Removal of melatonin receptor type 1 signalling induces dyslipidaemia and hormonal changes in mice subjected to environmental circadian disruption.

Background: Melatonin is a hormone secreted by the pineal gland in a circadian rhythmic manner with peak synthesis at night. Melatonin signalling was suggested to play a critical role in metabolism during the circadian disruption. Methods: Melatonin-proficient (C3H-f+/+ or WT) and melatonin receptor type 1 knockout (MT1 KO) male and female mice were phase-advanced (6 hours) once a week for 6 weeks. Every week, we measured weight, food intake and basal glucose levels. At the end of the experiment, we sacrificed the animals and measured the blood's plasma for lipids profile (total lipids, phospholipids, triglycerides and total cholesterol), metabolic hormones profiles (ghrelin, leptin, insulin, glucagon, glucagon-like-peptide and resistin) and the body composition. Results: Environmental circadian disruption (ECD) did not produce any significant effects in C3H-f+/+, while it increased lipids profile in MT1 KO with the significant increase observed in total lipids and triglycerides. For metabolic hormones profile, ECD decreased plasma ghrelin and increased plasma insulin in MT1 KO females. Under control condition, MT1 KO females have significantly different body weight, fat mass, total lipids and total cholesterol than the control C3H-f+/+ females. Conclusion: Our data show that melatonin-proficient mice are not affected by ECD. When the MT1 receptors are removed, ECD induced dyslipidaemia in males and females with females experiencing the most adverse effect. Overall, our data demonstrate that MT1 signalling is an essential modulator of lipid and metabolic homeostasis during ECD.

Melatonin receptor type 1 signals to extracellular signal-regulated kinase 1 and 2 via Gi and Gs dually coupled pathways in HEK-293 cells.

The pineal gland hormone melatonin exerts its regulatory roles in a variety of physiological and pathological responses through two G protein-coupled receptors, melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2), which have been recognized as promising targets in the treatment of a number of human diseases and disorders. The MT1 receptor was identified nearly 20 years ago; however, the molecular mechanisms by which MT1-mediated signaling affects physiology remain to be further elucidated. In this study, using HEK293 cells stably expressing the human MT1 receptor, melatonin induced a concentration-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). The melatonin-mediated phosphorylation of ERK1/2 at later time points (≥5 min) was strongly suppressed by pretreatment with pertussis toxin, but only a slight, if any, inhibition of ERK1/2 activation at early time points (≤2 min) was detected. Further experiments demonstrated that the Gßγ subunit, phosphoinositide 3-kinase, and calcium-insensitive protein kinase C were involved in the MT1-mediated activation of ERK1/2 at later time points (≥5 min). Moreover, results derived from cAMP assays combined with a MT1 mutant indicated that the human MT1 receptor could also couple to Gs protein, stimulating intracellular cAMP formation, and that the MT1-induced activation of ERK1/2 at early time points (≤2 min) was mediated by the Gs/cAMP/PKA cascade. Our findings may provide new insights into the pharmacological effects and physiological functions modulated by the MT1-mediated activation of ERK1/2.

MT1 and MT2 melatonin receptors: ligands, models, oligomers, and therapeutic potential.

Numerous physiological functions of the pineal gland hormone melatonin are mediated via activation of two G-protein-coupled receptors, MT1 and MT2. The melatonergic drugs on the market, ramelteon and agomelatine, as well as the most advanced drug candidates under clinical evaluation, tasimelteon and TIK-301, are high-affinity nonselective MT1/MT2 agonists. A great number of MT2-selective ligands and, more recently, several MT1-selective agents have been reported to date. Herein, we review recent advances in the field focusing on high-affinity agonists and antagonists and those displaying selectivity toward MT1 and MT2 receptors. Moreover, the existing models of MT1 and MT2 receptors as well as the current status in the emerging field of melatonin receptor oligomerization are critically discussed. In addition to the already existing indications, such as insomnia, circadian sleep disorders, and depression, new potential therapeutic applications of melatonergic ligands including cardiovascular regulation, appetite control, tumor growth inhibition, and neurodegenerative diseases are presented.

Melatonin receptors mediate improvements of survival in a model of polymicrobial sepsis.

