Figure 1.

The enhancing effects of AA in U937 cells exposed to low concentrations of peroxynitrite: evidence for a Ca²⁺-independent mechanism. (A–C) The cells were treated as shown in the figure and then exposed for: 10 min (A,B); or 30 min (C) to the indicated concentrations of peroxynitrite. AA (3 µM) was given to the cells 15 min prior to peroxynitrite. Pre-exposure to antimycin A (Ant A, 1 µM), or Cf (10 mM), was instead of only 5 min. In some experiments, rotenone (0.5 µM), myxothiazol (5 µM) or Ry (ryanodine) (20 µM) were added to the cells 5 min prior addition of AA, Ant A or Cf. After treatments, the cells were analyzed for: MitoSOX red-fluorescence (A); aconitase activity (B); and DNA strand scission (C); (D) the cells were exposed for 15 min to AA and immediately analyzed for their cellular and mitochondrial content of the vitamin; (E) Rhod 2-AM (acetoxymethyl) pre-loaded cells were treated for 10 min, as indicated in the figure, and analyzed as detailed in the Materials and Methods Section. The inset shows the Rhod 2-fluorescence response observed in cells exposed for 15 min to 0 or AA; (F) cells were permeabilized with digitonin and exposed for 10 min to the indicated concentrations of peroxynitrite alone or associated with rotenone, myxothiazol, Ry, EGTA (ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid) (10 µM), RR (ruthenium red) (200 nM) or LaCl₃ (100 µM). In some experiments, the cells were exposed to AA prior to permeabilization. In other experiments, the cells were supplemented with Ant A immediately after permeabilization. Results represent the means ± SD calculated from at least three separate experiments. * p < 0.001 as compared to untreated cells (one-way ANOVA followed by Dunnett’s test).