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.     

Melatonin and N-Acetylserotonin Stress Responses: Effects of Type of Stimulation and Housing Conditions

The effects of housing condition and type of stimulation on serum melatonin and N-acetylserotonin (NAS) were investigated. Male rats were housed under a 12/12-hour light-dark cycle, with ad libitum food and water, either individually or in groups of four. At the start of the light phase, separate groups were sacrificed at rest or subjected for 3 minutes to the stimulation of cold water, noise, novel environment, or ether vapour and then decapitated at 0, 5, 15, 30 or 60 minutes after the end of stimulation. Melatonin was measured by a modified radioimmunoassay and NAS by a specific radioimmunoassay. Melatonin levels responded to stimulation with an increase, while NAS levels responded with a decrease. Housing condition had no effect on hormone response. However, the pattern of response for each of the two hormones differed greatly among the stimuli. For melatonin, cold water was the most potent stimulus, followed by noise, novel environment, and ether. NAS responded most to ether, fleetingly to cold, and in a bimodal manner to noise. The data are interpreted as suggesting that separate mechanisms regulate serum melatonin and serum NAS is response to environmental stimulation and that under appropriate control conditions melatonin from the pineal is very responsive to environmental stimuli, in a manner similar to that of pituitary hormones.     

Secretory Patterns of Pineal Melatonin in the Rat

The release patterns of pineal melatonin were studied by continuously monitoring melatonin levels in the confluens sinuum plasma in sighted and bilaterally enucleated rats in the light and dark periods. Plasma melatonin was determined by radioimmunoassay, and the data were analyzed by a computerized algorithm developed in our laboratory. Pulsations of melatonin levels were found in the confluens sinuum plasma in all the animals studied, suggesting episodic secretion of pineal melatonin in rats. Because the minimum melatonin levels in the confluens sinuum were over three times the melatonin levels in the general circulation, it is postulated that 1) there is an episodic release pattern of pineal melatonin superimposed on a basal release pattern and 2) there are two pools of melatonin in the pineal gland, a readily releasible pool responsible for the basal release and a bound pool responsible for the pulsatile release. In the sighted rats, there was no diurnal difference in mean melatonin concentration, mean pulse amplitude, mean pulse rate, mean minimum melatonin level, and mean maximum melatonin level in the confluens sinuum. In the bilaterally enucleated rats, with the exception of the mean pulse amplitude, diurnal rhythms were demonstrated in all the other parameters studied with, higher values in the dark period. This experimental model should be employed in future investigations on the regulation of secretory patterns of pineal melatonin. Results of these studies may provide important insight into the regulation of pulsatile release of neuroendocrine secretions in general.     

The Effect of Serotonin, N-Acetylserotonin, and Melatonin on Spontaneous Contractions of Isolated Rat Intestine

A dose-dependent increase in tone and reduction in amplitude of contractions was observed after serotonin (5-HT) was administered to isolated segments of rat ileum, incubated in Locke's solution at 38 degrees C. Melatonin (M) reduced the tone but not the amplitude or frequency of contractions. Addition of M (administered in doses 20 to 100 X higher than 5-HT) relieved the spasm induced by 5-HT. Furthermore, pretreatment with M significantly reduced the 5-HT effect. N-acetylserotonin (NAS) exhibited delayed but similar effect to M. Neither M nor NAS could prevent or relieve acetylcholine-induced contractions or influence relief of intestinal contractions by adrenaline. This indicates that 5-HT and M act via a different mechanism than that of adrenaline and acetycholine system. Serotonin muscle receptor blocker methysergide reduced 5-HT effect but was not able to abolish it completely. As methysergide could not reduce the muscle tone and did not relieve spasm caused by 5-HT, it is speculated that M is not acting as antagonist of 5-HT-stimulatory receptors but rather as agonist of 5-HT-inhibiting neuronal receptors.     

