Figure 2

Ouabain increases the intracellular levels of ROS and Ca⁺⁺. (a) ROS/CDCF fluorescence as a function of OUA concentration. CDCFH-DA loaded cells were treated with OUA for 30 min. The data are the means ± S.D. of three independent experiments. Statistical analysis by Student’s t test is shown. (b) Ca⁺⁺/FLUO-3 fluorescence depends on the concentration of OUA and on the cellular entry of the ion. FLUO-3-AM loaded cells were treated with OUA at for 30 min. One cell sample was treated with OUA (1 μM) at the presence of EGTA (2 μM) to discriminate between Ca⁺⁺ entry and Ca⁺⁺ mobilization. The data are the means ± S.D. of five independent experiments. (*, P <0.05; **, P <0.005 in comparison with untreated cells). (c) Intracellular Ca⁺⁺ increase depends on the Na⁺/Ca⁺⁺-exchanger active in the Ca⁺⁺ influx mode. FLUO-3-AM loaded cells were either left untreated or treated with KBR (10 μM) to inhibit NCX or with Nifedipine (10 μM) for 30 min and then with OUA at the indicated concentrations for 30 min. The data are the means ± S.D. of four independent experiments. Statistical analysis by Student’s t test is shown. In all experiments the fluorescent signal of ≥ 10.000 events was evaluted under cytofluorimetry on a log scale (FL1) and recorded as MFI of the whole cell population. The results are expressed according to the formula (MFI in OUA treated cells)/(MFI in untreated cells) x 100.