文摘

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_2BCHO), in human embryonic kidney 293 (hA_2BHEK-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 [³H]-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 ([³H]-MRE 2029F20) revealed a single class of binding sites in hA_2BCHO, hA_2BHEK-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_2BHEK-293 cells using [³H]-MRE 2029F20, showed a rank order of potency typical of the A_2B receptors with K_i values in the range 3.2–28 nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA_2BCHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA_2BCHO 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_2BCHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC₅₀ value of 2.9 ± 0.3 nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A_2B receptors.