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Fig. 3. mtCa²⁺-dependent and mtCa²⁺-independent mechanisms promote toxicity mediated by mitochondrial permeability transition. (A) The cells were first treated for 5 minutes with 0.5 µM rotenone, 20 µM ryanodine (Ry), 10 U/ml catalase, 0.5 µM cyclosporin A (CsA) or 1 µM FK506, subsequently exposed for 3 minutes to peroxynitrite (40 or 200 µM) and finally incubated for a further 57 minutes in the absence or presence of 1 µM antimycin A or 10 µM or 2-hepthyl-4-hydroxyquinoline (HQNO). Toxicity induced by peroxynitrite in the absence or presence of antimycin A, or HQNO, was also tested in respiration-deficient (RD) cells. Cytotoxicity was then determined. Results represent the means ± s.e.m. calculated from three to five experiments. ^*P<0.001 compared with levels in untreated cells (unpaired Student's t-test). (B) Representative photomicrographs (magnification 400x) of cells treated for 3 minutes with 0 or 40 µM peroxynitrite and finally incubated for a further 7 minutes with MitoTracker Red CMXRos in the absence or presence of the indicated additions. (C) The cells were treated for 5 minutes with 0.5 µM CsA or 1 µM FK506, as detailed in the figure, subsequently exposed for 3 minutes to 40 µM peroxynitrite and finally incubated for a further 7 minutes with MitoTracker Red CMXRos in the absence or presence of 1 µM antimycin A or 10 µM HQNO. Fluorescence was then quantified as detailed in the Materials and Methods. Results represent the means ± s.e.m. calculated from three to five experiments. ^*P<0.001 compared with levels in untreated cells (unpaired Student's t-test). (D) Cells were exposed for 3 minutes to 40 µM peroxynitrite and finally incubated for a further 7 minutes in the absence or presence of various additions. Cells were then processed to obtain the mitochondrial and cytosolic fractions for western blot analysis using antibodies against cytochrome c, AIF or Bax. The blots were then washed and re-probed for actin or HSP-60.

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