OBJECTIVES: Melatonin has been demonstrated to improve survival after experimental sepsis via antioxidant effects. Yet, recent evidence suggests that this protective capacity may also rely on melatonin receptor activation. Therefore, the present study was designed to investigate whether selective melatonin receptor-agonist ramelteon may influence survival and immune response in a model of polymicrobial sepsis in rats, wild-type and melatonin receptor MT1/MT2 double knockout mice. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats (200-250 g) and male C3H/HeN wild-type and MT1/MT2 receptor knockout mice (20-22 g). INTERVENTIONS: Animals underwent cecal ligation and incision and remained anesthetized for evaluation of survival for 12 hours (rats: n = 15 per group) or 15 hours (mice: n = 10 per group). Analysis of immune response by means of enzyme-linked immunosorbent assay was performed before and 5 hours after cecal ligation and incision (rats only; n = 5 per group). After induction of sepsis, animals were treated IV with vehicle, different doses of melatonin (rats: 0.01/0.1/1.0/10 mg/kg; mice: 1.0 mg/kg), ramelteon, melatonin receptor-antagonist luzindole, ramelteon + luzindole, or melatonin + luzindole (each 1.0 mg/kg). Sham controls underwent laparotomy but not cecal ligation and incision. MEASUREMENTS AND MAIN RESULTS: Compared with vehicle, administration of ramelteon or melatonin significantly improved median survival time in rats (sepsis/melatonin [0.1 mg/kg], 554 min, [1.0 mg/kg] 570 min, [10 mg/kg] 579 min; sepsis/ramelteon, 468 min; each p < 0.001 vs sepsis/vehicle, 303 min) and wild-type mice (sepsis/melatonin, 781 min; sepsis/ramelteon, 701 min; both p < 0.001 vs sepsis/vehicle, 435 min). This effect was completely antagonized by coadministration of luzindole in all groups. Melatonin, ramelteon, or luzindole had no significant effect on survival time in knockout mice. Significantly elevated concentrations of tumor necrosis factor-α, interleukin-6, and interleukin-10 were observed 5 hours after cecal ligation and incision in rats (p < 0.05 vs baseline and corresponding sham); neither ramelteon nor melatonin treatment significantly affected immune response. CONCLUSIONS: Melatonin receptors mediate improvements of survival after polymicrobial sepsis in rats and mice; this effect appears to be independent from major alterations of cytokine release.

Molecular deficiency (ies) in MT1 melatonin signaling pathway underlies the melatonin-unresponsive phenotype in MDA-MB-231 human breast cancer cells.

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF-7 human breast cancer cells and several other estrogen receptor α (ERα)-positive human breast cancer cell lines. However, the MDA-MB-231 breast cancer cell line, an ERα-negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth-suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα-negative breast cancer cell lines including MDA-MB-231, BT-20, and SK-BR-3 cells. Although the MT1 G-protein-coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK-BR-3 cells without having any significant effect on the growth of MDA-MB-231 and BT-20 cells. We confirmed that the MT1-associated Gα proteins are expressed in MDA-MB-231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA-MB-231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.

Age-related decline in melatonin and its MT1 receptor are associated with decreased sensitivity to melatonin and enhanced mammary tumor growth.

The pineal hormone melatonin (MLT) has potent anti-breast cancer activity, its actions are heavily mediated via the MT1 receptor and subsequent modulation of downstream signaling pathways including cAMP/PKA, Erk/MAPK, p38, and Ca2+/calmodulin. Also, via the MT1 pathway, MLT can repress the transcriptional activity of some mitogenic nuclear receptors including ERα, GR, and RORα, while potentiating the activity of other receptors (RARα and RXRα) involved in differentiation, anti-proliferation, and apoptosis. A review of the literature supports the view that MLT, via its MT1 receptor, can suppress all phases of breast cancer including initiation, promotion, and progression. During the fifth and sixth decades of life, the production of MLT diminishes, concurrently with an increase in the incidence of breast cancer. Inasmuch as MLT has been demonstrated to have anti-cancer activity, we hypothesized that there may be a causal link between the reduction in MLT production in the pineal gland and the incidence of breast cancer which increases with age. We designed this study to establish whether a truly inverse relationship exists between tissue-isolated mammary tumor growth in young (2 months), adult (12 months), and old (20 months) female Buffalo rats and the decrease in both MLT and the MT1 receptor with age, such that a causal link could be found. Serum MLT levels were measured in both the light and dark phases. A significant 29% decrease in serum MLT levels, measured at the nocturnal peak, was found in the adult and senescent rats (75% decrease) in comparison to that in young rats. In young rats, the nocturnal pineal gland MLT content exceeded daytime levels by 19-fold compared to a sevenfold increase in old mice. Also, the MT1 receptor was found to be significantly lower in the nighttime and early morning in the senescent rat uterus as compared to uteri from young and adult rats. Analysis of the rate of growth in transplanted, tissue-isolated, mammary tumors induced by N-nitroso-n-methyl-urea (NMU) showed a significant increase in the senescent rats, but not in the young or adult rats Additionally, diminished response to the inhibitory action on tumor growth of exogenous MLT was noted in senescent rats such that tumor growth was suppressed by only 33% compared to 48% and 66% in adult and young rats, respectively. The diminution of the response of tumors to exogenous MLT was found to correlate with reduced MT1 receptor expression in senescent compared to young and adult rats. These data suggest that the observed age-associated enhanced growth of tumors is related to the much reduced levels of MLT and its receptor in aged animals which reduce the sensitivity of tumors to inhibition by exogenous MLT.

