Zygotic Exposure to Venlafaxine Disrupts the Circadian Locomotor Activity Behaviour in Zebrafish Larvae.

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly prescribed to treat major depressive disorder and is found at high concentrations in the aquatic environment. Concerns have been raised related to the health of aquatic organisms in response to this nontargeted pharmaceutical exposure. For instance, we previously demonstrated that exposure to venlafaxine perturbs neurodevelopment, leading to behavioural alterations in zebrafish (Danio rerio). We also observed disruption in serotonin expression in the pineal and raphe, regions critical in regulating circadian rhythms, leading us to hypothesize that zygotic exposure to venlafaxine disrupts the circadian locomotor rhythm in larval zebrafish. To test this, we microinjected zebrafish embryos with venlafaxine (1 or 10 ng) and recorded the locomotor activity in 5-day-old larvae over a 24-h period. Venlafaxine deposition reduced larval locomotor activity during the light phase, but not during the dark phase of the diurnal cycle. The melatonin levels were higher in the dark compared to during the light photoperiod and this was not affected by embryonic venlafaxine deposition. Venlafaxine exposure also did not affect the transcript abundance of clock genes, including clock1a, bmal2, cry1a and per2, which showed a clear day/night rhythmicity. A notable finding was that exposure to luzindole, a melatonin receptor antagonist, decreased the locomotor activity in the control group in light, whereas the activity was higher in larvae raised from the venlafaxine-deposited embryos. Overall, zygotic exposure to venlafaxine disrupts the locomotor activity of larval zebrafish fish during the day, demonstrating the capacity of antidepressants to disrupt the circadian rhythms in behaviour. Our results suggest that disruption in melatonin signalling may be playing a role in the venlafaxine impact on circadian behaviour, but further investigation is required to elucidate the possible mechanisms in larval zebrafish.

Melanogenesis Is Directly Affected by Metabolites of Melatonin in Human Melanoma Cells.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic (N1-acetyl-N2-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients.

Melatonin-mediated IGF-1/GLP-1 activation in experimental OCD rats: Evidence from CSF, blood plasma, brain and in-silico investigations.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric condition characterized by intrusive, repetitive thoughts and behaviors. Our study uses a validated 8-OH-DPAT-induced experimental model of OCD in rodents. We focus on the modulatory effects of Insulin-like growth factor-1 (IGF-1) and glucagon-like peptide-1 (GLP-1), which are linked to neurodevelopment and survival. Current research investigates melatonin, a molecule with neuroprotective properties and multiple functions. Melatonin has beneficial effects on various illnesses, including Alzheimer's, Parkinson's, and depression, indicating its potential efficacy in treating OCD. In the present study, we employed two doses of melatonin, 5 mg/kg and 10 mg/kg, demonstrating a dose-dependent effect on 8-OH-DPAT-induced rat changes. In addition, the melatonin antagonist luzindole 5 mg/kg was utilized to compare and validate the efficacy of melatonin. In-silico studies alsocontribute to understanding the activation of IGF-1/GLP-1 pathways by melatonin. Current research indicates restoring neurochemical measurements on various biological samples (brain homogenates, CSF, and blood plasma) and morphological and histological analyses. In addition, the current research seeks to increase understanding of OCD and investigate potential new treatment strategies. Therefore, it is evident from the aforementioned research that the protective effect of melatonin can serve as a strong basis for developing a new OCD treatment by upregulating IGF-1 and GLP-1 levels. The primary focus of current study revolves around the examination of melatonin as an activator of IGF-1/GLP-1, with the aim of potentially mitigating behavioral, neurochemical, and histopathological abnormalities in an experimental model of obsessive-compulsive disorder caused by 8-OH-DPAT in adult Wistar rats.

Effect of melatonin and luzindole antagonist on fipronil toxicity, detoxification and antioxidant enzyme system in different tissues of Helicoverpa armigera (Lepidoptera: Noctuidae).

