Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding by endogenous opioids acting at a cloned mu receptor.
Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding by endogenous opioids acting at a cloned mu receptor.
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发表时间：
1998-07
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通讯作者：
Andrew Alt;A. Mansour;Huda Akil;Fedor Medzihradsky;J. Traynor;James H. Woods
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作者：
Andrew Alt;A. Mansour;Huda Akil;Fedor Medzihradsky;J. Traynor;James H. Woods
内源性阿片样物质激活G蛋白的能力在来自稳定表达克隆的大鼠μ受体的C6大鼠神经胶质瘤细胞的膜中测量。研究了代表三个已知内源性阿片类物质家族（脑啡肽、内啡肽和强啡肽）的肽，以及两个最近发现的内源性阿片类物质，内吗啡肽-1和-2，它们被认为代表第四个内源性阿片肽家族。鸟苷-5 '-O-（3-[35 S]硫代）三磷酸（[35 S]GTP γ S）与膜结合的刺激被用作G蛋白活化的量度。有可能区分高功效化合物如Tyr-D-Ala-Gly-（Me）Phe-Gly-ol与低功效激动剂如吗啡或度冷丁。发现甲硫氨酸和亮脑啡肽、β内啡肽和强啡肽A都对μ受体具有高效力，肽片段β内啡肽-1（1-27）和强啡肽A-（1-13）也是如此。发现内吗啡肽-1和-2是部分激动剂，能够刺激[35 S]GTP γ S结合并拮抗由更高效激动剂Tyr-D-Ala-Gly-（Me）Phe-Gly-ol产生的刺激。通过放射性标记的拮抗剂的置换来测量阿片激动剂在克隆的μ受体处的结合亲和力。发现Ki值与[35 S]GTP γ S结合刺激的EC 50值密切匹配，表明在该系统中不存在用于G蛋白完全活化的大的受体储备。
The ability of endogenous opioids to activate G proteins was measured in membranes from C6 rat glioma cells stably expressing a cloned rat mu receptor. Peptides representing each of the three known families of endogenous opioids (enkephalins, endorphins and dynorphins) were studied, as well as two recently discovered endogenous opioids, endomorphin-1 and -2, which are thought to represent a fourth family of endogenous opioid peptides. Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding to membranes was used as a measure of G protein activation. It was possible to differentiate high efficacy compounds such as Tyr-D-Ala-Gly-(Me)Phe-Gly-ol from lower-efficacy agonists such as morphine or meperidine. Met- and leu-enkephalin, beta endorphin and dynorphin A were all found to have high efficacy at the mu receptor, as were the peptide fragments beta endorphin-1(1-27) and dynorphin A-(1-13). Endomorphin-1 and -2 were found to be partial agonists, capable of both stimulating [35S]GTP gamma S binding and antagonizing the stimulation produced by the higher-efficacy agonist Tyr-D-Ala-Gly-(Me)Phe-Gly-ol. Binding affinities for the opioid agonists at the cloned mu receptor were measured by the displacement of radiolabeled antagonist. It was found that the Ki values closely matched the EC50 values for [35S]GTP gamma S binding stimulation, indicating that a large receptor reserve does not exist for the complete activation of G proteins in this system.