Characterization of Melatonin Receptors in the Rat Area postrema: Modulation of Affinity with Cations and Guanine Nucleotides

Abstract

We have localized and characterized the binding of the melatonin agonist, 2-[¹²⁵I]iodomelatonin, in the rat area postrema (AP), by using quantitative autoradiography in vitro. At equilibrium conditions, Scatchard analysis revealed saturable high-affinity binding to a single class of sites (Ka 45.9 ± (SE) 6.0 pM and B_max 30.8 ± 4.6 fmol/mg protein, n = 4 experiments with a total of 18 rats). Melatonin and 6-hydroxymelatonin were potent displacers of 2-[¹²⁵I]iodomelatonin binding in the AP (IC50 20 and 500 pM, respectively) while N-acetylserotonin exhibited only a modest potency (IC50 25 nM). Micromolar concentrations of guanine nucleotides dose-dependently and specifically inhibited agonist binding at 22 ° C. Saturation studies revealed that this was due to a decrease in binding affinity. Divalent cations (4 mM CaCl₂ or 2 mM MgCl₂) had no detectable effect on the affinity of the binding site, whereas physiological concentrations of NaCl significantly decreased the binding affinity. These results demonstrate specific high-affinity binding sites for 2-[¹²⁵I]iodomelatonin in the rat AP and suggest coupling of these putative receptors to guanosine nucleotide binding regulatory protein(s).