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Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107.

1. We have examined whether the apparently ouabain-resistant fraction of cellular volume regulation in liver slices under isosmotic conditions is due to a failure of ouabain to cause complete inhibition of the coupled transport of Na+ and K+. The ion and water contents of rat and rabbit liver slices were altered by pre-incubation at 1 degree C and then allowed to recover at 38 degrees C, with or without ouabain or other inhibitors. The net movements of ions and water were determined during the recovery. The influx of 86Rb under steady-state conditions was taken as a measure of unidirectional influx of K+. 2. Concentrations of ouabain for half-maximal inhibition of 86Rb influx were 0.15 mM for rat and 0.15 microM for rabbit liver slices, with maximal inhibition at 2 mM and 10 microM respectively. Inhibition of net K+ reaccumulation closely followed inhibition of 86Rb influx. 3. The 86Rb influx persisting in the presence of maximally inhibiting concentrations of ouabain was not reduced by inhibitors of cellular respiration or glycolysis. 4. In rat liver slices, about 50% of net water extrusion was resistant to 2 mM-ouabain; rabbit liver showed a much smaller, but statistically significant, extrusion of water in the presence of 10 microM-ouabain. 5. In rat liver slices, a small, net uptake of K+ continued in the presence of amytal alone, when water extrusion was completely inhibited; by contrast, ouabain gave complete inhibition of K+ uptake while permitting 50% of the control water extrusion. 6. Isolated rat hepatocytes in primary culture were pre-incubated at 4 degrees C for 20 h. They recovered their original K+ content within 60 min of restoration to 37 degrees C. Ouabain, 1-2 mM, completely prevented this recovery. 7. The results imply that ouabain completely inhibits the coupled transport of Na+ and K+ in both rat and rabbit liver slices. Thus, the fraction of total water extrusion continuing in the presence of maximally inhibiting concentrations of ouabain is the consequence of a truly ouabain-resistant mechanism.