Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

14-3-3 proteins in pineal photoneuroendocrine transduction: how many roles?

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina.

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC ) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced arylalkylamine acetylation by S. cerevisiae extracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Regulation of arylalkylamine N-acetyltransferase-2 (AANAT2, EC 2.3.1.87) in the fish pineal organ: evidence for a role of proteasomal proteolysis.

In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.

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.

Melatonin synthesis: arylalkylamine N-acetyltransferases in trout retina and pineal organ are different.

Serotonin N-acetyltransferase (AANAT) is the first enzyme in the conversion of serotonin to melatonin. Changes in AANAT activity determine the daily rhythm in melatonin secretion. Two AANAT genes have been identified in the pike, pAANAT-1 and pAANAT-2, expressed in the retina and in the pineal, respectively. The genes preferentially expressed in these tissues encode proteins with distinctly different kinetic characteristics. Like the pike, trout retina primarily expresses the AANAT-1 gene and trout pineal primarily expresses the AANAT-2 gene. Here we show that the kinetic characteristics of AANAT in these tissues differ as in pike. These differences include optimal temperature for activity (pineal: 12 degrees C; retina: 25 degrees C) and relative affinity for indoleethylamines compared to phenylethylamines. In addition, retinal AANAT exhibited substrate inhibition, which was not seen with pineal AANAT. The kinetic differences between AANAT-1 and AANAT-2 appear to be defining characteristics of these gene subfamilies, and are not species specific.

Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities.

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 +/- 24.4 vs. H: 344.1 +/- 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 +/- 6.7 vs. H: 119.1 +/- 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

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.

Ovine arylalkylamine N-acetyltransferase in the pineal and pituitary glands: differences in function and regulation.

The enzyme arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) has been conventionally linked with the biosynthesis of melatonin within the pineal gland and retina. This study establishes that AANAT messenger RNA (mRNA) and functional enzyme occurs within the pars tuberalis (PT) and to a lesser degree within the pars distalis (PD) of the sheep pituitary gland; expression in these tissues is approximately 1/15th (PT) and 1/300th (PD) of that in the ovine pineal gland. AANAT mRNA in the PT appears to be expressed in the same cells as the Mel1a receptor. No evidence was obtained to indicate that either PT or PD cells have the ability to synthesize melatonin, suggesting that this enzyme plays a different functional role in the pituitary. We also found that cAMP regulation of the abundance of AANAT mRNA differs between the PT and pineal gland. Forskolin (10 microM) has no effect on pineal AANAT mRNA levels, yet represses expression in the PT. This suppressive influence could be mediated by ICER (inducible cAMP response early repressor), which is induced by forskolin in both tissues. Although it appears that the specific function and regulation of AANAT in the pituitary gland differ from that in the pineal gland, it seems likely that AANAT may play a role in the broader area of signal transduction through the biotransformation of amines.

Expression of melatonin synthesis genes is controlled by a circadian clock in the pike pineal organ but not in the trout.

The photosensitive teleost pineal organ exhibits a daily rhythm in melatonin production. In most teleosts, including the pike, this is driven by an endogenous pineal clock. An exception is the trout, in which the pineal melatonin rhythm is a direct response to darkness. This fundamental difference in the regulation of melatonin production in two closely related species provides investigators a novel opportunity to study the molecular mechanisms of vertebrate clock function. We have studied the circadian regulation of mRNA encoding two melatonin synthesis enzymes by Northern blot analysis. These two enzymes are serotonin N-acetyltransferase (AA-NAT), the penultimate enzyme in melatonin synthesis, and tryptophan hydroxylase (TPH), the first enzyme in melatonin synthesis. A clock controls expression of both AA-NAT and TPH mRNAs in the pineal organ of pike, but not that of trout, in which the levels of these mRNAs are tonically elevated. A parsimoneous explanation of this is that a single circadian system regulates the expression of both AA-NAT and TPH genes in most teleosts, and that in trout this system has been disrupted, perhaps by a single mutation.

Alternative splicing at the 3'-cDNA of human tryptophan hydroxylase.

Two alternatively spliced transcripts of human tryptophan hydroxylase (TPH) were identified that differed at the 3' end of the open reading frame. Comparison of the human TPH cDNA and genomic sequences revealed that an intron containing an in-frame stop codon could be alternatively spliced out of intron 11. This splicing would give rise to two human TPH isoforms with different C termini; the one that derives from the nonspliced intron contains a putative cyclic AMP-dependent protein kinase site, whereas the other one, which is 22 amino acids longer, does not. Analysis of various human tissues by RT-PCR revealed that the spliced TPH mRNA species was detected in all the postmortem tissues we tested, but the nonspliced species was expressed in only some tissues.

Transcripts encoding two melatonin synthesis enzymes in the teleost pineal organ: circadian regulation in pike and zebrafish, but not in trout.

In this report the photosensitive teleost pineal organ was studied in three teleosts, in which melatonin production is known to exhibit a daily rhythm with higher levels at night; in pike and zebrafish this increase is driven by a pineal clock, whereas in trout it occurs exclusively in response to darkness. Here we investigated the regulation of messenger RNA (mRNA) encoding serotonin N-acetyltransferase (AA-NAT), the penultimate enzyme in melatonin synthesis, which is thought to be primarily responsible for changes in melatonin production. AA-NAT mRNA was found in the pineal organ of all three species and in the zebrafish retina. A rhythm in AA-NAT mRNA occurs in vivo in the pike pineal organ in a light/dark (L/D) lighting environment, in constant lighting (L/L), or in constant darkness (D/D) and in vitro in the zebrafish pineal organ in L/D and L/L, indicating that these transcripts are regulated by a circadian clock. In contrast, trout pineal AA-NAT mRNA levels are stable in vivo and in vitro in L/D, L/L, and D/D. Analysis of mRNA encoding the first enzyme in melatonin synthesis, tryptophan hydroxylase, reveals that the in vivo abundance of this transcript changes on a circadian basis in pike, but not in trout. A parsimonious hypothesis to explain the absence of circadian rhythms in both AA-NAT and tryptophan hydroxylase mRNAs in the trout pineal is that one circadian system regulates the expression of both genes and that this system has been disrupted by a single mutation in this species.

Magnification of tributyl tin toxicity to oyster larvae by bioconcentration in biofilms of Shewanella colwelliana.

The toxic effects of dissolved versus bioconcentrated tributyl tin (TBT) on oyster larvae were compared. Water column TBT levels, which had no effect in solution, inhibited natural attachment and metamorphosis of oyster larvae on bottom surfaces due to bioconcentration by biofilms. This mechanism should be considered when evaluating heavy metal toxicity in the environment.

Catecholamines and dihydroxyphenylalanine in metamorphosing larvae of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).

The content of catecholamines and dihydroxyphenylalanine in larvae of the nudibranch Phestilla sibogae was analyzed by high-performance liquid chromatography with electrochemical detection. Dihydroxyphenylalanine, norepinephrine and dopamine were identified in larvae of all ages examined (5 through 12 days post-fertilization). Dihydroxyphenylalanine could be accurately quantified only in larvae of ages 8 through 12 days, when its average concentration increased from 0.62 to 6.71 x 10(-2) pmol micrograms protein-1. Between ages 5 and 12 days dopamine rose from 0.081 to 0.616 pmol microgram protein-1, and norepinephrine from 0.45 to 2.17 x 10(-2) pmol micrograms protein-1. Dihydroxyphenylalanine, dopamine and norepinephrine were also measured at different stages of metamorphic progress in 10- to 12-day larvae. Dihydroxyphenylalanine increased by a factor of 2.4 between the onset and completion of metamorphosis, but levels of dopamine and norepinephrine remained stable. One millimolar alpha-methyl-DL-m-tyrosine, an inhibitor of catecholamine synthesis, inhibited natural metamorphosis and depleted endogenous norepinephrine and especially dopamine, respectively, to 75% and 35% of control values. The existence of unexpectedly high levels of catecholamines in metamorphically competent larvae, and the association of catecholamine depletion with inhibition of metamorphosis, indicate that these compounds may participate in the control of gastropod development.

Regulation of pineal alpha1B-adrenergic receptor mRNA: day/night rhythm and beta-adrenergic receptor/cyclic AMP control.

Mammalian pineal function is regulated by norepinephrine acting through alpha1beta- and beta1-adrenergic receptors (ARs). Noradrenergic stimulation of alpha1beta-ARs potentiates the beta1-AR-driven increase in cAMP, serotonin N-acetyltransferase, and melatonin production. In the present study, we describe a 3-fold daily rhythm in mRNA-encoding alpha1beta-ARs in the pineal gland, with a peak at midnight. Pharmacological studies indicate that this increase in alpha1beta-AR mRNA is due to activation of beta-ARs. Second messenger studies indicate that alpha1beta-AR mRNA is increased by agents that increase cAMP, including dibutyryl cAMP, cholera toxin, forskolin, or vasoactive intestinal peptide. These observations indicate that alpha1beta-AR mRNA can be physiologically regulated by a beta-AR-dependent enhancement of cAMP. It also was observed that in vivo and in vitro changes in alpha1beta-AR mRNA are not accompanied by similar changes in alpha1beta-AR binding, indicating that turnover of alpha1beta-AR protein is significantly slower than that of alpha1beta-AR mRNA and that post-transcriptional mechanisms play an important role in regulating alpha1beta-AR binding.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel.

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.