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Horst-w. Korf - One of the best experts on this subject based on the ideXlab platform.
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Calcium oscillations in a subpopulation of S-antigen-immunoreactive Pinealocytes of the rainbow trout (Oncorhynchus mykiss).
Brain research, 1997Co-Authors: Susanne Kroeber, Christof Schomerus, Horst-w. KorfAbstract:Abstract By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca 2+ ] i was examined in isolated rainbow trout Pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the Pinealocytes exhibited spontaneous [Ca 2+ ] i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca 2+ or application of 10 μ M nifedipine caused a reversible breakdown of the [Ca 2+ ] i oscillations. Application of 60 mM KCl elevated [Ca 2+ ] i in 90% of the oscillating and 50% of the non-oscillating Pinealocytes. The effect of KCl was blocked by 50 μ M nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca 2+ ] i in trout Pinealocytes. Experiments with thapsigargin (2 μ M) revealed the presence of intracellular calcium stores in 80% of the trout Pinealocytes, but their role for regulation of [Ca 2+ ] i remains elusive. Treatment with norepinephrine (100 pM–50 μ M), previously shown to induce calcium release from intracellular calcium stores in rat Pinealocytes, had no apparent effect on [Ca 2+ ] i in any trout Pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.
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Regulation of the Intracellular Concentration of Free Calcium Ions in Pinealocytes of the Rainbow Trout and the Rat
Biological signals, 1997Co-Authors: Horst-w. Korf, Susanne Kroeber, Christof SchomerusAbstract:Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the Pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout Pinealocytes and in rat Pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated Pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout Pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout Pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout Pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout Pinealocyte. In rat Pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat Pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat Pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in Pinealocytes are complex and differ considerably between poikilothermic and mammalian species.
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Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat Pinealocytes: an immunocytochemical study
Cell and Tissue Research, 1995Co-Authors: Satoshi Tamotsu, Christof Schomerus, Jörg H. Stehle, Patrick H. Roseboom, Horst-w. KorfAbstract: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 α_1 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 α_1 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.
Regina P. Markus - One of the best experts on this subject based on the ideXlab platform.
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Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)
2016Co-Authors: Sanseray Da Silveira Cruz-machado, Luciana Pinato, Eduardo Koji Tamura, Emanuele Carvalho-sousa, Regina P. MarkusAbstract:The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or Pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or Pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (Pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in Pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-Pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our result
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glia Pinealocyte network the paracrine modulation of melatonin synthesis by tumor necrosis factor tnf
PLOS ONE, 2012Co-Authors: Sanseray Da Silveira Cruzmachado, Luciana Pinato, Eduardo Koji Tamura, Claudia Emanuele Carvalhosousa, Regina P. MarkusAbstract:The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or Pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or Pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (Pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in Pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-Pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.
David C Klein - One of the best experts on this subject based on the ideXlab platform.
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scRNA-seq reveals two transcriptionally distinct Pinealocyte populations.
2018Co-Authors: Joseph C. Mays, Martin F. Rath, Steven L Coon, Michael C. Kelly, Lynne Holtzclaw, Matthew W. Kelley, David C KleinAbstract:(A) Heatmap of expression values for top 10 most differential expressed genes (by effect size) for α- and β-Pinealocytes. Expression values are Z-scores of counts calculated between all cells of the two cell types. Each column represents one cell; random samples of 250 cells per cell type are shown. (B) Violin plots showing expression distribution differences between two Pinealocyte subtypes for three functional groups and one gene, Asmt. Y-Axis is either normalized counts or natural log (ln) of normalized counts. Horizontal lines represent the mean. (*) indicates p
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Differences in melatonin synthesis between α- and β-Pinealocytes inferred from scRNA-Seq.
2018Co-Authors: Joseph C. Mays, Martin F. Rath, Steven L Coon, Michael C. Kelly, Lynne Holtzclaw, Matthew W. Kelley, David C KleinAbstract:(A-B) Opacity indicates relative strength of the pathway module; greater opacity indicates a more active pathway. (A) Conversion of N-acetylserotonin (NAS) to melatonin in α-Pinealocytes is enhanced by increased ASMT activity and increased S-adenosyl methionine (SAM) availability, which is increased by greater ATP availability. ATP availability is increased by elevated ATP production from oxidative phosphorylation (OxPhos), as inferred by greater expression of mitochondrial genes in α-Pinealocytes. ATP availability is also increased by reduced consumption by protein synthesis, as inferred by decreased expression of ribosomal genes in α-Pinealocytes. (B) β-Pinealocytes also undergo melatonin synthesis, but do not have the same production increasing enhancements as α-Pinealocytes. (C) Melatonin (M) is synthesized in both Pinealocyte subtypes from N-acetylserotonin (NAS). NAS that is not converted to melatonin in β-Pinealocytes enters the α-Pinealocyte by passive diffusion through membranes and gap junctions (shown in blue). NAS is subsequently converted to melatonin by the high efficiency Asmt system in the α-Pinealocyte, thereby maximizing melatonin production.
