普卡因與安非他命引發動作電位猝發現象之藥理學研究

Pharmacological Study on Bursts Activity Elicited by Procaine and d-Amphetamine

林佳賢(Chia-Hsien Lin)

指導教授 ： 蔡明正

國立臺灣大學/醫學院/藥理學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.01250

摘要

本文利用procaine及d-amphetamine，以一般藥理學與電生理學的方法，探討procaine及d-amphetamine對於非洲大蝸牛(Achatina fulica Ferussac) 食道下神經節(suboesophageal ganglion)中的RP1神經元之藥理作用。在正常生理溶液灌流下，非洲大蝸牛食道下神經節 (suboesophageal ganglion) 的RP1神經元會產生規則的自發性動作電位。
細胞外投予procaine (10 mM) 能可逆性的誘發RP1神經元的動作電位由規律的自發性動作電位轉為猝發性 (burst firing) 動作電位。此猝發性 (burst firing) 的動作電位無法被(1) 事先投予prazosin (100 μM)、propranolol (100 μM)、d-tubocurarine (100 μM)、atropine (1 mM)，(2) 事先投予PKA 抑制劑KT-5720 (10 μM) 及H89 (10 μM)，(3) 事先投予PKC抑制劑chelerythrine chloride (10 μM) 及Ro 31-8220 (10 μM)，(4) 事先灌流缺鈣生理溶液 (Ca2+-free solution)，(5) 事先投予ryanodine (30 μM)，(6) 事先投予pentobarbital (30 μM)，及(7) 事先投予KB-R7943 (100 μM) 所抑制。這些結果顯示procaine所引起動作電位的猝發現象並非經由(1) cholinergic及adrenergic受體，(2) PKA及PKC相關的訊息傳遞路徑，(3) 細胞外鈣離子，(4) 對ryanodine 敏感的鈣離子通道，(5) 神經節中神經傳遞物質的釋放，及(6) 神經元上的鈉鈣交換而來。然而，動作電位的猝發現象可以被(1) 事先投予PLC 抑制劑U73122 (10 μM)，(2) 投予neomycin (3.5 mM)，(3) 投予高鎂 (30 mM) 生理溶液，(4) 灌流以鋰離子取代鈉離子之生理溶液，(5) 投予KCN (3 mM) 所抑制。低濃度的caffeine (10 mM) 及ruthenium red (100 μM) 能促進procaine 引發動作電位的猝發現象。m-3M3FBS (PLC activator) 會引起RP1神經元產生猝發現象。8-Bromo-cGMP (cGMP analogue) 並不會引起RP1神經元的猝發現象產生。細胞內注射鈣離子及procaine會引發動作電位的猝發現象產生。而在procaine (10 mM) 引起RP1神經元產生猝發現象後，細胞內注射高鎂離子，則會降低procaine所引起的猝發現象。而此猝發現象可能與phospholipase C活化及神經元內鈣離子的移動有關。
