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Vol. 59, Issue 6, 1497-1505, June 2001
Departments of Biochemistry (R.H., Z.B., B.S.) and Medical Chemistry (E.O.) and 1st Department of Medicine (K.T.), University of Pécs, Faculty of Medicine, Pécs, Hungary; and N-Gene Research Laboratories, Inc., Budapest, Hungary (P.L.-N.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Ischemia-reperfusion induces reactive oxygen species (ROS) formation, and ROS lead to cardiac dysfunction, in part, via the activation of the nuclear poly(ADP-ribose) polymerase (PARP, called also PARS and ADP-RT). ROS and peroxynitrite induce single-strand DNA break formation and PARP activation, resulting in NAD+ and ATP depletion, which can lead to cell death. Although protection of cardiac muscle by PARP inhibitors can be explained by their attenuating effect on NAD+ and ATP depletion, there are data indicating that PARP inhibitors also protect mitochondria from oxidant-induced injury. Studying cardiac energy metabolism in Langendorff heart perfusion system by 31P NMR, we found that PARP inhibitors (3-aminobenzamide, nicotinamide, BGP-15, and 4-hydroxyquinazoline) improved the recovery of high-energy phosphates (ATP, creatine phosphate) and accelerated the reutilization of inorganic phosphate formed during the ischemic period, showing that PARP inhibitors facilitate the faster and more complete recovery of the energy production. Furthermore, PARP inhibitors significantly decrease the ischemia-reperfusion-induced increase of lipid peroxidation, protein oxidation, single-strand DNA breaks, and the inactivation of respiratory complexes, which indicate a decreased mitochondrial ROS production in the reperfusion period. Surprisingly, PARP inhibitors, but not the chemically similar 3-aminobenzoic acid, prevented the H2O2-induced inactivation of cytochrome oxidase in isolated heart mitochondria, suggesting the presence of an additional mitochondrial target for PARP inhibitors. Therefore, PARP inhibitors, in addition to their important primary effect of decreasing the activity of nuclear PARP and decreasing NAD+ and ATP consumption, reduce ischemia-reperfusion-induced endogenous ROS production and protect the respiratory complexes from ROS induced inactivation, providing an additional mechanism by which they can protect heart from oxidative damages.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reactive
oxygen species (ROS) and peroxynitrite contribute to the ischemia- and
reperfusion-induced cardiac injury (Xie and Wolin, 1996
), and initiate
lipid peroxidation (Pan and Hori, 1994), protein oxidation (Butterfield
et al., 1997), and the formation of single-strand DNA breaks
(Zingarelli et al., 1997; Szabados et al., 1999a). Single-strand DNA
(ssDNA) breaks can activate the nuclear poly(ADP-ribose) polymerase
(PARP, called also PARS and ADP-RT) (Lindahl et al., 1995; Jacobson and
Jacobson, 1999), which ADP-ribosylates different nuclear proteins on
the expense of cleaving NAD+. If PARP activation
exceeds a certain limit it can lead to cellular NAD+ and ATP depletion, causing eventually cell
death (Radons et al., 1994; Gilad et al., 1997; Grupp et al., 1999;
Szabados et al., 1999a). Therefore, the inhibition of PARP can improve
the recovery of different cells from oxidative damages (Mizumoto et
al., 1993; Heller et al., 1995; Said et al., 1996; Thiemermann et al.,
1997).
Mitochondria play a pivotal role in oxidative cell damages because
oxidative damage can cause the release of cytochrome c from
mitochondrial intermembrane space and can induce apoptotic cell death
(Lenaz, 1998; Saikumar et al., 1998), or oxidant can partially
inactivate respiratory complexes (Turrens et al., 1991), resulting in
impaired energy metabolism and significant increase in mitochondrially
produced ROS, which eventually induce cell death (Takeyama et al.,
1993; Lemasters et al., 1998). The ROS-induced mitochondrial
permeability transition can lead to intramitochondrial NAD+ loss and the inhibition of mitochondrial
NAD+-linked substrate oxidation (Said et al.,
1996), further damaging the mitochondrial energy production (Takeyama
et al., 1993; Lemasters et al., 1998). It was reported previously that
PARP activation contributes to mitochondrial injury during
oxidant-induced cell death (Virag et al., 1998), and that a new PARP
inhibitor (BGP-15) decreased the oxidative damage of myocardium after
ischemia-reperfusion (Szabados et al., 2000). These data raise the
possibility that PARP inhibitors may interfere with endogenous
mitochondrial ROS formation by a different mechanism than antioxidants
(Szabados et al., 2000).
