Fig. 2. Suppression of the Na⁺, K⁺-pump activity by apoptotic insults. I_pump was recorded at –60 mV in cortical neurons during exposure to the apoptotic insult serum deprivation or staurosporine, which induced neuronal apoptosis in 24-48 hours. (A) I_pump was gradually suppressed after 5 hours in a serum-free medium; a 9-hour serum deprivation profoundly blocked the Na⁺, K⁺-pump (n=16). Pyruvate (5 mM) and succinate (5 and 20 mM) preserved the pump activity during serum deprivation. Notably, succinate showed less effect than pyruvate at 5 mM concentration; 20 mM succinate protected the pump current similarly as 5 mM pyruvate did. n=6-21 cells. (B) Incubation with 0.1 µM staurosporine for 30 minutes did not show any effect on the current; prolonged incubation, however, resulted in progressive depression of I_pump. By 12 hours, there was little strophanthidin-sensitive current detected. Pyruvate (5 mM) or succinate (20 mM), co-applied with staurosporine, was able to retain the pump current at around control levels even after 12-hour exposure. Succinate at 5 mM also attenuated the suppression of I_pump. n=7-17. (C) C₂-ceramide (25 µM) gradually suppressed I_pump; co-applied pyruvate (5 mM), however, could not prevent the Na⁺, K⁺-pump failure (n=8). Note that the effect of C₂-ceramide on the pump activity was slower and milder than that of serum deprivation and staurosporine, in agreement with less cell death induced by C₂-ceramide (see Fig. 7D). (D) Top: Representative inward Na⁺, K⁺-pump current induced by 500 µM strophanthidin in control cells and cells exposed to staurosporine (0.1 µM, 10 hours). Bar graph: control Na⁺, K⁺-pump current and the current in cells undergoing apoptosis (0.1 µM staurosporine, 9-10 hours); the Na⁺, K⁺-pump current in staurosporine-treated cells was significantly higher after 10-minute dialysis with an internal solution containing 10 mM ATP. n=5 for control group and the staurosporine group without ATP dialysis; n=6 for the staurosporine group with ATP dialysis. (E) Top: Representative outward current generated by activation of the Na⁺, K⁺-pump in control cells and cells exposed to 0.1 µM staurosporine (10 hours). Whole-cell recordings were established in regular external solution of 3 mM K⁺; cells were exposed to a K⁺-free extracellular solution for 10 seconds followed by 5 seconds exposure to 4 mM K⁺. The outward current triggered by external K⁺ was fully blocked by strophanthidin (data not shown), consistent with a pump current. Bar graph: The apoptotic insult staurosporine (0.1 µM, 9-10 hours) markedly suppressed the pump current (n=8). *P<0.01 compare with controls at time zero; #P<0.01 compare with apoptotic insult alone at same time point.

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