Receptor Antagonist Luzindole Research Articles

THE EFFECT OF CONSTANT DARKNESS, MELATONIN AND ITS ANTAGONIST LUZINDOLE ON RETINOL AND TOCOPHEROL IN RATS

Constant darkness stimulated the secretion of the pineal hormone melatonin. The intensity of melatonin synthesis, which has a strong antioxidant effect, can change significantly under changing light conditions. A free-running rhythm of melatonin secretion revealed in constant darkness affects the antioxidant system. In this study were investigated the effects of constant darkness, melatonin and its receptor antagonist luzindole on retinol and tocopherol contents in the tissues and organs of adult male rats. The rats (7 months old) were divided into two groups and kept in constant darkness (DD) or standard lighting (control, 12:12 LD) for 14 days. Animals of each group were divided into three subgroups: control, receiving melatonin 22 mg/kg of body weight or luzindole at a dose of 10 mg/l. The levels of retinol and tocopherol were determined by HPLC. The study showed that staying in the dark did not affect the tocopherol and retinol content in rat tissues and organs. The greatest changes in the content of vitamins A and E under the influence of all the studied factors were observed in the liver and skeletal muscle. The relationship of retinol and tocopherol with melatonin in most cases had a mutually compensatory nature, i. e. an increase in the content of one antioxidant induced a decrease in another so that the dynamic balance of the antioxidant system was maintained.

AGE-RELATED CHANGES IN THE BEHAVIOR AND PHOBIC ANXIETY REACTIONS IN RATS UNDER EXPOSURE TO LIGHT DEPRIVATION AND LUZINDOLE

The effect of both long-term stay of rats under the specific extreme conditions of light deprivation, and the administration of a melatonin receptor antagonist luzindole on the agerelated pattern of behavioral reactions and psycho-emotional manifestations was studied. The level of the emotional and phobic anxiety state of the animals was assessed in an “Open field” installation in combination with the test “Dark chamber with holes”. We found that in the process of aging, rats kept in standard light conditions (LD) demonstrated a decline in motor activity along with an increase in phobic anxiety reactions. The effect of a prolonged exposure to light deprivation on the psycho-emotional state and motor activity of rats varied depending on the ontogenetic period during which the animals were placed in these specific conditions. At an advanced age, the anxiety level increased in rats kept in the dark since birth (LD/DD) to a greater degree than in the animals exposed to such conditions since prenatal development (DD/DD). The administration of a melatonin receptor antagonist luzindole to animals kept in constant darkness led to an age-related increase in phobic anxiety in the open space (as shown in the “Open Field” test), but had a normalizing effect on the psycho-emotional state of the rats in the “Dark chamber with holes” installation, imitating the animals’ natural shelter. It is concluded that the melatoninergic system, including the pineal gland, melatonin and its receptors, is directly involved in the regulation of the behavior and psycho-emotional state of rats under specific lighting conditions, which are often extreme and harmful for their health.

Nickel-Catalyzed Regioselective C(2)-H Difluoroalkylation of Indoles with Difluoroalkyl Bromides.

Regioselective C(2)-H difluoroalkylation of C-3 unsubstituted indoles with commonly available fluoroalkyl bromides is successfully achieved employing a simple nickel catalyst system, (DME)NiCl2 /Xantphos. This methodology shows excellent regioselectivity and exhibits a broad substrate scope. Various functional groups, such as -OMe, -F, and -Br, are tolerated on the indole backbone to give the difluoroalkylated products in moderate to good yields. Preliminary mechanistic findings demonstrate that the reaction is homogeneous in nature and involves a radical manifold. Synthetic utility of this nickel-catalyzed method is demonstrated by synthesizing melatonin receptor antagonist Luzindole derivative.

