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Originally published In Press as doi:10.1074/jbc.M604123200 on October 6, 2006

J. Biol. Chem., Vol. 281, Issue 49, 37361-37371, December 8, 2006
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Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation

ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP RE-CYCLING ACTIVITY*Formula

Laudiene Evangelista Meyer{ddagger}1, Lilia Bender Machado{ddagger}1, Ana Paula S. A. Santiago{ddagger}§, Wagner Seixas da-Silva{ddagger}2, Fernanda G. De Felice{ddagger}, Oliver Holub{ddagger}, Marcus F. Oliveira{ddagger}13, and Antonio Galina{ddagger}14

From the {ddagger}Instituto de Bioquímica Médica, Programa de Biofísica e Bioquímica Celular and Programa de Biologia Molecular e Biotecnologia and the §Instituto de Biofísica Carlos Chagas Filho, Programa de Biologia Celular e Parasitologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Rio de Janeiro 21941-590, Brazil

Received for publication, May 1, 2006 , and in revised form, September 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846–39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H2O2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H2O2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H2O2 production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both {Delta}{Psi}m and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial electron transport chain is the major and continuous source of cellular reactive oxygen species (ROS),5 which are involved in several conditions, such as apoptosis, ischemia-reperfusion injury, neurodegenerative diseases, and toxicity induced by hyperglycemia (1–5). Electron leakage at the complexes I (6, 7) and III (6, 8–10) are the main sites for the monoelectronic reduction of oxygen, which results in superoxide (Formula) radical production in the respiratory chain. The rate of mitochondrial ROS production is highly dependent on the mitochondrial membrane potential ({Delta}{Psi}m) (9, 11) and evidence supporting these observations have long demonstrated (9) that pharmacological uncoupling of oxidative phosphorylation caused a drastic reduction in mitochondrial H2O2 formation. Similarly, activation of oxidative phosphorylation by ADP can also reduce the {Delta}{psi}m and ROS formation through activation of F1F0-ATP synthase complex by using the energy of the {Delta}{Psi}m to drive ATP synthesis (9, 11). On the other hand, when mitochondrial ADP levels drop, the respiratory rate is reduced, increasing the {Delta}{Psi}m levels, which ultimately leads to ROS generation. There is a clear link between the increased levels of oxidative stress markers and several neuropathies such as amyotrophic lateral sclerosis, Parkinson and Alzheimer disease and hyperglycemia-derived neuropathy (3, 12–15). However, if oxidative stress is a major cause or just a consequence of associated neuron cell loss remains elusive (3). Growing evidence indicates that ROS are involved in the propagation of cellular damage leading to neuropathy and thus, modulation of key enzymes that control oxidative stress is central for the development of drug therapies against these conditions. In support to this view, it has been well documented that creatine (Cr) exerts powerful protective effects in models of Huntington disease, Parkinson disease, amyotrophic lateral sclerosis, as well as in in vitro models of glutamate and beta-amyloid toxicity (12, 16–19). Moreover, neuronal ATP depletion is a feature of neurodegenerative diseases and the proposed mechanism for Cr protection has been attributed to a build-up of phosphocreatine (PCr) stores, which increase the efficiency of ATP regeneration (18, 19).

Hyperglycemia in animal and in vitro models of diabetes is associated with both enhanced production as well as decreased scavenging of ROS, leading to a cellular oxidative stress condition and impaired mitochondrial function, which ultimately leads to O2. overproduction by the mitochondrial electron transport chain (4, 5, 15, 20, 21). One of the hypotheses raised to explain the establishment of oxidative stress conditions in hyperglycemia is that excess glucose leads to an oversupply of electrons in the mitochondrial electron transport chain, resulting in mitochondrial membrane ({Delta}{Psi}m) hyperpolarization and ROS formation (12, 22). Glucose toxicity in chronic hyperglycemia is especially important in tissues where glucose uptake is independent of insulin such as hepatocytes, endothelial, epithelial, and immune cells as well as the cells from the central and peripheral nervous systems (4, 5, 20–24). In this regard, our group recently demonstrated that mitochondrial associated hexokinase (mt-HK) activity plays a central role on preventing mitochondrial ROS generation through steady-state ADP recycling in rat brain (25). However, when neurons were exposed to high glucose levels, it was observed that an increase not only in ROS production but also in the intracellular levels of glucose 6-phosphate (Glc-6-P) inhibits mt-HK activity impairing ADP re-cycling through inner mitochondrial membrane. Thus, ADP re-cycling enzymes would play a preventive antioxidant role in mitochondria by keeping lower {Delta}{Psi}m and ROS levels.

A search over other putative enzymes that would contribute to the ADP re-cycling mechanism revealed that brain has high levels of mitochondrial creatine kinase (mt-CK), which is located in the intermembranal space of mitochondria (26–33). CK (EC 2.7.3.2 [EC] ) comprise a group of isoenzymes that catalyze the following reversible reaction: Mg·ATP + Cr {leftrightarrow} PCr + Mg·ADP + H+. This enzyme performs a pivotal physiological role in high energy consuming tissues, by acting as an energy buffering and transport system between the sites of ATP production and consumption by ATPases (34, 35). The mt-CKs form octamers assembled as four dimers, but only the octameric form can interact with both inner and outer mitochondrial membranes through the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC) (36). The mt-CK activity couples the oxidative phosphorylation and mitochondrial PCr production by catalyzing the conversion of Cr to PCr at expenses of the intramitochondrially produced ATP. The PCr is exported to the cytosol, whereas the produced ADP is pumped back to the mitochondrial matrix via ANT, thus stimulating oxidative phosphorylation (37–39). In fact, Cr is an excellent stimulant for mitochondrial respiration during PCr generation (31, 40, 41). Besides its role on energy metabolism it has recently been demonstrated that activation of mt-CK inhibits the mitochondrial permeability transition (MPT), a process that is involved in apoptosis (42). The postulated protective mechanism of mt-CK activity against MPT pore opening lies on functional coupling between the mt-CK reaction and oxidative phosphorylation (42). Notwithstanding, MPT can be directly induced by mitochondrial ROS and it is conceivable that the protective role of mt-CK activity against MPT would occur through reduction of ROS generation by keeping ADP phosphorylation (43). Based on the fact that mt-HK activity exerts a key role on regulation of ROS generation in neurons, in the present work we propose that induction of mt-CK activity by Cr would play an even more important preventive antioxidant function by promoting re-cycling of mitochondrial ADP during hyperglycemia.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—ADP, ATP, NAD+, glucose, glucose 6-phosphate, 2-deoxyglucose (2-DOG), [3H]2-DOG, fatty acid-free bovine serum albumin, succinate, rotenone, antimycin A, atractyloside, safranine O, FCCP, Cr, Ap5A, polyornitine, RPMI 1640 medium, MM-CK, horseradish peroxidase, catalase, G6PDH from Leuconostoc mesenteroides, were all purchased from Sigma. Amplex Red was purchased from Invitrogen; Percoll was from Amersham Biosciences. Hydrogen peroxide was from Merck (Germany). Neurobasal medium was from Invitrogen. CM-H2DCFDA and JC-1 were obtained from Molecular Probes (Eugene, OR). All other reagents were analytical grade.

