species and tissue specific relationships between mitochondrial

1

SPECIES AND TISSUE SPECIFIC RELATIONSHIPS BETWEEN MITOCHONDRIAL

PERMEABILITY TRANSITION AND GENERATION OF ROS IN BRAIN AND LIVER

MITOCHONDRIA OF RATS AND MICE

1,2Alexander Panov, 3Sergey Dikalov, 2Natalia Shalbuyeva, 1Richelle Hemendinger, 2John T. Greenamyre,

1Jeffrey Rosenfeld

1Carolinas Neuromuscular/ALS-MDA Center, Carolinas Medical Center, Charlotte, NC 28203. 2Center for

Neurodegenerative Disease, 3Free Radicals in Medicine Core, Division of Cardiology, Emory University,

Atlanta, GA, 30322

Address correspondence to: Dr. Alexander V. Panov

Carolinas Neuromuscular/ALS-MDA Center1,

Carolinas Medical Center, 1000 Blythe Blvd,

Charlotte, North Carolina 28203

Tel: 704-355-5902

Fax: 704-446-6255

e-mail: [email protected]

Running title:

Brain and liver mitochondria in rats and mice

of 37Articles in PresS. Am J Physiol Cell Physiol (October 18, 2006). doi:10.1152/ajpcell.00202.2006

Copyright © 2006 by the American Physiological Society.

mailto:[email protected]

2

ABSTRACT

In animal models of neurodegenerative diseases pathological changes vary with the type of organ and

species of the animals. We studied differences in the mitochondrial permeability transition (mPT) and

reactive oxygen species (ROS) generation in the liver (LM) and brain (BM) of Sprague Dawley rats and

C57Bl mice. In the presence of ADP mouse LM and rat LM required 3 times less Ca2+ to initiate mPT than

the corresponding BM. Mouse LM and BM sequestered 70% and 50% more CaPi than the rat LM and BM.

MBM generated 50% more ROS with glutamate than the RBM, but not with succinate. With the NAD

substrates, generation of ROS do not depend on the energy state of the BM. Organization of the respiratory

complexes into the respirasome is a possible mechanism to prevent ROS generation in the BM. With BM

oxidizing succinate, 80% of ROS generation was energy dependent. Induction of mPT does not affect ROS

generation with NAD substrates and inhibit with succinate as a substrate. The relative insensitivity of the

liver to systemic insults is associated with its high regenerative capacity. Neuronal cells with low

regenerative capacity and a long life span protect themselves by minimizing ROS generation and by the

ability to withstand very large calcium insults. We suggest that additional factors, such as oxidative stress,

are required to initiate neurodegeneration. Thus the observed differences in the Ca2+-induced mPT and ROS

generation may underlie both the organ-specific and species-specific variability in the animal models of

neurodegenerative diseases.

Keywords: permeability transition, ROS generation, interspecies difference

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INTRODUCTION

Mitochondrial dysfunctions play important roles in pathogenesis of neurodegenerative diseases

(reviewed in (6, 26, 28). In many degenerative diseases cells die by an apoptotic mechanism (6, 24, 26, 31),

and mitochondria play a cardinal role in apoptotic or necrotic cell death (6, 15, 24, 26, 41). One mechanism

by which mitochondria may initiate cell death is associated with the phenomenon of permeability transition

followed by release of apoptosis-inducing factors (6, 18, 36, 52, 62). Mitochondria are also considered as

the major source of reactive oxygen species (ROS) (12), and increased ROS generation is regarded as an

important pathogenic mechanism in aging and neurodegeneration (40).

Experiments with isolated rat liver mitochondria (RLM) (12) and cultured hepatocytes (12, 31) have

shown that peroxides, such as tert-butyl hydroperoxide (t-BOOH), promote mPT even in the presence of

very small amounts of Ca2+. The current view is that oxidative stress plays an important role in promoting

the Ca2+ induced mPT (12, 62), and t-BOOH was regarded as a useful tool to study effects of oxidative

stress on mitochondrial functions (12, 31). It was suggested that increased oxidative stress may be an

important factor in pathogenesis of neurodegenerative diseases such as Parkinson’s disease (PD) (40)

amyotrophic lateral sclerosis (ALS) (47), and Huntington's disease (HD) (46).

The progress in studies of neurodegenerative diseases, such as HD and ALS, was also promoted by

creation of genetically engineered animal models of these diseases (reviewed in (65)), and by introduction

of the in vivo toxic models of PD (5, 14, 40) and HD (9). However, the animal models of the diseases

revealed a basic problem: different species respond differently to the pathological agents used in the in vivo

models of neurodegenerative diseases (20, 48). Besides, recent experiments have shown that during

systemic intoxication with rotenone, the brain mitochondria were severely damaged whereas liver

mitochondria remained virtually unaffected (40).

Thus, several fundamental questions arise that have to be answered in order to successfully use the

toxic and genetic animal models of neurodegenerative diseases. Some of the uncertainties stem from the

trivial fact that most of our current knowledge about mitochondrial functions, permeability transition in

particular, was obtained in experiments with mitochondria from rat tissues, predominantly liver. The

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genetic models of neurodegenerative diseases are, however, usually created with mice and involve studies

of tissues other than liver.

The first question to consider examines the extent to which brain mitochondria are different from

liver mitochondria. There is no consensus in this respect in the literature. The published respiratory

activities for brain mitochondria may differ more than 10 fold, while some authors failed to find any

difference in respiratory activity between liver and brain mitochondria (3, 64). However, it was also found

that in comparison with rat liver mitochondria, rat brain mitochondria are not sensitive to a damaging effect

of hydroperoxydes (27), do not produce ROS such as superoxide (15, 54) and nitric oxide (30), or, on the

contrary, generate more ROS than other tissue mitochondria (11). There are indications that brain

mitochondria are less sensitive to a damaging effect of Ca2+ (27), and are less sensitive to the protective

effect of cyclosporin A during calcium loads (39).

