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Vol. 282, Issue 3, 1600-1607, 1997
Brain Tumor Research Center and CNS Injury & Edema Research Center, Dept. of Neurological Surgery, University of California, San Francisco, California
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Nitric oxide and superoxide are free radicals that appear to contribute to the pathogenesis of a number of brain disorders, and cerebral endothelial cells are a potential target of these agents. Because of the capacity for these two agents to combine, it has been suggested that nitric oxide might either enhance or inhibit the toxic effects of superoxide. To establish the effect of the generation of superoxide and nitric oxide alone and in combination, cerebral endothelial cells were exposed to sodium nitroprusside, a source of nitric oxide, and/or paraquat, a source of superoxide. Paraquat enhanced the toxicity of sodium nitroprusside, as did diethyldithiocarbamate, an inhibitor of superoxide dismutase, which supports the hypothesis that enhanced levels of superoxide can combine with nitric oxide to form a more toxic product. Also, the toxicity of paraquat could be partially inhibited by blocking endogenous nitric oxide synthesis using NG-monomethyl-L-arginine. When ascorbate was administered along with sodium nitroprusside to increase nitric oxide generation, as little as 5 µM sodium nitroprusside was toxic when superoxide dismutase was inhibited. Whereas concentrations of 50 to 500 µM sodium nitroprusside and 0.4 mM ascorbate caused ~100% toxicity, there was no measurable toxicity when these doses were accompanied by 2 mM glutathione or 50 U/ml of catalase; this suggests that peroxides may also contribute to nitric oxide toxicity. These results suggest that the simultaneous generation of nitric oxide and superoxide is synergistic, resulting in enhanced toxicity.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Nitric
oxide and superoxide are free radicals that appear to play critical
roles in the development of brain injury after stroke (Chan et
al., 1987
; Chan et al., 1990; Kinouchi et
al., 1991; Lipton et al., 1993; Nishikawa et
al., 1994; Zhang et al., 1994) and may also contribute
to a number of other disorders of the CNS, including Parkinson's
disease, Alzheimer's disease and amyotrophic lateral sclerosis (Olanow
and Arendash, 1994). However, the specific effects of these free
radicals and the mechanism by which they modify CNS injury are not
fully understood. Superoxide generally appears to exacerbate ischemic
injury, and treatments that decrease the level of superoxide radicals
can effectively reduce ischemic brain injury (Chan et al.,
1987; Kinouchi et al., 1991; Yang et al., 1994).
The effects of nitric oxide on brain injury are less clear than those
of superoxide and seem to depend on whether the molecule is derived
from neurons or endothelial cells (Huang et al., 1994) and
on the exact chemical milieu in which the molecule resides and the
redox state of the molecule (Lipton et al., 1993). The
oxidized form of nitric oxide, the nitrosonium ion (NO+),
can protect neurons from NMDA-mediated toxicity, apparently by reacting
with a modulatory site on the NMDA receptor, whereas the reduced form
of nitric oxide is neurotoxic (Lipton et al., 1993). In
addition, nitric oxide can be beneficial after brain injury, apparently
because it can induce vasodilation (Palmer et al., 1987) and
might thereby increase flow to regions critically deprived of blood.
A critical question has been how the interaction of nitric oxide and
superoxide might affect the toxic properties of these free radicals.
Because nitric oxide can combine with superoxide, it has been suggested
that nitric oxide production might protect the brain from the damaging
effects of superoxide (Feigl, 1988). Nitric oxide can reduce the
production of cytotoxic superoxide radicals by leukocytes (Rubanyi
et al., 1991) and by endothelial cells (Heim et
al., 1991). However, the product of the combination of superoxide
and nitric oxide is peroxynitrite, a potentially toxic anion that is
capable of generating highly damaging hydroxyl radicals and nitration
of tyrosine residues, leading to protein damage (Beckman et
al., 1990; Ischiropoulos et al., 1992). Although it now
appears clear that nitric oxide and superoxide have the potential
in vitro to combine to form peroxynitrite, and although peroxynitrite is toxic to cells (Lipton et al., 1993), the
capacity of separate sources of nitric oxide and superoxide to combine in vivo and induce toxicity in a synergistic manner has not
been fully established.
It is also not clear which cells might play a critical role in the
response to superoxide and nitric oxide. However, it has been suggested
that cerebral endothelial cells may be an important target of these
free radicals and peroxynitrite (Beckman et al., 1990).