Studies have investigating the detoxification and antioxidant enzymes with melatonin under pesticide stress in many vertebrates, whereas no reports produced in invertebrates. In this study possible role of melatonin and luzindole effect on fipronil toxicity and the detoxification, antioxidant enzymes in H. armigera has been reported. Result showed high toxicity of fipronil treatment (LC50 4.24 ppm), followed by increased LC50 value with melatonin pretreatment (6.44 ppm). Whereas decreased toxicity was observed with melatonin and luzindole combination (3.72 ppm). The detoxification enzymes AChE, esterase and P450 were increased in larval head and whole body with exogenous melatonin level compared to control 1-1.5 µmol/mg of protein. The antioxidant levels of CAT, SOD and GST in whole body and head tissue had been increased by melatonin and fipronil combination 1.1-1.4 unit/mg of protein followed by GPx and GR in larval head (1-1.2 µmol/mg of protein). Mean while the luzindole antagonist inhibits CAT, SOD, GST and GR oxidative enzyme level (1-1.5 fold) in most of the tissue compared to melatonin and fipronil treatment (p < 0.01). Hence this study concludes that the melatonin pretreatment can reduce the fipronil toxicity by enhanced detoxification and antioxidant enzyme system in H. armigera.

Neural and molecular investigation into the paraventricular thalamic-nucleus accumbens circuit for pain sensation and non-opioid analgesia.

The paucity of medications with novel mechanisms for pain treatment combined with the severe adverse effects of opioid analgesics has led to an imperative pursuit of non-opioid analgesia and a better understanding of pain mechanisms. Here, we identify the putative glutamatergic inputs from the paraventricular thalamic nucleus to the nucleus accumbens (PVTGlut→NAc) as a novel neural circuit for pain sensation and non-opioid analgesia. Our in vivo fiber photometry and in vitro electrophysiology experiments found that PVTGlut→NAc neuronal activity increased in response to acute thermal/mechanical stimuli and persistent inflammatory pain. Direct optogenetic activation of these neurons in the PVT or their terminals in the NAc induced pain-like behaviors. Conversely, inhibition of PVTGlut→NAc neurons or their NAc terminals exhibited a potent analgesic effect in both naïve and pathological pain mice, which could not be prevented by pretreatment of naloxone, an opioid receptor antagonist. Anterograde trans-synaptic optogenetic experiments consistently demonstrated that the PVTGlut→NAc circuit bi-directionally modulates pain behaviors. Furthermore, circuit-specific molecular profiling and pharmacological studies revealed dopamine receptor 3 as a candidate target for pain modulation and non-opioid analgesic development. Taken together, these findings provide a previously unknown neural circuit for pain sensation and non-opioid analgesia and a valuable molecular target for developing future safer medication.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-ß superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-ß superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Immunolocalization of melatonin receptors in bovine ovarian follicles and in vitro effects of melatonin on growth, viability and gene expression in secondary follicles.

This study aims to investigate the (1) expression of melatonin receptors types 1A/B (MTNR1A/B) in bovine ovaries and (2) the in vitro effects of melatonin on secondary follicle development, antrum formation, viability, and expression of messenger ribonucleic acid (mRNA) for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase-1 (GPX1) and peroxiredoxin 6 (PRDX6). The expression of MTNR1A/B in bovine ovarian follicles was demonstrated by immunohistochemistry. To choose the most effective concentration of melatonin on follicular growth and viability, isolated secondary follicles were cultured individually at 38.5°C, with 5% CO2 in air, for 18 d in TCM-199+ alone or supplemented with 10-11, 10-9, 10-7 or 10-5 M melatonin. Then, melatonin receptor antagonist, luzindole, was tested to further evaluate the mechanisms of actions of melatonin, that is, the follicles were cultured in control medium alone or supplemented with 10-7 M melatonin, 10 µM luzindole and both 10-7 M melatonin and 10 µM luzindole. Follicular growth, morphology and antrum formation were evaluated at days 6, 12 and 18. At the end of culture, viability of secondary follicles was analyzed by calcein-AM and ethidium homodimer-1, and the relative levels of mRNA for SOD, CAT, GPX1 and PRDX6 were evaluated by real time polymerase chain reaction. Immunohistochemistry results showed expression of MTNR1A/B in oocyte and granulosa cells of primordial, primary, secondary and antral follicles. Secondary follicles cultured in medium supplemented with melatonin at different concentrations had well preserved follicles after 18 d of culture. Furthermore, follicles cultured in presence of 10-7 M melatonin presented significantly higher diameters than those cultured in other treatments. The presence of melatonin receptor antagonist, luzindole, blocked the effects of melatonin on follicular growth and viability. In addition, follicles cultured in medium containing only melatonin had significantly higher rates of antrum formation. Follicles cultured in medium containing only melatonin had higher relative levels of mRNA for CAT, SOD and PRDX-6 than those cultured with both melatonin and luzindole. Follicles cultured with luzindole only or both melatonin and luzindole had lower relative levels of mRNA for PRDX6 and GPX1 than those cultured control medium. In conclusion, melatonin promotes growth of bovine secondary follicles through its membrane-coupled receptors, while luzindole blocks the effects of melatonin on follicle growth and reduces the expression of antioxidant enzymes in cultured follicles.

