#Change password in timecamp Activator#

Buprenorphine was from the National Measurement Institute (Lindfield, Australia). Morphine was a kind gift from the Department of Pharmacology, University of Sydney. Tyr- d-Ala-Gly- N-MePhe-Gly-ol acetate (DAMGO) was purchased from Auspep (Tullamarine, Australia). Unless otherwise noted, tissue culture reagents and buffer salts were from Invitrogen or Sigma-Aldrich. The final concentration of DMSO was not more than 0.1%, and this concentration did not produce a signal in the assay.

Changes in fluorescence were expressed as a percentage of baseline fluorescence after subtraction of the changes produced by vehicle addition. The background fluorescence of cells without dye or dye without cells was negligible. Further additions were made in volumes of 20 µL, as indicated. Baseline readings were taken every 2 s for at least 2 min, at which time either drug or vehicle was added in a volume of 20 µL. Fluorescence was measured using a FlexStation 3 (Molecular Devices) microplate reader with cells excited at a wavelength of 530 nm and emission measured at 565 nm.

Plates were then incubated at 37 ☌ at ambient CO 2 for 60 min. Prior to the assay, cells were loaded with 90 µL/well of the dye solution without removal of the L-15, giving an initial assay volume of 180 µL/well. Membrane potential was measured using a FLIPR Membrane Potential Assay kit (blue) from Molecular Devices (Sunnyvale, CA). The cells were plated in a volume of 90 µL in black-walled, clear-bottomed 96-well microplates (Corning, Corning, NY) and incubated overnight at 37 ☌ in ambient CO 2. Human MOR (hMOR) receptor expression was induced with 2 µg/mL tetracyclin 20 h prior to the assay. The day before the assay, cells were detached from the flask with trypsin/EDTA (Sigma-Aldrich, Castle Hill, Australia) and resuspended in 10 mL of Leibovitz’s L-15 media supplemented with 1% FBS, 100 U penicillin/streptomycin, and 15 mM glucose. Cells for assays were grown in 75-cm 2 flasks and used at greater than 80% confluence. Assays were carried out on cells up to 30 passages. Cells were passaged at 80% confluency as required. Hygromycin concentration was reduced to 200 µg/mL beyond passage 5. The selected cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 U penicillin/streptomycin, and 500 µg/mL hygromycin B up to passage 5. Cells expressing MOR were selected using hygromycin B (500 µg/mL) and grown to confluency. The HA-tagged human µ-opioid receptor was synthesized by Genscript (Piscataway, NJ).

One CHO-FRT-TR cell line was chosen and stably transfected with a pcDNA5 construct encoding the hemagglutinin (HA)–tagged human µ-opioid receptor complementary DNA (cDNA) together with the pOG44 (Flp recombinase plasmid) using the transfectant Fugene (Promega).

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. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. The effects of DAMGO were blocked by pertussis toxin. Simultaneous application of the opioids DAMGO ( pEC 50, 7.4 E max, 56%), morphine ( pEC 50, 7.0 E max, 61%) and buprenorphine ( pEC 50, 8.6 E max, 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. CHO-MOR cells hyperpolarized in response to application of FSK ( pEC 50, 7.3) or calcitonin ( pEC 50, 9.4).

#Change password in timecamp Activator#

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. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential–sensitive fluorescent dye. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation.