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Inotek Corporation, Beverly, Massachusetts 01915, USA
1Correspondence: Inotek Corporation, 100 Cummings Center, Suite 419E, Beverly, MA 01915, USA. E-mail: szabocsaba{at}aol.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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+ endotoxin (IFN/LPS) -stimulated RAW macrophages from the
inhibition of mitochondrial respiration and inhibited nitrite
production from IFN/LPS-stimulated macrophages. We have selected
hypoxanthine as the most potent cytoprotective agent and PARP inhibitor
among the three purine compounds, and investigated the mechanism of its
cytoprotective effect. We have found that hypoxanthine protects
thymocytes from death induced by the cytotoxic oxidant peroxynitrite.
In line with the PARP inhibitory effect of purines, hypoxanthine has
prevented necrotic cell death while increasing caspase activity and DNA
fragmentation. As previously shown with other PARP inhibitors,
hypoxanthine acted proximal to mitochondrial alterations as
hypoxanthine inhibited the peroxynitrite-induced mitochondrial
depolarization and secondary superoxide production. Our data imply that
purines may serve as endogenous PARP inhibitors. We propose that, by
affecting PARP activation, purines may modulate the pattern of cell
death during shock, inflammation, and reperfusion injury.—Virág,
L., Szabó, C. Purines inhibit poly(ADP-ribose) polymerase
activation and modulate oxidant-induced cell death.
Key Words: nitric oxide • free radicals • antioxidants • shock • knockout • ADP-RT
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. An increased turnover of purines has also been
described in inflammation and shock (5
, 6)
. Adenosine and
its primary metabolite inosine are ubiquitous nucleosides that can be
released in substantial quantities from ischemic or inflamed tissue
(1
2
3
4
, 7
, 8)
. Adenosine is generally considered to act
primarily through cell surface adenosine receptors (A1, A2A, A2B, and
A3) (9)
and is considered a regulator of numerous
physiological and pathophysiological events in multiple tissues
(10
11
12
13
14
15
16
17
18)
. Adenosine and its analogs can alter the course
of a variety of inflammatory diseases such as rheumatoid arthritis,
nephritis, and uveitis (10
11
12
13
14
15
16
17
18
19
20
21)
. Inosine was generally
considered an inactive metabolite. However, recently we and others have
demonstrated that inosine can also exert antiinflammatory effects
(22
23
24)
. Very limited information is available on the
pharmacological effects or pathophysiological significance of
hypoxanthine, the purine base that is cleaved off from inosine.
Elevation of hypoxanthine levels in plasma, cerebrospinal fluid, and
urine reflects the severity of tissue injury so well that determination
of hypoxanthine in body fluids has been proposed to be a good clinical
marker of ischemic and inflammatory diseases (25
26
27
28
29
30)
.
Hypoxanthine may have a dual role in ischemia and inflammation
(31)
: it may promote tissue injury by serving as substrate
for the xanthine oxidase-mediated generation of superoxide radical and
thus contribute to tissue injury (32
33
34
35
36)
and may also
contribute to cell recovery after tissue injury by being used for the
regeneration of ATP (31)
.
Poly (ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme of
eukaryotic cells that has been implicated in the response to DNA injury
and oxidant-induced cell death (37
38
39
40)
. PARP is a nuclear
nick sensor enzyme that becomes activated in response to DNA damage
(37)
. Activated PARP cleaves NAD+
into nicotinamide and ADP-ribose, and catalyzes the latter on nuclear
acceptor proteins such as histones and PARP itself (37)
.
Excessive activation of PARP depletes the cellular
NAD+ and ATP pools and causes necrotic cell death
(38
39
40)
. PARP has also been implicated in the
transcriptional regulation of inflammatory mediators such as inducible
nitric oxide synthase (iNOS) (41
42
43)
. PARP inhibitors
provided remarkable protection from tissue damage, organ dysfunction,
and lethality in various forms of reperfusion injury, inflammation, and
shock (39
, 41
, 44
, 45)
.
Although the main effect of purines is mediated via cell surface purine
receptors, we hypothesized that purines may also exert cytoprotective
effects by interfering with the PARP activation pathway. Our assumption
was based on the structural similarity of hypoxanthine, inosine, and
adenosine to part of NAD+, the substrate of PARP.
The first aim of the current study was to investigate whether purines
affect oxidant-induced cell death. An additional aim was to investigate
whether selected purines are capable of suppressing the catalytic
activity of PARP. The potent cytotoxic oxidant peroxynitrite (the
reaction product of nitric oxide and superoxide) was used to trigger
cell death in these studies because 1) this species is known
to be produced in various forms of shock and inflammation;
2) it is responsible for some of the oxidant injury in these
conditions; and 3) it is a potent trigger of DNA single
strand breakage and PARP activation in vitro and in
vivo (40
, 41
, 46
, 47)
.
The results of the current study demonstrate that certain purines (chiefly, hypoxanthine) are relatively potent inhibitors of PARP in intact cells and markedly affect oxidant-mediated cell death. Purine-mediated inhibition of PARP may be a novel mechanism whereby purines affect cell death and organ injury in various pathophysiological conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxynitrite was diluted in phosphate-buffered saline (PBS) (pH 11.0)
and added to the cells in a bolus of 50 µl. Cells were then incubated
for various times (RAW cells: 20 min for PARP assay, 1 or 24 h for
MTT assay; thymocytes: 20 min for PARP assay, 3 h for the
measurement of mitochondrial parameters, 4 h for propidium iodide
staining, or 6 h for DNA fragmentation and caspase activation).
Decomposed peroxynitrite (incubated for 30 min at pH 7.0) served as
control and failed to influence any of the parameters studied (see also
ref 48
). In the RAW macrophages, bacterial
lipopolysaccharide (10 µg/ml) and murine interferon (50 U/ml)
were used to stimulate peroxynitrite generation from endogenous
sources. At 24 h, mitochondrial respiration was measured as
described below. Nitrite/nitrate production, an indicator of NO
synthesis, was measured in the supernatant, as described previously
(41)
. First, nitrate in the culture medium was reduced to
nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160
µM) at room temperature for 2 h. After 2 h, nitrite
concentration in the samples was measured by the Griess reaction by
adding 100 µl of Griess reagent (1% sulfanilamide and 0.1%
naphthylethylenediamide in 5% phosphoric acid) to 100 µl samples of
conditioned medium. The optical density at 550 nm
(OD550) was measured using a Spectramax 250
microplate reader (Molecular Devices, Sunnyvale, Calif.). Nitrate
concentrations were calculated by comparison with
OD550 of standard solutions of sodium nitrate
prepared in culture medium. Purines or other test compounds (up to 3
mM) did not scavenge nitrite or nitrate and did not interfere with the
activity of nitrate reductase.
Measurement of cellular PARP activity
RAW cells cultured in 6-well plates or thymocytes
(107 cells in 1 ml culture medium) were treated
with peroxynitrite (750 µM). After 20 min cells were spun, medium was
aspirated, and cells were resuspended in 0.5 ml assay buffer (56 mM
HEPES-pH 7.5, 28 mM KCl, 28 mM NaCl, 2 mM MgCl2,
0.01% w v-1 digitonin, and 0.125 µM
NAD+ and 0.5 µCi ml-1
3H-NAD+). PARP activity was then
measured as described previously (48)
. Briefly, after
incubation (10 min at 37°C), 200 µl ice-cold 50% w
v-1 trichloroacetic acid (TCA) was added and
samples incubated for 4 h at 4°C. Samples were spun (10,000
g, 10 min), then pellets were washed twice with ice-cold 5%
w v-1 TCA and solubilized overnight in 250 µl
2% w v-1 sodium dodecyl sulfate (SDS)/0.1 N
NaOH at 37°C. Contents of the tubes were added to 6.5 ml ScintiSafe
Plus scintillation liquid (Fisher Scientific, Pittsburgh, Pa.) and
radioactivity was determined using a liquid scintillation counter
(Wallach, Gaithersburg, Md.).
Measurement of PARP activity in vitro
To assess the direct effect of purines and test compounds on the
activity of the purified PARP enzyme, an in vitro assay was
carried out, as described (49)
. Briefly, 1 µg of bovine
PARP enzyme was added to 200 µl of buffer containing 100 mM Tris: 5
mM MgCl2, 1 mM DTT, pH 7.9, in the presence or
absence of purines or other test compounds. The mixture was allowed to
preincubate at room temperature for 30 min. After preincubation, 10
µl of sonicated DNA (32.5 µg/ml final concentration) and 10 µl
histone (4 mg/ml) was added to each tube along with 10 µl
3H-NAD+ (16 µCiµ/ml).
