A real-time, fluorescence-based assay for measuring µ-opioid receptor modulation of adenylyl cyclase activity in chinese hamster ovary cells

Knapman, A, Abogadie, F, McIntyre, P and Connor, M 2014, 'A real-time, fluorescence-based assay for measuring µ-opioid receptor modulation of adenylyl cyclase activity in chinese hamster ovary cells', Journal of Biomolecular Screening, vol. 19, no. 2, pp. 223-231.


Document type: Journal Article
Collection: Journal Articles

Title A real-time, fluorescence-based assay for measuring µ-opioid receptor modulation of adenylyl cyclase activity in chinese hamster ovary cells
Author(s) Knapman, A
Abogadie, F
McIntyre, P
Connor, M
Year 2014
Journal name Journal of Biomolecular Screening
Volume number 19
Issue number 2
Start page 223
End page 231
Total pages 9
Publisher Sage Publications
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.
Subject Basic Pharmacology
Keyword(s) cAMP
calcitonin
high throughput
membrane potential
opioid
DOI - identifier 10.1177/1087057113501391
Copyright notice © 2013 Society for Laboratory Automation and Screening.
ISSN 1087-0571
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Citation counts: TR Web of Science Citation Count  Cited 9 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 8 times in Scopus Article | Citations
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