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First published online 15 May 2007
doi: 10.1242/jcs.003228

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Inhibition of complex III promotes loss of Ca²⁺ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite

Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino "Carlo Bo", Via S. Chiara, 27-61029 Urbino (PU), Italy

View larger version (30K): [in this window] [in a new window]   Fig. 1. Antimycin A significantly lowers the threshold concentration and time necessary for maximal formation of ROS and DNA single-strand breaks in cells exposed to peroxynitrite. (A-D) The cells were first treated for 5 minutes with 0.5 µM rotenone, 5 µM myxothiazol or 20 µM ryanodine (Ry), as detailed in the figure, subsequently exposed for 3 minutes to increasing concentrations of peroxynitrite and finally incubated for a further 27 minutes in the absence (A,B) or presence (C,D) of 1 µM antimycin A. (E,F) Cells were exposed for 3 minutes to 0, 40 or 200 µM peroxynitrite and incubated for increasing time intervals in the absence or presence of 1 µM antimycin A. After treatment, cells were analyzed for DNA damage and dihydrorhodamine 123 (DHR)-fluorescence as detailed in the Materials and Methods. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.01; **P<0.001 compared with levels in untreated cells; (*)P<0.01; (**)P<0.001 compared with levels in cells treated with peroxynitrite alone (A,B) or associated with antimycin A (C,D) (ANOVA followed by Dunnett's test).

View larger version (21K): [in this window] [in a new window]   Fig. 2. The enhancing effects of antimycin A or 2-hepthyl-4-hydroxyquinoline (HQNO) are mediated by inhibition of electron transport through complex III and are independent on the accumulation of mtCa2+. (A) Representative microscope images (magnification 400x) of cells pre-loaded with Rhod-2 AM and MitoTracker Green and then treated for 10 minutes with 0 or 200 µM peroxynitrite. In the merged image (overlay) regions containing both Rhod-2 and MitoTracker Green fluorescence appear yellow. (B) Rhod 2-AM pre-loaded cells were first treated for 5 minutes with 20 µM ryanodine (Ry), subsequently exposed for 3 minutes to peroxynitrite (40 or 200 µM) and finally incubated for a further 7 minutes 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 untreated cells (unpaired Student's t-test). (C) Permeabilized cells were exposed for 10 minutes to 200 µM peroxynitrite (200), or 40 µM peroxynitrite/1 µM antimycin A (40 + Antimycin A), in the absence or presence of 10 µM ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 200 nM ruthenium red (RR), 100 µM LaCl3, 20 µM Ry, 0.5 µM rotenone, 5 µM myxothiazol or 10 U/ml catalase (enzymatically active or heat-inactivated). The DNA cleavage induced by peroxynitrite in the absence or presence of antimycin A was also assessed in respiration-deficient (RD) cells. The level of DNA single-strand breaks was measured immediately after the treatments. Results represent the means ± s.e.m. calculated from three to five experiments. *P<0.001 compared with levels in cells exposed to peroxynitrite alone or associated with antimycin A (unpaired Student's t-test).

View larger version (64K): [in this window] [in a new window]   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.