Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin a ssay, confocal laser scanning microscopy, and electronic cell sizing. A hyp otonic challenge (180 mOsm) was associated with a decrease in cellular F-ac tin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an in crease in cellular F-actin content at 1, 3, and 5 min, respectively, compar ed 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 cha nges in F-actin in other cell regions. The possible role of the F-actin cyt oskeleton in RVD was studied using 0.5 mu M cytochalasin B (CB), cytochalas in 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 pretre ated for 3 min with 0.5 mu M CB, but not in CD- and ChtC-treated cells, com pared to osmotically swollen control cells. Moreover, the maximal cell volu me after swelling was decreased in CB-treated, but not in CD- or Chtc-treat ed cells. Following a hypertonic challenge imposed using the RVD/RVI protoc ol, recovery from cell shrinkage was slower in CB-treated, but not in CD- o r Chtc-treated cells, whereas the minimal cell volume after shrinkage was u naltered by either of these treatments. It is concluded that osmotic cell s welling and shrinkage elicit a decrease and an increase in the F-actin cont ent in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 mu M CB, but not by 0.5 mu M CD, which is more specific for actin. (C) 1999 Academic Press.