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Brain Mitochondria Are Primed by Moderate Ca2+ Rise upon Hypoxia/Reoxygenation for Functional Breakdown and Morphological Disintegration

2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês

10.1074/jbc.m302743200

1083-351X

Lorenz Schild, Jens Huppelsberg, Stefan Kahlert, Gerburg Keilhoff, Georg Reiser,

Neuroscience and Neuropharmacology Research

In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo. In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo. Stroke commonly results in tissue infarction characterized by necrotic cell death of all cell types within the infarcted area of the brain. From investigations using animal models of stroke (1Sims N.R. Anderson M.F. Neurochem. Int. 2002; 40: 511-526Crossref PubMed Scopus (283) Google Scholar, 2Anderson M.F. Sims N.R. J. Neurochem. 1999; 73: 1189-1199Crossref PubMed Scopus (75) Google Scholar), it has become evident that mitochondria are injured during cerebral ischemia and post-ischemic reperfusion. Ischemia induces a decrease in the mitochondrial capacity for respiratory activity (1Sims N.R. Anderson M.F. Neurochem. Int. 2002; 40: 511-526Crossref PubMed Scopus (283) Google Scholar, 2Anderson M.F. Sims N.R. J. Neurochem. 1999; 73: 1189-1199Crossref PubMed Scopus (75) Google Scholar). Brain mitochondria undergo ultrastructural changes after transient and during permanent cerebral ischemia. After transient focal ischemia, cortical neuronal mitochondria become injured, which is manifested by condensation, increased matrix density, and deposits of electron-dense material, finally resulting in disintegration. In contrast, permanent ischemia causes increasing loss of matrix density, associated with mitochondrial swelling, which disappears after 24 h of focal ischemia (3Solenski N.J. diPierro C.G. Trimmer P.A. Kwan A.L. Helms G.A. Stroke. 2002; 33: 816-824Crossref PubMed Scopus (119) Google Scholar). A further response to cerebral ischemia is the permeabilization of at least the outer mitochondrial membrane. This results in the liberation of proapoptotic proteins such as cytochrome c, caspase 9, and second mitochondria-derived activator of caspases (4Namura S. Nagata I. Takami S. Masayasu H. Kikuchi H. Stroke. 2001; 32: 1906-1911Crossref PubMed Scopus (93) Google Scholar, 5Sugawara T. Noshita N. Lewen A. Gasche Y. Ferrand-Drake M. Fujimura M. Morita-Fujimura Y. Chan P.H. J. Neurosci. 2002; 22: 209-217Crossref PubMed Google Scholar). Elevation of cytosolic Ca2+ concentration during ischemia/reperfusion might be a signal for permeabilization of the mitochondrial membrane. In rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic treatment, an increase in cellular calcium was shown (6Zhang Y. Lipton P. J. Neurosci. 1999; 19: 3307-3315Crossref PubMed Google Scholar). Elevated cytosolic calcium concentrations favor the opening of the mitochondrial permeability transition pore (7Huser J. Rechenmacher C.E. Blatter L.A. Biophys. J. 1998; 74: 2129-2137Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 8Bernardi P. Broekemeier K.M. Pfeiffer D.R. J. Bioenerg. Biomembr. 1994; 26: 509-517Crossref PubMed Scopus (528) Google Scholar). In fact, it could be demonstrated that cyclosporin A as well as the non-immunosuppressive analogue N-methyl-Val-4-cyclosporin A, both of which are known to prevent opening of the mitochondrial permeability transition pore, diminished the infarct size, but they could not completely prevent tissue necrosis (9Friberg H. Wieloch T. Biochimie (Paris). 2002; 84: 241-250Crossref PubMed Scopus (174) Google Scholar, 10Matsumoto S. Friberg H. Ferrand-Drake M. Wieloch T. J. Cereb. Blood Flow Metab. 