Animals and Mitochondrial Isolation—Adult male Wistar rats weighting 200–230 g were fed overnight prior to killing by decapitation. Mitochondria were isolated from brain (RBM) and liver (RLM) by conventional differential centrifugation as described previously with small modifications (25, 44). Protein was determined by the Lowry (45) method using bovine serum albumin as standard. All experiments were carried out in a standard respiration buffer containing 10 mM Tris-HCl, pH 7.4, 0.32 M mannitol, 8 mM inorganic phosphate, 4 mM MgCl2, 0.08 mM EDTA, 1 mM EGTA, 0.2 mg/ml fatty acid-free bovine serum albumin, and 50 µM Ap5A (25).

Determination of Mitochondrial Hexokinase (mt-HK) Activity from Rat Brain—The radiochemical enzymatic activity assay for mt-HK activity was determined based on a previously described method with minor modifications (46). In this assay we used the respiration buffer plus 4.5 µM rotenone, 10 mM succinate, and 1 mM 2-DOG, after 0, 2, 5, 10, and 15 min of incubation in a final volume of 50 µl. Briefly, HK activity was measured by the selective adsorption of [3H]2-DOG-6P on DE81 paper discs using [3H]2-DOG as substrate. The reaction was stopped with 50 µl of ice-cold ethanol and dropped onto DE81 Whatman filters. The filters were washed with 10 ml of distilled water 10 times. The discs were dried and radioactivity was counted in a liquid scintillation counter.

Determination of Mitochondrial Creatine Kinase (mt-CK) Activity from Rat Brain—For this assay was used 0.1–0.15 mg/ml of the mitochondrial protein and the activity of mt-CK was determined by NADH formation following the absorbance at 340 nm at 37 °C. The assay medium contained: 50 mM Tris-HCl, pH 7.4, 10 mM glucose, 5 mM MgCl2, 2 mM ADP, 1 mM NAD+, 5 units/ml yeast hexokinase, and 1 unit/ml G6PDH. The reaction started when 5 mM PCr was added.

Oxygen Uptake Measurements—Oxygen uptake was measured in an oxymeter fitted with a water-jacket Clark-type electrode (Yellow Springs Instruments Co., model 5300). The RBM and RLM (0.15–0.25 mg/ml) were incubated with 1.5 ml of the standard respiration buffer described above.

Determination of Mitochondrial Membrane Potential ({Delta}{Psi}m)—The fluorescence signal changes of the cationic dye safranine O was monitored as previously described (25, 47). Data are reported as arbitrary fluorescence units. Other additions are indicated in the figure legends.

Determination of Mitochondrial Hydrogen Peroxide Generation—Mitochondrial release of H2O2 was determined by the Amplex Red oxidation method (48). Mitochondria (0.15 mg of protein/ml) were incubated in the standard respiration buffer supplemented with 10 µM Amplex Red and 4 units/ml horse-radish peroxidase. Fluorescence was monitored at excitation and emission wavelengths of 563 (slit 5 nm) and 587 nm (slit 5 nm), respectively. Calibration was performed by the addition of known quantities of H2O2. Other additions are indicated in the figure legends. In all experiments, small variations in the levels of H2O2 formation were observed with different preparations, but the overall pattern of response to different modulators was not changed.

Cortex Cell Cultures—Cortices from 14-day-old Wistar rat embryos were dissected and cultured as previously described (25, 49). Fluorescence images were acquired on a Nikon Eclipse TE 300 inverted light microscope (Nikon, Kanagawa, Japan), equipped with a Nikon CCD camera DXM 1200 controlled by Nikons image acquisition software ACT-1 and an Osram mercury lamp HBO103W/2 for epi-illumination.

Intracellular Determination of {Delta}{Psi}m in Neuronal Cells—To investigate the effects of glucose and Cr on the {Delta}{Psi}m, several aliquots of RPMI 1640 medium were supplemented with different solutions to achieve the following final concentrations: 10 or 40 mM glucose, or 40 mM glucose + 30 mM 2-DOG, or 40 mM glucose + 5 µM FCCP, or 40 mM glucose + 5 mM Cr. The cells were incubated 25 min at 37 °C with 4% CO2 and then the medium was supplemented with the dye, JC-1 (50), to achieve a final concentration of 5 µg/ml and the cells were incubated an additional 15 min. After that, each coverslip was washed and examined under the epifluorescence microscope using two standard filter combination sets for green and red fluorescence (B2 FITC blue filter combination: excitation 465–495 nm; dichroic filter 505 nm; emission 515–555 nm; and G-1B green filter combination; excitation 541–551 nm; dichroic filter 565 nm; emission 590 nm long-pass) and a x40 objective (Nikon Plan Fluor ELWD DM; N.A. 0.6; W.D. 3.7-2.7 mm; PH2) at fixed exposure times.

