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.
摘要
人细胞（HeLa）在含有或不含一系列浓度强心苷的生长培养基中培养长达48小时。在一些实验中，培养基中的钾浓度在0.3 mM至通常的5 mM之间变化。2. 在哇巴因中培养长达2小时，测量结合和解离速率常数并计算平衡结合常数（KD）；在哇巴因中生长1 - 2天后测量表观平衡结合常数（K'D）。3. 在5 mM K +生长培养基中，哇巴因在2天后的K'D为2 - 6 nM，2天后对钠泵的阻断作用比从未处理或先前用哇巴因处理的细胞中测得的结合和解离速率常数预期的大4倍。4. 这种效应是：（a）在0.3至5 mM的一系列外部钾浓度范围内大致相同，尽管哇巴因在该钾浓度范围内的绝对效应差异很大；（b）可能不是由于与未处理细胞相比，在哇巴因中生长的细胞中泵的同工型不同；（c）显然不是泵 - 糖苷复合物内化的结果。5. 我们得出结论，哇巴因在全细胞中对钠泵的作用有限；这可能是因为钠泵持续循环通过质膜的一个不可及区域。在评估强心苷对细胞的长期效应的大小以及测量泵的各种同工型的糖苷亲和力时都需要考虑这种效应。
