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Department of Clinical and Experimental Medicine, Pharmacology Unit (S.G., K.V., S.M., E.C., C.P., P.A.B.), Department of Diagnostic and Experimental Medicine, General Pathology Unit (R.R., Y.S.)
Department of Pharmaceutical Sciences (P.G.B.), University of Ferrara, Italy
Centro Nazionale di Eccellenza per lo Sviluppo di Metodologie Innovative per lo Studio ed il Trattamento delle Patologie Infiammatorie, Ferrara, Italy (S.G., K.V., S.M., E.C., C.P., P.A.B., R.R., Y.S.)
Institut fe Pharmakologie, Universitat Wezburg, Wezburg, Germany (K.N.K.)
King Pharmaceutical, Cary, North Carolina (E.L., S.M.)
Abstract
In this study, we compared the pharmacological and biochemical characteristics of A2B adenosine receptors in recombinant (hA2BHEK293 cells) and native cells (neutrophils, lymphocytes) by using a new potent 8-pyrazole xanthine derivative, [3H]N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide] ([3H]MRE 2029-F20), that has high affinity and selectivity for hA2B versus hA1,hA2A, and hA3 subtypes. [3H]MRE 2029-F20 bound specifically to the hA2B receptor stably transfected in human embryonic kidney (HEK) 293 cells with KD of 2.8 ± 0.2 nM and Bmax of 450 ± 42 fmol/mg of protein. Saturation experiments of [3H]MRE 2029-F20 binding in human neutrophils and lymphocytes detected a single high-affinity binding site with KD values of 2.4 ± 0.5 and 2.7 ± 0.7 nM, respectively, and Bmax values of 79 ± 10 and 54 ± 8 fmol/mg of protein, respectively, in agreement with real-time reverse transcription polymerase chain reaction studies showing the presence of A2B mRNA. The rank order of potency of typical adenosine ligands with recombinant hA2B receptors was consistent with that typically found for interactions with the A2B subtype and was also similar in peripheral blood cells. 5'-N-Ethyl-carboxamidoadenosine stimulated cAMP accumulation in both hA2BHEK293 and native cells, whereas phospholipase C activation was observed in recombinant receptors and endogenous subtypes expressed in neutrophils but not in lymphocytes. MRE 2029-F20 was revealed to be a potent antagonist in counteracting the agonist effect in both signal transduction pathways. In conclusion, [3H]MRE 2029-F20 is a selective and high-affinity radioligand for the hA2B adenosine subtype and may be used to quantify A2B endogenous receptors. In this work, we demonstrated their presence and functional coupling in neutrophils and lymphocytes that play a role in inflammatory processes in which A2B receptors may be involved.
Adenosine is a ubiquitous modulator that exerts its physiological functions through the interaction with four G protein-coupled receptors classified as A1, A2A, A2B, and A3 (Fredholm et al., 2001; Fredholm, 2003). In particular, A2B receptors are associated with stimulation of the adenylate cyclase and activation of phospholipase C through the coupling to Gs and Gq/11 proteins, respectively. A2B receptors have been found on practically every cell in most species. RT-PCR studies revealed their highest expression in cecum, large intestine, and urinary bladder, but lower levels in brain, spinal cord, lung, and vas deferens (Rivkees and Reppert, 1992). However, besides regulation at the level of gene expression, targeting of the receptor protein to specific cells or tissues is crucial in understanding their role in pathophysiological conditions. The lack of selective pharmacological tools has hampered research in this field; in particular, the lack of selective A2B receptor agonists has undoubtedly contributed to the general lack of information of their physiological functions. The characterization of A2B receptors, therefore, often relies on the lack of effectiveness of compounds that are potent and selective agonists of other receptor subtypes. The agonist CGS 21680, for example, has been useful in differentiating between A2A and A2B receptors (Feoktistov and Biaggioni, 1995; Fiebich et al., 1996, 1997). However, pharmacological characterization of receptors based on apparent agonist potencies is far from ideal, because it depends not only on agonist binding to the receptor but also on multiple processes involved in signal transduction (Feoktistov et al., 2001). Based on functional assays using novel A2B selective antagonists, significant progress has been made in the understanding of the molecular pharmacology and physiology of A2B adenosine receptors. They seem to be implicated in mast-cell secretion (Feoktistov et al., 2001), coronary flow regulation (Talukder et al., 2003), neoangiogenesis (Grant et al., 2001; Feoktistov et al., 2002, 2003; Afzal et al., 2003), cytokine release by bronchial smooth muscle cells (Zhong et al., 2004), and nociception (Abo-Salem et al., 2004), suggesting a possible role of the A2B subtype in the modulation of inflammatory processes involved in asthma, tumor growth, tissue injury, ischemia, and pain (Feoktistov et al., 2003; Livingston et al., 2004).
