Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: Optimization of assay conditions

The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu-selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314–316), and the binding of [ 3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [ 3H]bremazocine to BIT FIT -treated membranes was greatly increased by conducting the assay at 0° C in the presence of 0.4 M NaCl. Using this 0° C NaCl assay condition, the binding of [ 3H]bremazocine was best described by a one-site binding model with a K d of 0.45 nM and a B max of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [ 3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [ 3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0° C in the presence of 0.4 M NaCl using BIT FIT -treated membranes and that rat brain is endowed with a high level of kappa binding sites.