Pozzan T, Gatti G, Dozio N, Vicentini L M, Meldolesi J
J Cell Biol. 1984 Aug;99(2):628-38. doi: 10.1083/jcb.99.2.628.
The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.
在克隆的神经分泌细胞系PC12中研究了调节诱发神经递质释放过程的细胞内机制。使用了各种试剂，根据先前在其他系统中的研究，这些试剂已知以严格依赖或独立于细胞外Ca2+浓度（[Ca2+]o）的方式刺激释放。在悬浮于含Ca2+或无Ca2+的Krebs-Ringer培养基中的细胞中研究了三个参数：先前积累的[3H]多巴胺的释放；平均游离细胞质Ca2+浓度（[Ca2+]i，通过quin2技术测量）；以及细胞超微结构，特别关注分泌颗粒的数量和结构。运输单价阳离子的离子载体X537A和莫能菌素诱导的释放伴随着分泌颗粒结构的深刻改变（致密核心肿胀和溶解）而发生。这些结果表明，这些药物的作用主要是由于多巴胺从颗粒泄漏到细胞质和细胞外空间。相反，其他刺激药物引起的变化涉及的不是结构而是分泌颗粒的数量，表明使用这些药物时，胞吐作用的刺激是递质释放增加的潜在现象。Ca2+离子载体离子霉素诱导的释放反应依赖于[Ca2+]o，迅速发生，伴随着[Ca2+]i的显著升高，并且在1-2分钟后停止，即使[Ca2+]i在许多分钟内仍保持升高。12-O-十四烷酰佛波醇-13-乙酸酯和二酰甘油（两者均已知为蛋白激酶C的激活剂）诱导的反应缓慢，几乎完全独立于[Ca2+]o，并且不伴随[Ca2+]i的变化。蛋白激酶C激活剂与低浓度离子霉素的组合未能改变离子载体诱导的[Ca2+]i升高，但引发了释放反应的显著增强，比单独使用任何一种药物引发的反应之和大两到四倍。因此，蛋白激酶C的激活似乎在神经分泌细胞的胞吐作用调节中起重要作用，可能是通过增加和维持细胞内调节颗粒通过胞吐作用释放的装置对Ca2+的敏感性。
