Department of Cardiovascular Sciences, Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK.
Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy.
Br J Pharmacol. 2018 Dec;175(24):4496-4506. doi: 10.1111/bph.14504. Epub 2018 Nov 6.
The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQ ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression.
We assessed N/OFQ receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQ binding was measured in (i) HEK cells expressing NOP and NOP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).
N/OFQ bound to NOP receptor with nM affinity and high selectivity. N/OFQ activated NOP receptor by reducing cAMP formation and increasing Ca levels in CHO cells. N/OFQ was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQ was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOP receptor is activated by N/OFQ , movement of ligand and receptor from the cell surface to the cytosol can be measured.
In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQ to study a wide range of N/OFQ-driven cellular responses.
孤啡肽/N-末端前体（N/OFQ）受体（NOP）是阿片受体家族的成员，参与多种生理反应，如疼痛和免疫调节。在这项研究中，我们将红色荧光染料 ATTO594 与肽配体 N/OFQ（N/OFQ）缀合，用于 NOP 受体，并在高（在重组系统中）和低（在免疫细胞上）表达水平探索 NOP 受体功能。
我们评估了 N/OFQ 受体在重组（CHO）细胞系中的结合、选择性和功能活性。在（i）表达 NOP 和 NOP 受体的 HEK 细胞、（ii）表达 hNOPGαqi5 嵌合体（强制偶联到可测量的 Ca 反应）的 CHO 细胞和（iii）新鲜分离的人多形核细胞（PMN）中测量活细胞 N/OFQ 结合。
N/OFQ 以 nM 亲和力和高选择性结合 NOP 受体。N/OFQ 通过减少 CHO 细胞中的 cAMP 形成和增加 Ca 水平激活 NOP 受体。N/OFQ 还能够在 PMN 细胞的低表达水平上可视化 NOP 受体。在 NOP-GFP 标记的受体中，N/OFQ 用于 FRET 方案，其中 GFP 发射激活 ATTO，可视化配体-受体相互作用。当 NOP 受体被 N/OFQ 激活时，可以测量配体和受体从细胞表面向细胞质的运动。
在缺乏经过验证的 NOP 受体抗体和放射性标记物使用问题（尤其是在低表达系统中）的情况下，这些数据表明 N/OFQ 可用于研究广泛的 N/OFQ 驱动的细胞反应。
