Role of the F-actin Cytoskeleton in the RVD and RVI Processes in Ehrlich Ascites Tumor Cells

Abstract

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine–phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0.5 μM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin.Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 μM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 μM CB, but not by 0.5 μM CD, which is more specific for actin.