title: Characterization of nociceptin/orphanin FQ binding sites in dog brain membranes.
abstract: Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP), whose characteristics in the dog are unknown. We therefore compared [(3)H]N/OFQ binding in dog and rat brain membranes. Radioligand saturation/competition studies with these membranes and leucyl-[(3)H]N/OFQ(1-17)OH or the novel radioligand [(3)H]N/OFQ(1-13)NH(2) were performed to determine receptor density and ligand affinity. The density of classic opioid receptors was determined by using [(3)H]diprenorphine. Leucyl-[(3)H]N/OFQ(1-17)OH binding was concentration dependent and saturable in dog (maximum binding capacity [B(max)], 28.7 +/- 2.8 fmol/mg of protein; equilibrium dissociation constant as negative log [pK(d)], 10.27 +/- 0.11) and rat (B(max), 137.0 +/- 12.9 fmol/mg of protein; pK(d), 10.41 +/- 0.05). In comparison, the B(max) and pK(d) of [(3)H]diprenorphine were, respectively, 77.7 +/- 5.3 fmol/mg of protein and 9.74 +/- 0.09 in dog and 79.1 +/- 18.2 fmol/mg of protein and 9.51 +/- 0.04 in rat. In dog, [(3)H]N/OFQ(1-13)NH(2) binding to NOP receptors was also saturable (B(max), 23.7 +/- 2.0 fmol/mg of protein; pK(d), 10.16 +/- 0.12). In both species, leucyl-[(3)H]N/OFQ(1-17)OH was displaced by various NOP ligands. Dynorphin A, N/OFQ(1-5)NH(2), and nocistatin were essentially inactive. There was a significant positive correlation (r(2) = 0.95; P < 0.0001) between pK(i) values (an estimate of affinity) obtained in displacement studies in rat and dog. We have demonstrated a low density of NOP receptors, measured with two radioligands, in dog, and these receptors display a high degree of pharmacological similarity with those natively expressed in the rat.
authors.affiliation: *University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, United Kingdom; †Veterinary Medicine Research & Development, Pfizer Ltd., Sandwich, Kent, United Kingdom; ‡Isotope Laboratory, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary; and §Department of Pharmaceutical Sciences and Biotechnology Centre, University of Ferrara, Ferrara, Italy.
authors.forename: Emma E
authors.initials: EE
authors.lastname: Johnson
authors.forename: Helen
authors.initials: H
authors.lastname: Gibson
authors.forename: Beverley
authors.initials: B
authors.lastname: Nicol
authors.forename: Johannes
authors.initials: J
authors.lastname: Zanzinger
authors.forename: Peter
authors.initials: P
authors.lastname: Widdowson
authors.forename: Mark
authors.initials: M
authors.lastname: Hawthorn
authors.forename: Géza
authors.initials: G
authors.lastname: Toth
authors.forename: Judit
authors.initials: J
authors.lastname: Farkas
authors.forename: Remo
authors.initials: R
authors.lastname: Guerrini
authors.forename: David G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D005260:Female; D006439:Hemodynamics / Q000187:drug effects; D007700:Kinetics; D008297:Male; D018847:Opioid Peptides / Q000378:metabolism*; D010147:Pain Measurement / Q000187:drug effects; D019275:Radiopharmaceuticals; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000187:drug effects; D017473:Receptors, Opioid, kappa / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000187:drug effects; D000097629:Nociceptin
chemical_list: D018847:Opioid Peptides; D019275:Radiopharmaceuticals; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D000097629:Nociceptin