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Pathophysiology 4 (1997) 183 – 190

Respiratory activities of liver mitochondria, isolated from freshwater turtle Chinemys re6esii as an experimental anoxia-tolerant model system, determined by mitochondrial modifiers Hitoshi Hori *, Tatsuya Fujimoto, Hideakira Yokoyama, Ning Pan, Miki Kurosaki, Hideko Nagasawa Department of Biological Science and Technology, Faculty of Engineering, The Uni6ersity of Tokushima, Minamijosanjimacho-2, Tokushima 770, Japan Received 8 October 1996; received in revised form 12 February 1997; accepted 18 March 1997

Abstract We isolated liver mitochondria from the anoxia-tolerant, Chinese three keeled pond freshwater turtle (Chinemys re6esii ). We found that the turtle liver mitochondria (TLM) had slow respiratory activities, which were dependent upon the amounts of complexes in respiratory chain, and H + /O and VH + /VH + leak ratios, induced by proton leakage. All of these values were lower than those of rat liver mitochondria (RLM). When mitochondrial modifiers, such as oligomycin and the uncoupler TX-109, were added to TLM, respiratory substrates such as isocitrate, 2-oxoglutarate, and malate were not significantly different between TLM and RLM. Respiration inhibition experiments showed that the enhanced rate of glutamate-dependent state 4 respiration in RLM was sixteen times faster than that in TLM, and other substrate-dependent state 4 respiration in RLM was eight times faster than that of TLM. We originally identified ubiquinone-10 (UQ-10) in TLM. In UQ-depleted TLM, the synthetic UQ analog idebenone at 0.1 mM restored the respiratory activity to a maximum of 12.8 natom O/min per mg protein, while in native TLM, idebenone at 0.4 mM restored the respiratory activity to a maximum of 26.2 natom O/min per mg protein. These results indicated that the low respiratory activities of TLM are attributed to their low substrate oxidation rate. © 1997 Elsevier Science B.V. Keywords: Mitochondrial modifiers; TX-109; Idebenone; Freshwater turtle Chinemys re6esii; Mitochondrial respiration; Anoxiatolerance

1. Introduction Many invertebrates and some vertebrates can survive long periods of anoxia with minimal injurious effects [1]. The freshwater turtle species Pseudemys and Chrysemys are the premier vertebrate facultative anaerobe. The survival of a facultative anaerobe depends on the ability to capitalize on a combination of at least three strategies [2]: (1) an increased buffering capacity and compensatory ion changes to tolerate high concentrations of anaerobic end products and associated changes * Corresponding author. Tel.: +81 886 569514; fax: + 81 886 569514; e-mail: [email protected] 0928-4680/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 8 - 4 6 8 0 ( 9 7 ) 0 0 0 1 6 - 3

in pH; (2) storage of large reserves of anaerobic substrates; and (3) the ability to undergo coordinated metabolic down-regulation of metabolism. To study strategies of anoxia-tolerance an anoxia-tolerant model with functional homogeneity over several hours is received in which its cellular components, such as small organic molecules and macromolecules, are readily measurable by conventional analytical means such as spectroscopic methods, chromatographic and electrophoretic techniques. Currently, a complete vertebrate model of this type is not available. Turtle brain and heart have been extensively studied as anoxia-tolerant models [3–5]. Recently, Hochachka and coworkers described anoxia-tolerant turtle hepatocytes [2,6–10],

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which is an anoxia-tolerant-cell-model. There are few descriptions of an anoxia-tolerant-organelle-model, which is necessary for detailed bioenergetic studies. There are some reports on the metabolic nature of turtle heart and liver mitochondria [11 – 13]. However, these do not focus upon the respiratory activities in a stoichiometric manner. Thus, the molecular mechanisms responsible for coordinating their adaptation involved in anoxia-tolerance remain unknown. Generally, high anoxia-tolerant allotherms such as turtles have a lower metabolic rate than vertebrates. Recent experimental evidence shows that the inhibitory amino acids such as GABA, glycine and taurine mediate the depressed energy consumption displayed by anoxic tolerant vertebrates [14]. However, the underlying basis for these biochemical changes also remains unknown. We found that the Chinese three-keeled pond freshwater turtle Chinemys re6esii can survive without oxygen at 25°C for 8 h submerged in N2 – CO2 (95:5) bubbled water (data not shown). To study the strategies of anoxia-tolerance in the turtle at the molecular level, we used mitochondria, because they are implicated as important factors in the pathogenesis of irreversible ischemic or anoxic cell damage [15] and they are recognition organelles of cell stress [16]. We describe here the characteristics of respiratory activities of liver mitochondria, isolated from the Chinese three-keeled pond freshwater turtle Chinemys re6esii, determined by mitochondrial modifiers in comparison with those from rat liver mitochondria (RLM). We believe that a full understanding of the mechanisms by which anoxia-tolerant species cope without oxygen will be useful for providing alternative strategies for therapeutic intervention under conditions of hypoxia, asphyxia, cardiac arrest and stroke in humans.

