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Keywords:

  • brain mitochondria;
  • calcium;
  • cyclosporin A;
  • excitotoxicity;
  • free radicals;
  • mitochondrial permeability transition

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca2+ ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca2+-induced brain mitochondrial PT was stimulated by Na+ (10 mm) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca2+ from the mitochondrial suspension by EGTA or inhibition of Ca2+ uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca2+-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca2+ uptake. Ca2+-induced dichlorodihydrofluorescein oxidation was enhanced by Na+ and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca2+-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca2+-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.

Abbreviations used
AIF

apoptosis inducing factor

DCF

dichlorofluorescein

FCCP

carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone

H2-DCFDA

dichlorodihydrofluorescein diacetate

l-NAME

Nω-nitro-l-arginine methyl ester

PT

permeability transition

ROS

reactive oxygen species

TBARS

thiobarbituric acid reactive substances

ΔΨ

mitochondrial transmembrane electrical potential.

The maintenance of intracellular Ca2+ homeostasis is crucial for neuron survival, and its disruption may be involved in several central nervous system disorders including ischemic and hypoglycemic neuronal death and neurodegeneration in Huntington's disease (for a review see Choi 1995; Fiskum et al. 1999). During cytosolic Ca2+ overload, the mitochondrion is the main organelle responsible for calcium sequestration. Increased Ca2+ concentrations in the mitochondrial matrix may induce a phenomenon called mitochondrial permeability transition (PT), characterized by a cyclosporin A-sensitive non-selective permeabilization of the inner mitochondrial membrane (for a review see Gunter and Pfeiffer 1990; Zoratti and Szabò 1995; Smaili et al. 2000; Kowaltowski et al. 2001). Mitochondrial PT results in respiration uncoupled from ATP synthesis, organelle swelling, disruption of the outer membrane and release of different apoptogenic factors into the cytosol (Zoratti and Szabò 1995; Green and Reed 1998; Kroemer et al. 1998). These factors include cytochrome c, apoptosis inducing factor (AIF) and pro-caspases, which promote the execution of apoptosis. A PT-independent mechanism may be also responsible for release of the apoptogenic factor cytochrome c after brain mitochondrial Ca2+ accumulation (Andreyev and Fiskum 1999; Schild et al. 2001). Recent publications have indicated the participation of mitochondrial PT in neuronal death following hypoglycemia (Friberg et al. 1998), brain ischemia (Uchino et al. 1998; Matsumoto et al. 1999) and trauma (Okonkwo and Povlishock 1999; Sullivan et al. 2000). However, the participation of mitochondrial PT in excitotoxicity, i.e. glutamate receptor-mediated neuronal cell death, remains controversial (Nieminen et al. 1996; Castilho et al. 1998; Vergun et al. 1999; Brustovetsky and Dubinsky 2000b).

Recent work from our group using isolated rat liver mitochondria has demonstrated an important role for reactive oxygen species (ROS) in Ca2+-induced PT (for a review see Kowaltowski et al. 2001). Catalase, ebselen or thioredoxin peroxidase prevent the disruption of liver mitochondrial membrane potential and swelling caused by Ca2+ alone or in the presence of inducers such as t-butyl hydroperoxide, inorganic phosphate and fatty acids (Valle et al. 1993; Castilho et al. 1995; Kowaltowski et al. 1996, 1998; Catisti and Vercesi 1999). In addition, no PT occurs in liver mitochondria in the absence of molecular O2. It is proposed that Ca2+ ions are involved in the mechanism of mitochondrial PT pore opening by: (i) binding to the anionic head of membrane cardiolipins, stimulating the production of superoxide anion radicals and, hence, H2O2, by the respiratory chain (Valle et al. 1993; Castilho et al. 1995; Grijalba et al. 1999); (ii) stimulating the Fenton reaction through matrix iron mobilization (Castilho et al. 1995) and (iii) binding to membrane proteins that regulate PT pore opening (Bernardi et al. 1992; Zoratti and Szabò 1995).

