No abstract

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.

The pineal hormone melatonin has been shown to directly scavenge free radicals and to stimulate, in the mammalian brain, at least one enzyme, glutathione peroxidase, which reduces free radical generation. In the present studies, we examined the effect of melatonin on glutathione peroxidase activity in several tissues of an avian species. Melatonin (500 micrograms/kg), when injected into chicks, increased glutathione peroxidase activity within 90 min in every tissue examined. Tissue melatonin levels, measured by radioimmunoassay, also increased following its peripheral administration. Depending on the tissue, the measured increases in melatonin varied from 75% to 1,300% over the control values. The melatonin-induced increases in glutathione peroxidase activity varied with the tissue and were between 22% and 134%. These percentage increases in glutathione peroxidase activity were directly correlated with tissue melatonin content. These results suggest that melatonin induces the activity of the detoxifying enzyme, glutathione peroxidase, in several tissues in the chick. The findings also suggest that melatonin would reduce the generation of highly toxic hydroxyl radicals by metabolizing its precursor, hydrogen peroxide. Because of this ability to stimulate glutathione peroxidase activity, melatonin should be considered as a component of the antioxidative defense system in this avian species.

No abstract

High-affinity 2-125I-iodomelatonin binding sites in homogenates of purified cell nuclei from rat liver were localized and characterized using biochemical binding techniques. Binding at these sites was found to be rapid, reversible, saturable, and to demonstrate pharmacological selectivity. At 0 degrees C, binding reached equilibrium in about 10 min. Scatchard analysis of the data at equilibrium revealed a single class of binding sites with a dissociation constant of KD = 190 +/- 47 pM, Bmax = 9.8 +/- 0.6 fmol/mg protein, and a Hill coefficient of nH = 1.02 +/- 0.034. Kinetic analysis of the association and dissociation curves indicated a kinetic KD = 148 +/- 41 pM, which is in good agreement with the value obtained at equilibrium. The specific binding of 2-125I-iodomelatonin (45 pM) (0.51 +/- 0.04 fmol/mg protein) was significantly improved (0.79 +/- 0.04 fmol/mg protein) when the homogenates of purified liver cell nuclei were preincubated with DNase (2 micrograms/ml at 37 degrees C for 20 min) before being used in binding experiments. After the addition of either proteinase K or trichloroacetic acid to DNase-treated purified cell nuclear homogenates, the specific binding disappeared. This suggests that the specific binding of 2-125I-iodomelatonin in liver cell nuclei is associated with nuclear protein. Competition experiments show that N-acetyl-serotonin (Ki = 81.3 nM) was more potent than 5-hydroxytryptamine (Ki > 1 microM) and 5-methoxytryptamine (Ki >> 10 microM) in inhibiting 2-125I-iodomelatonin binding (Ki melatonin = 146 pM). These data indicate that specific 2-125I-iodomelatonin binding sites exist in the cell nuclei of rat liver, and that they may comprise a locus for the intracellular action of melatonin. The correlation between the KD and Bmax values with melatonin concentrations in nuclei suggest that these binding sites may be a physiological melatonin receptor, which could explain the described genomic effects of the pineal hormone.