A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.
A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.
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DOI：
10.1371/journal.pone.0268868
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
孤啡肽（N/OFQ）是N/OFQ受体或NOP的内源性阿片激动剂。该受体系统参与疼痛处理，但也在免疫调节中发挥作用。事实上，多形核细胞（PMN）表达N/OFQ前体的mRNA，是循环N/OFQ的潜在来源。目前的测量是基于ELISA和RIA技术。在这项研究中，我们设计了一种生物测定法来测量N/OFQ从单个PMN的释放。用人（h）NOP受体和Gα iq 5嵌合体转染的中国人卵巢（CHO）细胞在生物传感器细胞中迫使受体偶联以增加细胞内Ca 2 +;这可以用FLUO-4染料测量。如果将从健康人志愿者分离的PMN与CHOhNOPGα iq 5生物传感器细胞相邻分层，然后用化学引诱物N-甲酰基-甲硫氨酰基-亮氨酰基-苯丙氨酸（fMLP）刺激，我们假设释放的N/OFQ将激活生物传感器。中性粒细胞也释放ATP和CHO细胞表达嘌呤能受体耦合到升高的Ca 2+。在用高浓度PPADS和oATP阻断这些受体（P2 Y1、P2 Y2和P2 X7）的系统中，用fMLP刺激PMN会增加PMN中的Ca 2+，然后很快增加生物传感器细胞中的Ca 2+。因此，我们的数据报告了从免疫细胞释放单细胞N/OFQ的检测。当细胞与选择性NOP拮抗剂SB-612111预孵育时，这是不存在的。总的来说，这是单小区N/OFQ释放的第一个描述。我们将使用进一步纯化的单个细胞类型部署该测定，并使用其进一步研究N/OFQ-NOP系统在疾病中的作用;特别是脓毒症，其中有强有力的证据表明N/OFQ水平增加，结果恶化。
Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.
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影响因子：
8.1
作者：
Sueyoshi K;Sumi Y;Inoue Y;Kuroda Y;Ishii K;Nakayama H;Iwabuchi K;Kurishita Y;Shigemitsu H;Hamachi I;Tanaka H
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