HIOMT Activity Research Articles

Hydroxyindole-O-methyltransferase (HIOMT) activity in the retina of melatonin-proficient mice

Numerous pieces of evidence support the expression by the mammalian retina of Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4), the enzyme directly responsible for the biosynthesis of the pineal chronobiotic hormone melatonin (MLT). However, conflicting results obtained so far by enzyme-kinetic and immune-detection techniques still make HIOMT presence and relevance in the eye a matter of debate. This work aimed at evaluating unambiguously HIOMT activity in the mouse retina, a valuable model for studying the effects of MLT variations on ocular pathophysiology. Since laboratory mouse strains can bear genetic polymorphisms yielding defective enzymes of MLT biosynthesis, retinas and control pineal glands used in this study were obtained in a MLT-proficient crossing of A/J mice, the A/J/C57BL/10 strain. To improve the radiochemical reference assay, we tested different homogenization procedures coupled with HPLC detection. Concomitantly, we quantified MLT, and its precursor N-acetyl-serotonin (NAS) by HPLC coupled to electrochemical detection in retinas isolated from either light- or dark-adapted mice.Results showed that the standard radio-chemical assay was successful for pineal HIOMT only, whereas specific homogenization buffers and HPLC were required to detect retinal activity, presumably due to interfering methyl-transferases inhibited by NAS. Under present conditions, retinal HIOMT Vmax accounted for by ≈ 40 fmol/h/mg protein, 2.6-hundreds-fold lower than the pineal counterpart, displaying equivalent KMs (≈10 μM). Moreover, NAS and MLT rapidly decreased in light-exposed isolated retinas, corroborating light-sensitive in-situ MLT formation.Conclusively, we measured mouse retinal HIOMT kinetics under basal conditions, a useful result to elucidate the regulatory patterns, the possible impact on eye health, and therapeutic approaches related to this enzyme.

Study on regulatory effect of Kaixin San on endogenous melatonin biosynthesis in rat depression model

To study the effect of Kaixin San on the rate-limiting enzyme in biosynthesis of melatonin (MT) and pineal body in rat depression model. The unpredictable chronic mild stress was used to establish the rat depression model for 21 days. The rats were divided into the normal control group, the model group, Kaixin San low, medium and high dose groups (KXS 65, 130, 260 mg x kg x d(-1)) and the trazodone group. All groups were administered at 30 min after modeling each day. Rats were sacrificed and the pineal glands were isolated immediately after acquisition tail venous blood at 2:00a. m on the 22nd day. The plasma was analyzed for melatonin content by using a rat metabolic panel Milliplex kit. The pineal glands were analyzed for AANAT and HIOMT mRNA levels by Real-time quantitative PCR and for AANAT and HIOMT activity by a radiometric assay simultaneously. The plasma MT concentration, expression of AANT and HIOMT mRNA, activity of AANAT in rat pineal glands of the model group were significantly lower than the control group (P < 0.05), but the activity of HIOMT showed not change. Compared with the model group, all of Kaixin San groups showed increase in MT concentration in plasma (P <0. 05) , with the medium dose group revealing the highest level. Besides, the medium dose group displayed significant increase in AANAT, HIOMT mRNA level and AANAT activity (P < 0.05), but no increase in HIOMT activity. Kaixin San can regulate AANAT activity of pineal bodyand regulate MT biosynthesis in rat depression model.

Blocking of melatonin synthesis and MT(1) receptor impairs the activation of Jurkat T cells.

Melatonin has been proposed as regulating the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. To further investigate the melatonin's role in IL-2/IL-2R system, we established an inducible T-REx expression system in Jurkat cells in which the protein levels of HIOMT enzyme or MT(1) receptor were significantly down-regulated upon tetracycline incubation. We found that T-REx Jurkat cells with lower levels of HIOMT activity, and consequently lower content of endogenous melatonin, showed IL-2 production decrease after activation with lectin. Likewise, tetracycline-inducible stable cell line expressing MT(1) antisense produced decreased amounts of IL-2 (mRNA and protein levels) after stimulation. Moreover, in T-Rex-MT(1) cells incubated with tetracycline, a sub-optimal PHA dose failed to induce the early activation marker CD25 on the cell surface. The results shown here support the relevance of endogenous melatonin and its signaling in T cell activation.

