Cyclic AMP increases hydroxyindole-O-methyltransferase mRNA levels in the chicken pineal gland, but is not required for circadian rhythmicity of this transcript.

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In the chicken pineal gland, HIOMT mRNA concentration exhibits a circadian rhythm with a threefold peak at midday. The present study sought to evaluate the possible role of cyclic AMP in this transcriptional rhythm. In cultured pineal glands from 4-day-old chicks, cyclic AMP analogs and the adenylate cyclase activator, forskolin, increased HIOMT mRNA levels twofold to threefold in a dose-dependent manner. Vasoactive intestinal polypeptide increased HIOMT mRNA levels by 50%. Actinomycin-D chase experiments indicated that cyclic AMP did not affect the stability of HIOMT mRNA, thus providing indirect evidence that the effect of cyclic AMP was exerted at transcriptional level. In cultured pineal glands from 11 days embryos, HIOMT mRNA levels failed to respond to cyclic AMP. However, a daily rhythm of HIOMT mRNA, with an endogenous component in constant darkness was clearly observed at this developmental stage. This observation indicates that cyclic AMP is not required for circadian rhythmicity of HIOMT gene transcription.

Melatonin synthesis pathway: circadian regulation of the genes encoding the key enzymes in the chicken pineal gland and retina.

The mRNAs encoding three enzymes of the melatonin synthesis pathway (tryptophan hydroxylase (TPH), arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyl-transferase (HIOMT)) are expressed with a day/night rhythm in the chicken pineal gland and retina. TPH and AANAT mRNA levels reach their peak at night. HIOMT mRNA levels peak at night in the retina, but during the day in the pineal gland. In this tissue, the rhythm of TPH, AANAT and HIOMT mRNA levels persisted in constant darkness (DD), both in vivo and in vitro, indicating that the three genes are controlled by the circadian oscillator of the chicken pineal. In the retina, the rhythms of TPH and AANAT mRNA levels also persisted in DD in vivo, suggesting that they are driven by a circadian oscillator. In contrast, the rhythm of HIOMT mRNA in the retina appeared to be controlled only by light. The clones of chicken AANAT and HIOMT genes that we have isolated should help us to understand the molecular mechanisms of: 1) their transcriptional regulation by circadian oscillators and by light; 2) their tissue-specific expression in the pineal gland and the retina.

Circadian regulation of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland in vivo and in vitro.

The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.

Hydroxyindole-O-methyltransferase in the chicken retina: immunocytochemical localization and daily rhythm of mRNA.

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5-fold increase during the night. Light strongly suppressed the night-time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT-positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

Hydroxyindole-O-methyltransferase gene expression in the pineal gland of chicken embryo: development of messenger RNA levels and regulation by serum.

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controlled [correction of controled] by serum factors.

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.

Regulation of hydroxyindole-O-methyltransferase gene expression in the pineal gland and retina.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin biosynthesis and appears to be specifically expressed in the pineal gland and in the retina. This review deals with the regulation of HIOMT by environmental light and with the developmental aspects of HIOMT expression in chicken and rat. Early studies based on HIOMT activity measurements and more recent studies involving cDNA hybridization to HIOMT mRNA are taken into consideration. Together, the data reveal that long term regulation of HIOMT by light would rely on a day/night rhythm of HIOMT gene transcription, coupled to a slow turnover of the protein. Rapid changes in HIOMT mRNA levels and early expression during embryonic development suggest that further studies on this gene may shed light on the molecular mechanisms involved in the differentiation of the melatoninergic function and in its regulation by light, both in the pineal gland and in the retina.

Cellular localization of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland.

Hydroxyindole-O-methyltransferase (HIOMT), is the last enzyme of the melatonin biosynthesis pathway. We have applied immunocytochemistry and in situ hybridization to localize HIOMT producing cells, in the chicken epithalamus including the pineal gland. The selectivity of the hybridization signal over melatoninergic cells was ascertained by comparison with the immunolabelling. HIOMT transcripts could be observed in the two cell layers of the pineal follicles and in the pineal stalk, but not in other epithalamic regions. These results indicate that pineal modified photoreceptors and parafollicular pinealocyte-like cells represent the two populations of melatonin-producing cells of the chicken epithalamus.