1999; 19: 736-741Crossref PubMed Scopus (190) Google Scholar, 11Yoshimoto T. Siesjo B.K. Brain Res. 1999; 839: 283-291Crossref PubMed Scopus (138) Google Scholar). Thus, other mechanisms besides the opening of the mitochondrial permeability transition pore seem to be involved in tissue damage during ischemia/reperfusion. From in vitro studies using isolated brain mitochondria, it has become clear that the release of cytochrome c does not necessarily require the opening of the mitochondrial permeability transition pore (12Schild L. Keilhoff G. Augustin W. Reiser G. Striggow F. FASEB J. 2001; 15: 565-567Crossref PubMed Scopus (81) Google Scholar, 13Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (165) Google Scholar). Cytochrome c has been identified as an important modulator of death and survival in cells (14Reed J.C. Cell. 1997; 91: 559-562Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). The outcome for tissue survival after transient ischemia in in vivo models of stroke depends upon the interplay between a variety of factors, such as increased cytosolic Ca2+ concentration, reactive oxygen species, nitric oxide, and substrate supply. Therefore, in vitro models are required in order to delineate the effect of the distinct factors separately from their subsequent interaction. In this context, investigations on isolated brain mitochondria have revealed that Ca2+ accumulation, a hallmark of ischemia/reperfusion, can either inhibit or stimulate H2O2 production (15Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar). Moreover, experiments with isolated mitochondria demonstrated that the BH3 domain of Bax induces the release of cytochrome c from mitochondria (16Polster B.M. Kinnally K.W. Fiskum G. J. Biol. Chem. 2001; 276: 37887-37894Abstract Full Text Full Text PDF PubMed Google Scholar). The purpose of the present in vitro study was to use isolated brain mitochondria to elucidate the role played by hypoxia/ reoxygenation and elevated extramitochondrial Ca2+ concentration as signals for permeabilization of the mitochondrial membrane for matrix and intermembrane space proteins. Therefore, we subjected isolated rat brain mitochondria to hypoxia/reoxygenation and/or elevated extramitochondrial Ca2+ concentration and determined respiration, mitochondrial morphology, cytochrome c release, and membrane permeability by applying various complementary techniques. We found that in hypoxia/reoxygenation an elevated extramitochondrial Ca2+ concentration dramatically enhanced inhibition of active respiration. Moreover, Ca2+ had to enter the mitochondrial matrix to mediate this effect. Under these conditions there was a complete loss of mitochondrial integrity. The presence of ADP, but not AMP, during hypoxia/reoxygenation completely prevented mitochondrial damage. Permeabilization of the mitochondrial membrane did not depend on cyclosporin A, a compound known to keep the mitochondrial permeability transition pore in the closed state. Materials—Cyclosporin A was purchased from Sigma, cytochrome c from Roche Diagnostics, mouse monoclonal cytochrome c antibody from Pharmingen, and anti-mouse Ig and horseradish peroxidase from Roche Diagnostics. CGP37157 was from Tocris (Cologne, Germany). All other chemicals were of analytical grade. Preparation of Brain Mitochondria—Mitochondria were prepared from the brains of 220–240-g male Wistar rats in ice-cold medium containing 250 mm mannitol, 20 mm Tris, 1 mm EGTA, 1 mm EDTA, and 0.3% (w/v) bovine serum albumin at pH 7.4 (isolation medium) using a standard procedure (17Schild L. Westphal S. Scheithauer S. Holfeld M. Augustin W. Sabel B.A. Acta Ophthalmol. Scand. 1996; 74: 354-357Crossref PubMed Scopus (8) Google Scholar). After the initial isolation, Percoll was used for purification of mitochondria from a fraction containing some endoplasmic reticulum, Golgi apparatus, and plasma membranes. The mitochondria were well coupled, as indicated by a respiratory control index greater than 4 with glutamate plus malate as substrates. Protein content was measured according to the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as the standard. Incubation of Mitochondria—Mitochondria (0.5–1.0 mg of protein/ml) were incubated in a medium containing 10 mm KH2PO4, 0.5 mm EGTA, 60 mm KCl, 60 mm Tris, 110 mm mannitol, and 1 mm free Mg2+ at pH 7.4 and 30 °C. Extramitochondrial calcium was adjusted by using Ca2+-EGTA buffers. For calculating the concentration of free calcium, we used the complexing constants according to Fabiato