Introduction

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Of the four subtypes of adenosine receptors (ARs), A₁, A_2A, A_2B, and A₃ (Linden et al., 1994), radioligand binding assays have been extensively used for all except the A_2BAR. Consequently, the pharmacological characterization of A_2BARs has been based primarily on functional assays of cAMP accumulation in tissue slices or cultured cells. A_2BARs have been functionally evaluated in NIH3T3 cells (Brackett and Daly, 1994) and demonstrated in aorta (Martin, 1992), chromaffin cells (Casado et al., 1992), astrocytes (Peakman and Hill, 1994), erythroleukemia cells (Strohmeier et al., 1995), astroglioma cells (Fredholm and Altiok, 1994), mast cells (Auchampach et al., 1997), intestinal smooth muscle (Burnstock, 1978), and intestinal epithelia (Strohmeier et al., 1995). On cloning of the rat A_2BAR cDNA, highest levels of transcript were found in large intestine, cecum, and urinary bladder (Rivkees and Reppert, 1992). We have noted previously that a low-specific-activity radioligand, [1,3-³H]diethyl-8-phenylxanthine (DPX) (Fig. 1), binds to a saturable site on HEK-A_2B membranes (Robeva et al., 1996a). Here we describe a binding assay improved by the generation of new HEK-A_2B cell line that expresses a high density of A_2BARs. A second improvement in radioligand binding to A_2BARs derives from the use of a high-specific-radioactivity radioligand, ¹²⁵I-3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine (¹²⁵I-ABOPX).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   The structure of some xanthine antagonists of A2B adenosine receptors that are referred to in the text.

Antipeptide antibodies raised against the A_2B adenosine receptor recognize a putative nonglycosylated receptor with a molecular mass of 57 kDa (Puffinbarger et al., 1995). This differs from the 33-kDa glycoprotein predicted by the A_2BAR receptor cDNA. Here we show that a purified, recombinant human A_2B adenosine receptor extended on the amino terminus with hexahistidine and the FLAG epitope (DYKDDDDK) (H/F-A_2B) is a glycoprotein with an apparent molecular mass of 34.8 kDa.

Adenosine stimulates bronchoconstriction and the degranulation of mast cells in people with asthma, but not in normal subjects (Cushley and Holgate, 1985). The A₃AR has been implicated as the receptor that is activated by adenosine to trigger degranulation of rodent perivascular mast cells (Jin et al., 1997) and rat RBL-2H3 mast-like cells (Ramkumar et al., 1993). However, A_2BARs appear to play a predominant role in the regulation of canine BR mast cell degranulation (Auchampach et al., 1997) and the slow release of interleukin-8 from human HMC-1 mast cells (Feoktistov and Biaggioni, 1995). It is curious, however, that A_2B receptors are known to couple to cAMP accumulation, because the activation of protein kinase A is thought to inhibit mast cell degranulation (Lohse et al., 1987; Jin et al., 1997). Here we present evidence that in addition to coupling to G_s, A_2B receptors couple to G_q/11 and calcium mobilization in HEK-A_2B cells and HMC-1 mast cells. The antiasthma drugs theophylline and enprofylline effectively block human A_2BARs and receptor-mediated mast cell degranulation at therapeutic concentrations.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Wizard Megaprep columns and competent JM109 cells were obtained from Promega Corp. (Madison WI); R-N⁶-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), CPA, pepstatin A, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, benzamidine, and forskolin were obtained from Sigma Chemical Corp. (St. Louis, MO). 2-[4-(2-Carboxyethyl)phenethylamino]-5N-N-ethylcarboxamidoadenosine (CGS21680), 2-chloroadenosine, DPX, and 8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine (XAC) were obtained from Research Biochemicals International (Natick, MA); adenosine deaminase was obtained from Boehringer Mannheim (Indianapolis, IN); lipofectin, G418, tissue culture media and serum, were obtained from Gibco BRL (Gaithersburg, MD). N⁶-Iodoaminobenzyladenosine (I-ABA), 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BW-A1433), 8-cyclopentyl-1,3-dipropylxanthine (CPX) and ¹²⁷I-ABOPX (BW-A522) were gifts from Dr. Susan Daluge of Glaxo Wellcome (Research Triangle Park, NC). Aminobenzyl-5'-N-ethylcarboxamidoadenosine (AB-NECA) was a gift from Dr. Ray Olsson of the University of South Florida (Tampa, FL); anti-FLAG m2 antibodies were obtained from Kodak IBI (New Haven, CT).

Stable Transfection of HEK 293 Cells. cDNA for human A_2BARs was prepared by polymerase chain reaction of human brain cDNA (Clonetech, Palo Alto, CA) and sequenced on both strands. cDNAs encoding human A₁, A_2A, and A₃ ARs were gifts of Marlene Jacobson (Merck & Co, West Point, PA). DNA sequencing was carried out in the University of Virginia Biomolecular Research Facility with an ABI Prism 377 Automated DNA Sequencer. The four wild-type human adenosine receptor cDNAs were subcloned into the expression plasmid CLDN10B. To prepare H/F-A_2B, the cDNA was subcloned into the pDoubleTrouble plasmid (Robeva et al., 1996b. The plasmids were amplified in competent JM109 cells and plasmid DNA isolated by using Wizard Megaprep columns (Promega Corporation, Madison, WI). Recombinant receptors were introduced into HEK 293 cells by lipofectin. Colonies were selected by growth of cells in 0.6 mg/ml G418. Stably transfected cells were maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/F12 medium) with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.3 mg/ml G418. G418 was omitted from the last passage before harvest.

Radioligand Binding. To prepare ¹²⁵I-ABOPX, 10 µl of 1 mM ABOPX in methanol/1 M NaOH (20:1) was added to 50 µl of 100 mM phosphate buffer, pH 7.3. One or 2 mCi of Na¹²⁵I were added, followed by 10 µl of 1 mg/ml chloramine T freshly prepared in water. After incubating for 20 min at room temperature, 50 µl of 10 mg/ml Na-metabisulfite in water was added to quench the reaction. The reaction products were applied to a C18 HPLC column and eluted for 5 min with 4 mM phosphate, pH 6.0/methanol (65:35). The methanol concentration was then ramped to 100% over 15 min. ABOPX elutes in 11 to 12 min and ¹²⁵I-ABOPX elutes at 18 to 19 min in a yield of 50 to 60% of the initial ¹²⁵I. For equilibrium binding assays, the specific activity of ¹²⁵I-ABOPX was diluted 10- to 20-fold by the addition of ¹²⁷I-ABOPX . Radioligand binding assays were conducted at 21°C for 2 to 3 h in 100 µl of buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, 20 µg membrane protein, and 1 U/ml adenosine deaminase. Nonspecific binding was measured in the presence of 10 µM 8(4-((2-aminoethyl)-aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine (XAC). Competition binding experiments were carried out with 0.5 to 0.6 nM ¹²⁵I-ABOPX. To detect bound radioligand, membranes were filtered over Whatman GF/C filters by using a Brandel cell harvester (Gaithersburg, MD) and washed 3 times over 15 to 20 s with ice-cold buffer (10 mM Tris, 1 mM MgCl_2, pH 7.4).It is important to maintain the wash buffer in an ice-slurry and to prime the filtration apparatus with ice-cold wash buffer to prevent dissociation of specific binding during the wash of glass fiber filters. B_max and K_D values were calculated by nonlinear least-squares interpolation to a single-site binding model.

Western Blots. Membranes expressing H/F-A_2BARs were solubilized in digitonin and purified by anti-FLAG affinity chromatography (Robeva et al., 1996b). Purified receptors were subjected to SDS polyacrylamide gel electrophoresis. In some instances, receptors were incubated with 0.5 U of N-glycosidase F for 18 h at 37°C. Electrophoresed receptors were transferred to Westran polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C in 10% milk Blotto (50 mM Tris, 80 mM NaCl, 20% milk, 0.2% Tween 20, pH 8). Blots were rinsed with Tris-buffered saline/Tween 20 (TBS/T) buffer (20 mM Tris, 137 mM NaCl, 0.3% Tween 20, pH 7.6), incubated with anti-FLAG antibody (5 µg/ml) in TBS/T containing 1 mg/ml BSA for 1 h at room temperature, washed in TBS/T, and incubated for 1 h with horseradish peroxidase-conjugated sheep antimouse IgG F(ab')₂ fragments diluted 1:10,000. After washing in TBS/T, blots were visualized by enhanced chemiluminescence. If untransfected HEK cells replaced HEK-A_2B cells, no detectable protein or immunoreactivity was eluted from anti-FLAG affinity columns.

