Receptor binding of [3H]naloxone benzoylhydrazone: A reversible κ and slowly dissociable μ opiate

M. Price, M. A. Gistrak, Y. Itzhak, E. F. Hahn, G. W. Pasternak

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60 Scopus citations


In standard 3H-opioid binding assays, the benzoylhydrazone derivative of naloxone (6-desoxy-6-benzoylhydrazido-N-allyl-14-hydroxydihydronormorphinone; Nh alBzoH) inhibited μ, κ, and δ binding at nanomolar concentrations. At concentrations as low as 1 nM, it also produced a wash-resistant inhibition of opioid binding. [3H]NalBzoH binding typically gave a ratio of total to nonspecific binding of 8:1. Binding reached state levels by 1 hr and was linear with tissue concentration. [3H]NalBzoH labeled two classes of sites. The binding to one was easily reversible whereas the other was not and was termed pseudoir-reversible. At 25°, almost 90% of [3H]naloxone binding and approximately 60-75% of [3H]NalBzoH binding dissociated over 90 min. However, the remainder of [3H]NalBzoH binding, corresponding to pseudoirreversible binding, remained constant over the next 5 hr at 25° and additional studies suggested a dissociation half-life of approximately 24 hr. Competition studies indicated that the reversible binding corresponded to neither μ nor δ binding and may represent a novel subtype of κ receptor. Pseudoirreversible binding was predominantly to a combination of both μ1 and μ2 receptors. Despite its extremely slow rate of dissociation, pseudoirreversible binding was not covalent inasmuch as lowering the pH to 5 or adding the GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p] completely dissociated prebound [3H]NalBzoH. The ability of Gpp(NH)p to dissociate pseudoirreversible [3H]NalBsoH binding raised the possibility that the slow rate of dissociation was related to interactions with a guanine nucleotide-binding protein.

Original languageEnglish
Pages (from-to)67-74
Number of pages8
JournalMolecular Pharmacology
Issue number1
StatePublished - 1989
Externally publishedYes


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