RESUMEN
Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.
Asunto(s)
Estradiol/fisiología , Células de la Granulosa/metabolismo , Melatonina/biosíntesis , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Animales , N-Acetiltransferasa de Arilalquilamina/genética , N-Acetiltransferasa de Arilalquilamina/metabolismo , Células Cultivadas , Femenino , Células de la Granulosa/enzimología , OvinosRESUMEN
Melatonin (MLT) exerts its physiological effects principally through two high-affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a "knock-in" strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co-expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Melatonina/metabolismo , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Encéfalo/citología , Técnicas de Sustitución del Gen , Ratones , Ratones Noqueados , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genéticaRESUMEN
Clinical and experimental findings show that melatonin may be used as an adjuvant to the treatment of epilepsy-related complications by alleviates sleep disturbances, circadian alterations and attenuates seizures alone or in combination with AEDs. In addition, it has been observed that there is a circadian component on seizures, which cause changes in circadian system and in melatonin production. Nevertheless, the dynamic changes of the melatoninergic system, especially with regard to its membrane receptors (MT1 and MT2) in the natural course of TLE remain largely unknown. The aim of this study was to evaluate the 24-hour profile of MT1 and MT2 mRNA and protein expression in the hippocampus of rats submitted to the pilocarpine-induced epilepsy model analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases. Melatonin receptor MT1 and MT2 mRNA expression levels were increased in the hippocampus of rats few hours after SE, with MT1 returning to normal levels and MT2 reducing during the silent phase. During the chronic phase, mRNA expression levels of both receptors return to levels close to control, however, presenting a different daily profile, showing that there is a circadian change during the chronic phase. Also, during the acute and silent phase it was possible to verify MT1 label only in CA2 hippocampal region with an increased expression only in the dark period of the acute phase. The MT2 receptor was present in all hippocampal regions, however, it was reduced in the acute phase and it was found in astrocytes. In chronic animals, there is a reduction in the presence of both receptors especially in regions where there is a typical damage derived from epilepsy. Therefore, we conclude that SE induced by pilocarpine is able to change melatonin receptor MT1 and MT2 protein and mRNA expression levels in the hippocampus of rats few hours after SE as well as in silent and chronic phases.
Asunto(s)
Epilepsia/inducido químicamente , Epilepsia/metabolismo , Hipocampo/metabolismo , Pilocarpina/toxicidad , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Epilepsia/genética , Expresión Génica , Hipocampo/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genéticaRESUMEN
Melatonin is involved in the temporal organization of several physiological and behavioral events, controlled by hypothalamic nuclei, like sleep, feeding, reproduction and metabolic modulation and acts through two types of high-affinity G protein-coupled membrane receptors: MT1 and MT2. This study aimed to investigate the expression of MT1 and MT2 receptors proteins in four hypothalamic nuclei, i.e., SCN, supraoptic (SON), paraventricular (PVN) and anteroventral periventricular nuclei (AVPV), of the diurnal primate Sapajus apella using immunohistochemistry. Since these areas are involved in the expression of biological rhythms, they are candidates to have variations in their neurochemistry, so the MT1 and MT2 expression has been analyzed at a point in light and another in the dark phase. Both receptors were found to have day/night differences in the four hypothalamic nuclei with an apparent inverse expression in the SCN compared with the other areas. These differences could be related to the idea that the individual should be prepared to respond by different ways to melatonin signal within the several processes and can contribute to the efficacy of melatonin ligands or melatonin in therapies.
Asunto(s)
Ritmo Circadiano/fisiología , Núcleo Hipotalámico Paraventricular/metabolismo , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Cebus , Expresión Génica , Masculino , Primates , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genéticaRESUMEN
Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or "brake" of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.
Asunto(s)
Melatonina/metabolismo , Morfogénesis , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Glándulas Salivales/metabolismo , Animales , Apoptosis/genética , Adhesión Celular/genética , Forma de la Célula/genética , Desarrollo Embrionario , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica , Melatonina/genética , Ratones , Embarazo , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genética , Glándulas Salivales/embriología , Glándulas Salivales/ultraestructuraRESUMEN
Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.
