(3.238.174.50) 您好!臺灣時間:2021/04/20 20:21
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:林明忠
研究生(外文):Lin, Min-Jon
論文名稱:藥物及毒素對運動神經末梢離子電流及乙醯膽鹼釋放之影響
論文名稱(外文):Studies on ionic current and ACh release from motor nerve terminals
指導教授:蕭水銀蕭水銀引用關係
指導教授(外文):Lin-Shiau Shoei-Yn
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:175
中文關鍵詞:運動神經末梢離子電流乙醯膽鹼鈣電流鉀電流
外文關鍵詞:moter nerve terminalsionic currentsAChcalcium currentpotassium current
相關次數:
  • 被引用被引用:0
  • 點閱點閱:245
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
對於鈣管道的種類及特異性的研究  神經特異性的錐螺毒素MVIIC
(0.5-1μM)可以顯著地抑制神經引發的橫膈膜收縮、EPP以及末梢鈣電
流,但另一錐螺毒素GVIA,並無此作用。由於錐螺毒素MVIIC對於肌纖維
靜止膜電位、動作電位及MEPP振幅並不影響,因此它的作用是鍵前選擇性
的,此外,它對於神經鍵前鈉及鉀電流也不影響。在鉀管道抑制劑(3,4-
DAP)存在下,加入飽和濃度的濃度的錐螺毒素MVIIC(3-5 μM)只能抑
制EPP 38%左右,而剩餘不能抑制的部份可完全被鎘所阻斷,顯示有二種
或二種以上的鈣管道存在。  我們使用另一種P型選擇性的抑制劑蜘蛛
毒素ω-agatoxin IVA (0.3 μM),也發現其作用與錐螺毒素MVIIC(2
μM)一樣,可各別地阻斷EPP振幅。然而當我們一起加入nifedipine(50
μM)和錐螺毒素GVIA(2μM),作用20分鐘後,對神經引發肌肉的收縮
、EPP及神經鍵前鈣電流不會影響。由於鍵後EPP的大小與鍵前鈣離子訊號
為非線性的關係,只要阻斷小部份鈣離子進入細胞,即對鍵後EPP振幅有
很大的抑制。在此情況下,我們給予鉀管道抑制劑(3,4-DAP)或高鈣
(10 mM)來增加EPP和鈣電流,發現這個條件下的EPP振幅,不會被最大
抑制濃度的蜘蛛毒素IVA及錐螺毒素MVIIC所完全抑制,即使這二種毒素一
起加入,也只有抑制一部份神經引發的橫膜收縮及EPP振幅,剩餘的部份
,加入鎘(0.3 mM)之後,才完全阻斷,證實剩餘毒素不反應的部份,也
應該是鍵前神經機制所貢獻的。蜘蛛毒素IVA及錐螺毒素MVIIC即使一起加
入也不影響鍵前鈉、鉀電流以及MEPP振幅和頻率。相對地,nifedipine
(50μM)以及錐螺毒素GVIA(2 μM)並不會對鈣電流產生抑制作用。基
於這些實驗數據,我們推論在小鼠運動神經末梢共同存在多種的鈣離子管
道,一是錐螺毒素MVIIC敏感性的,一是蜘蛛毒素IVA敏感性的,另一則是
毒素不敏感性的,這些鈣管道共同來控制神經傳訊物質的釋出。  此外
重金屬離子如二價的Cd2+、Co2+、Ni2+及三價的Gd3+、La3+,它們對鈣電
流具有非選擇性的抑制作用。抑制神經傳訊的強度分別為Cd2+>Co2+、
Ni2+>Gd3+、La3+,然而在促進自發性神經傳訊的作用則是La3+>Gd3+>
Cd2+、Co2+、Ni2+。顯然二價及三價金屬離子具有不同的機制。由於Ni2+
須大於100μM才會對鈣離子電流有作用,因此T型鈣管道在神經末梢的角
色可能不重要。另一個鈣管道阻斷劑quinacrine,其抑制神經傳訊的作用
可能是經由抑制錐螺毒素敏感性的鈣管道而來的,它抑制此鈣管道的作用
與它本身具有的PLA2活性之抑制作用,並無關連。對於鉀管道的研究  
銣紅(10 μM)可以完全抑制神經引發的肌肉收縮及EPP振幅,但對於
MEPP只有部份的抑制作用。而其他的銣化合物(氯化銣及雙呲啶氯化銣)
,對於神經傳訊並無作用。在鍵前電流的分析,我們發現銣紅可增加快鉀
電流(IKf)以及鈣活化性鉀電流(IKca)。此外,銣紅可對抗β-
bungarotoxin所延長TEA不敏感的鈣電流,我們知道β-bungarotoxin可抑
制IKs而延長TEA不敏感的鈣電流,所以銣如有可能增加IKs而延長TEA不敏
感的鈣電流,所以銣紅有可能增加IKs來對抗β-bungarotoxin的作用。銣
紅對運動神經的這些作用,與其他典型鈣管道阻斷劑如蜘蛛毒素IVA(0.5
μM),鍺(0.5 mM)和鎘(0.3 mM)的作用相反,後者皆會抑制IKca。
雖然銣紅(1-30 μM)表面上似乎可以抑制神經鍵前鈣電流,但我們認為
銣紅增加鉀電流的作用,至少貢獻它抑制鈣電流及神經傳訊的作用。對尼
古丁自體受體的作用  Tripeptide,N-CBZ-Gly-Gly-Arg-β-
naphthylamide(Z-GGR-N)可抑制神經引發的橫膈膜收縮及EPP振幅,但
其它的蛋白質分解酵素受質(CBZ-Gly-Phe amide,CBZ-prolyl-leucyl-
glycinamide)及蛋白質分解酵素抑制劑aprotinin,並不具此作用。前處
理Z-GGR-N可以保護橫膈膜不受α-bungarotoxin不可逆的抑制。在電生理
研究方面,發現Z-GGR-N可以抑制EPP及MEPP的振幅,與d-tubocurarine相
似,它可造成高頻刺激時凋萎的現象(fading)。Suramin,一個已知可拮
抗非去極化肌肉鬆弛劑的藥物,卻可完全拮抗Z-GGR-N的所有抑制性作用
。在高頻神經刺激(20 Hz)下,Z-GGR-N抑制EPP振幅的作用比MEPP振幅
還明顯。因此基於這些實驗結果,我們首先發現Z-GGR-N是一個具有類
Curare作用的藥物,它可經由作用在鍵前自體受體及鍵後nACh受體來達到
它的作用,它對神經肌傳訊的機制作用與其蛋白質分解酵素的活化無關,
可能是它特異的化學結構所致。對於神經末梢ACh釋放池的作用  當我
們給予小鼠腹腔注射β-bungarotoxin(5-7 ng/每天) 2-6天後,發現從
第2天開始,MEPP頻率有顯著的抑制,但在1.5小時、3小時及1天,對於
MEPP頻率並沒有改變。以下以毒素處理4-6天做實驗,比較毒素處理後之
MEPP頻率受藥物之影響與對照組比較其異同,結果發現已降低頻率的MEPP
在細胞外鈣降低至1 μM左右,可再降低MEPP的頻率,而未處理毒素的小
鼠在細胞外鈣降低下,亦同樣可使MEPP頻率下降。另外高鉀(15 mM)及
Caffeine(5 mM)均可增加毒素處理及未處理兩組的MEPP頻率,後者增加
MEPP頻率是細胞外鈣依賴性。相反地,鑭(La3+)可增加對照組的,但不
影響毒素組的MEPP頻率(非細胞外鈣依賴性的)。在鍵前神經電流的分析
,毒素處理組鈉及鈣電流並不產生有意義的差別,但對於鉀電流有部份的
抑制。從以上結果,我們知道低濃度的β-bungarotoxin活體內給予,可
選擇性破壞La3+敏感性的ACh釋放池,來減少自發性ACh釋出。  綜合本
論文所研究的藥物,其作用在運動神經鍵前,經由不同的機制來影響神經
傳訊的作用。當神經末梢去極化時,鈣管道瞬間開啟,本論文證明鈣離子
可經由不同(二種或二種以上)的鈣管道進入細胞內,啟動細胞內釋放機
制而使ACh釋出,而一部份釋出的ACh作用到神經膜上的自體受體來調節並
維持ACh釋出。除此之外,在本論文的主要結果是(1)提供一個直接的證據
在神經末梢有多種鈣管道的存在來控制ACh的釋出。(2)銣紅增加運動神經
末梢鉀電流的作用,在過去從未有提出電位依賴性鉀電流增益劑的報告,
大部份所提出的是ATP(或鈣活化)-敏感性鉀電流的增益劑,因此這是
與過去所不同的。(3)我們找到一個作用在鍵前自體受體的藥物Z-GGR-N,
將來可作為自體受體更深入研究的工具。(4)低劑量β-bungarotoxin對於
La3+-敏感性的釋放池有選擇性的抑制,這個機制對於未來β-
bungarotoxin的研究將有很大的幫助。
Effect on Ca2+ currents A P/Q type Ca2+ channel blocker, ω-
conotoxin MVIIC (ω-CTx-MVIIC, 0.5-1 μM) but not a N-type Ca2+
channel blocker, ω-conotoxin GVIA (ω-CTx-GVIA, 1 μM) markedly
inhibits not only the nerve-evoked muscle contractions
accompanied with a decrease in the amplitude of endplate
potentials (EPP) in the mouse phrenic-nerve diaphragm but also
the Ca2+ - waveforms in the nerve terminals of triangularis
sterni. The inhibitory effects of ω-CTx-MVIIC were considered
to be specifically presynaptic rather than postsynaptic or
myogenic, since neither of the electrical properties of muscle
