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Treatment with forskolin for 8 hours during the day increases melatonin synthesis in the Syrian hamster pineal gland in organ culture: the long lag period is required for RNA synthesis.

The exposure of organ-cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in melatonin levels when glands were collected in the second half of the dark period and incubated for 4 h. However, when glands were collected at the beginning of the dark period and incubated with the same drug, a significant increase in melatonin content was observed only after 6-8 h. Likewise, when glands were collected at the beginning of the light period, forskolin stimulated melatonin synthesis only after 6-8 h of incubation with the drug. These results support the existence of a relatively long lag period necessary for the induction of melatonin production in the Syrian hamster pineal gland. Experiments with actinomycin D indicate that RNA synthesis occurs during the lag period; thus, actinomycin D blocks the induction of melatonin synthesis by forskolin in glands collected at the beginning of the dark period and incubated for 8 h. In contrast, when pineal glands were collected from hamsters killed in the second half of the dark period, and incubated with forskolin for either 4 or 8 h, actinomycin D was unable to block the induction of melatonin production. These data suggest that RNA, presumably messenger RNA, which is necessary for increasing hamster pineal melatonin synthesis, is synthesized and accumulates during the first half of the night.

Animals↗

Presynaptic effects of gamma-aminobutyric acid on norepinephrine release and uptake in rat pineal gland.

The effect of tau-aminobutyric acid (GABA) on pineal norepinephrine (NE) release was examined in vitro in the rat pineal gland. Exposure of pineal explants previously loaded with 3H-NE to 1-100 microM GABA caused a dose-dependent decrease of 3H-NE release triggered by 60 mM K+, with a threshold GABA concentration of 1 microM and IC50 of about 10 microM. The inhibitory effect of GABA was mimicked by the type B GABA agonist baclofen, displaying a similar dose-response relationship as GABA. The type A GABA agonist muscimol increased depolarization-induced 3H-NE release, while the co-incubation with GABA and the type A receptor antagonist bicuculline augmented significantly GABA's depressive effect on 3H-NE release. Bicuculline alone brought about a significant decrease of 3H-NE release. Neither GABA, nor baclofen, muscimol or bicuculline, modified the spontaneous pineal 3H-NE efflux. Assessment of 3H-NE uptake at a low NE concentration (0.5 microM) indicated that GABA decreased it in a dose-dependent manner (IC50 = 100 microM) through an effect blocked by bicuculline and mimicked by muscimol but not by baclofen; at a 5 microM-3H-NE concentration a bicuculline-sensitive GABA augmentation of uptake was found. A kinetic analysis study of the pineal NE uptake process indicated that GABA augmented both Vmax and Km of transmitter uptake. These results indicate that GABA may be a significant regulatory signal for rat pineal sympathetic synapses.

Animals↗

Cytology of the pineal gland: changes produced by various treatments.

The cytological appearance of the pineal body is related to stage of development or age, time of day, exposure to illumination, season of the year, and reproductive state. Experimental manipulations that alter the normal cytology of the pineal include hypophysectomy, administration of any of several hormones or drugs, blinding, denervation and exposure to stressful influences such as heat or cold. The effects of blinding are similar to the effects of continuous darkness on the pineal's cytology. Ultrastructural examinations reveal a number of changes in the organelles, especially an increased number of vesicles and complex membranous whorls. These features suggest heightened activity and generally are coincident with sexual quiescence. Membranous whorls also may be induced by cold exposure. Prolonged or continuous illumination leads to a reduction in the size of pinealocytes and to changes in their organelles that suggest reduced activity. Such alterations can often be correlated with heightened sexual activity. Sympathetic denervation of pineal glands also results in atrophy of the pinealocytes. Reproductive condition is reflected in the appearance of the pinealocytes. The estrous cycle, pregnancy, castration and the administration of gonadotropins or gonadal steroids affect pinealocyte structure. A feedback system involving the gonads and pineal gland is indicated.

Aging↗

Molecular cloning of the arylalkylamine-N-acetyltransferase and daily variations of its mRNA expression in the Syrian hamster pineal gland.

