Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation

The large daily rhythm in circulating melatonin levels is a highly conserved feature of vertebrate physiology: high values always occur at night. The dynamics of the rhythm are controlled by the next-to-last enzyme in melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin), arylalkylamine N-acetyltransferase (AANAT), the "melatonin rhythm enzyme". In vertebrate biology, AANAT plays a unique time-keeping role as the molecular interface between the environment and the hormonal signal of time, melatonin. This chapter describes the mammalian AANAT regulatory system, which includes the retina, neural structures, transsynaptic processes, and molecular events. In addition, special attention is paid to the functional characteristics of the systems which insure that the nocturnal increase in melatonin is an accurate and reliable indicator of the duration of the night, and why the melatonin rhythm is the most reliable output signal of the Mind's Clock.     

Involvement of the pineal gland and melatonin in murine analgesia

The data presented herein suggest that an intact pineal gland is required for the expression of the increased nocturnal sensitivity to morphine observed in mice. We report that the day/night rhythm of morphine analgesia was not evident in pinealectomized mice. Further, mice treated with melatonin exhibited a dose-related analgesic response. The decrease in sensitivity to pain was not observed in animals in which melatonin administration was followed by the opiate antagonist, naloxone. These data suggest that information derived from environmental lighting regulates sensitivity to pain via the pineal gland hormone melatonin, which is released and acts upon other areas of the CNS.     

The anatomy and innervation of the mammalian pineal gland

The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells. In addition, perivascular phagocytes are present. The phagocytes share antigenic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of the peptide. Nerve fibers innervating the mammalian pineal gland originate from perikarya located in the sympathetic superior cervical ganglion and the parasympathetic sphenopalatine and otic ganglia. The sympathetic nerve fibers contain norepinephrine and neuropeptide Y as neurotransmitters. The parasympathetic nerve fibers contain vasoactive intestinal peptide and peptide histidine isoleucine. Recently, neurons in the trigeminal ganglion, containing substance P, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating peptide, have been shown to project to the mammalian pineal gland. Finally, nerve fibers originating from perikarya located in the brain containing, for example, GABA, orexin, serotonin, histamine, oxytocin, and vasopressin innervate the pineal gland directly via the pineal stalk. Biochemical studies have demonstrated numerous receptors on the pinealocyte cell membrane, which are able to bind the neurotransmitters located in the pinealopetal nerve fibers. These findings indicate that the mammalian pinealocyte can be influenced by a plethora of neurotransmitters.     

Molecular components and mechanism of adrenergic signal transduction in mammalian pineal gland: regulation of melatonin synthesis

Rhythmic neural outputs from the hypothalamic suprachiasmatic nucleus (SCN), which programme the rhythmic release of norepinephrine (NE) from intrapineal nerve fibers, regulate circadian rhythm of melatonin synthesis. Increased secretion of NE with the onset of darkness during the first half of night stimulates melatonin synthesis by several folds. NE binds to both alpha1- and beta-adrenergic receptors present on the pinealocyte membrane and initiates adrenergic signal transduction via cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) generating pathways. The NE-induced adrenergic signal transduction switches 'on' melatonin synthesis during the early hours of night by stimulating expression of the rate-limiting enzyme of melatonin synthesis, N-acetyltransferase (AA-NAT) via cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-cAMP response element (CRE) pathway as well as by increasing AA-NAT activity via cAMP-PKA-14-3-3 protein pathway. Simultaneously, adrenergically-induced expression of inducible cAMP early repressor (ICER) negatively regulates aa-nat gene expression and controls the amplitude of melatonin rhythm. In the second half of night, increased release of acetylcholine from central pinealopetal projections, inhibition of NE secretion by SCN, withdrawal of adrenergic inputs and reversal of events that took place in the first half lead to switching 'off' of melatonin synthesis. Adrenergic signal transduction via cGMP-protein kinase G (PKG)-mitogen activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathway also seems to be fully functional, but its role in modulation of melatonin synthesis remains unexplored. This article gives a critical review of information available on various components of the adrenergic signal transduction cascades involved in the regulation of melatonin synthesis.     