Tubes were vortexed, then centrifuged at 10,000 rpm for 10 s, and
the reaction was allowed to proceed at 37°C for 15 min. Reactions
were stopped by the addition of 200 µl 50% TCA.
Reaction mixtures were kept at -20°C for 30 min. The TCA insoluble precipitate was collected by filtration through 0.2 µm nitrocellulose analytical test filter funnels under vacuum and washed five times with 3 ml of cold 5% TCA. The membranes were counted in 7 ml scintillation vials with Scintisafe mixture for 2 min beta spectrum in a Wallach 1409 scintillation counter.
Measurement of mitochondrial respiration in macrophages
Cell respiration was assessed by the mitochondrial-dependent
reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] to formazan (41)
. Cells in 96-well plates were
incubated at 37°C with MTT (0.2 mg/ml) for 1 h. Culture medium
was removed by aspiration and the cells were solubilized in DMSO (100
µl). The extent of reduction of MTT to formazan within cells was
quantitated by measurement of OD550.
Dihydrorhodamine assay
To test whether the purines used in our studies directly
scavenge peroxynitrite, the peroxynitrite-dependent oxidation of
dihydrorhodamine 123 to rhodamine 123 was measured based on the
principles of the method previously described (50)
.
Briefly, peroxynitrite (5 µM) was added into PBS containing 10 µM
dihydrorhodamine 123 and different concentrations of purines. After a
10 min incubation at 22°C, the fluorescence of rhodamine 123 was
measured using a Shimadzu RF-5301PC fluorometer at an excitation
wavelength of 500 nm, emission wavelength of 536 nm (slit widths 2.5
and 3.0 nm, respectively). In control, reverse-order experiments we
have confirmed that the compounds tested neither showed fluorescence at
the above wavelengths nor affected rhodamine 123 fluorescence (data not
shown).
Cytochrome c oxidation
To use another assay for testing whether the purines used in our
studies directly scavenge peroxynitrite, peroxynitrite-dependent
oxidation of cytochrome c2+,
was measured as described (50)
. Cytochrome c
was reduced by sodium dithionite immediately before use and purified by
chromatography on Sephadex G-25 using 100 mM potassium phosphate plus
0.1 mM DTPA, pH 7.2, as the elution buffer. The concentration of
cytochrome c2+ was determined
spectrophotometrically at 550 nm in the same buffer (
=21
mM-1 cm-1). Cytochrome
c2+ oxidation (50 µM) yields
upon addition of peroxynitrite (25 µM initial concentration after
mixing) were assessed by incubation of reaction mixtures in 100 mM
potassium phosphate plus 0.1 mM DTPA, pH 7.2, at 22°C for 3 min in
the absence or presence of purines. Oxidation of cytochrome
c2+ was followed at 550 nm
using a Shimadzu UV-Z401PC spectrophotometer. In control, reverse-order
experiments we have confirmed that the compounds tested do not
interfere with the spectrophotometric measurements at the above
wavelengths. Moreover, in control experiments we have confirmed that
the compounds tested do not reduce cytochrome
c3+.
Measurement of mitochondrial membrane potential, superoxide
production, and cardiolipin content in thymocytes
The mitochondrial membrane potential was quantitated by the flow
cytometric analysis of 3,3'dihexyloxacarbocyanine iodide [DiOC6(3)]
-stained cells (51)
. Intramitochondrial generation of
reactive oxygen intermediates was determined by analyzing with flow
cytometry the superoxide-induced conversion of the oxidant-sensitive
dye dihydroethidium to ethidium (51)
. Mitochondrial
membrane damage was determined by measuring the cardiolipin
degradation, as described (51)
.
Flow cytometry in thymocytes
Thymocytes were stained with 5 µg/ml PI, 40 nM DiOC6(3), 2
µM hydroethidine (HE) for 15 min at 37°C, washed once with PBS, and
analyzed with a FacsCalibur flow cytometer as described
(51)
. To measure mitochondrial parameters, forward and
side scatters were gated on the major population of normal-sized cells.
For the cytotoxicity assay, the percentage of PI-positive cells was
calculated from the total (ungated) population.
