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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2000 as doi:10.1096/fj.00-0151fje. |
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National Institute of Mental Health, NIH Clinical Center 10/3D-41, Bethesda, MD 20892-1264, USA
2Correspondence: National Institute of Mental Health, NIH Building 10, Room 3D-41, Bethesda MD 20892-1264, USA. E-mail: chiueh{at}helix.nih.gov
SPECIFIC AIMS
It has been reported that preconditioning stress induced by a transient ischemia can increase brain tolerance to oxidative stress, but the underlying neuroprotective mechanisms are not well understood. We first investigated the pathophysiology of hydroxyl (·OH) and nitric oxide (·NO) free radicals in apoptosis caused by serum deprivation in human SH-SY5Y cells. We further developed a preconditioning cell model for inducing stress genes such as redox factor 1 (Ref-1), Fos, and neuronal nitric oxide synthase (NOS1) for understanding the putative beneficial effects of ·NO and cGMP in the regulation of bcl-2 and p66shc, cell viability, and adaptive tolerance to lethal oxidative stress.
PRINCIPAL FINDINGS
1. Oxidative stress: ·NO vs. ·OH
To investigate the putative free radical mechanisms in oxidative
stress induced by serum deprivation, we measured both the generation of
·NO and the formation of ·OH in human brain-derived SH-SY5Y cells.
Serum deprivation for 24 h significantly increased the production
of both ·NO and ·OH and decreased cell viability. The concentration
of ·NO reached micromolar levels at least 100-fold greater than the
amounts of ·OH. ·OH scavenger but not NOS inhibitors protected
SH-SY5Y cells from serum deprivation-induced cell death. The
stress-increased ·OH generation and cell death were also completely
blocked by S-nitrosoglutathione (GSNO), an ·NO donor and a
potent antioxidant. Furthermore, there was a significant correlation
between the increase of ·OH but not ·NO with the severity of
cytotoxicity reflected by the population of cell death caused by serum
deprivation. In fact, serum can scavenge ·OH and protected cells from
oxidative stress. These observations indicate that ·OH rather than
·NO mediates lethal oxidative stress caused by serum deprivation,
confirming that ·NO is a potent antioxidant that can inhibit ·OH
generation and lipid peroxidation and enhance cell viability.
2. Redox regulation of the expression of stress proteins (Ref-1,
Fos, and NOS1)
The above data suggest that oxidative stress may
induce neuronal nitric oxide synthase (NOS1) and promote ·NO
formation. The expression of human NOS1 mRNA peaked within 2 h and
subsided thereafter during a continuous 24 h period of serum
deprivation stress. ·OH scavengers blocked the expression of
stress-induced NOS1 mRNA. Oxidative stress-induced expression of NOS1
mRNA can also be modified by freshly prepared but not oxidized
·NO-exhausted GSNO. In addition to the induction of NOS1 protein,
oxidative stress also evoked the synthesis of Ref-1 and Fos, but not
NF-
B. The induction of Ref-1 and Fos proteins occurred in the same
time frame as the expression of NOS1 mRNA whereas there was a
significant delay in the induction of NOS1 protein. The expression of
NOS1 protein was suppressed by the ·NO released from GSNO. This new
finding does not support a prominent theory that NOS1 is not inducible,
since it is a constitutive enzyme. It infers that the human isoform of
NOS1 can be inducible and is feedback regulated by ·NO. These results
suggest that the transcriptional expression of stress proteins (Ref-1,
Fos, NOS1) may be under a dynamic up- and down-regulation by oxidants
(i.e., ·OH or H2O2) and
antioxidants (·NO or GSNO), respectively.
3. Role of endogenously, newly synthesized ·NO in neuroprotective
mechanism of preconditioning stress
To investigate the putative neuroprotective role
of endogenous ·NO in cell survival and adaptive tolerance to lethal
oxidative stress, we used a nonlethal serum-free stress for 2 h
(preconditioning stress) to induce the expression of NOS1.
Preconditioning stress immediately induced the expression of NOS1 mRNA,
which peaked at the end of 2 h of preconditioning stress. There
was a delayed increase in the expression of NOS1 protein; the release
of newly synthesized ·NO and the accumulation of cGMP in SH-SY5Y
cells occurred 12 to 36 h after the prestress. Both the induction
of NOS1 mRNA and the expression of NOS1 protein were feedback regulated
by ·NO.
