Cell volume is an important parameter in many physiological processes, and is closely regulated in many cell types. In those cells, swelling induced by hypotonic media is followed by an ion-driven regulatory volume decrease. In many cell types, this regulatory volume decrease requires an intact actin cytoskeleton. Therefore, we investigated the changes in the structure and polymerization state of the actin cytoskeleton in HaCaT keratinocytes during cell swelling and regulatory volume decrease. Disruption of the actin cytoskeleton by 2μM cytochalasin D inhibits regulatory volume decrease in HaCaT cells. Cells swollen in the presence of low concentrations of cytochalasin D (0.8μM, 305–250mosM) keep the elevated volume even after cytochalasin D removal. A further decrease of tonicity (250–200mosM) is again counteracted by regulatory volume decrease reaching the volume, which has been established at 250mosM. In contrast, no visible changes occurred in actin cytoskeleton morphology of EGFP–actin-transfected HaCaT cells during swelling or regulatory volume decrease. However, biochemical analysis showed an increase in total F-actin levels 90s after the onset of hypotonicity. The ratio of Triton-soluble to -insoluble actin also increased after hypotonic shock, suggesting that the measured increase in F-actin is primarily due to de novo polymerization and formation of short actin filaments, i.e., actin oligomers. These results show that a rapid reorganization of the actin cytoskeleton takes place after hypotonic treatment. This reorganization can influence signaling in response to hypotonicity either indirectly by means of sequestering or releasing actin-associated proteins, or directly by the interaction of short actin filaments with plasma membrane ion channels, and may be involved in determining a new volume set point.

Tight regulation of the cell volume is important for the maintenance of cellular homeostasis. In a hypotonic environment, cells swell owing to osmosis. With many vertebrate cells, swelling is followed by an active reduction of volume, a process called regulatory volume decrease (RVD). A possible participant in RVD is the non-selective cation channel TRPV4, a member of the TRP superfamily that has been shown to react to hypotonic stimuli with a conductance for Ca2+. As a model for cell-volume regulation, we used a human keratinocyte cell line (HaCaT) that produces TRPV4 endogenously. When HaCaT cells were exposed to a hypotonic solution (200 mOsm) maximal swelling was followed by RVD. During swelling and volume regulation, a strong Ca2+ influx was measured. Gd3+, an inhibitor of TRPV4, blocked RVD of HaCaT cells and the accompanying rise of cytosolic Ca2+. To define the role of TRPV4 in volume regulation, a TRPV4-EGFP fusion protein was produced in CHO cells. CHO cells are unable to undergo RVD under hypotonic conditions and do not produce TRPV4 endogenously. Fluorescence imaging revealed that recombinant TRPV4 was localized to the cell membrane. Production of TRPV4 enabled CHO cells to undergo typical RVD after hypo-osmolarity-induced cell swelling. RVD of TRPV4-transfected CHO cells was significantly reduced by Gd3+ treatment or in Ca2+-free solution. Taken together, these results show a direct participation of TRPV4 in RVD.