A型肉毒毒素裂解SNAP-25在被川楝素阻断后仍抑制乙酰胆碱诱发离体幽门肌收缩
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摘要
背景与目的：A型肉毒毒素(botulinum toxin type A.BTX-A)选择性地裂解突触相关蛋白—25(synaptosomal-associated protein of 25 kDa，SNAP-25)，阻断突触囊泡中神经递质与突触前膜的融合、胞吐和释放。川楝素(toosendanin，TSN)近期被证实通过阻断BTX-A裂解SNAP-25而具有抗肉毒毒素功能。本研究依离体大鼠幽门平滑肌为实验对象，应用TSN阻断BTX-A裂解SNAP-25后，观察BTX-A是否仍对乙酰胆碱(acetylcholine，ACh)诱发的平滑肌收缩起抑制作用，以期揭示BTX-A对平滑肌存在的作用途径。
方法：三组Sprague-Dawley大鼠的幽门离体平滑肌条在Krebs液中1g的前负荷下孵育52分钟左右，待出现自发性收缩波且平稳后分别加入ACh，BTX-A，TSN和Atropine。第一组(n=10)：肌条对ACh 1001μM出现反应10分钟后，分别加入Atropine 1μM(n=5)或BTX-A 10 U/ml(n=5)记录4小时；第二组(n=10)：肌条对BTX-A 10U/ml(n=5)或AtropinelμM(n=5)出现反应30分钟后，加入ACh 100μM记录4小时；第三组(n=5)：肌条在含TSN 29.6μM溶液中孵育20分钟后，加BTX-A10U/ml，30分钟后再加ACh 100μM记录4小时。
结果：第一组：ACh 100μM显著地增强了幽门收缩的张力(P＜0.001)和频率(P＜0.001)，但振幅无显著性差异；后续加入BTX-A 10 U/ml抑制了ACh诱发的收缩作用(张力，P＜0.001：频率，P＜0.001；振幅，P=0.014)。然而，Atropine 1μM却完全性抑制了ACh诱发的收缩反应。这一结果显示BTX-A10 U/ml直接抑制了ACh诱发的收缩反应。
第二组：BTX-A 10 U/ml直接抑制了幽门自发性收缩(张力，P=0.006；频率，P＜0.001；振幅，P＜0.001)；AtropinelμM同样抑制了幽门肌的收缩。随后加入ACh100μM不能再激发幽门肌的收缩。
第三组：TSN 29.6μM孵育20分钟，幽门平滑肌的自发性收缩末发生变化；加入BTX-A 10 U/ml后，BTX-A降低幽门肌收缩反应(张力，P＜0.001；振幅，P＜0.001)。虽然这种抑制作用与未加TSN时BTX-A的效应不尽相似，但是BTX-A10 U/ml仍然抑制了幽门平滑肌收缩，即使ACh 100μM也同样不能激发它再收缩。
结论：A型肉毒毒素不仅抑制幽门平滑肌的自发性收缩，而且抑制了乙酰胆碱诱发的收缩；即使它介导SNAP-25裂解被川楝素阻断后，也同样抑制乙酰胆碱诱发的幽门部平滑肌收缩。这提示A型肉毒毒素不仅抑制胆碱能神经中乙酰胆碱的释放，而且抑制外源性乙酰胆碱与胆碱能毒蕈碱受体结合后产生的肌肉收缩反应。
Background and Aim: Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo fusion with presynaptic membrane, exocytosis and release. Toosendanin (TSN) was recently demonstrated a potential antibotulismic agent by antagonizing BTX-A-mediated cleavage of SNAP-25. The study was to decide whether BTX-A effected on the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin and investigate in which way BTX-A did.
Methods: Three groups of Sprague-Dawley rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Kerbs solution and its spontaneous contractile waves regularly emerging as its own control before adding respectively ACh, BTX-A, TSN and atropine. The first group (n=10), after initial response to ACh 100μM for 10 min, atropine 1μM (n=5) or BTX-A 10 U/ml (n=5) was respectively added for recording 4 h. The second group(n=10), after initial response to BTX-A 10 U/ml (n=5) or atropine 1μM (n=5) for 30 min. ACh 100μM was added for recording 4 h. The third group, after initial incubation of toosendanin (TSN) 29.6μM for 20 min, subsequent BTX-A 10 U/ml for 30 min and then ACh 100μM were added for recording 4 h.
Results: In the first group the addition of ACh 100μM into Krebs solution enhanced significantly pyloric muscle contractile tension (P<0.001) and frequency (P<0.001), but not contractile amplitude (P=0.769) in preparations. The further addition of BTX-A 10 U/ml suppressed ACh-induced contractile responses including tension (P<0.001), frequency (P<0.001) and amplitude (P=0.014). Atropine 1μM suppressed comparably ACh-induce contractile responses. Nevertheless, the inhibition of atropine was almost complete. The results showed that BTX-A 10 U/ml directly inhibits ACh-induced pyloric contractile response.
In the second group, BTX-A 10 U/ml inhibited directly pyloric muscle spontaneous contractions, such as contractile tension (P=0,006), frequency (P<0.001) and amplitude (P<0.001). Atropine 1μM similarly inhibited pyloric muscle contraction (unshown). Sequential addition of ACh 100μM did not agitate any more pyloric muscle contraction.
In the third group, the addition of TSN 29.6μM in initial period of 20 min did not influence pyloric muscle spontaneous contractility, subsequently BTX-A 10 U/ml addition still decreased pyloric muscle contractile (tension, P<0.001; amplitude, P<0.001). Though this inhibition was not similar to the one BTX-A did without TSN. BTX-A 10 U/ml still inhibited pyloric smooth muscle contractility so that ACh 100μM did no longer agitate it.
Conclusion: These data demonstrate that BTX-A inhibits not only pyloric smooth muscle spontaneous contraction but also ACh-induced contraction. Even after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin, BTX-A inhibits ACh-induced pyloric smooth muscle contraction. Such suggest that BTX-A inhibits not only acetylcholine release from cholinergic nerves but also cholinergic muscarinic muscular transmission.
方法：三组Sprague-Dawley大鼠的幽门离体平滑肌条在Krebs液中1g的前负荷下孵育52分钟左右，待出现自发性收缩波且平稳后分别加入ACh，BTX-A，TSN和Atropine。第一组(n=10)：肌条对ACh 1001μM出现反应10分钟后，分别加入Atropine 1μM(n=5)或BTX-A 10 U/ml(n=5)记录4小时；第二组(n=10)：肌条对BTX-A 10U/ml(n=5)或AtropinelμM(n=5)出现反应30分钟后，加入ACh 100μM记录4小时；第三组(n=5)：肌条在含TSN 29.6μM溶液中孵育20分钟后，加BTX-A10U/ml，30分钟后再加ACh 100μM记录4小时。
结果：第一组：ACh 100μM显著地增强了幽门收缩的张力(P＜0.001)和频率(P＜0.001)，但振幅无显著性差异；后续加入BTX-A 10 U/ml抑制了ACh诱发的收缩作用(张力，P＜0.001：频率，P＜0.001；振幅，P=0.014)。然而，Atropine 1μM却完全性抑制了ACh诱发的收缩反应。这一结果显示BTX-A10 U/ml直接抑制了ACh诱发的收缩反应。
第二组：BTX-A 10 U/ml直接抑制了幽门自发性收缩(张力，P=0.006；频率，P＜0.001；振幅，P＜0.001)；AtropinelμM同样抑制了幽门肌的收缩。随后加入ACh100μM不能再激发幽门肌的收缩。
第三组：TSN 29.6μM孵育20分钟，幽门平滑肌的自发性收缩末发生变化；加入BTX-A 10 U/ml后，BTX-A降低幽门肌收缩反应(张力，P＜0.001；振幅，P＜0.001)。虽然这种抑制作用与未加TSN时BTX-A的效应不尽相似，但是BTX-A10 U/ml仍然抑制了幽门平滑肌收缩，即使ACh 100μM也同样不能激发它再收缩。
结论：A型肉毒毒素不仅抑制幽门平滑肌的自发性收缩，而且抑制了乙酰胆碱诱发的收缩；即使它介导SNAP-25裂解被川楝素阻断后，也同样抑制乙酰胆碱诱发的幽门部平滑肌收缩。这提示A型肉毒毒素不仅抑制胆碱能神经中乙酰胆碱的释放，而且抑制外源性乙酰胆碱与胆碱能毒蕈碱受体结合后产生的肌肉收缩反应。
Background and Aim: Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo fusion with presynaptic membrane, exocytosis and release. Toosendanin (TSN) was recently demonstrated a potential antibotulismic agent by antagonizing BTX-A-mediated cleavage of SNAP-25. The study was to decide whether BTX-A effected on the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin and investigate in which way BTX-A did.
Methods: Three groups of Sprague-Dawley rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Kerbs solution and its spontaneous contractile waves regularly emerging as its own control before adding respectively ACh, BTX-A, TSN and atropine. The first group (n=10), after initial response to ACh 100μM for 10 min, atropine 1μM (n=5) or BTX-A 10 U/ml (n=5) was respectively added for recording 4 h. The second group(n=10), after initial response to BTX-A 10 U/ml (n=5) or atropine 1μM (n=5) for 30 min. ACh 100μM was added for recording 4 h. The third group, after initial incubation of toosendanin (TSN) 29.6μM for 20 min, subsequent BTX-A 10 U/ml for 30 min and then ACh 100μM were added for recording 4 h.
Results: In the first group the addition of ACh 100μM into Krebs solution enhanced significantly pyloric muscle contractile tension (P<0.001) and frequency (P<0.001), but not contractile amplitude (P=0.769) in preparations. The further addition of BTX-A 10 U/ml suppressed ACh-induced contractile responses including tension (P<0.001), frequency (P<0.001) and amplitude (P=0.014). Atropine 1μM suppressed comparably ACh-induce contractile responses. Nevertheless, the inhibition of atropine was almost complete. The results showed that BTX-A 10 U/ml directly inhibits ACh-induced pyloric contractile response.
In the second group, BTX-A 10 U/ml inhibited directly pyloric muscle spontaneous contractions, such as contractile tension (P=0,006), frequency (P<0.001) and amplitude (P<0.001). Atropine 1μM similarly inhibited pyloric muscle contraction (unshown). Sequential addition of ACh 100μM did not agitate any more pyloric muscle contraction.
In the third group, the addition of TSN 29.6μM in initial period of 20 min did not influence pyloric muscle spontaneous contractility, subsequently BTX-A 10 U/ml addition still decreased pyloric muscle contractile (tension, P<0.001; amplitude, P<0.001). Though this inhibition was not similar to the one BTX-A did without TSN. BTX-A 10 U/ml still inhibited pyloric smooth muscle contractility so that ACh 100μM did no longer agitate it.
Conclusion: These data demonstrate that BTX-A inhibits not only pyloric smooth muscle spontaneous contraction but also ACh-induced contraction. Even after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin, BTX-A inhibits ACh-induced pyloric smooth muscle contraction. Such suggest that BTX-A inhibits not only acetylcholine release from cholinergic nerves but also cholinergic muscarinic muscular transmission.
引文
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