Indian Journal of Biochemistry & Biophysics Vol 45, October 2008, pp. 289-304

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Melatonin: Fifty Years of Scientific Journey from the Discovery in Bovine Pineal Gland to Delineation of Functions in Human Indrajit Chowdhury1, Anamika Sengupta2 and Saumen Kumar Maitra3* 1

Department of Obstetrics and Gynecology, 2Department of Physiology, Morehouse School of Medicine, Atlanta, USA 3 Department of Zoology, Visva-Bharati University, Santiniketan, 731 235, India Received 24 November 2007; revised 01 July 2008

Melatonin (N-acetyl-5-methoxytryptamine) was first purified and characterized from the bovine pineal gland extract by Aron Lerner and co-workers in 1958. Since then, a plethora of information has piled up on its biosynthesis, metabolism, time-bound periodicity, physiological and patho-physiological functions, as well as its interactions with other endocrine or neuro-endocrine organs and tissues in the body. Melatonin has wide range of applications in physiology and biomedical fields. In recent years, a significant progress has been made in the understanding mechanism of its actions at the cellular and molecular levels. Consistent efforts have uncovered the mystery of this indoleamine, and demonstrated its role in regulation of a large as well as diverse body functions in different groups of animals in general, and in humans in particular. Current review, in commemoration of 50 years of discovery of melatonin, while revisiting the established dogmas, summarizes current information on biosynthesis, secretion, metabolism and molecular mechanism of action of melatonin at cellular level and highlights the recent research on its role in human physiology and clinical biology. Keywords: AA-NAT, Biological rhythm, Cancer, Melatonin, Pineal gland, Reproduction, Seasonal affective disorder Suprachiasmatic nucleus, Sleep, Jet-lag.

Until first half of the twentieth century, the pineal gland was considered as an epithalamic appendage of vertebrate brain with enigmatic functions. Discovery of a new biological substance from the bovine pineal gland extract by the dermatologist Aaron B Lerner at the Yale University School of Medicine was a major breakthrough in pineal research1. This purified pineal product was chemically identified as N-acetyl-5methoxy-tryptamine, and named melatonin (Fig. 1), in recognition to McCord and Allen’s2 observations as early as 1917 that bovine pineal extracts added to water caused the larvae to blanch due to aggregation _________________ *Corresponding author Tel: 91-3463-261268; Fax: 91-3463-261268 E-mail: [email protected], [email protected] Abbreviations: AA-NAT, arylalkylamine N-acetyl transferase; AMK, AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine; N1-acetyl-5-methoxykynuramine; c3OHM, cyclic3-hydroxymelatonin; camp, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CYp1A2, CYP1A1 or CYP1B1, cytochrome P450 mono-oxygenases; ER, estrogen receptor; GnRH, gonadotropin releasing hormone; HIOMT, hydroxyindole-Omethyl transferase; 5-HIAA, 5-hydroxy-indole acetic acid, IL, interleukin; LDL, low density lipoprotein, MT, melatonin receptor; NO, nitric oxide; NA, noradreneralin; REM, rapid eye movement; SAD, seasonal affective disorder; MT6s, 6-sulphatoxymelatonin; SCN, suprachiasmatic nucleus.

Fig. 1—Chemical structure of melatonin (N-acetyl-5-methoxy tryptamine)

of melanin granules within the skin cells. Though this indole hormone was first reported from the pineal gland, it was also found in retinae, Harderian gland and gastro-intestinal tracts in vertebrates, as well as in a wide variety of organisms, ranging from invertebrates to plants. One of the characteristic features of this tiny tryptophan derivative molecule is that its synthesis takes place during darkness in a light-dark cycle, irrespective of the habit of the concerned animals. Therefore, it is often described as ‘hormone of darkness’ or a ‘chronobiotic molecule’3.

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Past fifty years, since its discovery from the bovine pineal gland have witnessed accumulation of considerable data on different aspects of regulatory mechanism involved in the synthesis and release of melatonin, its role in the regulation of diverse body functions in vertebrates, human in particular. Studies in various animals, especially in mammals, have shown the importance of melatonin in regulation of a wide range of physiological functions, ranging from aging to aggression, hibernation to hypertension, sleep to stress, reproduction to tissue regeneration, scavenging of free radicals to synchronization of body functions with the environmental light-dark cycles. Now, it is clearly known that melatonin acts on its target cells/tissues through transmembrane receptors MT1, MT2 and MT3 or through orphan nuclear receptors of the retinoic acid receptor family4 Being a lipophilic molecule, it has free access to all cells, tissues, and organs of the body and thereby offers an additional receptor independent non-hormonal role of free radical scavenger in reducing the oxidative stress. The physiological and genetical aspects of the melatonin have been well-documented in recent years3-12. As a supplementary to the existing literature and to commemorate the golden jubilee year of the discovery of melatonin, this communication aims at summarizing the recent progress in the understanding of molecular mechanism of action and physiological role of melatonin with special emphasis on clinical significance and application in human subjects. Melatonin: A ubiquitously distributed biological molecule Melatonin is ubiquitously distributed in living system and is suggested to represent one of the most primitive biological signals appeared on earth13. It has been identified in all major taxa of organisms including bacteria, and unicellular eukaryotes, in different parts of plants including the roots, stems, flowers and seeds, and in invertebrate and vertebrate animals14,15. It is also present in different tissues, organs such as Harderian gland, extraorbital lacrimal gland, retina, bone marrow cells, platelets, lymphocytes, skin, enterochromaffin cells of gastrointestinal (GI) tract and in bile in a variety of animal species10, 16-18 . In fact, its concentration in the GI tract is greater than in the pineal gland or in the circulation and in the bile16. The concentration of melatonin in the GI tissues surpasses blood levels by 10–100 times and there is at least 400× more melatonin in the gastrointestinal tract than in the pineal gland16.