The second question regards the species differences in mitochondrial functions. There were reported

species-specific differences in State 4 (resting respiration) respiratory rates, which depend on intrinsic

proton conductivity of mitochondria, and thus determine the Standard Metabolic Rate of an animal. Some

of these differences were related to the size of the animals (8). In order to successfully use animal models of

diseases, we have to understand the nature of these species-specific differences in mitochondrial functions.

It is well established that responses of animals to pathological agents are genotype dependent (20, 48).

However, little is known how these diversities are translated at the functional level in different organs.

Because mPT and ROS production are thought to play important roles in neurodegeneration it was logical

to focus our attention on these two mitochondrial functions.

Thus the purpose of this work was to study organ-specific and species-specific differences between

rat and mouse liver and brain mitochondria in their major functions, namely permeability transition and

generation of ROS. The abnormalities of these functions may contribute to the pathogenesis of

neurodegenerative diseases, and the differences in the relationships between mPT and ROS generation in

rats and mice may underlie the species-specific diversity in animal models of neurodegenerative diseases.

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MATERIALS AND METHODS

Animals. 2-3 month old male Sprague Dawley rats and male C57Bl/6J mice were used for isolation of

the liver and brain mitochondria. The animals were housed and cared for in AAALAC-accredited facilities

at the Carolinas Medical Center and Emory University. All experiments involving animals were performed

in strict accordance with the NIH Guide for the Care and Use of laboratory animals.

Isolation of the liver and brain mitochondria. Both liver and brain mitochondria were isolated in medium

containing the following: (in mM) 225 mannitol, 75 sucrose, 20 MOPS (pH 7.2), 1 EGTA, and 0.1% BSA.

Liver mitochondria (LM) were isolated by conventional differential centrifugation with a final spin at 8600

g (40). Brain mitochondria were isolated from the pooled forebrains of three rats. We used the modified

method of Sims (53) to isolate and purify brain mitochondria (BM) in a Percoll gradient. The modifications

were as follows: brain tissue was homogenized with 15 strokes of a loose pestle in a Dounce homogenizer,

and 5 ml volumes per tube of 15%, 23% and 40% (v/v) of Percoll solutions were used to purify the brain

mitochondria. After the final sedimentation of mitochondria at 8600 g, the mitochondria were suspended in

250 mM sucrose and 10 mM MOPS (pH 7.2). Mitochondrial protein was determined using the Pierce

Coomassie Protein Assay Reagent Kit.

Registration of Permeability Transition and estimation of Calcium Retention Capacity (CRC).

Recently we introduced a quantitative parameter, CRC that allows a meaningful comparison of the

sensitivity to Ca2+ of mitochondria from different organs and species (39). CRC is the amount of calcium

that can be accumulated and retained by mitochondria until the permeability transition occurs. It is expressed

as nanomol of Ca2+ per mg of mitochondrial protein. Ca2+ was added to 2 ml of mitochondrial suspensions

(0.5 mg/ml) using aliquots of 5, 10 or 20 mM stock solutions of CaCl2 to achieve final concentrations of

Ca2+ of 12.5, 25, or 50 nmol/ml. At high CRC we switched to higher concentration of the CaCl2 stock

solutions in order to minimize volume changes. We utilized CaCl2 of very high purity – 99.99% from

Sigma.

Three different methods were used to estimate CRC and permeability transition.

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1) Potentiometric measurement of pH changes of the incubation medium during Ca2+ accumulation

and release by the mitochondria as described in (39). The pH measurements were performed using a Corning

pH meter model 440 equipped with a mono pH microelectrode from Lazar Co. and a Ag/AgCl reference

electrode connected to the incubation chamber by a KCl bridge.

2) Depolarization of mitochondria was registered by determining membrane potential (∆Ψ) with a

TPP+-sensitive electrode as described elsewhere (44). Because the volume of the matrix space and binding

constants for TPP+ in brain mitochondria and mouse liver mitochondria are unknown, the ∆Ψ values

presented are approximate, and show only the dynamics and direction of ∆Ψ changes.

3) Swelling of mitochondria, registered as a decrease in optical density, was recorded at 545 nm using

a Shimadzu Multispec-1501 model spectrophotometer. We measured simultaneously 2-3 parameters in

mitochondria in various combinations.

The mitochondrial CRC and swelling were estimated in a medium (Medium B) containing (in mM),

KCl 125, NaCl 10, MgCl2 0.5, glycyl-glycine 3, pH 7.2, KH2PO4 1, and glutamate 20 plus malate 2 for brain

and liver mitochondria. Mitochondrial protein was 0.5 mg/ml. The composition of medium B used in the

present study to measure CRC is different from the sucrose-based medium we used in our previous studies

(39, 40). We have found that the sucrose based medium with only 20 mM KCl and absence of Mg2+ may

inhibit maximal respiratory activities of the brain mitochondria (Panov et al., unpublished observations).

Therefore, the current composition of the medium for measuring CRC only minimally differs from the

medium A, which is optimal for maximal respiratory rates of brain mitochondria (40). We have found that

the current composition of medium B, as compared to the sucrose based medium, gives somewhat higher

and better reproducible values of CRC for the same type of brain mitochondria.

At the end of an experiment, addition of 125 nmol/ml H+, that caused a ∆pH of 0.07 pH units, served

as an internal calibration. This allowed a comparison of the total pH changes and calculation of the H+/Ca2+

ratios. Similarly, additions of 0.5 µM TPP+ aliquots served as internal calibration for membrane potential

changes. In experiments with liver mitochondria the final concentrations of TPP+ were 1.5 µM, with brain

mitochondria – 2 µM.

