Melatonin: presence and formation in invertebrates

In vertebrates, it is now clearly demonstrated that the pineal gland is implicated in conveying photoperiodic information via the daily pattern of melatonin secretion. Invertebrates, like vertebrates, use photoperiodic changes as a temporal cue to initiate physiological processes such as reproduction or diapause. How this information is integrated in invertebrates remains an unsolved question. Our review will be an attempt to evaluate the possible role of melatonin in conveying photoperiodic information in invertebrates. It is now well demonstrated in both vertebrates and invertebrates that melatonin as well as its precursors or synthesizing enzymes are present in various organs implicated in photoreceptive processes or in circadian pacemaking. Melatonin, serotonin or N-acetyltransferase have been found in the head, the eyes, the optic lobe and the brain of various invertebrate species. In some species it has also been shown that melatonin is produced rhythmically with high concentrations reached during the dark period. Moreover, the physiological effects of melatonin on various periodic processes such as rhythmic contractions in coelenterates, fissioning of asexual planarians or reproductive events in flies have been reported in the literature. All these results support the hypothesis (refs 36, 37) that melatonin is not solely a pineal hormone but that it may be an evolutionary conservative molecule principally involved in the transduction of photoperiodic information in all living organisms.     

Some reflections on the phylogeny and function of the pineal

The pineal gland is a universal feature of vertebrate organization and has been implicated in the control of rhythmic adaptations to daily and seasonal cycles. This paper considers three aspects of pineal function; the generation of a rhythmical endocrine signal (the nocturnal synthesis of melatonin) and the use of the signal in the regulation of circadian and photoperiodic functions. The shape of the nocturnal signal is determined by an interaction of afferent neural control and biochemical processes intrinsic to the pinealocyte. The nature of the effect of the signal upon circadian systems is unclear, and in adult mammals may not be a specific, direct influence upon the entrainment pathways of the oscillator. In the foetus, strong evidence exists for a physiological role of the maternal melatonin signal as a true internal zeitgeber, remnants of which may persist in the adult. Photoperiodic time measurement in adult and foetal mammals is critically dependent upon the melatonin signal. Indirect evidence indicates that several neural systems may be involved in the response to melatonin and consistent with this, a variety of central melatonin binding sites have been identified in the brain and pituitary. The intra-cellular actions of melatonin and the properties of melatonin responsive neural systems have yet to be identified, but in the context of photoperiodic time measurement, it is clear that the neural responses to melatonin are not dependent upon the circadian clock. The two central effects of melatonin; photoperiodic time measurement and circadian entrainment are probably mediated through completely separate mechanisms.     

Some reflections on the phylogeny and function of the pineal

Summary The pineal gland is a universal feature of vertebrate organization and has been implicated in the control of rhythmic adaptations to daily and seasonal cycles. This paper considers three aspects of pineal function; the generation of a rhythmical endocrine signal (the nocturnal synthesis of melatonin) and the use of the signal in the regulation of circadian and photoperiodic functions. The shape of the nocturnal signal is determined by an interaction of afferent neural control and biochemical processes intrinsic to the pinealocyte. The nature of the effect of the signal upon circadian systems is unclear, and in adult mammals may not be a specific, direct influence upon the entrainment pathways of the oscillator. In the foetus, strong evidence exists for a physiological role of the maternal melatonin signal as a true internal zeitgeber, remnants of which may persist in the adult. Photoperiodic time measurement in adult and foetal mammals is critically dependent upon the melatonin signal. Indirect evidence indicates that several neural systems may be involved in the response to melatonin and consistent with this, a variety of central melatonin binding sites have been identified in the brain and pituitary. The intra-cellular actions of melatonin and the properties of melatonin responsive neural systems have yet to be identified, but in the context of photoperiodic time measurement, it is clear that the neural responses to melatonin are not dependent upon the circadian clock. The two central effects of melatonin; photoperiodic time measurement and circadian entrainment are probably mediated through completely separate mechanisms.The Editors wish to thank Dr M. Hastings for coordinating this multi-author review.     

Neural systems underlying photoperiodic time measurement: a blueprint

This paper briefly reviews the formal properties of the photoperiodic time measurement apparatus of mammals and presents a hypothetical model for the operation of the neural systems responsible for reading and responding to the nocturnal pineal melatonin signal. The primary melatonin readout mechanism is held to be common to all species responsive to melatonin. It seems likely that this mechanism responds to relative changes in the duration and amplitude of the melatonin signal, rather than the absolute levels of melatonin encountered. A series of neural systems which exploit the calendar information provided by the primary readout is envisaged to vary between and within species, depending upon the neuroendocrine response under consideration. Of particular importance is a mechanism for comparing the relative duration of successive melatonin signals. These more complex elements are responsible for phenomena such as the effects of photoperiodic history and photorefractoriness. The brain may be able to encode an accumulated memory of melatonin signals and thereby define longer term intervals within the annual cycle. A series of response elements within the hypothalamus are engaged by the appropriately processed photoperiodic stimuli. For all elements of this model, their anatomical representations are poorly understood or, in certain cases, completely unknown.     