Biosynthesis, secretion and metabolism Biosynthesis

In all vertebrates investigated so far, melatonin is primarily synthesized within the pinealocytes of pineal gland10 during the night, regardless of the diurnal or nocturnal locomotor activity of the animals. Biosynthesis of melatonin is a four-step phenomenon. First, its precursor L-tryptophan is taken up from the circulation (blood) into the pinealocyte and converted to 5-hydroxytryptophan by tryptophan 5-monooxy genase/hydroxylase (L-tryptophan, tetra-hydropterindine: oxygen oxidoreductase, EC 1.14.16.4) and further decarboxylated by L-aromatic amino acid decarboxylase (aromatic L-amino acid carboxylase, EC 4.1.1.28) to form 5-hydroxytryptamine (5-HT) or serotonin. Serotonin is acetylated (N-acetylation), to form N-acetylserotonin using serotonin-Nacetyltransferase/arylalkylamine N-acetyltransferase (AA-NAT; acetyl CoA: aryl-amine N-acetyltransferase, EC 2.3.1.5). Finally, N-acetylserotonin is methylated by hydroxyindole-o-methyltransferase (HIOMT; S-adenosyl-L-methionine: N-acetylserotonin-o-methyltransferase, EC 2.1.1.4) to form melatonin11,19,20. AA-NAT is the rate-limiting enzyme in melatonin synthesis, but serotonin availability is one of the major factors that play an important regulatory role in this process9. The magnitude and duration of nocturnal increase in melatonin synthesis is dependent upon the length of the dark phase of photoperiodic cycle or external geophysical cycle and it acts as a “clock” and “calendar” for the entrainment of other biological activities21. The rhythm of melatonin synthesis is generated by interacting networks of circadian clock genes located in the suprachiasmatic nucleus (SCN), circadian oscillator/master pacemaker, of the hypothalamus in brain, which is considered as the major central rhythm-generating system or “clock” in mammals22,23. The pineal gland itself is a selfsustaining “clock” in some species, except in lower vertebrates24. The SCN clock is set to a 24-h day by the natural light-dark cycle via retinal light input which then sends circadian signals over a neural pathway including sympathetic nerve terminals that project from the superior cervical ganglia (SCG) to the pineal gland and thereby driving rhythmic melatonin synthesis. The main photoreceptor pigment for circadian timing appears to be melanopsin in the retinal ganglion cells25. Specifically, SCN is the major regulatory site of of AA-NAT activity, which is the

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penultimate and key enzyme in the synthesis of melatonin from tryptophan. The mechanism of biosynthesis of melatonin is presented in Fig. 2. There are notable species differences in the regulation of AA-NAT synthesis. In human and ovine system, the enzyme is regulated primarily at a posttranscriptional level, whereas in rodents, the key event appears to be cyclic AMP-dependent phosphorylation of a transcription factor that binds to the AA-NAT promoter20. The rapid decline in activity of AA-NAT with light treatment at night26 appears to be complex and associated with the control of catabolism through various phosphorylation of AA-NAT by protein kinase A (PKA), Rho kinase, checkpoint protein 1 (CHK1)19 and subsequent association of the phosphorylated AA-NAT with the chaperone-like 14.3.3 protein27. This dimerization either drives the complex to a proteosomal-mediated proteolysis or protects the complex from breakdown28. Catabolism of AA-NAT is limited to the pineal gland, whereas the retinal AA-NAT enzyme appears to be protected against breakdown during the day29.

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Post-ganglionic sympathetic nerve fibers that end at the pineal gland release noradrenalin (NA) or norepinephrine (NE) which also plays crucial role in the control of melatonin synthesis. The nocturnal elevation in noradrenergic stimulation via β and α1- adrenergic receptor of pinealocytes increase the intra-cellular concentration of cAMP, which in turn activates AANAT20 resulting in increased levels of melatonin at night. Stimulation of α-adrenergic receptors potentiates the βstimulation and requires participation of other molecules such as calcium ions, phosphatidylinositol, diacyl glycerol, and protein kinase C30. Thus, the synthesis and release of melatonin are stimulated by darkness (~80% synthesis) and are inhibited by light. The regulation of melatonin synthesis is also influenced by 5-HT, monoamine oxidase, cortisol, corticosterone, aldosterone, testosterone and estradiol, but their exact molecular mechanisms are not yet clearly known31,32. Secretion

The secretion of melatonin is related to the length of night. A single daily light pulse of suitable

Fig. 2—Diagrammatic presentation of the mechanism of biosynthesis and catabolism of melatonin [The hormone melatonin is synthesized from tryptophan under the control of the enzymes tryptophan 5-hydroxylase, 5-hydroxytryptophan-(5HTP)-decarboxylase, serotonin-N-acetyltransferase (NAT) or AANAT, and hydroxyindole-o-methyl transferase (HIOMT). The formation and secretion of AANAT and HIOMT are influenced by suprachiasmatic nucleus (SCN) activity via seasonal and circadian timing mechanisms. The catabolism of melatonin is through kynuric pathway, including metabolites formed by interaction of N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), glucuronides etc.]