Asunto(s)
Condrocitos/efectos de los fármacos , Placa de Crecimiento/patología , Melatonina/farmacología , Escoliosis/patología , Adolescente , Estudios de Casos y Controles , División Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Femenino , Proteínas de Unión al GTP/metabolismo , Humanos , Masculino , Procedimientos Ortopédicos , Cultivo Primario de Células , ARN Mensajero/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Receptor de Melatonina MT2/deficiencia , Receptor de Melatonina MT2/efectos de los fármacos , Receptor de Melatonina MT2/genética , Escoliosis/metabolismo , Escoliosis/cirugía , Transducción de Señal , Fusión VertebralRESUMEN
In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M) were incubated with fertilized embryos. Melatonin in the range of 10(-11) to 10(-5) M significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10(-9) and 10(-7) M. Using quantitative real-time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin-induced embryo development, while 4-P-PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up-regulated the expression of antioxidative (Gpx4, SOD1, bcl-2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down-regulating expression of pro-apoptotic genes (P53, BAX, and Caspase-3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Melatonina/farmacología , Receptor de Melatonina MT1/fisiología , Animales , Blastocisto/efectos de los fármacos , Bovinos , Glutatión Peroxidasa/biosíntesis , Receptor de Melatonina MT1/antagonistas & inhibidores , Receptor de Melatonina MT2/antagonistas & inhibidores , Receptor de Melatonina MT2/biosíntesis , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa-1 , Tetrahidronaftalenos/farmacología , Triptaminas/farmacologíaRESUMEN
In bile duct-ligated (BDL) rats, large cholangiocytes proliferate by activation of cAMP-dependent signaling. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell mitosis by interacting with melatonin receptors (MT1 and MT2) modulating cAMP and clock genes. In the liver, melatonin suppresses oxidative damage and ameliorates fibrosis. No information exists regarding the role of melatonin in the regulation of biliary hyperplasia. We evaluated the mechanisms of action by which melatonin regulates the growth of cholangiocytes. In normal and BDL rats, we determined the hepatic distribution of MT1, MT2, and the clock genes, CLOCK, BMAL1, CRY1, and PER1. Normal and BDL (immediately after BDL) rats were treated in vivo with melatonin before evaluating 1) serum levels of melatonin, bilirubin, and transaminases; 2) intrahepatic bile duct mass (IBDM) in liver sections; and 3) the expression of MT1 and MT2, clock genes, and PKA phosphorylation. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (MT1/MT2 antagonist) and 4-phenyl-2-propionamidotetralin (MT2 antagonist) before evaluating cell proliferation, cAMP levels, and PKA phosphorylation. Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM, serum bilirubin and transaminases levels, the expression of all clock genes, cAMP levels, and PKA phosphorylation in cholangiocytes. In vitro, melatonin decreased the proliferation, cAMP levels, and PKA phosphorylation, decreases that were blocked by luzindole. Melatonin may be important in the management of biliary hyperplasia in human cholangiopathies.
Asunto(s)
Conductos Biliares/patología , Melatonina/farmacología , Receptor de Melatonina MT1/efectos de los fármacos , Factores de Transcripción ARNTL/biosíntesis , Animales , Bicarbonatos/metabolismo , Conductos Biliares/citología , Proteínas CLOCK/biosíntesis , Proliferación Celular/efectos de los fármacos , Colestasis/patología , Criptocromos/biosíntesis , AMP Cíclico/metabolismo , Hiperplasia/tratamiento farmacológico , Hiperplasia/patología , Ligadura , Masculino , Melatonina/uso terapéutico , Ratones , Proteínas Circadianas Period/biosíntesis , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Ratas , Ratas Endogámicas F344 , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/biosíntesis , Receptor de Melatonina MT2/metabolismo , Secretina/farmacologíaRESUMEN
Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin â melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.