The arylalkylamine-N-acetyltransferase (AA-NAT) expressed in the vertebrate pineal gland catalyzes the N-acetylation of the serotonin into N-acetylserotonin and is considered to be the rate limiting enzyme of the pineal melatonin synthesis. Indeed, dramatic changes in its activity throughout the 24-h period drive the large day/night variations in plasma melatonin concentrations. Recently, AA-NAT was cloned in the rat pineal. In this species, AA-NAT mRNA variations were demonstrated to be responsible of the well known AA-NAT activity and plasma melatonin circadian fluctuations. In the Syrian hamster, the pineal melatonin secretion pattern is characterized by a late-night short-duration peak of melatonin synthesis. We investigated whether this typical pattern could be due to a late-night delayed pineal AA-NAT mRNA expression. The first part of our study was dedicated to the molecular cloning of a Syrian hamster AA-NAT cDNA. A PCR-generated clone of 1045 bp encoding the AA-NAT has been isolated and sequenced. In situ hybridization using an AA-NAT cRNA probe revealed that the AA-NAT mRNA expression undergoes strong daily fluctuations in the Syrian hamster pineal, with undetectable level in the second half of the light period and a dramatic increase at night. After lights off, the AA-NAT mRNA expression requires 6-7 h to reach its maximum expression. This result thus suggests that the transcription of the AA-NAT mRNA in the Syrian pineal gland determines the lag period in pineal responsiveness and melatonin synthesis to darkness.

Amino Acid Sequence↗

Expression of C-type natriuretic peptide in the bovine pineal gland.

Recent studies have pointed to membrane-bound guanylyl cyclases (GCs) type A and type B in the rat pineal gland, which augment levels of cyclic GMP (cGMP) in response to atrial natriuretic peptide (ANP), brain-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The present report demonstrates for the first time the expression of CNP in the bovine pineal gland. The CNP prohormone transcript (unlike pre-pro-ANP) was found by reverse transcriptase (RT)-PCR in bovine pineal extracts. CNP immunoreactivity (ir) was revealed in a subpopulation of pinealocytes in situ and in nearly all pinealocytes in culture. Electron microscopic immunohistochemical investigations showed the presence of CNP-ir in cytoplasmic vesicles, providing evidence for the potential secretion of this peptide by pineal cells. Furthermore, the CNP receptor (GC-B) and GC-A (receptor for ANP and BNP) were identified by RT-PCR. Although melatonin secretion was unaffected, natriuretic peptides were able to elevate markedly cGMP production in cultured bovine pinealocytes with a rank order of potency of CNP > BNP = ANP. These findings describe a tissue CNP system in the bovine pineal gland and suggest that CNP may be a local auto- or paracrine modulator of pineal function.

Animals↗

Characteristics of a prompt morphodynamic response of the pineal gland to an acute ACTH injection.

The morphodynamic response of the pineal gland in the period 5-30 min after a single ACTH injection was studied. In 5 min a saline injection, functioning as a control, elicited a prompt release of the pineal active compounds advocated to be contained in clear vesicles and structures traditionally labeled lipid droplets. In the subsequent time period, 15 min, a rise in the relative volume of the granular endoplasmic reticulum (GER) and the Golgi apparatus, as well as prosecretory granules, was interpreted as a sign of an increased pinealocyte activity which leads to the restoration of the pineal gland endocrine potential. A decline in the increased mass of this membranous elaborative compartment in 30 min could be understood as an illustration of the stabilisation of the pinealocyte stress-evoked response. The ACTH injection did not apparently change the pattern of the morphodynamic pinealocyte response promoted by the saline injection in 5 min. However, starting from 15 min, a significant increase of the mass of GER and the Golgi apparatus was found in both pinealocyte populations. The augmented presence of prosecretory granules and lipid droplets, in comparison with the saline-injected animals in the respective time period, as well as the absence of a clearly observable evidence of the pinealocyte secretory activity pointed out that the ACTH stimulative action on the pinealocyte membranous elaborative compartment rather facilitated the pinealocyte endocrine recovery than supported their current secretory activity. From the functional viewpoint, the results obtained suggest that a stress-induced ACTH burst could modulate the post-secretory restitution of the endocrine potential of previously stress-activated pinealocytes.

