Plasma melatonin concentration in neonatal northern elephant seals,Mirounga angustirostris

The development of pineal function in northern elephant seals was examined in an attempt to understand the physiological basis for previously observed high daytime levels of melatonin in neonatal southern elephant seals. Pineal glands from four northern elephant seal pups, estimated age less than 1 week, weighed 3.0 ± 0.80 g, which was significantly less than that previously found in southern elephant seals (4.6 ± 0.35 g). Midday concentrations of plasma melatonin in pups averaged more than 3000 pmol/l in the first 5 days post-partum, but declined rapidly to less than 400pmol/l after day 9. Daytime melatonin levels in northern elephant seals tended to be lower than in southern elephant seals, although they were very high compared with other species. A circadian cycle of plasma melatonin concentration was observed in newborn northern elephant seals, with levels of 3000–5000 pmol/1 during the day, rising to more than 10,000 pmol/1 late in the dark phase. Soon after weaning at 4 weeks of age, daytime and night-time levels were in the range 60–100 pmol/1 and 100–400 pmol/1, respectively. When approximately 10 weeks old, most samples were in the range 100–400 pmol/1 with no discernible difference between day and night levels. The results do not support the hypothesis that the pineal gland is involved in thermogenesis in new-born southern elephant seals. Instead, the very active pineal gland may contribute to energy conservation, by lowering body temperature, particularly at night. As physical insulation is acquired by the deposition of blubber, the mechanism is not required and melatonin falls to adult levels.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

σ Receptor Modulation of Noradrenergic-Stimulated Pineal Melatonin Biosynthesis in Rats

Abstract: Because σ receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the σ-receptor ligands 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine potentiated the enhancement of N -acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine enhanced significantly the physiological increases in both pineal N -acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific σ-receptor antagonist. These findings suggest that, in rats, the activation of pineal σ-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it pontentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.     

Microdialysis of Melatonin in the Rat Pineal Gland: Methodology and Pharmacological Applications

Abstract: The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluori-metric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day-and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the β-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of β-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of α₁-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.     

Melatonin and adrenal cortex steroid production: in vivo and in vitro studies.

The present study was designed to clarify the interaction between the pineal melatonin and adrenal cortex steroid production. Experiments with male rats under chronic stress conditions (sleep deprivation) revealed that melatonin circadian pattern was fully destroyed and daytime plasma concentration were significantly elevated. Constant illumination (2500 lux) during the nighttime was not able to suppress melatonin production in the stressed animals. Plasma concentration of corticosterone were increased in the stressed rats as well. The modulatory effect of melatonin on corticosterone and progesterone production by rat adrenals was studied in a superfusion system. During melatonin challenge progesterone secretion was two-three fold elevated with no effect on corticosterone content in the plasma samples. Pineal cytoplasmic glucocorticoid and progesterone receptors were investigated as well. A specific binding was not observed in that case. Presented data support the existence of direct communication between the pineal and adrenal glands.     

Vitamin A deficiency reduces the responsiveness of pineal gland to light in Japanese quail (Coturnix japonica)

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.     

Pineal melatonin in hibernating and aroused golden hamsters (Mesocricetus auratus)

Daily changes of pineal melatonin content were determined in warm-adapted nonhibernating and cold-adapted hibernating golden hamsters (Mesocricetus auratus). Pineal melatonin in nonhibernating golden hamsters showed marked daily rhythm with the night values about 20 times higher than the daytime ones. In hamsters hibernating for 2 and 3 days the melatonin rhythm was abolished, since no increase of pineal melatonin over basal levels occurred throughout 24 hr period. After arousal from hibernation melatonin increased rapidly regardless whether the hamsters were provoked to arousal during day or night.     

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)     

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.     

Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus Tridecemlineatus)

Pineal melatonin levels were compared in laboratory-raised or wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus) that were either exposed to 10 h of darkness at night or to light which had an irradiance of 400 μW/cm². In laboratory-born squirrels the period of darkness was associated with a gradual rise in pineal melatonin levels with peak values being reached at 0200 h, 6 h after darkness onset. Thereafter, melatonin levels decreased and were back to low daytime levels by 0800 h, 2 h after light onset. The exposure of laboratory-raised animals to an irradiance of 400 μW/cm² during the night totally prevented the nocturnal rise in pineal melatonin levels in these animals. In wild-captured ground squirrels the period of darkness at night was associated with a rapid rise in pineal melatonin such that by 2200 h, 2 h after lights out, peak melatonin values were already attained; additionally, melatonin levels remained high throughout the period of darkness but returned to daytime values by 0800 h. Exposure of wild-captured squirrels to a light irradiance of 400 μW/cm² during the normal dark period was completely incapable of suppressing pineal melatonin levels. The difference in the sensitivity of the pineal gland of laboratory-raised and wild-captured ground squirrels may relate to their previous lighting history.     

Nocturnal increase in plasma cGMP levels in humans.

The circadian dynamics of responses to cyclic guanosine 3',5'-monophosphate (cGMP) in in vitro experiments and the stimulating effects of the pineal hormone melatonin on cGMP levels both in vitro and in vivo provoked an investigation into the diurnal pattern of occurrence of this second messenger in human plasma and its correlation with plasma melatonin levels. Plasma cGMP levels were measured in 9 normal human subjects who were over 50 years of age. Samples were obtained hourly through a 20-h period (11 a.m. to 7 a.m.) that included the subjects' habitual hours of nocturnal sleep; physical activity was kept to a minimum during the daylight hours. The area under the time-plasma cGMP concentration curve showed a significant increase during the period of nocturnal sleep compared to that observed during the period of daytime wakefulness. The individual temporal pattern of the nocturnal rise in plasma cGMP differed among the subjects; however, the initial increase typically was observed soon after bedtime. No significant correlation was observed between individual nocturnal plasma melatonin levels and cGMP levels.     

Proteomic analysis of day-night variations in protein levels in the rat pineal gland

The pineal gland secretes the hormone melatonin. This secretion exhibits a circadian rhythm with a zenith during night and a nadir during day. We have performed proteome analysis of the superficial pineal gland in rats during daytime and nighttime. The proteins were extracted and subjected to 2-DE. Of 1747 protein spots revealed by electrophoresis, densitometric analysis showed the up-regulation of 25 proteins during nighttime and of 35 proteins during daytime. Thirty-seven of the proteins were identified by MALDI-TOF MS. The proteins up-regulated during the night are involved in the Krebs cycle, energy transduction, calcium binding, and intracellular transport. During the daytime, enzymes involved in glycolysis, electron transport, and also the Krebs cycle were up-regulated as well as proteins taking part in RNA binding and RNA processing. Our data show a prominent day-night variation of the protein levels in the rat pineal gland. Some proteins are up-regulated during the night concomitant with the melatonin secretion of the gland. Other proteins are up-regulated during the day indicating a pineal metabolism not related to the melatonin synthesis.     

Melatonin is rhythmic in newborn seals exposed to continuous light

We have investigated the effect of continuous light and darkness on plasma levels of melatonin in relation to the extremely large and active pineal gland typically found in newborn seals. Plasma levels of melatonin in captive newborn harp (Phoca groenlandica) and hooded seals (Cystophora cristata) were generally extremely high, with peak concentrations ranging from 0.8 ng/ml to 62.3 ng/ml. Moreover, plasma melatonin showed a similar, pronounced rhythmicity, both outdoors under natural light conditions (hooded seal only) and indoors under either 30 h of continuous light (490 lux) or 30 h of darkness (0 lux). In all animals, the melatonin rhythm was closely associated with the outdoor light-dark cycle. We suggest that the melatonin rhythmicity in newborn seals is mainly under circadian control and that it originates by maternal influence in the foetus. Daytime plasma concentrations of melatonin were also measured in foetal hooded seals and their mothers. The foetal melatonin level was similar to daytime levels in newborns and was about five times higher than in their mothers, which indicates a significant flow of foetal melatonin to the mother. We speculate that the large pineal gland and high melatonin levels in the newborn seals are temporary consequences of a foetal strategy to affect the maternal blood supply during diving.     

