Comparative Analysis of Nicotine-Like Receptor-Ligand Interactions in Rodent Brain Homogenate

Abstract

The effects of different variables such as incubation time, temperature, tissue protein content and pH on the interactions of various labeled nicotinic ligands with nicotine-like binding sites in vitro were studied in rodent brain preparations. The ligands tested were .alpha.-[3H]bungarotoxin (.alpha.-[3H]BTX), [3H]tubocurarine ([3H]TC) and [3H]nicotine ([3H]NIC). The regional distribution of the labeled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar with maximal binding within 5-10 min of incubation, followed by a continuous decrease. The binding of .alpha.-[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30-60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high-affinity site (Bmax, 60 pmol/g protein; KD, 6 nM) and a low-affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high-affinity [3H]NIC binding site are very similar to previously found data for the high-affinity binding site of [3H]TC and the binding site of .alpha.[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.