The implication of microfilament reorganization in cell volume regulation was initially studied by applying qualitative microscopic analysis (immunofluorescence and confocal laser-scanning microscopy). In cells exposed to hypotonic media, the majority of microscopic studies reported actin cytoskeleton disorganization and loss of microfilamentous structures such as stress fibers and formation of submembranous F-actin aggregations (Cornet et al., 1994; Dartsch et al., 1994; Hallows et al., 1991; Moran et al., 1996; Schwiebert et al., 1994). In specific cell types, such as astrocytes and pheochromocytoma PC12 cells, the reorganization of actin was a transient phenomenon (Cornet et al., 1994; Moran et al., 1996), whereas in renal epithelial OK cells, actin microfilament reorganization could not be detected by fluorescence microscopy (Dartsch et al., 1994). From these reports it became evident that cell volume changes result in reorganization of F-actin structures. It should be noted that although microscopic analysis provides an overall qualitative representation of cellular morphology, it could by no means provide specific insights on the dynamics of actin cytoskeleton organization. In addition, in cell systems expressing feeble microfilamentous structures, such as leukocytes, hepatocytes, or tumor cells, the evaluation of actin reorganization by fluorescence microscopy is problematical. Despite those limitations, however, microscopic approaches established a pivotal role of F-actin reorganization for the adjustment of cell volume (Hallows et al., 1991; Schwiebert et al., 1994).
An indirect approach in monitoring the dynamic state of actin polymerization during cell volume regulation was addressed by microscopic analysis (immunofluorescence and confocal laser-scanning microscopy) using various microfilament-disrupting agents. The existing literature reports that in various cell types, such as leukocytes, myocytes, Ehrlich ascites tumor cells, HEK 293 cells, and hepatocytes, cytochalasins inhibit RVD and RVI, indicating that an intact cytoskeleton is required for the mediation of cellular signals in response to cell volume alterations (Downey et al., 1995; Ebner et al., 2005; Hall et al., 1997; Jorgensen et al., 2003; Pedersen et al., 1999). It has been reported that involvement of the actin cytoskeleton in regulating RVD was directly verified in salivary gland cells (Liux et al., 2006). Indeed, by using N terminus-deleted aquaporin 5, the osmosensing transient receptor potential vanalloid 4 and RVD were suppressed and this effect was inhibited by actin depolymerization. However, cytochalasin-independent effects on regulated volume decrease have also been reported (Downey et al., 1995; Hallows et al., 1996; Moran et al., 1996), indicating that changes in the actin cytoskeleton are quite diverse in different cell types. Thus, evaluation of these results should be considered with caution. Although cytochalasins or phalloidins are well-studied drugs affecting actin organization and polymerization, their side effects on other aspects of cell physiology cannot be excluded. Complementary analysis of actin polymerization dynamics by quantitative biochemical analysis is needed to provide molecular insights into the regulatory role of the actin cytoskeleton in cell volume regulation.
A much more detailed analysis ofmicrofilament reorganization during the different phases of cell volume changes became possible by quantitative biochemical measurements of intracellular actin polymerization equilibrium, including assessment of cellular monomeric and polymerized actin levels using various techniques. In an initial study using the DNase I inhibition assay to assess the intracellular monomeric and total actin content, we determined actin polymerization dynamics in primary cultures of rat hepatocytes after hypotonic exposure (Theodoropoulos et al., 1992). Detailed analysis revealed rapid decrease of the monomeric to total actin ratio (G-/T-actin) upon induction of dramatic cell volume alterations. Interestingly, during the initial phase of cell swelling the total actin content of
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