title: Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: optimization of assay conditions.
abstract: 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 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax 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 degrees 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.
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D001575:Benzomorphans / Q000378:metabolism / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D017473:Receptors, Opioid, kappa; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D000477:Alkylating Agents; D001575:Benzomorphans; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D012965:Sodium Chloride; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; C027248:bremazocine