Menendez-Pelaez A, Poeggeler B, Reiter R J, Barlow-Walden L, Pablos M I, Tan D X
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio 78284.
J Cell Biochem. 1993 Dec;53(4):373-82. doi: 10.1002/jcb.240530415.
Melatonin was detected by an improved immunocytochemical technique in the cell nuclei of most tissues studied including several brain areas, pineal gland, Harderian gland, gut, liver, kidney, and spleen from rodents and primates. Cryostat sections from tissues fixed in Bouin's fluid, formalin, or acetone/ethanol were used. The nuclear staining appeared primarily associated with the chromatin. The nucleoli did not exhibit a positive reaction. The melatonin antiserum was used in the range of 1:500 to 1:5,000. Incubation of the antibody with an excess of melatonin resulted in the complete blockade of nuclear staining. Pretreatment of the sections with proteinase K (200-1,000 ng/ml) prevented the positive immunoreaction. In a second aspect of the study, we estimated the concentration of melatonin by means of radioimmunoassay in the nuclear fraction of several tissues including cerebral cortex, liver, and gut. The subcutaneous injection of melatonin (500 micrograms/kg) to rats resulted, after 30 min, in a rapid increase in the nuclear concentration of immunoreactive melatonin which varied in a tissue-dependent manner. However, samples collected 3 h after the injection showed that melatonin levels had decreased to control values. Pinealectomy in rats resulted in a clear reduction in the nuclear content of melatonin in the cerebral cortex and liver but not in the gut. The results of these studies suggest that melatonin may interact with nuclear proteins and that the indole may have an important function at the nuclear level in a variety of mammalian tissues.
采用改良免疫细胞化学技术在包括啮齿动物和灵长类动物的多个脑区、松果体、哈德氏腺、肠道、肝脏、肾脏和脾脏等大多数研究组织的细胞核中检测到了褪黑素。使用了用布安氏液、福尔马林或丙酮/乙醇固定的组织的冰冻切片。细胞核染色主要与染色质相关。核仁未呈现阳性反应。褪黑素抗血清的使用浓度范围为1:500至1:5000。用过量褪黑素孵育抗体导致细胞核染色完全被阻断。用蛋白酶K（200 - 1000 ng/ml）预处理切片可防止阳性免疫反应。在该研究的第二个方面，我们通过放射免疫测定法估计了包括大脑皮层、肝脏和肠道在内的几种组织的细胞核部分中褪黑素的浓度。给大鼠皮下注射褪黑素（500微克/千克），30分钟后，免疫反应性褪黑素的核浓度迅速增加，且这种增加因组织而异。然而，注射后3小时采集的样本显示褪黑素水平已降至对照值。大鼠松果体切除导致大脑皮层和肝脏中褪黑素的核含量明显降低，但肠道中未降低。这些研究结果表明，褪黑素可能与核蛋白相互作用，并且这种吲哚在多种哺乳动物组织的核水平上可能具有重要功能。
