Rate-limiting Enzyme In Melatonin Synthesis Research Articles

Two pathways regulate insulin-like growth factor genes in the brain and liver of the tropical damselfish Chrysiptera cyanea: A possible role for melatonin in the actions of growth and thyroid hormones

External and internal factors are involved in controlling the growth of fishes. However, little is known about the mechanisms by which external factors trigger stimulus signals. This study explored the physiological roles of melatonin in the transcription of growth-related genes in the brain and liver of Chrysiptera cyanea, a tropical damselfish with long-day preference. In brain samples of this species collected at 4-h intervals, the transcript levels of arylalkylamine N-acetyltransferase2 (aanat2), the rate-limiting enzyme of melatonin synthesis, and growth hormone (gh) peaked at 20:00 and 00:00, respectively. Concomitantly, the transcript levels of insulin-like growth factors (igf1 and igf2) in the brain and liver were upregulated during the scotophase. Levels of iodothyronine deiodinases (dio2 and dio3), enzymes that convert thyroxine (T4) to triiodothyronine (T3) and reverse T3, respectively, increased in the brain (dio2 and dio3) and liver (dio2) during the photophase, whereas dio3 levels in the liver showed the opposite trend. Fish reared in melatonin-containing water exhibited significant increases in the transcription levels of gh and igf1 in the brain and igf1 in the liver, suggesting that growth in this fish is positively regulated by the GH/IGF pathway on a daily basis. Melatonin treatment also stimulated the transcript levels of dio2 and dio3 in the liver, but not in the brain. Fish consuming pellets containing T3, but not T4, showed significant increases in gh and igf1 in the brain and igf1 and igf2 in the liver, suggesting that the intercellular actions of the TH/IGF pathway have an impact on growth on a daily basis. In summary, IGF synthesis and action in the brain and liver undergo dual regulation by distinct hormone networks, which may also be affected by daily, seasonal, or nutritional factors.

Evaluation of hippocampal arylalkylamine N-acetyltransferase activity in amyloid-β neurotoxicity.

Arylalkylamine N-acetyltransferase (AANAT), a rate-limiting enzyme in melatonin synthesis, is present in extra-pineal tissues such as the hippocampus. The hippocampal AANAT activity in amyloid β (Aβ) neurotoxicity has not been exactly defined. Adult male rats received bilateral intra-CA1 Aβ administration. The hippocampus tissue sampling was performed 2, 12, and 24 h after Aβ injection in the morning and night. The inflammation was monitored using tumor necrosis factor-alpha (TNF-α) immunohistochemistry. The AANAT enzyme activity and melatonin levels were measured using western blotting and high-performance liquid chromatography. The sampling in the morning vs night showed no significant differences in the AANAT activity. The Aβ increased the area of TNF-α positive staining 24 h after injection, which indicated the induction of an inflammatory context. It was accompanied by a significant reduction in AANAT activity and hippocampal melatonin. A reverse correlation was also detected between TNF-α and AANAT activity in the 24-h group. The TNF-α positive area was significantly increased in the 24-h group as compared to the 12-h group. Data showed that inflammatory processes began 12 h after the Aβ injection and augmented 24 h later. In the second experiment, the impact of Aβ injection on hippocampus AANAT activity was examined in the pinealectomized (PIN×) animals. The PIN× per se did not affect the hippocampal AANAT and melatonin levels. However, there was a significant decrease in hippocampal melatonin in the PIN×+Aβ group. The findings suggest the accompanying hippocampal inflammatory context and AANAT enzyme activity reduction in early stages after Aβ administration. Understanding the underlying mechanism of the decreased AANAT activity may suggest new treatment strategies.

Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus-oocyte complexes.

Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non-human primate cumulus-oocyte complexes (COCs). Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate-limiting enzyme in melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs. RT-PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)- and metaphase II (MII)-stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV- and MII-stage COCs. Our results provide the first evidence for the existence of the rate-limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non-human primate COCs.

