Receptor-Binding Assay.

[³H]DADLE, [³H]U69,593, and [³H]DAMGO were used to label the δ-, κ-, and µ-opioid receptors, respectively (Barrett and Vaught, 1983; Lahti et al., 1985; Onogi et al., 1995). The K_d and B_max values for the radioligands were determined using a saturation assay (Table 1). Monoclonal human opioid receptors were stably expressed in Chinese hamster ovary cell lines for δ- (provided by Dr. Stephen J. Cutler, University of South Carolina) and µ-opioid receptors (PerkinElmer) and in human embryonic kidney (HEK) cells for KOR (Dr. Stephen J. Cutler, University of South Carolina). The Bradford protein assay was used to determine and adjust the concentration of protein required for the assay (Tal et al., 1985). Ten micrograms of each membrane protein was separately incubated with the corresponding radioligand in the presence of different concentrations of test compounds in TME buffer [50 mM Tris (Sigma-Aldrich), 3 mM MgCl₂ (Sigma-Aldrich), and 0.2 mM EGTA (Sigma-Aldrich), pH 7.7] for 60 minutes at room temperature. The bound radioligand was separated by filtration using the Connectorate filtermat harvester for 96-well microplates (Dietikon, Switzerland) and counted for radioactivity using a Hidex sense β microplate reader (Hidex, Turku, Finland). Specific binding at δ-, κ-, and µ-opioid receptors was determined as the difference in binding obtained in the absence and presence of 10 µM SNC 80; 10 µM U69,593; and 10 µM naltrexone, respectively.

Summary of scintillation counting conditions employed for assessing affinity at various binding sites in competition for the radioligands labeling human opioid receptor subtypes

K_d and B_max values in parentheses are 95% CIs.