cAMP Measurements. HEK-A_2B or Chinese hamster ovary (CHO)-K1 cells were grown to near confluence on 150-mm diameter plates. The cells were removed by replacing the medium with PBS containing 5 mM EDTA for 5 min. Cells were pelleted by centrifugation at 250g for 5 min, washed once in DMEM, and resuspended in DMEM supplemented with 1 U/ml adenosine deaminase and 10 mM HEPES, pH 7.2, resulting in a cell density of 250,000 cells/ml. The cells were equilibrated at ambient temperature for 1 h. To initiate cAMP accumulation, 50 µl of test compound was added to 200 µl of suspended cells and incubated for 10 min at 37°C in a shaking water bath. The cells were lysed by the addition of 500 µl of 0.15 N HCl. After centrifugation at 2,000g for 10 min, 500 µl of supernatant was removed, acetylated, and acetyl-cAMP measured by automated radioimmunoassay. The pA₂ of antagonists was determined by the method of Schild (Schild, 1957).

Inositol Trisphosphate (IP₃) Measurements. HEK-A_2B or HEK-A₃ cells were grown on 100-mm tissue culture plates. The medium was removed and the cells were washed once with inositol-free DMEM/F12 and incubated for 24 to 48 h with 2.5 µCi/ml [myo-³H]inositol in inositol-free DMEM/F12 plus 2% fetal calf serum. For treatment of cells with pertussis toxin, 200 ng/ml toxin was added for 18 h. After tritium labeling, the cells were washed and resuspended in HEPES-buffered DMEM (20 mM HEPES, pH 7.2) plus 1 U/ml adenosine deaminase and 100 mM LiCl and pipetted into test tubes (10⁶ cells/0.2 ml). Cells were maintained at 37°C for 30 min in a shaking water bath. Assays were terminated by the addition of 400 µl of stop solution (0.5 M HClO₄, 5 mM EDTA, and 1 mM diethylenetriaminepentaacetic acid) and 1 mg/ml phytic acid. The tubes were placed on ice for 30 min and 5 M K₂CO₃ was added to raise the pH to 8 to 9. Samples were centrifuged to remove the KClO₄ precipitate and the supernatants were collected and filtered through 0.45-µm Gelman Acrodisc filters and then applied to columns containing 1 ml of anion exchange resin (AG 1-X8, chloride form, 200 to 400 mesh; Bio-Rad Laboratories, Richmond, CA). Columns were washed with 5 ml of water, then 5 ml of 40 mM HCl. IP₃ fractions were eluted with 5 ml of 170 mM HCl and radioactivity counted by liquid scintillation counting.

Measurement of Intracellular Ca²⁺. Cells on tissue culture plates were loaded with 1 µM 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester in a buffer containing 100 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM KH₂PO_4, 25 mM NaHCO₃, 0.5 mM CaCl₂, 2.7 g/liter D-glucose, 20 mM Na-HEPES, pH 7.4, and 0.25% BSA for 45 min. Cells were washed, detached, and resuspended in the same buffer without BSA, plus 1 U/ml adenosine deaminase to a density of 10⁶ cells/ml. Fluorescence was monitored in an SLM 1100 spectrofluorometer in a thermostated stirred cuvette at 37°C at an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Western Blotting of H/F-A_2B Adenosine Receptors. To estimate the molecular mass of H/F-A_2BARs, receptors were solubilized in digitonin, purified over anti-FLAG affinity columns and detected by anti-FLAG western blotting. To determine whether the A_2BAR was a glycoprotein, some of the purified receptors were incubated with N-glycosidase F before electrophoresis. The results are illustrated in Fig. 2. The H/F-A_2BAR had an apparent molecular mass of 34.8 kDa reduced to 31.3 kDa after enzymatic deglycosylation. Some purified deglycosylated receptors seemed to form dimers with an apparent molecular mass of 65 kDa.The amount of dimerized receptors detected in purified receptor preparations increased with incubation time, receptor concentration, and temperature and may be an artifact of receptor purification, without physiological significance. The properties of the H/F-A_2BAR monomeric glycoprotein are consistent with its expected size and the existence of consensus sites for N-liked glycosylation, based on the receptor cDNA sequence.

