Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors

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. [3H] 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 fmol/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] CGS 21680 binding site had a Kd of 17 nM and a Bmax of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 microM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N6-cyclohexyladenosine (CHA), 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10-25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl- 8-¿4-[(2-aminoethyl)amino]carbonylmethyl- oxyphenyl¿ xanthine (XAC), 8-(3-chlorostyryl)caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)- methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX > XAC >> CSC approximately KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA approximately NECA > or = CADO > APEC approximately CV1808 > CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2-200 nM) bound to a site (A2A-like) with a Kd of 20 nM and a Bmax of 56fmol/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax of 63fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680(0.2-200 nM) bound to a second high-affinity site with a Kd of 97 nM and a Bmax of 255 fmol/mg protein in the hippocampus and with a Kd of 112 nM and a Bmax of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A 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 t...

Involvement of Adenosine A1 and A2A Receptors in the Motor Effects of Caffeine after its Acute and Chronic Administration

“…In fact, like CPT, DPCPX penetrates into the brain in substantial amounts after systemic administration (Baumgold et al, 1992) and counteracts centrally mediated, motor-depressant effects of the A1 receptor agonist CPA (Marston et al, 1998). The difference in the motor-activating effects of these compounds could depend on the reported ability of DPCPX to bind with high affinity to binding sites other than A 1 receptors, such as a nonstriatal atypical A 2A receptor (Cunha et al, 1996) or the cystic fibrosis transmembrane conductance regulator (CFTR) (Cohen et al, 1997). As expected from its previously reported nonselective adenosine receptor antagonism in in vitro and in vivo radioligand binding experiments (Kaplan et al, 1996;Daly and Fredholm, 1998;Fredholm et al, 1999;El Yacoubi et al, 2001), caffeine also produced motor activation at doses that counteracted the motor depression induced by the adenosine agonists CPA and CGS 21680.…”

Selective inactivation of adenosine A2A receptors in striatal neurons enhances working memory and reversal learning

“…Total membranes were prepared from the striatum, the cortex, the hippocampus, and/or the olfactory bulb, and single-point saturation binding assays were performed in duplicate to quantify total A 2A R, A 1 R, D 1 R, and D 2 R levels as described earlier (Dewar et al 1989;Lidow et al 1989;Cunha et al 1996;Lopes et al 2004;Houchi et al 2005;Rebola et al 2005) with slight modifications. Each 300-mL binding assay, consisting of assay buffer (50 mL), radioligand (50 mL), and membranes (200 mL, 100-200 mg), was incubated at room temperature (or 30˚C for D 1 R) for 1 h (or 2 h for A 1 R).…”

Towards a revised nomenclature for P1 and P2 receptors

“…In conformity with the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) recommendations, the two A 2 receptors are now named A 2A and A 2B (rather than A 2a and A 2b ). Although there are reports of binding sites that suggest the existence of additional subtypes [7][8][9][10][11] , no evidence for new A 2 receptors has evolved from the extensive cloning efforts of several laboratories. Indeed at least one of these sites (denoted A 4 by the authors) may represent binding to a state of the A 2A receptor 12 , and a similar explanation may apply to the other putative receptor binding sites.…”

“…In order not to preempt the efforts of cloning, we recommend the term P2Y ADP rather than, e.g. P2Y 8 or P2Y 9 . The recently reported p2y 3 receptor from the chick 19 shows a preference for ADP, but is clearly different from the platelet ADP receptor.…”