The Role of Melatonin In Exercise Enhanced Endothelium-dependent Vasorelaxation In Mesenteric Arteries of SHR

PURPOSE: To determine if melatonin plays an important role in the hypotensive effects of exercise training. Further, to study the role of melatonin binding to melatonergic receptors in the vasorelaxation of small MAs in SHR. METHODS: Twelve-week-old male normotensive Wistar-Kyoto rats (WKY, n=18) and SHRs (n = 48) were used. SHRs were randomly divided into four groups: SHR sedentary group (SHR-SED, n = 18), SHR sedentary with melatonin receptor antagonist luzindole (Luz) injection group (SHR-SED + Luz, n = 6), SHR exercise group (SHR- EX, n = 18) , and SHR exercise with luzindole injection group (SHR-EX + Luz, n = 6). Luzindole injection groups were injected intraperitoneally (i.p.) with luzindole (1 mg/ kg/day in sterile saline) each day. Rats in the exercise groups were subjected to aerobic exercise. Blood pressure and heart rate were measured after exercise. Serum melatonin levels were examined by ELISA. The mechanical properties of small mesenteric arteries were studied with myograph. Western blot and immunofluorescence colocalization were performed to examine the protein expression and distribution of MT1, MT2 receptors and eNOS. RESULTS: Exercise training produced a significant reduction in blood pressure and heart rate in SHR, which was significantly attenuated by intraperitoneal administration of luzindole, a nonselective melatonin receptor (MT1/MT2) antagonist. Serum melatonin levels in the SHR group were significantly lower than those in the WKY group at 8:00-9:00 and 21:00-22:00, and exercise training reduced this difference. Endothelium-dependent vessel relaxation induced by acetylcholine was significantly blunted in SHR compared with age-matched WKY. Both exercise training and luzindole ameliorated this endothelium-dependent impairment of relaxation in hypertension. Immunohistochemistry and Western blotting showed that protein expression of the MT2 receptor and eNOS, as well as their colocalization in the endothelial cell layer in SHR, was significantly decreased; exercise training suppressed this reduction. CONCLUSIONS: These results provide evidence that regular exercise has a beneficial effect on improving endothelium-dependent vasorelaxation in MAs, in which melatonin plays a critical role by acting on MT2 receptor to increase NO production and/or NO bioavailability.

Melatonin mediates vasodilation through both direct and indirect activation of BKCa channels.

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BKCa channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of Nω-nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC50 The effect of melatonin was significantly attenuated in the presence of BKCa channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BKCa channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BKCa channels on mesenteric arterial myocytes.

Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells

The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells.

Nickel-Catalyzed C(sp2)–H/C(sp3)–H Oxidative Coupling of Indoles with Toluene Derivatives

Nickel-catalyzed oxidative C(sp2)–H/C(sp3)–H coupling of indoles with toluene derivatives is successfully achieved in the presence of 2-iodobutane as the oxidant. This method allows the selective C-2 benzylation of indoles with toluene derivatives over the alkylation with 2-iodobutane and permits the coupling of diversified indoles via the monochelation assistance. The reaction proceeded through a single-electron-transfer (SET) process, wherein both the C–H nickelation of indole and the C–H activation of toluene derivatives have a significant effect on the entire reaction rate. The synthetic utility of this nickel-catalyzed protocol is demonstrated by the facile removal of the directing group and by the convenient synthesis of the melatonin receptor antagonist Luzindole derivatives.

Ramelteon Improves Post-traumatic Stress Disorder-Like Behaviors Exhibited by Fatty Acid-Binding Protein 3 Null Mice.