Intracellular Determination of ROS in Neuronal Cells—After 96 h of culture, neurobasal medium was replaced with RPMI 1640 medium, supplemented with 2 µM CM-H2DCFDA to assess intracellular ROS formation. To investigate the effects of glucose, several aliquots of medium were supplemented with different solutions as described in the legend to Fig. 8. The cells were incubated during 40 min at 37 °C and 4% CO2 and, after that the cells were washed and examined under the epifluorescence microscope using a standard filter for green fluorescence (B2 FITC blue filter combination: excitation 465–495 nm; dichroic filter 505 nm; emission 515–555 nm) and a x40 objective (Nikon Plan Fluor ELWD DM; N.A. 0.6; W.D. 3.7-2.7 mm; PH2) at fixed exposure times. Fluorescence quantification was determined by using the Adobe® Photoshop software.

Image Analysis—The JC-1 red/green ratio was determined from each selected region of the cell culture, a set of two 24-bit RGB bitmap images was acquired (green and red fluorescence respectively) and both images were split into their corresponding three color channels. Images of green JC-1 fluorescence contained information in their green channel and images of red JC-1 fluorescence in the red channel only, which were extracted for further processing. The resulting two 8-bit images of red and green fluorescence intensity (denoted as red and green image) were exported separately by selecting them individually and subjecting them to further calculation of the integration image. A routine has been written in LabVIEW software (National Instruments, Austin, TX) and each image set was subjected to the following steps. 1) Loading of the green and red 8-bit bitmap image (defining red and green fluorescence levels in the range 0–255 for each pixel). 2) Determination of the background signal in each image: the program allows for the selection of a region of interest in the image. Using this option one selects a region free of any cellular structures in the image. The determined two average signals of the same region of interest for the red and green image are used for later background subtraction and the detected maximum background signal constitutes the threshold for background pixel removal. 3) For calculation of a division image, all background pixels of the two images determined by the previous step were excluded from analysis by the according threshold selection (as well as overexposed pixels with value 255 if present). If a pixel is excluded in the red image, the corresponding pixel in the green image is automatically also excluded, even if it would pass its own threshold settings and vice versa, a procedure ensuring that only pixel sets, which contain fluorescence signal in both images, are analyzed. From each of these pixels the average background is subtracted and the corresponding pixels of both images are divided by each other, resulting in a pixel wise division image, which allows for the visualization of the red/green ratio over the image. Automatically the division image is analyzed by calculation of the pixel average, standard deviation, minimum and maximum, and the plot of the histogram and probability distribution of pixel values with determination of mode, skewness, and kurtosis of the histogram.

Statistical Analysis—Data were plotted with GraphPad Prism 4.0 software (GraphPad) and analyzed by analysis of variance and a posteriori Tukey's test. p values <0.05 were considered statistically different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
mt-HK Activity Regulates Mitochondrial ROS Generation in RBM—Recently our group demonstrated that mt-HK plays a preventive antioxidant role in RBM through ADP re-cycling (25). Our first step was to investigate the role of mt-HK on regulation of the {Delta}{Psi}m in RBM (Fig. 1A). Induction of mt-HK activity by 2-DOG after ADP-induced state 3 led to a persistent depolarization of {Delta}{Psi}m, which was progressively reversed by Glc-6-P. ROS production in RBM is inversely related to the mt-HK activity using 2-DOG and intramitochondrially generated ATP as substrates (Fig. 1B). This occurs because Glc-6-P inhibits the mt-HK activity, thus increasing {Delta}{Psi}m and H2O2 generation, due to the blockage of ADP recycling through inner mitochondrial membrane. These findings are in agreement with our previous observation in RBM (25). In addition, it is important to note that the increments in {Delta}{Psi}m are strictly correlated with the inhibition of mt-HK activity (Fig. 1B, open circle and closed triangles), but the threshold to increase H2O2 generation is higher than those observed for {Delta}{Psi}m (Fig. 1B, closed circles). This observation is in accordance with Korshunov and co-workers (11), which demonstrated that large changes in H2O2 generation occurs only within a small range of {Delta}{Psi}m values near the maximum. Moreover, due to its localization, mt-HK is ready to use both intra- and extramitochondrial sources of ATP. mt-HK activation by glucose and intramitochondrially generated ATP leads to a stimulation of oxygen consumption, decrease in the {Delta}{Psi}m, and reduction in H2O2 generation (Fig. 2A). However, as previously described by our group, the effects of glucose in isolated RBM are transient, due to an increase in Glc-6-P accumulation and further inhibition of mt-HK. Thus, to overcome these effects, we evaluated the same parameters mentioned above in conditions where the mt-HK would be fully activated, by using 2-DOG and an external source of ATP. 2-DOG-6P, the product of this reaction has less ability to block mt-HK activity and consequently allows full activation of mt-HK led to a permanent stimulation of oxygen consumption rate, persistent {Delta}{Psi}m dissipation, and total blockage of H2O2 generation (Fig. 2B).


Figure 1
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  		FIGURE 1.
Glucose 6-phosphate induces {Delta}{Psi}m hyperpolarization and stimulates H2O2 production when mt-HK is phosphorylating 2-deoxyglucose in RBM. A shows {Delta}{Psi}m measured with safranine O. Numbers indicates the amount of Glc-6-P added to a final concentration of: 1,10 µM; 2,25 µM; 3,50 µM; 4, 200 µM, and 5, 350 µM. The dotted line represents the {Delta}{Psi}m measurement in which a single dose of 300 µM Glc-6-P was added. B, the mt-HK activity was measured, as described under "Experimental Procedures," by the amount of [3H]2-DOG-6-P formed from [3H]2-DOG phosphorylation using ATP synthesized by oxidative phosphorylation carried out by RBM (open circles). The H2O2 formation was measured in parallel reactions by the Amplex Red fluorescence method (closed circles) using 15 µM Amplex Red and 5 units/ml horseradish peroxidase. The increases in {Delta}{Psi}m induced by Glc-6-P are depicted as closed triangles. The reaction was measured using respiration buffer plus 4.5 µM rotenone, 10 mM succinate, 5 mM 2-DOG, 0.15 mM ADP, and 0.5 mg/ml Percoll-purified RBM. The reaction time was 10 min at the temperature of 28 °C. Similar results were obtained with at least five different independent mitochondrial preparations.

