Characterization of [ 3H]Met-enkephalin-Arg 6-Phe 7 binding to multiple sites in rat and guinea pig cerebellum

Abstract

[ 3H]Met-enkephalin-Arg 6-Phe 7 (MERF) has been shown to label opioid ( κ 2 and δ) and sigma 2 sites in rat and frog brain membrane preparations, and no specific binding to κ 1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in κ 2-sites, and in guinea pig cerebellar preparations where κ 1 opioid receptors are almost exclusively present. In accordance with our previous results, [ 3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a K d of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [ 3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (−)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma 2 ligand, displayed the highest affinities. Among opioid peptides β-endorphin and dynorphind (1–13) showed the highest potencies, displacing [ 3H]MERF also from its rion-opioid sites. It was concluded therefore that [ 3H]MERF does not bind to κ 1 sites, and besides κ 2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma 2 receptors also occurs.