In this paper, we study the involvement of oxidative stress in Ca2+-induced membrane PT in brain mitochondria. Our results indicate that ROS are implicated in Ca2+-induced brain mitochondrial PT. In addition, PT results in mitochondrial oxidative stress, represented by increased detection of ROS and secondary mitochondrial oxidative damage.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Isolation of rat brain mitochondria

Forebrain mitochondria were isolated as described by Brustovetsky and Dubinsky (2000a,b), with minor modifications. Wistar rats were fasted overnight prior to killing by decapitation. The brains were rapidly removed (within 1 min) and put into ice-cold isolation buffer I containing 225 mannitol, 75 mm sucrose, 1 mm K+-EGTA, 0.1% bovine serum albumin (BSA; free fatty acid) and 10 mm K+-HEPES pH 7.2. The cerebellum and underlying structures were removed and the remaining material was used as the forebrain. The tissue was minced using surgical scissors and then extensively washed. The tissue was then homogenized in a power-driven, tight fitting Potter-Elvehjem homogenizer with Teflon pestle. The resulting suspension was centrifuged for 7 min at 2000 g in a Beckman JA 20 rotor. After centrifugation the supernatant was recentrifuged for 10 min at 12 000 g. The pellet was resuspended in ‘isolation buffer II’ containing 225 mannitol, 75 mm sucrose, 1 mm K+-EGTA, and 10 mm K+-HEPES pH 7.2 and recentrifuged at 12 000 g for 10 min. The supernatant was decanted and the final pellet gently washed and resuspended in ‘isolation buffer II’ devoid of EGTA, at an approximate protein concentration of 30–40 mg/mL. The respiratory control ratio (state 3/state 4 respiratory rates) was over 3.0, measured using succinate and glutamate as substrates.

Preparation of dichlorodihydrofluorescein (H2-DCF)-loaded forebrain mitochondria

Rat forebrain mitochondria were obtained as described above, except that the pellet obtained after the second centrifugation was resuspended to a protein concentration of 10 mg/mL in ‘isolation buffer II’ containing 10 µm dichlorodihydrofluorescein diacetate (H2-DCFDA). The mitochondrial suspension was incubated at 30°C for 15 min and then recentrifuged at 12 000 g for 10 min. The supernatant was decanted and the pellet gently washed and diluted in isolation buffer II devoid of EGTA, at an approximate protein concentration of 20–30 mg/mL.

Standard incubation procedure

The experiments were carried out at 28°C, with continuous magnetic stirring, in a standard reaction medium containing 100 mm sucrose, 65 mm KCl, 10 mm K+-HEPES buffer (pH 7.2), 20 µm EGTA, 1 mm Pi, 5 mm glutamate and 5 mm succinate. Other additions are indicated in the figure legends. We used 20 µm EGTA to buffer contaminating free Ca2+ (8–10 µm) present in the reaction medium. Under our conditions, when 80 µm Ca2+ was added to the experiments, the free Ca2+ concentration in the medium was about 70 µm, as calculated according to Fabiato and Fabiato (1979). The results shown are representative of a series of at least four experiments, using different mitochondrial preparations. The results were reproduced within 15% of variation.

Measurements of mitochondrial transmembrane electrical potential (ΔΨ)

Mitochondrial ΔΨ was estimated through fluorescence changes of safranin O (5 µm) that were recorded on a model F-4010 Hitachi spectrofluorometer (Hitachi Ltd., Tokyo, Japan) operating at excitation and emission wavelengths of 495 and 586 nm, respectively, with slit widths of 3 nm (Åkerman and Wikström 1976).

Determination of Ca2+ movements

Variations in the concentration of free extramitochondrial Ca2+ were followed by measuring changes in the absorbance spectrum of arsenazo III (40 µm), using an SLM Aminco DW2000 spectrophotometer (SLM Instruments Inc., Urbana, IL, USA) set at the wavelength pair 665–685 nm (Scarpa 1979). The addition of mitochondria to reaction medium containing arsenazo III resulted in a rapid increase in absorbance (Figs 1C, 2B and 4C). This increase was observed even in the presence of a high EGTA concentration (500 µm; results not shown), indicating that was a phenomenon probably related to changes in medium turbidity and not due to the presence of contaminant Ca2+ in the mitochondrial suspension.

