Pineal hydroxyindole-O-methyltransferase Research Articles

Rumen-Protected 5-Hydroxytryptophan Improves Sheep Melatonin Synthesis in the Pineal Gland and Intestinal Tract.

BackgroundBased on the extensive biological effects of melatonin (MLT), it is beneficial to increase the MLT content in the bodies of animals at a specific physiological stage. This study was conducted to investigate the effect of a diet supplemented with rumen-protected (RP) 5-hydroxytryptophan (5-HTP) on the pineal gland and intestinal tract MLT synthesis of sheep.Material/MethodsEighteen Kazakh sheep were assigned randomly to 3 diet groups: control group (CT, corn-soybean meal basal diet), CT+111 group (111 mg/kg BW RP 5-HTP), and CT+222 group (222 mg/kg BW RP 5-HTP). The gene expressions of aromatic amino acid decarboxylase (AADC), arylalkylamine N-acetyltransferase (AA-NAT), hydroxyindole-O-methyltransferase (HIOMT), monoamine oxidase A (MAOA), and the intermediates of MLT synthesis were observed from the pineal gland and intestinal tract by the reverse transcription (RT)-PCR method. The 5-HTP, 5-HT, N-acetylserotonin (NAS), MLT, and 5-hydroxyindole acetic acid (5-HIAA) contents in the pineal gland and intestinal tract were analyzed by ultra-high-performance liquid chromatography–tandem mass spectrometry.ResultsThe study showed that the pineal gland HIOMT expression (P<0.05), MLT (P<0.05) and 5-HIAA (P<0.05) levels in the 222 mg/kg group significantly increased compared to those in the CT and CT+111 mg/kg groups. In addition, the AADC (P<0.01) and AA-NAT (P<0.05) gene expression levels in the duodenum and jejunum were increased by the supplementation of RP 5-HTP.ConclusionsRumen-protected 5-hydroxytryptophan promoted melatonin synthesis in the pineal gland and intestinal tract during the natural light period.

Possible involvement of neuropeptide Y in the seasonal control of hydroxyindole- O-methyltransferase activity in the pineal gland of the European hamster ( Cricetus cricetus)

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 established (1) that one of these neuropeptides, neuropeptide Y (NPY), stimulates specifically HIOMT activity in rat pinealocytes and (2) 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-methoxytryptophol (5-ML) content and of circulating MEL levels in European hamsters when NPYergic innervation is low (end of October) and when it is the highest (mid-December). We report in this study that HIOMT activity is significantly increased by 80% in mid-December 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.

Preliminary Evidence of a Synergistic α1- and β1-Adrenoceptor Regulation of Rat Pineal Hydroxyindole-O-methyltransferase

The activity of serotonin N-acetyltransferase in the rat pineal is regulated through a synergistic action of norepinephrine on α1- and β1-adrenoceptors. Rat pineal hydroxyindole-O-methyltransferase (HIOMT) activity is also responsive to the β1-adrenoceptor, while melatonin synthesis and indoleamine release in certain species is apparently under α1-adrenoceptor control. The present study reports that the simultaneous administration of doxazosin (a selective α1-adrenergic antagonist) and metoprolol (a selective β1-adrenergic antagonist) reduces peak HIOMT activity significantly more than either agent alone, indicating that rat pineal HIOMT is also subject to synergistic α1- and β1-adrenergic regulation. The relationship between α1-adrenoceptor stimulation, serotonin release, and HIOMT activity is considered, following the observation that nocturnal HIOMT activity is elevated in serotonin-depleted pineal glands.

Electric field exposure alters serum melatonin but not pineal melatonin synthesis in male rats.

