Measurement of Intracellular Cyclic Adenosine Monophosphate (cAMP)

cAMP measurements were conducted to investigate G protein pathway signal activation. We used cAMP-homogeneous time-resolved fluorescence kits for analysis. OPRM/OPRD bind Gα_i protein upon receptor activation, inhibiting intracellular cAMP accumulation induced by forskolin.⁵ Therefore, low cAMP levels are indicative of opioid receptor activation. To prevent degradation of intracellular cAMP, all experiments were conducted in the presence of 500 μM 3-isobutyl-1-methylxanthine (IBMX). The test compounds were diluted with dimethyl sulfoxide (DMSO) to a maximum final concentration of 1% (to prevent cell membrane disruption), or phosphate-buffered saline (PBS).³³ Irreversible antagonists (β-FNA for OPRM, β-CNA for OPRD) were used to reduce the population of receptors on the cell surface to diminish signaling amplification.³⁴ Cells were pretreated with various concentrations of irreversible antagonists for 20 min before cAMP measurement. All data were measured using a FlexStation 3 (Molecular Devices, CA) and normalized to the maximal assay response obtained using a full agonist (DAMGO for OPRM, SNC80 for OPRD). In assays using irreversible antagonist pretreatment, data were normalized to the maximal response of test compounds without pretreated irreversible antagonist group.