Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing

drlevebpment

Mechanisms of Ageing and Development

ELSEVIER

84 (1995) 139-150

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing Silvia Barogi”, Alessandra Baracca”, Giovanna Parenti Castelli”, Carla Bovina”, Gabriella Formiggini”, Mario Marchetti”, Giancarlo Solainib, Giorgio Lenaz*” “Diparfinzen/o

di Biothhicu

hScuo/a

Superiow

‘G. Moru-_zi’. di Studi

University

Ukwsituri

of’ Bolognn,

Via lrnerio

e di Perfe-_ionanmlio

48, 40126

S. Anna,

Piss,

Bolognu,

Im!,

Itah

Received 29 March 1995: revision received 20 July 1995; accepted 27 July 1995

Abstract ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slight!y decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF,. It is concluded that no activity changes to be directly ascribed to the catalytic sector F, of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F, occur as a consequence of mtDNA mutations.

Keynor&

Ageing: ATPase; Mitochondria

1. Introduction Mitochondria drial oxidative * Corresponding 0047-6374/95/$09.50

appear to be deeply involved in the ageing process [ 1,2]. Mitochonmetabolism is a major source of highly reactive radicals and other author, Tel.: + 39 51 351201~17: Fax: + 39 51 351217. 10 1995

SSDI 0047-6374(95)01640-L

Elsevier Science Ireland Ltd. All rights reserved

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toxic derivatives of oxygen [3]. The critical involvement of oxygen-radical mediated injury in cell ageing is of particular importance [4]: the target of reactive oxygen derivatives may be either lipids [5], proteins [6], or nucleic acids [7], resulting in serious risks to the function of mitochondria and other cellular structures [8]. A likely target of oxygen-mediated injury is mitochondrial DNA (mtDNA), whose localization in the mitochondrial matrix makes it adjacent to the main sources of oxygen radical generation [3]. It was proposed that accumulation of somatic mutations of mtDNA, induced by long-term exposure to free radical attack, mainly in postmitotic cells, leads to errors in the mtDNA-encoded polypeptide chains belonging to the proton translocating complexes of the mitochondrial inner membrane; the consequence of these alterations would result in defective electron transfer and energy conservation [2]. Unlike nuclear DNA, mutations of mtDNA are able to accumulate due to the intrinsic instability of the mitochondrial genome and lack of protection by proteins and efficient DNA repair mechanisms [91. Structurally, mtDNA is a single molecule of circular DNA encoding for 13 hydrophobic polypeptide chains of the four H + -translocating complexes of the inner mitochondrial membrane [lo]: in particular seven chains (the ND subunits) of Complex I (NADH: Coenzyme Q oxidoreductase), the cytochrome b subunit of Complex III (ubiquinol: cytochrome c oxidoreductase), three subunits (CO1 to III) of Complex IV (cytochrome c oxidase) and two subunits (ATPase 6 and 8) of the integral sector of the H +-ATPase (ATP synthase); moreover it encodes for 22 tRNAs and two rRNAs required for mitochondrial protein synthesis. A number of mtDNA deletions [l 1- 141 and point mutations [15] have been described in tissues from aged animals and humans. The ‘mitochondrial theory of ageing’ would predict that mutations affect Complex I, Complex IV, ATPase, and Complex III in decreasing order, owing to the decreased number and total size of genes affected; nevertheless, the deletions described to date to increase in ageing, as the 5 Kb ‘common’ deletion, usually encompass a specific region of the mitochondrial genome [16] starting at a repeated sequence near position 8 450; their occurrence would therefore specifically affect this region comprising four Complex I genes, one gene for cytochrome oxidase and both the ATPase 6 and ATPase 8 genes for the ATP synthase complex [17]. According to the literature [I 81, the T + G mutation at 8993 mtDNA of human lymphoblasts, occurring in a fairly conserved region of ATPase 6 gene coding sequence and causing a change of a leucine to an arginine residue [19], can induce a consistent decrease of the oxidative phosphorylation efficiency. A high percentage ( > 90%) of heteroplasmy of the 8993 mutation produces a disease phenotype characterized by a neurological deterioration known as Leigh’s disease [20]. Functional changes in the activity of mitochondrial enzymes have been described, but their presence and extent appear to be extremely variable [12,21-241; a ‘mosaic’ loss of cytochrome c oxidase, but not of succinate dehydrogenase, in skeletal and heart muscle mitochondria. as detected by histochemistry [25527], seems to be a consistent bioenergetic change found in ageing, and is compatible with the mitochondrial DNA theory.

