Duncan M J, Takahashi J S, Dubocovich M L
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Endocrinology. 1988 May;122(5):1825-33. doi: 10.1210/endo-122-5-1825.
Studies in a variety of seasonally breeding mammals have shown that melatonin mediates photoperiodic effects on reproduction. Relatively little is known, however, about the site(s) or mechanisms of action of this hormone for inducing reproductive effects. Although binding sites for [3H]melatonin have been reported previously in bovine, rat, and hamster brain, the pharmacological selectivity of these sites was never demonstrated. In the present study, we have characterized binding sites for a new radioligand, 2-[125I]iodomelatonin, in brains from a photoperiodic species, the Syrian hamster. 2-[125I]Iodomelatonin labels a high affinity binding site in hamster brain membranes. Specific binding of 2-[125I]iodomelatonin is rapid, stable, saturable, and reversible. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 3.3 +/- 0.5 nM and a total binding capacity (Bmax) of 110.2 +/- 13.4 fmol/mg protein (n = 4). The Kd value determined from kinetic analysis (3.1 +/- 0.9 nM; n = 5) was very similar to that obtained from saturation experiments. Competition experiments showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding site to hamster brain membranes was as follows: 6-chloromelatonin greater than or equal to 2-iodomelatonin greater than N-acetylserotonin greater than or equal to 6-methoxymelatonin greater than or equal to melatonin greater than 6-hydroxymelatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 5-methoxytryptophol greater than 5-methoxytryptamine greater than or equal to 5-methoxy-N,N-dimethyltryptamine greater than N-acetyltryptamine greater than serotonin greater than 5-methoxyindole (inactive). Compounds known to act at serotonergic, adrenergic, or dopaminergic receptors were either inactive or relatively ineffective as compared to melatonin. These results suggest that 2-[125I]iodomelatonin is a selective, high affinity probe for identifying melatonin receptor binding sites in rodent brain.
对多种季节性繁殖哺乳动物的研究表明，褪黑素介导光周期对繁殖的影响。然而，对于这种激素诱导繁殖效应的作用位点或机制，我们所知相对较少。尽管先前已报道在牛、大鼠和仓鼠脑中存在[3H]褪黑素的结合位点，但这些位点的药理学选择性从未得到证实。在本研究中，我们对一种光周期物种叙利亚仓鼠脑中一种新的放射性配体2-[125I]碘褪黑素的结合位点进行了表征。2-[125I]碘褪黑素在仓鼠脑膜中标记了一个高亲和力结合位点。2-[125I]碘褪黑素的特异性结合快速、稳定、可饱和且可逆。饱和研究表明，2-[125I]碘褪黑素与一类位点结合，其亲和力常数（Kd）为3.3±0.5 nM，总结合容量（Bmax）为110.2±13.4 fmol/mg蛋白质（n = 4）。通过动力学分析确定的Kd值（3.1±0.9 nM；n = 5）与从饱和实验获得的值非常相似。竞争实验表明，多种吲哚对抑制2-[125I]碘褪黑素与仓鼠脑膜结合位点的效力相对顺序如下：6-氯褪黑素≥2-碘褪黑素＞N-乙酰血清素≥6-甲氧基褪黑素≥褪黑素＞6-羟基褪黑素≥6,7-二氯-2-甲基褪黑素＞5-甲氧基色醇＞5-甲氧基色胺≥5-甲氧基-N,N-二甲基色胺＞N-乙酰色胺＞血清素＞5-甲氧基吲哚（无活性）。与褪黑素相比，已知作用于5-羟色胺能、肾上腺素能或多巴胺能受体上的化合物要么无活性，要么相对无效。这些结果表明，2-[125I]碘褪黑素是一种用于识别啮齿动物脑中褪黑素受体结合位点的选择性、高亲和力探针。