Ontogeny of Circadian Rhythmicity for Melatonin, Serotonin, and N-Acetylserotonin in Humans

The serum concentration of melatonin, serotonin, and N-acetylserotonin were measured by RIA procedures in 28 infants aged 1 week to 9 months. Blood specimens were obtained at 12:00 hr and 24:00 hr. A day-night difference in serum serotonin was present immediately after birth. A significant (P less than 0.001) decrease in serum serotonin concentrations at 12:00 hr and 24:00 hr was observed from the first month of age to the third to ninth month of age. A significant (P less than 0.05) difference in day-night N-acetylserotonin concentration is first seen at age 1-3 months. Serum melatonin concentrations, though detectable, did not show any day-night difference at birth. Melatonin concentrations progressively increased up to the third month of age, and a significant (P less than 0.01) day-night difference appeared thereafter. The results indicate that in humans the circadian organization for serotonin already exists at birth, and the circadian melatonin rhythm develops after birth.     

Effects of Melatonin and 5-Methoxytryptamine on Sleep-Wake Patterns in the Male Rat

Melatonin and 5-methoxytryptamine were administered continuously over a period of 1-3 months to adult male rats by means of subcutaneously implanted silastic capsules containing one or the other of these pineal hormones. Polygraphic recordings during several weeks following hormonal application showed an increase in the amount of time spent in both quiet and rapid eye movement sleep, during the light as well as the dark period; however, diurnal sleep-wake rhythmicity was not affected by either treatment. We conclude that pineal hormones have sleep-promoting effects, but that their correlation with the light-dark cycle does not imply that they are causal factors in generating diurnal rhythms of sleep and wakefulness.     

Pineal Gland Free Amino Acids and Indoles During Postnatal Development of the Rat: Correlations in Individual Glands

Free amino acids and indoles were measured by HPLC in single pineal glands of 5-, 10- and 20-day-old rats sacrificed during mid-light and mid-dark at each age. Melatonin was detectable in neonates (5-day-old), but day vs night differences in indole constituents did not occur until 10 days of age. Free amino acid steady state levels were high in neonates and there was a tendency for reversal of day vs night differences coinciding with the onset of circadian rhythmicity in indole biosynthesis. High correlations (r greater than 0.85) existed for taurine vs. glutamate in individual glands regardless of age and time of sacrifice. These findings suggest that taurine and glutamate are biochemically interrelated and that developmental changes in amino acid metabolic pools reflect functional innervation of the gland.     

Antihypertensive Action of Melatonin in the Spontaneously Hypertensive Rat

The effects of melatonin on blood pressure and heart rate were studied in 23-week-old male spontaneously hypertensive rats. Melatonin infused i.p. at a dose of 6 mg/rat per day for 5 days using an osmotic minipump produced a significant reduction of blood pressure and a slight but significant decrease of heart rate in the conscious and unrestrained state. These cardiovascular effects of melatonin developed gradually. Plasma renin concentration tended to decrease after melatonin treatment. These results demonstrate that melatonin has an antihypertensive action. The mechanism of the antihypertensive action of melatonin requires further study.     

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.     

Melatonin alleviates circadian system disruption induced by chronic shifts of the light-dark cycle in Octodon degus

Modern 24-h society lifestyle is associated with experiencing frequent shifts in the lighting conditions which can negatively impact human health. Here, we use the degus, a species exhibiting diurnal and nocturnal chronotypes, to: (a) assess the impact of chronic shifts of the light:dark (LD) cycle in the animal's physiology and behaviour and (b) test the therapeutic potential of melatonin in enhancing rhythmicity under these conditions. Degus were subjected to a “5d + 2d” LD-shifting schedule for 19 weeks. This protocol aims to mimic lighting conditions experienced by humans during shift work: LD cycle was weekly delayed by 8h during 5 “working” days (Morning, Afternoon and Night schedule); during weekends (2 days), animals were kept under Morning schedule. After 9 weeks, melatonin was provided daily for 6h in the drinking water. The “5d + 2d” shifting LD schedule led to a disruption in wheel-running activity (WRA) and body temperature (Tb) rhythms which manifested up to three separate periods in the circadian range. This chronodisruption was more evident in nocturnal than in diurnal degus, particularly during the Afternoon schedule when a phase misalignment between WRA and Tb rhythms appeared. Melatonin treatment and, to a lesser extent, water restriction enhanced the 24-h component, suggesting a potential role in ameliorating the disruptive effects of shift work.     

