Effects And Mechanisms Of Orexin-a On Cell Migration In Cultured Rat Astrocytes

Aim:orexin-A is an important hormone involved in feeding, arousal, energy expenditure and reward seeking regulation in the body. It was first discovered in a specific population of neurons in the lateral hypothalamic area in1998. It was widely studied in neurons but little is known in astrocytes. In this study, we aim to investigate the function of orexin-A on rat astrocytes migration.Methods:Use wound healing model to study cultured primary rat astrocytes migration and immunofluorescence to investigation the expression of orexin receptors.Results:Here, we report that orexin-A can promote rat primary cultured astrocytes migration significantly. The effect of orexin-A on astrocytes migration is time-and dose-dependent. It reached to the maximal level at10nM and24h treatment. Both OX1R and OX2R expressed in cultured astrocytes. OX1R exist in Cytoplasm and the nucleus, bu OX2R exist only in cytoplasm, very few in the nucleus. OX1R specific inhibitor SB334867could suppress orexin-A induced astrocytes migration. However, OX2R inhibitor TCS OX229had no effects.Conclusion: Orexin-A promote rat primary cultured astrocytes migration, OX1R mediated this effect. Aim:Previous studies have found that orexin-A can induce Ca2+influx and ERK1/2signal activaation in some cell types, but no report in astrocytes about these. So in this part we aim to investigate the action of orexin-A on astrocytes intracellular Ca2+and ERK1/2signal. Explore the mechanisms of orexin-A promoted astrocytes migration. Methods:Primary cortical astrocytes culture, western blotting, calcium imaging and wound healing cell migration model were used for our investigation.Results:To examine the relationship between orexin-A and ERK1/2activation, the primary cultured astrocytes were treated with orexin-A or vehicle for10min and the levels of ERK1/2phosphorylation were assayed by western blotting. The result shown orexin-A induced a significant phosphorylation of ERK1/2in primary cortical astrocytes, ERK1/2inhibitors PD98059and U0126could prevent orexin-A promote astrocyte migration. Next, we found that orexin-A induced ERK1/2activation was Ca2+dependent. Astrocytes were pretreated with a Ca2+chelator BAPTA-AM for30min, followed by application of orexin-A for10min. BAPTA-AM prevented ERK1/2phosphorylation induced by orexin-A. intracellular Ca2+concentration increased notably about500sec after orexin-A application. The orexin-A induced intracellular Ca2+increase could prevented by EGTA Ca2+free solution, CPA and2-APB pretreatment. Orexin-A induced astrocytes ERK1/2activation was prevented by EGTA,2-APB and U73122but not CPA. Cell migration assay show that BAPTA-AM,2-APB and U73122could surpress orexin-A induced astrocytes migration. Furthermore, both non-selective protein kinase C inhibitor and PKCa selective inhibitor, but not PKC8inhibitor, prevented the increase of ERK1/2phosphorylation and the migration of astrocytes. Orexin-A induced astrocytes migration was also prevented by PKCa inhibitor Go6976.Conclusion: Our results validate that ERK1/2signals participate orexin-A induced astrocytes migration. Orexin-A induced ERK1/2activation and cell migration were Ca2+dependent. Orexin-A induce a significant rise of astrocytes intracellular Ca2+levels. Store-operated calcium entry mediated orexin-A induced Ca2+influx. PLC-Ca2+-PKCa-ERK1/2signal mediated orexin-A induced astrocytes migration.