Responsiveness of Pineal N-Acetyltransferase and Melatonin in the Cotton Rat Exposed to Either Artificial or Natural Light at Night

In three separate experiments, the effect of acute exposure to either artificial or natural light during darkness of pineal N-acetyltransferase (NAT) activity and melatonin content was studied in the cotton rat (Sigmodon hispidus). The exposure of animals to an artificial-light irradiance of 160,000 microW/cm2 during darkness for either 1 s, 5 s, or 30 min was followed by a precipitous decline in pineal NAT activity and melatonin content when measured at either 15 or 30 min after light onset. When cotton rats were acutely exposed to light at night for 5 s, irradiances of either 3.2, 32, 320, and 3,200 did not suppress either pineal NAT or melatonin 30 min later; however, if the 5-s exposure had an irradiance of either 32,000 or 160,000 microW/cm2, the pineal enzyme activity and indole content were depressed. Moonlight, which had a maximal irradiance of 0.32 microW/cm2, was unable to suppress pineal NAT activity and melatonin content even when the animals were exposed to the moonlight for 30 min. The treatment of cotton rats with either norepinephrine or its agonist, isoproterenol, before their exposure to light at night retarded slightly the suppressive effect of light on the pineal constituents measured. Also, these drug treatments suppressed the pre-exposure levels of both NAT activity and melatonin content in the cotton rat pineal gland.     

Pharmacological Studies on the Regulation of N-Acetyltransferase Activity and Melatonin Content of the Pineal Gland of the Syrian Hamster

Thus far, all attempts to stimulate melatonin synthesis by beta-adrenergic receptor agonists in the Syrian hamster pineal gland have failed. Neither a wide range of dosages of isoproterenol (0.5 mg/kg to 24 mg/kg), nor prolonged treatment with norepinephrine, the natural neurotransmitter, increased N-acetyltransferase (NAT) activity or melatonin production. In the present study, the administration of isoproterenol at night was likewise ineffective in advancing or enhancing the normal nightly melatonin peak. Also, we did not find a delayed effect 7 or 8 h after the administration of the drug. Furthermore, we tested the idea of coneurotransmitters such as octopamine or dopamine being possibly necessary for stimulation, but could not find any effect of these substances on melatonin synthesis. In addition, a parasympatholytic agent, atropine, did not increase the responsiveness to sympathomimetic agents. Administration of a phosphodiesterase inhibitor was also ineffective in stimulating NAT activity. On the other hand, isoproterenol did retard the drop in NAT and melatonin after lights-on at night, indicating that beta-receptors are involved in maintaining elevated melatonin levels.     

Comparison of the Effects of β-Adrenergic Agents on Pineal Serotonin N-Acetyltransferase Activity and Melatonin Content in Two Species of Hamsters

The nighttime rise in pineal melatonin levels can be blocked by administration of the β-adrenergic receptor antagonist, propranolol, in both Syrian hamsters and rats. Although the administration of β-adrenergic receptor agonists such as norepinephrine or isoproterenol stimulates pineal melatonin production in the rat, these drugs are without apparent effect on indole production in the Syrian hamster. To determine whether this lack of stimulatory effect in the Syrian hamster is characteristic of this species, a comparison of the effects of norepinephrine and isoproterenol on pineal serotonin N-acetyltransferase activity and melatonin content was conducted. In contrast to their lack of effect in the Syrian hamster, norepinephrine and isoproterenol stimulated pineal serotonin N-acetyltransferase activity and melatonin content in the Djungarian hamster. Hourly injection of norepinephrine during a continuation of light into the normal dark period stimulated increases in the activity of serotonin N-acetyltransferase and melatonin content in the Djungarian hamster but was without effect on these pineal parameters in the Syrian hamster.     

No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands

As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.     