Transcriptional regulation of hydroxyindole O-methyltransferase in the chicken pineal gland: day/night changes and long-term effects of light and darkness.

The indolic hormone melatonin is produced by the pineal gland according to a daily rhythm. The terminal step of melatonin synthesis is catalysed by hydroxyindole O-methyltransferase (HIOMT, EC 2.1.1.4). Adaptation to constant light or darkness modifies HIOMT activity and concentration. Using a cDNA probe encoding HIOMT, we investigated the effect of environmental lighting on HIOMT gene expression in the chicken pineal gland. HIOMT mRNA levels increased by 100% in constant light as compared with constant darkness. In addition, the present study disclosed the existence of a day/night rhythm of HIOMT gene transcription, with 3-fold higher mRNA levels at midday than at midnight. This transcriptional rhythm was not accompanied by day/night changes in HIOMT concentration, probably due to a slow turnover of this protein. Unexpected darkness did not prevent the daytime rise in HIOMT mRNA levels, whereas unexpected light prevented the night-time fall in HIOMT mRNA levels. Together, the data would suggest that the day/night rhythm of HIOMT gene transcription in the chicken pineal gland involves both a response to light and the activity of a biological oscillator.

[Transcriptional regulation of an enzyme involved in the synthesis of melatonin]. / Régulation transcriptionnelle d'une enzyme impliquée dans la synthèse de mélatonine.

The indolic hormone melatonin is produced by the pineal gland according to a daily rhythm. The terminal step of melatonin synthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). Adaptation to constant light or darkness modifies HIOMT activity and concentration. Using a cDNA probe encoding HIOMT, we investigated the effect of environmental lighting on HIOMT gene expression in the chicken pineal gland. HIOMT mRNA levels increased by 100% in constant light as compared with constant darkness. In addition, the present study disclosed the existence of a day/night rhythm of HIOMT gene transcription, with 3-fold higher mRNA levels at midday than at midnight. Unexpected darkness did not prevent the daytime rise in HIOMT mRNA levels, whereas unexpected light prevented the night-time fall in HIOMT mRNA levels. Together, the data would suggest that the day/night rhythm of HIOMT gene transcription in the chicken pineal gland involves both a response to light and the activity of a biological oscillator.

Long-term effects of constant light or darkness on chicken pineal hydroxyindole-O-methyltransferase expression: biochemical and cellular aspects.

1. Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway. 2. Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ. 3. Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.

Molecular cloning and nucleotide sequence of a cDNA encoding hydroxyindole O-methyltransferase from chicken pineal gland.

Hydroxyindole O-methyltransferase (EC 2.1.1.4) is the enzyme that catalyses the synthesis of melatonin in the pineal gland and in the retina. Polyadenylated RNA from chicken pineal glands was used to prepare a cDNA library in lambda gt11. The library was screened with an antiserum directed against chicken hydroxyindole O-methyltransferase, and one cDNA clone was isolated. The fusion protein expressed by phage lysogens was identified on Western blots as a 165 kDA immunoreactive protein (beta-galactosidase, 110 kDa; hydroxyindole O-methyltransferase, 38 kDa). The fusion protein exhibited hydroxyindole O-methyltransferase activity. Its Km values for N-acetyl-5-hydroxytryptamine and S-adenosylmethionine were 5 times those of the natural enzyme. The intrinsic activity of the fusion protein was approx. 0.25% that of the natural enzyme. The cDNA consisted of 1436 nucleotides, including a 1038-nucleotide sequence encoding a full-length 346-amino-acid hydroxyindole O-methyltransferase. Comparison with bovine hydroxyindole O-methyltransferase [Ishida, Obinata & Deguchi (1987) J. Biol. Chem. 262, 2895-2899] revealed 52% identity in nucleotide sequences and 44% identity in peptide sequences. Northern-blot analysis revealed the presence of hydroxyindole O-methyltransferase mRNA transcripts in chicken pineal gland and retina, but not in the telencephalon.

Molecular and cellular aspects of hydroxyindole-O-methyltransferase expression in the developing chick pineal gland.