and Fabiato (19Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar). Hypoxia was produced by bubbling 2 ml of the incubation medium with N2 until oxygen was not detectable any more by means of a Clark-type electrode. Afterward, the mitochondria added to the medium further decreased the oxygen concentration via the respiratory chain. The final oxygen concentration was less than 2 nmol/ml. As the K m value of the mitochondrial cytochrome oxidase is about 0.3 μm, the limiting effect of oxygen was illustrated independently by the collapse of the membrane potential measured by means of a TPP+-sensitive electrode in separate experiments (data not shown). A 2-ml volume of air-saturated incubation medium was added to achieve reoxygenation. Measurement of Respiration—Oxygen uptake of the mitochondria was measured at 30 °C in a thermostat-controlled chamber equipped with a Clark-type electrode. For the calibration of the oxygen electrode, the oxygen content of the air-saturated incubation medium was taken to be 217 nmol/ml (20Reynafarje B. Costa L.E. Lehninger A.L. Anal. Biochem. 1985; 145: 406-418Crossref PubMed Scopus (196) Google Scholar). Measurement of Mitochondrial Swelling—The absorption of mitochondrial incubations was measured with a Varian spectrophotometer (Cary 1E) at 546 nm. Calcium-dependent swelling caused by the opening of the mitochondrial permeability transition pore was induced by adding 200 μm CaCl2. Immunoblotting for Detection of Cytochrome c—After 10 min of incubation, 2-ml samples of the incubation mixture were centrifuged at 12,000 × g for 10 min at 4 °C, and the resulting supernatants were centrifuged at 100,000 × g for 15 min at 4 °C. The supernatants were used for Western blot analysis as described by Ghafourifar et al. (21Ghafourifar P. Schenk U. Klein S.D. Richter C. J. Biol. Chem. 1999; 274: 31185-31188Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Electron Microscopy—For electron microscopy, three independent mitochondrial preparations were used for each incubation strategy. After sedimentation at 320 × g at 4 °C, the mitochondrial pellet was fixed with a mixture of 4% formaldehyde (freshly prepared from paraformaldehyde) and 0.4% glutaraldehyde for1hat4 °C. Thereafter, the pellet was rinsed thoroughly with phosphate-buffered saline (pH 7.4), postfixed in 1% osmium tetroxide for 1 h at 4 °C, dehydrated in a graded series of ethanol, en bloc contrasted with 1% uranyl acetate in 70% ethanol, and flat-embedded between two polyethylene sheets in Curcupan (Fluka/Sigma, Deisenhofen, Germany). Each washing and incubation step was followed by sedimentation at 320 × g at 4 °C to collect the mitochondria. Ultrathin sections (50–70 nm) were prepared with a Leica Ultracut UCT (Bensheim, Germany), mounted on Form-var-coated slot grids, and examined with a Zeiss transmission electron microscope 900 (Oberkochen, Germany). Influence of Extramitochondrial Ca2+ and Hypoxia/Reoxygenation on Mitochondrial Respiration—To investigate whether elevated extramitochondrial Ca2+ concentration plays a role in the impairment of mitochondrial function during ischemia/reperfusion, respiration of isolated rat brain mitochondria was analyzed. The influence of extramitochondrial Ca2+ alone and of the combination of a rise in Ca2+ with hypoxia/reoxygenation was tested (Fig. 1). First, at three different extramitochondrial Ca2+ concentrations (0, 1.5, and 3.5 μm) isolated rat brain mitochondria were subjected to 10 min of hypoxia and 5 min of reoxygenation in the absence of substrates. In general, during hypoxia/reoxygenation no substrates were present. After the addition of 5 mm glutamate plus 5 mm malate, active respiration (state 3) was induced by the addition of 200 μm ADP. At 3.5 μm extramitochondrial Ca2+, a decrease of about 30% in comparison with Ca2+-free incubation, was measured (Fig. 1A, black bars). The rate of active respiration of freshly isolated mitochondria was determined to be 71.0 ± 4.9 (n = 18) nmol of O2 × min–1 × mg–1 of mitochondrial protein. In Ca2+-free incubation, 10 min of hypoxia followed by 5 min of reoxygenation