Adrenergic and peptidergic regulations of hydroxyindole-O-methyltransferase activity in rat pineal gland (original) (raw)
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Developmental Brain Research, 1998
. Postnatal development of hydroxyindole-O-methyltransferase HIOMT mRNA expression, HIOMT activity and melatonin content Ž . was investigated in the rat pineal gland from birth to adulthood 62-day old . For each age, animals were sacrificed at two different time-points: midday and midnight. HIOMT mRNA was first detectable one day after birth and maximal diurnal levels were reached at the end of the first week. A 2-fold nocturnal increase appeared significantly 11 days after birth. HIOMT activity was detectable from 5 days of life and significant dayrnight variations did not appear before 21 days after birth. Appearance of melatonin synthesis and rhythm followed that of HIOMT mRNA. The 10-day delay observed between the appearance of the nocturnal increase in HIOMT activity and expression is discussed in term of differential regulation of both HIOMT mRNA expression and HIOMT activity. q 1998 Elsevier Science B.V. All rights reserved.
Ž. Hydroxyindole-O-methyltransferase HIOMT catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have Ž. Ž. Ž. established 1 that one of these neuropeptides, neuropeptide Y NPY , stimulates specifically HIOMT activity in rat pinealocytes and 2 Ž. Ž. that the density of the NPY-immunoreactive NPY-IR fibers innervating the pineal gland of the European hamster Cricetus cricetus displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryp-Ž. Ž. tophol 5-ML content and of circulating MEL levels in European hamsters when NPYergic innervation is low end of October and when Ž. it is the highest mid-December. We report in this study that HIOMT activity is significantly increased by 80% in mid-December Ž compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels with a. fourfold increase occurring in early dawn and decreasing slowly towards the end of the day. These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity. q
Brain Research, 1998
. Hydroxyindole-O-methyltransferase HIOMT catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have Ž . Ž . Ž . established 1 that one of these neuropeptides, neuropeptide Y NPY , stimulates specifically HIOMT activity in rat pinealocytes and 2 Ž . Ž . that the density of the NPY-immunoreactive NPY-IR fibers innervating the pineal gland of the European hamster Cricetus cricetus displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryp-Ž . Ž . tophol 5-ML content and of circulating MEL levels in European hamsters when NPYergic innervation is low end of October and when Ž . it is the highest mid-December . We report in this study that HIOMT activity is significantly increased by 80% in mid-December Ž compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels with a . fourfold increase occurring in early dawn and decreasing slowly towards the end of the day . These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity. q
Journal of Neurochemistry, 1980
Rat pineal hydroxyindole-0-methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N-methyltransferase. Sadenosylmethionine (SAM), the in v i w cofactor utilized by the enzyme to convert N-acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in i>iiw studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole-0-methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. I n llitro proteolytic studies further demonstrate that, although N-acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.
DNA and Cell Biology, 1993
Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of the pineal hormone melatonin. In this study, an HIOMT clone was isolated from a human pineal cDNA library using synthetic oligonucleotide probes based on the bovine HIOMT sequence. The human sequence is unusual because it contains a 3' fragment (84 bp) of the LINE-1 sequence, a highly repetitive sequence in the human genome and the genome of some primates and rodents. Exclusive of this LINE-1 fragment, the human HIOMT clone is 75% and 63% homologous to bovine and avian HIOMT sequences, respectively. The deduced amino acid sequence of the human cDNA clone encodes a 41.6-kD protein. In addition, the sequence is 70% and 57% identical and 81% and 73% similar to bovine and avian HIOMT, respectively. In agreement with the results of earlier studies, it was found that vertebrate HIOMT amino acid sequences are not homologous to any other vertebrate proteins, including several methyltransferases. However, HIOMT exhibits homology with a plant ('-methyl transí erase and an internal 120-am i no-acid region is approximately 35% identical to a region of four bacterial O-methyltransferases. The results of PCR and Southern blot analysis indicate that three species of HIOMT mRNA are typically present in the human pineal gland, only one of which contains the LINE-1 fragment. An antiserum was raised against a mixture of three synthetic peptides, corresponding to three regions of the deduced amino acid sequence of human HIOMT. This antiserum detected a single immunoreactive protein in Western blot analysis of human pineal glands. The size of the protein (-42 kD) is identical to that predicted from the HIOMT clone, including the LINE-1 fragment. The human HIOMT sequence should be useful in further studies of this enzyme and will also be of special importance in evaluating the functional significance of the inclusion of a fragment of the LINE-1 in an mRNA.
