Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.     

Effects of melatonin and antagonists of MT1 and MT2 receptors on somatic pain induced within the fixed circadian rhythm

We studied behavioral pain-related reactions (PRRs) induced in mice by subcutaneous injections of 5% formalin within different phases of the fixed circadian illumination rhythm under conditions of administration of exogenous melatonin and of blocking of MT1 and MT2 melatonin receptors. It was demonstrated that modulation of experimentally induced somatic pain depends considerably on the phase of the preset circadian rhythm. In the norm, the duration of PRRs in the middle of the dark phase was 30% smaller than that in the middle of the light phase. Administration of exogenous melatonin in the middle of the light phase decreased the duration of episodes of noxious behavior by 43%, on average. Injections of melatonin within the dark phase resulted in no significant changes in the duration of PRRs. In the dark phase, the blockade of MT1 receptors by luzindole led to an increase in the duration of PRRs by 45%, as compared with the norm, while in the light phase we observed no significant alterations of this duration under conditions of blocking of the above-mentioned receptors. The blockade of MT2 receptors by prazocine in the middle of dark and light phases increased the durations of PRRs by 92 and 28%, respectively. Our data indicate that the analgesic effect of melatonin depends significantly on the level of this hormone in the organism; in turn, such a level is determined by the illumination conditions. The antinoxious effect of melatonin is mediated by MT receptors, in particular by MT2 receptors. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 255–259, May–June, 2007.     

Restricted daytime feeding attenuates reentrainment of the circadian melatonin rhythm after an 8-h phase advance of the light-dark cycle

It is well established that in the absence of photic cues, the circadian rhythms of rodents can be readily phase-shifted and entrained by various nonphotic stimuli that induce increased levels of locomotor activity (i.e., benzodiazepines, a new running wheel, and limited food access). In the presence of an entraining light-dark (LD) cycle, however, the entraining effects of nonphotic stimuli on (parts of) the circadian oscillator are far less clear. Yet, an interesting finding is that appropriately timed exercise after a phase shift can accelerate the entrainment of circadian rhythms to the new LD cycle in both rodents and humans. The present study investigated whether restricted daytime feeding (RF) (1) induces a phase shift of the melatonin rhythm under entrained LD conditions and (2) accelerates resynchronization of circadian rhythms after an 8-h phase advance. Animals were adapted to RF with 2-h food access at the projected time of the new dark onset. Before and at several time points after the 8-h phase advance, nocturnal melatonin profiles were measured in RF animals and animals on ad libitum feeding (AL). In LD-entrained conditions, RF did not cause any significant changes in the nocturnal melatonin profile as compared to AL. Unexpectedly, after the 8-h phase advance, RF animals resynchronized more slowly to the new LD cycle than AL animals. These results indicate that prior entrainment to a nonphotic stimulus such as RF may "phase lock" the circadian oscillator and in that way hinder resynchronization after a phase shift.     

Molecular determinants for the differential coupling of Galpha(16) to the melatonin MT1, MT2 and Xenopus Mel1c receptors

The pineal neurohormone melatonin modulates a variety of physiological processes through different receptors. It has recently been reported that the cloned melatonin receptors (MT1, MT2 and Mel1c) exhibit differential abilities to stimulate phospholipase C (PLC) via G(16). Here we examined the molecular basis of such differences in melatonin receptor signaling. Coexpression of MT1 or MT2 with the alpha subunit of G(16) (Galpha(16) ) allowed COS-7 cells to accumulate inositol phosphates in response to 2-iodomelatonin. In contrast, Mel1c did not activate Galpha(16) even though its expression was demonstrated by radioligand binding and agonist-induced inhibition of adenylyl cyclase. As Mel1c possesses an exceptionally large C-terminal tail, we further asked if this structural feature prevented productive coupling to Galpha(16). Eleven chimeric melatonin or mutant receptors were constructed by swapping all or part of the C-terminal tail between MT1, MT2 and Mel1c. All chimeras were fully capable of binding 2-[(125) I]iodomelatonin and inhibiting adenylyl cyclase. Chimeras containing the full-length Mel1c tail were incapable of activating Galpha(16), while those that contained the complete C-terminal region of either MT1 or MT2 stimulated PLC. Incorporation of the extra portion of the C-terminal tail of Mel1c to either MT1 or MT2 completely abolished the chimeras' ability to stimulate PLC via Galpha(16). In contrast, truncation of the C-terminal tail of Mel1c allowed interaction with Galpha(16). Our results suggest that Galpha(16) can discern structural differences amid the three melatonin receptors and provide evidence for functional distinction of Mel1c from MT1 and MT2 receptors.     

