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.

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.

Inhibitors of messenger RNA and protein synthesis affect differently serotonin arylalkylamine N-acetyltransferase activity in clock-controlled and non clock-controlled fish pineal.

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.

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.

Photoreceptor cells of the pike pineal organ as cellular circadian oscillators.

In the pike pineal, the rhythm of melatonin (MEL) secretion is driven by a population of cellular circadian oscillators, synchronized by the 24 h light/dark (LD) cycle. Because the pineal photoreceptor contains both the input and output pathways of the clock, this cell is likely to be a cellular circadian system by itself. To support this idea, we have dissociated and cultured pike pineal cells as well as purified photoreceptors. In culture, the pineal cells reassociated in follicles, surrounded by collagen fibres. At the electron microscopic level, they appeared well preserved. Total cells consisted mainly of photoreceptors and glia. Purified cells corresponded exclusively to photoreceptors. Under LD, MEL production was rhythmic. Under constant darkness (DD), the rhythm was well sustained for at least six 24 h cycles (tau = 24/27 h) with 1 x 10(6) total cells/well or below; with 2 x 10(6) total cells/well, a strong damping occurred towards high levels as soon as after the second cycle. At the density of 0.5 x 10(6) cells/well, purified photoreceptors produced less MEL than an equivalent amount of total cells. However, the pattern of the oscillations was similar to that observed with 2 x 10(6) total cells, i.e. a damping occurred rapidly. Decreasing the density to 0.125 x 10(6) photoreceptors/well resulted in a loss of homogeneity among replicates. The production of melatonin by single photoreceptors was monitored by means of the reverse haemolytic plaque assay. Both under LD and under DD, the number of photoreceptors releasing melatonin was higher during the (subjective) dark than during the (subjective) light. The results provide strong support to the idea that the pike pineal photoreceptor is a cellular circadian system. Expression of the oscillations seemed to depend on several factors, including cell to cell contacts between photoreceptors. There is indication that also MEL and glia might be involved.

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.

Calciproteins regulate cyclic AMP content and melatonin secretion in trout pineal photoreceptors.

Photoreceptor cells of the fish pineal transduce photoperiodic information into the rhythmic secretion of melatonin. The nocturnal rise in melatonin secretion has been associated with an increase in cyclic AMP (cAMP) production and with an entry of Ca2+ ions through L-type voltage-dependent channels. It is shown here that two inhibitors of calciproteins, W7 and calmidazolium, inhibit melatonin secretion and, to a lesser extent, cAMP levels in cultured trout pineal photoreceptors. Kinetic studies indicated that melatonin secretion was affected earlier than cAMP in cells cultured in the presence of W7. The present results provide evidence that Ca2+ acts through one or more calciproteins to regulate melatonin production. It is suggested that Ca2+/calciprotein complexes might act at two different sites, one involving regulation of cAMP metabolism, and the other being independent from cAMP.

Calcium and melatonin production in dissociated trout pineal photoreceptor cells in culture.

Trout pineal cells maintained in primary culture produce melatonin in high amounts during night time and low amounts during daytime. The dark-induced increase in melatonin production was enhanced, in a dose-dependent manner, by elevating extracellular calcium concentration. Low external calcium concentration reduced nocturnal and diurnal melatonin production. Bay K 8644 increased, in a dose-dependent manner, the dark-induced rise in melatonin output, and this effect was antagonized by nifedipine and verapamil. This suggests a role for the dihydropyridine calcium channels in the regulation of the melatonin output. To confirm this, patch-clamp recordings (whole-cell perforated) were run in a 20 mmol/l barium medium at different holding potentials from -80 mV. A voltage-dependent inward current was activated from -30 mV to +40 mV with a maximal amplitude being observed at 0 mV. This current was drastically increased in the presence of Bay K 8644. Nifedipine inhibited the current both in the absence or in the presence of Bay K 8644. Our results are consistent with the idea that extracellular calcium participates in the control of melatonin secretion by photoreceptor cells. It is suggested that activation of the voltage-dependent L-type channel may modulate this secretion.

Immunocytochemical localisation of hydroxyindole-O-methyltransferase in pineal photoreceptor cells of several fish species.

Melatonin is an internal "Zeitgeber," involved in the timing and control of a number of rhythmic functions and behaviours. Its synthesising cells remain to be identified in the fish pineal. The last step in the melatonin biosynthetic pathway is catalysed by the enzyme hydroxyindole-O-methyltransferase. An affinity-purified antibody, directed against chicken pineal hydroxyindole-O-methyltransferase, was used in the present study to identify the melatonin synthesising cells in four fish species: a primitive chondrostean (sturgeon), a saltwater teleost (dorado), and two freshwater teleosts (pike, trout). Western blot immunolabeling of pike and trout pineal proteins revealed a single band at 38 KDa, which corresponds to the known molecular weight of the enzyme in bovine, rat, and chicken pineal. Regardless of the species, a specific immunocytochemical labeling, visualised by means of the peroxidase-antiperoxidase method, was exclusively associated with the photoreceptor cells. These results provide evidence that photoreceptors of the fish pineal are responsible for the biosynthesis of 5-methoxyindoles, including melatonin. In the pike, reactions were less intense in the distal portion of the pineal vesicle than in the other regions of the organ. It is questioned whether this might be related to the existence of a germinative zone, generating new photoreceptor cells in this distal portion. Hydroxyindole-O-methyltransferase has been previously demonstrated in mammalian pinealocytes, and modified photoreceptors of the avian pineal. It is now demonstrated in pineal photoreceptors of a primitive fish and of more evolved saltwater and freshwater fish. The results strengthen the view that these cells are related through phylogeny and that their well conserved melatoninergic function appears early in the course of evolution.

Multiple circadian oscillators in the photosensitive pike pineal gland: a study using organ and cell culture.

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.

Detection of estrogen receptor mRNA in trout pineal and retina: estradiol-17 beta modulates melatonin production by cultured pineal photoreceptor cells.

Pineal photoreceptor cells produce the neurohormone melatonin, a major "Zeitgeber" of the organism. This compound is involved in the control of development, growth, sexual maturation, and seasonal reproductive cycles. The present study reports that the photosensitive pineal organ and the retina of the trout express a 3.5-kb mRNA corresponding to the estradiol-17 beta receptor. The effects of estradiol-17 beta on melatonin production by cultured pineal photoreceptor cells were also studied. Under a light/dark (LD:12/12) cycle, these cells maintained a rhythmic secretion of melatonin, with higher amounts being released during the dark phase. The amplitude of the rhythm tended to increase with time in culture. Application of estradiol-17 beta during the dark phase of an LD cycle (i.e., for 12 hr) affected melatonin release in a dose-dependent manner; low concentrations (10(-10) to 10(-8) mol/liter) were inhibitory, whereas high concentrations (over 10(-7) mol/liter) were stimulatory (when compared to the values obtained at 10(-9) mol/liter). When estradiol-17 beta was given continuously for several 24-hr LD cycles, the inhibitory effect observed during the first dark phase disappeared in subsequent dark periods. In the presence of estradiol-17 beta, at concentrations ranging from 10(-10) to 10(-5) mol/liter, a high amplitude rhythm in melatonin secretion returned more rapidly than in controls. The present results suggest that estradiol-17 beta receptors are expressed in the fish pineal and retina and that estradiol-17 beta modulates melatonin secretion by cultured pineal photoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Photoreceptor cells of the pineal body in culture: effect of 17 beta-estradiol on the production of melatonin]. / Cellules photoréceptrices de l'organe pinéal en culture: effets du 17 beta-oestradiol sur la production de mélatonine.

