Influence of psoralen on NAT activity and melatonin levels in rat pineal gland during the daily period of darkness

A single injection of either 5 or 10 mg/kg 8-methoxypsoralen (8-MOP) was given intraperitoneally to male rats at the end of the 14 h light phase (at 2000 h). Two h later (at 2200 h), when the normal nocturnal surge of N-acetyltransferase (NAT) activity and melatonin content in the pineal gland had begun in vehicle-injected controls, mean pineal NAT after the 10 mg/kg 8-MOP was 1.8-fold higher than that after vehicle, though pineal melatonin content did not differ between vehicle- and drug-injected rats. By 4 h into the dark period (at 2400 h), the NAT activity in both 8-MOP injected groups of rats was greater than that in vehicle treated animals; again, however, 8-MOP treatment did not influence the pineal melatonin content. At 0200 h (6 h into the dark period), the difference between the NAT activity in pineals of rats treated with 5 mg/kg 8-MOP and the vehicle was not statistically significant, but the animals that received 10 mg/kg drug still had statistically elevated levels of the serotonin acetylating enzyme. At 0200 h the pineal melatonin levels were equivalent among the three treatment groups. Rats given 5mg/kg 8-MOP always had NAT values intermediate between those of rats injected with vehicle and those that received 10 mg/kg 8-MOP suggesting that the NAT response to the drug was dose related. These results show that the pineal response to psoralen involves an elevation of NAT activity without a commensurate change in the melatonin content of the gland.     

Melatonin secretion from goldfish pineal gland in organ culture

Pineal glands were removed from goldfish reared under 12L-12D at 25 degrees for 2 weeks. These were incubated for 6 days under (1) normal 12L-12D (lights on 0600-1800 hr), (2) reversed 12L-12D (lights on 1800-0600 hr), (3) continuous dark, or (4) continuous light condition at 25 degrees. The incubation medium was changed at 12-hr intervals (0600-1800 and 1800-0600 hr) and secreted melatonin (MLT) was measured by RIA. Under 12L-12D or reversed 12L-12D, MLT secretion was active in the dark phase and was suppressed in the light phase of a given photoperiod. Under a continuous dark condition, a large amount of MLT was secreted into the medium, although the amount gradually decreased. The MLT secretion was more active in the period corresponding to the dark phase of the acclimatory photoperiod than in the period corresponding to the light phase. This pattern in secretion remained for 4 days. Under a continuous light condition, MLT secretion was suppressed, but the secretion was rapidly increased after changing the photoperiod from the light to the dark condition. These findings clearly indicate that MLT secretion in the organ-cultured pineal gland is photosensitive. It is active under dark and inactive under light conditions. The existence of a circadian rhythm in MLT secretion is also suggested.     

Presynaptic effects of melatonin on norepinephrine release and uptake in rat pineal gland

The effect of melatonin injection on norepinephrine (NE) turnover rate in rat pineal gland was estimated from the decline of tissue NE levels after the injection of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. The administration of a single injection of 300 micrograms/Kg of melatonin at the beginning of the scotophase induced, 3 hr later, a significant decrease of pineal NE turnover. The possible direct effect of melatonin on pineal NE release was examined in vitro. Exposure of rat pineal explants previously loaded with 3H-NE to 10(-8)-10(-6) M melatonin decreased significantly 3H-NE release triggered by 60 mM K+. This activity of melatonin was revealed only in pineals excised at night (0000 and 0400, i.e., at the fourth or eighth hours of darkness) and not in those excised in the middle (1400) or late light phase of the daily photoperiod (2000). Melatonin did not modify the spontaneous pineal 3H-NE efflux. Melatonin decreased 3H-NE uptake at a low NE concentration (0.5 microM) in a dose-dependent manner (IC50 identical to 10(-10) M). A kinetic analysis of the pineal NE uptake process indicated that melatonin augmented both Vmax and Km of transmitter uptake. These results suggest that endogenously released melatonin may be a regulatory signal for rat pineal sympathetic synapses.     

Lack of effects of light on the rat pineal in organ culture

There were no significant differences in the incorporation of cytidine into RNA nor in the conversion of serotonin to melatonin, N-acetylserotonin, 5-hydroxytryptophol, 5-methoxytryptophol, and 5-methoxyindoleacetic acid between rat pineal explants exposed to either light or darkness for 24 hr. In contrast l-norepinephrine increased both pineal RNA and methoxyindole synthesis significantly.     

Immunohistological localization of melatonin in the pineal gland and retina of the rat

An incubation procedure for the demonstration of melatonin is described using freeze-dried, unfixed material embedded in vacuum directly in glycolmethacrylate. The results indicate the binding of melatonin in the nucleus of pineal parenchymal cells and in the outer nuclear layer of the retina of rats; binding is greater at night than during the day.     

Stimulatory effect of isoproterenol but not of dibutyryl cyclic AMP on N-acetyltransferase activity and melatonin content of Syrian hamster pineal gland in organ culture

The purpose of this study was to compare the response of Syrian hamster pineal glands in organ culture either to isoproterenol, a beta-adrenergic agonist, or to dibutyryl cyclic AMP. When pineal glands were collected at night, hamsters were exposed to light for 30 min to depress pineal N-acetyltransferase (NAT) activity and melatonin values to low levels. Pineal glands were removed and placed in organ culture containing either isoproterenol or dibutyryl cyclic AMP and subsequent changes in NAT activity and melatonin levels were measured. At night, isoproterenol (10(-7) or 10(-6) M) induced an increase in the NAT activity and melatonin levels in both pineals and culture media. However, dibutyryl cyclic AMP was either ineffective or minimally effective in stimulating these parameters at either different incubation times (2, 4, and 6 h) or drug concentrations (0.1, 0.5, and 1.0 mM). Conversely, when rat pineal glands were incubated with either isoproterenol (10(-7)) or dibutyryl cyclic AMP (0.5 mM) dramatic rises in NAT activity and melatonin levels were observed. In another experiment, hamster pineal glands were collected from animals killed either late in the light period (19.00 h) or in the latter half of the dark period. Isoproterenol promoted NAT activity and melatonin production only in glands collected in the latter half of the dark phase.     

