BENEXTRAMINE-NEUROPEPTIDE-Y (NPY) BINDING-SITE INTERACTIONS - CHARACTERIZATION OF H-3 NPY BINDING-SITE HETEROGENEITY IN RAT-BRAIN

Pre-incubation of rat brain membranes with 200 muM benextramine followed by extensive dilution and washing to remove unbound ligand reduced Bmax for N-[propionyl-H-3]NPY (H-3-NPY) specific binding by 61% relative to control membranes treated identically but in the absence of benextramine. When rat brain membranes were co-incubated with H-3-NPY and 57 muM benextramine, there was a significant shift to the right; the apparent Kd for H-3-NPY binding increased two-fold relative to control membranes. These data are consistent with the hypothesis that benextramine is a competitive and irreversible ligand for a population (60-65%) of rat brain NPY binding sites. 'Paired tube' assays were then used to determine the selectivity of these benextramine-sensitive and insensitive H-3-NPY binding site populations. PYY, NPY and NPY13-36 each displaced 100% of H-3-NPY from rat brain membrane binding sites both in the absence and presence of 1 mM benextramine. In contrast, [Leu31,Pro34]NPY displayed the same binding site selectivity as benextramine in displacing 65% of H-3-NPY from specific binding sites on untreated rat brain membranes, and it failed to displace H-3-NPY from membranes treated with 1 mM benextramine. Thus the selectivity of the benextramine-insensitive H-3-NPY binding site population-PYY >= NPY > NPY13-36 >>[Leu31,Pro34]NPY-is characteristic of a Y2-like NPY binding site population, while the benextramine-sensitive H-3-NPY binding sites appear to be a Y1-like binding site population.