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In order to determine maximal amplitudes of the mitochondrial swelling and complete release of CaPi

from the mitochondria after mPT, we added a bacterial toxin alamethicin (5 µg/mg mitochondrial protein).

Alamethicin forms a large conductance pore for low molecular mass hydrophilic substrates (21).

Measurements of hydrogen peroxide generation. H2O2 was determined using Amplex red (Molecular

Probes) method. In the presence of horse radish peroxidase (HRP) the following reaction occurs: Amplex

red + H2O2 � Resorufin + O2. Resorufin is a stable and highly fluorescent compound whose wavelength

spectra excitation/emission are 570/585 nm. The fluorescence of resorufin was determined in 1 ml

incubations in a medium (Medium A) containing (in mM): KCl 125, MOPS 10, pH 7.2, MgCl2 2, KH2PO4 2,

NaCl 10, EGTA 1, CaCl2 0.7, and 0.2 mg/ml mitochondrial protein, 5 µM Amplex red and 3 units of HRP as

described in (40). At the EGTA/Ca2+ ratio of 1/0.7 the concentration of [Ca2+]free was close to 1 µM as

determined with the Fura-2 method. We measured H2O2 production in the presence of glutamate 20 mM +

malate 2 mM, or succinate 5 mM. When present, the respiratory inhibitors and uncoupler were added to the

incubation media prior to addition of mitochondria. Additions of 50 nM of resorufin or correspondingly

diluted standard 3 mM solution of H2O2 (Fluka) were used for calibration of the fluorescence scale. The

background fluorescence was subtracted from experimental fluorescence, and the scale between 4000 and

6000 fluorescence arbitrary units, which corresponded to 44 nM of H2O2 or resorufin, was used in

calculations. There was a very close correlation between the fluorescence of freshly diluted H2O2 standard

solution and the resorufin standard. However, H2O2 solutions decayed rapidly while resorufin solutions

remained stable for a long time. Fluorimetric measurements were made using a fluorometer from C&L

Company, Middletown, Pennsylvania (www.fluorescence.com).

Data acquisition. The data acquisition was performed using hardware and software from C&L

Company, Middletown, Pennsylvania (www.fluorescence.com).

Chemicals. Chemicals were of highest purity available. All solutions were made using glass bidistilled

water.

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Statistics. Data are presented as mean ± standard error of 4-6 separate experiments. For comparison of

two groups, a two-tailed t-test was employed using Excel software. Statistical significance was assumed when

p < 0.05.

RESULTS

Permeability transition in liver mitochondria. Figure 1 shows changes in membrane potential (∆Ψ),

Ca2+ accumulation registered with the pH method (39) and optical density changes of rat (RLM) (Fig. 1A)

and mouse (MLM) (Fig. 1B) liver mitochondria during titration with calcium. The figures show that with

RLM and MLM depolarization and slow rate swelling began before the large pore opening registered as

alkalization of the medium (see also 39) clearly designating the moment of a large pore opening. These pH

changes are bound to a dissociation of calcium phosphate salts deposited in the matrix and binding of

protons to the released HPO42- and PO4

3- anions to accommodate the medium’s pH (15, 39).

Figures 5A and 5B summarize quantitatively the results of several experiments represented in figures

1, 2, 3 and 4. Figures 5A, B show that with LM the protective effect of CsA is much higher than that of ADP

+ oligomycin. With the rat and mouse LM Cyclosporin A is the most powerful known inhibitor of mPTP

opening (15, 39), and ADP enhances the ability of CsA to inhibit mPT only moderately. When RLM (Fig.

2A) and MLM (Fig. 2B) were protected by CsA and ADP plus oligomycin, the amounts of Ca2+ sequestered

by the mitochondria were the same (Figures 5A & 5B). In protected RLM and MLM (Figures 2A, 2B),

swelling, collapse of ∆Ψ and alkalization occurred simultaneously. This indicates that unlike unprotected

liver mitochondria that can acquire three conformational states: closed, low conductance and high

conductance states (39), the protected mitochondria have only closed and open high conductance states.

Permeability transition in brain mitochondria. Figure 3A shows that after the first two additions of

Ca2+ aliquots, the unprotected rat brain mitochondria (RBM) did not change their optical density. However,

membrane potential began to decline and then after further Ca2+ additions, slow swelling of RBM began.

The pH trace shows that RBM continued to accumulate and retain Ca2+ even after the swelling had begun.

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After addition of 4-5 Ca2+ aliquots (each 50 nmol Ca2+/mg) a large pore opened as registered by alkalization

of the medium. At this point the rate of swelling increased 2-3-fold, but soon the swelling stopped (Fig. 3A).

The unprotected mouse brain mitochondria (MBM) did not change their optical density during

incubation or addition of Ca2+ aliquots (Fig. 3B). When the large pore opened, as is indicated by the collapse

of ∆Ψ and alkalization of the medium, the swelling of MBM also began, but the amplitude of swelling was

small (0.2 OD). Unlike RBM, the opening of mPTP in MBM was transient and stopped spontaneously (see

Fig. 3B), and was reopened again after addition of CCCP. This effect of CCCP was not associated with

mitochondrial deenergization, but by the uncoupler directly promoting opening of mPTP (51). Figures 5A

and 5B show that unprotected MBM sequestered 2 times (p < 0.001) more Ca2+ before opening of mPTP

than RBM.

When RBM and MBM were protected with ADP (plus oligomycin) the RBM sequestered almost 5-

fold and MBM 3-fold more CaPi than the unprotected brain mitochondria. ADP-protected MBM

sequestered 70% (p < 0.001) more CaPi than the RBM (Fig. 5A, 5B). As with the rat and mouse liver

mitochondria, the RBM and MBM protected with CsA + ADP sequestered the same amounts of CaPi (Fig.