Asunto(s)
Acetilserotonina O-Metiltransferasa/biosíntesis , N-Acetiltransferasa de Arilalquilamina/biosíntesis , Colangiocarcinoma/fisiopatología , Neoplasias Hepáticas/fisiopatología , Melatonina/fisiología , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Apoptosis , Comunicación Autocrina , Neoplasias de los Conductos Biliares , Conductos Biliares Intrahepáticos/fisiología , Línea Celular Tumoral , Proliferación Celular , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/patología , Regulación hacia Abajo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Melatonina/uso terapéutico , Ratones , Ratones DesnudosRESUMEN
Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood-especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin-receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus-oocyte complexes (COCs) from abattoir ovaries were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT-PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin-treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis.
Asunto(s)
Acetilserotonina O-Metiltransferasa/metabolismo , Células del Cúmulo/enzimología , Melatonina , Oocitos/enzimología , Oogénesis/fisiología , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Bovinos , Núcleo Celular/metabolismo , Células del Cúmulo/citología , Citoplasma/metabolismo , Femenino , Melatonina/biosíntesis , Mitocondrias/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Melatonin is a potent free radical scavenger and antioxidant and has protective effects against ischemic damage. In the present study, we examined the relationship between the neuroprotective effects of melatonin and the activation of MT2 melatonin receptor in the hippocampal CA1 region (CA1) after transient cerebral ischemia. MT2 immunoreactivity and protein levels were increased in the CA1 after ischemic damage. Most of MT2-immunoreactive cells were colocalized with astrocytes, not microglia, in the ischemic CA1. In the melatonin-sham group, MT2 immunoreaction and protein levels were increased compared with the sham group, and MT2 immunoreactivity and its protein levels in the melatonin-ischemia group were similar to those in the melatonin-sham group. In addition, melatonin treatment attenuated the activation of astrocytes and microglia. These results indicate that MT2 are increased and expressed in astrocytes in the ischemic region after an ischemic insult. The activation of MT2 melatonin receptor in the CA1 after melatonin treatment may be involved in the neuroprotective effect associated with melatonin after ischemic injury.
Asunto(s)
Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/prevención & control , Melatonina/fisiología , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Receptor de Melatonina MT2/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Modelos Animales de Enfermedad , Gerbillinae , Ataque Isquémico Transitorio/patología , Masculino , Melatonina/metabolismo , Melatonina/farmacología , Fármacos Neuroprotectores/metabolismo , Resultado del TratamientoRESUMEN
An inverse relation exists between melatonin and androgen in most of the seasonally breeding rodents, but the regulation of their receptors in modulation of immune function has never been reported. The present study accessed the expression pattern of melatonin receptor types (mt1R & mt2R), immune parameters (lymphoid organs weight, leucocyte count, delayed type hypersensitivity and lymphocyte proliferation) in spleen and thymus whereas androgen receptor (AR) expression in thymus of Funambulus pennanti during reproductively active phase. In-vivo melatonin treatment (Mel) and castration (Cx) significantly increased mt1R expression, immune parameters in spleen and thymus but decreased AR expression in thymus only when compared with sham control (Con) squirrels as AR expression was not observed in spleen. Mel alone or in combination with testosterone (T) to Cx squirrels significantly increased mt1R expression, immune parameters in spleen and thymus but decreased AR expression in thymus. T alone in Cx squirrels significantly decreased mt1R expression, immune parameters in spleen and thymus but increased thymic AR expression significantly. In-vitro thymocyte culture supported our in-vivo findings. Mel significantly increased mt1R expression, lymphocyte proliferation, IL-2 secretion but decreased AR expression. T alone significantly decreased aforementioned three parameters but increased AR expression. Combined treatment of Mel and T bring back all parameters to control level. Though we found high mt2R expression, but no significant change has been observed. Thus, present study suggests a clear-cut trade-off relation between mt1R and AR expression that might be acting as an important mediator in seasonal adjustment of immune function in tropical rodents.
