Activation Of Melatonin Receptors Research Articles

Ramelteon protects against social defeat stress-associated abnormal behaviors

Psychological stress affects the neuroendocrine regulation, which modulates mental status and behaviors. Melatonin, a hormone synthesized primarily by the pineal gland, regulates many brain functions, including circadian rhythms, pain, sleep, and mood. Selective pharmacological melatonin agonist ramelteon has been clinically used to treat mood and sleep disorders. Posttraumatic stress disorder (PTSD) is a psychiatric condition associated with severe trauma; it is generally triggered by traumatic events, which lead to severe anxiety and uncontrollable trauma recall. We recently reported that repeated social defeat stress (RSDS) may induce robust anxiety-like behaviors and social avoidance in mice. In the present study, we investigated whether melatonin receptor activation by melatonin and ramelteon regulates RSDS-induced behavioral changes. Melatonin treatment improved social avoidance and anxiety-like behaviors in RSDS mice. Moreover, treatment of the non-selective MT1/MT2 receptor agonist, ramelteon, markedly ameliorated RSDS-induced social avoidance and anxiety-like behaviors. Moreover, activating melatonin receptors also balanced the expression of monoamine oxidases, glucocorticoid receptors, and endogenous antioxidants in the hippocampus. Taken together, our findings indicate that the activation of both melatonin and ramelteon regulates RSDS-induced anxiety-like behaviors and PTSD symptoms. The current study also showed that the regulatory effects of neuroendocrine mechanisms and cognitive behaviors on melatonin receptor activation in repeated social defeat stress.

Intermittent Restraint Stress and Recovery on GPCR Expression in Brain/Gut Axis of Mice: Activation of Adrenergic, Dopamine, Serotonin and Melatonin Receptors During Stress and Ease Response

Intermittent Restraint Stress and Recovery on GPCR Expression in Brain/Gut Axis of Mice: Activation of Adrenergic, Dopamine, Serotonin and Melatonin Receptors During Stress and Ease Response

Ramelteon attenuates hippocampal neuronal loss and memory impairment following kainate-induced seizures.

Evidence suggests that the neuroprotective effects of melatonin involve both receptor-dependent and -independent actions. However, little is known about the effects of melatonin receptor activation on the kainate (KA)neurotoxicity. This study examined the effects of repeated post-KAtreatment with ramelteon, a selective agonist of melatonin receptors, on neuronal loss, cognitive impairment, and depression-like behaviors following KA-induced seizures. The expression of melatonin receptors decreased in neurons, whereas it was induced in astrocytes 3 and 7 days after seizures elicited by KA (0.12 μg/μL) in the hippocampus of mice. Ramelteon (3 or 10 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) mitigated KA-induced oxidative stress and impairment of glutathione homeostasis and promoted the nuclear translocation and DNA binding activity of Nrf2 in the hippocampus after KA treatment. Ramelteon and melatonin also attenuated microglial activation but did not significantly affect astroglial activation induced by KA, despite the astroglial induction of melatonin receptors after KA treatment. However, ramelteon attenuated KA-induced proinflammatory phenotypic changes in astrocytes. Considering the reciprocal regulation of astroglial and microglial activation, these results suggest ramelteon inhibits microglial activation by regulating astrocyte phenotypic changes. These effects were accompanied by the attenuation of the nuclear translocation and DNA binding activity of nuclear factor κB (NFκB) induced by KA. Consequently, ramelteon attenuated the KA-induced hippocampal neuronal loss, memory impairment, and depression-like behaviors; the effects were comparable to those of melatonin. These results suggest that ramelteon-mediated activation of melatonin receptors provides neuroprotection against KA-induced neurotoxicity in the mouse hippocampus by activating Nrf2 signaling to attenuate oxidative stress and restore glutathione homeostasis and by inhibiting NFκB signaling to attenuate neuroinflammatory changes.