We previously reported that fatty acid-binding protein 3 (FABP3) knockout (Fabp3 -/-) mice exhibit abnormal dopamine-related behaviors such as enhanced dopamine D2 receptor antagonist-induced catalepsy behaviors. Here, we report that Fabp3 null mice exhibit cognitive deficits, hyperlocomotion and impaired fear extinction, and thus show post-traumatic stress disorder (PTSD)-like behaviors. Notably, chronic administration of ramelteon (1.0mg/kg, p.o.), a melatonin receptor agonist, improved all PTSD-like behaviors tested in Fabp3 -/- mice. Relevant to mechanisms underlying impaired fear extinction, we observed significantly reduced levels of Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation without changes in ERK phosphorylation in the anterior cingulate cortex (ACC). Inversely, CaMKII autophosphorylation increased in the basolateral amygdala (BLA) but remained relatively unchanged in hippocampus of Fabp3 -/- mice. Likewise, the number of c-Fos-positive neurons in BLA significantly increased after exposure to contextual fear conditions but remained unchanged in the central nucleus of the amygdala (CeA). Importantly, chronic ramelteon administration (1.0mg/kg, p.o.) restored abnormal c-Fos expression and CaMKII autophosphorylation in the ACC and BLA of Fabp3 -/- mice. Finally, the melatonin receptor antagonist luzindole (2.5mg/kg, i.p.) blocked ramelteon-dependent improvements. Taken together, Fabp3 -/- mice show PTSD-like behaviors, and ramelteon is a likely attractive candidate for PTSD therapy.

Mechanism of salutary effects of melatonin-mediated liver protection after trauma-hemorrhage: p38 MAPK-dependent iNOS/HIF-1α pathway.

Although melatonin attenuates the increases in inflammatory mediators and reduces organ injury during trauma-hemorrhage, the mechanisms remain unclear. This study explored whether melatonin prevents liver injury after trauma-hemorrhage through the p38 mitogen-activated protein kinase (MAPK)-dependent, inducible nitrite oxide (iNOS)/hypoxia-inducible factor (HIF)-1α pathway. After a 5-cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure ~40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), melatonin plus p38 MAPK inhibitor SB203580 (2 mg/kg), or melatonin plus the melatonin receptor antagonist luzindole (2.5 mg/kg). At 2 h after trauma-hemorrhage, histopathology score of liver injury, liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and asparate aminotransferase levels were significantly increased compared with sham-operated control. Trauma-hemorrhage resulted in a significant decrease in the p38 MAPK activation compared with that in the sham-treated animals. Administration of melatonin after trauma-hemorrhage normalized liver p38 MAPK phosphorylation and iNOS and HIF-1α expression and attenuated cleaved caspase 3 and receptor interacting protein kinase-1 levels. Coadministration of SB203580 or luzindole abolished the melatonin-mediated attenuation of the trauma-hemorrhage-induced increase of iNOS/HIF-1α protein expression and liver injury markers. Taken together, our results suggest that melatonin prevents trauma-hemorrhage-induced liver injury in rats, at least in part, through melatonin receptor-related, p38 MAPK-dependent iNOS/HIF-1α pathway.NEW & NOTEWORTHY Trauma-hemorrhage resulted in a significant decrease in liver p38 MAPK activation and increase in nitrite oxide synthase (iNOS) and hypoxia-inducible factor (HIF)-1α expression. Administration of melatonin after trauma-hemorrhage normalized liver p38 MAPK phosphorylation and iNOS and HIF-1α expression, which was abolished by coadministration of SB203580 or luzindole. Melatonin prevents trauma-hemorrhage-induced liver injury in rats via the melatonin receptor-related, p38 MAPK-dependent iNOS/HIF-1α pathway.

Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.

Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

Melatonin receptor activation increases glutamatergic synaptic transmission in the rat medial lateral habenula.

Melatonin (MLT) is secreted from the pineal gland and mediates its physiological effects through activation of two G protein-coupled receptors, MT1 and MT2 . These receptors are expressed in several brain areas, including the habenular complex, a pair of nuclei that relay information from forebrain to midbrain and modulate a plethora of behaviors, including sleep, mood, and pain. However, so far, the precise mechanisms by which MLT control the function of habenula neurons remain unknown. Using whole cell recordings from male rat brain slices, we examined the effects of MLT on the excitability of medial lateral habenula (MLHb) neurons. We found that MLT had no significant effects on the intrinsic excitability of MLHb neurons, but profoundly increased the amplitude of glutamate-mediated evoked excitatory post-synaptic currents (EPSC). The increase in strength of glutamate synapses onto MLHb neurons was mediated by an increase in glutamate release. The MLT-induced increase in glutamatergic synaptic transmission was blocked by the competitive MT1 /MT2 receptor antagonist luzindole (LUZ). These results unravel a potential cellular mechanism by which MLT receptor activation enhances the excitability of MLHb neurons. The MLT-mediated control of glutamatergic inputs to the MLHb may play a key role in the modulation of various behaviors controlled by the habenular complex.

