Neuropeptide FF receptors: structure-activity relationship and effect of morphine

Neuropeptide FF (FLFQPQRFamide, NPFF) is an octapeptide implicated in morphine analgesia, tolerance and dependence. Many of the behavioral effects of NPFF have also been observed with the invertebrate neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide), which binds to NPFF receptors because of its low homology to the C-terminal portion of NPFF. A competitive ligand binding assay was used to characterize NPFF receptors in rat spinal cord and a strong requirement was found for the C-terminal Arg-Phe-amide. It was found that FMRFamide (Ki = 1.8 nM) bound with lower affinity than NPFF (0.26 nM) but it was about 7-fold more potent than PQRFamide (12 nM). This finding explains the similar bioactivities of NPFF and FMRFamide. The Gln2 appeared to be the cause of the relatively low potency of PQRFamide, based on the binding specificity of NPFF receptors for a series of FMRFamide analogs. In contrast to the Arg-Phe-amide, substitutions at the first and second positions of FMRFamide were generally tolerated, with the most potent analogs being PMRFamide (Ki = 0.54 nM), FFRFamide (0.25 nM) and FWRFamide (0.42 nM). Among the most potent ligands was a pentapeptide containing a photoreactive Phe analog, D-Tyr-(p-benzoyl-Phe)-norLeu-Arg-Phe-amide (Ki = 0.23 nM). It was found that dansyl-PQRFamide and dansyl-RFamide also bound to NPFF receptors with Ki values of 6.1 and 73 nM, respectively. The radioligand binding and G-protein coupling of NPFF receptors were not altered by chronic morphine treatment.