Tocris). DPCPX was added to reduce the extensive basal activity of adenosine receptors [22]. The final 90-min incubation with GTPγ[35S] (54–68 pM; Perkin Elmer, Boston, USA) took place in the buffer supplemented with 2 mM GDP, 1 mM dithiothreitol, 1 µM DPCPX and various concentrations of DAMGO (0.1–10 µM, Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol) or morphine (10 µM), in the presence and absence of 10 µM naltrexone. Nonspecific binding was defined in the presence of 10 µM unlabeled GTPγS. The sections were washed twice for 5 min at 0°C in 50 mM Tris-HCl (pH 7.4) and 5 mM MgCl2, and dipped in distilled water at 0°C, before drying under a fan at room temperature. The sections were exposed to film (Kodak BioMax MR, Eastman Kodak, Rochester, USA) at 8°C for 4 days together with [14C]-standards (GE Healthcare, Buckinghamshire, UK), and thereafter films were developed. Films were digitized and binding densities of the sections quantified using Dage MTI camera system (DAGE-MTI Inc., Michigan City, USA) combined with MCID Elite M5+ 4.0 software (Imaging Research Inc., St. Catharines, Canada) with [14C]-standards, which were used to convert optical density values to radioactivity values. Non-specific binding values were subtracted from all other values. DAMGO-stimulation data were fitted for
