title: Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors.
abstract: The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.
authors.affiliation: Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Varani
authors.forename: Stefania
authors.initials: S
authors.lastname: Gessi
authors.forename: Stefania
authors.initials: S
authors.lastname: Merighi
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Vincenzi
authors.forename: Elena
authors.initials: E
authors.lastname: Cattabriga
authors.forename: Annalisa
authors.initials: A
authors.lastname: Benini
authors.forename: Karl-Norbert
authors.initials: KN
authors.lastname: Klotz
authors.forename: Pier Giovanni
authors.initials: PG
authors.lastname: Baraldi
authors.forename: Mojgan Aghazadeh
authors.initials: MA
authors.lastname: Tabrizi
authors.forename: Stephen Mac
authors.initials: SM
authors.lastname: Lennan
authors.forename: Edward
authors.initials: E
authors.lastname: Leung
authors.forename: Pier Andrea
authors.initials: PA
authors.lastname: Borea
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D058917:Adenosine A2 Receptor Antagonists; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D011485:Protein Binding; D043704:Receptor, Adenosine A2B / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: D058917:Adenosine A2 Receptor Antagonists; D043704:Receptor, Adenosine A2B; D011994:Recombinant Proteins; D000242:Cyclic AMP; D000241:Adenosine