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The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with Vmax values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Photoperiod is considered the most important factor entraining the circannual physiological rhythms through changing circadian patterns of melatonin (MEL) secretion from the pineal gland. The pineal gland of mammals does not respond directly to light but is controlled by light via neuronal phototransduction originating in the retina. In accordance with humoral phototransduction hypothesis, the aim of this study was to determine whether an increased concentration of CO, as a carrier of a light signal in pineal cell culture, affects the synthesis of melatonin. This study demonstrates that a commonly used carbon monoxide donor (CORM-2) markedly stimulated melatonin release from pineal cells incubated in vitro in a time-dependent manner, but the mechanism whereby CO modulates MEL release needs to be further explored.

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1β (75 pg/mL), or (4) NE + IL-1β. It was found that IL-1β abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1β also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1β treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

Helicobacter pylori colonization of gastric mucosa causes pain of unknown etiology in about 15-20% of infected subjects. The aim of the present work was to determine the level of expression of enzymes involved in the synthesis of melatonin in gastric mucosa of asymptomatic and symptomatic H. pylori infected patients. To diagnose H. pylori infection, histological analysis and the urea breath test (UBT C13) were performed. The levels of mRNA expression of arylalkylamine-N-acetyltransferase (AA-NAT) and acetylserotonin methyltransferase (ASMT) were estimated in gastric mucosa with RT-PCR. The level of AA-NAT expression and AMST was decreased in H. pylori infected patients and was increased after H. pylori eradication. We conclude that decreased expression of melatonin synthesizing enzymes, AA-NAT and ASMT, in patients with symptomatic H. pylori infection returns to normal level after H. pylori eradication.

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin → melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.

Depressive disorder (DD) is characterised by disturbances in blood melatonin concentration. It is well known that melatonin is involved in the control of circadian rhythms, sleep included. The use of melatonin and its analogues has been found to be effective in depression therapy. Melatonin synthesis is a multistage process, where the last stage is catalysed by acetylserotonin methyltransferase (ASMT), the reported rate-limiting melatonin synthesis enzyme. Taking into account the significance of genetic factors in depression development, the gene for ASMT may become an interesting focus for studies in patients with recurrent DD. The goal of the study was to evaluate two single-nucleotide polymorphisms (SNPs) (rs4446909; rs5989681) of the ASMT gene, as well as mRNA expression for ASMT in recurrent DD-affected patients. We genotyped two polymorphisms in a group of 181 recurrent DD patients and in 149 control subjects. The study was performed using the polymerase chain reaction/restriction fragment length polymorphism method. The distribution of genotypes in both studied SNPs in the ASMT gene differed significantly between DD and healthy subjects. The presence of AA genotype of rs4446909 polymorphism and of GG genotype of rs5989681 polymorphism was associated with lower risk for having recurrent DD. In turn, patients with depression were characterised by reduced mRNA expression for ASMT. In addition, ASMT transcript level in both recurrent DD patients and in healthy subjects depended significantly on genotype distributions in both polymorphisms. In conclusion, our results suggest the ASMT gene as a susceptibility gene for recurrent DD.

Pineal parenchymal tumours (PPT) are rare neoplasms and there have been few in vitro studies. Their capacity for synthesizing and secreting melatonin has been only partially examined. We investigated the presence of messenger RNA (mRNA) encoding tryptophan hydroxylase (TPH), arylalkylamine N-acetyltransferase (AANAT), hydroxyindol-O-methyltransferase (HIOMT), three enzymes involved in melatonin synthesis, and c-myc, a tumoural marker, in 10 PPT, one papillary tumour of the pineal region (PTPR), cell cultures derived from four PPTs and from three other tumours of the pineal region, and in normal pineal gland. Moreover, protein expression of TPH was investigated in three PPT and PTPR. Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry were used and the melatonin production by tumoural cells in vitro was analysed by radioimmunoassay. We showed that all the tumoural tissues and cells contained c-myc mRNA. mRNAs encoding TPH, AANAT and HIOMT were detected in all PPT, suggesting that tumour cells can synthesize melatonin. Only PPT expressed TPH protein. Cultured cells lost expression of transcripts throughout passages even if ultrastructural study revealed the presence of characteristic organelles in these tumoural cells. Nevertheless, the basal secretion of melatonin observed in one PPT culture is in favour of a maintained melatonin production and secretion by tumoural pinealocytes, but melatonin production was not stimulated by a beta noradrenergic agonist. Moreover, PTPR never expressed mRNA encoding TPH, AANAT and HIOMT. Our results may contribute to a better understanding of the biology of PTT and PTPR and may help to the diagnosis of these rare tumours.

