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  • 1
    ISSN: 1432-0568
    Keywords: Melatonin synthesis ; Pineal gland ; Rat ; “Synaptic” ribbons ; Sympathetic stimulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Melatonin metabolism in the mammalian pineal gland is under the clear influence of sympathetic fibers originating in the superior cervical ganglia (SCG). Previous studies suggested that pineal “synaptic” ribbons (SR) as well are regulated by the gland's sympathetic innervation. To gain more insight into the mechanisms involved, we examined the effects of sympathetic stimulation on SR number and on the activity of melatonin forming enzymes, serotonin N-acetyltransferase (NAT) and hydoxyindole-O-methyltransferase (HIOMT). The SCG in adult male rats were stimulated electrically during daytime for either 15 or 120 min. Immediately following stimulation, the glands were removed and processed for electron microscopy and for the determination of NAT and HIOMT activities. No differences in pineal SR number, size or location were found in rats stimulated with either parameters when compared with sham-stimulated or control animals. While the activity of HIOMT remained unchanged, the activity of NAT was also unaltered following 15 min of stimulation, but was augmented approximately three-fold in animals stimulated for 120 min. It is concluded that if SR in the rat pineal gland are under sympathetic control, the regulation is different from that involved in melatonin formation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The rat pineal gland is a suitable model to investigate neurotransmitter-controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N-acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE-stimulation for 8 h induced accumulation of PSP1-catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A-CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 19 (2004), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In vertebrates, the rhythmic transcription of clock genes, regulated by their own gene products, provides the basis for self-sustaining circadian clockworks. These endogenous clocks are lost in most mammalian tissues, but not in the central pacemaker of the hypothalamic suprachiasmatic nucleus (SCN). An interesting model system to understand this phylogenetic shift in function of clock gene products is the rodent pineal gland, as its intrinsic clockwork was replaced during evolution by an input-dependent oscillator. By means of immunohistochemistry, immunoblotting and real time PCR, we investigated the day/night expression profiles of all major clock genes and their products in the pineal gland of one melatonin-proficient and one melatonin-deficient mouse strain. All clockwork elements known to be indispensable for a sustained rhythm generation in the SCN were also found in the pineal organ of both mouse strains. Only mPer1 mRNA and PER1 protein accumulation coincides with timecourses of many other pineal genes and their products, which are cyclicAMP inducible. Here, presented data together with the known mechanisms for regulation of the mPer1 gene in the rodent pineal gland forward the idea that in this tissue PER1 may have a trigger function for initiating the cycles of the clockwork's transcriptional/translational feedback loops.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In the rat pineal gland noradrenaline is released in large quantities from sympathetic nerve endings at the onset of darkness, thereby driving rhythmic melatonin synthesis with elevated levels at night-time. Upon release, noradrenaline interacts with postsynaptic β1-adrenergic receptors to activate the cyclic AMP signalling pathway. Well characterized third messengers of this signalling cascade affect cyclic AMP-inducible genes that are crucially involved in initiation, maintenance and termination of hormone production. Among these third messengers are CREB (cyclic AMP responsive element binding protein) as an activating and ICER (inducible cyclic AMP early repressor) as an inhibitory transcription factor. Because a cyclic AMP-inducible promoter element is present on the β1-adrenergic receptor gene, the expression of the receptor itself may be under control of the cyclic AMP-signalling pathway. By in situ hybridization, Northern blot analysis and RT-PCR we demonstrate a day/night rhythm in β1-adrenergic receptor mRNA in the rat pineal gland with elevated levels during the dark period. As this rhythm persists, under constant darkness but is abolished upon removal of the sympathetic innervation, it is truly circadian. A marked day/night difference in the levels of β1-adrenergic receptor mRNA becomes evident only after postnatal day 10, coinciding with the appearance of a functional cyclic AMP signalling pathway in the rat pineal gland. Furthermore, targeting ICER expression by transfection of pinealocytes with an antisense ICER construct, clearly indicates that the levels of the β1-adrenergic receptor mRNA are regulated by the cyclic AMP-signalling pathway in a feedback mechanism.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 14 (2001), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In neurons, a temporally restricted expression of cAMP-inducible genes is part of many developmental and adaptive processes. To understand such dynamics, the neuroendocrine rodent pineal gland provides an excellent model system as it has a clearly defined input, the neurotransmitter norepinephrine, and a measurable output, the hormone melatonin. In this system, a regulatory scenario has been deciphered, wherein cAMP-inducible genes are rapidly activated via the transcription factor phosphoCREB to induce transcriptional events necessary for an increase in hormone synthesis. However, among the activated genes is also the inhibitory transcription factor ICER. The increasing amount in ICER protein leads ultimately to the termination of mRNA accumulation of cAMP-inducible genes, including the gene for the Aa-nat that controls melatonin production. This shift in ratio of phosphoCREB and ICER levels that depends on the duration of stimulation can be interpreted as a self-restriction of cellular responses in neurons and has also been demonstrated to interfere with cellular plasticity in many non-neuronal systems.