Melatonin as the most effective organizer of the rhythm of protein synthesis in hepatocytes in vitro and in vivo

Recent data has extended a large array of melatonin functions by the discovery of melatonin's involvement in the organization and regulation of the rhythm of intracellular protein synthesis. An ultradian rhythm in total protein synthesis has been detected in primary hepatocyte cultures 5 min after addition of 1-5 nM melatonin to the medium. The melatonin effect was mediated via its receptors (as shown in experiments with luzindole), leading to the cell synchronization as well as the mean rate of protein synthesis rate being increased. The chain of processes synchronizing the oscillation of the rate protein synthesis throughout the hepatocyte population includes Ca2+ fluxes {experiments with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetomethyl ester)]}. Inhibition of protein kinase activity (experiments with H7) inhibited the synchronizing function of melatonin. Activation of protein kinase activity results in a shift of the protein synthesis oscillation; the effect was the same as melatonin added to the culture medium. In another series of experiments, after melatonin was intraperitoneally injected to rat (0.015-0.020 μg/kg), hepatocytes were isolated and cultures established. A synchronizing effect of melatonin in vivo was detected as early as in the estimates from the direct action of melatonin on cell cultures. In the cultures obtained from old rats provided with melatonin, the amplitude of protein synthesis rhythm was enhanced, i.e. cell-cell interactions were increased, as well as rate of the protein synthesis being enhanced.     

Localization, physiological significance and possible clinical implication of gastrointestinal melatonin.

The gastrointestinal tract (GIT) is a major source of extrapineal melatonin. In some animals, tissue concentrations of melatonin in the GIT surpass blood levels by 10-100 times and the digestive tract contributes significantly to melatonin concentrations in the peripheral blood, particularly during the day. Some melatonin found in the GIT may originate from the pineal gland, as the organs of the digestive system contain binding sites, which in some species exhibit circadian variation. Unlike the production of pineal melatonin, which is under the photoperiodic control, release of GI melatonin seems to be related to periodicity of food intake. Melatonin and melatonin binding sites were localized in all GI tissues of mammalian and avian embryos. Postnatally, melatonin was localized in the GIT of newborn mice and rats. Phylogenetically, melatonin and melatonin binding sites were detected in GIT of numerous mammals, birds and lower vertebrates. Melatonin is probably produced in the serotonin-rich enterochromaffin cells (EC) of the GI mucosa and can be released into the portal vein postprandially. In addition, melatonin can act as an autocrine or a paracrine hormone affecting the function of GI epithelium, lymphatic tissues of the immune system and the smooth muscles of the digestive tube. Finally, melatonin may act as a luminal hormone, synchronizing the sequential digestive processes. Higher peripheral and tissue levels of melatonin were observed not only after food intake but also after a long-term food deprivation. Such melatonin release may have a direct effect on the various GI tissues but may also act indirectly via the CNS; such action might be mediated by sympathetic or parasympathetic nerves. Melatonin can protect GI mucosa from ulceration by its antioxidant action, stimulation of the immune system and by fostering microcirculation and epithelial regeneration. Melatonin may reduce the secretion of pepsin and the hydrochloric acid and influence the activity of the myoelectric complexes of the gut via its action in the CNS. Tissue or blood levels of melatonin may serve as a marker of GI lesions or tumors. Clinically, melatonin has a potential for a prevention or treatment of colorectal cancer, ulcerative colitis, irritable bowel syndrome, children colic and diarrhea.     

Photoperiodic control of melatonin synthesis in fish pineal and retina

Melatonin is the time-keeping molecule of vertebrates. The daily and annual variations of its rhythmic production allow synchronizing physiological functions and behaviours to the variations of the environment. In fish, melatonin is produced by the photoreceptor cells of the retina and pineal organ. It is also synthesized by other retinal cell types of the inner nuclear and ganglion cell layers. In most of the species investigated, the melatonin rhythm displays a high-at-night profile, resulting from the circadian control of the arylalkylamine N-acetyltranferase (AANAT) activity; AANAT is the penultimate enzyme in the melatonin biosynthesis pathway. Some fish species escape the high-at-night rule in the retina, and the rhythm displays a high-at-day profile, intermediate situations being sometimes observed. This review summarizes our current knowledge on the molecular and cellular mechanisms of the rhythmic control of production of an important circadian clock messenger, underlying their plasticity.     