Asunto(s)
Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Receptores Androgénicos/biosíntesis , Reproducción/inmunología , Sciuridae/inmunología , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Hipersensibilidad Tardía/inmunología , Inmunidad Celular/inmunología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Recuento de Leucocitos , Tejido Linfoide/inmunología , Masculino , Melatonina/farmacología , Receptor de Melatonina MT1/inmunología , Receptor de Melatonina MT2/inmunología , Receptores Androgénicos/inmunología , Sciuridae/metabolismo , Estaciones del Año , Testosterona/farmacología , Clima TropicalRESUMEN
Through inhibitory G protein-coupled melatonin receptors, melatonin regulates intracellular signaling systems and also the transcriptional activity of certain genes. Clock genes are proposed as regulatory factors in forming dopamine-related behaviors and mood and melatonin has the ability to regulate these processes. Melatonin-mediated changes in clock gene expression have been reported in brain regions, including the striatum, that are crucial for the development of dopaminergic behaviors and mood. However, it is not known whether melatonin receptors present in striatum mediate these effects. Therefore, we investigated the role of the melatonin/melatonin receptor system on clock gene expression using a model of primary neuronal cultures prepared from striatum. We found that melatonin at the receptor affinity range (i.e., nm) affects the expression of the clock genes mPer1, mClock, mBmal1 and mNPAS2 (neuronal PAS domain protein 2) differentially in a pertussis toxin-sensitive manner: a decrease in Per1 and Clock, an increase in NPAS2 and no change in Bmal1 expression. Furthermore, mutating MT1 melatonin receptor (i.e., MT1 knockouts, MT1(-/-)) reversed melatonin-induced changes, indicating the involvement of MT1 receptor in the regulatory action of melatonin on neuronal clock gene expression. Therefore, by controlling clock gene expression we propose melatonin receptors (i.e., MT1) as novel therapeutic targets for the pathobiologies of dopamine-related behaviors and mood.
Asunto(s)
Proteínas CLOCK/genética , Cuerpo Estriado/fisiología , Melatonina/fisiología , Neuronas/fisiología , Receptor de Melatonina MT1/genética , Análisis de Varianza , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas CLOCK/biosíntesis , Proteínas CLOCK/metabolismo , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Femenino , Masculino , Melatonina/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Circadianas Period/biosíntesis , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Toxina del Pertussis/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/biosíntesis , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Aging is a complex process associated with a diminished ability to respond to stress, a progressive increase in free radical generation and a decline in immune function. Melatonin, a molecule with a great functional versatility exerts anti-oxidant, oncostatic, immunomodulatory, and anti-aging properties. Melatonin levels drop during aging and it has been speculated that the loss of melatonin may accelerate aging. This study was designed to elucidate whether aging involves responsiveness to reduced melatonin. Melatonin membrane receptor (MT1 and MT2) expression and MT1 protein expression were analyzed in extrapineal tissues (thymus, spleen, liver, kidney, and heart) of 3- and 12-month-old rats using real time polymerase chain reaction and western blotting analysis. Moreover, melatonin in tissues was measured by high performance liquid chromatography. We report for the first time, an age-related reduction in mRNA MT1 and MT2 expression levels as well as MT1 protein expression in all tissues tested except the thymus, where surprisingly, both melatonin receptor levels were significantly higher in 12-month-old rats and MT1 protein expression maintained unchanged with age. Diminished melatonin concentrations were measured in spleen, liver, and heart during aging. As a conclusion, physiological aging seems to exert responsiveness to melatonin and consequently, the loss of this potent anti-oxidant may contribute to onset of aging.
