis. Dose-response ratios using EC50 values for CGS 21680 in the absence and presence of SCH58261 were calculated and used to a construct a Schild plot (Fig 5) and calculate Kb and pA2 (-log Kb) values. The EC50 values (concentrations of CGS 21680 that cause 50% of maximal response) were determined by fitting the experimental data with a multiparameter logistical equation using a nonlinear regression algorithm as described previously.12
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Results |
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Binding of [3H]SCH58261 (1 to 2 nmol/L) to membranes prepared from porcine coronary artery, porcine striatum, and PC12 cells was rapid, reached a maximum after 5 to 10 minutes of incubation at 21°C, and remained stable for at least 30 minutes (Fig 1
, top panels). Bound [3H]SCH58261 was promptly displaced upon the addition of a competing ligand; displacement was nearly complete 4 minutes after the addition of 50 µmol/L NECA (Fig 1, bottom panels). Both the association and dissociation data depicted in Fig 1 could be fit by monoexponential and biexponential curves; the biexponential model did not fit the data significantly better than the monoexponential model. Statistical analysis comparing single- and two-site models to describe KAssoc values of [3H]SCH58261 to coronary arteries, striatum, and PC12 cell membranes yielded P values that were .13, .56, and .83, respectively, whereas the P values for KDissoc were .17, .80, and .07, respectively. KAssoc and KDissoc for binding of [3H]SCH58261 are given in Table 1. The equilibrium dissociation constants (Kd values [KDissoc/KAssoc]) for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 2.42, 1.01, and 0.40 nmol/L, respectively.
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Binding of [3H]SCH58261 to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells was saturable and of high affinity. Results of equilibrium saturation binding experiments with [3H]SCH58261 are illustrated in Fig 2 and summarized in Table 2. The density of binding sites for [3H]SCH58261 was high (892 to 959 fmol/mg protein) in membranes prepared from all three sources (Table 2, Fig 2). Scatchard plots of the binding data were linear (Fig 2), consistent with the interpretation that a homogenous population of binding sites was present in each preparation. Kd values were 2.19 nmol/L and 1.20 nmol/L for [3H]SCH58261 binding to porcine coronary arteries and striatal membranes, respectively, and 0.81 nmol/L for binding to PC12 cell membranes (Table 2). The specific binding of [3H]SCH58261 was 
65% of total binding in all three preparations at a radioligand concentration equal to the Kd value (Table 2).
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Specific binding of [3H]SCH58261 to membranes prepared from porcine coronary artery, porcine striatum, and PC12 cells was reduced (displaced) in the presence of agonists (CGS 21680, HE-NECA, NECA, and R-PIA) and antagonists (SCH58261, CSC, and XAC) of adenosine receptors (Fig 3). Although displacement of [3H]SCH58261 binding by all adenosine receptor ligands was concentration dependent and complete (Fig 3), the ligand potencies were different (Tables 3 and 4 and Fig 4). The rank order of potency of the ligands to displace the specific binding of [3H]SCH58261 was as follows: for binding to membranes from coronary arteries, SCH58261>HE-NECA=CSC=CGS 21680=XAC>NECA=R-PIA; for binding to membranes from striatum, SCH58261>XAC=NECA=CGS 21680=HE-NECA=CSC>R-PIA; and for binding to membranes from PC12 cells, SCH58261>XAC=HE-NECA=CGS 21680=CSC=NECA>R-PIA. Analysis of the correlations among rank orders of ligand potencies by calculation of Spearman's rank correlation coefficients indicated that the rank orders for ligands to bind to pig coronary arterial and pig striatal membranes were not significantly correlated (r=.36; P=.39). Rank orders for ligands to bind to pig coronary arterial and PC12 cell membranes or for binding to pig striatal and PC12 cell membranes were, however, significantly correlated (r=.75 and .75, and P=.04 and .04, respectively). Comparison of the relative potencies of each ligand to compete with [3H]SCH58261 for binding sites in membranes from coronary arteries, striatum, and PC12 cells is depicted in Fig 4. The differences in pKi values of drugs to reduce binding of [3H]SCH58261 were to a significant degree (P<.001) dependent on the tissue source of the receptors. Except for SCH58261, CSC, and HE-NECA, all ligands had a significantly lower affinity for receptors in coronary artery membranes than for receptors in either brain striatal or PC12 cell membranes (Table 3 and Fig 4). When mean values of pKi for all drugs were analyzed across tissues by use of two-way ANOVA and Tukey's multiple comparison procedure, it was found that drug affinities for receptors in pig coronary arterial membranes were significantly different from drug affinities for receptors in either pig striatal or PC12 cell membranes.
