125I]EYF: a new high affinity radioligand to neuropeptide FF receptors

Abstract

[ 125I]EYF ([ 125I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [ 125I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K D = 0.041 and 0.019 nM, respectively). Competition studies showed that [ 125I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity. Autoradiographic studies demonstrated that [ 125I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [ 125I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn. Non specific binding reached 5–10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%. These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [ 125I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.