image

Figure 1. Ca2+-induced mitochondrial transmembrane electrical potential (ΔΨ) dissipation and Ca2+ release: effect of mitochondrial PT inhibitors. Isolated rat forebrain mitochondria (BM) (0.5 mg/mL) were incubated at 28°C in standard reaction medium containing 5 µm safranin O to estimate ΔΨ (panels A and B) or 40 µm arsenazo III to measure extramitochondrial free Ca2+ concentrations (panel C).In panel A, BM were added to reaction medium containing 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (line a), 200 µm ADP and 1 µg/mL oligomycin (line b), 1 µm cyclosporin A (line c) or no other additions (line d). Ca2+ (80 µm, lines a–d) and 1 µm FCCP (line d) were added where indicated by the arrows. In panel B, BM were added to reaction medium and 80 µm Ca2+ (lines b and c), 1 mm EGTA (line b), 1 µm ruthenium red (RR; line c), 200 µm ADP (lines a–c) and 1 µm FCCP were added where indicated by the arrows. In panel C, BM were added to reaction medium containing 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (line b) or no other additions (line a). Ca2+ (80 µm, lines a and b) and RR (1 µm, lines a and b) were added where indicated by the arrows. The dotted line represents standard additions of Ca2+ (10 µm) to the reaction medium in the absence of BM.

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image

Figure 2. Inhibitory effect of exogenous catalase on Ca2+-induced mitochondrial ΔΨ dissipation and Ca2+ release. BM (0.5 mg/mL) were incubated at 28°C in standard reaction medium containing 5 µm safranin O to estimate ΔΨ (panel A) or 40 µm arsenazo III to measure extramitochondrial free Ca2+ concentrations (panel B). In panel A, BM were added to reaction medium containing 2 µm catalase (lines a and c) or no other additions (lines b and d). Ca2+ (70 µm, lines a and b or 90 µm, lines c and d) and 1 µm FCCP were added where indicated by the arrows. In panel B, BM were added to reaction medium containing 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (line a), 2 µm catalase (line b) or no other additions (line c). Ca2+ (80 µm, lines a-c) was added where indicated by the arrow. The dotted line represents standard additions of Ca2+ (10 µm) to the reaction medium in the absence of BM.

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image

Figure 4. Effect of Na+ on Ca2+-induced ΔΨ dissipation (panel A), mitochondrial ROS generation (panel B) and mitochondrial Ca2+ release (panel C). In panel A, BM (0.5 mg/mL) were added to reaction medium at 28°C containing 5 µm safranine O in the presence of 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (lines a and b) and 10 mm NaCl (lines b and d). Ca2+ (80 µm) and 1 µm FCCP were added where indicated by the arrows (lines a-d). In panel B, BM (0.5 mg/mL) were added to reaction medium containing 1 µm H2-DCFDA at 28°C in the presence of 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (lines c and d) and 10 mm NaCl (line b, d and f). Ca2+ (80 µm) was added where indicated by the arrows (lines c-f). Lines a and b represent control experiments without the addition of Ca2+. In panel C, BM (0.5 mg/mL) were added to reaction medium containing 40 µm arsenazo III in the presence of 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (lines a and b) and 10 mm NaCl (lines b and d). Ca2+ (80 µm) and 1 µm RR were added where indicated by the arrows to the experiments represented by lines a-d. The dotted line represents standard additions of Ca2+ (10 µm) to the reaction medium in the absence of BM.

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Organelles in intact cells show a very slow accumulation of safranine (20–30 min; Åkerman and Jarvisalo 1980; Vercesi et al. 1991), and those within synaptosomes do not take up Ca2+ promptly under our experimental conditions. Thus, there would be no significant interference with the safranine and arsenazo measurements unless the synaptosomes were permeabilized.

Estimation of reactive oxygen species production

Mitochondrial generation of reactive oxygen species (H2O2) was determined spectrofluorometrically, using the membrane-permeable fluorescent dye H2-DCFDA (1 µm; LeBel et al. 1992; Garcia-Ruiz et al. 1997). Fluorescence was determined at 488 nm for excitation and 525 nm for emission, with slit widths of 3 nm. Calibration was performed by adding known concentrations of dichlorofluorescein (DCF), the product of H2-DCF oxidation.