Melatonin suppresses apoptosis and stimulates progesterone production by bovine granulosa cells via its receptors (MT1 and MT2).

Melatonin and its receptors have been detected in the ovary of many species, and mediate ovarian functions. The present study was designed to investigate the expression and subcellar location of melatonin receptors in bovine granulosa cells (GCs), using reverse transcription (RT) polymerase chain reaction, Western blot, and immunofluorescence analyses. Furthermore, expression level of melatonin receptors mRNA (real-time polymerase chain reaction) after treatment with various concentrations of melatonin, as well as its effects on cell apoptosis, proliferation, and steroidogenesis (by flow cytometry and RIA), were determined. In bovine GCs, melatonin receptors MT1 and MT2 were differentially located at the cell membrane, the cytoplasm, and nuclear membranes. The expression of MT1 and MT2 mRNA was regulated differently by melatonin in time- and dose-dependent manners. Exogenous melatonin suppressed cell apoptosis (P < 0.05) but not proliferation (P > 0.05). After 72 h, the apoptotic rate was significantly inhibited in all treatment groups. Meanwhile, melatonin supplementation stimulated progesterone production, but inhibited estradiol biosynthesis, in a time-dependent manner. Progesterone production was highest (P < 0.05) at 72 h. Estradiol concentrations were almost unaffected (P > 0.05) at 24 h, but were decreased (P < 0.05) at 48 h. In conclusion, exogenous melatonin acts via receptors and has important roles in regulation of development and function of bovine GCs.

Impaired mouse mammary gland growth and development is mediated by melatonin and its MT1G protein-coupled receptor via repression of ERα, Akt1, and Stat5.

To determine whether melatonin, via its MT(1) G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT(1) -Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT(1) receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT(1) expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins ß-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT(1) receptor expression and exogenous melatonin administration. These studies demonstrate that the MT(1) melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.

A new perspective in Oral health: potential importance and actions of melatonin receptors MT1, MT2, MT3, and RZR/ROR in the oral cavity.

BACKGROUND: Melatonin is involved in many physiological processes in mammals, amongst others; it is implicated in sleep-wake regulation. It has antioxidant and anti-inflammatory properties. It also acts as an immunomodulator, stimulates bone metabolism and inhibits various tumours. Additionally an abnormal melatonin rhythm may contribute to depression and insomnia. The mechanisms of action of melatonin include the involvement of membrane receptors (MT1, MT2), cytosolic binding sites (MT3 and calmodulin), and nuclear receptors of the RZR/ROR family. Melatonin also has receptor-independent activity and can directly scavenge free radicals. The current review addresses the functions of melatonin in the oral cavity in relation to its receptors. METHODS: An extensive search was conducted on the following scientific databases Pub Med, Science Direct, ISI Web of Knowledge and Cochrane database in order to review all pertinent literature. RESULTS: Melatonin from the blood into the saliva may play an important role in suppressing oral diseases. It may have beneficial effects in periodontal disease, herpes and oral cancer, amongst others. CONCLUSIONS: Melatonin contributes to protecting of oral cavity from tissue damage due to its action of different receptors. From the reviewed literature it is concluded that experimental evidence suggests that melatonin can be useful in treating several common diseases of the oral cavity. Specific studies are necessary to extend the therapeutic possibilities of melatonin to other oral diseases.

[The influence of melatonin on hair physiology]. / Einfluss von Melatonin auf die Physiologie des Haares.

Melatonin, the pineal gland hormone and a strong antioxidant, has long been known, particularly in animal-experiment based research and the wool-producing industry, to be a potent regulatory neuroendocrine substance in relation to hair growth, hair color and hair cycle, depending on light periods, seasonal rhythms, environmental factors and reproductive rhythms. Nevertheless, the biological mechanisms of this extremely versatile hormone, especially with regard to human hair follicles, are not fully understood. In recent years, however, essential knowledge has been gained on the melatoninergic system of the skin, melatonin levels in keratinocytes and hair follicles, extrapineal intrafollicular melatonin synthesis and noradrenalin-induced increase in synthesis, as well as hair cycle-dependent expression of the membrane-bound melatonin receptor MT2 and the nuclear receptor RORalpha. Functional data on the growth of human hair both in vitro and in vivo show that melatonin might play an essential role in hair physiology.

