In silico drug discovery of melatonin receptor ligands with therapeutic potential.

INTRODUCTION: The neurohormone melatonin (N-acetyl-5-methoxytryptamine) regulates circadian rhythms exerting a variety of effects in the central nervous system and in periphery. These activities are mainly mediated by activation of MT1 and MT2 GPCRs. MT1/MT2 agonist compounds are used clinically for insomnia, depression, and circadian rhythm disturbances. AREA COVERED: The following review describes the design strategies that have led to the identification of melatonin receptor ligands, guided by in silico approaches and molecular modeling. Initial ligand-based design, mainly relying on pharmacophore modeling and 3D-QSAR, has been flanked by structure-based virtual screening, given the recent availability of MT1 and MT2 crystal structures. Receptor ligands with different activity profiles, agonist/antagonist and subtype-selective compounds, are available. EXPERT OPINION: An insight on the pharmacological characterization and therapeutic perspectives for relevant ligands is provided. In silico drug discovery has been instrumental in the design of novel ligands targeting melatonin receptors. Ligand-based approaches has led to the construction of a solid framework defining structure-activity relationships to obtain compounds with a tailored pharmacological profile. Structure-based techniques could integrate previous knowledge and provide compounds with novel chemotypes and pharmacological activity as drug candidates for disease conditions in which melatonin receptor ligands are currently being investigated, including cancer and pain.

The effects of agomelatine on endoplasmic reticulum stress related to mitochondrial dysfunction in hippocampus of aging rat model.

BACKGROUND: Agomelatine, a novel antidepressant, is a melatonin MT receptor agonist and serotonin 5HT2C receptor antagonist. In this study, agomelatine was used to investigate the molecular mechanisms of hippocampal aging associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptosis, all of which led to short-term memory impairment. METHOD: Hippocampal aging was induced in male Wistar rats by d-galactose (D-gal) intraperitoneal injection (100 mg/kg) for 14 weeks. During the last 4 weeks of D-gal treatment, rats were treated with agomelatine (40 mg/kg) or melatonin (10 mg/kg). At the end of the experiment, all rats were assessed for short-term memory by using the Morris water maze test. Subsequently, rats were sacrified and the hippocampus was removed from each rat for determination of reactive oxygen species (ROS), malondialdehyde (MDA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays; and immunohistochemistry related to ER stress, mitochondrial dysfunction, and apoptosis. RESULTS: Agomelatine suppressed the expression of the aging-related proteins P16 and receptor for advanced glycation endproducts (RAGE), the expression of NADPH oxidase (NOX) 2 and 4, and ROS production. This treatment also shifted the morphology of astrocytes and microglia toward homeostasis. Furthermore, agomelatine decreased inositol-requiring enzyme 1 (pIRE1), protein kinase R-like endoplasmic reticulum kinase (pPERK), and chaperone binding immunoglobulin protein (BiP), leading to suppression of ER stress markers C/EBP homologous protein (CHOP) and caspase-12. Agomelatine reduced Ca2+ from the ER and stabilized the mitochondrial membrane stability, which was denoted by the BCL2 Associated X (Bax)/B-cell lymphoma 2 (Bcl2) balance. Agomelatine decreased cleaved caspase-3 production and the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive area, and glutamate excitotoxicity was prevented via suppression of N-methyl-d-aspartate (NMDA) receptor subunit expression. Agomelatine exhibited effects that were similar to melatonin. CONCLUSION: Agomelatine improved neurodegeneration in a rat model of hippocampal aging by attenuating ROS production, ER stress, mitochondrial dysfunction, excitotoxicity, and apoptosis.

Melatonin ameliorates aging-related impaired angiogenesis in gastric endothelial cells via local actions on mitochondria and VEGF-survivin signaling.

Tissue injury healing is impaired in aging, and this impairment is caused in part by reduced angiogenesis. Melatonin, a neuroendocrine hormone that regulates sleep and circadian rhythm, is also produced in the gastrointestinal tract. The expression of melatonin receptors MT1 and MT2 in gastric endothelial cells and their roles in aging-related impairment of gastric angiogenesis have not been examined. We hypothesized that MT1 and MT2 expression is reduced in gastric endothelial cells of aging rats and that melatonin treatment can upregulate their expression and improve angiogenesis. We examined the expression of MT1 and MT2 in gastric endothelial cells (GECs) isolated from young and aging rats. We also examined the effects of melatonin treatment on angiogenesis, GEC mitochondrial function, expression of vascular endothelial growth factor (VEGF), its signaling receptor (VEGFR-2), and the inhibitor of apoptosis protein, survivin. Young and aging GECs expressed MT1 (in the cytoplasm and mitochondria) and MT2 (in nucleus and mitochondria). In aging GECs, MT1 and MT2 levels, in vitro angiogenesis, and mitochondrial membrane potential were significantly reduced (by 1.5-fold, 1.9-fold, 3.1-fold, and 1.63-fold, respectively) compared with young GECs. Melatonin treatment of aging GECs significantly increased MT1 and MT2 expression compared with the controls, induced nuclear translocation of MT1, and significantly ameliorated the aging-related impairment of angiogenesis and mitochondrial function. Aging GECs have significantly reduced MT1 and MT2 expression, angiogenesis, and mitochondrial membrane potential compared with young GECs. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function.NEW & NOTEWORTHY This study showed reduced expression of melatonin receptors MT1 and MT2, angiogenesis, and mitochondrial function in gastric endothelial cells (GECs) isolated from aging rats. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function. These studies provide new insight into the mechanisms of the aging-related impairment of angiogenesis and delayed tissue injury healing and provide a rationale for melatonin treatment to reverse these abnormalities.

Melatonin regulates proliferation and apoptosis of endometrial stromal cells via MT1.

Endometrial dysfunction is an important factor for implantation failure. The function of the endometrium is regulated by multiple factors like sex hormones and circadian rhythms. Endometrial stromal cells (ESCs) are a major cellular component in the endometrium, which is essential for proper physiological activities of the endometrium and the establishment of pregnancy. Melatonin, as a circadian-controlled hormone, plays beneficial roles in the regulation of reproductive processes. MT1, a melatonin receptor, can regulate cell proliferation and apoptosis. Whether melatonin-MT1 signal affects biological function of ESCs remains unknown. Here, we showed that MT1 was expressed in human ESCs (hESCs), which could be regulated by estrogen and progesterone. MT1 knockdown inhibited proliferative activity and promoted apoptosis of hESCs by activating caspase-3 and upregulating the Bax/Bcl2 ratio. Melatonin could reverse the effect of MT1 knockdown on proliferative activity and apoptosis of hESCs. Melatonin could promote proliferative activity of hESCs via the JNK/P38 signal pathway and repress the apoptosis of hESCs via the JNK signal pathway. Moreover, in vivo experiments showed that MT1 expression was decreased in endometrial cells from mice with disrupted circadian rhythm, accompanied by increased apoptosis and suppressed proliferative activity, which could be alleviated by administration of melatonin. These results showed the regulatory effect of melatonin-MT1 signal on biological behaviors of ESCs, which might provide a novel therapeutic strategy for endometrial dysfunction induced by disrupted circadian rhythm.

Structure-based discovery of potent and selective melatonin receptor agonists.

Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.

Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT1 and MT2 receptors.

Melatonin is a neurohormone that translates the circadian rhythm to the peripheral organs through a series of binding sites identified as G protein-coupled receptors MT1 and MT2. Due to minute amounts of receptor proteins in target organs, the main tool of studies of the melatoninergic system is recombinant expression of the receptors in cellular hosts. Although a number of studies exist on these receptors, studies of several signaling pathways using a large number of melatoninergic compounds are rather limited. We chose to fill this gap to better describe a panel of compounds that have been only partially characterized in terms of functionality. First, we characterized HEK cells expressing MT1 or MT2, and several signaling routes with melatonin itself to validate the approach: GTPγS, cAMP production, internalization, ß-arrestin recruitment, and cell morphology changes (CellKey ® ). Second, we chose 21 compounds from our large melatoninergic chemical library and characterized them using this panel of signaling pathways. Notably, antagonists were infrequent, and their functionality depended largely on the pathway studied. This will permit redefining the availability of molecular tools that can be used to better understand the in situ activity and roles of these receptors.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Melatonin suppresses lung cancer metastasis by inhibition of epithelial-mesenchymal transition through targeting to Twist.

