Florio C, Traversa U, Vertua R, Puppini P
Institute of Pharmacology and Pharmacognosy, Faculty of Pharmacy, University of Trieste, Italy.
Neuropharmacology. 1988 Jan;27(1):85-94. doi: 10.1016/0028-3908(88)90205-5.
In the present study it is reported that [3H]NECA binds in a specific and saturable manner to membranes from the cerebral cortex of the rat. Scatchard analysis revealed two binding sites. The high affinity binding site (Kd 10.66 +/- 5 nM, Bmax 0.305 +/- 0.05 pmol/mg prot) was characterized by the following features: maximum binding at 25 degrees C, sensitivity to pretreatment with NEM and regulation by Gpp[NH]p, enhancing of binding in the presence of 1.0 mM MgCl2 and 1.5 mM CaCl2; the rank order of potency of several analogues of adenosine in competing with [3H]NECA for binding, was CHA greater than L-PIA greater than NECA greater than CADO. The low affinity binding site (Kd261.8 +/- 50 nM, Bmax 4.19 +/- 0.33 pmol/mg prot) showed maximum binding at 0 degrees C, insensitivity to pretreatment with NEM up to 1 mM and to regulation by Gpp[NH]p, and inhibition of binding in the presence of MgCl2 and CaCl2. The low affinity site was also present in membranes not pretreated with adenosine deaminase and, even in this condition, an IC50 of 188.5 +/- 36 nM for NECA and an IC50 of 4.35 +/- 0.20 microM for adenosine were found. It is concluded that the high affinity binding site is similar to the A1 adenosine receptors. The low affinity binding site is not classifiable among the A-type adenosine receptors, although it shows peculiar features shared both with the human platelet A2 receptor and the adenosine receptor formerly studied with [3H]adenosine in membranes from the brain of the rat; these results could reflect heterogeneity of adenosine receptors in central nervous system.
在本研究中，据报道[3H]NECA以特异性和可饱和的方式与大鼠大脑皮质膜结合。Scatchard分析显示有两个结合位点。高亲和力结合位点（解离常数Kd为10.66±5 nM，最大结合量Bmax为0.305±0.05 pmol/mg蛋白）具有以下特征：在25℃时结合量最大，对N-乙基马来酰亚胺（NEM）预处理敏感且受Gpp[NH]p调节，在1.0 mM MgCl2和1.5 mM CaCl2存在时结合增强；几种腺苷类似物与[3H]NECA竞争结合的效力顺序为：环已基腺苷（CHA）＞L-苯基异丙基腺苷（L-PIA）＞NECA＞环戊基腺苷（CADO）。低亲和力结合位点（解离常数Kd2为61.8±50 nM，最大结合量Bmax为4.19±0.33 pmol/mg蛋白）在0℃时结合量最大，对高达1 mM的NEM预处理不敏感且不受Gpp[NH]p调节，在MgCl2和CaCl2存在时结合受到抑制。即使未用腺苷脱氨酶预处理的膜中也存在低亲和力位点，在此条件下，发现NECA的半数抑制浓度（IC50）为188.5±36 nM，腺苷的IC50为4.35±0.20 μM。结论是高亲和力结合位点类似于A1腺苷受体。低亲和力结合位点不属于A型腺苷受体类别，尽管它显示出与人血小板A2受体以及先前在大鼠脑细胞膜中用[3H]腺苷研究的腺苷受体共有的独特特征；这些结果可能反映了中枢神经系统中腺苷受体的异质性。
