Williams M, Braunwalder A
J Neurochem. 1986 Jul;47(1):88-97. doi: 10.1111/j.1471-4159.1986.tb02835.x.
Adenine nucleotides displace the binding of the selective adenosine A-1 receptor ligand [3H]cyclopentyladenosine (CPA) to rat brain membranes in a concentration-dependent manner, with the rank order of activity being ATP greater than ADP greater than AMP. Binding was also displaced by GTP, ITP, adenylylimidodiphosphate (AppNHp), 2-methylthioATP, and the beta-gamma-methylene isostere of ATP, but was unaffected by the alpha-beta-methylene isosteres of ADP and ATP, and UTP. At ATP concentrations greater than 100 microM, the inhibitory effects on CPA binding were reversed, until at 2 mM ATP, specific binding of CPA was identical to that seen in controls. Concentrations of ATP greater than 10 mM totally inhibited specific binding. Inclusion of the catabolic enzyme adenosine deaminase in the incubation medium abolished the inhibitory effects of ATP, indicating that these were due to adenosine formation, presumably due to ectonucleotidase activity. The inhibitory effects were also attenuated by the alpha-beta-methylene isostere of ATP, an ectonucleotidase inhibitor. Adenosine deaminase, alpha-beta-methylene ATP (100 microM), and beta-gamma-methylene ATP (100 microM) had no effect on the "stimulatory" phase of binding, although GTP (100 microM) slightly attenuated it. Comparison of the binding of [3H]CPA in the absence and presence of 2 mM ATP by saturation analysis showed that the KD and apparent Bmax values were identical. Examination of the pharmacology of the control and "ATP-dependent" CPA binding sites showed slight changes in binding of adenosine agonists and antagonists. The responses observed with high concentrations of ATP were not observed with GTP, AppNHp, the chelating agents EDTA and EGTA, or inorganic phosphate. The divalent cations Mg2+ and Ca2+ at 10 mM attenuated the stimulatory actions of high (2 mM) concentrations of ATP, whereas EGTA and EDTA (10 mM) enhanced the "stimulatory" actions of ATP. EDTA (10 mM) abolished the inhibitory effects of ATP, indicating a specific dependence on Mg2+ for the inhibitory response. The effects of ATP on [3H]CPA binding were reversible for antagonists but not agonists. The mechanism by which ATP reverses its own inhibitory action on adenosine A-1 radioligand binding is unclear, and from the observed actions of the divalent cations and chelating agents probably does not involve a phosphorylation-dependent process.
腺嘌呤核苷酸以浓度依赖的方式取代选择性腺苷A - 1受体配体[3H]环戊腺苷（CPA）与大鼠脑膜的结合，活性顺序为ATP＞ADP＞AMP。GTP、ITP、腺苷酰亚胺二磷酸（AppNHp）、2 - 甲硫基ATP以及ATP的β - γ - 亚甲基类似物也能取代结合，但不受ADP和ATP的α - β - 亚甲基类似物以及UTP的影响。在ATP浓度大于100μM时，对CPA结合的抑制作用会逆转，直至在2 mM ATP时，CPA的特异性结合与对照组相同。ATP浓度大于10 mM时完全抑制特异性结合。在孵育介质中加入分解代谢酶腺苷脱氨酶可消除ATP的抑制作用，表明这些作用是由于腺苷形成，推测是由于外核苷酸酶活性。ATP的α - β - 亚甲基类似物（一种外核苷酸酶抑制剂）也可减弱抑制作用。腺苷脱氨酶、α - β - 亚甲基ATP（100μM）和β - γ - 亚甲基ATP（100μM）对结合的“刺激”阶段无影响，尽管GTP（100μM）会使其略有减弱。通过饱和分析比较在不存在和存在2 mM ATP时[3H]CPA的结合情况，发现KD和表观Bmax值相同。对对照和“ATP依赖”的CPA结合位点的药理学研究表明，腺苷激动剂和拮抗剂的结合有轻微变化。高浓度ATP所观察到的反应在GTP、AppNHp、螯合剂EDTA和EGTA或无机磷酸盐作用下未观察到。10 mM的二价阳离子Mg2 +和Ca2 +减弱高浓度（2 mM）ATP的刺激作用，而EGTA和EDTA（10 mM）增强ATP的“刺激”作用。EDTA（10 mM）消除ATP的抑制作用，表明抑制反应对Mg2 +有特异性依赖。ATP对[3H]CPA结合的影响对于拮抗剂是可逆的，但对于激动剂则不是。ATP逆转其自身对腺苷A - 1放射性配体结合抑制作用的机制尚不清楚，从二价阳离子和螯合剂的观察作用来看，可能不涉及磷酸化依赖过程。