fibers including action potentials, resting membrane potentials
nor the miniature endplate potential (MEPP) were affected.
Moreover, Na+ - and K+ -waveforms of the nerve terminals were
unaffected byω-CTx-MVIIC. At a saturating concentration of 3-5
μM, ω-CTx-MVIIC exerteda maximal inhibitory effect by 38% of
3-4-diaminopyridine-prolonged EPParea and inhibited only the
slow component of Ca2+ - current respectively and the remaining
fast component could be inhibited by subsequent addition of
cadmium chloride (Cd2+). A maximal concentration of either
ω-agatoxin IVA (ω-Aga-IVA 0.3 μM, a blocker of P-type Ca2+ -
channel) or ω-CTx-MVIIC 2 μM (P-and Q-type Ca2+-channel
blocker) can inhibit the nerve-evoked muscle contractions and
amplitude of EPP, respectively. In contrast, combined nifedipine
(50 μM, ablocker of L-type Ca2+ - channel) and ω-CTx-GVIA 2 μ
M can not elicitinhibitory effects on nerve-evoked muscle
contractions. EPP or the nerve terminal waveforms at all.
Because of the non-linear relationship betweenEPP and Ca2+
signals, a small decrease in the presynaptic Ca2+ entry
cansignificantly reduce the EPP amplitudes. Thus, we applied the
3,4-diaminopyridine (3,4-DAP, a K+ -channel blocker) or high
Ca2+ (10 mM) to accelerate and amplify the EPP and Ca2+
currents. The EPP amplified by 3,4-DAP or by high Ca2+
correspondingly proved to be quite resistantto both ω-Aga-IVA
and ω-CTx-MVIIC; ω-Aga-IVA resistant componentwas further
inhibited by ω-CTx-MVIIC. The component resistant to the two
toxins could be completely blocked by a non-selective Ca2+
channel blocker Cd2+ (300 μM). Combination of the two toxins
had no significant effects on either spontaneous transmitter
release or postsynaptic restingmembrane potentials of mouse
diaphragm or Na+ - and K+ - waveforms of triangularis sterni
preparations. This finding suggests a preferential inhibitory
effect at a presynaptic site. Measuring the Ca2+ currentsin
mouse triangularis sterni also revealed a partial inhibition by
ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA.