5A, B). In the RBM and MBM protected with ADP (not shown) or CsA + ADP (Figures 4A, 4B), collapse

of membrane potential, alkalization of the medium and swelling occurred simultaneously. Before opening of

mPTP, the optical density of RBM and MBM may be noticeably increased, evidently due to sequestration of

the optically dense CaPi salts. Membrane potentials remained relatively stable. The amplitudes and the rates

of swelling of the RBM and MBM after opening of mPTP were significantly larger than in the unprotected

mitochondria.

The H+/Ca2+ ratios during calcium and phosphate accumulation by mitochondria. Chalmers and

Nicholls (15) suggested that the H+/Ca2+ ratio, the ratio of the net H+ extruded to the number of Ca2+ ions

accumulated, reflects the type of the CaPi salt sequestered by the mitochondria. A decrease in the H+/Ca2+

ratio would mean that more CaPi is sequestered as CaHPO4 x 2H2O, which is relatively soluble. An increase

in the H+/Ca2+ ratio to 1.0 would indicate that more CaPi is sequestered as Ca3(PO4)2, which is 15.9 times

less soluble than the CaHPO4 salt (17).

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In the experiments presented in this paper for Sprague Dawley rats (Figs 1A-4A) and C57Bl/6J mice

(Figs 1B-4B), the H+/Ca2+ ratios for unprotected RLM and MLM were 0.77±0.01 and 0.79±0.01

correspondingly. When liver mitochondria were protected with CsA + ADP + oligomycin, the H+/Ca2+ ratio

of both RLM and MLM increased after the first few additions of calcium to 0.97±0.02. With unprotected

RBM and MBM, the H+/Ca2+ ratios were correspondingly 0.83±0.01 and 0.85±0.02. With protected brain

mitochondria, the H+/Ca2+ ratio during titration with calcium increased rapidly to 1.0 showing that CaPi was

sequestered as almost insoluble Ca3(PO4)2. Thus sequestration of CaPi is a highly dynamic process and can

vary depending on the type of mitochondria and incubation conditions. Our data show that relatively small

changes in the H+/Ca2+ ratios may result in large changes in the amounts of Ca2+ sequestered by the

mitochondria.

Effects of permeability transition modifiers on CRC. It is well established that addition of 200 µM tert-

Butyl hydroxyperoxide (tBOOH) to RLM significantly decreases the amount of Ca2+ necessary to open

mPTP (12, 61). However, we have found that tBOOH was ineffective with MLM. CRC of the MLM

remained at the control level even in the presence of 1 mM tBOOH (not shown). RBM and MBM were also

insensitive to the presence of this hydroperoxide (not shown). These results indicate that in RLM mPT is

radically different as compared to RBM, MBM and MLM.

To analyze the properties of mPTP in rat and mouse liver and brain mitochondria, we used various

compounds that are known to affect mPT. The figures 5A and 5B show that in the absence of mPT modifiers

mouse brain and liver mitochondria sequester considerably (p < 0.001) more CaPi than the corresponding rat

mitochondria. Both rat and mouse brain mitochondria were much more sensitive to the protective effect of

ADP than CsA alone. In the presence of ADP plus CsA, however, there was no difference in CRC values

between the rat and mouse mitochondria. Brain mitochondria sequestered two times more CaPi than the

liver mitochondria.

Mitochondrial generation of reactive oxygen species (ROS). Most mitochondria have active

superoxide dismutase both in the matrix (Mn2+-SOD) and in the intermembrane space (Cu2+-Zn2+-SOD),

therefore it is impossible to study intramitochondrial ROS production by following extramitochondrial O2

•

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released by intact mitochondria (40, 55). The best way to follow generation of O2

• in intact mitochondria is

to determine formation of H2O2 (40, 55). In this study we used the Amplex red method and a highly

sensitive fluorometer as described (40).

Generation of ROS by liver mitochondria. With RLM and MLM oxidizing succinate or glutamate +

malate, there was almost no difference in the rates of H2O2 generation (correspondingly 92.1 ± 4 and 80 ± 4

pmol H2O2/min/mg protein), which was in stark contrast to the 4-6-fold difference between the two

substrates in RBM and MBM shown in Figures 6 and 7. We have also found that with the exception of

antimycin A, addition of respiratory inhibitors rotenone, myxothiazol, and their combinations to LM had no

effect on the observed rates of H2O2 production (not shown, see also 40). As we have suggested and

explained earlier (40), measurements of the extramitochondrial H2O2 cannot be used for analysis of ROS

generation by the intact liver mitochondria.

Generation of ROS by brain mitochondria. Figures 6A and 7A show responses of ROS generation by

rat brain mitochondria, measured as H2O2, upon addition of respiratory chain inhibitors and CCCP with

glutamate + malate (Fig. 6A) and succinate (Fig. 7A) as substrates. Qualitatively, the responses of MBM

were similar to those of RBM. Quantitative comparisons of ROS generation by the RBM and MBM with the

two substrates are shown in figures 6B and 7B. Addition of 50 or 100 units of superoxide dismutase (Roche

Diagnostics GmbH) to the incubation medium did not affect the rates of ROS generation even in the

presence of antimycin A (not shown, see also 40), which is in contrast with the heart mitochondria (35).

Controls with addition of excess catalase, shown in Figures 6A and 7A, indicate that the traces in the figures

indeed represent changes in concentration of H2O2.

Figure 6A shows that with glutamate + malate, the rate of ROS generation by BM was the same in the

energized and in the uncoupled mitochondria, when membrane potential was collapsed. Thus with glutamate

or pyruvate (not shown) as a substrate generation of ROS does not depend on mitochondrial energization.