Detection of internucleosomal DNA fragmentation of thymocytes
Thymocytes were pretreated with purines for 30 min and then
treated with peroxynitrite. After 6 h, cells were washed once with
cold PBS and pellets were resuspended in sample buffer (10 mM Tris, pH
8.0, 5% v/v glycerol, 0.05% w/v- bromphenol
blue, 5 mg/ml RNase). DNA fragmentation was detected as described
(48)
. Agarose (2% w/v) was poured on a horizontal gel
support. After solidification of the gel, the top part (above the comb)
was replaced with 1% w/v agarose containing 2% w/v SDS and 64 µg/ml
proteinase K. Cells (2x106) were loaded in 20
µl sample buffer. Electrophoresis was carried out at 25 V for 12 h and the gel was stained with 2 µg/ml ethidium bromide for 1 h.
Measurement of caspase 3-like activity
Caspase activity was measured by the cleavage of the
fluorogenic tetrapeptide-amino-4-methylcoumarine conjugate (DEVD-AMC)
as described (52)
. Unless otherwise indicated, cells
(4–10x106) were harvested 6 h after
peroxynitrite treatment, washed once in PBS, and lysed in a lysis
buffer (10 mM HEPES, 0.1% w/v CHAPS, 5 mM dithiothreitol, 2 mM EDTA,
10 µg/ml aprotinin, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, and
1 mM PMSF, pH 7.25) for 10 min on ice. Cell lysates and substrates (50
µM) were combined in triplicate in the caspase reaction buffer (100
mM HEPES, 10% w/v sucrose, 5 mM dithiothreitol, 0.1% w/v CHAPS, pH
7.25) in the presence or absence of 10 µM of the tetrapeptide caspase
3 inhibitor N-acetyl-aspartyl-glutamyl-valyl-aspartyl-aldehyde
(DEVD-CHO) and samples were incubated at 37°C for 60 min. AMC
liberation was determined with a Perkin-Elmer fluorometer using 380 nm
excitation and 460 nm emission wavelength. Data are given as absolute
fluorescence units.
Materials
Peroxynitrite was a kind gift of Dr. H. Ischiropoulos (Inst.
Environmental Medicine, University of Pennsylvania, Philadelphia).
3-Morpholinosidnonimine (SIN-1) was purchased from Calbiochem (San
Diego, Calif.). HE, DIOC6(3), and propidium iodide were obtained from
Molecular Probes (Eugene, Oreg.). The tetrapeptide substrate (DEVD-AMC)
and inhibitor (DEVD-CHO) of caspase 3 and the purified PARP enzyme were
purchased from Biomol (Plymouth Meeting, Pa.). Proteinase K was
obtained from Life Technologies (Grand Island, N.Y). Tris, magnesium
chloride, analytical test filter funnels, and Scintisafe scintillation
mixture were from Fisher Scientific.
3H-NAD+ was purchased from
DuPont NEN (Boston, Mass.). Murine interferon was from Genzyme
(Cambridge, Mass.). Tissue culture medium and fetal calf serum were
from Life Technologies, Inc. Bacterial lipopolysaccharide
(Escherichia coli, serotype No. 0111:B4) and all other
reagents were obtained from Sigma (St. Louis, Mo.).
Statistical analysis
All values in the figures and text are expressed as mean ±
standard deviation (SD) of n observations;
n 
3. Data sets were examined by analysis of variance
and individual group means were then compared with Bonferroni’s
post hoc test. A P value of less than 0.05 was
considered statistically significant. When the results are presented as
representative gels or flow cytometry analyses, results similar to the
ones shown were obtained in at least three different experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Nevertheless, the difference between the potency of the
most potent purine tested (hypoxanthine) and the least potent
‘professional’ PARP inhibitor used (nicotinamide) was rather small
(EC50 values 
1 and 2 mM, respectively).
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Because differential cell uptake of the purines and PARP
inhibitors may influence the potency of PARP inhibition, we also
tested the effect of the purines and reference compounds in a cell-free
PARP assay. Again, the potency of the purines was
hypoxanthine>inosine>adenosine (Fig. 1B
), with
nicotinamide and 3-aminobenzamide becoming more potent relative to the
purines. These results indicate that limited cell uptake markedly
reduces the PARP inhibitory potency of 3-aminobenzamide and
nicotinamide, but only to a lesser degree in the case of the three
purines tested.
