Binding of the novel radioligand [3H]UFP-101 to recombinant human and native rat nociceptin/orphanin FQ receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-H-3]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [H-3]labeled NOP antagonist, [Nphe(1), 4'-H-3-Phe(4),Arg(14),Lys(15)]N/OFQ-NH2 ([H-3]UFP-101). We have characterized [H-3]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHOhNOP) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [H-3]UFP-101 (antagonist) was compared with [H-3]N/OFQ (agonist). The effects of GTP gamma S (10 mu M) and Na+ were investigated alone and in combination in competition experiments with both radioligands. In CHOhNOP, B (max), and pK (D), values were 561 and 580 fmol mg protein(-1) and 9.97 and 10.19 for [H-3]UFP-101 and [leucyl-H-3]N/OFQ, respectively. In rat cerebrocortex B (max) and pK (D), values were 65 and 88 fmol mg protein(-1) and 10.12 and 10.34 for [H-3]UFP-101 and [leucyl-H-3]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation (r(2) 0.92 in Rat and 0.97 in CHOhNOP). Naloxone was inactive. The binding of both radioligands was Na+ dependent with [H-3]UFP-101 being more sensitive (IC50 similar to 20 mM). Unlike the agonist [leucyl-H-3]N/OFQ, the antagonist [H-3]UFP-101 was unaffected by GTP gamma S. [H-3]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-H-3]N/OFQ in competition experiments. In addition, the binding of [H-3]UFP-101 is unaffected by GTP gamma S. This radioligand will be useful to further characterize NOP in a range of binding paradigms.