The melatonin rhythm: both a clock and a calendar

The paper briefly reviews the data which shows that the circadian production and secretion of melatonin by the pineal gland can impart both daily, i.e., clock, and seasonal, i.e., calendar, information to the organism. The paper summarizes the 3 patterns of nocturnal melatonin production that have been described. Clearly, regardless of the pattern of nocturnal melatonin production a particular species normally displays, the duration of nightime elevated melatonin is proportional to the duration of the night length. Since daylength under natural conditions changes daily the melatonin rhythm, which adjusts to the photoperiod sends time of year information to the organism. The melatonin receptors which subserve the clock message sent by the pineal gland in the form of a melatonin cycle may reside in the biological clock itself, namely, the suprachiasmatic nuclei (SCN). The melatonin receptors that mediate seasonal changes in reproductive physiology are presumably those that are located on the pars tuberalis cells of the anterior pituitary gland. Besides these receptors which likely mediate clock and calendar information, melatonin receptors have been described in other organs. Interestingly, the distribution of melatonin receptors is highly species-specific. Whereas the clock and calendar information that the melatonin cycle imparts to the organism relies on cell membrane receptors, a fact that is of some interest considering the high lipophilicity of melatonin, recent studies indicate that other functions of melatonin may require no receptor whatsoever.     

Melatonin and circadian control in mammals

Summary Although pinealectomy has little influence on the circadian locomotor rhythms of laboratory rats, administration of the pineal hormone melatonin has profound effects. Evidence for this comes from studies in which pharmacological doses of melatonin are administered under conditions of external desynchronization, internal desynchronization, steady state light-dark conditions, and phase shifts of the zeitgeber. Taken together with recent findings on melatonin receptor concentration in the rat hypothalamus, particularly at the level of the suprachiasmatic nuclei, these results suggest that melatonin is a potent synchronizer of rat circadian rhythms and has a direct action on the circadian pacemaker. It is possible, therefore, that the natural role of endogenous melatonin is to act as an internal zeitgeber for the total circadian structure of mammals at the level of cell, tissue, organ, whole organism and interaction of that organism with environmental photoperiod changes.     

Melatonin and circadian control in mammals

Although pinealectomy has little influence on the circadian locomotor rhythms of laboratory rats, administration of the pineal hormone melatonin has profound effects. Evidence for this comes from studies in which pharmacological doses of melatonin are administered under conditions of external desynchronization, internal desynchronization, steady state light-dark conditions, and phase shifts of the zeitgeber. Taken together with recent findings on melatonin receptor concentration in the rat hypothalamus, particularly at the level of the suprachiasmatic nuclei, these results suggest that melatonin is a potent synchronizer of rat circadian rhythms and has a direct action on the circadian pacemaker. It is possible, therefore, that the natural role of endogenous melatonin is to act as an internal zeitgeber for the total circadian structure of mammals at the level of cell, tissue, organ, whole organism and interaction of that organism with environmental photoperiod changes.     

Neural systems underlying photoperiodic time measurement: A blueprint

Summary This paper briefly reviews the formal properties of the photoperiodic time measurement apparatus of mammals and presents a hypothetical model for the operation of the neural systems responsible for reading and responding to the nocturnal pineal melatonin signal. The primary melatonin readout mechanism is held to be common to all species responsive to melatonin. It seems likely that this mechanism responds to relative changes in the duration and amplitude of the melatonin signal, rather than the absolute levels of melatonin encountered. A series of neural systems which exploit the calendar information provided by the primary readout is envisaged to vary between and within species, depending upon the neuroendocrine response under consideration. Of particular importance is a mechanism for comparing the relative duration of successive melatonin signals. These more complex elements are responsible for phenomena such as the effects of photopheriodic history and photorefractoriness. The brain may be able to encode an accumulated memory of melatonin signals and thereby define longer term intervals within the annual cycle. A series of response elements within the hypothalamus are engaged by the appropriately processed photoperiodic stimuli. For all elements of this model, their anatomical representations are poorly understood or, in certain cases, completely unknown.     

