Nociceptin binding sites in frog (Rana esculenta) brain membranes

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) s hares some homology with the opioid peptides but it binds to a distinct rec eptor type, termed nociceptin receptor. This study demonstrates the presenc e of specific nociceptin recognition sites in brain membrane fractions of a n amphibian, Rana esculenta. Para-iodo-Phe(1)-nociceptin-amide was radiolab elled by catalytic dehalotritiation, resulting in p[H-3]Phe(1)-nociceptin-a mide of 25 Ci/mmol specific radioactivity. Specific binding of [H-3]nocicep tin-amide to frog brain membranes was found to be saturable and of high aff inity with equilibrium K-d values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity wa s obtained in saturation and competition experiments. [H-3]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but no t by opioid ligands. Both sodium ions and 5'-guanylylimidodiphosphate decre ased the binding of the radioligand by transferring the receptor to a lower affinity state. Nociceptin dose-dependently stimulated the binding of the nonhydrolysable, radiolabeled GTP-analogue guanosine-5'-O-(3-thio) triphosp hate ([S-35]GTP gamma S) to G-proteins in frog brain membranes. Addition of 1 mu M naloxone caused no significant change in the curves, indicating tha t nociceptin-mediated activation of G-proteins occurred through nonopioid-m echanism. (C) 1999 Academic Press.