Sprague-Dawley male rats, maintained in a 14:10 h light:dark cycle were exposed for 30 days (starting at 56 days of age) to a 65 kV/m, 60 Hz electric field or to a sham field for 20 h/day beginning at dark onset. Pineal N-acetyltransferase (NAT), hydroxy-indole-o-methyl transferase (HIOMT), and melatonin as well as serum melatonin were assayed. Preliminary data on unexposed animals indicated that samples obtained 4 h into the dark period would reveal either a phase delay or depression in circadian melatonin synthesis and secretion. Exposure to electric fields for 30 days did not alter the expected nighttime increase in pineal NAT, HIOMT, or melatonin. Serum melatonin levels were also increased at night, but the electric field-exposed animals had lower levels than the sham-exposed animals. Concurrent exposure to red light and the electric field or exposure to the electric field at a different time of the day-night period did not reduce melatonin synthesis. These data do not support the hypothesis that chronic electric field exposure reduces pineal melatonin synthesis in young adult male rats. However, serum melatonin levels were reduced by electric field exposure, suggesting the possibility that degradation or tissue uptake of melatonin is stimulated by exposure to electric fields.

A Distinct Diurnal Rhythm in Hydroxyindole-O-methyltransferase (HIOMT) Activity in the Male Albino Rat

Pineal hydroxyindole-O-methyltransferase (HIOMT) has been reported to show a diurnal rhythm activity in the rat, although this is not fully confirmed. The present study reexamines this issue and reports a distinct diurnal rhythm in rat pineal HIOMT. Peak enzyme activity occurs 5 hr after the onset of darkness, followed by two lesser bursts of activity preceeding each change in the photoperiod. The approximately 18-fold increase in methylating capacity at night represents the greatest variation in HIOMT activity thus far reported in the rat and may be attributed to the age, weight, and housing regimen of the animals used.

Carbaryl-induced changes in indoleamine synthesis in the pineal gland and its effects on nighttime serum melatonin concentrations

The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole- O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion.

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.

Probable mechanism of catalysis of pineal gland hydroxyindole-O-methyltransferase (HIOMT) from rainbow trout (Salmo gairdneri).

Activity of trout pineal HIOMT was found to increase with increase in incubation temperature from 5 to 40 degrees C although the activation energy remained constant over this range. From examination of the effects of the products of HIOMT catalysis on the enzyme it was apparent that the catalytic mechanism was ordered Bi-Bi with S-adenosylmethionine as the obligatory first substrate. Trout HIOMT was found to methylate all the common pineal hydroxyindoles with hydroxytryptophol having the greatest affinity for the enzyme. The pH optimum for trout HIOMT was found to be about pH 9.0 although routine use of a pH of 7.9 is recommended to limit potentially deliterious effects caused by degradation of S-adenosylmethionine at elevated pHs.

Pineal gland hydroxyindole-O-methyl transferase (HIOMT) from rainbow trout (Salmo gairdneri) subject to temperature-dependent substrate inhibition by N-acetylserotonin.

The effect of different incubation temperatures on the activity of trout hydroxyindole-O-methyltransferase (HIOMT) was determined in order to study the potential effects of changes in environmental temperature on production of melatonin. In the study, N-acetylserotonin (NAS) was found to inhibit trout pineal HIOMT, inhibitory concentration decreasing with a decrease in incubation temperature. An increase in incubation temperature was accompanied by an increase in maximal velocity (Vm) and a decrease in affinity of the substrate NAS for the enzyme, while the affinity of the inhibitor NAS remained more or less constant with increase in temperature up to 30 degrees C. It was interesting to note that at subinhibitory concentrations of NAS, possibly concentrations within the normal physiological range, there was a balance between increased velocity and decreased affinity for substrate, with increase in temperature resulting in a constant rate of melatonin production. On the basis of these results it is suggested that substrate inhibition might be involved in modulation of melatonin production and that trout pineal HIOMT can maintain a constant rate of melatonin production over a wide range of temperatures by a combination of compensatory changes in enzyme kinetic parameters.

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.

Day-night differences in the response of the pineal gland to swimming stress.