 


Figure 2
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  		FIGURE 2.
H2O2 production, oxygen consumption, and membrane potential ({Delta}{Psi}m) in RBM are regulated by hexose phosphorylation by mt-HK. A represents the effect of glucose phosphorylation using low concentrations of ATP formed by RBM on H2O2 generation, oxygen consumption, and {Delta}{Psi}m. The reactions were measured using the respiration buffer described under "Experimental Procedures" with 0.2 mg/ml Percoll-purified RBM. The arrows indicate sequential additions of: Suc, 10 mM succinate; ADP, 0.15 mM ADP; Glc, 5 mM glucose; and FCCP,5 µM FCCP. The trace–Glc indicates that glucose was omitted from the reaction. B represents the effect of 2-DOG phosphorylation using a high concentration of ATP added to the medium on H2O2 generation, oxygen consumption, and on {Delta}{Psi}m in RBM. The arrows indicate sequential additions as described for A, except that ATP was used instead of ADP and 2-DOG replaces Glc: ATP, 1 mM ATP; 2-DOG, 10 mM 2-DOG. The trace–2-DOG represents that 2-DOG was omitted from the reaction. C shows a control in which addition of 1000 units of catalase removed H2O2 formed in state 4 respiration. The temperature was 28 °C. Similar results were obtained with at least five different independent mitochondrial preparations.

 
mt-CK Activity Also Regulates Mitochondrial H2O2 Generation in RBM—Besides mt-HK, it is very well known that a large portion of the total creatine kinase activity is compartmentalized in RBM between VDAC and ANT, supporting ADP recycling (51). Several lines of evidence have shown that Cr supplementation prevented induction of mitochondrial permeability transition and ROS formation (52, 53). As an attempt to gain insight into other mechanisms involved in ADP recycling in mitochondria, in subsequent experiments we investigated whether mt-CK activation by Cr would also affect physiological functions of RBM. Fig. 3A shows that mt-CK activation by Cr is able to impair H2O2 production of RBM by simultaneously accelerating the oxygen consumption rate and decreasing {Delta}{Psi}m. Identical results were obtained when Cr and low (0.15 mM) or high (1 mM) ATP were used (Fig. 3B). Interestingly, a transient blockage of H2O2 generation was achieved when limiting amounts (0.2 mM) of Cr was added, resulting in a subsequent increase in H2O2 production after all Cr is converted to PCr (Fig. 3A, trace 2). A simultaneous transitory acceleration in respiration rate and a decrease in {Delta}{Psi}m of RBM were also observed after inclusion of the limiting Cr concentration (data not shown). These observations indicate that the electron transport chain responds to Cr immediately when the [ATP]/[ADP] ratio is high. All three parameters analyzed were not affected by Cr and ATP supplementation in RLM, which are devoid in mt-CK activity (Fig. 3C).


Figure 3
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  		FIGURE 3.
H2O2 production, oxygen consumption, and membrane potential ({Delta}{Psi}m) in RBM are regulated by Cr phosphorylation by mt-CK. A represents the effect of Cr phosphorylation using a high concentration of ATP added to the medium on H2O2 generation, oxygen consumption, and {Delta}{Psi}m in RBM. The reactions were measured using the respiration buffer as described under "Experimental Procedures" with 0.2 mg/ml Percoll-purified RBM. The arrows indicate the sequential additions of: Suc, 10 mM; ATP, 1 mM; Cr, 10 mM; and FCCP,5 µM. The trace–Cr represents that the Cr was omitted from the reaction. Trace 1 shown the effect of ATP on H2O2 generation in the absence (–Cr) or presence (+Cr) of 10 mM Cr. Trace 2 shows the effect of different concentrations of Cr (arrow) on H2O2 generation in the presence of 1 mM ATP. The dashed line represents the rate of H2O2 generation after addition of 0.2 mM Cr. Panel B represents the effect of Cr phosphorylation using a low concentration of ATP formed by RBM on ROS generation, oxygen consumption, and {Delta}{Psi}m. The arrows indicate the sequential additions of: Suc, 10 mM; ADP, 0.15 mM; Cr, 10 mM; and FCCP,5 µM. C shows the effect of Cr supplementation on H2O2 generation, oxygen consumption, and {Delta}{Psi}m in RLM. The arrows indicate the sequential additions of: Suc, 10 mM; ATP, 1 mM + Cr = 5 mM; ADP, 1 mM; and Atrac, 0.1 mM (only in {Delta}{Psi}m). In the O2 consumption trace of RLM the additions of ADP were: 0.15 and 0.35 mM. The dotted line in C means the basal rate of H2O2 production in RLM. The temperature was 28 °C. Similar results were obtained with at least five different independent mitochondrial preparations.

 
H2O2 Generation in RBM Is Controlled by the Steady-state Kinetics of Mitochondrial Kinases—In an attempt to evaluate whether regulation of mitochondrial H2O2 generation by either mt-HK or mt-CK activities obeys the steady-state kinetics of these two enzymes, we next measured ROS production using different amounts of substrates (glucose, 2-DOG, or Cr) in two ATP concentrations (0.15 and 1.0 mM). Fig. 4 shows that activation of mt-kinases through their substrates reduces H2O2 formation in a substrate concentration-dependent manner. Interestingly, at 0.15 mM ATP (open circles), the effect of different concentrations of glucose on H2O2 generation exhibited a biphasic pattern, reducing the initial rate of H2O2 production at low glucose concentrations, in the 40–90 µM range and reaching the lowest value near 5 mM glucose. On the other hand, H2O2 production was only modestly affected in higher glucose concentrations (above 10 mM) (Fig. 4A, open circles). A possible explanation for this effect would be that, above 10 mM glucose, the ratio of [Glc-6-P]/[ATP] was high, causing competition between the sugar-phosphate and the ATP for the catalytic site of the mt-HK (54). Nevertheless, the general effect of 1.0 mM ATP (closed circles), on H2O2 production was more pronounced than at 0.15 mM ATP (open circles). Also, at 1.0 mM ATP, the inhibition of ROS production was detected with lower amounts of glucose, 2-DOG, or Cr than in 0.15 mM ATP (Fig. 4, closed circles).