Melatonin plays a critical role in a variety of mammalian reproductive processes not only acting on the central nervous system but also behaving as a peripheral physiologic regulator. To address the relevance of melatonin to the maintenance of pregnancy at the feto-maternal interface, we investigated the expression of two types of membrane melatonin receptors, MT1 and MT2, as well as arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), the two enzymes required for the conversion of serotonin to melatonin, in the human placenta and the effect of melatonin on the release of human chorionic gonadotropin (hCG) from cultured human trophoblast cells. RT-PCR analysis and DNA sequencing revealed that transcripts of MT1, MT2, AA-NAT, and HIOMT were present in the first-trimester human placenta. We also found that melatonin significantly potentiated hCG secretion at optimal concentrations. These results suggest that melatonin may regulate human placental function in a paracrine/autocrine manner, providing evidence for a novel role in human reproduction.

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.

We characterized the expression levels of the retinoid Z receptor alpha (RZR alpha), RORalpha mRNA isoforms (RORalpha1, RORalpha2, and RORalpha3), and both melatonin receptor MT1 and hydroxindole-O-methyltransferase (HIOMT) genes. For this purpose, the following human peripheral blood mononuclear cells populations were isolated: monocytes (CD14+ cells), B lymphocytes (CD19+ cells), T helper lymphocytes (CD14(-) CD4+), cytotoxic T lymphocytes (CD56(-) CD8+ cells), and natural killer (NK) lymphocytes (CD56+ cells). PBMCs subsets were obtained by Dynabeads M-450 (Dynal) isolation procedure. We observed a strong gene expression signal for RZRalpha in all subpopulations studied, whereas both RORalpha1 and RORalpha2 transcripts were amplified only in CD8+ cells. Specific signal for RORalpha2 was obtained in all subpopulations studied, but we were not able to detect the RORalpha3 mRNA transcript in human immune cells studied. A weaker signal (especially in CD19+ cells) was also detected in all subsets of cells for the MT1 gene. With regard to HIOMT, a strong signal was achieved among all but one subpopulation of cells; the only exception was CD14+ cells. Thus, in addition to its classical function in the nervous and endocrine system, melatonin could act directly as a paracrine and/or autocrine agent in the human immune system.

Purpose: To examine whether magnetic fields (MF) affect N-acetyltransferase (NAT) and hydroxy-indole-O-methyltransferase (HIOMT) activity directly or exert their effect through a cellular pathway that indirectly regulates the activity of these enzymes and melatonin release., Materials and Methods: The pineal glands from Wistar rats were isolated at 10:00 h and exposed to MF (50 Hz, 1 mT) for 4 h in vitro, with or without 1 micro M norepinephrine. An additional group of pineals was exposed to MF 30 min before norepinephrine addition. The direct effect of MF on the activity of the enzymes was studied in sonicated glands exposed to MF. NAT activity, HIOMT activity and melatonin release were determined., Results: In pineal glands isolated in the morning, 4-h in vitro exposure did not affect the basal release of melatonin from the pineal gland as well as the basal NAT and HIOMT activities. Pineal gland exposure to MF 30 min before norepinephrine addition significantly (p<0.05) increased NAT activity, HIOMT activity and melatonin release (p<0.05). These effects were not observed in pineals co-treated with MF and norepinephrine or in sonicated glands exposed to MF., Conclusions: The results suggest that in pineals isolated in the morning, 4-h MF exposure changes melatonin release by affecting the signal transduction pathway leading from the norepinephrine receptor to NAT and HIOMT and not via a direct effect at the enzyme levels.

A central function of chicken photoreceptors is the rhythmic synthesis of the neurohormone melatonin; however, it is unclear how they can achieve--already before hatching (i.e. without light exposure)--a circadian pacemaker system. Here we studied melatonin synthesis and secretion in rosetted spheroids, which are de novo histotypic spheres, re-aggregated from dissociated retinal cells of chicken embryos. Spheroids were cultured during a 12-hour light/12-hour dark cycle. Shortly after the onset of photoreceptor differentiation in spheroids near day 5 in culture, melatonin secretion set in and, by day 8 in culture, showed distinct dark-light oscillations, with high values during the dark and lower ones during the light period. Concomitantly, expression of two key enzymes of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected by northern blot analysis from day 6 onwards, increasing until day 8. Only HIOMT, but not AA-NAT levels were 2- to 3-fold higher at the end of the light compared with the dark phase. Even under permanent darkness, a slight melatonin oscillation was revealed after 6 days. In conclusion, photoreceptors in de novo regenerated retinal spheroids become light-responsive, establish appropriate pathways for melatonin synthesis and secrete more melatonin in the dark. The underlying circadian pacemaker seems to depend on photoreceptor differentiation, not on previous light-dark entrainment., (Copyright 2002 S. Karger AG, Basel)

The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors.

