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

[Phe(1)psi(CH2-NH) Gly(2)]noc/OFQ(1-13)-amide {[F/G]NC(1-13) NH2} and acety l-RYYRIK-amide (Ac-RYYRIK-NH2), two peptidic ligands of the nociceptin/orph anin FQ (noc/OFQ) receptor, have been shown to exert both agonist and antag onist activity in different in vitro and in vivo systems. This is despite t he observation that both peptides competitively antagonized the coupling of the activated receptor to G-proteins in brain preparations, measured in GT P gamma(35)S binding assays. In this study, [Nphe(1)]NC(1-13)-amide ([Nphe( 1)]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 GTP gam ma(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 sele ctive because the peptide inhibited the noc/OFQ-evoked GTP gamma(35)S bindi ng to rat brain membranes but not that evoked by selective agonists of the mu-, delta-, and kappa-opioid receptors. In rat cortical membranes, the eff ects of [F/G] NC(1-13) NH2 and Ac-RYYRIKN-H-2 on the binding of GTP gamma(3 5)S were clearly differentiated from the effect of [Nphe(1)]NC(1-13) NH2 wh en the concentration of GDP, competing with GTP gamma S for binding, was lo wered from 100 mu M (assay optimum) to 5 mu M. At 5 mu M GDP, the former pe ptides showed clear partial agonist activity, whereas [Nphe(1)]NC(1-13) NH2 did not. These data indicate that only [Nphe(1)]NC(1-13) NH2 was a pure an tagonist of noc/OFQ receptor G-protein coupling. Furthermore, it is suggest ed that the variable behavior of [F/G] NC(1-13)NH2 and Ac-RYYRIK-NH2 (agoni st, partial agonist, and antagonist) in different in vitro and in vivo syst ems may be explained by different partial GTP binding agonism and the exist ence of a GTP binding stimulus/response reserve (coupling reserve).