In heart tissue, a dominant fraction of energy production occurs in the
mitochondria, therefore protection against oxidative damage of
mitochondria can be very important step in the normalization of cardiac
energy production. In this work, we investigated the effect of PARP
inhibitors and 3-aminobenzoic acid (an inactive chemical analog of the
best studied PARP inhibitor) (Fig. 1) on the energy metabolism of heart during ischemia-reperfusion cycle by
31P NMR spectroscopy. Furthermore, we
investigated the effect of PARP inhibitors on the
ischemia-reperfusion-induced oxidative cardiac damages (lipid
peroxidation, protein oxidation, and ssDNA break formation),
inactivation of respiratory enzymes, and direct effect of PARP
inhibitors on isolated mitochondria to extend our knowledge about the
molecular mechanisms by which PARP inhibitors can protect mitochondria
and cardiomyocytes from ischemia-reperfusion-induced damages.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. 3-Aminobenzamide, 3-aminobenzoic acid, 4-hydroxyquinazoline, nicotinamide, H2O2, NAD+, and dihydrorhodamine123 were purchased from Sigma-Aldrich Chemical Co. (Budapest, Hungary); malondialdehyde-bis(diethylacetal) was obtained from Merck (Darmstadt, Germany). BGP-15 was a gift from N-Gene Research Laboratories, Inc. (Budapest, Hungary). All other reagents were of the highest purity commercially available.
Animals. The hearts of adult male Wistar rats weighing 300 to 350 g were used for Langendorff heart perfusion experiments. All animal experiments were conducted in conformity with the guiding principles in the care and use of animals.
Heart Perfusion.
Rats were anesthetized with 200 mg/kg
ketamine intraperitoneally and heparinized with sodium heparin (100 IU/rat i.p.). Hearts were perfused via the aorta according to the
Langendorff method at a constant pressure of 70 mm Hg, at 37°C as
described before (Szabados et al., 1999a,b). The perfusion medium was a
modified phosphate-free Krebs-Henseleit buffer consisting of 118 mM
NaCl, 5 mM KCl, 1.25 mM CaCl2, 1.2 mM
MgSO4, 25 mM NaHCO3, 11 mM
glucose, and 0.6 mM octanoic acid without or together with PARP
inhibitors [3 mM nicotinamide, 3 mM 3-aminobenzamide, or 40 mg/l
(113.9 µM) BGP-15]. The perfusate was adjusted to pH 7.4 and bubbled
with 95% O2, 5% CO2
through a glass oxygenator. After a washout (nonrecirculating period of
15 min), hearts were either perfused under normoxic conditions for the
given time, or were subjected to 25-min global ischemia by closing the
aortic influx and reperfused for 15 min. During ischemia, hearts were
submerged into perfusion buffer at 37°C. At the end of the perfusion,
hearts were freeze-clamped.
Assay of NAD+.
The concentration of
NAD+ in the neutralized perchloric acid extract
of the cardiac muscle was measured by using alcohol dehydrogenase reaction as described previously (Skuta et al., 1999).
Determination of DNA Single-Strand Breaks.
Single-strand DNA
breaks were determined by the alkaline fluorescence analysis of DNA
unwinding as described previously (Birnboim and Jevcak, 1981). DNA
samples were prepared from normoxic and ischemic perfused hearts. To
estimate the quantity of undamaged double-stranded DNA, samples were
divided into three sets of tubes. DNA fluorescence was determined under
different conditions. To determine F values, DNA was kept at
pH 12.4 to permit partial unwinding of DNA. To determine
Fmin, DNA was kept at pH 12.4, but at the
beginning of the incubation period the DNA sample was sonicated for
60 s. To determine Fmax the DNA sample
was kept at pH 11.0, which is below the pH needed to induce unwinding. Solutions were incubated for 30 min at 0°C followed by 15-min incubation at 15°C. Unwinding was stopped by adjusting the pH to pH
11.0. Fluorescence was measured after the addition of the dye ethidium
bromide (0.67 µg/ml), with an excitation wavelength of 520 nm and an
emission wavelength of 590 nm by a PerkinElmer luminescence
spectrometer. Results are expressed as D (percentage of double-stranded
DNA) = (F 
Fmin)/(Fmax Fmin) × 100.
