Nociceptin binding sites in frog (Rana esculenta) brain membranes.
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-Phe(1)-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[(3)H]Phe(1)-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [(3)H]nociceptin-amide to frog brain membranes was found to be saturable and of high affinity 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 was obtained in saturation and competition experiments. [(3)H]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 ([(35)S]GTPgammaS) to G-proteins in frog brain membranes. Addition of 1 microM naloxone caused no significant change in the curves, indicating that nociceptin-mediated activation of G-proteins occurred through nonopioid mechanism.
Benyhe
S
S
Institute of Biochemistry and Isotope Laboratory, Biological Research Centre, Hungarian Academy of Sciences, Szeged, H-6701, Hungary. benyhe@nucleus.szbk.u-szeged.hu
Monory
K
K
Farkas
J
J
Tóth
G
G
Guerrini
R
R
Salvadori
S
S
Orosz
G
G
Wollemann
M
M
Borsodi
A
A
D000577:Amides / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D009294:Narcotics / Q000378:metabolism; D018847:Opioid Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011893:Rana esculenta; D011957:Receptors, Opioid / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013816:Thermodynamics; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D000577:Amides; D019204:GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006165:Guanylyl Imidodiphosphate; D008024:Ligands; D009270:Naloxone; D009292:Narcotic Antagonists; D009294:Narcotics; D018847:Opioid Peptides; D011957:Receptors, Opioid; D012964:Sodium; D000094942:Nociceptin Receptor; D000097629:Nociceptin