When mt-HK was activated by the glucose analog 2-DOG, a similar pattern of inhibition of H2O2 formation was achieved, but the concentration range of 2-DOG necessary to reach the lowest rates was higher (500–600 µM) than for glucose (40–90 µM) (Fig. 4B). These values are in agreement with the expected concentration ranges of the HK activities using these sugars as substrates (55).


Figure 4
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  		FIGURE 4.
H2O2 generation in RBM is controlled by the steady-state kinetics of mt-kinases. The initial rates of ROS production were measured using the respiration buffer described under "Experimental Procedures" with 0.2 mg/ml Percoll-purified RBM induced by 10 mM succinate. For mt-HK (A and B) or mt-CK (C) reactions, different concentrations of glucose, 2-DOG, or Cr were added before the inclusion of 1 mM ATP (closed circles) or after the conversion of 0.15 mM ADP to ATP by RBM (open circles). The transition to a state 3-like respiration was started by the activation of mt-kinase substrates and the decreased initial rate was monitored at 28 °C. Similar results were obtained with at least five different independent mitochondrial preparations.

 


Figure 5
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  		FIGURE 5.
Initial rate of H2O2 production depends on the [PCr]/[Cr] ratio. The initial rate of H2O2 production was measured in respiration buffer as described under "Experimental Procedures" with 0.15 mg/ml Percoll-purified RBM induced by 10 mM succinate. Different ratios of [PCr]/[Cr] were obtained varying the concentration of either PCr or Cr from 0 to 7 mM. The total creatine pool ([PCr] + [Cr]) was maintained at 7 mM. The reaction mixture was incubated with different [PCr]/[Cr] ratios and a steady rate of H2O2 production was obtained. After this period, 0.4 mM ATP was added to activate mt-CK, inducing a state 3-like respiration.

 
The kinetics of CK can be explained as a random-order, rapid equilibrium kinetic mechanism (56). The result shown in Fig. 4C indicates that mt-CK catalyzing the forward reaction (i.e. in the direction of PCr formation) is able to substantially lower the rate of H2O2 production at the physiologically relevant Cr concentration ranges in brain (1–5 mM Cr) (57). Similarly to the mt-HK, inhibition of H2O2 generation performed with mt-CK was also achieved regardless of the ATP concentration (Fig. 4C). At 1.0 mM ATP, the half-maximum inhibition of H2O2 formation for Cr was detected near 100 µM, which is in the same concentration range of the estimated Km for the mammalian ubiquitous mt-CK activity found in humans or mouse (58). The half-maximal inhibition of H2O2 production at lower ATP concentrations (0.15 mM) was observed at a much higher Cr concentration, near 700 µM (Fig. 4C, open circles). This difference in apparent affinity of mt-CK for Cr is due in part to the low ATP concentration present in the medium, which tends to increase the apparent Km value for Cr owing of the synergistic properties of mt-CK (56, 58).

The Equilibrium of mt-CK Reaction Sets the Rate of H2O2 Production in Isolated RBM—The previous experiments (Figs. 1 and 2) demonstrated that when mt-HK activity is directed to Glc-6-P or 2-DOG-6-P formation, the production of H2O2 is lowered (25). Similar results were obtained when mt-CK activity uses Cr and ATP to form PCr (Fig. 3). Nevertheless, the reaction catalyzed by CK is reversible and this property is central to cellular energy buffering (59). The interplay between the mitochondrial and cytosolic CK isoenzymes allows the maintenance of high local [ATP]/[ADP] ratios in the vicinity of cellular ATPases for a maximal {Delta}G of ATP hydrolysis, whereas in the mitochondrial matrix, relatively low [ATP]/[ADP] ratios are found stimulating the oxidative phosphorylation (59). Thus, in Fig. 5, we evaluated whether changes in the [PCr]/[Cr] ratio would modulate H2O2 generation due to influences on the [ATP]/[ADP] ratios by RBM. Setting the total amount of [PCr] + [Cr] at 7 mM, it was observed that at a very low ratio, [PCr]/[Cr] (close to zero; i.e. 7 mM Cr), the ROS production was completely abolished. When the PCr/Cr ratio approaches 2, the rate of H2O2 generation is close to 50% of maximum, whereas the maximum rate is detected at a [PCr]/[Cr] ratio of 7 (i.e. 7 mM PCr).

Microcompartmentation of mt-CK Regulates H2O2 Generation—To evaluate the effect of the specific location and mt-CK activity levels on H2O2 production by RBM, we investigated the effect of an externally added cytosolic MM-CK on H2O2 generation in RLM. This was proposed based on the fact that liver mitochondria is almost devoid of CK activity and as ADP recycling plays an important role on mitochondrial H2O2 generation, any changes in ADP levels due to the externally added cytosolic MM-CK activity would impact H2O2 production (Fig. 6). In fact, the CK activity levels in RLM are much lower than in RBM (Fig. 6A and Ref. 60). In Fig. 6B it was shown that H2O2 production by RLM was also inhibited by activation of the cytosolic MM-CK reaction working in the PCr formation (Fig. 6B, closed triangles). Noteworthy is that even using an order of magnitude more cytosolic MM-CK activity levels, as those present in native RBM, the rate of H2O2 production was not fully inhibited, suggesting that localization of the mitochondrial isoform of CK in brain plays an important preventive antioxidant role.


Figure 6
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  		FIGURE 6.
Localization of mt-CK activity is important to control H2O2 production. A, mt-CK activity was measured in Percoll-purified RBM (open bar) or RLM (closed bar). In B, the initial rate of ROS production was measured in respiration buffer described under "Experimental Procedures" with 0.3 mg/ml Percoll-purified RBM (open circle) or 0.5 mg/ml RLM (closed triangles) induced by 10 mM succinate. The reaction mixture of RLM was supplemented with increasing amounts of exogenous cytosolic MM-CK (closed triangles) plus 5 mM Cr and a steady rate of H2O2 production was obtained. After this period, 0.4 mM ATP was added to activate mt-CK, inducing a state 3-like respiration. The maximal rate of H2O2 generation (state 4 respiration) in mt-CK-depleted RLM or RBM was used as 100%. Bars represent mean ± S.E. of four independent preparations and similar results were obtained with at least four different independent mitochondrial preparations.

