RT Journal Article
SR Electronic
T1 Expression, Pharmacological Profile, and Functional Coupling of A<sub>2B</sub> Receptors in a Recombinant System and in Peripheral Blood Cells Using a Novel Selective Antagonist Radioligand, [<sup>3</sup>H]MRE 2029-F20
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 2137
OP 2147
DO 10.1124/mol.104.009225
VO 67
IS 6
A1 Gessi, Stefania
A1 Varani, Katia
A1 Merighi, Stefania
A1 Cattabriga, Elena
A1 Pancaldi, Cecilia
A1 Szabadkai, Youri
A1 Rizzuto, Rosario
A1 Klotz, Karl-Norbert
A1 Leung, Edward
A1 Mac Lennan, Stephen
A1 Baraldi, Pier Giovanni
A1 Borea, Pier Andrea
YR 2005
UL http://molpharm.aspetjournals.org/content/67/6/2137.abstract
AB 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.