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intensity and duration26 in otherwise constant darkness is sufficient to phase shift and to synchronize the melatonin rhythm in animals to 24 h via SCN21. There are clear indications that prolonged duration of the night leads to longer duration of secretion of melatonin in most animal species3. Ocular light serves to entrain/synchronize the rhythm to 24 h and to suppress secretion at the beginning and/or the end of the dark phase. The amount of light required to suppress melatonin secretion during the night varies from species to species with the time of night and with the history of previous light exposure31,32. The amplitude of nocturnal melatonin secretion exhibits considerable inter-individual differences and is possibly genetically determined. In humans, serum levels of melatonin is low during the day (10-20 pg/ml), but significantly higher at night (80-120 pg/ml) with peak between 24:00 h and 03:00 h. The onset of secretion usually takes place during 22.0002:00 h and the offset between 07:00-09:00 h31,32. Melatonin concentration varies in relation to the stage of development, puberty, menstrual cycle and age of the individuals. Its peak values in blood may also vary from one individual to the other depending on their age, sex and disease10. Just after birth, very little melatonin or 6-sulphatoxymelatonin (aMT6s) is detectable in the body fluids. A robust melatonin rhythm appears in 6 to 8 weeks of age33. The serum melatonin level increases rapidly thereafter and reaches a lifetime peak in between 3-5 yr of age34. The increment is much greater at night (54-75 pg/ml). Subsequently, a steady decrease occurs reaching to a mean adult concentration in mid to late teens with the major decline before puberty, with relatively stable until 35 to 40 yrs, and further reach to low levels (16-40 pg/ml) in old age31,32,35. However, elderly women show higher levels of melatonin than in elderly men. Although melatonin concentration is low in precocious puberty, higher melatonin is found in delayed puberty and hypothalamic amenorrhea, compared to age-matched control34 and abnormal melatonin secretion in patients with pre-menstrual tension36. Low melatonin is associated with cardiovascular diseases and diabetic autonomic neuropathology37. Metabolism

Pineal melatonin is released in the cerebrospinal fluid in the third ventricle via pineal recess and attains up to 20-30 times higher levels than in blood, but rapidly diminishes with increasing distance from the

pineal gland38 suggesting that melatonin is taken up by brain tissues. In the blood, 50-75% of total melatonin is bound reversibly to albumin and glycoproteins. The half-life of melatonin is bi-exponential, with a first distribution half-life of 2 min and a second of 20 min13. The endogenous halflife of melatonin in serum is about 30-60 min and exogenous melatonin has even shorter half-life, 12-48 min39. The mechanism of melatonin catabolism is less understood with the exception of conjugation steps that account for ~70% of the ingested dose. Melatonin is metabolized non-enzymatically in all cells, and by free radicals as an oxidant. It is converted into cyclic 3-hydroxymelatonin (c3OHM), when it directly scavenges two hydroxyl radicals40. The circulating melatonin is primarily metabolized through classical hydroxylation pathway in human and rodent liver by microsomal enzymes, cytochrome P450 monooxygenases (isoenzymes CYP1A2, CYP1A1 or CYP1B1) to 6-hydroxymelatonin which further conjugates with either sulphate (catalyzed by sulphotransferase ST1A3) to form 6-sulphatoxy melatonin (MT6s), or glucuronic acid (catalyzed by UDP-glucuronosyltransferase) to form 6-hydroxy melatonin glucuronide13. Several other metabolites (approximately 30% of overall melatonin) are also formed by pyrrole ring cleavage19. This oxidative catabolism by indoleamine-2, 3-dioxygenase and/or myeloperoxidase leads to the formation of unstable intermediary kynuramine derivative N1-acetyl-N2formyl-5-methoxy-kynuramine (AFMK), which is further deformylated to more stable N1-acetyl-5methoxy-kynuramine (AMK) by kynuramine formamidase. Thus, the clearance rate of melatonin is predominantly associated with excretion in the urine in metabolized form and in small quantities in unmetabolized form41. Mechanism of action at cellular level The development of high affinity radioligand binding assay of 2-[125I] iodomelatonin, autoradio graphy and studies with putative melatonin receptor agonists and antagonists have allowed to identify, characterize and demonstrate distribution patterns of melatonin receptors in various central and peripheral tissues42,43. The first melatonin receptor was cloned from Xenopus laevis immortalized melanophore mRNAs44, but expressed only in non-mammalian species (birds and fish). Subsequently, human melatonin receptors have also been cloned45.

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Two forms of high-affinity melatonin receptors (MT1 and MT2) and a low-affinity receptor (MT3) have been identified based on pharmacological and kinetic differences in 2-[125I] iodomelatonin binding assay46,47. In human, MT1 receptor consists of 350 amino acids, and is mapped to chromosome 4q35.148 while gene for MT2 receptor is located in chromosome 11q21-22 and cDNA encodes a protein containing 365 amino acids with 60% homology with MT1 receptor45. These membrane-bound receptors (MT1 and MT2) belong to the superfamily of guanidine triphosphate binding proteins or G-protein coupled receptors containing the typical seven transmembrane domains7,46,49,50. Studies on immunolocalization, 2-[125I] iodomelatonin binding assay and cloning of mRNA from different tissues have shown that these receptors are widely distributed in the peripheral tissues, viz. heart, arteries, kidney, liver, gall bladder, intestine, adipocytes, granulosa cells of ovarian follicles, uterus, breast cells, prostate and skin7, besides retina, cornea, ciliary body, lens, choroids and sclera51. Even MT1 receptor is expressed in the SCN, hypophyseal pars tuberalis and other parts of the brain including hypothalamus, thalamus, hippocampus, cerebellum and cerebral cortex52. . The density of melatonin receptors not only varies with species and location, but also with the lighting regime, time of the day, tissues, and developmental or endocrine status of the concerned animals53. The SCN is the putative site of circadian action of melatonin, and the hypophyseal pars tuberalis is the putative site for its reproductive effects. Melatonin binding with MT1 modulates intracellular signal by inhibiting adenylate cyclase and stimulating phosphoinositide hydrolysis, while activation of MT2 receptor inhibits formation of two second messengers namely, cAMP and cGMP at cellular levels13. The decrease in cAMP production reduces the uptake of linoelic acid, an essential and major fatty acid by specific fatty acid transporters54 MT1 acts by suppressing neuronal firing activity by mediating vasoconstriction and MT2 acts mainly by inducing phase shifts by mediating vasodilation46. A third mechanism of the biological effects of melatonin is through MT3 receptor, quinine reductase 2 (QR2), a known detoxifying enzyme55 which is identified with lower melatonin affinity, very rapid ligand association/dissociation kinetics and widely distributed in various tissues of the body55. The MT3 receptor modulates calcium and calmodulin activity5,