Pineal photoreceptor cells produce the neurohormone, melatonin, a major "Zeitgeber" of the organism. This compound has been involved in the control of development, growth, sexual maturation, and seasonal reproductive cycles. We investigated, here, the effects of estradiol-17 beta on melatonin production by cultured pineal photoreceptor cells. Under a light/dark (LD: 12/12) cycle, cultured trout pineal cells maintained a rhythmic secretion of melatonin with higher amounts being released during the dark phase. The amplitude of the rhythm tended to increase with time spent in culture. Application of estradiol-17 beta during the dark phase of a LD cycle (i.e., for 12 h) affected melatonin release in a dose-dependent manner: low concentrations (10(-10) to 10(-8) mol/l) were inhibitory whereas high concentrations (over 10(-7) mol/l) were stimulatory. When estradiol-17 beta was applied continuously for several 24 h LD cycles, the inhibitory effect observed during the first dark phase disappeared later on. Rather, in the presence of estradiol-17 beta, at the concentrations of 10(-9) and 10(-6) mol/l, a high amplitude rhythm in melatonin secretion was recovered faster, when compared to controls. Replacement of 10% fetal calf serum by 0.1% bovine serum albumin did not affect the responses of the photoreceptor cells to 10(-6) mol/l of estradiol-17 beta. The present results bring the first evidence that estradiol-17 beta modulates melatonin secretion by cultured pineal photoreceptors. They further support the idea according to which sexual hormones exert a feedback regulation on the pineal. Pineal photoreceptors appear as multieffector cells which transduce information from, both, physical (photoperiod) and internal (chemical) factors.

Pineal photoreceptor cells: photoperiodic control of melatonin production after cell dissociation and culture.

Trout pineal cells were dissociated using a trypsin-DNase digestion technique. An enriched population of photoreceptor cells was selected from a Percoll gradient centrifugation. The ability of cultured photoreceptor cells (selected or not on a Percoll gradient) to produce melatonin rhythmically was investigated during seven 24 h light/dark cycles. During each cycle, trout pineal photoreceptor cells released low amounts of melatonin during daytime and high amounts during night-time. Under continuous darkness, melatonin release was continually high. The profile of its rhythm and that of the activity of the hydroxyindole-O-methyltransferase-the last enzyme of the melatonin biosynthetic pathway-depended on the substrates and on the culture media used. Some of them appear suitable for short- or long-term culture of photoreceptor cells permitting the study of their neuroendocrine properties.

Pineal photoreceptor cells in culture: fine structure, and light/ dark control of cyclic nucleotide levels and melatonin secretion.

Trout pineal photoreceptor cells were dissociated by trypsin-DNase digestion and further purified by a Percoll gradient centrifugation. Total cells or purified photoreceptor cells were then embedded in a collagen gel, or layered on culture-treated polycarbonate membranes, or maintained in suspension, with RPMI 1640 medium or BGjb medium. It has been shown that cells maintain a rhythmic production of melatonin for at least seven 24 h light/dark cycles under these conditions. In this complementary study, the morphofunctional state of the photoreceptor cells was examined 1) by electron (transmission, scanning) microscopy, and 2) by pharmacological tests under different lighting conditions. Using polycarbonate membranes together with RPMI 1640 medium appeared the most suitable. The segmented organization of photoreceptor cells was well preserved when using the culture-treated membranes. It tended to disappear in cells embedded in the collagen gel and was lost after passage through the Percoll gradient. However, this one allowed obtention of an homogeneous population of photoreceptors, as recognized by their intracellular components. Intracellular organelles were rather well preserved in the embedded photoreceptors. The study also provides novel information on the nature of second messengers involved in the photoperiodic control of melatonin production in photoreceptor cells. From the effects of an adenylyl cyclase activator and a phosphodiesterase inhibitor it appeared that 1) total cells and Percoll-selected cells behaved similarly, 2) the nocturnal rise in melatonin secretion was associated with an increase in cAMP content, and 3) a fall in cAMP may be a mechanism through which light reduces melatonin secretion by photoreceptor cells. Cyclic GMP, the metabolism of which also appeared to be controlled by light, did not seem involved in the photoperiodic control of melatonin production. The method proposed herein offers interesting perspectives for the study of the photoneuroendocrine properties of isolated photoreceptor cells.

Synthesis and actions of a melanotropin conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, on melanocytes and melanoma cells in vitro.

L-Glutamic acid (gamma-4'-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4'-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, D-Phe7]alpha-MSH4-10-NH2. The melanotropin:anilide conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than alpha-MSH or Ac-[Nle4, D-Phe7]alpha-MSH4-10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.