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.     

Influence of acute stress on the pineal activity during day- and nighttime

Ovariectomized, steroid implanted female ewes were used as a model for studying the effect of acute isolation and confinement stress on the pineal activity during day and nighttime under artificial luteal phase conditions. Male and female intact buffaloes were employed as well, with the aim to establish the influence of another perturbation (venous catheter insertion) on the melatonin levels during daytime. Stress appeared to influence pineal melatonin secretion in controversial manner, namely, decreasing further the low indole levels during the day, while elevating the peripheral concentrations at night, though the initial response to stress during daytime was a transient elevation in melatonin levels. There are no indications that the adrenals are directly involved in the changes observed. Possibilities for different mechanisms of melatonin secretion and release in different species are considered.     

Ovine Pineal Indoles: Effects of l-Tryptophan or l-5-Hydroxytryptophan Administration

L-5-Hydroxytryptophan (L-5-HTP) (20 or 200 mg/kg i.p.) but not L-tryptophan (500 mg/kg i.p.) loading substantially increases serum melatonin in sheep. In the present study we examined the effects of these compounds on pineal serotonin and six serotonin metabolites. L-Tryptophan failed to increase 5-hydroxytryptamine (5-HT; serotonin) or any of its metabolites despite a five-fold increase in pineal tryptophan. In contrast, L-5-HTP loading produced a marked increase in pineal 5-HT and its metabolites, including N-acetylserotonin (NAS) and melatonin, indicating that an increased synthesis of melatonin is responsible for the increased serum melatonin concentration after loading with this precursor. No change in pineal indoleamine N-acetyltransferase (NAT) activity was seen. These results are consistent with the suggestion that, during daytime in the sheep, 5-HT availability may limit the production of melatonin.     

Correlations of Melatonin Content in Pineal Gland, Blood, and Brain of Some Birds and Mammals

Diurnal, rhythmic variation of melatonin content in the pinealgland, blood serum, and brain have been found in chickens, withgreater amounts present in all tissues during nighttime thanduring daytime. Similar daily rhythms appear to occur in thepineal gland and serum of rats and in the serum and urine ofhumans. It is proposed that these correlated fluctuations inmelatonin levels are causally related, elevated pineal contentresulting in increased melatonin content of the blood and increasedaccumulation by the brain. The brain of chickens, especiallythe hypothalamus, appears to accumulate melatonin and is probablya primary site of action of melatonin.     

Rhythmic control of endocannabinoids in the rat pineal gland

Endocannabinoids modulate neuroendocrine networks by directly targeting cannabinoid receptors. The time-hormone melatonin synchronizes these networks with external light condition and guarantees time-sensitive and ecologically well-adapted behaviors. Here, the endocannabinoid arachidonoyl ethanolamide (AEA) showed rhythmic changes in rat pineal glands with higher levels during the light-period and reduced amounts at the onset of darkness. Norepinephrine, the essential stimulus for nocturnal melatonin biosynthesis, acutely down-regulated AEA and other endocannabinoids in cultured pineal glands. These temporal dynamics suggest that AEA exerts time-dependent autocrine and/or paracrine functions within the pineal. Moreover, endocananbinoids may be released from the pineal into the CSF or blood stream.     

Pineal and serum melatonin at midday and midnight following pinealectomy or castration in male rats.

Melatonin has been extractkd by chloroform from rat serum and quantified by the Rana pipiens tadpole bioassay. Like pineal melatonin, serum melatonin was high at mid-dark and low at mid-light. This finding suggests a diurnal rhythm of serum melatonin in the rat. Serum samples obtained at mid-dark had melatonin levels ranging from 0.02 to 0.05 ng per ml of serum. When rats were pinealectomized, melatonin activity in the serum was abolished. Thus, rat serum melatonin may originate primarily from the pineal. There was no significant change in pineal melatonin content nor in serum melatonin concentrations 7 or 12 days following orchidectomy.