ums. Wistar male rats, weighing 150–250 g, were purchased from Japan SLC (Shizuoka, Japan).

2.2. Li6er mitochondria 2.2.1. Isolation of turtle li6er mitochondria A male Chinese three-keeled pond freshwater turtle Chinemys re6esii, weighing 300–400 g, was killed by a subcutaneous injection of pentobarbital sodium (200 mg/kg body weight). After 30 min, the plastron was removed with a bone saw. Livers were rapidly excised, placed in 20 ml ice-cold isolation medium, and minced with surgical steel scissors. The isolation medium consisted of 220 mM mannitol, 70 mM sucrose, 2 mM HEPES (pH 7.4). The solution was decanted to remove blood and free lipid. Tissues were re-suspended in 10 volumes of ice-cold isolation medium, disrupted in homogenizer with a loosely fitting Teflon pestle in a glass vessel and centrifuged for 10 min at 600 × g. The pellets were discarded, and the supernatant was centrifuged for 20 min at 5500× g. The pellets were further centrifuged twice for 15 min at 6000× g. The resulting pellets were divided into the three portions, the light and heavy upper-precipitated pellets (ppt. 1), and the lower-precipitated pellet (ppt. 2). The mitochondrial fraction was identified based on the respiratory control ratio (RCR), the specific activity of cytochrome c oxidase and the influence of the respiratory chain using respiratory chain inhibitors and the cytochrome c oxidase reducing agent TMPD. The mitochondrial fraction obtained as the heavy upper-precipitated pellets were re-suspended in isolation medium at a final concentration of 24 mg mitochondrial protein/ml. The amount of mitochondrial protein was determined by the biuret method [18] using BSA as a standard.

2. Materials and methods

2.1. Materials The uncoupler of oxidative phosphorylation, TX-109 was synthesized in our laboratory (Fig. 1) [17]. Idebenone (Fig. 1) was a gift from Takeda Chemical Industries Ltd., (Osaka, Japan). Ubiquinone-10 (UQ10), ubiquinone-9 (UQ-9), N,N,N%,N%-tetramethyl-pphenylenediamine dihydrocloride (TMPD), ADP, ATP, rotenone, oligomycin, antimycin A, EDTA, and BSA were purchased from Sigma Chemical Co., (St. Louis, MO). HEPES, NADH, cytochrome c from horse heart, ascorbate, KCN and all other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Male Chinese three-keeled pond freshwater turtle Chinemys re6esii, weighing 330 – 420 g, were purchased from TSUTSUMI-TANSUI (Tokushima, Japan) and maintained indoors at 15 – 20°C in aquari-

Fig. 1. Chemical structures of mitochondrial modifiers. Uncoupler TX-109 and UQ analog idebenone.