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    European journal of neuroscience 22 (2005), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Melatonin provides a rhythmic neuroendocrine output, driven by a central circadian clock that encodes information about phase and length of the night. In the hypophyseal pars tuberalis (PT), melatonin is crucial for rhythmic expression of the clock genes mPer1 and mCry1, and melatonin acting in the PT influences prolactin secretion from the pars distalis. To examine further the possibility of a circadian clockwork functioning in the PT, and the impact of melatonin on this tissue, we assessed circadian clock proteins by immunohistochemistry and compared the diurnal expression in the PT of wild type (WT), and MT1 melatonin receptor-deficient (MT1–/–) mice. While in the PT of WT mice mPER1, mPER2, and mCRY1 showed a pronounced rhythm, mCRY2, CLOCK, and BMAL1 were constitutively present. Despite reported differences in maximal levels and timing of mCry1, mPer1, and mPer2 RNAs, the corresponding protein levels peaked simultaneously during late day, suggesting a codependency for their stabilization and/or nuclear entry. MT1–/– mice had reduced levels of mPER1, mCRY1, CLOCK and BMAL1, consistent with the earlier reported reduction in mRNA expression of these clock genes. Surprisingly, mPER2-immunoreaction was constitutively low, although mPer2 was rhythmically expressed in the PT of MT1–/– mice. This suggests that mPER2 is degraded due to the reduced levels of its stabilizing interaction partners mPER1 and mCRY1. The results show that melatonin, acting through the MT1, determines availability of the circadian proteins mPER1, mPER2 and mCRY1 and thus plays a crucial role in regulating rhythmicity in PT cells.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1600-079X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: There is ever-increasing evidence that intrapineal peptides have an important role in the modulation of pineal melatonin synthesis. In the pineal gland of the Mongolian gerbil (Meriones unguiculatus), we have previously shown the presence of VIP-immunoreactive nerve fibers as well as pinealocytic VIP receptors. To assess the functional significance of these findings, 10 μl of a 1 μM or 1 nM solution of VIP were injected into the lateral ventricle of gerbils over a period of 10 min. Animals were killed 1.5 hr after injection, and the superficial pineal glands were excised and assayed for N-acetyltransferase (NAT) activity. Injection of the 1 μM VIP solution stimulated the NAT activity to values four times the control values. The results are compatible with an in vivo influence on the pineal gland indole metabolism of the nonsympathetic VIP-containing nerve fibers via VIP-receptors present in the gland.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1600-079X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In the rat pineal gland neuronal signals determine the rhythmic synthesis of the hormone melatonin. Norepinephrine (NE) is the principal neurotransmitter that drives hormone synthesis by activating the cAMP signaling pathway. This activation depends on transcriptional and posttranscriptional regulatory mechanisms. The cAMP-dependent transcriptional regulation of the rate-limiting enzyme of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) involves the activating transcription factor (TF) CREB and the inhibitory TF ICER. By silencing elements of this cAMP-dependent neuroendocrine transduction cascade we wished to gain further insight into the role of ICER in the regulation of gene expression in rat pineal gland. Inhibition of specific kinases in primary pinealocyte cultures showed that ICER induction depends pivotally on the activation of cAMP-dependent protein kinase II. Eliminating ICER's impact by transfecting antisense constructs into pinealocytes revealed a predominant β-adrenergic mechanism in regulating a cotransfected CRE-inducible reporter gene and notably, also the endogenous AA-NAT gene. Deciphering molecular details of the cAMP-dependent gene expression in mammalian pinealocytes provides a basis for understanding the general architecture of this signaling pathway that serves adaptive processes ubiquitously in the organism.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0878
    Keywords: Key words: Pinealocytes ; Cyclic AMP responsive element binding protein (CREB) ; Phosphorylated CREB ; β-Adrenergic receptor ; S-antigen (arrestin) ; Transcription factors ; Cell culture (Rat ; Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. In the present study we investigated whether norepinephrine, which stimulates melatonin biosynthesis in the mammalian pineal organ, causes phosphorylation of the cyclic AMP responsive element binding protein (CREB) in rat pinealocytes. Cells isolated from the pineal organ of adult male rats and cultured on coated coverslips were treated with norepinephrine, β- or α1-adrenergic agonists for 1, 5, 10, 20, 30, 60 or 300 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (p-CREB). Treatment with norepinephrine or β-adrenergic agonists resulted in a similar, time-dependent induction of p-CREB immunoreactivity, exclusively found in cell nuclei. The α1-adrenergic agonist phenylephrine did not induce p-CREB immunoreactivity at low doses (0.1 μM) or when high doses (10 μM) were applied in combination with a β-antagonist (propranolol, 0.1 μM). This indicates that induction of CREB phosphorylation is elicited by β-adrenergic receptor stimulation. The response was first seen after 10 min and reached a maximum after 30 to 60 min when more than 90% of the cells displayed p-CREB immunoreactivity. The intensity of the p-CREB immunoreactivity showed marked cell-to-cell variation, but nearly all immunoreactive cells were identified as pinealocytes by double-labeling with an antibody against the S-antigen, a pinealocyte-specific marker. The results show that norepinephrine stimulation induces p-CREB immunoreactivity by acting upon β-adrenergic receptors in virtually all rat pinealocytes. The findings support the notion that phosphorylation of CREB is a rather rapid and uniform response of pinealocytes to noradrenergic stimulation and thus is an important link between adrenoreceptor activation and subsequent gene expression in the rat pineal organ.
    Type of Medium: Electronic Resource
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