Arylalkylamine N-acetyltransferase mRNA Expression Research Articles

The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Effect of Monochromatic Light on Melatonin Secretion and Arylalkylamine N‐Acetyltransferase mRNA Expression in the Retina and Pineal Gland of Broilers

The goal of this study is to investigate the effects of various monochromatic lights on plasma melatonin (MT) levels and the expression of arylalkylamine N-acetyltransferase (AANAT) mRNA in the pineal gland and retina. A total of 160 newly hatched (posthatching day 1, P1) broilers, including intact, sham-operated, and pinealectomized groups were exposed to blue light (BL), green light (GL), red light (RL), and white light (WL) by light emitting diode (LED) system for short term (24 hr) or long term (2 weeks), separately. For intact and sham-operated birds, the plasma MT level exhibited marked circadian rhythms at P7 and P14 regardless of short-term and long-term exposure to four monochromatic lights. However, WL and BL showed a faint suppression of MT secretion in contrast to GL and RL at either light or dark time points, with the following rank order: GL < RL < WL < BL. Larger circadian amplitude of MT levels was observed in GL group versus BL group (at P14: 87.70 pg/mL vs. 19.85 pg/mL, respectively). Pinealectomy disturbed the MT rhythm under different light colors, especially in RL. Additionally, consistent with the alteration of plasma MT levels, we observed increased AANAT mRNA expression and immunoreactive cell numbers of proliferating cell nuclear antigen (PCNA) and c-Fos in the pineal gland or retina in GL than that of BL, whereas 5-HT immunoreactive cell number was significantly decreased in GL. These data suggested that GL enhanced chick pinealocytes and retinal cells to express AANAT mRNA and to secrete MT, which may be depended on promoting c-Fos expression and cell proliferation.

Melatonin Plays a Protective Role in Postburn Rodent Gut Pathophysiology

Melatonin is a possible protective agent in postburn gut pathophysiological dynamics. We investigated the role of endogenously-produced versus exogenously-administered melatonin in a major thermal injury rat model with well-characterized gut inflammatory complications. Our rationale is that understanding in vivo melatonin mechanisms in control and inflamed tissues will improve our understanding of its potential as a safe anti-inflammatory/antioxidant therapeutic alternative. Towards this end, we tested the hypothesis that the gut is both a source and a target for melatonin and that mesenteric melatonin plays an anti-inflammatory role following major thermal injury in rats with 3rd degree hot water scald over 30% TBSA. Our methods for assessing the gut as a source of melatonin included plasma melatonin ELISA measurements in systemic and mesenteric circulation as well as rtPCR measurement of jejunum and terminal ileum expression of the melatonin synthesizing enzymes arylalkylamine N-acetyltransferase (AA-NAT) and 5-hydroxyindole-O-methyltransferase (HIOMT) in sham versus day-3 postburn rats. Our melatonin ELISA results revealed that mesenteric circulation has much higher melatonin than systemic circulation and that both mesenteric and systemic melatonin levels are increased three days following major thermal injury. Our rtPCR results complemented the ELISA data in showing that the melatonin synthesizing enzymes AA-NAT and HIOMT are expressed in the ileum and jejunum and that this expression is increased three days following major thermal injury. Interestingly, the rtPCR data also revealed negative feedback by melatonin as exogenous melatonin supplementation at a dose of 7.43 mg (32 micromole/kg), but not 1.86 mg/kg (8 micromole/kg) drastically suppressed AA-NAT mRNA expression. Our methods also included an assessment of the gut as a target for melatonin utilizing computerized immunohistochemical measurements to quantify the effects of exogenous melatonin supplementation on postburn gut mucosa barrier inflammatory profiles. Here, our results revealed that daily postburn intraperitoneal melatonin administration at a dose of 1.86 mg/kg (8 micromole/kg) significantly suppressed both neutrophil infiltration and tyrosine nitrosylation as revealed by Gr-1 and nitrotyrosine immunohistochemistry, respectively. In conclusion, our results provide support for high mesenteric melatonin levels and dynamic de novo gut melatonin production, both of which increase endogenously in response to major thermal injury, but appear to fall short of abrogating the excessive postburn hyper-inflammation. Moreover, supplementation by exogenous melatonin significantly suppresses gut inflammation, thus confirming that melatonin is protective against postburn inflammation.

Retinal Ganglion Cells Are Autonomous Circadian Oscillators Synthesizing N-Acetylserotonin during the Day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

Rhythmic melatonin secretion does not correlate with the expression of arylalkylamine N-ACETYLTRANSFERASE , inducible cyclic amp early repressor, period1 or cryptochrome1 mRNA in the sheep pineal

