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 co mbination of physiological and morphological approaches under conditio ns 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 v esicles was often observed. By itself, replacement of medium Cl- by NO 3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and tbe numb ers and size of vesicles were much reduced. The vesicles accumulating in the pres ence of ouabain showed a yellow fluorescence after the cel ls were loaded with acridine orange, implying that the vesicular conte nts were acidic. Total fluid-phase endocytosis, determined by uptake o f Lucifer yellow, was not affected by ouabain or the absence of CP, Ho wever, 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 ves icles, an explanation consistent with the vesicle fusion seen by elect ron microscopy. The Cl- free medium also retarded Lucifer yellow efflu x, to the same extent as ouabain, and the effects of the two treatment s were not additive. These observations are consistent with the activi ty in hepatocytes of an ouabain-resistant, Cl--dependent mechanism for cell volume control. It is suggested that this depends on the accumul ation of water into acidic vesicles, which is driven by the Cl--couple d activity of the vacuolar ATPases of the organelles, followed by exoc ytotic expulsion of their contents.