The characterization of A2B receptors through radioligand binding studies has been performed, until now, by using low-affinity and nonselective antagonists, such as [3H]-DPCPX, [3H]ZM 241385, and 3-(3-[125I]iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine, that, as a consequence of their low affinity, display a rapid dissociation rate from the receptor. In addition, because these ligands are nonselective, their utility in native systems is hampered because many tissues and cell lines express several adenosine subtypes. Based on these considerations, it is evident that, to obtain more information about the physiological role of A2B receptors, new radiolabeled compounds with high affinity and selectivity should be synthesized. High-affinity radioligands for A2B receptors, [3H]MRS 1754, and [3H]-OSIP339391 have been introduced (Ji et al., 2001; Stewart et al., 2004), but they were not commercially available at the time of this study. Our group has identified a series of 8-pyrazole xanthine derivatives as potent and selective human A2B adenosine antagonists (Baraldi et al., 2004a,b), and a radiolabeled form of one compound of this series was used as a new pharmacological tool to describe the comparison between human recombinant A2B receptors stably transfected in HEK 293 cells (hA2BHEK 293 cells) and endogenous receptors present in neutrophils and lymphocytes, which represent inflammatory cells potentially involved in the exacerbation of asthma and other inflammatory processes in which A2B receptors are thought to be involved. In this study, we demonstrated that A2B receptors are strongly linked to adenylyl cyclase in both transfected and native cells, whereas for phospholipase C activity, they are well coupled in transfected cells, less in neutrophils, and not at all in lymphocytes, where the A2B expression is quite low.
Materials and Methods
Materials. [3H]MRE 2029-F20 (specific activity, 123 Ci/mmol) and [3H]MRE 3008-F20 (specific activity, 67 Ci/mmol) were synthesized at Amersham Biosciences (Little Chalfont, Buckinghamshire, UK); [3H]ZM 241385 (specific activity, 20 Ci/mmol) was purchased from Tocris (Boston, MA). [3H]DPCPX (specific activity, 120 Ci/mmol) and [3H]Ins(1,4,5)P3 (specific activity, 21 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). CHA, NECA, (R)-PIA, (S)-PIA, CGS 21680, IB-MECA, CGS 15943, and DPCPX were obtained from Sigma/RBI (Natick, MA). MRS 1754 was obtained from Sigma-Aldrich (Milano, Italy). SCH 58261, MRE 3008-F20, MRE 2029-F20, AS 16, and AS 100 were synthesized by Prof. P. G. Baraldi (Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy). All other reagents were of analytical grade and obtained from commercial sources.
Synthesis of [3H]MRE 2029-F20. The synthesis of [3H]MRE 2029-F20 was prepared through custom synthesis at Amersham Bioscience from tritium gas by a method developed by Nycomed Amersham plc. The product was purified by reversed-phase high-performance liquid chromatography using an acetonitrile/trifluoroacetic acid gradient (Baraldi et al., 2004a).
Stable Transfection of HEK 293 Cells. cDNA encoding human A2B receptors was a gift of Prof. Karl-Norbert Klotz (Institut fe Pharmakologie und Toxikologie, Universitat Wezburg, Wezburg, Germany) and subcloned into the expression plasmid pcDNA 3.1 (Invitrogen). The plasmid was amplified into a competent Escherichia coli strain and plasmid DNA isolated by using QIAGEN Maxiprep columns (plasmid purification kit; QIAGEN, Valencia, CA). cDNA was then sequenced on both strands in the University of Padova, DNA Sequence Service CRIBI, and transfected into HEK 293 cells by using the calcium phosphate precipitation method (Chen and Okayama, 1987). Colonies were selected by growth of cells on 0.8 mg/ml G-418. Stably transfected cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Dulbecco's modified Eagle's medium/F-12 medium) with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.3 mg/ml G-418, at 37° in 5% CO2/95% air.
Membrane Preparation. For membrane preparation, the culture medium was removed. The cells were washed with phosphate-buffered saline and scraped off T75 flasks in ice-cold hypotonic buffer (5 mM Tris HCl and 2 mM EDTA, pH 7.4). The cell suspension was homogenized using a Polytron homogenizer, and the homogenate was spun for 30 min at 36,000g. The membrane pellet was resuspended in 50 mM Tris-HCl buffer, containing 10 mM MgCl2, 1 mM EDTA, and 0.1 mM benzamidine, pH 7.4, and incubated with 2 IU/ml adenosine deaminase for 30 min at 37°C. The protein concentration was determined according to a Bio-Rad method (Bradford, 1976) with bovine serum albumin as reference standard.