Circadian regulation of hydroxyindole-O-methyltransferase mRNA levels in rat pineal and retina
Brain Research, 1996
Hydroxyindole-O-methy ltransfcrase (HIOMT, EC 2.1. 1.4) catalyzes the methykttion of acetylserotonin to complete the synthesis of melatonin in the pineal and retina. A complete 1728 nucleotidc cDNA encoding rat pineal HIOMT was isolated, characterized, and used to evaluate day/night levels of H1OMT mRNA. As previously reported for HIOMT enzyme activity, HIOMT mRNA levels were also greater in the pineal than in the retina. Northern blo{ analysis and in situ hybridization were useful for detection of HIOMT mRNA in the pineal but not the retina, whereas the reverse transcriptase-poly rnerase chain reaction or RNaseprotectionassay revealedtranscriptsfor HIOMTboth in the pineal and retina. InvestigatingHIOMTmRNAIcvelsin rat pineal and retina at 6 time-pointsthroughouta 24 h periodrevealedhigherlevelsof HIOMTmessageduringdarkness.The daily fluctuation in HIOMT mRNA persisted in constantdarkness, verifying an endogenous circadian rhythm both in the pined and retina. In mammalian pineals, sympathetic innervation, synthesizing norepinephrine that activates beta ((3) adrcnergic receptors, entrain several circadian bodily functions through the synthesis and release of melatonin. A single injection of the (hdrenergic agonist, isoprotcrenol, induced a dramatic increase of HIOMT mRNA levels in the light-adapted pineal, in vivo. Moreover. a single injection of the~-adrencrgic antagonist, propranolol, prevented the nocturnal increase of pineal HIOMT mRNA. Using a combination of'mc{huds, it has been shown that the level of H1OMT mRNA fluctuates daily in both the pineal gland and retina. This day/nigh[ rhythm can be modula[ed either by (3receptor agonists or antagonists when applied appropriately during the circadian cycle, suggesting that the mRNAchangesin HIOMTmay be controlledat tbe transcriptional level.
Noradrenergic control of the synthesis of two rat pineal proteins
Brain Research, 1990
Pineal physiology is controlled by norepinephrine released from sympathetic nerves terminating in the gland. In the present study, the effect of norepinephrine on the labelling of specific proteins was investigated by incubating glands with [35S]methionine and then resolving the proteins by two-dimensional polyacrylamide gel electrophoresis; the patterns were analyzed by computer-assisted image analysis. The most prominent effects of norepinephrine were distinct and consistent increases in the labelling of two proteins (37 kDa, pI = 6.0, 50 kDa, pI = 6.0), designated adrenergically induced protein (AIP 37/6 and AIP 50/6). In both cases, norepinephrine was effective at low concentrations (EC50 = 10 nM). Pharmacological studies indicated that the effects of norepinephrine on both proteins involved a beta-adrenergic receptor, and that cyclic AMP was the second messenger. Pulse-chase labelling experiments revealed that these effects of norepinephrine did not involve post-translational modification of previously labelled precursor proteins, but depended upon de novo synthesis of protein. An inhibitor of mRNA synthesis, actinomycin-D, was found to block the effect of norepinephrine on AIP 50/6 but not on AIP 37/6, suggesting that norepinephrine acted on AIP 50/6 via a transcriptional mechanism and on AIP 37/6 via a translational mechanism. These in vitro studies were extended into in vivo investigations by measuring silver-stained AIP 37/6 in the two-dimensional gels. Changes in the amount of AIP 37/6 in pineal glands were studied in response to treatments which block the adrenergic stimulation of the gland, including exposure to constant lighting or removal of the superior cervical ganglia. Both treatments reduced AIP 37/6 by 50-75% in 8 weeks. These observations, together with those from in vitro studies, suggest that the amount of AIP 37/6 in the pineal gland is regulated by norepinephrine; and further, that norepinephrine acts through a beta-adrenergic-cyclic AMP mechanism to control AIP 37/6 synthesis at a translational level.
Journal of Pineal Research, 1990
A naturally occurring compound, 6-methoxybenzoxazolinone (6-MBOA), present in grasses, has been shown to induce sexual maturation in a number of rodent species. The structural similarity of 6-MBOA and melatonin has led researchers to suspect that 6-MBOA might induce its progonadal effects by directly altering pineal function. Previous studies have shown that 6-MBOA has the properties of a weak β-adrenergic agonist capable of stimulating rat pineal N-acetyltransferase (NAT) activity at pharmacological concentrations of 10−3 M. In the present study we have examined the effect of 6-MBOA on both the pineal melatonin synthesizing enzymes, namely. NAT and hydroxyindole-O-methyltransferase (HIOMT) as well as on melatonin production, in organ cultured rat pineal glands. In addition, we have also examined the ability of 6-MBOA to displace a ligand from rat brain α-and β-adrenoceptors. Our results confirm that 6-MBOA stimulates NAT activity and melatonin production at the high concentration of 10−3 M. It appears to have no effect on HIOMT activity. A competition study shows that 6-MBOA is able of displacing ligands at the α-and β-adrenoceptors but only at concentrations greater that 10−4 M. Whether such high concentrations of 6-MBOA reach the pineal of rodent in their natural habitat is unknown. However, if 6-MBOA does mediate progonadal effects by altering pineal function it would be expected that 6-MBOA would ultimately inhibit the effects of melatonin. The possibilities are that the high melatonin levels induced by 6-MBOA cause desensitization of melatonin receptors or that 6-MBOA is an antagonist at the level of the melatonin receptor, thus restricting the inhibitory effects of melatonin on the reproductive system.
Molecular Brain Research, 1988
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. fied melatonin-secreting cells by immunocytochemical labeling of HIOMT.