Action of Morphine on Melatonin Release in the Rat

Some data from the literature raised the possibility of an interaction between the opioidergic system and pineal secretion. The present study was undertaken in order to investigate the acute influence exerted by opioids upon plasma melatonin levels in the albino rat. Different doses of morphine hydrochloride were injected (1, 1.5, 2, 3 mg/kg) intraperitoneally into anaesthetized adult male rats bearing a cannula previously inserted into the carotid. Blood samples were collected subsequently at 30-min intervals, within a period of 90 min following drug administration. Plasma melatonin contents were determined by a radioimmunoassay (RIA) method. Acute administration resulted in a dose-dependent increase in plasma melatonin concentration when compared to the respective controls. This effect is blocked by pretreatment with Naloxone. The present result seem to support the hypothesis that the opioidergic system, in certain circumstances, might contribute to the activation of melatonin secretion.     

Pineal, Retinal, Serum Melatonin, and N-Acetylserotonin Rhythms in Chicks on Reversing Light Regimen Eye Covering

After reversing a 12:12-h light-dark regimen of environmental lighting, pineal and retinal melatonin levels of white leghorn chicks recorded at the first mid-darkness were greatly enhanced and reached maximum levels after three more days; those recorded during the light periods indicate gradual decline but were far from the nadir of the original light period, even at the end of the experiment. The first mid-darkness serum melatonin levels recorded after photoperiod reversal were not much different from their original mid-light values. However, on continuing with the reversed regimen, the next mid-darkness levels were sharply increased, and maximum levels were reached after a further 2 days. Under the same experimental conditions light had much more drastic effects, and 6 h on the reversed regimen were sufficient to bring down completely the high value of the original mid-darkness period to the level of the starting mid-light nadir. Retinal N-acetylserotonin (NAS) measured simultaneously had a pattern similar to that of melatonin, but the pineal NAS rhythm did invert completely, albeit gradually. Eye covering did not prevent inversion of pineal and serum melatonin rhythms, which were identical in eye-covered and sighted control chicks on the reversed regimen of light. However, retinal melatonin values of the light periods were significantly less depressed in eye-covered than in sighted control chicks. Moreover, eye covering completely prevented the retinal NAS depression under light but did not affect pineal NAS. During darkness retinal and pineal NAS elevation was sluggish in the eye-covered chicks.     

Melatonin Concentration in the Cerebral Vascular Sinuses of Sheep and Evidence for Its Episodic Release

Blood was collected from the cerebral sinuses and from the jugular vein of 5 ewes during both the day and night. Cerebral sinus samples were collected by means of a permanently indwelling cannula (roughly every 5 min) while jugular vein samples were collected by venipuncture (roughly every 10 min). In each of the 5 animals mean nighttime melatonin concentrations were greater at night than during the day. In 2 animals, cerebral sinus plasma melatonin concentrations were greater than in the jugular vein; in 2 animals the sinus and jugular plasma had similar melatonin levels; in 1 ewe jugular vein blood melatonin levels exceeded those in the cerebral sinus plasma. These differences among animals are presumably due to slight positional differences in the cerebral venous cannula placement. In several animals episodic release of melatonin was apparent. Whereas the episodes were most obvious in the cerebral venous blood at night, they were also apparent in 1 case in the jugular vein plasma and in 1 animal during the day. When episodes appeared they occurred about every 15-20 min.     

