Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina 27695, United States.
J Am Chem Soc. 2023 Nov 8;145(44):24071-24080. doi: 10.1021/jacs.3c07487. Epub 2023 Oct 19.
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.
内源性阿片肽系统通常是疼痛治疗的靶点，但由于缺乏直接检测特定阿片肽神经肽原位释放的方法，神经肽释放的基本性质仍未得到很好的理解。这些肽类物质集中在嗜铬细胞的大致密核心囊泡中，并从其中释放出来。虽然这些神经内分泌细胞的儿茶酚胺释放已经得到很好的描述，但以前尚未实现阿片肽胞吐事件的直接定量。在这项工作中，平面碳纤维微电极作为一种“突触后”传感器，通过安培监测来探测儿茶酚胺和神经肽释放动力学。采用 500 mV 的恒定电位来定量儿茶酚胺释放，而 1000 mV 的更高电位用于驱动从嗜铬细胞释放的阿片肽神经肽中酪氨酸的氧化，酪氨酸是阿片肽神经肽的 N 末端氨基酸。通过区分在两个电位下收集的数据，可以在单个囊泡水平上揭示这两种神经化学物质类别的独特动力学。肽信号的幅度随着重复刺激而降低，而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。嗜铬颗粒致密核心被确定为一个重要的机制手柄，通过它可以在从同一囊泡群体释放时对不同类别的递质进行动力学调节。总的来说，这些数据提供了前所未有的见解，揭示了从分离的嗜铬细胞中释放儿茶酚胺和阿片肽神经肽之间的关键差异。