Asunto(s)
Envejecimiento/metabolismo , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Envejecimiento/genética , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Expresión Génica , Hígado/metabolismo , Masculino , Melatonina/metabolismo , Miocardio/metabolismo , Glándula Pineal/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genética , Bazo/metabolismo , Estadísticas no Paramétricas , Timo/metabolismoRESUMEN
AIM: To determine the receptor subtypes of melatonin in estrogen receptor-positive endometrial cancer cell line, Ishikawa, and the influence of melatonin on chemosensitivity. METHODS: To confirm the subtype of melatonin on Ishikawa cells, cells were treated with melatonin alone and with antagonists against melatonin receptor luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT). Expression of MT1/MT2 mRNA was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Immunocytochemistry of MT1/MT2 was also performed. The effect of melatonin against expression of MT1, MT2, and ERalpha-receptors mRNA was compared with RT-PCR. To determine whether melatonin enhances the effect of anticancer agents, chemosensitivity test was performed with or without melatonin. RESULTS: Our study revealed that Ishikawa cells express MT1 by both RT-PCR and immunocytochemistry. In contrast, expression of MT2 mRNA was not found. Furthermore, ERalpha mRNA expression was attenuated at melatonin level of 1 x 10(-9) M. Chemosensitivity test revealed that melatonin enhanced anti-tumor effects of paclitaxel among anticancer drugs tested. CONCLUSION: Based on the above results, MT1 receptor, but not MT2, is expressed in Ishikawa cells. It was also revealed that the cytostatic effect of melatonin is partly an action mediated by MT1 receptor, and attenuation of ERalpha expression was predicted as the mechanism of action. Clinical application of melatonin to biochemotherapy might be also expected.
Asunto(s)
Neoplasias Endometriales/metabolismo , Estrógenos/metabolismo , Melatonina/metabolismo , Receptor de Melatonina MT1/biosíntesis , Línea Celular Tumoral , Neoplasias Endometriales/genética , Femenino , Humanos , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/biosíntesis , Receptor de Melatonina MT2/genéticaRESUMEN
We investigated the effect of 2 wk continuous postinfarction subcutaneous melatonin supply on the expression of the rat left ventricular (LV) dihydropyridine receptor (DHPR), ryanodine receptor (RyR(2)), and sarco-endoplasmic reticulum Ca(2+)-ATPase2 (SERCA2), as they are fundamental proteins in cardiac contractility. The levels of plasma and LV atrial (ANP) and brain natriuretic peptide and melatonin were also measured, as was the expression of LV MT(1) and MT(2) receptors and pineal arylalkylamine N-acetyltransferase. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery and vehicle or melatonin (4.5 mg/kg per day) was administered by subcutaneous osmotic pumps. Echocardiography, real-time quantitative reverse transcription-polymerase chain reaction, and western blotting were used to analyze the samples. Echocardiography revealed that MI induced serious systolic LV dysfunction. The expression of DHPR, RyR(2), and SERCA2 mRNAs was significantly lower in the LVs of melatonin-treated MI rats compared with vehicle-treated rats (P < 0.01 for DHPR and P < 0.05 for RyR(2) and SERCA2). Melatonin also elevated the amount of LV MT(2) receptors to 1.9-fold (P < 0.05) and the concentration of LV ANP to over fivefold (P < 0.05) compared with vehicle rats after MI. Therefore, the results suggest that melatonin may influence the cardiac contractility after MI by regulating the expression of DHPR, RyR(2), and SERCA2, and melatonin receptors, particularly MT(2)s, might contribute to the postinfarction cardioprotective actions of melatonin. Furthermore, the finding of the relationship between melatonin and ANP suggests a novel mechanism for melatonin in protecting the heart after MI.
Asunto(s)
Factor Natriurético Atrial/biosíntesis , Canales de Calcio Tipo L/biosíntesis , Ventrículos Cardíacos/metabolismo , Melatonina/fisiología , Infarto del Miocardio/metabolismo , Receptor de Melatonina MT2/biosíntesis , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Animales , Factor Natriurético Atrial/metabolismo , Canales de Calcio Tipo L/genética , Masculino , Melatonina/administración & dosificación , Péptido Natriurético Encefálico/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Melatonina MT2/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.