Recently, it was demonstrated that inbred strains of mice have a clearcut circadian rhythm of pineal and serum melatonin. Moreover, it is known that melatonin is involved in many immunoregulatory functions. Among them, hematopoiesis is influenced by the action of melatonin via melatonin-induced opioids on kappa-opioid receptors, which are present on stromal bone marrow cells. Therefore, the present study was carried out to investigate the presence of melatonin in the bone marrow in which immunocompetent cells are generated. Specifically, we aimed at answering the following question: are bone marrow cells involved in melatonin synthesis? In the present study, we demonstrate that (1) bone marrow cells contain high concentrations of melatonin; (2) bone marrow cells have a N-acetyltransferase activity and they express the mRNA encoding hydroxy-O-methyltransferase and (3) bone marrow cells cultured for a prolonged period exhibited high levels of melatonin. Results presented here suggest that mouse and human bone marrow and bone marrow cells are capable of de novo synthesis of melatonin, which may have intracellular and or paracrine functions.

The mRNAs encoding three enzymes of the melatonin synthesis pathway (tryptophan hydroxylase (TPH), arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyl-transferase (HIOMT)) are expressed with a day/night rhythm in the chicken pineal gland and retina. TPH and AANAT mRNA levels reach their peak at night. HIOMT mRNA levels peak at night in the retina, but during the day in the pineal gland. In this tissue, the rhythm of TPH, AANAT and HIOMT mRNA levels persisted in constant darkness (DD), both in vivo and in vitro, indicating that the three genes are controlled by the circadian oscillator of the chicken pineal. In the retina, the rhythms of TPH and AANAT mRNA levels also persisted in DD in vivo, suggesting that they are driven by a circadian oscillator. In contrast, the rhythm of HIOMT mRNA in the retina appeared to be controlled only by light. The clones of chicken AANAT and HIOMT genes that we have isolated should help us to understand the molecular mechanisms of: 1) their transcriptional regulation by circadian oscillators and by light; 2) their tissue-specific expression in the pineal gland and the retina.

Postnatal development of hydroxyindole-O-methyltransferase (HIOMT) mRNA expression, HIOMT activity and melatonin content was investigated in the rat pineal gland from birth to adulthood (62-day old). For each age, animals were sacrificed at two different time-points: midday and midnight. HIOMT mRNA was first detectable one day after birth and maximal diurnal levels were reached at the end of the first week. A 2-fold nocturnal increase appeared significantly 11 days after birth. HIOMT activity was detectable from 5 days of life and significant day/night variations did not appear before 21 days after birth. Appearance of melatonin synthesis and rhythm followed that of HIOMT mRNA. The 10-day delay observed between the appearance of the nocturnal increase in HIOMT activity and expression is discussed in term of differential regulation of both HIOMT mRNA expression and HIOMT activity., (Copyright 1998 Elsevier Science B.V.)

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes.

The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of melatonin. In the present study, the regulation of HIOMT expression was examined in the human Y-79 retinoblastoma cell line. Cells were grown in suspension culture using medium supplemented with 10% fetal calf serum (FCS). HIOMT activity and mRNA were strongly reduced when FCS was substituted with 0.1% bovine serum albumin (BSA), and were restored by addition of FCS. The effect of FCS on HIOMT expression was relatively selective, because the abundance of mRNA encoding actin, G3PDH or interphotoreceptor retinoid-binding protein did not change following serum deprivation. However, S-antigen (arrestin) mRNA was regulated by serum coordinately with HIOMT mRNA, suggesting that S-antigen expression is also controlled by a serum factor. The effect of serum on HIOMT expression was not duplicated by treatment with a series of known differentiating factors, nor was it reduced by dialysis or stripping procedures which remove steroids, growth factors and thyroid hormones.

