Kisspeptin is a 54 amino acid peptide that stimulates Gonadotrophin-Releasing-Hormone (GnRH) release from the hypothalamus and consequently activates the Hypothalamic-Pituitary-Gonadal (HPG) axis. Recent studies have suggested that Kisspeptin also binds and activates Neuropeptide FF (NPFF) receptors. The C-terminal ten residues of Kisspeptin are capable of activation of both GnRH release and NPFF receptors. The aim of the current study was to determine which amino acids of Kisspeptin were involved in NPFF activation and whether the NPFF activation was independent of GnRH releasing activity. A series of fragments of the C-terminus of human Kisspeptin 54 were prepared and tested for NPFF activity using the ND7/23 neuroblastoma, which expresses NPFF receptors, and the LA-N-1 neuroblastoma that releases GnRH. For NPFF activation the effects on forskolin stimulated cyclic adenosine monophosphate (cAMP) were determined and compared with NPFF inhibition of release. The NPFF receptor antagonist RF9 was used to block NPFF receptor mediated actions. The GnRH release was measured by immunoassay and compared to Kisspeptin 54 stimulated release. The Kisspeptin receptor antagonist Peptide 234 was used to block Kisspeptin mediated actions. Results showed that the minimal Kisspeptin sequence required to inhibit forskolin stimulated cAMP via NPFF receptors was Trp-Asn-Ser-Phe-NH2, with the hexapeptide Tyr-Asn-Trp-Asn-Ser-Phe-NH2 showing maximal activity. Neither of these peptides activated GnRH release. The hexapeptide Tyr-Asn-Trp-Asn-Ser-Phe-NH2 was termed Kissorphin and its actions could be blocked by the NPFF receptor antagonist RF9. The Kisspeptin receptor antagonist Peptide 234 had no effect on Kissorphin inhibition of forskolin stimulated cAMP. Kissorphin and NPFF were also able to reduce forskolin neuroprotection against H2O2 toxicity in ND7/23 neurones. Kissorphin comprises residues 4550 of Kisspeptin 54 and has NPFF-like activity without showing any GnRH releasing activity.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.