caused a significant decrease in active respiration amounting to about 50%. The Ca2+ rise enhanced the inhibition of respiration by hypoxia/reoxygenation in a dose-dependent manner. In the case of 3.5 μm extramitochondrial Ca2+, active respiration finally decreased down to about 15% of active respiration of freshly isolated mitochondria (Fig. 1A, gray bars). In the next series of experiments we investigated the influence of the duration of hypoxia on active respiration (state 3) of isolated rat brain mitochondria. Therefore, the time of hypoxia was varied in the absence of extramitochondrial Ca2+ or in the presence of 3.5 μm Ca2+, and then active respiration was measured immediately after reoxygenation. In Ca2+-free incubation, active respiration decreased within a hypoxic period of 15 min to 58% of initial state 3 respiration (Fig. 1B, black bars). At any hypoxic period, 3.5 μm extramitochondrial Ca2+ caused a substantial additional reduction of respiration (Fig. 1B, gray bars). To investigate whether the deleterious effect on active respiration found after hypoxia/reoxygenation by Ca2+ is exerted from the inside or outside of mitochondria, we repeated the experiments in the presence of ruthenium red in combination with CGP37157. Under these conditions both Ca2+ uptake by the electrogenic uniporter and Ca2+ efflux by the Na+-Ca2+-exchanger is blocked. In Fig. 2, oxygen traces after 10 min of hypoxia and 5 min of reoxygenation (trace a), after 10 min of hypoxia and 5 min of reoxygenation in the presence of 3.5 μm Ca2+ (trace b), and in the additional presence of 2 μm ruthenium red and 25 μm CGP37157 (trace c) are presented. 3.5 μm extramitochondrial Ca2+ caused a decrease in active respiration (14.6 ± 3.4 (n = 4) versus 21.9 ± 2.8 (n = 4) nmol of O2/min/mg of mitochondrial protein). The block of the mitochondrial Ca2+ transport system not only prevented Ca2+-induced decrease in active respiration but also partially protected mitochondria from hypoxia/reoxygenation-mediated damage of active respiration (32.0 ± 4.4 (n = 4) versus 21.9 ± 2.3 (n = 4) nmol of O2/min/mg of mitochondrial protein). Thus, protection could be partial because small amounts of endogenous Ca2+ are present during exposure of mitochondria to hypoxia/reoxygenation. In conclusion, Ca2+ exerts its deleterious effect in the mitochondrial matrix. Adenine nucleotides are present commonly within mammalian cells. In the ischemic phase, ATP is converted first into ADP and subsequently into AMP to maintain energy-consuming processes for as long as possible. In a separate series of experiments, we tested whether extramitochondrial ADP or extramitochondrial AMP affects the decrease in active respiration by hypoxia/reoxygenation. Fig. 3 shows the oxygen traces of a normoxic control (trace a), of mitochondria after 10 min of hypoxia followed by 5 min of reoxygenation in the presence of 3.5 μm extramitochondrial Ca2+ (trace b) and in the additional presence of 5 mm ADP (trace c) or 5 mm AMP (trace d). The presence of ADP during hypoxia/reoxygenation almost completely protected brain mitochondria from decrease in active respiration (62.0 ± 5.7 (n = 5) versus 19.2 ± 3.6 (n = 5) and 73.0 ± 4.4 (n = 5) nmol of O2/min/mg of mitochondrial protein). Under this condition, the addition of 200 μm ADP did not result in any further stimulation of respiration because the ADP added previously was not completely consumed by the mitochondria. In contrast, AMP had no protective effect (11.7 versus 19.2 nmol of O2/min/mg). Mitochondrial Morphology Is Influenced by Extramitochondrial Ca2+ and Hypoxia/Reoxygenation—To elucidate the role of hypoxia/reoxygenation and elevated extramitochondrial Ca2+ concentration on mitochondrial morphology, we performed electron microscopy analyses. The electron micrographs were inspected by two independent investigators blinded to the treatment group. Typical structures corresponding to distinct incubation conditions are presented at a magnification of 1:20,000 in Fig. 4. In each image in Fig. 4 (A–D) an inset illustrates detailed structures at the magnification of 