Effects of ouabain and chloride-free medium on isoosmotic volume control and ultrastructure of hepatocytes in primary culture.

Rat hepatocytes in primary monolayer culture have been studied by a combination of physiological and morphological approaches under conditions affecting ion transport and cell volume. A concentration of ouabain completely inhibiting the coupled transport of Na+ and K+ had little effect on cell volume, as indicated by cell water content, but induced the formation of many vesicles in the cytoplasm. Apparent fusion of vesicles was often observed. By itself, replacement of medium Cl by NO3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and the numbers and size of vesicles were much reduced. The vesicles accumulating in the presence of ouabain showed a yellow fluorescence after the cells were loaded with acridine orange, implying that the vesicular contents were acidic. Total fluid-phase endocytosis, determined by uptake of Lucifer yellow, was not affected by ouabain or the absence of Cl-. However, ouabain considerably retarded the subsequent release of Lucifer yellow; this suggests that the dye originally taken into endocytotic vesicles became diluted by mixing with contents of ouabain-induced vesicles, an explanation consistent with the vesicle fusion seen by electron microscopy. The Cl-free medium also retarded Lucifer yellow efflux, to the same extent as ouabain, and the effects of the two treatments were not additive. These observations are consistent with the activity in hepatocytes of an ouabain-resistant, Cl(-)-dependent mechanism for cell volume control. It is suggested that this depends on the accumulation of water into acidic vesicles, which is driven by the Cl(-)-coupled activity of the vacuolar ATPases of the organelles, followed by exocytotic expulsion of their contents.