37 research outputs found
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-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