The effect of swimming stress on pineal N-acetyltransferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content was studied during the day and night in adult male rats. At night, elevated pineal activity was suppressed by light exposure before the animals swam. During the day, swimming for 2 hr did not stimulate NAT activity unless the animals were pretreated with desmethylimipramine (DMI), a norepinephrine uptake blocker. Pineal melatonin content after daytime swimming exhibited a weak rise, unless DMI was injected, in which case melatonin levels showed a highly significant increase. Swimming at night caused a greater (compared to daytime levels) increase in NAT activity in both noninjected and DMI-injected rats. Melatonin levels at night were highly significantly stimulated (compared to daytime values) even without pretreatment of the rats with DMI. The greater response of the rat pineal to swimming stress at night may relate either to an increase in the number of beta-adrenergic receptors in the pinealocyte membrane at night or to a reduced capacity of the sympathetic neurons in the pineal to take up excess circulating catecholamines. Pineal HIOMT activity was not influenced by swimming (with or without DMI) either during the day or at night.

Seasonal changes in pineal melatonin content and hydroxyindole- O-methyltransferase activity in juvenile chinook salmon, Oncorhynchus tshawytscha

Juvenile fall and spring chinook salmon were examined for seasonal changes in pineal hydroxyindole- O-methyltransferase (HIOMT) activity. Numerous small peaks in pineal HIOMT specific activity were found in fall chinook salmon and HIOMT activity per pineal gland increased linearly with age and/or size. In spring chinook salmon, cyclic changes in pineal HIOMT specific activity occurred in midsummer and midwinter, independent of the photoperiod under which the fish were reared. HIOMT activity per pineal gland showed a peak in midwinter. Activity increased with age and/or size of the fish during the following spring. Melatonin content of the pineal gland of spring chinook salmon did not change seasonally in fish reared under a normal photoperiod. Fish reared under constant illumination had about 50% higher pineal melatonin content that those under a normal photoperiod until November. At that time, pineal melatonin content decreased to the same level as that of fish reared under a normal photoperiod.

Regulation of biogenic amine methyltransferases by glucocorticoids via s-adenosylmethionine and its metabolizing enzymes, methionine adenosyltransferase and s-adenosylhomocysteine hydrolase

This report examines the possibility that glucocorticoids control the degradation of adrenal phenylethanolamine n-methyltransferase and pineal hydroxyindole O-methyltransferase by regualting endogenous concentrations of the cosubstrate, S-adenosylmethionine, via its metabolic enzymes, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase. Assays for these latter enzymes were established and optimized in the adrenal and pineal glands. The effects of hypophysextomy and dexamethasone or ACTH treatment on these enzymes were monitored along with concomitant changes in methyltransferase activity. Hypophysectomy simultaneously decreases methionine adenosyltransferase and S-adenosylhomocysteine hydrolase activity in both tissues. Dexamethasone administration to hypophysectomized animals does not alter either methionine adenosyltransferase activity of S-adenosylhomocysteine hydrolase activity in the adrenal gland. However, it does increase S-adenosylhomocysteine hydrolase activity in the pineal gland. In contrast, ACTH administration restores both enzymes in the adrenal while being ineffective in the pineal. These results suggests that glucocorticoids may be regulating S-adenosylmethionine levels and methyltransferase activity via the metabolic enzymes, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase, but that glucocoticoid control may be both tissue- and drug-specific.

Beta-adrenergic receptor control of rat pineal hydroxyindole-O-methyltransferase.