Melatonin Attenuates Early Brain Injury via the Melatonin Receptor/Sirt1/NF-κB Signaling Pathway Following Subarachnoid Hemorrhage in Mice.

Melatonin (Mel) has been reported to alleviate early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of silent information regulator 1 (Sirt1), a histone deacetylase, has been suggested to be beneficial in SAH. However, the precise role of Sirt1 in Mel-mediated protection against EBI following SAH has not been elucidated. The present study aims to evaluate the role of melatonin receptor/Sirt1/nuclear factor-kappa B (NF-κB) in this process. The endovascular perforation SAH model was used in male C57BL/6J mice, and melatonin was administrated intraperitoneally (150mg/kg). The mortality, SAH grade, neurological score, brain water content, and neuronal apoptosis were evaluated. The expression of Sirt1, acetylated-NF-κB (Ac-NF-κB), Bcl-2, and Bax were detected by western blot. To study the underlying mechanisms, melatonin receptor (MR) antagonist luzindole and Sirt1 small interfering RNA (siRNA) were administrated to different groups. The results suggest that Mel improved the neurological deficits and reduced the brain water content and neuronal apoptosis. In addition, Mel enhanced the expression of Sirt1 and Bcl-2 and decreased the expression of Ac-NF-κB and Bax. However, the protective effects of Mel were abolished by luzindole or Sirt1 siRNA. In conclusion, our results demonstrate that Mel attenuates EBI following SAH via the MR/Sirt1/NF-κB signaling pathway.

A Dual Role for Melatonin in Medaka Ovulation: Ensuring Prostaglandin Synthesis and Actin Cytoskeleton Rearrangement in Follicular Cells.

Understanding the direct effects of melatonin on vertebrate ovulation remains a challenge. The present study provides the first characterization of the role of melatonin in ovulation using the teleost medaka. The melatonin receptor antagonist luzindole inhibited in vitro follicle ovulation. In the preovulatory follicles, arylalkylamine N-acetyltransferase 1a and hydroxyindole-O-methyltransferase 2, the enzymes responsible for melatonin synthesis, were expressed in the granulosa cells throughout the 24 h spawning cycle. The granulosa cells of the follicle also expressed the melatonin receptor 1a-a. An in vitro characterization study using medaka OLHNI-2 cells revealed that melatonin and luzindole act as an agonist and an antagonist, respectively, of the melatonin receptor. The intracellular cAMP levels in these cells were reduced after melatonin treatment. The expression of cytosolic phospholipase A2 group 4a (Pla2g4a), the enzyme producing arachidonic acid (cyclooxygenase-2 substrate), was inhibited in the granulosa cells in luzindole-treated follicles. Follicular prostaglandin E2 levels and in vitro follicle ovulation were suppressed in follicles isolated at 12 h prior to ovulation and incubated with the Pla2g4a inhibitor AACOCF3. The G-actin:F-actin ratios in follicular cells increased with approaching ovulation, but this increase was suppressed after luzindole treatment. The phosphorylation of moesin, an ezrin-radixin-moesin protein, was inhibited in the follicular cells in luzindole-treated follicles. These results indicate a dual role for melatonin in medaka ovulation: melatonin ensures prostaglandin E2 synthesis throughout the spawning cycle and induces actin cytoskeleton rearrangement in the follicular cells at ovulation.

Melatonin receptor signaling contributes to neuroprotection upon arousal from torpor in thirteen-lined ground squirrels.

