An electrophysiological technique to measure change in hepatocyte water volume.
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 cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA 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 alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less 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 X 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 less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 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 K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.
Khalbuss
W E
WE
Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614.
Wondergem
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D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008815:Mice, Inbred Strains; D009761:Nystatin / Q000494:pharmacology; D009997:Osmotic Pressure; D015221:Potassium Channels / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014867:Water / Q000378:metabolism*; D014882:Water-Electrolyte Balance
D015221:Potassium Channels; D000644:Quaternary Ammonium Compounds; D014867:Water; D009761:Nystatin; D001464:Barium; D000666:Amphotericin B; C027917:tetramethylammonium