(Received for publication, April 4, 1997, and in revised form, June 6, 1997)
From the Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275
Rates of mitochondrial superoxide anion radical
(O2) generation are known to be inversely correlated with the
maximum life span potential of different mammalian species. The
objective of this study was to understand the possible mechanism(s)
underlying such variations in the rate of O
2 generation. The
hypothesis that the relative amounts of the ubiquinones or coenzyme Q
(CoQ) homologues, CoQ9 and CoQ10, are
related with the rate of O
2 generation was tested. A
comparison of nine different mammalian species, namely mouse, rat,
guinea pig, rabbit, pig, goat, sheep, cow, and horse, which vary from
3.5 to 46 years in their maximum longevity, indicated that the rate of
O
2 generation in cardiac submitochondrial particles (SMPs) was
directly related to the relative amount of CoQ9 and
inversely related to the amount of CoQ10, extractable from
their cardiac mitochondria. To directly test the relationship between
CoQ homologues and the rate of O
2 generation, rat heart SMPs,
naturally containing mainly CoQ9 and cow heart SMPs, with high natural CoQ10 content, were chosen for
depletion/reconstitution experiments. Repeated extractions of rat heart
SMPs with pentane exponentially depleted both CoQ homologues while the
corresponding rates of O
2 generation and oxygen consumption
were lowered linearly. Reconstitution of both rat and cow heart SMPs
with different amounts of CoQ9 or CoQ10 caused
an initial increase in the rates of O
2 generation, followed by
a plateau at high concentrations. Within the physiological range of CoQ
concentrations, there were no differences in the rates of O
2
generation between SMPs reconstituted with CoQ9 or
CoQ10. Only at concentrations that were considerably higher than the physiological level, the SMPs reconstituted with
CoQ9 exhibited higher rates of O
2 generation than
those obtained with CoQ10. These in vitro
findings do not support the hypothesis that differences in the
distribution of CoQ homologues are responsible for the variations in
the rates of mitochondrial O
2 generation in different
mammalian species.
A current hypothesis of aging postulates that oxidative
stress/damage is a major causal factor in the attrition of functional capacity occurring during the aging process (1-6). The basic tenet of
this hypothesis is that there is an intrinsic imbalance between the
reactive oxygen species
(ROS),1 that are incessantly
generated in the aerobic cells and the antioxidative defense against
them, thereby resulting in the accrual of steady-state levels of
oxidative molecular damage. The direct evidence in support of this
hypothesis is that the augmentation of antioxidative defenses by
simultaneous overexpression of Cu/Zn superoxide dismutase, which
converts superoxide anion radicals (O2) into
H2O2, and catalase, which removes
H2O2, retards the age-associated increase in
the levels of molecular oxidative damage and extends the life span of
Drosophila melanogaster by one-third (7, 8).
Although there are several intracellular loci for the generation of
O2 (the first molecule in the ROS series), it is widely accepted that the mitochondrial electron transport chain is the main
source of O
2 (9, 10). Previous studies in this laboratory have
indicated that the rate of mitochondrial O
2 generation varies greatly, even in the same type of tissue, among different mammalian species and is inversely related to the maximum life span potential (MLSP) of the species (11, 12). The inverse relationship between the
rate of O
2 generation and MLSP was found to hold in a sample of mammalian species as well as a group of dipteran insect species (11-13).
The question that arose out of these studies and that is also the
subject of this investigation is what is the underlying mechanism for
the variations in the rates of mitochondrial O2 generation in
different species? Although opinions vary (14), a number of
experimental studies in the literature suggest that ubiquinones
modulate the rate of mitochondrial
O
2/H2O2 generation (10, 15-18).
