Melatonin induces alterations in protein expression in the Xenopus laevis retina

The hormone melatonin is synthesized by pinealocytes and retinal photoreceptors with a diurnal rhythm. Melatonin produced in the retina at night is thought to exert local modulatory effects by binding to specific receptors in several different retinal cell types. The mechanisms by which melatonin influences circadian activity in retinal cells is poorly understood. Suppression of cyclic AMP synthesis appears to be a major signaling pathway in response to melatonin receptor binding in many tissues. A potential downstream consequence of melatonin-induced changes in cyclic AMP concentrations and protein phosphorylation is the up- or down-regulation of expression of specific genes. In this report, we examined the changes in expression levels of specific proteins in the neural retina and retinal pigment epithelium (RPE) in response to melatonin treatment, because both of these tissues express melatonin receptors. Neural retina and RPE isolated from the eyes of Xenopus laevis were treated with or without 1 microM melatonin for 6 hr, then the rapidly synthesized tissue proteins were radiolabeled by a 15 min incubation with 35S-methionine, and the proteins were subsequently analyzed by two-dimensional gel electrophoresis and autoradiography. In both the neural retina and RPE, the densities of some specific proteins were altered in response to melatonin treatment, and the few protein spots that were altered were distinct between the two tissues. These results support the concept that one function of melatonin may be to regulate the expression of specific genes and the consequent protein levels, and that the target genes may differ according to the cell or tissue type.     

Daily variation in the concentration of melatonin and 5-methoxytryptophol in the goose pineal gland, retina, and plasma

The goose pineal gland rhythmically produces two 5-methoxyindole compounds, namely melatonin and 5-methoxytryptophol. Melatonin concentrations were high at night and low during the day, while in contrast 5-methoxytryptophol levels were markedly higher during the day compared to the night-time values. Rhythmic oscillations in melatonin content, with high night-time values, have also been found in plasma and the retina of goose. The pineal and retinal melatonin rhythm mirrored oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). Acute exposure of geese to light at night markedly decreased melatonin levels in the pineal, plasma, and retina. In addition, this light exposure resulted in a significant increase in pineal 5-methoxytryptophol content. Our results demonstrate, for the first time, the ability of the goose pineal gland and retina to synthesise melatonin and 5-methoxytryptophol in a rhythmic manner.     

Effect of light and darkness on the in vivo release of N-acetylserotonin and melatonin by the retina of guinea pigs

The effect of light and darkness on the in vitro release of N-acetylserotonin and melatonin by the retina of male pigmented guinea pigs was studied. One group of the retina was incubated in light and the other group in dark for 12 h. Histological examination of the cultured retinal tissues indicated that the retinal cells appeared intact and healthy. N-acetylserotonin and melatonin released into the medium was extracted and then quantified by radioimmunoassay. The release of N-acetylserotonin and melatonin by retinas cultured in dark was significantly higher than those in light, indicating that the in vitro release of N-acetylserotonin and melatonin in increased by darkness or reduced by light. This supports the notion that the retina may be an important extrapineal source of melatonin in the circulation and may at least help to sustain diurnal rhythms of serum N-acetylserotonin and melatonin.     

Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro.

The pineal gland of birds contains one or more circadian oscillators that play a major role in overall temporal organization. We have developed a flow-through culture system for the isolated pineal by which we can measure the release of melatonin continuously from superfused glands over long periods of time. Chicken pineals release melatonin rhythmically, and these rhythms persist in vitro with a circadian oscillation. In light cycles the release of melatonin is strongly rhythmic; however, in constant conditions the amplitude of the rhythm is lower and appears to be damping. Light has at least two effects upon the isolated pineal: cyclic light input synchronizes the rhythm, and acute light exposure at night rapidly inhibits melatonin release. The cultured avian pineal clearly offers great potential as a model system for the study of vertebrate circadian oscillators and may open the way for an analysis of mechanism.     