Adrenocorticotropic Hormone↗

Light and electron microscopic examination of pineal gland in rats exposed to constant light and constant darkness.

OBJECTIVE: This study was aimed to examine the pineal gland of rats exposed to constant light and darkness at light and electron microscopic level. DESIGN: For this purpose 18 male Wistar rats were used. Animals were divided into three groups. Rats in group I (Control) were kept under 12 hrs light: 12 hrs dark conditions. Rats in group II were exposed to constant darkness, while rats in group III were exposed to constant light for 6 weeks. At the end of the experiment, all animals were killed by decapitation. The pineal glands of rats were removed, then processed for light and electron microscopy. RESULTS: In our study, extensive number of pinealocytes was observed in the structure of pineal gland of rats exposed to constant darkness and some of the observed pinealocytes were determined to contain double nucleoli. Furthermore, mitochondria and lipid droplets in the cytoplasm of pinealocytes were increased and rough endoplasmic reticulum sacs were enlarged in this group. Whereas, in rats those exposed to the constant light, a decrease in pinealocyte intensity was associated with increase in the connective tissue between parenchymal cells. Additionally, mitochondria and lipid droplets in the cytoplasm of cells were decreased. CONCLUSIONS: It was observed that the pinealocyte cell activity of rats exposed to constant darkness was increased but decreased in rats exposed to constant light.

Adaptation, Physiological↗

Melatonin-like immunoreactivity in the pineal gland of the cow: an immunohistochemical study.

With a view to checking the presence of melatonin in the pineal gland of the cow, in the present work we used six adult animals, ranging in age from one to six years, which were sacrificed at dawn. Sections of 6 micro m thickness of Bouin-fixed and paraffin-embedded pineal glands were incubated in an anti-melatonin serum, which was provided by the Institute for Molecular and Cellular Recognition, Gunma University, Maebshi, Japan. After incubation and successive washings in PBS, some of the sections were treated with the avidin-biotin-peroxidase complex (ABC) technique using antisera from Sigma, and developed with the method of Graham and Karnovsky (which employs 3,3'-diaminobenzidine and H2O2 as developer). Other sections were incubated in a goat-anti-rabbit IgG (H+L) bound to fluorochrome Cy5 for immunofluorescence studies. An intense reaction for melatonin was observed in the cytoplasm but not in the nucleus of melatonin secreting pinealocytes located in peripheral and intermediate zones of the pineal gland. Immunoabsorption of the antimelatonin primary antibody with melatonin at a dilution of 10 mM per 0.1 ml of serum prevented the reaction, as happened when any of the antisera used in the procedure were used. Immunoabsorption of anti-melatonin serum with different amounts of bovine albumin (ranging between 1/5 to 1/50) failed to inhibit the immunoreactivity. When a bovine anti-albumin antibody was employed, working with the above methods, no immunoreaction was detected. Our data suggest that the pinealocytes of cows sacrificed at dawn contain immunoreactive melatonin.

Animals↗

Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock.

In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine-N-acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3-day-old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock-controlled rhythmic expression of zfaanat2, and that a 1-h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up-regulated by light but is not regulated by the circadian oscillator. To establish the association between light-induced zper2 expression and light-induced clock-controlled zfaanat2 rhythm, zPer2 knock-down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1-h light pulse, was abolished in zPer2 knock-down embryos. These experiments indicated that light-induced zper2 expression is crucial for establishment of the clock-controlled zfaanat2 rhythm in the zebrafish pineal gland.

Animals↗

Atypical 24-hour rhythms of serotonin N-acetyltransferase activity in the rat pineal gland.