The epithelial-mesenchymal transition (EMT) phenotype, whereby mature epithelial cells undergo phenotype transition and differentiate into motile, invasive cells, has been indicated in tumor metastasis. The melatonin hormone secreted by the pineal gland has an antioxidant effect and protects cells against carcinogenic substances that reduce tumor progression. However, the effects of melatonin in EMT and lung cancer metastasis are largely unknown. We found that melatonin down-regulated EMT by inhibiting Twist/Twist1 (twist family bHLH transcription factor 1) expression. This effect was mediated by MT1 receptor, PLC, p38/ERK and ß-catenin signaling cascades. Twist expression was positively correlated with tumor stage and negatively correlated with MT1 expression in lung cancer specimens. Furthermore, melatonin inhibited EMT marker expression and lung cancer metastasis to liver in vivo Finally, melatonin shows promise in the treatment of lung cancer metastasis and deserves further study.

O-linked melatonin dimers as bivalent ligands targeting dimeric melatonin receptors.

A series of dimeric melatonin analogues 3a-e obtained by connecting two melatonin molecules through the methoxy oxygen atoms with spacers spanning 16-24 atoms and the agomelatine dimer 7 were synthesized and characterized in 2-[125-I]-iodomelatonin binding assays, bioluminescence resonance energy transfer (BRET) experiments, and in functional cAMP and ß-arrestin recruitment assays at MT1 and MT2 receptors. The binding affinity of 3a-e generally increased with increasing linker length. Bivalent ligands 3a-e increased BRET signals of MT1 dimers up to 3-fold compared to the monomeric control ligand indicating the simultaneous binding of the two pharmacophores to dimeric receptors. Bivalent ligands 3c and 7 exhibited important changes in functional properties on the Gi/cAMP pathway but not on the ß-arrestin pathway compared to their monomeric counterparts. Interestingly, 3c (20 atoms spacer) shows inverse agonistic properties at MT2 on the Gi/cAMP pathway. In conclusion, these findings indicate that O-linked melatonin dimers are promising tools to develop signaling pathway-based bivalent melatonin receptor ligands.

Melatonin receptor activation provides cerebral protection after traumatic brain injury by mitigating oxidative stress and inflammation via the Nrf2 signaling pathway.

Traumatic brain injury (TBI) is a principal cause of death and disability worldwide. Melatonin, a hormone made by the pineal gland, is known to have anti-inflammatory and antioxidant properties. In this study, using a weight-drop model of TBI, we investigated the protective effects of ramelteon, a melatonin MT1/MT2 receptor agonist, and its underlying mechanisms of action. Administration of ramelteon (10 mg/kg) daily at 10:00 a.m. alleviated TBI-induced early brain damage on day 3 and long-term neurobehavioral deficits on day 28 in C57BL/6 mice. Ramelteon also increased the protein levels of interleukin (IL)-10, IL-4, superoxide dismutase (SOD), glutathione, and glutathione peroxidase and reduced the protein levels of IL-1ß, tumor necrosis factor, and malondialdehyde in brain tissue and serum on days 1, 3, and 7 post-TBI. Similarly, ramelteon attenuated microglial and astrocyte activation in the perilesional cortex on day 3. Furthermore, ramelteon decreased Keap 1 expression, promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation, and increased levels of downstream proteins, including SOD-1, heme oxygenase-1, and NQO1 on day 3 post-TBI. However, in Nrf2 knockout mice with TBI, ramelteon did not decrease the lesion volume, neuronal degeneration, or myelin loss on day 3; nor did it mitigate depression-like behavior or most motor behavior deficits on day 28. Thus, timed ramelteon treatment appears to prevent inflammation and oxidative stress via the Nrf2-antioxidant response element pathway and might represent a potential chronotherapeutic strategy for treating TBI.

Effects of Ramelteon and Other Sleep-Promoting Drugs on Serum Low-Density Lipoprotein and Non-high-density Lipoprotein Cholesterol: A Retrospective Comparative Pilot Study.