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As for ATPase, Guerrieri et al. [28] described a decrease of ATP hydrolytic activity and an increase of passive proton conduction in brain and heart of aged rats; concomitant immunoblot analyses revealed a significantly greater age-dependent decrease of F, subunits than the decrease of the F, subunits. These findings suggest that the changes are independent of mtDNA and may be related to age-dependent altered import of nuclear-encoded F, subunits into the mitochondrion, membrane enzyme assembly and/or turn-over of F, subunits [28,29]. The high sensitivity of F, subunits to oxidative stress in vitro [30] and the preferential inactivation of F, subunits in comparison to F, subunits [31] may also be due to age-dependent changes resulting directly from enzyme protein inactivation by free radical attack. In a previous study in our laboratory on mitochondrial enzymes in rat heart, Castelluccio et al. [22] had failed to detect any significant age-related change of mitochondrial ATPase; we have therefore re-examined the activity of the enzyme in liver, heart and skeletal muscle of adult (6 months) and aged rats (24 months), attempting to find an explanation of the discrepancy between the results of Guerrieri et al. [28,29] and Castelluccio et al. [22] by a thorough kinetic analysis of the activation parameters of the enzyme. Nevertheless the study has again failed to detect any major change, suggesting that ATPase alterations in ageing, when present, are not a required pathogenetic feature. 2. Materials and methods All chemicals used were purchased from Sigma Chemical Co., St. Louis. MO, and all solvents were pure reagents from Merck, Darmstadt, Germany. Two groups of male albino rats of the Wistar strain, aged 6 and 24 months, respectively, purchased from Charles River Italia S.p.A., Milano, were kept for one week under constant environmental conditions [22] and fed a normal laboratory diet; each group was comprised of eight animals. All procedures involving animals were performed according to the ethical guidelines for animal experimentation issued by the University of Bologna. Mitochondria were prepared from liver, heart, and skeletal muscle (gastrocnemius) immediately after killing the animals by decapitation, according to the method of Kun et al. [32] slightly modified avoiding the digitonin treatment. Essentially tissue homogenate (0.22 M Mannitol, 0.07 M Sucrose, 2 mM Tris, 1 mM EDTA and 20 mM Hepes, pH 7.2 at room temperature, containing 0.4% albumin) was centrifuged at 2000 rev./min for 80 s. in a rotor JA20-Beckman (5000 rev./min for 1.5 min., for liver tissue only) to remove the fraction containing nuclei and plasma membrane fragments. The crude nuclear fraction was re-extracted by the same technique and the two supernatants were combined. Then it was centrifuged at 12 000 rev./min for 2.5 min. in a JA20-Beckman rotor to obtain the mitochondrial pellet. Finally it was washed twice in buffer solution (0.22 M Mannitol, 0.07 M Sucrose, 2 mM Tris, 1 mM EDTA and 20 mM Hepes, pH 7.2 at room temperature) to avoid any albumin contamination. Mitochondrial protein was determined by a biuret method [33] with the addition of 10% Na deoxycholate

