Gil A, Rueda J, Viniegra S, Gutiérrez L M
Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, Campus de San Juan, 03550, Alicante, Spain.
Neuroscience. 2000;98(3):605-14. doi: 10.1016/s0306-4522(00)00132-9.
Adrenal chromaffin cells were used to test the role of the peripheral cytoskeleton of F-actin in controlling different vesicle pools. Phorbol 12-myristate 13-acetate and calyculin A, two substances affecting phosphorylation-dephosphorylation cycles, produced different degrees of F-actin reorganization, inducing the partial and the almost total disassembly of this structure, respectively, as visualized using rhodamine-phalloidin staining. Consequently, electron microscopy studies revealed the higher efficiency of calyculin-A over phorbol 12-myristate 13-acetate in promoting vesicle access to the plasmalemma boundary. Surprisingly, only the phorbol ester enhanced fast kinetics and the population of rapidly releasable vesicle pools as studied by single-cell amperometry, whereas both agents, as well as the F-actin severing compound, Latrunculin A, promoted an increase in the population of vesicles recruited in response to prolonged or repetitive stimulations. Taken together, our data support the notion that the F-actin peripheral barrier controls primary granule recruitment from reserve vesicle pools, whereas the phorbol ester effect on the rapidly releasable pools might be related to the alteration of late secretory stage through protein kinase C-dependent phosphorylation of an unidentified target.
肾上腺嗜铬细胞被用于测试F-肌动蛋白外周细胞骨架在控制不同囊泡池方面的作用。佛波醇12-肉豆蔻酸酯13-乙酸酯和花萼海绵诱癌素A这两种影响磷酸化-去磷酸化循环的物质，产生了不同程度的F-肌动蛋白重组，分别诱导了该结构的部分和几乎完全解体，这通过罗丹明-鬼笔环肽染色得以可视化。因此，电子显微镜研究显示，在促进囊泡接近质膜边界方面，花萼海绵诱癌素A比佛波醇12-肉豆蔻酸酯13-乙酸酯更有效。令人惊讶的是，通过单细胞安培法研究发现，只有佛波醇酯增强了快速动力学和快速可释放囊泡池的数量，而这两种物质以及F-肌动蛋白切断化合物Latrunculin A，都促进了响应长时间或重复刺激而募集的囊泡数量的增加。综上所述，我们的数据支持这样一种观点，即F-肌动蛋白外周屏障控制着储备囊泡池中初级颗粒的募集，而佛波醇酯对快速可释放池的影响可能与通过蛋白激酶C依赖的未知靶点磷酸化对晚期分泌阶段的改变有关。
