title: Expression, pharmacological profile, and functional coupling of A2B receptors in a recombinant system and in peripheral blood cells using a novel selective antagonist radioligand, [3H]MRE 2029-F20.
abstract: In this study, we compared the pharmacological and biochemical characteristics of A(2B) adenosine receptors in recombinant (hA(2B)HEK293 cells) and native cells (neutrophils, lymphocytes) by using a new potent 8-pyrazole xanthine derivative, [(3)H]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] ([(3)H]MRE 2029-F20), that has high affinity and selectivity for hA(2B) versus hA(1),hA(2A), and hA(3) subtypes. [(3)H]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 [(3)H]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(2B)HEK293 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, [(3)H]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.
authors.affiliation: Faculty of Medicine, Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Gessi
authors.forename: Katia
authors.initials: K
authors.lastname: Varani
authors.forename: Stefania
authors.initials: S
authors.lastname: Merighi
authors.forename: Elena
authors.initials: E
authors.lastname: Cattabriga
authors.forename: Cecilia
authors.initials: C
authors.lastname: Pancaldi
authors.forename: Youri
authors.initials: Y
authors.lastname: Szabadkai
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Karl-Norbert
authors.initials: KN
authors.lastname: Klotz
authors.forename: Edward
authors.initials: E
authors.lastname: Leung
authors.forename: Stephen
authors.initials: S
authors.lastname: Mac Lennan
authors.forename: Pier Giovanni
authors.initials: PG
authors.lastname: Baraldi
authors.forename: Pier Andrea
authors.initials: PA
authors.lastname: Borea
mesh_terms: D000081:Acetamides / Q000494:pharmacology*; D058917:Adenosine A2 Receptor Antagonists; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011687:Purines / Q000494:pharmacology*; D011720:Pyrazoles / Q000494:pharmacology*; D043704:Receptor, Adenosine A2B / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000081:Acetamides; D058917:Adenosine A2 Receptor Antagonists; C495752:N-benzo(1,3)dioxol-5-yl-2-(5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy)-acetamide; D011687:Purines; D011720:Pyrazoles; D043704:Receptor, Adenosine A2B; D011994:Recombinant Proteins; D019830:Adenosine-5'-(N-ethylcarboxamide)