Asunto(s)
Islotes Pancreáticos/fisiología , Receptores de Melatonina/fisiología , Animales , Línea Celular Tumoral , Colforsina/farmacología , AMP Cíclico/metabolismo , Glucagón/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Ratones , ARN Mensajero/metabolismo , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Receptores de Melatonina/biosíntesis , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
We examined the time course of changes in the synthesis and levels of endogenous melatonin and in the expression of MT(1) and MT(2) melatonin receptors 1 day, 2 and 4 wk after myocardial infarction (MI) in rats. MI was produced by ligation of the left anterior descending coronary artery. Transthoracic echocardiography was performed to characterize structural and functional changes after MI. mRNA levels were measured by real-time quantitative reverse transcription-polymerase chain reaction and proteins by Western blotting. One day after infarction, MI rats had 4.3 times (P < 0.001) higher pineal melatonin synthesis, than sham-operated animals, which was associated with the increased concentration of melatonin in plasma (P < 0.001) and left ventricle (LV) (P = 0.01). The amount of MT(1) receptor protein decreased significantly in MI LVs compared with control LVs 1 day after infarction (P < 0.01), followed by recovery during the next 2 wk. Furthermore, the expression of MT(1) receptor mRNA of the MI LVs was elevated 2 wk after infarction (P < 0.01) compared with control LVs. The amount of MT(2) receptor proteins in MI LVs was higher than in sham-operated LVs 1 day (P < 0.05) and 4 wk (P < 0.01) after MI. In conclusion, melatonin synthesis in the pineal gland increased rapidly in response to the MI, supporting an important role for endogenous melatonin in protecting the heart after MI. The observed changes in the expression of MT(1) and MT(2) receptors suggest that melatonin receptors may be involved in mediating, at least, in part, the protective effects of melatonin in the heart after infarction.
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N-Acetiltransferasa de Arilalquilamina/metabolismo , Melatonina/biosíntesis , Infarto del Miocardio/metabolismo , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Animales , Western Blotting , Vasos Coronarios/cirugía , Ventrículos Cardíacos/metabolismo , Ligadura , Masculino , Melatonina/sangre , Melatonina/metabolismo , Glándula Pineal/metabolismo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Melatonin crosses the placenta and enters the fetal circulation. Moreover, experimental data suggest a possible influence of melatonin on placental function and fetal development in humans. To date, the expression and role of melatonin receptors in human placenta choriocarcinoma cell lines and in human term placental tissues remain to be elucidated. METHODS AND RESULTS: Results from RT-PCR, western blotting and confocal microscopy demonstrated that the MT1, MT2 and RORalpha1 melatonin receptors are expressed in the human term placental tissues and in choriocarcinoma cell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbent assay showed that 6-chloromelatonin (a melatonin agonist) inhibits, in a dose-dependent manner, forskolin-stimulated hCG-beta secretion in JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but had no effect on basal human chorionic gonadotrophin (hCG-beta) levels. This effect of 6-chloromelatonin on forskolin-stimulated HCG-beta secretion was abolished by pertussis toxin (PTX), suggesting that melatonin regulates hCG-beta production by an action involving an inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatonin stimulated basal hCG-beta secretion (P < 0.001). CONCLUSION: These results demonstrate, for the first time, the expression of melatonin receptors in human term placental tissues and in choriocarcinoma cells and suggest a possible paracrine/autocrine function for melatonin in human placenta.
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Coriocarcinoma/metabolismo , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Placenta/metabolismo , Receptores de Melatonina/biosíntesis , Receptores de Ácido Retinoico/biosíntesis , Línea Celular Tumoral , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Humanos , Melatonina/análogos & derivados , Melatonina/antagonistas & inhibidores , Melatonina/farmacología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Toxina del Pertussis/farmacología , Receptor de Melatonina MT1/biosíntesis , Receptor de Melatonina MT2/biosíntesis , Receptores Citoplasmáticos y Nucleares , TransactivadoresRESUMEN
We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.