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and thereby play a role in the initiation of S and M phases of the cell cycle, cell cycle-related gene expression, and the re-entry of quiescent cells from G0 back into the cell cycle55. Becker-Andre and co-workers56 demonstrated that beside MT receptors, melatonin has genomic action through novel class of orphan nuclear receptors of the retinoic acid receptor family. These receptors have been cloned and named as retinoid Z receptor (RZR) and retinoid acid receptor-related orphan receptor (ROR)56,57. The RZR/ROR family consists of three subtypes: α, β and γ. The RORα1, RORα2 and RZRβ have low affinity in normal and cancer tissues59. RORα1 and RORα2 receptors are involved in immune modulation, whereas the receptor RZRβ is expressed in the CNS, including pineal gland. Melatonin also acts in an intracrine, paracrine or autocrine manner in the pineal gland, eye, lymphocytes, gut, bone marrow, skin, and gonads for the local coordination7,16,17,52,58,60,61. The mechanism of actions of melatonin at cellular level is schematically presented in Fig. 3. Functions: A testimony of physiological diversity Due to pleiotropic nature of melatonin, a wide range of its physiological or patho-physiological functions is ascribed to human or other mammalian species. Most of the studies have used pharmacological doses of melatonin (1 µM and above) and a few studies have confirmed these functions either clinically or experimentally as opposed to physiological doses (below the nanomolar range) of melatonin49,62. Several recent 7,8,11,12,63,64 have reviewed melatonin publications involvement in multiple physiological functions, but only a few of them have shown its importance in the context of human health. Synchronization of rhythmic body functions with the environment

In all living organisms, circadian periodicity (for human 24.2 h/cycle) in many of the body functions is an inherited characteristic which appears to be closely related to diurnal preference and the early or late timing of the circadian system (cBT) in a normal entrained situation65. Melatonin acts as an endogenous synchronizer either in stabilizing rhythms (circadian) of body functions or in reinforcing them, hence, called a ‘chronobiotic’ molecule66. Melatonin is also considered as a “neuroendocrine transducer” or “hormone of darkness” or “biological night” which is

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Fig. 3—Mechanism of action of melatonin at cellular level: A schematic presentation

exclusively involved in signaling the “length of night” or “time of day” and “time of year” to all tissues. In mammalian species and humans, exogenous administration of melatonin is reported to change the timing of rhythms by increasing sleepiness, wake EEG theta activity, REM (rapid eye movement) sleep propensity, non-REM sleep propensity, endogenous melatonin and cortisol levels; and decreasing core body temperature that ultimately leads to sleep4,67,68. This phase shifting effect of melatonin depends upon its time of administration. It causes phase-advance of the circadian clock when given during the evening and the first half of the night, and phase-delays when given during the second half of the night or at early daytime. The magnitude of phase-advance or phasedelay depends on the dose of melatonin69. As melatonin crosses the placenta, it plays an active role in synchronizing the fetal biological clock13. The chronobiotic effect of melatonin is caused by its direct influence on the electrical and metabolic activity of the SCN as shown by in vitro as well as in vivo experiments71-73. Melatonin through various receptors induces a differential influence on clock genes. The currently known mammalian clock genes, which have been cloned only during the last onedecade include three mammalian homologues of period (Per1, 2, 3), clock (circadian locomotor output

cycles kaput), Bmal1 (Brain and Muscle ARNT-like 1, Mop3), the two homologues of Drosophila cryptochrome (Cry1 and Cry2) and casein kinase 1ε (CK1ε). Human clock genes show high similarity to those of other mammalian clock genes. The Per1, 2, 3; clock, Bmal1 (Mop3), Cry1 and Cry2 genes are expressed in all peripheral tissues74,75. The clock genes are conserved from flies to human. Nevertheless, the functional roles played by some of their corresponding products differ between insects and mammal. In human, the pars tuberalis and SCN clock genes expression pattern show 24 h rhythmicity. Per1 is activated at the beginning of light phase and Cry1 at the onset of dark phase. Long or short photoperiod information is encoded within the SCN. Thus, synthesis of melatonin is driven by the SCN to convey the photoperiodic information to the pars tuberalis by virtue of its secretion pattern. This phenomenon, in turn, influences the pattern of expression of the clock genes Per1 and Cry1 within the pars tuberalis, providing a means of translating the melatonin signal for the control of body rhythm or rhythmic synthesis and secretions of hormones42. However, amplitude modulation is unrelated to clock gene expression in the SCN42,76. The circadian regulation is also determined by the interactions with neurotransmitter functions. The

