研究Dynasore對牛腎上腺嗜鉻細胞胞吐與 胞吞作用之影響

Abstract

Dynamin為一GTP分解酵素，在細胞的胞吞作用中扮演關鍵的角色，其功能為負責將吞入的小泡從細胞膜上分離下來。而dynasore為一新發現之dynamin活性抑制劑，相較於過去其它抑制dynamin活性的方法，dynasore可直接穿透細胞膜，且對於dynamin具有較好的專一性。在我們的實驗當中，利用10 ~ 100 μM的dynasore 抑制dynamin後，細胞在5 Hz，2秒鐘的去極化刺激之下，膜電容值下降之速率遭到 抑制。同時，我們也觀察膜電容上昇值的變化，可以發現dynasore的抑制對於在相 同的刺激條件下，膜電容值之上昇沒有造成顯著地改變，但在休息2分鐘後的第二 次刺激，在50 μM的dynasore處理狀況中，膜電容值的上昇就被抑制了。利用碳纖 維電極觀察胞吐作用過程，嗜鉻細胞胞吐作用之機制並不會因100 μM dynasore之 處理而遭到抑制。另一方面，在50 μM dynasore的條件中，dynasore可能改變細胞 內鈣離子濃度的恆定性，使鈣離子濃度在60 mM高鉀溶液地刺激之下無法造成一樣 地上昇。綜觀我們的實驗結果，我們認為10~100 μM dynasore會抑制胞吐作用，然 而可能並不直接影響胞吐作用的進行，而由於dynasore也會影響細胞中鈣離子濃度 之變化，而這也可能對胞吞與胞吐作用造成影響，是否dynasore只是透過抑制 dynamin的方式，抑或是透過影響鈣離子反應之機制，來影響胞吐胞吞作用，則需 要透過更多的實驗來釐清其中的機制。

Dynamin is a GTPase, and it plays a very important role in the process of endocytosis. When the vesicles are budded into the cell, it cannot be severed from the membrane without the working of dynamin. Dynasore is a novel inhibitor of the activities of dynamin. Compared with other dynamin inhibitor, dynasore is membrane permeable, and it has higher specificity than most inhibitors that have been used. In our experiments, the decreasing rate of membrane capacitance is inhibited significantly under 5 Hz, 2 seconds depolarization after the treatment of 10 ~ 100 μM dynasore. However, dynasore has no effects on the increasing rate of membrane capacitance under the same depolarization, but after 2 minutes resting, the increasing of capacitance is inhibited under the same depolarization in 50 μM dynasore. Using amperometric method to assess the mechanism of exocytosis, 100 μM dynasore does not interfere on the mechanism of exocytosis. Besides, the excitability of calcium homeostasis is altered. When the high potassium (60 mM) solution is used to stimulate the cells, it causes the smaller raising of calcium concentration. Above all, the endocytosis is inhibited under 10 ~ 100 μM dynasore, but it seems no effects on exocytosis. However, dynasore affects the homeostasis of calcium, which plays a key role on exo-endocytoisis. In terms of the direct inhibition of dynamin, or in terms of the alteration of calcium concentration to affect the exo-endocytosis, it needs more experiments to elucidate the detail mechanism.