COMMUNICATION
Nitric Oxide Inhibits Caspase-3 by S-Nitrosation
in Vivo*
Lothar
Rössig
,
Birgit
Fichtlscherer§,
Kristin
Breitschopf
,
Judith
Haendeler
,
Andreas M.
Zeiher
,
Alexander
Mülsch§, and
Stefanie
Dimmeler
¶
From the
Molecular Cardiology, Department of Internal
Medicine IV and the § Institute of Cardiovascular
Physiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
 |
ABSTRACT |
In cultured human endothelial cells,
physiological levels of NO prevent apoptosis and interfere with the
activation of the caspase cascade. In vitro data have
demonstrated that NO inhibits the activity of caspase-3 by
S-nitrosation of the enzyme. Here we present evidence for
the in vivo occurrence and functional relevance of this
novel antiapoptotic mechanism. To demonstrate that the cysteine residue
Cys-163 of caspase-3 is S-nitrosated, cells were
transfected with the Myc-tagged p17 subunit of caspase-3. After
incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by
electron spin resonance spectroscopy after liberation and spin trapping of NO by
N-methyl-D-glucamine-dithiocarbamate-iron
complex. Transfection of cells with a p17 mutant, where the essential
Cys-163 was mutated into alanine, completely prevented
S-nitrosation of the enzyme. As a functional correlate, in
human umbilical vein endothelial cells the NO donors sodium
nitroprusside or PAPA NONOate (50 µM) significantly
reduced the increase in caspase-3-like activity induced by
overexpressing caspase-3 by 75 and 70%, respectively. When human
umbilical vein endothelial cells were cotransfected with
-galactosidase, morphological analysis of stained cells revealed
that cell death induction by overexpression of caspase-3 was completely
suppressed in the presence of sodium nitroprusside, PAPA NONOate, or
S-nitroso-L-cysteine (50 µM). Thus, NO
supplied by exogenous NO donors serves in vivo as an
antiapoptotic regulator of caspase activity via
S-nitrosation of the Cys-163 residue of caspase-3.
 |
INTRODUCTION |
Apoptosis is central to the regulation of tissue homeostasis but
also contributes to cancer, neurodegenerative diseases, and inflammation. Morphologically, the programmed form of cell death is
characterized by cytoplasmic membrane blebbing, chromatin condensation, and fragmentation into apoptotic bodies. The apoptotic process is under
control of a highly conserved signaling network mainly discovered in
Caenorhabditis elegans, with at least three families of
genes being involved termed ced-3
(C.
elegans death gene
3), ced-4, and ced-9 (1). The
mammalian homologues of ced-3 encode for the family of
cysteine proteases, caspases, that constitute an enzyme cascade
culminating in activation of caspase-3 (2). Thus, caspase-3 represents
the execution enzyme of the caspase cascade that cleaves the DNase
inhibitor ICAD (inhibitor of
caspase-activated
deoxyribonuclease) to activate DNA-degrading DNases (3).
Within the molecular structure of caspase-3, the catalytic cysteine
group that accounts for the proteolytic activity of the enzyme is
located at position 163 of the p17 subunit (4).
NO has been implicated to be involved in regulating apoptosis in a
variety of tissues (5, 6). In addition to the well established
proapoptotic effects of NO (6-8), a growing body of evidence indicates
that low levels of NO function as an important inhibitor of apoptosis
by interference with signal transduction pathways that control
apoptotic cell death (9-12). Several studies suggested that NO may
inhibit apoptosis via interacting with the caspase cysteine proteases
(13, 14). As a general feature of its biochemical properties, NO is
known to be capable of modifying proteins that contain cysteine
residues by S-nitrosation of the thiol group of the
respective cysteine (15-17). Indeed, the decrease in caspase-3-like
enzyme activity by in vitro incubation of purified enzyme
with NO donors could be specifically assigned to
S-nitrosation of the essential cysteine residue at the
active site of caspase-3 (11, 18). This could represent a potential
molecular mechanism underlying the functional relationship between NO
and inhibition of apoptosis signaling observed in cell culture.
However, taking into account that NO could also interfere with upstream
cell death signals that result in caspase activation (19-21), it still
has to be proven whether caspases are directly targeted by NO in intact cells and whether an S-nitrosation-mediated inhibitory
effect of NO on caspase-3-driven apoptosis does occur in
vivo. Thus, in the present study we sought to demonstrate that
intracellular caspase-3 is subjected to S-nitrosation of
cysteine residue 163 following incubation of cells with NO donors, and
we determined the functional impact of exogenous NO on caspase-3-like
activity and caspase-3-induced apoptotic cell death in
vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials--
SNP was bought from Sigma (Munich, Germany), PAPA
NONOate was from Alexis (Läufeling, Switzerland), and
S-nitrosopenicillamine was from Biomol (Hamburg, Germany).