Daily rhythms of norepinephrine, 𝛃1-adrenoceptor mRNA, serotonin, arylalkylamine N-acetyltransferase mRNA, arylalkylamine N-acetyltransferase and hydroxyindol-O-methyltransferase activities, and melatonin in the pineal gland of viscacha

We described the daily rhythms of parameters involved in melatonin production in the pineal of viscacha. Norepinephrine showed the highest levels at night. A peak in β1-adrenoceptor mRNA was observed at the end of the light phase. Serotonin exhibited higher values during the light period. AA-NAT mRNA showed the highest expression at Zeitgeber time 0 (ZT0), reaching a minimum at night. AA-NAT activity and melatonin content exhibited a nocturnal peak at ZT16, a lower diurnal increase during the light period and minimal level at ZT8. HIOMT activity showed a peak at ZT16. The daily pattern in norepinephrine content was similar to that of AA-NAT activity and melatonin levels. Our data suggest that the melatonin synthesis in viscacha might be activated via adrenergic induction; the differences observed in the daily rhythms of AA-NAT activity and its mRNA suggest that post-transcriptional mechanisms may be involved, and other Zeitgeber and other mechanisms might also control melatonin production.

Red light suppresses N-acetylserotonin but does not elevate 5-HT and 5-HIAA of the rat pineal glands at night.

HPLC with EC detector was used to study the red light effect on the rhythmicity of catecholamines (NE, DOPAC and dopamine) and indoleamines (NAS, 5-HT, 5-HIAA) in the pineal glands and retinas of the Sprague-Dawley rat. We found the prominent nocturnal elevations of NAS in rat pineal glands and the suppression of the NAS but not the elevation of 5-HT and 5-HIAA after red light exposure at night. Our data show two findings: metabolic pathway of the indoleamines (5-HT, 5-HIAA and NAS) after red light exposure at night may be different from those by white light. Second, the increased HIOMT activity that effects on 5-HT and 5-HIAA may also be involved in the indoleamine formation after red light exposure at night.

Hydroxyindole- O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl- l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole- O-methyltransferase (HIOMT; S-adenosyl- l-methionine: N-acetylserotonin- O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin ( N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl- l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

RNA synthesis inhibitors increase melatonin production in Y79 human retinoblastoma cells

Y79 human retinoblastoma cells synthesize melatonin in cell culture thus providing a unique preparation for studying the regulation of melatonin biosynthesis in mammalian retinas. We have previously demonstrated that Y79 cells express NAT and HIOMT activity and produce melatonin in a cAMP- and protein synthesis-dependent manner by increasing NAT, and not HIOMT activity, as has been demonstrated in other retinal and pineal melatonin synthesizing systems. We have extended these studies to investigate the role of RNA synthesis in melatonin regulation, and report here that RNA synthesis inhibitors do not suppress melatonin production in Y79 retinoblastoma cells. Rather, at intermediate concentrations, the inhibitors actinomycin D and camptothecin increase melatonin levels. Camptothecin, a topoisomerase I inhibitor, also increased NAT activity and accumulated cAMP levels in a calcium-dependent manner. This effect on cAMP did not appear to occur through phosphodiesterase, and other regulators of retinal melatonin such as melatonin degradation or components of the dopamine system were unaffected. These results are in contrast with the suppression of melatonin synthesis by RNA synthesis inhibitors observed in rat and chick pineal glands and in chick retinas.

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.