Circadian 5-HT production regulated by adrenergic signaling

Using on-line microdialysis, we have characterized in vivo dynamics of pineal 5-hydroxytryptamine (5-HT; serotonin) release. Daily pineal 5-HT output is triphasic: (i) 5-HT levels are constant and high during the day; (ii) early in the night, there is a novel sharp rise in 5-HT synthesis and release, which precedes the nocturnal rise in melatonin synthesis; and (iii) late in the night, levels are low. This triphasic 5-HT production persists in constant darkness and is influenced strongly by intrusion of light at night. We demonstrate that both diurnal 5-HT synthesis and 5-HT release are activated by sympathetic innervation from the superior cervical ganglion and show that these processes are controlled by distinct receptors. The increase in 5-HT synthesis is controlled by beta-adrenergic receptors, whereas the increase in 5-HT release is mediated by alpha-adrenergic signaling. On the other hand, the marked decrease in 5-HT content and release late at night is a passive process, influenced by the extent of melatonin synthesis. In the absence of melatonin synthesis, the late-night decline in 5-HT release is prevented, reaching levels roughly twice as high as that of the day value. In summary, our results demonstrate that 5-HT levels display marked circadian rhythms that depend on adrenergic signaling.     

Melatonin regulates circadian electroretinogram rhythms in a dose- and time-dependent fashion

The circadian rhythm of the chick electroretinogram (ERG) is regulated by the indoleamine hormone melatonin. To determine if the concentration of melatonin or the time at which it was administered would have differential effects on ERG parameters, we conducted experiments analyzing the effects of melatonin at different times of the day. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed in both light:dark (LD) and in continuous darkness (DD). Intramuscular melatonin administration of 1 mg/kg and 100 ng/kg decreased a- and b-wave amplitudes and increased a- and b-wave implicit times. This effect was significantly greater than that observed for 1 ng/kg melatonin, which had little to no effect over the saline controls. The effect of 1 mg/kg and 100 ng/kg melatonin on a- and b-wave amplitude in LD and on b-wave amplitude in DD was greater during the night (ZT/CT 17) than during the day (ZT/CT 5). The fold change in b-wave implicit time over that of controls was greater during the day (ZT/CT 5) than during the night (ZT/CT 17). These data indicate that melatonin may play a role in regulating a day and night functional shift in the retina, and that it does so via regulation of a retinal clock.     

Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity.

There is a large-amplitude circadian rhythm of indoleamine metabolism in the retina-pigment epithelium of the chicken. N-Acetyltransferase activity (arylamine acetyltransferase; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) and melatonin content are 15-fold higher at night than during the day in a cycle of a 4-fold increase during the subjective night. Light at midnight inactivates N-acetyltransferase and lowers melatonin. N-Acetyltransferase activity is found predominantly in the retina. The circadian rhythm of this enzyme activity persists in pinealectomized chicks. Thus the pineal is not responsible for retinal indoleamine rhythms. Retinal and pineal levels of N-acetyltransferase activity behave similarly under several conditions. In the chicken, the eye is a major site of rhythmic indoleamine metabolic activity.     

Circadian rhythms of ocular melatonin in the wrasse Halichoeres tenuispinnis, a labrid teleost

Using in vivo and in vitro methods we studied the regulation of ocular melatonin rhythms in the wrasse Halichoeres tenuispinnis, by either light or the circadian clock. Rhythmic changes in ocular melatonin levels under light-dark (LD) cycles were persistent under constant darkness (DD), and had a circadian periodicity of approximately 24h. However, ocular melatonin levels remained low under constant light conditions. When wrasse were exposed to a single 6-h light pulse at three different circadian phases under DD, phase-dependent phase shifts in the circadian rhythms of ocular melatonin were observed. When eyecups were prepared during mid-light periods or at the onset of darkness, and incubated in vitro in either light or dark periods, both time and light conditions affected melatonin release. These results indicate that the melatonin rhythms in the wrasse eye are driven by an ocular circadian clock that is entrained to LD cycles via local photoreceptors.     

Daily rhythm in pineal phosphodiesterase (PDE) activity reflects adrenergic/3',5'-cyclic adenosine 5'-monophosphate induction of the PDE4B2 variant

The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold; doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling.     

Time keeping by the quail's eye: circadian regulation of melatonin production

Previous studies have shown that eye removal disrupts the circadian body temperature and activity rhythms of Japanese quail supporting the hypothesis that the eyes act as pacemakers within the quail circadian system. Furthermore, the putative ocular pacemakers are coupled to the rest of the circadian system via neural and hormonal outputs. Although the neural pathway has yet to be identified, experiments suggest that the daily rhythm of ocular melatonin synthesis and release is the hormonal output. We sought to strengthen the hypothesis that the eyes are the loci of circadian pacemakers, and that melatonin output is involved, by examining melatonin secretion in cultured quail retinas. Using an in vitro flow-through system we demonstrated that (1) isolated retinal tissue could exhibit a rhythm of melatonin release, (2) the rhythm of melatonin synthesis is directly entrainable by 24-h light-dark cycles, and (3) supplementation of the culture medium with serotonin is necessary for robust, rhythmic production of melatonin in constant darkness. These results show definitively that the eyes are the loci of a biological clock and, in light of previous studies showing the disruptive effects of blinding on the circadian system, strengthen the hypothesis that the ocular clock is a circadian pacemaker that can affect the rest of the circadian system via the cyclic synthesis and release of melatonin. The quail retina is proving to be a valuable in vitro model for investigating properties of circadian pacemakers.     