Ubiquinones (2,3-dimethoxy-5-methyl-6-multiprenyl-1,4-benzoquinone), or
coenzyme Q (CoQ), is a quinone derivative with a chain of 1-12 isoprene units in the different homologue forms (CoQn) occurring in nature. Relatively short-lived mammalian species such as
the mouse and the rat primarily contain CoQ9, whereas the
larger long-lived mammals such as man predominantly exhibit CoQ10 (19). The present study tests the hypothesis that
variations in the rate of O
2 by cardiac submitochondrial
particles (SMPs) in different mammalian species are related to the
relative CoQ9 and/or CoQ10 content. The
hypothesis was prompted by the fact that longevity of non-primate
mammalian species tends to be inversely correlated with the rate of
mitochondrial O
2 generation and directly correlated with the
body mass.
All solvents used were of HPLC grade (Fisher). Ubiquinone-9, ubiquinone-10, (±)-tocopherol and ferricytochrome c (Type Vl), superoxide dismutase, rotenone, antimycin A, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTFA) were purchased from Sigma.
AnimalsHearts were obtained from mouse (Swiss), rat
(Harlan Sprague Dawley), guinea pig (Hartley Albino), rabbit (New
Zealand White), pig (Yorkshire), goat (Angora), sheep (Rambouillet),
cow (Holstein), and horse (mixed) which range from 3.5 to 46 years in
MLSP (21, 29). All the animals were young, healthy, sexually mature
adult males. The approximate ages of the animals were: mouse, rat, and guinea pig, 4 months; rabbit, 7 months; pig, 6-7 months; goat and
sheep, 1 year; cow and horse, 3 years. In smaller animals the entire
heart was processed; however, in the pig, cow, and horse the hearts
were cut into smaller pieces, and representative samples were selected.
The values for the rates of O2 generation in different species
are partially based on the result of previous studies in this
laboratory (12, 22). MLSP values for different species, obtained from
the literature (20, 21), in years, are: mouse, 3.5; rat, 4.5; guinea
pig, 7.5; rabbit 13; goat, 18; sheep, 20; pig, 27; cow, 30; horse,
46.
Mitochondria were isolated by differential centrifugation as described by Arcos et al. (23). Briefly, pieces of the heart were homogenized in 10 volumes (w/v) of isolation buffer containing 180 mM KCl, 0.5% bovine serum albumin, 10 mM MOPS, 10 mM EGTA Tris base, pH 7.2, at 4 °C. The homogenate was centrifuged at 1,000 × g for 10 min, and the supernatant was recentrifuged at 17,500 × g. The resulting mitochondrial pellet was washed and resuspended in 0.25 M sucrose, 1 mM EGTA, 10 mM MOPS, pH 7.2. To prepare SMPs, the mitochondrial pellet was resuspended in 30 mM potassium phosphate buffer, pH 7.0, and sonicated three times, each consisting of a 30-s pulse burst, at 1-min intervals at 4 °C. The sonicated mitochondria were centrifuged at 8,250 × g for 10 min to remove the unbroken organelles; the supernatant was recentrifuged at 80,000 × g for 45 min, and the resulting pellet was washed and resuspended in 0.1 M phosphate buffer, pH 7.4, as described previously (12).
Extraction and Quantitation of Coenzyme QCoQ was extracted
from mitochondria using a hexane:ethanol mixture as described by Takada
et al. (24). Briefly, 50 µl of mitochondrial suspension,
containing ~100 µg of protein and 50 µl of double-distilled
H2O were mixed with 750 µl of hexane:ethanol (5:2) for 1 min using a vortex mixture. The mixture was centrifuged for 3 min at
1,200 × g, and 450 µl of the hexane layer was
collected, dried under helium, and dissolved in 100 µl of ethanol.
Quantitation of ubiquinones was performed by HPLC by the method of
Katayama et al. (25). The ethanol extract (10-20 µl) was
chromatographed on a reverse phase C18 HPLC column
(25.0 × 0.46 cm, 5 µm, Supelco), using a mobile phase
consisting of 0.7% NaClO4 in ethanol:methanol:70% HClO4 (900:100:1) at a flow rate of 1.2 ml. The
electrochemical and UV detectors consisted of an ESA Coulochem II and a
Waters Associates Model 440 absorbance detector at a wavelength of 280 nm. The setting of the electrochemical detector was as follows: guard
cell (upstream of the injector) at +200 mV, conditioning cell at 550
mV (downstream of the column), followed by the analytical cell at +150
mV. The concentrations of ubiquinones were estimated by comparison of
the peak area with those of standard solutions of known
concentration.
