Characterization and maturational differences of melatonin binding sites in the masu salmon brain

To obtain a better understanding of the roles of melatonin in the mediation of photoperiodic signaling, we have examined the pharmacological characteristics, guanine nucleotide modulation, and maturational differences of melatonin binding sites in the brain of masu salmon Oncorhynchus masou by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The specific binding of 2-[125I]iodomelatonin was rapid, stable, saturable, and reversible. Saturation experiments demonstrated that 2-[125I]iodomelatonin binds to a single class of receptor sites with an affinity constant (K(d)) of 6.3+/-0.5 pM and a total binding capacity (B(max)) of 15.18+/-0.22 fmol/mg protein in underyearling precocious males in July. Competition experiments revealed that the binding sites are highly specific for melatonin and related analogues. Treatment with guanosine 5(')-O-(3-thiotriphosphate) significantly reduced the specific binding, indicating that melatonin binding sites in the masu salmon brain are coupled to G protein. Significant differences were seen in B(max), but not K(d), among the fish groups differing in maturity. In the underyearling fish in July, the B(max) of precocious males and immature males was significantly higher than that of immature females. Then, the B(max) of precocious males decreased in October, when the fish spermiated. In the 2-year-old fish, B(max) was significantly higher in spermiating males than ovulated females. These results indicate that melatonin plays neuromodulatory roles in the central nervous system through specific receptors. Furthermore, gonadal maturation affects the density of melatonin binding sites in the masu salmon brain by an unknown mechanism.