1:30,000. Increasing the extramitochondrial Ca2+ concentration from zero up to 3.5 μm did not significantly modify the mitochondrial structure (Fig. 4, B versus A). We were not able to detect changes in the cristae structure. Moreover, intact outer membrane structures were clearly maintained. Hypoxia/reoxygenation by itself (Fig. 4C) led to several changes in mitochondrial morphology. Besides a population of mitochondria with normal morphology, mitochondria with dented (bleb-like) outer membranes and another population of mitochondria with loss of cristae were found. The combination of 3.5 μm extramitochondrial Ca2+ with hypoxia/reoxygenation caused dramatic changes in mitochondrial morphology. Mitochondrial integrity nearly completely disappeared (Fig. 4D). Instead, parts of the mitochondrial structures and conglomerates were found (mitochondrial debris). The loss of mitochondrial structure caused by hypoxia/reoxygenation seems to illustrate an irreversible and uncontrolled breakdown of mitochondria. Mitochondrial disruption was also studied by light scattering experiments. The protocol of typical traces is shown in Fig. 5A. Isolated rat brain mitochondria, which were preincubated at 3.5 μm Ca2+, could be forced to swell by an extramitochondrial Ca2+ stimulus only when Ca2+ reached very high concentrations, such as 200 μm (trace 3). This low sensitivity to Ca2+ has already been reported previously (12Schild L. Keilhoff G. Augustin W. Reiser G. Striggow F. FASEB J. 2001; 15: 565-567Crossref PubMed Scopus (81) Google Scholar). In trace 1, the presence of 2 μm cyclosporin A prevented mitochondrial swelling after stimulation with 200 μm Ca2+. Cyclosporin A keeps the mitochondrial permeability transition pore in the closed state. Thus, Ca2+ concentrations in the high micromolar range induce opening of the mitochondrial permeability transition pore in intact brain mitochondria. However, in mitochondria that previously had been exposed to 10 min of hypoxia and 5 min of reoxygenation in the continuous presence of 3.5 μm Ca2+, the basal value of absorbance was already increased, most likely because of the condensation seen in the electron micrographs of mitochondria (see Fig. 4). Moreover, we could not find any decrease in absorbance even after adding a stimulus of 200 μm Ca2+ (trace 2). This observation confirms our conclusion that mitochondria lose their morphological integrity during hypoxia/reoxygenation in the presence of 3.5 μm Ca2+, because intact brain mitochondria would be able to swell. Furthermore, the involvement of the permeability transition pore in mitochondrial rupture because of hypoxia/reoxygenation in the presence of Ca2+ was studied by respiration experiments (Fig. 5B). Cyclosporin A, applied at a concentration which was sufficient to prevent intact mitochondria from Ca2+-induced swelling, could not prevent decrease in active respiration. Respiration measurements (Fig. 5B) show that for active respiration of brain mitochondria subjected to substrate-free 10 min of hypoxia and 5 min of reoxygenation in the presence of 3.5 μm Ca2+, no significant difference was found between the values either with or without 2 μm cyclosporin A. Thus, we conclude that mitochondrial rupture did not require the opening of the mitochondrial permeability transition pore. A further series of experiments was performed to test whether mitochondrial constituents are lost during hypoxia/reoxygenation in the presence of low micromolar Ca2+, to substantiate our conclusion of mitochondrial disintegration under these conditions. It is well known that the active respiration of intact brain mitochondria is insensitive to extramitochondrial cytochrome c and NADH, because neither of these compounds can permeate through the mitochondrial membrane system. Therefore, we analyzed whether active respiration after 10 min of hypoxia and 5 min of reoxygenation in the presence of 3.5 μm Ca2+ becomes sensitive to extramitochondrial cytochrome c and NADH. This approach was used to demonstrate membrane permeabilization. Respiration of intact mitochondria is shown in Fig. 