Abstract

The binding in vitro of an opiate agonist, 3H-dihydromorphine, was studied using a particulate fraction obtained from rat brain homogenates and compared with that of an opiate antagonist, 3H-naloxone. The binding of 3H-dihydromorphine may be separated into two components: one a saturable component and the other nonsaturable. The saturable binding may be calculated from the differences in binding observed in the absence and presence of high concentrations of levorphanol. The use of dextrorphan results in an artifactual separation of this component, although relative stereospecificity was observed for levorphanol and dextrorphan. There were marked regional differences in the distribution of saturable 3H-dihydromorphine binding in the brain. These were primarily due to the difference in the concentration of the saturable binding sites within various brain regions. It appeared that the saturable binding sites from various brain regions had similar affinities for dihydromorphine except for the binding site from cerebral cortex which had a higher affinity. In contrast, saturable binding sites for naloxone in various brain regions had different affinities for naloxone. It appears that naloxone has at least two types of saturable binding sites, one of which is not available to dihydromorphine. This is based on observations 1) that the total concentration of saturable binding sites for naloxone was greater than that for dihydromorphine in each brain region studied irrespective of the assay medium used and 2) that unlabeled dihydromorphine inhibited the 3H-naloxone binding in striatum but failed to alter it significantly in cerebellum, whereas unlabeled naloxone reduced 3H-naloxone binding significantly in both brain regions. The difference in concentrations of saturable binding sites for naloxone and dihydromorphine was relatively small in striatum but larger in cerebellum, indicating that the saturable binding sites in cerebellum are predominantly naloxone-specific, whereas those in striatum are capable of binding both naloxone and dihydromorphine. In cerebrospinal fluid or in simulated intracellular fluid, the apparent affinity for dihydromorphine was lower and that for naloxone was higher than in Tris-HCl buffer. It is concluded that naloxone binds to dihydromorphine binding site and to another site, which has a different affinity for naloxone and is not available to dihydromorphine. Studies in which opiate receptor binding was assayed in Tris-HCl buffer may need to be re-evaluated. Further, in studies where opiate binding in vitro is assessed following pharmacologic intervention, such binding should be estimated in a relevant physiological medium rather than in Tris-HCl buffer.