RVD in ZR-75-1 cells, epithelial-derived human breast cancer cells, is HCO₃^– dependent and involves the parallel activation of the K⁺ and volume-regulated anion channel (VRAC; Nicholl et al. 2001). Preliminary data indicated that the mechanisms of RVD were calcium (Ca²⁺) dependent, although the exact nature is unknown. This study investigated the Ca²⁺ dependency of RVD in ZR-75-1 cells using the video imaging technique.

ZR-75-1 cells were bathed in isotonic solution at 37 °C for 10 min and allowed to equilibrate. They were subsequently exposed to a 23 % hypotonic solution for 15 min and then returned to isotonic solution for 12 min. Photographs were taken every minute and the area in pixels measured using Scion Image computer software. Cell volume was calculated and expressed as relative volume (mean ± S.E.M.) with respect to control values. The role of Ca²⁺ was investigated by exposure to Ca²⁺-free solution and Ca²⁺ entry blockers. The significance of changes in cell volume during hypotonic exposure was tested using ANOVA followed by the Tukey HSD test.

In hypotonic solution control cells swelled to 1.27 ± 0.016 (n = 8) in 3 min and recovered to 1.03 ± 0.015 during the 15 min. The role of external Ca²⁺ was investigated using a Ca²⁺-free hypotonic solution; cells swelled to 1.27 ± 0.004 in 4 min and after 15 min in hypotonic solution the volume was 1.26 ± 0.002 (n = 3). In the absence of extracellular Ca²⁺ the mean volume change was significantly less than the control (P ▓le│ 0.01). The absence of RVD suggests a role for external Ca²⁺.

The role of Ca²⁺ entry was investigated using 20 mM cadmium (Cd²⁺), a non-specific voltage-gated Ca²⁺ channel blocker. Cells swelled to 1.27 ± 0.004 (n = 5) and after 15 min in hypotonic solution the volume was 1.19 ± 0.013. The type of Ca²⁺ entry pathway was investigated using 50 mM nifedipine, an L-type voltage-gated Ca²⁺ channel blocker. Cells swelled to 1.30 ± 0.018 (n = 6), and after 15 min in hypotonic solution the volume was 1.24 ± 0.048. Ca²⁺ entry was also investigated using 50 mM flunarizine, a specific T-type voltage-gated Ca²⁺ channel blocker. Cells swelled to 1.29 ± 0.029 (n = 5) and after 15 min in hypotonic solution the volume was 1.16 ± 0.038. In each case the volume change was significantly less than in the control (P ▓le│ 0.01).

These results support preliminary experiments indicating that RVD in ZR-75-1 cells is Ca²⁺ dependent. RVD is inhibited in both the absence of extracellular Ca²⁺ and in the presence of Ca²⁺ entry blockers, Cd²⁺, nifedipine and flunarizine. These results suggest that external Ca²⁺ enters into the cell via voltage-gated Ca²⁺ channels and favours entry via L-type voltage-gated Ca²⁺ channels.