Previous long-term studies have shown that in the pineal gland of rats melatonin synthesis is subject to infradian rhythms with periods between 4 and 7 days. Since in these studies melatonin-related parameters were measured at one timepoint of a 24-hr cycle only, the aim of the present investigation was to extend these experiments by more frequent sampling, to characterize the infradian rhythmicity in more detail. Male Sprague-Dawley rats kept under a light schedule of LD 12:12 (lights on at 0700) were killed at 6-hr intervals on 8 consecutive days. After decapitation the pineal gland was rapidly dissected out, followed by measurements of one of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) activity. It was found that pineal NAT activity exhibited the well known day/night rhythm, i.e. low activity during daytime and strikingly enhanced activity at night, during the first 4 days of the experiment. On the fifth night (from Saturday to Sunday) an unusually high NAT peak occurred at 2400 hr, followed by two atypical 24-hr cycles. In the first cycle the midnight and 0600 hr values were equal and in the second cycle the 0600 hr value was significantly higher than the midnight value. To investigate whether the unusually high NAT peak was a single event or not, four additional short-term experiments were carried out at 2400 hr on 4 consecutive weekends, from Friday to Monday. In each of the four 4-day experiments a distinctly higher peak of NAT activity was found on Saturday, but with time the peaks became less prominent.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗

Influence of dehydration on activity changes of pineal gland and subcommissural organ cells in Rana temporaria L. in annual cycle.

The karyometric analysis of the cells of the pineal gland and subcommissural organ in Rana temporaria L. was carried out under the influence of dehydration in the annual cycle. The dehydration stress was found to retard the activity of the pineal gland and at the same time to stimulate the activity of the subcommissural organ apart from the breeding period and the middle period of active life on land. The results showed that both the pineal gland and the subcommissural organ take part in the regulation of water balance in the species under study.

Animals↗

A synergistic interaction between histamine and vasoactive intestinal peptide (VIP) on cyclic AMP production in the chick pineal gland.

The effects of vasoactive intestinal peptide (VIP) and histamine, alone and in combination, on cyclic AMP formation have been studied in selected tissues of the central nervous system (CNS) of chick. VIP strongly stimulated cyclic AMP production in the pineal gland, moderately in the retina (maintained in "eye-cup" preparations), and had no effect in cerebral cortical slices. Combination of VIP with forskolin produced a synergistic response in the pineal gland; in the cerebral cortex VIP did not influence the elevation of cyclic AMP production evoked by forskolin, histamine and isoprenaline. Histamine stimulated cyclic AMP synthesis in the all tested CNS tissues, showing the following order of sensitivity to the amine: pineal gland > cerebral cortex > retina. The effects of histamine were stronger in the presence of forskolin. A combination of VIP and histamine produced the cyclic AMP response in the pineal gland clearly more than additive; such a synergistic interaction was antagonized by aminopotentidine, an accepted in mammals H2-histamine receptor blocker. It is concluded that both VIP and histamine can be considered as functionally important neuromodulators in avians (similar to mammalian species). These two substances may play in concert to regulate the pineal physiology in avian species.

Amino Acid Sequence↗

The melatonin generating system in the rat retina and pineal gland: effect of single and repeated electroconvulsive shock (ECS).

N-Acetyltransferase (NAT), a rate-limiting enzyme in the melatonin synthesis which converts serotonin to N-acetylserotonin, shows a distinct circadian rhythm in the rat pineal gland and retina, with low activities during the light phase and peak activities during the dark phase. Hydroxyindole-O-methyltransferase (HIOMT), an enzyme which methylates N-acetylserotonin to melatonin, did not show any significant diurnal variations in both analyzed tissues. Isoproterenol, a selective beta-adrenoceptor agonist, when administered during morning hours of the light phase, markedly increased NAT activity in the pineal gland, but not in the retina. Electroconvulsive shock (ECS), especially when applied repeatedly (ECS x 10, once daily) significantly increased NAT activity in the retina and tended to decrease the enzyme activity in the pineal gland in isoproterenol-treated rats. ECS x 10 slightly increased and decreased the nocturnally-stimulated NAT activity in the rat retina and pineal gland, respectively.

Acetylserotonin O-Methyltransferase↗

Day/night changes of pineal gland volume and pinealocyte nuclear size assessed over 10 consecutive days.

