Met 5 ‐enkephalin‐Arg 6 ‐Phe 7 , an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain

Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium‐labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad‐spectrum peptidase inhibitors at 0°C. Under these conditions, the equilibrium binding was achieved in 30–40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (K d value) of [ 3 H]Met‐enkephalin‐Arg 6 ‐Phe 7 , calculated from saturation binding data, was 10.2 ± 2.5 nM, and the maximal number (B max ) of the heptapeptide binding sites was found to be 468 ± 43 fmol/mg protein. About half the sites represent nonopioid sites because the B max was only 255 ± 30 fmol/mg, when the nonspecific binding was measured with 1 μM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [ 3 H]Metenkephalin‐Arg 6 ‐Phe 7 recognition sites are probably not μ and certainly not κ 1 sites, whereas these sites are characterized by a κ 2 ‐like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF‐selective “heptapeptide” binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high‐affinity stereospecific component and a substantially lower‐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)Phe 4 ‐Gly 5 ‐ol]enkephalin, morphine, or naloxone. [ 3 H] Met‐enkephalin‐Arg 6 ‐Phe 7 binding can also be inhibited by FMRF‐amide analogs and sigma receptor ligands, such as (+) N ‐allyl‐normetazocine and haloperidol, 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‐insensitive nonopioid sites. J. Neurosci. Res. 48:249–258, 1997. © 1997 Wiley‐Liss, Inc.