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2.2.2. Rat li6er mitochondria Mitochondria were isolated from the liver of Wistar rats as described [19]. The mitochondrial fraction was suspended in isolation medium at a final concentration of 1 –2 mg mitochondrial protein/ml. The amount of mitochondrial protein was determined using the biuret method [18] with BSA as a standard. 2.2.3. Ubiquinone-depleted mitochondria Ubiquinone-depleted mitochondria from turtle liver mitochondria (TLM) and rat liver mitochondria (RLM) were extracted from TLM and RLM with acetone, as described Morimoto and Imada [20]. 2.2.4. Identification of ubiquinone [21–23] TLM suspension was lyophilized and disrupted with n-hexane (20 ml per 50 mg protein/ml) in a Teflon homogenizer. The extract was filtered and evaporated under nitrogen. The residue was dissolved in 2 ml of n-hexane and mixed with 8 ml of methanol, then extracted three times with 4 ml of n-hexane. The combined extracts were extracted twice with 2 ml of methanol and evaporated to dryness under nitrogen. The amount of UQ of TLM (TLM-UQ) in the original pentane extract was determined by UV spectrophotometry at 275 nm (o:12.2 mM − 1 cm − 1). The ethanol solution of UQ was passed through a SEP-PAC-C18 (octadecylsilane; ODS cartridge, MILLIPORE) and collected by HPLC (ODS, 5 mm, 4.6× 150 mm; MeOH: H2O = 99.5:0.5) using authentic UQ-10 and UQ-9 as the internal references. The eluate was evaporated under nitrogen and the residue was stored at −80°C. The residue was identified by fast atom bombardment mass (FAB-MS) spectroscopy and its high resolution FAB-MS (FAB-HR-MS) spectroscopy (JOEL JMS-SX 102A instrument, Tokyo, Japan). 2.3. Respiratory acti6ities of TLM and RLM Oxygen uptake was monitored polarographically with a Clark-type oxygen electrode (Yellow Spring, YSI 5331) at 25°C. The incubation medium consisted of 200 mM sucrose, 1 mM EDTA and 2 mM MgCl2 in 10 mM potassium phosphate buffer (pH 7.2, 25°C). Mitochondria were added at 1 mg protein/ml in a total volume of 2.5 ml. Succinate at 10 mM was the respiratory substrate. The RCR (Respiratory control ratio= ratio of state 3 respiration rate to state 4 respiration rate) was calculated by comparing the states 3 and 4 respiration rates. The ADP/O ratio was calculated as the ratio of the added amount of ADP to the total oxygen consumption during state 3 respiration. The H + /O ratio [24] was calculated from the extrapolated of H + ejected (nmol) and the amount of oxygen added. VH + , the amount of H + ejected for an equivalent of oxygen atoms consumed at the state 4 respiration, is given by

Fig. 2. Schematic isolation procedure for TLM.

VH + = (H + /O)× Vs4 (dimensions: nmol H + /min per mg protein), where Vs4 is the state 4 respiratory rate (natom O/min per mg protein). VH + leak (nmol H + /min per mg protein) is expressed as the rate of H + leak during the state 4 respiration.

2.4. Specific acti6ity of complex IV, cytochrome c oxidase, in TLM [25] Cytochrome c oxidase specific activity was measured by following the oxidation of cytochrome c (II) at 550 nm (o: 19.1 mM − 1 cm − 1) with 580 nm as the reference wavelength. All assays are performed at 25°C in a final volume of 1 ml. Cytochrome c (II) at 300 mM was added to assay medium consisting of 0.1 mM potassium phosphate (pH 7.0 at 20°C) and 1 mM EDTA, and the nonenzymatic rate was recorded. Mitochondria at 0.38–1.92 mg of protein were added, and complex IV activity was measured as the initial rate. Cytochrome c (II) was prepared immediately before use by reducing cytochrome c (III) with ascorbate following purification by Sephadex G-25 chromatography.

3. Results We isolated TLM from the Chinese three-keeled pond freshwater turtle (Chinemys re6esii ) by the modified isolation of RLM as shown in Fig. 2. We confirmed that this turtle survived without oxygen at 25°C for 8 h submerged in water bubbled with N2 –CO2 but responded slowly to exogenous action (data not shown). The quality of fresh TLM was estimated as

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respiratory activities such as RCR, using complex IV (cytochrome c oxidase) as the mitochondrial marker enzyme, and the activities of respiratory chain enzymes. As shown in Fig. 3, the heavy fraction consisted of good quality mitochondria. Thus, Fig. 3(a, b) shows that ppt. 1 contained a high concentration of mitochondria because of the high specific activity (89× 103 U/ mg) of complex IV, cytochrome c oxidase, and a higher RCR (RCR=1.5) among the fractions separated. Thus, ppt. 1 was separated into light and heavy fractions. The quality of these two fractions was estimated by comparing respiratory activities using several mitochondrial modifiers such as rotenone, ADP,