Submitochondrial
particles were depleted of native CoQ by pentane extraction, as
described by Maguire et al. (26) and selectively repleted
with exogenous CoQ9 or CoQ10. Aliquots of SMPs
(100 µl containing ~250 µg of protein) were freeze-dried and
extracted 6 times, each for 45 min at 4 °C, with 1 ml of pentane
containing 15 µM -tocopherol (for the last extraction
pure pentane was used). The pentane layer was removed by centrifugation
and discarded. In some cases the pentane layer was collected and
brought to dryness under a stream of helium and resuspended in 100 µl
of ethanol, and the CoQ content was measured by HPLC. Various amounts
of CoQ9 or CoQ10 were added to
pentane-extracted and/or freeze-dried SMPs, dried under helium,
resuspended in 100 mM potassium phosphate buffer (pH 7.4),
and sonicated for up to 3 s in a Branson 2200 sonicator. CoQ
depletion and incorporation of exogenous CoQ9 or CoQ10 into SMPs membranes were confirmed by hexane:ethanol
extraction and HPLC analysis, as described above.
Rate of O2 generation by SMPs was measured
as superoxide dismutase-inhibitable reduction of acetylated
ferricytochrome c (27), as described previously (12). The
reaction mixture contained 10 µM acetylated
ferricytochrome c, 6 µM rotenone, 1.2 µM antimycin A, 100 units of superoxide dismutase/ml (in
the reference cuvette), and 10-100 µg of SMP protein in 100 mM potassium phosphate buffer, pH 7.4. The reaction was
started by adding 7.5 mM succinate, and the reduction of
acetylated ferricytochrome c was followed at 550 nm.
The rate of respiration of submitochondrial particles was measured polarographically using a Clark-type electrode at 37 °C. The incubation mixture, to measure state 4 respiration, consisted of buffer (154 mM KCl, 3 mM MgCl2, 10 mM KPO4, 0.1 mM EGTA, pH 7.4) and 30-100 µg of SMP protein; 7 mM succinate and/or 7 mM NADH were used as substrates, and 1.2 µM antimycin A, 6 µM rotenone, or 0.5 mM TTFA were employed as specific respiratory inhibitors.
Comparisons of the concentrations of CoQ9 and CoQ10 extracted from the heart mitochondria were made in nine different mammalian species, namely mouse, rat, guinea pig, rabbit, pig, goat, sheep, cow, and horse. The data, presented in Table I and Fig. 1, indicate that both the total as well as the relative concentrations of CoQ9 and CoQ10 in heart mitochondria vary greatly in different species. The total concentration of mitochondrial CoQ, i.e. CoQ9 + CoQ10, varied about 2-fold in different species with the rank order: horse > mouse > cow > sheep = goat > rat > pig > rabbit > guinea pig. Although all nine species examined in this study contained both CoQ9 and CoQ10 the ratio of CoQ10/CoQ9 varied > 600-fold. In species such as the mouse and the rat almost 90% of mitochondrial CoQ occurred as CoQ9 while in the guinea pig CoQ9 and CoQ10 were present in roughly equal amounts. In mitochondria from rabbit, pig, goat, sheep, cow, and horse, CoQ10 was the predominant form, with CoQ9 constituting ~1.3 to 4.0% of the total CoQ content.
|
Correlation between CoQ and Superoxide Anion Radical Generation
To determine the relationship between mitochondrial
CoQ content and the rate of O2 generation in different
species, the amounts of CoQ9 and of CoQ10 were
plotted against the average rates of O
2 generation by SMPs,
partially determined in the context of previous studies (12). As shown
in Fig. 1A, the amount of
CoQ9 was directly correlated and that of CoQ10
was inversely correlated (Fig. 1B) with the rate of
O
2 generation in different species. There was no correlation
between the total amount of CoQ content and the rate of O
2
generation (data not presented).
