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.     

Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation

The large daily rhythm in circulating melatonin levels is a highly conserved feature of vertebrate physiology: high values always occur at night. The dynamics of the rhythm are controlled by the next-to-last enzyme in melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin), arylalkylamine N-acetyltransferase (AANAT), the "melatonin rhythm enzyme". In vertebrate biology, AANAT plays a unique time-keeping role as the molecular interface between the environment and the hormonal signal of time, melatonin. This chapter describes the mammalian AANAT regulatory system, which includes the retina, neural structures, transsynaptic processes, and molecular events. In addition, special attention is paid to the functional characteristics of the systems which insure that the nocturnal increase in melatonin is an accurate and reliable indicator of the duration of the night, and why the melatonin rhythm is the most reliable output signal of the Mind's Clock.     

Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.     

cAmp regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87): a new cell line (1E7) provides evidence of intracellular AANAT activation

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.     

Daily oscillation in melatonin synthesis in the Turkey pineal gland and retina: diurnal and circadian rhythms

The aim of the present study was to examine arylalkylamine N-acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light-dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night-time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high-amplitude melatonin rhythms in the turkey.     

Neural regulation of dark-induced abundance of arylalkylamine N-acetyltransferase (AANAT) and melatonin in the carp (Catla catla) pineal: an in vitro study

In all the vertebrates, synthesis of melatonin and its rhythm-generating enzyme arylalkylamine N-acetyltransferase (AANAT) reaches its peak in the pineal during the night in a daily light-dark cycle, but the role of different neuronal signals in their regulation were unknown for any fish. Hence, the authors used specific agonist and antagonists of receptors for different neuronal signals and regulators of intracellular calcium (Ca(2+)) and adenosine 3',5'-cyclic monophosphate (cAMP) in vitro to study their effects on the abundance of AANAT and titer of melatonin in the carp (Catla catla) pineal. Western blot analysis followed by quantitative analysis of respective immunoblot data for AANAT protein, radioimmunoassay of melatonin, and spectrophotometric analysis of Ca(2+) in the pineal revealed stimulatory effects of both adrenergic (α(1) and β(1)) and dopaminergic (D(1)) agonists and cholinergic (both nicotinic and muscarinic) antagonists, inhibition by both adrenergic and dopaminergic antagonists and cholinergic agonists, but independent of the influence of any agonists or antagonists of α(2)-adrenergic receptors. Band intensity of AANAT and concentration of melatonin in the pineal were also enhanced by the intracellular calcium-releasing agent, activators of both calcium channel and adenylate cyclase, and phophodiesterase inhibitor, but suppressed by inhibitor of calcium channel and adenylate cyclase as well as activator of phophodiesterase. Moreover, an inhibitory effect of light on the pineal AANAT and melatonin was blocked by both cAMP and proteasomal proteolysis inhibitor MG132. Collectively, these data suggest that dark-induced abundance of AANAT and melatonin synthesis in the carp pineal are a multineuronal function, in which both adrenergic (α(1) and β(1), but not α(2)) and dopaminergic signals are stimulatory, whereas cholinergic signals are inhibitory. This study also provides indications, though arguably not conclusive evidence, that in either case the neuronal mechanisms follow a signal-transduction pathway in which Ca(2+) and cAMP may act as the intracellular messengers. It also appears that proteasomal proteolysis is a conserved event in the regulation of AANAT activity in vertebrates.     

The 2004 Aschoff/Pittendrigh lecture: Theory of the origin of the pineal gland--a tale of conflict and resolution

A theory is presented that explains the evolution of the pinealocyte from the common ancestral photoreceptor of both the pinealocyte and retinal photoreceptor. Central to the hypothesis is the previously unrecognized conflict between the two chemistries that define these cells-melatonin synthesis and retinoid recycling. At the core of the conflict is the formation of adducts composed of two molecules of retinaldehyde and one molecule of serotonin, analogous to formation in the retina of the toxic bis-retinyl ethanolamine (A2E). The hypothesis argues that early in chordate evolution, at a point before the genes required for melatonin synthesis were acquired, retinaldehyde--which is essential for photon capture--was depleted by reacting with naturally occurring arylalkylamines (tyramine, serotonin, tryptamine, phenylethylamine) and xenobiotic arylalkylamines. This generated toxic bis-retinyl arylalkylamines (A2AAs). The acquisition of arylalkylamine N-acetyltransferase (AANAT) prevented this by N-acetylating the arylalkylamines. Hydroxyindole-O-methyltransferase enhanced detoxification in the primitive photoreceptor by increasing the lipid solubility of serotonin and bis-retinyl serotonin. After the serotonin --> melatonin pathway was established, the next step leading toward the pinealocyte was the evolution of a daily rhythm in melatonin and the capacity to recognize it as a signal of darkness. The shift in melatonin from metabolic garbage to information developed a pressure to improve the reliability of the melatonin signal, which in turn led to higher levels of serotonin in the photodetector. This generated the conflict between serotonin and retinaldehyde, which was resolved by the cellular segregation of the two chemistries. The result, in primates, is a pineal gland that does not detect light and a retinal photodetector that does not make melatonin. High levels of AANAT in the latter tissue might serve the same function AANAT had when first acquired- prevention of A2AA formation.     

