[H-3] DYNORPHIN(1-8) BINDING-SITES IN FROG (RANA-ESCULENTA) BRAIN MEMBRANES

Opioid binding sites specific for [H-3]dynorphin(1-8) were characteriz ed 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 [ H-3]dynorphin(1-8) to frog brain membranes was stereoselective, revers ible, saturable, and displaceable by a series of opioid ligands includ ing dynorphin(1-13), bremazocine, levorphanol and naloxone. The specif ic binding of [H-3]dynorphin(1-8) can be significantly inhibited by Na + ions and/or guanine nucleotides confirming the agonist property of t he ligand in vitro. A single set of high affinity opioid binding sites with a K-d approximate to 7.5 nM is present in the membranes. The max imum density of binding sites (B-max approximate to 1.1 pmol [H-3]dyno rphin(1-8) per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated op ioid peptides selective for the mu- and delta-types of opioid receptor showed that in the frog brain most of the sites labelled by [H-3]dyno rphin(1-8) are kappa-sites and that this is a rich source of such site s.