Sites and mechanisms of action of melatonin in mammals: the MT1 and MT2 receptors

The rhythmic secretion of melatonin by the pineal gland plays a key role in the synchronisation of circadian and seasonal functions with cyclic environmental variations. The biological effects of this neurohormone are relayed mainly by G-protein-coupled seven-transmembrane receptors. These receptors, known as MT1 and MT2, are present in a large number of central and peripheral structures in mammals, with considerable inter-species variations. However, only the suprachiasmatic nuclei of the hypothalamus, the site of the master circadian biological clock, and the pars tuberalis of the adenohypophysis contain melatonin receptors in the majority of species. Inhibition of the production of AMPc by a Gi/Go protein is one of the principal signalling pathways of the MT1 and MT2 receptors, although many other signal transduction pathways are also brought into play according to the cell type studied (PKC, Ca2+, K+ channels or GMPc in the case of MT2, etc.). Numerous factors or physiological stimuli are capable of influencing the number and functional status of the MT1 and MT2 receptors, such as melatonin, the photoperiod, the circadian clock or the phenomena of receptor dimerisation. Melatonin has numerous physiological effects for which the mechanisms of action and the specific role of the MT1 and MT2 receptors have not yet been clearly elucidated. However, selective pharmacological tools for each of the two receptor subtypes are currently being identified, notably in the Servier Group, for the purpose of furthering our knowledge of the functionality and physiological role of the MT1 and MT2 receptors in the central and peripheral structures.     

Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats

In vivo pharmacological effects of ramelteon (TAK-375), a novel, highly MT1/MT2-selective receptor agonist, were studied in rats to determine ramelteon's ability to reentrain the circadian rhythm after an abrupt phase advance. Experiments were also conducted to assess the potential cognitive side effects of ramelteon and its potential to become a drug of abuse. After an abrupt 8-h phase shift, ramelteon (0.1 and 1 mg/kg, p.o.) and melatonin (10 mg/kg, p.o.) accelerated reentrainment of running wheel activity rhythm to the new lightdark cycle. Ramelteon (3-30 mg/kg, p.o.) and melatonin (10-100 mg/kg, p.o.) did not affect learning or memory in rats tested by the water maze task and the delayed match to position task, although diazepam and triazolam impaired both of the tasks. Neither ramelteon (3-30 mg/kg, p.o.) nor melatonin (10-100 mg/kg, p.o.) demonstrated a rewarding property in the conditioned place-preference test, implying that MT1/MT2 receptor agonists have no abuse potential. In contrast, benzodiazepines and morphine showed rewarding properties in this test. The authors' results suggest that ramelteon may be useful for treatment of circadian rhythm sleep disorders without adverse effects typically associated with benzodiazepine use, such as learning and memory impairment, and drug dependence.     

Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice

Melatonin has many protective effects against ischemic stroke, but the underlying neuroprotective mechanisms are not fully understood. Our aim was to explore the relationship between melatonin's neuroprotective effects and activation of the MT2 melatonin receptor in a murine ischemic-stroke model. Male ICR mice were subjected to a transient middle cerebral ischemic/reperfusional injury, and melatonin (5 and 10 mg/kg, ip) was administrated once daily starting 2 h after ischemia. More than 80% of the mice died within 5 days after stroke without treatment. Melatonin treatment significantly improved the survival rates and neural functioning with modestly prolonged life span of the stroke mice by preserving blood-brain barrier (BBB) integrity via a reduction in the enormous amount of stroke-induced free radical production and significant gp91(phox) cell infiltration. These protective effects of melatonin were reversed by pretreatment with MT2 melatonin receptor antagonists (4-phenyl-2-propionamidotetralin (4P-PDOT) and luzindole). Moreover, treatment with melatonin after stroke dramatically enhanced endogenous neurogenesis (doublecortin positive) and cell proliferation (ki67 positive) in the peri-infarct regions. Most ki67-positive cells were nestin-positive and NG2-positive neural stem/progenitor cells that coexpressed two neurodevelopmental proteins (adam11 and adamts20) and the MT2 melatonin receptor. RT-PCR revealed that the gene expression levels of doublecortin, ki67, adamts20, and adam11 are markedly reduced by stroke, but are restored by melatonin treatment; furthermore, pretreatment with 4P-PDOT and luzindole antagonized melatonin's restorative effect. Our results support the hypothesis that melatonin is able to protect mice against stroke by activating MT2 melatonin receptors, which reduces oxidative/inflammatory stress. This results in the preservation of BBB integrity and enhances endogenous neurogenesis by upregulating neurodevelopmental gene/protein expression.     

