[Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain.

[Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-amide ([F/G]NC(1-13)NH(2)) and acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)), 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 GTPgamma(35)S binding assays. In this study, [Nphe(1)]NC(1-13)-amide ([Nphe(1)]NC(1-13)NH(2)), 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 GTPgamma(35)S binding to rat cerebral cortex membranes with pA(2) of 7.76 (Schild analysis). This antagonism of noc/OFQ receptor G-protein coupling was selective because the peptide inhibited the noc/OFQ-evoked GTPgamma(35)S binding to rat brain membranes but not that evoked by selective agonists of the mu-, delta-, and kappa-opioid receptors. In rat cortical membranes, the effects of [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) on the binding of GTPgamma(35)S were clearly differentiated from the effect of [Nphe(1)]NC(1-13)NH(2) when the concentration of GDP, competing with GTPgammaS for binding, was lowered from 100 microM (assay optimum) to 5 microM. At 5 microM GDP, the former peptides showed clear partial agonist activity, whereas [Nphe(1)]NC(1-13)NH(2) did not. These data indicate that only [Nphe(1)]NC(1-13)NH(2) 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)NH(2) and Ac-RYYRIK-NH(2) (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).