The melatonin MT2 receptor is involved in the anti-apoptotic effects of melatonin in rats with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a widely prevalent chronic disease and risk factor for several other diseases, such as cardiovascular diseases, neuropathy, nephropathy, and retinopathy. Apoptosis is a homeostatic mechanism to maintain cell numbers at a certain level in tissues. Chronic high blood glucose levels might lead to mitochondrial dysfunction and trigger undesirable apoptosis in T2DM. The pineal hormone melatonin has been shown to regulate apoptosis. The aim of this study was to investigate the impact of the melatonin MT2 receptor in the role of melatonin to prevent undesirable apotosis in different tissues of diabetic rats. Male Sprague Dawley rats were randomly divided into 4 groups; 1. Control group (only vehicle), 2. Diabetic group (streptozotozin/nicotinamide treated), 3. Diabetic group treated with melatonin (500µg/kg/day), and 4. Diabetic group treated with melatonin (500 µg/kg/day for 6 weeks) and the selective MT2 receptor antagonist luzindole (0.25 g/kg/day for 6 weeks). Various tissue samples (kidney, liver, adipose tissue, pancreas) were removed after 6 weeks for immunohistochemistry and western blot analysis. Our results demonstrated an increased rate of apoptosis in different tissues of diabetic rats compared to controls with melatonin reducing the apoptotic rate in the tissues of rats with T2DM. Furthermore, the anti-apoptotic effects of melatonin were partly mediated by the melatonin MT2 receptor.

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.

Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach, Periplaneta americana, via melatonin/MT2-like receptor.

Melatonin (MEL) orchestrates daily and seasonal rhythms (eg, locomotion, sleep/wake cycles, and migration among other rhythms) in diverse organisms. We investigated the effects of pharmacological doses (0.03-1 mM) of exogenous MEL intake in the cockroach, Periplaneta americana, on locomotor activity. As per os MEL concentration increased, cockroach locomotor rhythm in light-dark (LD) cycles became more synchronized. The ratio of night activity to 24-h activity increased and the acrophase (peak) slightly advanced. MEL application also influenced total activity bouts in the free-running rhythm. Since MEL slightly influenced τ in the free-running rhythms, it is not a central element of the circadian pacemaker but must influence mutual coupling of multi-oscillatory system components. Arylalkylamine N-acetyltransferase (aaNAT) regulates enzymatic production of MEL. aaNAT activities vary in circadian rhythms, and the immunoreactive aaNAT (aaNAT-ir) is colocalized with the key clock proteins cycle (CYC)-ir and pigment-dispersing factor (PDF)-ir These are elements of the central pacemaker and its output pathway as well as other circadian landmarks such as the anterior and posterior optic commissures (AOC and POC, respectively). It also partially shares immunohistochemical reactivity with PER-ir and DBT-ir neurons. We analyzed the role of Pamericana aaNAT1 (PaaaNAT1) (AB106562.1) by injecting dsRNAaaNAT1 . qPCR showed a decrease in accumulations of mRNAs encoding PaaaNAT1. The injections led to arrhythmicity in LD cycles and the arrhythmicity persisted in constant dark (DD). Continuous administration of MEL resynchronized the rhythm after arrhythmicity was induced by dsRNAaaNAT1 injection, suggesting that PaaaNAT is the key regulator of the circadian system in the cockroach via MEL production. PaaaNAT1 contains putative E-box regions which may explain its tight circadian control. The receptor that mediates MEL function is most likely similar to the mammalian MT2, because injecting the competitive MT2 antagonist luzindole blocked MEL function, and MEL injection after luzindole treatment restored MT function. Human MT2-ir was localized in the circadian neurons in the cockroach brain and subesophageal ganglion. We infer that MEL and its synthesizing enzyme, aaNAT, constitute at least one circadian output pathway of locomotor activity either as a distinct route or in association with PDF system.