6A. The oxygen consumption after hypoxia/reoxygenation is depicted in Fig. 6B. In intact mitochondria, ADP caused a 4.6-fold increase in respiration (stimulation of active respiration) to 87 ± 5.8 (n = 5) nmol of O2/min/mg of mitochondrial protein, whereas after hypoxia/reoxygenation the rate of active respiration was as low as 15.2 ± 1.9 (n = 5) nmol of O2/min/mg of mitochondrial protein. The application of 30 μm cytochrome c after hypoxia/reoxygenation in the presence of 3.5 μm Ca2+ only very moderately accelerated state 3 respiration (19.6 ± 2.2 (n = 5) versus 15.2 ± 1.9 (n = 5) nmol of O2/min/mg of mitochondrial protein). In contrast, a nearly 4-fold increase in the rate of oxygen consumption was found in the presence of 5 mm NADH in the incubation medium (55.3 ± 3.3 (n = 5) versus 15.2 ± 1.9 (n = 5) nmol of O2/min/mg of mitochondrial protein). This oxygen consumption was not associated with ATP synthesis, because oligomycin was without influence. In intact mitochondria, however, the inhibition of ATP synthesis by oligomycin slowed down the rate of active respiration to resting (state 4) level (18.7 ± 2.3 (n = 5) versus 87 ± 5.8 (n = 5) nmol of O2/min/mg of mitochondrial protein). Stimulation of oxygen consumption by extramitochondrial NADH clearly demonstrates that the inner mitochondrial membrane becomes permeabilized, because under physiological conditions the mitochondrial membrane is impermeable to NADH. The moderate increase in respiration after cytochrome c addition proves that most of the cytochrome c was still associated with parts of the respiratory chain. In fact, Western blot analysis revealed that only 15% of the cytochrome c pool was released into the incubation medium during 10 min of hypoxia and 5 min of reoxygenation in the presence of 3.5 μm Ca2+ (data not shown). Impairment of Mitochondria upon Ischemia/Reperfusion— Animal models of stroke have revealed that mitochondria are impaired upon ischemia/reperfusion in brain tissue. Decrease in respiratory capacity (1Sims N.R. Anderson M.F. Neurochem. Int. 2002; 40: 511-526Crossref PubMed Scopus (283) Google Scholar), change in mitochondrial morphology (3Solenski N.J. diPierro C.G. Trimmer P.A. Kwan A.L. Helms G.A. Stroke. 2002; 33: 816-824Crossref PubMed Scopus (119) Google Scholar), and permeabilization of the mitochondrial membrane system have been reported (4Namura S. Nagata I. Takami S. Masayasu H. Kikuchi H. Stroke. 2001; 32: 1906-1911Crossref PubMed Scopus (93) Google Scholar). A further hallmark of ischemic neuronal insults is the disturbance of cellular Ca2+ homeostasis, characterized by increased cytosolic Ca2+ concentration. Elevation of the cytosolic Ca2+ concentration is a well known trigger of mitochondrial damage (6Zhang Y. Lipton P. J. Neurosci. 1999; 19: 3307-3315Crossref PubMed Google Scholar). Hypoxia/reoxygenation caused impairment of mitochondrial function in cultured astrocytes (22Makarov P.R. Wiswedel I. Augustin W. Schild L. Brain Res. 2002; 933: 91-97Crossref PubMed Scopus (21) Google Scholar) and isolated mitochondria (23Schild L. Reinheckel T. Wiswedel I. Augustin W. Biochem. J. 1997; 328: 205-210Crossref PubMed Scopus (85) Google Scholar). Here we provide evidence that hypoxia/reoxygenation induces decrease in active respiration and changes in mitochondrial structure, which is characterized by a subpopulation of mitochondria with dented (bleb-like) outer membranes and another subpopulation of mitochondria with diminished number of cristae. Mitochondria isolated from brain display high resistance to relatively high extramitochondrial Ca2+ concentrations in comparison with mitochondria from other tissues such as liver (13Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (165) Google Scholar). Also, recently published data from our own studies (12Schild L. Keilhoff G. Augustin W. Reiser G. Striggow F. FASEB J. 2001; 15: 565-567Crossref PubMed Scopus (81) Google Scholar) have shown that Ca2+ in the micromolar range induces only a moderate decrease in active respiration and the release of a small amount of cytochrome c through the outer membrane