代謝性抑制劑對安非他命及普卡因引起之猝發動作電位影響之研究

Effects of metabolic inhibitors on the potential changes elicited by d-amphetamine and procaine

盧冠伶(Guan-Ling Lu)

指導教授 ： 蔡明正

國立臺灣大學/醫學院/藥理學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02022

摘要

1.本論文主要探討代謝性抑制劑 (metabolic inhibitors, NaN3、rotenone及KCN) 對於非洲大蝸牛 (Achatina fulica) 之RP4神經元之 (1) 自發性動作電位的影響； (2) d-amphetamine引發RP4神經元產生猝發現象之相關性。

2.正常生理溶液灌流下，RP4神經元會產生規律的自發性動作電位(spontaneous action potential)。當投予低濃度的代謝性抑制劑 NaN3 (30、100、300 μM、1及3 mM)、rotenone (3、10及30 μM)及KCN (0.3、1 及3 mM) 經灌流60分鐘後，對RP4神經元的自發性動作電位並無影響。

3.當投予高濃度的代謝性抑制劑 NaN3 (30 mM) 20分鐘、rotenone (100 μM) 10分鐘及KCN (10 mM) 10分鐘後，RP4神經元的自發性動作電位會被抑制且靜止膜電位有去極化的現象。

4.NaN3 (30 mM) 會使RP4神經元自發性動作電位逐漸消失且有去極化的現象。利用電流箝制 (current clamp) 的方法給予一個過極化的電流後，可以觀察到動作電位有部分恢復的現象，表示NaN3 (30 mM) 導致RP4神經元之自發性動作電位消失與去極化有關。

5.高濃度的NaN3 (30 mM) 會使RP4神經元自發性動作電位的振幅逐漸消失。利用膜電位箝制 (voltage clamp) 發現高濃度的NaN3 (10及30 mM) 會減少RP4神經元之總內向電流，推論此總內向電流的減少與RP4神經元自發性動作電位的振幅逐漸消失有關。為了了解低濃度的NaN3 (30 μM) 是否經由影響離子電流來促進低濃度的d-amphetamine (135 μM) 產生猝發現象。利用膜電位箝制 (voltage clamp) 發現NaN3 (30 μM) 對總內向及總外向電流的影響皆不顯著。顯示NaN3 在30 μM時，並不經由離子電流的改變來促進低濃度d-amphetamine (135 μM) 產生猝發現象。

6.NaN3 (10及30 mM) 對RP4神經元input resistance的影響並不顯著；且NaN3 (10及30 mM) 對總外向電流的影響也不顯著，表示NaN3 (10及30 mM) 對RP4神經元細胞膜鉀離子的permeability無顯著之影響。

7.投予較低濃度之d-amphetamine (135 μM) 60分鐘後，不會引起RP4神經元產生猝發性動作電位，然而若投予較高濃度的d-amphetamine (270 μM) 經30分鐘，即可誘發猝發現象產生。顯示d-amphetamine引起的猝發性動作電位具有濃度依賴性。

8.使用低濃度的代謝性抑制劑NaN3 (30 μM)、rotenone (10 μM) 及KCN (1 mM) 灌流60分鐘後，再給予較低濃度的d-amphetamine (135 μM) 60分鐘，則RP4神經元會產生猝發性動作電位。結果顯示低濃度的代謝性抑制劑有促進低濃度的d-amphetamine (135 μM) 產生猝發性動作電位的能力。

9.使用低濃度的代謝性抑制劑 NaN3 (30 μM)、rotenone (10 μM) 及KCN (1 mM) 灌流60分鐘後，再給予較低濃度的procaine (5 mM) 60分鐘，則RP4神經元會產生猝發性動作電位。結果顯示低濃度的代謝性抑制劑有促進低濃度的procaine (5 mM) 產生猝發性動作電位的能力。