Melatonin action in neonatal gonadotrophs

Neonatal pituitary cells express MT1 and MT2 subtype of melatonin receptors that are coupled to pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings have revealed that a reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores. Inhibition of calcium influx, probably in a cAMP/protein kinase C-dependent manner, and the accompanying calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. Melatonin-induced attenuation of GnRH-induced and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular pools attenuates the amplitude of calcium signal. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. During the development, such tonic inhibitory effects of melatonin on GnRH action gradually decline due to a decrease in expression of functional melatonin receptors. In adult animals, melatonin does not have obvious direct effects on pituitary functions, whereas the connections between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.     

Melatonin synchronizes protein synthesis rhythm in hepatocyte cultures as an agonist of intercellular calcium and protein kinases

Melatonin in nanomolar concentrations synchronizes protein synthesis in primary cultures of hepatocytes through calcium-dependent activation of protein kinases. The synchronizing effect of melatonin is blocked by the cytoplasmic calcium chelating agent BAPTA-AM (20 μM) as well as by the inhibitor of protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (40 μM). Thus, protein phosphorylation is the key event in hepatocyte synchronization by melatonin, as we have demonstrated previously for gangliosides and biogenic amines. The antagonist of melatonin receptors luzindole (20 nM) blocks the synchronizing function of melatonin.     

Model of control of diurnal melatonin secretion by the solar radiation

The mathematical model of the control process of diurnal melatonin secretion under the influence of solar radiation on retina photoreceptors is proposed. Invariant relations for calculating melatonin secretion rate and its concentration in blood plasma are obtained. Spectral, time and energy characteristics of solar radiation synchronizing diurnal melatonin secretion and circadian rhythms in human are defined. A possibility of using the relations obtained is shown for arbitrary combination of calendar dates, local time of any time zone and geographical coordinates of a calculated point on earth surface. The adequacy of model is confirmed by coincidence of the calculation data with the results of independent experimental studies on diurnal secretion of melatonin and circadian rhythm in human. The model proposed can be used during investigation of diurnal secretion of melatonin and circadian rhythm in human.     

Mechanism of melatonin action

Melatonin transduces the effect of photoperiod on the neuroendocrine system. Synthesis of melatonin in the pineal gland is well described, but the location of its target(s) and the mechanism of its action are little known. In attempt to localize melatonin target(s), the presence of high affinity binding sites in rat brain was determined. Such sites were detected in discrete brain areas, including the hypothalamus and anterior pituitary. Subcellular analysis indicated these binding sites were on plasma membranes, which suggests that melatonin modulates cell functions through intracellular second messengers. The effects of melatonin on second messengers were studied using the neonatal anterior pituitary, in which melatonin is known to inhibit the LHRH-induced release of LH. Studies on the effects of melatonin on second messenger indicated [corrected] that melatonin inhibits accumulation of cAMP and cGMP as well as synthesis of diacylglycerol and release of arachidonic acid. Time-course analysis indicates that inhibition by melatonin of the LHRH-induced release of LH increases following long preincubation. Since the effect of melatonin on LHRH-induced release of LH is prevented by dibutyryl cAMP, we conclude that melatonin might act by inhibiting production of cAMP.     

Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

A novel metabolic pathway of melatonin: oxidation by cytochrome C

The indoleamine melatonin is ubiquitously distributed, and because of its small size and amphiphilic nature, it is able to reach easily all cellular compartments. The highest intracellular melatonin concentrations are found in the mitochondria, suggestive of local metabolism and/or direct participation in organelle function. In mitochondria cytochrome c (cyt c) could represent a melatonin target since it has the capability to oxidize organic molecules in the presence of H2O2, and mitochondria are the main site of H2O2 production in nonphagocytic cells. Therefore, we investigated oxidation of melatonin by cyt c/H2O2 couple as a potential pathway for its metabolism in the mitochondria. We found melatonin conversion into N(1)-acetyl-N(2)-formyl-5-methoxykynuramine via sequential steps that generate the intermediates 2-hydroxymelatonin and 2,3-dihydroxymelatonin. We experimentally excluded mediation by a Fenton/Haber-Weiss-type reaction and documented the dependence on oxoferryl heme for melatonin oxidation. Given the high mitochondrial concentrations of both melatonin and cyt c as well as the continuous generation of H2O2 during respiration, it is entirely possible that mitochondrial cyt c-mediated oxidation of melatonin may be a plausible pathway of its biotransformation in vivo.     

