title: Autoradiography in opioid triple knockout mice reveals opioid and opioid receptor like binding of naloxone benzoylhydrazone.
abstract: Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.
authors.affiliation: Pharmacology Group, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
authors.forename: Veronica
authors.initials: V
authors.lastname: Cox
authors.forename: Siân
authors.initials: S
authors.lastname: Clarke
authors.forename: Tracy
authors.initials: T
authors.lastname: Czyzyk
authors.forename: Micheal
authors.initials: M
authors.lastname: Ansonoff
authors.forename: Joshua
authors.initials: J
authors.lastname: Nitsche
authors.forename: Ming-Sing
authors.initials: MS
authors.lastname: Hsu
authors.forename: Anna
authors.initials: A
authors.lastname: Borsodi
authors.forename: Csaba
authors.initials: C
authors.lastname: Tömböly
authors.forename: Géza
authors.initials: G
authors.lastname: Tóth
authors.forename: Ray
authors.initials: R
authors.lastname: Hill
authors.forename: John
authors.initials: J
authors.lastname: Pintar
authors.forename: Ian
authors.initials: I
authors.lastname: Kitchen
mesh_terms: D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009270:Naloxone / Q000031:analogs & derivatives* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017465:Receptors, Opioid, delta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; C058380:naloxone benzoylhydrazone; D009270:Naloxone