Lipid Peroxidation.
Lipid peroxidation was estimated from
the formation of thiobarbituric acid reactive substances (TBARS). TBARS
were determined using a modification of a described method (Serbinova
et al., 1992). Cardiac tissue was homogenized in 6.5% trichloroacetic acid and a reagent containing 15% trichloroacetic acid, 0.375% TBA,
and 0.25% HCl was added, mixed thoroughly, heated for 15 min in a
boiling water bath, cooled, centrifuged, and the absorbance of the
supernatant was measured at 535 nm against a blank that contained all
the reagents except the tissue homogenate. Using malondialdehyde
standard, TBARS were calculated as nanomoles per gram of wet tissue.
Determination of Protein Carbonyl Content.
Fifty milligrams
of freeze-clamped perfused heart tissue was homogenized with 1 ml of
4% perchloric acid and the protein content was collected by
centrifugation. The protein carbonyl content was determined by using
the 2,4-dinitrophenylhydrazine method (Butterfield et al., 1997;
Szabados et al., 2000).
Isolation of Mitochondria. The hearts of sacrificed rats were quickly removed and immersed in an ice-cold isolation buffer containing 150 mM KCl, 5 mM Tris, 1 mM EDTA, pH 7.4. Hearts were cut into small pieces, homogenized in a Teflon homogenizer in the isolation buffer, and centrifuged for 10 min at 500g. The supernatant was then centrifugated for 10 min at 18,000g. After removing the supernatant, the pellet was carefully suspended in 1 ml of isolation buffer.
Incubation of Mitochondria with Hydrogen Peroxide and PARP
Inhibitors.
Aliquots of the suspended mitochondria were incubated
in a buffer containing 150 mM KCl, 5 mM 4-morpholinepropanesulfonic acid, 1 mM EDTA, 1 mM succinate, pH 7.4 with 0.5 mM
H2O2 and in the treated
groups with a PARP inhibitor for 15 min at 37°C. To stop the hydrogen
peroxide-induced injury of mitochondria, dithioerythritol (final
concentration 4 mM) was added to the medium. The samples were then
centrifuged for 5 min at 15,000g. The pellet was suspended in 400 µl of isolation buffer and sonicated for 15 s. The
samples were frozen at 80°C.
Measurement of Mitochondrial Enzyme Activity.
Citrate
synthase (Sumegi et al., 1985), NADH:cytochrome c
oxidoreductase (Sumegi et al., 1990), and cytochrome oxidase (Sumegi et
al., 1990) were measured as described previously.
Determination of Mitochondrial ROS Production.
Mitochondria
were incubated in a buffer containing 150 mM KCl, 1 mM EDTA, 5 mM
4-morpholinepropanesulfonic acid, 1 mM succinate, pH 7.4 and in the
presence of PARP inhibitors and 3-aminobenzoic acid (0.2, 1, 3 mM). The
mitochondrial suspension was stirred and the ROS production was
continuously monitored by following the oxidation of
dihydrorhodamine123 to rhodamine123 in a PerkinElmer fluorescence
spectroscope at an excitation wavelength of 496 nm and an emission
wavelength of 536 nm (Szabados et al., 1999).
NMR Spectroscopy. NMR spectra were recorded with a Varian UNITY INOVA 400 WB instrument. 31P measurements (161.90 MHz) of perfused hearts were run at 37°C in a Z·SPEC 20-mm broadband probe (Nalorac Co., Martinez, CA), applying proton decoupling during acquisition. Field homogeneity was adjusted by following the proton signal (w1/2 = 10-15 Hz). Spectra were collected with a time resolution of 3 min by accumulating 120 transients in each free induction decay. Pulses of 45° flip angle were used after a 1.25-s recycle delay, and transients were acquired over a 10-kHz spectral width in 0.25 s, and the acquired data points (5000) were zero-filled to 16,384.
Under the above-described circumstances the relative concentrations of the species can be taken proportional to the peak areas, because interpulse delays exceeded 4 to 5× the T1 values of the metabolites to be analyzed in 31P experiments.Statistical Analysis. Statistical analysis was performed by analysis of variance and all of the data were expressed as the mean ± S.E.M. Significant differences were evaluated by use of unpaired Student`s t test and p values below 0.05 were considered to be significant.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of the PARP Inhibitors on Ischemia-Reperfusion-Induced Lipid
Peroxidation.