Gene expression profile associated with Asmt knockout-induced depression-like behaviors and exercise effects in mouse hypothalamus.

Sleep disorder caused by abnormal circadian rhythm is one of the main symptoms and risk factors of depression. As a known hormone regulating circadian rhythms, melatonin (MT) is also namely N-acetyl-5-methoxytryptamine. N-acetylserotonin methyltransferase (Asmt) is the key rate-limiting enzyme of MT synthesis and has been reportedly associated with depression. Although 50–90% of patients with depression have sleep disorders, there are no effective treatment ways in the clinic. Exercise can regulate circadian rhythm and play an important role in depression treatment. In the present study, we showed that Asmt knockout induced depression-like behaviors, which were ameliorated by swimming exercise. Moreover, swimming exercise increased serum levels of MT and 5-hydroxytryptamine (5-HT) in Asmt knockout mice. In addition, the microarray data identified 10 differentially expressed genes (DEGs) in KO mice compared with WT mice and 29 DEGs in KO mice after swimming exercise. Among the DEGs, the direction and magnitude of change in epidermal growth factor receptor pathway substrate 8-like 1 (Eps8l1) and phospholipase C-β 2 (Plcb2) were confirmed by qRT-PCR partly. Subsequent bioinformatic analysis showed that these DEGs were enriched significantly in the p53 signaling pathway, long-term depression and estrogen signaling pathway. In the protein–protein interaction (PPI) networks, membrane palmitoylated protein 1 (Mpp1) and p53-induced death domain protein 1 (Pidd1) were hub genes to participate in the pathological mechanisms of depression and exercise intervention. These findings may provide new targets for the treatment of depression.

Differential Role of cAMP, cGMP, and Ca2+ and Involvement of Kinases and CBP-CREB CRE Pathway in Regulation of Arylalkylamine N-acetyltransferase 2 mRNA Levels in the Pineal Organ of an Air-Breathing Catfish, Clarias gariepinus

Transcription of arylalkylamine N-acetyltransferase 2 (aanat2) gene leads to the formation of AANAT2 - the rate-limiting enzyme in melatonin synthesis in the photosensitive fish pineal. Unlike in mammalian pineal, there is practically no information on signal transduction pathway(s) involved in the regulation of aanat2 gene transcription in the fish pineal. Therefore, we investigated the role of important molecular components of signalling via cAMP, cGMP, and Ca2+ involving cAMPdependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), Ca2+-dependent protein kinase (PKC), mitogen-activated protein kinase (MAPK) kinase (MEK) and p38 MAPK (p38 MAPK) as well as the possible role of serine/threonine phosphatases (PPs), cAMP response element binding protein (CREB) and CREB binding protein (CBP) using their specific inhibitors in regulation of aanat2 transcripts in the fish pineal under in vitro conditions. db-cAMP and db-cGMP increased AANAT2 mRNA levels. db-cAMP- and db-cGMP-induced increase in AANAT2 mRNA levels was significantly reduced in the presence of H-89 (PKA inhibitor), KT5823 (PKG inhibitor), chelerythrine chloride (PKC inhibitor), U0126 (MEK inhibitor), and SB 202190 (p38 MAP kinase inhibitor). Inhibitors of PP1 and PP2A significantly increased AANAT2 mRNA as well as significantly reduced db-cAMP and db-cGMP induced increase in AANAT2 mRNA levels. Inhibitors of both CREB and CBPCREB interaction completely blocked the cAMP-induced increase in AANAT2 mRNA levels. Based on these findings, we suggest that cAMP, cGMP, and Ca2+ increase AANAT2 mRNA levels via PKA, PKG, and PKC, respectively. Further, protein phosphatases and the CBP-CREB-CRE pathway are actively involved in the regulation of AANAT2 mRNA levels in the fish pineal.

