Age-related differences in serum melatonin and pineal NAT activity and in the response of rat pineal to a 50-Hz magnetic field.

In a previous study we have shown that exposure to a 50-Hz sinusoidal magnetic field decreased serum melatonin concentration and pineal enzyme activities in young rats (9 weeks). In the present study we looked for the effect of a magnetic field of 100 microT on serum melatonin and pineal NAT activity in aged rats and compared them to young rats. We hypothesized that aging may change sensitivity of rats to a magnetic field. Two groups of Wistar male rats [aged rats (23 months) and young rats (9 weeks)] were exposed to 50-Hz magnetic fields of 100 microT for one week (18h/day). The animals were kept under a standard 12:12 light: dark cycle with a temperature of 25 degrees C and a relative humidity of 45 to 50%. Control (sham-exposed) animals were kept in a similar environment but without exposure to a magnetic field. The animals were sacrificed under red dim light. Serum melatonin concentration and pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities were studied. Our results showed that sinusoidal magnetic fields altered the production of melatonin (28% decrease; P <0.05) through an inhibition of pineal NAT activity (52% decrease; P <0.05) in the young rats whereas no effect was observed in aged ones. On the other hand, when comparing data from control animals between young and aged rats, we observed that serum melatonin level and NAT activity, but not HIOMT activity, decreased in aged rats (decrease by about 38% and 36% respectively). Our data strongly suggest that old rats are insensitive to the magnetic field.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Static and extremely low frequency electromagnetic field exposure: Reported effects on the circadian production of melatonin

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (5HT) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 μtesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of 5HT and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Two-phase response of rat pineal melatonin to lethal whole-body irradiation with gamma rays.

Male Wistar rats adapted to artificial light:dark (LD) regimen 12:12 h were whole-body irradiated with a single dose of 9.6 Gy of gamma rays and sham/irradiated in the night in darkness. The rats were examined 60 min, 1, 3 and 5 days after exposure between 22:00 and 01:30 h in the darkness. The results obtained indicate a two-phase reaction of pineal melatonin after the lethal irradiation of rats: the decline of melatonin concentration early after the exposure (at 60 min) with unchanged serotonin N-acetyltransferase (NAT) activity followed by an increase of melatonin synthesis, accompanied by an increase of pineal and serum melatonin on day 5 after the exposure. NAT activity was increased on day 3 after the exposure. Serum corticosterone concentrations in irradiated rats were increased 60 min and 3 days after exposure. With respect to the antioxidant, immunomodulating and stress-diminishing properties of melatonin, we consider the increase in melatonin synthesis during later periods after irradiation as part of adaptation of the organism to overcome radiation stress.     

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.     

Elevated daytime rat pineal and serum melatonin levels induced by isoproterenol are depressed by swimming

Isoproterenol (1 mg/kg) was subcutaneously injected into adult male rats during the day to stimulate pineal N-acetyltransferase (NAT) activity and pineal and serum melatonin levels. Two hours after isoproterenol administration when levels of each of these variables had increased significantly, the experimental animals swam for 10 min in 22 degrees C water. At 15 min after swimming onset, pineal and serum melatonin levels were highly significantly depressed compared to those in control animals that did not swim. The high NAT level was not influenced by swimming. In a second study, isoproterenol injected rats swam for either 1, 3, 6 or 10 min and were sampled 15 min after the onset of swimming. The reduction in the elevated pineal melatonin in these animals was correlated with the length of the swim, i.e., as the duration of swim increased the percent reduction in pineal melatonin also increased. Neither pineal NAT nor hydroxyindole-O-methyltransferase (HIOMT) activities were influenced by swimming. The results suggest that elevated pineal and serum melatonin induced by isoproterenol can be depressed with no effect on the activity of the enzymes which convert serotonin to melatonin.     

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.     

Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase activity, and melatonin content in the pineal gland of the Syrian hamster

The activities of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content were measured in Syrian hamster pineal glands at 2-hr intervals over a period of 24 hr. NAT and HIOMT are the two enzymes which catalyze the formation of melatonin from serotonin. The use of micromethods for determination of the enzyme activities allowed concurrent measurement of NAT and melatonin or HIOMT and melatonin in the same gland. HIOMT activity showed no significant diurnal rhythm whereas NAT activity and melatonin content exhibited distinct peak values late in the dark phase as described previously. Despite an apparent parallelism between the NAT activity rhythm and melatonin content, no correlation exists between these parameters in single pineal glands.     

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.     

Inconsistent suppression of nocturnal pineal melatonin synthesis and serum melatonin levels in rats exposed to pulsed DC magnetic fields

The purpose of these experiments was to determine whether the exposure of rats at night to pulsed DC magnetic fields (MF) would influence the nocturnal production and secretion of melatonin, as indicated by pineal N-acetyltransferase (NAT) activity (the rate limiting enzyme in melatonin production) and pineal and serum melatonin levels. By using a computer-driven exposure system, 15 experiments were conducted. MF exposure onset was always during the night, with the duration of exposure varying from 15 to 120 min. A variety of field strengths, ranging from 50 to 500 μT (0.5 to 5.0 G) were used with the bulk of the studies being conducted using a 100 μT (1.0 G) field. During the interval of DC MF exposure, the field was turned on and off at 1-s intervals with a rise/fall time constant of 5 ms. Because the studies were performed during the night, all procedures were carried out under weak red light (intensity of <5 μW/cm²). At the conclusion of each study, a blood sample and the pineal gland were collected for analysis of serum melatonin titers and pineal NAT and melatonin levels. The outcome of individual studies varied. Of the 23 cases in which pineal NAT activity, pineal melatonin, and serum melatonin levels were measured, the following results were obtained; in 5 cases (21.7%) pineal NAT activity was depressed, in 2 cases (8.7%) studies pineal melatonin levels were lowered, and in 10 cases (43.5%) serum melatonin concentrations were reduced. Never was there a measured rise in any of the end points that were considered in this study. The magnitudes of the reductions were not correlated with field strength (i.e., no dose-response relationships were apparent), and likewise the reductions could not be correlated with the season of the year (experiments conducted at 12-month intervals under identical exposure conditions yielded different results). Duration of exposure also seemed not to be a factor in the degree of melatonin suppression. The inconsistency of the results does not permit the conclusion that pineal melatonin production or release are routinely influenced by pulsed DC MF exposure. In the current series of studies, a suppression of serum melatonin sometimes occurred in the absence of any apparent change in the synthesis of this indoleamine within the pineal gland (no alteration in either pineal NAT activity or pineal melatonin levels). Because melatonin is a direct free radical scavenger, the drop in serum melatonin could theoretically be explained by an increased uptake of melatonin by tissues that were experiencing augmented levels of free radicals as a consequence of MF exposure. This hypothetical possibly requires additional experimental documentation. Bioelectromagnetics 19:318–329, 1998. © 1998 Wiley-Liss, Inc.     

The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Sigmodon hispidus

Wild-captured cotton rats (Sigmodon hispidus) trapped and tested in September and October exhibited a rapid reduction in pineal N-acetyltransferase (NAT) activity and melatonin levels after exposure to a light irradiance of 300 ωW/cm² during the dark period. The half-time for the depression of both NAT and melatonin was on the order of 2 min. The exposure of cotton rats during darkness to much lower irradiances of light, i.e., 5.0, 0.04, 0.03 or 0.01 W/cm², for 32 min also greatly diminished pineal NAT activity and radioimmunoassayable melatonin levels; however, a light irradiance of 0.005 ωW/cm² failed to significantly depress either the acetylating enzyme or the melatonin content of the pineal gland. The results show that the pineal gland of the wild-captured cotton rat, as judged by NAT activity and melatonin levels, is inhibited even by very low irradiances of light.     

Regulation of pineal enzymes by photoperiod, gonadal hormones and melatonin in Coturnix quail.