Regulation of gene expression by melatonin: a microarray survey of the rat retina

The pineal secretory product melatonin is synthesized by pinealocytes and retinal photoreceptors on a cyclic rhythm, with highest levels occurring at night. Our previous work has demonstrated that melatonin treatment increases the sensitivity of the rat retina to light-induced photoreceptor cell death. This raises the possibility that inappropriate exposure of photoreceptors to melatonin may result in visual impairment, caused by a loss of retinal photoreceptors. We hypothesize that retinal genes whose expression levels are altered in response to melatonin may be involved in processes that contribute to light-induced photoreceptor cell death. To identify retinal genes that are up- or down-regulated in response to melatonin receptor binding, rats were treated with or without melatonin, and the RNA from the neural retinas and retinal pigment epithelium (RPE) were analyzed for differential gene expression by hybridization of labeled cRNA probes to an Affymetrix rat genome microarray set. GeneChip algorithms were applied to measured hybridization intensities of compared samples and showed that in the neural retina, six genes were up-regulated, and eight were down-regulated. In the RPE, 15 genes were up-regulated, and two genes were down-regulated. The protein products of these specific genes are potentially involved in the molecular mechanism of melatonin action in the retina, and may play a role in the effect of melatonin on light-induced photoreceptor cell death. Identification of these candidate genes and their response to melatonin administration may provide a foundation for further studies on gene regulation by melatonin, the function of melatonin in the retina, and the role of circadian signaling in inherited and environmentally induced photoreceptor degenerations.     

Regulation of melatonin production by catecholamines and adenosine in a photoreceptive pineal organ. An in vitro study in the pike and the trout

The pineal organ of fish contains photoreceptor cells with structural and functional analogies to retinal photoreceptors. In these cells, the light/dark (LD) cycle influences the production of melatonin by controlling the activity of one of its synthetizing enzymes, serotonin N-acetyltransferase (NAT). The daily rhythm in NAT activity is generated endogenously in the pike but not in the trout pineal. We report here that in addition to the LD information, chemical factors are also involved in the control of melatonin production. Adenosine and two of its analogs stimulated or inhibited NAT activity and melatonin release in cultured pike and trout pineals, depending on the experimental conditions. It is believed that the nucleoside, produced locally, exerts a modulatory role on the neurohormonal output via still enigmatic mechanisms, involving a transmembranous carrier. Nocturnal melatonin production in cultured pike pineals was inhibited by alpha-adrenergic agonists and stimulated by a beta-adrenergic agonist. No effect could be induced in trout pineals cultured under similar conditions. Because melatonin production by pineal photoreceptors is apparently regulated by both light and chemical inputs, we propose they might be multieffector cells.     

Lower Tryptophan:Phenylalanine Ratios in Culture Media Increase Medium: Pineal Melatonin Ratios in Early Dark but not Late Light Phase

Pineals from male Long-Evans rats (60-65 days old; adapted to a 0700-1900 photoperiod) were cultured for 6 h either in light (1200-1800) or in dark (1800-2400). The objective was to ascertain the effects of tryptophan (trp) and phenylalanine (phe) levels and ratios in the culture medium on melatonin levels in the pineals and their respective media. Total culture (pineal + medium) melatonin levels, determined by RIA, were similar under all conditions. However, in cultures during the early dark phase (1800-2400) lower trp:phe ratios in the medium led to lower pineal:medium ratios of melatonin content. In cultures during the late light phase (1200-1800) the trp:phe ratio had little impact on the pineal:medium distribution of melatonin. Trp:phe ratio rather than absolute level of either amino acid appeared responsible for this effect. Functionally this means that during early dark phase, but not late light phase, movement of melatonin from cultured pineal to medium is progressively facilitated by lower trp:phe ratios. It remains to be determined to what extent darkness per se and/or endogenous pineal rhythmic mechanisms have a permissive role in the action of trp:phe ratio on pineal melatonin release. A melatonin compartmentalization/release effect of these or other amino acids, or their ratios, has not been reported previously and may possibly contribute to mechanisms for melatonin's transport or release at night.     

