Respiration-dependent removal of exogenous H2O2 in brain mitochondria: inhibition by Ca2+.
In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect approximately 40%, IC50, 12 microm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60-70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 microm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased DeltaPsi, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks.
Zoccarato
Franco
F
Department of Biological Chemistry and Consiglio Nazionale delle Ricerche, Unit for the Study of Biomembranes, University of Padova, Viale G. Colombo 3, Padova 35121, Italy. franco.zoccarato@unipd.it
Cavallini
Lucia
L
Alexandre
Adolfo
A
D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D001921:Brain / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D019802:Succinic Acid / Q000378:metabolism
D018053:Arsenites; D004533:Egtazic Acid; D009249:NADP; D019802:Succinic Acid; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; C015001:arsenite; D002118:Calcium