Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF1 and NPFF2) receptors

The binding characteristics of [3H]-NPVF and [3H]-EYF, the two first tritiated probes for the respective labelling of NPFF1 and NPFF2 receptors, are presented. In membranes from CHO cells transfected with the human NPFF1 receptor, [3H]-NPVF labelled one class of binding sites with a high affinity (Bmax = 4 pmol/mg protein, Kd = 2.65 nM). In membranes from CHO cells transfected with the human NPFF2 receptor, [3H]-EYF labelled one class of binding sites with a high affinity (Bmax = 16 pmol/mg protein, Kd = 0.54 nM). Both radioligands exhibited time-dependent binding, low (10–20%) non-specific binding and poor cross-reactivity towards the related receptor subtype. The potency of different NPFF ligands to displace [3H]-NPVF and [3H]-EYF binding profiles was in good agreement with the profile previously measured by using 125I-probes (NPFF1 receptor: NPVF ≥ 1DMe = SPA-NPFF > NPFF = SQA-NPFF = QFW-NPSF > NPSF > RF9; NPFF2 receptor: SPA-NPFF > > SQA-NPFF = QFW-NPSF = 1DMe = NPFF ≫ NPSF = NPVF > RF9). Therefore, [3H]-NPVF and [3H]-EYF are new valuable tools for performing binding on NPFF receptors.