Effect of prolonged ouabain treatment on Na, K, Cl and Ca concentration and fluxes in cultured human cells

Abstract

1. Girardi and Hela cells (derived from human heart and cervix respectively) were grown as monolayer cultures in B.M.E. (Eagles basal medium) containing concentrations of ouabain up to 5 x 10(-8)M for periods ranging up to 5 days. The cell sizes, numbers, Na, K, Cl, and Ca concentrations and fluxes were then measured.2. Twenty-four hours incubation in ouabain concentrations equal to or less than 5 x 10(-8)M caused a rise in [Na](i) and an almost equal fall in [K](i) to new steady levels. The concentrations so reached were linearly related to the ouabain concentrations, such that in 5 x 10(-8)M ouabain [Na](i) rose to 124 m-mole/l. intracellular water and [K](i) fell to 55 m-mole/l. i.c. water in Girardi cells. In Hela cells the changes were smaller at any particular ouabain concentration. These levels were maintained constant for at least 5 days.3. In cells in the logarithmic phase of growth, raising [Na](i) and lowering [K](i) by ouabain caused a slowing of growth rate proportional to the ouabain concentration used. In cells in the stationary phase there was no change in the cell numbers over 24 hr. The volume of the cells was not directly affected by the treatment.4. Reducing [K](o) from the normal value of 5.4 to 2.5 mM increased the effect of any ouabain concentration, whereas increasing [K](o) to 7.5 decreased the effect of ouabain.5. Reduction of [K](o) to 2.5 mM had no effect on the [K](i) or [Na](i) but halved the cell numbers, probably by a reduction in the growth rate. The mechanism of this effect is obscure.6. In Girardi cells raising [Na](i) and lowering [K](i) by prolonged treatment increases the total Na fluxes and decreases the total K fluxes but keeps the total Na + K flux constant. High-Na, low-K cells had a reduced Na:K exchange compared to fresh cells and also had a Na:K pumped ratio nearer 4:1 than the 3:2 normally found.7. These cells also show ouabain-sensitive and ouabain-insensitive Na:Na exchanges. In high-Na, low-K cells the ouabain sensitive Na:Na exchange is the same as in fresh cells. The effect of treatment on the ouabain insensitive Na:Na exchanges has not been elucidated.8. The Cl content and fluxes are not altered by prolonged ouabain treatment. From this it is inferred that the membrane potential in high-Na, low-K cells is the same as in normal cells.9. High-Na, low-K cells have the same calcium content and fluxes as fresh cells. From this it is concluded that there is no Na:Ca coupling in these cells.