CHARACTERISTICS OF 2-[I-125]IODOMELATONIN BINDING-SITES IN THE PIGEONSPLEEN AND MODULATION OF BINDING BY GUANINE-NUCLEOTIDES

2-[I-125]Iodomelatonin binding sites in membrane preparations of pigeo n spleen have been characterized. The binding was stable, saturable, r eversible, and of high affinity. Rosenthal and Hill analyses showed th at the radioligand-receptor interaction involved a single class of bin ding sites. Analysis of the binding results of spleens collected durin g 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 t he Kd (46.9 +/- 5.0 pmol/1) or the Bmax values (2.4 +/- 0.3 fmol/mg pr otein) 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 showe d 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-acetylseroton in, the other indoles or neurotransmitters tested have little inhibiti on on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTPg ammaS), a nonhydrolysable analog of GTP, was found to inhibit the bind ing in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding s ites. These data suggest that a single type of melatonin receptor is f ound in the pigeon spleen and that the site is coupled to a guinine nu cleotide binding protein (G-protein). Our findings support a direct pi neal melatonin action on the immune system.