Investigation for morphology and function of astrocyte end-food membrane

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The aim of this study was to demonstrate the expression of aquaporin4 (AQP4), aquaporin5 (AQP5), aquaporin9 (AQP9) and Kir4.1 in rat primary astrocytes using Western blot.Trypsinized new-born rat brain was harvested into culture flasks. After 3 to 4 passages, the cell was solubilized with sodium dodecyl sulfate (SDS) and dithiothreitol. The lysate was separated with SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies and the secondary antibody (Horseradish peroxidase labeled IgG), then generated chemiluminescence was detected with X-ray film.Glial fibrillary acidic protein (GFAP) specific for astrocytes showed was strongly expressed at about 50KDa. According to the protein database SWISS-PROT, the molecular size of GFAP is 49.9KDa, and then we identified the band as GFAP. AQP4 showed dual band at about 30KDa and 60KDa. According to SWISS-PROT, AQP4 is produced two different molecular size of protein, because two initiation codons in AQP4 mRNA called M1 and M23, then produced protein were 34.5KDa and 32.5KDa.AQP5 and AQP9 showed a very weak band at each 60KDa. According to SWISS-PROT, molecular size of AQP5 and AQP9 are 28.4KDa and 31.4KDa, respectively. As a result, this may be dimer of AQP5 and AQP9 in our experiments.Kir4.1 showed a positive band at about 50KDa. SWISS-PROT showed that the molecular size of Kir4.1 was 42.4KDa. It was possible to detect Kir4.1 in cultured astrocytes because this discrepancy between our finding and SWISS-PROT data was very little.The next plans are to make a quantitative analysis of aquaporin expression in cell fraction and a morphological analysis of localization of aquaporin in astrocytes under brain edema.