Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [3H]naloxone binding sites in rat brainstem synaptosomal preparations at 23 degrees C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [3H]naloxone gave pseudo-first order association rate of 0.9 +/- 0.04, 1.23 +/- 0.08 and 1.06 +/- 0.08 min-1, respectively. The dissociation of a 1 nM [3H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min-1. On the other hand, dissociation of 10 nM [3H]naloxone was monophasic with a kd of 1.1 min-1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [3H]naloxone binding sites could be seen by displacement studies performed with opioid peptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.