Melatonin has been suggested to play important roles in lipid metabolism as well as circadian rhythm; however, very few studies explored the effects of ramelteon, a selective melatonin receptor agonist, on serum lipid profiles. In this study effects of ramelteon on serum lipid profiles were explored, comparing to those of other sleep-promoting drugs including benzodiazepines and non-benzodiazepines, in patients with insomnia. We retrospectively reviewed medical charts of outpatients who were treated with ramelteon (8 mg/d) or other sleep-promoting drugs for no less than 8 weeks during the period between October 1st, 2011 and September 30th, 2014, and compared the changes in serum lipid profiles between the two groups. Patients with regular dialysis or malignant diseases treated with cytotoxic anti-cancer drugs, or whose lipid-lowering drugs were altered during the study period, were excluded. Among 365 or 855 outpatients treated with ramelteon or other sleep-promoting drugs, 35 or 46 patients, respectively, had complete serum low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (non-HDL-C) data. Serum LDL-C was significantly reduced from 103.1±4.4 to 94.6±4.2 mg/dL (8.2% reduction, p<0.05, n=31) in the ramelteon group, and was not significantly changed (p=0.23, n=40) in the other sleep-promoting drug group. Non-HDL-C was significantly decreased from 138.8±6.0 to 130.6±4.9 mg/dL (5.9% reduction, p<0.05, n=32) in the ramelteon group, and was not significantly altered (p=0.29, n=42) in the other sleep-promoting drug group. Ramelteon, but not other sleep-promoting drugs, specifically lowers serum LDL-C and non-HDL-C levels.

Melatonin prevents senescence of canine adipose-derived mesenchymal stem cells through activating NRF2 and inhibiting ER stress.

Transplantation of adipose-derived mesenchymal stem cells (ADMSCs) can aid in the treatment of numerous diseases in animals. However, natural aging during in vitro expansion of ADMSCs prior to their use in transplantation restricts their beneficial effects. Melatonin is reported to exert biorhythm regulation, anti-oxidation, and anti-senescence effects in various animal and cell models. Herein, by using a senescent canine ADMSCs (cADMSCs) cell model subjected to multiple passages in vitro, we investigated the effects of melatonin on ADMSCs senescence. We found that melatonin alleviates endoplasmic reticulum stress (ERS) and cell senescence. MT1/MT2 melatonin receptor inhibitor, luzindole, diminished the mRNA expression levels and rhythm expression amplitude of Bmal1 and Nrf2 genes. Nrf2 knockdown blocked the stimulatory effects of melatonin on endoplasmic reticulum-associated degradation (ERAD)-related gene expression and its inhibitory effects on ERS-related gene expression. At the same time, the inhibitory effects of melatonin on the NF-κB signaling pathway and senescence-associated secretory phenotype (SASP) were blocked by Nrf2 knockdown in cADMSCs. Melatonin pretreatment improved the survival of cADMSCs and enhanced the beneficial effects of cADMSCs transplantation in canine acute liver injury. These results indicate that melatonin activates Nrf2 through the MT1/MT2 receptor pathway, stimulates ERAD, inhibits NF-κB and ERS, alleviates cADMSCs senescence, and improves the efficacy of transplanted cADMSCs.

Ramelteon, a selective MT1/MT2 receptor agonist, suppresses the proliferation and invasiveness of endometrial cancer cells.

The incidence of endometrial cancer is increasing, making it the fifth most common cancer worldwide. To date, however, there is no standard therapy for patients with recurrent endometrial cancer. Melatonin, a hormone secreted by the pineal gland, has been shown to have anti-tumor effects in various tumor types. Although melatonin is available as a supplement, it has not been approved for cancer treatment. Ramelteon, a selective melatonin receptor type 1 and 2 (MT1/MT2) receptor agonist, has been approved to treat sleep disorders, suggesting that ramelteon may be effective in the treatment of endometrial cancer. To determine whether this agent may be effective in the treatment of endometrial cancer, this study investigated the ability of ramelteon to suppress the proliferation and invasiveness of HHUA cells, an estrogen receptor-positive endometrial cancer cell line. Ramelteon at 10-8 M maximally suppressed the proliferation of HHUA cells, reducing the percentage of Ki-67 positive proliferating cells. This effect was completely blocked by luzindole, a MT1/MT2 receptor antagonist. Furthermore, ramelteon inhibited HHUA cell invasion and reduced the expression of the MMP-2 and MMP-9 genes. These results suggested that ramelteon may be a candidate for the treatment of recurrent endometrial cancer, with activity similar to that of melatonin.

Melatonin receptors as therapeutic targets in the suprachiasmatic nucleus.

INTRODUCTION: Disorders of rhythmicity can cause a variety of pathologies and are known to impair processes involved in metabolism, as well as in cardiovascular disease and cancer. Developing strategies to treat or prevent such diseases is a new challenge for medicine. Rhythms depend on a complex multi-oscillatory circadian network which, in mammals, is hierarchically organized with the suprachiasmatic nuclei (SCN) as master clock. The SCN, thus form an ideal structure for target discovery in circadian pathologies. AREAS COVERED: The development of strategies to treat or prevent disorders of rhythmicity is a new challenge for medicine. Several pharmacological approaches have been suggested, but until now, it has been mostly melatonin (MTL) or MTL-agonists which have demonstrated usefulness in modulating clock activities in vivo. A great number of structurally different MTL receptor ligands have been developed, some of which are already approved and marketed as drugs. The MTL receptor involved in phase-shifting circadian rhythms (chronobiotic effect) is the MT1 subtype. EXPERT OPINION: As the two receptor subtypes for MTL may have divergent functions, the development of highly selective MT1 and MT2 agonists (and antagonists) is key for the discovery of novel therapeutic agents in specifically defined circadian pathologies. The identification of cells expressing the different MTL receptor subtypes should also permit a better understanding of MLT physiology/pharmacology.

Influences of melatonin treatment, melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) gene polymorphisms on first conception in Sarda ewe lambs.

In order to investigate if the melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) genes influence the reproductive response to melatonin treatment, 510 Sarda ewe lambs were divided into groups C (control) and M; Group M received one melatonin implant (18mg). After 35 days rams were introduced for 40 days and subsequent lambing dates and number of newborns were recorded. The MTNR1A gene Exon II and KiSS-1 gene Exon I were amplified and genotyped by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism analysis. Two single nucleotide polymorphisms (SNPs; C606T and G612A) in MTNR1A and one (G1035A) in KiSS-1 were found. The most frequent genotypes were G/G (63%) and C/C (53%) for MTNR1A and G/G (92%) for KiSS-1. Treated animals showed a higher lambing rate (P<0.05) and an advanced lambing date (P<0.05) compared with controls. The three SNPs did not influence the onset of reproductive activity. The majority of the G/G animals of Group M lambed before 190 days after ram introduction (P<0.05), while in Group C a higher number of G/G animals lambed after this date. Data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that the G/G genotype of the MTNR1A gene is able to influence the reproductive response to melatonin treatment.

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.

Melatonin receptor activation suppresses adrenocorticotropin production via BMP-4 action by pituitary AtT20 cells.

The role of melatonin, a regulator of circadian rhythm, in adrenocorticotropin (ACTH) production by corticotrope cells has not been elucidated. In this study, we investigated the effect of melatonin on ACTH production in relation to the biological activity of bone morphogenetic protein (BMP)-4 using mouse corticotrope AtT20 cells that express melatonin type-1 (MT1R) but not type-2 (MT2R) receptors. We previously reported that BMP-4 inhibits corticotropin-releasing hormone (CRH)-induced ACTH production and proopiomelanocortin (POMC) transcription by inhibiting MAPK signaling. Both melatonin and an MT1R/MT2R agonist, ramelteon, suppressed CRH-induced ACTH production, POMC transcription and cAMP synthesis. The inhibitory effects of ramelteon on basal and CRH-induced POMC mRNA and ACTH levels were more potent than those of melatonin. Treatment with melatonin or ramelteon in combination with BMP-4 additively suppressed CRH-induced ACTH production. Of note, the level of MT1R expression was upregulated by BMP-4 stimulation. The suppressive effects of melatonin and ramelteon on POMC transcription and cAMP synthesis induced by CRH were not affected by an MT2R antagonist, luzindole. On the other hand, BMP-4-induced Smad1/5/8 phosphorylation and the expression of a BMP target gene, Id-1, were augmented in the presence of melatonin and ramelteon. Considering that the expression levels of BMP receptors, ALK-3/BMPRII, were increased by ramelteon, MT1R action may play an enhancing role in BMP-receptor signaling. Among the MT1R signaling pathways including AKT, ERK and JNK pathways, inhibition of AKT signaling functionally reversed the MT1R effects on both CRH-induced POMC transcription and BMP-4-induced Id-1 transcription. Collectively, MT1R signaling and BMP-4 actions were mutually augmented, leading to fine-tuning of ACTH production by corticotrope cells.

Melatonin receptor agonist-induced reduction of SNP-released nitric oxide and cGMP production in isolated human non-pigmented ciliary epithelial cells.