View larger version (62K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of purified H/F-A2B adenosine receptors. Purified receptors (50 ng) were subjected to western blotting with anti-FLAG antibodies without () or with (+) prior incubation overnight at 37°C with N-glycosidase F (N-Gly-F). The apparent molecular masses of the glycosylated and deglycosylated receptors, respectively, are 34.8 and 31.3 kDa. The upper band in the deglycosylated lane has a molecular mass of 65 kDa.

Radioligand Binding Studies. Clonal lines of HEK-A_2B cells grown in G418 were screened for maximal ¹²⁵I-ABOPX binding. Figure 3 shows equilibrium binding of ¹²⁵I-ABOPX to membranes derived from the clone that was found to have the highest level of expression of the wild-type A_2BAR, with a B_max in excess of 20,000 fmol/mg protein. A single saturable binding site was detected in transfected, but not in untransfected cells. There is an endogenous A_2BAR that stimulates cAMP production in untransfected HEK 293 cells, but the density of this receptor is too low to be detected by radioligand binding with ¹²⁵I-ABOPX. The pharmacological properties of the ¹²⁵I-ABOPX binding site were characterized in competition binding studies with a series of 11 agonists and eight antagonists, as summarized in Table 1. The binding affinity of agonists was reduced by substitutions on either the N⁶- or the C2 position of adenine. NECA, which is unsubstituted on the N⁶ or the C2 position, binds to A_2BARs with more than 60-fold higher affinity than any of the other agonists tested.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Equilibrium binding of 125I/127I-ABOPX to membranes derived from HEK-A2B cells. A, Specific () and nonspecific equilibrium binding () were determined with isotope dilution as described in the methods. B, Scatchard transformation of specific binding. The Bmax value is 21,400 fmol/mg protein; the KD value is 37 nM. Each point is the mean ± S.E.M. of triplicate determinations. The results are typical of quadruplicate experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   A, competition by the enprofylline and theophylline for 125I-ABOPX binding to HEK-A2B membranes. Each tube contained 208,000 cpm (0.56 nM) 125I-ABOPX and 20 µg of membrane protein. Each point is the mean ± S.E.M. of triplicate determinations; where omitted, the S.E.M. is smaller than the symbols. KI values derived from triplicate experiments are summarized in Table 1. B, competition by CPX to inhibit NECA-stimulated cyclic AMP accumulation in CHO-A2B cells. The concentration of CPX (nm) is O (), 30 (), 150 (), and 750 (). The inset is a Schild plot, the pA2 is 7.66.

Schild Analyses of Theophylline and Enprofylline Effects. We next set out to estimate K_I values from pA2 values for enprofylline and theophylline as antagonists of NECA-stimulated cAMP accumulation in HEK-A_2B cells to determine whether this estimate of binding affinity agrees with K_I values derived by radioligand binding assays. As illustrated in Fig. 5, K_I values for theophylline and enprofylline are both 10 to 17 µM, in reasonably good agreement with the binding constants reported in Table 1. Enprofylline and theophylline caused an increase in the slope of NECA does response curves. It is notable that basal cAMP in HEK-A_2B cells was dose-dependently decreased by either theophylline or enprofylline from a level of about 20 pmol/ml to about 6 pmol/ml. This may be because of a small degree of constitutive activity by the overexpressed receptor, or the effects of endogenous adenosine released by the cells and not degraded by added adenosine deaminase. Inhibition of phosphodiesterase by these xanthines may also contribute to this effect on the slope of the NECA dose-response curve.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Schild analysis of theophylline and enprofylline to inhibit NECA-stimulated cAMP accumulation in HEK-A2B cells. A, competition by theophylline. B, competition by enprofylline. Each point is the mean ± S.E. of triplicate determinations. Basal levels for cAMP ranged from 3 to 20 pmol/ml; the maximum response to NECA was 240-270 pmol/ml. C, Schild analyses of the data derived from A and B; pA2 values for theophylline and enprofylline ranged from 10 to 17 µM in triplicate experiments.

