Mundell S J, Kelly E
Department of Pharmacology, School of Medical Sciences, University of Bristol, UK.
Biochem Pharmacol. 1998 Mar 1;55(5):595-603. doi: 10.1016/s0006-2952(97)00466-8.
Using receptor-selective agonists and antagonists, the possible presence of both A2a and A2b adenosine receptor subtypes coupled to activation of adenylyl cyclase was investigated in NG108-15 neuroblastoma x glioma hybrid cells. The relatively non-selective adenosine receptor agonist 5'-(N-ethyl carboxamido)-adenosine (NECA; 1 nM-300 microM) produced a biphasic increase in adenylyl cyclase activity in cell homogenates, best fitted to two components with high (EC50 0.7 microM) and low (EC50 16.0 microM) potency, respectively. The selective adenosine A2a receptor agonist CGS-21680 (1 nM-300 microM) also produced a biphasic increase in adenylyl cyclase. The NECA-dependent increase in adenylyl cyclase activity was almost completely inhibited by the non-selective adenosine receptor antagonist xanthine amine congener (XAC; 30 microM), but only partially inhibited by the selective A2a adenosine antagonist 8-(3-chlorostyryl)caffeine (CSC; 1 microM). Experiments were also performed to investigate the time course of NECA-induced desensitization of putative A2a and A2b receptor responses. The A2a-response was quantified using 10 microM CGS-21680, whilst the A2b response was quantified using 100 microM NECA in the presence of 1 microM CSC. The t0.5 for desensitization for each subtype was found to be around 20 min. Neither activation (with dibutyryl cAMP; 1 mM) nor inhibition (with H-89; 10 microM) of cyclic AMP-dependent protein kinase altered the ability of NECA pretreatment to desensitize A2a or A2b receptor-activated adenylyl cyclase. However zinc (200 microM), an inhibitor of G-protein coupled receptor kinase 2 (GRK2), significantly reversed the agonist-induced desensitization of A2a and A2b receptor-activated adenylyl cyclase. These experiments suggest the co-existence of A2a and A2b receptors coupled in a stimulatory fashion to adenylyl cyclase in NG108-15 cells. Furthermore desensitization of A2a and A2b responses occurs at the same rate and may involve a G-protein-coupled receptor kinase.
利用受体选择性激动剂和拮抗剂，在NG108 - 15神经母细胞瘤x胶质瘤杂交细胞中研究了与腺苷酸环化酶激活相关的A2a和A2b腺苷受体亚型的可能存在情况。相对非选择性的腺苷受体激动剂5' -（N - 乙基甲酰胺基） - 腺苷（NECA；1 nM - 300 microM）使细胞匀浆中的腺苷酸环化酶活性呈双相增加，最适合分别用高（EC50 0.7 microM）和低（EC50 16.0 microM）效力的两个成分来拟合。选择性腺苷A2a受体激动剂CGS - 21680（1 nM - 300 microM）也使腺苷酸环化酶呈双相增加。NECA依赖性的腺苷酸环化酶活性增加几乎被非选择性腺苷受体拮抗剂黄嘌呤胺同类物（XAC；30 microM）完全抑制，但仅被选择性A2a腺苷拮抗剂8 -（3 - 氯苯乙烯基）咖啡因（CSC；1 microM）部分抑制。还进行了实验以研究NECA诱导的假定A2a和A2b受体反应脱敏的时间进程。使用10 microM CGS - 21680对A2a反应进行定量，而在存在1 microM CSC的情况下使用100 microM NECA对A2b反应进行定量。发现每个亚型脱敏的t0.5约为20分钟。环磷酸腺苷依赖性蛋白激酶的激活（用二丁酰环磷酸腺苷；1 mM）或抑制（用H - 89；10 microM）均未改变NECA预处理使A2a或A2b受体激活的腺苷酸环化酶脱敏的能力。然而，锌（200 microM），一种G蛋白偶联受体激酶2（GRK2）的抑制剂，显著逆转了激动剂诱导的A2a和A2b受体激活的腺苷酸环化酶的脱敏。这些实验表明在NG108 - 15细胞中存在以刺激方式与腺苷酸环化酶偶联的A2a和A2b受体。此外，A2a和A2b反应的脱敏以相同速率发生，并且可能涉及一种G蛋白偶联受体激酶。
