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

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, [Leu³¹,Pro³⁴]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 ≫[Leu³¹,Pro³⁴]NPY-is characteristic of a Y₂-like NPY binding site population, while the benextramine-sensitive ³H-NPY binding sites appear to be a Y₁-like binding site population.