Vol. 294, Issue 2, 428-433, August 2000

[Nphe¹]NC(1-13)NH₂ Selectively Antagonizes Nociceptin/Orphanin FQ-Stimulated G-Protein Activation in Rat Brain

Hartmut Berger, Girolamo Calo', Erika Albrecht, Remo Guerrini and Michael Bienert

Institute of Molecular Pharmacology (H.B., E.A., M.B.), Berlin, Germany; and Department of Experimental and Clinical Medicine, Section of Pharmacology (G.C.) and Department of Pharmaceutical Sciences and Biotechnology Center (R.G.), University of Ferrara, Ferrara, Italy

[Phe¹psi(CH₂-NH)Gly²]noc/OFQ(1-13)-amide {[F/G]NC(1-13)NH₂} and acetyl-RYYRIK-amide (Ac-RYYRIK-NH₂), two peptidic ligands of the nociceptin/orphanin FQ (noc/OFQ) receptor, have been shown to exert both agonist and antagonist activity in different in vitro and in vivo systems. This is despite the observation that both peptides competitively antagonized the coupling of the activated receptor to G-proteins in brain preparations, measured in GTP³⁵S binding assays. In this study, [Nphe¹]NC(1-13)-amide ([Nphe¹]NC(1-13)NH₂), a new noc/OFQ analog recently characterized as a pure and selective noc/OFQ receptor antagonist in several in vitro and in vivo assay systems, was shown to competitively inhibit the noc/OFQ-stimulated GTP³⁵S binding to rat cerebral cortex membranes with pA₂ of 7.76 (Schild analysis). This antagonism of noc/OFQ receptor G-protein coupling was selective because the peptide inhibited the noc/OFQ-evoked GTP³⁵S binding to rat brain membranes but not that evoked by selective agonists of the µ-, -, and -opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH₂ and Ac-RYYRIK-NH₂ on the binding of GTP³⁵S were clearly differentiated from the effect of [Nphe¹]NC(1-13)NH₂ when the concentration of GDP, competing with GTPS for binding, was lowered from 100 µM (assay optimum) to 5 µM. At 5 µM GDP, the former peptides showed clear partial agonist activity, whereas [Nphe¹]NC(1-13)NH₂ did not. These data indicate that only [Nphe¹]NC(1-13)NH₂ was a pure antagonist of noc/OFQ receptor G-protein coupling. Furthermore, it is suggested that the variable behavior of [F/G]NC(1-13)NH₂ and Ac-RYYRIK-NH₂ (agonist, partial agonist, and antagonist) in different in vitro and in vivo systems may be explained by different partial GTP binding agonism and the existence of a GTP binding stimulus/response reserve (coupling reserve).