MT2-like melatonin receptor modulates amplitude receptor potential in visual cells of crayfish during a 24-hour cycle

Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2 h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT₂ melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT₂ receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [¹²⁵I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [¹²⁵I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (K_d) of 1.1 nM and a total number of binding sites (B_max) of 6 fmol/mg protein at CT 6, and a K_d of 1.46 nM and B_max of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT₂-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells.     

Therapeutic Potential of Melatonin Ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT₁ and MT₂ receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT₁ and MT₂ agonist and 5‐HT_2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to “protective” effects and (2) that over‐expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia—effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.     

Melatonin Signaling Controls the Daily Rhythm in Blood Glucose Levels Independent of Peripheral Clocks

Melatonin is rhythmically secreted by both the pineal gland and retina in a circadian fashion, with its peak synthesis occurring during the night. Once synthesized, melatonin exerts its effects by binding to two specific G-protein coupled receptors–melatonin receptor type 1(MT₁) and melatonin receptor type 2(MT₂). Recent studies suggest the involvement of MT₁ and MT₂ in the regulation of glucose homeostasis; however the ability of melatonin signaling to impart timing cues on glucose metabolism remains poorly understood. Here we report that the removal of MT₁ or MT₂ in mice abolishes the daily rhythm in blood glucose levels. Interestingly, removal of melatonin receptors produced small effects on the rhythmic expression patterns of clock genes within skeletal muscle, liver, and adipose tissue. Taken together, our data suggest that the loss of the daily rhythm in blood glucose observed in MT₁^-/- and MT₂^-/- mice does not occur as a consequence of ‘disrupted’ clocks within insulin sensitive tissues. Finally our results highlight a diurnal contribution of melatonin receptor signaling in the daily regulation of blood glucose levels.     

Role of the MT1 and MT2 melatonin receptors in mediating depressive‐ and anxiety‐like behaviors in C3H/HeN mice

Melatonin is a neurohormone primarily synthesized by the pineal gland following a circadian rhythm with a high level during the night and a low level during the day. Alterations in the synthesis and secretion of melatonin have been reported in various mood disorders, including major depressive disorder. However, the role of endogenous melatonin in the pathophysiology of depressive disorder is unclear. Melatonin primarily acts through two G protein‐coupled receptors, termed MT₁ and MT₂. The present study investigated the effect of genetic deletion of the MT₁ and/or MT₂ receptors on tests associated with depression‐ and anxiety‐like behaviors in C3H/HeN mice. Deletion of the MT₁ and/or MT₂ receptors caused a deficit in hedonic and social interaction behavior, and increased anxiety‐like behavior. It is likely that dysregulations of the MT₁ and/or MT₂ melatonin receptors could be involved in the pathophysiology of depression and anxiety.     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

The Presence and Localization of Melatonin Receptors in the Rat Aorta

Melatonin is involved in blood pressure modulation in rats and humans. Some of the effects of melatonin are presumably mediated via two G-protein-coupled receptors (MT₁ and MT₂), but the distribution of MT₁ and MT₂ in the cardiovascular system remains to be explored comprehensively. We investigated the expression of both the receptors in the rat aorta on mRNA level by RT-PCR and real time RT-PCR as well as on protein level via western blotting and immunofluorescence microscopy. We verified MT₁ mRNA expression in the rat aorta and demonstrated the absence of MT₂ mRNA in this vessel type. MT₁ receptors were confirmed also at the protein level, and surprisingly they were preferentially localized to the tunica adventitia. Since no daily changes in MT₁ mRNA expression were detected, we suppose that the circadian changes in circulating melatonin concentrations are sufficient to mediate circadian effects of melatonin in the aorta. The localization of MT₁ in the tunica adventitia suggests an influence of melatonin on vasa vasorum function and signal transduction in the aorta wall.     

Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT₁ and MT₂ receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatonin-related receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT₁-I49N, which is devoid of any melatonin binding and cell surface expression, and MT₁-G166E and MT₁-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, Δ502–505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients.     