The results of the correlational study, presented in Fig. 1, led to the
question of whether the relationship between mitochondrial CoQ
homologues and the rate of O2 generation was purely
coincidental or causally related. To investigate the possible existence
of a causal relationship, SMPs were experimentally depleted of native CoQ and reconstituted with either CoQ9 or
CoQ10. These studies were conducted on cardiac SMPs of rat,
a short-lived species containing relatively high amounts of
CoQ9, and of cow, a representative of the long-lived
species containing relatively high CoQ10 content.
Repeated extractions with pentane were found to exponentially deplete the amount of native CoQ9 from the rat heart SMPs (Fig. 2, inset); the amount remaining after six serial extractions was about 4.5% of the total amount extractable by hexane. In contrast, apparently due to the much lower natural content of CoQ10, only three extractions with pentane were sufficient to deplete SMPs of CoQ10 to a level below the detection threshold of 0.2 µM (i.e. 0.015 nmol/mg of SMP protein).
To determine the effect of pentane extractions on the functional state
of the SMPs, rates of oxygen consumption and O2 generation were determined after each extraction procedure. The rate of
succinate-supplemented oxygen consumption was highest in the
unextracted SMPs, decreasing linearly following each extraction
procedure, reaching 25% of the initial value after seven successive
extraction procedures (Fig. 2). Addition of antimycin A and TTFA
greatly reduced (to <2%) the rate of oxygen consumption by the
depleted SMPs, whereas rotenone had no effect, indicating that
O2 consumption observed was specifically due to succinate
oxidase activity. NADH did not, in most instances, stimulate the rate
of oxygen consumption by the depleted SMPs.
A similar study was conducted on the effect of various pentane
extractions on the rate of O2 generation by the SMPs. Again, the rate of O
2 generation was highest in the unextracted SMPs and progressively declined with each sequential pentane extraction, reaching 45% of the control value after six extraction procedures, where less than 5% of the original CoQ was present (Fig.
3).
Overall the results of the depletion experiments indicated that even
after six or seven serial extractions with pentane the SMPs exhibited
succinate oxidase activity and were able to generate O2 albeit
at rates lower than the unprocessed SMPs.
Rat heart SMPs that had been extracted with pentane
six times, as described above, were reconstituted with different
amounts of CoQ9 and CoQ10. Reconstitution with
increasing amounts of CoQ9 or CoQ10 caused an
initial steep increase in the succinate-supplemented rate of oxygen
consumption, which was followed by a plateau (Fig. 4B, inset). No
significant differences in the rates of oxygen consumption were
observed between the SMPs reconstituted with equal amounts of
CoQ9 or CoQ10.
Reconstitution of the depleted rat heart SMPs with increasing amounts
of CoQ9 resulted in an initial sharp rise in the rate of
O2 generation, followed by a more gradual increase (Fig.
4A, inset). At the highest concentration of
repleted CoQ9 used in this study, the rate of O
2
generation increased about 2-fold as compared with the depleted SMPs.
Addition of increasing amounts of CoQ10 to the depleted
SMPs also caused an initial steep rise in the rate of O
2
generation, but unlike CoQ9 further additions did not
result in correspondingly increased rates of O
2
generation (Fig. 4A). For example, rat heart SMPs
reconstituted with 50 nmol of CoQ9 exhibited a rate of
O
2 generation that was 40% higher than when an equal amount
of CoQ10 was used for reconstitution. The rate of
succinate-supplemented oxygen consumption and O
2 generation by
SMPs, reconstituted with different amounts of CoQ9 or
CoQ10, were virtually undetectable following the addition
of TTFA, demonstrating the involvement of electron transport chain and
succinate oxidase activity in these two functions. It should however be
noted that up to and within the physiological range of CoQ
concentrations there were no differences in the rates of O
2
generation between SMPs reconstituted with CoQ9 or
CoQ10 (Fig. 4A, inset). The
differences between CoQ9- and
CoQ10-reconstituted SMPs emerged only at concentrations
considerably greater than the in vivo level (Table I).
