Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathway

Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1-100 micromol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P < 0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.     

A molecular perspective of the genetic relationships of G-protein coupled melatonin receptor subtypes

Abstract: Successful cloning of melatonin receptors from various target tissues in the past few years has increased our understanding of the molecular signal transduction mechanisms of G-protein coupled melatonin receptors, of which three subtypes (MEL-1A, MEL-1B, and MEL-1C) have been reported in different vertebrates. Based upon melatonin receptor sequences available in the Genbank database, we have performed phylogenetic analyses of the nucleotide and encoded amino acid sequences of G-protein-coupled melatonin receptors, and determined the range of amino acid identities between melatonin receptors of the same and different subtypes. Besides the three well-known subtypes, a potential novel subtype of MEL-1D, as exemplified by unique separation of Xenopus X2.0 sequence (Genbank accession No. U31826) from the others in the protein phylogenetic tree, possibly exists. In addition, one of the chicken brain melatonin receptor sequences has been identified as belonging to the MEL-1B subtype. Our analyses showed that melatonin receptors of the same subtype and different subtypes are likely to share >75% and <65% amino acid identities, respectively. Phylogenetic analysis based on amino acid comparisons will be needed to determine the subtype status of any pair of melatonin receptor sequences that exhibit ≥65% to <75% amino acid identity. Despite the usefulness of genetic relatedness in the subtype classification of G-protein-coupled melatonin receptors, functional correlation of molecular structure may ultimately prove the most comprehensive approach in melatonin receptor classification.     

Melatonin receptor potentiation of cyclic AMP and the cystic fibrosis transmembrane conductance regulator ion channel

We have used the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as a model system to study the cAMP signal transduction pathways coupled to the Xenopus melatonin receptor. During forskolin (Fsk) stimulation, melatonin reduced the amplitude of the CFTR currents in oocytes injected with in vitro transcribed cRNAs for the Xenopus melatonin receptor and CFTR. Pertussis toxin (Ptx) treatment eliminated melatonin inhibition of Fsk stimulated CFTR currents. In oocytes injected with cRNA for melatonin receptors, serotonin receptors (5-HT7), and CFTR Cl- channels, application of melatonin together with serotonin (5-HT) activated an additional inward current showing potentiation of adenylyl cyclases by melatonin receptors. Subthreshold activation of 5-HT7 receptors was sufficient and necessary to permit activation of CFTR channels by melatonin. Preexposure to melatonin desensitized the melatonin receptor mediated response. Therefore, based on this model system, the effects of melatonin in vivo could be either positive or negative modulation of other neuronal inputs, depending on the mode of adenylyl cyclase stimulation.     

Minireview: Cell protective role of melatonin in the brain

Abstract: In recent years an increasing amount of data has been published involving melatonin in the control of brain function. The pineal gland exerts a depressive influence on CNS excitability. This activity is linked to melatonin, since pharmacological doses of the hormone prevent seizures in several animal models. In addition, melatonin also has analgesic properties in these species. However, the sites and mechanism of melatonin action are not known. A role for the pineal gland and its hormone melatonin as a homeostatic system controlling brain excitability has been proposed, and GABA-containing neurons may be involved in some central action of melatonin. There is evidence supporting a role of melatonin in the regulation of the GABA-benzodiazepine receptor complex, and it appears that melatonin potentiates this inhibitory neurotransmitter system in brain. Melatonin does not bind to GABA or benzodiazepine binding sites themselves, because in vitro binding data showed that melatonin is a weak competitor of benzodiazepine binding in brain membranes at concentrations greater than 10^?5 M. The effect of melatonin on brain activity also involves the participation of corticotropic and opioid peptides, and the existence of an opioid-antiopioid homeostatic system is proposed, with the GABA-benzodiazepine receptor complex as an effector. Moreover, the interaction of melatonin with corticotropic peptides and mitochondrial benzodiazepine receptors may result in a participation of neurosteroids in the control of GABA activity and function. The most recently available data from biochemical and electrophysiological studies support the possibility that the anticonvulsant and depressive effects of melatonin on neuron activity may depend on its antioxidant and antiexcitotoxic roles, acting as a free radical scavenger and regulating brain glutamate receptors. The full characterization of the nuclear melatonin receptor explains the genomic effects of melatonin, opening a new perspective regarding actions and roles of melatonin as a cellular protector.     

Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation

Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re‐oxygenation‐induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pm –1 mm ) increases the fusion index (syncytium formation; 21% augmentation at 1 mm melatonin vs. vehicle) and β‐hCG secretion (121% augmentation at 1 mm melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well‐being.     

Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). Cell bodies and terminals containing the dodecapeptide GnIH are localized in the paraventricular nucleus (PVN) and median eminence, respectively. To understand the physiological role of GnIH, we investigated the mechanisms that regulate GnIH expression. In this study, we show that melatonin originating from the pineal gland and eyes induces GnIH expression in the quail brain. Pinealectomy (Px) combined with orbital enucleation (Ex) (Px plus Ex) decreased the expression of GnIH precursor mRNA and content of mature GnIH peptide in the diencephalon, which includes the PVN and median eminence. Melatonin administration to Px plus Ex birds caused a dose-dependent increase in expression of GnIH precursor mRNA and production of mature peptide. The expression of GnIH was photoperiodically controlled and increased under short-day photoperiods, when the duration of melatonin secretion increases. To identify the mode of melatonin action on GnIH induction, we investigated the expression of Mel(1c), a melatonin receptor subtype, in GnIH neurons. In situ hybridization of Mel(1c) mRNA combined with immunocytochemistry for GnIH revealed that Mel(1c) mRNA was expressed in GnIH-immunoreactive neurons in the PVN. Melatonin receptor autoradiography further revealed specific binding of melatonin in the PVN. These results indicate that melatonin is a key factor for GnIH induction. Melatonin appears to act directly on GnIH neurons through its receptor to induce GnIH expression. This is the first demonstration, to our knowledge, of a direct action of melatonin on neuropeptide induction in any vertebrate class.     