10.若以KT5720 (10 μM) 灌流RP4神經元60分鐘，再加入 NaN3 (30 μM) 60分鐘及d-amphetamine (135 μM) 60分鐘後，RP4神經元無猝發現象產生。此結果顯示KT5720 (10 μM) 可以抑制NaN3 (30 μM) 促進低濃度的d-amphetamine (135 μM) 產生猝發性動作電位，所以經NaN3 (30 μM) 促進低濃度的d-amphetamine (135 μM)產生猝發性動作電位的現象，極有可能與cAMP-dependent protein kinase system的路徑有關。

11.預先以NaN3 (30 μM) 促進低濃度的d-amphetamine (135 μM) 產生猝發性動作電位後，接著投予phospholipase C抑制劑U73122 (10

關鍵字

代謝性抑制劑 ； 安非他命 ； 猝發現象

並列摘要

The roles of metabolic inhibitors (including NaN3, rotenone and KCN) on (1) spontaneous action potential and (2) bursts of potential elicited by d-amphetamine were studied on RP4 neurons of snail, Achatina fulica Ferussac in vitro.
Metabolic inhibitors altered the spontaneous action potential in a concentration dependant manner. Lower concentrations of NaN3 (30, 100, 300 μM, 1 and 3 mM), rotenone (3, 10 and 30 μM) and KCN (0.3, 1 and 3 mM), did not affect the resting membrane potential, amplitude and frequency on RP4 neurons, while higher concentrations of NaN3 (30 mM), rotenone (100 μM) and KCN (10 mM) did abolish the spontaneous action potential on RP4 neurons and depolarize the RP4 neurons. The action potential was partially recovered that if injected a hyperpolarized current into the depolarized RP4 neurons. NaN3 (10 and 30 mM) did not alter the input resistance of the RP4 neurons. Voltage clamp studies revealed that NaN3 (10 and 30 mM) decreased the total fast inward currents (70 ms) while the steady-state outward currents (5 s) were not altered. These results suggested that NaN3 (10 and 30 mM) abolished the spontaneous action potential on RP4 neurons by depolarizing RP4 neurons and decreasing the total fast inward currents, while the input resistance and the permeability of potassium ion on RP4 neurons were not involved.
Interactions between metabolic inhibitors and d-amphetamine or procaine were studied. No bursts of potential were found 60 min after administration of d-amphetamine (135 μM) or 120 min after administration of metabolic inhibitors, NaN3 (30 μM), rotenone (10 μM) or KCN (1 mM) on RP4 neurons. However, bursts of potential were found 60 min after d-amphetamine (135 μM) administration if NaN3 (30 μM), rotenone (10 μM) or KCN (1 mM) was treated 60 min prior to d-amphetamine administration. No burst of potential was found 60 min after administration of procaine (5 mM) on RP4 neurons. However, bursts of potential were found 60 min after procaine (5 mM) administration if NaN3 (30 μM), rotenone (10 μM) or KCN (1 mM) was treated 60 min prior to procaine administration. These results suggested that lower concentrations of metabolic inhibitors could facilitate the bursts elicited by d-amphetamine (135 μM) or procaine (5 mM).
The bursts of potential elicited by NaN3 (30 μM) and d-amphetamine (135 μM) decreased following treatment with KT5720 (protein kinase A inhibitors) or intracellular injection of EGTA. However, the bursts of potential were not affected by applying U73122 or neomycin (phospolipase inhibitors). Voltage clamp studies revealed that NaN3 (30 μM) didn’t significantly alter total fast inward and steady-state outward currents. These results suggested that the bursting activity elicited by NaN3 (30 μM) and d-amphetamine (135 μM) was mainly due to PKA related messenger system and intracellular calcium, while phospholipase activity and change of ion current were not involved.
It is concluded that metabolic inhibitors abolished the spontaneous action potential on the RP4 neurons. The effect was associated with depolarizing RP4 neurons and decrease of fast total inward currents, while it was not associated with input resistance and the permeability of potassium ion on RP4 neurons. The lower concentrations of metabolic inhibitors could facilitate the bursts of potential elicited by lower concentrations of d-amphetamine and procaine. The effect of NaN3 and d-amphetamine may be related to PKA and intracellular calcium, while it was not related to phospholipase and change of ion currents.

並列關鍵字

metabolic inhibitor ； d-amphetamine ； bursting

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