Characterization of nociceptin/orphanin FQ binding sites in dog brain membranes.
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.
Johnson
Emma E
EE
*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.
Gibson
Helen
H
Nicol
Beverley
B
Zanzinger
Johannes
J
Widdowson
Peter
P
Hawthorn
Mark
M
Toth
Géza
G
Farkas
Judit
J
Guerrini
Remo
R
Lambert
David G
DG
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
D018847:Opioid Peptides; D019275:Radiopharmaceuticals; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D000097629:Nociceptin