DHEA-sulfate causes a phase-dependent increase in melatonin secretion: a study of perifused rat pineal glands

Steroid hormones affect various metabolic activities, including melatonin synthesis, in mammals and nonmammals. We report here the effects of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), two steroids with weak androgen potency, on the levels of isoproterenol-stimulated melatonin released by perifused rat pineal glands removed in the middle of the light and dark spans [7 and 19 Hours After Light Onset (HALO), respectively] in a L/D 12:12 regimen. DHEA-S but not DHEA was found to have a direct action on beta-adrenergic-stimulated melatonin release. DHEA-S increased melatonin secretion (by 50-80%) dose-dependently in pineals obtained during the light span. This effect depended on the circadian stage, because at night (19 HALO), only the highest concentration (10(-3) M) of DHEA-S increased melatonin secretion (by 25%). In contrast, DHEA had no effect on melatonin release in pineals obtained during the light span. This work shows that DHEA-S but not DHEA was able to stimulate melatonin secretion by adrenergic-stimulated pineals removed during the light phase. It also suggests that the effects observed, or their intensity, or both depend on the circadian stage.     

Age-related changes in 24-hour rhythms of norepinephrine content and serotonin turnover in rat pineal gland: effect of melatonin treatment

The 24-hour rhythms of pineal norepinephrine (NE) content and serotonin (5-HT) turnover [estimated from the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT] were studied in young (2 months) and aged (18-20 months) Wistar rats killed at 6 different time points throughout a 24-hour cycle. In the first study, significant changes dependent on the time of day were identified, with acrophases in the first half of the activity span for both parameters. Old rats showed significantly smaller mesor and amplitude of the 24-hour rhythm of pineal NE content. They also showed decreased amplitude of the pineal 5-HT turnover rhythm, in the absence of changes in mesor. In old rats, pineal 5-HT and 5-HIAA concentrations were 41-47% of those found in young rats. In a second study, young and old rats received daily intraperitoneal injections of melatonin (30 microg) or vehicle for 11 days at 19.00 h (i.e. 11 h after light on). Analyzed as a main factor in a factorial analysis of variance, both pineal NE content and 5-HT turnover decreased in old rats while pineal 5-HT turnover increased after melatonin treatment. Melatonin treatment augmented the amplitude of the 24-hour rhythm of pineal NE content by 120 and 52% in young and old rats, respectively. The amplitude of the 24-hour rhythm of pineal 5-HT turnover almost doubled after melatonin treatment in young rats and did not change in old rats. Melatonin injection did not modify the rhythm's acrophase. The results indicate that old rats had lower amplitude and lower mesor values of 24-hour variations in pineal NE content and 5-HT turnover. Melatonin treatment only partly restored pineal NE content and was devoid of activity on pineal 5-HT turnover and 5-HT and 5-HIAA concentration in old rats. Impairment of pineal melatonin synthesizing capacity and intrapineal responses to melatonin may underlie pineal aging in rats.     

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.     

Lack of effect of ghrelin treatment on melatonin production in rat pineal and Harderian glands

The effects of ghrelin, a peptide hormone secreted from the stomach, on melatonin remain unknown. The aim of the study was to investigate possible ghrelin-melatonin interactions by studying the effect of ghrelin treatment on melatonin production in rat pineal and Harderian glands. Young (9 weeks) and old (20 months) male Wistar rats, maintained under a light:dark cycle regimen of 12:12, were assigned randomly to either a single subcutaneous (s.c.) injection of saline or ghrelin (1 microg/rat or 15 microg/rat) 1 h before sacrifice in the middle of the dark phase, or repeated s.c. saline or ghrelin injections (15 microg/rat), 3, 2 and 1 h before sacrificed in the middle of the dark phase. Neither ghrelin doses (1 microg/rat or 15 microg/rat) nor type of treatment (acute or repeated) influenced melatonin levels or the melatonin synthesizing enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase activities, either in pineal gland or in Harderian glands. At the concentrations used, ghrelin does not influence melatonin production in rat pineal and Harderian glands, and therefore is not involved in the regulation of melatonin secretion, at least under our experimental conditions.     

Age-Associated Changes in Pineal Adrenergic Receptors and Melatonin Synthesizing Enzymes in the Wistar Rat

The nocturnal stimulation of pineal melatonin synthesis and elevation of serum melatonin is known to be reduced in old age in several species. In Wistar rats the capacity of the beta-adrenoceptor to develop supersensitivity (increase in Bmax) during the light period of the diurnal light/dark cycle is lost during maturation (3-6 months) rather than old age. Further, the present study shows that neither the alpha 1- nor beta-adrenoceptor density of the pineal declines as rats age. Pineal hydroxyindole-O-methyltransferase activity does fall (17-55%) in rats after 18 months of age, but nocturnal pineal arylalkylamine N-acetyltransferase activity is not significantly altered. Thus, from examination of these parameters across the life span of the rat, it seems likely that the reported reduction in serum melatonin in old animals is related to a reduced capacity of the pineal to synthesize melatonin, rather than an altered responsiveness of the gland to neural stimulation.     