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highest concentration of serotonin in the CNS is in the SCN77,78 and its turnover exhibits marked circadian and seasonal rhythmicity, which is rapidly stimulated by light exposure77. At the cellular level, circadian rhythms of other clock genes are driven by the interlocking self-regulatory interaction. The positive arm of the circuit is the heterodimer of the proteins CLOCK:BMAL1. This complex binds E-box elements (CACGTG/T) at the promoter region of Per1-2 and Cry1-2, including their transcription. The negative regulators are the translated CRY and PER proteins that complex with CK1ε to translocate into the nucleus, interacting with the CLOCK:BMAL1 complex and thus blocking their own transcription. Phosphorylation of PER and CRY proteins by CK1ε controls their proteosomal degradation, delaying the formation of CRY:PER complex and determines the length of the cycle. Additional regulators are negative loop generated by the transcription of the dec1 (sharp2/stra13) and dec2 (sharp1) genes, which are also driven by CLOCK: BAML1 via E-boxes in their promoters. DEC1 and DEC2 proteins may block circadian gene expression, in part by forming a non-functional heterodimer with BMAL1, which inhibits the expression of all genes dependent on an E-box as well as play a role in light induction of genes in the SCN. The circadian oscillation of clock gene expression controls the expression of genes involved in multiple cellular functions in the 24 h period (clock control genes, CCGs) by at least two mechanisms: direct interaction with E-boxes in the promoters of these genes; and through the regulation of other CCGs that in turn are influenced by transcription factors like DBP79. These studies reveal that melatonin is an effective chronobiotic molecule that synchronizes rhythmic body functions with the environmental variables and clock genes. Regulation of sleep-wakefulness cycle

Two-process model of sleep regulation considers the timing and architecture of sleep to be a consequence of a homeostatic process of rising sleep pressure and the duration of prior wakefulness that is dissipated during the sleep period, and is a function of circadian pacemaker80. Over the last decade, two important protocols have been developed to investigate circadian and sleep homeostatic processes in human81. A strong relationship has been found between sleep and melatonin levels67. Both, nocturnal

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melatonin levels and the quality of sleep, decline at puberty and in elderly82. The period of sleep tends to become shorter and the quality of sleep poorer with decreased amplitude of the circadian rhythm and waking in a 12 h light/12 h dark cycle, and sometimes with phase advancement of circadian rhythm called delayed sleep phase syndrome (DSPS)81. Cross meridian flights involve disorganization of biological rhythm caused by the rapid change of environment and associated light/dark cues. The clock genes in the SCN gradually adapt to phase-shift of the light dark cycle (as found in shift work, transmeridian flight). Peripheral clocks in the muscle, liver, pancreas, kidney, heart, lung and mononuclear leukocytes are entrained directly by the SCN through some neurohormonal signals, glucocorticoids, retinoic acid, growth factors and by other Zeitgebers, such as body temperature and feeding time, which resynchronize their clock genes at their own rates83,84. Circadian clocks in peripheral tissues/cells also exhibit identical features. This results in “double desynchronisation”-“internal desynchronisation” between different clocks in the body and brain, and “external desynchronisation” between the timing of body rhythm with respect to the light-dark cycle. This temporal orchestra of ‘jet lags’ (sleep disturbance, mental inefficiency or daytime fatigue) can be corrected by melatonin given at local bedtime i.e., between 10 p.m. and mid-night85. Moreover, patient or animal model with primary insomnia (wakefulness and inability to fall asleep before 2.00 to 3.00 a.m.), narcolepsy (a disorder of disturbed circadian sleep/wake rhythm and REM sleep deficit) and sleep disorders in children (hyperactivity disorder) can be successfully corrected with pharmacological doses (5-50 mg) of melatonin86. Thus, melatonin can be used at local bedtime to resynchronize the circadian oscillator with the new environment for coordination of circadian rhythms and sleep function86,87. However, higher secretion of melatonin causes maximum sleep and fatigue at night87. These studies indicate that application of melatonin in optimum dose and schedule attenuates sleep related problems in different human age groups. Regulation of mental state, behaviour and brain functions

The pineal gland promotes homeostatic equilibrium through melatonin, acts as a “tranquilizing organ” in stabilizing electrical activity of the CNS and causes rapid synchronization of the EEG88. The classic endogenous or non-seasonal depression is

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characterized by the insomnia (early morning awakening), appetite suppression, weight loss and advanced onset of nocturnal melatonin release that begins in the spring and persists through the summer or winter during the period of light-phase shortening89. Similarly, seasonal affective disorder (SAD) is characterized by late sleep, morning hypersomnia, increased appetite, and retarded onset of nocturnal melatonin release90 which peaks in the autumn and spring68. Similar phenomena are associated with individuals with low nocturnal melatonin levels and major depressive/panic disorders91. The epidemiologic and chronobiological evidences strengthen the link between melatonin levels, pineal function and mood disorders. Administration of different doses (>1 g/day) of melatonin in these individuals at night prolongs the nocturnal melatonin rise and helps in recovering SAD by changing the expression of clock gene and by changing the expression of Per2 gene in bipolar or classic depression91,92. However, large doses in the morning or early afternoon failed to show any clear effect93, though phototherapy as an adjuvant may accelerate response to antidepressants among patients with depression94. Melatonin secretion has been shown to be wavelength-dependent as exposure to mono chromatic light at 460 nm produces a 2-fold greater circadian phase-delay95. These results are further confirmed by measuring the brain serotonin and tryptophan levels that rise after melatonin administration ,and directly linked with an array of neuropsychiatric phenomenon96. The diminished central serotonin, as indicated by low levels of serotonin marker 5-hydroxy indole acetic acid (5-HIAA) in cerebrospinal fluid is associated with impulsiveness, aggression and auto-aggregation, alcoholism, compulsive gambling, overeating, and other obsessive-compulsive behaviours97. Moreover, the requirement of intact β-receptor function for melatonin synthesis and stimulatory effect of norepinephrine on melatonin synthesis and release demonstrate a direct relation of melatonin to depression98. Administration of tricyclic antidepressants (TCA) at night also exerts sedative effect with increased melatonin synthesis through binding with β-adrenergic receptors of pinealocyte and increased cAMP production, which in turn, activates AA-NAT and enhances melatonin rhythm amplitude. TCA also

