Abstract
We have applied an electrophysiologic technique (Reuss L.(1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cell to nystatin. Intracellular TMA+ activity (αiTMA) was measured with TMA+ -sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions αiTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased αiTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(αiTMA)O/(αiTMA)4min] - 1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less than predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 × 286 mosmol (control), cell water volume increased by a factor of 1.44 ± 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 ± 0.02, P< 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 ± 0.04 to 1.24 ± 0.09, P < 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of the K+ -channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.