Molecular mechanisms of melatonin anticancer effects.

The authors have shown that, via activation of its MT1 receptor, melatonin modulates the transcriptional activity of various nuclear receptors and the proliferation of both ER alpha+ and ER alpha- human breast cancer cells. Employing dominant-negative (DN) and dominant-positive (DP) G proteins, it was demonstrated that G alpha i2 proteins mediate the suppression of estrogen-induced ER alpha transcriptional activity by melatonin, whereas the G alpha q proteins mediate the enhancement of retinoid-induced RAR alpha transcriptional activity by melatonin. In primary human breast tumors, the authors' studies demonstrate an inverse correlation between ER alpha and MT1 receptor expression, and confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that its expression can be repressed by estrogen and melatonin. Melatonin, via activation of its MT1 receptor, suppresses the development and growth of breast cancer by regulation of growth factors, regulation of gene expression, regulation of clock genes, inhibition of tumor cell invasion and metastasis, and even regulation of mammary gland development. The authors have previously reported that the clock gene, Period 2 (Per2), is not expressed in human breast cancer cells but that its reexpression in breast cancer cells results in increased expression of p53 and induction of apoptosis. The authors demonstrate that melatonin, via repression of ROR alpha transcriptional activity, blocks the expression of the clock gene BMAL1. Melatonin's blockade of BMAL1 expression is associated with the decreased expression of SIRT1, a member of the Silencing Information Regulator family and a histone and protein deacetylase that inhibits the expression of DNA repair enzymes (p53, BRCA1 & 2, and Ku70) and the expression of apoptosis-associated genes. Finally, the authors developed an MMTV-MT1-flag mammary knock-in transgenic mouse that displays reduced ductal branching, ductal epithelium proliferation, and reduced terminal end bud formation during puberty and pregnancy. Lactating female MT1 transgenic mice show a dramatic reduction in the expression of beta-casein and whey acidic milk proteins. Further analyses showed significantly reduced ER alpha expression in mammary glands of MT1 transgenic mice. These results demonstrate that the MT1 receptor is a major transducer of melatonin's actions in the breast, suppressing mammary gland development and mediating the anticancer actions of melatonin through multiple pathways.

The Galphai and Galphaq proteins mediate the effects of melatonin on steroid/thyroid hormone receptor transcriptional activity and breast cancer cell proliferation.

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ERalpha) and retinoic acid receptor alpha (RARalpha), but not ERbeta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the G(alphai2), G(alphai3), G(alphaq), and G(alphall) proteins, and via activation of G(alphai2) proteins, melatonin suppresses forskolin-induced 3',5'-cyclic adenosine monophosphate production, while melatonin activation of G(alphaq), is able to inhibit phospholipid hydrolysis and ATP's induction of inositol triphosphate production in MCF-7 breast cancer cells. Employing dominant-negative and dominant-positive) forms of these G proteins, we demonstrate that G(alphai2) proteins mediate the suppression of estrogen-induced ERalpha transcriptional activity by melatonin, while the G(q) protein mediates the enhancement of retinoid-induced RARalpha transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both G(alphai2) and G(alphaq) proteins.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors.

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Frequent silencing of a putative tumor suppressor gene melatonin receptor 1 A (MTNR1A) in oral squamous-cell carcinoma.

Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous-cell carcinoma (OSCC) cell lines for genome-wide copy-number aberrations by array-CGH using our in-house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A (MTNR1A) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2'-deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor-suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis.

C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes.

Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT(1) and MT(2). In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT(1)C7.72A and MT(2)C7.77A) and (ii) the C-terminal tail in the MT(1) and MT(2) receptors was truncated, removing the putative phosphorylation and beta-arrestin binding sites (MT(1)Y7.64 and MT(2)Y7.64). These mutations did not alter the affinity of 2-[(125)I]-iodomelatonin binding to the MT(1) or MT(2) receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3',5'-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT(1)Y7.64 and MT(2)Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions.

Melatonin inhibits prostaglandin E2- and sodium nitroprusside-induced ion secretion in rat distal colon.

Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

[Sites and mechanisms of action of melatonin in mammals: the MT1 and MT2 receptors]. / Sites et mécanismes d'action de la mélatonine chez les Mammifères: les récepteurs MT1 et MT2.

