Comparison of the binding of [(3)H]nociceptin/orphaninFQ(1-13)NH(2), [(3)H]nociceptin/orphaninFQ(1-17)OH and [(125)I]Tyr(14)nociceptin/orphaninFQ(1-17)OH to recombinant human and native rat cerebrocortical nociceptin/orphanin FQ receptors.
Nociceptin/orphanin FQ (N/OFQ) is a 17-amino acid endogenous neuropeptide ligand for the nociceptin receptor (NOP). We have prepared a [(3)H]-labelled truncated N/OFQ peptide, [(3)H]N/OFQ(1-13)NH(2) and compared its binding characteristics with [(3)H]N/OFQ(1-17)OH and [(125)I]Y(14)N/OFQ(1-17)OH in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHO(hNOP)) and the rat cerebrocortex. [(3)H]N/OFQ(1-13)NH(2), [(3)H]N/OFQ(1-17)OH and [(125)I]Y(14)N/OFQ(1-17)OH binding to CHO(hNOP) was concentration dependent and saturable with receptor density (B(max)) and radioligand equilibrium dissociation constant (pK(d)) values (mean +/- SEM) of 1043 +/- 58 fmol/mg protein and 10.35 +/- 0.03, 1348 +/- 44 fmol/mg protein and 10.06 +/- 0.04, and 1169 +/- 76 fmol/mg protein and 10.45 +/- 0.06, respectively. In the rat, B(max) and pK(d) values for [(3)H]N/OFQ(1-13)NH(2) and [(3)H]N/OFQ(1-17)OH were 130 +/- 1 fmol/mg protein and 10.70 +/- 0.03, and 157 +/- 4 fmol/mg protein and 10.34 +/- 0.02, respectively. The binding of all radioligands was displaced by a range of peptide and non-peptide ligands. There was a strong correlation (r(2) = 0.92, P = 0.002) between pK(i) values estimated with [(3)H]N/OFQ(1-13)NH(2) and [(3)H]N/OFQ(1-17)OH. No such correlation was observed in comparison with the [(125)I]-labelled peptide (poor agreement with low affinity N/OFQ(1-9)NH(2), Dynorphin-A and Naloxone benzoylhydrazone). We suggest that [(3)H]N/OFQ(1-13)NH(2) may be a useful alternative to [(3)H]N/OFQ(1-17)OH.
University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK.
E
E
Hashiba
D G
DG
Lambert
J
J
Farkas
G
G
Toth
G
G
Smith
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D007457:Iodine Radioisotopes; D009474:Neurons / Q000378:metabolism*; D018847:Opioid Peptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D014316:Tritium; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D007457:Iodine Radioisotopes; D018847:Opioid Peptides; D010446:Peptide Fragments; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D014316:Tritium; D000094942:Nociceptin Receptor; D000097629:Nociceptin; C439964:nociceptin (1-13) amide; C466980:nociceptin orphanin FQ(1-17)OH; C106528:nociceptin, Tyr(1)-