Peripheral Blood Cells Isolation. Lymphocytes and neutrophils were isolated from buffy coats kindly provided by the Blood Bank of the University Hospital of Ferrara, according to Gessi et al. (2002, 2004). In brief, the blood was centrifuged on Ficoll-Hypaque density gradients. The human peripheral blood mononuclear cells were isolated and removed from the Ficoll-Hypaque gradients. Thereafter, they were washed in 0.02 M phosphate-buffered saline at pH 7.2. Further purification of lymphocytes from peripheral blood mononuclear cells was performed by adhesion of monocytes to plastic plates for 2 h at 37°C. To obtain neutrophils, the lower phase of Ficoll-Hypaque gradients was washed and supplemented with 20 ml of 6% Dextran T500. After gentle mixing, erythrocytes were allowed to settle at 20°C for 60 min. The remaining erythrocytes were lysed by suspending the cell pellet in 10 ml of distilled water at 4°C under gentle agitation. After 30 s, isotonicity was restored by adding 3 ml of a solution containing 0.6 M NaCl. Neutrophils were sedimented by centrifugation at 20°C for 20 min at 250g. This procedure resulted in approximately 95% neutrophils, and the cell viability was more than 95% as detected by trypan blue exclusion test. Rat neutrophils and lymphocytes were isolated from male Sprague-Dawley rats (300eC350 g; Stefano Morini, Reggio Emilia, Italy). Blood samples from the inferior vena cava were collected in an EDTA-anticoagulated tube, diluted in phosphate-buffered saline, and isolated by density gradient centrifugation (Ficoll-Hypaque) as described above for human blood cells. Membrane preparations were performed essentially according to Gessi et al. (2002; 2004).
Real-Time RT-PCR Experiments. Total cytoplasmic RNA was extracted from HEK 293 and peripheral blood cells by the acid-guanidinium-thiocyanate-phenol method (Chomczynski and Sacchi, 1987). Quantitative real-time RT-PCR assay (Higuchi et al., 1993) of A2B mRNA transcript was carried out using gene-specific double fluorescently labeled TaqMan MGB probe (minor groove binder) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington Cheshire, UK). For the real-time RT-PCR of the A2B gene, the assays-on-demand gene expression product (GenBank accession no. NM_0006756) was used with the fluorescent reporter 6-carboxy fluorescein and the quencher 6-carboxy-N,N,N',N'-tetramethylrhodamine. For the real-time RT-PCR of the reference gene, the endogenous control human -actin kits was used, and the probe was fluorescence-labeled with VIC (Applied Biosystems, Monza, Italy). Moreover, a curve of A2B cDNA plasmid standards with a range spanning at least 6 orders of magnitude (10eC11eC10eC16 g/e蘬) was generated. This standard curve displayed a linear relationship between Ct values and the logarithm of plasmid amount. Therefore, quantification of A2B message in blood cells was made by interpolation from standard curve of Ct values generated from the plasmid dilution series.
Binding Assays to Human Cloned Adenosine Receptors. Binding assays to human cloned A1, A2A, and A3 receptors were performed at 4°C using [3H]DPCPX, [3H]ZM 241385, and [3H]MRE 3008-F20, respectively, as described by Varani et al. (2000).
[3H]MRE 2029-F20 Binding Assays. Kinetic studies of [3H]MRE 2029-F20 (2.5 nM) were performed incubating membranes obtained by HEK 293 cells transfected with the human A2B receptors in a thermostatic bath at 4°C. A total assay volume of 250 e蘬 was employed in which the final protein level was 70 e蘥 per well. For the measurement of the association rate, the reaction was terminated at different times (from 5 to 180 min) by rapid filtration under vacuum, followed by washing with 5 ml of ice-cold buffer four times. For the measurement of dissociation rate, the samples were incubated at 4° for 90 min, and then 1 e MRE 2029-F20 was added to the mixture. The reaction was terminated at different times from 5 to 100 min. Saturation binding experiments of [3H]MRE 2029-F20 (0.3 to 30 nM) to hA2B HEK293 cell membranes were performed by incubation for 90 min at 4°C. Competition experiments of 3 nM [3H]MRE 2029-F20 were performed in duplicate in a final volume of 250 e蘬 in test tubes containing 50 mM Tris HCl buffer, 10 mM MgCl2, 1 mM EDTA, and 0.1 mM benzamidine, pH 7.4, 100 e蘬 of membranes, and at least 12 to 14 different concentrations of typical adenosine receptor agonists and antagonists. Nonspecific binding was defined as binding in the presence of 1 e MRE 2029-F20 and, at the KD value of the radioligand, was approximately 30 to 35% and 40 to 45% of total binding in HEK 293 cells and in blood cells, respectively. Similar results were obtained in the presence of 10 e DPCPX and 100 e NECA. Bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/B glass-fiber filters using a Micro-Mate 196 cell harvester (PerkinElmer Life and Analytical Sciences). The filter bound radioactivity was counted with a Top Count microplate scintillation counter (efficiency, 57%) with MicroScint 20. Ki values were calculated from IC50 values according to the Cheng and Prusoff equation, Ki = IC50/(1 + [C]/KD), where [C] is the concentration of the radioligand and KD is its dissociation constant. A weighted nonlinear least-squares, curve-fitting program LIGAND (Munson and Rodbard, 1980) was used for computer analysis of saturation and inhibition experiments.