inhibits cytochrome p450 enzyme CYP1A2, which metabolizes the melatonin in hepatocytes, leading to an increase in endogenous melatonin levels99,100. Thus, it finally supports serotonergic system to change or elevate mood, reduce aggression, increase the pain threshold, reduce anxiety, relieve insomnia, improve impulse control and ameliorate obsessivecompulsive syndromes91. However, higher doses or long-term use of TCA has side effects by blocking muscarinic, histaminergic and adrenergic receptors, because of its joined benzene ring structure. In isolated cases, a long-term use of melatonin has been found to cause psychomotor disturbance, increased seizure risk and blood clotting abnormalities. Collectively, these findings suggest that appropriate exogenous melatonin administration can restore human neurological disorders with direct impact on general health of elderly people64. Scavenger of free-radicals

Melatonin, because of its lipophilic nature13, crosses all morphological, physical and hematoencephalic barriers (blood brain barrier, placenta) and reaches all tissues of the body within a short period of time101. As a result, it exhibits antioxidant effect and performs a very important receptor-independent metabolic function, i.e., multifaceted scavenger of free radicals. The antioxidant effects of melatonin have been well described, and are known to include both direct and indirect effects with equal efficiency in multiple sites (nucleus, cytosol and membranes) of the cell78. It detoxifies a variety of free radicals and reactivates oxygen intermediates, including the hydroxyl radical/hydrogen peroxide, peroxy radicals, peroxynitrite anion, singlet oxygen, nitric oxide and lipid peroxidation. Melatonin is a more potent antioxidant than vitamin C and E102,103. The antioxidant property of melatonin is shared by two of its major metabolites, namely N1-acetylN2-formyl-5-methoxy-kynuramine (AFMK)41 and a higher efficient, N1-acetyl-5-methoxykynuramine 104 (AMK) . AFMK is produced by both enzymatic and non-enzymatic mechanisms105 and mainly by myeloperoxidase19. The potent scavenger AMK consumes additional radicals in primary and secondary reactions106. Interestingly, AMK interacts not only with reactive oxygen, but also with reactive nitrogen species107. It exerts its effects on electron flux through the respiratory chain and improves ATP synthesis in conjugation with the rise in complex I and IV activities108. The broad-

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spectrum antioxidant activity of melatonin also includes indirect effect of upregulating several antioxidative enzymes and downregulating pro-oxidant enzymes, in general, and 5- and 12-lipo-oxygenases109, glutathione peroxidase, glutathione reductase, glucose-6-phospahte dehydrogenase, superoxide dismutase, catalase110 and nitric oxide (NO) synthase40, in particular. Due to strong antioxidant property, melatonin protects lipid in membranes, proteins in cytosol, DNA in nucleus and mitochondria from free radical damages. Thus, it can be used in the treatment of several neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s chorea and amyotrophic lateral sclerosis, which are caused by interrelated processes namely glutamate excitotoxicity, free radical-mediated nerve injury and mitochondrial dysfunction111-114. Studies on disruption of nocturnal surge of melatonin in ischaemic stroke patients and patients with acute cerebral haemorrhage115 and reduction in the degree of tissue damage against ischaemic injury through direct free radical scavenging or by indirect antioxidant activities in these exogenously melatonin administered patients suggest the neuroprotective role of melatonin in strokes116. The oxidative stress is an important hallmark in disorganizing the cortical actin cytoskeletal assembly and disruption of accompanied sub cellular intricate fibrous network composed of microtubules, microfilaments and intermediate filaments (IF) as well as by associated proteins117. Melatonin prevents cytoskeletal structure disruption, followed by cell shape changes and increased lipid peroxidation or apoptosis induced by okadaic acid of physiological plasma and cerebrospinal fluid118. In this process, participation of PKC as an important signaling molecule has been evident from the studies that a PKC inhibitor (bisindolylmaleimide) abolishes cytoskeletal reestablishment elicited by melatonin, while the PKC agonist (PMA or phorbol 12-myristate 13-acetate) reorganizes microtubules and microfilaments119. Thus, melatonin may be used in the treatment of neurodegenerative diseases as a cytoskeletal modulator as well as a free radical scavenger5,96,120. The anti-oxidant properties of melatonin also help in reducing blood cholesterol (~38%), mainly by inhibiting copper-induced oxidation of low-density lipoprotein (LDL) with reduction in blood pressure and catecholamine levels via relaxation of smooth muscles in aortic walls and potentially contributing to an antiatherosclerotic effect on cardiovascular

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system12. Likewise, exogenous administration of melatonin causes vasodilatation, decreasing internal artery pulsatile index, increases the cardiac vagal tone, decreases circulating norepinephrine levels and finally reduces blood pressure in hypertensive patients121. Antioxidant properties of melatonin can also be additive to the treatment of gastric ulcer, bowel syndrome, ulcerative colitis, diarrhea, glycerolinduced renal failure122,123. Thus, melatonin can act as a potent free radical scavenger, neutralizing hydroxyl and peroxyl radicals among others, preventing lipid membrane peroxidation, apoptosis and protecting the DNA from the damage induced by free radicals124. Modulation of immune function

