Abstract
Four adenosine receptor subtypes of the family of G protein-coupled
receptors, designated A1, A2A, A2B and A3 are currently known. In
this study all human subtypes were stably transfected into Chinese
hamster ovary (CHO) cells in order to be able to study their pharmacological
profile in an identical cellular background utilizing radioligand
binding studies (A1, A2A, A3) or adenylyl cyclase activity assays
(A2B). The A1 subtype showed the typical pharmacological profile
with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with
the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine
(PIA) diastereomers. In competition with antagonist radioligand biphasic
curves were observed for agonists. In the presence of GTP all receptors
were converted to a single low affinity state indicating functional
coupling to endogenous G proteins. For A2A adenosine receptors CGS
21680 (2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamidoadeno
sine) 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 A2B adenosine receptor
could only be tested by measurement of receptor-stimulated adenylyl
cyclase activity. NECA was the most potent agonist with an EC50-value
of 2.3 microM whereas all other compounds tested were active at concentrations
in the high micromolar range. Inhibition of NECA-stimulated adenylyl
cyclase identified xanthine amino congener (XAC; 8-4-(2-aminoethyl)amino-carbonylmethyloxyphenyl-1,3-dipropylxa
nthine) as the most potent antagonist at this receptor subtype. The
A3 receptor was characterized utilizing the nonselective agonist
3HNECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide
(MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity
of the A3 receptor should be dropped at least for the human receptor
as xanthines with submicromolar affinity were found. Overall, the
pharmacological characteristics of the human receptors are similar
to other species with some species-specific characteristics. In this
study we present for the first time the comparative pharmacology
of all known human adenosine receptor subtypes. The CHO cells with
stably transfected adenosine receptors provide an identical cellular
background for such a pharmacological characterization. These cells
are valuable systems for further characterization of specific receptor
subtypes and for the development of new ligands.
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