The apparent discrepancy of ouabain inhibition of cation transport and of lymphocyte proliferation is explained by time‐dependency of ouabain binding

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Abstract
Mitogenesis of human blood lymphocytes in culture is inhibited by concentrations of ouabain that are approximately one order of magnitude lower than those that block Na and K transport. For example, the 50% inhibition (ID50) of Na-K transport, 280 nM, is seven-fold greater than the ID50 for RNA synthesis, DNA synthesis, or blastogenesis, ˜40 nM. Yet, inhibition of transport and consequent reduction in cell K is considered responsible for the effects of ouabain on mitogenesis. Since synthetic processes are assessed at least 24 hours after lymphocyte stimulation, this discrepancy could be explained by either 1) a progressive increase in K leak, or 2) a progressive inhibition of Na-K transport by ouabain during 24 hours of PHA treatment. We found that the lymphocyte membrane leak rate of K increased immediately after PHA treatment but did not increase further from 4 to 24 hours. In contrast, the ouabain sensitivity of 42K uptake was markedly increased with time: ID50 for 42K uptake of 35 nM at 24 hours as compared to 280 nM at 30 minutes. Measurement of ouabain binding revealed a seven-fold increase in the lymphocyte-associated ouabain after 24 hours compared to binding at 1 hour. These data indicate that the dose response of ouabain inhibition of active K transport and lymphocyte proliferation are closely correlated if one considers the slow membrane binding of ouabain at low concentrations.