The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5-15 mu M) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca2+-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEES Lett, 423 (1998) 339-342], Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (Delta Psi) in Ca2+-loaded rat liver mitochondria respiring on succinate, The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of Delta Psi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of Delta Psi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP, These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening. (C) 1999 Federation of European Biochemical Societies.464416719710

The Participation Of Pyridine Nucleotides Redox State And Reactive Oxygen In The Fatty Acid-induced Permeability Transition In Rat Liver Mitochondria.

The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.46497-10

Permeability Transition Pore Closure Promoted By Quinine.

The mitochondrial membrane permeability transition induced by Ca2+ is inhibited by quinine in a dose-dependent fashion. Competition experiments strongly suggest that quinine displaces Ca2+ bound to the inner membrane. This is supported by experiments showing that quinine inhibits Ca2+-dependent but not Ca2+-independent mitochondrial swelling induced by phenylarsine oxide. As with Ca2+ chelators, quinine induces permeability transition pore closure preventing the contraction induced by poly(ethylene glycol) 2000 in mitochondria preswollen by incubation in KSCN medium containing Ca2+ and inorganic phosphate. These results suggest that quinine dislodges Ca2+ bound to the protein site, which triggers pore opening.31153-

Calcium mobilization by arachidonic acid in trypanosomatids

A recent report (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A(2) and arachidonic acid, Biochem J 1998;336:659-66) provided evidence that calcium entry in Trypanosoma brucei bloodstream trypomastigotes is regulated via a signaling pathway involving phospholipase A(2)-mediated generation of arachidonic acid and stimulation of a plasma membrane-located calcium channel. Here we show that Ca2+ influx in T. brucei procyclic trypomastigotes, Leishmania donovani promastigotes and T. cruzi amastigotes was also stimulated in a dose-dependent manner (50-400 nM) by the amphiphilic peptide melittin. This effect was blocked by the phospholipase A(2) inhibitor 3-(4-octadecyl)-benzoylacrylic acid. The unsaturated fatty add arachidonic acid, in the range of 10-75 mu M, induced Ca2+ entry by a mechanism sensitive to LaCl3. However, both melittin and arachidonic acid induced an increase in [Ca2+](i) in T. brucei procyclic trypomastigotes incubated in Ca2+-free medium implying Ca2+ mobilization from intracellular stores. This hypothesis was supported by experiments showing that arachidonic acid promoted Ca2+ release from the acidocalcisomes of these cells. The results showing changes in mitochondrial membrane potential. release of acridine orange and Ca2+ from the acidocalcisomes and Ca2+ transport across the plasma membrane suggest that in addition to the possible stimulation of a Ca2+ channel-mediated process, arachidonic acid, in the range of concentrations used here: have other nonspecific effects on the trypanosomatids membranes. (C) 2000 Elsevier Science B.V. All rights reserved.105226127

Presence of a Na+/H+ exchanger in acidocalcisomes of Leishmania donovani and their alkalization by anti-leishmanial drugs

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain most of the cellular calcium. The data presented here demonstrate that Leishmania donovani acidocalcisomes possess a Na+/H+ exchanger. 3,5-Dibutyl-4-hydroxytoulene, in the concentration range of 0-20 mu M, inhibited the Na+/H+ exchanger, and strongly stimulated the activity of the vacuolar H+-ATPase responsible for vacuolar acidification. As occurs with Na+, the cationic anti-leishmanial drugs pentamidine, WR-6026, and chloroquine promoted a fast and extensive alkalization of the L, donovani acidocalcisomes. (C) 2000 Federation of European Biochemical Societies.473220320

Ca2+ induces a cyclosporin A-insensitive permeability transition pore in isolated potato tuber mitochondria mediated by reactive oxygen species

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mirochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, acid is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.331435

Low temperature and aging-promoted expression of PUMP in potato tuber mitochondria

In this communication, we show that the plant uncoupling mitochondrial protein (PUMP) present in potato tuber mitochondria is induced by aging at 28 degrees C and that this induction is strongly stimulated when the potato tubers are stored at low temperature (4 degrees C). PUMP activity was detected by the degree of linoleic acid (LA)-induced ATP-sensitive mitochondrial uncoupling measured as a function of the decrease in membrane potential(Delta Psi). The PUMP content was evaluated by immunoblot analysis using polyclonal antibodies raised against potato PUMP that specifically detected a 32 kDa band. In agreement with the effect of LA on AY, the content of the 32 kDa band increased during storage and was stimulated by low temperature. These results support the proposed role of PUMP in plant thermogenesis and possibly in fruit ripening and senescence. (C) 1999 Federation of European Biochemical Societies.457110310

P-type proton ATPases are involved in intracellular calcium and proton uptake in the plant parasite Phytomonas francai

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca2+ compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca2+-ATPase activity was found to be responsible for intracellular Ca2+ transport in these cells, with no evidence of a mitochondrial Ca2+ transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A,, and stimulated by Na+ ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca2+ and H+ transport in promastigotes of P. francai.521556

Ca2+ Induces A Cyclosporin A-insensitive Permeability Transition Pore In Isolated Potato Tuber Mitochondria Mediated By Reactive Oxygen Species.

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.3343-5

Low Temperature And Aging-promoted Expression Of Pump In Potato Tuber Mitochondria.

In this communication, we show that the plant uncoupling mitochondrial protein (PUMP) present in potato tuber mitochondria is induced by aging at 28 degrees C and that this induction is strongly stimulated when the potato tubers are stored at low temperature (4 degrees C). PUMP activity was detected by the degree of linoleic acid (LA)-induced ATP-sensitive mitochondrial uncoupling measured as a function of the decrease in membrane potential (delta psi). The PUMP content was evaluated by immunoblot analysis using polyclonal antibodies raised against potato PUMP that specifically detected a 32 kDa band. In agreement with the effect of LA on delta psi, the content of the 32 kDa band increased during storage and was stimulated by low temperature. These results support the proposed role of PUMP in plant thermogenesis and possibly in fruit ripening and senescence.457103-