Age-related characteristics of the effects of epithalamin on serotonin metabolism in the pineal gland of rats]

The biosynthesis of serotonin into melatonin was decreased in old (18-20-month) in comparison to young (4-5-month) male Wistar rats. 5-day morning injections to young and old rats with polypeptide pineal preparation (epithalamin) in a dose of 2.5 mg/kg of body weight induced the increase in the night peak of serotonin, N-acetylserotonin and melatonin in young and melatonin alone in old rats and did not influence 5-methoxytryptamine, 5-oxy- and 5-methoxyindoleacetic acids level. These data support suggestion of ultrashort loop between pineal peptides and indoles and that epithalamin increases the metabolism of serotonin into melatonin.     

Modification of physical movement in old C57BL/6 mice by DHEA and melatonin supplementation.

As old age results in reduced physical activity as well as less dehydroepiandrosterone (DHEA) and melatonin (MLT) production, low hormone levels may be a component of inactivity. Therefore, we studied the effects of DHEA and/or MLT supplementation on movement and resting in young and old female C57 black mice. Our results showed for the first time that old female C56BL/6 mice have significantly decreased physical activity. Their average speed and resting time were significantly higher than in young mice, whereas ambulatory time, distance traveled, and body movements when stationary (stereo time) were lower. DHEA supplementation significantly increased stereo time in old mice, while decreasing ambulatory time and distance traveled. MLT supplementation of old mice decreased average speed, resting time, and stereo time compared to untreated, old mice. Supplementation with MLT or DHEA, whose production is reduced in aging, restored physical activity levels in old mice. MLT also increased ambulatory time and distance traveled while reducing body movements of young mice.     

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.     

Some Perturbations That Disturb the Circadian Melatonin Rhythm

The circadian melatonin rhythm is highly reproducible and generally not easily altered. The few perturbations that are capable of significantly changing either the amplitude or the pattern of the 24-h melatonin rhythm are summarized herein. Aging alters cyclic melatonin production by decreasing the amplitude of the nocturnal melatonin peak in all species in which it has been studied. The best known acute suppressor of nocturnal melatonin is light exposure. The brightness of light required to acutely depress pineal melatonin production is species dependent; of the visible wavelengths, those in the blue range (∼500-520 nm) seem most effective in suppressing melatonin production. Nonvisible, nonionizing radiation in the extremely low frequency range (e.g., 60 Hz) seems also capable of altering pineal melatonin synthesis. Hormones have relatively little influence on the circadian production of melatonin, although either adrenalectomy or hypo-physectomy does attenuate the amplitude of the melatonin cycle. Exercise at the time of high melatonin production rapidly depresses pineal concentrations of the indole without influencing its synthesis; the mechanism of this suppression remains unknown.     

Some Perturbations That Disturb the Circadian Melatonin Rhythm

The circadian melatonin rhythm is highly reproducible and generally not easily altered. The few perturbations that are capable of significantly changing either the amplitude or the pattern of the 24-h melatonin rhythm are summarized herein. Aging alters cyclic melatonin production by decreasing the amplitude of the nocturnal melatonin peak in all species in which it has been studied. The best known acute suppressor of nocturnal melatonin is light exposure. The brightness of light required to acutely depress pineal melatonin production is species dependent; of the visible wavelengths, those in the blue range (～500-520 nm) seem most effective in suppressing melatonin production. Nonvisible, nonionizing radiation in the extremely low frequency range (e.g., 60 Hz) seems also capable of altering pineal melatonin synthesis. Hormones have relatively little influence on the circadian production of melatonin, although either adrenalectomy or hypo-physectomy does attenuate the amplitude of the melatonin cycle. Exercise at the time of high melatonin production rapidly depresses pineal concentrations of the indole without influencing its synthesis; the mechanism of this suppression remains unknown.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

The effects of periodic alteration of the temperature on the rhythmic melatonin release of explanted chicken pineals

The melatonin rhythm of cultured chicken pineal cells can be synchronized by cyclic environmental effects. Unlike the effects of light on the melatonin secretion, those of the temperature changes are much less known. Similarly, only a few data are available on the interactions between environmental illumination and periodic temperature changes and on the sensitivity of the pineal gland to temperature changes in different ages of animals. We monitored the effects of temperature on chicken pineals for several days in vitro, in a perifusion system under different illumination patterns. The effects of temperature on pineals from chicken of different age were also compared. The phase of the melatonin rhythm was controlled by periodic elevations of temperature under both constant darkness and continuous illumination. These results show that rhythmic changes of temperature prevent desynchronization induced by constant light. Following elevation of the temperature, the melatonin rhythm of pineals of young chickens (less, than 14 weeks old) was altered for 16 - 18 hours. Similar changes in melatonin rhythm were not found in older animals. It is concluded that the sensitivity for temperature changes of the pineal cells is varying with age.     

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 cycl 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. © 1994 Wiley-Liss, Inc.     