The effects of benzodiazepines on basal and isoproterenol‐stimulated N‐acetyltransferase activity by the rat pineal gland, in vivo and in vitro

The present study examined the effects of "peripheral," "central," and "mixed" benzodiazepine agonists and antagonists on the nocturnal rise in rat pineal NAT activity in vivo and the isoproterenol-stimulated NAT activity of pineals in organ culture. Administration of the central agonist clonazepam or the mixed agonist-antagonist diazepam, 4 hr after dark, at a dose of 25 mg/kg each, inhibited nocturnally elevated NAT 20 min later, while this same dose of the peripheral agonist RO 54864 elevated NAT activity. In a second study these agents were administered in vivo 1 hr before dark, at a dose of 3, 10, or 25 mg/kg i.p. and tested 4 hr after dark for in vitro rat pineal NAT activity. None of these agents affected NAT activity at the 3- or 10-mg/kg dose, but RO 54864 25 mg/kg did induce elevated activity. In a third study, all of these agents prolonged the time period for NAT induction by isoproterenol in rat pineals cultured for 48 hr before stimulation. The data suggests that benzodiazepine stimulation of NAT activity in vitro is not specific to "central" or "peripheral" benzodiazepine receptors and that inhibition of melatonin production in vivo occurs either at some step before NAT induction or is involved with the inhibition of pineal HIOMT activity.

Hydroxyindole- O-methyltransferase in rat retinal bipolar cells: persistence following photoreceptor destruction

The presence ofhydroxyindole- O-methyltransferase (HIOMT) activity and localization of HIOMT immunoreactivity was examined in albino rat retinas following photoreceptor destruction. Male Sprague-Dawley rats were exposed to high intensity flourescent light for 4 consecutive days, then placed on a 14:10 h light:dark cycle for two weeks to allow for phagocytic removal of damaged cells from the retina. Histologic examination revealed almost complete destruction and removal of all photoreceptors. The damaged retinas exhibited an increase in HIOMT activity relative to controls, when expressed as activity per mg of protein. HIOMT activity in the pineal glands was not affected. When control and light damaged retinas were examined for HIOMT localization by immunocytochemistry, the control retinas displayed intense HIOMT immunoreactivity in all photoreceptors, and a somewhat lighter labeling in a population of bipolar cells, whereas the light damaged retinas (lacking photoreceptors) showed intense HIOMT immunoreactivity in bipolar cells. These results suggest that the increase in HIOMT activity following photoreceptor destruction is due to increased synthesis of this enzyme in a population of bipolar cells. Thse HIOMT-immunoreactive bipolar cells may perhaps respond in a compensatory manner to changing levels of melatonin in the retina.

Melatonin in the lacrimal gland: First demonstration and experimental manipulation

NAT, HIOMT and melatonin are described in the extra-orbital lacrimal glands. The extra-orbital lacrimal glands of female Syrian hamsters contain higher NAT activity and melatonin levels than those in male glands, while male glands have higher HIOMT activity. Castration did not change melatonin in the lacrimal glands, although NAT and HIOMT activities were altered. The exposure of female hamsters to light in the morning (0600h) was associated with a reduction in both NAT activity and melatonin levels. Porphyrins were not detected in the lacrimal glands of either male or female hamsters.

Pregnancy outcome in heat-exposed hamsters; the involvement of the pineal.

The effect of high ambient temperature (34 degrees C) on the function of the female reproductive system, on embryonic development and on outcome of pregnancy, was investigated in heat-exposed sham-operated (Sh) and pinealectomized (Px) golden hamsters maintained under short photoperiod. Plasma prolactin levels were reduced in both heat-exposed groups (ShH and PxH) but pituitary prolactin was increased in the pinealectomized groups irrespective of ambient temperature (21 or 34 degrees C). Pituitary weights and LH contents were not affected in any test group. Heat exposure brought about a reduction in the number of corpora lutea and of pups born, the latter being more drastically reduced in absence of the pineal; the depressant effect of heat on ovarian weight was evident only in the pinealectomized animals. Progesterone levels were not affected in any test group and pregnancy was not prolonged, thus, it would seem that pregnant hamsters adapt themselves well to heat. Moreover, high ambient temperature promoted a rise in pineal. HIOMT activity and boosted cortisol levels in presence of the pineal gland only, which, together with the above findings, shows that the pineal can provide protection for pregnant hamsters against adverse effects of high ambient temperature.

