Characterization of membrane melatonin receptor in mouse peritoneal macrophages: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.

Melatonin binding sites were characterized in mouse peritoneal macrophages. Binding of 2-[125I]melatonin by macrophages fulfills all criteria for binding to a receptor site. Thus, binding was dependent on time, temperature and cell concentration, stable, reversible, saturable and specific. Stoichiometric studies showed a high-affinity binding site with a Kd of 0.58-0.71 nM. These data are in close agreement with data obtained from kinetic studies (Kd = 0.29 nM). The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, binding experiments using macrophage crude membranes showed that melatonin bound specifically to the membranes. Additionally, in competition studies we observed a low-affinity binding site (Kd = 2.02 microM). Melatonin inhibited significantly forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner. This effect was blocked by luzindole, an antagonist of the melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybosilation and Western blot experiments demonstrated both alpha(i1/2) and alpha(i3/o) G protein subunits expression in mouse peritoneal macrophages membranes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal transduction pathway that involves the inhibition of adenylyl cyclase.

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.

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.

Differential adrenergic regulation of rat pineal cyclic AMP production and N-acetyltransferase activity during postnatal development: involvement of G alpha s and G alpha i1-2 proteins.

We have studied why rat pineal N-acetyltransferase (NAT) activity is relatively insensitive to isoproterenol in young rats when compared with adult rats. We report that activation by isoproterenol of pineal cyclic AMP production and NAT activity is higher in adult than in 2-week-old rats. However, the effect of dibutyryl cyclic AMP, which enters the pinealocyte and duplicates the effect of cyclic AMP, on NAT activity was similar at both ages. Moreover, we found that both alpha- and beta-adrenergic receptors are highly specific at both ages, since the binding of the specific radioligands used to their receptors could be displaced only by their corresponding agonists and antagonists. However, we observed differences between pineals from young and adult rats when several families of the alpha subunit of G-proteins were studied in cell membranes. ADP-ribosylation and immunoblot studies have shown clear differences in both 42 and 45 kDa forms of the Gs alpha Both forms exhibit low values in pineals from 2-week-old animals when compared with 6-week-old. We also show that the later appearance of both Gs alpha forms is roughly similar to the potent activation of cyclic AMP production and NAT activity in adult rats when compared with young rats. In conclusion, the results presented suggest that the relative lack of sensitivity of rat pineal gland to beta-adrenergic receptor agonists early in the postnatal development may be explained by the low levels of membrane Gs alpha, rather than postreceptor-mediated mechanisms or changes in the characteristics of the beta-adrenergic receptors on the pinealocyte membrane.

Binding of [125I]iodocyanopindolol by rat harderian gland crude membranes: kinetic characteristics and day-night variations.

The Harderian glands are innervated by sympathetic fibers originating in the superior cervical ganglia. The aim of this study is to characterize the beta-adrenergic receptors in the rat Harderian gland. The characteristics of beta-adrenergic receptors were determined in crude membrane preparations from rat Harderian gland, using [125I]iodocyanopindolol ([125I]CYP) as radioligand. The binding of the ligand to the receptor is rapid, reversible, saturable, specific and dependent on time, temperature and membrane concentration. At 30 degrees C, stoichiometric data suggest the presence of one binding site with a Kd value of 0.29 nM and Bmax of 32 pmol/L. The interaction shows a high degree of specificity for beta-adrenergic agonists and blockers, as suggested by competitive displacement experiment with isoproterenol (IC50 = 19.1 nM), propranolol (IC50 = 28.1 nM), and norepinephrine (IC50 = 96.3 nM). Clonidine, yohimbine, methoxamine, and prazosin are ineffective at concentrations up to 1 microM. In the other hand, binding of [125I]CYP by Harderian gland membranes exhibits day-night variations. Binding values are low during the daytime and increase progressively late in the evening to reach a maximum at 2200 h (2 h after the onset of dark period), but decreased to the end of the dark period (0600 h). In conclusion, the results presented in this paper show the functional and pharmacological characterization of beta-adrenergic receptors in the rat Harderian gland. This neurotransmitter may play a physiological role at this level regulating, at least, processes such as a thyroid hormone metabolism.

Characterization of binding sites for beta-adrenergic agonists and vasoactive intestinal peptide in the rat harderian gland.

Vasoactive intestinal peptide (VIP) receptors and beta-adrenergic receptors were investigated in rat Harderian gland membranes using 125I-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The receptor bindings were rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP as follows: VIP > helodermin > rGRF > PHI > > secretin. Glucagon, somatostatin, insulin, and pancreastatin were ineffective at concentrations up to 1 microM. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site was 290 pM with a binding capacity of 32 pmol/L. The pharmacological characterization of 125I-CYP binding to membranes showed that only isoproterenol, a beta-adrenergic agonist, and norepinephrine, an alpha beta-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, alpha 1- and alpha 2-adrenergic agonists and blockers such as methoxamine, prazosin, clonidine, and yohimbine were shown to be ineffective. These results demonstrate the presence of specific VIP and beta-adrenergic receptors in the Harderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland.

Specific binding of 2-[125I]iodomelatonin by rat spleen crude membranes: day-night variations and effect of pinealectomy and continuous light exposure.

Melatonin binding sites were characterized in rat spleen crude membranes. The specific binding of 2-[125I]iodomelatonin by spleen crude membranes fulfills all the criteria for binding to a receptor site. Thus, binding was dependent on time and temperature, stable, specific, and increased under constant light exposure and after pinealectomy. In competition studies, the specific binding of 2-[125I]iodomelatonin to spleen crude membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that the data were compatible with the existence of two classes of binding sites: a high affinity site with a Kd of 0.53 nM and a binding capacity of 2.52 pM, and a low-affinity site with a Kd of 374 nM and binding capacity of 820 pM. Moreover, binding of 2-[125I]iodomelatonin exhibited day-night variations with the highest binding observed late during the light period, and the lowest binding was observed late at night. However, binding of 2-[125I]iodomelatonin to membranes remained high when animals were kept under light exposure at night. Results support the hypothesis of a regulatory role of melatonin on the immune system in which melatonin downregulates its own binding site.

Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells.

This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1-1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.

Specific binding of 2-[125I]iodomelatonin by rat splenocytes: characterization and its role on regulation of cyclic AMP production.

In the present paper we show that pineal hormone melatonin interacts with rat splenocytes through high-affinity binding sites. Binding of 2-[125I]iodomelatonin ([125I]MEL) by splenocytes 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, specificity, and increased under constant light exposure. Results suggest binding to a single class of binding sites without cooperative interactions. The dissociation constant (Kd) for the single site was 0.34 nM with a binding capacity of 2.25 fmol/10(7) cells. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived value of the dissociation constant was 0.20 nM. The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, pharmacological doses of melatonin also inhibited cyclic AMP production stimulated by forskolin, a potent activator of adenylate cyclase system. The demonstration of [125I]MEL binding sites in the spleen, in addition to those described in blood mononuclear cells and thymus, provides evidence to support a direct mechanism of action of melatonin on immune system.