Mansvelder H D, Kits K S
Research Institute Neurosciences Vrije Universiteit, Faculty of Biology, Membrane Physiology Section, 1081 HV, Amsterdam, The Netherlands.
J Neurosci. 1998 Jan 1;18(1):81-92. doi: 10.1523/JNEUROSCI.18-01-00081.1998.
Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.
促黑素细胞会响应通过电压门控钙通道进入的钙离子，释放预先停靠的、含有α-黑素细胞刺激素的大的、有致密核心的囊泡。我们的首要目标是研究在单个动作电位（AP）持续时间量级的短程去极化所诱发的胞吐作用、快速内吞作用和钙离子内流之间的关系。通过电容测量来监测胞吐作用和快速内吞作用。我们发现短程去极化（40毫秒）仅诱发约3%的预先停靠的可释放囊泡池的快速释放。其次，我们通过调节细胞内缓冲能力来询问这些细胞中电压门控钙通道与预先停靠的囊泡之间的距离是多少。低浓度的钙螯合剂乙二醇双四乙酸（EGTA）对胞吐作用和快速内吞作用有不同影响。相对于50微摩尔，100微摩尔的EGTA使胞吐作用略有减弱，但在400微摩尔时胞吐作用则被强烈抑制，这表明钙离子必须移动较长距离才能刺激胞吐作用。然而，对于2至40毫秒之间的脉冲持续时间，钙离子刺激胞吐作用的效力是恒定的，这表明在促黑素细胞中，胞吐作用与单个AP期间钙离子内流的量和持续时间呈线性相关。在100微摩尔EGTA时快速内吞作用就已被强烈抑制，这表明在促黑素细胞中内吞作用本身是依赖钙离子的。此外，在33摄氏度时快速内吞作用的时间常数约为116毫秒，这比室温下快三倍。内吞作用的幅度与紧接内吞作用之前的胞吐作用幅度之间存在很强的相关性。这种相关性以及快速的内吞作用时间常数都表明胞吐囊泡能被快速回收。