In contrast to the rat,
bovine cardiac SMPs contain a relatively high amount of
CoQ10 and a small amount of CoQ9 (see Table I).
Depletion of bovine SMPs by six serial extractions with pentane achieved a 96% extraction of CoQ10 and virtually the
entire amounts of the detectable CoQ9. Reconstitution of
these depleted SMPs with varying concentrations of CoQ9 or
CoQ10 indicated different patterns for the two homologues
for the rate of oxygen consumption and of O2 generation.
Augmentation of SMPs with relatively low amounts of CoQ9 or
CoQ10 caused a sharp increase in both the rate of oxygen
consumption (Fig. 5B,
inset) and O
2 generation (Fig. 5A,
inset), but at higher concentrations these rates leveled
off. Within the physiological range of CoQ content (Table I), there were no differences in the rates of O
2 generation between SMPs reconstituted with CoQ9 or CoQ10 (Fig.
5A, inset). However, the maximal rates of
O
2 generation were greater for CoQ9 than for CoQ10. For example, in the bovine SMPs, reconstituted with
50 nmol of CoQ9, the rate of O
2 generation was
35% greater than in the SMPs reconstituted with an equal amount of
CoQ10 (Fig. 5A).
To further determine whether CoQ9 and CoQ10
content above the in vivo level had a different effect on
the rate of O2 generation, freeze-dried unextracted bovine
SMPs were augmented with CoQ9 or CoQ10.
As shown in Fig. 6, the rates of
O
2 generation were stimulated to a greater extent by the
addition of CoQ9 than CoQ10. The differences in
the rate of O
2 generation tended to increase with the
augmented amounts of CoQ9 or CoQ10 in the
SMPs.
Results of this study indicated that rates of O2
generation in different mammalian species are directly correlated with
the amounts of mitochondrial CoQ9 and are inversely related
to the CoQ10 content. The in vitro
depletion/reconstitution studies do not, however, support the
hypothesis that variations in the relative concentrations of
CoQ9 and CoQ10 are directly involved in the modulation of rates of O
2 generation in mitochondria. Although it is widely believed that the components of the mitochondrial respiratory chain, located within the domains of NADH- and
succinate-cytochrome c reductase, are the main sites of
O
2 generation (9, 10), there are at least two different
schools of thought about whether or not autoxidation of ubisemiquinone
is indeed the actual source of O
2 generation. The view,
implicating ubisemiquinone (15-18), is based on the following
evidence: (i) rates of O
2/H2O2
generation by SMPs and/or mitochondria are highest in the presence of
rotenone and antimycin A with succinate as the substrate (9); (ii)
bovine heart SMPs, depleted of endogenous CoQ and reconstituted with variable amounts of exogenous CoQ, exhibit a linear relationship with
the amount of quinone added (15, 16); (iii) rates of O
2
generation by isolated NADH-ubiquinone reductase particles, supplemented with different CoQ homologues, were found to be linearly dependent on the amount of exogenous CoQ (17); and (iv) in
reconstituted SMPs, activities of succinate dehydrogenase and
succinate-cytochrome c reductase reached a plateau at
relative low concentrations of reducible CoQ, whereas the rate of
H2O2 generation was linearly related to a
higher range of CoQ concentration (16). In the opposing view (14), such
observations do not necessarily establish a direct association between
autoxidation of ubisemiquinone and O
2 generation. It is argued
that such findings are also compatible with the interpretation that the
iron-sulfur centers in mitochondrial respiratory complex I (NADH-
ubiquinone reductase), II (succinate-ubiquinone reductase), or III
(ubiquinol-cytochrome c reductase) may be the sources of
O
2 generation. While the results of the present study are
insufficient to resolve this controversy, they however support the view
that CoQ is directly or indirectly associated with the modulation of
the rates of O
2 generation, since experimental variations in
its content have a clear effect on the rate of O
2 generation.