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Melatonin Synthesis: Acetylserotonin O-Methyltransferase (ASMT) Is Strongly Expressed in a Subpopulation of Pinealocytes in the Male Rat Pineal Gland
Endocrinology, 2016Co-Authors: Martin F. Rath, Fernanda Gaspar Do Amaral, Morten Møller, Steven L Coon, Joan L Weller, David C KleinAbstract:The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase (TPH)-1 and aralkylamine N-acetyltransferase. In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat, and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in Pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive Pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocalize with S-antigen, a widely used Pinealocyte marker; this colocalization was seen in cells throughout the pineal complex and also in displaced Pinealocyte-like cells of the medial habenular nucleus. Inconsistent colocalization between ASMT and TPH protein was also detected in the pineal gland. ASMT protein was not detected in extraepithalamic parts of the central nervous system or in peripheral tissues. The findings in this report are of special interest because they provide reason to suspect that melatonin synthesis varies significantly among individual Pinealocytes.
Christof Schomerus - One of the best experts on this subject based on the ideXlab platform.
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Calcium oscillations in a subpopulation of S-antigen-immunoreactive Pinealocytes of the rainbow trout (Oncorhynchus mykiss).
Brain research, 1997Co-Authors: Susanne Kroeber, Christof Schomerus, Horst-w. KorfAbstract:Abstract By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca 2+ ] i was examined in isolated rainbow trout Pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the Pinealocytes exhibited spontaneous [Ca 2+ ] i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca 2+ or application of 10 μ M nifedipine caused a reversible breakdown of the [Ca 2+ ] i oscillations. Application of 60 mM KCl elevated [Ca 2+ ] i in 90% of the oscillating and 50% of the non-oscillating Pinealocytes. The effect of KCl was blocked by 50 μ M nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca 2+ ] i in trout Pinealocytes. Experiments with thapsigargin (2 μ M) revealed the presence of intracellular calcium stores in 80% of the trout Pinealocytes, but their role for regulation of [Ca 2+ ] i remains elusive. Treatment with norepinephrine (100 pM–50 μ M), previously shown to induce calcium release from intracellular calcium stores in rat Pinealocytes, had no apparent effect on [Ca 2+ ] i in any trout Pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.
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Regulation of the Intracellular Concentration of Free Calcium Ions in Pinealocytes of the Rainbow Trout and the Rat
Biological signals, 1997Co-Authors: Horst-w. Korf, Susanne Kroeber, Christof SchomerusAbstract:Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the Pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout Pinealocytes and in rat Pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated Pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout Pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout Pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout Pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout Pinealocyte. In rat Pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat Pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat Pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in Pinealocytes are complex and differ considerably between poikilothermic and mammalian species.
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Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat Pinealocytes: an immunocytochemical study
Cell and Tissue Research, 1995Co-Authors: Satoshi Tamotsu, Christof Schomerus, Jörg H. Stehle, Patrick H. Roseboom, Horst-w. KorfAbstract: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 α_1 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 α_1 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.
Morten Møller - One of the best experts on this subject based on the ideXlab platform.
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Melatonin Synthesis: Acetylserotonin O-Methyltransferase (ASMT) Is Strongly Expressed in a Subpopulation of Pinealocytes in the Male Rat Pineal Gland
Endocrinology, 2016Co-Authors: Martin F. Rath, Fernanda Gaspar Do Amaral, Morten Møller, Steven L Coon, Joan L Weller, David C KleinAbstract:The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase (TPH)-1 and aralkylamine N-acetyltransferase. In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat, and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in Pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive Pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocalize with S-antigen, a widely used Pinealocyte marker; this colocalization was seen in cells throughout the pineal complex and also in displaced Pinealocyte-like cells of the medial habenular nucleus. Inconsistent colocalization between ASMT and TPH protein was also detected in the pineal gland. ASMT protein was not detected in extraepithalamic parts of the central nervous system or in peripheral tissues. The findings in this report are of special interest because they provide reason to suspect that melatonin synthesis varies significantly among individual Pinealocytes.
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Peptidergic cells in the mammalian pineal gland. Morphological indications for a paracrine regulation of the Pinealocyte.
Biology of the cell, 1997Co-Authors: Morten MøllerAbstract:Several neuropeptides are present in the mammalian pineal gland. Most of these peptides, eg neuropeptide Y, vasoactive intestinal peptide, and peptide histidine isoleucine, are located in nerve fibres innervating the gland. In some mammalian species, neuropeptides are also found in cells scattered in the pineal parenchyma. In the rat, bipolar cells immunoreactive for somatostatin are present, just as cells containing mRNA encoding somatostatin can be detected in the gland by in situ hybridisation. In the pineal gland of the European hamster, many cells are immunoreactive for enkephalin. Ultrastructural cytochemical analysis of these cells reveals a Pinealocyte morphology. Processes from the opioidergic Pinealocytes terminate in the parenchyma between the non-immunoreactive Pinealocytes. Some of the processes contain small clear and large dense core vesicles and end in club shaped swellings which make synapse-like contacts with other Pinealocytes. The ultrastructural morphology suggests that the opioidergic cells exert a paracrine regulation on other Pinealocytes.