臺灣博碩士論文加值系統

本文主旨利用一般藥理學及電生理學的方式，探討三氧化二砷(arsenic trioxide, As2O3)對於非洲大蝸牛(Achatina fulica)食道下神經節中RP4神經元之電生理作用，進一步了解arsenic trioxide引起神經毒性的機轉。

正常生理溶液灌流下，RP4神經元會產生規則的自發性動作電位。細胞外投予低劑量的arsenic trioxide (1~3 mM)時，並不會影響RP4神經元膜電位(resting membrane potential, RMP)及動作電位振幅(amplitude)，但動作電位頻率(frequency)會減少。然而投予高劑量arsenic trioxide (10 mM)時，則會影響RP4神經元產生兩種階段的膜電位、動作電位頻率及振幅變化。投予藥物20鐘後，膜電位產生去極化現象(depolarization)，此時動作電位的頻率加快，動作電位的振幅減少(階段1)。然而投予藥物30分鐘後，膜電位產生過極化現象(hyperpolarization)，此時動作電位變成猝發形式(burst firing)(階段2) ，猝發現象在投予藥物60分鐘後仍存在。投予arsenic trioxide (10 mM) 60分鐘後，使用正常生理溶液灌洗藥物60分鐘，細胞無法回復至未加藥前狀態，顯示(10 mM) arsenic trioxide是不可逆的（irreversible）影響細胞電生理特性。以更高濃度arsenic trioxide (30 mM)投予RP4神經元時，10分鐘後，膜電位比起加藥前快速地偏正(去極化)，動作電位頻率加快，振幅變小，最後動作電位消失，沒有過極化發生。投予arsenic trioxide (30 mM) 10分鐘後，使用正常生理溶液灌洗藥物60分鐘，無法回復至未加藥前狀態，顯示(30 mM) arsenic trioxide是不可逆的(irreversible)影響細胞電生理特性。

以鋰離子取代鈉離子的細胞外液灌流RP4神經元40分鐘，此時神經元膜電位產生去極化，接著投予arsenic trioxide (10~30 mM)則不會影響神經元膜電位。以Tris取代鈣離子的細胞外液灌流RP4神經元30分鐘，此時神經元膜電位產生去極化，接著投予arsenic trioxide (10~30 mM)，arsenic trioxide (10 mM)並不影響神經元膜電位，然而投予arsenic trioxide (30 mM) 10分鐘後，膜電位仍產生去極化現象。以高鉀 (25 mM)溶液灌流RP4神經元30分鐘，此時神經元膜電位產生去極化現象，接著投予arsenic trioxide (10~30 mM)， arsenic trioxide (10 mM)仍影響神經元膜電位產生去極化及過極化現象，投予arsenic trioxide (30 mM) 10分鐘後，膜電位仍產生去極化現象。以乙酸根取代氯離子的細胞外液灌流RP4神經元40分鐘，此時神經元膜電位產生過極化，接著投予arsenic trioxide (10~30 mM)， arsenic trioxide (10 mM)仍影響神經元膜電位產生去極化現象，投予arsenic trioxide (30 mM)10分鐘後，膜電位仍產生去極化現象。顯示細胞外溶液以鋰離子取代鈉離子可拮抗arsenic trioxide (10~30 mM)引起的膜電位(resting membrane potential)變化現象。

預先投予細胞U73122 (10 μM) (phospholipase C, PLC 抑制劑) 60分鐘，再投予arsenic trioxide (10 mM)，20分鐘後並無沒有膜電位去極化，30分鐘後也沒有過極化的現象。顯示arsenic trioxide引起的膜電位變化與PLC可能有關。

預先細胞內注射或細胞外投予維他命C (100 μM)，再投予arsenic trioxide (10 mM)，20分鐘後膜電位仍去極化(階段 1)，30分鐘後膜電位過極化，此時產生猝發現象(階段2)。顯示維他命C不可影響arsenic trioxide引起的膜電位變化現象，arsenic trioxide引起的膜電位變化與活性氧分子(reactive oxygen species，ROS)可能無關。

預先投予verapamil (60 μM) (voltage-dependent Ca2+ channel 抑制劑) 60分鐘，再投予arsenic trioxide (10 mM)，20分鐘後膜電位仍去極化(階段 1)，30分鐘後膜電位過極化，此時產生較嚴重的猝發現象(階段 2)，60分鐘後投予細胞 neomycin (3 mM)(PLC抑制劑)，能減緩這嚴重猝發現象。顯示抑制voltage-dependent Ca2+channel會促使 arsenic trioxide 引起的反應變得劇烈，抑制PLC能減緩這劇烈反應。