Agomelatine decreases cocaine-induced locomotor sensitisation and dopamine release in rats

Background Agomelatine is a melatoninergic antidepressant approved to treat the major depressive disorder. Agomelatine exerts its behavioural, pharmacological, and physiological effects through the activation of MT1 and MT2 melatonin receptors and the blockade of 5-HT 2B and 5-HT 2C serotonin receptors. Some studies have reported that the activation of the MT1 and MT2 melatonin receptors decreased cocaine-induced locomotor activity and cocaine self-administration. These findings from another study showed that agomelatine decreased alcohol consumption. This study aimed to evaluate the effects of agomelatine administration on cocaine-induced behavioural (cocaine-induced locomotor activity and cocaine-induced locomotor sensitisation) and neurochemical (dopamine levels) effects. Methods Male Wistar rats (250–280 g) received cocaine (10 mg/kg) during the induction and expression of locomotor sensitisation. Agomelatine (10 mg/kg) was administered 30 minutes before cocaine. After each treatment, locomotor activity was recorded for 30 minutes. Dopamine levels were determined in the ventral striatum, the prefrontal cortex (PFC), and the ventral tegmental area (VTA) by high-performance liquid chromatographic (HPLC) in animals treated with agomelatine and cocaine. Luzindole (30 mg/kg) was administered to block the agomelatine effect. Results In this study, we found that agomelatine decreased cocaine-induced locomotor activity and the induction and expression of locomotor sensitisation. In addition, agomelatine decreased cocaine-induced dopamine levels. Luzindole blocked the agomelatine-induced decrease in the expression of locomotor sensitisation in rats. Conclusion Our results suggest (1) that agomelatine showed efficacy in decreasing cocaine psychostimulant effects and (2) that agomelatine can be a useful therapeutic agent to reduce cocaine abuse.

Sex-Dependent Effects of Piromelatine Treatment on Sleep-Wake Cycle and Sleep Structure of Prenatally Stressed Rats.

Prenatal stress (PNS) impairs the circadian rhythm of the sleep/wake cycle. The melatonin (MT) analogue Piromelatine (Pir) was designed for the treatment of insomnia. The present study aimed to explore effects of Pir on circadian rhythmicity, motor activity, and sleep structure in male and female rats with a history of prenatal stress (PNS). In addition, we elucidated the role of MT receptors and brain-derived neurotrophic factor (BDNF) to ascertain the underlying mechanism of the drug. Pregnant rats were exposed to different stressors from day seven until birth. Piromelatine (20 mg/kg/day/14 days) was administered to young adult offspring. Home-cage locomotion, electroencephalographic (EEG) and electromyographic (EMG) recordings were conducted for 24 h. Offspring treated with vehicle showed sex-and phase-dependent disturbed circadian rhythm of motor activity and sleep/wake cycle accompanied by elevated rapid eye movement (REM) pattern and theta power and diminished non-rapid eye movement (NREM) sleep and delta power. While Pir corrected the PNS-induced impaired sleep patterns, the MT receptor antagonist luzindol suppressed its effects in male and female offspring. In addition, Pir increased the BDNF expression in the hippocampus in male and female offspring with PNS. Our findings suggest that the beneficial effect of Pir on PNS-induced impairment of sleep/wake cycle circadian rhythm and sleep structure is exerted via activation of MT receptors and enhanced BDNF expression in the hippocampus in male and female offspring.

Melatonin inhibits angiotensin II–induced atrial fibrillation through preventing degradation of Ang II Type I Receptor–Associated Protein (ATRAP)

Angiotensin II (Ang II) induced Atrial fibrillation (AF) often accompanied with reduced ATRAP which is a negative modulator of Ang II type 1 receptor (AT1R). Melatonin can protect against AF, but the underlying molecular mechanism remains poorly understood. In this study, Ang II was used to induce AF, and AF inducibility and duration were documented telemetrically. Ang II-infused mice had a higher AF incidence, which was associated with atrial fibrosis, inflammation, and oxidative stress. Melatonin partially inhibited these effects, and enforced expression of siRNA-ATRAP in atria counteracted the beneficial role of melatonin. Specifically, melatonin inhibited expression of Ang II-induced proteasome and immunoproteasome subunits β2, β2i, β5, and β5i as well as their corresponding trypsin-like and chymotrypsin-like activities and blocked ATRAP degradation. In turn, this inhibited AT1R-mediated NF-κB signaling, transforming growth factor (TGF)-β1/Smad signaling in the atria, and thereby affected atrial remodeling and AF. Melatonin receptor inhibition by the chemical inhibitor luzindole partially inhibited the inhibitory effects of melatonin on proteasome activity and also Ang II-induced pathological changes in the atria. Overall, our study demonstrates that melatonin protects against Ang II-induced AF by inhibiting proteasome activity and stabilizing ATRAP expression, and these effects are partially dependent on melatonin receptor activation.