Adrenalectomy Prevents Changes in Rat Pineal Melatonin Content N-Acetyltransferase Activity Induced by Acute Insulin Stress

The activity of N-acetyltransferase (NAT) the content of melatonin (MEL) in the rat pineal have been shown to be sensitive to several types of stressors. This study was designed to assess the role of the adrenals in mediating the effect of one such stressor, insulin-induced hypoglycemia, on pineal synthetic activity. Intact bilaterally adrenalectomized (ADX) adult male rats were kept under light:dark cycles of 14:10 (lights on 0600 h) injected intraperitoneally with 10 IU insulin at 1300 h, groups (n = 8) were killed 2, 3, or 4 h postinjection. Plasma catecholamines were assayed by means of high performance liquid chromatography radioimmunoassay was used to assess pineal NAT activity MEL content. All injected groups were rendered hypoglycemic by insulin administration. Compared to uninjected controls, plasma epinephrine in hypoglycemic intact rats rose after 2 h, whereas epinephrine did not change in hypoglycemic ADX animals. The increase in epinephrine in intact animals was correlated with a rise in NAT activity at 2 h. Moreover, pineal MEL content at 2, 3, 4 h was significantly greater than control values. In contrast, no changes in pineal biosynthetic function were found in ADX rats. This differential response by intact ADX rats suggests that an adrenal product (possibly epinephrine) is responsible for mediating the stimulatory effects of acute insulin-induced hypoglycemic stress on the rat pineal.     

The Influence of Various Irradiances of Artificial Light, Twilight, and Moonlight on the Suppression of Pineal Melatonin Content in the Syrian Hamster

The purpose of the present studies using artificial light was to determine how the timing and duration of exposure influence the light-induced suppression of pineal melatonin levels in hamsters. An 8-min exposure to 0.186 microW/cm2 of cool white fluorescent light caused a continued depression of pineal melatonin even when animals were returned to darkness. In addition, the pineal gland does not appear to change its sensitivity to light throughout the night. A 20-min exposure to 0.019 microW/cm2 of cool white fluorescent light did not significantly suppress pineal melatonin during any time of the melatonin peak, whereas a 20-min exposure to 0.186 microW/cm2 was capable of always suppressing melatonin. Furthermore, increasing the duration of 0.019-microW/cm2 exposure to 30, 60, 120, or 180 min does not increase the capacity of this irradiance to depress melatonin. Similar to artifical light, natural light has a variable capacity for suppressing nocturnal levels of pineal melatonin. Twilight irradiances of 0.138 microW/cm2 or less did not suppress nocturnal melatonin whereas twilight irradiances of 3.0 microW/cm2 or greater did suppress pineal melatonin. A few animals did have lower melatonin after a 40-min exposure to full moonlight during July (0.045 microW/cm2) or January (0.240 microW/cm2). However, pineal melatonin levels remained high in the majority of animals exposed to full moonlight.     

Inhibition of Pineal Type-II 5''-Deiodinase Does Not Affect the Nocturnal Increase of N-Acetyltransferase Activity and Melatonin Content in Either Euthyroid or Thyroidectomized Rats

The presence of type-II thyroxine 5'-deiodinase (5'-D) activity in rat pineal gland has been previously described. In the present paper, 5'-D activity, N-acetyltransferase (NAT) activity, and melatonin content were measured in the same rat pineal. Each of these constituents exhibits a nocturnal increase with peak values at 0100 h for melatonin (1.20 +/- 0.12 ng/gland) and at 0300 h for both 5'-D (39.5 +/- 11.9 fmol/gland/h) and NAT (8.38 +/- 1.04 nmol/gland/h) activities. In vivo treatment with iopanoic acid (IOP) completely prevented the nocturnal increase in 5'-D activity (14.1 +/- 2.6 fmol/gland/h at 0300 h) with no modification in either the NAT activity or melatonin content. Thyroidectomy greatly enhanced the 5'-D activity during the dark period (102.9 +/- 10.2 vs. 31.6 +/- 4.2 fmol/gland/h), reaching a peak at 0200 h; thyroidectomy, however, did not affect daytime pineal 5'-D activity (3.11 +/- 0.78 vs. 2.5 + 0.92 fmol/gland/h). Treatment of rats with IOP acid completely inhibited the pineal 5'-D activity in both control (7.86 +/- 0.88 fmol/gland/h) and thyroidectomized animals (2.24 +/- 1.10 fmol/gland/h) with no change in the melatonin content of the gland (1.21 +/- 0.32 vs. 0.99 +/- 0.18 ng/gland).     