Purines inhibit the suppression of cell viability in
immunostimulated macrophages
Stimulation of macrophages with bacterial lipopolysaccharide and
-interferon results in the production of peroxynitrite from
endogenous sources, which leads to a decrease in cellular viability
that is mediated in part by PARP (47)
. Here we tested
whether adenosine, inosine, or hypoxanthine affect the decrease in cell
viability in response to immunostimulation in RAW macrophages. Exposure
of the macrophages to bacterial lipopolysaccharide and interferon
resulted in production of nitrite/nitrate (oxidized products of NO) and
a decrease in mitochondrial respiration (Fig. 2
). Pretreatment of the cells with hypoxanthine (and, to a lesser extent,
inosine), provided significant protection against the suppression of
cell viability (Fig. 2A
). Furthermore, hypoxanthine
(potently) and the other purines tested (weakly) inhibited the
production of nitrite/nitrate in response to immunostimulation (Fig. 2B
). A similar suppression of immunostimulation-induced NO
production and iNOS expression has previously been reported with
3-aminobenzamide, nicotinamide, and other PARP inhibitors, as well as
by the PARP negative phenotype (41
42
43
, 53)
.
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Purines do not scavenge peroxynitrite
To test whether the purines used exert a direct scavenging effect
on peroxynitrite or on the peroxynitrite-generating agent SIN-1 (rather
than a direct inhibitory effect on the catalytic activity of PARP), we
tested, in two different assays, whether the purines used interfere
with peroxynitrite- or SIN-1-mediated oxidations (and thus act as
scavengers of peroxynitrite). Adenosine, inosine, or hypoxanthine (at
100 µM–3 mM) failed to inhibit the peroxynitrite- and SIN-1-induced
dihydrorhodamine 123 oxidation or cytochrome c oxidation
(n=9, not shown). The positive control N-acetylcysteine
exhibited a dose-dependent inhibitory effect on the
peroxynitrite-induced oxidations, with EC50
values of 9 µM and 100 µM in the dihydrorhodamine and cytochrome
assays, respectively (n=6–9).
Effect of hypoxanthine on peroxynitrite-induced thymocyte death and
mitochondrial alterations
Since hypoxanthine was the most potent PARP inhibitor from the
group of purines tested, we selected this agent for detailed evaluation
on the mode and mechanism of peroxynitrite-induced cell death in
thymocytes. Authentic peroxynitrite (20 µM) caused thymocyte death,
as assessed by the uptake of propidium iodide (Fig. 3A
). Since we have demonstrated previously that PARP
activation mediates necrotic thymocyte death in this system (48
, 51)
, we subsequently investigated the effect of hypoxanthine on
peroxynitrite-induced necrosis. In line with its PARP inhibitory
effect, hypoxanthine (300 µM–3 mM) inhibited peroxynitrite-induced
cytotoxicity in a dose-dependent manner (Fig. 3
, top panel). In
addition to bolus addition of authentic peroxynitrite, we also used
SIN-1, a peroxynitrite-generating agent. Hypoxanthine has also provided
concentration-dependent protection against SIN-1-induced cytotoxicity
(Fig. 3
, bottom panel).
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Mitochondria maintain a negative transmembrane potential that
dissipates during various forms of cell death, both apoptosis and
necrosis. The mitochondrial transmembrane potential can be quantitated
by using cationic fluorescent dyes such as
DiOC(6)3, which are accumulated by the
mitochondria. Peroxynitrite-induced thymocyte necrosis is also
accompanied by a decrease of the mitochondrial transmembrane potential
and increased secondary superoxide production (51)
. These
mitochondrial alterations have also been found to be dependent on PARP
activation (51)
. Consistent with the PARP inhibitory
effect of hypoxanthine, the compound reduced the collapse of
mitochondrial membrane potential as indicated by
DiOC(6)3 uptake (Fig. 4A
). Furthermore, hypoxanthine also inhibited
peroxynitrite-induced secondary superoxide production (Fig. 4B
).
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Effect of hypoxanthine on the mode of peroxynitrite-induced cell
death
We have previously shown that peroxynitrite causes both apoptotic
and necrotic cell death (48)
. Furthermore, our group and
others have also shown that PARP activation diverts oxidant-induced
cell death from the default apoptosis toward necrosis (48
, 54
, 55)
. Accordingly, in the presence of PARP inhibitors or in
PARP-deficient cells, an increased output of apoptotic parameters such
as caspase activation and DNA fragmentation can be observed (48
, 54)
. We have investigated the effect of hypoxanthine on these
apoptotic parameters. Hypoxanthine (3 mM) had no effect on the
spontaneous DNA fragmentation of thymocytes (Fig. 5A
), which reflects the spontaneous apoptosis that thymocytes
are known to undergo in culture. Peroxynitrite (20 µM) inhibited the
spontaneous DNA fragmentation of thymocytes, an effect that has
previously been demonstrated to be mediated by PARP activation
(48)
. Hypoxanthine (3 mM) prevented the
peroxynitrite-induced decrease of oligonucleosomal DNA cleavage (Fig. 5A
).
|
Peroxynitrite-induced cell death is also accompanied by the
activation of caspase-3 like proteases, which have been shown to
mediate DNA fragmentation (48
, 52)
. Small concentrations
of peroxynitrite (10–30 µM) induce caspase activation, whereas at
higher concentrations (40–80 µM) of peroxynitrite caspase activity
declines (49)
. At the concentration used in the current
experiment (30 µM), peroxynitrite induced a small degree of caspase
activation as measured by the cleavage of the fluorochrome-conjugated
tetrapeptide substrate DEVD-AMC. In the presence of hypoxanthine,
however, significantly higher caspase activity has been detected (Fig. 5B
). The effect of hypoxanthine was dose dependent. Taken
together, the results in thymocytes demonstrated that hypoxanthine,
similar to the prototypical PARP inhibitors 3-aminobenzamide and very
similar to the findings obtained in PARP-deficient thymocytes exposed
to cytotoxic oxidants (48
, 51)
, dose-dependently
suppresses peroxynitrite-induced thymocyte necrosis (and related
parameters), increases the fraction of cells exhibiting normal
phenotype, but also simultaneously increases apoptotic DNA
fragmentation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8
, 30
, 56
, 57)
. Normal values of
hypoxanthine in the serum range between 1 and 11 µM (3)
.
However, increases of serum hypoxanthine concentration by two orders of
magnitude have been reported in endotoxemic dogs (58)
and
during the resuscitation of a newborn baby with cardiac arrest
(59)
. Most studies report normal values between 0 and 5
µM in the cerebrospinal fluid, whereas after severe hypoxia levels up
to 700 µM have been found (3)
. Considering the generally
held view that tissue levels of purine metabolites far exceed values
measured in bodily fluids, tissue hypoxanthine levels are generally
estimated to be in the millimolar range, especially under certain
pathophysiological conditions such as shock, ischemia, and
inflammation. Indeed, sporadic attempts made to determine tissue levels
of hypoxanthine support our estimation. For example, normal
hypoxanthine levels in rat heart or in human placenta were found to be
138 µM and 284 µM, respectively (3)
. In bovine
chromaffin cells, intracellular concentrations of 2.1, 3.8, and 1.5 mM
have been reported for adenosine, inosine, and hypoxanthine,
respectively (60)
. Thus, it is most likely that millimolar
concentrations of hypoxanthine occur in vivo, and therefore
hypoxanthine may well act as an inhibitor of PARP activation in
ischemic or inflamed tissues. This PARP inhibitory and cytoprotective
effect of hypoxanthine may be further increased by a possible synergism
between inosine, adenosine, and hypoxanthine. A synergistic or
additional action is also possible between purines and nicotinamide,
another endogenous PARP inhibitor whose tissue level also increases
during hypoxia due to increased catabolism of nicotinamide coenzymes
(61
62)
.
Cytoprotective effect of purines by PARP inhibition
Hypoxanthine, and to a lesser extent inosine and adenosine,
prevented cytotoxicity induced either by bolus addition of authentic
peroxynitrite, continuous generation of peroxynitrite from SIN-1, or
endogenous production of peroxynitrite by activated macrophages. The
cytotoxic effect of peroxynitrite has previously been shown to be
mediated in part by PARP activation (40
, 41
, 48
, 63)
.
