Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors.
The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.
Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK.
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L
Yates
J H
JH
Clark
T J
TJ
Martin
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S
James
K J
KJ
Broadley
E J
EJ
Kidd
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008024:Ligands; D015636:Magnesium Chloride; D011799:Quinazolines / Q000494:pharmacology; D011869:Radioligand Assay; D043684:Receptor, Adenosine A3 / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014230:Triazoles / Q000494:pharmacology
C110041:9-chloro-2-(2-furyl)-5-phenylacetylamino(1,2,4)triazolo(1,5-c)quinazoline; D007457:Iodine Radioisotopes; D008024:Ligands; C087339:N(6)-(4-amino-3-iodophenyl)methyl-5'-N-methylcarboxamidoadenosine; D011799:Quinazolines; D043684:Receptor, Adenosine A3; D011994:Recombinant Proteins; D014230:Triazoles; D015636:Magnesium Chloride; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C048599:N(6)-cyclopentyladenosine; D000242:Cyclic AMP; D000241:Adenosine