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-³H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [³H]labeled NOP antagonist, [Nphe¹,4’-³H-Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ ([³H]UFP-101). We have characterized [³H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHO_hNOP) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [³H]UFP-101 (antagonist) was compared with [³H]N/OFQ (agonist). The effects of GTPγS (10 µM) and Na⁺ were investigated alone and in combination in competition experiments with both radioligands. In CHO_hNOP, B _max, and p K _D, values were 561 and 580 fmol mg protein⁻¹ and 9.97 and 10.19 for [³H]UFP-101 and [leucyl-³H]N/OFQ, respectively. In rat cerebrocortex B _max and p K _D, values were 65 and 88 fmol mg protein⁻¹ and 10.12 and 10.34 for [³H]UFP-101 and [leucyl-³H]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 CHO_hNOP). Naloxone was inactive. The binding of both radioligands was Na⁺-dependent with [³H]UFP-101 being more sensitive (IC₅₀ ~20 mM). Unlike the agonist [leucyl-³H]N/OFQ, the antagonist [³H]UFP-101 was unaffected by GTPγS. [³H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-³H]N/OFQ in competition experiments. In addition, the binding of [³H]UFP-101 is unaffected by GTPγS. This radioligand will be useful to further characterize NOP in a range of binding paradigms. [ABSTRACT FROM AUTHOR]