Role of melatonin in the dynamics of acute spinal cord injury in rats.

Melatonin is well-documented to have the ability of reducing nerve inflammation and scavenging free radicals. However, the therapeutic effect of melatonin on spinal cord injury has not been fully described. In this study, we assessed the effect of melatonin on T9 spinal cord injury established by Allen method in rats. Melatonin deficiency significantly delayed the recovery of sensory and motor functions in SCI rats. Treatment with melatonin significantly alleviated neuronal apoptosis and accelerated the recovery of spinal cord function. These results suggest that melatonin is effective to ameliorate spinal cord injury through inhibition of neuronal apoptosis and promotion of neuronal repair.

Melatonin promotes cardiomyocyte proliferation and heart repair in mice with myocardial infarction via miR-143-3p/Yap/Ctnnd1 signaling pathway.

The neonatal heart possesses the ability to proliferate and the capacity to regenerate after injury; however, the mechanisms underlying these processes are not fully understood. Melatonin has been shown to protect the heart against myocardial injury through mitigating oxidative stress, reducing apoptosis, inhibiting mitochondrial fission, etc. In this study, we investigated whether melatonin regulated cardiomyocyte proliferation and promoted cardiac repair in mice with myocardial infarction (MI), which was induced by ligation of the left anterior descending coronary artery. We showed that melatonin administration significantly improved the cardiac functions accompanied by markedly enhanced cardiomyocyte proliferation in MI mice. In neonatal mouse cardiomyocytes, treatment with melatonin (1 µM) greatly suppressed miR-143-3p levels. Silencing of miR-143-3p stimulated cardiomyocytes to re-enter the cell cycle. On the contrary, overexpression of miR-143-3p inhibited the mitosis of cardiomyocytes and abrogated cardiomyocyte mitosis induced by exposure to melatonin. Moreover, Yap and Ctnnd1 were identified as the target genes of miR-143-3p. In cardiomyocytes, inhibition of miR-143-3p increased the protein expression of Yap and Ctnnd1. Melatonin treatment also enhanced Yap and Ctnnd1 protein levels. Furthermore, Yap siRNA and Ctnnd1 siRNA attenuated melatonin-induced cell cycle re-entry of cardiomyocytes. We showed that the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration was impeded by the melatonin receptor inhibitor luzindole. Silencing miR-143-3p abrogated the inhibition of luzindole on cardiomyocyte proliferation. In addition, both MT1 and MT2 siRNA could cancel the beneficial effects of melatonin on cardiomyocyte proliferation. Collectively, the results suggest that melatonin induces cardiomyocyte proliferation and heart regeneration after MI by regulating the miR-143-3p/Yap/Ctnnd1 signaling pathway, providing a new therapeutic strategy for cardiac regeneration.

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.

Acute blockade of endogenous melatonin by Luzindole, with or without peripheral LPS injection, induces jejunal inflammation and morphological alterations in Swiss mice

This study investigated the acute blockade of endogenous melatonin (MLT) using Luzindole with or without systemic lipopolysaccharide (LPS) challenge and evaluated changes in inflammatory and oxidative stress markers in the mouse jejunum. Luzindole is an MT1/MT2 MLT receptor antagonist. Both receptors occur in the small intestine. Swiss mice were treated with either saline (0.35 mg/kg, ip), Luzindole (0.35 mg/kg, ip), LPS (1.25 mg/kg, ip), or Luzindole+LPS (0.35 and 1.25 mg/kg, ip, respectively). Jejunum samples were evaluated regarding intestinal morphometry, histopathological crypt scoring, and PAS-positive villus goblet cell counting. Inflammatory Iba-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, nuclear factor (NF)-kB, myeloperoxidase (MPO), and oxidative stress (NP-SHs, catalase, MDA, nitrate/nitrite) markers were assessed. Mice treated with Luzindole, LPS, and Luzindole+LPS showed villus height shortening. Crypt damage was worse in the LPS group. Luzindole, LPS, and Luzindole+LPS reduced the PAS-goblet cell labeling and increased Iba-1-immunolabelled cells compared to the saline group. Immunoblotting for IL-1β, TNF-α, and NF-kB was greater in the Luzindole group. The LPS-challenged group showed higher MPO activity than the saline and Luzindole groups. Catalase was reduced in the Luzindole and Luzindole+LPS groups compared to saline. The Luzindole group showed an increase in NP-SHs, an effect related to compensatory GSH activity. The acute blockade of endogenous MLT with Luzindole induced early changes in inflammatory markers with altered intestinal morphology. The other non-detectable deleterious effects of Luzindole may be balanced by the unopposed direct action of MLT in immune cells bypassing the MT1/MT2 receptors.