The brain of mammalian hibernators is naturally protected. Hibernating ground squirrels undergo rapid and extreme changes in body temperature and brain perfusion as they cycle between lengthy torpor bouts and brief periods of euthermia called interbout arousals (IBAs). Arousal from torpor to IBA occurs rapidly, but there is no evidence of brain injury accompanying this extreme physiological transition. Production of the hormone melatonin accompanies arousal, suggesting that it plays a protective role at this time. Here, we investigated mechanisms of melatonin receptor-mediated protection in the brain of the hibernating ground squirrel. We administered the competitive melatonin receptor antagonist luzindole (30 mg/kg ip) to ground squirrels at the predicted end of a torpor bout, triggering an arousal. We found that luzindole-treated animals exhibited caspase-3 activity two times higher than vehicle-treated animals in the hypothalamus at midarousal (P = 0.01), suggesting that melatonin receptor signaling is important for protection in this brain region. We also found a 30% decline in succinate-fueled mitochondrial respiration in luzindole-treated animals compared with vehicle-treated animals (P = 0.019), suggesting that melatonin receptor signaling is important for optimal mitochondrial function during arousal from torpor. The mitochondrial effects of luzindole treatment were seen only during the hibernation season, indicating that this effect is specifically important for arousal from torpor. These data provide evidence for the protective role of melatonin receptor signaling during the extreme physiological transition that occurs when a hibernating mammal arouses from torpor and provide further evidence for regional and seasonal changes in the hibernator brain.

Abstract 3573: Novel G protein-coupled receptor targets in HER2+ breast cancer

Abstract Background: G protein-coupled receptors (GPCRs) are excellent drug targets. However, not many GPCRs have been identified as potential drug targets in breast cancer (BC). This may be due to two factors: (1) The primary mode of GPCR regulation is at the level of signaling rather than gene expression, which is the common method for large-scale target identification. (2) GPCR expression in BC stem cells or drug-resistant cells could have been overlooked because these cells exist in small fractions. In this study, we aimed to identify novel GPCR targets based on large panel functional screening and differential gene expression in HER2+ BC stem cells and drug resistant cells. Methods: (I) For the functional screening, each of the 370 GPCR genes was knocked down using targeted Silencer® Select siRNA library comprising of 3 individual siRNAs per target in BT474 and SKBR3 cell lines (N = 3). Candidate GPCRs were selected based on at least 20% alteration in cell growth by at least 2 out of 3 siRNAs. (II) For the expression screening, gene expression of 343 GPCRs was measured in ALDH+ BC stem cells vs. ALDH- cells as well as in anti-HER2 drug-resistant derivatives vs. parental cells of BT474 cells using Taqman RT-PCR arrays (N = 2-4). Melatonin MT1 receptor (MTNR1A) gene knockdown and pharmacological modulation were employed to functionally validate its role in anchorage-dependent (MTT assay) and -independent (soft agar colony formation assay) cell growth. Results: Gene knockdown of 11 GPCRs (ADORA, ADRA1D, CCR8, GNRHR, GPR26, GPR113, GPR120, LPAR3, MC5R, MTNR1A, NTSR2) altered cell growth of HER2+ BC cells by at least 20%. The MT1 receptor was selected as a candidate GPCR for this study because (1) its knockdown resulted in at least 20% inhibition of cell growth in both BT474 and SKBR3 cells, and (2) its expression was increased in ALDH+ vs. ALDH- as well as in drug-resistant cells vs. parental BT474 cells. MTNR1A knockdown resulted in 25-35% inhibition in the anchorage-dependent cell growth of both BT474 and SKBR3 cells. Furthermore, MTNR1A knockdown caused a marked (3-fold) decrease in the number of colonies in soft agar assay. MT1 receptor antagonist luzindole reduced the anchorage-dependent cell growth of BT474 cells in a concentration-dependent manner (IC50 = 20-55 nM). However, the efficacy of luzindole was only ∼50% of lapatinib efficacy in reducing cell growth. Luzindole (1μM) also significantly reduced (by 2-fold) anchorage-independent cell growth of BT474 cells. Neither MTNR1A knockdown nor luzindole significantly improved the efficacy of lapatinib (1nM) in inhibiting cell growth of BT474 cells. Conclusion: In summary, functional knockdown screening and differential gene expression of GPCRs is a powerful tool for identifying GPCRs as candidate targets in BC. Using the combination of these two approaches, we have discovered that melatonin MT1 receptor as a potential drug target in HER2+ BC cells. The place in therapy for MT1 receptor antagonists, however, still remains to be determined. Citation Format: Raksha Bhat, Puja Yadav, Pavel Christiny, Rachel Schiff, Meghana V. Trivedi. Novel G protein-coupled receptor targets in HER2+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2015-3573

