[Nphe(1)]NC(1-13)NH2 selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain

[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 GTPg35S 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 GTPg35S 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 GTPg35S binding to rat brain membranes but not that evoked by selective agonists of the m-, d-, and k-opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH2 and Ac-RYYRIKNH 2 on the binding of GTPg35S were clearly differentiated from the effect of [Nphe1]NC(1-13)NH2 when the concentration of GDP, competing with GTPgS for binding, was lowered from 100 mM (assay optimum) to 5 mM. At 5 mM 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).