Cyclic AMP Assay. hA2BHEK-293 and peripheral blood cells were suspended in 0.5 ml of Krebs-Ringer phosphate buffer (KRPG) (136 mM NaCl, 5 mM KCl, 0.67 mM Na2HPO4, 0.2 mM KH2PO4, 3 mM NaHCO3, 1 mM CaCl2, 5 mM glucose, 5 mM HEPES, 10 mM MgCl2, and 2.0 IU/ml adenosine deaminase, pH 7.4) containing 0.5 mM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as phosphodiesterase inhibitor, and preincubated for 10 min in a shaking bath at 37°C. Then the nonselective adenosine agonist NECA was added to the mixture, and the incubation continued for a further 10 min. The reaction was terminated by the addition of ice-cold 6% trichloroacetic acid. The trichloroacetic acid suspension was centrifuged at 2000g for 10 min at 4°C, and the supernatant was extracted four times with water-saturated diethyl ether. The final aqueous solution was tested for cyclic AMP levels by a competition binding assay carried out essentially according to Varani et al. (1997). Samples of cyclic AMP standards (0eC10 pmol) were added to each test tube containing the incubation buffer (0.1 M Trizma base, 8.0 mM aminophylline, and 6.0 mM 2-mercaptoetanol, pH 7.4) and [3H]cyclic AMP in a total volume of 0.5 ml. The binding protein, previously prepared from beef adrenal glands, was added to the samples and incubated at 4° for 150 min. After the addition of charcoal, samples were centrifuged at 2000g for 10 min. The clear supernatant (0.2 ml) was mixed with 4 ml of Atomlight and counted in a LS-1800 Beckman scintillation counter.
Ins(1,4,5)P3 Binding Assay. IP3 generation in hA2BHEK 293 and peripheral blood cells was measured by [3H]Ins(1,4,5)P3 competition assay to IP3 binding protein according to the method described by Challiss et al. (1990). hA2BHEK 293 cells (2 x 106 cells in each tube) and peripheral blood cells (8 x 106 cells in each tube) were suspended in KRPG buffer and stimulated with agonists at 37°C. The reaction was terminated by addition of 0.5 M trichloroacetic acid. Acidified samples were left on ice for 15 min, centrifuged at 3000g for 15 min at 4°C and trichloroacetic acid was extracted with five 2-vol washes with water-saturated diethyl ether. Finally, 125 e蘬 of 30 mM EDTA and 125 e蘬 of 60 mM NaHCO3 were added to 500 e蘬 of cell extract, and samples were taken for analysis. Buffer samples were also taken through the acidification/extraction protocol to provide diluent for the Ins(1,4,5)P3 assay standard curve. A 30-e蘬 portion of sample or of trichloroacetic acid-extracted buffer containing standard amounts of Ins(1,4,5)P3 (0.12eC12 pmol) or DL-Ins(1,4,5)P3 (0.3 nmol to define nonspecific binding) was added to 30 e蘬 of assay buffer (25 mM Tris HCl and 1 mM EDTA, pH 8) and 30 e蘬 of [3H]Ins(1,4,5)P3 (7000 dpm/assay). Then, 30 e蘬 (0.4 mg of protein) of the adrenal-cortical binding protein preparation was added and incubation continued for 30 min. Bound and free [3H]Ins(1,4,5)P3 were separated by rapid filtration through Whatman GF/B glass fiber filters with four 3-ml washes of ice-cold 25 mM Tris HCl, 1 mM EDTA, and 5 mM NaHCO3, pH 8. Scintillator was added to the filter discs, and radioactivity was determined after a 12-h extraction period by scintillation counting.
Measurement of Cytosolic Ca2+ Concentration. [Ca2+]i was evaluated by incubating hA2BHEK 293 cells with the Ca2+-sensitive fluorescent dye Fura 2-acetoxymethyl ester (5 e) in KRPG buffer for 45 min at 37°C. Alternate excitation at 340 nM and 380 nM was supplied, and the F340/F380 emission ratio was recorded with a dynamic image analysis system (Laboratory Automation 2.0; RCS, Florence, Italy). For calcium measurements in human lymphocytes and neutrophils, cells were loaded with fura 2-acetoxymethyl ester (2 e) in KRPG buffer, for 30 min at 37°C, according to Gessi et al. (2002). The cells were centrifuged at 1000g for 10 min to remove extracellular dye and were resuspended in KRPG solution at 4 x 106 cells/ml. Fluorescence was monitored with a LS50 spectrofluorometer (PerkinElmer Life and Analytical Sciences), at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm, in cuvettes thermostatically controlled at 37°C and continuously stirred.
