Use of 2-[125I]iodomelatonin to characterize melatonin binding sites in chicken retina.

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Abstract
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 .gtoreq. melatonin .gtoreq. 6,7-dichloro-2-methylmelatonin > 6-hydroxymelatonin .gtoreq. 6-methoxymelatonin > N-acetyltryptamine > N-acetyl-5-hydroxtryptamine > 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.