Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

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Dysregulation of Ca2+ has long been implicated to be important in cell injury. A Ca2+-linked process important in necrosis and apoptosis (or necrapoptosis) is the mitochondrial permeability transition (MPT). In the MPT, large conductance permeability transition (PT) pores open that make the mitochondrial inner membrane abruptly permeable to solutes up to 1500 Da. The importance of Ca2+ in MPT induction varies with circumstance. Ca2+ overload is sufficient to induce the MPT. By contrast after ischemia-reperfusion to cardiac myocytes, Ca2+ overload is the consequence of bioenergetic failure after the MPT rather than its cause. In other models, such as cytotoxicity from Reye-related agents and storage-reperfusion injury to liver grafts, Ca2+ appears to be permissive to MPT onset. Lastly in oxidative stress, increased mitochondrial Ca2+ and ROS generation act synergistically to product the MPT and cell death. Thus, the exact role of Ca2+ for inducing the MPT and cell death depends on the particular biologic setting.

We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously. After addition of 100 ,M t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition.

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Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to <1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained >90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na+-free buffer at pH 7.4, pHi did not rise and cell viability remained >80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5–1 μM) at pH 7.4, the MPT was prevented and cell viability remained >80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30–40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.

Cell surface 'blebbing' is an early consequence of hypoxic and toxic injury to cells. A rise in cytosolic free Ca2+ has been suggested as the stimulus for bleb formation and the final common pathway to irreversible cell injury. Here, using digitized low-light video microscopy, we examine blebbing, cytosolic free Ca2+, mitochondrial membrane potential and loss of cell viability in individual cultured hepatocytes. Unexpectedly, we found that after 'chemical hypoxia' with cyanide and iodoacetate, cytosolic free Ca2+ does not change during bleb formation or before loss of cellular viability. Cell death was precipitated by a sudden breakdown of the plasma membrane permeability barrier, possibly caused by rupture of a cell surface bleb.