Effect of hypotonic medium on aquaporin-4 expression in cultured astrocytes

摘要

BACKGROUND: Aquaporin-4 (AQP4) is abundant in astrocytes, ependymal cells, and the choroid plexus, and is associated with cerebrospinal fluid formation and osmoregulation. AQP4 is speculated to be the hypothalamic osmoreceptor and regulator of water balance. OBJECTIVE: To examine AQP4 expression and its role in cultured rat astrocytes after exposure to hypotonic medium. DESIGN, TIME AND SETTING: Randomized control experiment. This experiment was carried out in the Research Room of Neurobiology, Chongqing University of Medical Science, China, between April and October 2003. MATERIALS: Two-day-old newborn Wistar rats (n =20), weighing 10-15 g, were purchased from the Experimental Animal Center of Chongqing University of Medical Science, China. METHODS: Purified rat cerebral cortical astrocytes were isolated from Wistar rats for in vitro cell culture experiments. The cells were randomly divided into control and hypotonic groups. The in vitro cell edema model was established by exposing astrocytes to hypotonic medium (268, 254, or 240 mmol/L). Cells in the control group were cultured in normal culture medium. MAIN OUTCOME MEASURES: Morphological changes in astrocytes were observed under an inverted microscope and a transmission electron microscope after cells were cultured for 3, 6, 12, or 24 hours with hypotonic medium or normal culture medium. In each group, AQP4 protein and mRNA expression were assessed by immunocytochemistry, in situ hybridization, and reverse transcription polymerase chain reaction at the different time points. RESULTS: After astrocytes were cultured for 3, 6, 12, or 24 hours with hypotonic medium (268, 254, 240 mmol/L), they showed typical features of cell edema. In the control group, no astrocytes developed pathological changes. There were no significant changes in the AQP4 mRNA and protein expression in the control group at any of the time points after astrocytes were cultured with normal culture medium (P > 0.05). Compared with the control group, AQP4 mRNA and protein expression in the hypotonic group were remarkably increased at all time points after astrocytes cultured with hypotonic medium (268, 254, 240 mmol/L; P < 0.05). AQP4 mRNA and protein expression increased with increasing exposure time and with decreasing concentration of the hypotonic medium. CONCLUSION: Hypotonic medium induced cell edema and increased AQP4 mRNA and protein expression. Up-regulated expression of AQP4 was correlated with hypotonic medium concentration in a time dependent manner.