3H]dynorphin 1–8 binding sites in frog ( Rana esculenta) brain membranes

Abstract

Opioid binding sites specific for [ 3H]dynorphin 1–8 were characterized in the particulate membrane fraction of frog ( Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [ 3H]dynorphin 1–8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin 1–13, bremazocine, levorphanol and naloxone. The specific binding of [ 3H]dynorphin 1–8 can be significantly inhibited by Na + ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a K d ≈ 7.5 nM is present in the membranes. The maximum density of binding sites (B max ≈ 1.1 pmol [ 3H]dynorphin 1–8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the μ- and σ-types of opioid receptor showed that in the frog brain most of the sites labelled by [ 3H]dynorphin 1–8 are κ-sites and that this is a rich source of such sites.