Fig. 3. (a) Cytochrome c oxidase activity and RCR of TLM fractions. (b) Respiration rates of heavy and light fractions of TLM by mitochondrial modifiers.

oligomycin, TX-109 (an uncoupler of oxidative phosphorylation as mitochondrial modifier [17]), malonate, ascorbate and TMPD. The heavy fraction contained higher-quality mitochondria than the light fraction. We therefore used the heavy fraction of ppt. 1 as TLM for our study. The respiratory activity of TLM was evaluated by RCR, ADP/O ratio, H + /O ratio, proton ejection rate VH + , and VH + leak and compared with those of RLM, as shown in Table 1. The ADP/O ratio of TLM was markedly lower (ADP/O : 1) than that of RLM (ADP/ O= 2–3). The RCR of TLM was also lower than that of RLM regarding substrates other than malate. The ADP/O ratio of TLM corrected by subtracting the oxygen consumed for proton leak (ADP/O%) were comparable to that of RLM. In TLM, both the low H + /O and the low VH + /VH + leak ratios can be explained qualitatively by proton leakage. Citric acid cycle enzymes in TLM were determined by monitoring changes in the respiration rate using citric acid cycle intermediates and mitochondrial inhibitors and comparing them with those of RLM. As shown in Fig. 4(a, b), each citric acid cycle intermediate initiated state 4 respiration. ADP then stimulated state 3 respiration, oligomycin induced the state 4, and the inhibited respiration was released by the uncoupler TX-109. The succinate dehydrogenase inhibitor, malate, decreased or stopped the respiration. The effects of all substrates tested were essentially the same upon which TLM- and RLM-respiration except for pyruvate and isocitrate, had different profiles in the presence of either oligomycin or TX-109 on TLM. As shown in Table 2, the reaction of TLM with all the complex I–V inhibitors as well as RLM indicated the participation of all complexes (complex I–V) in TLM. From the ratio of ICmax,RLM to ICmax,TLM for the inhibitors, we estimated the stoichiometric ratio (index) of the complex between TLM and RLM to be 0.5 for rotenone, 1 for malonate, 0.25 for TTFA (thenoyltrifluoroacetone), 0.25 for antimycin, 0.5 for KCN, and 1 for oligomycin. From this stoichiometric ratio, the calculated rate of succinate-dependant state 4 respiration in RLM was estimated to be 16 times lower than that in TLM, and the rates of the other substrate-dependent state 4 respiration in RLM were eight times higher than that of TLM. As shown in Table 3, the ratio of rateRLM to rateTLM was 15.42 for succinate, 8.085 for pyruvate and 9.568 for isocitrate. These values were close to those calculated, except for the observed ratios of isocitrate and 2-oxoglutarate. The ubiquinone in TLM was extracted and HPLC and FAB-HR-MS identified it as ubiquinone 10 (UQ10). The estimated amount of UQ in TLM was 1.58 nmol/mg TLM protein, compared with amount of 0.93 nmol/mg RLM protein of UQ-9 in RLM. To check the contribution of UQ to the low respiratory activity of

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Table 1 Respiratory activities of RLM and TLM Substrate

Vs4

RCR

ADP/O

ADP/O%

H+/O

2.640 3.450 1.663 1.477 4.057

1.599 4.227 2.898 1.126 1.071

3.148 9.599 2.060 3.723 0.649

5.574 1.407 0.870 0.635 0.911

0.565 6.822 2.368 5.863 0.712

4.460 8.227 3.214 3.061 4.065

42.109 111.39 87.034 184.54 26.42

16.622 22.864 12.056 8.923 10.760

2.533 4.872 7.2191 20.681 2.455

TLM Pyruvate Isocitrate 2-Oxoglutarate Succinate Malate

1.969 2.271 0.711 3.305 0.606

1.795 1.444 1.390 1.680 1.545

0.491 1.182 0.527 0.818 0.509

RLM Pyruvate Isocitrate 2-Oxoglutarate Succinate Malate

9.629 13.539 27.079 62.281 6.499

3.122 2.755 3.024 1.690 1.496

2.215 2.144 2.362 1.851 2.917

VH+

VH+leak

UNCVH+/VH+leak

Vs4, state 4 respiration rate (natom O/min per mg protein); RCR, respiratory control ratio; ADP/O, the number of molecules of ADP phosphorylated to ATP when two electrons are transferred from a substrate through a respiratory chain to reduce one ‘O’; ADP/O%, ADP/O corrected by substrating the oxygen consumed for proton leak; H+/O, the number ot protons translocated by a respiratory chain during the passage of two electrons from substrate to oxygen; VH+, proton flux rate (Vs4×H+/O, nmol H+/min per mg protein); VH+leak, proton leak rate (nmol H+/min per mg protein).

