Cunha R A, Johansson B, Constantino M D, Sebastião A M, Fredholm B B
Laboratory of Pharmacology, Gulbenkian Institute of Science, Oeiras, Portugal.
Naunyn Schmiedebergs Arch Pharmacol. 1996 Feb;353(3):261-71. doi: 10.1007/BF00168627.
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 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.
研究了腺苷A2A受体选择性激动剂2-[4-(2-对羧乙基)苯基氨基]-5'-N-乙基羧酰胺基腺苷（CGS 21680）与大鼠海马和大脑皮质膜的结合情况，并与纹状体膜进行了比较。在测试的浓度范围（0.2 - 200 nM）内，[3H]CGS 21680在海马中以单一位点结合，解离常数（Kd）为58 nM，最大结合容量（Bmax）为353 fmol/mg蛋白质；在皮质中，Kd为58 nM，Bmax为264 fmol/mg蛋白质；在纹状体中，单一高亲和力的[3H]CGS 21680结合位点的Kd为17 nM，Bmax为419 fmol/mg蛋白质。鸟苷酰亚胺二磷酸（100 μM）和Na+（100 mM）均使[3H]CGS 21680在纹状体中的结合亲和力降低一半，并几乎完全消除其在海马和皮质中的结合。[3H]CGS 21680与1,3 - 二丙基 - 8 - 环戊基黄嘌呤（DPCPX）、N6 - 环己基腺苷（CHA）、5'-N - 乙基羧酰胺基腺苷（NECA）和2 - 氯腺苷（CADO）的结合曲线在海马、皮质以及纹状体中均为双相。纹状体中占主导的[3H]CGS 21680结合位点（80%）具有与A2A受体相符的药理学特征，在海马和皮质中也存在，占[3H]CGS 21680结合的10 - 25%。海马和皮质中占主导的[3H]CGS 21680结合位点具有与A2A受体不同的药理学特征：作为[3H]CGS 21680（5 nM）在海马和大脑皮质中结合的置换剂，腺苷拮抗剂DPCPX、1,3 - 二丙基 - 8 - [4 - [(2 - 氨基乙基)氨基]羰基甲基 - 氧苯基]黄嘌呤（XAC）、8 - (3 - 氯苯乙烯基)咖啡因（CSC）和(E) - 1,3 - 二丙基 - 8 - (3,4 - 二甲氧基苯乙烯基)甲基黄嘌呤（KF 17,837）的相对效价顺序为DPCPX > XAC >> CSC ≈ KF 17,837；腺苷激动剂CHA、NECA、CADO、2 - [(2 - 氨基乙氨基)羰基乙基苯乙氨基] - 5'-N - 乙基羧酰胺基腺苷（APEC）和2 - 苯氨基腺苷（CV 1808）的相对效价顺序为CHA ≈ NECA ≥ CADO > APEC ≈ CV1808 > CGS 21680。在DPCPX（20 nM）存在下，[3H]CGS 21680（0.2 - 200 nM）在海马中以解离常数为20 nM、最大结合容量为56 fmol/mg蛋白质的位点（类A2A）结合，在皮质中Kd为22 nM，Bmax为63 fmol/mg蛋白质。在CSC（200 nM）存在下，[3H]CGS 21680（0.2 - 200 nM）在海马中以解离常数为97 nM、最大结合容量为255 fmol/mg蛋白质的第二个高亲和力位点结合，在皮质中Kd为112 nM，Bmax为221 fmol/mg蛋白质。在海马、皮质和纹状体的突触体膜中发现了两个药理学上不同的[3H]CGS 21680结合位点，一个对应于A2A受体，另一个对应于第二个高亲和力的[3H]CGS 21680结合位点。相比之下，[3H]CHA在三个脑区突触体膜中的药理学相似。目前的结果证实了中枢神经系统中至少存在两个高亲和力的[3H]CGS 21680结合位点，并表明在海马和大脑皮质中占主导的[3H]CGS 21680结合位点具有与在纹状体中占主导的经典A2A腺苷受体不同的结合特性。