Upon addition of rotenone there was a 4-5-fold increase in ROS generation (Figure 6A, B). Upon

addition of antimycin A or myxothiazol there was correspondingly a 5.5-fold and 3.3-fold increase in

generation of ROS. A significantly larger effect of antimycin A indicates that some of the ROS was

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generated on complex III. With myxothiazol, which prevents reduction of the CoQ centers on complex III,

and thus abolishes the effect of antimycin A, evidently all of the ROS was generated on Complex I. MBM

generate (80 pmol H2O2/mg protein), that is 62% more ROS (p < 0.001) than RBM (49.3 pmol H2O2/mg

protein) in metabolic State 4 (Figure 6B); however, there was no difference in ROS production between

MBM and RBM in the presence of rotenone or uncoupler CCCP. In the presence of myxothiazol or

antimycin A, MBM generated more ROS than the RBM. The mechanism of this difference is unclear.

Figure 7A shows representatively how brain, or spinal cord, mitochondria respond to CCCP and

respiration inhibitors when ROS generation was supported by succinate. Figure 7B gives a quantitative

comparison of ROS generation by RBM and MBM. Figure 7B shows that under all conditions there was no

difference between the two species in the rates of ROS production by BM oxidizing succinate. Addition of

rotenone to BM inhibited ROS production by about 80%. However energization of the mitochondria was

preserved. Antimycin A also inhibits respiration and energization of the mitochondria and thus prevents

backward electron flow. However, in the presence of antimycin A, the CoQ sites of complex III become

reduced and thus increase O2

• generation (35). Because the CoQO site of complex III is located close to the

outer surface of the inner mitochondrial membrane, a large portion of O2

• is released from the mitochondria

(35, 60). This is one reason why antimycin A dramatically increases generation of ROS with succinate as a

substrate. The other reason for the 4-5-fold increase in ROS generation may be explained by the fact that

mitochondria have 3 times more complexes III than complexes I (23, 49). Thus in the presence of antimycin

A most of O2

• was generated on complex III.

Myxothiazol, which inhibits respiration and prevents reduction of the complex III CoQ sites, also

caused deenergization of the mitochondria and thus inhibited the energy dependent backward electron flow

driven by succinate. As a result, production of ROS was at the same level as with rotenone. In the presence

of rotenone and myxothiazol, the electrons from the membrane pool of CoQ reduced by succinate cannot go

downstream to complex III, or upstream because mitochondria are de-energized and rotenone would block

reduction of the complex I redox sites. Therefore, in the presence of rotenone + myxothiazol generation of

ROS may occur only on complex II (40). Figures 7A and 7B show that with rotenone, myxothiazol, or

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rotenone + myxothiazol the rates of ROS production were practically the same, and thus complex II was

responsible for this basic rate generation of ROS. When CCCP was added to mitochondria oxidizing

succinate, the rate of ROS production was the same or lower than that observed in the presence of rotenone

and myxothiazol.

Taken together, the data show that generation of ROS is strongly substrate dependent. With the NAD-

dependent substrates, generation of ROS does not depend on mitochondrial energization or the functional

state of the mitochondria, whereas with succinate it does. Importantly, with the NAD-dependent substrates

MBM during resting respiration generate 62% more ROS than RBM (Figure 6B).

Simultaneous measurements of ROS generation and mPTP opening. It was suggested by a number of

researchers that mPT stimulates ROS generation by the mitochondria (14, 32). Figure 8 shows simultaneous

registration of ROS generation and the Ca2+-induced mPT in RBM oxidizing either glutamate + malate

(Figure 8A) or succinate (Figure 8B). Similar results were obtained with MBM (not shown). With glutamate

+ malate there was a slight inhibition of ROS generation during active Ca2+ consumption, which again

slightly increased to the initial level upon opening of mPTP. These changes in the rate of ROS generation

were small. Thus with the NAD-dependent substrates mPT does not affect dramatically the rate of ROS

generation.

With succinate as a substrate, the initial rate of ROS generation in State 4 was high, and upon

additions of Ca2+, the ROS production was quickly inhibited and remained low after mPTP opening. This

completely agrees with the data presented in Figure 7 that most of the succinate associated ROS production

was caused by the energy-dependent backward electron flow. As soon as the backward electron flow was

inhibited by partial de-energization during CaPi sequestration, and then by the depolarization caused by

mPT, the generation of ROS dropped to the low basal level as in the presence of an uncoupler shown in

figure 7B. Thus opening of the mitochondrial mPTP by itself may not change dramatically the rate of ROS

generation in brain mitochondria.

DISCUSSION

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Permeability transition in brain and liver mitochondria. We employed simultaneously several

methods to register mPT because mPTP opening is always accompanied by a collapse of ∆Ψ, but

mitochondrial deenergization does not always results in opening of mPTP (4, 39, 42). Regarding brain

mitochondria, there is still controversy as to whether mPT causes mitochondrial swelling or not, and the role

of swelling in cytochrome c release (10, 15, 28, 64). We studied brain mitochondria from several strains of

rats and mice and found that in the absence of mPT modifiers, BM from several species do not

spontaneously open mPTP and do not undergo swelling (39). In general, the amplitudes of swelling of the

unprotected brain mitochondria were not large, evidently not enough to rupture the outer membrane (39).

This suggestion agrees with the observation made by Andreev & Fiskum (2) that unlike RLM, in RBM

cytochrome c was released by the mPT-independent mechanism. In the presence of ADP, which is a more

physiological condition because ADP is always present in a cell, brain mitochondria sequester several times

more CaPi (see Fig. 5A, B) and always undergo mPT and large amplitude of swelling. We believe that in

this case cytochrome c release may be associated with mPT as suggested in (10). Thus, it is very likely that

the roles of mPT in cytochrome c release and initiation of apoptosis may depend on the species specific

intrinsic properties of BM, as well as on assay conditions.

