Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Dark Pulses (III)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought to be responsible for melatonin circadian rhythms. The enzyme has circadian properties--its rhythm persists in constant conditions, and it is precisely controlled by light and dark. Experiments are reported in which 4-h light or dark pulses were imposed on chicks (Gallus domesticus) over a 24-h period. Pineal NAT profiles were measured during and subsequent to the pulses. The phase of the NAT cycle following pulses was plotted to obtain phase-response curves. Light pulses produced a maximum phase shift (advance of 5 h) 8 h after the expected time of lights-out; dark pulses produced a maximum phase shift (advance of 4 h) 3 h after the expected time of lights-out. Maximum phase delays (-2 h) occurred 1-2 h after the expected lights-out for light pulses and 8 h after expected lights-on for dark pulses.     

Occurrence and regional distribution of striated muscle fibers in the rat pineal gland

Summary In a total of 96 rat pineals studied 31 were found to contain striated muscle fibers or their precursors. The muscle fibers were most frequently present in the stalk region and more frequently found in the left than in the right hemisphere. Size measurements revealed that the lengths of pineal muscle cell nuclei differ only slightly from those of the sphincter muscle of the iris. However, the yellowish appearance of pineal muscle cell nuclei under darkfield investigation, a phenomenon observed in all muscular tissues of mesenchymal origin and connective tissue cells, may support the hypothesis that pineal musculature is of mesenchymal rather than ectodermal origin.Supported by a grant (Vo 135/4) of the Deutsche Forschungsgemeinschaft within the Schwerpunktprogramm Neuroendokrinologie     

Immunohistochemical evidence for the presence of melatonin in the pineal gland,the retina and the Harderian gland

Summary The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20–25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10–16, 1980 (Vivien-Roels et al. 1980)The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates     

Occurrence and regional distribution of calcareous concretions in the rat pineal gland

Summary Serial sections of 90 Sprague-Dawley rat brains with the pineal in situ were scanned to determine the occurrence and regional distribution of calcareous concretions within the pineal gland and its surrounding leptomeningeal tissue. In 90 % of the cases examined concretions were found in varying number and appearance, predominantly lying in the dorsal region of the pineal gland and in the distal portion of the pineal stalk.Discussing the hypothesis advanced by Lukaszyk and Reiter (1975) that the origin of pineal concretions may be related to a neurosecretory process involving a pineal carrier protein, called neuroepiphysin, it is thought that, in view of the intra- and extra-pineal occurrence of concretions, processes other than secretion should be considered. Since in the pineal organ lymphatics are lacking it may well be that, due to a reduced drainage of tissue fluid, the coagulation of intercellular organic debris mingled with minerals increases with age.Supported by a grant (Vo 135/4) of the Deutsche Forschungsgemeinschaft within the Schwerpunktprogramm Neuroendokrinologie     

松果体昼夜节律生物钟分子机制的研究进展

在各种非哺乳类脊椎动物中 ,松果体起着中枢昼夜节律振荡器的作用。近来 ,在鸟类松果体中相继发现了几种钟基因 ,如Per、Cry、Clock和Bmal等 ,其表达的时间变化规律与哺乳类视交叉上核 (SCN)的非常相似。钟的振荡由其自身调控反馈环路的转录和翻译组成 ,鸟类松果体和哺乳类SCN似乎具有共同的钟振荡基本分子构架 ;若干钟基因产物作为正向或负向调节子影响钟的振荡 ;昼夜性的控时机制同时也需要翻译后事件的参与。这些过程对钟振荡器的稳定性和 /或钟导引的光输入通路有着重要的调控作用     

Acetylation of Serotonin in the Rabbit Pineal Gland: An N-Acetyltransferase with Properties Distinct from NAT1 and NAT2 Is Responsible

Two rabbit arylamine N-acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, p-aminosalicylic acid, and p-aminobenzoic acid. In the pineal gland, serotonin is metabolized to N-acetylserotonin by an unknown N-acetyl-transferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N-acetyltransferase (AA-NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as p-phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N-acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross-hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA-NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N-acetylation of serotonin.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24-Hour Cycles (IV)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Gallus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1-3 days of light-dark treatments during which NAT activity was measured in their pineal glands. (a) In LD12:12, NAT activity rose from less than 4.5 nmol/pineal gland/h during the light-time to 25-50 nmol/pineal gland/h in the dark-time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26-45% of the NAT activity cycle in LD12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12-h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator. (c) In less than 24-h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4:4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks' previously scheduled dark-time but not the previously scheduled light-time of LD12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8-h dark periods, even though the second one fell in the light-time of the prior LD12:12 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphetamine-induced changes in immunoreactive NPY in rat brain, pineal gland and plasma

Acute injection of d-amphetamine (10 mg/kg), administered to rats 60 minutes prior to sacrifice, induced a doubling of immunoreactive NPY (NPY-IR) in pineal gland. No changes, however, could be detected in levels of NPY-IR in grossly dissected or microdissected regions of rat brain, nor were changes evident in plasma level concentrations of NPY-IR following acute amphetamine pretreatment. When amphetamine was injected twice daily for six days and once more 60 minutes prior to sacrifice, levels of NPY-IR were decreased in caudate putamen and the paraventricular and dorsomedial nuclei of the hypothalamus, while concentrations of NPY-IR were increased in medial preoptic nucleus, pineal gland, and plasma. These data indicate that levels of NPY-IR are susceptible to manipulation by amphetamine, where the extent and direction of change (increase or decrease) depends on both the frequency of drug administration and the nature of the sampled tissue. Based on the effects of amphetamine on central and peripheral norepinephrine and epinephrine disposition observed in other studies, the data also suggest that NPY-IR and catecholamine dispositions are not directly correlated and may be inversely related in some tissue.     

