title: A real-time, fluorescence-based assay for measuring μ-opioid receptor modulation of adenylyl cyclase activity in Chinese hamster ovary cells.
abstract: Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential-sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK (pEC₅₀, 7.3) or calcitonin (pEC₅₀, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO (pEC₅₀, 7.4; E(max), 56%), morphine (pEC₅₀, 7.0; E(max), 61%); and buprenorphine (pEC₅₀, 8.6; E(max), 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.
authors.affiliation: 1Australian School of Advanced Medicine, Macquarie University, NSW Australia.
authors.forename: Alisa
authors.initials: A
authors.lastname: Knapman
authors.forename: Fe
authors.initials: F
authors.lastname: Abogadie
authors.forename: Peter
authors.initials: P
authors.lastname: McIntrye
authors.forename: Mark
authors.initials: M
authors.lastname: Connor
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D037342:Pertussis Toxin / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases
keywords: cAMP; calcitonin; high throughput; membrane potential; opioid