The protective effect of melatonin on methamphetamine‐induced calpain‐dependent death pathway in human neuroblastoma SH‐SY5Y cultured cells

Abstract: Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH‐induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain‐dependent cascades in methamphetamine‐induced toxicity in human dopaminergic neuroblastoma SH‐SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH‐induced calpain‐dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH‐SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH‐SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH‐SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain‐dependent death pathways in the processes of METH‐induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH‐SY5Y cultured cells.     

Daily and light-at-night induced variations of circulating 5-methoxytryptophol (5-ML) in ewes with respectively high and low nocturnal melatonin secretion.

The aim of the present study was to determine whether the genetic differences previously reported in ewe plasma melatonin concentrations were correlated with differences in the synthesis and release of other 5-methoxyindoles. To determine if 5-methoxytryptophol (5-ML), which is known to be present in large amounts in the sheep pineal gland, is released, as is melatonin, into the general circulation, and if some temporal relationships between 5-ML and melatonin release could be observed, two groups of ewes were selected with respect to their endogenous melatonin secretion: in the first experiment, ten ewes from the low melatonin group (low group) and ten ewes from the high melatonin group (high group). 5-ML was measured every hour during a 24-hr period by radioimmunoassay. In all ewes, 5-ML was released during day-time, the rhythm of 5-ML concentrations being inversely related with the melatonin rhythm. Both day-time and night-time 5-ML concentrations were higher in the ewes from the high group than in the ewes from the low group (14.7 +/- 1.0 pg/mL plasma versus 6.4 +/- 0.3 pg/mL plasma during the day, 3.1 +/- 0.2 pg/mL plasma versus 1.9 +/- 0.2 pg/mL plasma during the night). The 5-ML/melatonin ratio appeared much higher during the day than during the night but was very similar in both groups (day-time: 1.03 in the high group versus 1.16 in the low group, night-time: 0.01 in both groups). In a second experiment, six low group and seven high group ewes were submitted to 1 hr of extra light at night. 5-ML increased and melatonin decreased during extra light. Our results clearly show for the first time a daily variation in circulating 5-ML, and that the strong genetic contribution in the variability in melatonin concentrations in sheep are clearly correlated with a similar variability in 5-ML concentrations. Whether 5-ML, like melatonin, plays a physiological role in the different adaptation processes to the environment remains to be determined.     

Retinal melatonin is not involved in corneal mitotic rhythms in the Japanese quail: Effects of formoguanamine hydrochloride and eye-lid suture

Abstract: Relation between retinal melatonin and corneal mitotic rhythms in the Japanese quail was investigated in experiments manipulating the ocular physiology by treatments with formoguanamine hydrochloride (FG) and eye-lid suture. In experiment 1, we investigated the effects of FG, which is known to induce photoreceptor degeneration, on retinal melatonin and corneal mitotic rhythms. FG-treatment completely abolished the retinal melatonin rhythms in both LD 12: 12 and constant darkness (DD), but the corneal mitotic rhythm was maintained with high mitotic rate in darkness under a LD cycle and subjective night under DD. The result suggests that 1) the photoreceptor cells in the retina are the site for melatonin production and/or for the oscillator which drives the circadian rhythm in retinal melatonin, and 2) melatonin is not involved in generation of the corneal mitotic rhythm. In experiment 2, we investigated the effects of eye-lid suture, which is known to induce eye enlargement and bulgy cornea, on the retinal melatonin and corneal mitotic rhythms. Eye-lid suture abolished the corneal mitotic rhythm in both LD and DD, with a high mitotic rate being maintained throughout 24 hr. But retinal melatonin maintained its rhythm with high levels in darkness under a LD cycle and in subjective night under DD. The result suggests that 1) bulgy cornea in the sutured eye was induced by the increase in mitotic rate in the light period, and 2) disappearance of the corneal mitotic rhythm does not have a relation to retinal melatonin. These results suggest that retinal melatonin is not involved in generation of the corneal mitotic rhythm and that there are two circadian clock systems in the eye.     

