Activity of Na⁺ / K⁺ ATPases sustains K⁺ and Na⁺ gradients across the plasma membrane. The Na⁺ gradient facilitates cellular uptake of glucose and amino acids. Consequently, blockade of Na⁺ / K⁺ ATPase activity not only causes a collapse of the Na⁺ gradient but also impairs transport of nutrients into the cell. Nutrient deprivation initializes autophagic processes, which may promote oxidative stress and, finally, cell death. In Jurkat cells, CD-95-induced apoptosis is associated with an inhibition of the Na⁺ / K⁺ ATPase (Nobel et al., Apoptosis, 2000).

In the present study, we used ouabain to block Na⁺ / K⁺ ATPase activity and an antibody to 4-hydroxynonenal (HNE)-modified proteins to visualize oxidative stress in microglial cells. HNE is a toxic metabolite of lipid peroxidation. We identified discrete immune-labeled patches in control as well as in ouabain-exposed cells. However, ouabain-exposed cells revealed larger and more immune-labeled patches compared to control cells. In some ouabain-exposed cells, immune-labeling was concentrated around the nucleus. To investigate whether immune-labeling depends on starvation-induced oxidative stress, we pre-incubated cells with the anti-oxidant, a-tocopherol, before exposure to ouabain. In these experiments, cells had smaller and fewer immune-labeled patches. Furthermore, we studied the distribution of condensed chromatin in DAPI-labeled cells using confocal microscopy. We found that ouabain-exposed preparations contained more cells showing crescent-like chromatin condensation at the nuclear periphery, which is a hall mark of apoptosis. Therefore, our study indicates that ouabain exposure increases oxidative stress, which may ultimately lead to apoptosis in microglial cells.