Properties of Opioid Receptor Binding in the Rat Brain

Abstract

The properties of specific binding of ³H-naloxone, opiate antagonist, were studied in a particulate fraction from a rat brain. The specific binding was time and pH dependent, saturable with respect to ³ H-naloxone and tissue concentration. The binding of ³ H-naloxone could be described on the basis of two independent binding sites with apparent Kd of 3 nM and 57 nM, respectively. Receptor binding of ³H-naloxone was enhanced by Na⁺ and Li⁺ but not by K⁺, and the divalent cations depressed the specific binding. When 100 mM sodium was included in the incubation medium, the number of apparent high affinity binding sites was almost doubled, but the number of low affinity sites was unaffected, and there was no significant effect on the dissociation constants. Non radioactive opiates and synthetic endorphins were incubated with ³H-naloxone and rat-brain homogenate in the absence and presence of 100 mM NaCl, and the concentration of the drug required to give 50% inhibition of binding (IC₅₀) was determined by a log-probit analysis. When the bound radioactivity was separated by filtration through a glass-fiber filter, the sodium index (IC₅₀ + NaCl/IC₅₀-NaCl) for naloxone was 1, while levallorphan and pentazocine, mixed agonist-antagonists, exhibited the sodium index of 2.2 and 3.3, respectively. The sodium index for agonists, morphine and synthetic endorphins, ranged between 50-307. When the centrifugation method was used to isolate the ³ H-naloxone-receptor complex, the sodium index for mixed agonist-antagonists and agonists were 2-fold and 4-10-fold, respectively, while the sodium index for the antagonist was unaffected.
Opioid receptor distribution, determined by the specific binding of ³H-naloxone, was examined in the 8 regions of a rat brain. Opioid receptors were highly localized in the corpus striatum, thalamus plus subthalamus and hypothalamus, and negligible in the cerebellum. Brain enkephalin activity, determined by an opioid radioreceptor assay with methionine-enkephalin as standard, had a similar regional distribution to that of the opioid receptors, with the highest level in the corpus striatum and the lowest level in the cerebellum. However, there were some discrepancies between regional variations for enkephalin activity and the opioid receptor, which may be ascribed to variable composition and characteristics of endogenous opioid substances in various brain regions.