Role of AQP2 in activation of Calcium Entry by Hypotonicity: Implications in cell volume regulation.

GALIZIA L; FLAMENCO P; RIVAROLA V; CAPURRO C; FORD P

Salzburgo

Simposio; 6th International Symposium on Cell Volume Regulation in Health and Disease; 2007

Paracelsus Medical University. Salzburg, Austria

The ability of cells to regulate their volume is essential for maintenance of cellular homeostasis under anisotonic environmental conditions. Most cells respond to decrease in tonicity first by swelling and second by initiating mechanisms that allow them to recover their original volume. Such regulatory volume decrease changes (RVD) depend on the activation of different ion permeabilities that reverse the osmotic gradient and direction of water flow. However, the underlying mechanism(s) that senses the change in osmolarity and/or cell volume to initiate RVD is not completely understood. We have previously reported in a cortical collecting duct cell line that the presence of a water channel (aquaporin 2) in the cell membrane is critical for the rapid activation of RVD mechanisms (Ford et al. Biol Cell 2005; 97: 687-697). The investigation of whether AQPs may form part of the cellular device for volume sensing and regulation is our present goal. Therefore we investigated the signaling pathway that links Aqp2 to the rapid RVD activation. In different cell types hypotonic conditions induce intracellular calcium [Ca2+]i increases, and this Ca2+ entry has been reported to be critical for the volume recovery process. Using a fluorescent probe technique, we have studied intracellular calcium (FURA2) and cell volume changes (BCECF) in response to a hypotonic shock in two renal cell lines, one not expressing aquaporins (WT-RCCD1) and other displaying a constitutive expression of AQP2 in the apical plasma membrane (AQP2-RCCD1). We have found that hypotonicity increases intracellular calcium only in AQP2-RCCD1 cells. This [Ca2+]i increase is strongly dependent on extracellular Ca2+ and is partially inhibited by gadolinium (Gd3+). Furthermore, exposure of cells to Gd3+ or to a free calcium solution significantly reduces the RVD while not affecting the rate of cell swelling in response to hypotonic shock. Incubation of the cells with thapsigargin also reduces the [Ca2+]i increase and the subsequent regulatory mechanism, suggesting the involvement of Ca2+ mobilization from intracellular stores. In conclusion, we have found that, in RCCD1 cells, the presence of AQP2 is crucial for the activation of [Ca2+]i increase by hypotonicity, which is necessary to turn on the transporters involved in cell volume regulation.