Determination of NAD(P) redox state

The oxidation or reduction of pyridine nucleotides in the mitochondrial suspension was followed using a Hitachi F-4010 spectrofluorometer operating at excitation and emission wavelengths of 366 and 450 nm, respectively, with slit widths of 5 nm. Diamide (1 mm) was added at the end of each experiment to fully oxidize the pyridine nucleotides which remained in the reduced state.

Determination of thiobarbituric acid-reactive reactive substances (TBARS)

TBARS production by mitochondria was measured according to Buege and Aust (1978). Briefly, 0.4 mL samples were taken after 15 min incubation at 30°C in standard reaction medium and mixed with 0.4 mL of 1% thiobarbituric acid in 50 mm NaOH, 0.2 mL de 20% of H3PO4 and 40 µL of 10 N NaOH. The mixture was heated at 90°C for 20 min in the presence of 1 mm butylated hydroxytoluene. After cooling, 1.5 mL of butanol was added to the solution. The mixture was shaken and centrifuged at 2000 r.p.m. during 3 min. The optical density of the organic layer was determined at 535 nm. Under these conditions, the molar extinction coefficient used to calculate TBARS concentrations is 1.56 × 105 M-1cm-1 (Buege and Aust 1978).

Chemicals

Most chemicals, including ADP, arsenazo III, catalase (10 000 units/mg), cyclosporin A, diamide, dithiothreitol, ebselen, EGTA, FCCP, glutamic acid, HEPES, safranine O, succinic acid, ruthenium red and thiobarbituric acid, were obtained from Sigma Chemical Company (St Louis, MO, USA). H2-DCFDA was purchased from Molecular Probes (Eugene, OR, USA). All other reagents were commercial products of the highest purity grade available.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present work, PT in isolated brain mitochondria was assessed by measuring the transmembrane electrical potential (ΔΨ) and mitochondrial Ca2+ release (Brustovetsky and Dubinsky 2000a,b). The results in Fig. 1(A) show that the classical PT inhibitors (Zoratti and Szabò 1995) cyclosporin A (line c) or ADP plus oligomycin (line b) only partially inhibit ΔΨ dissipation caused by Ca2+ (line d). A complete inhibition of Ca2+-induced ΔΨ dissipation was only obtained when ADP, oligomycin and cyclosporin A were present simultaneously (line a). Under this last condition, only a transient decrease of ΔΨ, due to electrophoretic mitochondrial Ca2+ uptake, was observed. These results are in accordance with previous publications showing only partial inhibition of brain mitochondrial PT by cyclosporin A (Kristal and Dubinsky 1997; Brustovetsky and Dubinsky 2000a,b; Kristian et al. 2000). Figure 1(B) shows that removal of Ca2+ from the brain mitochondrial suspension by EGTA (line b) or inhibition of mitochondrial Ca2+ uptake by ruthenium red (line c) partially reverts the Ca2+-induced ΔΨ dissipation. ΔΨ reversibility by EGTA or ruthenium red was not complete and a further increase in ΔΨ was obtained by the addition of ADP. In contrast, the addition of ADP to a control experiment (line a) caused a transient decrease in ΔΨ related to ADP phosphorylation. In Fig. 1(C), measuring the release of intramitochondrial Ca2+ determined mitochondrial membrane permeabilization. Ca2+ addition is followed by a fast mitochondrial Ca2+ uptake, and subsequent release of mitochondrial Ca2+ to the reaction medium (line a). Mitochondrial Ca2+ release was inhibited by cyclosporin A, ADP and oligomycin (line b), indicating the participation of PT in this process. Since mitochondrial Ca2+ release occurs simultaneously with Ca2+ uptake, blocking mitochondrial Ca2+ uptake with ruthenium red resulted in a faster increase of extramitochondrial Ca2+ in the absence of PT inhibitors (line a).

In Fig. 2, the effect of the antioxidant catalase was tested on brain mitochondrial PT. The presence of catalase significantly inhibited ΔΨ dissipation (Fig. 2A, line a) when a lower Ca2+ concentration was added (70 µm; line b). However a smaller inhibition of ΔΨ dissipation was obtained by catalase (line c) when a higher Ca2+ concentration was added (90 µm; line d). The presence of the nitric oxide inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 3–5 mm) resulted in no inhibition of Ca2+-induced ΔΨ dissipation (result not shown). The results depicted in Fig. 2(B) show that catalase significantly inhibited (line b) Ca2+-induced mitochondrial Ca2+ release in the absence of PT inhibitors (line c).