Arylamine N-acetyltransferase and arylalkylamine N-acetyltransferase in the mammalian pineal gland

Amine N-acetylation in the pineal gland is of special importance because it is the first step in the synthesis of melatonin from serotonin. In the present study the N-acetylation of arylamines and arylalkylamines by homogenates of rat and sheep pineal glands was investigated. The arylamines studied were p-phenetidine and aniline; the arylalkylamines studied were tryptamine, serotonin, 5-methoxytryptamine, 6-fluorotryptamine, and phenylethylamine. These amines were acetylated by pineal homogenates of both species, although marked interspecies differences in apparent Km and Vmax values were found. A series of observations in both species indicate that aromatic amine N-acetylation is catalyzed by two distinct enzymes; one preferentially acetylates arylamines and the other preferentially acetylates arylalkylamines. First, isoproterenol treatment of the rat increased arylalkylamine N-acetylation 100-fold without increasing arylamine N-acetylation. Second, cycloheximide treatment in sheep reduced arylalkylamine N-acetylation at night to one-tenth control values, without altering arylamine N-acetylation. Third, arylamine N-acetyltransferase and arylalkylamine N-acetyltransferase inactivated at different rates at 4 degrees C. Fourth, the two enzymes were resolved by size exclusion chromatography. These results clearly establish that the pineal gland contains an arylamine N-acetyltransferase and a second, independently regulated arylalkylamine N-acetyltransferase which appears to be primarily responsible for the physiological conversion of serotonin to melatonin via the intermediate N-acetylserotonin.     

A role of melatonin in the initial stage of photoperiodism in the Japanese quail

To estimate whether melatonin is involved in gonadal activity in the male quail, the dynamics of plasma melatonin at an early stage of the photoperiodic response were investigated. Nocturnal levels of melatonin were manipulated by treatment with anti-melatonin (anti-M). By means of 4 additional hours of photic stimulation of the brain (provided by a red light-emitting diode inserted through the back of the head) after the environmental lights (8L:16D, lights-on, 1000 h) were turned off, the elevation of levels of melatonin after lights-off was significantly suppressed on Days 1 and 2 (p less than 0.01); after 5 days of brain-lighting, gonadal growth first became noticeable. However, 4 h of brain-lighting before lights-on elicited no change in levels of melatonin or in gonadal growth. The injections of anti-M just before lights-off (at 1800 h) for the first 3 days caused significant gonadal growth (p less than 0.01), whereas injections at 2200, 0200, or 0600 h were without effect. In addition, 4 h of brain-lighting before lights-on became gonadostimulatory (p less than 0.01) when it was accompanied by the injection of anti-M at 1800 h, but remained without effect when anti-M was injected at 0600 h. These results suggest that melatonin is involved in the initial stage of photoperiodism in birds, and the timing of suppression of the elevation of melatonin levels is critical in gonadal development.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

Cellular and molecular mechanisms controlling melatonin release by mammalian pineal glands

1. The pineal gland is regulated primarily by photoperiodic information attaining the organ through a multisynaptic pathway initiated in the retina and the retinohypothalamic tract. 2. Norepinephrine (NE) released from superior cervical ganglion (SCG) neurons that provide sympathetic innervation to the pineal acts through alpha1- and beta 1- adrenoceptors to stimulate melatonin synthesis and release. 3. The increase in cyclic AMP mediated by beta 1-adrenergic activation is potentiated by the increase in Ca2+ flux, inositol phospholipid turnover, and prostaglandin and leukotriene synthesis produced by alpha 1-adrenergic activation. 4. Central pinealopetal connections may also participate in pineal control mechanisms; transmitters and modulators in these pathways include several neuropeptides, amino acids such as gamma-aminobutyric acid (GABA) and glutamate, and biogenic amines such as serotonin, acetylcholine, and dopamine. 5. Secondary regulatory signals for pineal secretory activity are several hormones that act on receptors sites on pineal cells or at any stage of the neuronal pinealopetal pathway.     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

Immunohistochemical evidence for the presence of melatonin in the pineal gland,the retina and the Harderian gland

Summary The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20–25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10–16, 1980 (Vivien-Roels et al. 1980)The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates     

Determination of rat pineal gland melatonin content by a radioimmunoassay.

Melatonin content in individual rat pineal glands was measured by radioimmunoassay (RIA). The RIA used can very reliably detect as little as 50 pg of melatonin. The various precursors, analogues, and the metabolite of melatonin (6-hydroxymelatonin) which were tested for cross-reactivity were not recognized by the antibody. The effects on melatonin levels in rat pineal glands following the administration of L-tryptophan, 5-hydroxy-L-tryptophan, serotonin, N-acetylserotonin, melatonin and pargyline are also presented.     

Synaptic ribbons of a mammalian pineal gland circadian changes

Summary Synaptic ribbons (SR) of the mammalian pineal gland are functionally enigmatic. In the present study it is shown that in male guinea-pigs kept under a lighting regimen of 12 hrs illumination (7.00–19.00) and 12 hrs darkness (19.00–7.00) the SR of pinealocytes vary about 25-fold in number over a period of 24 hrs. An increase is found between 15.00 and 6.00 and a decrease between 6.00 and 15.00. Analysis of the intracellular localization and the topographical relationships indicate that SR lie near the cell membrane of pinealocytes throughout the 24 hr period and that they are related to adjacent pinealocytes. A working hypothesis put forward implies that SR represent cell organelles involved in intercellular communication and that it is their function to either enhance the secretory activity of the pineal gland or to establish circuits within the gland between adjacent pinealocytes, similar to neuronal circuits.The skilled technical assistance of Mrs. C. Howe is gratefully acknowledged.     

des-Tyr1-gamma-endorphin and haloperidol increase pineal gland melatonin levels in rats

The effect of subcutaneously injected DT gamma E (beta-endorphin, (beta E)2-17) on the pineal melatonin level was compared with that of closely related peptides and the neuroleptic drug haloperidol. As found previously, DT gamma E (3 ng/rat and 300 ng/rat) increased the melatonin levels. Similar doses of DT alpha E (beta E 2-16), DT beta E (beta E 2-31), gamma E (beta E 1-17), alpha E (beta E 1-16) and beta E failed to significantly change the melatonin levels in both the dark and the light phase. Haloperidol in a dose of 300 ng/rat exhibited a similar effect as DT gamma E.     

No short-term effects of high-frequency electromagnetic fields on the mammalian pineal gland

There is ample experimental evidence that changes of earth-strength static magnetic fields, pulsed magnetic fields, or alternating electric fields (60 Hz) depress the nocturnally enhanced melatonin synthesis of the pineal gland of certain mammals. No data on the effects of high-frequency electromagnetic fields on melatonin synthesis is available. In the present study, exposure to 900 MHz electromagnetic fields [0.1 to 0.6 mW/cm², approximately 0.06 to 0.36 W/kg specific absorption rate (SAR) in rats and 0.04 W/kg in Djungarian hamsters; both continuous and/or pulsed at 217 Hz, for 15 min to 6 h] at day or night had no notable short-term effect on pineal melatonin synthesis in male and female Sprague-Dawley rats and Djungarian hamsters. Pineal synaptic ribbon profile numbers (studied in rats only) were likewise not affected. The 900 MHz electromagnetic fields, unpulsed or pulsed at 217 Hz, as applied in the present study, have no short-term effect on the mammalian pineal gland. Bioelectromagnetics 18:376–387, 1997. © 1997 Wiley-Liss, Inc.