Comparison of the binding of [3H]nociceptin/orphaninFQ(1–13)NH2, [3H]nociceptin/orphaninFQ(1–17)OH and [125I]Tyr14nociceptin/orphaninFQ(1–17)OH to recombinant human and native rat cerebrocortical nociceptin/orphanin FQ receptors

Abstract

Nociceptin/orphanin FQ (N/OFQ) is a 17-amino acid endogenous neuropeptide ligand for the nociceptin receptor (NOP). We have prepared a [3H]-labelled truncated N/OFQ peptide, [3H]N/OFQ(1–13)NH2 and compared its binding characteristics with [3H]N/OFQ(1–17)OH and [125I]Y14N/OFQ(1–17)OH in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP) and the rat cerebrocortex. [3H]N/OFQ(1–13)NH2, [3H]N/OFQ(1–17)OH and [125I]Y14N/OFQ(1–17)OH binding to CHOhNOP was concentration dependent and saturable with receptor density (Bmax) and radioligand equilibrium dissociation constant (pKd) values (mean±SEM) of 1043±58 fmol/mg protein and 10.35±0.03, 1348±44 fmol/mg protein and 10.06±0.04, and 1169±76 fmol/mg protein and 10.45±0.06, respectively. In the rat, Bmax and pKd values for [3H]N/OFQ(1–13)NH2 and [3H]N/OFQ(1–17)OH were 130±1 fmol/mg protein and 10.70±0.03, and 157±4 fmol/mg protein and 10.34±0.02, respectively. The binding of all radioligands was displaced by a range of peptide and non-peptide ligands. There was a strong correlation (r2=0.92, P=0.002) between pKi values estimated with [3H]N/OFQ(1–13)NH2 and [3H]N/OFQ(1–17)OH. No such correlation was observed in comparison with the [125I]-labelled peptide (poor agreement with low affinity N/OFQ(1–9)NH2, Dynorphin-A and Naloxone benzoylhydrazone). We suggest that [3H]N/OFQ(1–13)NH2 may be a useful alternative to [3H]N/OFQ(1–17)OH.