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Expression, Pharmacological Profile, and Functional Coupling of A_2B Receptors in a Recombinant System and in Peripheral Blood Cells Using a Novel Selective Antagonist Radioligand, [³H]MRE 2029-F20

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.), and 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 für Pharmakologie, Universitat Würzburg, Würzburg, Germany (K.N.K.); and King Pharmaceutical, Cary, North Carolina (E.L., S.M.)

In this study, we compared the pharmacological and biochemical characteristics of A_2B adenosine receptors in recombinant (hA_2BHEK293 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 hA_2B versus hA₁,hA_2A, and hA₃ subtypes. [³H]MRE 2029-F20 bound specifically to the hA_2B receptor stably transfected in human embryonic kidney (HEK) 293 cells with K_D of 2.8 ± 0.2 nM and B_max 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 K_D values of 2.4 ± 0.5 and 2.7 ± 0.7 nM, respectively, and B_max 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 A_2B mRNA. The rank order of potency of typical adenosine ligands with recombinant hA_2B receptors was consistent with that typically found for interactions with the A_2B subtype and was also similar in peripheral blood cells. 5'-N-Ethyl-carboxamidoadenosine stimulated cAMP accumulation in both hA_2BHEK293 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 hA_2B adenosine subtype and may be used to quantify A_2B 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 A_2B receptors may be involved.

Address correspondence to: Prof. Dr. Pier Andrea Borea, Chair of Pharmacology, Faculty of Medicine, University of Ferrara, Department of Clinical and Experimental Medicine, Pharmacology Unit, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail: bpa{at}dns.unife.it

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