Characteristics of 2-[125I]iodomelatonin binding sites in the pigeon spleen and modulation of binding by guanine nucleotides

Abstract

2-[125I]Iodomelatonin binding sites in membrane preparations of pigeon spleen have been characterized. The binding was stable, saturable, reversible, and of high affinity. Rosenthal and Hill analyses showed that the radioligand-receptor interaction involved a single class of binding sites. Analysis of the binding results of spleens collected during mid-light revealed an equilibrium dissociation constant (Kd) of 36.6 ± 4.8 pmol/l (mean ± sem, n = 10) and a maximum density (Bmax) of 2.3 ± 0.2 fmol/mg protein. There was no significant difference in the Kd (46.9 ± 5.0 pmol/l) or the Bmax values (2.4 ± 0.3 fmol/mg protein) for spleens collected during mid-dark (n = 9), although the mid-dark serum and pineal melatonin levels were significantly higher (P < 0.05) than the corresponding mid-light values. Kinetic analysis showed a Kd of 8.6 ± 2.0 pmol/l (n ± 4), in agreement with that derived from the saturation studies. Except for inhibition by 2- iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N- acetylserotonin, the other indoles or neurotransmitters tested have little inhibition on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTPγS), a nonhydrolysable analog of GTP, was found to inhibit the binding in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding sites. These data suggest that a single type of melatonin receptor is found in the pigeon spleen and that the site is coupled to a guinine nucleotide binding protein (G-protein). Our findings support a direct pineal melatonin action on the immune system.