Untapped Potential: Real-Time Measurements of Opioid Exocytosis at Single Cells.
Untapped Potential: Real-Time Measurements of Opioid Exocytosis at Single Cells.
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DOI：
10.1021/jacs.3c07487
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发表时间：
2023-10
影响因子：
15
通讯作者：
J. Denison;A. C. De Alwis;Ruby Shah;G. McCarty;Leslie A. Sombers
中科院分区：
文献类型：
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作者：
J. Denison;A. C. De Alwis;Ruby Shah;G. McCarty;Leslie A. Sombers
内源性阿片类系统通常是疼痛治疗的目标，但由于缺乏原位直接检测特定阿片类神经肽的方法，神经肽释放的基本性质仍然知之甚少。这些肽集中在嗜铬细胞的大致密核心囊泡中并从中释放。尽管这些神经内分泌细胞释放的儿茶酚胺已得到很好的表征，但阿片肽胞吐作用事件的直接定量此前尚未实现。在这项工作中，平面碳纤维微电极充当“突触后”传感器，通过电流监测探测儿茶酚胺和神经肽的释放动态。采用 500 mV 的恒定电势来定量儿茶酚胺释放，并使用 1000 mV 的较高电势来驱动酪氨酸（从嗜铬细胞释放的阿片样神经肽中的 N 端氨基酸）的氧化。通过区分在两个电位下收集的结果，数据揭示了这两种神经化学物质在单囊泡水平上的独特动力学。肽能信号的幅度随着重复刺激而降低，因为这些信号的半宽度同时增加。相比之下，儿茶酚胺释放事件的幅度随着重复刺激而增加，但每个事件的半宽度没有变化。嗜铬粒蛋白致密核心被认为是一个重要的机械手柄，当从同一囊泡群中释放时，可以通过该手柄对不同类别的递质进行动力学调节。总体而言，这些数据为了解分离的嗜铬细胞释放的儿茶酚胺和阿片类神经肽之间的关键差异提供了前所未有的见解。
The endogenous opioid system is commonly targeted in pain treatment, but the fundamental nature of neuropeptide release remains poorly understood due to a lack of methods for direct detection of specific opioid neuropeptides in situ. These peptides are concentrated in, and released from, large dense-core vesicles in chromaffin cells. Although catecholamine release from these neuroendocrine cells is well characterized, the direct quantification of opioid peptide exocytosis events has not previously been achieved. In this work, a planar carbon-fiber microelectrode served as a "postsynaptic" sensor for probing catecholamine and neuropeptide release dynamics via amperometric monitoring. A constant potential of 500 mV was employed for quantification of catecholamine release, and a higher potential of 1000 mV was used to drive oxidation of tyrosine, the N-terminal amino acid in the opioid neuropeptides released from chromaffin cells. By discriminating the results collected at the two potentials, the data reveal unique kinetics for these two neurochemical classes at the single-vesicle level. The amplitude of the peptidergic signals decreased with repeat stimulation, as the halfwidth of these signals simultaneously increased. By contrast, the amplitude of catecholamine release events increased with repeat stimulation, but the halfwidth of each event did not vary. The chromogranin dense core was identified as an important mechanistic handle by which separate classes of transmitter can be kinetically modulated when released from the same population of vesicles. Overall, the data provide unprecedented insight into key differences between catecholamine and opioid neuropeptide release from isolated chromaffin cells.