通过对大鼠脑中[3H]α,β-亚甲基-ATP结合进行定量放射自显影研究P2X受体上的配体识别位点。

Ligand recognition sites on P2X receptors studied by quantitative autoradiography of [3H]alpha,beta-methylene-ATP binding in rat brain.

作者信息

Worthington R A, Hansen M A, Bennett M R, Barden J A, Balcar V J

机构信息

Department of Anatomy and Histology, University of Sydney, NSW, Australia.

出版信息

Biochem Biophys Res Commun. 1998 Aug 10;249(1):166-71. doi: 10.1006/bbrc.1998.8968.

DOI:10.1006/bbrc.1998.8968

PMID:9705850

Abstract

The specificity of alpha, beta-methylene-ATP for P2X receptor binding sites in the CNS has been examined by testing the effects of several ATP analogues and other ATP-related substances on the binding of 10 nM [3H]alpha,beta-methylene-ATP to 20 microns thick sections of fresh-frozen rat brain. The labelling of the putative P2X receptor binding sites by [3H]alpha,beta-methylene-ATP was evaluated by quantitative densitometry. [3H]alpha,beta-methylene-ATP binding was strongly inhibited by two close ATP analogues, 3'-O-(trinitrophenyl)-adenosine-5'-triphosphate and beta,gamma-imido-ATP (IC50 2.5 microM). beta,gamma-Methylene-ATP was, however, less potent (< 50% inhibition at 25 microM). Inosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate, and cytidine-5'-triphosphate were practically inactive up to concentrations of 100 microM. Periodate oxidised ATP and 1, N6-etheno-ATP produced < 50% inhibition at 100 and 500 microM concentrations, respectively. Cations (K+, Rb+, Cs+, and Mg2+ at 5 mM and Na+ at 150 mM) reduced [3H]alpha,beta-methylene-ATP binding by no more than 50%. Several agents known to interact with Ca2+- and/or ATP-related cationic channels (Cd2+, glibenclamide, dantrolene, nifedipine, and thapsigargin) had no effect. We conclude that [3H]alpha,beta-methylene-ATP at low nanomolar concentrations binds to a site that has very strict structural requirements and is pharmacologically similar to ATP P2X receptors.

摘要

通过测试几种ATP类似物和其他与ATP相关的物质对10 nM [3H]α,β-亚甲基-ATP与新鲜冷冻大鼠脑20微米厚切片结合的影响，研究了α,β-亚甲基-ATP对中枢神经系统中P2X受体结合位点的特异性。通过定量光密度测定法评估[3H]α,β-亚甲基-ATP对假定的P2X受体结合位点的标记。[3H]α,β-亚甲基-ATP结合受到两种紧密的ATP类似物3'-O-（三硝基苯基）-腺苷-5'-三磷酸和β,γ-亚氨基-ATP的强烈抑制（IC50为2.5 microM）。然而，β,γ-亚甲基-ATP的效力较低（在25 microM时抑制率<50%）。肌苷-5'-三磷酸、鸟苷-5'-三磷酸、尿苷-5'-三磷酸和胞苷-5'-三磷酸在浓度高达100 microM时实际上没有活性。高碘酸盐氧化的ATP和1,N6-乙烯基-ATP在100 microM和500 microM浓度下分别产生<50%的抑制作用。阳离子（5 mM的K+、Rb+、Cs+和Mg2+以及150 mM的Na+）使[3H]α,β-亚甲基-ATP结合减少不超过50%。几种已知与Ca2+和/或ATP相关阳离子通道相互作用的药物（Cd2+、格列本脲、丹曲林、硝苯地平和毒胡萝卜素）没有作用。我们得出结论，低纳摩尔浓度的[3H]α,β-亚甲基-ATP与一个具有非常严格结构要求且在药理学上与ATP P2X受体相似的位点结合。