Harderian gland N-acetyltransferase activity in the male Syrian hamster: effects of gonadectomy, short photoperiod exposure, or subcutaneous melatonin implants.

The activities of NAT and HIOMT and the melatonin concentration in the Harderian glands of intact, gonadectomized, and gonadally-regressed male Syrian hamsters were studied. To produce gonadal regression, hamsters were exposed to either artificial or naturally short photoperiods. NAT activity of castrated and gonadally-regressed hamsters was always less in comparison to that of animals with intact gonads. Castrated hamsters exposed to long days showed higher NAT activity than that of castrated animals exposed to short photoperiods indicating that light may have some influence on Harderian NAT independent of the gonadal status. Also, gonadally-regressed hamsters exposed to long photoperiods exhibited higher NAT activity in comparison to gonadally-regressed animals exposed to short days. The HIOMT activity and melatonin content of Harderian glands in all these groups of male Syrian hamster were very low.

Pineal indole metabolism in the mouse

The activity of two pineal enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole- O-methyltransferase, (HIOMT) and the pineal content of serotonin (5-HT) and N-acetylserotonin (NAS) were measured in several strains of mice ( Mus domesticus) in order to compare melatonin synthetic pathways among them. Of the strains we examined, C57BL/6J, AKR/J, BALB/c, NZB/BLNJ and wild mice, only wild mice synthesize pineal melatonin. Wild mice had high activity of both SNAT and HIOMT; NZB had SNAT activity but did not have HIOMT; the three other strains had neither SNAT nor HIOMT; the activity of SNAT in wild mice showed a clear daily rhythm but HIOMT activity did not show any significant daily changes. The pineal content of 5-HT in wild and NZB mice was higher during the day than during the night, however in C57BL and AKR, 5-HT levels were higher during the night than during the day. As expected NAS was found in wild and NZB mice.

N-Acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin levels in the Harderian glands of the female Syrian hamster: Changes during the light: Dark cycle and the effect of 6-parachlorophenylalanine administration

The activities of NAT and HIOMT and the melatonin content of the Harderian glands of female Syrian hamsters were studied. When hamsters were kept under a light:dark cycle of 14:10 (lights on at 06.00 h), NAT activity exhibited a sharp, short term rise at one hour after lights on. Simultaneously, the activity of HIOMT, which forms melatonin, exhibited a rapid decline. Melatonin levels, like HIOMT activity, also showed a precipitous drop at one hour after light onset. After the respective changes, both NAT and HIOMT activity reverted back to night time levels. Melatonin levels remained depressed for several hours but by 1400 h (8 hours after lights on), nighttime melatonin values were re-established. Treatment of female hamsters with PCPA, a trytophan hydroxylase inhibitor, led to depressed levels of Harderian melatonin without affecting the activities of either NAT or HIOMT.

Changes in the circadian rhythmicity of N- and O-acetyltransferase activities in the pineal gland of 38 day old male wistar rats when examined under white, red and green light.

N-acetyltransferase activity was quantified in the pineal gland of 38 day-old male Wistar rats under different experimental conditions. Under daily white light/dark photoperiods (12 L : 12 D), N-acetyltransferase activity showed a peak of activity at midnight for the synthesis of both N-acetylserotonin and melatonin. Using red light instead of white light a shift of the enzyme activity involved in the production of both compounds towards the preceding light period was observed. Using green light instead of white light the enzyme activity shifted to later periods. Under white light/dark conditions pineal O-acetyltransferase activity when quantified was maximal during the early hours of the night. With periodic red light this maximal activity was extended over a longer period including late hours of the preceding day and later hours of the night. Periodic illumination with green light caused a peak of activity at midnight and an increase of activity at then end of the dark period. A possible relationship between previous results obtained on the influence of pterins on HIOMT activity and the effects of light on reproduction is discussed.