Cellular stability of serotonin N-acetyltransferase conferred by phosphonodifluoromethylene alanine (Pfa) substitution for Ser-205

Large changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) in the pineal gland control the rhythmic production of the time-keeping hormone melatonin. The activity of AANAT reflects changes in the amount and activation state of the AANAT protein, both of which increase at night. The molecular basis of this regulation is now becoming known, and recent data indicate that this involves phosphorylation-dependent binding to the 14-3-3 protein at two sites, one of which, Ser-205, is located several residues from the C terminus. In this study, we determined whether substitution of this residue with a non-hydrolyzable the phosphoserine/phosphothreonine mimetic would promote binding to the 14-3-3 protein and enhance cellular stability. To accomplish this, a C-terminal AANAT peptide containing the phosphonodifluoromethylene alanine at Ser-205 was synthesized and fused to bacterially expressed AANAT(30-199) using expressed protein ligation. The resulting semisynthetic protein has enhanced affinity for the expressed 14-3-3 protein and exhibits greater cellular stability in microinjection experiments, as compared with the unmodified AANAT. Enhanced 14-3-3 binding was also observed using humanized ovine AANAT, which has a different C-terminal sequence (Gly-Cys) than the ovine enzyme (Asp-Arg), indicating that that characteristic is not unique to the ovine enzyme. These studies provide the first evidence that substitution of Ser-205 with the stable phosphomimetic amino acid phosphonodifluoromethylene alanine enhances binding to 14-3-3 and the cellular stability of AANAT and are consistent with the view that Ser-205 phosphorylation plays a critical role in the regulation of AANAT activity and melatonin production.     

Analysis of serotonin N-acetyltransferase regulation in vitro and in live cells using protein semisynthesis

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3zeta recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3zeta. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with 14-3-3zeta that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3zeta heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3zeta-AANAT interaction using 14-3-3zeta fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3zeta interaction.     

Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout ( Oncorhynchus mykiss ). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l -tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.     

The arylalkylamine-N-acetyltransferase (AANAT) acetylates dopamine in the digestive tract of goldfish: A role in intestinal motility

Melatonin has been found in the digestive tract of many vertebrates. However, the enzymatic activity of the arylalkylamine-N-acetyltransferase (AANAT) and the hydroxindole-O-methyltransferase (HIOMT), the last two enzymes of melatonin biosynthesis, have been only measured in rat liver. Therefore, the first objective of the present study is to investigate the functionality of these enzymes in the liver and gut of goldfish, analyzing its possible daily changes and comparing its catalytic properties with those from the retina isoforms. The daily rhythms with nocturnal acrophases in retinal AANAT and HIOMT activities support their role in melatonin biosynthesis. In foregut AANAT activity also show a daily rhythm while in liver and hindgut significant but not rhythmic levels of AANAT activity are found. HIOMT activity is not detected in any of these peripheral tissues suggesting an alternative role for AANAT besides melatonin synthesis. The failure to detect functional HIOMT activity in both, liver and gut, led us to investigate other physiological substrates for the AANAT, as dopamine, searching alternative roles for this enzyme in the goldfish gut. Dopamine competes with tryptamine and inhibits retinal, intestinal and hepatic N-acetyltryptamine production, suggesting that the active isoform in gut is AANAT1. Besides, gut and liver produces N-acetyldopamine in presence of acetyl coenzyme-A and dopamine. This production is not abolished by the presence of folic acid (arylamine N-acetyltransferase inhibitor) in any studied tissue, but a total inhibition occurs in the presence of CoA-S-N-acetyltryptamine (AANAT inhibitor) in liver. Therefore, AANAT1 seems to be an important enzyme in the regulation of dopamine and N-acetyldopamine content in liver. Finally, for the first time in fish we found that dopamine, but not N-acetyldopamine, regulates the gut motility, underlying the broad physiological role of AANAT in the gut.     

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.