Melatonin-Loaded Nanocarriers: New Horizons for Therapeutic Applications.

The use of nanosized particles has emerged to facilitate selective applications in medicine. Drug-delivery systems represent novel opportunities to provide stricter, focused, and fine-tuned therapy, enhancing the therapeutic efficacy of chemical agents at the molecular level while reducing their toxic effects. Melatonin (N-acetyl-5-methoxytriptamine) is a small indoleamine secreted essentially by the pineal gland during darkness, but also produced by most cells in a non-circadian manner from which it is not released into the blood. Although the therapeutic promise of melatonin is indisputable, aspects regarding optimal dosage, biotransformation and metabolism, route and time of administration, and targeted therapy remain to be examined for proper treatment results. Recently, prolonged release of melatonin has shown greater efficacy and safety when combined with a nanostructured formulation. This review summarizes the role of melatonin incorporated into different nanocarriers (e.g., lipid-based vesicles, polymeric vesicles, non-ionic surfactant-based vesicles, charge carriers in graphene, electro spun nanofibers, silica-based carriers, metallic and non-metallic nanocomposites) as drug delivery system platforms or multilevel determinations in various in vivo and in vitro experimental conditions. Melatonin incorporated into nanosized materials exhibits superior effectiveness in multiple diseases and pathological processes than does free melatonin; thus, such information has functional significance for clinical intervention.

STAT1-NFκB crosstalk triggered by interferon gamma regulates noradrenaline-induced pineal hormonal production.

Melatonin production by pineal glands is modulated by several immune signals. The nuclear translocation of nuclear factor kappa-B (NFκB) homodimers, lacking transactivation domains, once induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), inhibits the expression of Aanat gene and the synthesis of noradrenaline (NA)-induced melatonin. Interferon gamma (IFN-γ), on the other hand, increases melatonin synthesis. Furthermore, this cytokine activates the signal transducer as well as the activator of transcription 1 (STAT1) pathway, which was never evaluated as a melatonin synthesis modulator before. Reports demonstrated that IFN-γ might also activate NFκB. The present study evaluated the role of STAT1-NFκB crosstalk triggered by IFN-γ regarding the regulation of NA-induced pineal glands' hormonal production. Moreover, IFN-γ treatment increased NA-induced Aanat transcription, in addition to the synthesis of N-acetylserotonin (NAS) and melatonin. These effects were associated with STAT1 nuclear translocation, confirmed by the co-immunoprecipitation of STAT1 and Aanat promoter. Pharmacological STAT1 enhancement augmented NA-induced Aanat transcription as well as NAS and melatonin production. Additionally, IFN-γ induced the nuclear translocation of RelA-NFκB subunits. The blockade of this pathway prevented IFN-γ effects on the pineal function. The present data show that STAT1 and NFκB crosstalk controls melatonin production through a synergistic mechanism, disclosing a new integrative mechanism regarding pineal hormonal activity control.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV.

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Melatonin promotes reduction in TNF levels and improves the lipid profile and insulin sensitivity in pinealectomized rats with periodontal disease.

AIM: This study aimed to investigate the effects of melatonin (ME) on insulin resistance (IR) and signaling (IS), proinflammatory cytokine levels, and lipid profiles in pinealectomyzed (PNX) rats with periodontal disease (PD). MAIN METHODS: One hundred and forty-four rats (age = 40 days) were distributed into 8 groups: 1) control (CN); 2) PD only; 3) PNX only; 4) PNX and PD (PNXPD); 5) CN treated with ME (CNM); 6) PD treated with ME (PDM); 7) PNX treated with ME(PNXM); 8) PNX and PD treated with ME(PNXPDM). The PNX groups were subjected to pinealectomy at 40 and at 60 days of age. The animals were then subjected to PD induction in the mandibular first molars. After PD induction, the ME replacement therapy (MERT-5 mg/kg body weight) was performed using water for 28 days. After this period, the plasma concentration of glucose, insulin, TNF, IL-6, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, and VLDL-cholesterol and the HOMA-IR index were determined. Akt serine phosphorylation status in the white adipose tissue, gastrocnemius muscle, and rat liver were also evaluated. KEY FINDINGS: PD, PNX, and PNXPD groups showed an increase in IR with elevated plasma levels of insulin and TNF compared to CN group. PNX and PNXPD groups presented alteration in lipid profile compared to CN group. MERT improved all of the analyzed parameters. No difference was observed in the IS among different groups. SIGNIFICANCE: The results suggest that MERT efficiently prevents IR, improves lipid profile, and increases plasma levels of insulin and TNF in PD and PNX rats.