Status of Dopamine in Bovine Pineal Glands and the Stimulation of N-Acetyltransferase Activity by D2-Dopaminergic Receptor Agonists in the Rat Pineal Glands in Culture

In a previous study, we identified in the bovine pineal gland two [3H]spiroperidol-binding sites with KD values of 0.18 and 2.1 nM and Bmax values of 37 and 630 fmol/mg protein, respectively. In this study, the status of dopamine in the bovine pineal glands was delineated further by measuring the relative concentrations of dopamine and norepinephrine and the relative concentrations of serotonin and melatonin. Furthermore, the presence of 4.0 +/- 0.6 micrograms/dopamine/gm tissue encouraged us to delineate the effects of select dopaminergic receptor agonists and antagonists on the synthesis of melatonin in vivo and on the activity of N-acetyltransferase in the rat pineal gland in culture. The acute administration of haloperidol (3 mg/kg intraperitoneally [ip]) or sulpiride (200 mg/kg ip) increased the concentration of melatonin in the pineal gland from 160.6 +/- 8.18 to 327.6 +/- 45.43 and 306.5 +/- 40.53 pg/gland, respectively. Dopamine exhibited dual effects on the activity of N-acetyltransferase, inhibiting the basal activity at 0.1 microM and stimulating it at 10 microM, and the later effect was blocked by propranolol. D2-dopaminergic receptor agonists such as bromocriptine (4.0 microM) or LY-171555 (10.0 microM) partially attenuated the norepinephrine-induced stimulation of N-acetyltransferase, and these attenuating effects were reversed by D2-dopaminergic antagonists such as haloperidol (10 microM) or domperidone (10 microM). The results of these studies are interpreted to indicate that for the synthesis of melatonin, the pineal D2-dopaminergic receptors may function independently from those of the beta 1-adrenergic receptor sites. Furthermore, the said D2-dopaminergic receptor are amenable to down regulation since the activity of N-acetyltransferase remained unaltered (0.0717 vs. 0.0729 nmol/gland/h) following chronic treatment (4 mg/kg ip/day for 30 days) with bromocriptine.     

Cytochemical Analysis of Calcium Distribution in the Superficial Pineal Gland of the Mongolian Gerbil

The potassium pyroantimonate-osmium method was employed to determine the distribution of cations in the superficial pineal gland of the Mongolian gerbil. The reaction product in the glands was presumed to be calcium following the use of both the chelator EGTA and x-ray microanalysis. A gradient of reaction product was found in the pineal with the highest concentrations occurring in and around the pineal concretions (PC). The periphery of mature PC demonstrated a heavy precipitate as did the surrounding pinealocytes. PC-associated pinealoeytes contained reaction product in many organelles, including small vesicles that appeared to be opening in the direction of the concretions. The vacuoles of the pinealocytes contained various concentrations of precipitate. The results obtained in the present investigation have led to a hypothesis on the formation of pineal concretions: (1) a pinealocyte vacuole is formed, (2) calcium is transported to the vacuole, (3) the cation content of the vacuole increases, and (4) the cations precipitate into an immature concretion. Fibrillar material and an amorphous substance located within the pinealocyte vacuole appear to form cation containing vesicles that contribute to the periphery of the developing PC, resulting in the continued growth of the concretion.     

Diurnal Changes in Serum Melatonin Concentrations Under Indoor and Outdoor Environments and Light Suppression of Nighttime Melatonin Secretion in the Female Japanese Monkey

To examine whether artificial light with the intensity commonly used for animal experimentation can mimic natural sunlight with respect to diurnal changes in serum melatonin, and to determine the minimum light intensity required to suppress nocturnal melatonin, serum melatonin profiles were examined in groups of female Japanese monkeys (Macaca fuscata fuscata). Under outdoor environment, light intensities at the level of the monkey's eyes varied during daytime (0900-1500 h) depending on weather conditions (minimum and maximum on particular experimental days: 170 lux at 0900 h on a rainy day and 9500 lux at 0900 h on a slightly cloudy day); under indoor environment, light was provided by ordinary fluorescent bulbs that resulted in intensities of 400-500 lux at the level of monkey's eyes. No difference was found in diurnal changes in serum melatonin concentrations regardless of weather or housing conditions: Serum melatonin remained low during daytime and increased during nighttime. Following exposure to light, irradiances of 10,000, 400-500, 100-140, 50-100, and 10-30 lux at midnight resulted in a rapid decrease in serum melatonin to daytime levels within 1 to 2 h. After the onset of dark, serum melatonin reverted to previous nighttime levels within 2 h. Exposure to a light irradiance of 2-5 lux, however, did not suppress nocturnal melatonin secretion. It is concluded that artificial light can mimic natural sunlight with respect to melatonin secretion in the female Japanese monkey, and that light of 10-30 lux irradiance was sufficient to suppress serum melatonin to near daytime levels.     

