Abstract

The study of [³H]naltrexone binding in a membrane preparation from rat brain revealed that experimental conditions of the opiate receptor assay markedly influenced the outcome of the corresponding Scatchard analysis. The use of inappropriate concentrations of the unlabeled displacing drugs to assess stereospecific [³H]naltrexone interaction resulted in Scatchard plots which mimicked cooperativity in binding. These monophasic plots also indicated that sodium affects antagonist binding by changing receptor affinity (K_D) but not the density of sites. When assessed under correct experimental conditions the Scatchard plots for specific [³H]naltrexone binding were biphasic. Sodium, without affecting the K_D, increased and decreased the number of high- and low-affinity sites, respectively. Thus, at 25° the total number of opiate receptor sites for [³H]naltrexone binding in the absence and presence of sodium was statistically indistinguishable. Initial membrane incubation competed with sodium in increasing the density of high-affinity [³H]naltrexone-binding sites. After kinetic resolution and computer analysis considering several binding models, the data for specific [³H]naltrexone binding provided best fit for two saturable sites. At 25° and in the absence of sodium the respective approximate K_D values were 0.4 and 30 nM, with densities of 200 and 350 fmoles/mg of protein. Under a variety of experimental conditions, including different temperatures and exposure of the membranes to trypsin or freezing, some of these binding parameters differed, but the biphasic nature of specific [³H]naltrexone binding was unaltered. Identical biphasic Scatchard plots were obtained if specific binding of [³H]naltrexone was determined with excess unlabeled naltrexone, morphine, ethylketocyclazocine, or SKF 10047; with dextrorphan and levorphanol; or with enantiomers of naloxone as displacing ligands. Terminating the binding assay by rapid centrifugation yielded results identical with those obtained when quick filtration was used. The dissociation of bound [³H]naltrexone was resolved into a rapid component and a slow component. In the presence of high concentrations of the drug an additional rapid component of dissociation became apparent. The K_D values calculated from the two rapid components of dissociation and the corresponding rate constants of association agreed well with those determined for the high- and low-affinity [³H]naltrexone-binding sites by Scatchard analysis. The nature of the slow dissociation component with markedly high affinity has to await further clarification. The results of this study characterize the interaction of naltrexone with opiate receptor, contribute to the understanding of the mechanism of the sodium effect, and describe the role of methodology in evaluating ligand binding.