Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture.

1. Human cells (HeLa) were cultured for periods up to 48 h in growth medium in the absence or presence of a range of concentrations of cardiac glycosides. In some experiments the potassium concentration of the medium was varied between 0.3 mM and the usual 5 mM. 2. For periods up to 2 h in ouabain the association and dissociation rate constants were measured and the equilibrium binding constant (KD) calculated; the apparent equilibrium binding constant (K'D) was measured after 1-2 days growth in ouabain. 3. Ouabain had a K'D after 2 days of 2-6 nM in 5 mM K+ growth medium, a 4 fold greater blocking effect on sodium pumps after 2 days than expected from the association and dissociation rate constants measured in untreated or previously ouabain-treated cells. 4. This effect was: (a) approximately the same over a range of external potassium concentrations from 0.3 to 5 mM, although the absolute effect of ouabain over this range of potassium was much different; (b) probably not due to different isoforms of pumps in cells grown in ouabain compared to untreated cells; (c) apparently not a consequence of internalisation of pump-glycoside complexes. 5. We conclude that ouabain has only a limited access to sodium pumps in whole cells; this could be because sodium pumps cycle continuously through an inaccessible region of the plasma membrane. This effect needs to be considered both in the assessment of the magnitude of the long term effects of cardiac glycosides on cells, and in the measurement of the glycoside affinities of various isoforms of the pump.