Indeed, there are several sources of both superoxide and nitric oxide
in these cells. For example, endothelial cells synthesize superoxide in
response to stimulation by bradykinin (Holland et al.,
1990), during enzymatic activity of xanthine oxidase (Terada et
al., 1991) and during normal respiration. In addition, endothelial cells can synthesize nitric oxide directly, and adjacent cells, such as
smooth muscle cells, may also synthesize nitric oxide as a response to
inflammatory mediators (Koide et al., 1993). Endothelial
cell damage certainly occurs after a variety of insults, including
cerebral ischemia (Kuroiwa et al., 1988; Yang and Betz, 1994), brain trauma (Cortez et al., 1989) and radiation
(Gobbel et al., 1991), resulting in breakdown of the
blood-brain barrier, edema, tissue swelling and exacerbation of injury.
The finding that reduction of superoxide levels during and after
cerebral ischemia leads to a reduction in blood-brain barrier breakdown and cerebral edema (Chan et al., 1987; Kinouchi et
al., 1991) supports the assertion that endothelial cells are a
probable target of free radicals.
In the present study, we have measured the effects of superoxide and nitric oxide generation on cerebral endothelial cells, and we have determined the role of the interaction of these free radicals in endothelial cell injury. We postulated that the simultaneous administration of independent sources of these two free radicals might have synergistic toxic effects. The goals of this study were to determine 1) whether the severity of injury induced by a nitric oxide generator, SNP, is altered by a superoxide source, paraquat, 2) whether the severity of injury due to superoxide or nitric oxide production is modulated by alteration of the endogenous production of nitric oxide or superoxide, and 3) whether the severity of injury due to nitric oxide is modulated by alteration of the concentration or activity of antioxidant enzymes, such as cytosolic SOD or catalase.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation of cerebral endothelial cells.
Cerebral
endothelial cells were isolated from 14-day-old Sprague-Dawley rats of
both sexes as previously described (Gobbel et al., 1994).
Cells were grown on plastic, 24-well dishes coated with collagen (25 µg/cm2 of type VII collagen from rat tail, Sigma Chemical
Co., St. Louis, MO) in M199 with Earle's salts supplemented with 10%
horse serum, heat-inactivated 5% fetal calf serum, 20 mM HEPES buffer,
0.1 mg/ml heparin sulfate, 0.1 mg/ml endothelial cell growth supplement (Sigma Chemical Co.), 2.0 mM glutamine, 2 mg/ml glucose, 2.5 µg/ml fungizone and 0.1 mg/ml gentamycin. The cells were incubated at 37°C
in 5% CO2, and the cultivation medium was changed every 2 to 3 days. Only primary cultures were used at confluence, 5 to 9 days
after isolation.
Drug treatment. Paraquat, SNP, carbachol, NMMA, DDC, ascorbic acid, SOD, catalase, GSH and BCA, a copper chelator, were obtained from Sigma Chemical Co. SIN-1 was a gift from Dr. Joseph Beckman. All agents were dissolved directly in a solution of M199 with Earle's salts, 5% horse serum, 2.5 µg/ml fungizone and 0.1 mg/ml gentamycin just before treatment, and the solution was passed through a 0.22-µ filter to remove any bacterial contamination. To expose the cells to the various agents, the cultivation medium was changed to M199 with Earle's salts, 5% horse serum, 2.5 µg/ml fungizone and 0.1 mg/ml gentamycin, and the cells were incubated for 18 hr in the various drug-containing solutions.
Determination of cell viability. Cell viability was determined by measuring the release of the intracellular enzyme LDH by nonviable cells with increased membrane permeability. The amount of LDH released was determined by measuring the concentration of LDH in an aliquot of the medium overlying the cells. The LDH was measured spectrophotometrically using a commercially available kit (Sigma Chemical Co.) that is based on the increase in absorption at 340 nm due to the reduction of NAD by LDH. The amount of LDH remaining in the cells was determined by replacing the treatment medium of the cells with an equal volume of PBS, disrupting the cells by sonication for 10 sec and measuring LDH within the PBS. The amount of LDH released was normalized to the total LDH present in the culture (released and intracellular) to quantify the fraction of LDH released, which should approximate the fraction of nonviable cells in the culture. The LDH concentration in untreated control cells is typically around 0.7 U/mg protein, and each well of a 24-well plate contains approximately 0.06 mg of protein when the cells reach confluence.
Statistical analysis. The results are presented as the mean of the data ± S.D. One-way ANOVA followed by Dunnett's test was used to compare the viability of cells treated with multiple doses of SNP or paraquat to the viability of cells treated with no drug (controls). Two-way ANOVA was used to evaluate the effect of various drugs or drug combinations on the viability of cells after treatment with SNP, paraquat or both paraquat and DDC. The factors included in the two-way ANOVA were dose of paraquat or SNP and dose of additional drugs or drug combinations. Analyses with P < .05 were considered significant.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Enhancement of toxicity by simultaneous generation of superoxide and nitric oxide. Treatment of cerebral endothelial cells with SIN-1, which simultaneously generates both superoxide and nitric oxide radicals, caused a pronounced increase in toxicity as measured by LDH release, and the effect was dose-dependent over a narrow range of concentrations. The percentages of LDH released after 18 hr of treatment with 0, 1 and 5 mM SIN-1 were 12.6 ± 1.4, 13.9 ± 1.8 and 99.4 ± 0.7 (n = 4), respectively.
To determine the effects of nitric oxide and superoxide alone and in combination, we added SNP and/or paraquat as separate sources of these two free radicals (fig. 1). Cerebral endothelial cells were adversely affected by increasing doses of either paraquat (fig. 2A) or SNP (fig. 2B). There was a significant increase in the percentage of LDH released by the cells after exposure to >0.2 mM paraquat, and 96.7 ± 0.6% of the LDH was released after treatment with 5 mM paraquat, which indicated that virtually all cells were killed by this dose. A significant increase in LDH release relative to untreated controls was detected after exposure to 1 mM SNP, and the high level of LDH release after exposure to 10 mM (100% for all samples) indicated virtually complete killing by these doses.
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Effect of modulation of endogenous superoxide or nitric oxide
production on toxicity of exogenous superoxide or nitric oxide.
The effect of DDC alone on the toxicity of SNP was tested to determine
whether increases in endogenous superoxide production resulting from
inhibition of SOD might enhance nitric oxide toxicity and partially
account for the results shown in figure 4. There was a significant
increase in SNP toxicity to cerebral endothelial cells in the presence
of DDC (ANOVA; P < .0001; fig. 5).
In contrast, modification of endogenous production of nitric oxide did
not substantially affect the toxicity of paraquat as measured by LDH release. Endogenous nitric oxide production was stimulated using the
ACh receptor agonist carbachol at 0.1 mM and inhibited using the nitric
oxide synthase blocker NMMA at 0.1 mM (fig. 1). There was no apparent
significant effect of NMMA (fig. 6A).
Although the presence of carbachol did slightly alter the toxicity due to paraquat (ANOVA; P < .05), the only notable increase in
paraquat toxicity was at 0.2 mM with slightly greater amounts of LDH
release (28.3 ± 7.0%) in the presence of carbachol compared with
the LDH release in the absence of carbachol (21.4 ± 0.4%) (fig.
6A). This effect was not present when the cells were treated with
paraquat and DDC in combination (fig. 6B), and NMMA also had no impact on the effect of paraquat and DDC in combination.
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). However, BCA had no effect on the apparent synergism
of paraquat and SNP in the presence of DDC.
Effect of a reducing agent and antioxidant enzymes on SNP
toxicity.
Ascorbic acid was added to our cultures to act as a
reducing agent and increase the generation of nitric oxide (Harrison
and Bates, 1993; Lipton et al., 1993) (fig. 1). This caused
a significant (ANOVA; P < .0001) and relatively dramatic increase
in the toxicity of SNP, which suggested that the release of nitric
oxide was important for the toxicity of SNP. In the absence of ascorbic
acid, the addition of 0.5 mM SNP increased LDH release only from
22.8 ± 1.5% to 25.9 ± 1.2%; in the presence of 0.4 mM
ascorbate, the addition of 0.5 mM SNP increased LDH release from
18.8 ± 3.3% to 89.7 ± 5.6%. Similar increases in LDH
release in response to the addition of ascorbate were seen after
treatment with 2 mM SNP.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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An unanswered question has been whether the co-production of
superoxide and nitric oxide would reduce or enhance the toxic effects
of these individual free radicals. This is important to consider,
because the inadvertent combination of nitric oxide and superoxide from
diverse sources might exacerbate certain diseases. For example, there
may be excess production of both superoxide and nitric oxide after
cerebral ischemia; superoxide might be formed because of increased
metabolism of ATP and breakdown of the resultant hypoxanthine by
xanthine oxidase (Terada et al., 1991). Simultaneously,
excess production of nitric oxide could occur either in an attempt to
bring about vasodilation or because of cytokine stimulation of smooth
muscle cells (Koide et al., 1993). In the present
experiments, we used SNP and paraquat to generate nitric oxide and
superoxide, respectively. Each molecule of SNP generates one molecule
of nitric oxide after activation by light or chemical reduction
(Harrison and Bates, 1993), whereas each molecule of paraquat can
generate many superoxide molecules by a redox cycle involving oxidation
of an agent, such as NADPH, followed by reduction of O2
(Bus and Gibson, 1984). The concentrations of nitric oxide and
superoxide achieved in the cells by given concentrations of SNP and
paraquat, respectively, thus depend on a number of factors, including
the availability of reducing agents and the rate of subsequent reaction
and degradation of the free radicals generated. Because the
availability of reducing agents and the rate of degradation are
unknown, it is impossible to predict accurately the actual
concentration of free radicals generated by SNP and by paraquat.
However, in the present experiments, it appears that the toxicity of
SNP and paraquat is due to generation of nitric oxide and superoxide
rather than to some other secondary reaction. The reason we draw this
conclusion is that the toxicity of SNP was enhanced by a reducing agent
to increase the rate of nitric oxide release. In addition, toxicity of
paraquat was enhanced by DDC, an inhibitor of the SOD enzyme
responsible for superoxide degradation. Our results strongly support
the concept that independent sources of nitric oxide and superoxide can
act in a synergistic fashion to enhance rather than ameliorate
toxicity. In the first place, the toxic effects of SNP and paraquat
were significantly and consistently exacerbated by the presence of the
other molecule, even when SNP was present at concentrations that would
cause minimal toxicity when administered alone (fig. 4). Second, merely
inhibiting SOD enhanced the toxicity of SNP (fig. 5).
One reason why nitric oxide and superoxide could be synergistic is that
they can combine to form peroxynitrite, a highly toxic compound (Lipton
et al., 1993). We would predict that, if superoxide is
combining with nitric oxide to form peroxynitrite, then treatment with
paraquat and SNP should increase nitrotyrosine formation (Beckman
et al., 1992). In a preliminary experiment examining the
effect of the treatment of endothelial cells with SNP and paraquat,
there was no evidence of proteins containing nitrotyrosine, judging on
the basis of Western blotting. Our ability to detect nitrotyrosine may
have been hampered by a currently poor understanding of the lifetime of
nitrotyrosine-containing proteins and of the amount of such proteins
that may be formed. Nevertheless, this result suggests that it is
important to consider whether reactions other than the formation of
peroxynitrite might account for our results. For example, we considered
whether the enhancement of the synergism between paraquat and SNP by
DDC might be related to the reported ability of DDC to increase free
copper concentrations by transport of this metal ion across the cell.
Copper could then catalyze a Haber-Weiss type of reaction to produce
increased levels of highly damaging hydroxyl radicals:
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; Nobel et al., 1995). BCA does not readily cross cell
membranes (Harmon and Crane, 1976), so it should not interfere with SOD
activity, but it should interfere with transport of copper into the
cell by DDC. Although the concentration of BCA that we used has been
reported to be effective in other systems (Mohindru et al.,
1983), this concentration did not alter the DDC-mediated enhancement of
paraquat and SNP toxicity in our studies.
We also considered whether superoxide might act as ascorbate does to reduce SNP and increase the release of nitric oxide (fig. 1). However, if superoxide does act in this fashion, it appears to do so rather poorly in that there was enhancement of the toxicity of SNP only when paraquat was added at toxic or near toxic concentrations (figs. 2 and 4). If the reactivity of superoxide with SNP is lower than its reactivity with SOD, then the ability to generate nitric oxide may be limited unless SOD activity is inhibited. This could explain why inhibiting SOD to increase superoxide formation increases the toxicity of SNP down to concentrations of around 5 µM (fig. 4). Although we cannot completely rule out the possibility that superoxide increases nitric oxide generation by SNP and that nitric oxide alone is toxic, another finding strongly supports the hypothesis that the combination of nitric oxide and superoxide is more toxic than either molecule alone. This finding is that inhibition of SOD by DDC enhanced toxicity even when ascorbate was already present to effectively enhance the production of nitric oxide from SNP (figs. 7 and 8).
