Membrane preparations of brain regions were incubated at room temperature for 30 min with 100 mM NaCl and 100 μM GDP in 50 mM Tris–HCl buffer (pH 7.4) containing 1 mM EDTA (TE buffer), centrifuged, and washed three times with the TE buffer by resuspension and centrifugation. Membranes were incubated with ~3 nM [3H]DAMGO in TE buffer with 5 mM MgCl2. Such pretreatment and binding conditions have been shown to convert the MOPR to high agonist affinity states (Nishino et al., 1990; Wong et al., 1994). Nonspecific binding was measured in the presence of naloxone (1 μM). Each binding assay was carried out in duplicate in a final volume of 0.5 ml with 0.1–0.3 mg protein/tube to yield 1500 –3000 dpm. Incubations were terminated by filtration through Whatman GF/B filters under vacuum. Radioactivity in filters was measured by liquid scintillation counting.
