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.     

Melatonin in rat milk and the likelihood of its role in postnatal maternal entrainment of rhythms

The rhythm of melatonin in rat milk and the capacity of pups to synthesize and metabolize melatonin were studied. Melatonin was undetectable in milk in the light (< 21 pM), but increased rapidly 2-4 h after dark to peak at 357 +/- 66 pM at mid-dark. Oral or subcutaneous administration of melatonin to 5- and 10-day-old pups resulted in peak plasma melatonin levels 30 min after administration and rapid metabolism. Increases in pineal and plasma melatonin levels at night were detected at 5 and 6 days of age, respectively. Isoproterenol administration (2 microg/g body wt) at mid-light to day 10 pups increased plasma melatonin from 312 +/- 40 pM to 1,298 +/- 160 pM, whereas propranolol (2 microg/g body wt) suppressed nocturnal melatonin secretion from 1,270 +/- 128 pM to 395 +/- 66 pM. The rise of pineal and plasma melatonin in day 10 pups occurred 1 and 2 h after dark onset, respectively, preceding the onset in dams by 3 and 4 h, respectively. Propranolol administration to 2- and 5-day lactating dams inhibited plasma and milk melatonin at night but had no effect on their suckling pups. Transfer of melatonin via the milk is unlikely to provide an entraining signal for rat pups.     

Daily and circadian variations of the pineal and ocular melatonin contents and their contributions to the circulating melatonin in the Japanese newt, Cynops pyrrhogaster

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LD 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px+Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.     

Light at night cannot suppress pineal melatonin levels in the lizard Anolis carolinensis

Up to 2 hr exposure of anoles to high intensity natural or artificial illumination at mid-dark does not suppress pineal melatonin levels. The results support the hypothesis that the lizard pineal is completely insensitive to acute exposure to light at night which is in direct contrast to the effects of light in higher vertebrates.     

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.     

Serum melatonin and pineal indoleamine metabolism in a species with a small day/night N-acetyltransferase rhythm

Ovine serum and pineal melatonin levels are low during the day, increase five to ten-fold at night, decrease during a light pulse at night, and rapidly increase to night levels following the light-dark transition. N-Acetyltransferase activity increases three-fold at night, falls significantly in response to the light pulse, but does not increase following the light pulse. No significant change in N-acetylserotonin occurs under these conditions. These results suggest that the biochemical mechanisms controlling pineal melatonin synthesis in the sheep pineal gland may be different from those in the rat.     

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

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

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

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.     

Pineal melatonin rhythms in female Turkish hamsters: effects of photoperiod and hibernation

Daily rhythms of pineal and serum melatonin content were characterized for adult female Turkish hamsters (Mesocricetus brandti) exposed to long days (16L:8D, 22 degrees C) or after transfer to short days (10L:14D, 22 degrees C). The nocturnal peak of pineal melatonin content was found to be approximately 3 b greater in duration on short than on long days. Changes in levels of serum melatonin closely paralleled those of pineal melatonin. Thus, an effect of photoperiod on synthesis and secretion of pineal melatonin was demonstrated. In a separate experiment, female hamsters were induced to hibernate by exposure to a short-day, cold environment (10L:14D, 6 degrees C). During the 4 to 5-mo hibernation season, Turkish hamsters are known to display 4 to 8-day hours of torpor (body temperature = 7-9 degrees C) alternating with 1 to 3-day intervals of euthermia (body temperature = 35-37 degrees C). Little evidence of nocturnal synthesis or secretion of pineal melatonin was detected in females sampled during torpor. However, animals sampled during the first day after arousal from a torpor bout displayed melatonin rhythms no different in phase or amplitude from those seen in females held at 22 degrees C. Thus, despite the absence of pineal melatonin output during torpor, the pineal gland of hibernating Turkish hamsters produces an appropriately phased, rhythmic melatonin signal during intervals of euthermia.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