Figures 5A and 5B show that both unprotected and ADP protected MBM require correspondingly 2

times and 70% more Ca2+ to open mPTP than RBM under the same conditions. This might be associated

with the fact that MBM have slightly higher H+/Ca2+ ratios than RBM during CaPi sequestration. We have

shown that even small changes in the H+/Ca2+ ratios dramatically change the CRC values (see also 7). A

very large effect of ADP (plus oligomycin) on the CRC of BM (see figures 5A and 5B) is bound to a

decrease in the mitochondrial conductivity for H+ and K+ ions due to a change in the conformational state of

ANT (39, 41). Decreased proton conductivity promotes mitochondrial energization, increases the H+/Ca2+

ratio, and thus increases the ability of mitochondria to sequester more CaPi (39, 41). The liver mitochondria

have a relatively small amount of ANT (reviewed in 38) in comparison with mitochondria from other

organs, and therefore the protective effect of ADP is small.

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Brain mitochondria require additional insults to induce mPT. Because ADP is always present in a

cell, we suggest that under in situ conditions the BM may sequester very large amounts of CaPi. In the

presence of ADP, RBM sequester 867 ± 33, and MBM sequester 1473 ± 79 nmol Ca2+/mg protein (see

Figures 5A and 5B). Thus normally BM can withstand very large calcium insults without detrimental

consequences for the neurons. However, if the cell and/or mitochondria were predisposed to mPT by some

other hazardous effect, such as inhibition of the respiratory chain or oxidative stress, the detrimental events

may occur. This ability of normal BM to withstand large calcium insults may explain the fact that in most

neurodegenerative diseases clinical manifestations of a disease develop well after midlife when

mitochondria have undergone significant oxidative stress associated with the aging process (34).

We have found that both rat and mouse BM and, surprisingly, MLM are insensitive to the oxidative

stress caused by a peroxide t-BOOH, which is in stark contrast with RLM (14). The mechanism of this BM

and MLM resistance to this peroxide remains unclear. However, this fact suggests that the damaging effects

of peroxides are much less general than was indicated earlier (14). Evidently different species of ROS

responsible for oxidative stress may vary in their mechanisms, and their adverse effects depend on the

animal species and the type of mitochondria.

Because neuronal cells have very high respiratory activity and have a large life span (59), prevention

of increased ROS generation by brain mitochondria is of paramount biological importance. There have to be

evolutionary mechanisms present to lessen or prevent oxidative stress. One of these mechanisms is

represented by the cytoplasmic and mitochondrial enzymatic and metabolic antioxidant defenses, such as

catalase, SODs, GSH-reductases, and vitamin E, which vary from tissue to tissue (33). Because liver

mitochondria have a relatively high antioxidant system, including catalase, this may explain why it is

difficult to study generation of ROS in intact liver mitochondria (39).

Here we will outline the intrinsic mitochondrial structural mechanisms to minimize ROS production.

For some reasons, these mechanisms have missed the attention of researchers who study oxidative stress, but

the factual basis for our discussion was laid down by researchers in other fields of mitochondrial biology

(23, 49).

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Generation of ROS in brain mitochondria. In mitochondria, ROS can potentially be formed

spontaneously at any protein involved in redox reactions, that may have a group capable of one electron reduced

state such as ubiquinone (UQ) and transition metals (1, 25). Currently many authors consider

semiubiquinones (UQ•-) of the respiratory chain Complex I and Complex III as the major sources of O2•- (13,

25). Recently, it was shown that mitochondrial α-ketoglutarate dehydrogenase can generate ROS (56).

However, there is no consensus regarding both the sites and regulation of ROS generation under normal in

situ conditions.

In many studies the authors designate the role of a respiratory chain component in oxidative stress by

the ability of this component to generate ROS in the presence of a specific inhibitor. For example, antimycin

A dramatically increases generation of O2•- at Complex III in mitochondria oxidizing succinate, therefore

some authors consider Complex III as one of the major sites of ROS production (35). The inhibitors show us

only a potential capacity of a given respiratory component to generate ROS in the presence of this inhibitor.

Some pathological situations may be compared to the effects of inhibitors. For example, hypoxia may

resemble effects of rotenone or antimycin A, and the loss of cytochrome c after opening of mPTP may

resemble the effect of antimycin A on ROS generation (29). But these are not normal, physiological

situations. Under “normal or physiological conditions” we understand situations when mitochondria

function in the absence of added inhibitors at and between the metabolic State 3 in an activated neuron, and

closer to metabolic State 4 in a quiescent cell.

Supercomplexes of the mitochondrial respiratory chain and ROS generation. From our earlier

experiments we have concluded that pyruvate and also glutamate are the best substrates for brain

mitochondria (39). This agrees with the current view that the major energy substrate for the brains of adult

mammals is glucose (19). Moreover, recent research showed that there are complex interactions between

astroglia and neuronal cells designed to optimize energy supply in the form of lactate for activated neurons

(reviewed in 19, 45).

In figure 6 we have shown that both in the energized and de-energized brain mitochondria the rates of

ROS generation were at the same relatively low level (50-60 pmol/min/mg protein measured as H2O2). Thus

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with the NAD-dependent substrates the rate of ROS generation does not depend on the energy state of

mitochondria. Only when the electron flow was prevented by any of the respiratory inhibitors did the

generation of ROS increase several folds due to increased reduction of the CoQ sites of Complex I.

That ROS generation in BM oxidizing NAD-dependent substrates is at minimum and does not depend

on the energy state of the mitochondria can be explained by the assumption that all mitochondrial

components, which potentially can generate ROS, are maintained in the oxidized state. Evidently, the steady

low level of ROS generation occurs at the initial rate-limiting step which is more likely either FMN or the

(2Fe-2S)N1a center of Complex I. We suggest that oxidation of the Fe-S clusters and CoQ centers in Complexes

I and III is ensured by the superstructure of the respiratory chain.

