Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [(3)H]Ac-RYYRIK-ol.
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 with exceptionally high specific radioactivity of 94Ci/mmol. The radioprobe is chemically stable even at 24 degrees C in ethanol solution for at least 4 days. No significant decomposition of the [(3)H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ((h)NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing (h)NOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [(3)H]Ac-RYYRIK-ol recognition sites were 237 and 749fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K(d)s) were determined by three independent way providing identical results. In rat brain membranes K(d)s of 0.3-1.3nM were found depending upon the assay type. In homologous competition studies performed on (h)NOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [(3)H]Ac-RYYRIK-ol (K(i) 2.8nM) or with [(3)H](Leu(14))nociceptin (2.3nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [(3)H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Temesvari, Hungary.
Engin
E
Bojnik
Judit
J
Farkas
Anna
A
Magyar
Csaba
C
Tömböly
Umit
U
Güçlü
Ozge
O
Gündüz
Anna
A
Borsodi
Maïthe
M
Corbani
Sándor
S
Benyhe
D000345:Affinity Labels; D000465:Algorithms; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D009474:Neurons / Q000378:metabolism*; D009842:Oligopeptides; D011869:Radioligand Assay; D019275:Radiopharmaceuticals; D051381:Rats; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000094942:Nociceptin Receptor
C512381:Ac-RYYRIK-ol; D000345:Affinity Labels; D008024:Ligands; D009842:Oligopeptides; D019275:Radiopharmaceuticals; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor