From the Departamento de Bioquímica y
Biología Molecular, Universidad de Salamanca, and the
¶ Unidad de Investigación, Hospital Universitario de
Salamanca, 37007 Salamanca, Spain
Received for publication, July 9, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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Peroxynitrite is thought to be a nitric
oxide-derived neurotoxic effector molecule involved in the disruption
of key energy-related metabolic targets. To assess the consequences of
such interference in cellular glucose metabolism and viability, we
studied the possible modulatory role played by peroxynitrite in glucose
oxidation in neurons and astrocytes in primary culture. Here, we report
that peroxynitrite triggered rapid stimulation of pentose phosphate pathway (PPP) activity and the accumulation of NADPH, an essential cofactor for glutathione regeneration. In contrast to peroxynitrite, nitric oxide elicited NADPH depletion, glutathione oxidation, and
apoptotic cell death in neurons, but not in astrocytes. These events
were noticeably counteracted by pretreatment of neurons with
peroxynitrite. In an attempt to elucidate the mechanism responsible for
this PPP stimulation and neuroprotection, we found evidence consistent
with both exogenous and endogenous peroxynitrite-mediated activation of
glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the
first rate-limiting step in the PPP. Moreover, functional
overexpression of the G6PD gene in stably transformed PC12 cells
induced NADPH accumulation and offered remarkable resistance against
nitric oxide-mediated apoptosis, whereas G6PD gene-targeted antisense
inhibition depleted NADPH levels and exacerbated cellular vulnerability. In light of these results, we suggest that G6PD activation represents a novel role for peroxynitrite in neuroprotection against nitric oxide-mediated apoptosis.
Nitric oxide (·NO) is a short-lived physiological
neural messenger involved in diverse biologically relevant
functions (see Ref. 1 for a review). In neurons and astrocytes,
·NO is produced through the activation of a constitutive
calcium-dependent neuronal nitric-oxide synthase and
participates in the signaling pathway, leading to rises in cGMP levels
(2-4). In addition, astrocytes have the ability to form ·NO in
a calcium-independent fashion that requires prior transcriptional expression of the inducible nitric-oxide synthase isoform by
lipopolysaccharide (LPS)1
and/or certain cytokines (5, 6). Under certain neuropathological situations, however, excessive or inappropriate ·NO
biosynthesis (nitrosative stress) is followed by a reaction of
·NO with superoxide (O With respect to the neurotoxicity elicited by inappropriate ·NO
biosynthesis, the intracellular content of glutathione (GSH) appears to
play a key role in dictating cellular vulnerability. Thus, ·NO
and peroxynitrite potently oxidize sulfhydryls, including GSH (11, 12).
Accordingly, neuronal GSH oxidation has been proposed to be a
contributing factor leading to the mitochondrial damage and
neurotoxicity associated with nitrosative stress (13-15). Unlike neurons, astrocytes are cells that efficiently maintain GSH in its
reduced redox status, even under conditions of excessive endogenous ·NO formation (16, 17). However, the mechanism(s) involved in
such different cellular abilities to restore GSH levels is an issue
that remains to be elucidated.
Hepatocytes are cells prone to hydrogen peroxide
(H2O2)-mediated activation of
glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the
first rate-limiting step in the oxidative branch of the pentose
phosphate pathway (PPP) (18, 19). Furthermore, stimulation of this
pathway in neurons (20) and astrocytes (21) has been proposed to elicit
a protective action against H2O2 toxicity through the PPP activity-mediated production of NADPH, a cofactor necessary for GSH regeneration from oxidized glutathione (GSSG) (22,
23). A similar protective mechanism has also been reported for Jurkat
human T cells, where targeted inactivation of transaldolase, i.e. the rate-limiting step in the non-oxidative branch of
the PPP, prevents the GSH oxidation and apoptosis caused by a number of
pro-oxidant compounds, including ·NO (24).
In keeping with the above-mentioned studies, we reported previously
that glucose utilization through the PPP in LPS-stimulated astrocytes
offers self-protection against endogenous ·NO-mediated GSH
oxidation (25). Because LPS-stimulated astrocytes synthesize
O Materials
Peroxynitrite was synthesized and quantified
spectrophotometrically ( Cell Cultures
Astrocyte-rich primary cultures derived from neonatal
1-day-old Wistar rats were prepared as previously described (28). Cell
suspensions were plated in culture medium supplemented with 10% (v/v)
fetal calf serum at a density of 1.25 × 105
cells/cm2 in 175-cm2 flasks. Cells were
maintained in a humidified incubator under an atmosphere of 5%
CO2 and 95% air at 37 °C with a change of medium twice
a week. After 12-14 days, cells were collected by trypsinization and
reseeded in culture medium at a density of 2.5 × 105
cells/cm2 on appropriate plastic dishes. For the
experiments, astrocytes were used 24 h after reseeding.
Cerebral cortex neurons in primary culture were prepared from fetal
rats at 16-17 days of gestation (29). Dissociated cell suspensions
were plated at a density of 2.5 × 105
cells/cm2 on appropriate plastic dishes previously coated
with poly-D-lysine (15 µg/ml) in culture medium
supplemented with 10% fetal calf serum. Cells were incubated at
37 °C in a humidified atmosphere containing 5% CO2 and
95% air. Forty-eight hours after plating, the culture medium was
replaced with DMEM supplemented with 5% horse serum and 20 mM D-glucose. On day 4 of culture, cytosine arabinoside was added (10 µM) to prevent non-neuronal
proliferation. For the experiments, neurons were used on day 9.
PC12 cells were kindly provided by Dr. Dionisio
Martín-Zanca (Universidad de Salamanca, Salamanca,
Spain) and were maintained in DMEM supplemented with 10% fetal calf
serum, 6% horse serum, and 20 mM glucose on
collagen-coated plastic dishes. For selection and growth of stably
transfected cells, this medium was supplemented with G418 (500 µg/ml).
G6PD Plasmid Constructs and PC12 Stable Transfection
Full-length rat G6PD cDNA was subcloned into the
EcoRI site of the mammalian expression vector pEGFP
(Clontech) and sequenced. Constructs with the G6PD
gene inserted in the sense (pEGFP-G6PD-sense) or antisense
(pEGFP-G6PD-antisense) orientation were selected. PC12 cells were
seeded at a density of 1 × 105 cells/cm2
and transfected by lipofection (TransFast) with
pEGFP-G6PD-sense, pEGFP-G6PD-antisense, or pEGFP plasmid constructs
following the manufacturer's instructions. Forty-eight hours after
transfection, cells were passaged and incubated in culture medium
containing G418 (500 µg/ml), with a medium change twice a week to
allow the selection of stably expressing clones. Isolated colonies were transferred to 12-well plates and expanded. Different clones were subjected to Northern blotting for G6PD mRNA identification as well
as G6PD activity determination. Clones showing maximum (sense) or
minimum (antisense) G6PD activities were used for the experiments.
Measurement of Glucose Oxidation through the Pentose Phosphate
Pathway
The activity of the PPP was measured essentially as described by
Hothersall et al. (30) based on the determination of the difference in 14CO2 production from
[1-14C]glucose (decarboxylated by the 6-phosphogluconate
dehydrogenase-catalyzed reaction and by the Krebs cycle) and from
[6-14C]glucose (decarboxylated only by the Krebs cycle).
Astrocytes and neurons grown in 175-cm2 flasks were
collected by trypsinization. Cell pellets were resuspended in
O2-saturated incubation buffer (11 mM sodium
phosphate, 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4, 1.2 mM
MgSO4, and 1.3 mM CaCl2, pH 7.4).
Aliquots (500 µl) of the suspension were placed in Erlenmeyer flasks
containing 2 µCi of D-[1-14C]glucose or 8 µCi of D-[6-14C]glucose (5.5 mM
D-glucose) in either the absence (control) or presence of
SIN-1 at the indicated concentrations. Each Erlenmeyer flask was
equipped with a central well containing an Eppendorf tube containing
benzethonium hydroxide. The flask atmosphere was flushed with
O2 for 20 s, after which the flask was sealed with a
rubber cap and incubated for 5 min at 37 °C in a water bath with
shaking. Incubations were stopped by injection into the main well of
0.2 ml of 1.75 M HClO4, although shaking was
continued for a further 20 min to facilitate the entrapment of
14CO2 by benzethonium hydroxide. The
radioactivity trapped by benzethonium hydroxide was measured in
scintillation fluid (Universol, ICN Biomedicals Inc., Irvine, CA) by
liquid scintillation counting (98% efficiency; LS 6500, Beckman
Instruments). Blanks without cells were used in parallel to measure
background radioactivity, which was subtracted from the sample values.
Preliminary experiments showed that, under these conditions, the
14CO2 generated was linear with time, at least
up to 30 min, in both control and SIN-1 (up to 1 mM)-treated cells. Protein contents were determined in
10-µl aliquots of the cell suspensions. For the calculations, the
specific radioactivity (dpm/mol) of glucose was used, and the results
are expressed as pmol of glucose transformed into
CO2/min/mg of protein.
Metabolite Determinations
Total and Oxidized Glutathione--
For glutathione
determinations, cells grown in 8-cm2 wells were washed with
ice-cold phosphate-buffered saline (PBS) and immediately collected by
scraping off with 1% (w/v) sulfosalicylic acid. Cell lysates were
centrifuged at 13,000 × g for 5 min at 4 °C, and the supernatants were used for glutathione determinations on the same
day as previously described (25, 31, 32). Total glutathione contents
(GSx) were determined in comparison with GSSG standards (0-50
µM) treated in the same way as the samples. GSSG was
quantified after derivatization of GSH with 2-vinylpyridine using
similarly treated GSSG standards (0-5 µM). Results are
expressed as the oxidized glutathione status ((GSSG/GSx) × 100)
(33).
Nicotinamide-adenine Dinucleotides--
Cells seeded on
4-cm2 plates were washed with ice-cold PBS, and 250 µl of
0.5 M KOH in 50% (v/v) ethanol was immediately added, as
described by Stocchi et al. (34) for the stable extraction of both reduced and oxidized nicotinamide-adenine dinucleotides. Aliquots (200 µl) of the cell lysates were transferred to Eppendorf tubes, neutralized to a pH of 7.8 with 200 µl of 0.5 M
triethanolamine and 0.5 M potassium phosphate, and
centrifuged at 13,000 × g for 2 min at 4 °C.
Fifty-microliter aliquots of the supernatant were immediately used for
NADPH plus NADH determination by chemiluminescence as previously
described (35). For NADPH determinations, NADH was previously oxidized
by incubating the samples with 0.5 milliunits/µl lactate
dehydrogenase and 1 mM pyruvate (36). For NADP+
determinations, NADP+ was previously reduced by incubating
the samples with 5 milliunits/ml G6PD, 5 mM glucose
6-phosphate, and 5 mM MgSO4 (36). For
NAD+ determinations, NAD+ was previously
reduced by incubating the samples with 5 milliunits/µl alcohol
dehydrogenase and 172 mM ethanol (36). NADPH, NADH, NADP+, and NAD+ concentrations were calculated
by extrapolation of the sample values to their respective appropriate
standard curves obtained with pure standards (Sigma).
Glucose 6-Phosphate and 6-Phosphogluconate--
Cells were
washed with ice-cold PBS and deproteinized with 0.6 M
HClO4. Cells were scraped off the plastic dishes and
centrifuged at 20,000 × g for 10 min at 4 °C. After
neutralization of the supernatants with 5 M
K2CO3, the neutralized extracts were
lyophilized and resuspended in 250 µl of water. Glucose 6-phosphate
(37) and 6-phosphogluconate (38) concentrations were determined in these samples as described.
G6PD Activity Determinations
Cells were resuspended in 500 µl of 0.1 M
potassium phosphate buffer, pH 7.0, to give a final protein
concentration of ~1 mg/ml and homogenized with a Ultraturrax T-8
homogenizer (Ika, Staufen, Germany), and G6PD activity was determined
spectrophotometrically as previously described (39). Enzyme activity is
expressed as nmol of glucose 6-phosphate transformed per min/mg of protein.
Northern Blotting
Northern blot analysis was carried out on total RNA samples
isolated from the cells by the guanidinium isothiocyanate method as
previously described (40). The samples were electrophoresed (15 µg of
RNA/line) on a formaldehyde-containing 1% (w/v) agarose gel. After
transfer to a GeneScreen Plus membrane (PerkinElmer Life Sciences) and
cross-linking with ultraviolet irradiation (UV Stratalinker Model 2400, Genetic Research Instruments, Essex, UK), membranes were hybridized for
18 h at 65 °C in the presence of the appropriate random-primed
[ Western Blotting
Cells were scraped off the plastic dishes with lysis buffer
(12.5 mM Na2HPO4, 116 mM NaCl, 0.5 M EDTA, 1% (v/v) Triton
X-100, 0.1% (w/v) SDS, 100 µM
N G6PD Release Experiments
Astrocytes or neurons were removed from the flasks by mild
trypsinization and resuspended at a density of 20 × 106 cells/ml in prewarmed (37 °C) buffered Hanks'
solution (5.26 mM KCl, 0.43 mM
KH2PO4, 132.4 mM NaCl, 4.09 mM NaHCO3, 0.33 mM Na2HPO4, 20 mM glucose, 2 mM CaCl2, and 20 mM HEPES, pH 7.4)
containing streptolysin O (1 kilounit/ml) (42) and the protease
inhibitor mixture (see above). Cells were incubated in either the
absence (control) or presence of SIN-1 (1 mM) at 37 °C
in a water bath with shaking for 30 min (astrocytes) or 5 min
(neurons). When appropriate, some incubations were performed in the
presence of a degraded SIN-1 solution (72 h at 37 °C). After the
incubation period, aliquots of the cell suspension were centrifuged at
500 × g, and both the supernatants and pellets were
subjected to anti-G6PD antibody Western blotting as described above.
Films were scanned; the intensity of the bands was quantified; and the
values were subtracted from the background intensity values using an
image analyzer system (NIH Image), kindly supplied by Wayne Rasband (National Institutes of Health).
Formation of Endogenous Peroxynitrite
To estimate the endogenous formation of peroxynitrite from
astrocytes (43), cells were treated with LPS (1 µg/ml) for 18 h
in either the absence or presence of the peroxynitrite scavenger methionine (10 mM) (44). Peroxynitrite generation was shown by the ability of these cells to oxidize dihydrorhodamine 123 as
previously described (45). Thus, cells were incubated with dihydrorhodamine 123 (1 µg/ml) in buffered Hanks' solution for 1 h at 37 °C, and rhodamine 123 fluorescence microphotographs were taken with an inverted microscope using a fluorescein filter (excitation filter, 480-490 nm; and emission filter, 510-560 nm).
Flow Cytometric Analysis of Apoptosis
Early apoptotic cell death was determined after staining with
Alexa Fluor 488-conjugated annexin V and propidium iodide using a
commercially available kit (Molecular Probes, Inc.) following the
manufacturer's instructions. Data acquisition (~10,000 cells) was
carried out in a FACSCalibur flow cytometer (BD Biosciences) equipped
with a 15-milliwatt argon ion laser tuned at 488 nm using CellQuest
software (BD Biosciences). The analyzer threshold was adjusted on the
FSC channel to exclude noise and most of the subcellular debris.
Annexin V-stained cells that were propidium iodide-negative (annexin
V+/propidium iodide Protein Determinations
Proteins were determined in the cell lysates by the method of
Lowry et al. (46) or Bradford (41) using bovine serum
albumin as a standard.
Statistical Analysis
Results are expressed as the means ± S.E. for the number
of culture preparations indicated in the figure legends. Statistical significance was evaluated by one-way analysis, followed by the least
significant difference multiple range test. In all cases, p < 0.05 was considered significant.
Peroxynitrite Triggers a Rapid and Persistent Increase in PPP
Activity and NADPH Concentrations in Astrocytes and Neurons--
To
address the potential involvement of peroxynitrite in the regulation of
PPP activity, the differences in the rates of oxidation of
D-[1-14C]glucose and
D-[6-14C]glucose to
14CO2 (30) during a 5-min incubation period
were assessed in astrocytes and neurons in primary culture. Because of
the extreme instability of authentic peroxynitrite solutions at
physiological pH (half-life of ~1.7 s), we used SIN-1 as the
peroxynitrite donor (47, 48). Preliminary experiments using the
·NO-sensitive electrode (ISO-NO) (data not shown) suggested an approximate peroxynitrite formation from SIN-1 (1 mM) of
~20 µM, as previously reported (48). To ensure
immediate maximum peroxynitrite release from SIN-1, all
SIN-1-containing solutions were always preincubated in DMEM at 37 °C
for 20 min before addition to the cells. As shown in Fig.
1A, the PPP activity found in
control astrocytes was ~2.6-fold higher than that found in control
neurons. Incubation of both cell types with SIN-1
dose-dependently increased the PPP activity to values
ranging between ~2- or ~1.5-fold at 0.25 mM SIN-1 and
up to ~3.5- or ~2.5-fold at 1 mM SIN-1 in astrocytes or
neurons, respectively (Fig. 1A). In all cases, the observed enhancement of PPP activity by peroxynitrite treatment was due to
increased [1-14C]glucose oxidation, whereas
[6-14C]glucose oxidation remained unaltered (data not
shown).
To investigate whether the observed activation of PPP activity by
peroxynitrite was associated with NADPH generation, the intracellular
concentrations of this dinucleotide were assessed by chemiluminescence
after exposure of the cells to SIN-1. As shown in Fig. 1B,
NADPH concentrations were ~4-fold higher in control astrocytes than
in control neurons. Incubation of the cells with SIN-1 for 10 min
dose-dependently increased NADPH concentrations by ~1.8-
and ~4.4-fold at 1 mM SIN-1 in astrocytes and neurons, respectively (Fig. 1B). To test whether peroxynitrite was
the molecule responsible for the observed SIN-1-mediated increase in
NADPH concentrations, a solution of SIN-1 (1 mM) was
preincubated in DMEM at 37 °C for 72 h to ensure complete
degradation of SIN-1 before addition to neurons. Under these
conditions, incubation of the neurons at 37 °C for 5 min with
degraded solutions of SIN-1 showed no effects on NADPH levels compared
with control (untreated) cells, whereas "active" SIN-1 triggered an
~4-fold increase (Fig. 1C). To further confirm that
peroxynitrite formation from SIN-1 was responsible for the observed
effects on NADPH concentrations, we used TEMPOL (500 mM) or
hemoglobin (50 µM) to scavenge O
Measurements of NADPH levels for short incubation periods revealed that
the increase in the concentrations of this dinucleotide by SIN-1 was
very rapid (within 1 min) in both cell types (Fig. 1D).
Furthermore, incubation of the cells in the presence of SIN-1 for
longer times revealed that the increase in NADPH concentrations observed at 10 min was transient in astrocytes, but not in neurons (Fig. 1, B-D). Thus, after 30 min of incubation, the
SIN-1-mediated rise in NADPH concentrations was progressively
attenuated, with a modest, although statistically significant,
~1.2-fold value being reached at 24 h (Fig. 1E). In
contrast to astrocytes, neuronal NADPH concentrations progressively
increased after the first minute of incubation with SIN-1, reaching up
to ~5.2-fold at 30 min and ~11-fold at 4 h of incubation (Fig.
1F). Furthermore, neuronal NADPH concentrations were
maintained at high levels along during the incubation time in the
presence of SIN-1, for at least up to 24 h (Fig.
1F).
Peroxynitrite Triggers Activation of the G6PD-catalyzed
Reaction--
To investigate the mechanism through which peroxynitrite
triggered the rapid activation of PPP activity and NADPH
concentrations, we determined the levels of nicotinamide-adenine
dinucleotides in peroxynitrite-treated astrocytes. As shown in Fig.
2A, the increase in NADPH
concentrations observed after 30 min of incubation with SIN-1 (1 mM) was accompanied by a parallel decrease in
NADP+ levels, suggesting that at least some of the NADPH
produced would be obtained from NADP+ reduction. However,
the decreased amount of NADP+ (~50 pmol/mg of protein)
per se could not account for the increased amount of NADPH
(~130 pmol/mg of protein) (Fig. 2A). NAD+
concentrations were noticeably decreased by ~100 pmol/mg of protein, and NADH levels by ~40 pmol/mg of protein following peroxynitrite treatment (Fig. 2A). Furthermore, SIN-1 treatment (1 mM, 30 min) elicited a decrease in glucose 6-phosphate
concentrations (by 40%) and an increase in 6-phosphogluconate
concentrations (by ~5-fold) (Fig. 2B), strongly suggesting
that peroxynitrite activates the G6PD-catalyzed reaction,
i.e. the first rate-limiting step in the PPP (22). To
investigate the possible activation of G6PD by peroxynitrite directly,
the activity of G6PD was determined spectrophotometrically in the
homogenates obtained from control (untreated) or SIN-1-treated
astrocytes. The results showed that, despite the biochemical evidence
for G6PD activation (Fig. 2B), total G6PD was unmodified by
peroxynitrite treatment (Fig. 2C). In this context, previous
observations by Stanton et al. (42) have demonstrated that
inactive, structural intracellular element-bound G6PD would be
prone to rapid activation through the release of G6PD into the
cytosolic fraction by treatment of renal cortical cells with growth
factors. Accordingly, we decided to investigate whether
peroxynitrite-mediated G6PD activation could be due to possible enzyme
release. Thus, astrocytes were collected, and the cell suspension was
permeabilized by mild treatment with streptolysin O (42) and exposed or
not (control) to active SIN-1 (1 mM) or degraded
SIN-1. After 30 min, the cell suspensions were centrifuged, and both
the cytosolic supernatants and pellets were separately subjected to
Western blotting using anti-G6PD antibody. The results showed that,
whereas most of the G6PD remained in the pellets, a significant amount
of it appeared in the supernatant of SIN-1-treated cells compared with
control or degraded SIN-1-treated cells (band intensity in the
supernatants, 16.6 ± 0.2 and 36.6 ± 0.1 arbitrary units for
degraded SIN-1 and SIN-1, respectively) (Fig. 2D). These results suggest that peroxynitrite would activate G6PD activity by
promoting the release of G6PD to the cytosol from a structural intracellular element.
Evidence for Activation of the G6PD-catalyzed Reaction by
Endogenous Peroxynitrite in Astrocytes--
To assess whether
endogenous peroxynitrite promoted G6PD activation, astrocytes were
incubated with LPS (1 µg/ml) for 18 h as previously reported
(43). Endogenous peroxynitrite production was evidenced by the ability
of LPS-treated cells to oxidize dihydrorhodamine 123 to rhodamine 123 (45). Thus, LPS increased rhodamine 123 fluorescence, an effect that
was prevented by co-incubation with the peroxynitrite scavenger
methionine (10 mM) (Fig.
3A) (44). As shown in Fig.
3B, LPS elicited a decrease in glucose 6-phosphate concentrations (by ~3-fold) and an increase in 6-phosphogluconate concentrations (by ~2.5-fold), strongly suggesting G6PD activation. Furthermore, these effects were counteracted by scavenging
peroxynitrite with methionine (Fig. 3B).
Evidence for G6PD Activation by Peroxynitrite in Neurons--
To
further confirm that peroxynitrite was responsible for the increase in
neuronal NADPH concentrations, we used a solution of authentic
peroxynitrite (ONOO Overexpression of the G6PD Gene Increases and Inhibition of G6PD
Gene Expression Decreases NADPH Concentrations in PC12 Cells--
To
assess the essential role played by G6PD activity in generating
intracellular NADPH, we used a transgene approach to endogenously modulate G6PD gene expression. Thus, we obtained stable G6PD
gene-expressing cells by transfecting PC12 cells with pEGFP mammalian
plasmid vectors into which the full-length G6PD gene had been inserted in either the sense (PC12-pEGFP-G6PD-sense) or antisense
(PC12-pEGFP-G6PD-antisense) orientation. Transfection of PC12 cells
with the vector alone (PC12-pEGFP) was also carried out as a control.
Stably expressing cells were selected and analyzed for G6PD
mRNA levels, G6PD-GFP fusion protein levels, G6PD activity, and
intracellular NADPH concentrations. Northern blot analyses showed that
control cells (untransfected PC12 and transfected PC12-pEGFP)
expressed endogenous G6PD mRNA (Fig.
5A). Furthermore, Western blot
analyses using anti-GFP antibody revealed an ~27-kDa band in
PC12-pEGFP cells that was not present in PC12 cells, confirming
successful expression of the GFP protein in the former cells (Fig.
5A). Northern blot analyses of sense G6PD
gene-overexpressing cells (PC12-pEGFP-G6PD-sense) revealed an
additional mRNA band corresponding to the G6PD-GFP mRNA
transcript (Fig. 5A). Western blot analyses of
PC12-pEGFP-G6PD-sense cell protein extracts using anti-GFP antibody
disclosed an ~86-kDa band, confirming successful expression of the
G6PD-GFP fusion protein (Fig. 5A). Analysis of these protein
extracts by Western blotting failed to detect any anti-G6PD
immunopositive fusion band, probably due to the interference of the
fused GFP moiety in antibody recognition. Northern and Western blot
analyses of antisense G6PD gene-overexpressing cells
(PC12-pEGFP-G6PD-antisense) revealed a weak G6PD-GFP mRNA
band and undetectable GFP or G6PD-GFP protein (Fig. 5A),
probably due to the increased degradation of newly synthesized G6PD
mRNA, as judged by the proposed mechanism for antisense
gene-silencing methods (50, 51). Interestingly, both G6PD activity and
NADPH concentrations were significantly higher (~1.5-fold) in
PC12-pEGFP-G6PD-sense cells compared with control (untransfected) and
pEGFP-transfected cells (Fig. 5B). In contrast, G6PD
activity and NADPH concentrations in PC12-pEGFP-G6PD-antisense cells
were significantly lower (by ~24%) (Fig. 5B). These
results strongly confirm the functional efficiency of G6PD transgene
expression in these cells and point to the essential role played by
G6PD activity in supporting an enhanced intracellular pool of
NADPH.
Peroxynitrite Transiently Prevents Nitric Oxide-mediated Neuronal
NADPH Depletion, Glutathione Oxidation, and
Apoptosis--
Previous results from our laboratory (52)
demonstrated that the exposure of neurons to ·NO causes
apoptosis, whereas astrocytes remain unaffected. Among other possible
mechanisms, this differential intercellular susceptibility appears to
involve the higher antioxidant reserve, such as glutathione concentrations, found in astrocytes compared with neurons (17, 53). We
were therefore prompted to investigate whether the
peroxynitrite-mediated activation of PPP activity and increased NADPH
concentrations observed here might serve as a potential neuroprotective
mechanism against ·NO-mediated glutathione oxidation and
cytotoxicity. Astrocytes and neurons were incubated in the presence of
the ·NO donor DETA-NO (1 mM), a compound widely used
as a long-term chemical source of ·NO (54). DETA-NO (1 mM) was seen to continuously release ~2.8 µM ·NO for ~24 h in DMEM at 37 °C as measured
by an ·NO-sensitive electrode. To ensure immediate maximum
·NO release from DETA-NO, all DETA-NO-containing solutions were always preincubated in DMEM at 37 °C for 20 min before addition to
the cells. Exposure of astrocytes to ·NO for 4 h had no
detectable effect on NADPH concentrations, the GSSG/GSx ratio, or
apoptotic cell death (Table I). In
contrast to astrocytes, neurons were highly susceptible. Thus, exposure of neurons to ·NO triggered an ~50% decrease in NADPH
concentrations, a 2-fold increase in the GSSG/GSx ratio, and a 1.7-fold
increase in the proportion of apoptotic cells (Table I). Neuronal NADPH
depletion by DETA-NO (1 mM) treatment occurred very
rapidly, as judged by the 30% decrease in NADPH concentrations
observed after a 5-min incubation period (Table
II). Furthermore, a solution of degraded DETA-NO (incubation of 1 mM DETA-NO in DMEM at 37 °C for
48 h) had no effect, and a bolus of authentic ·NO (10 µM) decreased NADPH concentrations in neurons by
23% after a 5-min incubation period (Table II).
We next investigated whether the stimulation of PPP activity brought
about by peroxynitrite might protect neurons against ·NO-mediated oxidative damage. To perform these experiments,
neurons were incubated under four different conditions, (a)
control (untreated) neurons incubated in DMEM for 9 h;
(b) neurons preincubated with SIN-1 for 1 h, followed
by washing with PBS, and then incubated in DMEM for a further 8 h;
(c) neurons incubated with DETA-NO for 8 h; and
(d) neurons preincubated with SIN-1 for 1 h, followed by washing with PBS, and incubated with DETA-NO for a further 8 h.
As shown in Fig. 4A, incubation of neurons with DETA-NO
markedly and persistently decreased neuronal NADPH concentrations over the 8-h incubation period. By contrast, treatment of neurons with SIN-1
for 1 h increased NADPH concentrations (Fig.
6A; see also Fig.
1F). This effect was transient, as judged by the observed progressive decrease in the concentrations of this dinucleotide after
SIN-1 removal. Noticeably, despite this progressive decrease, NADPH
concentrations remained at levels that were significantly higher than
those observed in control neurons, at least up to the end of the 8-h
incubation period (Fig. 6A). Interestingly, preincubation of
neurons with SIN-1 for 1 h fully or partially prevented the
DETA-NO-mediated decrease in NADPH concentrations, at least during the
following 8-h incubation period (Fig. 6A).
To assess the glutathione redox status in neurons subjected to an
identical treatment, we determined oxidized (GSSG) and total (GSx)
glutathione concentrations in these cells, expressing the values as the
GSSG/GSx ratio (oxidized glutathione status) (33). As shown in Fig.
6B, the neuronal oxidized glutathione status remained
unaltered over the 8-h incubation period following SIN-1 pretreatment,
although it increased after 24 h (control, 5.2 ± 0.5; and
SIN-1, 4.8 ± 0.2; n = 3). SIN-1 pretreatment
noticeably prevented the long-term oxidation (8 h) of glutathione
observed in control cells. In contrast to SIN-1, the exposure of
neurons to DETA-NO significantly increased the oxidized glutathione
status from 1 h up to 8 h of incubation (Fig. 6B).
Also, pretreatment of neurons with SIN-1 for 1 h partially
prevented DETA-NO-mediated glutathione oxidation during the incubation
period between 1 and 4 h, but failed to protect glutathione
oxidation after 8 h (Fig. 6B). It should be mentioned
that the changes in the GSSG/GSx ratio observed were due to changes in
GSSG (not GSx) concentrations (values for control (2 h), SIN-1
(1 h) plus none (1 h), DETA-NO (1 h), and SIN-1 (1 h) plus DETA-NO (1 h) were 0.30 ± 0.03, 0.30 ± 0.02, 0.45 ± 0.02 (p < 0.05 versus control), and 0.34 ± 0.02 nmol/mg of protein for GSSG and 13.1 ± 0.6, 12.9 ± 0.3, 12.9 ± 0.6, and 13.1 ± 1.0 nmol/mg of protein for GSx, respectively).
To investigate whether the peroxynitrite-mediated protection of
cellular oxidation dictated cellular survival, we determined the
proportion of apoptotic cells, i.e. propidium
iodide-negative neurons showing a positive immunoreaction to annexin V
(% of annexin V+/propidium iodide Overexpression of the G6PD Gene Protects and Inhibition of G6PD
Gene Expression Enhances Nitric Oxide-mediated Apoptotic Death in
PC12 Cells--
In view of the biochemical evidence suggesting a key
role for G6PD activity in peroxynitrite-mediated protection against
·NO, we investigated whether the endogenous modulation of G6PD transgene expression might be responsible for ·NO-mediated
neurotoxicity. Accordingly, PC12-pEGFP cells were incubated in either
the absence (control) or presence of DETA-NO (0.5 mM;
~1.4 µM ·NO) for 60 min. At 10-min intervals,
aliquots from the cell suspension were analyzed for annexin
V+/propidium iodide The correlation between rapid activation of PPP activity and NADPH
accumulation described here upon peroxynitrite treatment confirms the
well known NADPH-generating function of the PPP (22). Due to its
relatively low concentrations, NADP+ availability
alone could not account for NADPH accumulation. However, the
decrease in NAD+ concentrations brought about by
peroxynitrite suggests that NAD+ phosphorylation may
provide sufficient NADP+ for ready conversion into NADPH
through G6PD activity (55-57). Accordingly, our data are compatible
with the notion that peroxynitrite would stimulate G6PD activity, as
demonstrated by the increase in the 6-phosphogluconate/glucose
6-phosphate ratio by either exogenous or endogenous peroxynitrite. This
hypothesis was further examined in PC12 cells stably expressing plasmid
constructs into which the G6PD gene had been inserted in either the
sense or antisense orientation. As judged by the good correlation
between the G6PD activity and NADPH concentrations observed in these
cells, our results confirm the idea that the modulation of G6PD
activity does dictate endogenous NADPH concentrations (58, 59).
Previous studies carried out on several cell types have already
suggested the essential role of PPP and/or G6PD activity in cellular
protection against H2O2-induced glutathione
oxidation (18-21, 23). Furthermore, our previous work carried out in
LPS-activated astrocytes (25) suggested a critical role for G6PD
induction in astrocyte autoprotection. However, the rapid activation of G6PD activity by peroxynitrite observed here rules out any possible transcriptional effect. Because peroxynitrite failed to alter total
G6PD activity, we investigated whether any changes in subcellular enzyme localization might be responsible for the rapid activation. In
this context, previous work focused on studying the regulation of G6PD
activity by growth factors in renal cells (42) has elegantly demonstrated that a rapid stimulation of G6PD activity occurs through
enzyme release from a structural intracellular element. Our data from
Western blot analyses demonstrated that peroxynitrite triggers G6PD
release from both astrocytes and neurons, suggesting that such a
mechanism would be a feasible explanation for the observed
peroxynitrite-mediated G6PD activation.
Astrocytes have efficient self-protective systems, such as active
glycolytic (52, 60) and de novo glutathione-synthesizing (13, 33, 53, 61) pathways or higher superoxide dismutase expression
(62), which account for the resistance shown by glial cells to a wide
range of insults, including ·NO exposure (this work). In
contrast to astrocytes, neurons are vulnerable cells that, upon
exposure to either endogenous (9, 63) or exogenous (52) excess
·NO, rapidly (within 1 h) undergo apoptotic death (this
study). Pretreatment of neurons with peroxynitrite fully prevented
apoptosis shortly (1 h) after ·NO treatment, although this
protection progressively decreased thereafter (to ~60% protection
after 4 h and to ~25% after 8 h). Furthermore, these
changes in peroxynitrite-mediated neuroprotection showed a good time
course correlation with the observed changes in the glutathione redox
status. Because glutathione oxidation has been implicated in
·NO-mediated neuronal apoptosis (13, 63-65), it is tempting to speculate that the neuroprotection exerted by peroxynitrite would be
associated with its ability to activate PPP activity and to generate
NADPH. Thus, during the period of maximum protection (i.e.
from 1 to 4 h after peroxynitrite treatment), neuronal NADPH levels were ~200-350 pmol/mg of protein. Assuming a cell volume of
~4 µl/mg of protein (66), the concentrations of NADPH would be
~50-100 µM, i.e. within the published
glutathione reductase km values for NADPH (~8-61
µM for bovine brain; reviewed in Ref. 67). Accordingly,
it is likely that the maintenance of the reduced status of GSH after
peroxynitrite treatment would occur at the expense of increased NADPH
availability to serve as the cofactor for glutathione reductase activity.
Because peroxynitrite-mediated neuroprotection against ·NO would
be associated with its ability to stimulate G6PD activity, we were
prompted to investigate the possible role played by the modulation of
G6PD gene expression in ·NO-mediated apoptosis. Our results
showed that PC12 cells expressing the sense G6PD gene offered
remarkable resistance to ·NO-mediated apoptosis, whereas cells
expressing the antisense G6PD gene showed a considerably higher
susceptibility. These results confirm the notion that G6PD activity
would play an essential role in preventing oxidative/nitrosative
cellular damage (23, 58, 59) and suggest a key role for G6PD in
neuroprotection. However, it should be noted that, besides G6PD, other
well known NADPH-generating systems have also been proposed to
contribute to the protection against oxidative damage, such as
cytosolic NADP+-dependent isocitrate
dehydrogenase (68), the PPP non-oxidative branch rate-limiting enzyme
transaldolase (24), and NADP+-dependent malic
enzyme (22). Whether peroxynitrite would be involved in the modulation
of such antioxidant systems is unknown and deserves further research.
The transient neuroprotective role for peroxynitrite described here is
in apparent contradiction with the widely held assumption that
peroxynitrite would be the ·NO-derived neurotoxic effector
molecule (7-9, 17). Indeed, peroxynitrite interferes with key
energy-related targets, such as aconitase (69), poly(ADP-ribose)
synthetase (70-74), and mitochondrial respiratory chain complexes (16,
17, 75, 76). Because neurons are strong energy-demanding cells, these
phenomena would affect neuronal survival. In our hands, peroxynitrite
failed to cause neuronal apoptotic death up to 8 h. However, it
was highly cytotoxic when the incubation time was extended up to
24 h, in good agreement with previous reports (9, 77). Therefore, the relationship between peroxynitrite-mediated interference with key
energy-related metabolic targets and the time course of the observed
neurotoxicity is an issue that should be revisited. It is also
interesting to note that Clancy et al. (12) previously found
that exposure of human neutrophils to ·NO is associated with
O In conclusion, we have show here that peroxynitrite plays a novel
neuroprotective role against ·NO-mediated apoptotic cell death.
The mechanism responsible for this phenomenon would involve G6PD
release from a structural intracellular element, leading to enzyme
activation, followed by NADPH generation through PPP activity. The G6PD
transgene approach confirmed the critical neuroprotective role played
by this enzyme against ·NO-mediated apoptosis. Furthermore,
given the essential role of NADPH as a cofactor for glutathione
regeneration, our results may contribute to the elucidation of
mechanisms through which neurons would be able to protect themselves
against ·NO-mediated glutathione oxidation and apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), a pro-oxidant molecule thought to execute the
neurotoxic ·NO-mediated responses (7-10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
302 = 1670 M
1 cm
1) as previously described
(27). Alkaline stock solutions with an approximate ONOO
concentration of 0.5 M were stable at
70 °C for at
least 3-4 months. Nitric oxide was purchased from Al Air Liquide
(Madrid, Spain) and extemporarily dissolved in O2-free
water by 20-min bubbling until saturation. The concentration of
·NO solutions was estimated with an ·NO electrode
(ISO-NO, World Precision Instruments, Inc.) using the
·NO-saturated solution as a standard (~2 mM
·NO). Dulbecco's modified Eagle's medium (DMEM), LPS,
dihydrorhodamine 123, TEMPOL
(4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), and hemoglobin were
obtained from Sigma. Fetal calf serum was purchased from Roche
Diagnostics (Heidelberg, Germany). Type I rat tail collagen was from BD
Biosciences. D-[1-14C]Glucose and
D-[6-14C]glucose were obtained from
ARC Inc. (St. Louis, MO), and [
-32P]dCTP and
Hybond® nitrocellulose membranes were from Amersham
Biosciences (Buckinghamshire, UK). The peroxynitrite donor SIN-1
(3-morpholinosydnonimine) and the ·NO donor diethylenetriamine
(DETA)-NO were purchased from Alexis Corp. (San Diego, CA).
2-Vinylpyridine was from Aldrich (Gillingham-Dorset, UK). Plastic
tissue culture dishes were purchased from Nunc (Roskilde, Denmark).
Anti-G6PD antiserum was a generous gift from Prof. Matilde V. Ursini
(Instituto Internazionale di Genetica e Biofisica, Consiglio Nazionale
delle Ricerche, Naples, Italy). Anti-green fluorescent protein (GFP)
antibody was obtained from Clontech (Palo Alto, CA), and anti-rabbit secondary antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA). The Vybrant apoptosis assay kit was
purchased from Molecular Probes, Inc. (Eugene, OR), and TransFast
transfection reagent and the antibiotic G418 were from Promega
(Madison, WI). Other substrates, enzymes, and coenzymes were purchased
from Sigma, Roche Diagnostics, or Merck (Darmstadt, Germany).
-32P]dCTP-radiolabeled cDNA probes and exposed to
Eastman Kodak XAR-5 film. As cDNA probes, we used either the 2.2-kb
cDNA encoding the full-length rat G6PD gene (a generous gift from
Dr. Ye-Shih Ho, Wayne State University) or a 0.7-kb cDNA fragment
of the rat cyclophilin gene (generously donated by Dr. Dionisio
Martín-Zanca). Cyclophilin mRNA was used as a control of
the amount of total RNA loaded in each lane.
-p-tosyl-L-lysine
chloromethyl ketone, 100 µM phenylmethylsulfonyl fluoride, 1 mM phenanthroline, 10 µg/ml pepstatin A, 100 µM N-tosyl-L-phenylalanine chloromethyl ketone, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml soybean trypsin inhibitor, pH 7), and aliquots containing 100 µg of protein from each sample, extemporarily determined following the method of Bradford (41) using ovalbumin as a standard and the
BenchMarkTM prestained protein ladder (Invitrogen), were
electrophoresed on 10% acrylamide gel (MiniProtean®,
Bio-Rad) and transferred to a Hybond® nitrocellulose
membrane. Membranes were blocked with 5% (w/v) low-fat milk/TBST
buffer (20 mM Tris, 500 mM NaCl, and 0.1%
(w/v) Tween 20, pH 7.5) for 1 h and then incubated in the presence
of the appropriate antibody (either anti-G6PD at 1:1000 dilution or
anti-GFP at 1:250 dilution) at 4 °C overnight. After washing, membranes were further incubated in 3% (w/v) low-fat milk/TBST buffer
for 45 min at room temperature in the presence of horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody at 1:40,000 dilution and immediately incubated with the luminol chemiluminescence reagent (Santa Cruz Biotechnology) for 1 min before being exposed to
HyperfilmTM chemiluminescence film for 3 min (anti-G6PD
antibody) or 25 min (anti-GFP antibody).
) were considered to be apoptotic.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Peroxynitrite triggers a rapid and persistent
increase in PPP activity and NADPH concentrations in astrocytes and
neurons. A, for the measurement of PPP activity, cell
suspensions (15 × 106 cells/ml) were incubated in the
presence of appropriate concentrations of SIN-1 in
O2-saturated incubation buffer, pH 7.4, containing either 2 µCi of D-[1-14C]glucose or 8 µCi of
D-[6-14C]glucose (5.5 mM
D-glucose) at 37 °C for 5 min. The
14CO2 released by the cells was trapped and
quantified for the estimation of PPP activity as described under
"Experimental Procedures." B-F, for the determination
of NADPH concentrations, cells were incubated in DMEM in the absence
(control) or presence of SIN-1 at the indicated concentrations and with
TEMPOL or hemoglobin when appropriate (C) for 1 min to
24 h (10 min in B and 5 min in C) and
digested in KOH/ethanol for NADPH determination by chemiluminescence as
described under "Experimental Procedures." Data are the means ± S.E. obtained from three to four different experiments. *,
p < 0.05 compared with the corresponding control (0 mM SIN-1) values; #, p < 0.05 compared
with the corresponding values found in neurons.
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Fig. 2.
Peroxynitrite triggers activation of the
G6PD-catalyzed reaction. Astrocytes were incubated at 37 °C for
30 min in either the absence (control) or presence of SIN-1 (1 mM) as well as, where indicated, a degraded solution of
SIN-1. A, for the determination of NAD concentrations, cells
were digested in KOH/ethanol and used for the NADPH, NADH,
NADP+, and NAD+ chemiluminescence assays
as described under "Experimental Procedures." B, glucose
6-phosphate and 6-phosphogluconate concentrations were determined in
the neutralized perchloric acid cell extracts. C, G6PD
activity was determined spectrophotometrically in the cell homogenates.
D, for the assessment of G6PD release, cell suspensions
(20 × 106 cells/ml) were incubated in Hanks'
solution containing streptolysin O (1 kilounit/ml) plus SIN-1 (1 mM) at 37 °C for 30 min, and aliquots of these
suspensions were centrifuged before anti-G6PD antibody Western blot
analyses of both the supernatant and pellets. Data are the means ± S.E. obtained from three different experiments. *, p < 0.05 compared with the corresponding control values.
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Fig. 3.
Evidence for activation of the G6PD-catalyzed
reaction by endogenous peroxynitrite in astrocytes. Astrocytes
were incubated at 37 °C for 18.5 h in either the absence
(control) or presence of LPS (1 µg/ml) alone or in combination with
methionine (10 mM). A, dihydrorhodamine 123 oxidation was examined by fluorescence microscopy as indicated under
"Experimental Procedures." B, glucose 6-phosphate and
6-phosphogluconate concentrations were determined in the neutralized
perchloric acid cell extracts. Data are the means ± S.E. obtained
from three different experiments. *, p < 0.05 compared
with the corresponding control values.
, stock alkaline solutions of ~0.5
M, synthesized in our laboratory). Due to its extreme
instability (half-life of ~1.7 s at pH 7.4), neurons were exposed to
repeated pulses (10 pulses, one pulse every 30 s) of peroxynitrite
(final concentrations of 50 and 100 µM) during the 5-min
incubation period. Under these conditions, our results showed that
exposure of neurons to authentic peroxynitrite increased NADPH
concentrations by ~1.8-fold (50 µM ONOO
)
or ~4-fold (100 µM ONOO
) (Fig.
4), whereas a degraded ONOO
solution (1 mM, 30 min, 37 °C) had no effect on NADPH
concentrations (Fig. 4A). To investigate whether the
peroxynitrite-mediated G6PD enzyme release observed in astrocytes also
occurred in neurons, these cells were collected, and the cell
suspension was exposed to active SIN-1 (1 mM) or degraded
SIN-1. After 5 min, the cell suspensions were centrifuged, and the
cytosolic supernatants were subjected to Western blotting using
anti-G6PD antibody. The results showed a significant increase in G6PD
protein in the supernatant of SIN-1-treated neurons compared with
degraded SIN-1-treated cells (band intensity in the supernatants,
20.8 ± 0.1 and 32.6 ± 0.1 arbitrary units for degraded
SIN-1 and SIN-1, respectively) (Fig. 4B).
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Fig. 4.
Evidence for G6PD activation by peroxynitrite
in neurons. A, neurons were incubated for 5 min in the
presence of peroxynitrite, which was added to the cells in 10 pulses
(one pulse every 30 s) of either 50 or 100 µM (final
concentration, in Hanks' solution) from a 0.5 M stock
solution. Cells were digested in KOH/ethanol for NADPH determination by
chemiluminescence as described under "Experimental Procedures."
B, for the assessment of G6PD release, cell suspensions
(20 × 106 cells/ml) were incubated in Hanks'
solution containing SIN-1 (1 mM) at 37 °C for 5 min, and
aliquots of these suspensions were centrifuged before anti-G6PD
antibody Western blot analyses of the supernatant. Data are the
means ± S.E. obtained from three to four different experiments.
*, p < 0.05 compared with control or degraded
ONOO values.
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Fig. 5.
Overexpression of G6PD increases and
inhibition of G6PD expression decreases NADPH concentrations in PC12
cells. Cells were stably transfected with pEGFP plasmid vectors
into which the G6PD gene had been inserted in either the sense
(PC12-pEGFP-G6PD-sense) or antisense (PC12-pEGFP-G6PD-antisense)
orientation. As transfection controls, cells were also transfected with
pEGFP alone (PC12-pEGFP). A, total RNA and protein samples
were extracted from these clones and subjected to Northern (15 µg/lane using either a G6PD or cyclophilin cDNA probe) and
Western (100 µg/lane using anti-GFP antibody) blot analyses,
respectively. B, G6PD activity and NADPH concentrations were
determined in either cell homogenates (in 0.1 M phosphate
buffer, pH 7.0) or alkaline extracts (KOH/ethanol), respectively, as
described under "Experimental Procedures." *, p < 0.05 compared with the corresponding PC12 and PC12-pEGFP values.
