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Screening for the smooth muscle relaxants

平滑肌松弛剂的筛选

基本信息

批准号：
07557310
负责人：
KOHAMA Kazuhiro
金额：
$ 1.15万
依托单位：
Gunma University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557310/
关键词：
purinergic receptor capsicin receptor patch clamp Cytoskeleton PC-12 Cell actin nerve cell recombinant protein 創薬血管 ATPase アクトミオシン

项目摘要

This research aims to examine the effects of cytoskeletal proteins on the activity of ionotropic receptor. It has been known for many years that Cytochalasin and Colchicine, which destroy cytoskeltons, affected some receptor activities, when they were applied extracellularly. Therefore, we expected that actin and tubilin and their associated proteins may exert regulatory roles to the ionotropic channels.In 1996, we applied cytochalasin (which destroys actin filaments) and phalloidin (which stabilizes actin filaments) and recorded the changes in the ionic currents of purinergic P_2x_2 by the patch clamp method. Unfortunately we failed to detect any significant effects of the agents.In 1997, we obtained cDNA coding P_2x_2 receptor by subjecting PC-12 cells to RT-PCR method. We then tried to express the receptor in E.Coli by the pET expression system so that the interaction between P_2x_2 and cytoskeletal proteins was examined. However, E.Coli transfected with the system did not produce P_2x_2.In 1998. we obtained cDNA coding a capsicin receptor by subjecting dorsal root ganglion to RT-PCR, and inserted the PCR product to pET expression system. E.Coli transfected the system fortunately produced the capsicin receptor. We purified the receptor protein by the column chromatographies in a large amount. Actin-binding activity was detected for the receptor protein by precepitating it together with actin filaments. This preliminary experiment will provide a clue to examine the effect of cytoskeletal proteins on P_2x_2 activity.

本研究旨在研究细胞骨架蛋白对离子型受体活性的影响。多年来人们都知道，细胞松弛素和秋水仙碱可以破坏细胞骨架，当它们应用于细胞外时，会影响一些受体的活性。因此，我们预计肌动蛋白和微管蛋白及其相关蛋白可能对离子通道发挥调节作用。 1996年，我们应用细胞松弛素（破坏肌动蛋白丝）和鬼笔环肽（稳定肌动蛋白丝），通过膜片钳方法记录了嘌呤能P_2x_2离子电流的变化。遗憾的是我们未能检测到药物的任何显着作用。1997年，我们通过RT-PCR方法对PC-12细胞获得了编码P_2x_2受体的cDNA。然后我们尝试通过pET表达系统在大肠杆菌中表达该受体，以便检查P_2x_2和细胞骨架蛋白之间的相互作用。然而，转染该系统的大肠杆菌并没有产生P_2x_2。1998年，我们通过对背根神经节进行RT-PCR，获得了编码辣椒素受体的cDNA，并将PCR产物插入pET表达系统中。幸运的是，大肠杆菌转染系统产生了辣椒素受体。我们通过柱层析大量纯化了受体蛋白。通过将受体蛋白与肌动蛋白丝一起沉淀来检测其肌动蛋白结合活性。该初步实验将为检查细胞骨架蛋白对 P_2x_2 活性的影响提供线索。