Preconditioning stress decreased both the ·OH generation and cell death evoked by the lethal 24 h serum deprivation, which can be prevented by 7-nitroindazole. Furthermore, preconditioning stress and GSNO inhibited caspase-3 catalytic activity through the modification of the cysteine residues of caspases-3 because the anti-protease activity of GSNO can be reversed by dithiothreitol. The anti-protease activity of GSNO is at least 100-fold more potent than other ·NO donors and thiol-modifying reagents. These results indicate that preconditioning stress increases the expression of NOS1 and that the generation of ·NO, which is feedback regulated, can only be produced up to micromolar concentrations. Newly synthesized ·NO initiated a cGMP-independent antioxidative process: 1) scavenging free radicals (·OH, peroxyl lipids, thionyl radicals), 2) inhibiting caspase-3, and 3) protecting human brain cells against oxidative stress.
4. The cGMP-dependent cytoprotective processes: up-regulation of
the anti-apoptotic protein bcl-2 and down-regulation of the
p66shc adaptor protein
It has been reported that cGMP can promote cell survival. To study
whether the preconditioning stress also induces a cGMP-dependent
cytoprotective process, we further investigated the effects of
inhibitors of NOS1 and guanylyl cyclase on cGMP formation, stress
protein expression, and cell viability in the prestressed SH-SY5Y
cells. The inhibition of cGMP formation during the induction of NOS1 by
7-nitroindazole (a NOS1 inhibitor), methylene blue (a ·NO
scavenger), and LY-83,583 (a guanylyl cyclase inhibitor) significantly
blunted preconditioned stress-enhanced tolerance to oxidative stress.
Prestress also increased the expression of anti-apoptotic bcl-2 protein
and concomitantly down-regulated the expression of p66shc
adaptor protein during the period from 24 to 36 h after
application of the brief 2 h serum-free stress. NOS1 and guanylyl
cyclase inhibitors blocked this preconditioning regulation of bcl-2 and
p66shc. Thus, stress-induced NOS1 can control apoptosis and
enhance cell viability through a cGMP-dependent mechanism of up- and
down-regulation of the expression of bcl-2 and p66shc,
respectively.
5. Role of endogenous and exogenous ·NO on apoptosis caused by
serum deprivation
To investigate whether apoptotic cell death induced by serum
deprivation can be prevented by ·NO generated by either
preconditioning induction of NOS1 or GSNO (a donor of ·NO and GS·),
oxidative stress-induced apoptosis was imaged by Hoechst 33258
fluorescent nuclear dye. The 24 h lethal serum-free stress
significantly increased the apoptotic cell population. This
stress-induced apoptotic cell death was prevented by endogenous ·NO
generated by a brief 2 h preconditioning stress and also by
exogenous ·NO released from the freshly prepared GSNO. The
photodegraded, ·NO-exhausted GSNO can no longer protect human brain
cells against oxidative stress. These results confirmed that the
oxidative stress-induced apoptosis in human brain cells can be
prevented by ·NO generated either endogenously or exogenously.
CONCLUSIONS
Serum deprivation increased the generation of ·NO and ·OH in the cell cultures of human neuroblastoma cells. These human brain-derived neurotrophic SH-SY5Y cells can synthesize dopamine and serotonin. The notion of ·OH- rather than ·NO-mediated neurotoxicity caused by serum deprivation-induced oxidative stress is supported by the fact that serum-free stress-induced cell death can be prevented by ·OH scavenger, but not NOS inhibitors. The present in vitro and prior in vivo observations indicate that micromolar endogenous and exogenous ·NO act like atypical antioxidants. The observed beneficial neuroprotective effects of ·NO synthesized from newly induced NOS1 are consistent with earlier studies using exogenous administered GSNO but somewhat at odds with other reports. Species differences (murine vs. human), multiple nonselective actions of ·NO donors and NOS inhibitors, dynamic regulation of isoforms of NOS in different cell types, and lack of proper sham controls may have contributed to this controversy. The present results indicate that the neuronal subtype of NOS1 is dynamically feedback regulated by ·NO and can generate only micromolar (not millimolar) concentrations of ·NO. At micromolar concentrations, ·NO can nitrosylate iron complexes, inhibit Fenton reaction or ·OH generation, scavenge reactive oxygen, peroxyl lipid, and thionyl radicals, and inactivate caspase-3, resulting in cytoprotection instead of neurotoxicity.