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and using bovine serum albumin as standard; lipid phosphorus was assayed by the micromethod of Marinetti [34]. To evaluate the microsome contamination of rat liver mitochondrial preparations we have assayed the 5’-nucleotidase activity measuring spectrophotometricaily the inorganic phosphate released from AMP during the reaction [35] as previously described [36]. According to this method 600 ~1 of the reaction mixture contained 10 mM MgCl,, 100 mM Glycine, pH 9.1 and 5 mM AMP (30°C) the reaction was started by addition of 1 mg protein and stopped 10 min later by adding 60 ~1 50% trichloroacetic acid. The specific activity of liver mitochondrial preparations was 0.010 f 0.004 and 0.008 + 0.002 U/mg f S.D. in adult (6 months) and aged (24 months) rats, respectively. The ATPase activity of mitochondria was assayed after four freezing and thawing cycles followed by incubation for 10 min. at a protein concentration of 2 mg/ml in an hypotonic medium (10 mM Tris/CI, pH 7.4). The ATP-hydrolytic activity was measured at room temperature (25°C) with an ATP-regenerating system [22,37] by following the decrease of NADH absorption at 340 nm with a 7850 model Jasco spectrophotometer equipped with a thermostat. The reaction mixture (1 ml) contained 25 pmol Tris/acetate (pH 7.4), 25 pmol KCL, 5 pmol MgCl, 160 nmol NADH, 1.5 pmol phospho(enol)pyruvate, 5 units of lactate dehydrogenase, 3.5 units of pyruvate kinase and 1 pg of rotenone. All assays contained 4 mM ATP, a saturating substrate concentration (steady state), and the reaction was initiated by addition of 20 jig of mitochondrial protein to the medium. The oligomycin sensitivity and the bicarbonate stimulation of the ATPase activity were determined measuring the ATP hydrolytic activity in the presence of 2 ,uM oligomycin or 20 mM NaHCO,, respectively. The ATPase inhibitor protein release was induced according to Rouslin and Broge [38]. Essentially, intact mitochondria (10 mg/ml) were incubated for 10 min at 37°C in an energizing buffer (6 mM Potassium Glutamate, 6 mM Potassium Malate, 2.5 mM Potassium Phosphate, 0.25 M Sucrose, 1 mM EGTA, 10 mM Tris/Cl pH 7.2) and centrifuged (5°C) for 10 min. at 10 000 g; the resulting mitochondrial pellets were resuspended to approximately 2 mg/ml in 0.25 M Sucrose, 1 mM EGTA, 10 mM Tris/Cl, pH 8.2 and exposed to a brief sonication at 5°C. The control sample was similarly processed but in order to avoid the inhibitor protein release, the procedure was performed at low temperature (S’C) using buffers at pH 6.4 in the presence of 2 ,uM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and 2 mM Mg-ATP. The same percentage of enzyme activation, due to the inhibitor protein release, was observed using as samples either sonicated mitochondrial suspensions or submitochondrial particles prepared therefrom according to Lee and Ernster [39]. All data are presented as means f SD.; the significance of differences was evaluated by the unpaired t-test. 3. Resuits The yield of mitochondrial protein was roughly the same comparing each tissue from adult and aged animals.

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The mitochondrial ATPase activities of liver, heart and skeletal muscle of adult (6 months) and aged (24 months) rats are reported in the histograms in Fig. 1. The heart and skeletal muscle enzyme activities are lower in aged rats (82% and 65% of the control, respectively), nevertheless the statistical analysis does not reveal significant changes of the mitochondrial ATPase activity in any tissue investigated with respect to adult rats. Since the activity of mitochondrial ATPase can be modulated by several endogenous and exogenous effecters [40-421 we also assayed the ATPase activity under different experimental conditions. Thus, as a first approach to evaluate whether the ATPase activity of the mitochondrial samples was catalyzed by the coupled F,F,-complex we analyzed the oligomycin sensitivity of the reaction. Fig. 2 shows the results obtained with mitochondria from liver and heart of adult and aged rats: the enzyme from tissues was well coupled, showing an oligomycin sensitivity above 90%, but with a slight statistically significant decrease in heart mitochondria from aged rats (P < 0.05).

