TRP 通道為一種非專一性陽離子通道，在視覺、味覺、嗅覺、聽覺或觸覺等感覺生理上扮演重要角色，協助生物體偵測周圍環境或壓力的變化。2-APB是大部份TRP通道的抑制劑，但在我們爪蟾神經肌-突觸培養中，利用whole-cell patch clamp記錄方式發現，單獨施予2-APB反而造成運動神經自發性神經傳遞物質釋放量的上升。進一步將細胞外溶液置換成不含鈣離子的生理緩衝液(Ca2+ free Ringer)、加入廣效性鈣離子通道抑制劑Cd2+或膜通透性高的鈣離子螯合劑BAPTA-AM，再觀察2-APB的作用情形，發現皆能有效地抑制2-APB促進神經傳遞物質釋放作用，顯示2-APB促進神經活性與細胞外鈣離子有關。於SKF96365、flufenamic acid或RuR (TRP 通道的抑制劑)存在時，2-APB的促進作用亦消失。利用cell-attached patch-clamp單一離子通道電流紀錄方法觀測2-APB開啟離子通道後所造成的電流訊號，但是當廣效性TRP通到抑制劑SKF96365存在的情況下，2-APB所活化的離子通道活性明顯減小。因此我們認為2-APB影響突觸前神經傳遞物質的釋放，與TRP通道活性有極大的關係。
而當我們進行實驗前，事先投予各種不同訊息傳遞路徑的抑制劑至培養基中，再觀察2-APB的作用，發現當PLC抑制劑（U73122)，tyrosine kinase 抑制劑(Genistein)或DAG類似物(PMA)存在的情況下，2-APB促進神經活性的作用受到抑制。將培養液置換成不含胎小牛血清（fetal calf serum）的生理緩衝溶液，2-APB的促進作用不再產生。總括以上結果我們認為在爪蟾神經肌細胞培養中，培養液中的血清含有成長因子，能活化膜上受體使tyrosine kinase活化，啟動PI3K和PLC訊息路徑產生DAG，進而打開TRP通道，加入2-APB可能增強TRP通道的活化作用，造成神經細胞末梢內的鈣離子濃度上升，而促進神經傳遞物質大量釋放。

The transient receptor potential (TRP) channel superfamily is a non-selective Ca2+-permeable cation channels involved in sensory physiology. Here we show that 2-aminoethoxydiphenyl borate (2-APB), a compound commonly used as TRP channel inhibitor, dose-dependently induce a significant facilitation on the frequency of spontaneous neurotransmitter release at developing Xenopus neuromuscular junction through, surprisingly, TRP channel activation. Bath application of universal TRP channel inhibitors either SKF96365, flufenamic acid or RuR cease the 2-APB-induced synaptic facilitation. Exclusion of Ca2+ from culture medium or bath application of the pharmacological Ca2+ channel inhibitor cadmium, membrane-permeable Ca2+ chelator BAPTA-AM, effectively hampered the facilitation of neurotransmitter release induced by 2-APB, suggesting Ca2+ influx is requisite for 2-APB-induced synaptic facilitation. Blockade of the voltage-dependent Ca2+ channel with either nifedipine, verapamil or ω-CTX failed to abolish the SSC facilitating effect of 2-APB. Electrophysiological recording of 2-APB induced single channel currents by using cell-attached patch-clamp technique reveals 2-APB evoked a robust single channel activity recorded at different pipette voltages. Furthermore, the 2-APB-evoked single-channel events are significantly abolished in the presence of SKF96365.
Either pretreatment of the cultures with inhibitor of phospholipase C (U73122) or tyrosine kinase (Genistein) abolishes 2-APB induced potentiation of synaptic transmission. The structure of PMA is analogous to diacylglycerol (DAG), which abolishes 2-APB induced synaptic facilitation. 2-APB no longer elicited any changes in SSC frequency when serum is eliminated from culture medium. Overall, results from our current study provide evidences that 2-APB induces the opening of TRP channels and Ca2+ influx which resulting in facilitation of spontaneous neurotransmitter release at developing Xenopus neuromuscular synapse. Serum may activate tyrosine kinase to turn on PI3K and phospholipase C. Then phospholipase C cleavage PIP2 to IP3 and diacylglycerol, and diacylglycerol induced TRP channel opening. 2-APB potentiates and sensitizes the TRP channel, increasing Ca2+ inffux. Elevated [Ca2+]i resulted in enhancement of neurotransmitter release from presynaptic nerve terminal.

縮寫表 1
中文摘要 2
英文摘要 4
緒論 6
實驗材料 13
實驗方法 15
§ 電生理紀錄方法 15
§ 單一離子通道電流紀錄方法 16
§ 量測細胞內鈣離子濃度 17
§ 胚胎顯微注射 18
結果 21
討論 35
參考文獻 44
圖表 52
圖 表 順 序
附圖1 52
附圖2 53
附圖3 54
附圖4 55
Fig 1. 57
Fig 2. 58
Fig 3. 60
Fig 4. 62
Fig 5. 64
Fig 6. 67
Fig 7. 69
Fig 8. 71
Fig 9. 73
Fig 10. 75
Fig 11. 78
Fig 12. 80
Fig 13. 82
Fig 14. 84
Fig 15. 85

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