·NO donors at high concentrations (0.5 mM) may induce both necrotic and apoptotic cell death. However, emerging evidence suggests that the cytotoxic effects of sodium nitroprusside may be not mediated by ·NO, since ·NO-exhausted sodium nitroprusside still causes the same or a greater degree of cell death that can be completely blocked by GSNO. The present in vitro results confirm our prior reports that freshly prepared, but not photodegraded, ·NO-exhausted GSNO protects brain dopamine neurons against oxidative stress stimulated by reactive free radicals in vivo. Our observations indicate that we can use a brief 2 h preconditioning oxidative stress to maximally induce NOS1 mRNA in human brain cells in order to generate micromolar concentrations of ·NO without causing appreciable cell death. In fact, ·NO produced by preconditioning stress enhances cell survival and tolerance to oxidative stress. These preconditioning-induced antioxidative, anti-apoptotic, and cytoprotective mechanisms are mostly mediated by the delayed induction of NOS1, ·NO, and cGMP since the prestress enhanced cell survival was significantly reduced by the inhibition of NOS1 by 7-nitroindazole and the blockade of guanylyl cyclase by LY-83,583.
The observed antioxidative effects of endogenous ·NO are consistent with previous in vivo findings that exogenous ·NO or GSNO terminates oxidative chain reactions, brain lipid peroxidation, and neurotoxicity caused by pro-oxidants such as peroxynitrite, sodium nitroprusside, hemoglobin, and ferrous citrate. In addition to scavenging superoxide anions, ·NO may also scavenge highly reactive glutathionyl radicals (GS·), yielding a potent antioxidant GSNO in endothelial and astroglial cells that contains millimolar glutathione. In fact, GSNO is at least 100-fold more potent than GSH in suppressing iron-evoked ·OH generation and peroxynitrite-produced brain lipid peroxidation. The present evidence indicates that GSNO—a putative GS·and ·NO donor—inhibits the proapoptotic protein caspase-3 via a cGMP-independent, dithiothreitol-sensitive S-nitrosylation and S-glutathionylation mechanism. Due to its additional S-glutathionylation, the anti-caspase effect of GSNO is at least 100-fold more potent than other ·NO donors. These anti-caspase and free radical scavenging properties of ·NO may partially explain why the induction of NOS1 increases resistance of human brain cells against oxidative stress caused not only by serum deprivation, but also by 1-methyl-4-phenylpyridinium (unpublished results). It may also answer the question of why the NOS1-containing neurons can tolerate oxidative damage induced by ischemia/reperfusion injury and viral encephalitis.
The present observations indicate that the low output activity of
the constitutive isoform of human NOS1 can be dynamically induced to
meet both functional and compensatory demands for cGMP activation, gene
regulation, antioxidative defense, and tolerance to oxidative stress.
Serum deprivation-induced oxidative damage can be prevented by a brief
2 h preconditioning stress through the compensatory activation of
cellular antioxidative defense systems including the induction of Ref-1
and NOS1, but not NF-B-related anti-apoptotic proteins (Fig. 1
). Furthermore, the present results indicate that endogenous ·NO
synthesized by the newly induced NOS1 may inhibit apoptosis in
cells/neurons through 1) the inhibition of caspase-3,
2) the up-regulation of bcl-2, and 3) the
ablation of p66shc. In addition to up-regulating the
expression of anti-apoptotic bcl-2, the present observation indicates
that the induction of NOS1 also mediates the down-regulation of
p66shc(Fig. 1)
. The ablation of p66shc could
enhance cellular resistance to oxidative stress because the
p66shc adaptor protein is a cytoplasmic signal transducer
involved in the transmission of mitogenic signals, leading to oxidative
damage and ultimately apoptosis. This is consistent with prior reports
that the ablation of p66shc, induction of Ref-1 and bcl-2,
and inhibition of caspase-3 can protect cells and neurons from
oxidative damage in cell cultures and increase life span in animals.
The present findings that endogenous ·NO/cGMP regulates bcl-2,
p66shc, and caspase-3 may explain prior unsolved
observations as to why cGMP and ·NO support neurite outgrowth,
inhibit apoptosis, and increase cell viability. It is concluded that
preconditioning stress-induced Ref-1 and NOS1 may mediate adaptive
mechanisms leading to brain tolerance against lethal oxidative stress
through both cGMP-dependent and -independent processes.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0151fje To cite this article, use (September 8, 2000) FASEB J. 10.1096/fj.00-0151fje 
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