Results
Real-Time RT-PCR Experiments. HEK 293 cells are recognized as cells expressing native A2B receptors. Therefore, the mRNA presence of this adenosine subtype was investigated in both wild-type and hA2BHEK 293 cells. Transfection of A2B receptors in HEK 293 cells produced a 350 ± 30-fold increase of A2B mRNA accumulation with respect to the corresponding wild-type cells; the expression level of A2B receptors was normalized to the expression level of the endogenous reference (-actin) in each sample (Fig. 1, A and B). In addition, the mRNA level of A2B receptors was investigated in primary cells in which this adenosine subtype is supposed to play an important regulatory role, such as peripheral blood cells. The amount of product was expressed as the ratio of mRNA (determined by interpolation from standard curve of Ct values generated from the plasmid dilution series) and total RNA and indicated the following rank order: monocytes < lymphocytes neutrophils, with monocytes expressing approximately 4-fold less mRNA compared with the other blood cells (Student's t test; , P < 0.01 versus monocytes) (Fig. 1C).
Radioligand Binding Studies in Recombinant and Native Cells. Fig. 2A shows that [3H]MRE 2029-F20 binding to hA2BHEK 293 cells reached equilibrium after approximately 45 min and was stable for at least 3 h. [3H]MRE 2029-F20 binding was rapidly reversed by the addition of 1 e MRE 2029-F20 as shown in Fig. 2B. A one-component model fit association and dissociation curves significantly better than a two-component model (P < 0.05). The rate constants were: kobs = 0.046 ± 0.002 mineC1 and keC1 = 0.025 ± 0.003 mineC1. From these values, a kinetic dissociation constant (KD) of 2.98 nM was calculated. Figure 3 shows a saturation curve of [3H]MRE 2029-F20 to adenosine A2B receptor, and the linearity of the Scatchard plot in the inset is indicative, in our experimental conditions, of the presence of a single class of binding sites with a KD value of 2.8 ± 0.2 nM and a Bmax value of 450 ± 42 fmol/mg of protein. In contrast to the presence of mRNA message, no specific binding of [3H]MRE 2029-F20 was detectable in HEK 293 wild-type cells (data not shown). Table 1 shows the affinities, expressed as Ki values, of selected adenosine receptor agonists and antagonists to hA2B receptors expressed in HEK 293 cells using [3H]MRE 2029-F20. The order of potency in [3H]MRE 2029-F20 displacement assays for adenosine receptor agonists was NECA > (R)-PIA > CHA > IB-MECA > (S)-PIA > CGS 21680 (Fig. 4A). The order of potency of the receptor antagonists was: MRE 2029-F20 > MRS 1754 > AS 100 > ZM 241385 > CGS 15943 > AS 16 > DPCPX > enprofylline > theophylline (Fig. 4B). The selectivity of MRE 2029-F20 for the human A2B over A1, A2A, and A3 receptors was evaluated in radioligand binding assays by using [3H]DPCPX, [3H]ZM 241385, and [3H]MRE 3008-F20, respectively. MRE 2029-F20 displays low affinity for the human A1 receptor (Ki = 245 ± 31 nM) and no significant affinity for the human A2A and A3 subtypes (Ki > 1000 nM). This indicates that MRE 2029-F20 is 88-fold selective for A2B over A1 receptors and more than 300-fold selective for A2B over A2A and A3 subtypes, which means a range of selectivity similar to [3H]MRS 1754 (selectivity of 210-, 260-, and 290-fold for A2B over A1, A2A, and A3 subtypes, respectively) and better than [3H]OSIP339391 (selectivity of 70-fold for A2B over A1, A2A, and A3 subtypes) (Ji et al., 2001; Stewart et al., 2004). Saturation experiments of [3H]MRE 2029-F20 binding performed at 21 and 37°C revealed KD values of 3.5 ± 0.4 and 6.5 ± 0.7 nM and Bmax values of 460 ± 50 and 430 ± 48 fmol/mg of protein, respectively, thus suggesting that dissociation constants increased with temperature, whereas Bmax data were largely independent of it. This binding behavior has previously been found to be typical of adenosine A1, A2A, and A3 subtypes (Borea et al., 1996; Varani et al., 2000). To investigate whether MRE 2029-F20 would be a useful tool to detect A2B receptors in primary cells, we isolated monocytes, lymphocytes, and neutrophils from peripheral blood. As for human monocytes, it was not possible to detect A2B receptors, even though the mRNA was present, suggesting that possibly in these cells their level was extremely low. On the other hand, in agreement with real-time RT-PCR experiments, neutrophils and lymphocytes both express A2B subtype with the following binding parameters: KD of 2.4 ± 0.5 and 2.7 ± 0.7 nM and Bmax of 79 ± 10 and 54 ± 8 fmol/mg of protein, respectively (Fig. 5, A and B). The pharmacological profile of selected adenosine agonists and antagonists obtained from inhibition binding experiments in peripheral blood cells revealed a rank order of potency strictly similar to that obtained in transfected cells: NECA > (R)-PIA > CHA> IB-MECA > (S)-PIA >CGS 21680 and MRE 2029-F20 > MRS 1754 > AS100 > ZM 241385 > AS16 > DPCPX > enprofylline > theophylline (Table 1). Specific binding of [3H]MRS 2029-F20 (55eC60%) was also detected in rat neutrophils and lymphocytes.