Lipid peroxidation induced by ischemia-reperfusion
in Langendorff perfused heart was characterized by the formation of TBA reactive substances. Under our experimental conditions,
ischemia-reperfusion increased the amount of TBA reactive substances
compared with the normoxic conditions (p < 0.01)
(Table 1). In normoxic hearts, PARP
inhibitors did not have significant effects on TBA-reactive substance
formation (Table 1). When ischemia-reperfusion occurred in the
presence of PARP inhibitors, the formation of TBA reactive substances
was significantly lower than in the hearts subjected to
ischemia-reperfusion without PARP inhibitors (Table 1), indicating that
PARP inhibitors prevented the ischemia-reperfusion-induced lipid
peroxidation. However, 3-aminobenzoic acid, an inactive structural
analog of the PARP inhibitor 3-aminobenzamide, could not prevent the
ischemia-reperfusion-induced lipid peroxidation (Table 1).
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Effect of PARP Inhibitors on Ischemia-Reperfusion-Induced Protein
Oxidation.
ROS formation in ischemia-reperfusion cycle can induce
the oxidation of proteins in the cardiomyocytes, which can be
characterized by the quantity of protein-bound aldehyde groups
(Butterfield et al., 1997; Skuta et al., 1999). Table
2 shows that ischemia-reperfusion significantly increased the quantity of protein-bound aldehyde groups.
However, the presence of PARP inhibitors during ischemia-reperfusion cycle prevented the increase in the quantity of protein-bound aldehyde
groups (Table 2). However, 3-aminobenzoic acid could not prevent the
ischemia-reperfusion-induced protein oxidation (Table 2).
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Effect of PARP Inhibitors on Ischemia-Reperfusion-Induced
Single-Strand DNA Breaks Formation and NAD+
Catabolism.
Ischemia-reperfusion increased ROS formation in
perfused hearts, which can contribute to the formation of single-strand
DNA breaks. Under normoxic conditions, most of the DNA was undamaged, but ischemia-reperfusion induced large amounts of single-strand DNA
breaks, and the quantity of undamaged DNA decreased to under 30% (Fig.
4). In the presence of PARP inhibitors,
ischemia-reperfusion increased only slightly the amount of ssDNA breaks
(Fig. 4), and the amount of undamaged DNA was significantly higher than
in postischemic hearts and not significantly lower than the normoxic
values (Fig. 4). The inactive analog of 3-aminobenzamide,
3-aminobenzoic acid, could not decrease the
ischemia-reperfusion-induced ssDNA breaks (Fig. 4).
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Effect of PARP Inhibitors on Energy Metabolism of Perfused Hearts
during Ischemia-Reperfusion.
Energy metabolism of Langendorff
perfused hearts was monitored in the magnet of NMR spectroscope, making
it possible to detect changes in high-energy phosphorus intermediates
(Fig. 6). Ischemia induced a rapid
decrease in ATP and creatine phosphate levels and a fast evolution of
inorganic phosphate. Under our experimental conditions, high-energy
phosphate intermediates recovered only partially in 15-min reperfusion
phase, and 3-aminobenzamide facilitated the recovery of ATP and
creatine phosphate (Fig. 6). However, 3-aminobenzoic acid did not
improve the recovery of ischemic heart (data not shown), indicating
that the inhibition of PARP was responsible for the improved recovery.
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Protecting Effect of PARP Inhibitors against
Ischemia-Reperfusion-Induced Damage of Respiratory Complexes.
Under our experimental conditions, ischemia-reperfusion caused a
partial inactivation of respiratory complexes (Table
3), which could be the consequence
of mitochondrial ROS formation during reperfusion (Ambrosio et al.,
1993; Vanden Hoek et al., 1998; Szabados et al., 1999b). Our data
(Figs. 2 and 3; Tables 1 and 2) indicated that PARP inhibitors
decreased the oxidative damage for different components of heart during
ischemia-reperfusion cycle; therefore, it is reasonable to assume that
PARP inhibitors may attenuate the inactivation of respiratory
complexes. Using four different PARP inhibitors, we found that
cytochrome oxidase activity was almost completely protected from the
ischemia-reperfusion-induced partial inactivation (Table 3).
Ischemia-reperfusion also partially inactivated the NADH:cytochrome
c oxidoreductase activity (complex I-III), and PARP
inhibitors could partially protect NADH:cytochrome c
oxidoreductase activity in postischemic heart mitochondria (Table 3).