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT₁ and MT₂ membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT₁ and ROR receptor mRNA and protein are expressed in both thymus and spleen, while MT₂ receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.Received 2 June 2003; received after revision 6 August 2003; accepted 14 August 2003     

Pineal melatonin rhythms and the timing of puberty in mammals

Summary The direction of change in daylength provides the seasonal time cue for the timing of puberty in many mammalian species. The pattern of melatonin secretion from the pineal gland transduces the environmental light-dark cycle into a signal influencing the neuroendocrine control of sexual maturation. The change in duration of nocturnal melatonin secretion is probably the key feature of the melatonin signal which conveys daylength information. This information may also be used by neuroendocrine axes controlling seasonal changes in pelage colour, growth and metabolism. The mechanism of action of melatonin on neuroendocrine pathways is unknow. Although the ability to synthesize and secrete melatonin in a pattern that reflects the duration of the night may not occur until the postnatal period, the rodent and ovine foetus has the ability to respond in utero to photoperiodic cues to which its mother is exposed in late gestation. Transplacental passage of maternal melatonin is likely to be the mechanism by which photoperiodic cues reach the foetus. Species which do not exhibit seasonal patterns of puberty, such as the human, also secrete melatonin in a pattern which reflects the environmental light-dark cycle, but they do not respond reproductively to the seasonal melatonin information.     

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the “triple membrane bypass” pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.     

Extrapineal melatonin: sources,regulation, and potential functions

Endogenous melatonin is synthesized from tryptophan via 5-hydroxytryptamine. It is considered an indoleamine from a biochemical point of view because the melatonin molecule contains a substituted indolic ring with an amino group. The circadian production of melatonin by the pineal gland explains its chronobiotic influence on organismal activity, including the endocrine and non-endocrine rhythms. Other functions of melatonin, including its antioxidant and anti-inflammatory properties, its genomic effects, and its capacity to modulate mitochondrial homeostasis, are linked to the redox status of cells and tissues. With the aid of specific melatonin antibodies, the presence of melatonin has been detected in multiple extrapineal tissues including the brain, retina, lens, cochlea, Harderian gland, airway epithelium, skin, gastrointestinal tract, liver, kidney, thyroid, pancreas, thymus, spleen, immune system cells, carotid body, reproductive tract, and endothelial cells. In most of these tissues, the melatonin-synthesizing enzymes have been identified. Melatonin is present in essentially all biological fluids including cerebrospinal fluid, saliva, bile, synovial fluid, amniotic fluid, and breast milk. In several of these fluids, melatonin concentrations exceed those in the blood. The importance of the continual availability of melatonin at the cellular level is important for its physiological regulation of cell homeostasis, and may be relevant to its therapeutic applications. Because of this, it is essential to compile information related to its peripheral production and regulation of this ubiquitously acting indoleamine. Thus, this review emphasizes the presence of melatonin in extrapineal organs, tissues, and fluids of mammals including humans.     

Vertebrate circadian rhythms: Retinal and extraretinal photoreception

Summary ERRs Both the pineal and the SCN are elements of the vertebrate multioscillator system although the relative importance of these 2 areas probably varies between, and possibly within, the different vertebrate classes. Extraretinal photoreception is a universal feature of submammalian vertebrates, and possibly of neonatal mammals, but is absent in adult mammals. Although the pineal systems of sumammalian vertebrates are photosensitive, the pineal system has been directly implicated as an extraocular site for the perception of entraining light cycles only in amphibians. In all other submammalian vertebrates extraretinal entrainment can occur in the absence of the pineal system although it is certainly conceivable that the pineal system may act as an alternate route of photoreception. These extraretinal-extrapineal receptors are located within the brain but the exact location(s) of these receptors within the brain is unknown. The hypothalamus would be likely area for this extraretinal photoreception, however, for several reasons: 1. Neurophysiological studies have identified light sensitive neurons in the frog's hypothalamus⁴³. 2. The avian hypothalamus is a site of photoperiodic photoreception^100–103. 3. The only other light sensitive structures known in vertebrates—the pineal system and the lateral eyes—are all derived embryologically from the hypothalamus. 4. The hypothalamus appears to be the site of a circadian clock and there may be advantages in having the photoreceptors and the clock anatomically close to one another. These considerations, of course, do not exclude the possibility that other brain areas may be involved as well. The reason behind the loss of extraretinal photoreception in mammals is uncertain. The shift to exclusive retinal photoreception in mammals may have been dictated by the extensive reorganization that occurred during the evolution of the mammalian brain. Or, perhaps, the increased size of the mammalian skull and overlying tissue made direct photoreception difficult and necessitated a shift to retinal photoreception. The persistence of extraretinal photoreceptors in submammalian vertebrates, however, underscores their importance in the sensory repertoire of vertebrates.     

