title: Receptor binding of [3H]naloxone benzoylhydrazone: a reversible kappa and slowly dissociable mu opiate.
abstract: In standard 3H-opioid binding assays, the benzoylhydrazone derivative of naloxone (6-desoxy-6-benzoylhydrazido-N-allyl-14-hydroxydihydronormorphi none; NalBzoH) inhibited mu, kappa, and delta 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 steady 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 pseudoirreversible. At 25 degrees, 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 degrees and additional studies suggested a dissociation half-life of approximately 24 hr. Competition studies indicated that the reversible binding corresponded to neither mu nor delta binding and may represent a novel subtype of kappa receptor. Pseudoirreversible binding was predominantly to a combination of both mu 1 and mu 2 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]NalBzoH binding raised the possibility that the slow rate of dissociation was related to interactions with a guanine nucleotide-binding protein.
authors.affiliation: Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York, New York.
authors.forename: M
authors.initials: M
authors.lastname: Price
authors.forename: M A
authors.initials: MA
authors.lastname: Gistrak
authors.forename: Y
authors.initials: Y
authors.lastname: Itzhak
authors.forename: E F
authors.initials: EF
authors.lastname: Hahn
authors.forename: G W
authors.initials: GW
authors.lastname: Pasternak
mesh_terms: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D009270:Naloxone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D014316:Tritium
chemical_list: D011759:Pyrrolidines; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D014316:Tritium; C058380:naloxone benzoylhydrazone; D006165:Guanylyl Imidodiphosphate; D009270:Naloxone; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer