Regulatory mechanisms in melatonin biosynthesis in retina.

The vertebrate retina produces melatonin in a light-dependent rhythmic fashion, synchronized with, but independent from the rhythm of the hormone formation in the pineal gland. This review summarizes the current status of our knowledge on regulatory mechanisms involved in controlling the retinal melatonin biosynthesis. Special emphasis is given to the role and mode of action of dopamine and GABA, two established retinal neurotransmitters, as well as that of second messengers (cyclic AMP, calcium ions). Comparisons are made between lower vertebrates and mammals.     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

Melatonin circadian rhythm in the retina of mammals

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood.     

Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

Solubilization and Biochemical Characterization of the Melatonin Deacetylase from Xenopus laevis Retina

Abstract: Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N -acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.     

MELATONIN CIRCADIAN RHYTHM IN THE RETINA OF MAMMALS

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood. (Chronobiology International, 17(5), 599–612, 2000)     

Photoperiodic control of melatonin synthesis in fish pineal and retina

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

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Effects of melatonin and 6-methoxy-tetrahydro-beta-carboline in light induced retinal damage: a computerized morphometric method

The effects of melatonin and a related 5-methoxy-indole, 6-methoxy-1,2,3,4-tetrahydro-beta-carboline (6-MeO-THBC) were investigated in rats on the development of retinal degeneration in presence of high intensity illumination (HII). A morphometric method is used in which the degree of degeneration was evaluated by a computer-coupled graphical analyzer. Instead of measuring individual thicknesses of different retinal layers at various loci we measured large areas of retinal light microscopic sections. Thus the influence of sporadic artefactual and other fluctuations in the thickness of various layers of the retina can be essentially reduced. Continuous light produced significant degeneration of the retina and the degree of degeneration was further increased by both studied compounds and even more by 6-MeO-THBC. The role of melatonin and 6-MeO-THBC in retinal physiology is discussed.     

Pertussis toxin blocks melatonin-induced inhibition of forskolin-stimulated adenylate cyclase activity in the chick brain.

The high-affinity guanine nucleotide-sensitive receptor sites for melatonin in the mammalian hypothalamus and pars tuberalis mediate inhibition of adenylate cyclase (AC) activity. Therefore, we have examined whether similar sites in the chick brain and retina also modulate AC activity. Melatonin did not alter basal or forskolin-stimulated AC activity in whole forebrain or retinal homogenates. In contrast, melatonin significantly inhibited forskolin-stimulated AC activity in forebrain synaptosomal membranes and partially purified retinal membranes in a concentration-dependent manner. Maximal inhibition (approximately 25-30%) of stimulated AC activity was observed at 10-100nM melatonin, while the concentrations (EC50's) which caused half-maximal effects were 22 +/- 6 pM and 30 +/- 5 pM in the brain and retina respectively. Pretreatment of forebrain slices with pertussis toxin abolished the inhibitory effect of melatonin on stimulated AC activity. These data provide the first evidence that melatonin suppresses AC activity in the chick CNS via a pertussis toxin-sensitive G-protein.     

Cyclic AMP-Dependent Melatonin Production in Y79 Human Retinoblastoma Cells

Abstract: Melatonin is rhythmically synthesized in some vertebrate retinas and has been implicated in the regulation of key rhythmic events in the photoreceptor-pigment epithelial complex. In human retina, melatonin is present; however, no information exists on the cellular regulation of this hormone. We report here that the established human retinoblastoma cell line Y79 synthesizes and releases melatonin. Treatments that elevate cyclic AMP (cAMP) levels (forskolin, 8-Br-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1 -meth-ylxanthine) all stimulate melatonin release from static cultures of Y79 cells. Other 8-bromo nucleotide analogues (cyclic GMP, ATP, and AMP) are not effective. These results suggest that Y79 human retinoblastoma cells require a cAMP-dependent mechanism for melatonin biosynthesis similar to that described previously in other vertebrates. This is the first demonstration of melatonin release from a cultured human cell line. These results support the idea that human retinal cells share homologies with pineal cells, as suggested by the condition trilateral retinoblastoma.     

Inferior retinal light exposure is more effective than superior retinal exposure in suppressing melatonin in humans

Illumination of different areas of the human retina elicits differences in acute light-induced suppression of melatonin. The aim of this study was to compare changes in plasma melatonin levels when light exposures of equal illuminance and equal photon dose were administered to superior, inferior, and full retinal fields. Nine healthy subjects participated in the study. Plexiglass eye shields were modified to permit selective exposure of the superior and inferior halves of the retinas of each subject. The Humphrey Visual Field Analyzer was used both to confirm intact full visual fields and to quantify exposure of upper and lower visual fields. On study nights, eyes were dilated, and subjects were exposed to patternless white light for 90 min between 0200 and 0330 under five conditions: (1) full retinal exposure at 200 lux, (2) full retinal exposure at 100 lux, (3) inferior retinal exposure at 200 lux, (4) superior retinal exposure at 200 lux, and (5) a dark-exposed control. Plasma melatonin levels were determined by radioimmunoassay. ANOVA demonstrated a significant effect of exposure condition (F = 5.91, p < 0.005). Post hoc Fisher PLSD tests showed significant (p < 0.05) melatonin suppression of both full retinal exposures as well as the inferior retinal exposure; however, superior retinal exposure was significantly less effective in suppressing melatonin. Furthermore, suppression with superior retinal exposure was not significantly different from that of the dark control condition. The results indicate that the inferior retina contributes more to the light-induced suppression of melatonin than the superior retina at the photon dosages tested in this study. Findings suggest a greater sensitivity or denser distribution of photoreceptors in the inferior retina are involved in light detection for the retinohypothalamic tract of humans.     

Rhythmic regulation of retinal melatonin: Metabolic pathways,neurochemical mechanisms,and the ocular circadian clock

1. Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions. 2. Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species. 3. The development of suitable in vitro retinal preparations, particularly the eyecup from the African clawed frog, Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation. 4. Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotonin N-acetyltransferase, and a novel pathway for degradation of melatonin within the retina. 5. Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator. 6. A recently developed in vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms. 7. A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.     

Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the first enzyme in the conversion of serotonin to melatonin. Large changes in AANAT activity play an important role in the daily rhythms in melatonin production. Although a single AANAT gene has been found in mammals and the chicken, we have now identified two AANAT genes in fish. These genes are designated AANAT-1 and AANAT-2; all known AANATs belong to the AANAT-1 subfamily. Pike AANAT-1 is nearly exclusively expressed in the retina and AANAT-2 in the pineal gland. The abundance of each mRNA changes on a circadian basis, with retinal AANAT-1 mRNA peaking in late afternoon and pineal AANAT-2 mRNA peaking 6 h later. The pike AANAT-1 and AANAT-2 enzymes (66% identical amino acids) exhibit marked differences in their affinity for serotonin, relative affinity for indoleethylamines versus phenylethylamines and temperature-activity relationships. Two AANAT genes also exist in another fish, the trout. The evolution of two AANATs may represent a strategy to optimally meet tissue-related requirements for synthesis of melatonin: pineal melatonin serves an endocrine role and retinal melatonin plays a paracrine role.     

The arylalkylamine-N-acetyltransferase (AANAT) acetylates dopamine in the digestive tract of goldfish: A role in intestinal motility

Melatonin has been found in the digestive tract of many vertebrates. However, the enzymatic activity of the arylalkylamine-N-acetyltransferase (AANAT) and the hydroxindole-O-methyltransferase (HIOMT), the last two enzymes of melatonin biosynthesis, have been only measured in rat liver. Therefore, the first objective of the present study is to investigate the functionality of these enzymes in the liver and gut of goldfish, analyzing its possible daily changes and comparing its catalytic properties with those from the retina isoforms. The daily rhythms with nocturnal acrophases in retinal AANAT and HIOMT activities support their role in melatonin biosynthesis. In foregut AANAT activity also show a daily rhythm while in liver and hindgut significant but not rhythmic levels of AANAT activity are found. HIOMT activity is not detected in any of these peripheral tissues suggesting an alternative role for AANAT besides melatonin synthesis. The failure to detect functional HIOMT activity in both, liver and gut, led us to investigate other physiological substrates for the AANAT, as dopamine, searching alternative roles for this enzyme in the goldfish gut. Dopamine competes with tryptamine and inhibits retinal, intestinal and hepatic N-acetyltryptamine production, suggesting that the active isoform in gut is AANAT1. Besides, gut and liver produces N-acetyldopamine in presence of acetyl coenzyme-A and dopamine. This production is not abolished by the presence of folic acid (arylamine N-acetyltransferase inhibitor) in any studied tissue, but a total inhibition occurs in the presence of CoA-S-N-acetyltryptamine (AANAT inhibitor) in liver. Therefore, AANAT1 seems to be an important enzyme in the regulation of dopamine and N-acetyldopamine content in liver. Finally, for the first time in fish we found that dopamine, but not N-acetyldopamine, regulates the gut motility, underlying the broad physiological role of AANAT in the gut.     

Circadian Regulation of Melatonin in the Retina of Xenopus laevis: Limitation by Serotonin Availability

Treatments expected to increase retinal serotonin levels were found to stimulate melatonin production by cultured eyecups from Xenopus laevis. The monoamine oxidase inhibitor pargyline (100 microM) caused a sixfold increase in melatonin release, and the serotonin precursor 5-hydroxy-L-tryptophan (100 microM) caused a 70-fold increase. Both acted synergistically with eserine, an inhibitor of melatonin deacetylation in the retina. The effect of 5-hydroxytryptophan was dose dependent, with effects increasing from 1 to 100 microM. Increasing the tryptophan level in the culture medium had no effect on melatonin release. These results indicate that the rate-limiting step in retinal melatonin synthesis is 5-hydroxylation of tryptophan. Melatonin released from individual eyecups in superfusion culture in constant darkness with and without added 5-hydroxy-L-tryptophan was monitored over a 5-day period. Control eyecups released low levels of melatonin, with circadian rhythmicity persisting for 1-3 days. With 5-hydroxy-L-tryptophan added, melatonin levels were elevated 10-20-fold at all times, and rhythmicity was apparent for as long as five cycles. This provides a model system for studies of the circadian clock in the eye.     

Analysis of pore-rich areas on the nuclear envelope of spermatocytes

The S-antigen is a protein of photoreceptors, mainly known for its autoantigenic properties in mammals, which is widely distributed in the retina of vertebrates and in photoreceptor organs of invertebrates. Using three monoclonal antibodies specific for different epitopes of S-antigen, this study complements our previous data on retinal rods and cones and presents new results on the photosensory cells of the pineal complex. Immunoreactivity was found in (i) retinal rods and cones, (ii) cone-like and modified photoreceptor cells, and pinealocytes of the pineal organ of vertebrates, (iii) cone-like photoreceptors of the frontal organ of the frog and of the third eye of the lizard. According to the species and the antibody used, some differences were found at the level of the cellular compartments of the pineal photoreceptor cells.