Ultrastructural observations on the central innervation of the guinea-pig pineal gland

Summary In the present study the central innervation of the guinea-pig pineal gland was investigated. The habenulae and the pineal stalk contain myelinated and non-myelinated nerve fibres with few dense-cored and electron-lucent vesicles. Some myelinated fibres leave the main nerve fibre bundles, lose their myelin-sheaths and terminate in the pineal gland. Although direct proof is lacking, the non-myelinated fibres appear to end near the site where the bulk of the myelinated fibres are located. Here a neuropil area exists where synapses between non-myelinated fibre elements are abundant. Neurosecretory fibres were also seen. The results support the concept of functional interrelationships between hypothalamus, epithalamus and the pineal gland.     

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.     

High Accumulation of Calcium and Phosphorus in the Pineal Bodies with Aging

To elucidate compositional changes of the pineal body with aging, the authors investigated age-related changes of elements in the pineal body. After the ordinary dissection by medical students was finished, the pineal bodies and seven arteries were resected from the subjects ranging in age from 58 to 94 years. The element contents were determined by inductively coupled plasma atomic emission spectrometry. It was found that a high accumulation of Ca and P occurred in the pineal bodies with aging. Regarding the relationships among the elements, it was found that there were significant direct correlations among the contents of Ca, P, and Mg. With regard to the relationships between the pineal body and the arteries, no significant correlations were found in the Ca content between the pineal body and the arteries, such as the thoracic and abdominal aortas and the coronary, common carotid, pulmonary, splenic, and common iliac arteries.     

Diurnal Variations in [125I]Melatonin Binding by Rat Thymus Membranes: Effects of Continuous Light Exposure and Pinealectomy

Binding of melatonin by rat thymus membranes exhibited diurnal changes. Binding increased during the daytime and reached maximal values before entering the dark period. Then, binding decreased rapidly during the dark phase. In rats kept in light at night, binding of [¹²⁵I]melatonin by membranes was significantly higher than in animals that entered the normal dark period. Neonatal pinealectomy, which suppresses the circadian rhythm of melatonin, led to an increase in melatonin binding of 106%. Moreover, in animals maintained under continuous light exposure, which corresponds to functional pinealectomy, binding of melatonin by thymus membranes also increased in a time-dependent manner. The results support the hypothesis of a regulatory role of melatonin in the thymus in which melatonin downregulates its own binding sites.     

Determination of endogenous melatonin in the individual pineal glands of inbred mice using precolumn oxidation reversed-phase micro-high-performance liquid chromatography

The amount of endogenous melatonin in the individual pineal glands of inbred mice has been determined using reversed-phase micro-high-performance liquid chromatography after precolumn oxidation of melatonin to a compound having strong fluorescence. The fluorescent compound was identified as N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide. The excitation and emission wavelengths of this compound are 245 and 380 nm, respectively, and the fluorescence intensity is 6.8 times greater than that of melatonin. Molar absorptivity and fluorescence quantum yield of this compound are 46,300[L mol(-1)cm(-1)] and 0.31 (245 nm), respectively. The lower quantification limit of melatonin in biological samples using this precolumn oxidation method is 200 amol, and the calibration curve of spiked melatonin is linear from 200 amol to 50 fmol (r>0.999). The sensitivity of the present method is almost 10 times higher than that of the previous method. The values of endogenous melatonin obtained for ICR, C57BL, BALB/c, and AKR mice are 4.7, 6.1, 7.4, and 18.8 fmol/pineal gland, respectively. The amounts of endogenous pineal melatonin of these strains had not been clearly reported due to the poor enzymatic activities for melatonin biosynthesis; this is the first report that clearly demonstrates the existence of endogenous melatonin in these inbred mice.     

The synaptic ribbons of the guinea-pig pineal gland under normal and experimental conditions

Summary Synaptic ribbons (SR), functionally enigmatic structures of mammalian pinealocytes, were studied electron microscopically with regard to number, intracellular localization and topographical relationships, both under normal and experimental conditions. Pineal glands of guinea-pigs serving as controls contained 1.75 ribbon fields/unit area in the males and 2.58 in the females. In animals subjected to continuous illumination for 64 days the number of ribbon fields increased 20-fold in the males and 9-fold in the females. Continuous darkness (26 to 70 days) had varying effects; in some animals SR increased either strongly or moderately, in others they appeared unchanged. Under continuous illumination a higher percentage of ribbon fields bordered the cell membrane than in the controls. Moreover, paired ribbon fields occurred. The topographical analysis revealed that 98 % of the ribbon fields bordering the cell membrane lay opposite another pinealocyte and the remainder opposite nerve fibres, blood vessels and collagenous fibres. It is suggested that SR of mammalian pinealocytes do not represent non-functioning phylogenetic relics but true organelles possibly involved in coupling adjacent pinealocytes functionally.This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn.     

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.