In order to further investigate the participation of oxidative stress in Ca2+-induced brain mitochondrial PT, we measured mitochondrial reactive oxygen species (ROS) production (Fig. 3). H2-DCFDA is oxidized mainly by H2O2 and peroxynitrite (Reynolds and Hastings 1995; LeBel et al. 1992; Jakubowski and Bartosz 2000) generating DCF, which is highly fluorescent. A fast increase in the rate of DCF production was observed 3–4 min after the addition of Ca2+(Fig. 3A, line a), indicating a higher detection of ROS under this condition. This increase in mitochondrial ROS production was completely inhibited by the PT inhibitors cyclosporin A, ADP and oligomycin (line d). It is important to emphasize that under our experimental conditions, no significant mitochondrial swelling, measured by changes in absorbance at 520 nm, was observed in the presence of Ca2+ (results not shown). This excludes the possibility that the increase in DCF fluorescence could be an experimental artefact related to a decrease in light scattering of the mitochondrial suspension. The presence of synaptosomes in our mitochondrial suspension may mask absorbance changes related to Ca2+-induced PT (Brustovetsky and Dubinsky 2000a,b). Ca2+ removal by EGTA (line c) and the antioxidants ebselen plus glutathione (line b) blocked Ca2+-induced increase in mitochondrial ROS detection. Using H2-DCF-loaded mitochondria (Fig. 3B), we obtained similar results to those observed when H2-DCFDA was initially present only in the extramitochondrial medium (Fig. 3A). These results suggest that Ca2+-induced increase in mitochondrial ROS production is not related to PT-promoted access of H2-DCFDA to intramitochondrial ROS production sites.

image

Figure 3. Ca2+-induced increased detection of mitochondrial generation of reactive oxygen species. In panel A, BM (0.5 mg/mL) were added to standard reaction medium at 28°C containing 1 µm H2-DCFDA in the presence of 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (line d) or no other additions (lines a-c). Ca2+ (80 µm), EGTA (1 mm), GSH (200 µm) and ebselen (10 µm) were added where indicated by the arrows (lines a-d). Line e represents a control experiment without addition of Ca2+. In panel B, H2-DCF-loaded BM (see Materials and methods) were added to reaction medium at 28°C in the presence of 200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A (line b) or no other additions (line a and c). Ca2+ (80 µm) was added where indicated by the arrow (lines b and c). Line a represents a control experiment without the addition of Ca2+.

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As, under some pathological conditions, increase in cytoplasmic free Ca2+ in neurons is accompanied by an increase in Na+ concentrations (e.g. excitotoxicity; Choi 1987), the effect of Na+ on Ca2+-induced brain mitochondrial PT and ROS generation was studied (Fig. 4). We found that Na+ stimulates Ca2+-induced ΔΨ dissipation (panel A, line d compared with line c), mitochondrial ROS detection (panel B, line f compared with line e) and mitochondrial Ca2+ release sensitive to PT inhibitors (panel C, line d compared with line c). As expected, the presence of Na+ increased the release of intramitochondrial Ca2+, even in the presence of PT inhibitors (line b), due to the activation of the Na+/Ca2+ exchanger in brain mitochondria (Crompton et al. 1978; Nicholls and Scott 1980). However, the rate of mitochondrial Ca2+ release observed in line d was faster than the rate of mitochondrial Ca2+ release observed in line c (PT-mediated Ca2+ release in the absence of Na+) plus line b (Na+/Ca2+ exchanger-mediated Ca2+ release). These results suggest that Na+ increases PT-mediated mitochondrial Ca2+ release.

