HepG2 cells undergo regulatory volume decrease by mechanically induced efflux of water and solutes

This study challenges the conventional belief that animal cell membranes lack a significant hydrostatic gradient, particularly under anisotonic conditions, as demonstrated in the human hepatoma cell line HepG2. The Boyle van’t Hoff (BvH) relation describes volumetric equilibration to anisotonic conditions for many cells. However, the BvH relation is simple and does not include many cellular components such as the cytoskeleton and actin cortex, mechanosensitive channels, and ion pumps. Here we present alternative models that account for mechanical resistance to volumetric expansion, solute leakage, and active ion pumping. We found the BvH relation works well to describe hypertonic volume equilibration but not hypotonic volume equilibration. After anisotonic exposure and return isotonic conditions cell volumes were smaller than their initial isotonic volume, indicating solutes had leaked out of the cell during swelling. Finally, we observed HepG2 cells undergo regulatory volume decrease at both 20 °C and 4 °C, indicating regulatory volume decrease to be a relatively passive phenomenon and not driven by ion pumps. We determined the turgor-leak model, which accounts for mechanical resistance and solute leakage, best fits the observations found in the suite of experiments performed, while other models were rejected.