預先投予KB-R7943 (30 μM) (Na+/Ca2+ exchanger抑制劑) 60分鐘，再投予arsenic trioxide (10 mM)，20分鐘後膜電位仍去極化(階段1)，30分鐘後膜電位過極化，此時產生較嚴重的猝發現象(階段2)。顯示抑制Na+/Ca2+ exchanger會促使arsenic trioxide引起的反應變得劇烈。

預先投予glybenclamid (100 μM) (KATP 通道抑制劑) 60分鐘，再投予arsenic trioxide (10 mM)，20分鐘後膜電位仍去極化(階段 1)，30分鐘後膜電位過極化，但此時產生較嚴重的較猝發現象(階段 2)，60分鐘後给予細胞 neomycin (3 mM) (PLC抑制劑)，能有效的抑制這嚴重猝發現象。顯示抑制KATP 通道會促使arsenic trioxide 引起的反應變得劇烈，抑制PLC能拮抗這劇烈反應。

預先投予細胞d-amphetamine (135 μM) 60分鐘(此濃度為不產生猝發反應的濃度)再投予低濃度arsenic trioxide (3 mM)，90分鐘後即可見RP4神經元產生猝發現象。顯示低濃度arsenic trioxide (3 mM)能夠促進低濃度d-amphetamine (135 μM)引起神經細胞產生猝發現象。

利用膜電位箝制(voltage clamp)的實驗方式，測試arsenic trioxide 10 mM對於總內向離子電流和steady-state外向離子電流的影響，並作成電流與電位的關係圖(current-voltage relationship)，發現投予arsenic trioxide時，對內向電流產生抑制作用，但會增加細胞steady-state外向電流，此增加現象是不可逆的。若預先投予細胞KATP通道的抑制劑glybenclamid (100 μM) 60分鐘後，再投予arsenic trioxide (10 mM)，並沒有增加其外向電流。顯示arsenic trioxide能增加steady-state外向電流，抑制KATP通道可拮抗arsenic trioxide引起的外向電流增加。

為了探討arsenic trioxide (10 mM)對RP4神經元細胞膜電阻(total input resistance)的影響。投予arsenic trioxide (10 mM)後，分別在30分鐘以及60分鐘時，注射-1nA、-2nA電流，觀察膜電位變化，最後以數學算式計算 RP4神經元膜電阻(membrane resistance)變化。結果顯示雖然arsenic trioxide (10 mM)會隨著給藥的時間而增加細胞膜的電阻(total input resistance)，然而統計上並沒有顯著差異。

本論文實驗結果發現在非洲大蝸牛之RP4神經元上，投予arsenic trioxide (10 mM)可引起RP4神經元產生細胞膜電位去極化(階段1)，接著過極化(階段2)的反應。此膜電位變化可能與細胞內外鈉、鈣離子有關，並可能減少細胞內ATP而活化KATP 通道，保護細胞免於太過興奮。Arsenic trioxide (10 mM)的細胞膜電位變化可能有PLC訊息途徑參與作用，與ROS可能無關。

Effects of arsenic trioxide on excitable membrane were tested on isolated RP4 neuron from subesophageal ganglia of snail, Achatina fulica Ferussac elctrophysiologically.

Spontaneously generated action potential was observed in isolated RP4 neuron. Arsenic trioxide affected the excitable membrane by a concentration dependent manner. Arsenic trioxide (1-3 mM) did not alter the resting membrane potential, amplitudes of the spontaneously generated action potential on the RP4 neuron even after 1 hr of perfusion, while the frequency of the spontaneously generated action potential was decreased. Twenty min after administration of arsenic trioxide (10 mM), the resting membrane potential of RP4 neuron was depolarized and the frequency of the action potential was increased and the amplitude of action potential was decreased (phase 1). Thirty min after arsenic trioxide (10 mM) administration, the resting membrane potential was hyperpolarized and it also elicited bursts of potential (phase 2). The bursts of potential lasts for more than 60 min. Ten min after arsenic trioxide (30min) administration, the resting membrane potential was depolarized and the action potential was disappeared. After a sixty min wash, the RMP, frequency and amplitude of action potential did not recover to control level from arsenic trioxide.

The effects of arsenic trioxide (10 mM) on the RMP were not altered in high potassium solution but altered in chloride free solution. Arsenic trioxide (10 mM) elicited bursts of potential in chloride free solution, while arsenic trioxide (10 mM) did not elicit bursts of potential in high potassium solution. Arsenic trioxide (10-30 mM) did not depolarize or hyperpolarize the RP4 neuron in lithium substituted for sodium solution. Arsenic trioxide (10 mM) did not depolarize or hyperpolarize the RP4 neuron in calcium free solution and also did not elicit bursts of potential of the RP4 neuron.While the effects of arsenic trioxide (30 mM) on the RMP were not altered in calcium free solution.

