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

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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.