The nature of the postsynaptic adrenergic receptor on rat pinealocytes which controls hydroxyindole-O-methyltransferase (HIOMT) activity was studied. This enzyme is involved in the synthesis of the pineal hormone melatonin. Adrenergic drugs were administered continuously for a 7-day period to rats in which neural stimulation of the pineal gland was blocked by either superior cervical ganglionectomy or exposure to constant light. l-Isoproterenol, a beta-adrenergic agonist, prevented the fall in enzyme activity that occurs when neural stimulation is interrupted; d-isoproterenol was ineffective. The potency order of different adrenergic agonists was d,l-isoproterenol greater than l-norepinephrine greater than l-epinephrine. Terbutaline, a selective beta 2-adrenergic agonist, was ineffective. The selective alpha 1-adrenergic agonists phenylephrine and methoxamine and the alpha 2-agonist clonidine were also ineffective. High doses of the beta-adrenergic blocker propranolol antagonized the effect of isoproterenol and caused a fall in HIOMT activity in normal rats housed under normal diurnal lighting. This in vivo evidence is consistent with the hypothesis that the neural control of pineal HIOMT is mediated via a beta-adrenergic receptor.

Inhibition of avian and mammalian hydroxy-indole-o-methyl-transferase (HIOMT) with low molecular weight fractions of mammalian pineal glands

Partially purified fractions from sheep pineal gland extracts, which show a fluorescence like pteridines, were shown to suppress the enzyme hydroxy-indole-o-methyltransferase (HIOMT) isolated from avian pineals and retinas. The results described demonstrate that the mechanism of inhibition of HIOMT activity is independent of the species and organ. A blocking effect was found in experiments whether retinal or pineal HIOMT were used and the effect was found as well with bovine HIOMT. It is shown that low molecular weight molecules show inhibitory activity and accompany the high molecular weight enzyme HIOMT.

Human post-mortem pineal enzyme activity.

The synthetic enzymes for melatonin production, serotonin-N-acetyltransferase (SNAT) and hydroxy-indole-O-methyltransferase (HIOMT) were measured in post-mortem pineal organs from eighty-eight men and women. The activity of each enzyme was correlated with age and sex. No difference was seen in either parameter when the maximum or minimum activities were measured in the death time periods of the diurnal rhythm established earlier. However, an annual bimodal rhythm was seen in HIOMT activity with maximal values occurring in January and July and minimum activities in March and October. The SNAT and HIOMT enzymes have previously been shown to be stable when measured in pineal organs held under the usual post-mortem conditions. Pineal HIOMT has also been measured in twenty-one psychotic individuals. Whilst the schizophrenic group showed significantly increased values, the schizophrenic-like group had normal values. The results were not affected by ante-mortem or post-mortem states or by drug treatment. Any clinical interpretation of pineal dysfunction in disease states need not, therefore, take into consideration age, or possible sex, but should be aware of the normal diurnal rhythm as well as the bimodal annual rhythm.

Circadian rhythmicity of the activity of hydroxyindole-O-methyl transferase (HIOMT) in the formation of melatonin and 5-methoxytryptophol in the pineal, retina, and harderian gland of the golden hamster.

The day and night rhythms in the activity of HIOMT in the formation of melatonin of 5-methoxytryptophol have been determined in the pineal, retina and Harderian gland of the adult male golden hamster. In all hamsters used there was no detectable HIOMT activity in the deep pineal. In the superficial pineal HIOMT activity, involved in the synthesis of melatonin (Mel), was observed to be high at the end of the dark period and at the middle of the light period. Considering the HIOMT activity involved in the production of 5-methoxytryptophol (5-MTL), an increase in 5-MTL synthesis was observed only during the light period. Comparing the peak of Mel-production with that of 5-MTL it appears that during the light period the pineal produces more 5-MTL than Mel. In the Harderian glands, the circadian course of HIOMT activity involved in the synthesis of Mel seems to run parallel to that of the enzyme implicated in 5-MTL synthesis, both being stimulated at the end of the dark period. The activity of HIOMT in 5-MTL production is, however, always approximately 2 times higher than for Mel synthesis. In the retina the synthesis of Mel and 5-MTL is not significantly higher during the dark period than during the light period. However, the production of 5-MTL is larger than that of Mel. It appears that (1) with the exception of the end of the dark period, the extra-pineal synthesis of Mel and 5-MTL is always higher than that in the pineal; (2) the carcadian synthesis of 5-methoxyindoles is different in each organ, and (3) in the pineal the carcadian activity of HIOMT involved in 5-MTL formation is different from that of the same enzyme involved in the formation of Mel. The results are discussed.