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Antagonism by SCH58261 of the A2AAdoR-Mediated Increase in cAMP Accumulation in PC12 Cells
The potency of SCH58261 to antagonize the action of CGS 21680 to increase accumulation of cAMP in PC12 cells was determined. As illustrated in Fig 5, CGS 21680 stimulated cAMP accumulation in a concentration-dependent manner, with an EC50 value of 30 nmol/L and a maximal increase of 420-fold above a basal value of 2.1 pmol/mg per minute. This action of CGS 21680 to increase cellular cAMP content was competitively attenuated by SCH58261 (Fig 5). As the concentration of SCH58261 was increased from 10 to 20, 50, and 100 nmol/L, the relationship between CGS 21680 concentration and cAMP accumulation was progressively shifted to the right in a nearly parallel fashion (Fig 5A). Schild analysis of these data is illustrated in panel B of Fig 5. The slope of the Schild plot was near unity (-1.1±0.1), and the pA2 and Kb values were 8.55±0.12 and 2.8 nmol/L, respectively.
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Discussion |
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The major and novel finding of the present study is that the radiolabeled A2AAdoR antagonist [3H]SCH58261 identifies a large number (900 fmol/mg protein) of high-affinity homogeneous binding sites in porcine coronary artery membranes that appear by pharmacological criteria to be A2AAdoRs. The lack of available radioligands has until now hindered characterization of the A2AAdoR in arterial tissues. Thus, this newly developed A2AAdoR antagonist radioligand, which was recently shown to label A2AAdoRs in rat striatum8 and human platelets,13 represents a major contribution to the study of A2AAdoRs.
The results of equilibrium saturation binding assays revealed that [3H]SCH58261 binds to a single class of high-affinity (low Kd) and high-density (high Bmax) sites in crude membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells (Table 2). The specific binding of [3H]SCH58261 to membranes was rapid, saturable, and reversible. The Kd values obtained from the equilibrium saturation binding assays were in excellent agreement with those calculated from the results of kinetic experiments (compare Kd values in Tables 1 and 2). Also of note was the finding that the Kd and Bmax values for [3H]SCH58261 binding to coronary artery, striatum, and PC12 cell membranes were not significantly different from each other (Table 2). However, the nonspecific binding of [3H]SCH58261 to coronary artery membranes was significantly greater than the nonspecific binding of [3H]SCH58261 to membranes prepared from striatum or PC12 cells. Regardless, the specific binding of [3H]SCH58261 to porcine coronary artery membranes as a percentage of total binding (Table 2) is markedly higher than that recently reported for 125I-APE in the same membrane preparation5 (65% versus 25% to 30% at Kd) and similar to that reported for specific binding of [3H]SCH58261 to human platelet membranes.13 Furthermore, the nonspecific binding of [3H]SCH58261 to striatum and PC12 cells (Table 2) appears to be lower than that reported for other A2AAdoR antagonists in similar preparations.6 7 The specific binding of [3H]SCH58261 to crude membranes prepared from porcine striatum and PC12 cells was 85% and 90% of total binding at the Kd value, respectively, whereas the specific binding of [3H]KF 178375 and [125I]ZM 241385 to membranes prepared from rat and bovine striatum was 60% to 70% of total binding.6 7 Thus, with regard to the percentage of specific binding, [3H]SCH58261 appears to be superior to other currently available A2AAdoR antagonist radioligands. Of interest, no specific binding of [3H]SCH58261 to membrane preparations from porcine cortex and aorta was detected; ie, none of the [3H]SCH58261 bound to these membranes was displaced by 50 µmol/L NECA (unpublished data from our laboratory, 1996). This finding is consistent with the observation that SCH58261 does not antagonize NECA-induced relaxation of guinea pig aortic rings14 and that this radioligand selectively labels A2AAdoRs.8
Evidence in support of the conclusion that the [3H]SCH58261 binding site in coronary artery membranes is an A2AAdoR comes from the results of competition binding assays and functional studies with unlabeled SCH58261. As illustrated in Figs 3 and 4 and summarized in Tables 3 and 4, the agonist and antagonist affinities for [3H]SCH58261 binding sites are consistent with the identification of these sites as A2AAdoRs. CGS 21680 and HE-NECA, selective A2AAdoRs agonists, were significantly more potent than the A1AdoR agonist R-PIA to compete with [3H]SCH58261 for binding sites. Also, unlabeled SCH58261 was significantly more potent than the unselective AdoR antagonist XAC. The rank order of potency of ligands to compete with [3H]SCH58261 for binding to coronary artery membranes was, in part, different from the rank order of potency of ligands to compete for binding to striatum or PC12 cell membranes (Fig 3 and Table 4). Statistical analysis (see "Materials and Methods") revealed that the differences in pKi values for drugs to reduce binding of [3H]SCH58261 were to a significant (P<.001) degree dependent on the tissue source of receptors. Differences in the rank order of potencies of ligands to compete with [3H]SCH58261 for binding sites in these different tissues may reflect the existence of distinct subtypes of A2AAdoRs.
