Characterization of Opioid Receptor Activation

[35 ]GTPγS binding was performed on membranes prepared from stably opioid receptor transfected cells in the presence and absence of the indicated compound for 60 min at 30 °C in the assay bufier (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 10 mM NaCl) containing 0.05 nM [³⁵S]GTPγS, 2 μg/mL each leupeptin, pepstatin, aprotinin, and bestatin, and 30 μM GDP, as previously described.⁴⁰ After the incubation, the reaction was filtered through glass-fiber filters (Whatman Schleicher & Schuell, Keene, NH) and washed three times with 3 mL of ice-cold bufier (50 mM Tris-HCl, pH 7.4) on a semiautomatic cell harvester. Filters were transferred into vials with 3 mL of Liquiscent (National Diagnostics, Atlanta, GA), and the radioactivity in vials was determined by scintillation spectroscopy in a Tri-Carb 2900TR counter (PerkinElmer Life and Analytical Sciences). Basal binding was determined in the presence of GDP and the absence of drug. Data was normalized to 100 nM DAMGO, DPDPE, and U50,488 for MOR-1, DOR-1, and KOR-1 binding, respectively. EC₅₀ and %E_max values were calculated by nonlinear regression analysis (GraphPad Prism, San Diego, CA).