Potency Order of Agonists in cAMP Assays. As a further validation of the new radioligand binding assay, we determined the potencies of three agonists in cAMP assays. The absolute potencies of all three agonists is greater in cAMP than in radioligand binding assays, but the ratio of affinities derived from functional and binding assays is similar. Thus, IB-MECA is 169- and 162-fold less potent than NECA in functional and binding assays, respectively. CGS21680 is 530- and 1090-fold less potent than NECA.

Coupling of A_2BARs to Phospholipase C. The activation of A_2BARs in HEK-A2B cells was found to increase the cellular content of IP₃. This response was not affected by pretreating the cells with pertussis toxin (Fig. 6A). In contrast, pertussis intoxication to inactivate G_i and G_o family G proteins abolished the ability of IB-MECA to stimulate phospholipase C in HEK-A₃ cells (Fig. 6B). The data are consistent with the idea that A₃ receptors are coupled to phospholipase C via the subunits of G_i/o G proteins, whereas A_2B receptors are coupled to phospholipase C via G_q/11. A dose-response curve for NECA to stimulate IP₃ in HEK-A_2B cells is shown in Fig. 6C. The ED₅₀ value for NECA, 19 nM, is close to its ED₅₀ to stimulate cAMP accumulation (29 nM; Fig. 7). We considered the possibility that there might be cross-talk between the cAMP and IP₃ pathways in HEK cells. However, as illustrated in Fig. 8, two activators of adenylyl cyclase, isoproterenol and forskolin, increase cAMP levels in HEK-A_2B cell and minimally influence IP₃ levels, whereas UTP increases IP₃ but not cAMP. The effects of UTP to stimulate phospholipase C or NECA to stimulate cAMP accumulation are not affected by pretreatment of cells with pertussis toxin (data not shown). The data are consistent with the interpretation that A_2BARs are dually coupled to G_s and to G_q/11.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Stimulation of phospholipase C by recombinant adenosine receptors. HEK-A2B (A) or HEK-A3 (B) cells were pretreated for 16 h without or with 100 ng/ml pertussis toxin (PTX) and then stimulated with NECA or IB-MECA. Control concentrations of [3H]IP3 were 500-600 cpm/tube. C, dose-response curve for NECA to stimulate [3H]IP3 accumulation in HEK-A2B cells. The ED50 value of NECA is 19 nM. Each bar or point is the mean ± S.E. of triplicate determinations. The results are typical of three to five experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Dose-response curves of agonists to stimulate cAMP accumulation in HEK-A2B cells. Each point is the mean ± S.E. of triplicate determinations. ED50 values are: NECA, 28.6 nM; IB-MECA, 4.8 µM; CGS21680, 15.2 µM. The results are typical of triplicate experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Stimulation of IP3 (A) and cAMP (B) accumulation by various compounds in HEK-A2B cells. Cells were treated with 1 µM isoproterenol (ISO), 10 µM forskolin (Forsk), 10 µM UTP, or 10 µM NECA, and cAMP and IP3 accumulation were determined as described in Experimental Procedures. Each bar is the mean ± S.E. of duplicate (IP3) or triplicate (cAMP) determinations. The results are typical of three to four replicate experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   NECA stimulation of calcium mobilization in HEK-A2B cells. Cells in suspension (106/ml) were stimulated with 1 µM NECA (arrows). A, control cells or cells pretreated with 200 ng/ml pertussis toxin (PTX) for 18 h. B, control cells or cells pretreated with enprofylline or theophylline for 5 min. The results are typical of triplicate experiments.

Coupling of A_2BARs to Calcium Mobilization in Human Mast Cells. Adenosine stimulates the degranulation of rodent and canine mast cells by activating A₃ and A_2B receptors, respectively (Ramkumar et al., 1993; Auchampach et al., 1997; Jin et al., 1997). A_2BARs are thought to be responsible for triggering interleukin-8 release in the human mast cell tumor line, HMC-1 (Feoktistov and Biaggioni, 1995). Figure 10 shows that calcium mobilization and cAMP accumulation in HMC-1 cells are stimulated with a potency order of NECA > IB-MECA. The response to NECA was not affected by pretreating HMC-1 cells with pertussis toxin. These data are consistent with the involvement of an A_2BAR in human mast cell calcium mobilization. However, we could not detect specific ¹²⁵I-ABOPX binding to HMC-1 cell membranes, which suggests that A_2B receptor density is too low to be detected with this radioligand.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Stimulation of calcium and cAMP accumulation in HMC-1 mast cells. A, calcium mobilization in response to NECA or IB-MECA added at the arrow. B, concentration response curves for cAMP accumulation. Each point is the mean ± S.E. of triplicate determinations. The results are typical of triplicate experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Physical Properties of A_2B Adenosine Receptors. In this study, we have characterized the physical, pharmacological, and coupling characteristics of recombinant human A_2BARs. Based on their cDNA sequences, the wild-type and H/F-A_2BARs are predicted to have molecular masses of 36 and 38.5 kDa, respectively. There are consensus sites for N-linked glycosylation on asparagines 153 and 163 in the second extracellular loop of the human receptor. The first site is conserved in the A_2B sequences of all species that have been cloned to date (Linden and Jacobson, 1998). In a previous study, Western blots conducted with antipeptide antibodies derived from the human A_2BAR sequence detected immunoreactivity in 50- to 55-kDa nonglycoproteins found in various human tissues (Puffinbarger et al., 1995). Hence, we considered the possibility that the antipeptide immunoreactivity might detect a cross-reacting epitope on a nonreceptor protein. To explore this possibility, the physical properties of the purified H/F-A_2BAR were investigated. Figure 1 indicates that the purified H/F-A_2BAR has characteristics close to those expected from the cDNA sequence (i.e., by western blotting with anti-FLAG antibodies it appears as a 35-kDa glycoprotein). These data imply that the immunoreactivity previously detected in tissues may not represent the A_2BAR.

¹²⁵I-ABOPX Binding to A_2BARs. ¹²⁵I-ABOPX is described here as a new and improved radioiodinated ligand for the detection recombinant A_2BARs. It is nonselective; hence, it cannot be used to detect A_2BAR on cells or tissues that possess multiple subtypes of adenosine receptors. It has an affinity (K_D = 37 nM) that is low enough to require isotope dilution to saturate binding sites. The radioligand cannot be used to detect low levels of endogenous receptors on untransfected HEK cells or HMC-1 mast cells. Nevertheless, the results indicate that ¹²⁵I-ABOPX binds with a high signal-to-noise ratio to recombinant A_2BARs on HEK-A_2B cells. When used in conjunction with a new HEK-A_2B cell line that has a high density of A_2BARs (in excess of 20,000 fmol/mg protein), specific binding of ¹²⁵I-ABOPX is >80% of total binding (Fig. 4). Hence this compound can used efficiently to screen competing ligands in competition radioligand binding assays.

Agonist Binding to A_2BARs. The results of competition binding assays generally confirm the assessment, based on functional data, that A_2BARs have a relatively low affinity for agonists (Daly et al., 1983). The affinity of agonists for the A_2BAR is reduced by the presence of bulky substituents on either the N⁶- or the C2-position of the adenine ring. Thus, N⁶-aminobenzyl-NECA binds with 65-fold lower affinity than NECA. The introduction of a relatively small chlorine atom at the C2-position has a minor effect on affinity, thus 2-Cl-CPA binds with only 1.17-fold lower affinity than CPA. However, CGS21680, which consists of NECA with a bulky 2-aralkyl substituent, binds with an affinity more than 1000 times lower than NECA. The data also are consistent with the previous observation that 2-phenylethoxy-9-methyladenine is a 2-substituted adenine-analog antagonist that discriminates well between A_2A receptors on coronary arteries and A_2B receptors on the aorta (Martin et al., 1993). The introduction of N-ethylcarboxamide on the 5'-position of the ribose ring of adenosine moderately enhances agonist affinity for A_2BARs inasmuch as the affinity of N⁶-aminobenzyl-5'-NECA (AB-NECA) is 5.8-fold higher than the affinity of the same compound lacing the 5' substitution, ABA.