Melatonin biological activity and binding sites in human melanoma cells

The effects of melatonin, N-acetylserotonin and serotonin on the growth and tyrosinase activity of SK-Mel 23 and SK-Mel 28 human melanoma cell lines were investigated. Binding assays were also performed to establish the nature of the binding site. SK-Mel 28 cells were responsive to melatonin and its precursors, exhibiting a decrease in growth and an increase in tyrosinase activity after a 72 hr treatment. N-acetylserotonin was as potent as melatonin, the minimal effective concentration (MEC, which is defined as the smallest concentration that elicits a measurable biological response, significantly different from control) being 10-8 m. Serotonin was the least potent (MEC = 10-6 m). Both melatonin antagonists, prazosin and luzindole, exhibited no effect per se and reversed both responses to melatonin. SK-Mel 23 cells, however, showed no significant responses to the indoleamines. Competition binding assays in SK-Mel 28 cells demonstrated the presence of binding sites to 2-[125 I]-iodomelatonin, which was displaced by the unlabelled hormone, by both antagonists, and by N-acetylserotonin. The curve adjustment of the displacement values with melatonin suggests the existence of two binding sites, with the following Ki values: 1.0 x 10-10 m and 6.5 x 10-6 m. Ki values for acetylserotonin, prazosin and luzindole were, respectively, 3.8 x 10-8 m, 1.2 x 10-8 m, and 8.3 x 10-6 m. Surprisingly, in SK-Mel 23 cells, melatonin and luzindole were able to compete with the radioligand, with Ki values of 3.1 x 10-8 and 2.4 x 10-8 m, respectively. Our data suggest that SK-Mel 28 cells probably possess high affinity binding sites to melatonin and, in addition, MT3 low affinity binding sites, because N-acetylserotonin was as effective as the native hormone, and prazosin effectively blocked the actions of melatonin. Both sites are functional as demonstrated by the blockade promoted by both luzindole and prazosin on the proliferative and melanogenic responses. Although growth and tyrosinase activity of SK-Mel 23 cells were not affected by melatonin or its precursors, this cell line possesses high affinity binding sites, which may be non-functional, or trigger responses other than the ones herein investigated.     

Melatonin Inhibits Mitotic Activity of Adrenocortical Cells In Vivo and in Organ Culture

The goal of this study was to examine the effects of melatonin, as well as those of melatonin and corticotropin (1-24 adrenocorticotrophic hormone (ACTH); Synacthen Depot) administered together, on the mitotic activity of adrenocortical cells in male and female mice. Melatonin was given subcutaneously once daily, in late-afternoon injections (between 16:00 and 18:00) in doses of 1 microgram, 10 micrograms, and 100 micrograms, and ACTH in a dose of 0.1 mg (10 U) daily for 10 days. Additionally, the highest dose of melatonin (100 micrograms daily) was administered together with ACTH. The metaphase-arrest technique using colchicine as a stathmokinetic agent was employed in the study. Melatonin, in all the examined doses, significantly decreased mean mitotic activity rate (MMAR) of the adrenal cortex in both male and female mice. Moreover, in a dose of 100 micrograms, melatonin suppressed the mitogenic effect of ACTH on the adrenal cortex. Furthermore, the present study investigated the effects of melatonin (5 x 10(-7)M), N-acetylserotonin (NAc-5HT) (5 x 10(-7)M), and ACTH (250 mU/ml or 1,000 mU/ml) alone as well as the effect of ACTH (250 mU/ml) applied jointly with melatonin on the mitotic activity of adrenocortical cells in rat adrenal explants incubated in vitro. Both pineal indoleamines (melatonin and NAc-5HT) significantly decreased the MMARs of adrenocortical cells. Corticotropin, as well as ACTH and melatonin applied together, also reduced the MMAR of adrenocortical cells. The present data suggest that melatonin may be directly involved in the inhibitory control of adrenocortical cell proliferation.     

Melatonin Content of the Pineal Gland in Different Mouse Strains

Pineal melatonin content at several times during the day and night was measured in 36 inbred strains of mice (Mus musculus) kept under LD 12:12 cycles. The results have indicated that only five inbred strains have pineal melatonin content, with higher levels during the night and lower levels during the day; the other 31 strains do not contain detectable melatonin in their pineal gland at any of times examined. The former group includes two commonly used strains (C3H/He and CBA/Ms) and three wild-derived strains (Mol-A, Mol-Nis, MOM). C3H and CBA mice showed a similar pattern of pineal melatonin rhythm with a peak at 2 hours before lights on. The peak levels were about 150 pg/gland in both strains. The rhythmic patterns of melatonin content in Mol-A, Mol-Nis, and MOM were slightly different from those in CBA and C3H. In the wild-derived strains, the peak of melatonin content did not occur at 2 hours before lights on but tended to occur at midnight. The peak levels were 67-91 pg/gland at the highest point in these strains.     