Exl6

months 24 months

LIVER

T

HEART

MUSCLE

Fig. I. Mitochondrial ATPase activity of liver, heart and skeletal muscle of adult (6 months) and aged (24 months) rats. The assay was carried out as detailed in materials and methods. The reported values are means of eight different individuals f S.D.

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months

LIVER Fig. 2. Percentage of oligomycin

and Deurlopmenr

sensitive ATPase

HEART activity evaluated

in liver and heart mitochondria

of

adult (6 months) and aged (24 months) rats. The reported values are means of eight different individuals f

S.D.

The decrease of the oligomycin sensitivity of ATPase in aged rat heart mitochondria suggests that partial uncoupling of the F,F, -ATPase complex may mask a more pronounced decrease of activity than that observed in total hydrolytic activity. ATPase is activated by a number of anions [43] apparently because they can increase the rate of ADP product release from the catalytic site, a rate-limiting step of the ATPase reaction in the absence of anion [44,45]. When we assayed the enzyme activity in the presence of 20 mM bicarbonate, which is among the most efficient activating anions, we observed that in heart and skeletal muscle tissues the ATPase activity was similar whether mitochondria were isolated from 6 or 24 months old-rats (Fig. 3). On the contrary, a statistically significant difference (P < 0.05) was observed in liver mitochondria: the ATP hydrolytic activity of adult rat liver mitochondria was nearly 50% higher than in the absence of HCO,, whereas the activity in aged rats was found to be only 32% higher than in the absence of the anion. Finally, we investigated the binding capability of the endogenous inhibitor protein (IF,) to the ATPase complex in mitochondria from adult and aged rats. The IF, protein, which has been implicated in one of the most important regulation

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mechanisms of the ATP synthase activity [46], was released from energized sonicated mitochondria as detailed in materials and methods. The ATPase activity of the mitochondrial samples increased in livers of adult and aged rats by 70% and nearly SO%, respectively, without a statistical difference between the two groups (Fig. 4). Similarly, the release of IF, from heart mitochondria increased by 40% and 43%, respectively, the ATP hydrolytic activity of adult and aged rats. 4. Discussion In principle, all cellular macromolecules are susceptible to the damaging action of oxygen radicals [21,47]: however the mechanism of accumulation of enzymatic defects in mitochondria in ageing organisms would differ depending upon the type of molecule being the primary target of oxidative injury. Accumulation of primary damage to non-information-containing molecules such as enzymic proteins and membrane lipids can be sustained either by a decreased catabolic rate of the target molecules [48] or alternatively by an increased steady 200

r

Es36

months

24 months

T

T /

HEART Fig. 3. Percentage of bicarbonate activation of the mitochondrial ATPase activity in liver, heart, skeletal muscle of adult (6 months) and aged (24 months) rats. The reported values are means of eight different individuals k S.D.

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250

lsso

months 24 months

E 4 200 .r( 3 .rl 3 0 cd a, z 5

150

100

LIVER

HEART

Fig. 4. Percentage of activation of the mitochondrial ATPase activity induced by the inhibitor protein (IF,) release evaluated in liver and heart of adult (6 months) and aged (24 months) rats. The reported values are means of eight different individuals + SD.

state concentration of the damaging species, which could result from an enhanced production or a decreased scavenging activity by antioxidants. On the other hand, accumulation of genetic damage in the form of deletions and/or point mutations, in somatic cells could result from steady-state oxygen radical concentrations, as the accumulating lesions would be maintained during mitochondrial replication. The changes observed in respiratory enzymes accompanying ageing are mostly compatible with a mitochondrial genetic origin, as they appear to affect only those enzymes having subunits encoded by mtDNA [lo] and their severity is related to the number of such subunits [23]. In the contrary, results of studies on ATPase suggest that the situation is more complex; Guerrieri et al. [28] reported that the catalytic sector F,, in which all subunits are encoded by nuclear DNA, is affected in ageing more severely than the membrane sector F,, in which two subunits are encoded by mtDNA [49,50]. On the other hand, Castelluccio et al. 2221 and the present study have been unable to observe any major age-related change of the enzyme.