Cd2+ (300 μM)did abolish the toxin-resistant component of CTx-
GVIA (2 μM) were without inhibitory effect. We conclude that
multiple types of Ca2+ channels, e.g.,ω-Aga-IVA-sensitive, ω-
CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels,co-exist
in mouse motor nerve terminals. Heavy metal, divalent metals
(Cd2+, Co2+, Ni2+) and trivalent metals (Gd3+, La3+) all can
non-selectively inhibit the Ca2+ currents at motor nerve
terminals.The relative potency of metal ions were Cd2+>Co2+,
Ni2+>Gd3+, La3+. However, the relative potency in increasing
MEPP frequency were La3+>Gd3+>Cd2+, Co2+, Ni2+. Hence the action
mechanisms of divalent and trivalent metal ions are different.
Since the Ca2+ current can not be inhibited by a low
concentration of Ni2+ (<50μM), thus T-type current might not
play a role at motor nerve terminals. Heavy metal, divalent
metals (Cd2+, Co2+, Ni2+) and trivalent metals (Gd3+, La3+) all
can non-selectively inhibit the Ca2+ currents at motor nerve
terminals.The relative potency of metal ions were Cd2+ > Co2+,
Ni2+>Gd3+, La3+. However, the relative potency in increasing
MEPP frequency were La3+ > Gd3+ > Cd2+, Co2+, Ni2+. Hence the
action mechanisms of divalent and trivalent metal ions are
different. Since the Ca2+ current can not be inhibited by a low
concentration of Ni2+ (< 50 μM), thus T-type current might not
play a role at motor nerve terminals. An another Ca2+ channel
blocker, quinacrine, inhibited the nerve-evoked muscle
contractions and EPPs due to an inhibitory effect on ω-
conotoxinMVIIC-sensitive Ca2+ channel. The inhibitory effect on
neuromuscular transmissionby quinacrine was considered to be
irrelevant to its PLA2 activity.Effect on K+ current RR (10
μM) was shown not only in the complete suppression of nerve-
evokedmuscle contractions associated with the depression of EPP
amplitudes but alsopartial inhibition on the amplitudes of MEPP.
However, the other ruthenium compounds, ruthenium chloride and
tris (2,2-bipyridyl) ruthenium chloride didnot significantly
affect the neuromuscular transmission. In presynaptic waveform
studies, the fast K+ - current (IK(f))as well as Ca2+ -
activated K+-current (IK(Ca)) was significantly enhanced by 10
μM RR. Furthermore, 10 μM RR antagonized the β-bungarotoxin
(a blocker of slow K+ - channel (IK(s)) in enhancing presynaptic
Ca2+ currents. By contrast, the typical Ca2+ -channel blockers,
ω-agatoxin IVA (0.5 μM), Gd3+ (0.5 mM) and Cd2+ (0.3 mM) all
suppressed the IK(Ca). Altheough RR (1-30 μM) inhibited the
Ca2+ -currentsof the nerve terminals induced by the combined
treatment with K+ - channel blockers, 3,4-diaminopyridine plus
tetraethylammonium chloride in a concentration-dependent manner,
it is considered that RR-enhanced K+ - currentswere responsible
for, at least in part, the observed inhibition of Ca2+ - current
which led to the blockade of transmitter release.Effect on
nicotinic autoreceptor Tripeptide, N-CBZ-Gly-Gly-Arg-β-
naphthylamide (Z-GGR-N) but none of other protease substrates
(CBZ-Gly-Phe-amide and CBZ-prolyl-leucyl-glycinamide) nor
inhibitor (aprotinin) depresses the nerve-evoke muscle
contractions. In addition, Z-GGR-N protects the diaphragm muscle
from the inhibitory effect of α-bungarotoxin. By means of
electrophysiological studies. Z-GGR-N inhibitsthe amplitudes of
both EPP and MEPP but increases the frequencies of MEPP.Similar
to d-tubocurarine, tetanic fading can be induced by Z-GGR-N both
innerve-evoke muscle contraction and EPP. Suramin, a competitive
reversible inhibitor of non-depolarizing relaxants (e.g. d-
tubocurarine), antagonized all of the inhibitory effects of Z-
GGR-N. Furthermore, Z-GGR-N exerted a greater depression on EPP
amplitudes than that on MEPP amplitudes. All of these findings
suggest that Z-GGR-N is a novel polypeptide possessing curare-
likeaction on presynaptic and postsynaptic sites, possibly
mediated by its specific chemical structure rather than its
protease substrate property.Effect on intracellular releasing
pool of acetylcholine Treatment with β-bungarotoxin (5-7 ng/
g per day) for 2-6 days, produced a significant reduction of the
MEPP frequency, but not treatment for 1.5 hr, 3hr and 24 hr.
