Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [3H]Ac-RYYRIK-ol

摘要: Abstract The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [ 3 H]Ac-RYYRIK-ol exceptionally high specific radioactivity of 94 Ci/mmol. radioprobe is chemically stable even at 24 °C in ethanol solution least 4 days. No significant decomposition H]ligand occurred under condition binding experiments indicating fine enzymatic stability peptide. Radioreceptor studies were conducted using native neuronal NOPr preparation rat brain membrane fractions recombinant human nociceptin ( h NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing NOPr. Specific compound was reversible, saturable affinity. cross-reaction opioid receptors observed suggesting superior selectivity ligand. Monophasic isotherm curves obtained radioligand saturation homologous displacement indicated presence single sites both preparations. Average densities recognition 237 749 fmol/mg protein transfected cells, respectively. Equilibrium affinity values K d s) determined by three independent way providing identical results. In membranes s 0.3–1.3 nM found depending upon assay type. competition performed on NOP-CHO cell almost same affinities measured either i 2.8 nM) or H](Leu 14 )nociceptin (2.3 nM). A number ligands screened heterologous displayed rank order profile being consistent fairly good H]Ac-RYYRIK-ol. Taken together, molar activity, improved chemical biological capability labeling make this novel available further exploring biochemical pharmacology receptor-ligand interaction NOP receptor.