Unknown

Hartley-VAF Plus guinea pigs were killed by decapitation and the brains removed. The brains, less cerebellum, were homogenized in six volumes of 0.05 M Trizma buffer (pH 7.4) with a Virtrishear homogenizer at a control setting of 70 for three 5 s intervals. The homogenate was centrifuged at 25000g at 40C for 20 min. The pellet was resuspended in six volumes of aqueous 0.32 M sucrose and stored at -70 0C until needed. Frozen homogenate was thawed at room temperature and diluted with 0.05 M Trizma buffer (pH 7.4) to give a final dilution ratio of 1:60 (initial brain weight to total solution volume). Radioligands used were [3H]bremazocine (0.5 nM) for total ligand binding, [3H]DAMG0 (1.0 nM) for preceptor binding, [3H]DPDPE (1.0 nM) for delta- receptor binding, and [3H]U-69,593( 1.0 nM) for kapa-receptor N-MPPP was tested against bremazocine and DPDPE in duplicate at nine concentrations between 1.0 and 1000 nM, N-MPPP tested against [3H]U-69,593 in triplicate at five concentrations between 0.22 and 4.7 nM. Nonspecific binding was measured in the presence of 10 mkM naloxone