 
Activation of mt-CK Reduces H2O2 Generation in Conditions Mimicking Hyperglycemia in RBM—Several lines of evidence show that, in hyperglycemic conditions, there is an overshooting of intracellular Glc-6-P levels due to GLUT and HK activities in a variety of cell types (25, 61–63). We have previously demonstrated that accumulation of Glc-6-P levels in neurons promotes mitochondrial H2O2 production as mt-HK-dependent ADP recycling becomes impaired (25). As the experiments presented in Figs. 2 and 3 show that activation of either mt-HK or mt-CK consumes the {Delta}{Psi}m and causes a decrease in H2O2 generation, we evaluated whether activation of mt-CK would release ADP recycling from the impairment observed in hyperglycemic conditions where mt-HK was inhibited (high Glc-6-P). Fig. 7 (trace 1) shows that after addition of succinate, there is a progressive and steady accumulation of H2O2 that was transiently blocked by 0.2 mM ADP. In agreement with our previous experiments, when 2-DOG was added to the assay medium, a sustained blockage of H2O2 production was achieved by 0.2 mM ADP (Fig. 7, trace 2), which was only reversed when the mt-HK was inhibited by 1 mM Glc-6-P inclusion. Interestingly, ROS generation due to interference with ADP recycling performed by mt-HK was reversed when 5 mM Cr was added after Glc-6-P inclusion, causing an immediate fall in H2O2 production (Fig. 7, trace 3). Moreover, this result also indicates that the ATP pool formed by oxidative phosphorylation is promptly available for mt-CK even when mt-HK is inhibited by Glc-6-P (Fig. 7, trace 3). Finally, when both kinases substrates, 2-DOG and Cr, were present in the assay medium, we observed a sustained blockage of H2O2 production upon 0.2 mM ADP addition (Fig. 7, trace 4), which was unaffected by mt-HK inhibition by 1 mM Glc-6-P. It is also important to note that the addition of 2-DOG and Cr to the reaction medium did not modify the rate of H2O2 formation after the addition of succinate (Fig. 7, trace 4), suggesting that both substrates do not have intrinsic antioxidant properties under our assay conditions. These results indicate that mt-CK is able to support ADP recycling, thus reducing H2O2 formation, even when mt-HK activity is impaired due to an accumulation of Glc-6-P in hyperglycemic conditions.


Figure 7
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  		FIGURE 7.
mt-CK activity prevents H2O2 generation due to inhibition of mt-HK. H2O2 production was measured in respiration buffer as described under "Experimental Procedures" with 0.2 mg/ml Percoll-purified RBM induced by succinate. Trace 1 shows the effect of ADP on H2O2 production. In trace 2, the reaction medium contained 10 mM 2-DOG before the start of the reaction with succinate. The dashed line indicates the time course of the reaction in the absence of Glc-6-P. Trace 3 shows H2O2 production as in trace 2, but after the addition of Glc-6-P (G6P), it was added to Cr. In trace 4, the reaction medium contained 10 mM 2-DOG and Cr before the start of the reaction with succinate. The arrows indicate the sequential additions of: 10 mM Suc, 0.2 mM ADP, 1 mM Glc-6-P, and 5 mM Cr. The figure shows a representative experiment. Similar results were obtained with at least four different independent mitochondrial preparations.

 
mt-CK Activity Prevents {Delta}{Psi}m Hyperpolarization and ROS Formation in Hyperglycemic Embryonic Rat Cortical Neurons— It has been reported that hyperglycemia induces intracellular ROS formation in different cell cultures (4, 20–22) that, in cortical neurons, is related to an impairment of ADP recycling through mt-HK (25). If this possibility was correct, this would imply that in hyperglycemic conditions the intracellular {Delta}{Psi}m would be increased causing an elevation in ROS production due to an imbalance of the mt-HK activity. To confirm this, primary cultures of embryonic rat cortical neurons were incubated in different concentrations of glucose (10 or 40 mM) with or without the mitochondrial kinases substrates (2-DOG or Cr) and the evaluated intracellular {Delta}{Psi}m measured by the fluorescence ratio of JC-1 and ROS levels by the CMH2-DCFDA fluorescence. Indeed, we found a pronounced increase in the intracellular levels of {Delta}{Psi}m in 40 mM glucose, whereas simultaneous addition of 40 mM glucose + 30 mM 2-DOG did not cause mitochondrial hyperpolarization (Fig. 8A). Likewise, activation of mt-CK in hyperglycemia, by previous incubation with 5 mM Cr, prevented the increase in {Delta}{Psi}m levels, showing a similar effect to 5 µM FCCP addition (Fig. 8A, see also supplemental materials Fig. 1).


Figure 8
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  		FIGURE 8.
Mitochondrial hyperpolarization and ROS formation induced by high glucose concentrations are prevented by mt-CK activity in rat cortical neurons. A, {Delta}{Psi}m was measured by the ratio between red/green fluorescence of the probe JC-1 of cultured cortical neurons from 14-day-old Wistar rat embryos incubated with RPMI medium containing 10 mM (10 Glc) or 40 mM (40 Glc) glucose; 40 mM Glc plus 30 mM 2-DOG (40 Glc 30 DOG); 40 mM Glc plus 5 µM FCCP (40 Glc FCCP); and 40 mM Glc plus 5 mM Cr (40 Glc 5 Cr). B, quantification of green fluorescence microscopy images (arbitrary fluorescence intensity (AUF)) of CM-H2DCFDA staining reflecting the intracellular ROS levels. The glucose and Cr concentrations are the same as for A. When antimycin A was used (Ant A) it was added to a final concentration of 5 µM. Data represent mean ± S.E. corresponding to four different experiments. The fluorescence microscopy images utilized to quantify {Delta}{Psi}m and ROS formation in A and B are available as supplemental figures S1–S2.