Considering that superoxide appears to enhance nitric oxide toxicity,
the potentially high endogenous production of superoxide by cerebral
endothelial cells could make them particularly susceptible to nitric
oxide toxicity. For example, the mitochondrial content of cerebral
endothelial cells is 2- to 5-fold higher than that of other cells
(Oldendorf et al., 1977), and mitochondria are a potential
source of superoxide (Sanders et al., 1993). Furthermore, the activity of xanthine oxidase, a superoxide-generating enzyme, is
relatively high within these cells (Terada et al., 1991).
Indeed, the data that exist support the notion that cerebral
endothelial cells are sensitive to nitric oxide. Treatment with 150 µM SNP in the presence of 0.4 mM ascorbate caused a >90% decrease
in viability of the endothelial cells in our experiments (fig. 7A). In
comparison, treatment of cortical neurons with 0.4 mM SNP and 0.4 mM
ascorbate for 18 hr produced only a 20% to 30% decrease in viability
(Lipton et al., 1993). However, it may be difficult to
compare directly results taken from individual studies done in separate
laboratories. Furthermore, the role of xanthine oxidase in nitric oxide
toxicity is not clear. Inactivation of xanthine oxidase activity within
endothelial cells does not change the basal level of production of
oxygen-derived free radicals (Paler-Martínez et al.,
1994) and does not reduce the amount of damage induced by cerebral
ischemia (Lindsay et al., 1991). Nevertheless, our findings
suggests that further investigation into the role of supporting cells
such as endothelial cells may be useful in exploring the pathogenesis
of disorders in which nitric oxide appears to play a role.
The observation that inhibition of SOD by DDC enhanced the toxicity of
nitric oxide suggested that exogenous application of SOD might modulate
the toxicity of exogenous nitric oxide. However, SOD was ineffective,
possibly because the large size of SOD (Mr = 32,000) may limit its ability to cross the cell membrane and enter the
cytoplasm and because the negative charge on the superoxide anion may
limit its movement out of the cell. We also found that blockade of
nitric oxide synthetase by NMMA failed to ameliorate paraquat toxicity
judged on the basis of LDH release. This result is in contrast to the
report that NMMA can block death in PC12 cells due to superoxide
produced after SOD down-regulation (Troy et al., 1996).
However, cell viability in that report was based on the number of cells
remaining on the plate, whereas LDH release, as used in our study, was
determined by changes in membrane permeability. When we reevaluated our
data and used total LDH remaining on the plate as a measure of number
of cells remaining after treatment, there was significantly greater
viability in the NMMA-treated population (ANOVA; P < 0.005). In
the absence of NMMA, after treatment with 20, 50 and 100 µM paraquat,
the remaining amounts of LDH were 78.6 ± 4.8, 60.8 ± 12.0 and 40.4 ± 19.5 mU, respectively; in the presence of 0.1 mM µM
NMMA, the remaining amounts of LDH were 90.4 ± 2.1, 76 ± 2.7 and 55.2 ± 9.8 mU, respectively. Although we cannot rule out
the possibility that the increase in remaining LDH was due to
protection of the enzyme from inactivation by free radicals, the result
is consistent with a 15% to 35% sparing of cells by NMMA. This effect
of NMMA on remaining LDH was observed only when DDC was present. In
contrast to the apparent effects of NMMA on paraquat toxicity in the
presence of SOD inhibition, stimulation of nitric oxide synthesis by
carbachol did not induce any changes in survival as evaluated either by
percentage of LDH released or by amount of remaining LDH.
The inability of carbachol to enhance the toxicity of paraquat may be
related to the concentrations of endogenous nitric oxide and the degree
of change in nitric oxide production induced by carbachol. Although we
used a dose that should effectively stimulate nitric oxide production
(Dowell et al., 1993), the change in concentrations may be
too low to have a measurable effect on toxicity. In addition, endogenous nitric oxide concentrations are reported to be around 100 nM
(Shibuki, 1990), which is 50-fold less than the 5 µM concentration of
nitric oxide generators that our results suggest is necessary to
enhance toxicity.
