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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.

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.

The pineal organ of fish contains photoreceptor cells. In some species (e.g., pike) each photoreceptor is a cellular circadian system which contains a photoreceptive unit, the clock and an output unit. In others (e.g., trout) the clock is lacking. The main rhythmic output of the pineal photoreceptor is melatonin, an internal 'zeitgeber' of the organisms. The nocturnal rise in melatonin secretion results from an increase in the activity of the arylalkylamine-N-acetyltransferase (AA-NAT) which converts serotonin to N-acetylserotonin. In the present study we investigated the effects of transcription and translation inhibitors on AA-NAT activity in pike and trout pineal organs in culture. Cycloheximide, anisomycin, and puromycin inhibited the rise in AA-NAT activity observed during the first 2, 4 or 6 h of the dark phase, in both species. Actinomycin D was active only in the pike. Six hours of treatment during the first half of the night induced inhibition of AA-NAT activity, providing that forskolin (an adenylyl cyclase stimulator) was present in the culture medium. When the treatment was run for 3, 6 or 12 h, starting at midday of a 12L/12D cycle, basal and forskolin-stimulated AA-NAT activity (measured at midnight) were dramatically reduced. Such a treatment had no effect on trout AA-NAT activity. It is concluded that: (1) the dark-induced rise in AA-NAT activity and melatonin secretion are dependent on newly synthesized protein in both pike and trout pineal; (2) AA-NAT regulation takes place at the translational and post-translational levels in both species; (3) AA-NAT regulation occurs also at the transcriptional level in the pike, but not in the trout; and (4) the cAMP-dependent activation of AA-NAT requires transcription in the pike, not in the trout. The presence of a cell population acting as a circadian clock in the pike pineal, but not in the trout pineal, can explain the difference between these two species. Thus, we suggest that the clock mechanism operates at the genetic level in these cells. Further comparative studies between clock-controlled and non-clock-controlled pineals might prove interesting to demonstrate the difference between these two regulatory pathways., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

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.

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.