Melatonin modulates M4-type ganglion-cell photoreceptors

In the retina, melatonin is secreted at night by rod/cone photoreceptors and serves as a dark-adaptive signal. Melatonin receptors have been found in many retinal neurons including melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), suggesting it could modulate the physiology of these inner retinal photoreceptors. Here, we investigated whether melatonin modulates the alpha-like M4-type ipRGCs, which are believed to mediate image-forming vision as well as non-image-forming photoresponses. Applying melatonin during daytime (when endogenous melatonin secretion is low) caused whole-cell-recorded M4 cells’ rod/cone-driven depolarizing photoresponses to become broader and larger, whereas the associated elevation in spike rate was reduced. Melanopsin-based light responses were not affected significantly. Nighttime application of the melatonin receptor antagonist luzindole also altered M4 cells’ rod/cone-driven light responses but in the opposite ways: the duration and amplitude of the graded depolarization were reduced, whereas the accompanying spiking increase was enhanced. These luzindole-induced changes confirmed that M4 cells are modulated by endogenous melatonin. Melatonin could induce the above effects by acting directly on M4 cells because immunohistochemistry detected MT1 receptors in these cells, although it could also act presynaptically. Interestingly, the daytime and nighttime recordings showed significant differences in resting membrane potential, spontaneous spike rate and rod/cone-driven light responses, suggesting that M4 cells are under circadian control. This is the first report of a circadian variation in ipRGCs’ resting properties and synaptic input, and of melatoninergic modulation of ipRGCs.

Environmental Circadian Modulators Target Melatonin Receptors in Pancreatic β‐Cells

In silico 3D modelling and 2-[125I]-iodomelatonin binding to human MT1 and MT2 melatonin receptors identified the carbamate insecticides, carbaryl and carbofuran, as potential melatonin receptor circadian modulators (M P-G et al., EB Abstract, 2014). In humans, signaling through MT1 and MT2 melatonin receptors modulates insulin secretion from pancreatic β-cells (Int J Mol Sci 14: 6981, 2013). This study determined the pharmacological profile of melatonin receptors and the effect of carbamate insecticides in rat insulinoma cells (INS-1), a model for pancreatic β-cells. Saturation binding with 2-[125I]-Iodomelatonin (1 to 1000 pM) to INS-1 cell membranes yielded a KD of 50.2 ± 13.2 pM and a Bmax of 10.8 ± 2.8 fmol/mg protein (n=5). Competition binding of melatonin and the competitive melatonin receptor antagonist luzindole for 2-[125I]-iodomelatonin binding to INS-1 cell membranes yielded Ki of 83 ± 18 pM (n=9) and 0.50 ± 0.14 µM (n=4), respectively. The affinity of luzindole and the reported low to undetectable levels of MT2 receptor expression in INS-1 cells, suggest that 2-[125I]-iodomelatonin binds primarily to MT1 receptors. The Ki for carbaryl was 2.59 ± 0.54 µM (n=7) while concentrations as high as 1 mM of carbofuran did not compete for 2-[125I]-iodomelatonin binding to INS-1 cell membranes. The presence of GTP (100 µM) did not alter the competition of carbaryl for 2-[125I]-iodomelatonin binding (Ki = 5.39 ± 0.19 µM, n=4). These results suggest that carbaryl may potentially block melatonin receptors in INS-1 cells. Supported by ES 023684 to MLD and RVR.

