臺灣博碩士論文加值系統

一、本文利用isoquinolinone之衍生物BDPBI [ 7-bromo-1,4-dihydro- 2-phenyl- 4,4-bis(4-pyridinylmethyl)-2H-isoquinolin-3-one ]，以一般藥理學與電生理學的方法，探討BDPBI對於非洲大蝸牛（Achatina fulica Ferussac）食道下神經節（suboesophageal ganglion）中的RP4, RP2及LP4神經元之藥理作用。
二、正常生理溶液灌流下，非洲大蝸牛之食道下神經節的RP4神經元會產生規則的自發性動作電位(spontaneous action potential)。BDPBI ( >150 μM )投予40分鐘後，可觀察到RP4神經元的動作電位由規律自發性的動作電位轉為猝發性(burst firing)動作電位。此猝發現象可持續3~4小時。然而在另外兩個具有規則自發性動作電位的LP4和RP2神經元上，以BDPBI (150 μM )投予40分鐘後，只會稍微減小LP4和RP2神經元的動作電位振幅和膜電位，不影響動作電位之頻率，並且無法引起猝發現象。
三、給予BDPBI（50 μM）40分鐘後，並不會引起RP4神經元產生明顯的猝發現象，此時再以高鉀離子生理溶液（high-K+ physiological solution, 12 mM）灌流RP4神經元20分鐘後，即產生明顯的猝發現象。
四、BDPBI (150 μM)使RP4神經元產生猝發現象後，再以高鎂離子生理溶液(high-Mg2+ physiological solution, 30 mM)灌流20分鐘，此猝發現象會受到抑制，形成單一波峰的動作電位。
五、BDPBI (150 μM)使RP4神經元產生猝發現象後，分別投予AP-5 (DL-2-amino-5-phosphonopentanoic acid, NMDA受體的抑制劑; 1mM)或是hexamethonium (100 μM)、d-tubocurarine (100 μM)、atropine (1 mM)、prazosin (100 μM)、propranolol (100 μM)和haloperidol（260 μM），經過40分鐘後，BDPBI所引起的猝發現象仍然存在。
六、BDPBI（150 μM）於RP4神經元引起猝發現象時，換置成缺鈣離子且含EGTA (2 mM)之生理溶液(Ca2+-free physiological solution contained EGTA 2 mM)灌流20分鐘後，BDPBI所引起的猝發性動作電位的波峰數目變少，且振幅變小，但是並無法完全抑制猝發現象。
七、BDPBI（150 μM）於RP4神經元引起猝發現象後，以鋰離子取代鈉離子之生理溶液(sodium replaced by lithium physiological solution)灌流或是直接投予LiCl (50 mM)，皆可以抑制BDPBI所引起的猝發現象，形成單一波峰的動作電位。
八、在BDPBI（150 μM）引起RP4神經元產生猝發現象後，投予phospholipase C抑制劑neomycin (3.5 mM)經40分鐘，可以抑制BDPBI所引起的猝發現象，形成單一波峰的動作電位。
九、在BDPBI（150 μM）引起RP4神經元產生猝發現象後，投予phospholipase C抑制劑U73122 (6 μM)經40分鐘，可以抑制BDPBI所引起的猝發現象，形成單一波峰的動作電位；若以U73122 (6 μM)預先處理RP4神經元40分鐘，再給予BDPBI (150 μM) 40分鐘，BDPBI並無法引起RP4神經元產生猝發現象。
十、在BDPBI（150 μM）引起RP4神經元產生猝發現象後，再投予protein kinase C抑制劑chelerythrine (30 μM)經40分鐘，無法抑制BDPBI所引起的猝發現象。
十一、在BDPBI（150 μM）引起RP4神經元產生猝發現象後，再投予protein kinase A抑制劑KT5720 (20 μM)經40分鐘，無法抑制BDPBI所引起的猝發現象。
十二、BDPBI（150 μM）於RP4神經元引起猝發現象時，投予U73122 (6 μM) 40分鐘，在猝發現象被抑制住之後，再投予d-amphetamine (270 μM)，60分鐘後可引起另一猝發現象。
十三、利用膜電位箝制(voltage clamp)的實驗方式，測試BDPBI對於steady-state外向離子電流、總內向離子電流和鈣離子電流的影響，並做成電流與電位的關係圖(current-voltage relationship)，發現BDPBI對於外向離子電流有抑制的現象，但是在steady-state外向離子電流的I-V圖上，並不會引起negative slope resistance (NSR)；對於鈣離子電流則也有些微的抑制作用。
十四、BDPBI (150 μM)在RP4神經元引起猝發現象後，再分別投予diphenhydramine (500 μM)或chlorpheniramine (500 μM)經60分鐘後，BDPBI引起的猝發現象受到抑制，形成單一波峰的動作電位。另外BDPBI（150 μM）於RP4神經元引起猝發現象後，再投予cimetidine (500 μM)經60分鐘，並不會抑制BDPBI所引起的猝發現象。
十五、以HTMT ( 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide, H1受體的活化劑; 2 mM)在RP4神經元引起猝發現象後，再分別投予diphenhydramine (500 μM)或chlorpheniramine (500 μM) 60分鐘後，HTMT引起的猝發現象並不會受到抑制。
十六、本論文實驗結果發現在非洲大蝸牛的RP4神經元上，細胞外給予BDPBI( >150 μM )會引起明顯的猝發現象，此一猝發現象會受到細胞外鉀離子與鈣離子的影響，且並非經由興奮cholinergic, adrenergic, dopaminergic, histaminergic, NMDA受體而產生作用，而是經由活化phospholipase C的訊息傳遞路徑，以及抑制外向離子電流的表現，使細胞趨於興奮而產生猝發現象，且此過程中並不經由PKC以及PKA之作用。