TLM, we examined the effects of the synthetic UQ analog, idebenone, to the ubiquinone-depleted (acetone-extracted) TLM. The addition of idebenone in ubiquinone-depleted TLM restored the respiratory activity of TLM to reach the maximum of 12.8 natom O/min per mg protein at a concentration of 0.1 mM, in comparison with the maximum of 26.2 natom O/min

Fig. 4. Changes in respiration rates of TLM and RLM by TCA cycle intermediates and mitochondrial modifiers.

per mg protein at 0.4 mM idebenone in native TLM, as shown in Fig. 5.

4. Discussion To understand the anoxic tolerance of turtles at the molecular level, we investigated the mitochondrial anoxia-tolerance that is implicated as an important factor in the pathogenesis of irreversible ischemic (anoxic or hypoxic) cell damage. We first established a means of isolating liver mitochondria from the Chinese three-keeled pond freshwater turtle Chinemys re6esii. These TLM were of good quality based on their respiratory activities such as RCR and complex IV activity (cytochrome c oxidase), as shown in Fig. 3(a). RCR is responsible for the potency of ATP synthesis. We selected complex IV (cytochrome c oxidase) activity, because it is the terminal respiratory enzyme, which is responsible for oxygen consumption and relatively simple to measure. There were differences between the RCR of liver mitochondria of the Chinese three-keeled pond freshwater turtle Chinemys re6esii and that of the turtle Chrysemys picta bellii reported by Almeida-Val et al. [13]. The low H + /O ratio on TLM, which is not consistent with the chemosmotic hypothesis, suggests proton leakage during the state 3, as well as state 4 respiration, as shown in Table 1. Among all the substrates on both TLM and RLM, the highest VH + NH + leak ratio for succinate was due to the high rate of state 4 respiration caused by the high stoichiometric amount of complex II. However, the VH + /VH + leak ratio on TLM, as a whole, was two-fold lower than that of RLM, because of the lower rate of state 4 respiration on TLM.

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Table 2 Inhibition of respiratory-chain complex inhibitors on RLM and TLM respiration Respiratory-chain complex

Inhibitor

Complex I Complex II

Rotenone (nM) Malonate (mM) TTFA (mM) Antimycin A (ng/ml) KCN (mM) Oligomycin (ng/ml)

Complex III Complex IV Complex V

RLM

TLM

Index

IC50

Cmax

IC50

Cmax

57.5 0.416 2.16 11.5 3.6 55.8

340 16.0 56.0 22.0 84.4 218

18.8 0.25 12.5 4.0 2.25 63.9

200 16.6 13.8 10.0 40.0 240

0.5 1 0.25 0.5 0.5 1

Values are means (n =3) in concentrations/mg mitochondrial protein. Cmax, concentration of maximum inhibition. Index, Cmax (TLM)/Cmax (RLM).

Although there was a similar preference for citric acid cycle intermediates, TLM oxidized all citric acid cycle intermediates at a rate 50 times slower than RLM. Among citric acid cycle intermediates succinate was oxidized at the highest rate in TLM. One notable difference between TLM and RLM was that adding oligomycin during state 3 respiration induced TLM to use citric acid cycle intermediates such as pyruvate and isocitrate at higher respiratory rates, while in RLM, the respiration decreased for most of the citric acid cycle intermediates. This is probably due to the continuous proton leak [26] during state 3 respiration. The proton leak seemed to be caused by oligomycin inhibiting complex IV, followed by symport with pyruvate and isocitrate to increase the respiratory rate on TLM. RLM metabolized all of the citric acid cycle intermediates except malate at a higher rate, when the uncoupler TX-109 was added after the oligomycin stimulated transfer from state 3, to state 4 respiration. In contrast, TLM metabolized 2-oxoglutarate and succinate and pyruvate and isocitrate at significantly higher rate. TLM metabolized malate at a similar rate to that of RLM under the condition described. A possible cause of the lower uncoupler stimulated respiration rate by TLM than RLM, is that proton gradient affected TLM more than RLM. From the stoichiometric ratios (index) shown in Table 2 (0.5 for complex I, 0.25 for complex II, 0.5 for complex III, 0.5 for complex IV, and 1 for complex V),