Ultracytochemical Localization of Calcium in the Superficial Pineal Gland of the Mongolian Gerbil

In order to demonstrate fine localization of Ca2+ in the superficial pineal gland of Meriones unguiculatus, the pyroantimonate technique was employed. Control experiments were performed with EGTA and analysis of obtained reaction product using an energy-dispersive x-ray microanalysis. Precipitates of calcium antimonate were formed almost exclusively in swollen clear pinealocytes, in and along their cell membranes, over their nuclei, in mitochondria, the Golgi apparatus, endoplasmic and integrade reticulums, acervuli, in vesicles surrounding synaptic bars, cytoplasmic matrix, and flocculent extracellular material. It has been concluded that the swollen pinealocytes represent a degenerative cell form probably occurring by impairment of plasmalemmal Ca2+-ATPase's ability to eliminate Ca2+ ion from the cell, with consecutive increase of calcium in the cytoplasmic matrix, followed by increase of intramitochondrial Ca2+ concentration, gradual arrest of mitochondrial function, depolymerization of cytoskeletal microtubuli, loss of the cell form, and cell death. Decrease of function of pinealocyte plasmalemma seems to be related to aging.     

Stimulation of Serotonin-N-Acetyltransferase Activity in the Pineal Gland of the Mongolian Gerbil (Meriones unguiculatus) by Intracerebroventricular Injection of Vasoactive Intestinal Polypeptide

There is ever-increasing evidence that intrapineal peptides have an important role in the modulation of pineal melatonin synthesis. In the pineal gland of the Mongolian gerbil ( Meriones unguiculatus ), we have previously shown the presence of VIP-immunoreactive nerve fibers as well as pinealocytic VIP receptors. To assess the functional significance of these findings, 10 μl of a 1 μM or 1 nM solution of VIP were injected into the lateral ventricle of gerbils over a period of 10 min. Animals were killed 1.5 hr after injection, and the superficial pineal glands were excised and assayed for N-acetyltransferase (NAT) activity. Injection of the 1 μM VIP solution stimulated the NAT activity to values four times the control values. The results are compatible with an in vivo influence on the pineal gland indole metabolism of the nonsympathetic VIP-containing nerve fibers via VIP-receptors present in the gland.     

Pineal rhythm of N-acetyltransferase activity and melatonin in the male badger, Meles meles L, under natural daylight: Relationship with the photoperiod

The rhythmicity of melatonin secretion and of pineal NAT activity was compared in male badger kept in natural daylight during two distinctly different photoperiods (January and June). The hormone and its enzyme follow the same pattern with a nighttime elevation and a low level during the day, demonstrating the presence of a nyctohemeral rhythm. The high correlation found between the NAT activity and the melatonin concentration suggests that NAT is the rate-limiting enzyme in melatonin synthesis in the badger. Peak amplitudes were similar under the two photoperiods. Melatonin secretion occurred in the first part of the night irrespective of the photoperiod. The rhythm of melatonin secretion is modified by the photoperiod. The duration of high nighttime levels varies; it is longer (8 h) when the night is long (16 h) in January, and shorter (6 h) when the night is short (8 h) in June. In the badger, differences in the duration of high level melatonin at night may reflect variations in day length and convey to the animal the photoperiodic information.     

