Bai and A.I. during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-XLin cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of LRIG2 antibody Bcl-XL, failed to show protection against I/R. These Coelenterazine H findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial Coelenterazine H apoptosis appears to represent a redundant death pathway in this model of simulated I/R. == INTRODUCTION == Tissue ischemia is characterized by severe hypoxia, acidosis, energy depletion and cell death. Although timely restoration of blood flow is currently the most effective means of minimizing ischemic injury, reperfusion of ischemic tissue triggers a paradoxical increase in cell death [6]. Excessive oxidant stress is well accepted as an important component of ischemia-reperfusion (I/R) injury [5,6]. ROS production begins early in ischemia, and is followed by a large burst of oxidant stress during the first few minutes of reperfusion [2,30,41,46]. Potential sources of ROS include mitochondria, NAD(P)H oxidases, nitric oxide (NO) synthase, and xanthine oxidase, and the critical targets of oxidant stress may include proteins, membrane lipids, and DNA [7,14,25,37]. Although many of the details regarding the sources and targets of oxidant stress during I/R are not known, a consensus regarding the importance of ROS in I/R injury has developed, based on studies showing that cells are protected during I/R by pretreatment with antioxidants or by over-expression of antioxidant enzymes [8,9,16,34]. Other reports have implicated mitochondrial apoptosis in the cell death triggered by I/R. This highly conserved process can be initiated by the activation of BH3-containing pro-death proteins in response to a variety of stimuli including hypoxia, nutrient deprivation, ROS and DNA damage [20]. Apoptotic triggers cause the Bcl-2 family members Bax and Bak to translocate to the mitochondria and Coelenterazine H trigger the release of cytochrome c to the cytosol [19]. This leads to the activation of caspases and subsequent cell death through an ATP-dependent pathway [42,49]. Consistent with that model, transgenic mice that over-express Bcl-2, a Bax/Bak suppressor, or that are deficient in the Bax protein, have been reported to exhibit smaller infarcts compared with wild-type mice subjected to I/R [23,26,43]. Other studies report that pharmacological inhibition of caspases is protective against I/R injuryin vitro[36,40] although there is debate as to whether caspase inhibition can prevent cell death following cytochrome c release to the cytosol, due to the existence of redundant effector pathways of apoptosis that act downstream of the mitochondria. Although oxidant stress and apoptosis have both been implicated in I/R-induced cell death, the relationship between these processes is not clearly established. One possibility is that ROS generation during I/R leads to the BH3-dependent activation of mitochondrial apoptosis, cytochrome c release, and caspase-mediated cell death. Alternatively, oxidant stress generated during I/R could trigger opening of the mitochondrial permeability transition pore, leading to cytochrome c release to the cytosol, bioenergetic failure, and cell death by a necrotic rather than an apoptotic pathway [11,12]. In the former case cytochrome c (cyt-c) release would mediate cell death, whereas in the latter case the release of cyt-c would represent only a marker of lethal cell damage. The present study examined the relationship between oxidant stress during I/R and mitochondria-mediated cell death, using a model of simulated I/R in which the cells were superfused with hypoxic, hypercarbic and acidotic medium lacking glucose. The data show that oxidant stress generated during simulated ischemia triggers oscillation of the mitochondrial permeability transition pore, and that this response precedes the irreversible opening of the pore and cyt-c release during reperfusion. Although cytochrome c release during reperfusion leads to caspase activation, interventions that inhibit BH3-dependent apoptosis.