The results of the biochemical and functional studies of Zocchi et al14 revealed that SCH58261 does not interact with A2BAdoR or A3AdoR and is 53- and 100-fold selective for A2AAdoR versus A1AdoR in rat and bovine brains, respectively. SCH58261 antagonized the vasorelaxant effects of NECA or CGS 21680 on bovine and porcine coronary arteries14 15 and inhibited the effects of NECA or CGS 21680 on platelet aggregation.13
The results of radioligand binding studies were consistent with the results of functional assays of PC12 cell responses. SCH58261 attenuated CGS 21680–induced cAMP accumulation in PC12 cells in a concentration-dependent and competitive manner with a pA2 value of 8.55±0.12 (Fig 5) and competed with [3H]SCH58261 for binding to PC12 cell membranes with a pKi value of 9.17±0.09 (Fig 3 and Table 3). The greater potency of CGS 21680 to stimulate accumulation of cAMP in PC12 cells (EC50, 30 nmol/L; Fig 5) than to compete with [3H]SCH58261 for binding to PC12 cell membranes (Ki, 219 nmol/L; Fig 3 and Table 3) may indicate that the relationship between A2AAdoR occupancy and cAMP accumulation in PC12 cells is nonlinear, indicating that the A2AAdoR in this cell line has a high receptor coupling efficiency (that is, a large receptor reserve). Similar conclusions may also apply for A2AAdoRs of porcine coronary artery. The EC50 values for CGS 21680–induced relaxation of porcine coronary artery rings with or without endothelium are reported to be 34 to 60 nmol/L14 16 and 500 nmol/L,16 respectively, whereas the mean Ki value for CGS 21680 to displace binding of [3H]SCH58261 to membranes from porcine coronary artery is 1148 nmol/L (Fig 3 and Table 3).
The low potencies of the agonists NECA and CGS 21680 and of the antagonist XAC to compete with [3H]SCH58261 for binding to coronary artery membranes may in part explain why [3H]NECA, [3H]CGS 21680, and [3H]XAC do not label A2AAdoRs of coronary artery membranes2 3 4 but have been successfully used to label A2AAdoRs in membranes of other tissues.17 18 19 20 21 22 In keeping with this explanation is the finding that NECA, CGS 21680, and XAC are more potent to compete with [3H]SCH58261 for binding to membranes prepared from striatum or PC12 cells, respectively, than to compete for binding to membranes from porcine artery (Table 3).
Recently Hussain et al5 reported that the maximum specific binding (Bmax) of the agonist radioligand 125I-APE to porcine coronary artery membranes was 7 fmol/mg protein, a value 
128-fold less than the 900 fmol/mg protein of specific binding of [3H]SCH58261 to the same preparation (Table 2). One possible explanation for this difference is that 125I-APE is an agonist, whereas [3H]SCH58261 is an antagonist radioligand. Agonists are known to preferentially recognize the high-affinity state of the receptor, whereas antagonists can bind to both low- and high-affinity states of the receptor with equal affinity. Hence, the possibility exists that the majority of A2AAdoRs in membranes prepared from coronary arteries are in a low-affinity state for agonists and thus are poorly coupled to G proteins. Alternatively, a low density of high-affinity agonist binding sites may be an artifact of the method of membrane preparation or assay. The existence of 7 fmol of high-affinity agonist binding sites per milligram of coronary artery membranes5 would not be detectable in our experiments and would not significantly alter the Hill coefficients calculated from results of competitive binding assays using [3H]SCH58261. Zocchi et al8 and Dionisotti et al13 also found that the Hill coefficients for competitive displacement of [3H]SCH58261 binding to membranes of rat striatum and human platelets by several adenosine receptor agonists and antagonists were not significantly different from unity, which argues against the existence of multiple affinity states of the A2AAdoR in these preparations. In addition, GTP (100 µmol/L) did not affect either the potency or the Hill coefficients of A2AAdoR agonists to compete with [3H]SCH58261 for binding sites.8 On the other hand, investigators using other radioligands have obtained evidence in favor of the existence of more than one affinity state for the A2AAdoR in the striatum,21 23 24 and because A2AAdoRs are thought to belong to the G protein–coupled receptor superfamily, two affinity states for agonists are expected.
In summary, [3H]SCH58261 is the first A2AAdoR radioligand to be shown to detect sites in membranes from coronary arteries that exhibit a pharmacological profile consistent with that of A2AAdoRs. The significant selectivity8 9 14 and high affinity for A2AAdoRs, combined with low nonspecific binding, makes [3H]SCH58261 a suitable radioligand for pharmacological and biochemical characterization of A2AAdoRs expressed in various tissues.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors thank Kathleen Duvall and Peggy Ramsey for assistance in preparation of the manuscript.
Received May 7, 1996; accepted September 17, 1996.
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References |
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