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1.在神經系統,細胞內Ca2+濃度的平衡是維持神經活性相當重要的機制, 而細胞內鈣離子儲存處在此扮演了相當重要的角色.粒腺體為鈣離子儲存 處之一,而且也是主要能量生產工廠,而CCCP(carbonyl cyanide m- chlorophenyl-hydrazone)其為氧化磷酸化抑制劑,會抑制ATP的產生及粒 腺體對鈣離子的回收.因此在本論文利用CCCP來研究胚胎期運動神經的活 性並與蛋白激脢丙活化劑TPA比較其作用.2.Glutamate是中樞神經興奮性 傳遞物質,在本細胞培養,給予運動神經glutamate,NMDA和kainate均可以 促使ACh自發性的釋放增加.但在CCCP存在下,這些藥物的作用只有短暫的 增加SSC頻率,隨即沒有任何的SSCs出現.而高鉀也有類似的作用,但是TPA 仍然可以促進ACh的釋放,顯示在短期內synaptic vesicles並未完全遭到 排空,堆測可能是因為glutamate受體激活劑或高鉀在CCCP存在下使細胞內 鈣離子增加過多造成神經的傷害,使SSCs消失.3.在鈣離子測定實驗中,得 知CCCP可以促使胞內的鈣離子儲存處釋放鈣離子至細胞質.而在CCCP存在 下,由glutamate受體激活劑所引起細胞內鈣離子濃度的增加可以被促進, 更顯示所造成SSCs消失,應與胞內鈣離子有密切關係.4.由此論文得知TPA 與CCCP增加神經活性可以經不同的機制;TPA的部份作用是經由活化PKC,促 使與exocytosis有關之蛋白磷酸化,即使在細胞內鈣離子濃度沒有增加之 情況均可以增加自發性ACh的釋放,而CCCP主要是經由增加鈣離子儲存處釋 放鈣離子.此外,CCCP也會加速glutamate受體激活劑或高鉀對神經的毒性, 不過,TPA卻不受影響.顯示CCCP促使神經毒性的產生與胞內鈣離子有關. 1. Intracellular calcium homeostasis is very important for the regulation of neuronal activity in nervous system. The intracellular calcium stores play important role in maintaining neuronal function. Mitochondria is one of calcium stores and also the factory of energy's source. CCCP (carbony1 cyanide m-chlorophenylhydrazone) inhibits oxidative phosphorylation and the uptake of calcium by mitochondria. We investigated the effects of CCCP on the synaptic transmission and neuronal toxicity and used TPA (12- o- tetradecanoy1-phorbol-13-acetate) for comparision.2. Glutamate is an important excitatory neurotransmitter in central nervous system. In this developing motoneuron, application of glutamate(30uM), NMDA(100 uM), or kainate (100 uM) markedly increased spontaneous Ach release and persisted more than ten minutes. However in the presence of CCCP, glutamate, NMDA, or kainate increased SSC frequency only transiently and SSC then disappeared. High k+ (20 mM) but not TPA exerted similar effects to glutamate in the presence of CCCP.3. Direct measurements of [Ca2+]i in the single neuronal soma was carried out using fura-2 unagubg. CCCP and glutamate receptor agonists additively or synergistically increased [Ca2+]i.4. In conclusion, TPA and CCCP may enhance the spontaneous ACH release via the different mechanisms. Increase of [Ca2+]i is essential for the action of CCCP. Whereas TPA can increase ACH release without the elevation of [Ca2+]i , possibly by the phosphorylation of synaptic proteins related to the exocy- tosis of neurotransmitter. CCCP potentiated the neurotoxicity of glutamate receptor agonists or high K+. Intracellular depletion of ATP and [Ca2+]i increase may contribute the neurotoxicity caused by CCCP plus glutamate receptor agonists or high or high K+.
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