The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa： differences from the zona pellucida

The acrosome reaction （AR）, an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca2＋ into the spermatozoa through voltage-dependent Ca2＋ channels and store-operated channels. Maitotoxin （MTx）, a Ca2＋-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida （ZP）, possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 （rhZP3）, mouse ZP and two MTx channel blockers （U73122 and U73343）, we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca2＋ （[Ca2＋]~） in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 （inactive analogue）, can block phospholipase C （PLC）. Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

Caribbean maitotoxin elevates [Ca^(2+)]i and activates non-selective cation channels in HIT-T15 cells

AIM:To investigate the cytotoxic mechanism of caribbean maitotoxin(MTX-C) in mammalian cells.METHODS:We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells,which is a system where the effects of MTX have been observed.HIT-T15 cells stably express L-type calcium current,making it a suitable model for this study.Using the fluorescence calcium indicator Indo-1 AM,we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.RESULTS:About 3 min after perfusion of MTX-C,a gradual increase in free calcium concentration was observed.This elevation was sustained throughout the entire recording period.Application of MTX-C did not elicit the L-type calcium current,but large cationiccurrents appeared after applying MTX-C to the extracellular solution.The current-voltage relationship of the cation current is approximately linear within the voltage range from-60 to 50 mV,but flattened at voltages at-80 and-100 mV.These results indicate that MTX-C induces a non-voltage activated,inward current under normal physiological conditions,which by itself or through a secondary mechanism results in a large amount of cationic influx.The biophysical mechanism of MTX-C is different to its isoform,pacific maitotoxin(MTX-P),when the extracellular calcium is removed.CONCLUSION:We conclude that MTX-C causes the opening of non-selective,non-voltage-activated ion channels,which elevates level of intracellular calcium concentration and leads to cellular toxicities.

刺尾鱼毒素对大鼠神经肌接头突触传递的影响

目的和方法 :在大鼠不均匀牵张膈肌标本 (INSMP)上 ,用传统的微电极胞内记录方法。研究MTX对大鼠膈肌膈神经突触传递的影响。结果 :①浴槽给予MTX(10 μg/L) ,18.0min后 ,串刺激神经突然不能产生串终板电位(EPP)。随后 ,突触后膜开始逐渐去极化 ,最大去极化 2 7.0mV。6 2 .7min后小终板电位 (mEPP)频率逐渐增高 ,到70 .3min达到最高频率 ,比给药前增加了 32倍。这种高频的MEPP可以持续 2 0～ 30min ;②提前 2 0min浴槽给予2 0 μmol/L的L 型Ca2 +通道阻断剂异搏定 (Veranpamil) ,然后给于MTX(10 μg/L ,78.5min后串刺激神经不能产生串EPP ,与单给MTX相比时间明显延长 (P <0 .0 0 1)。而突触后膜最大去极化幅度、mEPP频率增高时间及最高频率与单给MTX相比没有明显区别。结论 :MTX对神经肌接头突触传递的阻断作用首先表现在神经纤维不能兴奋 ,这种作用可以部分被L 型Ca2 +通道阻断剂Verapamil所对抗。随后出现突触后膜去极化、mEPP频率显著增高 。

刺尾鱼毒素对NG108细胞的毒性效应

目的 :研究刺尾鱼毒素 (maitotoxin ,MTX)对神经胶质瘤细胞 (NG10 8)毒性作用机制 ,为探讨可能的防治途径提供理论依据。方法 :采用噻唑蓝 (MTT)比色法检测MTX对细胞的毒性。结果 :NG10 8细胞对MTX的作用比较敏感。MTX作用 4h内细胞存活率明显呈剂量、时间依赖性下降 ;毒素作用 1h的LD50 值为 0 .5 3ng/ml。MTX的细胞毒性与胞外Ca2 + 浓度关系密切 ,随着胞外Ca2 + 的升高 ,MTX的毒性增大 ,胞外无Ca2 + 时 ,观察不到MTX对NG10 8细胞的毒性作用。硝苯地平可以明显抑制MTX的细胞毒性。结论 :MTX是一种作用强烈的生物毒素 ,它对NG10 8细胞的毒性效应与胞内Ca2 + 超载有关 ,Ca2 + 通道阻断剂硝苯地平可在一定程度上抑制MTX的毒性效应。

刺尾鱼毒素研究进展

刺尾鱼毒素(maitotoxin,MTX)是由冈比甲藻属和福冈属的鞭毛藻产生,经食物链蓄积于刺尾鱼体内的一类结构独特的聚醚类海洋生物毒素。MTX是1种电压依赖型Ca2+通道激活剂,在作用于生物体细胞后,会引起一系列钙离子相关的生理反应,最终导致细胞凋亡或死亡,因此MTX对Ca2+通道及相关机制研究具有重要作用。MTX作为药理学研究工具药在国内外应用广泛,但对其毒理作用研究较少,激活相应Ca2+通道的具体机制尚不明确。目前MTX的检测方法主要有生物检测法、细胞毒性实验检测法、色谱检测法等。其检测方法大多应用于毒性评价、产毒藻种筛选以及MTX的分离纯化等方面,但对于生物体MTX中毒,迄今仍缺少快速简便的检测方法。因此,发展MTX快速检测技术对公共卫生事业意义重大。本文就MTX毒性机制和MTX检测方法进行了综述,总结了其研究现状和进展,为MTX毒性作用的进一步研究和毒性的检测防护提供了依据。