Antagonist Binding to A_2BARs. K_I values for antagonists noted in this study generally agree with literature K_I estimates for human receptors based on functional assays (Brackett and Daly, 1994; Alexander et al., 1996; Cooper et al., 1997). We found in this study that A_2BARs have a high affinity for CPX and ZM241385. It is significant in this regard that CPX or ZM241385 are often used in the concentration range of 0.1 to 1 µM to selectively block A₁ or A_2A receptors, respectively. At 1 µM, either of these antagonists would occupy more than 90% of human A_2BARs. The K_I value of enprofylline for binding to A_2B receptors, 6 µM, agrees closely with functional estimates of K_I value based in inhibition of agonist-stimulated cAMP accumulation in NIH-3T3 cells (Brackett and Daly, 1994) and HEL cells (Feoktistov and Biaggioni, 1995). It was noted in 1982 that the K_I value of enprofylline needed to inhibit cAMP accumulation in rat hippocampal slices, approximate 7 µM, is 20 times more potent than the K_I for binding to rat A₁ receptors. Hence, it seems likely that the receptor responsible for cAMP accumulation in hippocampal slices is A_2B.

Coupling Characteristics of A_2BARs. We demonstrate in this study that the over-expressed recombinant human A_2BARs in HEK 293 cells couple not only to cAMP accumulation, but also to phospholipase C activation and calcium mobilization. It is possible that the coupling of recombinant receptors to one or both of these signaling pathways could be an artifact of receptor over-expression, resulting in indiscriminate coupling of recombinant receptors to various HEK cell G proteins. However, there is an endogenous A_2BAR on HEK 293 cells (Townsend-Nicholson, 1997) that also couples to the accumulation of cAMP and calcium as well as to the activation of the mitogen-activated protein kinase isoform extracellular-signal regulated kinase (Gao et al., 1999). We provide evidence here that calcium mobilization in response to NECA in HEK-A_2B cells is not a result of cAMP accumulation (Fig. 8); rather, it is probably caused by the direct coupling of A_2BARs to G_q/11.

A_2BARs and Mast Cell Function. The A_2BAR has been implicated as the adenosine receptor that is responsible for stimulating the degranulation of canine BR mastocytoma cells (Auchampach et al., 1997) and for triggering the slow release of interleukin-8 from human HMC-1 mast cells (Feoktistov and Biaggioni, 1995). In this study, we show that A_2BARs also trigger a rapid mobilization of calcium in HMC-1 cells. The agonist potency order (NECA > IB-MECA) is consistent with calcium mobilization in HMC-1 cells being mediated by an A_2BAR. It is notable that a different AR subtype, the A₃AR, mediates the degranulation of hamster perivascular mast cells (Shepherd et al., 1996; Jin et al., 1997) and rat RBL-2H3 mast-like cells (Ramkumar et al., 1993). It is calcium mobilization rather than cAMP accumulation that is likely to trigger mast cell degranulation in response to A_2BAR activation, because cAMP seems to inhibit mast cell degranulation (Church and Hughes, 1985; Hughes and Church, 1986; Hughes et al., 1987; Jin et al., 1997).

Xanthines in the Treatment of Asthma. We found that enprofylline and theophylline block human A_2BARs with K_I values in the range of 6 to 8 µM. Optimal plasma levels of theses compounds for the treatment of asthma have been reported to be 4 µg/ml (19 µM) for enprofylline, and 10 µg/ml (55 µM) for theophylline (Vilsvik et al., 1990). Both enprofylline and theophylline have lower affinities for the A₃AR than for the other human adenosine receptor subtypes. Theophylline is nonselective among A₁, A_2A, and A_2B subtypes, whereas enprofylline is somewhat selective for the A_2BAR subtype (Table 2). Blockade of A_2AARs by theophylline may be counterproductive in the treatment of asthma because activation of A_2AARs has inhibitory effects on mast cells and several other types of inflammatory cells (Sullivan and Linden, 1998). Adverse side effects of theophylline that are not shared by enprofylline may be mediated principally by A₁AR blockade. These include diuresis, free fatty acid release, gastric secretion, and central nervous system stimulation (Persson, 1986).