Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

1 The present study describes the labelling of the nociceptin (NC) receptor, ORL 1 , in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C‐terminal; the ligand, [ 3 H]‐NC‐NH 2 , has a specific activity of 24.5 Ci mmol −12 Saturation experiments revealed a single class of binding sites with a K D value of 0.55 n m and B max of 94 fmol mg −1 of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: K obs =0.104 min −1 ; K 1 =0.034 min −1 ; T 1/2 =20 min; K +1 =0.07 min n m −1 . 3 Thermodynamic analyses indicated that [ 3 H]‐NC‐NH 2 binding to the mouse ORL 1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ). 4 Receptor affinities of several NC fragments and analogues, including the newly discovered ORL‐1 receptor antagonist [Phe 1 ψ(CH 2 ‐NH)Gly 2 ]NC(1–13)‐NH 2 ([F/G]NC(1–13)‐NH 2 ), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC‐OH=NC‐NH 2 =NC(1–13)‐NH 2 >> NC(1–12)‐NH 2 > NC(1–13)‐OH >> NC(1–11)‐NH 2 , and for NC and NC(1–13)‐NH 2 analogues: [Tyr 1 ]NC‐NH 2 [Leu 1 ]NC(1–13)‐NH 2 [Tyr 1 ]NC(1–13)‐NH 2 [F/G]NC(1–13)‐NH 2 >> [Phe 3 ]NC(1–13)‐NH 2 > [ D F/G]NC(1–13)‐NH 2 . 5 Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [ 3 H]‐NC‐NH 2 binding when applied at concentrations up to 10 μ m indicating that this new radioligand interacts with a non opioid site, probably the ORL 1 receptor.British Journal of Pharmacology (1998) 125 , 1485–1490; doi: 10.1038/sj.bjp.0702226