Sheridan R E, Deshpande S S
Neurotoxicology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, USA.
Toxicon. 1995 Apr;33(4):539-49. doi: 10.1016/0041-0101(94)00185-b.
Exposure of isolated mouse hemidiaphragms to botulinum neurotoxins, 0.1 nM BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak isometric twitch tension to 50% of control values at 55 min (BoNT-A) to 68 min (BoNT-B) after application. Either coincubation of BoNT with the heavy metal chelator TPEN, preincubation with TPEN followed by BoNT, or application of TPEN after BoNT but before neuromuscular block, delayed the onset of muscle failure in a dose-dependent manner by up to five-fold. TPEN doses between 2 and 10 microM were required to antagonize significantly the muscle block produced by BoNT, and the delay in onset was maximal between 10 and 50 microM TPEN. Treatment of muscles with a Zn(2+)-TPEN coordination complex, rather than TPEN alone, eliminated any beneficial effects of TPEN on BoNT intoxication, indicating that these effects were mediated by chelation of Zn2+. Other metal chelators that were not as membrane permeant as TPEN were ineffective in delaying BoNT paralysis, suggesting that TPEN acts by chelating intraterminal Zn2+. In the absence of BoNT, TPEN caused a dose-dependent increase in nerve-elicited twitch tension with a half-maximal concentration at 8 microM. There was no corresponding change in twitches from direct electrical stimulation of the muscle. After BoNT (A or B serotype) had reduced the muscle twitch by 20 to 70%, however, subsequent application of TPEN rapidly depressed nerve-elicited twitches. The shift from potentiation to depression after BoNT treatment suggests that presynaptic vesicle mobilization and/or release involve Zn(2+)-dependent enzymes and that BoNTs interact with these enzyme pathways.
将分离的小鼠半膈肌在36℃下暴露于肉毒杆菌神经毒素（0.1 nM的BoNT-A或BoNT-B），在施加毒素后55分钟（BoNT-A）至68分钟（BoNT-B）时，神经诱发的等长收缩峰值张力降低至对照值的50%。BoNT与重金属螯合剂TPEN共同孵育、先与TPEN预孵育再加入BoNT，或在加入BoNT后但在神经肌肉阻滞前加入TPEN，均可使肌肉衰竭的发生延迟，且呈剂量依赖性，延迟倍数最高可达5倍。需要2至10 microM的TPEN剂量才能显著拮抗BoNT引起的肌肉阻滞，且在10至50 microM的TPEN之间延迟作用最大。用Zn(2+)-TPEN配位复合物而非单独的TPEN处理肌肉，消除了TPEN对BoNT中毒的任何有益作用，表明这些作用是由Zn2+螯合介导的。其他不如TPEN具有膜通透性的金属螯合剂在延迟BoNT麻痹方面无效，这表明TPEN通过螯合终末内的Zn2+起作用。在没有BoNT的情况下，TPEN导致神经诱发的抽搐张力呈剂量依赖性增加，半数最大浓度为8 microM。直接电刺激肌肉引起的抽搐没有相应变化。然而，在BoNT（A或B血清型）使肌肉抽搐降低20%至70%后，随后加入TPEN会迅速抑制神经诱发的抽搐。BoNT处理后从增强到抑制的转变表明，突触前囊泡的动员和/或释放涉及Zn(2+)依赖性酶，且BoNTs与这些酶途径相互作用。
