Saito I, Dozio N, Meldolesi J
Neuroscience. 1985 Apr;14(4):1163-74. doi: 10.1016/0306-4522(85)90285-4.
Neurosecretory PC12 cells differentiated in vitro by prolonged (at least 2 weeks) treatment with nerve growth factor were exposed to alpha-latrotoxin and studied by morphological and biochemical techniques. Cell monolayers or suspensions responded to the toxin with a prompt and massive release of neurotransmitter. The dose dependency (Km approximately 5 X 10(-10)M) and the maximum release effect (approximately 60% of the stored [3H]dopamine released within 8 min) were not appreciably different from the values found in non-differentiated PC12 cells. Moreover, the concentration dependency of the release was found to correspond closely to that of the [125I]alpha-latrotoxin binding to its specific sites (the alpha-latrotoxin receptors). The number of these receptors was over two-fold higher in differentiated than in undifferentiated cells. Since, however, differentiation implies a large increase in cell size and surface area, the receptor density (number/unit area) remained virtually unchanged. By radioautography the alpha-latrotoxin receptors were found to remain diffusely distributed at the entire surface of differentiated cells even when these were allowed to form synapses with myotubes. This situation is at variance with that demonstrated recently at the frog neuromuscular junction, where alpha-latrotoxin receptors are exclusively localized at the nerve terminal plasmalemma. Scanning and transmission electron microscopy revealed that the enlargements of neurites where dense granules are preferentially accumulated--the varicosities and terminals--underwent swelling and extensive disorganization within a few minutes after the application of alpha-latrotoxin, whereas the cell bodies and the tracts of neurites occupied primarily by microtubules were less severely affected. The greater sensitivity of varicosities and terminals with respect to the other parts of the differentiated cells, rather than the consequence of a specific addressing of the toxin to these structures, might be due to their vulnerability by toxin-induced events, such as the uncontrolled activation of ion fluxes.
用神经生长因子进行长时间（至少2周）处理在体外分化的神经分泌型PC12细胞，使其暴露于α- latrotoxin，并通过形态学和生化技术进行研究。细胞单层或悬浮液对该毒素的反应是迅速且大量地释放神经递质。剂量依赖性（Km约为5×10⁻¹⁰M）和最大释放效应（8分钟内约60%储存的[³H]多巴胺被释放）与未分化的PC12细胞中的值没有明显差异。此外，发现释放的浓度依赖性与[¹²⁵I]α- latrotoxin与其特异性位点（α- latrotoxin受体）的结合密切相关。这些受体的数量在分化细胞中比未分化细胞中高出两倍多。然而，由于分化意味着细胞大小和表面积大幅增加，受体密度（数量/单位面积）实际上保持不变。通过放射自显影发现，即使分化细胞与肌管形成突触，α- latrotoxin受体仍分散分布在分化细胞的整个表面。这种情况与最近在青蛙神经肌肉接头处所证明的情况不同，在那里α- latrotoxin受体仅定位在神经末梢质膜上。扫描和透射电子显微镜显示，在应用α- latrotoxin后几分钟内，神经突中密集颗粒优先积累的部位——曲张体和终末——会发生肿胀和广泛的紊乱，而主要由微管占据的细胞体和神经突束受影响较小。曲张体和终末相对于分化细胞的其他部分具有更高的敏感性，这可能不是毒素特异性作用于这些结构的结果，而是由于它们对毒素诱导的事件（如离子通量的失控激活）的脆弱性。