Under the same experimental conditions,
H2O2 or PARP inhibitors did
not affect citrate synthase activity (data not shown).
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Effect of PARP Inhibitors on Oxidative Inactivation of Cytochrome
Oxidase.
Hydrogen peroxide (0.5 mM) induced a relatively fast
inactivation of cytochrome oxidase in isolated mitochondria (Fig.
9), showing that ROS can indeed
inactivate respiratory complexes. In the same system, PARP inhibitors
(3-aminobenzamide, nicotinamide, BGP-15, and 4-hydroxyquinazoline)
could almost completely protect cytochrome oxidase from
H2O2-induced inactivation,
but the chemical analog of 3-aminobenzamide, 3-aminobenzoic acid,
failed to do so (Fig. 9). At the same time, PARP inhibitors did not
affect the amount of
H2O2-induced ROS production
as determined by the oxidation of dihydrorhodamine123 to rhodamine123
(data not shown), so the protection was not due to a decreased amount
of ROS in the presence of PARP inhibitors. Dose response of PARP
inhibitors in the protection of respiratory complexes against
H2O2-induced inactivation
is shown on Fig. 9. The IC50 values were 394, 238, 38, and 14 µM for 3-aminobenzamide, nicotinamide, BGP-15, and 4-hydroxyquinazoline, respectively.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Langendorff heart perfusion system has been successfully used
for studying ischemia-reperfusion-induced cardiac damages. In this
model, PARP inhibitors were reported to improve the recovery of heart
function and ATP level, as well as decrease the infarct size (Bowes et
al., 1999; Docherty et al., 1999). Similar protective effects of PARP
inhibitors were also observed in local cardiac ischemia in living
animals (Zingarelli et al., 1997; Bowes et al., 1998). It is known that
ROS are mainly produced by mitochondrial respiratory complexes in
postischemic heart (Ambrosio et al., 1993; Vanden Hoek et al., 1998),
and that ROS-induced oxidative damages represent an important mechanism
of the pathological processes in postischemic heart. Our previous data
indicated that a new PARP inhibitor (BGP-15) decreased the
ischemia-reperfusion-induced oxidative damages in Langendorff perfused
heart model system without having any obvious antioxidant property
(Szabados et al., 2000). These experiments raise an interesting
question whether this is a special property of the compound BGP-15 or
other PARP inhibitors have similar characteristics. To answer this
question we investigated the effect of well-established PARP inhibitors
(3-aminobenzamide and nicotinamide) together with BGP-15 on the
oxidative cell damage induced by either ischemia-reperfusion or
hydrogen peroxide in Langendorff heart perfusion system.
Under our experimental conditions, PARP inhibitors abrogated the
ischemia-reperfusion-induced lipid peroxidation (Fig. 2; Table 1) and
protein oxidation (Fig. 3; Table 2), and significantly decreased ssDNA
break formation (Fig. 4), suggesting that PARP inhibitors somehow
reduced the ischemia-reperfusion-induced mitochondrial ROS production
and ROS-related oxidative damages. When the oxidant was given to the
heart externally (1 mM
H2O2), it induced lipid peroxidation and protein oxidation, but PARP inhibitors only partially protected heart tissue from the lipid peroxidation and protein oxidation (Figs. 1 and 2). These observations could not be explained by
a simple antioxidant effect because the studied compounds could not
entrap ROS generated chemically as shown in this article and by
Szabados et al. (2000). In perfused heart, most of the ROS is produced
in the mitochondria during ischemia-reperfusion cycle (Ambrosio et al.,
1993; Vanden Hoek et al., 1998); therefore, our data (Figs. 1-3)
indicate that PARP inhibitors probably decrease the
ischemia-reperfusion-induced increase of mitochondrial ROS production,
and so all oxidative damages related to enhanced ROS production.
Because the blocking of normal electron flow generally activates the
formation of partially reduced reactive oxygen intermediates (Turrens
et al., 1991), the observation that PARP inhibitors prevent the
ischemia-reperfusion-induced inactivation of respiratory complexes (Fig. 9) supports this argument.
In the case of externally added
H2O2, PARP inhibitors could
not prevent the direct oxidative damage caused by the
H2O2 (Tables 1 and 2) but
could decrease the
H2O2-induced inactivation
to the components of the mitochondrial respiratory chain (Fig.
