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.     

Norepinephrine Release in the Rat Pineal Gland: The Input from the Biological Clock Measured by In Vivo Microdialysis

Abstract: The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on-line in vivo microdialysis. NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringer's solution at concentrations of 10⁻⁶ and 10⁻⁵ M . This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments. Perfusion with 10⁻⁶ M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the α₂-receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic α₂-receptors. Long-term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.     

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.     

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.     

Nyctohemeral Rhythm in the Levels of S-Adenosylmethionine in the Rat Pineal Gland and Its Relationship to Melatonin Biosynthesis

Abstract: Liquid chromatographic techniques that permit the simultaneous analysis of S -adenosylmethionine, melatonin, and its intermediary metabolites N -acetyl-5-hydroxytryptamine and 5-hydroxytryptamine within individual pineal glands have been developed. S -Adenosylmethionine has been shown to undergo a marked nyctohemeral rhythm in the pineal gland of the rat, with maximal levels occurring during the light period and minimal levels during the dark period. Detailed studies of the temporal relationships between the levels of S -adenosylmethionine and those of melatonin and its intermediary metabolites suggest that an association exists between the levels of S -adenosylmethionine and the status of the biosynthesis of melatonin. Exposure of animals to continuous light and the administration of the β-adrenoreceptor antagonist propranolol were both found to inhibit the induction of melatonin synthesis and prevent the reduction in the levels of S -adenosylmethionine during the dark period. As a corollary the induction of melatonin biosynthesis following the administration of the β-adrenoreceptor agonist isoproterenol during the light period was accompanied by a marked decrease in the levels of S -adenosylmethionine in the pineal gland. The significance of the link between the nyctohemeral rhythms in the levels of S -adenosylmethionine and the biosynthesis of melatonin in the pineal gland is discussed in the context of the therapeutic efficacy of S -adenosylmethionine as an antidepressant.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland

Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC₅₀ of 50 n M , the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α₂-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC₅₀ of 5 n M , the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 m M ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.     

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.     

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.     

Neuroendocrine mediated effects of electromagnetic-field exposure: possible role of the pineal gland

Reports from recent epidemiological studies have suggested a possible association between extremely low frequency (ELF; including 50- or 60-Hz) electric- and magnetic-field exposure, and increased risk of certain cancers, depression, and miscarriage. ELF field-induced pineal gland dysfunction is a possible etiological factor in these effects. Work in our laboratory and elsewhere has shown that ELF electromagnetic-field exposure can alter the normal circadian rhythm of melatonin synthesis and release in the pineal gland. Consequences of reduced or inappropriately timed melatonin release on the endocrine, neuronal, and immune systems are discussed. Laboratory data linking ELF field exposure to changes in pineal circadian rhythms in both animals and humans are reviewed. The authors suggest that the pineal gland, in addition to being a convenient locus for measuring dyschronogenic effects of ELF field exposure, may play a central role in biological response to these fields via alterations in the melatonin signal.     

Progesterone inhibits, on a circadian basis, the release of melatonin by rat pineal perifusion

The effects of 10(-6) and 10(-9) M of progesterone were documented on isoproterenol-stimulated melatonin release by perifused pineal glands removed from female rats in diestrous at two different times of a 12 : 12 h light/dark cycle, 7 and 19 h after light onset (which corresponds to daytime and nighttime, respectively), to look for the existence of a circadian stage-dependence of the hormone effects. Three weeks before the experiment, the rats were synchronized with a 12 : 12 lighting regimen. Progesterone decreased by approximately 50% the release of melatonin during the light span, but not during the dark span. These results show the direct effects of this ovarian hormone on pineal melatonin release and strongly suggest a time-related effect of progesterone on pineal function.     

Does the circadian pacemaker act through cyclic AMP to drive the melatonin rhythm in chick pineal cells?