The present study was designed to determine the effects of melatonin and its receptor agonists on SNP-released nitric oxide (NO) and cGMP production in aqueous humor producing cells of the ciliary body because these effects may play a role in melatonin receptor-mediated regulation of intraocular pressure (IOP). NO release protocols were carried out using human non-pigmented ciliary epithelial (hNPCE) cells treated in dye free DMEM containing l-arginine (10(-3) M). The cGMP experimental protocols were performed using dye free DMEM containing 3-isobutyl-1-methylxanthine (IBMX, 10(-4) M). The effects of varying concentrations (10(-13), 10(-11), 10(-9), 10(-7), and 10(-5) M) of melatonin, 5-MCA-NAT (putative MT(3) agonist), N-butanoyl-2-(2-methoxy-6H-isoindolo[2, 1-a]indol-11-yl)ethanamine (IIK7; selective MT(2) agonist) or S-27633-1 (selective MT(1) agonist) on sodium nitroprusside (SNP)-released NO or cGMP production were determined in separate experiments. NO and cGMP levels were measured using a colorimetric assay or enzyme immunoassay (EIA), respectively. Melatonin receptor selectivity was evaluated using luzindole (LUZ; nonselective MT(1)/MT(2) antagonist) or 4-phenyl-2-propionamidotetralin (4P-PDOT; selective MT(2) antagonist). Melatonin, 5-MCA-NAT, and IIK7 all caused concentration-dependent reduction of SNP-released NO and cGMP production. The inhibitory actions of melatonin, 5-MCA-NAT and IIK7 were either completely blocked at 10(-13), 10(-11), and 10(-9) M concentrations of the agonists or partially at 10(-7) and 10(-5) M in the presence of luzindole or 4P-PDOT. Results from this study suggest that melatonin and its analogs, 5-MCA-NAT and IIK7 inhibit SNP-released NO and cGMP production via activation of MT(2) receptors in human NPCE cells. These actions may play a role in melatonin agonist-induced regulation of aqueous humor secretion and IOP.

Gender-related invasion differences associated with mRNA expression levels of melatonin membrane receptors in colorectal cancer.

Melatonin inhibits growth and invasive capacity of colon cancer cells in vitro through its membrane (MT1 and MT2) and/or nuclear receptors (RORα). Previous studies showed that this indoleamine is present in both the normal and colon cancer at similar levels. Therefore, we analyzed MT1, MT2, and RORα expression in tumor samples versus normal mucosa (NM) from patients suffering from colorectal cancer (CRC). Given the existence of sex differences in the incidence and pathology of CRC and the involvement of steroid receptors in the oncostatic actions of melatonin in some types of cancer, we also analyzed the expression of androgen (AR) and estrogen receptor (ER) α and ERß. Finally, we conducted some experiments in colon cancer cell lines to corroborate the experiments carried out in human tumors. We found a decreased expression of MT1, MT2, AR, ERα, and ERß in tumor samples versus NM, but no changes in RORα expression in the whole cohort of patients. Classifying tumors by stage and gender, MT1, MT2, AR, ERα, and ERß expression decreased in both early stage and advanced tumors, but only in male patients. On the other hand, MT1 and MT2 expression correlated positively with AR, ERα, and ERß expression in male patients and with ERα or ERß in female patients. In vitro, the invasive capacity was higher in cells with the least expression of MT1, MT2, and AR, and nonselective MT1/MT2 agonists inhibited cell growth and invasion. These results could indicate a possible interaction of these pathways.

Description of the constitutive activity of cloned human melatonin receptors hMT(1) and hMT(2) and discovery of inverse agonists.

Melatonin receptors have been described to activate different G protein-dependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G protein-coupled receptors has been shown to be key in physiological and pathological conditions. In the case of melatonin receptors, information is rather scare and concerns only MT1 receptors. In the present report, we show that the G protein-coupled melatonin receptors do have a constitutive, nonmelatonin-induced signaling activity using two cellular models of different origins, the Chinese hamster ovary cell line and Neuro2A, a neuroblastoma cell line. Furthermore, we show that this constitutive activity involves mainly Gi proteins, which is consistent with the common knowledge on the melatonin receptors. Importantly, we also describe, for the first time, inverse agonist properties for melatonin ligands. Although it is clear than more in-depth, biochemistry-based studies will be required to better understand by which pathway(s) the constitutively active melatonin receptors transfer melatonin information into intracellular biochemical events; our data open interesting perspectives for understanding the importance of the constitutive activity of melatonin receptors in physiological conditions.