Swimming depresses nighttime melatonin content without changing N-acetyltransferase activity in the rat pineal gland

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.     

Influence of prolactin on pineal gland activity in chicks

The aim of the present investigation is to ascertain the effects of prolactin on the pineal gland activity in chicks. Ovine prolactin treatment at all the dosages (total dosage: 15, 50, 100 IU per bird in 10 days) increased the mitotic frequency and nuclear size in the pineal gland of chicks. The rate of increase of these manifestations was seen in proportion to the amount of hormone administered. It is suggested that prolactin can perhaps stimulate the pineal gland function in chick and that it has a dose-dependent action in this gland.     

The human pineal gland and melatonin in aging and Alzheimer's disease

The pineal gland is a central structure in the circadian system which produces melatonin under the control of the central clock, the suprachiasmatic nucleus (SCN). The SCN and the output of the pineal gland, i.e. melatonin, are synchronized to the 24-hr day by environmental light, received by the retina and transmitted to the SCN via the retinohypothalamic tract. Melatonin not only plays an important role in the regulation of circadian rhythms, but also acts as antioxidant and neuroprotector that may be of importance in aging and Alzheimer's disease (AD). Circadian disorders, such as sleep-wake cycle disturbances, are associated with aging, and even more pronounced in AD. Many studies have reported disrupted melatonin production and rhythms in aging and in AD that, as we showed, are taking place as early as in the very first preclinical AD stages (neuropathological Braak stage I-II). Degeneration of the retina-SCN-pineal axis may underlie these changes. Our recent studies indicate that a dysfunction of the sympathetic regulation of pineal melatonin synthesis by the SCN is responsible for melatonin changes during the early AD stages. Reactivation of the circadian system (retina-SCN-pineal pathway) by means of light therapy and melatonin supplementation, to restore the circadian rhythm and to relieve the clinical circadian disturbances, has shown promising positive results.     

GABAergic signaling in the rat pineal gland

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ‐aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABA_A receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABA_B1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABA_A receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage‐gated Ca²⁺ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72‐h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine‐induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.     

Development of the rhythmic melatonin secretion in the embryonic chicken pineal gland

In order to elucidate details on the development of the circadian clock, the effects of light on the in vitro melatonin (MT) release and the presence of mRNAs of several clock genes in the embryonic chicken pineal gland were investigated. Chicken embryos of various developmental stages were exposed to stimuli of light in vitro in dynamic, four day long bioassay (perifusion). MT secretion and mRNA levels of Cry1, Cry2, Clock and Bmal2 clock genes were determined. Our conclusions: (1) environmental illumination modified MT secretion from explanted embryonic pineal glands as early as on the 13th embryonic day, (2) daily rhythm of MT release develops between embryonic days 16 and 18 under periodic environmental illumination. (3) Chicken Cry1, Cry2, Clock and Bmal2 clock gene mRNAs were also detected in glands of animals of 15th embryonic day. Although both MT secretion and clock genes have been developed by then, the circadian MT rhythm appears first on the 17th embryonic day. Either the mechanisms coupling the clock with the melatonin output or the synchronization of the individual pinealocytes develop around this age. Rhythmic MT release in the embryonic chicken pineal gland evolves only if the egg is exposed to rhythmic environmental stimuli.     

Daytime dark exposure increases pineal melatonin in rat pups

Abstract: Photic regulation of the pineal melatonin synthesis was studied in 3- to 21-day-old rat pups by exposing the animals to light at night (30–40 min) or to darkness during the day (30–240 min). The pineal melatonin contents were measured by radioimmunoassay. A significant day/night difference in the melatonin content and the nocturnal light-induced decrease were not found until second postnatal week. A novel finding was that at the age of 13–17 days a daytime dark exposure elevated the pineal melatonin content; it was twofold as compared with the normal daytime level and about half of the nocturnal peak level. In 21-day-old rats the response had disappeared, while the nocturnal suppression by light persisted. The dark-induced increase of the melatonin synthesis was independent of the opening of the eyelids which occurs in pups at the age of two weeks, but it was greater in maternally isolated than non-isolated pups. The results suggest that one component of the circadian regulatory system matures at the end of the third postnatal week. This mechanism inhibits the elevation of the melatonin synthesis by darkness during the daytime.     

Induction of photosensitivity in neonatal rat pineal gland

Pineal glands removed from neonatal rats at 5, 7, and 9 days of age and explanted into short-term culture, synthesized melatonin when stimulated with norepinephrine (NE); their melatonin synthesis could not be suppressed with bright white light. Dispersed pineal cell cultures or pineal explants prepared from 1-day-old neonates and held in culture for 7 or 9 days also synthesized melatonin when stimulated with NE, but in these cases melatonin synthesis was significantly suppressed by light, demonstrating that the pineals had become photosensitive while in culture. The development of photosensitivity in culture could be partially or completely abolished by the continuous presence of 1 or 10 microm of NE in the culture medium. The pineals of all nonmammalian vertebrates are photoreceptive, whereas those of mammals do not normally respond to light. We hypothesize that a mechanism to suppress pineal photosensitivity by using NE released from sympathetic nerve endings evolved early in the history of mammals.