Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro

In order to understand the molecular mechanism underlying astroglial swelli ng, we studied primary astrocyte cultures from newborn mouse and analyzed t hem for expression of functional water channels. Immunocytochemical analysi s of mouse brain confirms the presence of AQP4 location in astrocytic endfe et with a polarized pattern, as found in rat. Using Southern blot PCR and W estern blot analysis, we demonstrate that primary astrocyte cultures from m ouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Den sitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong s urface membrane expression of AQP4 protein in cultured cells, even though t he polarity of the expression is not maintained. Furthermore, functional st udies indicate that cultured astrocytes manifest rapid and temperature-inde pendent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl2 in sensitive, suggesting AQP4 and AQP7 as putative water channels. Using Weste rn blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Alt ogether, the results indicate that primary astrocyte cultures are a valid c ell model for further investigation of the molecular mechanism of water mov ement in the brain and its physiological regulation. (C) 2000 Wiley-Liss, I nc.