TEMPORALLY RESOLVED CATECHOLAMINE SPIKES CORRESPOND TO SINGLE VESICLE RELEASE FROM INDIVIDUAL CHROMAFFIN CELLS
TEMPORALLY RESOLVED CATECHOLAMINE SPIKES CORRESPOND TO SINGLE VESICLE RELEASE FROM INDIVIDUAL CHROMAFFIN CELLS
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DOI：
10.1073/pnas.88.23.10754
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发表时间：
1991-12-01
影响因子：
11.1
通讯作者：
VIVEROS, OH
中科院分区：
文献类型：
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作者：
WIGHTMAN, RM;JANKOWSKI, JA;VIVEROS, OH
从含有尼古丁、氨基甲酰胆碱或钾离子的微管中短暂的压力喷射或机械刺激，可引起培养中单个牛嗜铬细胞分泌儿茶酚胺。用放置在电池附近的碳纤维微电极进行电化学监测。循环伏安法用于鉴定分泌物，而恒电位安培法用于提高儿茶酚胺检测的时间分辨率(毫秒范围)。在分泌过程中，观察到短暂的电流尖峰，这被证明是由于通过电氧化检测到儿茶酚胺。这些尖峰具有在随机时间和位置从单细胞表面释放的儿茶酚胺的多分子包的物理特征。研究发现，随着电池-电极间距的增加，尖峰的半宽也随之增大。儿茶酚胺的特性与基于单个储存囊分泌的预期很好地相关。在缓冲液中没有钙离子的情况下不会出现尖峰，大多数尖峰分布在0.2到2个皮库仑之间，对应于检测到的1-10个儿茶酚胺的attomoles。刺激物的频率随着刺激强度的增加而增加，但每个刺激物中儿茶酚胺的平均含量与刺激物无关。因此，这些测量代表了儿茶酚胺的量子分泌的时间分辨观察，并为胞吐假说提供了直接证据。
Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoyl-choline, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.