Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

August 1990

Abstract

The electrophysiologic technique (Reuss, L., Proc. Natl. Acad. Sci. USA 82:6014, 1985) was modified to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA$\sp{+}$) during transient exposure of cells to nystatin. Intracellular TMA$\sp{+}$ activity (a$\sp{\rm i}\sb{\rm TMA}$) was measured with TMA$\sp{+}$-sensitive, double-barreled microelectrodes. Loading hepatocytes with TMA$\sp{+}$ did not change their membrane potential (V$\sb{\rm m}$), and under steady-state conditions a$\sp{\rm i}\sb{\rm TMA}$ remained constant over 4 min in single impalements. Hyperosmotic solutions (50, 100, & 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 & 100 mM) increased and decreased a$\sp{\rm i}\sb{\rm TMA}$, respectively, which suggested transmembrane water movements. The regression coefficient of the ratio of control a$\sp{\rm i}\sb{\rm TMA}$/experimental a$\sp{\rm i}\sb{\rm TMA}$ versus the relative osmolality of media (experimental mOsm/control mOsm) was -0.34 $\pm$ 0.03, p $<$ 0.001, which is less than expected for a perfect osmometer. Corresponding measurements of V$\sb{\rm m}$ showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba$\sp{2+}$ (2 mM) was present during hyposmotic stress of 0.66 $\times$ 286 mOsm (control), cell water volume increased by a factor of 1.44 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 $\pm$ 0.02, p $<$ 0.001. Ba$\sp{2+}$ also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.62 $\pm$ 0.04 to 1.24 $\pm$ 0.09, p $<$ 0.01. When verapamil (50 $\mu$M) was present during hyposmotic stress of 0.69 $\times$ 292 mOsm (control), cell water volume increased by a factor of 1.42 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.19 $\pm$ 0.02, p $<$ 0.001. Verapamil also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.34 $\pm$ 0.07 to 1.08 $\pm$ 0.08, p $<$ 0.05. Similar results were obtained when exposing hepatocytes to Ca$\sp{2+}$-free medium plus EGTA (5 mM). It was concluded that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of V$\sb{\rm m}$ in the presence of the K$\sp{+}$-channel blocker, Ba$\sp{2+}$, the Ca$\sp{2+}$-channel blocker, verapamil and the Ca$\sp{2+}$-chelator, EGTA. This indicated that cell calcium and membrane potassium conductance (g$\sb{\rm K}$) were involved in hepatocyte volume regulation mechanism and that variation in V$\sb{\rm m}$ provides an intercurrent, electromotive force for hepatocyte volume regulation.

Document Type

Dissertation - unrestricted

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