COMPARATIVE PHARMACOLOGY OF HUMAN ADENOSINE RECEPTOR SUBTYPES - CHARACTERIZATION OF STABLY TRANSFECTED RECEPTORS IN CHO CELLS

Four adenosine receptor subtypes of the family of G protein-coupled re ceptors, designated A(1), A(2A), A(2B) and A(3) are currently known. I n this study all human subtypes were stably transfected into Chinese h amster ovary (CHO) cells in order to be able to study their pharmacolo gical profile in an identical cellular background utilizing radioligan d binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity ass ays (A(2B)). The Al subtype showed the typical pharmacological profile with 2-chloro-N-6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N-6-phenyliso propyladenosine (PIA) diastereomers. In competition with antagonist ra dioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state ind icating functional coupling to endogenous G proteins. For A(2A) adenos ine receptors CGS 21680 )phenylethylamino]-5'-N-ethylcarboxamidoadenos ine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R-and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A(2B) adenosine receptor co uld only be tested by measurement of receptor-stimulated adenylyl cycl ase activity. NECA was the most potent agonist with an EC50-value of 2 .3 mu M whereas all other compounds tested were active at concentratio ns in the high micromolar range. Inhibition of NECA-stimulated adenyly l cyclase identified xanthine amino congener (XAC; ]carbonyl]methyl]ox y]phenyl]-1,3-dipropylxanthine) as the most potent antagonist at this receptor subtype. The. A(3) receptor was characterized utilizing the n onselective agonist [H-3]NECA. The N-6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most pot ent agonists. The notion of xanthine-insensitivity of the A(3) recepto r should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological charac teristics of the human receptors are similar to other species with som e species-specific characteristics.In this study we present for the fi rst time the comparative pharmacology of all known human adenosine rec eptor subtypes. The CHO cells with stably transfected adenosine recept ors provide an identical cellular background for such a pharmacologica l characterization. These cells are valuable systems for further chara cterization of specific receptor subtypes and for the development of n ew ligands.