2-[¹²⁵I]Iodomelatonin labels sites with identical pharmacological characteristics in chicken brain and chicken retina

The binding and pharmacological characteristics of the melatonin site labeled by the radioligand 2-[¹²⁵I]iodomelatonin in chicken brain membranes were determined and compared with those of the melatonin site of chicken retinal membranes. The specific binding of 2-[¹²⁵I]iodomelatonin to chicken brain membranes was found to be stable, saturable, reversible and of high affinity. Scatchard analysis of the binding revealed an affinity constant (K_d) of 344 ± 24pM (n = 4) and a total number of binding sites (B_max) of 57.6 ± 10.1fmol/gm protein (n = 4). The K_d value correspond closely with that found in kinetic studies (K_d = 407 pM) and that reported in chicken retinal membranes. Competition experiments were carried out with various compounsd revealing the following order of pharmacological affinities: 6-chloromelatonin ≥22-iodomelatonin > melatonin > 2-methyl-6,7-dichloromelatonin > 6-hydroxymelatonin > N-acetyl-5-hydroxytryptamine > luzindole > N-acetyl-5-methoxykynurenamine > 6-methoxymelatonin > N-acetyl-tryptamine > 5-methoxytryptamine > 5-hydroxytryptamine > 5-methoxy-N,N-dimethyltryptamine > 5-methoxytryptophol. This order of pharmacological affinities is identical to that found in chicken retinal membranes. Correlation between affinity constants for various melatonin receptor agonists and putative melatonin receptor antagonists obtained in chicken brain and retinal membranes yielded a correlation coefficient (r) of 0.966 (slope = 0.652, n = 14, P < 0.01). We conclude that the high affinity site labeled by 2-[¹²⁵I]iodomelatonin in chicken brain membranes has identical binding and pharmacological characteristics to the ML-1 melatonin receptor site previously described in chicken retinal membranes.