Intracellular infusion of high molecules by treatment of cell membrane with saponin

本课题的目的是使子宫肌细胞膜高通透，然后在细胞内注入camodulin或aequorin等分子，封闭膜，恢复其兴奋性和收缩性。首先，按照Endo et al.（1977）描述的方法，用皂苷处理组织，并证明兴奋性的恢复是徒劳的。摩根和摩根（1984）将含20 mM EGTA的高渗溶液用于雪貂门静脉后，肌肉在生理盐水溶液中恢复了膜的兴奋性。在EGTA溶液处理期间，钙调蛋白（一种钙受体蛋白）的应用增强了强直性收缩，表明细胞膜的高渗透和封闭成功。赠款购买的主要设备（NF数据记录仪）用于记录和再现电气和机械活动。最终目的是在细胞中装载对钙敏感的发光蛋白aequorin，以研究钙瞬态尚未成功。在实验中发现了一个有趣的结果，即在无ca的沐浴液中加入三磷酸腺苷（ATP）会产生强直性收缩。在高K介质中加入Ca或Ba诱导的收缩受ATP的抑制，而依赖于外源Mg、Mn和La的存在，因此证明ATP诱导的收缩与外源Ca的存在无关。罂粟碱对钙诱导的收缩有抑制作用，而对镁诱导的收缩无抑制作用。鉴于上述结果，这种不依赖钙的收缩的结构基础是由于肌动球蛋白系统，还是由于细胞骨架等其他系统，仍有待澄清。

The aim of this project is to hyperpermealize the cell membrane of uterine muscle, then to load intracellulary with molecules such as camodulin or aequorin, and to seal the membrane to recover the excitability and contractility. To begin with, the tissue was treated with saponin after the method described by Endo et al.(1977), and the recovery of excitability was proved in vain. The membrane excitability was recovered in the normal saline solution, after the muscle wastreated with the hyperpermealizing solution containing 20 mM EGTA, which was used for the ferret portal vein by Morgan and Morgan (1984). During the treatment with the EGTA solution, the tonic contraction was potentiated by the application of calmodulin, a Ca-receptor protein, indicating a success of the hyperpermealization and sealing of cell membrane. The main apparatus (NF data recorder) purchased by the grant-in-aid was used to stock and reproduce the electrical and mechanical activities. The final purpose that the cells are loaded with aequorin, a Ca-sensitive luminescent protein, to investigate the Ca-transient has not yet been successful.An interesting result was found during the experiment that a tonic contraction was generated by adenosine triphosphate (ATP) added to the Ca-free bathing solution. The ATP-induced contraction was proved to be independent of external Ca contained in the bathing solution, because the contraction induced by Ca or Ba added to high K medium was suppressed by the application of ATP, and dependent on the presence of external Mg, Mn and La. The Ca-induced contraction was suppressed by papaverine, but the Mg-induced contraction was not. In view of above results, whether the structural basis for such a Ca-independent contraction is due to actomyosin system, or due to another system such as cytoskeleton remains to be clarified.