The photoperiodic and hormonal regulation of melatonin-synthesizing enzymes was determined in pineals of Coturnix quail. N-Acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT) were twofold higher in pineals of female and male Coturnix quail during exposure to darkness (16L:8D). Castration decreased pineal HIOMT activity in both female and male Coturnix, while selective gonadal steroids restored activity. NAT was not affected by castration or gonadal steroids. Implantation of melatonin into female Coturnix decreased both HIOMT and NAT activities. These results suggest that NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of the external perceptive environment and the internal hormonal milieu, with both enzymic activities modulated by the feedback inhibitory influence of endogenous melatonin.     

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.     

Pineal melatonin: circadian rhythm and variations during the hibernation cycle in the ground squirrel, Spermophilus lateralis

Variations in pineal melatonin content throughout a 24-hour period and during different phases of the hibernation bout cycle were studied in the golden-mantled ground squirrel (Spermophilus lateralis). In addition to pineal melatonin, the circadian variation in the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were also investigated in summer animals maintained at 22 +/- 2 degrees C, on a light:dark (L:D) schedule of 12:12 hr for 1 month (lights on at 08.00 hr). Pineal glands were collected from six animals in each group at 1200, 1600, 2000, 2400, 0200, 0400, and 0800 hr. Changes in pineal melatonin content during the hibernation bout cycle were investigated in ground squirrels housed at 4 +/- .05 degrees C in relative darkness (1.9-3.4 lux; 10:14 LD). Pineal glands were obtained between 12:00 and 18:00 hr from 30 animals during one of three phases of the cycle (deep hibernation, euthermic interbout, and entrance into hibernation). Pineal melatonin was also measured for comparison in six winter euthermic animals that were housed at 22 +/- 2 degrees C, on a L:D schedule of 10:14 hr. Melatonin was measured in individual pineal glands by radioimmunoassay. The daily melatonin rhythm in S. lateralis was characterized by a marked increase in pineal melatonin during the dark phase, in which peak nighttime values were nearly 20-fold greater than daytime basal levels. The daily rhythm for NAT activity paralleled the changes in melatonin, showing a peak activity at 0200 hr that was 45 times greater than mean daytime values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative correlation of age and the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin in male rats

Pineal concentrations of N-acetylserotonin and melatonin and serum levels of melatonin were studied in 3-wk-old (prepubertal), 8-wk-old (adult), and 17-mo-old (senile) male rats. They were adapted to a photoperiod of 12 h light/12 h darkness for a minimum of 1 wk and killed at mid-light and mid-dark. Melatonin and N-acetylserotonin were determined by radioimmunoassay. The concentrations of pineal N-acetylserotonin and melatonin were high in the dark period and low in the light period. Statistical analysis indicated that pineal N-acetylserotonin and melatonin levels per 100 gm body weight declined with age. Similarly, serum melatonin demonstrated diurnal changes in all the age groups studied. In addition, there was a significant reduction in the levels of serum melatonin with age. The parallel patterns of decrease in pineal and serum melatonin levels with age suggest a decline in pineal secretion of melatonin in the older animals.     

Swimming-induced suppression of rat pineal melatonin is prevented by pretreatment with calcium channel blockers

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22 degrees C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Stimulatory effects of LH on release of melatonin and activities of its synthesizing enzymes NAT and HIOMT in organ-cultured pineal glands of female rats.

The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) exerts antigonadotropic effects in some mammalian species. To evaluate the effect of luteinizing hormone (LH) on melatonin release and its synthesizing enzyme activities in pineal glands, pineals of adult female rats undergoing diestrus were organ-cultured in a medium containing 10(-12), 10(-10) or 10(-8) M LH for 6 h. Melatonin release increased significantly in pineals cultured with 10(-12) and 10(-10) M LH, as compared to control values. Similarly, the activity of arylalkylamine N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, was significantly higher in pineals cultured with 10(-12) and 10(-10) M LH for 6 h, while LH at 10(-8) M had no effect. Although LH at 10(-10) M increased pineal hydroxyindole-O-methyltransferase (HIOMT) activity, which catalyzes the final step of melatonin biosynthesis, LH at 10(-12) and 10(-8) M had no effect. These results demonstrate that at relatively low physiological levels, LH stimulates pineal melatonin synthesis and release, mainly by increasing NAT activity.