Results of this study should be interpreted in light of the fact that
the preparatory procedures involving freeze-drying and pentane
extractions, although widely employed (15-17, 26, 27), have an
irreversible effect on the functional state of SMPs. For example,
freeze-drying of SMPs followed by depletion and reconstitution with the
original (natural) amount of CoQ decreased the rate of O2
generation by about 30% and of oxygen consumption by about 60%.
However, the rates of O
2 generation and oxygen consumption of
pentane-extracted SMPs, reconstituted with relatively high concentrations of CoQ9 or CoQ10, reached the
level comparable to the unextracted freeze-dried preparations. In both
the rat and the bovine heart SMPs there were no differences in the
rates of O
2 generation between SMPs reconstituted with the
in vivo amounts of CoQ9 or CoQ10.
Only at concentrations higher than those present under physiological
conditions relatively higher rates of O
2 generation were
obtained with CoQ9 than with CoQ10. The same
tendency was observed when unextracted bovine SMPs were augmented with
CoQ9 or CoQ10. Altogether results of
depletion/reconstitution and/or augmentation studies suggest that
CoQ9 and CoQ10 differ in their in
vitro effect on the rate of O
2 generation only at concentrations that exceed the in vivo amounts.
A question arising from this study and also having some bearing on the
interpretation of the present results concerns the nature of the
structural differences between CoQ9 and CoQ10
molecules that apparently exert an effect on the rate of O2
generation, albeit at high concentrations. Indeed, the molecular
structural differences between CoQ9
(C54H82O4;
Mr 794; melting point 44-45 °C) and
CoQ1O (C59H90O4;
Mr 862; melting point 49 °C) (28) are
relatively minor. Nevertheless, the relative length of the
polyisoprenoid chain and the resultant effects on the hydrophobicity of
the molecule have been shown to have an effect on the location of the
molecule within the phospholipid bilayer of the cell membrane. Although the relative position of CoQ9 and CoQ10 in the
phospholipid bilayer has not been precisely determined, the CoQ
homologues with relatively short polyisoprenoid chains are believed to
lie closer to the surface of the bilayer, whereas the long chained ones
are thought to be nearer to the center of the bilayer (29-32). For
example, studies by Kagan et al. (33) have shown that short
chain ubiquinols are relatively more efficient in inhibiting
Fe2+-ascorbate-induced lipid peroxidation, suggesting that
the polyisoprenoid chain length has an effect on the interaction
between the quinols and the ROS present in the aqueous phase.
Studies by Matsura et al. (34) on rat and guinea pig
hepatocytes also implicate major differences in antioxidant efficiency between the reduced CoQ9 and the reduced CoQ10.
In response to the hydrophilic radical initiator,
2-2-azobis-(2-amidinopropane) dihydrochloride, CoQ9 was
found to be preferentially oxidized as compared with CoQ10
homologue and thus may be more accessible to ROS present in the
surrounding aqueous phase. This mechanism, albeit hypothetical, may
underlie the relatively higher rates of O2 generation in
highly CoQ9-rich SMPs observed in this study.
Functional differences between CoQ9 and CoQ10
have also been reported by Edlund et al. (35), who found
that treatment of mumps virus-infected cultured neurons with
CoQ10 protected the cells from degeneration, whereas no
effects were observed in response to CoQ9 treatment.
Results of the present in vitro studies demonstrate that
CoQ9 and CoQ10 homologues can differentially
affect the rate of O2 generation at high concentrations;
however, the in vivo variations in rates of mitochondrial
O
2 generation among different mammalian species cannot be
explained on the basis of relative CoQ9 or
CoQ10 content.