Prenatal exposure to lipopolysaccharide induces changes in the circadian clock in the SCN and AA-NAT activity in the pineal gland

The mammalian circadian pacemaker in the suprachiasmatic nucleus (SCN) regulates behavioral and physiological processes in a 24-h cycle. During its development, the SCN can be sensitive to external stimuli which may change the circadian phenotypes in adulthood. Here, we investigated the effects of prenatal exposure to endotoxin lipopolysaccharide (LPS) on the developing rhythms in expression of Per1, Per2, Nr1d1 and Rasd1 along the rostrocaudal axis of the SCN, and on the rhythm of the rate-limiting enzyme in melatonin synthesis, pineal alkylamine N-acetyltransferase (AA-NAT). The prenatal LPS treatment induced anxiety-like behavior in adulthood as shown before and affected the rhythmicity of clock genes in the SCN. The major effect was observed for Nr1d1 expression; the least affected gene was Per2. The Nr1d1 in the LPS-treated group was arrhythmic at postnatal day 3, but showed significantly higher amplitude at postnatal day 20 at all SCN parts, similarly to the AA-NAT activity in pineal glands, thus suggesting adaptive flexibility of the developing SCN to immune challenges in early development.

Melatonin in Mitochondria: Mitigating Clear and Present Dangers.

In cancer cells, glucose is primarily metabolized to pyruvate and then to lactate in the cytosol. By allowing the conversion of pyruvate to acetyl-CoA in mitochondria, melatonin reprograms glucose metabolism in cancer cells to a normal cell phenotype. Acetyl-CoA in the mitochondria also serves as a necessary co-factor for the rate-limiting enzyme in melatonin synthesis, thus ensuring melatonin production in mitochondria of normal cells.

Adenosine triphosphate inhibits melatonin synthesis in the rat pineal gland.

Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects.

Melatonin synthesis follows a daily cycle in Daphnia

In freshwater systems, Daphnia has been demonstrated to show adaptive responses following the light-dark cycle. The adjustment of these responses to the change of day and night is probably transmitted via the hormone melatonin. The rate-limiting enzyme in melatonin synthesis is the arylalkylamine N-transferase (AANAT). We identified three genes coding for insect-like AANATs in Daphnia, of which we measured the gene expression in an ecologically relevant light-dark cycle. We demonstrated that Daphnia's insect-like AANAT gene expression oscillated in a daily manner, and that the highest peak of expression after the onset of darkness was followed by a peak of melatonin production at midnight. Moreover, we could show an oscillation of endogenous melatonin synthesis in Daphnia. In most organisms, melatonin synthesis is due to rhythmic expression of genes of the circadian clock, since transcription of aanats is directly linked to a circadian transcription factor. We could demonstrate that putative clock genes and insect-like AANAT genes of Daphnia were equally expressed. Therefore, we propose that melatonin synthesis is coupled to the expression of Daphnia clock genes, and that insect-like AANATs of crustaceans have a similar function as AANATs of vertebrates: The initiation of melatonin synthesis. In future studies with Daphnia, it will be necessary to take the time of day into account since melatonin concentrations might influence stress responses.

Melatonin and its potential biological functions in the fruits of sweet cherry

Melatonin is a well-known molecule which possesses many beneficial effects on human health. Many agriculture products provide natural melatonin in the diet. Cherry is one such fruit as they are rich in melatonin. In order to understand the biological roles of melatonin in cherry fruit, melatonin synthesis and its changes over 24hr period were systematically monitored both during their development and in the ripe cherries in two cultivars, 'Hongdeng' (Prunus avium L. cv. Hongdeng) and 'Rainier' (Prunus avium L. cv. Rainier). It was found that both darkness and oxidative stress induced melatonin synthesis, which led to dual melatonin synthetic peaks during a 24hr period. The high levels of malondialdehyde induced by high temperature and high intensity light exposure were directly related to up-regulated melatonin production. A primary function of melatonin in cherry fruits is speculated to be as an antioxidant to protect the cherry from the oxidative stress. Importantly, plant tryptophan decaboxylase gene (PaTDC) was identified in cherry fruits. Our data shows that PaTDC expression is positively related to the melatonin production in the cherry. This provides additional information to suggest that tryptophan decaboxylase is a rate-limiting enzyme of melatonin synthesis in plants.

Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin's primary function and evolution in eukaryotes

Mitochondria and chloroplasts are major sources of free radical generation in living organisms. Because of this, these organelles require strong protection from free radicals and associated oxidative stress. Melatonin is a potent free radical scavenger and antioxidant. It meets the criteria as a mitochondrial and chloroplast antioxidant. Evidence has emerged to show that both mitochondria and chloroplasts may have the capacity to synthesize and metabolize melatonin. The activity of arylalkylamine N-acetyltransferase (AANAT), the reported rate-limiting enzyme in melatonin synthesis, has been identified in mitochondria, and high levels of melatonin have also been found in this organelle. From an evolutionary point of view, the precursor of mitochondria probably is the purple nonsulfur bacterium, particularly, Rhodospirillum rubrum, and chloroplasts are probably the descendents of cyanobacteria. These bacterial species were endosymbionts of host proto-eukaryotes and gradually transformed into cellular organelles, that is, mitochondria and chloroplasts, respectively, thereby giving rise to eukaryotic cells. Of special importance, both purple nonsulfur bacteria (R.rubrum) and cyanobacteria synthesize melatonin. The enzyme activities required for melatonin synthesis have also been detected in these primitive species. It is our hypothesis that mitochondria and chloroplasts are the original sites of melatonin synthesis in the early stage of endosymbiotic organisms; this synthetic capacity was carried into host eukaryotes by the above-mentioned bacteria. Moreover, their melatonin biosynthetic capacities have been preserved during evolution. In most, if not in all cells, mitochondria and chloroplasts may continue to be the primary sites of melatonin generation. Melatonin production in other cellular compartments may have derived from mitochondria and chloroplasts. On the basis of this hypothesis, it is also possible to explain why plants typically have higher melatonin levels than do animals. In plants, both chloroplasts and mitochondria likely synthesize melatonin, while animal cells contain only mitochondria. The high levels of melatonin produced by mitochondria and chloroplasts are used to protect these important cellular organelles against oxidative stress and preserve their physiological functions. The superior beneficial effects of melatonin in both mitochondria and chloroplasts have been frequently reported.

Diurnal gene expression of Period2, Cryptochrome1, and arylalkylamine N-acetyltransferase-2 in olive flounder, Paralichthys olivaceus

The suprachiasmatic nucleus (SCN) of the teleost hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light-dark cycles. It has been shown that exposure to darkness during the day causes phase shifts in circadian rhythms. In this study, we examined the effect of exposure to darkness on the mRNA expression levels of two circadian clock genes, namely, Period2 (Per2) and Cryptochrome1 (Cry1), and the rate-limiting enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase-2 (Aanat2), in the pineal gland of olive flounder, Paralichthys olivaceus. The expression of these genes showed circadian variations and was significantly higher during the dark phase. These changes may be involved in the mechanism of dark-induced phase shifts. Furthermore, this study suggests that olive flounder may be a teleost model to investigate the localization and function of circadian oscillators.

Role of calcium in the gating of isoproterenol‐induced arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals.

N‐acetyltransferase is not the rate‐limiting enzyme of melatonin synthesis at night

Circadian melatonin production in the pineal gland and retina is under the control of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase. Because NAT activity varies diurnally, it has been considered both the melatonin rhythm-generating enzyme and the rate-limiting enzyme of melatonin synthesis. In rats with dramatically reduced NAT activity due to a H28Y mutation in NAT, melatonin levels remained the same as in wildtype controls, suggesting that NAT does not determine the rate of melatonin production at night. Using a combination of molecular approaches with a sensitive in vivo measurement of pineal diurnal melatonin production, we demonstrate that (i) N-acetylserotonin (NAS), the enzymatic product of NAT, is present in vast excess in the night pineals compared with melatonin; (ii) the continuous increase in NAT protein levels at late night does not produce a proportional increase in melatonin; and (iii) an increase in NAS in the same animal over several circadian cycles do not result in corresponding increase in melatonin output. These results strongly suggest that NAT is not the rate-limiting enzyme of melatonin formation at night.

