Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria

In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O2−) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. Addition of the reactive oxygen species (ROS) generator KO2 produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca2+ independent. Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O2− production detected with hydroethidine in mitochondrial preparations exposed to Ca2+ and the fact that ROS were required for Ca2+-induced mitochondrial swelling. Superoxide anions, but not Ca2+, decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. Calcium, but not O2−, triggered a rapid loss of mitochondrial potential. Calcium-induced Δψm dissipation was inhibited by Ruthenium Red, but not by CSA. Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization. Keywords: Caspase, mitochondrion, excitotoxicity, apoptosis, ROS, brain Introduction Mitochondrial dysfunctions, because of DNA defects, protein mutations or alterations in electron transport (Koutnikova et al., 1997) have been linked to neurodegenerative diseases (Sheehan et al., 1997; Brown et al., 2000) and aging processes (Wallace, 2001). Mitochondria play a central role in different cell death pathways leading to either apoptosis or necrosis (Green & Reed, 1998; Kroemer & Reed, 2000). The first evidence that mitochondria play an important role in cellular death mechanisms came from the observation that ‘in vitro' models required an organelle fraction enriched in mitochondria to induce apoptosis (Newmeyer et al., 1994). Apoptosis might involve changes in respiration-dependent mitochondrial potential (Δψm) and, in some models, formation of a voltage-dependent high conductance multiproteic channel, referred to as mitochondrial permeability transitory pore (MPTP) (Bernardi, 1999). However, there is increasing evidence suggesting that MPTP activation is also important for necrosis activation (Kroemer & Reed, 2000). The Δψm role in apoptosis is still an enigma, as either preservation (Prehn et al., 1996) or dissipation (Stout et al., 1998) has been shown to block neuronal death. Moreover, the relation between MPTP and Δψm remains unclear. There are reports suggesting that for some apoptotical stimuli, MPTP formation is independent of mitochondrial transmembrane depolarization (Madesh & Hajnoczky, 2001). As a result of MPTP opening, mitochondria might lose their membrane permeability control, releasing internal proteins (cytochrome c, apoptosis-inducing factor (AIF), SMAC/Diablo, caspase family members, etc.) to the cytoplasm. This release results in the activation of different pathways that lead to cell death. For example, cytochrome c participates in the apoptosome formation, and AIF results in chromatin condensation and DNA fragmentation (Susin et al., 1999). Although the physiological role of MPTP has not yet been determined, its role as mediator of cell injury and death is generally accepted. Calcium influx and O2− production within the mitochondria prior to cell death are events shared by cells treated with neurotoxic agents like NMDA, veratridine and β-amyloid (Bindokas et al., 1996; Jordan et al., 2000, 2002; Longo et al., 2000). Overexpression of proteins that regulate Ca2+ and O2− levels protect neuronal cultures from neurotoxic stimuli (Jordan et al., 1995; Prehn et al., 1997). Evidence has been presented suggesting that intracellular O2− production during apoptosis injury is Ca2+ dependent and from mitochondrial origin (Ankarcrona et al., 1995; Bindokas et al., 1996; Jordan et al., 2000) and either stimuli result in MPTP formation (for review, see Bernardi, 1999). In the present study, we have analyzed the plausible effects of O2− and Ca2+ on mitochondrial swelling and Δψm dissipation using isolated rat brain mitochondria.