The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe1-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[3H]Phe1-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [3H]nociceptinamide to frog brain membranes was found to be saturable and of high affinity with equilibrium Kd values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [3H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5*- guanylylimidodiphosphate decreased 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)triphosphate ([35S]GTPgS) to G-proteins in frog brain membranes. Addition of 1 mM naloxone caused no significant change in the curves, indicating that nociceptinmediated activation of G-proteins occurred through nonopioid mechanism.

Nociceptin binding sites in frog (Rana esculenta) brain membranes

GUERRINI, Remo;SALVADORI, Severo;
1999

Abstract

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe1-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[3H]Phe1-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [3H]nociceptinamide to frog brain membranes was found to be saturable and of high affinity with equilibrium Kd values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [3H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5*- guanylylimidodiphosphate decreased 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)triphosphate ([35S]GTPgS) to G-proteins in frog brain membranes. Addition of 1 mM naloxone caused no significant change in the curves, indicating that nociceptinmediated activation of G-proteins occurred through nonopioid mechanism.
Benyhe, S; Menory, K; Farkas, J; Toth, G; Guerrini, Remo; Salvadori, Severo; Orosz, G; Wollemann, M; Borsodi, A.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/470868
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