Therefore, we investigated whether or not these selected purines
interfere with PARP activation. In the millimolar range, the purines
tested inhibited PARP activation in RAW macrophages with a potency
comparable to the prototypical ‘benchmark’ PARP inhibitors
3-aminobenzamide and nicotinamide. The purines tested also exerted
inhibitory effect on the purified PARP enzyme. However, in this
cell-free assay, purines proved to be considerably weaker inhibitors
than nicotinamide or 3-aminobenzamide. The better performance of
purines in the cellular PARP assay is likely to be explained by the
superior membrane permeability/cell uptake of purines as compared to
3-aminobenzamide or nicotinamide. We have found a good correlation
between PARP inhibitory and cytoprotective potency of
purines—hypoxanthine being the most potent and adenosine being the
least potent in both assays.
Effect of purines on NO production
Nitric oxide and NO-derived oxidants such as peroxynitrite are
important mediators of various forms of inflammation, shock, and
ischemia-reperfusion injury. In our current work we have found that in
activated macrophages, purines inhibit NO production, as assessed by
the determination of nitrite, the stable end product of NO metabolism.
This is in line with the PARP inhibitory effect of purines, as we and
others have reported earlier that PARP inhibition reduces NO production
by inhibition of iNOS mRNA expression (43
, 53)
.
Furthermore, we have previously published that fibroblasts derived from
PARP-/- mice showed reduced expression of iNOS
mRNA and protein, as compared to fibroblasts derived from wild-type
mice (PARP+/+) (41)
. As the
transcription factor NF
B is known to be a crucial regulator of iNOS
gene expression and PARP has been shown to interact with NFB and
other transcription factors as well (42
, 64)
, inhibition
by purines of NO production may be the consequence of an interference
by PARP inhibition of NFB activation. Thus, we hypothesize that, in
addition to cytoprotection (i.e., direct protection against oxidant
induced necrosis by PARP inhibition), some of the antiinflammatory
effect of purines may also be explained by the reduced production of
inflammatory mediators such as NO.
Regulation of the mode of cell death (apoptosis vs. necrosis by
hypoxanthine
The role of PARP in mediating the cytotoxic effect of
peroxynitrite is conveniently characterized in thymocytes, a frequently
used model system for cell death. Using a multiparametric approach, we
have previously shown that 1) low concentrations of
peroxynitrite causes apoptotic cell death (caspase activation, DNA
fragmentation); 2) high concentrations of peroxynitrite
cause necrosis (plasma membrane disruption, mitochondrial alterations,
inhibition of apoptotic parameters); and 3) activation of
PARP and consequent ATP depletion divert the default apoptotic death
pathway toward necrosis (48
, 51)
. The same pattern of
modulation of cell death is seen with agents that indirectly interfere
with PARP activation, such as cellular calcium chelators or zinc
chelators (49
, 50)
. In our current work we have shown that
hypoxanthine, in line with its PARP inhibitory properties,
dose-dependently inhibited the peroxynitrite and SIN-1-induced necrosis
of thymocytes. Similar to PARP inhibitors such as 3 aminobenzamide,
nicotinamide, or 5-iodo-6-amino 1,2-benzopyrone, hypoxanthine diverted
peroxynitrite-induced necrosis toward apoptosis, as indicated by
increased caspase activation and DNA fragmentation. This effect of PARP
inhibitors can be explained by the preservation of cellular ATP
required for the energy-demanding apoptotic process. We have also shown
before that oxidant-induced and PARP-mediated rapid necrotic cell death
is associated with mitochondrial alterations such as mitochondrial
membrane depolarization and secondary superoxide production
(51)
, similar to perturbations characterizing prolonged
apoptotic cell death. Hypoxanthine also inhibited peroxynitrite-induced
and PARP-mediated mitochondrial alterations, providing further support
for PARP being the primary target of hypoxanthine in its cytoprotective
effect. Thus, taken together, purines appear to work, at least in the
currently used model systems, as nonprofessional but reasonably potent
inhibitors of PARP activation; they modulate cell death in a fashion
that is entirely consistent with their PARP inhibitory effect.
The current results are the first to demonstrate that an endogenous molecule (other than nicotinamide) inhibits PARP activation and therefore modulates oxidant-induced cell death in a physiologically or pathophysiologically relevant concentration range. We propose the hypothesis that endogenously produced purines (such as hypoxanthine) inhibit PARP activation in various forms of reperfusion injury and inflammation, and thereby modulate the fate of the oxidant-exposed parenchymal cells.
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ACKNOWLEDGMENTS |
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Received for publication May 9, 2000.
Revision received June 15, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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