Analysis of global gene expression: Studies are in progress which have characterized gene expression in the pineal gland. The first stage has involved analysis of the rat pineal gland: """"""""The rodent pineal transcriptome was investigated ..... using microarray gene expression. Comparison of midday and midnight expression profiles revealed that a global >2-fold change in the expression of 1000 genes, 2/3 of which increase at night. Among these, 400 increase >4- fold in expression;studies in organ culture reveal that in nearly all cases, the expression of the highly upregulated genes is induced by treatment with NE or cyclic nucleotide analogs. These findings are consistent with the conclusion that NE-cyclic nucleotide signaling is the primary mechanism responsible for the nocturnal increase in gene expression. However, it is also clear that other mechanisms are involved, because a small number of highly rhythmic genes are not induced or are weakly induced by NE treatment. Comparison of the level of gene expression in the pineal gland to the median expression in other tissues indicates that a set of >300 genes are expressed >8- fold higher in the pineal gland. A significant subset of the most highly expressed genes encode proteins involved in melatonin synthesis and the control of this process, including signalling via adrenergic receptors and second messengers including cyclic nucleotides, Ca++ and phospholipids. Clusters of highly expressed genes are associated with the cellular biology of thyroid hormone, retinoid acid, glutamate biology;and, with metal ion homeostasis, membrane trafficking, and the immune response. Other highly and/or rhythmically expressed genes also encode transcription factors, ion channels, transporters, receptors, regulatory molecules and secreted products that have not previously appeared in the pineal literature. Comparison of the pineal gene expression profile to that of several other tissues adds to the evidence that the pineal gland is most similar to the retina by expanding the number of genes that are highly expressed exclusively in these two tissues. This study indicates that control of pineal biology is significantly more complex than previously thought, that the number of highly expressed genes in the pineal gland and retina is higher than previously thought, and also provides molecular evidence to suspect that the gland might function outside of the highly conserved role it plays in melatonin production."""""""" From (Bailey et al, in preparation). The work on the rodent pineal gland is being followed up with similar work on the pineal gland of the monkey and human, so as to determine the similarity of the patterns of gene expression in these three tissues. The results of the analysis of the rodent pineal gland has triggered a number of studies, some of which have been published, which have focused on genes that have been highlighted by the microarray studies. An example is detailed in HD000095-37. Control of dopamine signal transduction in the pineal gland: Dopamine plays a broad role in biology through actions mediated by specific G-protein coupled receptors. """"""""We have discovered that the expression of the gene that encodes the dopamine D4 receptor (Drd4), can change rapidly. Drd4 mRNA increases 20-fold at night in the pineal gland and retina to levels that are >10-fold higher than those in other tissues The abundance of pineal Drd4 transcripts is controlled by the well described circadian regulatory system that controls pineal function. In vitro studies indicate that Drd4 is induced by an And gate mechanism which is activated by adrenergic /cyclic AMP signaling and is dependent on thyroid hormone (T3). These findings point to an important role of dopamine/Drd4 signalling in the pineal gland and retina. On a more general level, it appears reasonable to consider that dopamine/D4Rsignaling in other tissues could reflect the interaction of cyclic AMP and T3."""""""" (From Kim et al,) Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina: """"""""Levels of dopamine and melatonin exhibit diurnal rhythms in the rat retina. Dopamine ishigh during daytime adapting the retina to light, whereas melatonin is high during nighttimeparticipating in the adaptation of the retina to low light intensities. Dopamine inhibits the synthesis of melatonin in the photoreceptors via Drd4-receptors located on the cell membrane of these cells. In this study, we show by semiquantitative in situ hybridization a prominent day/night variation in Drd4 expression in the retina of the Sprague Dawley rat with a peak during the nighttime. Drd4 expression is seen in all retinal layers but the nocturnal increase is confined to the photoreceptors. Retinal Drd4 expression is not affected by removal of the sympathetic input to the eye, but triiodothyronine treatment induces Drd4 the expression in the photoreceptors. In a developmental series, we show that the expression of Drd4 is restricted to postnatal stages with a peak at postnatal day 12. The high Drd4 expression in the rat retinal photoreceptors during the night supports physiological and pharmacologic evidence that the Drd4 receptor is involved in the dopaminergic inhibition of melatonin synthesis upon light stimulation. The sharp increase of Drd4 expression at a specific postnatal time suggests that dopamine is involved in retinal development."""""""" Control of cyclic AMP degradation: """"""""The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold;doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling."""""""" From (1) Control of circadian rhythms by the Ptprn and Ptprn2. We have participated in an effort to describe the role that two synaptic vesicle proteins play in circadian biology. """"""""We have found that synaptic vesicle proteins islet antigen 2(IA-2, Ptprn) and islet antigen 2-beta (IA-2-B, Ptprn2) are essential for circadian rhythms in activity, blood pressure, heart rate and temperature. IA-2 and IA-2-B are expressed in the suprachiasmatic nucleus (SCN), the master circadian oscillator in mammals the SCN of animals lacking these genes exhibit patterns of electrical activity which indicate that electrical activity of the SCN is not coherently rhythmic and that total activity is markedly reduced. IA-2 and IA-2-B may act in the SCN to facilitate cell-cell communication.
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