離子電流在蝸牛中樞神經元經由procaine 所引發動作電位猝發現象的角色以雙電極膜電位箝制電生理記錄法測量。膜電位箝制的研究顯示在RP1神經元上procaine能降低鈣電流、鈉電流、瞬間外向鉀電流(transient outward K+ current, IA)及steady-state 鉀電流。4-AP (IA的抑制劑)對蝸牛的RP1神經元並不會對動作電位產生猝發現象，然而，TEA (延遲外向鉀電流抑制劑)會造成RP1神經元產生猝發現象，因此認為procaine對RP1神經元產生猝發性的動作電位可能經由抑制RP1神經元延遲外向鉀電流所造成。Diazoxide (ATP敏感性鉀離子通道活化劑) 並不會對procaine在RP1神經元所產生動作電位的猝發現象造成抑制，且不會對procaine所引起的鉀離子電流抑制情形造成影響。Procaine並不會影響Xenopus oocyte上ROMK1離子通道內流型整流電流及Shaker鉀離子電流。此外，U73122 (PLC inhibitor)可以減少procaine抑制延遲外向鉀電流。這些結果顯示procaine 經由活化phospholipase C來抑制延遲外向鉀電流。
細胞外投予d-amphetamine (270 μM) 能可逆性的引發猝發性的 動作電位。此猝發性的動作電位無法被(1) 事先投予prazosin (100 μM)、propranolol (100 μM)、d-tubocurarine (100 μM)、atropine(1 mM)，(2) 事先投予PKC 抑制劑chelerythrine chloride (10 μM)及Ro 31-8220 (10 μM)，(3) 事先投予U73122 (10 μM) (PLC inhibitor)，(4) 投予neomycin (3.5 mM)，(5) 灌流缺鈣生理溶液 (Ca2+-free solution)，及(6) 投予高鎂 (30 mM) 生理溶液所抑制。這些結果顯示d-amphetamine所引起動作電位的猝發現象並非經由(1) cholinergic及adrenergic受體，(2) PKC及PLC相關的訊息傳遞路徑，(3) 細胞外鈣離子而來。然而，動作電位的猝發現象可以被事先投予PKA 抑制劑KT-5720 (10 μM) 及H89 (10 μM) 所抑制。Forskolin (10 μM) (AC activator) 能促進d-amphetamine引起動作電位的猝發現象，而高濃度之forskolin (100 μM) 本身即會引起RP1神經元產生猝發現象。細胞內注射d-amphetamine會引發RP1神經元的動作電位產生猝發現象。而在d-amphetamine引起RP1神經元產生猝發現象後，細胞內注射高鎂離子，則會降低d-amphetamine所引起的猝發現象。d-Amphetamine引起蝸牛中樞神經元動作電位的猝發現象可能與cAMP第二傳訊物質路徑有關。
在灌流池中含有d-amphetamine 及KT-5720，procaine仍會在相同神經元中引發猝發性的動作電位。而在灌流池中含有procaine 及U73122，d-amphetamine仍會在相同神經元中引發猝發性的動作電位。這些結果顯示d-amphetamine及procaine會在同一個非洲大蝸牛的RP1神經元上分別經由不同的訊息傳遞系統而產生猝發性動作電位。d-Amphetamine所產生的猝發現象可能與活化cAMP-dependent protein kinase system的路徑有關，導致細胞內鈣離子移動及延遲鉀電流受到抑制而引發動作電位的猝發現象產生，而procaine所產生的猝發現象則可能是經由活化PLC的訊息傳遞路徑來造成細胞內鈣離子移動及抑制部分的延遲鉀電流，而造成RP1神經元動作電位的猝發現象。因此，認為在相同細胞上所產生的猝發現象可能會經由各種不同的訊息傳遞過程所調控。

