Evidence for the genetic control of the sodium pump density in HeLa cells

1. HeLa cells were grown in normal and altered growth solutions; the ion contents, volumes, K sensitive ouabain binding, the Na-K-ATPase and the Na and K transport measured.2. Cells grown in 1 x 10(-4)M ethacrynate or low-K media for 24 hr have a raised [Na](i), a decreased [K](i), and an increased ouabain binding. Those grown in low-K also have an increased Na-K-ATPase activity.3. When cells are put into low-K solutions the [Na](i) initially rises to a high value, and then starts to fall some 8 hours later as the ouabain binding increases, suggesting that these additional sites represent working Na pumps. Flux measurements on low-K cells provide some support for this view.4. Experiments in which sorbitol replaced [Na](o) showed that the increased ouabain binding and Na-K-ATPase was related to the increase in [Na](i) rather than the decrease in [K](i) and was not due to a non-specific effect of [K](o) change.5. The protein synthesis inhibitors cycloheximide and puromycin stopped the effect of ethacrynate and low-K solutions on increased ouabain binding. They also decreased the ouabain binding and K influx in normal cells over 24 hr. Cycloheximide had similar effects on Na-K-ATPase in low-K treated and normal cells. These results suggest that protein synthesis is required for the appearance of more ouabain sensitive sites in the cell membrane, both in response to ethacrynate and low-K treatment and for normal replacement during the cell's life.6. The RNA synthesis inhibitors actinomycin D (AMD) and cordycepin had complex effects on ouabain binding in fresh and ethacrynate treated cells. These inhibitors increased the ouabain binding but decreased the K influx. This discrepancy was due to the appearance of ouabain binding sites with different characteristics from normal sites. A limited investigation of this phenomenon was carried out. Probably AMD stops the normal replacement of sites in the membrane.7. These results are consistent with the hypothesis that HeLa cells have a system for controlling the number of Na pumps in their membranes. This system responds to the level of [Na](i) within the cell and involves protein synthesis. It is not clear to what extent the nucleus is normally involved in this process.