Melatonin Act as an Antidepressant via Attenuation of Neuroinflammation by Targeting Sirt1/Nrf2/HO-1 Signaling.

Physical or psychological stress can cause an immunologic imbalance that disturbs the central nervous system followed by neuroinflammation. The association between inflammation and depression has been widely studied in recent years, though the molecular mechanism is still largely unknown. Thus, targeting the signaling pathways that link stress to neuroinflammation might be a useful strategy against depression. The current study investigated the protective effect of melatonin against lipopolysaccharide (LPS)-induced neuroinflammation and depression. Our results showed that LPS treatment significantly induced depressive-like behavior in mice. Moreover, LPS-treatment enhanced oxidative stress, pro-inflammatory cytokines including TNFα, IL-6, and IL-1ß, NF-κB phosphorylation, and glial cell activation markers including GFAP and Iba-1 in the brain of mice. Melatonin treatment significantly abolished the effect of LPS, as indicated by improved depressive-like behaviors, reduced cytokines level, reduced oxidative stress, and normalized LPS-altered Sirt1, Nrf2, and HO-1 expression. However, the melatonin protective effects were reduced after luzindole administration. Collectively, it is concluded that melatonin receptor-dependently protects against LPS-induced depressive-like behaviors via counteracting LPS-induced neuroinflammation.

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells.

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca2+ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 µm) evoked a slow and progressive increase in intracellular free Ca2+ ([Ca2+ ]i ) towards a plateau. The effect of the compound on Ca2+ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin (1 µm), in the absence of Ca2+ in the extracellular medium, induced a transient increase in [Ca2+ ]i . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca2+ . Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca2+ . In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.

Melatonin decreases cocaine-induced locomotor activity in pinealectomized rats

Objective: Several studies have shown that the time of day regulates the reinforcing effects of cocaine. Additionally, melatonin and its MT1 and MT2 receptors have been found to participate in modulation of the reinforcing effects of such addictive drugs as cocaine. Loss of the diurnal variation in cocaine-induced locomotor sensitization and cocaine-induced place preference has been identified in pinealectomized mice. In addition, several studies in rodents have shown that administration of melatonin decreased the reinforcing effects of cocaine. The objective of this study was to evaluate the effect of melatonin on cocaine-induced locomotor activity in pinealectomized rats at different times of day (zeitgeber time [ZT]4, ZT10, ZT16, and ZT22). Methods: Naïve, pinealectomized Wistar rats received cocaine at different times of day. Melatonin was administered 30 min before cocaine; luzindole was administered 15 min prior to melatonin and 45 min before cocaine. After administration of each treatment, locomotor activity for each animal was recorded for a total of 30 min. Pinealectomy was confirmed at the end of the experiment through melatonin quantitation by ELISA. Results: Cocaine-induced locomotor activity varied according to the time of day. Continuous lighting and pinealectomy increased cocaine-induced locomotor activity. Melatonin administration decreased cocaine-induced locomotor activity in naïve and pinealectomized rats at different times of day. Luzindole blocked the melatonin-induced reduction in cocaine-induced locomotor activity in pinealectomized rats. Conclusion: Given its ability to mitigate various reinforcing effects of cocaine, melatonin could be a useful therapy for cocaine abuse.

Luzindole attenuates LPS/d-galactosamine-induced acute hepatitis in mice.