Previous studies aimed at elucidating day/night changes of pineal gland size and pinealocyte nuclear volume have yielded contradictory results. The aim of the present investigation was to examine the above parameters over a period of 10 successive days, at 6-hour intervals under a lighting regimen of LD 12:12 (7 a.m. to 7 p.m.). It was found that changes in pineal gland volume could not be correlated with the light/dark phases; instead, a rhythm with a two-day period was encountered. Pinealocyte nuclear volume, by contrast, showed clear circadian changes, exhibiting troughs during photophase and peaks during scotophase. In this experimental series, the karyometric results conformed to the melatonin forming rhythm in the rat pineal gland as reported in the literature.

Animals↗

Extraretinal mechanisms mediate light-induced changes in neonatal rat pineal gland N-acetyltransferase activity.

Nocturnal light-induced changes in pineal gland N-acetyltransferase activity were investigated in intact or enucleated 4-, 6-, or 8-day-old maternally isolated rats maintained at different ambient temperatures. Nighttime enzyme activity was increased markedly in rats maintained in 23 degrees C compared to 35 degrees C environments. Four hours of nocturnal light significantly reduced N-acetyltransferase activity in intact rats at all ages studied when the animals were maintained at 35 degrees C. Shorter duration (1 min) nocturnal light reduced N-acetyltransferase activity in intact rats 6 days of age. However, the enzyme activity suppression observed after the 4 hr of nocturnal light was still present in 4-day-old rats (but not older animals) even when the younger animals had been made blind by bilateral orbital enucleation. Taken together, these data suggest that an extraretinal mechanism may mediate light-induced changes in some aspects of pineal gland indoleamine biochemistry in newborn rats.

Acetyltransferases↗

Analysis of gene expression following norepinephrine stimulation in the rat pineal gland using DNA microarray technique.

Several studies have demonstrated that norepinephrine (NE) is the critical neurotransmitter for the regulation of gene expression in the pineal gland. We studied the acute effect of NE stimulation in cultured rat pineal glands using Affymetrix rat genome microarray GeneChip probe arrays. Our data demonstrate that NE stimulation affects regulation of several genes; 44 and 29 genes were up- or down-regulated more than 2.5-fold, respectively. As shown in previous studies, arylalkylamine N-acetyltransferase, cyclic AMP responsive element modulator, jun-B and c-fos mRNA levels were increased by NE stimulation. Genes that were not previously reported and increased by NE stimulation in the pineal gland were protein tyrosine phosphatase, nuclear receptors, and activity and neurotransmitter-induced early genes. Unlike up-regulated genes, most of the down-regulated genes were not reported previously. Genes encoding enzymes involved in metabolism and structural proteins were decreased following NE stimulation. Identification of genes affected by NE stimulation would provide valuable information to understanding pineal biology fully.

Adrenergic alpha-Agonists↗

In vivo administration of isoproterenol or forskolin during the light phase induces increases in the melatonin content of the Syrian hamster pineal gland without a rise in N-acetyltransferase activity.

In vivo melatonin production was stimulated during the daytime in pineal glands of female Syrian hamsters following the administration of several injections of either isoproterenol, a beta-receptor agonist, or forskolin, an adenylate cyclase stimulator. The large increase in melatonin following either isoproterenol or forskolin administration was not accompanied by significant changes in N-acetyltransferase (NAT) activity. The results suggest that the Syrian hamster pineal gland, as in other species, responds by producing melatonin during the light phase if the stimulus is adjusted to its particular and specific regulatory mechanisms, i.e., if beta-adrenergic stimulation is continued for 4-8 h. The lack of a commensurate increase in NAT activity raises the question of the need of maximal enzymatic activity for a significant rise in melatonin production in the Syrian hamster pineal gland.

Acetyltransferases↗

[Development of the views on the function of the pineal gland].

Physiology has been collecting the knowledge about the functional significance of the pineal gland during the last 30 years. The recent scientific knowledge about the physiological functions of this gland is compared with the historical development of the views about the role of the pineal gland in the human body.

Animals↗