MET(5)-ENKEPHALIN-ARG(6)-PHE(7), AN ENDOGENOUS NEUROPEPTIDE, BINDS TOMULTIPLE OPIOID AND NONOPIOID SITES IN RAT-BRAIN

Receptor binding properties of the naturally occurring opioid heptapep tide MERF were studied in rat brain membrane preparations using tritiu m-labeled derivative of the peptide with 40 Ci/mmol specific radioacti vity, Binding assays were performed in the presence of broad-spectrum peptidase inhibitors at 0 degrees C, Under these conditions, the equil ibrium binding was achieved in 30-40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis , The apparent affinity (Kd value) of [H-3]Met-enkephalin-Arg(6)-Phe(7 ), calculated from saturation binding data, was 10.2 +/- 2.5 nM, and t he maximal number (B-max) of the heptapeptide binding sites was found to be 468 +/- 43 fmol/mg protein, About half the sites represent nonop ioid sites because the B-max was only 255 +/- 30 fmol/mg, when the non specific binding was measured with 1 mu M naloxone, The rank order pot encies of the examined compounds revealed that the opioid component of [H-3]Met-enkephalin-Arg(6)-Phe(7) recognition sites are probably not mu and certainly not K-1 sites, whereas these sites are characterized by a K-2-like binding profile, Considering the discrepancies between r at and frog brain found in the affinity of some compounds, including n altrindole and norbinaltorphimine, the presence of a novel, MERF-selec tive ''heptapeptide'' binding site in rat brain membranes is also sugg ested, A number of the heterologous competition curves could be descri bed by a high-affinity stereospecific component and a substantially lo wer-affinity binding element, which could completely be displaced with several peptide ligands such as Met(5)-enkephalin, dynorphin((1-13)), and unlabeled MERF but not by other compounds such as [D-Ala(2)-(Me)P he(4)-Gly(5)-ol]enkephalin, morphine, or naloxone, [H-3] Met-enkephali n-Arg(6)-Phe(7) binding can also be inhibited by FMRF-amide analogs an d sigma receptor ligands, such as (+)N-allyl-normetazocine and haloper idol, although with moderate affinity, It is concluded that the stereo specific high-affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone-in sensitive nonopioid sites. (C) 1997 Wiley-Liss, Inc.