Results are presented as the mean ± S.E.M. of four independent experiments.
Evaluation of cAMP Levels. To investigate the functional coupling of native and recombinant A2B receptors to adenylyl cyclase, we evaluated the stimulation of cAMP production in hA2BHEK 293 cells and in peripheral blood cells. Our results show that in hA2BHEK 293 cells, NECA showed a potency greater in cAMP (EC50 value of 4.5 ± 0.4 nM) than in radioligand binding studies (Ki value of 262 ± 30 nM) (Fig. 6A). We were surprised to find that this effect was not potently antagonized by MRE 2029-F20 that showed an IC50 value of 150 ± 18 nM. Because of the low potency of MRE 2029-F20 in inhibiting cAMP accumulation induced by NECA, we suspected the presence of basal levels of A2A receptors in addition to A2B in wild-type HEK 293 cells. This was assessed by real-time RT-PCR experiments revealing a similar amount of A2A and A2B mRNA expression (data not shown) and confirmed by saturation binding experiments of [3H]ZM 241385 binding (0.1eC10 nM) showing an affinity of 1.04 ± 0.2 nM and a density of 35 ± 4 fmol/mg of protein. This value was possibly not caused by a contaminating presence of endogenous A2B receptors, as demonstrated by the lack of [3H]MRE 2029-F20 specific binding in untransfected HEK 293 cells. CGS 21680, an A2A-selective agonist, stimulated cAMP levels with an EC50 of 410 ± 52 nM, but it was less efficacious than NECA. The unstimulated level of cAMP was 15 ± 3 pmol/106cells; at concentrations producing maximal effects (10 e), CGS 21680 induced a 4-fold increase, whereas NECA caused an 8-fold increase in cAMP (to 60 ± 8 and 120 ± 15 pmol/106cells, respectively), suggesting the activation, in the first case, of only A2A receptors and, in the second, of both A2A and A2B subtypes (Fig. 6A). Therefore to evaluate the potency of MRE 2029-F20 at A2B receptors, a Schild analysis of this compound was performed in the presence of the A2A blocker, SCH 58261 (100 nM). The pA2 value of MRE 2029-F20 was 7.81 ± 0.10 nM, in agreement with its affinity in binding experiments performed at 37°C (KD = 6.5 ± 0.7 nM) (Fig. 6B). In human neutrophils and lymphocytes expressing different adenosine subtypes, the nonselective agonist NECA, through the activation of both A2A and A2B receptors, showed EC50 values of 147 ± 16 and 220 ± 20 nM, respectively. The unstimulated levels of cAMP were 20 ± 5 and 16 ± 4 pmol/106cells and were increased by NECA stimulation to 80 ± 15 and 60 ± 9 in neutrophils and lymphocytes, respectively. To distinguish the role played by the two stimulatory adenosine subtypes, we repeated the concentration-response curve of NECA in the presence of 100 nM MRE 2029-F20 or 100 nM SCH 58261. In the first case, the EC50 values related to the A2A component were 39 ± 6 and 70 ± 10 nM in neutrophils and lymphocytes, respectively. In the second condition, the EC50 values of NECA related to the A2B receptor stimulation were 2100 ± 220 and 2640 ± 240 nM in neutrophils and lymphocytes, respectively (Fig. 7, A and B).
Phosphoinositide Turnover. It is well known that A2B receptors, in addition to adenylyl cyclase, are also coupled to phospholipase C and stimulate phosphoinositide production. In hA2BHEK 293 cells, 1 e NECA induced a significant stimulation of IP3 generation raising from basal values of 1.0 ± 0.3 to 15 ± 2 pmol of IP3 from 1 x 106 cells (P < 0.01, n = 3). Pretreatment of the cells with 100 nM MRE 2029-F20 and the aminosteroid PLC inhibitor U73122 (5 e) antagonized the NECA-mediated IP3 generation, suggesting the involvement of A2B receptors and PLC, respectively (Fig. 8A). When IP3 generation was evaluated in human lymphocytes, we could not observe any coupling to PLC. In contrast, in human neutrophils, it was possible to detect IP3 turnover induced by classic stimuli linked to PLC activation, such as 10 e formyl-methionyl-leucyl phenylalanine and 100 e NECA. This dose of NECA induced an increase from 0.45 ± 0.15 to 5.94 ± 0.5 pmol of IP3 from 1 x 106 cells that was significantly reduced in the presence of 100 nM MRE 2029-F20 and 5 e U73122 (2.5 ± 0.5 and 0.7 ± 0.2 pmol/106 cells, respectively) (P < 0.01, n = 3) (Fig. 8B).
Intracellular Calcium Measurements. To better characterize A2B coupling to PLC activation, intracellular calcium levels were determined. NECA revealed a marked concentration-dependent increase of intracellular Ca2+ in hA2BHEK 293 cells with an EC50 value of 312 ± 30 nM (Fig. 9A). This effect was potently reversed by increasing concentrations of MRE 2029-F20, showing an IC50 value of 12 ± 2 nM (Fig. 9B). Treatment with 5 e U73122 completely abrogated NECA-induced calcium increase, whereas no changes in calcium levels were observed in the presence of pertussis toxin that inactivate Gi and Go family G proteins (Fig. 9C). These data suggest that A2B receptors in hA2BHEK 293 cells signal through the activation of Gq/11 protein and PLC enzyme. To investigate the coupling of A2B receptors with this pathway also in native cells, we performed the same experiments in human lymphocytes and neutrophils. Consistent with IP3 assay, in human lymphocytes, A2B receptors did not seem to stimulate intracellular calcium levels. As for human neutrophils, we observed that 100 e NECA increases intracellular calcium levels (Fig. 10A). This effect was antagonized by MRE 2029-F20 with an IC50 value of 125 ± 23 nM and completely abrogated in the presence of the PLC inhibitor U73122 indicating the involvement of A2B receptors and PLC enzyme (Fig. 10, A and B).
Discussion
In this work, we have compared the pharmacological behavior and the functional coupling of recombinant A2B adenosine receptors expressed in transfected HEK 293 cells with endogenous receptors present in peripheral blood cells by using the novel radiolabeled receptor antagonist [3H]MRE 2029-F20. In saturation assays, this radioligand shows a KD value of 2.8 nM, in agreement with data obtained from kinetic experiments, and a receptor density of 450 fmol/mg of protein. The pharmacology of A2B receptors shows that reference adenosine ligands bound to human A2B receptors with a rank order of potency typical of the A2B subtype. Adenosine antagonists show affinity values similar to those reported in literature. In particular, MRE 2029-F20 has affinity and selectivity values comparable with those of MRS 1754 and better than OSIP339391, ZM 241385, and DPCPX. In competition experiments performed in cells transfected with A1, A2A, and A3 receptors, MRE 2029-F20 displayed a selectivity of 88-fold and >300-fold for A2B than for A1 (Ki value of 245 ± 31 nM), A2A, and A3 receptors (Ki > 1000 nM), respectively. Taken together, these binding characteristics make [3H]MRE 2029-F20 a useful tool for detection of A2B receptors, not only in transfected cells but also in native systems. [3H]MRE 2029-F20 was used in this work to investigate the presence of A2B receptors in peripheral blood cells chosen because 1) they represent primary cells expressing endogenous adenosine receptors; 2) they are important inflammatory cells, potentially involved in the exacerbation of asthma and other inflammation conditions; and 3) they contain more than one adenosine receptor subtype and are therefore difficult to study without the availability of selective radioligands.
First of all, the presence of A2B mRNA message in lymphocytes, neutrophils, and monocytes was assessed through real-time RT-PCR assays revealing the presence of A2B transcript with the following increasing rank order: monocytes < lymphocytes neutrophils. Therefore, the A2B message was investigated at the protein level by using [3H]MRE 2029-F20 binding, which shows a single class of high-affinity binding sites with KD values of 2.4 ± 0.5 and 2.7 ± 0.7 nM and Bmax values of 79 ± 10 and 54 ± 8 fmol/mg of protein in neutrophils and lymphocytes, respectively. The pharmacological profile of A2B ligands in these cells confirmed the data obtained in transfected hA2BHEK 293 cells; MRE 2029-F20 was the most potent compound followed by MRS 1754, AS 100, ZM 241385, and DPCPX. As for agonists, they all confirm a low affinity for the A2B site. In contrast to mRNA presence of A2B receptors, in human monocytes it was not possible to detect any specific binding of [3H]MRE 2029-F20, and this is in agreement to findings reported by Thiele et al. (2004) showing an increase of A2B message in cultured human monocytes in comparison with freshly isolated cells but not A2B receptor functionality.