we calculated that the state 4 respiration rate of RLM was 16 times lower than that of TLM with FADH-generating-substrate succinate and eight times higher than that of TLM with other NADH-generating substrates. As shown in Table 3, the observed ratio of RLM state 4 respiration rate to that of TLM was similar. If both complexes have similar structures, these ratios would correspond to the stoichiometric ratios of RLM to TLM complexes. If these structures are not similar, these ratios correspond to the stoichiometric ratio of the number of reaction sites of each complex among them. We discovered that the UQ involved in TLM is UQ-10 [27,28]. We also estimated that the amount of UQ-10 in TLM was 1.58090.868 nmol/mg mitochondrial protein, and that of UQ-9 in RLM was 0.9339 0.067 nmol/mg mitochondrial protein. We calculated that the total amount of UQs, such as UQ-9, 7, 8, and 10, in RLM from the data reported by Battino et al. [28], was 1.661 9 0.119 nmol/mg mitochondrial protein, which is similar to the amount to TLM. However, the published data describing the ubiquinone concentration in tissues are not always easily compared because of

Table 3 Comparison of the rates of state 4 respiration of RLM and TLM Substrate

RateRLM

RateTLM

RateRLM/RateTLM

Pyruvate Isocitrate 2-Oxoglutarate Succinate Malate

15.92 21.73 16.72 50.95 13.00

1.969 2.271 0.711 3.305 0.606

8.085 9.568 23.52 15.42 21.45

Rate, natom O/min per mg protein.

Fig. 5. Effect of idebenone upon respiration rates of TLM with or without acetone.

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different experimental conditions. Further efforts in improving the means of measuring the ubiquinone levels are required. We examined the extent to which UQ-10 contributes to the low respiratory activities of TLM, using idebenone as a synthetic UQ-10 analog in ubiquinonedepleted TLM. The succinate oxidation in the UQ-10 depleted TLM was restored by idebenone. These findings are consistent with those of Imada [29], obtained in UQ-depleted canine brain mitochondria, except for the low restoration of maximum respiratory rate (12.8 natom O/min per mg protein) in UQ-depleted TLM. This failure of idebenone to stimulate the maximum respiratory rate in UQ-depleted TLM may be related mainly to the following. Because idebenone is less lipophilic than UQ-10, it cannot permeate the lipophilic region in the inner mitochondrial membrane sufficiently to transfer electrons. If an excess of idebenone is added, it might disturb the inner mitochondrial membrane. ln fact, an idebenone concentration of 0.4 mM or above weakened the respiratory rate. In summary, we showed that the respiratory function of liver mitochondria from the Chinese three-keeled pond freshwater turtle Chinemys re6esii is unlike that of RLM, as it was low stoichiometric amounts of respiratory chain enzymes. We postulate that the low respiratory activities of TLM, attributed to the low substrate oxidation rate, are significant for their high anoxic tolerance. Furthermore, it is of interest that the increased respiration throughout the evolution of mammals is not associated with major changes in the composition of mitochondria, especially of the enzymes involved in the respiratory chain.

Acknowledgements The authors thank Mrs M. Nakamura, Mrs E. Okayama, Mrs K. Yamashita of our faculty, Dr H. Satake, Center for Corporative Research at our university, and Dr H. Terada, Dr Y. Shinohara and Mrs Y. Yoshioka of the Faculty of Pharmaceutical Sciences at our university, for spectral measurements. The authors also thank Dr H. Terada for helpful discussions. We also thank Takeda Chemical Industries Ltd. for idebenone. This work was supported by a Grant-in-Aid for Scientific Research (No. 02671001) from the Ministry of Education, Science and Culture of Japan.

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