Cholesterol Depletion Facilitates Recovery from Hypotonic Cell Swelling of CHO

Abstract

The maintenance of cell volume homeostasis prevents pathological cell swelling that can lead to severe cellular dysfunction or death. A key step in maintaining cell volume in many cell types is activation of volume-regulated anion channels (VRAC). Our earlier studies showed that activity of VRAC is facilitated by a decrease in cellular cholesterol (Levitan et al 2000). These observations suggest that lowered cholesterol should also facilitate regulatory volume decrease (RVD), a process used by cells to recover from hypotonic swelling. The main constraint in testing this prediction, however, has been the lack of adequate methods to rapidly and reproducibly measure changes in cell volume of substrate-attached cells. In this study, we address this question using a novel microfluidic methodology from Reichert Inc. (CVC-7000), to measure cell volume response to hypotonic challenges (30% osmotic gradient) in real time. Cholesterol depletion facilitated the recovery from swelling via a more rapid onset of RVD (∼130 s vs. 215 s in control and cholesterol depleted cells, respectively) and a higher degree of volume recovery after 10 min (41%±6% vs. 65%±6% in control and cholesterol depleted cells, respectively). In contrast, enriching the cells with cholesterol had no effect on RVD. These observations are consistent with our previous studies showing that while cholesterol depletion increases cell stiffness, cholesterol enrichment has no effect (Byfield et al 2004). These observations suggest that cholesterol depletion, and the consequent increase in cell stiffness, facilitates RVD by enhancing the activity of VRAC.