Benextramine-neuropeptide Y (NPY) binding site interactions: Characterization of 3H-NPY binding site heterogeneity in rat brain

Abstract

Pre-incubation of rat brain membranes with 200 μM benextramine followed by extensive dilution and washing to remove unbound ligand reduced B max for N-[propionyl- 3H]-NPY ( 3H-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 3H-NPY and 57 μM benextramine, there was a significant shift to the right; the apparent Kd for 3H-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 3H-NPY binding site populations. PYY, NPY and NPY 13–36 each displaced 100% of 3H-NPY from rat brain membrane binding sites both in the absence and presence of 1 mM benextramine. In contrast, [Leu 31,Pro 34]NPY displayed the same binding site selectivity as benextramine in displacing 65% of 3H-NPY from specific binding sites on untreated rat brain membranes, and it failed to displace 3H-NPY from membranes treated with 1 mM benextramine. Thus the selectivity of the benextramine-insensitive 3H-NPY binding site population—PYY >= NPY > NPY 13–36 ⪢[Leu 31,Pro 34]NPY—is characteristic of a Y 2-like NPY binding site population, while the benextramine-sensitive 3H-NPY binding sites appear to be a Y 1-like binding site population.