Functional consequences of nociceptin receptor activation

Nociceptin orphanin FQ (N/OFQ) is the 17 amino acid endogenous ligand for the Gi-coupled N/OFQ-receptor (NOP). In vivo administration produces a wide range of physiological responses including; analgesia, hyperalgesia and anti-opioid actions.;In a series of in vitro assays including [leucyl -3H]N/OFQ binding, GTPgamma[35S] binding and inhibition of cAMP formation the following linked studies were performed; (1)N/OFQ structure-activity relationships (SAR) in cells (CHO) stably expressing human NOP (2)evaluation of receptor density on efficacy using the ecdysone inducible expression system and native tissues, (3)an investigation of NOP/G-protein coupling efficiency.;SAR studies can be summarized as combining arginine14, lysine15 repeat in N/OFQ (increase affinity/potency) with C-terminal [F/G]N/OFQ(1--13)NH2([F/G]) and [Nphe1]N/OFQ(1--13)NH 2([Nphe1]) modifications (reduce/eliminate efficacy). Arg14/Lys15 increased the affinity (pKi:10.31--11.16) and potency (pEC50:8.98--9.85) of N/OFQ. [F/G] and [Nphe 1] reduced the efficacy of N/OFQ from 1.0 to 0.44 and 0. Combination of Arg14/Lys15 and [Nphe1] in UFP-101 produced the highest affinity peptide antagonist available (pA 2:9.13, [Nphe1] alone:7.54). Using the ecdysone inducible expression system it was possible to vary the efficacy of [F/G], between antagonist (alpha:0 upstream/low density), partial agonist (alpha:0.3--0.75) and full agonist (alpha:1). In the mouse vas deferens and colon [F/G] displayed varying degrees of partial agonism possibly indicating differences in NOP receptor density. The binding of GDP and GTP in CHOhNOP was to high and low affinity sites. The fraction of GDP binding to the high affinity site was reduced by N/OFQ (77%, under basal to 32%). The reduction in high affinity binding of GDP appeared dependent on the efficacy of the ligand.;This thesis has identified several new peptides of varying efficacy/potency for use in defining the pathophysiological role(s) of N/OFQ-NOP. Efficacy is not just a property of the ligand but of the assay systems and its input receptor density. A cautious approach to new ligands characterized at a single endpoint is advocated.