In Fig. 5, we studied the effect of Ca2+ and PT on endogenous reduced pyridine nucleotides (NAD(P)H) in brain mitochondria. Incubation of brain mitochondria in the presence of Ca2+, during 15 min, resulted in approximately 40% loss of reduced pyridine nucleotides. Interestingly, the PT inhibitors cyclosporin A, ADP and oligomycin completely prevented the Ca2+-induced loss of reduced pyridine nucleotide. The addition of the Ca2+ chelator EGTA or the NAD(P) reductants isocitrate and β-hydroxybutyrate at 5 min incubation partially prevents the effect of Ca2+ on reduced pyridine nucleotides.

image

Figure 5. Ca2+-induced brain mitochondrial PT and NAD(P)H oxidation. BM (0.5 mg/mL) were added to reaction medium at 28°C in the presence of 80 µm Ca2+ and PT inhibitors (200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A), as indicated in the figure. At 5 min, 1 mm EGTA, 100 µm isocitrate, 4 µm rotenone and/or 5 mmβ-hydroxybutyrate were added to the experiments as indicated. At 18 min, the remaining reduced pyridine nucleotides were estimated by the addition of the oxidant diamide (1 mm). The data are presented as percent of control experiments in which BM were incubated in the presence of 1 mm EGTA and diamide was added after 18 min. Values represent averages of 4 experiments (± SD), using different mitochondrial preparations. *p < 0.01, post hoc Bonferroni's test compared with the experiment in which only Ca2+ was added.

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Mitochondrial lipid oxidation was quantified in order to study a possible correlation between PT-induced increased detection of ROS and membrane damage (Fig. 6). Incubation of brain mitochondria in the presence of Ca2+ increases the basal content of TBARS 3–4 fold, in a process completely prevented by the PT inhibitors cyclosporin A, ADP and oligomycin. PT inhibitors did not inhibit mitochondrial lipid oxidation induced by Fe(II)-citrate (Castilho et al. 1994), indicating that these compounds do not have direct antioxidant properties.

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Figure 6. Ca2+-induced brain mitochondrial PT and TBARS formation. BM (1.0 mg/mL) were incubated at 30°C in standard reaction medium for 15 min in the presence of 160 µm Ca2+ and/or PT inhibitors (200 µm ADP, 1 µg/mL oligomycin and 1 µm cyclosporin A) as indicated in the figure. In the experiments of this figure, as in others (Figs 1 and 3,4,5), 160 nmol Ca2+/mg protein was used. A larger mitochondrial protein concentration was used in this experiment (1 mg/mL) to increase the sensitivity of TBARS detection. Fe2+ (50 µm) plus 2 mm citrate were used to induce lipid peroxidation independent of mitochondrial PT. Values represent averages of 4 experiments (± SD), using different mitochondrial preparations. *p < 0.01, post hoc Bonferroni's test compared with control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The PT in rat liver and heart mitochondria can be mediated by a concerted action between Ca2+ and ROS leading to oxidation of membrane protein thiols (Valle et al. 1993; Castilho et al. 1995; Grijalba et al. 1999; Kowaltowski et al. 2001). We have previously shown that Ca2+ stimulates mitochondrial ROS production (Valle et al. 1993; Castilho et al. 1995; Grijalba et al. 1999), which promotes the oxidation and cross-linkage of mitochondrial membrane protein thiol groups, leading to PT in liver and heart mitochondria (Fagian et al. 1990; Valle et al. 1993; Castilho et al. 1995; Kowaltowski et al. 1996). In the present work, the participation of oxidative stress in Ca2+-induced brain mitochondrial PT was evidenced by: (i) a significant inhibition of both Ca2+-induced ΔΨ dissipation and mitochondrial Ca2+ release in the presence of catalase (Fig. 2), (ii) an increased detection of ROS following PT (Fig. 3) and (iii) depletion of endogenous reduced pyridine nucleotides and oxidation of membrane lipids associated with PT (Figs 5 and 6).

The measurements of H2-DCFDA oxidation detected an oxidative stress situation that was totally inhibited by PT pore inhibitors (Fig. 3). It is possible that Ca2+ stimulates the generation of ROS at specific sites of the inner mitochondrial membrane, where they attack protein thiols opening PT pore (Valle et al. 1993; Castilho et al. 1995; Kowaltowski et al. 1996; Kowaltowski et al. 1998). These ROS may not be detected before mitochondrial PT by H2-DCFDA oxidation measurements due to their local and instantaneous effect on membrane proteins (Stadtman 1990).