The characteristics of the synchronizing action of melatonin on the dynamics of circadian mobility in rats

Morning (9.00-10.00 h), but not noon, repeated injections of the pineal hormone melatonin (1 or 10 mg/kg daily, during 2 weeks) synchronize the circadian locomotor rhythm of rats. As shown with inverted photoperiod, melatonin facilitates off and on light reactions. Some animals have low and some high hormonal susceptibility. This indicates the role of injections regime and rat's individual reactivity for expressiveness of melatonin synchronizing effect.     

Circadian regulation of bird song, call, and locomotor behavior by pineal melatonin in the zebra finch

As both a photoreceptor and pacemaker in the avian circadian clock system, the pineal gland is crucial for maintaining and synchronizing overt circadian rhythms in processes such as locomotor activity and body temperature through its circadian secretion of the pineal hormone melatonin. In addition to receptor presence in circadian and visual system structures, high-affinity melatonin binding and receptor mRNA are present in the song control system of male oscine passeriform birds. The present study explores the role of pineal melatonin in circadian organization of singing and calling behavior in comparison to locomotor activity under different lighting conditions. Similar to locomotor activity, both singing and calling behavior were regulated on a circadian basis by the central clock system through pineal melatonin, since these behaviors free-ran with a circadian period and since pinealectomy abolished them in constant environmental conditions. Further, rhythmic melatonin administration restored their rhythmicity. However, the rates by which these behaviors became arrhythmic and the rates of their entrainment to rhythmic melatonin administration differed among locomotor activity, singing and calling under constant dim light and constant bright light. Overall, the study demonstrates a role for pineal melatonin in regulating circadian oscillations of avian vocalizations in addition to locomotor activity. It is suggested that these behaviors might be controlled by separable circadian clockworks and that pineal melatonin entrains them all through a circadian clock.     

Melatonin in sleep disorders and jet-lag

In elderly insomniacs, melatonin treatment decreased sleep latency and increased sleep efficiency. This is particularly marked in Alzheimer's disease (AD) patients. Melatonin is effective to reduce significantly benzodiazepine use. In addition, melatonin administration synchronizes the sleep-wake cycle in blind people and in individuals suffering from delayed sleep phase syndrome or jet lag. Urinary levels of 6-sulphatoxymelatonin decrease with age and in chronic diseases like AD or coronary heart disease. The effect of melatonin on sleep is probably the consequence of increasing sleep propensity (by inducing a fall in body temperature) and of a synchronizing effect on the circadian clock (chronobiotic effect).     

Methoxyindoles of the retina

Melatonin and other 5-methoxyindoles are compounds usually associated with the pineal gland. Research is expanding from studies of pineal melatonin to studies of extrapineal organs and of other 5-methoxyindoles besides melatonin. Research in recent years has shown that the retina also contains and synthetises 5-methoxyindoles. The biochemical modes of action are still unclear. Nevertheless, they seem to have physiological roles in the pineal gland and the retina. These compounds are thought to participate in the regulation of the cyclic metabolism of the retina. Melatonin and other 5-methoxyindoles are often classified as neuromodulators.     