Pineal circadian clocks gate arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

Effect of dark exposure in the middle of the day on Period1, Period2, and arylalkylamine N-acetyltransferase mRNA levels in the rat suprachiasmatic nucleus and pineal gland

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light–dark cycles. It has been shown that dark exposure in the day causes phase shifts in circadian rhythms, but it does not induce changes in the melatonin levels in the pineal gland. In this study, we examined the effect of dark exposure on two “circadian clock” genes Period1 and Period2 mRNA levels in the rat SCN, and on Period1, Period2, and arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) gene expression in the pineal gland. Period1 and Period2 mRNA levels were significantly decreased in the SCN after 0.5 and 2 h, respectively, therefore suggesting that changes in those mRNA levels may be the part of the mechanisms of dark-induced phase shifts. Period1 and Aa-Nat mRNA levels in the pineal gland were not affected by darkness, but Period2 was moderately affected. Since Period1 and Aa-Nat mRNA levels in the pineal gland did not respond to dark stimulation, we further examined whether the pineal gland itself is capable of responding to adrenergic stimulation at this time of the day. Isoproterenol significantly induced Period1 and Aa-Nat mRNA levels; however, it did not affect Period2. Although previous studies have reported that during the day the SCN “gates” the dark information reaching the pineal, our data demonstrate that dark information may reach the pineal during the daytime.

Embryonic and post-embryonic expression of arylalkylamine N-acetyltransferase and melatonin receptor genes in the eye and brain of chum salmon ( Oncorhynchus keta)

Melatonin and arylalkylamine N-acetyltransferase (AANAT), the rate-limiting enzyme in melatonin synthesis, have taken on special importance in vertebrate circadian biology. Recent identification of genes encoding two AANAT (AANAT 1 and AANAT 2) and two subtypes of melatonin receptor (Mel-R; Mel 1a and Mel 1b) in several fish species has led to rapid advances in characterizing the physiological roles of melatonin. In the present study, partial cDNAs encoding these four genes were cloned from the eye and brain of chum salmon ( Oncorhynchus keta). Based on the nucleotide sequences, we developed highly sensitive real-time PCR systems for these four mRNAs. The development of daily rhythmicity in AANAT 1, AANAT 2, Mel 1a, and Mel 1b transcript levels was examined in the eye and brain of chum salmon during embryonic and post-embryonic stages (from day −9 to day +180). In a parallel experiment, ocular and brain melatonin levels were measured by radioimmunoassay. Parallelism in developmental changes and in circadian rhythms of AANAT mRNAs and melatonin levels in the eye and the brain supports a hypothesis that the developmental increases of nocturnal melatonin levels results partly from the elevated transcription of AANAT genes. Moreover, abundant expression of AANAT and Mel-R mRNAs in the optic tectum, thalamus, hypothalamus, cerebellum, and eye indicates possible roles of melatonin in visual processing and neuroendocrine regulation, through which melatonin might be involved in migratory behavior of chum salmon.

The phototransduction cascade in the isolated chick pineal gland revisited

It is well established that the isolated chick pineal gland is directly light sensitive and that melatonin synthesis of the gland can be inhibited by exposing the gland to light during scotophase. Since not all the steps of the phototransduction cascade have been clarified to the same extent as in the retina, we have treated isolated chick pineal glands with 90 min of light during scotophase and with drugs that affect key-components of vertebrate phototransduction, i.e., cyclic guanosine monophosphate (cGMP) phosphodiesterase 6 (PDE6), cGMP levels and cGMP-gated calcium channels. The endpoint measured was the activity of the rate-limiting enzyme of melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), which is inhibited by light. The effects on AA-NAT activity of light were negated by addition of dipyridamol and zaprinast, either of which inhibits the light-induced activation of PDE6. The effect of light was also counteracted by the nitric oxide donor sodium nitroprusside and C-type natriuretic peptide, both of which increase cGMP levels, and by the calcium channel agonist Bay K 8644, which prevents the cGMP-decrease-induced closure of cGMP-gated calcium channels. Inhibition of nitric oxide synthase (NOS) by N G-nitro- l-arginine did not influence the inhibitory effect of light, suggesting that the NOS pathway does not play a role. Since the light effect on AA-NAT activity involves both cGMP and cyclic adenosine monophosphate (cAMP) hydrolysis, we have also studied whether the cGMP-inhibited cAMP phosphodiesterase 3 (PDE3) is involved. As the specific PDE3 inhibitor cilostamide is without effect, we assume that the light-induced decrease of cAMP levels does not involve PDE3. These results taken together strongly suggest that the investigated steps of the phototransduction cascade in the isolated chick pineal gland are basically similar to those in the retina.