Melatonin is a well-documented hormone that plays central roles in the regulation of sleep-wake cycles. There is cumulative evidence to suggest that melatonin is also a pleiotropic regulator of inflammation, and luzindole has been widely used as a melatonin receptor antagonist. This study investigated the potential effects of luzindole on LPS/d-galactosamine (d-GalN)-induced acute hepatitis. The results indicated that treatment with luzindole alleviated histological damage in the liver, reduced the level of transaminases in plasma and improved the survival of LPS/d-GalN-exposed mice. Treatment with luzindole also suppressed the production of the pro-inflammatory cytokines TNF-α and IL-6 in LPS/d-GalN-exposed mice. In addition, treatment with luzindole inhibited the activation of caspase-3, -8 and -9, and suppressed the cleavage of caspase-3 and poly(ADP-ribose) polymerase. Therefore, treatment with luzindole attenuates LPS/d-GalN-induced acute liver injury, suggesting that luzindole might have potential value for the intervention of inflammation-based hepatic disorders.

The melatonin receptor antagonist luzindole induces Ca2+ mobilization, reactive oxygen species generation and impairs trypsin secretion in mouse pancreatic acinar cells.

BACKGROUND: In this work we studied the effects of the melatonin receptor-antagonist luzindole (1 µM-50 µM) on isolated mouse pancreatic acinar cells. METHODS: Changes in intracellular free-Ca2+ concentration, reactive oxygen species production and trypsin secretion were analyzed. RESULTS: Luzindole induced increases in [Ca2+]i that diminished CCK-8 induced Ca2+ mobilization, compared with that observed when CCK-8 was applied alone. Treatment of cells with thapsigargin (1 µM), in the absence of Ca2+ in the extracellular medium, evoked a transient increase in [Ca2+]i. The additional incubation of cells with luzindole (10 µM) failed to induce further mobilization of Ca2+. In the presence of luzindole a concentration-dependent increase in ROS generation was observed that decreased in the absence of Ca2+ or by pretreatment of cells with melatonin (100 µM). Incubation of pancreatic acinar cells with luzindole (10 µM) impaired CCK-8-induced trypsin secretion. Melatonin was unable to revert the effect of luzindole on CCK-8-induced trypsin secretion. CONCLUSION: The melatonin receptor-inhibitor luzindole induces Ca2+-mediated pro-oxidative conditions and impairment of enzyme secretion, which creates a situation in pancreatic acinar cells that might compromise their function. GENERAL SIGNIFICANCE: The effects of luzindole that we have observed, might be unspecific and could mislead the observations when it is used to study the actions of melatonin on the gland. Another possibility is that melatonin receptors exhibit a basal or agonist-independent activity in pancreatic acinar cells, which might be modulated by melatonin or luzindole.

Role of Melatonin Receptors in Hyperthermia-Induced Acute Seizure Model of Rats.

Melatonin is a neurohormone that has anticonvulsant activity in different experimental seizure models including hyperthermic febrile seizure. However, the mechanisms of this effect are not clear at the receptor level. The aim of the study was to determine which melatonin receptors involve in the hyperthermic febrile seizure model. 22-30 days Wistar male rats were used, and in children, it corresponds to 1.5-2 years. Groups were performed as (1) control, (2) ethanol/saline, (3) DMSO, (4) melatonin (MT), (5) MT + luzindole (LUZ), (6) MT + K-185, (7) MT + prazosin (PRZ), (8) MT + LUZ + K-185, (9) MT + LUZ + PRZ, (10) MT + K-185 + PRZ, and (11) MT + LUZ + PRZ + K-185. The hyperthermic febrile seizure pattern was established by keeping the rats in 45 °C hot water, and the latency, duration, and severity of seizures were determined in all groups. MT, LUZ, K-185, and PRZ were given 15, 45, 15, and 30 min before the induction of seizure, respectively. It was observed that melatonin shortened the duration of seizure, reduced the severity, and did not affect latency and that these effects were not completely blocked by receptor antagonists when compared with control, ethanol/saline, and DMSO groups. In conclusion, the fact that the anticonvulsant effect of melatonin is not completely blocked by all melatonin receptor antagonists. We can conclude that a multimodal mechanism may be responsible for the effect of melatonin receptors alone on the anticonvulsant effect of melatonin. It will be useful to design new pharmacological studies to make the subject clear.