Biochemical characterization of a synthetic NPFF agonist with high affinity and resistance to brain peptidase inactivation

Abstract

NPFF, the brain mammalian FMRFamide-like peptide inhibits the analgesic effects of both exogenous and endogenous opiates. We have previously demonstrated the existence of specific NPFF binding sites, suggesting that these opiate-modulating effects are mediated by receptors, different from opiate receptors. Therefore, NPFF may be classified in the family of endogenous substances generically termed “antiopiates” which could contribute to opiate tolerance and dependence. When injected into CNS, NPFF induces a short-lasting effect. With the aim to obtain an agonist with longer-lasting effects, we have synthesized a new N-terminal substituted analog. As compared to NPFF which was fully hydrolysed by membrane peptidases by 150 min at 25°C, (1DMe)Y8Fa exhibited a marked resistance to enzymatic breakdown. In binding assay, [ 125I](1DMe)Y8Fa was able to bind to rat spinal cord membranes in time-dependent, dose-dependent, saturable and reversible manners (Kd = 0.05 nM; Bmax = 14.7 fmol/mg protein). Electrophysiological studies showed that (1DMe)Y8Fa was about ten times more potent than NPFF in preventing the morphine inhibition of C fibre-evoked activity of dorsal horn neurones.