U73122 (10 μM) did not alter the RMP, frequency and amplitudes of RP4 neurons 60 min after incubation. In the presence of U73122, arsenic trioxide (10 mM) neither depolarized nor hyperpolarized the RMP of RP4 neuron. Arsenic trioxide (10 mM) altered neither the frequency nor the amplitude of action potential of RP4 neuron. No bursts of potential were found in arsenic trioxide (10 mM) and U73122 treated preparation. Intra-cellular injection or extra-cellular application of ascorbic acid (vitamine C) did not alter the RMP, frequency and amplitudes of RP4 neurons elicited by arsenic trioxide (10 mM). The effect of arsenic trioxide (10 mM) on RMP, action potential of RP4 neuron was not altered in intra-cellular ascorbic acid injected neuron or extra-cellular application of ascorbic acid (100 μM ).

Verapamil (60 μM) did not alter the RMP, frequency and amplitudes of RP4 neurons 60 min after incubation. In the presence of verapamil, the resting membrane potential of RP4 neuron was depolarized and the frequency of the action potential was increased and the amplitude of action potential was decreased if arsenic trioxide (10 mM) was added for 20 min, and the RMP was hyperpolarized 30 min after arsenic trioxide administration. Bursts of potential were also found in verapamil and arsenic trioxide treated preparation, and this duration of bursts of potential was prolonged. Neomycin decreased the bursts of potential elicited by arsenic trioxide.

KB-R7943 (30 μM) did not alter the RMP, frequency and amplitudes of RP4 neurons after 60 min of incubation. In the presence of KBR-7943 (30 μM), the resting membrane potential of RP4 neuron was depolarized and the frequency of the action potential was increased and the amplitude of action potential was decreased if arsenic trioxide (10 mM) was added for 20 min, and the RMP was hyperpolarized 30 min after arsenic trioxide administration. Bursts of potential was also found in KBR-7943 (30 μM) and arsenic trioxide treated preparation, and this duration of bursts of potential was prolonged.

Glybenclamid (100 μM) did not alter the RMP, frequency and amplitudes of RP4 neurons after 60 min of incubation. In the presence of glybenclamid (100 μM), the resting membrane potential of RP4 neuron was depolarized and the frequency of the action potential was increased and the amplitude of action potential was decreased if arsenic trioxide (10 mM) was added for 20 min, and the RMP was hyperpolarized 30 min after arsenic trioxide administration. Bursts of potential were also found in glybenclamid and arsenic trioxide 10 mM treated preparation, and the duration of bursts was prolonged. Neomycin blocked the bursts of potentials elicited by glybenclamid and arsenic trioxide.

d-Amphetamine (135 μM) or arsenic trioxide (3 mM) did not elicit bursts of potential on RP4 neuron even after 180 min of treatment. However, bursts of potential were found if arsenic trioxide(3 mM) was added to preparation incubated with amphetamine (135 μM.).

Voltage clamped studies revealed that the fast inward current of RP4 neuron was decreased 20, 30 and 60 min after arsenic trioxide (10 mM) administration, while the steady outward current of RP4 neuron was increased 20, 30 and 60 min after arsenic trioxide (10 mM) administration. Glybenclamid (100μM) did not alter the fast inward and steady outward currents 60 min after incubation. However, arsenic trioxide (10 mM) did not increase the steady outward current in the presence of glybenclamid (100μM).

It is concluded that arsenic trioxide elicited electro-physiological changes on the excitable membrane. The effects of arsenic trioxide on the RMP of RP4 neuron may partially relate to the PLC activity of the neuron and the effect may not relate to the ROS of the neuron. The duration of bursts of potential elicited by arsenic trioxide was prolonged in the presence of verapamil and KB-R7943. The results suggest that intracellular calcium ion may in part contribut to the bursts of potential elicited by arsenic trioxide. Glybenclamide can block the arsenic trioxide-increased steady outward currents. The results suggest that arsenic trioxide may decrease the ATP to result in activating the KATP channel.

縮寫表 ( Abbreviations ) ------------------------------ i
一、中文摘要( Abstract in Chinese ) ------------------- 1
二、英文摘要( Abstract in English ) ------------------- 5
三、緒論( Introduction ) ------------------------------ 8
四、實驗方法與材料( Materials and Methods ) ---------- 12
五、結果( Result ) ----------------------------------- 17
六、討論與結論( Discussion and Conclusion ) ---------- 33
七、參考文獻( References ) --------------------------- 41
八、圖表( Figures and Tables ) ------------------------47

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