9). The endogenous ROS production could thereby decrease the
total amount of oxidative damages seen in Figs. 2 and 3 and Tables 1 and 2. Biochemical effects of
H2O2 can be different
depending on the concentration applied. High concentration (higher than 1 mM) of H2O2 induces DNA
breaks, PARP activation, NAD+ depletion, ATP
depletion, cell membrane damage, and necrotic cell death, processes
that can be attenuated by PARP inhibitors (Gardner et al., 1997; Virag
et al., 1998; Filipovic et al., 1999). H2O2 can also induce
apoptic cell death in several cell types, which can also be prevented
under certain conditions by PARP inhibitors (Hivert et al., 1998). In
contrast to these, very low concentrations of
H2O2 can be regarded as a
life signal, and help proliferation and protect against apoptosis
possibly by preventing caspase activation and PARP cleavage (delBello
et al., 1999).
Under our experimental conditions, PARP inhibitors decreased the
ischemia-reperfusion-induced NAD+ depletion. This
obviously PARP-dependent nuclear process through the modulation of
NAD+ level (normoxic 0.45 mg/g of wet tissue,
ischemia-reperfusion 0.24 mg/g of wet tissue, and ischemia-reperfusion
in the presence of PARP inhibitors 0.35-0.41 mg/g wet tissue) (Fig. 5)
could contribute to the better recovery of the high-energy phosphate
intermediates (Figs. 6-8). The PARP inhibitors decreased the rate of
NAD+ cleavage, and so reduced the ATP consumption
for the resynthesis of NAD+ in postischemic
hearts. Our data show that PARP inhibitors uniformly promoted the
recovery of ATP and creatine phosphate levels and resulted in not only
higher percentage of recovery but also the a significantly faster rate
of recovery (Fig. 7). In addition, the consumption rate of inorganic
phosphate was increased during the recovery period by PARP inhibitors.
Because Ca2+ and inorganic phosphate can induce
mitochondrial permeability transition (Javadov et al., 2000), the
faster and more complete utilization of inorganic phosphate can be
advantageous for the preservation of metabolically active, coupled
mitochondria in postischemic heart. Hearts predominantly produce energy
by mitochondrial oxidation, therefore the faster recovery seen in the
presence of PARP inhibitors (Fig. 7) can be, at least in part, the
consequence of protected mitochondrial energy metabolism. It is known
that ROS can inactivate respiratory complexes (Turrens et al., 1991; Fig. 9), therefore, protection against ischemia-reperfusion-induced inactivation of cytochrome oxidase and NADH:ubiquinone oxidoreductase can also be a factor in addition to the inhibition of nuclear PARP in
the faster and more complete recovery of high-energy phosphate intermediates.
According to one possible argument, PARP inhibition by moderating
cytoplasmic NAD+ loss can help to retain
mitochondrial NAD+, and may prevent the decrease
of the mitochondrial NAD+-linked substrate
oxidation and ROS formation between NADH-dehydrogenase and ubiquinone.
Therefore, the protective effect of PARP inhibitors against oxidative
damages (lipid peroxidation, protein oxidation, single-strand DNA
breaks, and the inactivation of respiratory complexes) in postischemic
heart may not rely exclusively on the inhibition of PARP. It required
further confirmation that the inhibition of a nuclear enzyme (PARP) can
result in such a good protection against mitochondrial damages.
Therefore, we investigated whether PARP inhibitors can have a
protective effect against
H2O2-induced inactivation
of cytochrome oxidase in isolated rat heart mitochondria (Fig. 9). Our
data showed the
H2O2-induced inactivation
of cytochrome oxidase was almost completely protected by PARP
inhibitors in isolated mitochondria (Fig. 9). In the same experimental
system, 3-aminobenzoic acid, an inactive chemical homolog of the PARP inhibitor 3-aminobenzamide, did not protect cytochrome oxidase against
H2O2-induced inactivation
(Fig. 9). In isolated mitochondria, nuclear PARP cannot play any
possible role (PARP activity was not detectable in our isolated
mitochondria with autoradiography, data not shown); therefore, we have
to assume that PARP inhibitors bind to another mitochondrial protein
that plays a role in the oxidative damage of mitochondria. It is known
that several PARP inhibitors can also inhibit mono-ADP-ribose
transferase or NAD+ glycohydrolase, which are
present in the mitochondria (Ziegler et al., 1997; Jorcke et al.,
1998); therefore, it is possible that PARP inhibitors by decreasing the
activity of any of these enzymes prevent the inactivation of
respiratory complexes.