Effect of stimulation of endogenous melatonin secretion during constant light exposure on 6-sulphatoxymelatonin rhythmicity in rats

When light is presented unexpectedly at night to rats, melatonin production and secretion is acutely inhibited and the time of onset of production on the subsequent night is altered. In a series of experiments, we examined the effects of 6-12 hr light (200 lux) at night on melatonin metabolite excretion (6-sulphatoxymelatonin, aMT.6S). During the light exposure, we administered isoproterenol to stimulate endogenous production of melatonin by the pineal gland to determine if replacement of melatonin would block any phase shifting effects of the light. Exposure to 6 hr of light either during the first or second half of the night suppressed aMT.6S excretion during the light treatment and delayed the onset of melatonin secretion by 3.7 +/- 0.6 and 2.5 +/- 0.6 hr, respectively, compared to a change of 0.5 +/- 0.1 hr in animals maintained in darkness. Twelve hours light exposure (i.e. one night of continuous light) suppressed aMT.6S excretion completely and resulted in a delay in the onset the next night of 2.1 +/- 0.7 hr. When propranolol (10 mg/kg) was administered at 2-hr intervals during darkness, aMT.6S excretion was suppressed throughout the night, but on the subsequent release into constant darkness the onset of excretion was not delayed (0.6 +/- 0.1 hr delay). Administration of isoproterenol (10 mg/kg) to animals in constant light, at the time of expected lights off (CT12), and 5 hr later (CT17) resulted in an increase in melatonin production and aMT.6S excretion that was similar in duration and amount to the control night. The stimulation of endogenous melatonin production failed to block the phase shifting effects of the light exposure and, in fact, appeared to potentiate the delay at least on the first night (4.2 +/- 0.9 hr). The timing of the release into constant darkness following the light treatment had an unexpected effect on melatonin production on the cycle after treatment. Thus, animals exposed to 12 hr light and released into darkness at the normal time of lights off as above had a delay of about 2 hr and excreted 71 +/- 18% of the aMT.6S excreted on a control night. Animals released into darkness at the expected time of lights on failed to excrete more than 20 pmol/hr(i.e. no onset of excretion could be determined) at any time on the first subjective night after light treatment, which was no different from the excretion during the light treatment. On the next subjective night, the onset was delayed as expected and the amount of aMT.6S produced was restored. Treatment with isoproterenol at CT12 and CT17 failed to affect either the amount of aMT.6S excreted on the first subjective night after light treatment or the phase delay on the second night after treatment. The failure to produce melatonin on the first subjective night after 12 hr light exposure and release into darkness at CTO was not due to failure at the level of the pineal gland since injection ofisoproterenol at CT12 and CT17 on the first subjective night after light restored the normal amount of melatonin production. These results suggest that the absence of melatonin during light stimulation at night is not responsible for the phase delay in melatonin production and excretion on subsequent nights. The basis of the failure of the rats to commence melatonin production following 12 hr extended light exposure followed immediately by continuous darkness is not known.     

Vasoactive intestinal polypeptide and alpha 2-adrenoceptor agonists regulate adenosine 3',5'-monophosphate accumulation and melatonin release in chick pineal cell cultures

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate melatonin synthesis in mammalian pineal; however, a regulatory role for VIP in the avian pineal has not been explored. Immunocytochemical and physiological response experiments were performed to investigate whether 1) immunoreactive VIP fibers innervated the avian pineal gland; 2) VIP had a specific effect on melatonin release that was mediated by cAMP stimulation; and 3) alpha 2-adrenergic signal transduction was associated with a reduction in cAMP levels. Immunocytochemical experiments demonstrated the presence of both tyrosine hydroxylase- and VIP-immunoreactive fibers in the avian pineal gland. Treatment of dispersed chick pineal cell cultures with VIP stimulated melatonin release (maximum 6-fold increase; EC50 = 1.8 nM) when administered during the 12-h light period of a 12-h light, 12-h dark cycle. Of the other four peptides tested [porcine VIP-(10-28), porcine peptide histidine isoleucine, porcine secretin, and human glucagon), only peptide histidine isoleucine stimulated melatonin release (EC50 = 30 nM). The effect of VIP was mediated by a time- and dose-dependent increase in cAMP accumulation (maximum 4-fold increase). The specific alpha 2-agonist UK-14,304 reduced cAMP accumulation (maximum 43% reduction) and inhibited melatonin release (EC50 = 19 nM) in the presence of 3 X 10(-8) M VIP. Norepinephrine-induced inhibition of nocturnal melatonin release was blocked by the elevation of cAMP achieved through the administration of forskolin (EC50 = 0.2 microM), isobutylmethylxanthine (EC50 = 112 microM), or 8-bromo-cAMP (EC50 = 166 microM). Collectively, these results demonstrate the presence and functional significance of VIP in the avian pineal gland, and the interaction of VIP and norepinephrine at the level of cAMP in the regulation of melatonin biosynthesis.