It was shown that for the heart mitochondria the ratio for oxidative phosphorylation (OXPHOS)

Complexes I:II:III:IV:V is 1:2:3:6-7:3-5 (23), or more recently determined as 1:1.5:3:6:3 (37). The

respiratory complexes interact with each other to form a supercomplex named the respirasome (49). Based

on the above ratios of the OXPHOS complexes, Schägger et al. (49) suggested that the respirasome exists

as a mixture of two large supercomplexes and one smaller complex. Each of the two large supercomplexes

are comprised of a Complex I monomer, a Complex III dimer, and four copies of Complex IV. The smaller

supercomplex contains two Complexes III and four Complexes IV (49, 50). The major advantages of the

supercomplex structure of the mitochondrial respiratory chain are substrate channeling, catalytic

enhancement, sequestration of reactive intermediates and structural stabilization (49, 50). The advantage of

the substrate channeling is the use of localized substrate molecules, for example, quinone and cytochrome

c, which can react independently of bulk properties of a quinone or cytochrome c pool (49).

We suggest that in addition to the benefits listed above, the respirasome is also an evolutionary

adaptive mechanism designed to prevent excessive production of ROS. Evidently, this mechanism

developed early during the evolution of the aerobic organisms because it is available in aerobic bacteria and

yeast (49, 50). The initial reaction of NADH with the FMN of the Complex I is the rate limiting step in

oxidation of the NAD-dependent substrates. The suggested composition of the respirasome ensures that all

components proximal to Complex IV, which is in a 4-fold excess of Complex I, are kept oxidized

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regardless of the energy state of the mitochondria. In brain mitochondria, which respire at rates similar or

even higher than heart mitochondria, the composition of the respirasome should be similar to that reported

for beef heart mitochondria (49, 50).

Recently we have found that unlike RLM, brain mitochondria have no intramitochondrial substrate

storage pool (A.V. Panov, unpublished observations). This may represent yet another adaptive mechanism to

minimize generation of ROS when brain mitochondria are inactive or de-energized.

Possible control of the succinate-driven ROS generation. In the figure 7 we have shown that in the

energized brain mitochondria the rate of ROS generation with succinate is 4-6 times higher than with an

NAD-dependent substrate. Inhibitor analysis showed that approximately 80% of the total ROS with

succinate is generated on Complex I. This conclusion agrees with previously published papers (40, 60, 63).

Approximately 20% of the total ROS produced by the BM oxidizing succinate was independent of

mitochondrial energization, and occurred on Complex II, presumably on the FAD moiety of the enzyme

(40). As is shown in figures 6 and 7, in the absence of antimycin A there was no generation of ROS on

Complex III whether BM oxidized glutamate or succinate.

Because in the presence of rotenone, rotenone + myxothiazol, and CCCP the rates of ROS production

are virtually the same, we may conclude that this basic level of superoxide generation occurs on Complex II,

which does not depend on the energy state of the mitochondria. Based on the results of inhibitor analysis we

can also conclude that Complex III does not generate O2•- in energized and de-energized BM. Complex III

generates ROS only in the presence of antimycin A (Figure 7A, 7B see also 37). The general agreement is

that when energized mitochondria oxidize succinate, most of the ROS is generated on CoQ centers of

Complex I that become reduced due to the energy-dependent backward electron flow (40, 61, 63).

In situ, when brain mitochondria utilize pyruvate or glutamate as respiratory substrates, succinate is

formed during the functioning of the Krebs cycle. Succinate dehydrogenase (SDH) is the only Krebs cycle

enzyme that is also part of the mitochondrial electron transport chain, designated as Complex II. During

oxidation of succinate to fumarate complexes II feed electrons into the mitochondrial pool of CoQ with such

a force (redox potential E = +90 mV, ref. 37) that electrons can go not only downstream to Complex III, but

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also upstream reducing components of the Complex I (40, 61, 63). This catalyzed Complex II oxidation of

succinate is an irreversible reaction, at least in mammalian mitochondria. Coupling of the SDH/Complex II

to the thermodynamically irreversible electron transport reactions of the respiratory chain makes the Krebs

cycle also work irreversibly in the clockwise direction to ensure utilization of pyruvate and other

intermediates as the source of hydrogen. However, the stoichiometric formation of succinate as a Krebs

cycle intermediate raises the question of how brain mitochondria protect themselves from excessive ROS

generation when a neuronal cell is at rest and mitochondria become more energized?

Schagger et all. (49, 50), using a BN-PAGE method, and Bianchi et al. (7), using a flux control

analysis, did not observe stable binding of Complex II to Complexes III and IV. However, other researchers,

using different methods, isolated functional Complexes II and III (62). In submitochondrial particles

prepared by sonication of the mitochondria, the activity of Complexes II-III can be readily assessed (58),

although the SDH activity becomes almost totally inhibited (43). Schagger (49) suggested that Complex II

and other FAD-dependent dehydrogenases can contact the smaller supercomplex comprising Complexes III

and IV, which functions as a sink for electrons provided by the membrane pool of reduced CoQ.

We suggest further that because brain mitochondria do not have a storage pool of the Krebs cycle

intermediates (A.V. Panov, unpublished data), in active mitochondria the steady-state concentrations of the

mitochondrial substrates, including succinate, must be also low. Thus unlike artificial in vitro experimental

conditions, when brain mitochondria oxidize mM concentrations of succinate, the in vivo succinate

concentrations must be low, and therefore the rates of reduction of the membrane pool of CoQ may be

lower, as compared with the rate of its oxidation by the smaller superstructure of Complexes II, III and IV.

Therefore the succinate-driven backward electron flow could be prevented. In addition, brain mitochondria in

vivo may never be in the highly energized state as in the in vitro State-4. Thus channeling of the substrate

utilization by the respirasome increases the efficiency of mitochondria and prevents excessive production of

ROS. In addition, our preliminary data have shown that there may be other means of suppression of the

succinate-supported ROS generation that involve metabolic control over SDH activity. This suggestion

requires further investigation.