Acute Cerebral Hemorrhage Changes the Nocturnal Surge of Plasma Melatonin in Humans

The diurnal rhythm of plasma melatonin was studied in 46 Chinese patients with acute cerebral hemorrhage. The state of consciousness of each patient was assessed clinically. The individual sites of lesion were determined by computerized tomography scanning. One to five days after stroke, blood samples were collected by venipuncture at 1000 and 1400 h in the daytime and 0200 and 0400 h at night. Plasma melatonin was extracted by dichloromethane and determined by radioimmunoassay. It was found that patients with lesions in the brain stem or in the third and lateral ventricles had melatonin levels significantly different from the other subjects in that these values were lower and lacking a nocturnal rise. These results are consistent with the presumptive retina-pineal pathway proposed in humans. Dramatic blunting or obliteration of the nocturnal melatonin surge in the blood was also observed in some patients with lesions in the frontal lobe, fronto-parietal lobe, parieto-temporal lobe, and basal ganglia. These brain regions are not involved in the retina-pineal pathway described in rodents or humans. Thus, our results suggest that brain regions other than the presumptive retina-pineal neural pathway may play an important role in the generation and/or regulation of the diurnal production and/or secretion of pineal melatonin in humans. However, a global functional disturbance caused by cerebral hemorrhage cannot be ruled out in some cases. It should be noted that many of the lesions leading to a change in the nocturnal rise of plasma melatonin were unilateral lesions. The significance of this finding is presently unknown. In addition, patients without a nocturnal rise of plasma melatonin were mostly comatose. They had lesions in the basal ganglion, fronto-parietal lobe, brain stem, and lateral and third ventricles. The latter findings suggest that in the brain, certain regions responsible for the state of consciousness of the individual may also be important to the dirunal rhythm of pineal melatonin secretion.     

Melatonin and its potential biological functions in the fruits of sweet cherry

Melatonin is a well‐known molecule which possesses many beneficial effects on human health. Many agriculture products provide natural melatonin in the diet. Cherry is one such fruit as they are rich in melatonin. In order to understand the biological roles of melatonin in cherry fruit, melatonin synthesis and its changes over 24 hr period were systematically monitored both during their development and in the ripe cherries in two cultivars, ‘Hongdeng’ (Prunus avium L. cv. Hongdeng) and ‘Rainier’ (Prunus avium L. cv. Rainier). It was found that both darkness and oxidative stress induced melatonin synthesis, which led to dual melatonin synthetic peaks during a 24 hr period. The high levels of malondialdehyde induced by high temperature and high intensity light exposure were directly related to up‐regulated melatonin production. A primary function of melatonin in cherry fruits is speculated to be as an antioxidant to protect the cherry from the oxidative stress. Importantly, plant tryptophan decaboxylase gene (PaTDC) was identified in cherry fruits. Our data shows that PaTDC expression is positively related to the melatonin production in the cherry. This provides additional information to suggest that tryptophan decaboxylase is a rate‐limiting enzyme of melatonin synthesis in plants.     

Melatonin and Pituitary-Gonadal Function in Disorders of Eating Behavior

In order to study the possible relationships between melatonin secretion and pituitary-gonadal function, the circadian rhythm of plasma melatonin, the basal levels of estradiol-17beta and testosterone and the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to luteinizing hormone-releasing hormone (LH-RH) stimulation were evaluated in normally cycling healthy women and in two groups of women with menstrual dysfunctions related to eating disorders (19 patients with anorexia nervosa and 16 with primary obesity). The circadian rhythm of plasma melatonin reached statistical significance in anorectic patients but not in obese patients. The mean 24 h melatonin level was significantly higher in anorectic than in obese patients and in control subjects. However, both groups of patients shared some abnormalities of melatonin circadian pattern, such as increased ratio between day and night melatonin levels, abnormal secretory peaks during the light hours and great interindividual variability for timing, amplitude, and duration of melatonin nocturnal peak. A selective impairment of LH secretion was observed in both anorectic and obese patients. By considering together the two groups of patients and controls, a linear inverse correlation between the circadian mesor of plasma melatonin and the basal and LH-RH stimulated LH levels was found. The persistence of a certain melatonin secretion during the light hours in both anorectic and obese patients could play an inhibitory role on the pituitary gonadal function in these subjects.