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A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

Bird, M F ; Hebbes, C P ; Scott, S W M ; Willets, J ; Thompson, J P ; Lambert, D G Vaudry, Hubert

PloS one, 2022-05, Vol.17 (5), p.e0268868-e0268868 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells
Autor: Bird, M F ; Hebbes, C P ; Scott, S W M ; Willets, J ; Thompson, J P ; Lambert, D G
Vaudry, Hubert
Assuntos: Analysis ; Animals ; Asthma ; Bioassays ; Biological Assay ; Biology and Life Sciences ; Biosensors ; Calcium ; Calcium (intracellular) ; Calcium ions ; Cell culture ; CHO Cells ; Cricetinae ; Cricetulus ; Detectors ; Engineering and Technology ; Enzyme-linked immunosorbent assay ; Evaluation ; Formyl peptides ; Humans ; Immune system ; Immunoregulation ; Kinases ; Lasers ; Leukocytes (polymorphonuclear) ; Ligands ; Medicine and Health Sciences ; mRNA ; Narcotics ; Neutrophils ; Nociceptin ; Opioid Peptides ; Opioid receptors ; Pain ; Peptides ; Phenylalanine ; Purine receptors ; Receptors ; Receptors, Opioid - metabolism ; Research and analysis methods ; Sepsis
É parte de: PloS one, 2022-05, Vol.17 (5), p.e0268868-e0268868
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
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MFB and CPH are joint first authors on this work.
Competing Interests: DGL is a scientific adviser to Cellomatics, a SME-CRO. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Descrição: 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.
Editor: United States: Public Library of Science
Idioma: Inglês

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A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells

Bird, M F ; Hebbes, C P ; Scott, S W M ; Willets, J ; Thompson, J P ; Lambert, D G Vaudry, Hubert

United States: Public Library of Science

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