Production and characterization of antibodies directed against the human melatonin receptors Mel-1a (mt1) and Mel-1b (MT2)

We report the production of polyclonal antibodies directed against the human melatonin receptors Mel-1a (mt1) and Mel-1b (MT2) by means of antigenic synthetic peptides with sequences unique to these proteins. Immunostaining on NIH3T3 cells stably transfected with Mel-1a and Mel-1b cDNA gave intense reactions. Neither the preimmune serum nor cross-tested antisera showed any reactivity. These polyclonal antibodies will be essential immunocytochemical tools to study the human melatonin receptors distribution at subcellular level.     

Effects of macromolecule synthesis inhibitors on light-induced phase shift of the circadian rhythm in melatonin release from the cultured pineal organ of a teleost, ayu (Plecoglossus altivelis)

Effects of macromolecule synthesis inhibitors on the light-induced phase shift of the circadian clock in the photoreceptive pineal organ of a teleost, ayu (Plecoglosus altivelis) were investigated using melatonin release as an indicator. A single light pulse during the early- and late-subjective night delayed and advanced the phase of the circadian rhythm in melatonin release, respectively. During the late subjective-night, protein synthesis inhibitor cycloheximide (CHX) delayed the rhythm while RNA synthesis inhibitor 5,6-dichlorobenzimidazole riboside (DRB) had little effect. Light-induced phase advance was diminished by the treatment of CHX but not by DRB. During the early subjective-night, DRB, CHX, light and combination of these (DRB+light, CHX+light) all phase-delayed the rhythm. There were no additive effects of light and DRB or CHX. These results indicate that macromolecule synthesis is somehow involved in generation of circadian oscillation, and that de novo protein synthesis is required for light-induced phase shift of the circadian clock in the ayu pineal organ.     

Influence of altered photoperiods on serum melatonin and its receptors (MT1 and MT2) in the brain, retina, and ovary in carp Catla catla

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues-brain, retina, and ovary-and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ～2-3?h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ～37?kDa and a band at ～36?kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp.     

Bivalent ligand approach on N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide: synthesis, binding affinity and intrinsic activity for MT(1) and MT(2) melatonin receptors

We report the synthesis, binding properties and intrinsic activity at MT(1) and MT(2) melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimerization of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity for the MT(1) receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was the most selective for the MT(1) subtype (112-fold selectivity) and compound 3d, characterized by a hexamethylene spacer, had the highest MT(1) binding affinity (pK(iMT1)=8.47) and 54-fold MT(1)-selectivity. Dimerization through the aniline nitrogen of 1 abolished MT(1) selectivity, leading to compounds with either a full agonist or an antagonist behavior depending on the nature of the linker.     

Eventual entrainment of the human circadian pacemaker by melatonin is independent of the circadian phase of treatment initiation: clinical implications

About 15% of the legally blind completely lack light perception. Most of these individuals have abnormally phased circadian rhythms and many free-run. Light treatment is not an option for them. However, melatonin treatment can be highly effective. A daily dose of 0.5 mg of melatonin usually results in entrainment. It has been suggested that treatment in individuals with circadian periods > 24 h should be initiated on the advance zone of the melatonin phase response curve, which was based on findings in which melatonin initiated on the delay zone were less likely to result in entrainment, even though treatment continued across all circadian phases. In the present study, 7 totally blind people started low-dose melatonin treatment (0.5 mg; 1 person was given 0.05 mg) on the delay zone. All entrained as circadian phase free-ran and the advance zone of the melatonin phase response curve coincided with the time of melatonin administration. These results are consistent with studies in other mammals. It does not appear that low-dose melatonin treatment needs to be initiated on the advance zone to induce eventual entrainment in blind people with free-running rhythms > 24 h. Therefore, it is not essential that circadian phase be ascertained before starting low-dose melatonin treatment of blind people.     