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A Summary of the species-specific differences in the brain and liver mitochondria.

Altogether, the data presented show that the observed differences in the Ca2+-induced mPT and ROS

generation may underlie the organ and species-specific differences observed in animal models of

neurodegenerative diseases. Interestingly, short living liver cells and long living neuronal cells have

different strategies towards the Ca2+-induced mPT and ROS generation. Liver cells have strong antioxidant

defense against ROS that were formed in the mitochondria (16). Whole liver mitochondria release very little

O2•- and H2O2 (40). Because liver mitochondria have much lower calcium capacities, they evidently undergo

mPT followed by apoptosis much easier than brain mitochondria. Elimination of damaged hepatocytes and

replacement with the new healthy cells is evidently the reason why during systemic intoxication of animals

with a toxin the liver mitochondria remain almost normal (40). This probably reflects the high regenerative

potential of the liver. Brain mitochondria, on the other hand, have “chosen” a strategy to prevent generation

of ROS. In the brain, which has negligible regenerative potential and a very long life-span, mitochondria can

withstand enormous calcium insults (see Figs. 5A, 5B). Therefore, we suggest that interactions between

ROS generation and mechanisms of CaPi sequestration determine responses to pathological situations in

animal models of neurodegenerative diseases.

However, the relationships between these functions require further investigation. Mouse brain

mitochondria, for example, generate 50% more ROS with the NAD-dependent substrates than RBM, but

required 70% more calcium in order to undergo mPT. Quite unexpected was the insensitivity of MLM to t-

BOOH, which dramatically decreases the amount of Ca2+ that cause mPT in RLM. Evidently, other strains

of mice and rats may have different parameters of intrinsic mitochondrial functions. Further quantitative

studies may help to reveal the subtle functional mechanisms that determine the animal’s sensitivity to a

pathogenic factor when modeling a neurodegenerative disease.

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ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health NIEHS Grant 12068 and a Grant from the

Picower foundation.

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FIGURE LEGENDS

Figure 1. Changes in optical density, membrane potential and medium pH during titration of mitochondria

with calcium. (A) Rat liver mitochondria. (B) Mouse liver mitochondria. Incubation conditions: Medium B,

final volume 2 ml. Additions: TPP+ was added in 0.5 µM aliquots to achieve final concentration of 1.5 µM,

mitochondria 0.5 mg/ml, Ca2+ was added in 12.5 nmol/ml aliquots, alamethicin 5 µg/ml;. HCl 125 nmol/ml

caused ∆pH of 0.07. See Methods for other experimental details.


with calcium in the presence of cyclosporin A 500 nM, ADP 50 µM and oligomycin 2 µg/mg

mitochondrial protein. (A) Rat liver mitochondria. (B) Mouse liver mitochondria. Additions: Ca2+ was

added in 25 nmol/ml aliquots. Other additions as in Fig. 1.


with calcium. (A) Rat brain mitochondria. (B) Mouse brain mitochondria. Incubation conditions as in

figure 1. Additions: TPP+ was added in 0.5 µM aliquots to achieve final concentration of 2 µM, brain

mitochondria 0.5 mg/ml, Ca2+ was added in 25 nmol/ml aliquots. Other additions as in Fig. 1.


with calcium in the presence of cyclosporin A 500 nM, ADP 50 µM and oligomycin 2 µg/mg

mitochondrial protein. (A) Rat brain mitochondria. (B) Mouse brain mitochondria. Additions: Ca2+ was

added in 50 nmol/ml aliquots. Other additions as in Fig. 1.

Figure 5. Effects of mPTP inhibitors on calcium retention capacity of mitochondria. Open bars - liver

mitochondria. Black bars - brain mitochondria. Incubation conditions as in Figures 1, 2, 3, 4. A. Rat liver

and brain mitochondria. B. Mouse liver and brain mitochondria. (1) Control; (2) ADP (50 µM) +

oligomycin (2 µg/ml); (3) CsA (500 nM); (4) CsA + ADP + oligomycin.

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Asterisks denote a statistically significant difference of changes related to the corresponding controls for

liver or brain: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. The “p” number over the control columns in A

and B shows a statistical comparison between liver and brain mitochondria.

Figure 6. Generation of H2O2 by rat brain and mouse brain mitochondria with glutamate as a substrate.

Incubation conditions: Medium A (see Methods), Glutamate 20 mM + malate 2 mM, 5 µM Amplex Red,

3U HRP, 0.2 mg rat brain mitochondria, volume 1 ml. Additions: Catalase (Roche Diagnostics GmbH)

30,000, in experiments when Antimycin A was present - 130,000 U; Antimycin A 5 µM, Rotenone 5 µM,

Myxothiazol 5 µM, CCCP 0.5 µM

A. Response of H2O2 production by RBM to additions of CCCP and respiratory chain inhibitors. B. A

quantitative comparison of ROS generation by RBM and MBM oxidizing glutamate. Asterisks denote a

statistically significant difference between rat and mouse brain mitochondria: * = p < 0.05, ** = p < 0.01,

*** = p < 0.001.

Figure 7. Generation of H2O2 by rat brain and mouse brain mitochondria with succinate as a substrate (5

mM). Other incubation conditions and additions were as in Fig. 6.

A. Response of H2O2 production by RBM to additions of CCCP and respiratory chain inhibitors. B. A

quantitative comparison of ROS generation by RBM and MBM oxidizing succinate.

Figure 8. Simultaneous measurements of H2O2 generation and mPT registered with membrane potential

and pH method.

Incubation conditions and additions as in figure 3A. A. RBM oxidizing 20 mM glutamate + 2 mM malate.

B. RBM oxidizing succinate 5 mM.

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species and tissue specific relationships between mitochondrial

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