Design,synthesis and pharmacological evaluation of novel naphthalenic derivatives as selective MT1 melatoninergic ligands

Novel heterodimer analogues of melatonin were synthesized, when agomelatine (1) and various aryl units are linked via a linear alkyl chain through the methoxy group. The compounds were tested for their actions at melatonin receptors. Several of these ligands are MT₁-selective with nanomolar or subnanomolar affinity. In addition, while most of the derivatives behave as partial agonists on one or both receptor subtypes, N-[2-(7-{4-[6-(1-methoxycarbonylethyl)naphthalen-2-yloxy]butoxy}naphthalen-1-yl)ethyl]acetamide (36), a subnanomolar MT₁ ligand with an 11-fold preference over MT₂ receptors, is a full antagonist on both receptors. Our results also confirm that the selectivity seen for the MT₁ receptor arises predominantly from steric factors and is not a consequence of the bridging of melatonin receptor dimers.     

Human Gastroenteropancreatic Expression of Melatonin and Its Receptors MT1 and MT2

Background and Aim

The largest source of melatonin, according to animal studies, is the gastrointestinal (GI) tract but this is not yet thoroughly characterized in humans. This study aims to map the expression of melatonin and its two receptors in human GI tract and pancreas using microarray analysis and immunohistochemistry.

Method

Gene expression data from normal intestine and pancreas and inflamed colon tissue due to ulcerative colitis were analyzed for expression of enzymes relevant for serotonin and melatonin production and their receptors. Sections from paraffin-embedded normal tissue from 42 individuals, representing the different parts of the GI tract (n=39) and pancreas (n=3) were studied with immunohistochemistry using antibodies with specificity for melatonin, MT₁ and MT₂ receptors and serotonin.

Results

Enzymes needed for production of melatonin are expressed in both GI tract and pancreas tissue. Strong melatonin immunoreactivity (IR) was seen in enterochromaffin (EC) cells partially co-localized with serotonin IR. Melatonin IR was also seen in pancreas islets. MT₁ and MT₂ IR were both found in the intestinal epithelium, in the submucosal and myenteric plexus, and in vessels in the GI tract as well as in pancreatic islets. MT₁ and MT₂ IR was strongest in the epithelium of the large intestine. In the other cell types, both MT₂ gene expression and IR were generally elevated compared to MT₁. Strong MT₂, IR was noted in EC cells but not MT₁ IR. Changes in gene expression that may result in reduced levels of melatonin were seen in relation to inflammation.

Conclusion

Widespread gastroenteropancreatic expression of melatonin and its receptors in the GI tract and pancreas is in agreement with the multiple roles ascribed to melatonin, which include regulation of gastrointestinal motility, epithelial permeability as well as enteropancreatic cross-talk with plausible impact on metabolic control.     

Are G Protein‐Coupled Receptor Heterodimers of Physiological Relevance?—Focus on Melatonin Receptors

In mammals, the circadian hormone melatonin targets two seven‐transmembrane–spanning receptors, MT₁ and MT₂, of the G protein‐coupled receptor (GPCR) super‐family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT₁ and MT₂ homodimers and MT₁/MT₂ heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT₁/MT₂ heterodimers remains to be shown in native tissues but is suggested by the documented co‐expression of MT₁ and MT₂ in many melatonin‐sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.     

Polybenzyls from Gastrodia elata,their agonistic effects on melatonin receptors and structure-activity relationships

Gastrodia elata is a famous traditional Chinese herb with medicinal and edible application. In this study, nine polybenzyls (1?9), including six new ones (2?5, 7 and 9), were isolated from the EtOAc extract of G. elata. Five compounds 1, 3, 4, 6 and 8 were found to activate melatonin receptors. Especially, compound 1 showed agonistic effects on MT₁ and MT₂ receptors with EC₅₀ values of 237 and 244 μM. For better understanding their structure-activity relationships (SARs), ten polybenzyl analogs were further synthesized and assayed for their activities on melatonin receptors. Preliminary SARs study suggested that two para-hydroxy groups were the key pharmacophore for maintaining activity. Molecular docking simulations verified that compound 1 could strongly interact with MT₂ receptor by bonding to Phe 118, Gly 121, His 208, Try 294 and Ala 297 residues.     

