(46) PROOXIDANTS OPEN TWO DIFFERENT PORES IN THE INNER MITOCHONDRIAL MEMBRANE

Yulia E. Kushnareva and Patricia M. Sokolove
Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201

Prooxidants are generally included among the agents that trigger the mitochondrial permeability transition (MPT). The effects of the prooxidants diamide and t-butylhydroperoxide on the permeability of isolated rat liver mitochondria were studied with the following results: In the absence of exogenous inorganic phosphate (1) prooxidants caused a collapse of the membrane potential that preceded the onset of mitochondrial swelling; (2) substantially more cyclosporin A was required to inhibit the decrease in the membrane potential than to prevent swelling, with the result that collapse of the membrane potential could be observed in the complete absence of swelling; (3) swelling was inhibited by spermine and magnesium, whereas the collapse of the membrane potential was not; (4) collapse of the membrane potential was associated with ruthenium red-insensitive calcium efflux but not with glutathione release; (5) collapse of the membrane potential induced by t-butylhydroperoxide could be restored upon addition of EGTA; dithiothreitol reversed the collapse of the memrane potential induced by both t-butylhydroperoxide and by diamide. When inorganic phosphate ( >1 mM) was added to the assay medium, the onsets of swelling and dissipation of the membrane potential were coordinately speeded, i.e., in the presence of the phosphate levels generally used to assay the MPT, no separation of the collapse of the membrane potential and swelling was observed. Our data suggest that prooxidants open two different pores or pore substates in the inner mitochondrial membrane, which can be distinguished on the basis of permeating species and inhibitor sensitivity: the classic MPT which allows passage of solutes with molecular masses up to 1500 Da (high conductance state) and a pore permeable only to protons/calcium (low conductance state).