Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation.

We previously reported in a rat cortical collecting duct cell line (RCCD(1)) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 97: 687-697, 2005). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium (Ca(2+)) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we studied Ca(2+) and cell volume changes in response to a hypotonic shock in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD(1) cells exhibit a substantial increase in Ca(2+). This Ca(2+) increase is strongly dependent on extracellular Ca(2+) and is partially inhibited by thapsigargin (1 muM) indicating that the rise in Ca(2+) reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD(1) cells to 100 muM gadolinium reduced the increase in Ca(2+) suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all of the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in Ca(2+) which is necessary to activate RVD in RCCD(1) cells.