These data do not conflict with the previous observation using
PARP-1-deleted cells or PARP-1 knockout mice (Endres et al., 1998;
Zingarelli et al., 1998; Grupp et al., 1999; Yang et al., 2000), but
indicate that the PARP inhibitors besides their inhibitory effect on
nuclear PARP, which results in significant protection against oxidative
damage, can have an additional mitochondrial target (binding site for
these molecules) and this direct mitochondrial effect can play a
protecting role in oxidative mitochondria damage.
In conclusion, the studied PARP inhibitors, besides their primary effect of decreasing the activity of nuclear PARP and therefore decreasing NAD+ consumption and ATP consumption, protect mitochondrial energy metabolism (Figs. 5-8), decrease the ischemia-reperfusion-induced mitochondrial ROS formation (Figs. 2 and 3), and protect the respiratory complexes from ROS-induced inactivation (Fig. 9). The mitochondrial protective effect of PARP inhibitors, at least in partially, is independent of nuclear PARP activity because it can be observed in isolated mitochondria. Therefore, it represents a novel mechanism for the mitochondrial protective effects of PARP inhibitors.
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Acknowledgments |
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We thank Laszlo Giran, Bertalan Horvath, Peter Deres, and Botond Literati-Nagy for their excellent technical help.
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Footnotes |
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Received September 18, 2000; Accepted February 26, 2001
This work was supported by the N-Gene Research Laboratories, Inc., Hungarian Science Foundation Grant T023076, the Ministry of Health and Welfare Grant ETT 35/2000, and the Ministry of Education Grant FKFP 1393/1997.
Send reprint requests to: Balazs Sumegi, University of Pécs, Faculty of Medicine, Department of Biochemistry, Szigeti út 12, 7624-Pécs, Hungary. E-mail: sumegib{at}apacs.pote.hu
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Abbreviations |
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ROS, reactive oxygen species; ssDNA, single-strand DNA; PARP, poly(ADP-ribose) polymerase; BGP-15, O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime; TBARS, thiobarbituric acid reactive substances; TBA, thiobarbituric acid.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 M. Chen, Z. Zsengeller, C.-Y. Xiao, and C. Szabo Mitochondrial-to-nuclear translocation of apoptosis-inducing factor in cardiac myocytes during oxidant stress: potential role of poly(ADP-ribose) polymerase-1 Cardiovasc Res, September 1, 2004; 63(4): 682 - 688. [Abstract] [Full Text] [PDF]
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 K. Yamazaki, S. Miwa, K. Ueda, S. Tanaka, S. Toyokuni, O. Unimonh, K. Nishimura, and M. Komeda Prevention of myocardial reperfusion injury by poly(ADP-ribose) synthetase inhibitor, 3-aminobenzamide, in cardioplegic solution: in vitro study of isolated rat heart model Eur. J. Cardiothorac. Surg., August 1, 2004; 26(2): 270 - 275. [Abstract] [Full Text] [PDF]
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B. Veres, B. Radnai, F. Gallyas Jr., G. Varbiro, Z. Berente, E. Osz, and B. Sumegi Regulation of Kinase Cascades and Transcription Factors by a Poly(ADP-Ribose) Polymerase-1 Inhibitor, 4-Hydroxyquinazoline, in Lipopolysaccharide-Induced Inflammation in Mice J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 247 - 255. [Abstract] [Full Text] [PDF]
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) at 0.25 mM,
p < 0.05, at higher concentrations,
p < 0.01; nicotinamide (
) at 1 and 2 mM,
p < 0.01; BGP-15
(
) at 0.025 mM,
p < 0.05, at higher concentrations,
p < 0.01; and 4-hydroxyquinazoline (
) at 0.01 mM and higher concentrations, p < 0.01.
p < 0.01.
); IR + NA,
ischemia-reperfusion in the presence of 3 mM nicotinamide (
); IR + 3-AB, ischemia-reperfusion in the presence of 3 mM 3-aminobenzamide
(
); and IR + 4HQ, ischemia-reperfusion in the
presence of 0.1 mM 4-hydroxyquinazoline (
) at 0.01 mM, p < 0.05 and at higher concentrations,
p < 0.01.
