study about melatonin
Use of 2-[125I]iodomelatonin to Characterize Melatonin Binding Sites in Chicken Retina
Margarita L. Dubocovich and Joseph S. Takahashi
2-[125I>Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-[125I>iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-[125I>iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 ± 56 pM and a total number of binding sites (Bmax) of 74.0 ± 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-[125I>iodomelatonin binding with a pharmacological order of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin > 6-chloromelatonin melatonin 6,7-dichloro-2-methylmelatonin > 6-hydroxymelatonin 6-methoxymelatonin > N-acetyltryptamine > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine >>> 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-[125I>iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of [3H>dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-[125I>iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.