Effects of the 5HT1A agonist/antagonist BMY 7378 on light-induced phase advances in hamster circadian activity rhythms during aging

The entrainment of some circadian rhythms in rodents and humans to the environmental light-dark cycle deteriorates during aging. Recent evidence suggests that the time-keeping ability of the circadian pacemaker maintains its endogenous period in both hamsters and humans. This suggests that any changes in the coupling between environmental cues and the circadian pacemaker are not due to changes in "clock speed," but rather due to a weakened coupling between the afferent systems relaying environmental information and the circadian pacemaker located in the suprachiasmatic nucleus. The suprachiasmatic nucleus receives serotonergic input from the raphe nuclei, and serotonergic 5HT1A,7 agonists have been reported to lose their circadian phase-adjusting efficacy during aging in hamsters. In the present study, the authors report the effects of a novel serotonergic agonist BMY 7378 on light-induced phase advances during aging in the hamster. The present report demonstrates that BMY 7378 is a highly efficacious chronobiotic that more than doubles the magnitude of light-induced phase shifts in hamster wheel-running activity rhythms. Light-induced phase advances in hamster wheel-running activity of at least 6 h following a single systemic dose of BMY 7378 are routinely observed. Furthermore, BMY 7378 potentiation of phase shifts is maintained in old hamsters, suggesting that BMY 7378 has a different site of activity than previously reported 5HT1A,7 agonists that have a diminished effect on circadian phase during aging.     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

Diurnal and circadian regulations by three melatonin receptors in the brain and retina of olive flounder Paralichthys olivaceus: profiles following exogenous melatonin

To establish the molecular basis of circadian rhythm control by melatonin receptors (MTs), we investigated the mitochondrial ribonucleic acid (mRNA) expressions of three types of MTs in different tissues of the olive flounder (Paralichthys olivaceus). All three types of MT mRNAs were expressed in the neural tissues, while MT1 mRNA was expressed in the peripheral tissues and MT2 and MT3 mRNAs were weakly expressed or undetected in these tissues. We observed increased MT mRNA expression in the neural tissues at night under both light–dark (LD) and constant dark (DD) conditions. Although the melatonin-treated cultured pineal gland samples showed similar diurnal variations with high-MT mRNA expression levels at night compared to those of untreated cultured pineal gland samples, the expression levels were considerably higher in the melatonin-treated samples. The plasma melatonin level also significantly increased at night. Under DD conditions, the expression patterns of MT mRNAs were similar to those under the LD photocycle, but the peak was lower and the circadian change patterns were less clear. These findings reinforce the hypothesis that MTs are active in processing light information, and that these genes are regulated by the circadian clock and light, thus suggesting that MTs play an important role in daily and circadian variations in the brain and retina of olive flounders.     

New melatonin (MT1/MT2) ligands: Design and synthesis of (8,9-dihydro-7H-furo[3,2-f]chromen-1-yl) derivatives

Herein we describe the synthesis of novel tricyclic analogues issued from the rigidification of the methoxy group of the benzofuranic analogue of melatonin as MT₁ and MT₂ ligands. Most of the synthesized compounds displayed high binding affinities at MT₁ and MT₂ receptors subtypes. Compound 6b (MT₁, K_i = 0.07 nM; MT₂, K_i = 0.08 nM) exhibited with the vinyl 6c and allyl 6d the most interesting derivatives of this series. Functional activity of these compounds showed full agonist activity with EC₅₀ in the nanomolar range. Compounds 6a (EC₅₀ = 0.8 nM and E_max = 98%) and 6b (EC₅₀ = 0.2 nM and E_max = 121%) exhibited good pharmacological profiles.     

Retinal GABA(A) receptors participate in the regulation of circadian responses to light

A role for retinal gamma-aminobutyric acid Type A (GABA(A)) receptors in the regulation of circadian responses to light was examined. Intraocular injections of the GABA(A) antagonist, bicuculline, were performed during the early (Circadian Time [CT] 13.5) and late subjective night (CT 20), followed by a light pulse. Bicuculline significantly decreased the magnitude of phase delays induced by light to 65%, whereas it had no effect on phase advances. To explore the nature of the inhibition elicited by bicuculline, an intensity-response curve was performed. Intraocular injections of bicuculline inhibited phase delays only when induced by high-saturating light illuminances (20 and 100 lux). No effect was observed at light intensities < or = 5 lux. These results suggest that retinal GABA(A) receptors modulate the responsivity of the circadian system to light.     

The stereochemistry of hydrogen transfer to NADP+ by enzymes acting upon stereoisomeric substrates

The stereochemistry of hydrogen transfer from estradiol-17α and estradiol-17β to NADP⁺ in the presence of chicken liver estradiol-17α and estradiol-17β dehydrogenases was found in both cases to involve the 4-pro-S proton of the pyridine nucleotide. One of these enzymes must therefore use the stereochemically less favorable mode of interaction of steroid with coenzyme.