from brain mitochondria with still intact morphology. Ca2+ has to enter the mitochondrial matrix via the mitochondrial Ca2+ transport system in order to affect the permeability of the mitochondrial membrane. It is known that alterations in the Ca2+ concentration can modify the number of contact sites between the inner and outer membrane, possibly by unmasking nonspecific channels of the mitochondrial outer membrane such as the voltage dependent anion channel (24Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar, 25Doran E. Halestrap A.P. Biochem. J. 2000; 348: 343-350Crossref PubMed Scopus (127) Google Scholar, 26Bakker A. Bernaert I. De Bie M. Ravingerova T. Ziegelhoffer A. Van Belle H. Jacob W. Biochim. Biophys. Acta. 1994; 1224: 583-588Crossref PubMed Scopus (29) Google Scholar). This might explain the Ca2+-mediated change in the permeability of the mitochondrial membrane system. At the cellular level, an increase in cytosolic Ca2+ concentration has been identified as the major cause for mitochondrial impairment, such as in striatal neurons (27Alano C.C. Beutner G. Dirksen R.T. Gross R.A. Sheu S.S. J. Neurochem. 2002; 80: 531-538Crossref PubMed Scopus (63) Google Scholar) and in hippocampal astrocytes (28Kahlert S. Reiser G. FEBS Lett. 2002; 529: 351-355Crossref PubMed Scopus (19) Google Scholar). Here we demonstrate that the application of hypoxia/reoxygenation sensitizes mitochondria to moderately elevated extramitochondrial Ca2+ concentration. Then, even low micromolar Ca2+ causes dramatic functional and morphological changes in isolated brain mitochondria. These include permeabilization and breakdown of the mitochondrial membrane. Again, Ca2+ has to enter the mitochondrial matrix to exert the deleterious effect, because blocking the Ca2+ transport system completely protected mitochondria from Ca2+-mediated damage. Transient hypoxia in combination with Ca2+ elevated into the low micromolar range most appropriately mimics the in vivo situation during ischemia/reperfusion. Interestingly, in our experiments, the impairment of mitochondria was not sensitive to cyclosporin A. In vivo studies, however, using animal models of stroke indicate the involvement of the mitochondrial permeability transition pore (10Matsumoto S. Friberg H. Ferrand-Drake M. Wieloch T. J. Cereb. Blood Flow Metab. 1999; 19: 736-741Crossref PubMed Scopus (190) Google Scholar). Remarkably, the latter seems to be true only for a part of the brain cells localized within the infarct area, because cyclosporin A only diminished the size of the infarct but could not completely prevent necrosis (10Matsumoto S. Friberg H. Ferrand-Drake M. Wieloch T. J. Cereb. Blood Flow Metab. 1999; 19: 736-741Crossref PubMed Scopus (190) Google Scholar, 29Kuroda S. Janelidze S. Siesjo B.K. Brain Res. 1999; 835: 148-153Crossref PubMed Scopus (49) Google Scholar, 30Vachon P. Beaudry F. Marier J.F. Ste-Marie L. Montgomery J. Brain Res. 2002; 943: 1-8Crossref PubMed Scopus (15) Google Scholar). It still remains unclear which Ca2+-mediated mechanism is responsible for the cyclosporin A-independent rupture of the mitochondrial membrane. Investigations on isolated mitochondria reveal that elevated Ca2+ concentration, hypoxia/reoxygenation, or the combination of both treatments clearly induces distinct effects. These observations suggest that, depending on the degree of hypoxia and the level of the cytosolic Ca2+, mechanisms other than the opening of the mitochondrial permeability transition pore may be involved in the process of mitochondrial damage during ischemia/reperfusion. Impact of Mitochondrial Deterioration after Ischemia/Reperfusion on Brain Cell Fate—A complete breakdown of mitochondrial ATP production is a prerequisite of necrotic cell death in brain (31Martin L.J. Brambrink A.M. Price A.C. Kaiser A. Agnew D.M. Ichord R.N. Traystman R.J. Neurobiol. Dis. 2000; 7: 169-191Crossref PubMed Scopus (119) Google Scholar). ATP is required for the maintenance of cellular morphology, ion homeostasis, protein synthesis, and many other cellular functions. Moreover, ATP is even necessary to