The role of melatonin as an immunomodulator in the regulation of development, differentiation, and functions of lymphoid tissues is known for nearly past three decades58,125-127. In rodents, post-natal pinealectomy suppresses immunity and thymic atrophy, whereas exogenous melatonin treatment increases cell-mediated immune function by increasing natural killer cell activity104. Immunomodulatory effects of melatonin are observed in human with bronchial asthma. The nocturnal rise in blood melatonin levels in human is associated with increased production of interleukins (IL1, IL-2, IL-6, IL-12), thymosin 1a, thymulin and tumor necrosis factor α (TNF α). On the other hand, exogenous melatonin has an adverse effect in patients with asthma104. The nocturnal asthma is associated with elevation and phase-delay of serum melatonin levels128. In adjuvant-induced arthritis, both prophylactic and therapeutic melatonin administration inhibit the inflammatory response129. Melatonin implants enhance a defined T helper 2-based immune response under in vivo conditions, suggesting potential role of melatonin as a novel adjuvant immunomodulatory agent130. Melatonin acts on immunocompetent cells (monocytes, B-lymphocytes, natural killer lymphocytes, T-helper lymphocytes, cytotoxic T lymphocytes) through MT1 and RZR/RORα orphan nuclear receptor family and enhances cytokine production/secretions, cell proliferation or oncostasis131,132. In B-lymphocytes, it binds to the RZR/RORα receptors to downregulate 5-lipoxy-genase expression, which is an important enzyme in allergic and inflammatory diseases like asthma and arthritis133. Thus, melatonin and the immune system are linked by complex bidirectional communication. The immune system, in turn, reciprocally regulates pineal gland functions, mainly via cytokines produced by activated immune cells and depends on age, sex and species58,125-127. The precise

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mechanisms involved in autoimmune diseases are yet largely unknown and need further elucidation. Effects on endocrine network

Melatonin affects the synthesis, secretion and action of steroid as well as non-steroid hormones. In vitro and in vivo experiments have shown that effects of melatonin on adenohypophysial activity vary in different animal species with their age and sex, and in relation to the concentration of the hormone and experimental conditions. It modifies synthesis and secretion of different adenohypophysial hormones (growth hormone/somatotrophin, GH; thyrotropin, TSH; adrenocorticotropin, ACTH) either directly by influencing the secretory activity of the cells in the anterior pituitary, or indirectly by influencing the hypothalamic neurons producing the respective neurohormones which stimulate or inhibit the release of adequate adenohypophysial hormones134,135. Melatonin has a positive phase relation with prolactin synthesis, and negative with GH134-136; it is involved in the regulation of calcium and phosphorus metabolism by stimulating parathyroid gland or inhibiting calcitonin release and prostaglandin synthesis137. A similar change in melatonin levels is observed with the nocturnal increase and morning decrease in prolactin levels, whereas melatonin administration stimulates prolactin secretions138. The relationship between melatonin and GH is poorly understood. Melatonin affects the activity of pituitary-adrenal axis by modulating the peripheral action of corticoids134,135. It possibly acts as a corticotrophinreleasing factor inhibitor with dis-inhibition of the pituitary-adrenal axis in major depression, when pineal melatonin levels are low and unable to modulate functions of adrenal gland139. Low melatonin levels are also found in patients with Cushing disease (hyperadrenocorticism)140 and hypercortisolemia, which have been linked to several aspects of aging and age-associated phenomena, including glucose intolerance, atherogenesis, impaired immune function, and cancer141. The nocturnal melatonin levels decline or almost completely lost with aging in humans35,142. This close reciprocal relation of melatonin and corticoids or loss of melatonin rhythmicity may be responsible for the pituitary/adrenal axis dis-inhibition, characteristic of aging. The adrenal of elderly human is apparently hypersensitive to adrenocorticotropic hormone, and midnight corticoid levels (low in young) are markedly elevated at old age143. Thus, melatonin has phasic

inhibitory effects on both the release of corticoids and their peripheral actions by immune depression, hypercatabolism, thymic involution and adrenal suppression129, which finally unequivocally delays aging. However, direct experimental evidences are not sufficient to demonstrate a relationship between melatonin and hypothalamic-pituitary-adrenal-axis. A strong relationship is known between the functions of pineal gland and hypothalamo-hypophysial-thyroid axis in rodents and several non-mammalian species144. Nevertheless, such relationship is yet to be functionally characterized in humans. Effects on reproduction

The role of melatonin in regulation of reproduction is one of the major areas to have received serious attention for studies in animals145 as well as in human146. Melatonin has a strong influence on the reproductive function in seasonal mammals by its inhibitory action at various levels of the hypothalamic-pituiatry-gonadal axis147. It downregulates gonadotropin-releasing hormone (GnRH) gene expression in a cyclical pattern over a 24 h period and exerts an inhibitory effect by acting on G-protein coupled melatonin receptors MT1 and MT2 and nuclear orphan receptors RORα and RZRβ148. In neonatal pituitary cells, it inhibits GnRH-induced calcium signaling and gonadotropin secretion through MT1 and MT2 receptors. This inhibitory role of melatonin on the hypothalamus is responsible for pubertal maturation. The decline of serum melatonin below a threshold value (~115 pg/ml) may constitute the activating signal for hypothalamic pulsatile secretion of GnRH and subsequent onset of pubertal changes149. The hypothalamic-pituitary-gonadal axis is active during fetal life, but remains quiescent until the age of ~10 yr due to high levels of melatonin and is reactivated thereafter with increase in the amplitude and frequency of GnRH pulses. Stimulation of pulsatile secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are crucial for pubertal changes and, therefore, the decline in melatonin concentration below the threshold value is very important for the initiation of puberty as a central rhythm generator134,135. The inhibitory effects of melatonin on GnRH action gradually decline due to decreased expression of functional melatonin receptors150. The low concentration of melatonin would result in premature activation of the hypothalamic GnRH secretion and the occurrence of precocious puberty151. These effects

