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標題: | 鈉鈣交換蛋白在大鼠胚胎神經細胞上功能之研究 Functions of Na+/Ca2+ exchanger in rat embryonic neuronal cells |
作者: | Meng-Pei Wu 吳孟蓓 |
指導教授: | 潘建源 |
關鍵字: | 鈉鈣交換蛋白,神經,鈣庫,神經傳導物質, Na+/Ca2+ exchanger,neuron,intracellular store,neurotransmitter, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 細胞膜上的鈉鈣交換蛋白可分為K+-dependent (NCKX)及K+-independent (NCX)兩種,對調控胞內鈣離子濃度([Ca2+]i) 的恆定,扮演重要的角色。根據細胞膜內外的鈉鈣電化學濃度梯度,鈉鈣交換蛋白有將Ca2+打出細胞外(正轉) 及將Ca2+送入細胞內(反轉) 兩種模式。當胞內鈉離子濃度提高以及細胞去極化時,會傾向於引發鈉鈣交換蛋白的反轉,但鈉鈣交換蛋白反轉對神經細胞活性的調控,目前並不清楚。在本實驗中我們以Ca2+影像以及RT-PCR的方法,來偵測初級培養的大鼠胚胎大腦神經細胞中,[Ca2+]i變化及鈉鈣交換蛋白的表現。結果顯示NCX1~3及NCKX2~4皆表現於大鼠胚胎神經細胞中。使用ouabain處理細胞後,以不含Na+的NMG溶液刺激,能引發鈉鈣交換蛋白的反轉使[Ca2+]i上升,且NCX及NCKX皆參與此[Ca2+]i上升。Tg、2-APB及ryanodine分別可將[Ca2+]i 的上升抑制到對照組的30.94 ± 5.23, 31.89 ± 5.47, 及 53.78 ± 5.47 %,顯示胞內鈣庫參與在其中。為瞭解此[Ca2+]i上升是否牽涉到神經傳導物質的釋放,進一步以CNQX、TTX處理細胞,結果顯示兩種處理皆可抑制約35 %的[Ca2+]i 上升。此外,U73122將[Ca2+]i 上升抑制到對照組50.04 ± 7.46 %,顯示有PLC途徑的參與。且CNQX可幾乎完全抑制位於被刺激細胞下游的細胞之[Ca2+]i上升。最後,我們進一步以AMPA刺激細胞,使得[Ca2+]i上升,此[Ca2+]i上升可被Tg所抑制。相對的,高鉀溶液所引發的[Ca2+]i上升卻無法被Tg所抑制。這些結果顯示鈉鈣交換蛋白反轉可能在正常生理狀態下經由AMPA的開啟所引發,進一步使胞內鈣庫上的離子通道開啟並釋放出神經傳導物質,提高[Ca2+]i 。 Na+/Ca2+ exchanger is an antiporter on plasma membrane which transports Ca2+ out (forward mode) of or into (reverse mode) the cell depending on the electrochemical gradients of Na+ and Ca2+. The exchanger can be either K+-dependent (NCKX) or -independent (NCX). However, the roles of the reverse mode activity in the stimulus-secretion coupling are not clear in rat embryonic neuronal cells. Therefore, we monitored the intracellular Ca2+ concentration ([Ca2+]i) response by fura-2 fluorescence dye. The reverse mode activity can be activated by treating the cell with ouabain to increase intracellular Na+ concentration and replacing extracellular Na+ by NMG. This elevation in [Ca2+]i was inhibited by KB-R7943 and K+ free NMG.. Furthermore, we confirmed NCX1~3, and NCKX2~4 were expressed in rat embryonic neurons by RT-PCR technique. These results show that both NCX and NCKX are involved in this elevation. We also found this elevation in [Ca2+]i was inhibited by thapsigargin, 2-APB or ryanodine to 30.94 ± 5.23, 31.89 ± 5.47, or 53.78 ± 5.47 % of control cells, respectively. This elevation in [Ca2+]i could elevate [Ca2+]i in downstream postsynaptic neurons but blocked by CNQX. About 35 % of the elevation is also suppressed by U73122. These results suggest that the elevation in [Ca2+]i is facilitated by intracellular Ca2+ stores and can induce neurotransmitter release When neuron was stimulated by AMPA, the elevation in [Ca2+]i was significantly inhibited by thapsigargin; on the contrary, the elevation in [Ca2+]i elicited by high K+ stimulation was slightly enhanced by thapsigargin. We propose that Na+ influx through ionotropic receptors may not only depolarize the cell but also activate the reverse mode of Na+/Ca2+ exchanger to induce Ca2+ release from the intracellular stores and in turn release neurotransmitters to facilitate synaptic transmission. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34771 |
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