Modulation of Neuropeptide FF Receptors by Guanine Nucleotides and Cations in Membranes of Rat Brain and Spinal Cord

Abstract: Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF(NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl₂ increased specific ¹²⁵I-YLFQPQRFamide (¹²⁵I-Y⁸Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 m M NaCl and 1 m M MgCl₂. Guanine nucleotides dose-dependently inhibited specific ¹²⁵I-Y⁸Fa binding to rat brain and spinal cord membranes with maximal effects of 64 ± 6 and 71 ± 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl₂. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control K_D and B_max values were 0.068 n M and 7.2 fmol/mg of protein, respectively, and with 0.1 μ M guanylylimidodiphosphate the respective values were 0.081 n M and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-0-(3-thiotriphosphate). Our data suggest that ¹²⁵I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.