Activation of melatonin receptor 1 by CRISPR-Cas9 activator ameliorates cognitive deficits in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of neurotoxic beta-amyloid (Aβ) in the brain. Melatonin receptors have been reported to associate with aging and AD, and their expression decreased with the progression of AD. As an alternative to AD treatment, overexpression of melatonin receptors may lead to melatonin-like effects to treat alleviate the symptoms of AD. Here, we successfully activated the type 1 melatonin receptor (Mt1) in vivo brain using a Cas9 activator as a novel AD therapeutic strategy. The Cas9 activator efficiently activated the endogenous Mt1 gene in the brain. Activation of Mt1 via Cas9 activators modulated anti-amyloidogenic and anti-inflammatory roles in 5xFAD AD mice brain. Moreover, activation of Mt1 with the CRISPR/Cas9 activator improved cognitive deficits in an AD model. These results demonstrated the therapeutic potential of melatonin receptor activation via CRISPR/Cas9 activator for AD.

Functional Investigation of Melatonin Receptor Activation by Homogenous cAMP Assay.

Cyclic adenosine monophosphate (cAMP) is an important ubiquitous second messenger and one of the major pathways transducing the activation of G protein-coupled receptors (GPCRs). Quantifying intracellular levels of cAMP in an accurate and high-throughput manner is, therefore, of high interest to access functional responses of GPCRs. The neurohormone melatonin is selectively recognized by two GPCRs in mammals, named MT1 and MT2. Both have an inhibitory action on intracellular cAMP levels. Here, we describe a homogeneous high-throughput-compatible methodology routinely used in our laboratory to measure cAMP levels following activation of melatonin receptors.

Melatonin-Activated Receptor Signaling Pathways Mediate Protective Effects on Surfactant-Induced Increase in Jejunal Mucosal Permeability in Rats.

A well-functional intestinal mucosal barrier can be compromised as a result of various diseases, chemotherapy, radiation, and chemical exposures including surfactants. Currently, there are no approved drugs targeting a dysfunctional intestinal barrier, which emphasizes a significant medical need. One candidate drug reported to regulate intestinal mucosal permeability is melatonin. However, it is still unclear if its effect is primarily receptor mediated or antioxidative, and if it is associated with enteric neural pathways. The aim of this rat intestinal perfusion study was to investigate the mechanisms of melatonin and nicotinic acetylcholine receptors on the increase in intestinal mucosal clearance of 51Cr-labeled ethylenediaminetetraacetate induced by 15 min luminal exposure to the anionic surfactant, sodium dodecyl sulfate. Our results show that melatonin abolished the surfactant-induced increase in intestinal permeability and that this effect was inhibited by luzindole, a melatonin receptor antagonist. In addition, mecamylamine, an antagonist of nicotinic acetylcholine receptors, reduced the surfactant-induced increase in mucosal permeability, using a signaling pathway not influenced by melatonin receptor activation. In conclusion, our results support melatonin as a potentially potent candidate for the oral treatment of a compromised intestinal mucosal barrier, and that its protective effect is primarily receptor-mediated.

Selective MT 2 Melatonin Receptor Antagonist Modulates Circadian Activity via Inhibition of the Endogenous Melatonin Signal in the East Bound Jet Lag Model

Activation of melatonin receptors facilitates re-entrainment of circadian activity rhythms following an advance of the dark onset. In an eastbound jet lag model, administration of exogenous melatonin, at the new dark onset, accelerates re-entrainment via activation of the MT1 receptor (Dubocovich et al., 2005). Conversely, melatonin proficient mice (C3H/HeN) with a targeted deletion of the MT2 receptor (MT2KO), but not the MT1 receptor (MT1KO), experience a deceleration in re-entrainment, probably due to impairment of the endogenous melatonin facilitated signaling mediated by activation of the MT2 receptor (Pfeffer et al., 2012). To further elucidate the functional modulatory role of melatonin receptors on the synchronization of the circadian clock, we assessed locomotor activity rhythms in C3H/HeN mice in response to pharmacological intervention to determine the ability of the MT2 selective antagonist, ATBT-23 (hMT1/hMT2 Ki=190), to decelerate re-entrainment by blocking the MT2 receptor, thus mimicking the MT2KO. Male C3H/HeN mice in a 12/12 L/D cycle were subjected to an eastbound jet lag paradigm in which Vehicle (30% ethanol/saline, s.c) or ATBT-23 (1 mg/kg in Vehicle, s.c) were administered on three consecutive days at the new dark onset following an abrupt 6-hour advance of the L/D cycle. The number of days to re-entrain and re-entrainment rates (hrs/day) were analyzed by two-way ANOVA (Tukey's post hoc test) and a mixed effect two-way repeated measure ANOVA (Sidak's post hoc test), respectively. WT (7.54±0.33 days, n=28) and MT1KO (7.36±0.54 days, n=14) mice treated with vehicle did not show a significant difference in the number of days necessary to attain re-entrainment. However, MT2KO (11.92±0.40 days, n=13) mice, treated with vehicle, took significantly longer to re-entrain when compared to WT and MT1KO mice (p<0.0001). This deceleration of re-entrainment in the MT2KO was reproduced by ATBT-23, which decelerated the rate of re-entrainment in both WT (p<0.0001, n=25) and MT1KO (p<0.01, n=12) males, increasing the number of days to re-entrain to 9.76±0.46 (p<0.01) and 10.00±0.63 (p<0.05) days, respectively. ATBT-23 (n=14) did not significantly affect the number of days to re-entrain in MT2KO mice, suggesting deceleration of re-entrainment is not mediated by the MT1 receptor. The present study suggests that ATBT-23 decelerates re-entrainment of circadian activity rhythms by blocking the potent acceleration of re-entrainment by endogenous melatonin via action on the MT2 receptor. Therefore, selective MT2 melatonin receptor antagonists can potentially be used to delay advanced circadian rhythms, as observed in depression and advance sleep phase disorders, when administered at proper circadian times.

A Circadian Clock in the Retina Regulates Rod-Cone Gap Junction Coupling and Neuronal Light Responses via Activation of Adenosine A2A Receptors.

Adenosine, a major neuromodulator in the central nervous system (CNS), is involved in a variety of regulatory functions such as the sleep/wake cycle. Because exogenous adenosine displays dark- and night-mimicking effects in the vertebrate retina, we tested the hypothesis that a circadian (24 h) clock in the retina uses adenosine to control neuronal light responses and information processing. Using a variety of techniques in the intact goldfish retina including measurements of adenosine overflow and content, tracer labeling, and electrical recording of the light responses of cone photoreceptor cells and cone horizontal cells (cHCs), which are post-synaptic to cones, we demonstrate that a circadian clock in the retina itself—but not activation of melatonin or dopamine receptors—controls extracellular and intracellular adenosine levels so that they are highest during the subjective night. Moreover, the results show that the clock increases extracellular adenosine at night by enhancing adenosine content so that inward adenosine transport ceases. Also, we report that circadian clock control of endogenous cone adenosine A2A receptor activation increases rod-cone gap junction coupling and rod input to cones and cHCs at night. These results demonstrate that adenosine and A2A receptor activity are controlled by a circadian clock in the retina, and are used by the clock to modulate rod-cone electrical synapses and the sensitivity of cones and cHCs to very dim light stimuli. Moreover, the adenosine system represents a separate circadian-controlled pathway in the retina that is independent of the melatonin/dopamine pathway but which nevertheless acts in concert to enhance the day/night difference in rod-cone coupling.

Melatonin membrane receptor 2 activation is a key determinant for melatonin-mediated cardioprotection in cardiac ischaemia-reperfusion injury

Abstract Background Cardiac ischaemia/reperfusion (I/R) injury has been an economic and health burden worldwide. Previous studies have reported the beneficial effects of melatonin when given prior to cardiac ischaemia in animals with cardiac I/R injury. However, the effects of melatonin on the hearts when it is given after ischaemia or at the onset of reperfusion, which is more relevant to the clinical setting, is not known. Moreover, the mechanisms responsible for the potential benefits of melatonin and the roles of melatonin receptors on the heart during cardiac I/R injury have not been fully investigated. Purpose We tested the hypothesis that in rats with cardiac I/R injury, melatonin exerts cardioprotective effects even when it is given after ischaemia via an activation of both melatonin receptors 1 (MT1) and 2 (MT2), leading to decreased mitochondrial dysfunction, mitochondrial dynamics imbalance, excessive mitophagy, cardiomyocyte death and finally resulting in decreased infarct size and improved left ventricular (LV) function. Methods Male Wistar rats were subjected to cardiac I/R (30 min of LAD ligation and 120 min of reperfusion). These rats were divided into 4 interventions (n=12/group) including vehicle, pretreatment with melatonin, melatonin treatment during ischaemia, or at the onset of reperfusion. Melatonin was given to the rats at the dose of 10 mg/kg via intravenous injection. In addition, either a non-specific melatonin receptor blocker (Luzindole) or specific MT2 blocker (4-PPDOT) at 1 mg/kg was given intravenously to 2 additional sets of rats (n=12/set) prior to melatonin and cardiac I/R induction. At the end of cardiac I/R, infarct size, LV function, and molecular mechanisms were determined. Furthermore, in vitro experiment was conducted in MT1 or MT2 silenced H9C2 cell with hypoxia/reoxygenation (H/R) to investigate the mechanism underlying cardioprotective effects of melatonin during cardiac I/R. Results Rats in all melatonin-treated groups had similarly reduced cardiac I/R injury as indicated by reduced infarct size (Fig. 1A), arrhythmia score. Melatonin-treated rats also had decreased mitochondrial ROS production, mitochondrial depolarization and swelling, decreased p-Drp1/Drp1 ratio (Fig. 1B) and increased Mfn1, Mfn2, and OPA1, and decreased apoptosis, leading to increased %LVEF. Luzindole and 4-PPDOT abolished these protective effects of melatonin (Fig. 1A). In in vitro study, melatonin increased %cell viability (Fig. 1C), reduced mitochondrial dynamics imbalance and cardiomyocyte apoptosis in H9C2 cells with H/R. However, these beneficial effects of melatonin were abrogated only in MT2 silenced H9C2 cell with H/R. Conclusion Melatonin exerted both preventive and treatment effects in reducing cardiac I/R injury. Its cardioprotective effects were dependent upon the activation of MT2 receptor. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Science and Technology Development Agency of Thailand

Melatonin decreases cocaine-induced locomotor sensitization and cocaine-conditioned place preference in rats

Melatonin is a hormone that produces behavioral, pharmacological, and physiological effects through the activation of MT1 and MT2 melatonin receptors. Melatonin receptors participate in the modulation of the reinforcing effects of cocaine. Some studies report that dosing of melatonin decreases cocaine-induced locomotor activity and cocaine self-administration and that luzindole, an MT1, and MT2 melatonin receptor antagonist, blocks the melatonin-dependent decrease in cocaine-induced locomotor activity. The objective of this study was to evaluate the effect of acute or chronic dosing of melatonin on the induction and expression of cocaine-induced locomotor sensitization and cocaine-CPP in rats.Male Wistar rats received cocaine during the induction and expression of locomotor sensitization. Melatonin was administered 30 min before cocaine. After each treatment, locomotor activity was recorded for 30 min. Additionally, dopamine levels were determined in the ventral striatum, the prefrontal cortex (PFc), and the ventral tegmental area (VTA) by HPLC in animals treated with melatonin and cocaine.Melatonin decreased cocaine-induced locomotor sensitization and intracellular dopamine levels, as well as cocaine-CPP. Luzindole blocked the melatonin-induced decrease in the expression of locomotor sensitization in rats.These data suggest that melatonin may be a useful therapeutic agent to reduce cocaine abuse; additionally, they suggest that MT1 and MT2 receptors could be therapeutic targets, useful for the treatment of drug abuse disorder.

Melatonin receptor agonist protects against acute lung injury induced by ventilator through up-regulation of IL-10 production

BackgroundIt is well known that ventilation with high volume or pressure may damage healthy lungs or worsen injured lungs. Melatonin has been reported to be effective in animal models of acute lung injury. Melatonin exerts its beneficial effects by acting as a direct antioxidant and via melatonin receptor activation. However, it is not clear whether melatonin receptor agonist has a protective effect in ventilator-induced lung injury (VILI). Therefore, in this study, we determined whether ramelteon (a melatonin receptor agonist) can attenuate VILI and explore the possible mechanism for protection.MethodsVILI was induced by high tidal volume ventilation in a rat model. The rats were randomly allotted into the following groups: control, control+melatonin, control+ramelteon, control+luzindole, VILI, VILI+luzindole, VILI + melatonin, VILI + melatonin + luzindole (melatonin receptor antagonist), VILI + ramelteon, and VILI + ramelteon + luzindole (n = 6 per group). The role of interleukin-10 (IL-10) in the melatonin- or ramelteon-mediated protection against VILI was also investigated.ResultsRamelteon treatment markedly reduced lung edema, serum malondialdehyde levels, the concentration of inflammatory cytokines in bronchoalveolar lavage fluid (BALF), NF-κB activation, iNOS levels, and apoptosis in the lung tissue. Additionally, ramelteon treatment significantly increased heat shock protein 70 expression in the lung tissue and IL-10 levels in BALF. The protective effect of ramelteon was mitigated by the administration of luzindole or an anti-IL-10 antibody.ConclusionsOur results suggest that a melatonin receptor agonist has a protective effect against VILI, and its protective mechanism is based on the upregulation of IL-10 production.

Melatonin receptor protects cardiomyocyte against oxidative stress-induced apoptosis through the MAPK-ERK signaling pathway

Context: The role of melatonin receptor in cardiomyocyte oxidative stress has not been investigated.Aim: The aim of our study is to verify the beneficial effects of melatonin receptors on cardiomyocyte viability under oxidative stress.Methods: Cardiomyocytes were treated with hydrogen peroxide. Cell viability was measured via MTT assay and TUNEL staining. Then, anti-oxidative factors measurement and signaling pathway analysis were performed via qPCR and ELISA.Results: Melatonin receptor activation could attenuate hydrogen peroxide-mediated cardiomyocyte death via reducing apoptosis. At the molecular levels, melatonin receptor activation reduced inflammation response and maintained mitochondrial membrane potential. In addition, melatonin receptor activation is associated with decreased oxidative stress and increased anti-oxidative factors. Finally, we found that melatonin receptor activation triggered an elevation in the activity and expression of ERK pathway and blockade of ERK pathway would abolish the beneficial effects exerted by melatonin receptors activation on cardiomyocyte survival under oxidative stress.Conclusions: Our data suggest that melatonin receptor could attenuate oxidative stress injury in cardiomyocyte through regulation of the ERK signaling pathway.

Melatonin receptor activation protects against low potassium-induced ventricular fibrillation by preserving action potentials and connexin-43 topology in isolated rat hearts.

Hypokalemia prolongs the QRS and QT intervals, deteriorates intercellular coupling, and increases the risk for arrhythmia. Melatonin preserves gap junctions and shortens action potential as potential antiarrhythmic mechanisms, but its properties under hypokalemia remain unknown. We hypothesized that melatonin protects against low potassium-induced arrhythmias through the activation of its receptors, resulting in action potential shortening and connexin-43 preservation. After stabilization in Krebs-Henseleit solution (4.5mEq/L K+ ), isolated hearts from Wistar rats underwent perfusion with low-potassium (1mEq/L) solution and melatonin (100μmol/L), a melatonin receptor blocker (luzindole, 5μmol/L), melatonin+luzindole or vehicle. The primary endpoint of the study was the prevention of ventricular fibrillation. Electrocardiography was used, and epicardial action potentials and heart function were measured and analyzed. The ventricular expression, dephosphorylation, and distribution of connexin-43 were examined. Melatonin reduced the incidence of low potassium-induced ventricular fibrillation from 100% to 59%, delayed the occurrence of ventricular fibrillation and induced a faster recovery of sinus rhythm during potassium restitution. Melatonin prevented QRS widening, action potential activation delay, and the prolongation of action potential duration at 50% of repolarization. Other ECG and action potential parameters, the left ventricular developed pressure, and nonsustained ventricular arrhythmias did not differ among groups. Melatonin prevented connexin-43 dephosphorylation and its abnormal topology (lateralization). Luzindole abrogated the protective effects of melatonin on electrophysiological properties and connexin-43 misdistribution. Our results indicate that melatonin receptor activation protects against low potassium-induced ventricular fibrillation, shortens action potential duration, preserves ventricular electrical activation, and prevents acute changes in connexin-43 distribution. All of these properties make melatonin a remarkable antifibrillatory agent.

Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin-conferred cardioprotection against myocardial ischemia/reperfusion injury.

Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin-conferred cardioprotection remains unclear. In this study, we employed both loss-of-function and gain-of-function approaches to reveal the answer. Mice (wild-type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild-type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R-initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2-silenced heart, but not the MT1 subtype. Furthermore, AAV9-mediated cardiomyocyte-specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.

Carbaryl Modulates Chronobiological Behaviors via Melatonin Receptors

Humans are exposed to low levels of carbaryl and other carbamate insecticides found in household products, food, and water due to persistent and widespread use. Environmental and occupational exposure to carbamate pesticides is linked to metabolic disorders and sleep apnea, though it is not clear which biological targets contribute to disease pathology (Montgomery et al. 2008; James‐Todd et al., 2016; Baumert et al. 2017). Carbaryl binds to MT1 and MT2 G protein‐coupled receptors with varying efficacies and potencies as demonstrated by our team via in‐silico molecular modeling and in‐vitro bioassays (Popovska‐Gorevski et al. 2017; Jones et al. 2017 EB Abst; Glatfelter et al. 2017 EB Abst). We hypothesize that carbaryl may produce alterations in circadian rhythms through interactions with melatonin receptors that could contribute to the aforementioned disease symptomology, progression, and/or pathologies. Previous in‐vitro (1–100μM) and ex‐vivo (10 mg/kg, i.p.) experiments demonstrated specificity for competition of carbaryl for 2‐[125I]‐iodomelatonin binding to brain melatonin receptors and its ability to reach target brain areas controlling circadian rhythms in‐vivo (e.g. suprachiasmatic nucleus: SCN; Glatfelter et al. 2017 EB Abs). We tested the ability of carbaryl (10 mg/kg, i.p.) to phase shift onset of circadian running wheel activity when given at circadian time (CT) 10 (CT12 = onset of running wheel activity in constant dark) for 3 days in wild‐type C3H/HeN (C3H) mice. Our results indicate that carbaryl (0.81h ± 0.1; n=12; p&lt;0.0001) and positive control melatonin (3mg/kg s.c.; 0.97h ± 0.2; n=4; p&lt;0.001) significantly phase advance running wheel activity onset compared to vehicle (0.03h ± 0.04; n=7) treated controls (F2,20=18.46; p&lt;0.05; Dunnet's Post Test). In the same paradigm, carbaryl (10 mg/kg i.p.) given 10 hrs before onset of running wheel activity in constant dark at CT 2 showed a phase delay (−1.2h ± 0.11; n=14; p&lt;0.0001) similar to positive control melatonin (3mg/kg s.c. 1.3h ± 0.14; n=4; p&lt;0.001) compared to vehicle (−0.12h ± 0.02; n=19) treated controls (F2,34=75.96; p&lt;0.05; Dunnet's Post Test). These data demonstrate that carbaryl has the same periods of circadian sensitivity for phase shift at CT2 &amp; CT10 as melatonin (Benloucif &amp; Dubocovich 1996). A dose response curve for carbaryl (0.01 – 10 mg/kg i.p.) to phase advance circadian running wheel activity rhythms at CT10 reveals an ED50 value of 0.019 mg/kg, i.p. which is similar in magnitude to melatonin (ED50=0.024 mg/kg s.c. Dubocovich et al. 1998). Carbaryl (10 mg/kg i.p.) did not phase advance circadian running wheel activity rhythms in C3H MT1KO mice (−0.04h ± 0.04; n=18; n.s.) at CT10 compared to vehicle controls (−0.04h ± 0.03; n=19; t=0.12; n.s.) suggesting an MT1 dependent mechanism. We conclude that carbaryl phase shifts circadian activity rhythms via activation of MT1 melatonin receptors suggesting environmental or occupational exposure to this insecticide in the tested dose range could influence disease pathologies via modulation of circadian biology.Support or Funding InformationSupported by ES 023684 and UB Jacobs School of Medicine funds to MLD and RVR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relationship between endogenous melatonin concentrations and uterine contractions in late third trimester of human pregnancy.