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes: significance in IL-2 production

Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE₂ in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE₂-diminished IL-2 production. The decrease in IL-2 production after PGE₂ and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE₂ in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT₁ antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology. Received 23 September 2008; received after revision 19 November 2008; accepted 21 November 2008     

Persistent 24-h variations of urinary 6-hydroxy melatonin sulphate and cortisol in Antarctica

Bright light (2000-3000 lux) of sufficient intensity to suppress human melatonin secretion, acts as a strong zeitgeber in the entrainment of circadian rhythms in man. In polar conditions, light of this intensity is not experienced for several weeks during the winter. The entrainment of human circadian rhythms, in particular that of melatonin, is clearly of interest in these circumstances. Urinary 6-hydroxy melatonin sulphate (aMT6s) is a good index of melatonin secretion in man. In a limited study of seven male volunteers living on an Antarctic base the overall 24-h rhythm of aMT6s excretion was maintained at four different times of year (spring, summer, autumn and winter) and no significant seasonal effects were noted. Cortisol excretion, appeared to be markedly affected by the season although other factors such as social and environmental stress cannot be discounted. These observations suggest that in the absence of a strong light-dark cycle melatonin production may be entrained by other factors.     

Persistent 24-h variations of urinary 6-hydroxy melatonin sulphate and cortisol in Antarctica

Summary Bright light (2000–3000 lux) of sufficient intensity to suppress human melatonin secretion, acts as a strong zeitgeber in the entrainment of circadian rhythms in man. In polar conditions, light of this intensity is not experienced for several weeks during the winter. The entrainment of human circadian rhythms, in particular that of melatonin, is clearly of interest in these circumstances. Urinary 6-hydroxy melatonin sulphate (aMT6s) is a good index of melatonin secretion in man. In a limited study of seven male volunteers living on an Antarctic base the overall 24-h rhythm of aMT6s excretion was maintained at four different times of year (spring, summer, autumn and winter) and no significant seasonal effects were noted. Cortisol excretion, appeared to be markedly affected by the season although other factors such as social and environmental stress cannot be discounted. These observations suggest that in the absence of a strong light-dark cycle melatonin production may be entrained by other factors.     

Chronobiology of sleep in humans

Periodic circadian (24-h) cycles play an important role in daily hormonal and behavioural rhythms. Usually our sleep/wake cycle, temperature and melatonin rhythms are internally synchronized with a stable phase relationship. When there is a desynchrony between the sleep/wake cycle and circadian rhythm, sleep disorders such as advanced and delayed sleep phase syndrome can arise as well as transient chronobiologic disturbances, for example from jet lag and shift work. Appropriately timed bright light is effective in re-timing the circadian rhythm and sleep pattern to a more desired time, ameliorating these disturbances. Other less potent retiming effects may also be obtained from the judicious use of melatonin and exercise.     

Circadian rhythmicity is involved in photoperiodic time measurement in the aphidMegoura viciae

The photoperiodic clock in the vetch aphidMegoura viciae is generally accepted to be based on a non-circadian mechanism or hourglass, as no evidence has been found for the involvement of the circadian system in the photoperiodic response. By using a recently-devised protocol which discriminates between single and repeated night length measurement, we demonstrate here that long-night measurement inMegoura is executed in a repetitive way, and thus that its photoperiodic clock is based on a circadian oscillator after all. However, it is also apparent that the determination of short nights is not repetitive.Dedicated to our dear friend, the late Professor A. D. (tony) Lees who, sadly, died before we had a chance to discuss the data reported here.     

Pineal melatonin rhythms and the timing of puberty in mammals

The direction of change in daylength provides the seasonal time cue for the timing of puberty in many mammalian species. The pattern of melatonin secretion from the pineal gland transduces the environmental light-dark cycle into a signal influencing the neuroendocrine control of sexual maturation. The change in duration of nocturnal melatonin secretion is probably the key feature of the melatonin signal which conveys daylength information. This information may also be used by neuroendocrine axes controlling seasonal changes in pelage colour, growth and metabolism. The mechanism of action of melatonin on neuroendocrine pathways is unknown. Although the ability to synthesize and secrete melatonin in a pattern that reflects the duration of the night may not occur until the postnatal period, the rodent and ovine foetus has the ability to respond in utero to photoperiodic cues to which its mother is exposed in late gestation. Transplacental passage of maternal melatonin is likely to be the mechanism by which photoperiodic cues reach the foetus. Species which do not exhibit seasonal patterns of puberty, such as the human, also secrete melatonin in a pattern which reflects the environmental light-dark cycle, but they do not respond reproductively to the seasonal melatonin information.