Study of the effects of the casein derived bitter tastant on the melanophores in milieu with the melatonin receptors

The present study was undertaken to ascertain whether the casein derived bitter tastant Cyclo (Leu-Trp) [CLT] has an affinity or not for the particular receptors of the pineal hormone, melatonin, on the melanophores of a major carp Labeo rohita (Ham.). The bitter tastant CLT, in the dose range of 3.34?×?10^?16 M to 3.34?×?10^?4 M, has induced an aggregatory effect but not in a dose dependent manner. Binding of CLT with the receptors may vary at different concentrations. Denervation of the melanophores has shown a complete inhibition of the CLT mediated aggregation. Prazosin has partially inhibited the aggregatory effect of CLT. Moreover, the bitter tastant’s response is mediated through the α₂ adrenoceptors only at particular dose ranges. The MT₁ and MT₂ melatonin receptor antagonist luzindole and the MT₂ specific antagonist K185 have perfectly blocked the aggregatory effects of CLT. We have found that the CLT mediated aggregatory effect is dependent upon the release of neurotransmitters and the two subtypes of melatonin (MT) receptors (MT₁ and MT₂) possess a perfect affinity towards the bitter tastant CLT. Our study demands a need to further make a clinical research on the effects of bitter tastants on the physiology of the biological rhythm maintaining hormone melatonin.     

Design and synthesis of 3-phenyltetrahydronaphthalenic derivatives as new selective MT2 melatoninergic ligands. Part II

Following our studies of the melatoninergic receptors, we have developed new tetrahydronaphthalenic derivatives of melatonin that have been tested as selective melatonin receptors ligands. Regarding the role of the phenyl substituent to obtain selective ligands, modulation of selectivity and activity have been achieved by modifications of the acyl group and substitutions on the phenyl ring. Ten of the seventeen evaluated derivatives have MT₂ receptor affinity similar to that of melatonin. Moreover, we have achieved remarkable MT₂ selectivity over MT₁ (selectivity >100) and have been able to further extend the RSA of the tetrahydrophthalenic series. However, the compounds presented here display partial agonist or antagonist behavior instead of full agonist.     

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y₁R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y₁R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09792-9.     

Synthesis of new melatoninergic hexahydroindenopyridines

Hexahydroindenopyridine (HHIP) is an interesting heterocyclic framework that contains an indene core similar to ramelteon. This type of tricyclic piperidines aroused our interest as potential melatoninergic ligands. Melatonin receptor ligands have applications in insomnia and depression. We report herein an efficient two-step method to prepare new HHIP by the reaction of an enamine with 3-bromopropylamine hydrobromide. Some synthesized compounds showed moderate affinity for melatonin receptors in the nanomolar or low micromolar range. Furthermore, the methylenedioxy HHIPs 2d (N-phenylacetamide) and 2f (N,N-diethylacetamide), exhibited high selectivity at MT₁ or MT₂ receptors, respectively, when compared with melatonin. It seems that the methylenedioxy group on the indene ring system and the N-acetamide substituent are important structural features to bind selectively MT₁ or MT₂ subtypes.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

Melatonin protects diabetic heart against ischemia-reperfusion injury,role of membrane receptor-dependent cGMP-PKG activation

It has been demonstrated that the anti-oxidative and cardioprotective effects of melatonin are, at least in part, mediated by its membrane receptors. However, the direct downstream signaling remains unknown. We previously found that melatonin ameliorated myocardial ischemia-reperfusion (MI/R) injury in diabetic animals, although the underlying mechanisms are also incompletely understood. This study was designed to determine the role of melatonin membrane receptors in melatonin's cardioprotective actions against diabetic MI/R injury with a focus on cGMP and its downstream effector PKG. Streptozotocin-induced diabetic Sprague-Dawley rats and high-glucose medium-incubated H9c2 cardiomyoblasts were utilized to determine the effects of melatonin against MI/R injury. Melatonin treatment preserved cardiac function and reduced oxidative damage and apoptosis. Additionally, melatonin increased intracellular cGMP level, PKGIα expression, p-VASP/VASP ratio and further modulated myocardial Nrf-2-HO-1 and MAPK signaling. However, these effects were blunted by KT5823 (a selective inhibitor of PKG) or PKGIα siRNA except that intracellular cGMP level did not changed significantly. Additionally, our in vitro study showed that luzindole (a nonselective melatonin membrane receptor antagonist) or 4P–PDOT (a selective MT₂ receptor antagonist) not only blocked the cytoprotective effect of melatonin, but also attenuated the stimulatory effect of melatonin on cGMP-PKGIα signaling and its modulatory effect on Nrf-2-HO-1 and MAPK signaling. This study showed that melatonin ameliorated diabetic MI/R injury by modulating Nrf-2-HO-1 and MAPK signaling, thus reducing myocardial apoptosis and oxidative stress and preserving cardiac function. Importantly, melatonin membrane receptors (especially MT₂ receptor)-dependent cGMP-PKGIα signaling played a critical role in this process.     

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT₁ and MT₂ receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.     

GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.