When a cell is exposed to hypotonic conditions it increases in size through osmotic swelling, activating a mechanism known as regulatory volume decrease (RVD) which returns the cell back to its original volume mainly through KCl efflux. This adaptive response is particularly important in the brain, as swelling can lead to ischaemic and anoxic brain damage.

This study investigates the conditions necessary for cell volume regulation in a neuronal cell line CAD (Qi et al. 1997), and suggests a role for chloride (Cl^–) channels in RVD. Cells were perfused with a Hepes-buffered isotonic solution (300 mosmol kgH₂O^-1) for 3 min followed by a 30 % hypotonic solution (210 mosmol kgH₂O^-1) for up to 47 min at 37 °C unless otherwise stated. Digital photographs were taken every minute and cell volume measurements were calculated using the Scion Image computer software package. All cell volume measurements are expressed as relative cell volumes with respect to control values. Changes in cell volumes during hypotonic exposure (mean volume change ± S.E.M.) were compared using a Student’s unpaired t test or ANOVA followed by the Tukey HSD test.

On exposure to a 30 % hypotonic solution cells swelled to 1.32 ± 0.013 in the first 4 min and recovered to 1.23 ± 0.017 (n = 9) within 15 min. Cells exposed to a 23 % hypotonic solution (231 mosmol kgH₂O^-1) swelled to 1.24 ± 0.007 and recovered to 1.17 ± 0.003 (n = 3) within 15 min. Although cell swelling is affected by the degree of hypotonicity (P < 0.001) there was no difference in the mean cell volume changes after 15 min hypotonic exposure (P > 0.10). However, RVD is temperature dependent as a significant difference in mean cell volume change was observed when cells were exposed to a 23 % hypotonic solution for 15 min at 21 or 37 °C (n = 3, P < 0.005).

The role of Cl^– channels in RVD was investigated using the volume-regulated anion channel (VRAC) antagonists tamoxifen and 4,4Ô-diisothiocyanatostilbene-2,2Ô-disulphonic acid (DIDS). On exposure to a 30 % hypotonic solution containing 10 mM tamoxifen, cells swelled to 1.40 ± 0.012 and recovered to 1.38 ± 0.023 (n = 5) within 15 min, and 1.29 ± 0.015 (n = 3) within 47 min. On exposure to a 30 % hypotonic solution containing 100 mM DIDS, cells swelled to 1.35 ± 0.012 and recovered to 1.32 ± 0.012 (n = 5) within 15 min, and 1.29 ± 0.025 (n = 4) within 47 min. In both cases changes in mean cell volume at 15 and 47 min were significantly different from control values (P < 0.01).

In conclusion CAD cells act as near perfect osmometers and are capable of regulating their volume on exposure to a hypotonic medium. Furthermore, these results suggest that a volume-regulated Cl^– channel is important for RVD in CAD cells.