In 1999, Linden et al. reported that in transfected HEK 293 cells, A2B receptors seem to be coupled to adenylyl cyclase and phospholipase C activity; in the same work, coupling to Gq proteins was also observed in HMC-1 cells. However, it is well known that recombinant receptors that are expressed in very high concentrations may produce different coupling in comparison with natural systems in which they are present at a lower level. Therefore, we compared the intracellular signaling pathways regulated by A2B adenosine receptors in both recombinant systems and native cells. To achieve this, we investigated the regulation of adenylyl cyclase and phospholipase C activity by A2B receptors, using NECA as the most potent nonselective A2B agonist available and MRE 2029-F20 as a highly selective A2B antagonist in HEK 293 cells, neutrophils, and lymphocytes. It is interesting that in the cAMP accumulation assay, NECA exhibited a potency higher than that observed in binding experiments, according to Linden et al. (1999). Because this effect was not potently reversed by MRE 2029-F20 (IC50, 150 nM), we investigated the possible presence of A2A receptors in these cells. Even though previous studies reported the absence of A2A transcript and protein in HEK 293 cells (Cooper et al., 1997; Gao et al., 1999), in our conditions, real-time RT-PCR and binding studies revealed the presence of A2A receptors (Bmax of 35 fmol/mg of protein). Therefore, new experiments to determine the potency of MRE 2029-F20 to counteract the NECA-induced cAMP levels were performed in the presence of a fixed concentration of the A2A blocker SCH 58261. With this experimental approach, the A2B antagonist showed a pA2 of 7.8, which was consistent with its binding affinity. The discrepancy between our data and previous data might be related either to a different method used to reveal A2A receptors or to a different strain of HEK 293 cells. In native cells, the coexpression of all adenosine subtypes coupled to the same effector system makes it impossible to study the contribution of each receptor subtype without the availability of selective ligands. Because of the lack of selectivity of NECA, cAMP accumulation in peripheral blood cells was evaluated by using the same pharmacological approach described above and previously reported in HMC-1 cells (Feoktistov and Biaggioni, 1998). The stimulatory effect on cAMP levels caused by the A2A receptor was distinguished from an A2B effect through the use of SCH 58261 and MRE 2029-F20 as A2A and A2B selective antagonists, respectively. Functional A1 and A3 subtypes are also present together with A2A and A2B in both neutrophils and lymphocytes (Gessi et al., 2002, 2004). In most cases, a selective blockade of inhibitory adenosine receptors is required to unmask functional stimulatory subtypes (Feoktistov and Biaggioni, 1997). However, under our conditions, as already reported in various glial cells (Fiebich et al., 1996), it was not necessary to block A1 and A3 receptors to observe either A2A or A2B cAMP stimulation. The simultaneous expression of different adenosine subtypes in a single cell is more of a rule than an exception. Because A2B receptors have low affinity for adenosine it is possible that their role become important in pathologic conditions, when adenosine levels increase. However it has to be noted that other intracellular signaling pathways have been associated with the A2B stimulation.
In agreement with literature data showing A2B/Gq coupling in a variety of cells (Feoktistov and Biaggioni, 1995; Strohmeier et al., 1995; Linden et al., 1998, 1999; Gao et al., 1999; Grant et al., 2001; Feoktistov et al., 2002; Rees et al., 2003), we also found that A2B receptors in hA2BHEK 293 cells activate PLC, as demonstrated by inositol phosphate generation and calcium mobilization experiments. NECA induced a significant increase in IP3 production that was counteracted by the A2B antagonist MRE 2029-F20 and also by the PLC inhibitor U73122 . In calcium mobilization studies, NECA had an EC50 in the high nanomolar range, in agreement with its affinity in binding experiments, and MRE 2029-F20 was able to potently antagonize this effect. A2B regulation of intracellular calcium increase was dependent on Gq/11 and PLC as demonstrated in experiments carried out in the presence of pertussis toxin and U73122 . Stimulation of intracellular calcium levels was not observed in wild-type HEK 293 cells, according to Ryzhov et al. (2004), but in contrast with Gao et al. (1999). Again, a different strain of cells might be responsible for these discrepancies, and under our experimental conditions, one could hypothesize that the increase in levels of receptor expression obtained by transfection technique led to a gain in coupling of A2B receptors to PLC signaling pathway. As for neutrophils, both IP3 production and calcium increase were observed after 100 e NECA stimulation. Because of the presence of A3 receptors in human neutrophils that might be coupled to PLC (Gessi et al., 2002), we verified the role of A2B receptors in the NECA-induced effect. MRE 2029-F20 reduced both IP3 production and intracellular calcium increase, showing a potency in the nanomolar range that was consistent with the involvement of the A2B subtype. The coupling to PLC was not observed in human lymphocytes (Mirabet et al., 1999), where the amount of A2B receptors was slightly lower compared with human neutrophils, suggesting that receptor density might be important to establish the signaling pathways activated by receptor agonist stimulation.
In conclusion, in this work, we have demonstrated the presence and functional coupling of human A2B adenosine receptors in different peripheral blood cells that play a role in immune and inflammatory processes in which A2B receptors are thought to be involved. This analysis was made possible by [3H]MRE 2029-F20, a new selective and high-affinity antagonist radioligand for human A2B adenosine receptors that has been successfully employed to detect and quantify the amount of this adenosine subtype in native cells. Our data reveal that recombinant A2B receptors expressed in HEK 293 cells show binding characteristics similar to those presented by A2B subtypes expressed in peripheral blood cells but might differ from these in terms of signal transduction coupling as seen in lymphocytes. Further studies might elucidate other effects mediated by A2B receptors in neutrophils and lymphocytes, such as their involvement in superoxide anion generation or in cytokine release. The appreciation of the pathways activated by A2B receptors in native cells raises the possibility that selective antagonists may become useful tools for the pharmacological characterization of diseases in which A2B receptors may be involved and possibly a basis for drug development.
doi:10.1124/mol.104.009225.
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