Our results showed an increase in mitochondrial generation of ROS following membrane depolarization, thus indicating a condition of oxidative stress. This contrasts with the expected decrease in ROS production after membrane depolarization under control conditions (Boveris and Chance 1973; Skulachev 1996). Mitochondrial membrane depolarization increases the rate of electron transfer in the respiratory chain, thereby decreasing the steady state reduction of electron carriers and the oxygen tension. This minimizes superoxide formation at the level of complexes I and III (Boveris and Chance 1973; Skulachev 1996).

The PT-induced oxidative stress observed in the present work may be the result of either a stimulation of mitochondrial ROS production or a failure of mitochondrial antioxidant systems. Mitochondrial PT may result in structural alterations of the inner mitochondrial membrane that affect respiratory chain function, including coenzyme Q mobility (Nohl et al. 1996; Grijalba et al. 1999), and favor monoelectronic oxygen reduction (superoxide radical generation) at intermediate steps of the respiratory chain. In addition, PT-induced cytochrome c release from the mitochondrial intermembrane space results in stimulation of mitochondrial generation of superoxide (Cai and Jones 1998). PT is also expected to impair the mitochondrial antioxidant systems glutathione reductase/peroxidase and thioredoxin reductase/peroxidase that depend on NADPH to reduce H2O2 to water. Mitochondrial PT results in dissipation of the transmembrane proton electrochemical gradient and, at low membrane potentials, the NADP transhydrogenase cannot sustain high levels of mitochondrial reducing power (NADPH, reduced glutathione and thioredoxin), favoring oxidative stress (Vercesi 1987; Hoek and Rydstrom 1988). Moreover, the opening of mitochondrial PT pores results in loss of endogenous NAD(P)H and glutathione to the extramitochondrial medium (Igbavboa et al. 1989). Indeed, our results showed a decrease, partially due to oxidation, in the concentration of reduced pyridine nucleotides following brain mitochondrial PT (Fig. 5). Higher generation of ROS following mitochondrial membrane depolarization in neurons exposed to excitotoxic conditions has also been reported (Tenneti et al. 1998; Luetjens et al. 2000). A similar phenomenon was recently described in intact cardiac myocytes exposed to photoactivated tetramethylrhodamine derivatives (Zorov et al. 2000). PT-induced brain mitochondrial oxidative stress, with consequent peroxidation of membrane lipids (Fig. 6), may result in the impairment of mitochondrial oxidative phosphorylation and in irreversible inner membrane permeabilization.

Interestingly, Ca2+-induced brain mitochondrial PT was stimulated by Na+ (10 mm; Fig. 4). This suggests that, under conditions in which an increase in cytoplasmic free Ca2+ is accompanied by an increase in Na+ concentration, such as that found in excitotoxicity (Choi 1987), there is a stimulation of Ca2+-induced brain mitochondrial PT and oxidative stress. This result is in accordance with a previous report (Dykens 1994) showing that Na+ increases ROS generation by cerebral and cerebellar isolated mitochondria in the presence of Ca2+. On the other hand, Kristal et al. (2000) did not observe a significant stimulatory effect of Na+ on brain mitochondrial PT, estimated by mitochondrial swelling. This observation is in apparent contrast with our results showing that Na+ potentiates Ca2+-induced brain mitochondrial ΔΨ dissipation and Ca2+ release. Probably, the swelling measurements secondary to the entry of the osmotic support can not detect small alterations in inner mitochondrial membrane permeability (Gunter and Pfeiffer 1990), explaining why Kristal et al. (2000) did not observe a stimulatory effect of Na+ on Ca2+-induced brain mitochondrial PT.

Finally, we suggest that PT-induced brain mitochondrial oxidative stress and dysfunction may participate, together with the release of mitochondrial apoptogenic signal molecules into the cytosol, in the cascade of events (Tenneti et al. 1998; Andreyev and Fiskum 1999; Castilho et al. 1999; Luetjens et al. 2000; Petersen et al. 2000) that determine neuronal cell death under conditions associated with cytosolic Ca2+ overload.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr Alicia J. Kowaltowski for critical reading of the manuscript and Mrs Elisangela J. Silva for the preparation of rat forebrain mitochondria. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Fundo de Apoio ao Ensino e Pesquisa (FAEP-UNICAMP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). ENM is supported by a FAPESP fellowship.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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