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The discrepancy between the results by Guerrieri et al. [28,29] and ours may have two alternative bases: some parameters of the enzyme activity may be different in the two studies leading to rate-limiting steps which are differently affected by ageing; alternatively the changes observed are a non-obligated consequence of the senescence process which depends upon additional factors, such as the lipid composition of the membrane or the extent of antioxidant defenses, as we had postulated in the previous publication by Castelluccio et al. [22]. In this study we have modulated enzymic activity by procedures designed to obviate possible rate-limiting steps. Repeated freezing and thawing of the mitochondrial membranes prior to assays abolished permeability barriers to substrate as shown by lack of further stimulation by either detergents or sonic irradiation. It may be argued that a population of damaged mitochondria deficient in various functions is lost during isolation preventing to observe consistent changes. Even if this is a possibility, we judge it rather remote, because the yield of mitochondria was approximately the same in adult and old animals. The small but significant decrease of oligomycin sensitivity in heart mitochondria from aged rats may have counterbalanced and masked a decrease of coupled activity; even if so, the change is so small as to represent a minor effect. The extent of HCO; activation is an important parameter for removing product release as a rate-limiting step of the ATP hydrolytic reaction [44,45]; despite the lower bicarbonate activation in aged mitochondria, this effect was again of minor extent and found significant only in liver mitochondria. The interference of the inhibitor protein seems to represent a more serious problem, and was considered as a possible cause of discrepancy, as the results of Guerrieri et al. [28,29] were obtained in EDTA submitochondrial particles in which the IF, may be significantly reduced. Nonetheless, in our study, IF, removal induces no significant difference in ATPase stimulation in the two groups. The results therefore suggest that ageing does not affect the stability of the ATPase-IF, complex, indicating that changes of the interaction between the enzyme and the inhibitor protein are not responsible for counterbalancing phenomena related to the enzyme activity during aging. Furthermore, IF,, at least in cardiac muscle, is considered to be an endogenous factor contributing to the defense from cell injury in situations of anoxia or ischemia by reducing the possible ATP hydrolytic activity of mitochondria and consequent decrease of the energy charge and the pH of the cell [46,51,52]. Our data indicate a comparable capability of adult and aged animals to contrast the above situations through the mechanism involving the ATPase-IF, system. The involvement of ATPase seems therefore to represent a variable feature during the progression of senescence, at least when investigated as ATP hydrolytic activity, to be mainly referrable to the F, catalytic sector. The degree of damage, interpreted as direct damage to the F, subunits [28,30], may vary depending on factors such as extent of antioxidant power; in addition, when present, the damage may be secondary to ageing and unrelated to the pathogenic events of senescence. It is significant that the same aged rats used in the present study developed severe decreases of Complex I activity in all three tissues investigated (M.L. Genova et al.,

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unpublished observations), indicating that age-related changes in mitochondria were present and detectable and were likely to be ascribed to mtDNA genetic defect. If primary changes in the F, subunits encoded by mtDNA are also present in ageing, they are unable to affect maximal ATPase activity and its gross oligomycin sensitivity. It is not excluded, however that more subtle kinetic changes to be ascribed to the mitochondrially synthesized subunits may be revealed by inhibitor titration of either enzymatic activity or proton conduction or by kinetic analysis of ATP synthesis in coupled membranes. Acknowledgements We thank Dr R.E. Beyer, Dep. of Biology, University of Michigan, Ann Arbor, for critically reading the manuscript. This study was supported by the Target Project on Ageing of C.N.R. Rome (Code No. 951609). References [I] J. Miquel, Geronrol.,

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