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1

Effects of orexins on survival of the primary neuronal and glial cell cultures

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Sokolowska P., Urbanska A., Bieganska K., Namiecinska M., Zawilska J.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Orexins (hypocretins) are hypothalamic peptides present in neurons that project throughout the brain. They act through two receptors: OX1 and OX2. Recently, it has been demonstrated that orexins exert potent proapoptotic activity in various cancer cell lines. On the other hand, little is known about the role of these peptides in survival of neurons and their supportive cells in the brain. Therefore, the aim of our study was to investigate whether orexins are implicated in receptor-mediated survival- or deathpromoting effects in cultured neurons and astrocytes derived from rat cerebral cortex. Real-time PCR experiments indicated that both types of orexin receptors were expressed in rat neurons and astrocytes. In cultured neurones OX2R expression was considerably higher than that of OX1R. In astrocytes similar expressions of both types of orexin receptors were identified but they were markedly lower compared to neurons. Incubation of primary neuronal cultures with orexin A, orexin B and [Ala11-D-Leu15]orexin B (a selective agonist of OX2R) resulted in a marked increase of cells viability and a parallel reduction of apoptotic cells as assessed by MTT test and caspase-3 assay kit. In cultured astrocytes the tested neuropeptides increased cell viability (MTT) and stimulated 3 H-thymidine incorporation but had no effect on caspase-3 activity, an observation indicating that orexins may affect astrocytes survival by enhancing cell proliferation. In the next set of experiments cultured neurons were subjected to hypoxia induced chemically by iron chelator cobalt chloride. Orexins A and B, and [Ala11-D-Leu15]orexin B effectively protected neuronal cells, suggesting that the peptides may be endowed with neuroprotective potential in the brain. Supported by MNiSW (grant No 4254/B/ PO1/2010/38) and InterMolMed (grant No POIG.01.01.02-10- 107/09).
2

The orexin-induced release of cytokines in primary microglial cell cultures

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Namiecinska M., Sokolowska P., Bieganska K., Urbanska A., Andrzejczak D., Zawilska J. B.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Orexin A and B (hypocretin 1 and 2) are hypothalamic neuropeptides that have been implicated in a variety of physiological functions, including sleep and arousal, reaction to stress, regulation of energy homeostasis and hypothalamo-pituitary-adrenal axis. The neuropeptides exert their numerous actions by interacting with two specific, membrane-bound, G-protein-coupled receptors, OX1R and OX2R. We have recently demonstrated that orexin receptors are expressed in rat astrocytes and modify their function. Thus, searching for new orexins roles, in this work we investigated whether these peptides can affect the production of cytokines by microglial cells. Studies were performed on primary microglial cultures from rat cerebral cortex. Orexin A and orexin B stimulated the release of IL-6 as well as TNF-α from microglial cells, as measured by rat IL-6 and TNF-α immunoassay ELISA kits. Interestingly, orexins did not affect the release of IL-1. The obtained results might be important in the aspect of interaction between microglia and neurons/astrocytes in the central nervous system. Supported by MNiSW (grant No 4254/B/PO1/2010/38) and InterMolMed (grant No POIG.01.01.02-10-107/09).
3

Effects of orexins on survival of rat C6 glioma cells. A comparison to the primary rat astrocyte cell cultures

86%
Bieganska K., Sokolowska P., Namiecinska M., Urbanska A., Sobczak E., Zawilska J. B.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Orexin-A and orexin-B, also named hypocretin-1 and hypocretin-2, are hypothalamic neuropeptides which are important regulators of sleep-wake cycles, reward-seeking, and body energy balance. These neuropeptides bind to two specific, membranebound receptors: OX1R and OX2R, members of the GPCR superfamily. A potent proapoptotic activity of orexins has recently been demonstrated in colon cancer cell lines and human colorectal tumor. In our studies we investigated effects of orexins on survival of rat C6 glioma cells, an experimental model for studies on glioblastoma multiforme, and compared them with those exerted on cultured astocytes from rat cerebral cortex. Orexins A and B decreased the number of surviving C6 glioma cells after 48 h of treatment (MTT test), and reduced [3H]thymidine incorporation into proliferating C6 cells. On the contrary, 48 h incubation of cultured astrocytes with orexins increased astrocyte viability and moderately stimulated their proliferation. Taken together, our results suggest that effects of orexins on cell survival depend on the cell type (normal versus cancer). It can be speculated that orexins may serve as potential anticancer factors in therapy of brain tumors. Supported by MNiSW (grant No 4254/B/PO1/2010/38) and InterMolMed (grant No POIG.01.01.02-10-107/09).
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