Seasonal relationship between pineal hydroxyindole-O-methyltransferase (HIOMT) activity and reproductive status in the pony

Pony pineal glands and female reproductive tracts were collected monthly for 1 year from a local slaughterhouse. Pineal gland weights did not change significantly throughout the year. Pineal gland tissue homogenates were assayed for hydroxyindole-O-methyltransferase (HIOMT) activity with N-acetylserotonin as the primary substrate. The greatest HIOMT activity was obtained with N-acetylserotonin as substrate, but three other related 5-OH indole substrates (5-hydroxytryptophol, serotonin, and 5-hydroxy-2-methylindole) were also methylated. HIOMT activity with all substrates was highest during the late fall and winter (November–February) and lowest during the spring, summer, and early fall (April–October). Percentage of mares ovulatory was inversely related to pineal HIOMT activity, except that there was a 1- to 2-month lag phase. These findings are consistent with a hypothesized antigonadal activity of both melatonin and the pineal in mammals.

Pineal hydroxyindole-O-methyltransferase: mechanism, and inhibition by scotophobin A.

We had shown that the behaviorally active peptide, scotophobin A, a synthetic analogue of native scotophobin, acted to increase dark avoidance in goldfish by inhibiting pineal hydroxyindole-O-methyltransferase (HIOMT), the enzyme which converts N-acetylserotonin (NAS) to melatonin (MEL). Here we determine the reaction sequence of bovine pineal HIOMT and the mechanism whereby scotophobin A inhibits this enzyme. Initial rate studies in which the substrates NAS and the methyl donor, S-adenosylmethionine SAM), were independently varied indicated the enzyme reacted by a sequential mechanism. With the product, S-adenosylhomocysteine (SAH), included in the reaction mixtures, data were obtained consistent with the following order of substrate addition and product discharge: (see text). The turnover number was 5.7 moles melatonin formed/mole HIOMT/min. The substrate KMs were 4.2 x 10(-4) M for NAS and 4.9 x 10(-5) M for SAM. Further studies showed that scotophobin A is an inhibitor (KI = 7 x 10(-7) M) competitive with NAS, indicating that this peptide combines with the enzyme-SAM complex. The structural similarity of the tyrosinamide end of scotophobin A to NAS and several other HIOMT inhibitors, including two antischizophrenic drugs, is consistent with these observations.

Scotophobin A causes dark avoidance in goldfish by elevating pineal N-acetylserotonin.

We had shown that synthetic rat scotophobin A caused several effects upon goldfish, apparently mediated by the pineal gland. Here we report that norepinephrine decreased goldfish dark avoidance in a manner that was blocked by scotophobin or pinealectomy. Increased dark avoidance was caused by either propranolol or scotophobin alone. Certain components of the pineal melatonin pathway also affected goldfish light-dark preference: serotonin, and especially N-acetylserotonin, increased dark avoidance, as did the hydroxyindole-O-methyl-transferase (HIOMT) product inhibitor, S-adenosyl-homocysteine. Melatonin and S-adenosyl-methionine were without effect in this regard. Pinealectomy prevented the dark avoidance increase caused by serotonin and N-acetylserotonin. These data suggested that increased dark avoidance behavior in goldfish was correlated with N-acetylserotonin buildup in the pineal, and that scotophobin could cause this, if it were to inhibit pineal HIOMT. To test this hypothesis the effect of various agents upon pineal melatonin levels was determined. Scotophobin was found to both reduce pineal melatonin and to block the melatonin-increasing effect of N-acetylserotonin. This led to the discovery that, indeed, scotophobin was an effective inhibitor (KI50, 6 x 10(-7) M) of purified bovine HIOMT.