Naunyn-Schmiedeberg's Arch Pharmacol (1996) 353:261 271 © Springer-Verlag 1996 Rodrigo A. Cunha • Bjiirn Johansson M. Dolores Constantino • Ana M. Sebastifio Bertil B. Fredholm Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3HI CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors Received: 13 Juty 1995/Accepted: 11 October 1995 Abstract The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl) phenylamino] -5'-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3HI CGS 21680, in the concentration range tested (0.2-200 nM), bound to a single site with a Kd of 58 nM and a Bmax of 353 fmo1/mg protein in the hippocampus, and with a Kd of 58 nM and a Bmax of 264 fmol/mg protein in the cortex; in the striatum, the single high- affinity [3H l CGS 21680 binding site had a Ka of 17riM and a Bma x of 419fmol/mg protein. Both guanylylimidodiphosphate (100 ~tM) and Na ÷ (100 mM) reduced the affinity of [3HI CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [-3HI CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N6-cyclohexyladenosine (CHA), Y-N-ethyl- carboxamidoadenosine (NECA) and 2-chloro- adenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3HlCGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A recep- tors and was also present in the hippocampus and cortex, representing 10-25% of [3H]CGS 21680 bind- ing. The predominant [-3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency R.A. Cunha ([E~) • M.D. Constantino • A.M. Sebasti~o Laboratory of Pharmacology, Gulbenkian Institute of Science, P-2781 Oeiras codex, PortugaI B. Johansson • B.B. Fredholm Section of Molecular Neuropharmacology, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden order of adenosine antagonists DPCPX, 1,3-dipropyl- 8-{4-[(2-aminoethyl)amino]carbonylmethyl- oxyphenyl} xanthine (XAC), 8-(3-chlorostyryl) caffeine (CSC), and (E)-l,3-dipropyl-8-(3,4-dimethoxystyryl)- methylxanthine (KF 17,837) as displacers of ]-3HI CGS 21680 (5 riM) binding in the hippocampus and cerebral cortex was DPCPX > XAC >> CSC ~ KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethy- lamino)carbonylethylphenylethylamino]-5'-N-ethylcar- boxamidoadenosine (APEC), and 2-phenylaminoade- nosine (CV 1808) was CHA ~ NECA > CADO > APEC ~ CV1808 > CGS 21680. In the presence of DPCPX (20nM), [3HI CGS 21680 (0.2 200nM) bound to a site (A2a-like) with a Kd of 20 nM and a Bmax of 56 fmo1/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax of 63 fmol/mg protein in the cortex. In the presence of CSC (200 riM), [-3H]CGS 21680 (0.2-200 nM) bound to a second high-affinity site with a Kd of 97 nM and a Bmaxof 255 fmol/mg protein in the hippocampus and with a Kd of 112 nM and a Bm~xof 221 fmol/mg protein in the cortex. Two phar- macologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hip- pocampus and cortex and in the striatum, one corres- ponding to AZA receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum. Key words Adenosine receptors • Hippocampus • Cerebral cortex • Striatum • CGS 21680 • Radioligand assay