Goodall A R, Danks K, Walker J H, Ball S G, Vaughan P F
Institute for Cardiovascular Research, University of Leeds, England.
J Neurochem. 1997 Apr;68(4):1542-52. doi: 10.1046/j.1471-4159.1997.68041542.x.
Western blot analysis showed that the human neuroblastoma SH-SY5Y expresses the proteins synaptotagmin l, synaptobrevin, synapsin-l, rab3a, syntaxin, SNAP-25, NSF, alpha-SNAP, and munc-18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin l, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine-beta-hydroxylase on sucrose-D2O gradient fractions indicates the presence of a population of noradrenaline-containing large dense-cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48-kDa synaptophysin antigen absent from the large dense-cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin l contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH-SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization-evoked release of noradrenaline from SH-SY5Y occurs by LDCV exocytosis. This enhances the value of SH-SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.
蛋白质印迹分析表明，人神经母细胞瘤SH-SY5Y细胞表达突触结合蛋白l、突触小泡蛋白、突触素-1、rab3a、 syntaxin、SNAP-25、 NSF、α-SNAP和munc-18等蛋白质，这些蛋白质与其他神经内分泌细胞胞吐过程中囊泡的移动、对接和融合有关。通过免疫荧光显微镜研究了分泌粒蛋白I和II、突触结合蛋白l、神经肽Y、rab3a、突触小泡蛋白、突触素和syntaxin的亚细胞定位，结果显示出分泌囊泡特有的点状染色模式。去甲肾上腺素、分泌粒蛋白II和多巴胺-β-羟化酶在蔗糖-D2O梯度级分上的共迁移表明存在一群含去甲肾上腺素的大致密核心囊泡（LDCV）。此外，还存在一种较轻的囊泡群体，它似乎不是去甲肾上腺素能的，并且含有大致密核心囊泡中不存在的48 kDa突触素抗原。免疫细胞化学实验表明，并非所有表达突触结合蛋白l的囊泡都含有分泌粒蛋白II。因此，我们的研究表明SH-SY5Y细胞中存在两种类型的囊泡，其中一种是LDCV，含有去甲肾上腺素。这些发现证实了我们之前的研究，即SH-SY5Y细胞中去甲肾上腺素的去极化诱发释放是通过LDCV胞吐作用发生的。这提高了SH-SY5Y作为一种细胞系的价值，可用于研究去甲肾上腺素释放的调节机制。