 
Regarding the cellular ROS formation, we noticed that 40 mM glucose in the medium increased ROS levels as indicated by CMH2-DCFDA fluorescence, which was reduced by simultaneous addition of 5 mM Cr (Fig. 8B, see also supplemental materials Fig. 2). This result raises the possibility that the presence of Cr per se in the cells may be acting as an antioxidant scavenger. To test this hypothesis, we simulated a pro-oxidant condition by adding 2.5 µM antimycin A, an inhibitor of the electron transport chain at complex III and a known powerful inducer of superoxide formation, to the low glucose medium (10 mM). As expected, antimycin A caused a significant raise in ROS formation that was not affected by the presence of 5 mM Cr, indicating that the observed effect of Cr on intracellular ROS generation was not derived from scavenger activity of this molecule (Fig. 8B, see also supplemental materials Fig. 2). Taken together, the results presented in Fig. 8 indicate that activation of rat brain mt-CK in hyperglycemia is sufficient to avoid hyperpolarization, preventing electron leakage and ROS formation due to a steady-state ADP recycling through the inner mitochondrial membrane in embryonic rat cortical neurons.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Hyperglycemia is associated with several metabolic dysfunctions, such as diabetes and sepsis (22, 23), and excessive glucose can be harmful to tissues (3–5). ROS production by mitochondria through the electron transport chain is a causal link between high glucose and the main pathways responsible for hyperglycemic damage (4, 8, 20, 21). Even a short-term exposure of neurons to hyperglycemia produces oxidative damage and apoptosis in nervous cells and these effects can be prevented by antioxidants (5). Although the mechanism of glucose toxicity is not completely understood, it has been proposed that increased pyruvate oxidation would stimulate mitochondrial respiratory chain and O2. radicals production by an excess of NADH availability to the electron transport chain (4, 5, 20–22, 65). In previous work (25), our group showed that ADP re-cycling through the inner mitochondrial membrane, performed by mt-HK, plays an essential preventive antioxidant role by decreasing {Delta}{Psi}m. In a high glucose medium condition, we observed an accumulation of intracellular Glc-6-P (25), indicating that excess pyruvate has reached the mitochondria to be oxidized. However, as Glc-6-P inhibits mt-HK, this would disrupt ADP recycling, as demonstrated previously (25), favoring the increase in {Delta}{Psi}m. Thus, both increased pyruvate oxidation and the impairment of mitochondrial ADP re-cycling, due to a blockage of mt-HK activity, would lead to {Delta}{Psi}m hyperpolarization and reduce electron flux in the electron transport chain, inducing ROS generation. In the present work we demonstrate that the activities of mitochondrial kinases (mt-HK and mt-CK) in RBM contribute to regulation of ROS generation through the ADP re-cycling mechanism. Particularly, this mechanism is relevant in high glucose medium, where the mt-HK is not fully active (Figs. 1, 2, 4, 8, and 9 and Ref. 25) but the impairment of ROS generation can be achieved by mt-CK activity. In fact in RLM, which is devoid of mt-CK, Cr had no effect on {Delta}{Psi}m,O2 consumption, or H2O2 generation (Fig. 3C), suggesting that the preventive antioxidant role of Cr depends on the presence of mt-CK. Thus, the effects observed in Fig. 8A (see also Ref. 25), indicating that 2-DOG decreases cellular {Delta}{Psi}m and prevents ROS generation, could be interpreted either by decreasing pyruvate oxidation, because 2-DOG competes for glucose phosphorylation by mt-HK, or by activating the ADP re-cycling through the ANT-VDAC complex. In our previous work (25), we could not distinguish clearly between these two possibilities. However, the main contribution of the present work is that when pyruvate oxidation is high (high glucose medium) the activation of ADP re-cycling by mt-CK activity, induced by Cr, is sufficient to reduce both the {Delta}{Psi}m and ROS generation. These observations can be concluded based on experiments where Cr supplementation abolishes ROS generation in high glucose medium (Figs. 3, 7, and 8). One would speculate that Cr might cause an inhibition of glucose metabolism resulting in a decrease in both {Delta}{Psi}m and ROS production. Based on the literature, we suggest that Cr does not change the glycolytic flux and so the pyruvate levels. Conversely, conditions where glucose metabolism are impaired would reduce pyruvate oxidation in mitochondria, avoiding {Delta}{Psi}m hyperpolarization and ROS generation. Noteworthy, if Cr directly decreased glucose metabolism, we would expect a reduction in ROS production independently from the CK activity. However, several reports showed that Cr has no inhibitory effects on glucose metabolism in skeletal muscle and brain, because: 1) dietary supplementation of Cr to rats caused no changes in basal nor insulin-glucose uptake in rats (64). 2) Brain glycolytic flux is enhanced in hyperglycemia, not affecting PCr to Cr conversion under ischemia (65). 3) Stimulation in brain functional activity led to an increase of glycolytic flux, parallel to PCr utilization and Cr accumulation (66). Thus, we cannot support the hypothesis that decrease of {Delta}{Psi}m and ROS production in our conditions (at 40 mM glucose + 5 mM Cr) is due to reduced pyruvate levels (Fig. 8). Also, succinate-induced ROS generation in isolated brain mitochondria is not affected by Cr (Figs. 3B and 7) and is drastically reduced when ADP re-cycling is performed by mt-CK activation through ATP addition, simulating a state 3 respiration. These observations reinforce the concept that the preventive antioxidant role of Cr is mediated by mt-CK activation, allowing the ADP re-cycling. The possibility that Cr itself would be exerting scavenger antioxidant effects seems not to be the case, as state 4 H2O2 generation in isolated brain mitochondria (Figs. 3B and 7) and neuronal ROS generation induced by antimycin A (Fig. 8B) were both unaffected by Cr supplementation. Altogether, these data support the notion that mitochondrial ROS production induced by high glucose oxidation can be related to the rate of ADP re-cycling. Therefore, any event that regulates, directly or indirectly, the electron flux, would modulate ROS generation. In this way, Nishikawa and colleagues (4) showed that pharmacological uncoupling or overexpression of uncoupling proteins in hyperglycemia abrogated ROS generation. Taking into account this information, the main contribution of the present work is not only to support the Nishikawa proposal but also to suggest that regulation of {Delta}{Psi}m through mitochondrial kinase activation leads to impairment of hyperglycemia-induced oxidative stress.