Hydroxyindole O-methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the final step in the synthesis of melatonin in the pineal gland and retina. HIOMT mRNA was localized by in situ hybridization in the chicken retina to some, but clearly not all, photoreceptors, while in the pineal gland, most pinealocytes displayed a positive hybridization signal. The in situ hybridization localization was confirmed by immunocytochemistry, using an antibody directed against a synthetic chicken HIOMT peptide. Western blot analysis demonstrated an immunoreactive protein of about 40 kilodaltons in the pineal, but the HIOMT protein was below detectable levels in the retina. However, the HIOMT-peptide antibody did identify a modestly immunoreactive subpopulation of retinal photoreceptors. These observations suggest that, in the chicken, melatonin biosynthetic activity is located mainly in a subpopulation of retinal photoreceptors and in most pinealocytes.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin biosynthesis and appears to be specifically expressed in the pineal gland and in the retina. This review deals with the regulation of HIOMT by environmental light and with the developmental aspects of HIOMT expression in chicken and rat. Early studies based on HIOMT activity measurements and more recent studies involving cDNA hybridization to HIOMT mRNA are taken into consideration. Together, the data reveal that long term regulation of HIOMT by light would rely on a day/night rhythm of HIOMT gene transcription, coupled to a slow turnover of the protein. Rapid changes in HIOMT mRNA levels and early expression during embryonic development suggest that further studies on this gene may shed light on the molecular mechanisms involved in the differentiation of the melatoninergic function and in its regulation by light, both in the pineal gland and in the retina.

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme that catalyzes the final step in the synthesis of the hormone melatonin. We have examined the localization of expression of the mRNA encoding HIOMT by in situ hybridization in the 3-day-old and adult chicken retina and pineal gland. The riboprobe utilized for this study was transcribed from the complete coding region of HIOMT cDNA synthesized from retina RNA and amplified by polymerase chain reaction (PCR). High levels of HIOMT mRNA were present in the pinealocytes of the pineal gland. In the retina, the hybridization signal was localized to the photoreceptors. The retinal photoreceptors of the 3-day-old chick displayed a much lower level of hybridization than did the photoreceptors of the adult chicken. This study strongly suggests that the photoreceptors are the sites of melatonin synthesis in the retina.

A gene for sex-linked imperfect albinism in the chicken (s(al)) has been associated with increased egg production with an implication that environmental light may play a role. In this study, levels of melatonin and hydroxyindole O-methyltransferase (HIOMT) and N-acetyltransferase (NAT), two enzymes leading to melatonin production, were studied in young albino and nonalbino chickens in relation to the daily light cycle, and after 19 days of constant light or dark. Differences between genotypes were found in the levels of HIOMT activity in the pineals and retinas of birds kept in constant light for 19 days. Other measurements were not significantly different. This study would appear to show that the visual system of imperfect albino chickens reacts differently to light than that of nonalbinos, but not with changes in the daily cycle of plasma melatonin or in NAT activity, which is the enzyme primarily responsible for the control of the melatonin level in the body.

1. Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway. 2. Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ. 3. Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.

The presence of immunoreactive LH-releasing hormone (LH-RH) was revealed in the epiphysis of rats by radioimmunochemical method. The content of this hormone displayed circadian variations: it was maximum at 6 p.m. and minimum at 6 a.m. Intravenous injection of synthetic LG-RH to infantile and sexually mature rats of both sexes induced in the epiphysis the activity of hydroxyindol-O-methyl transpherase (HIOMT), the key enzyme of melatonin synthesis. Increased activity of this enzyme in the epiphysis persisted for at least 5 hours after the administration of LH-RH. HIOMT activation is preceded by a rapid fall (in 5 minutes) of cyclic adenosine-3', 5'-monophosphate in the epiphysis. The data obtained permitted to suppose that feed-back of the hypothalamus with the epiphysis could be realized with the aid of LH-RH.

1. Hydroxyindole-O-methyltransferase and DNA-dependent RNA polymerase activities were determined in the pineal gland removed from the ovariectomised rat and cultured under various experimental conditions. 2. The transferase activity declined very slowly during 24 h of incubation. 17beta-Oestradiol significantly increased the transferase activity within 2h after the addition, and the extent of increase was dose-dependent within the concentration range from 0.1 to 15 nM, being increased by 80% at 15 nM. Enhancement of the transferase activity by oestradiol was abolished not only by inhibitors of protein synthesis (cycloheximide and puromycin), but also by those of RNA synthesis (actinomycin D and alpha-amanitin). It was also blocked by clomiphene citrate, an agent which is known to inhibit the binding of steroid hormones to their respective receptors. 3. RNA polymerase activity (forms A and B) declined rapidly during the initial period of pineal culture. Oestradiol (15 nM) increased the RNA polymerase B activity by 50% within 2 h after the addition. The increase was dose-dependent within the concentration range from 0.1 to 15 nM, and was abolished by clomiphene citrate. 4. The possibility is suggested that the pineal is a target organ of oestradiol, and that the steroid-induced reaction sequence in the pineal conforms to that is known in other target organs.