Voltammetric and pharmacological characterization of dopamine release from single exocytotic events at rat pheochromocytoma (PC12) cells
Voltammetric and pharmacological characterization of dopamine release from single exocytotic events at rat pheochromocytoma (PC12) cells
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DOI：
10.1021/ac980129f
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发表时间：
1998-08-01
影响因子：
7.4
通讯作者：
Ewing, AG
中科院分区：
文献类型：
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作者：
Kozminski, KD;Gutman, DA;Ewing, AG
虽然大鼠嗜铬细胞瘤（PC 12）神经递质储存囊泡中含有多种神经递质，包括儿茶酚胺，很少有证据表明，在电流监测胞吐过程中检测到的分子种类是儿茶酚胺。相反，由于这些是含儿茶酚胺的细胞，因此假设释放了儿茶酚胺。另外，尽管释放的递质的总量可以定量，但是对于每个胞吐事件，评价释放点的浓度是极其困难的。解释伏安图中获得的受影响的电极和细胞之间的体积和胞吐过程中的胞吐孔的大小定义的阿升是一个极端的分析挑战。在这里，我们使用伏安法从单个胞吐事件释放类似于10（-19）mol来鉴定，沿着药理学证据，在PC 12细胞中释放的化合物为儿茶酚胺，最可能是多巴胺。在胞吐事件之后发生氧化的电极面积与获取伏安图之前的时间延迟成比例。该模型允许确定来自各个释放事件的相对浓度，并已用于检查对照细胞和与多巴胺前体L-3，4-二羟基苯丙氨酸（L-DOPA）孵育的细胞的事件，暴露于L-DOPA（100 μ M，1小时）导致11个细胞的145个可检测事件，而29个对照细胞的77个事件，清楚地表明囊泡可以“装载”多巴胺。然而，在电极表面处测量的浓度为L-DOPA处理的细胞和对照细胞提供了相似的分布。循环伏安法测量的相对浓度为zeptomole水平的发射器在阿升体积提供的证据表明，加载囊泡通过增加发射器合成不会导致在个别释放位点的浓度升高。
Although rat pheochromocytoma (PC12) neurotransmitter storage vesicles are known to contain a variety of neurotransmitters including catecholamines, there is little evidence that the molecular species detected during amperometric monitoring of exocytosis is a catecholamine. Rather, as these are catecholamine-containing cells, one assumes catecholamines are released, Additionally, although the total amount of transmitter released can be quantified, it has been extremely difficult to evaluate the conentration at the point of release for each exocytosis event. Interpreting voltammograms obtained in the attoliter volume affected between the electrode and the cell and defined by the size of the exocytosis pore during exocytosis is an extreme analytical challenge. Here we use voltammetry of similar to 10(-19) mol released from individual exocytosis events to identify, along with pharmacological evidence, the released compound at PC12 cells as a catecholamine, most likely dopamine. The area of the electrode at which oxidation occurs following an exocytosis event is proportional to the temporal delay prior to acquisition of a voltammogram, This model allows determination of relative concentrations from individual release events and has been used to examine events at control cells and cells incubated with the dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), Exposure to L-DOPA (100 mu M for 1 h) results in 145 detectable events for 11 cells compared to 77 events for 29 control cells, clearly indicating that vesicles can be "loaded" with dopamine. However, the concentrations measured at the electrode surface provide similar distributions for both L-DOPA-treated and control cells. Cyclic voltammetric measurements of relative concentration for zeptomole levels of transmitter in attoliter volumes provide evidence that loading vesicles by increased transmitter synthesis does not lead to elevated concentrations at individual release sites.