Membrane binding was assessed in preparations from CHO-K1 cells transfected with OPRM1 receptor cDNA and incubated with 2nM [3H]-D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO; 37Ci/mmol; NEN, Boston, MA) and nonradioactive naloxone, morphine, and β-endorphin (β-End) ranging from 0.01nM to 1000 nM. The mixture was incubated at room temperature, filtered through Whatman GF/B filters (FP-100, Brandel), washed with ice-cold Tris-HCl, and assayed for radioactivity by liquid scintillation. Data were analyzed by nonlinear analysis using the Prism computer program (GraphPad Software, Inc.).
