Previously, we showed that the oxidant chemical, tertbutylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca 2؉ in t-BuOOH cytotoxicity to 1-daycultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 mol/L) caused an early increase of mitochondrial free Ca 2؉ , as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca 2؉ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca 2؉ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca 2؉ chelation also prevented the increase of mitochondrial Ca 2؉ , generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca 2؉ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca 2؉ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca 2؉ uptake, leading to increased matrix Ca 2؉ , in- Steady-state mitochondrial free Ca 2ϩ concentration results from the balance of membrane potential (⌬⌿)-driven Ca 2ϩ uptake by the mitochondrial Ca 2ϩ uniporter and Ca 2ϩ efflux by concerted 3Na ϩ /Ca 2ϩ and Na ϩ /H ϩ exchange driven by both mitochondrial ⌬⌿ and pH gradient (⌬pH). 1-2 These transport systems maintain resting mitochondrial free Ca 2ϩ close to cytosolic free Ca 2ϩ in most cell types. [3][4][5][6][7] Although early modeling suggested that mitochondrial Ca 2ϩ responds slowly to physiological changes of cytosolic free Ca 2ϩ , several recent studies show that mitochondrial Ca 2ϩ can increase rapidly in response to cytosolic Ca 2ϩ transients caused by hormone stimulation, muscle contraction, and other stimuli. [4][5][6][7][8][9] Increases of mitochondrial Ca 2ϩ activate Ca 2ϩ -dependent dehydrogenases that are responsible for mitochondrial adenosine triphosphate production. [10][11][12] Although it has been widely proposed that mitochondria release Ca 2ϩ under conditions of cellular stress, this is unlikely for the simple reason that mitochondria do not have the Ca 2ϩ to release. [3][4][5]7 Rather, mitochondrial Ca 2ϩ concentration may increase as a consequence of cellular stress. 3,[13][14][15] One possible consequence of mitochondrial Ca 2ϩ loading is onset of the mitochondrial permeability transition (MPT). Onset of the MPT is the result of the opening of highconductance permeability transition pores in the mitochondrial inner membrane. These pores conduct both neutral and charged solutes of molecular weights up...

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