The pineal gland, through nocturnal melatonin, acts as a neuroendocrine transducer of daily and seasonal time. Melatonin synthesis is driven by rhythmic activation of the rate-limiting enzyme, arylalkylamine N-acetyltransferase (AA-NAT). In ungulates, AA-NAT mRNA is constitutively high throughout the 24-h cycle, and melatonin production is primarily controlled through effects on AA-NAT enzyme activity; this is in contrast to dominant transcriptional control in rodents. To determine whether there has been a selective loss of circadian control of AA-NAT mRNA expression in the sheep pineal, we measured the expression of other genes known to be rhythmic in rodents (inducible cAMP early repressor ICER, the circadian clock genes Period1 and Cryptochrome1, as well as AA-NAT). We first assayed gene expression in pineal glands collected from Soay sheep adapted to short days (Light: dark, 8-h: 16-h), and killed at 4-h intervals through 24-h. We found no evidence for rhythmic expression of ICER, AA-NAT or Cryptochrome1 under these conditions, whilst Period1 showed a low amplitude rhythm of expression, with higher values during the dark period. In a second group of animals, lights out was delayed by 8-h during the final 24-h sampling period, a manipulation that causes an immediate shortening of the period of melatonin secretion. This did not significantly affect the expression of ICER, AA-NAT or Cryptochrome1 in the pineal, whilst a slight suppressive effect on overall Per1 levels was observed. The attenuated response to photoperiod change appears to be specific to the ovine pineal, as the first long day induced rapid changes of Period1 and ICER expression in the hypothalamic suprachiasmatic nuclei and pituitary pars tuberalis, respectively. Overall, our data suggest a general reduction of circadian control of transcript abundance in the ovine pineal gland, consistent with a marked evolutionary divergence in the mechanism regulating melatonin production between terrestrial ruminants and fossorial rodents.

Elevated arylalkylamine- N-acetyltransferase ( AA-NAT) gene expression in medial habenular and suprachiasmatic nuclei of hibernating ground squirrels

Hibernation, an adaptive response for energy conservation in mammals, involves a variety of physiological changes. Melatonin is linked with the regulation of core body temperature and intervenes in generating circadian cycles; its role in seasonal (circannual) rhythms of hibernation is explored here. Melatonin is primarily produced in the pineal gland. Since arylalkylamine- N-acetyltransferase (AA-NAT) is the rate-limiting enzyme for synthesizing melatonin, AA-NAT gene expression was investigated to assess the possible role of melatonin in hibernation. The findings presented here utilized combined in situ hybridization and immunohistochemistry methodologies to evaluate the AA-NAT mRNA expression in brains of both hibernating and non-hibernating ground squirrels. Brains were examined for the expression of AA-NAT mRNA using a oligonucleotide AA-NAT probe; antibody against neurofilament-70 (NF-70) was used as a neuronal marker. All hibernating animals expressed significantly ( P<0.01) elevated levels of AA-NAT mRNA in both the epithalamic medial habenular nuclei (MHb) area and the hypothalamic suprachiasmatic nuclei (SCN), which is also known as the master biologic clock. These findings represent the first demonstration of the expression of mRNA encoding for AA-NAT in the extra-pineal (i.e. SCN and MHb) sites of thirteen-lined ground squirrels and indicate that the habenular nucleus may be an important supplementary location for melatonin biosynthesis. The data presented here indicate that AA-NAT gene is one of the few specific genes up-regulated during hibernation and suggest that elevation of its expression in SCN and MHb may play an essential role in the generation and maintenance of hibernation.

Rhythms of the pineal N-acetyltransferase mRNA and melatonin concentrations during embryonic and post-embryonic development in chicken

Development of a daily rhythmicity in transcription of a gene encoding a rate-limiting enzyme of melatonin biosynthesis, the arylalkylamine- N-acetyltransferase (AA-NAT) was studied by northern blot analysis in pineal glands of 16 and 19-day-old embryos and 1, 4, 8, 11, and 14-day-old chicks. In a parallel experiment, melatonin content in pineal glands and plasma was measured. A significant rhythm of AA-NAT expression was found at embryonic day (ED) 16, the earliest day assayed in this experiment. Expression was low during the daytime and a clear signal was found in the middle of the darktime. The intensity of the signal was increasing during the ontogeny. The nocturnal pineal melatonin concentrations were increasing over the studied period (from ED 19 until post-embryonic day 21). Midnight plasma melatonin concentrations increased from ED19 to PD 3 and oscillated around this value afterwards. Data show that rhythmic expression of AA-NAT mRNA starts very early in development of chicken and plays a major role in melatonin rhythm generation during embryonic development.

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): II. Effect of vitamin A deficiency.

Levels of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A-free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A-deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light.

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.

Molecular cloning of the arylalkylamine- N-acetyltransferase and daily variations of its mRNA expression in the Syrian hamster pineal gland

The arylalkylamine- N-acetyltransferase (AA-NAT) expressed in the vertebrate pineal gland catalyzes the N-acetylation of the serotonin into N-acetylserotonin and is considered to be the rate limiting enzyme of the pineal melatonin synthesis. Indeed, dramatic changes in its activity throughout the 24-h period drive the large day/night variations in plasma melatonin concentrations. Recently, AA-NAT was cloned in the rat pineal. In this species, AA-NAT mRNA variations were demonstrated to be responsible of the well known AA-NAT activity and plasma melatonin circadian fluctuations. In the Syrian hamster, the pineal melatonin secretion pattern is characterized by a late-night short-duration peak of melatonin synthesis. We investigated whether this typical pattern could be due to a late-night delayed pineal AA-NAT mRNA expression. The first part of our study was dedicated to the molecular cloning of a Syrian hamster AA-NAT cDNA. A PCR-generated clone of 1045 bp encoding the AA-NAT has been isolated and sequenced. In situ hybridization using an AA-NAT cRNA probe revealed that the AA-NAT mRNA expression undergoes strong daily fluctuations in the Syrian hamster pineal, with undetectable level in the second half of the light period and a dramatic increase at night. After lights off, the AA-NAT mRNA expression requires 6–7 h to reach its maximum expression. This result thus suggests that the transcription of the AA-NAT mRNA in the Syrian pineal gland determines the lag period in pineal responsiveness and melatonin synthesis to darkness.