Schäfer T, Karli U O, Schweizer F E, Burger M M
Dept. of Biochemistry, Biocenter of the University, Basel.
Biosci Rep. 1987 Apr;7(4):269-79. doi: 10.1007/BF01121448.
Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by increasing the free Ca2+-concentration to microM levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca2+: a short (2 min) pulse of Ca2+ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca2+ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasin B or colchicine. The permeabilized cell system is a valuable technique for the in vitro study of interaction between secretory vesicles and their target membrane.
对分泌小泡的假定对接进行了研究，该对接包括识别和附着于质膜上未来的融合位点，研究对象为牛肾上腺髓质嗜铬细胞和大鼠嗜铬细胞瘤（PC 12）细胞。在用洋地黄皂苷通透细胞后，通过将游离Ca2+浓度提高到微摩尔水平，可在这两种细胞类型中刺激分泌。在开始通透处理后长达1小时都能引发分泌活动，尽管可溶性细胞质成分有所损失，这表明颗粒稳定附着于质膜上，等待融合触发信号。在通透处理的PC 12细胞中，可在电子显微镜下观察到对接的颗粒，其中很大一部分颗粒排列在质膜下方。由于颗粒在整个细胞质中分散，嗜铬细胞中假定对接颗粒的群体不太容易辨别。进一步比较PC 12细胞和嗜铬细胞的实验表明，在存在Ca2+的情况下，通透细胞仍可进行颗粒的主动对接但不能进行运输：PC 12细胞中短时间（2分钟）的Ca2+脉冲会导致在15分钟的观察期内几乎所有可释放激素的分泌，而在嗜铬细胞中，只有一小部分颗粒对接，去除Ca2+会导致分泌立即停止。嗜铬颗粒从高尔基体运输到质膜对接位点似乎依赖于一种对通透敏感的机制。这通过两种通透细胞类型释放的激素量的差异得以体现，反映了PC 12细胞或嗜铬细胞中对接至质膜的颗粒比例的差异。细胞松弛素B或秋水仙碱均不影响对接或对接状态。通透细胞系统是体外研究分泌小泡与其靶膜之间相互作用的一种有价值的技术。
