Hermans E, Jeanjean A P, Fardin V, Pradier L, Garret C, Laduron P M, Octave J N, Maloteaux J M
Laboratoire de Neurochimie, Université Catholique de Louvain, Brussels, Belgium.
Eur J Pharmacol. 1993 Mar 15;245(1):43-50. doi: 10.1016/0922-4106(93)90167-8.
In the present study, we describe the effects of RP 67580, a substance P non-peptide antagonist, in binding and second messenger experiments performed using transfected Chinese hamster ovary cells expressing the rat NK1 receptor. The cDNA sequence encoding the rat brain substance P receptor was transfected in Chinese hamster ovary cells, and cellular clones which stably express the corresponding protein were isolated. [3H]Substance P binding was performed in homogenates of these transfected cells and revealed the presence of NK1 receptors in displacement experiments, using peptide analogs of three mammalian tachykinins (substance P, neurokinin A, neurokinin B). Scatchard analysis indicated a KD value of 0.33 +/- 0.13 nM and a Bmax value of 5.83 +/- 1.16 pmol/mg of protein. RP 67580, a selective NK1-receptor antagonist was found to displace the specific binding of [3H]substance P. When [3H]RP 67580 was used as a ligand, it displayed a high affinity (KD value: 1.22 +/- 0.27 nM) in transfected cell homogenates and only competed with NK1 receptor ligands. Substance P stimulated the hydrolysis of phosphoinositide in a time- and concentration-dependent manner and this effect was mimicked by selective agonists of the NK1 receptor ([Pro9]SP and septide). RP 67580 did not induce any accumulation of inositol phosphates, but was found to inhibit the inositol phosphate increase mediated by substance P, without affecting the maximal response. From these results, one may conclude that the receptor expressed by the transfected Chinese hamster ovary cells revealed similar binding characteristics as the NK1 receptor present in the rat brain and also confirmed the high affinity and the antagonist properties of RP 67580.
在本研究中，我们描述了P物质非肽拮抗剂RP 67580在使用表达大鼠NK1受体的转染中国仓鼠卵巢细胞进行的结合和第二信使实验中的作用。编码大鼠脑P物质受体的cDNA序列被转染到中国仓鼠卵巢细胞中，并分离出稳定表达相应蛋白的细胞克隆。在这些转染细胞的匀浆中进行了[3H]P物质结合实验，在置换实验中使用三种哺乳动物速激肽（P物质、神经激肽A、神经激肽B）的肽类似物揭示了NK1受体的存在。Scatchard分析表明解离常数（KD）值为0.33±0.13 nM，最大结合容量（Bmax）值为5.83±1.16 pmol/mg蛋白。发现选择性NK1受体拮抗剂RP 67580能置换[3H]P物质的特异性结合。当使用[3H]RP 67580作为配体时，它在转染细胞匀浆中表现出高亲和力（KD值：1.22±0.27 nM），并且仅与NK1受体配体竞争。P物质以时间和浓度依赖性方式刺激磷酸肌醇的水解，这种作用被NK1受体的选择性激动剂（[Pro9]SP和septide）模拟。RP 67580未诱导任何肌醇磷酸的积累，但被发现可抑制由P物质介导的肌醇磷酸增加，而不影响最大反应。从这些结果可以得出结论，转染的中国仓鼠卵巢细胞表达的受体显示出与大鼠脑中存在的NK1受体相似的结合特性，并且也证实了RP 67580的高亲和力和拮抗剂特性。