Rhythm Development in Pineal and Circulating Serotonin, N-Acetylserotonin, and Melatonin in Syrian Hamsters

The ontogeny of diurnal rhythm patterns in the pineal and serum levels of melatonin, serotonin, and N-acetylserotonin was studied in Syrian hamsters (Mesocricetus auratus) from birth to adulthood. The pineal and blood specimens were collected at 1100 h and 0200 h, and the compounds were measured by radioimmunoassay (RIA) procedures. Pineal melatonin and serotonin did not show any circadian rhythm at day 5 of postnatal age. At this age N-acetylserotonin was undetectable in the light phase but became manifest at night. By 10 days of age pineal serotonin registered an established rhythm pattern, with a higher level during the day. The occurrence of circadian rhythm in pineal melatonin was delayed and manifested first at 25 days of age. At this age, the first detectable daytime level of N-acetylserotonin also occurred. Circadian rhythm in serum melatonin was also established at this age. The serum serotonin did not evince any rhythm pattern throughout the observation period, except at day 17 of postnatal age. The massive concentration of daytime serotonin in the pineal was not reflected in the circulatory system. For serum N-acetylserotonin there was no discernable day-night rhythm in all age groups, except at 25 days of age. The results show that the timing of the appearance of various compounds in the neonatal pineal is variable; the release of the substances does not always reflect their synthesis; the ontogenesis of circadian rhythm is a part of the maturational process; and 25 days of age is a rather critical time in development.     

Opposite Effects of EGTA and Neutral Surfactants on the Loss of Chicken Pineal Serotonin N-Acetyltransferase Activity

The effects of some general purpose drugs on the deactivation and activity measurement of the chicken pineal gland enzyme serotonin N-acetyl transferase (EC 2.3.1.5.) were studied. The drugs used were EGTA and two neutral surfactants, Nonidet P40 and Triton X-100. Enzyme activity showed significant variations ranging from 2.8 +/- 1.3 nmol/gland/h when Nonidet P40 was added to the homogenate buffer, to 31.8 +/- 1.7 nmol/gland/h when EGTA was present. This striking variation seemed to be caused by the ability of these compounds to modify the rate of NAT deactivation acting either as accelerating agents, as in the case of the detergents or as braking agent, as in the case of EGTA.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.     

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

Abstract:  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.     

A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro

Nocturnal melatonin production is reportedly controlled by the rhythms of serotonin N-acetyltransferase (NAT, or arylalkylamine N-acetyltransferase). While analyzing the melatonin synthetic pathways of Long Evans cinnamon (LEC) rats mutant for PINA, a pineal night-specific ATPase defective in Wilson disease, we discovered that NAT activity and protein levels are greatly reduced in LEC rats, and that the highly conserved histidine 28 is mutated to tyrosine. To study the effect of H28Y, we isolated a new strain of rat termed LPN that is mutant for NAT but wildtype for both PINA and coat color. Compared with control rats, the LPN rats displayed low NAT protein levels and enzyme activities. These results suggest that the H28Y mutation in NAT is the cause of reduced NAT levels in vivo. The identical H28Y mutation was also found in Sprague-Dawley rats from Zivic-Miller, suggesting it may be a common mutation in rodents. When analyzed in bacterial cells and HEK293 cells, the mutation resulted in reduction of both NAT protein stability and catalytic activity, confirming that the in vivo NAT phenotype in LPN rats was due to the H28Y mutation. Further analysis of the NAT-H28Y will focus on the mechanisms of the increased degradation both in vitro and in vivo, which will facilitate our understanding of how melatonin synthesis is controlled at the molecular level.     

Immunohistochemical Assessment of Melatonin Binding in the Pineal Gland

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.     

Alloxan-Induced Diabetes and the Pineal Gland: Differential Effects on the Levels of Pineal N-Acetylserotonin, Pineal Melatonin, and Serum Melatonin

Effects of alloxan treatment on the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin were investigated. Male rats were housed under a photoperiod of 12 h light: 12 h darkness and a temperature of 23 +/- 3 degrees C. Three weeks after alloxan (170 mg/kg) or carrier injection (s.c.), the animals were killed at mid-light (1200 h) and mid-dark (2400 h). Pineal and serum indoles were extracted and quantified by radioimmunoassays. It was found that pineal levels of N-acetylserotonin in the diabetic rats were significantly higher (P less than 0.05) than those of the controls. Conversely, pineal and serum levels of melatonin in the control rats were significantly higher (P less than 0.05) than those of the alloxan-induced diabetics. Our results suggest that alloxan-induced diabetes may decrease pineal melatonin synthesis in rats by reducing the activity of hydroxyindole-O-methyltransferase, resulting in a decrease in pineal melatonin secretion.