Thus 4-6 days after toxin-treatment were adapted through the
whole experiments. Lowering the external Ca2+ concentration to
∼1 μM decreased MEPP frequency both in the toxin-treated and
control mouse diaphragms.On the other hand, perfusing high K+
(15 mM) significant elevated the MEPP frequency to a similar
extent in toxin-treated and control mouse diaphragms.Caffeine (5
mM), an enhancer of MEPP frequency dependent on external Ca2+,
also increased the MEPP frequency on both control and toxin-
treated diaphragms.However, La3+, a potent Ca2+ - independednt
transmitter releaser which canonly increase the MEPP frequency
in the control but not in the toxin-treated diaphragms. Analysis
of the presynaptic waveforms reveled that both the Na+ and Ca2+
- current remained unaffected but K+ - current was significantly
depressed in the toxin treated preparations. Thus, the lower
concentrationof β-bungarotoxin appears to be specifically in
impairing La3+ - sensitive releasing pooly and also the
presynaptic K+ -current. In summary, it was concluded that:
(1) We provide a direct evidence thatmultiple subtypes of Ca2+
channels co-exist at the same motor nerve terminal.(2) We first
report that RR can enhance the voltage-dependent K+ channel.this
effect of RR is quite different from the previous reported ATP -
sensitiveK+ channel opener. (3) Z-GGR-N, a tripeptide acting on
presynaptic nicotinicautoreceptor, can provide a research tool
for the study on nicotinic autoreceptor.(4) β-Bungarotoxin (5-7
ng/g) at low concentration in vivo can specifically impair the
La3+ - sensitive releasing pool. It is possible that action
mechanismof β-bungarotoxin is related to the La3+ binding
synaptic protein.
封面
目錄(Contents)
中文摘要(Abstract in Chinese)
第一章 文獻回顧
第二章 實驗方法與材料(Materials And Methods)
第三章 錐螺毒素MVIIC與隔對運動神緀末梢離子道道的影響
(Functional assessment of Ca2+ current in the mouse motor nerve terminals)
第四章 多型式離子管道存在於運動神經末梢之證實
(Multiple types of Ca2+ current at the mouse motor nerve terminals)
第五章 重金屬離子對於運動神經末梢鈣離子管道之影響
(Effect of heavy metals on Ca2+ current at mouse motor nerve terminals)
第六章 金雞鈉鹼(Quinacrine)對於運動神經末梢鈣離子管道的影響
(Effect of quinacrine of Ca2+ current of mouse motor nerve)
第七章 銣紅對於鈣離子管道與鉀離子管道之影響
(Ruthenium red, a novel K+ current enhancer at mouse motor nerve terminals)
第八章 Tripetide對於鍵前及鍵後乙醯膽鹼受體之研究
(Evidence for antagonism on nicotinicACh receptor by a tripeptide
CBZ-Gly-Gly-Arg-B-naphthylamide in mouse diaphragm)
第九章 B-雨傘節毒素低劑量活體實驗對於非鈣型自發性乙醯膽鹼釋出特異作用之研究
(Effect of B*-bungarotoxin in vivo on spontaneous ACh release from the mouse motor nerve terminals)
第十章 結論與未來展望(Conclusions and Perspective)
英文摘要(Abstract in English)
發表與摘要(Publications and Abstracts)
參考文獻(References)
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