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ANESTHETIC PHARMACOLOGY

Characterization of Nociceptin/Orphanin FQ Binding Sites in Dog Brain Membranes

Emma E. Johnson, BSc(Hons)^*, Helen Gibson, BSc(Hons)^*, Beverley Nicol, PhD, Johannes Zanzinger, PhD, Peter Widdowson, PhD, Mark Hawthorn, PhD, Géza Toth, PhD, Judit Farkas, PhD, Remo Guerrini, PhD, and David G. Lambert, PhD^*

*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

Address correspondence and reprint requests to D. G. Lambert, University Department of Anesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK. Address e-mail to DGL3{at}le.ac.uk

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 [³H]N/OFQ binding in dog and rat brain membranes. Radioligand saturation/competition studies with these membranes and leucyl-[³H]N/OFQ(1–17)OH or the novel radioligand [³H]N/OFQ(1–13)NH₂ were performed to determine receptor density and ligand affinity. The density of classic opioid receptors was determined by using [³H]diprenorphine. Leucyl-[³H]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 [³H]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, [³H]N/OFQ(1–13)NH₂ 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-[³H]N/OFQ(1–17)OH was displaced by various NOP ligands. Dynorphin A, N/OFQ(1–5)NH₂, and nocistatin were essentially inactive. There was a significant positive correlation (r² = 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.

IMPLICATIONS: Experimentally, the dog represents a species used in various anesthetic studies, yet little is known regarding the expression of nociceptin receptors (with opioid-like activity) in this model. In comparison with rat, dog membranes express a small density of pharmacologically identical binding sites whose functional activity remains to be determined.