EFFECT OF INORGANIC-PHOSPHATE CONCENTRATION ON THE NATURE OF INNER MITOCHONDRIAL-MEMBRANE ALTERATIONS MEDIATED BY CA2- A PROPOSED MODEL FORPHOSPHATE-STIMULATED LIPID-PEROXIDATION( IONS )

Addition of high concentrations (>1 mM) of inorganic phosphate (P-i) o r arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitoch ondrial swelling was only partially prevented either by butylhydroxyto luene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibi tor of the mitochondrial permeability transition pore. This swelling w as totally prevented by the simultaneous presence of these compounds. At lower P-i concentrations (1 mM), mitochondrial swelling is reversib le and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prev ented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest th at, at low P-i concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high P-i concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a pu tative quencher of triplet carbonyl species. This suggests that high P -i or arsenate concentrations stimulate propagation of the peroxidativ e reactions initiated by mitochondrial-generated ROS because these ani ons are able to catalyze C-n-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower C-n-1-aldehy de in the triplet state. This proposition was also supported by experi ments using a model system consisting of phosphatidyl-choline/dicethyl phosphate liposomes and the triplet acetone-generating system isobutan al/horseradish peroxidase, where phosphate and Ca2+ cooperate to incre ase the yield of thiobarbituric acid-reactive substances.