The rhythmic secretion of melatonin by the pineal gland plays a key role in the synchronisation of circadian and seasonal functions with cyclic environmental variations. The biological effects of this neurohormone are relayed mainly by G-protein-coupled seven-transmembrane receptors. These receptors, known as MT1 and MT2, are present in a large number of central and peripheral structures in mammals, with considerable inter-species variations. However, only the suprachiasmatic nuclei of the hypothalamus, the site of the master circadian biological clock, and the pars tuberalis of the adenohypophysis contain melatonin receptors in the majority of species. Inhibition of the production of AMPc by a Gi/Go protein is one of the principal signalling pathways of the MT1 and MT2 receptors, although many other signal transduction pathways are also brought into play according to the cell type studied (PKC, Ca2+, K+ channels or GMPc in the case of MT2, etc.). Numerous factors or physiological stimuli are capable of influencing the number and functional status of the MT1 and MT2 receptors, such as melatonin, the photoperiod, the circadian clock or the phenomena of receptor dimerisation. Melatonin has numerous physiological effects for which the mechanisms of action and the specific role of the MT1 and MT2 receptors have not yet been clearly elucidated. However, selective pharmacological tools for each of the two receptor subtypes are currently being identified, notably in the Servier Group, for the purpose of furthering our knowledge of the functionality and physiological role of the MT1 and MT2 receptors in the central and peripheral structures.

[Melatonin binding site MT3 is QR2: state of the art]. / Le récepteur MT3: où en sommes-nous?

Melatonin is a neurohormone primarily synthesized in the pineal gland at night. It has numerous functions in various pathophysiological situations, including anti-oxidant properties at pharmacological concentrations (1 microM and above). It is believed that melatonin acts through three main targets: two 7TM receptors (MT1 and MT2) and one atypical binding site called MT3. This last binding site has been purified in our laboratory and is designated as quinone reductase 2 (QR2, E.C. 1.10.99.2). This enzyme has several individualistic features. It does not recognize standard nicotinamide derivatives as co-substrates, but rather, it recognizes rare ones such as N-ribosylnicotinamide. Among other features of this enzyme, two are of major importance: 1) experiments from Dr Jaiswal (Houston, Texas) laboratory with QR2-/- mice and with cells derived from them demonstrated that this enzyme is implicated in the toxicological activation of menadione, and thus, may have an activation rather than a detoxification role, as formerly believed, and 2) the polyphenol resveratrol, a molecule with anti-oxidant properties, is a potent inhibitor of QR2 ( approximately 30 nM). This talk will briefly summarize these findings, and will present our working hypotheses, molecular tools and findings on several aspects of the possible relationship between QR2 and melatonin, in particular those suggesting a mechanism for the anti-oxidant activity of melatonin.

Functional MT1 and MT2 melatonin receptors in mammals.

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT1 and MT2 G protein-coupled receptors viewed as potential targets for drug discovery. MT1 melatonin receptors modulate neuronal firing, arterial vasocon-striction, cell proliferation in cancer cells, and reproductive and metabolic functions. Activation of MT2 melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT1 and MT2 native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.

17Beta-estradiol modulates hMT1 melatonin receptor function.

Estrogen modulates expression and function of G-protein-coupled receptors. The goal of this study was to assess the effect of 17beta-estradiol (10 nM) exposure for 1 (E1) or 6 (E6) days on density and function of hMT1 and hMT2 melatonin receptors expressed in Chinese hamster ovary (CHO) cells (CHO-MT1/CHO-MT2 cells). This strain of CHO cells expressed both estrogen receptor alpha and beta mRNAs, as determined by RT-PCR amplification. 17beta-Estradiol treatment did not modify the affinity of either receptor; however, it significantly increased the density of 2-[125I]iodomelatonin-binding sites in CHO-MT2 cells. 17beta-Estradiol treatment (1-6 days) did not affect the potency of melatonin to inhibit forskolin stimulation of cAMP formation through activation of either MT1 or MT2 receptors; however, it significantly attenuated the maximal inhibition of forskolin-stimulated cAMP formation induced by melatonin (0.01-1 microM) in CHO-MT1 cells. Melatonin stimulation of [35S]GTPgammaS binding to CHO-MT1 cell membranes was also attenuated following estradiol treatment. The inverse agonist luzindole reduced basal [35S]GTPgammaS binding in estradiol-treated cells but not in control CHO-MT1 cells, suggesting that estradiol promotes constitutive activity of MT1 melatonin receptors. We suggest that 17beta-estradiol differentially affects MT1 and MT2 melatonin receptor functions, attenuates melatonin responses through activation of MT1 receptors, and increases the MT2 receptors density.