Characterization and messenger ribonucleic acid distribution of a cloned pituitary adenylate cyclase-activating polypeptide type I receptor in the frog Xenopus laevis brain

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to modulate neuroendocrine functions in the frog brain and pituitary, but the nucleotide sequence and brain distribution of messenger RNA (mRNA) for the selective type I receptor for PACAP (PAC1) in the frog are still unknown. Here, we report the isolation and characterization of a PAC1 receptor complementary DNA (cDNA) clone from a frog (Xenopus laevis) tadpole brain cDNA library. This cDNA encoded a 466-amino acid protein that has 74% homology with human PAC1 receptor and 48% homology to the frog vasoactive intestinal peptide/PACAP receptor. Injection of in vitro synthesized mRNA of the cloned cDNA into Xenopus oocytes resulted in expression of selective high affinity PACAP receptors (Kd = 47 pM). IC50 values for PACAP-38, PACAP-27, and VIP were 27 pM, 110 pM and >1 microM, respectively. These results indicated that the cloned cDNA represents a Xenopus PACAP-preferring PAC1 receptor. Northern hybridization revealed that PAC1 receptor mRNA was present at high levels in the brain. In situ hybridization showed that the PAC1 receptor gene was expressed highly in the pallium, preoptic nucleus, and nucleus of cerebellum, and moderately in the Purkinje cell layer of the cerebellum. Moderate PAC1 receptor mRNA signals were detected in the distal lobe of the pituitary. A knowledge of the molecular structure and expression pattern of the PAC1 receptor will facilitate further investigation of the physiological roles of PAC1 receptor in the frog brain.     

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aβ) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aβ levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.     

Melatonin induces alterations in protein expression in the Xenopus laevis retina

The hormone melatonin is synthesized by pinealocytes and retinal photoreceptors with a diurnal rhythm. Melatonin produced in the retina at night is thought to exert local modulatory effects by binding to specific receptors in several different retinal cell types. The mechanisms by which melatonin influences circadian activity in retinal cells is poorly understood. Suppression of cyclic AMP synthesis appears to be a major signaling pathway in response to melatonin receptor binding in many tissues. A potential downstream consequence of melatonin-induced changes in cyclic AMP concentrations and protein phosphorylation is the up- or down-regulation of expression of specific genes. In this report, we examined the changes in expression levels of specific proteins in the neural retina and retinal pigment epithelium (RPE) in response to melatonin treatment, because both of these tissues express melatonin receptors. Neural retina and RPE isolated from the eyes of Xenopus laevis were treated with or without 1 microM melatonin for 6 hr, then the rapidly synthesized tissue proteins were radiolabeled by a 15 min incubation with 35S-methionine, and the proteins were subsequently analyzed by two-dimensional gel electrophoresis and autoradiography. In both the neural retina and RPE, the densities of some specific proteins were altered in response to melatonin treatment, and the few protein spots that were altered were distinct between the two tissues. These results support the concept that one function of melatonin may be to regulate the expression of specific genes and the consequent protein levels, and that the target genes may differ according to the cell or tissue type.     

Expression of cardiac Na channels with appropriate physiological and pharmacological properties in Xenopus oocytes.

The objective of this study was to determine whether the Xenopus laevis oocyte can express an exogenous cardiac Na channel that retains its normal physiological and pharmacological properties. Cardiac Na channels were expressed in oocytes following injection of RNA from guinea pig, rat, and human heart and detailed analysis was performed for guinea pig cardiac Na channels. Average current amplitudes were -351 +/- 37 nA with peak current observed at -8 +/- 1 mV. Steady-state inactivation was half-maximal at -49 +/- 0.6 mV for the expressed channels. All heart Na currents were resistant to block by tetrodotoxin compared to Na currents expressed from brain RNA with IC50 values for guinea pig, rat, and human heart of 651 nM, 931 nM, and 1.3 microM, respectively. In contrast, rat brain Na channels were blocked by tetrodotoxin with an IC50 value of 9.1 nM. In addition, the effects of the cardiac-selective agents lidocaine and DPI 201-106 were examined on Na currents expressed from brain and heart RNA. Lidocaine (10 microM) blocked cardiac Na current in a use-dependent manner but had no effect on brain Na currents. DPI 201-106 (10 microM) slowed the rate of cardiac Na channel inactivation but had no effect on inactivation of brain Na channels. These results indicate the Xenopus oocyte system is capable of synthesizing and expressing cardiac Na channels that retain normal physiological and pharmacological properties.     

Melatonin receptors in human fetal brain: 2-[(125)I]iodomelatonin binding and MT1 gene expression

The purpose of this study was to identify sites of action of melatonin in the human fetal brain by in vitro autoradiography and in situ hybridization. Specific, guanosine triphosphate (GTP) sensitive, binding of 2-[(125)I]iodomelatonin was localized to the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalalmus, specific binding was present in the suprachiasmatic nuclei (SCN) as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei. The localization of MT1 receptor subtype gene expression as determined by in situ hybridization matched the localization of 2-[(125)I]iodomelatonin binding. No MT2 receptor subtype gene expression was detected using this technique. Thus, melatonin may act on the human fetus via the MT1 receptor subtype at a number of discrete brain sites. A major site of action of melatonin in both fetal and adult mammals is the pars tuberalis of the pituitary gland. However, no 2-[(125)I]iodomelatonin binding or melatonin receptor gene expression was detected in the pituitary gland in the present study, indicating that the pituitary, particularly the pars tuberalis, is not a site of action of melatonin in the human fetus.