Differential regulation of arylalkylamine N-acetyltransferase activity in chicken retinal ganglion cells by light and circadian clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30-60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar ).     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

Effects of time of l-ornithine administration on the diurnal rhythms of plasma growth hormone,melatonin, and corticosterone levels in mice

The synthesis and secretion of many hormones such as growth hormone (GH), melatonin, and corticosterone, exhibit temporal variations over each day and night. Oral administration of several nutritional factors, including l-ornithine, modulates these hormonal secretions and induces an acute increase in plasma GH levels. However, the impact of l-ornithine on the diurnal rhythms of hormone secretion remains unclear. In this study, we evaluated whether the diurnal rhythms of plasma GH, melatonin, and corticosterone secretion were altered by the daily administration of l-ornithine as well as the timing of the administration, in CBA/N mice. Our results showed that the plasma GH levels that peaked at light phase were amplified by l-ornithine (500?mg/kg) administered at Zeitgeber time (ZT) 22, but not at ZT10. Additionally, l-ornithine (1000?mg/kg) administered at ZT22 advanced the onset of the nocturnal rise of melatonin, which resulted in the elongation of the melatonin peak. On the other hand, l-ornithine (500 and 1000?mg/kg) administered at ZT10, but not at ZT22, suppressed the diurnal rhythm peaks of plasma corticosterone. The effects of l-ornithine on plasma GH rhythms lasted for at least 2 days after cessation of the daily administration. Running wheel activity during the active phase was slightly elevated by l-ornithine administration at ZT22, but the overall patterns were only slightly affected. l-Ornithine levels in the plasma and hypophysis after a single administration of l-ornithine at ZT22 were lower than those after administration at ZT10, suggesting that the metabolic rate of l-ornithine differs between day and night. In conclusion, our data suggest that a daily administration of l-ornithine regulates the diurnal rhythms of GH, melatonin, and corticosterone in a manner dependent on administration time, which might be related to the diurnal rhythms of l-ornithine metabolism.     

Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase

Circadian rhythms might be involved in addictive behaviors. The pineal secretory product melatonin decreases cocaine sensitization in rats; mice mutant for the critical melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase (AANAT), exhibit altered behaviors. We hypothesized that AANAT/melatonin system, which is up-regulated at night, affects cocaine sensitization in mice. Intraperitoneal cocaine treatment (10 and 20 mg/kg) dose-dependently increased locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice; this effect was similar during the day and at night. Injections of cocaine during the day for three days resulted in behavioral sensitization in normal and AANAT mutant mice whereas treatment at night triggered sensitization in AANAT-deficient mice only. AANAT expression and synthesis of N-acetylserotonin/melatonin could play a role in addictive properties of cocaine.     

Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish

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

Effects of L-serine supplementation on the daily rhythms of growth hormone and corticosterone concentrations in mice

The impact of L-serine on the daily rhythms of growth hormone (GH) and corticosterone remains unknown. We explored whether the daily rhythms of these hormones were affected by L-serine supplementation as well as the supplementation time. The results showed that plasma GH concentration at Zeitgeber time (ZT)4 and 8 were significantly increased by L-serine supplementation at ZT22, while the diurnal rhythms peaks of plasma corticosterone at ZT12 were suppressed by L-serine supplementation at ZT10. After the supplementation was stopped, the effects of L-serine on the diurnal rhythms of plasma GH and corticosterone lasted for 2 days then they were fading on day 4. L-serine concentrations in plasma and hypothalamus after supplementation at ZT22 was lower than those after supplementation at ZT10. In conclusion, L-serine modulates the daily rhythms of GH and corticosterone depending on its supplementation time. The modulation effect might be association with the daily rhythms of L-serine metabolism.     

Differential Regulation of Arylalkylamine N-Acetyltransferase Activity in Chicken Retinal Ganglion Cells by Light and Circadian Clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30–60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar)     

Effect of Lithium on the Rhythms of Melatonin in the Pineal Gland, Serum and Retina of Viscacha (Lagostomus maximus maximus)

The aim of this work was to describe the pineal, serum and retinal melatonin daily rhythms in viscacha (Lagostomus maximus maximus), a nocturnal and subterranean rodent. A daily rhythm with a nocturnal peak and an increase during the light phase was found in all cases, with the seric rhythm being delayed with respect to the pineal one. Chronic lithium administration to viscachas suppressed the diurnal increase of pineal melatonin, abolished the retinal rhythm and had no effect on seric melatonin.     

Circadian rhythms of pineal N-acetyltransferase activity in the Djungarian hamster,Phodopus sungorus,in response to seasonal changes of natural photoperiod

Summary The aim of this study was to describe the regular annual pattern of the daily melatonin synthesis in Djungarian hamsters,Phodopus sungorus sungorus. The hamsters were maintained from birth in natural photoperiodic conditions and in bimonthly intervals the day/night rhythms of pineal N-acetyltransferase (NAT) were measured. Analysis of the circadian profiles of NAT activity showed that the duration of elevated melatonin synthesis closely reflects the duration of the scotophase throughout the seasons. Thus the duration of elevated melatonin seems to represent a direct humoral signal transmitting the photoperiodic message. The duration of the nightly melatonin pulse appears to be influenced mainly by the time of dawn rather than by the time of dusk. Additional information about the time of year might be encoded in the total amount of melatonin synthesized per day, whereas the amplitude of the nightly melatonin peak seems to be of minor importance.Abbreviation NAT N-acetyltransferase Dedicated to Dr. Klaus Hoffmann on the occasion of his 60th birthday     

Changes in light and dark periods affect the arylalkylamine N-acetyl transferase,melatonin activities and redox status in the head and hemolymph of nocturnal insect Spodoptera litura

The present study was conducted to describe the impact of circadian rhythm on melatonin levels and redox statusunder three photoperiods (12L:12D, 0L:24D, and 24L:0D) in head and hemolymph of Spodoptera litura. Melatonin is an powerful antioxidant and controls the reproduction of organisms. In this study, melatonin levels, Arylalkylamine N-acetyltransferase(AA-NAT), and antioxidant enzyme activities were analyzed. Results showed melatonin, AA-NAT levels in hemolymph were significantly (p < 0.05) higher during the dark period than during LL regime. HPLC chromatogram of the insect head and hemolymph showed 5 peaks while hemolymph showed 6 peaks in LD, and LLregimes. The day–night changes of melatonin increased the antioxidant enzymes (GST, CAT, POX) persisted in the insect hemolymph, but were suppressed by constant light. The present study leads us to speculate that synthesis and release of melatonin in the S.litura head occur as circadian rhythm and light has an inhibitory effect on melatonin synthesis.