Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors |
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| Authors: | Varani Katia Gessi Stefania Merighi Stefania Vincenzi Fabrizio Cattabriga Elena Benini Annalisa Klotz Karl-Norbert Baraldi Pier Giovanni Tabrizi Mojgan Aghazadeh Lennan Stephen Mac Leung Edward Borea Pier Andrea |
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| Affiliation: | Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy. |
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| 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. |
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| Keywords: | AS16, 2-(4-benzyloxy-phenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl]-acetamide AS70, 8-[2-methyl-5-[2-oxo-2(4-phenyl-piperazin-1-yl)-ethoxy]-2H-pyrazol-3-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione AS74, N-benzo-[1,3]-dioxol-5-yl-2[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-isoxazol-3-yloxy]-acetamide AS94, N-[4-(ethoxycarbonyl)phenyl]2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide AS95, N-(4-carboxy-phenyl)-2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide AS96, 1,3-diallyl-8-[2-methyl-5-[2-oxo-2(4-phenyl-piperazin-1-yl)-ethoxy]-2H-pyrazol-3-yl]-3,7-dihydro-purine-2,6-dione AS99, N-[3,4] (dimethyl-phenyl)2-[5-(2,6-dioxo-1,3dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide AS100, N-2,3,4 dichlorophenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide AS101, N-(3,4-dimethoxy-phenyl)-2-5-(2,6-di-oxo-1,3-dipropyl-2,3,6,7-tetra-hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide cAMP, cyclic AMP CGS 21680, 2-[p-(2-carboxyethyl)-phenetyl-amino]-5′-N-ethyl-carboxamidoadenosine DPCPX, 1,3-dipropyl-8-cyclopentyl-xanthine HE-NECA, 2-hexynyl-5′-N-ethylcarboxamidoadenosine [3H]-MRE 2029F20, [3H]-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 [3H]-MRE 3008F20, [3H]-5-n-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine [3H]-ZM 241385, [3H]-4-(2-[7-amino-2-[furyl] [1,2,4] triazolo [2,3-a] [1,3,5]triazin-5-ylamino]ethyl] phenol [3H]-DPCPX [3H]-1,3-dipropyl-8-cyclopentyl-xanthine hA2BCHO, human A2B adenosine receptor in Chinese hamster ovary hA2BHEK-293, human A2B adenosine receptor in human embryonic kidney 293 HMC-1, human mast cells MRE 3008F20, 5-n-(4-methoxy phenyl-carbamoyl) amino-8-propyl-2-(2-furyl) pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine MRE 2029F20, 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 NECA, 5′-N-ethylcarboxamidoadenosine R-PIA, R(−)-N6(2-phenyl-isopropyl)-denosine S-PIA, S(−)-N6(2-phenylisopropyl)-adenosine SCH 58261, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine |
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