Pharmacological characterization of adenosine A 2B receptors : Studies in human mast cells co-expressing A 2A and A 2B adenosine receptor subtypes

Abstract

Characterization of A 2B receptors is hampered by the lack of selective pharmacological probes and often relies on their relative affinity to agonists that are selective at other receptor types. This approach is limited because the affinity of A 2B receptors for putative A 3 agonists has not been determined. Using the human erythroleukemia cell line HEL as a cellular model for A 2B-mediated adenylate cyclase activation, we found the following potencies (pD 2) for the non-selective agonist 5′- N-ethylcarboxamidoadenosine (NECA) (5.65 ± 0.04), the putative A 3 agonists N 6-benzyl-NECA (4.17 ± 0.06) and N 6-(3-iodobenzyl)- N-methyl-5′-carbamoyl-adenosine (IB-MECA) (3.7 ± 0.02), and the A 2A agonist 4-[( N-ethyl-5′-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS21680) (2.8 ± 0.1). Because of the lack of a selective agonist, characterization of A 2B receptor function is difficult in cells co-expressing A 2A receptors. In the human mast cell line HMC-1, NECA induced cAMP accumulation with a concentration-response relationship best fitted to a two-sited model (pD 2 7.69 ± 0.42 and 5.92 ± 0.21 for high- and low-affinity sites), suggesting the presence of both A 2A and A 2B receptors in these cells. We demonstrated that A 2B receptors can be selectively activated with NECA in the presence of the selective A 2A antagonist 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5- c] pyrimidine (SCH 58261). Under these conditions, the concentration-response relationship of NECA for cyclic AMP accumulation was now best fitted to a one-site model (pD 2 5.68 ± 0.03, Hill slope 0.93 ± 0.06, 95% confidence intervals 0.8 to 1.06)corresponding to selective activation of A 2B receptors. Using the approaches developed in this study, we determined that A 2B, and not A 2A or A 3, receptors account for all the calcium mobilization induced by NECA in HMC-1 cells.