Effect of Inorganic Phosphate Concentration on the Nature of Inner Mitochondrial Membrane Alterations Mediated by Ca2+ Ions

Abstract

Addition of high concentrations (>1 mM) of inorganic phosphate (P i ) or arsenate to Ca 2؉ -loaded mitochondria was followed by increased rates of H 2 O 2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower P i concentrations (1 mM), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, 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 putative quencher of triplet carbonyl species. This suggests that high P i or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze C n -aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower C n ؊ 1 -aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca 2؉ cooperate to increase the yield of thiobarbituric acid-reactive substances.The decrease in ATP levels that occurs under pathological conditions such as prolonged anoxia or ischemia/reperfusion results in inorganic phosphate (P i ) accumulation and alterations in intracellular Ca 2ϩ homeostasis (1-7). It is argued that high P i concentrations mimic the metabolic conditions prevalent during ischemia and that, at concentrations higher than 10 mM, P i inhibits mitochondrial oxidative phosphorylation (8). Indeed, it is well known (see Refs. 9 and 10, and references therein) that accumulation of Ca 2ϩ and P i by mitochondria results in increased permeability of the inner mitochondrial membrane. This is proposed to be a key step in the pathogenesis of cell injury that occurs during ischemia and reperfusion (8 -11). Different mechanisms such as lipid peroxidation, phospholipid hydrolysis by phospholipase A 2 , or opening of the mitochondrial permeability transition pore (MTP) 1 have been proposed to take place under these conditions (9 -14). In this regard, P i concentration, which is reported to increase up to 20 mM in cells exposed to prolonged anoxia (6), has a profound effect in the rate, extent, and nature of mitochondrial membrane altera...

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“…Moreover, the MPT possesses at least two redox-sensitive sites, one of them in equilibrium with GSH of mitochondrial matrix and another that can be directly activated by ROS [30]. Therefore, in addition to the oxidation of membrane protein sulfhydryl groups, the oxidation of both GSH and NAD(P)H of mitochondrial matrix [30], followed closely by an increase in the mitochondrial Ca 2 + and ROS generation within mitochondria (or a decreasing in their detoxification), contributes to the MPT induction, as shown by studies with several MPT inducers, including tert-butylhydroperoxide [39][40][41][42] and inorganic phosphate [31,43]. Besides the generation of ROS, NO can also act on mitochondria amplifying the signals promoting the MPT [44,45].…”

“…3, trace 7) required to trigger the MPT induction. In addition, it should be taken into account its effects either on the thiol redox status [18,42,43,50] of the MPT or on the mitochondrial bioenergetics, as previously reported [22].…”

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