perform the apoptotic cell death program (32Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Another factor in inducing neuronal demise is the change of the permeability of the mitochondrial membrane. Depending on the mode of activation, permeabilization can cause either apoptosis or necrosis. Distinct permeabilization of the mitochondrial outer membrane can be achieved in brain mitochondria by increased cytosolic Ca2+ concentrations or by members of the Bcl-2 family in cooperation with cardiolipin (33Zamzami N. Kroemer G. Curr. Biol. 2003; 13: R71-R73Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). This type of permeabilization causes a partial release of proapoptotic factors such as cytochrome c (12Schild L. Keilhoff G. Augustin W. Reiser G. Striggow F. FASEB J. 2001; 15: 565-567Crossref PubMed Scopus (81) Google Scholar, 13Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (165) Google Scholar). The members of the Bcl-2 family also interact with the mitochondrial permeability transition pore (34Vieira H.L. Kroemer G. Cell. Mol. Life Sci. 1999; 56: 971-976Crossref PubMed Scopus (40) Google Scholar). Reversible permeabilization of the mitochondrial membrane by opening of the permeability transition pore also causes the release of proapoptotic factors from mitochondria. If sufficient ATP is available within the cell, apoptosis is initiated. In fact, signs of apoptosis and cyclosporin A sensitivity in brain injury have been demonstrated in animal models of stroke (35Noshita N. Sugawara T. Fujimura M. Morita-Fujimura Y. Chan P.H. J. Cereb. Blood Flow Metab. 2001; 21: 557-567Crossref PubMed Scopus (96) Google Scholar). In contrast, the disruption of the mitochondrial membrane causes necrotic cell death (36Li P.A. Kristian T. He Q.P. Siesjo B.K. Exp. 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This finding may contribute further to the fact that longer periods of ischemia are required to achieve tissue damage in comparison with isolated mitochondria that have to be exposed only for a short period of time to hypoxia in order to induce mitochondrial damage. Pathological Consequences in Brain Mitochondria Caused by Ischemia/Reperfusion—From both the present data and the observations obtained with animal models of stroke, we conclude that three qualitatively different situations of stroke injury have to be distinguished. (i) Increased cytosolic Ca2+ concentrations into the low micromolar range induce permeabilization of the mitochondrial outer membrane and release of proapoptotic factors such as cytochrome c, resulting in the induction of apoptosis. (ii) Short periods of hypoxia or moderate elevation of the extramitochondrial Ca2+ concentration cause reversible opening of the mitochondrial permeability transition pore followed by the release of proapoptotic factors from morphologically intact mitochondria, also resulting in the induction of apoptosis. (iii) Long-lasting hypoxia, followed by reoxygenation at moderately elevated cytosolic Ca2+ concentration, leads to permanent opening of the permeability transition pore or the permeability transition pore-independent disruption of the mitochondrial membrane. The resulting failure of mitochondrial energy metabolism causes necrotic cell death. In conclusion, we provide evidence that isolated rat brain mitochondria are highly vulnerable to hypoxia/reoxygenation applied in combination with the rise of the extramitochondrial Ca2+ concentration into the low micromolar range. However, each stimulus, Ca2+ or hypoxia/reoxygenation, applied individually can be well tolerated by brain mitochondria. Thus, our data explain the apparent discrepancy between in vivo and in vitro data concerning the harm exerted by cytosolic Ca2+ concentrations in the low micromolar range. When observed in vitro, high resistance of mitochondria is seen, whereas in vivo, upon ischemia/reperfusion cell death occurs. Therefore, in cells of the nervous tissue therapeutic concepts aimed at preventing neural damage after ischemia (stroke) should focus on the prevention of pathological elevations of cytosolic Ca2+.