Key aspects of melatonin physiology: thirty years of research

Numerous studies of melatonin, by now widely acknowledged as a circadian rhythm-affecting neurohormone, also describe its anti-oxidant, anti-cytotoxic or immune-modulating activity. While emphasizing the multifunctional aspect of melatonin action, this review presents the results of our thirty years of research, which point to the following conclusions: melatonin is capable of promoting platelet production by megakaryocytes, of acting on the latter's ion channels by way of the outward currents, and of performing a physiological anti-aggregation function thus lengthening platelet life span. Melatonin can be transported everywhere by platelets and, thanks to its lipophilicity, can cross cellular membranes easily, thus regulating blood-tissue exchanges and ensuring an improved haematic crisis. It interacts with endothelial cells by regulating their release of both relaxing-factor and contracting-factor, and with platelets by affecting their discharge of dense-body components. Finally, platelets could behave as mobile and itinerant serotonergic and/or melatonergic elements, a function comparable to the release of neurotransmitters by neurons of the central nervous system. This dynamism in melatonin physiology could prove to be a key in approaching tumour aetiopathogenesis.     

Encystment of Gonyaulax polyedra: Dependence on Light

The unicellular alga Gonyaulax polyedra reacts to short days and low temperatures by forming asexual cysts. Its photoperiodic response is elicited via the physiological mediation of melatonin. This indoleamine known as a dark signal in vertebrates is also synthetised by this dinophyt attaining concentrations as high as in the mammalian pineal gland. Its level varies in a circadian fashion, showing a steep increase after the onset of darkness, followed by a gradual decline towards the beginning of photophase. The critical photoperiod of the encystment response shows in Gonyaulax polyedra the remarkably high precision of about half an hour. Under otherwise non-inducing conditions, a single addition of 10 –4 M melatonin, given 1 h before the onset of darkness, elicits encystment as much, and with similar kinetics, as in short-days. The effect of melatonin action during long-day conditions (11:13) and low temperature (15°C) has been investigated. After addition of 10 –4 M or 7x10 –5 M melatonin each 3 h, the cyst-inducing capacity depends on the circadian phase of treatment. The differences in efficiency of melatonin observed are negatively correlated with the endogenous melatonin production of Gonyaulax polyedra which is higher at the begining of darkness. These results lead to novel consequences relating to the rapid catabolism of this substance and the dependence of its efficiency on light.     

Encystment of Gonyaulax polyedra: Dependence on Light

The unicellular alga Gonyaulax polyedra reacts to short days and low temperatures by forming asexual cysts. Its photoperiodic response is elicited via the physiological mediation of melatonin. This indoleamine known as a dark signal in vertebrates is also synthetised by this dinophyt attaining concentrations as high as in the mammalian pineal gland. Its level varies in a circadian fashion, showing a steep increase after the onset of darkness, followed by a gradual decline towards the beginning of photophase. The critical photoperiod of the encystment response shows in Gonyaulax polyedra the remarkably high precision of about half an hour. Under otherwise non-inducing conditions, a single addition of 10 -4 M melatonin, given 1 h before the onset of darkness, elicits encystment as much, and with similar kinetics, as in short-days. The effect of melatonin action during long-day conditions (11:13) and low temperature (15°C) has been investigated. After addition of 10 -4 M or 7x10 -5 M melatonin each 3 h, the cyst-inducing capacity depends on the circadian phase of treatment. The differences in efficiency of melatonin observed are negatively correlated with the endogenous melatonin production of Gonyaulax polyedra which is higher at the begining of darkness. These results lead to novel consequences relating to the rapid catabolism of this substance and the dependence of its efficiency on light.     

Melatonin's Role in Vertebrate Orcadian Rhythms

The circadian secretion of melatonin by the pineal gland and retinae is a direct output of circadian oscillators and of the circadian system in many species of vertebrates. This signal affects a broad array of physiological and behavioral processes, making a generalized hypothesis for melatonin function an elusive objective. Still, there are some common features of melatonin function. First, melatonin biosynthesis is always associated with photoreceptors and/or cells that are embryonically derived from photoreceptors. Second, melatonin frequently affects the perception of the photic environment and has as its site of action structures involved in vision. Finally, melatonin affects overt circadian function at least partially via regulation of the hypothalamic suprachiasmatic nucleus (SCN) or its hofnologues. The mechanisms by which melatonin affects circadian rhythms and other downstream processes are unknown, but they include interaction with a class of membrane-bound receptors that affect intracellular processes through guanosine triphosphate (GTP)-binding protein second messenger systems. Investigation of mechanisms by which melatonin affects its target tissues may unveil basic concepts of neuromodulation, visual system function, and the circadian clock.