Of rodents and ungulates and melatonin: creating a uniform code for darkness by different signaling mechanisms.

Melatonin synthesis in the mammalian pineal gland is one of the best investigated output pathways of the circadian clock because it can be readily measured and is tightly regulated by a clearly defined input, the neurotransmitter norepinephrine. In this system, a regulatory scenario was deciphered that is centered around the cyclic AMP pathway but shows peculiar species-specific differences. In rodents, the cyclic AMP-mediated, temporally sequential up-regulation of two transcription factors, the activator CREB (cyclic AMP-responsive element-binding protein) and the inhibitor ICER (inducible cyclic AMP-dependent early repressor), is the core mechanism to determine rhythmic accumulation of the mRNA encoding for the rate-limiting enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase (AA-NAT). Thus, in rodents, the regulation of melatonin synthesis bears an essential transcriptional component, which, however, is flanked by posttranscriptional mechanisms. In contrast, in ungulates, and possibly also in primates, AA-NAT appears to be regulated exclusively on the posttranscriptional level. Here, increasing cyclic AMP levels inhibit the breakdown of constitutively synthesized AA-NAT protein by proteasomal proteolysis, leading to an elevated enzyme activity. Thus, self-restriction of cellular responses, as a reaction to external cues, is accomplished by different mechanisms in pinealocytes of different mammalian species. In such a temporally gated cellular adaptation, transcriptionally active products of clock genes may play a supplementary role. Their recent detection in the endogenously oscillating nonmammalian pineal organ and, notably, also in the slave oscillator of the mammalian pineal gland underlines that the mammalian pineal gland will continue to serve as an excellent model system to understand mechanisms of biological timing.

Expression and role of phosphodiesterase 6 in the chicken pineal gland.

The chicken pineal gland is directly photosensitive, with light causing an inhibition of melatonin synthesis. A possible role of phosphodiesterase 6 (PDE6, the primary effector of retinal phototransduction) in mediating this response was investigated. RT-PCR, DNA sequencing and northern blots revealed the presence of RNA encoding both catalytic and regulatory subunits of PDE6 in the chicken pineal gland. Both rod and cone forms of PDE6 subunits mRNA were detected. The concentration of the transcripts encoding PDE6 catalytic subunits peaked at night. Western blot analysis of chicken pineal proteins with an antibody directed against the catalytic subunits of bovine rod PDE6 identified a single immunoreactive protein of 97 kDa. Anion exchange chromatography of chicken pineal soluble proteins revealed a peak of PDE6 activity that accounted for about 30% of cyclic GMP-hydrolysis. In cultured chick pineal glands, arylalkylamine N-acetyltransferase (AA-NAT), the rate-limiting enzyme of melatonin synthesis, was protected from inhibition by light when selective PDE5/6 inhibitors (zaprinast, DMPPO) were added to the culture medium. PDE5/6 inhibitors did not affect AA-NAT activity in the dark. In contrast, a general PDE inhibitor (IBMX) increased AA-NAT in a light-independent manner. Together, the data indicate that rod and cone forms of PDE6 are expressed in chick pineal cells and that this enzyme plays a role in the inhibition of melatonin synthesis by light.