A thin-layer chromatographic assay for measuring pineal hydroxyindole-O-methyltransferase activity

A thin-layer chromatographic procedure for measuring pineal HIOMT activity is described, based on the methylation of NAS to melatonin. The method enables simple and accurate determination in small aliquots from a single pineal organ homogenate.

Effect of 6-hydroxydopamine on pineal norepinephrine content and enzyme activity in the cyclic female rat.

The response of two pineal enzymes, serotonin N-acetyltransferase (SNAT; E.C.2.3.1.5) and hydroxyindole-O-methyltransferase (HIOMT; E.C.2.1.1.4) to the reduction of pineal norepinephrine (NE) content produced by systemic injections of 6-hydroxydopamine (6-OHDA) was examined in the present study twice daily, at 1200 and 2400 h, in the cyclic female rat. In rats sacrificed 3 days postinjection during the dark period (2400 h), injections of 6-OHDA that produced maximal inhibition of SNAT activity (58% of control values) also caused significant decreases in NE content in pineal, hypothalamic and cardiac tissues (48, 75 and 12% of control values, respectively). In rats sacrificed 17 days postinjection at 2400 h, SNAT activity and pineal NE content had returned to control levels, while hypothalamic and cardiac NE content remained significantly depressed (70 and 22% of control levels, respectively). These results confirm the role of NE in the induction of SNAT activity and reveal that 6-OHDA is more effective in decreasing cardiac than pineal NE content. In a second experiment, HIOMT activity was measured throughout the estrous cycle in both treated and control animals. Reduction of hypothalamic NE content by 6-OHDA did not block ovulation or disrupt the estrous cycle, although the initial cycle was prolonged by 1 day in some animals. Although average HIOMT activity during the estrous cycle in 6-OHDA treated animals did not differ from control levels, the amplitude of the circadian alteration in HIOMT activity as well as the relationship of this pineal enzyme to the estrous cycle was altered in animals receiving the drug. These findings suggest that the nocturnal rise in HIOMT activity and the relationship of this pineal enzyme to the estrous cycle are regulated, in part, by a NE sensitive component in either the pineal gland, hypothalamus or both.

Day and night rhythms in the methylation of N-acetylserotonin/5-hydroxytryptophol in the pineal gland of males rats of different ages.

Next to a night rhythm of the methylation of N-acetylserotonin/5-hydroxytryptophol the presence of a daytime rhythm could also be established. Rhythmicity was studied in May during the night and in June during daytime in 21, 42 and 70 days old male Wistar rats. In 21 days old rats, moderate HIOMT activity was observed from 12 p.m.--4 a.m. In rats aged 42 and 70 days HIOMT activity was increased showing a peak at 4 a.m. In September this night maximum is observed at 12 p.m. in rats, aged 42 days. This points to the presence of a seasonal change in HIOMT rhythmicity during the night. During daytime a moderate HIOMT activity is present, which reaches a maximum at 2 p.m. in the 21 and 42 day old animals, while in the adult 70 days old rats activity starts to increase at 2 p.m. probably reaching a maximum at 6 p.m.

Circadian rhythmicity in the methylation of 5-hydroxy-indoles and norepinephrine in the pineal gland of 10 day old rats

In pineals of 10 day old rats 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, N-acetylserotonin/5-hydroxytryptophol and norepinephrine are methylated following a circadian rhythm. During the night HIOMT and COMT activities were measured for the above mentioned substrates, while HIOMT activity for 5-hydroxytryptophan and N-acetylserotonin/5-hydroxytryptophol was also determined during daytime.