Further characterization of the events involved in mitochondrial Ca 2+ release and pore formation by prooxidants

Abstract

Addition of the prooxidant 3,5-dimethyl- N-acetyl- ρ-benzoquinone imine (3,5(Me) 2NAPQI) to Ca 2+-loaded mitochondria caused a rapid and extensive release of the sequestered Ca 2+. Ca 2+ release was accompanied by irreversible NAD(P)H oxidation and was followed by the release of adenine and pyridine nucleotides into the extramitochondrial medium; this is evidence of the opening of the pore in the inner mitochondrial membrane. Preincubation of the mitochondria with ADP, cyclosporin A (CSA), m-iodobenzylguanidine (MIBG) or Mg 2+ inhibited the prooxidant-induced Ca 2+ release and prevented pore-opening. When mitochondria were preincubated with ruthenium red, Ca 2+ release was only minimally stimulated by 3,5(Me) 2NAPQI. However, increasing the concentration of the prooxidant caused release of an increasing fraction of the sequestered Ca 2+. Alternatively, increasing the intra-mitochondrial Ca 2+ load resulted in a lowering of the concentration of 3,5(Me) 2NAPQI required for near complete Ca 2+ release to occur. In the presence of ruthenium red, 3,5(Me) 2NAPQI-induced Ca 2+ release was accompanied by irreversible pyridine nucleotide oxidation and followed by the release of nucleotides into the extramitochondrial medium, events which were prevented on preincubation with CSA. Similarly, the addition of CSA, ADP or MIBG during 3,5(Me) 2+NAPQI-induced Ca 2+ release arrested further Ca 2+ release. In addition to their inhibitory effect on the 3,5(Me) 2NAPQI-induced Ca 2+ release, CSA, ADP or MIBG also decreased the rate of the basal, ruthenium red-induced mitochondrial Ca 2+ release by 45–70%. It is proposed that the basal, ruthenium red-induced and the prooxidant-induced mitochondrial Ca 2+ release occur through a common component that is sensitive to inhibition by CSA, ADP and MIBG and that is involved in mitochondrial pore formation. Furthermore, 3,5(Me) 2NAPQI-induced pore opening does not involve Ca 2+-cycling, but rather involves a site(s) that is (are) synergistically activated by Ca 2+ and the prooxidant.