Cyclic AMP is a key regulator of melatonin production in the chick pineal gland. Agents that raise cyclic AMP levels (such as forskolin), or cyclic AMP analogues (such as 8-bromocyclic AMP), increase melatonin synthesis and release, whereas agents that lower cyclic AMP levels (including light) decrease melatonin synthesis and release. A circadian oscillator in these cells also raises and lowers melatonin output. We have been investigating the relationships between cyclic AMP and the circadian pacemaker in the regulation of melatonin production. In the chick pineal (unlike certain neuronal systems), the weight of the evidence indicates that cyclic AMP is not on an entrainment pathway to the circadian pacemaker. Instead, cyclic AMP appears to act downstream from the pacemaker. The pacemaker might itself act directly through cyclic AMP, regulating melatonin content by raising and lowering cyclic AMP levels. If this were the case, and if the effects of cyclic AMP levels on melatonin output are saturable (as they must be), then, in the face of such saturating levels of cyclic AMP, the pacemaker should no longer raise or lower melatonin output. To test this prediction, maximally effective concentrations of forskolin and 8-bromocyclic AMP were determined. Both agents markedly increased melatonin output. After 36 hr, cells were refractory to additional stimulation of melatonin output by addition of both agents together, or by higher concentrations of forskolin (although cyclic AMP levels could still be raised further). Nonetheless, the circadian pacemaker continued to raise and lower melatonin output: The rhythm persisted in the face of saturating levels of cyclic AMP. It is therefore suggested that the circadian pacemaker in chick pineal cells acts with, not through, cyclic AMP to regulate melatonin synthesis. Cyclic AMP and the pacemaker act synergistically to regulate serotonin N-acetyltransferase activity and the melatonin rhythm, with cyclic AMP mediating acute effects and amplitude regulation.     

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.     

Differential daily rhythms of melatonin in the pineal gland and gut of goldfish Carassius auratus in response to light

The objectives of this study were to test the effects of light on melatonin rhythms in the pineal gland and gut of goldfish Carassius auratus and to investigate whether melatonin function differed in these two tissues, which are photosensitive and non-photosensitive respectively. Rhythms were evaluated by measuring arylalkylamine N-acetyltransferase (AANAT2) and melatonin receptor 1 (MT-R1) mRNA expression and melatonin concentration in the pineal gland, gut (in vivo), and cell cultures of the two tissues (in vitro). Compared to control, pineal gland melatonin secretion was higher at night, whereas the 24-h dark and ophthalmectomy groups maintained higher AANAT2 and MT-R1 mRNA expression during the day. Melatonin levels and AANAT2 and MT-R1 mRNA expression in the gut were also the highest at night, but the 24-h light, dark, and ophthalmectomy groups did not significantly differ from control. Furthermore, we measured AANAT2 and MT-R1 mRNA expression in high temperature water (30 °C) to investigate differences in the antioxidant capacity of pineal gland vs. gut melatonin. Melatonin and H₂O₂ levels, as well as AANAT2 and MT-R1 mRNA expression, were all higher in the two tissues under thermal stress, compared with their levels at 22 °C. Taken together, our results suggest that light has no effect on melatonin patterns in the gut, which appears to exhibit its own circadian rhythm, but both gut and pineal gland melatonin exhibit similar antioxidant function.     

高原鼠兔松果腺褪黑激素含量昼夜节律的研究

自然光照条件下,高原鼠兔（Ｏｃｈｏｔｏｎａｃｕｒｚｏｎｉａｅ）松果腺褪黑激素（Ｍｅｌａｔｏｎｉｎ,ＭＬＴ）含量呈现明显的昼夜节律（Ｐ＜０．００１,夜间组含量均值与白天组含量均值差异显著性比较）。在２月份的实验中,对１８只鼠兔（体重１２２—１６４克）松果腺的采样时间分别为０２．００,０９．００,１２．００,１８．００,２２．００和２４．００时。白天ＭＬＴ含量波动为５６—６４微微克／松果腺,夜间波动为１１３—１７０微微克／松果腺。夜间ＭＬＴ含量高峰值出现在２４．００时。在１０月份的实验中,对６０只鼠兔（体重１０２—１５３克）松果腺的采样时间分别为０３．００,０６．００,０９．００,１２．００,１５．００,１８．００,２１．００和２４．００时。白天ＭＬＴ含量波动为７７—１１９微微克／松果腺,夜间波动为１３９—５０５微微克／松果腺。夜间ＭＬＴ含量高峰值出现在０３．００时。将２月份和１０月份高原鼠兔松果腺ＭＬＴ含量进行差异显著性比较,１０月组显著高于２月组（Ｐ＜０．０５）。结果表明,该动物的松果腺本身对光周期具有敏感性,它能够感知环境光周期的变化,成功地完成神经内分泌的转换。     

Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.     

Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

Static and extremely low frequency electromagnetic field exposure: Reported effects on the circadian production of melatonin

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (5HT) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 μtesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of 5HT and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.