Day-night changes in plasma melatonin levels,synaptophysin expression and ultrastructural properties of pinealocytes in developing female sheep under natural long and short photoperiods

The aim of the present study was to analyze the 24-h rhythm in plasma melatonin concentration and the day-night differences in synaptophysin expresion and ultrastructural characteristics of the pinealocytes in developing female sheep. Ewes of three different ages were examined: infantile (1-6 months old), pubertal and early fertile age (9-24 months old) and adult (36-60 months old). Experiments were conducted under natural non-stimulatory (long) and stimulatory (short) photoperiods. The obtained results were similar for both analyzed photoperiods. Plasma melatonin concentration, measured in samples obtained every 4 h, showed a similar pattern in the three age groups, with peak values at 02:00 h and troughs at 14:00 h. Mean value of plasma melatonin levels in 9-24 month-old sheep was significantly greater than that in younger or older sheep. The weight of pineal glands obtained at night (02:00 h) was significantly higher than in daylight (14:00 h). Pubertal and early fertile sheep had the largest pineal glands. The pineal volume, and the total number of pinealocytes per gland of 9-24 months-old sheep differed significantly from that of younger or older sheep. The pineal volume, and the mean volume of pinealocytes was significantly greater in animals killed at night. Number of pinealocytes did not vary between animals killed during daylight or at night. The mean volumen of pinealocytes did not show statistical differences between the age groups. In quantitative ultrastructural analysis of pinealocyte cells, the relative volume of mitochondria, rough endoplasmic reticulum and Golgi complexes was significantly greater in 9-24 month-old sheep and in animals killed at night. The relative volume of lipid droplets was highest in older sheep. Collectively, the data support the existence of developmental changes in pinealocyte morphology and quantity, partially in coincidence with a higher melatonin secretion rate.     

Effect of exogenous melatonin on viability, ingestion capacity, and free-radical scavenging in heterophils from young and old ringdoves (Streptopelia risoria)

The decrease of melatonin production with aging contributes to the decline in immune function as organisms age. Treatment with the exogenously administered indoleamine restores the reduced immunological functions. Therefore, we investigated the effect of melatonin on viability, phagocyte ingestion capacity, and free radical generation levels of heterophils from young and old ringdove (Streptopelia risoria) aged 3–4 and 11–13 years, respectively. Animals received a single oral dose of melatonin 1 h before lights off for three consecutive days. Experiments were performed at the acrophases and nadirs of melatonin. Melatonin treatment significantly increased serum melatonin levels at the acrophases, but not at the nadirs of the two age groups. In both young and old animals there was increased heterophil viability at acrophases with respect to nadirs, and also increased cell resistance to oxidative stress in the old animals after the melatonin treatment. At acrophases, the index, percentage and efficiency of phagocytosis all increased significantly, and superoxide anion levels decreased significantly with respect to the nadir values of vehicle and melatonin-treated animals, the effect being greater in young than in old ringdoves. At the nadirs, no change was observed in any parameter analyzed. In both young and old animals, phagocytosis and melatonin were positively correlated, while superoxide anion levels and melatonin were negatively correlated. In conclusion, exogenous melatonin enhanced heterophil viability in old animals as well as increasing phagocytosis and free-radical scavenging in both age groups during the nocturnal period, accompanied by an increase in the levels of the indoleamine.     

Comparative Study of the Activity/Rest Rhythms in Young and Old Ringdove (Streptopelia Risoria): Correlation with Serum Levels of Melatonin and Serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2–3 and 10–12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12∶12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09∶00 to 18∶00 h) and 1 h (from 21∶00 to 06∶00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02∶00 and 14∶00 h in the young and 01∶00 and 13∶00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one‐third that of the young animals. The acrophase and nadir were at 15∶00 and 03∶00 h in the young and 16∶00 and 04∶00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age‐associated sleep.     

Ageing,opioid analgesia and the pineal gland

The effects of ageing on day-night rhythms of analgesia was examined with young (1–2 months), mature (8–12 months) and old (20–30 months) mice. Significant age-related declines were observed both in the absolute levels and diel rhythms of morphine analgesia, with the most pronounced changes occuring at night. Administration of the pineal hormone, melatonin, augmented day-time levels of analgesia in all age classes and reversed the age-related decline in nocturnal morphine analgesia in old mice. Inhibition of pineal function in young mice by either exposure to light pulses or treatment with benserazide mimicked the effects of ageing on nocturnal morphine analgesia. These findings suggest that the pineal gland and melatonin are involved in modulating diel rhythms of analgesia and have an influential role on age-related changes in opioid responses.     

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.     

Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associated with increases in catalytically defective forms

Erythrocytes from young (6–8 month old) and old (28–31 month old) WF rats were separated into four age fractions by centrifugation on a discontinuous, isotonic, arabinogalactan density gradient. Specific activities of super-oxide dismutase (SOD) with respect to activity per unit hemoglobin (Hb) and activity per cell were determined for each cell age group. SOD activity was found to diminish with respect to erythrocyte age. More significantly, it was found that young erythrocytes of old animals already contain considerably reduced SOD activity as compared to cells of young animals. The level of SOD catalytic activity per unit enzyme antigen was also found to decrease with both increasing cell and animal age. Young cells of old animals contain significant amounts of catalytically altered molecules.