The pineal gland influences circadian activity and seasonal breeding through the production of an indolic hormone, melatonin. The terminal step of melatonin biosynthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Using an antibody directed against HIOMT, we examined the differentiation of the melatoninergic phenotype in the developing chick pineal gland. HIOMT first appeared 4 days before hatch and rose linearly until the 7th day posthatch. This was correlated with an increased immunoreactivity of the 38 kDa enzyme on Western blots and with an accelerated rate of HIOMT biosynthesis as demonstrated by [35S]methionine labeling. Immunocytochemistry revealed a growing number of HIOMT-positive cells between day 2 before hatch and day 15 posthatch. Until hatching HIOMT was expressed almost exclusively in modified photoreceptors. Parafollicular pinealocytes became HIOMT-positive mostly after hatching. Their different timings of functional differentiation emphasize the existence of two populations of melatonin-producing cells in the chick pineal gland.

An antiserum against chicken hydroxyindole-O-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors.

Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole-O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4-5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.

5'-Nucleotidase activity in the pineal organ of the pike. An electron-microscopic study.

To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5'-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5'-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear "synaptic" vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.

Synthesis of melatonin by the pineal modified photoreceptors of birds immunocytochemical localization of hydroxyindole-O-methyltransferase.

Photoperiodic control of several biological rhythms is exerted through the inhibitory effect of light on melatonin synthesis in the pineal organ. Hydroxyindole-O-methyltransferase (HIO-MT), the last acting-enzyme in melatonin biosynthesis, constitutes a specific marker of melatoninergic cells. In the present study, an antibody directed against chicken HIOMT was affinity-purified and used to identify melatoninergic cells in the pineal organ of chicken, quail, sparrow and blackbird. Regardless of the species, intense immunocytochemical reactions were observed in modified photoreceptors, whereas other cellular constituents (mostly glial cells) remained unlabeled. We conclude that modified photoreceptors synthesize melatonin in the avian pineal gland and are thus accountable for the translation of the photoperiodic input into hormonal output.

Rhythmic melatonin biosynthesis in a photoreceptive pineal organ: a study in the pike.

Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase, involved in the biosynthesis of melatonin from serotonin, were assessed over 24 h in the pineal organ of pikes (Esox lucius, L.; teleosts) entrained to natural (winter) environmental conditions. Only NAT activity exhibited daily changes, rising at the onset of darkness and resuming low values shortly before the end of the scotophase. The rhythm was damped under constant darkness, lower and higher values being shifted towards intermediate ones. NAT activity was lowered under constant light; however, a significant increase was seen at the end of the subjective night, suggesting the existence of a low-amplitude rhythm. Illumination of the pikes at the middle of the scotophase induced inactivation of roughly half of the maximal NAT activity, possibly indicating the existence of one photolabile and one photostable enzymatic component. Under natural conditions, radioassayable melatonin of the pike pineal organ displayed daily variations which paralleled those of NAT activity. Melatonin production thus appears to reflect the daily changes of NAT, synchronized to the light/dark cycle.

Pineal-retinal molecular relationships: distribution of "S-antigen" in the pineal complex.

The immunocytochemical localization of S-antigen, a specific protein first discovered in retinal photoreceptors, was studied in the pineal complex of vertebrates (eel, pike, frog, lizard, passerines, mouse, hamster) using monoclonal antibody immunofluorescence. S-antigen immunoreactivity was demonstrated concurrently in retinal photoreceptors and in most pineal phototransducers of all species, i.e. in pineal cells of the receptor series (cone-like, modified photoreceptor cells, pinealocytes) and in cone-like photoreceptors of the frog frontal organ and lizard parietal eye. The labelling was distributed either in all compartments of these cells, or restricted to outer segments. The functional significance of the S-antigen as well as some phylogenetic and ontogenic implication of this marker are discussed.

Indoles in the photoreceptor cells of the lamprey pineal complex.

In the pineal complex of the brook lamprey, the hydroxyindole-0-methyltransferase (involved in the biosynthesis of 5-methoxyindoles) displays a daily rhythm of activity with a nocturnal peak, during a light/dark cycle and in constant darkness. Immunoreactive indoles were found in both varieties of photoreceptor cells (P1 and P2): serotonin and N-acetylserotonin-like substances in P2 and a melatonin-like compound in P1. It is suggested that P1 and P2 are involved in indole metabolism.