Salt stress increases the Ca2+-independent phosphoenolpyruvate carboxylase kinase activity in Sorghum leaves.

In C4 plants, the photosynthetic enzyme phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) is subjected to a phosphorylation process via the light-dependent up-regulation of a Ca2+-independent PEPCase-kinase. The present work aimed to study the effect of salt stress on PEPCase phosphorylation in Sorghum vulgare Pers. leaves. The growth of salt-treated plants was reduced compared with that of the control plants. PEPCase activity modestly increased (around 20-40%) whereas PEPCase phosphorylation was markedly enhanced, on a protein basis, in extracts from illuminated leaves. The enhanced protein kinase activity was found to display a low molecular mass in the range 32-35 kDa, to be independent of Ca2+ and to be up-regulated by light. Furthermore, up-regulation was blocked in vivo by the cytosolic protein synthesis inhibitor cycloheximide. Collectively, these data demonstrated that salinity stress altered the Ca2+-independent PEPCase-kinase, presumably by increasing the mesophyll content of the enzyme. Potassium chloride, but not abscisic acid, mimicked the effect of NaCl on PEPCase-kinase activity.

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines.

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Nuclear receptors are involved in the enhanced IL-6 production by melatonin in U937 cells.

This report shows that melatonin enhances IL-6 production by U937 cells via a nuclear receptor-mediated mechanism. Resting U937 cells only express membrane (mt1) melatonin receptors. In these cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus lipopolysaccharide, a treatment that downregulates the expression of mt1 receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the ROR alpha 1 and ROR alpha 2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor but not membrane receptors is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Involvement of nuclear receptors in the enhanced IL-2 production by melatonin in Jurkat cells.

This report shows that melatonin enhances IL-2 production by Jurkat cells via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZR alpha, ROR alpha 1, and ROR alpha 2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL-2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL-2 production by Jurkat cells.

Melatonin activates Th1 lymphocytes by increasing IL-12 production.

Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-gamma and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.

Physiological levels of melatonin contribute to the antioxidant capacity of human serum.

This work evaluates whether physiological concentrations of the pineal secretory product melatonin contribute to the total antioxidant status (TAS) of human serum. Day and nighttime serum samples were collected from healthy volunteers ranging from 2 to 89 years of age and used to measure melatonin and TAS. Results showed that both melatonin and TAS in human serum exhibited 24 hr variations with nocturnal peak values at 01:00 hr. Moreover, exposure of volunteers to light at night resulted in clear decreases of both TAS and melatonin. Furthermore, when melatonin was removed from sera collected at night, the TAS value of the sample was reduced to basal daytime values. In aging studies, it was found that nocturnal serum values of TAS and melatonin exhibited maximal values during the first four decades; thereafter, these values decreased as age advanced. In 60-year-old individuals, day/night differences in serum melatonin and TAS levels were clearly diminished, by more than 80%, with these differences being completely abolished in older individuals. Our results suggest that melatonin contributes to the total antioxidative capability of human serum. This antioxidant contribution of melatonin is reduced as age advances correlating with the age-related reduction of melatonin.

Specific binding of melatonin by purified cell nuclei from spleen and thymus of the rat.

In the present paper, we show that pineal hormone melatonin interacts with purified cell nuclei from rat spleen and thymus. Binding of 2-[125I]iodomelatonin ([125I]melatonin) by cell nuclei fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, and specificity. Results suggested binding to single classes of binding sites. The dissociation constants (Kd) for the binding sites in the spleen and thymus nuclei were 68 and 102 pM, respectively. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived values of the dissociation constant in the spleen and thymus nuclei were 166 and 537 pM, respectively. The affinities for melatonin of these nuclear binding sites suggest that they may recognize the physiological concentrations of melatonin in the tissues. Finally, we have demonstrated that binding of [125I]melatonin by the nuclei is displaced by CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR. Results strongly suggest that in addition to membrane receptor-related mechanisms, nuclear receptors may be involved in the regulation of immune system by melatonin.

Involvement of nuclear binding sites for melatonin in the regulation of IL-2 and IL-6 production by human blood mononuclear cells.

Many functional studies show that melatonin plays a fundamental role in neuroimmunomodulation. In this paper, we have extended our studies on the influence of melatonin on IL-2 and IL-6 production by human peripheral blood mononuclear cells (PBMCs) by comparing the effects of the specific membrane receptor agonist S 20098, the RZR/ROR(alpha) receptor agonist CGP 52608, and structurally related thiazolidinediones. Melatonin bound to membranes as well as to nuclei of human PBMCs with about the same affinity (IC50 values around 5 nM). S 20098 bound to PBMC membranes but not to PBMC nuclei, although the affinity was at least 100 times lower than that of melatonin; this compound did not stimulate cytokine production. In contrast, all four CGP compounds did not bind to PBMC membranes, while binding to nuclei exhibited IC50 values comparable to those of melatonin. The thiazolidinediones activating the RZR/ROR(alpha) receptor (CGP 52608, CGP 53079) also increased IL-2 and IL-6 production. CGP 55644 had no effect on cytokine production and antagonized the effects of CGP 52608 on IL-2 and IL-6 production; moreover, CGP 55644 decreased the enhanced IL-2 production caused by melatonin. Results obtained in monocyte cultures resembled closely those shown in PBMCs. The results reported in this paper confirm the involvement of a nuclear mechanism in the melatonin effects on cytokine production in human PBMCs. We have also shown a synergistic effect of S 20098 and CGP 52608, suggesting a possible link between nuclear and membrane melatonin receptors in PBMCs.

Melatonin enhances IL-2, IL-6, and IFN-gamma production by human circulating CD4+ cells: a possible nuclear receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes.

This paper shows that melatonin is able to activate human Th1 lymphocytes by increasing the production of IL-2 and IFN-gamma in vitro. Th2 cells appear not to be affected by melatonin, since IL-4, which is mostly produced by Th2 cells, is not modified by the hormone. Melatonin also enhances IL-6 production by PBMCs. The activation by melatonin of IL-6 production is apparently related to the presence of monocytes, rather than to Th2 cells, in the cell preparation, since PBMCs depleted of monocytes (CD14+ cells) were not activated. Activation of PBMCs by melatonin was dependent on the dose and, measured by cytokine production, was observed only when cells were either not activated or only slightly activated by low concentrations of PHA, or when cell activation was achieved by incubating the cells with previously irradiated cells. Using a different approach to identify what type of cells among the PBMC subsets was activated by melatonin, the expression of CD69, a marker of cell activation, was studied. Melatonin increased the percentage of cells expressing the CD69 Ag in CD4+ but not in CD8+ cells. We have also achieved enhanced production of IL-2 and IL-6 using CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR, raising the possibility of direct effects of melatonin on gene regulation in both Th1 cells and monocytes. The results suggest that melatonin may be involved in the regulation of human immune functions by modulating the activity of Th1 cells and monocytes via nuclear receptor-mediated transcriptional control.

High-affinity binding of melatonin by human circulating T lymphocytes (CD4+).

This paper shows the presence of high-affinity binding sites for melatonin in human circulating T lymphocytes, but not in B lymphocytes. The binding of melatonin to T cells was dependent on time, stable, reversible, saturable, specific, and inversely correlated to the production of melatonin, expressed as the nocturnal 12 h production of its urinary metabolite 6-sulfatoxymelatonin. The affinity of these binding sites (Kd = 0.27 nM) suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, among the lymphocyte subpopulations studied, binding of melatonin was mostly found in CD4+ cells rather than in CD8+ cells. Results suggest that CD4+ cells may be the target of melatonin among the human circulating lymphocytes.