Effect of nitric oxide on NADPH levels, oxidized glutathione status,
and apoptotic cell death in astrocytes and neurons in primary culture
Rapid NADPH depletion by nitric oxide in neurons in primary culture
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Fig. 6.
Peroxynitrite transiently prevents nitric
oxide-mediated neuronal NADPH depletion, glutathione oxidation, and
apoptosis. Neurons were preincubated in the absence (control) or
presence of SIN-1 (1 mM) at 37 °C for 1 h in DMEM.
After removal of the SIN-1 solutions, the cells were washed with PBS
and then further incubated in either the absence (SIN-1 + none) or presence (SIN-1 + DETA-NO) of DETA-NO (1 mM) in DMEM at 37 °C for 8 h. One group of cells
was not preincubated with SIN-1 and was incubated only in the presence
of DETA-NO (1 mM) (none + DETA-NO) for 8 h.
NADPH concentrations (A), total (GSx) and oxidized (GSSG)
glutathione concentrations (B), and the proportion of
apoptotic cells (% of annexin V+/propidium
iodide (PI
)) (C) were
determined as described under "Experimental Procedures." Data are
the means ± S.E. obtained from three to four different
experiments. *, p < 0.05 compared with the
corresponding control values; #, p < 0.05 compared
with the corresponding none + DETA-NO values.
cells), by
flow cytometry. As shown in Fig. 6C, preincubation of
neurons with SIN-1 for 1 h had no effect on the proportion of
apoptotic neurons compared with control cells, at least up to 8 h
of incubation. In contrast, incubation of neurons with DETA-NO
triggered cellular apoptosis in a time-dependent fashion (Fig. 6C). However, a 1-h pretreatment of neurons with SIN-1
fully (>1 h) or partially (4-8 h) prevented DETA-NO-mediated
neurotoxicity (Fig. 6C). Because a considerable body of
earlier work (e.g. Refs. 8, 9, and 17) has consistently
reported the long-term neurotoxicity (~24 h) of peroxynitrite, we
also investigated this possibility. Indeed, both a 1-h pretreatment of
neurons with SIN-1 (1 mM), followed by a 23-h incubation in
DMEM, and a continuous exposure (24 h) of neurons to SIN-1 (1 mM) significantly increased apoptotic cell death (control
at 24 h, 19.0 ± 0.6%; a 1-h incubation with SIN-1, followed
by a 23-h incubation in DMEM, 50.6 ± 5.1%; and a 24-h incubation
with SIN-1, 57.6 ± 0.7%). Finally, to further confirm that
ONOO
was the species responsible for the protection shown
by the neurons against ·NO-mediated apoptosis, the cells were
treated with ONOO
(10 pulses of 50 µM
ONOO
for 5 min) before exposure to DETA-NO (1 mM, 30 min). As shown in Table
III, the ~1.4-fold increase in
apoptotic neurons observed after 30 min of incubation with DETA-NO was
prevented by pretreatment with ONOO
.
Peroxynitrite pretreatment prevents nitric oxide-mediated neuronal
apoptotic death
(10 pulses of 50 µM for 5 min) or with a degraded solution of
ONOO
(0.5 mM; control) at 37 °C for 5 min in
buffered Hanks' solution. After removal of the buffer, the cells were
washed with PBS and then further incubated in the presence of DETA-NO
(1 mM) at 37 °C in DMEM for 30 min. The proportion of
apopotic cells (% of annexin V+/propidium iodide
(PI
)) was determined as described under "Experimental
Procedures." Data are the means ± S.E. from three different
experiments.
cells by flow cytometry.
As shown in Fig. 7A, DETA-NO
caused a rapid (20 min) and time-dependent increase (by
~3-fold at 60 min) in the proportion of apoptotic cells. To compare
the differential susceptibility of G6PD modulation to
·NO-mediated apoptosis, these cells were incubated in the
presence of DETA-NO (0.5 mM) for 30 and 60 min and
subjected to annexin V+/propidium iodide
assessment. As shown in Fig. 7B, the proportion of apoptotic PC12 cells increased by ~1.9-fold after 30 min and by ~1.5-fold after 60 min of incubation in the presence of DETA-NO. Intriguingly, cells transfected with the vector alone (PC12-pEGFP) showed higher susceptibility to DETA-NO (~2.5- and ~3-fold increase in
apoptotic cells after 30 and 60 min, respectively). However, cells
overexpressing the sense G6PD gene (PC12-pEGFP-G6PD-sense) showed a
remarkable resistance to DETA-NO-mediated apoptotic cell death (Fig.
7B). In contrast, cells overexpressing the antisense G6PD
gene (PC12-pEGFP-G6PD-antisense) showed marked enhanced susceptibility
to DETA-NO-mediated apoptotic cell death (~3.8- and ~5.6-fold
increase in apoptotic cells after 30 and 60 min, respectively) (Fig.
7B). Intriguingly, expression of the vector alone
(PC12-pEGFP) slightly increased the proportion of apoptosis (Fig.
7B).
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Fig. 7.
Overexpression of G6PD protects and
inhibition of G6PD enhances nitric oxide-mediated apoptotic death in
PC12 cells. PC12 cells or PC12 cells stably expressing the vector
alone (PC12-pEGFP) or pEGFP mammalian plasmid vectors with the G6PD
gene inserted in either the sense (PC12-pEGFP-G6PD-sense) or antisense
(PC12-pEGFP-G6PD-antisense) orientation were incubated in the absence
(control) or presence of DETA-NO (0.5 mM) in Hanks'
solution at 37 °C for the time periods indicated. The proportion of
apoptotic cells (% of annexin V+/propidium
iodide (PI
)) was determined by
flow cytometry as described under "Experimental Procedures." Data
are the means ± S.E. obtained from three to four different
experiments. *, p < 0.05 compared with the
corresponding control values; #, p < 0.05 compared
with PC12-pEGFP cells.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We are especially grateful to Dr. Ralf Dringen (University of Tübingen, Tübingen, Germany) for excellent critical reading of the manuscript and advice.
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FOOTNOTES |
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* This work was supported in part Ministerio de Ciencia y Tecnología Grant SAF2001-1961, Junta de Castilla y León Grant SA065/01, and the Fundación Ramón Areces, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a predoctoral fellowship from the Fundación Ramón Areces.
To whom correspondence should be addressed: Dept. de
Bioquímica y Biología Molecular, Universidad de
Salamanca, Edificio Departamental, Plaza Doctores de la Reina s/n,
37007 Salamanca, Spain. Tel.: 34-923-294526; Fax: 34-923-294579;
E-mail: jbolanos@usal.es.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206835200
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; G6PD, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway; DMEM, Dulbecco's modified Eagle's medium; DETA, diethylenetriamine; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GSx, amount of GSH plus 2 times the amount of GSSG.
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