A New Role for 5‐methoxytryptophol On Bone Cells Function in Vitro

The present study investigates the direct action of 5-methoxytryptophol (5-MTX) in both MC3T3-E1 and RAW264.7 cells and compares it with melatonin (MEL), another 5-methoxyindol known to play a significant role on bone metabolism. We first screened increasing doses of both 5-MTX and MEL to determine their effect on metabolic activity and viability of preosteoblastic MC3T3-E1 cells. The optimal dose was used to determine its effect on differentiation of MC3T3-E1 cells and preosteoclastic RAW264.7 cells. Finally, we investigated the mechanism of action by adding the melatonin receptor antagonist luzindole (LUZ) and detecting the immunostaining of phospho-ERK. In MC3T3-E1 cells, most of the 5-MTX doses reduced slightly the metabolic activity of osteoblasts compared with the control, while MEL only decreased it for the highest dose (2.5 mM). As regards to cytotoxicity, low doses (0.001-0.1 mM) of both indoles showed a protective effect on osteoblasts, while the highest dose of MEL showed a higher cytotoxicity than the 5-MTX one. After 14 days of cell culture, Rankl mRNA levels were decreased, especially for 5-MTX. 5-MTX also induced a higher osteocalcin secretion and mineralization capacity than MEL. In RAW264.7 cells, 5-MTX decreased the number of osteoclast formed and its activity whereas MEL did not affect significantly the number of multinucleated TRAP-positive cells formed and showed a lower activity. Finally, MEL and 5-MTX promoted activation of the ERK1/2 pathway through the phosphorylation of ERK, while LUZ addition suppressed this effect. In conclusion, the present study demonstrates a new role of 5-MTX inhibiting osteoclastogenesis and promoting osteoblast differentiation.

Melatonin influences somatostatin secretion from human pancreatic δ-cells via MT1 and MT2 receptors.

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor-transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α-cells. Virtually, nothing is known concerning the melatonin-mediated effects on islet δ-cells. Analysis of a human pancreatic δ-cell model, the cell line QGP-1, and the use of a somatostatin-specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ-cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose-dependently stimulating somatostatin secretion, in experiments employing the membrane-permeable 8-Br-cAMP. 8-Br-cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1nm melatonin was reversed after incubation of QGP-1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2-selective antagonist 4-P-PDOT (4-phenyl-2-propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ-cells at low glucose concentrations was significantly inhibited during co-incubation with 1nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ-cells and on somatostatin release.

Effects of melatonin on the proliferation and apoptosis of sheep granulosa cells under thermal stress.

The cross-talk between oocyte and somatic cells plays a crucial role in the regulation of follicular development and oocyte maturation. As a result, granulosa cell apoptosis causes follicular atresia. In this study, sheep granulosa cells were cultured under thermal stress to induce apoptosis, and melatonin (MT) was examined to evaluate its potential effects on heat-induced granulosa cell injury. The results demonstrated that the Colony Forming Efficiency (CFE) of granulosa cells was significantly decreased (heat 19.70% ± 1.29% vs. control 26.96% ± 1.81%, p < 0.05) and the apoptosis rate was significantly increased (heat 56.16% ± 13.95%vs. control 22.80% ± 12.16%, p < 0.05) in granulosa cells with thermal stress compared with the control group. Melatonin (10−7 M) remarkably reduced the negative effects caused by thermal stress in the granulosa cells. This reduction was indicated by the improved CFE and decreased apoptotic rate of these cells. The beneficial effects of melatonin on thermal stressed granulosa cells were not inhibited by its membrane receptor antagonist luzindole. A mechanistic exploration indicated that melatonin (10−7 M) down-regulated p53 and up-regulated Bcl-2 and LHR gene expression of granulosa cells under thermal stress. This study provides evidence for the molecular mechanisms of the protective effects of melatonin on granulosa cells during thermal stress.