In humans, circulating levels of the hormone melatonin and the initiation of spontaneous labor are both higher at night than during the day. Since activation of uterine melatonin receptors can stimulate human in vitro uterine contractions and these receptors are only expressed on the uterine tissue of women in labor, we hypothesized that circulating melatonin concentrations would affect uterine contractions in vivo. We evaluated the impact of light-induced modulation of melatonin secretion on uterine contractions in women during late third trimester (~36-39weeks) of pregnancy in two inpatient protocols. We found a significant (P<0.05) positive linear association between circulating melatonin concentrations and the number of uterine contractions under both protocols. On average, uterine contractions increased between 1.4 and 2.1 contractions per 30minutes for every 10pg/mL*h increase in melatonin concentration. These findings have both basic science and clinical implications for pregnant women, since endogenous melatonin levels and melatonin receptor activity can be altered by light and/or pharmaceutical agents.

Melatonin selectively influences the transcription of pluripotency and differentiation markers in human non-cancer cells

Melatonin (MEL) may influence the efficiency of reprogramming both by somatic cell nuclear transfer and by direct induction of pluripotent stem cells (iPSC) through a yet unidentified mechanism. Transcription factors linked to cell reprogramming and cell signalling may be differentially expressed according to cell differentiation status. To address the effect of MEL on the expression of transcription factors linked to reprogramming, we used two distinct in vitro models of cellular plasticity: human foreskin fibroblasts (HFF) and primary human granulosa-lutein cells (GLC). Real-time quantitative polymerase chain reaction (qRT-PCR) analysis revealed amplification of transcripts for KLF4, MYC and NANOG in both cell types. In GLC, treatment with 10 nmol/L of MEL provoked significant up-regulation of the expression of MYC and NANOG compared to controls. KLF4 expression was not altered in GLC but was significantly down-regulated in MEL-treated HFF cells. Alterations in the expression of ERK1/2 and pERK1/2 in GLC as analyzed by Western blot were not observed regardless of the MEL treatment. On the contrary, HFF cells responded to MEL treatment with 1.6-fold higher levels of pERK1/2 compared to the non-treated controls. Our data suggest that the activation of MT1 melatonin receptor is probably related to phosphorylation of ERK1/2 at least in expanding HFF, which subsequently may act to alter gene expression and regulate cell fate. In conclusion, we demonstrated for the first time, the selective effect of MEL in vitro at physiological concentration on transcription factors regulating pluripotency and differentiation in human non-cancer cells according to cell differentiation status.