Radioimmunoassay for melatonin and its application to fowl pineal research

A radioimmunoassay for melatonin has been developed after raising anti-melatonin antibodies in rabbit. Melatonin was extracted from serum or pineal gland of chickens (Gallus domesticus). The radioimmunoassay was performed by using 3H-melatonin as tracer. The standard curve covered the range 0.022-0.345 pmol/vial and the KD value for melatonin was estimated at 1.37 x 10(10) l/mol. The antiserum specificity has been analysed, none of the common melatonin analogues influencing this method of melatonin measurement. The intra-assay variability was 7.2% for serum samples and 8.6% for pineal extract. The inter-assay variability for this biological sample was 15.3% and 6.4% respectively.     

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.     

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

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

LH inhibitory properties of aqueous extracts of rat pineal glands

Crude aqueous extracts of rat pineal glands markedly inhibited the 24 and 48 hour postcastration rise of serum LH in mature male rats. After partial purification by exclusion chromatography on Sephadex G-25 the LH inhibitory activity was found to reside in a volume not coinciding with that of melatonin, indicating that some substance other than this indole may be responsible for the anti-LH property in aqueous extracts of rat pineal glands.     

Melatonin binding sites in senegal sole: day/night changes in density and location in different regions of the brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B(max)) at mid-light (ZT06) and mid-dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2-[(125)I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid-light (189.5+/-46 pg/ml) than mid-dark (455.5+/-163 pg/ml). Values of B(max) were statistically significantly higher in the optic tectum (5.6+/-0.6 and 12.3+/-1 fmol/mg prot, at mid-light and mid-dark, respectively) and in the cerebellum (7.7+/-1.1 and 10.6+/-1.3 fmol/mg prot, at mid-light and mid-dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down-regulation.     

Antigonadal effects of blinding in the presence of antibody against circulating melatonin

The effect of melatonin on reproductive function in the rat was studied. Reproductive organ weights and sex hormone levels were compared between sighted controls and animals which were either blinded, blinded and pinealectomized or blinded and immunized against circulating melatonin. Circulating androgens as measured by accessory sexual organ weights were significantly reduced by blinding. This effect was reversed by pinealectomy but not by immunization. Blinding also increased pineal melatonin levels but there were no differences in the plasma levels of luteinizing hormone. Circulating testosterone levels and pineal melatonin levels of immunized animals did not differ from those of blinded controls. These findings confirm reports that pineal stimulation by blinding enhances pineal melatonin content and inhibits accessory sex organ development. Circulating melatonin does not appear to be the mediator of the stimulated pineal's antigonadal effects in the rat since, in contrast to pinealectomy, neutralization of circulating melatonin failed to reverse accessory organ regression.     

Vasoactive Intestinal Peptide Stimulates Chick Pineal Melatonin Production and Interacts with Other Stimulatory and Inhibitory Agents but Does Not Show α1-Adrenergic Potentiation

Vasoactive intestinal peptide (VIP) is known to mimic the effects of beta-adrenergic receptor stimulation in the rat pineal, including marked potentiation by alpha 1-adrenergic receptor stimulation, and to cause increased melatonin synthesis. In contrast, the chick pineal does not respond to beta-adrenergic stimulation, and melatonin synthesis is inhibited by norepinephrine via an alpha 2-adrenergic receptor. The present experiments show that chick pineal cells in primary culture do, however, respond to VIP with increased melatonin production. The effect of VIP was inhibited by addition of norepinephrine or of nitrendipine or by exposing the cells to "unexpected" white light. Stimulation by VIP was enhanced by addition of forskolin or Bay K 8644 but not by alpha 1-adrenergic receptor stimulations. Although stimulation by VIP appears similar in the chick pineal to that seen in the rat pineal and other systems, "dual-receptor regulation," at least with alpha 1-adrenergic receptors, appears to be absent.     

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction with alpha 1-adrenoceptors; (c) GABA inhibits melatonin production in vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.