Differential response of pineal microglia to surgical versus pharmacological stimuli.

Microglial cells are one of the interstitial elements of the pineal gland (PG). We recently reported the pattern of microglia colonization and activation, and microglia-Pax6+ cell interactions during normal pineal ontogeny. Here, we describe the dynamics of microglia-Pax6+ cell associations and interactions after surgical or pharmacological manipulation. In adult rats, the superior cervical ganglia (SCG) were exposed, and either bilaterally excised (SCGx) or decentralized (SCGd). In the SCGx PGs, the density of Iba1+ microglia increased after surgery and returned to sham baseline levels 13 days later. Pineal microglia also responded to SCGd, a more subtle denervation. The number of clustered Iba1+ /PCNA+ /ED1+ microglia was higher 4 days after both surgeries compared to the sham-operated group. However, the number of Pax6+ /PCNA- cells and the percentage of Pax6+ cells contacted by and/or phagocytosed by microglia increased significantly only after SCGx. Separate groups of rats were treated with either bacterial lipopolysaccharides (LPS) or doxycycline (DOX) to activate or inhibit pineal microglia, respectively. Peripheral LPS administration caused an increase in the number of clustered Iba1+ /PCNA+ /ED1+ microglial cells, and in the percentage of Pax6+ cells associated with and/or engulfed by microglia. In the LPS-treated PGs, we also noted an increase in the number of PCNA+ cells that were Iba1- within the microglial cell clusters. The density of Pax6+ cells did not change after LPS treatment. DOX administration did not influence the parameters analyzed. These data suggest that pineal microglia are highly receptive cells capable of rapidly responding in a differential manner to surgical and pharmacological stimuli.

Minimal disseminated disease evaluation and outcome in trilateral retinoblastoma.

Trilateral retinoblastoma (TRb) presents a management challenge, since intracranial tumours are seldom times resectable and quickly disseminate. However, there are no risk factors to predict the final outcome in each patient. OBJECTIVE: To evaluate minimal disseminated disease (MDD) in the bone marrow (BM) and the cerebrospinal fluid (CSF) at diagnosis and during follow-up and reviewing its potential impact in the outcome of patients with TRb. METHODS AND ANALYSIS: We evaluated MDD in five patients with TRb, detecting the mRNA of CRX and/or GD2, in samples from BM and CSF, obtained at diagnosis, follow-up and relapse. RESULTS: Treatment involved intensive systemic chemotherapy in four patients, one did not receive this treatment and died of progression of the disease. Two patients underwent stem cell rescue. Three patients had leptomeningeal relapse and died. One patient remains disease-free for 84 months. RB1 mutations were identified in the five patients, all of them were null mutations. At diagnosis, one patient had tumour cells in the CSF, and none had the BM involved. Only one case of four presented MDD during follow-up in the CSF, without concomitant detection in the BM. On leptomeningeal relapse, no case had MDD in the BM. In all these cases, cells in the CSF were positive for GD2 and/or CRX. CONCLUSION: CSF dissemination always concluded in the death of the patient, without concomitant systemic dissemination denoting the importance of increasing treatment directed to the CSF compartment. The MDD presence could indicate a forthcoming relapse.

Dual Effect of Catecholamines and Corticosterone Crosstalk on Pineal Gland Melatonin Synthesis.

BACKGROUND/AIM: The nocturnal production of melatonin by the pineal gland is triggered by sympathetic activation of adrenoceptors and may be modulated by immunological signals. The effect of glucocorticoids on nocturnal melatonin synthesis is controversial; both stimulatory and inhibitory effects have been reported. During pathophysiological processes, an increased sympathetic tonus could result in different patterns of adrenoceptor activation in the pineal gland. Therefore, in this investigation, we evaluated whether the pattern of adrenergic stimulation of the pineal gland drives the direction of the glucocorticoid effect on melatonin production. METHODS: The corticosterone effect on the pineal hormonal production induced by ß-adrenoceptor or ß+α1-adrenoceptor activation was evaluated in cultured glands. We also investigated whether the in vivo lipopolysaccharide (LPS)-induced inhibition of melatonin is dependent on the interaction of glucocorticoids and the α1-adrenoceptor in adrenalectomized animals and on the in vivo blockade of glucocorticoid receptors (GRs) or the α1-adrenoceptor. RESULTS: Corticosterone potentiated ß-adrenoceptor-induced pineal melatonin synthesis, whilst corticosterone-dependent inhibition was observed when melatonin production was induced by ß+α1-adrenoceptors agonists. The inhibitory effect of corticosterone is mediated by GR, as it was abolished in the presence of a GR antagonist. Moreover, LPS-induced reduction in melatonin nocturnal plasma content was reversed by adrenalectomy and by antagonizing GR or α1-adrenoceptors. CONCLUSIONS: The dual effect of corticosterone on pineal melatonin synthesis is determined by the activation pattern of adrenoceptors (ß or ß+α1) in the gland during GR activation, suggesting that increased activation of the sympathetic system and the hypothalamic-pituitary-adrenal axis are necessary for the control of melatonin production during defense responses.

Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and ß-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

Selective protection of the cerebellum against intracerebroventricular LPS is mediated by local melatonin synthesis.

Although melatonin is mainly produced by the pineal gland, an increasing number of extra-pineal sites of melatonin synthesis have been described. We previously demonstrated the existence of bidirectional communication between the pineal gland and the immune system that drives a switch in melatonin production from the pineal gland to peripheral organs during the mounting of an innate immune response. In the present study, we show that acute neuroinflammation induced by lipopolysaccharide (LPS) injected directly into the lateral ventricles of adult rats reduces the nocturnal peak of melatonin in the plasma and induces its synthesis in the cerebellum, though not in the cortex or hippocampus. This increase in cerebellar melatonin content requires the activation of nuclear factor kappa B (NF-κB), which positively regulates the expression of the key enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Interestingly, LPS treatment led to neuronal death in the hippocampus and cortex, but not in the cerebellum. This privileged protection of cerebellar cells was abrogated when G-protein-coupled melatonin receptors were blocked by the melatonin antagonist luzindole, suggesting that the local production of melatonin protects cerebellar neurons from LPS toxicity. This is the first demonstration of a switch between pineal and extra-pineal melatonin production in the central nervous system following a neuroinflammatory response. These results have direct implications concerning the differential susceptibility of specific brain areas to neuronal death.

The in vitro maintenance of clock genes expression within the rat pineal gland under standard and norepinephrine-synchronized stimulation.

Although the norepinephrine (NE) synchronization protocol was proved to be an important procedure for further modulating in vitro pineal melatonin synthesis, the maintenance of clock genes under the same conditions remained to be investigated. The aim of this study was to investigate the maintenance of the clock genes expression in pineal gland cultures under standard and NE-synchronized stimulation. The glands were separated into three experimental groups: Control, Standard (acute NE-stimulation), and NE-synchronized. The expression of Bmal1, Per2, Cry2, Rev-erbα, the clock controlled gene Dbp and Arylalkylamine-N-acetyltransferase were investigated, as well as melatonin content. No oscillations were observed in the expression of the investigated genes from the control group. Under Standard NE stimulation, the clock genes did not exhibit a rhythmic pattern of expression. However, in the NE-synchronized condition, a rhythmic expression pattern was observed in all cases. An enhancement in pineal gland responsiveness to NE stimulation, reflected in an advanced synthesis of melatonin was also observed. Our results reinforce our previous hypothesis that NE synchronization of pineal gland culture mimics the natural rhythmic release of NE in the gland, increasing melatonin synthesis and keeping the pineal circadian clock synchronized, ensuring the fine adjustments that are relied in the clockwork machinery.

Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.

AIMS: The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with ß1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. MAIN METHODS: Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. KEY FINDINGS: Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. SIGNIFICANCE: Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis.

Modulation of pineal melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through NF-κB activation.

The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF- κ B activation were analyzed in astrocytes and pinealocytes. TNF- α 's possible mediation of the effect of glutamate was also investigated. The results showed that glutamate's inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF- α . Moreover, the activation of the astrocytic NF- κ B seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF- κ B transcription factor and possibly by subsequent TNF- α release.

Relevance of the chronobiological and non-chronobiological actions of melatonin for enhancing therapeutic efficacy in neurodegenerative disorders.

Melatonin is an indolamine with a large spectrum of functions that can be divided into chronobiotic and nonchronobiotic. Chronobiotic effects are mediated by the daily rhythm of melatonin in the plasma due to nocturnal pineal synthesis, whereas the melatonin produced by other cells, such as gastrointestinal and immune competent cells, is independent of the light/dark cycle and exert non-chronobiotic effects. The concentrations achieved by the two sources are significantly different, varying in the pM-nM range in the plasma, and may achieve concentrations in the mM range when released locally by activated immune-competent cells. Consequently, the effects of the melatonin produced in these two situations are distinct. Much has been reported about the beneficial response to exogenous melatonin administration in several pathological conditions. However, the relationship between the establishment of a disease and the state of the physiological activity of the pineal gland is still poorly understood. Here, we review the state of art in the modulation of pineal melatonin synthesis, relevant patents, and discuss its relationship with neurodegenerative disorders that involve a central inflammatory response, such as Alzheimer's disease, to suggest the putative relevance of new therapeutic protocols that replace this pineal hormone.

Ethanol consumption and pineal melatonin daily profile in rats.

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in ß1 and α1 adrenergic receptors' mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland.

AIMS: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. MAIN METHODS: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3:AANAT complex, respectively. KEY FINDINGS: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced "night". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IRbeta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. SIGNIFICANCE: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland.

Acute increase in urinary 6-sulfatoximelatonin after clomipramine, as a predictive measure for emotional improvement.

Nocturnal melatonin pineal output is triggered by sympathetic outflow. Antidepressants that block norepinephrine neuronal uptake should increase pineal function. This can be monitored by measuring 6-sulfatoximelatonin (aMT6s), the main melatonin metabolite, in the urine. In this study, we compared the excretion of aMT6s before (baseline), one, and 21 days after administration of clomipramine to healthy subjects (n = 32). At the end of treatment, subjects were divided into responders (n = 12) and non-responders (n = 20) according to the improvement in their emotional state in three out of four domains (interpersonal tolerance, efficiency, well-being and feeling different from the usual self). There was no difference in aMT6s before clomipramine between responders and non-responders in any of the time intervals analysed (06:00-12:00, 12:00-18:00, 18:00-24:00 and 24:00-06:00 hours). At day one, but not at day 21, the fraction of aMT6s excreted during the time interval 24:00-06:00, relative to the total amount excreted by each subject per day, was significantly higher (P = 0.0287) than baseline (0.57 +/- 0.04) in responders. No significant difference was observed in non-responders. The increase in pineal function induced by clomipramine was restricted to day one, indicating that long-lasting adaptation restores pineal function. In addition, the day one increase in aMT6s was significantly increased only in the responders group, raising the possibility that the blocking of neuronal uptake is predictive of emotional improvement.

Local corticosterone infusion enhances nocturnal pineal melatonin production in vivo.

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-alpha decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N-acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland.

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

Insulin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland.

The mammalian pineal gland synthesizes melatonin in a circadian manner, peaking during the dark phase. This synthesis is primarily regulated by sympathetic innervations via noradrenergic fibers, but is also modulated by many peptidergic and hormonal systems. A growing number of studies reveal a complex role for melatonin in influencing various physiological processes, including modulation of insulin secretion and action. In contrast, a role for insulin as a modulator of melatonin synthesis has not been investigated previously. The aim of the current study was to determine whether insulin modulates norepinephrine (NE)-mediated melatonin synthesis. The results demonstrate that insulin (10(- 8)M) potentiated norepinephrine-mediated melatonin synthesis and tryptophan hydroxylase (TPOH) activity in ex vivo incubated pineal glands. When ex vivo incubated pineal glands were synchronized (12h NE-stimulation, followed by 12h incubation in the absence of NE), insulin potentiated NE-mediated melatonin synthesis and arylalkylamine-N-acetyltransferase (AANAT) activity. Insulin did not affect the activity of hydroxyindole-O-methyltranferase (HIOMT), nor the gene expression of tpoh, aanat, or hiomt, under any of the conditions investigated. We conclude that insulin potentiates NE-mediated melatonin synthesis in cultured rat pineal gland, potentially through post-transcriptional events.

Melatonin formation in pineal gland from rats with hexachlorobenzene experimental porphyria.

Hexachlorobenzene produces an experimental hepatic porphyria in rats, which is similar to human porphyria cutanea tarda, with hyperpigmentation as one of its characteristic features. Alterations in tryptophan metabolism have been previously observed in this chronic porphyria. Melatonin formation from tryptophan via serotonin shows diurnal rhythmicity in the pineal gland, and higher values are observed during the dark phase of an imposed light-dark cycle. The purpose of this study was to determine the contents of tryptophan and its metabolites in pineal gland of normal and hexachlorobenzene-treated rats in order to find alterations potentially related to porphyria cutanea tarda. Results show that in animals with this experimental porphyria some tryptophan metabolite levels (serotonin and 5-hydroxyindoleacetic acid) increase only during the light period, whereas tryptophan content remained equal to the controls. Hydroxyindole-O-methyltransferase activity also increases by light in pineal gland from hexachlorobenzene-treated rats. On the other hand, tryptophan is converted to melatonin in the dark period, but this route is not exacerbated in hexachlorobenzene porphyria. The relevance of these alterations is discussed in relation to hyperpigmentation, neoplastic and oxidative stress processes associated with this porphyria.