Although exogenous SOD had no detectable effect on toxicity of SNP,
catalase alone had rather dramatic protective effects. This result
extends the report that SOD and catalase in combination can reduce the
damage induced in cortical neurons by nitric oxide- and
peroxynitrite-generating chemicals, because our results suggest that
much of the protection may be due to catalase (Lipton et al., 1993). This finding is consistent with the report that
catalase can protect against the toxicity of the combination of SNAP, a nitric oxide generator, and hypoxanthine/xanthine oxidase, a
superoxide-generating system (Volk et al., 1995). That
report suggested that the toxicity of nitric oxide might be due to an
interaction with hydrogen peroxide resulting from the dismutation of
superoxide. Our results show that catalase is protective even when a
superoxide generator is not present, which suggests that endogenous
production of peroxides due to treatment with nitric oxide generators
is sufficient to cause toxicity. We tested the possibility that
catalase, by enzymatically scavenging the peroxides formed upon
dismutation of superoxide (fig. 1), might be enhancing SOD activity and
reducing superoxide concentrations. However, catalase was protective
even when SOD was inhibited by DDC, which suggests that peroxides
themselves are responsible for the toxicity. Asahi and colleagues have
indicated that SNAP can inactivate GSHpx and that administration of
SNAP can enhance peroxide formation (Asahi et al., 1995).
Thus the primary cause of excess peroxide generation may be
inactivation of GSHpx. Interestingly, the IC50 for
inactivation of GSHpx by SNAP is reported to be 2 µM (Asahi et
al., 1995), which is close to the minimal toxic concentration of
SNP found in our experiments. Nitric oxide may also deplete GSH, a
substrate in the GSHpx reaction (fig. 1), by reacting with the
sulfhydryl group to form S-nitrosoglutathione (Clancy
et al., 1994). Indeed, our results show that adding
exogenous GSH can block the toxicity of sodium nitroprusside (fig. 9).
Catalase may protect against excess peroxides generated upon
inactivation of GSHpx and/or depletion of glutathione, because unlike
GSHpx, catalase is not affected by nitric oxide (Asahi et
al., 1995) and does not require GSH as a substrate (fig. 1).
In conclusion, our findings support the hypothesis that independent sources of nitric oxide and superoxide may act in concert in a synergistic fashion to produce toxicity. They further suggest that cerebral endothelial cells are particularly susceptible to toxicity induced by nitric oxide when SOD activity is inhibited. Finally, considering that peroxide-scavenging molecules can block damage produced by nitric oxide, the toxicity appears to be mediated by the generation of peroxides.
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Acknowledgments |
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The authors would like to thank Dr. Robert Maiwald for his helpful suggestions on this manuscript.
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Footnotes |
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Accepted for publication May 27, 1997.
Received for publication November 18, 1996.
1 This work was supported by the American Brain Tumor Association and by grants CA 13525, NS 14543, NS 25372 and AG 08938 from the National Institutes of Health.
Send reprint requests to: Glenn T. Gobbel, D.V.M., Ph.D., Brain Tumor Research Center, Dept. of Neurological Surgery, Box 0520, 505 Parnassus Ave., University of California, San Francisco, CA 94143.
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Abbreviations |
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ANOVA, analysis of variance; BCA, bathocuproinedisulfonic acid; DDC, diethyldithiocarbamate; GSHpx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; LDH, lactate dehydrogenase; NMDA, N-methyl-D-aspartate; NMMA, NG-monomethyl-L-arginine; PBS, phosphate-buffered saline; SIN-1, 3-morpholinosydnonimine; SNP, sodium nitroprusside; SOD, Cu,Zn-superoxide dismutase; SNAP, S-nitroso-N-acetyl-DL-penicillamine.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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a protective factor?
Nature (Lond.)
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, respectively. SNP generates nitric oxide (NO) after reduction with ascorbate or another agent. NO can also be generated
physiologically by the conversion of arginine to citrulline by nitric
oxide synthase. Nitric oxide synthase activity is stimulated by
carbachol and inhibited by the arginine analog NMMA. Reduced paraquat
reacts with oxygen to generate superoxide
(O2·
). Superoxide can potentially react
with NO to generate peroxynitrite (ONOO