關鍵字

普卡因 ； 安非他命 ； 神經元 ； 動作電位猝發現象 ； 突觸傳導 ； 第二傳訊物質

並列摘要

The modulating effects of d-amphetamine and procaine on the spontaneously generated action potentials were studied pharmacologically and electrophysiologically on the RP1 central neuron of giant African snails (Achatina fulica Ferussac). The RP1 neuron showed spontaneous firing of action potentials.
Extra-cellular application of procaine (10 mM) reversibly elicited bursts of potential. The bursts of potential were not affected by (1) pretreatment with prazosin (100 μM), propranolol (100 μM), d-tubocurarine (100 μM), atropine (1 mM), (2) pretreatment with KT-5720 (10 μM) and H89 (10 μM), the PKA inhibitors, (3) pretreatment with chelerythrine chloride (10 μM) and Ro 31-8220 (10 μM), the PKC inhibitors, (4) perfusion with calcium-free solution, (5) treatment with ryanodine (30 μM), (6) treatment with pentobarbital (300 μM), and (7) pretreatment with KB-R7943 (100 μM). These results suggest that the bursting activity elicited by procaine is not due to (1) the cholinergic and adrenergic receptor of the excitable membrane, (2) the PKA, PKC related messenger systems, (3) the extra-cellular calcium ion, (4) the ryanodine sensitive calcium channels, (5) the transmitter releasing of ganglia, and (6) Na+-Ca2+ exchanger in the neuron. However, the bursts of potential were decreased following pretreatment with U73122 (10 μM) (PLC inhibitor), or treatment with neomycin (3.5 mM), a high-magnesium (30 mM), physiological sodium ion was replaced with lithium ion, KCN (3 mM). Lower doses of caffeine (10 mM) or ruthenium red (100 μM) facilitated the procaine-elicited bursts of potential. m-3M3FBS, a PLC activator, elicited bursts of potential. 8-Bromo-cGMP, a cGMP analogue did not elicit bursts of potential in the RP1 neuron. Intracellular injection of calcium ions or procaine elicited bursts of potential in the RP1 neuron. The bursts of potential elicited by procaine were decreased following intracellular injection with a high-magnesium solution. The procaine-elicited bursts of potential might dependent on the phospholipase activity and the calcium mobilization in the neuron.
The role of the ionic currents on the bursts of potential in central snail neurons elicited by procaine was studied in the identified RP1 neuron using two-electrode voltage-clamp method. Voltage-clamped studies revealed that procaine decreased the Na+ currents, Ca2+ currents, transient outward K+ currents and steady-state K+ currents of the RP1 neuron. 4-AP, the inhibitor of transient outward K+ current (IA), did not elicit any bursts of potential on the RP1 neuron. However, TEA (tetraethylammonium), a blocker of the delayed outward K+ current, elicited action potential burst in the RP1 neuron. It is suggested that the inhibitory effects of procaine on delayed outward K+ currents may be associated with the generation of bursts of potential. Diazoxide, a KATP opener, did not prevent procaine-induced reduction in delayed outward K+ current. Procaine did not affect the ROMK1 and Shaker K+ currents in Xenopus oocyte. In addition, procaine decreased the delayed outward K+ current and the effect was prevented by pretreatment with U73122, a PLC inhibitor. These results demonstrated that procaine decreased the delayed outward K+ current through phospholipase C.
Extra-cellular application of d-amphetamine (270 μM) reversibly elicited bursts of potential. The bursts of potential were not affected by (1) pretreatment with prazosin (100 μM), propranolol (100 μM), d-tubocurarine (100 μM), atropine (1 mM), (2) pretreatment with chelerythrine chloride (10 μM) and Ro 31-8220 (10 μM), the PKC inhibitors, (3) pretreatment with U73122 (10 μM) (PLC inhibitor), (4) treatment with neomycin (3.5 mM), (5) perfusion with calcium-free solution, (6) treatment with a high-magnesium (30 mM) solution. These results suggest that the bursting activity elicited by d-amphetamine is not due to (1) the cholinergic and adrenergic receptor of the excitable membrane, (2) the PKC and PLC related messenger systems, (3) the extra-cellular calcium ion in the neuron. The bursts of potential elicited by d-amphetamine was decreased following extracellular application of specific PKA inhibitors, H89 (10 μM) and KT-5720 (10 μM). Forskolin (10 μM), an activator of adenylyl cyclase, facilitated the generation of the bursts of potential elicited by d-amphetamine. However, a higher concentration of forskolin (100 μM) elicited bursts of potential in the snail RP1 neuron. Intracellular injection of d-amphetamine elicited bursts of potential in the RP1 neuron. The bursts of potential elicited by d-amphetamine were decreased following intracellular injection with a high-magnesium solution. These results suggest that the bursts of potential elicited by d-amphetamine are associated with the cyclic adenosine monophosphate (cAMP) second messenger system in the central neuron.
In the medium containing d-amphetamine and KT-5720, procaine still elicited bursts of potential in the same neuron. In the preparations containing U73122 and procaine, d-amphetamine still elicited bursts of potential in the same neuron. These results suggest that d-amphetamine and procaine elicited bursts of potential in the same neuron through separated messenger systems. The bursts of potential elicited by d-amphetamine were dependent on the cAMP activity then induced calcium mobilization and decreased the delayed outward K+ current in the neuron while the bursts of potential elicited by procaine were dependent on the phospholipase activity then induced calcium mobilization and decreased the delayed outward K+ current in the neuron. It is concluded that various second messenger systems separately elicited bursts of potential in the same neuron.

並列關鍵字

Procaine ； d-Amphetamine ； neuron ； bursts of potential ； synaptic transmission ； second messengers

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