Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Hodgkin Building, Leicester LE1 9HN. UK.
Department of Chemical, Pharmaceutical and Agricultural Sciences and LTTA, University of Ferrara, Italy.
J Pharmacol Toxicol Methods. 2022 Jan-Feb;113:107132. doi: 10.1016/j.vascn.2021.107132. Epub 2021 Oct 30.
Opioid receptors are divided into the three classical types: MOP(μ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; Dermorphin (MOP) and N/OFQ (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQ we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels.
MOP（μ：mu）、DOP（δ：delta）和 KOP（κ：kappa），它们对纳洛酮敏感，还有一种额外的纳洛酮不敏感的痛敏肽/孤啡肽 FQ（N/OFQ）肽受体（NOP）。确定阿片受体位置和周转率的研究各不相同，依赖于：（i）测量受体 mRNA，（ii）遗传标记受体，（iii）用放射性配体标记受体，（iv）在免疫组织化学/免疫印迹中使用抗体，或（v）测量与选择性拮抗剂结合的受体功能。所有这些都有其缺点，包括 mRNA 不一定能预测蛋白质、抗体选择性差以及放射性标记在低表达系统中的应用有限等问题。在这篇小综述中，我们讨论了使用荧光标记的阿片受体配体。为了保持相应母体配体的药理学性质，荧光标记配体必须考虑荧光团（亮度和漂白倾向）、接头长度和化学性质，以及接头（因此探针）将连接的位置。使用具有光谱重叠的供体和受体荧光团有助于在 FRET 型测定中使用，以确定配体或标记受体对的接近程度。所有四种阿片受体类型都有广泛的激动剂和拮抗剂探针；由于内吞作用的可能性，需要对激动剂探针保持谨慎。我们已经制备了两种新型 ATTO 基探针；Dermorphin（MOP）和 N/OFQ（NOP）。这些探针在一系列制剂中标记 MOP 和 NOP，并使用 N/OFQ 我们通过 FRET 证明了内化和配体-受体相互作用。荧光阿片受体探针相对于更传统的抗体和放射性标记物具有潜在的方法学优势。