Figure 9
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  		FIGURE 9.
Schematic representation of the proposed mechanism by which mt-CK regulates oxygen consumption, {Delta}{Psi}m, and ROS production in mitochondria during hyperglycemic conditions. In brain and other tissues, HK is bound to the outer mitochondrial membrane through an association with the VDAC. The octameric form of mt-CK localizes in the intermembrane space, through an association to VDAC and ANT. The figure represents mitochondria under a hyperglycemic condition and Cr supplementation, in which mt-HK is inhibited by Glc-6-P accumulation (Fig. 1 and Ref. 25) but ADP re-cycling is maintained by mt-CK activity, regulating oxygen consumption, {Delta}{Psi}m, and ROS generation. Bold arrows and solid lines indicate a high flux of metabolites, whereas gray dashed lines represent low flux. Numbers represent the complexes of respiratory electron chain. UQ, ubiquinone; Cyt c, cytochrome c; SOD, superoxide dismutase; GPx, glutathione peroxidase.

 
It is known that several neurological disorders, with different primary defects, often converge to display similar impairments in cellular energy metabolism in the brain (33). In these instances, the intracellular ATP concentration is decreased, resulting in cytosolic accumulation of Ca2+ and ROS formation. A common feature among these disorders is the impairment of brain Cr metabolism, i.e. decrease in total Cr and PCr concentration, CK activity, and/or Cr transporter content (67, 68). In cultured rat neurons, as well as in astrocytes, Cr protected against glutamate, beta-amyloid, and 3-nitropropionic acid toxicity (18, 19, 69). Furthermore, reduced neuronal damage and ROS formation were observed when cultures were administered with Cr at least 6 h before 3-hydroxyglutarate treatment (53). In isolated mitochondria, the inhibition of the MPT by CK substrates seems to be an important blocker of both necrotic and apoptotic cell death (42, 52). Although Cr protected the brain against malonate-induced hydroxyl radical generation, due to increased high energy phosphate reserves (17), a direct involvement of Cr with cellular and mitochondrial ROS generation was not yet established. Therefore, the neuroprotection of Cr against Huntington disease could involve the partial restoration of neuronal ROS homeostasis mediated by mt-CK activity. The involvement of mt-CK as modulator of the MPT by Cr was challenged by data showing that Cr still exerts neuroprotective effects in mt-CK knock-out mice, suggesting that these effects are not mediated by mt-CK to inhibit the MTP (70). However, these data may be explained by the fact that enough levels of cytosolic CK activity were found in mt-CK knock-out mice to support Cr phosphorylation. The cytosolic isoform would compensate the absence of the mitochondrial enzyme assuring the maintenance of Cr phosphorylation and ADP production, supporting the reduction of ROS generation (Fig. 6B and Ref. 71). Supporting this idea is that the double knock-out mice lacking both isoforms of CK caused cognitive dysfunctions and spatial learning. Interestingly, the absence of the mt-CK isoform caused a compensatory increase in the PCr/Cr ratio induced by Cr supplementation in both cortex and cerebellum (70). Together with our results, the change in the PCr/Cr ratio in the mt-CK knock-out mice indicates that the cytosolic CK would be sufficient to allow mitochondrial ADP recycling. A common explanation of the protective effects of Cr preventing or ameliorating the features of neurodegenerative diseases is based on the improvement of the energy charge of the neural cells, evaluating the PCr/Cr ratio. Thus, based in our results, we propose in Fig. 9 that mt-CK controls mitochondrial ROS generation through the ADP cycling system. This mechanism may be supported by other kinases, such as mt-HK and probably mt-adenylate kinase providing steadily an ADP flux through the mitochondrial inner membrane. In hyperglycemia, mt-HK is not fully active, because Glc-6-P accumulates intracellularly because other metabolic pathways cannot use all of this metabolite (25), leading to an impairment of ADP cycling. However, in the presence of Cr, mt-CK assures the ADP shuttle coupling the oxidative phosphorylation and PCr formation, causing a decrease in the {Delta}{Psi}m and thus ROS formation. This cycling accelerates the respiration rates, which, in turn, diminish the electron leak, producing less O2. radicals that are further converted to H2O2 by superoxide dismutase. In conclusion, the maintenance of ADP shuttle in mitochondria, performed by mitochondrial kinases, is a key preventive antioxidant system, which would complement other classic antioxidant defenses, such as superoxide dismutase and catalase. It remains to be determined how the suppression of ROS generation under Cr treatment would impact the redox balance and if this correlates with the neuroprotective effects of Cr in brain.


   FOOTNOTES
 
* This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Fundação Universitária José Bonifácio, and the Third World Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S2. Back

1 These authors contributed equally to the results of this work. Back

2 Present address: Thyroid Section, Division of Endocrinology, Diabetes, and Hypertension, Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. Back

3 Research fellow of CNPq. To whom correspondence may be addressed: Av. Brigadeiro Trompowsky, s/n, CCS, Bloco D, sub-solo sala D-013 and D-005, Laboratory of Bioenergetic and Mitochondrial Physiology Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil. E-mail: maroli{at}bioqmed.ufrj.br. 4 Research fellow of CNPq. To whom correspondence may be addressed: E-mail: galina{at}bioqmed.ufrj.br.

5 The abbreviations used are: ROS, reactive oxygen species; mt-CK, mitochondrial-associated creatine kinase; PCr, phosphocreatine; Cr, creatine; mt-HK, mitochondrial-associated hexokinase; MM-CK, cytosolic rabbit muscle creatine kinase; VDAC, voltage-dependent anion channel; ANT, adenine nucleotide transporter; {Delta}{Psi}m, mitochondrial membrane potential; 2-DOG, 2-deoxyglucose; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Ap5A, P1,P5-di(adenosine 5')-pentaphosphate; Glc-6-P, glucose 6-phosphate; JC-1, 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolylcarbocyanine iodide; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; G6PDH, glucose-6-phosphate dehydrogenase; RBM, rat brain mitochondria; RLM, rat liver mitochondria; MTP, mitochondrial permeability transitition. Back


   ACKNOWLEDGMENTS
 
We express our gratitude to Dr. Roger Castilho (Unicamp, SP, Brasil) for valuable contributions and helpful discussions as well as for the kind supply of Amplex Red. We also thank Dr. Leopoldo de Meis for the laboratory facilities.



   REFERENCES
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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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