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

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse 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. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.