Abstract
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.
Funder
Biotechnology and Biological Sciences Research Council
Publisher
Public Library of Science (PLoS)
Reference31 articles.
1. The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential;D.G. Lambert;Nat Rev Drug Discov,2008
2. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems;L. Toll;Pharmacological Reviews,2016
3. Nociceptin/orphanin FQ-NOP receptor system in inflammatory and immune-mediated diseases;E.C. Gavioli;Vitam Horm,2015
4. Evidence for nociceptin/orphanin FQ (NOP) but not micro (MOP), delta (DOP) or kappa (KOP) opioid receptor mRNA in whole human blood.;M. Al-Hashimi;Br J Anaesth,2016
5. The nociceptin/orphanin FQ-NOP receptor antagonist effects on an animal model of sepsis;D. Carvalho;Intensive Care Med,2008
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