title: A continuous, fluorescence-based assay of μ-opioid receptor activation in AtT-20 cells.
abstract: Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.
authors.affiliation: Macquarie University, Sydney, NSW, Australia.
authors.forename: Alisa
authors.initials: A
authors.lastname: Knapman
authors.forename: Marina
authors.initials: M
authors.lastname: Santiago
authors.forename: Yan Ping
authors.initials: YP
authors.lastname: Du
authors.forename: Philip R
authors.initials: PR
authors.lastname: Bennallack
authors.forename: Macdonald J
authors.initials: MJ
authors.lastname: Christie
authors.forename: Mark
authors.initials: M
authors.lastname: Connor
mesh_terms: D000701:Analgesics, Opioid / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D004361:Drug Tolerance; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009020:Morphine / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D015221:Potassium Channels / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000701:Analgesics, Opioid; D008024:Ligands; D015221:Potassium Channels; D017450:Receptors, Opioid, mu; D009270:Naloxone; D009020:Morphine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins