Madeddu L, Saito I, Hsiao T H, Meldolesi J
J Neurochem. 1985 Dec;45(6):1719-30. doi: 10.1111/j.1471-4159.1985.tb10527.x.
Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)
将豚鼠脑皮质突触体和神经分泌性PC12细胞用[3H]3,4 - 二羟基苯乙胺（[3H]DA，[3H]多巴胺）加载，然后暴露于瘦素毒素 - h（LPTx）（从Leptinotarsa haldemani血淋巴中获得的纯化和部分纯化制剂）。在含Ca2 +的林格氏培养基中，该毒素诱导神经递质迅速大量释放。对于突触体，在估计约为3×10（-11）M的浓度下获得半数最大效应，对于PC12细胞则为1.5×10（-10）M。在所研究的两个实验系统中，释放反应在不同程度上依赖于培养基中的Ca2 +浓度。在突触体中，即使在无Ca2 +、含EGTA的培养基中，若使用高浓度（10（-10）M范围），毒素也能引发[3H]DA的明显释放，尽管速度较慢；在10（-5）M Ca2 +时观察到接近最大反应。相比之下，毒素诱导的PC12细胞释放仅在3×10（-5）M Ca2 +时明显，在2×10（-4）M及以上时达到最大。在突触体和PC12细胞中，Sr2 +和Ba2 +均可替代Ca2 +；Co2 +具有抑制作用，而Mn2 +未能改变含Ca2 +培养基中毒素诱导的释放。电压依赖性Ca2 +通道的有机阻滞剂（维拉帕米和尼群地平）以及钙调蛋白阻断药物（三氟拉嗪和平痛新）未能抑制毒素诱导的[3H]DA释放。LPTx诱导了深刻的形态学效应。在含Ca2 +的培养基中处理的突触体表现出突触小泡融合、形成大量内褶和大池以及线粒体改变。在无Ca2 +的培养基中，效应相似，只是出现延迟，线粒体保存良好。在PC12细胞中观察到肿胀，伴有高尔基体区域扩大、多囊泡体积累、线粒体改变以及分泌颗粒数量减少（含Ca2 +培养基）。形态计量分析显示，突触小泡（突触体）和神经分泌颗粒（PC12细胞）数量的减少与生化测定的[3H]DA释放之间存在良好相关性。这有力地表明毒素的释放效应是由于对胞吐作用的刺激。总体而言，这些结果证实了LPTx与配套文章中提到的黑寡妇蜘蛛毒液的α - 拉托毒素作用的相似性。然而，效应和靶标特异性的差异表明这两种毒素作用于不同的结合位点。（摘要截断于400字）