Effects of BDPBI [ 7-bromo-1,4-dihydro-2-phenyl-4,4-bis( 4-pyridinylmethyl )-2H-isoquinolin-3-one] on the spontaneous action potentials of central neuron of giant African snails (Achatina fulica Ferussac) were studied pharmacologically and electrophysiologically. The RP4, RP2 and LP4 neurons showed a tendency to have a spontaneous firing of action potentials. Extracellular application of BDPBI (>150 μM) elicited burst firing of action potentials in RP4 neuron, but not in LP4 and RP2 neuron. BDPBI (50 μM) did not alter the spontaneous action potentials of RP4 neuron, while high-K+ solution (12 mM) facilitated the effects of BDPBI (50 μM) and induced the burst firing of action potential in RP4 neuron. The burst firing of action potentials elicited by BDPBI (150 μM) were inhibited by high-Mg2+ solution (30 mM), but not by d-tubocurarine (100 μM), hexamethonium (100 μM), atropine (1 mM), prazosin (100 μM), propranolol (100 μM), haloperidol (260 μM). Moreover, Ca2+-free solution contained EGTA (2 mM) did not completely inhibit the bursting activity elicited by BDPBI (150 μM). These results suggested that the bursting activity elicited by BDPBI (150 μM) was not due to the synaptic effect of neurotransmitters.
The BDPBI-elicited bursting activity was inhibited after extracellular administration with (1) phospholipase C inhibitor, neomycin (3.5 mM) and U73122 (6 μM) (2) sodium replaced by lithium solution and high concentration LiCl (50 mM). However, the burst firing of action potentials elicited by BDPBI were not affected after extracellular application of chelerythrine (30 μM, protein kinase C inhibitor) and KT5720 (20 μM, protein kinase A inhibitor).These results suggested that BDPBI induced bursting activity of action potential may be through phospholiapse C signal pathway in RP4 neuron.
The ionic currents of RP4 neuron were measured under voltage clamping. BDPBI significantly decreased the amplitude of total outward currents, but did not elicit a negative slope resistance (NSR) in I-V relationships of steady-state outward currents. Moreover, BDPBI also slightly decreased the amplitude of calcium currents.
It is concluded that BDPBI-elicited burst firing of action potential associated with the activity of phospholipase C and inhibition of outward currents.

目錄 ( Contents )
頁數
一、 中文摘要 ( Abstract in Chinese ) ----------------- 1
二、 英文摘要 ( Abstract ) ---------------------------- 5
三、 緒論 ( Introduction ) ---------------------------- 7
四、 實驗方法與材料 ( Materials and Methods ) --------- 13
五、 實驗結果 ( Results ) ----------------------------- 17
六、 討論 ( Discussion ) ------------------------------ 30
七、 參考文獻 ( References ) -------------------------- 37
八、 圖表 ( Figures ) --------------------------------- 45

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