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are further clinically demonstrated in humans, showing that acute oral doses of melatonin amplify LH pulses in early follicular phase, and stimulate prolactin and vasopressin secretions152. Evidences show that the melatonin can also exert effects on reproductive axis by directly binding to granulosa cells in the ovary153. In human granulosa cells, both MT1 and MT2 are present and melatonin upregulates LH mRNA-receptor too154. LH is essential for the initiation of leuteinization. Furthermore, melatonin treatment enhances human chorionic gonadotropin (hCG) stimulated progesterone secretion with an inhibition of GnRH and GnRH receptor expression. GnRH in the ovary is suggested to be an important paracrine and/or autocrine regulator, and may be involved in the regression of corpus luteum134,135,155. These studies indicate involvement of melatonin in the maintenance of corpus luteum during pregnancy. Effects on cancer

The study on the effects of melatonin on cancer has a long history. Cohen et al.156 first indicated the possible role of pineal gland on the etiology of breast cancer and suggested that a decrease in pineal function (i.e. reduction in melatonin secretion) could induce a state of relative hyper-estrogenism, and the early and prolonged exposure of the breast tissue to estrogens could be involved in the etiology of breast carcinogenesis. Melatonin is known to shift forskolin and estrogen-induced elevation of cAMP levels by 57% and 45% respectively, thereby affecting signal transduction mechanisms in human breast cancer cells157. It is proposed that melatonin through the action on neuroendocrine reproductive axis may downregulate the expression of estrogen receptor α (ERα), and finally inhibit the binding of estradiol-ER complex to the estrogen response element158. Subsequent studies have shown reduced levels of melatonin in patients with certain types of cancers, compared to normal healthy people of the same age159,160. Melatonin has shown an antiproliferative effect through receptors MT1 and MT2 on various types of cancers (breast, lung, metastatic renal cell carcinoma, hepatocellular carcinoma, brain metastases from solid tumors, ovarian carcinoma, human neuroblastoma cells, bladder carcinoma and erythroleukemia) with tumor growth, and the incidence of metastases has shown physiological to pharmacological effects of melatonin161,162. Moreover, melatonin treatment has shown MT1/MT2-dependent inhibition of uptake of fatty acids, in general, and of linoleic acid in particular,

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thereby preventing the formation of its mitogenic metabolite 13-hydroxyoctadecadienonic acid55. In addition, melatonin also inhibits the fatty acid growth factor uptake by cancer cells, inhibits telomerase activity by reducing telomere length, which causes apoptosis in cancer cells. It also inhibits endothelin-1 synthesis, an angiogenic factor that promotes blood vessel growth in tumors, and finally modulates the expression of tumor suppressor gene TP53 or inhibits transcriptional expression of cyclin D1163. The action at different levels of signaling pathways in a tumor cell collectively suggests melatonin to be a supportive anticancer drug in cancer prevention and treatment. However, exact mechanism of melatonin action on cancer remains to be understood properly. Conclusion The scientific journey initiated 50 years ago with the isolation, purification and characterization of melatonin from the bovine pineal gland has already opened up a new vista of understanding on the pleiotropic nature of the functions of this small tryptophan derivative molecule. The information summarized in this communication by no means describe all actions of melatonin. However, it illustrates briefly the versatile functions of melatonin in humans. This physiologically wonder molecule is unique in sharing the properties of receptor-mediated actions of hormone as well as of the receptorindependent actions of an antioxidant. In view of the data presented herein, one feels compelled to surmise that multiple functions of melatonin may be brought forth by primary and secondary effects. Melatonin may interact with its receptors in different parts of nervous system and peripheral organs, and produce its primary effects as a hormone. Because of its lipophilic nature, it may also interact with numerous receptors for amino acids, biogenic amines, and peptides and produce its secondary effects as a co-hormone, or co-transmitter. However, its anti-oxidant actions are relatively recent addition to its functional repertoire, which because of lipophilic and hydrophilic properties is readily accessible to all cells of the body, and thereby does not require any receptors for carrying out the scavenging actions on free radicals. Collectively, the functions of melatonin would be to orchestrate, synchronize, and refine the multiplicity of biological functions, which make life possible. The research for and discovery of how melatonin with its apparent omnipotent effects brings forth these

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multiple functions may raise the exciting prospect of providing new avenues of treating numerous diseases, thus replacing old treatments which sustain life, but diminish its quality164. Nevertheless, there are several practical impediments to routine clinical use of melatonin. The optimal dose and timing of administration remain to be clearly defined, particularly for various human populations in different geographical locations having different photoperiodic conditions. There is an urgent need of further research for clarification of its clinical uses and side effects, especially with prolonged use. Obviously, there is a long way to go, before we arrive at a firm conclusion on the physiological and clinical significance of melatonin in human. In the context of existing situation, it “may be more exciting to travel than to land.” Acknowledgement The authors are thankful to their colleagues, especially Ms. Mohua Seth for extending helpful hands in preparation of the manuscript. Financial assistance by DAAD (Germany), DBT, DST, CSIR (Govt. of India) greatly nurtured the interest of corresponding author in melatonin research. References 1

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