Cell Culture--
Human umbilical vein endothelial cells (HUVEC;
Cell Systems/Clonetics, Solingen,
Germany)1 were cultured in
endothelial basal medium (Cell Cystems/Clonetics) supplemented with
hydrocortisone (1 µg/ml), bovine brain extract (3 µg/ml),
gentamicin (50 µg/ml), amphotericin B (50 µg/ml), epidermal growth
factor (10 µg/ml), and 10% fetal calf serum (Life Technologies, Inc., Berlin, Germany) until the third passage. After detachment with
trypsin, cells were grown in culture dishes for 18 h before experiments were performed. COS-7 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with glutamine (40 mM), penicillin-streptomycin, and 10% fetal calf serum.
Plasmids and Transfection--
Caspase-3 was amplified by
polymerase chain reaction with oligonucleotides, which were synthesized
to contain BamHI and EcoRV restriction sites and
subsequently cloned into the respective sites of the
pcDNA3.1-MycHis vector (InVitrogen, Leek, The Netherlands). The
catalytically active p17 subunit of caspase-3 was amplified by
polymerase chain reaction to contain BamHI and
EcoRV restriction sites and then was cloned into the
respective sites of pcDNA3.1-MycHis vector. The p17 mutant, where
Cys-163 was replaced by alanine (p17-C163A), was generated by
site-directed mutagenesis (Stratagene, Heidelberg, Germany). Transient
transfection of HUVEC was performed by incubation of 3.0 × 105 cells/6-cm well with 3 µg of plasmid as described
previously (22). For cotransfection experiments, HUVEC were incubated
with pcDNA3.1-lacZ (1 µg) and either pcDNA3.1-caspase-3 (2 µg) or the pcDNA3.1 control vector (2 µg), followed by
-galactosidase staining of cotransfected cells according to our
previously published method (22). To transiently transfect COS-7 cells,
7 µg/3.0 × 105 cells of pcDNA3.1 plasmid
containing the respective insert were employed using the Superfect
method (Qiagen, Hilden, Germany).
Immunoprecipitation of p17-Myc--
Transfected cells were grown
for 36 h to allow for protein expression before cell lysis by
incubation with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM KCl, 5 mM
MgCl2, 1% Nonidet P-40 (AppliChem, Darmstadt, Germany), 1 mM phenylmethylsulfonyl fluoride, 10 mM
N-ethylmaleimide, 1 mM BCS, and 1 mM
DTPA (Sigma). To isolate Myc-tagged protein, whole cell lysates of
COS-7 cells (10 mg of protein) overexpressing p17-wt-Myc or the
p17-C163A mutant were incubated with anti-Myc antibody (Santa Cruz
Biotechnology) overnight at 4 °C. After incubation with 30 µl of
protein A/G-agarose beads (Santa Cruz Biotechnology)/mg protein for
1.5 h at 4 °C, the p17-Myc-anti-Myc immunocomplexes attached to
beads were washed extensively in a modified lysis buffer with reduced
concentrations of BCS (50 µM) and DTPA (50 µM) to remove contaminating S-nitrosothiols and other low molecular mass NO donors.
Western Blot Analysis--
To probe immunoprecipitates for
Myc-tagged protein, 30 µl of SDS sample dye were added to agarose
beads, and the precipitated protein was released into the supernatant
by 5 min of incubation at 100 °C, followed by centrifugation (2000 rpm, 5 min). Then samples were run on a 13% SDS-polyacrylamide gel and
blotted onto polyvinylidene fluoride membranes, which were blocked in
5% milk powder for 2 h and probed with mouse anti-Myc antibody,
1:200, 1% milk powder followed by an incubation with anti-mouse
antibody linked to horseradish peroxidase.
NO Spin Trap and ESR Analysis--
The washed beads were
resuspended in 400 µl of modified washing buffer lacking metal
chelators (BCS and DTPA) and N-ethylmaleimide. A freshly
prepared aqueous solution (20 µl) of FeSO4/sodium citrate (final concentration, 0.1 mM/0.4 mM) was added
to the samples, followed 1 min later by an aqueous solution (20 µl)
of N-methyl-D-glucamine-dithiocarbamate (MGD;
final concentration, 24 mM). MGD served to release NO from S-nitrosothiol (23) and to generate the water-soluble NO
trapping agent Fe(MGD)2, which avidly binds NO to form a
paramagnetic mononitros-iron complex (NOFe(MGD)2) (24).
After 5 min, the samples were centrifuged (200 rpm, 5 min), and
Na2S2O4 was added to the
supernatant (final concentration, 200 mM) to prevent
oxidation of the spin adduct (25, 26) and to convert all nitrite
eventually formed during decomposition of S-nitrosothiol
into NO. The solution was then filled into teflon tubes (5-mm inner
diameter; 40-mm length) and shock frozen in liquid nitrogen. The frozen
samples were expelled from the teflon tubes by means of a glass rod and
inserted into the finger of a fingertip-shaped quartz Dewar, which was
filled with liquid nitrogen. The concentration of the
NOFe(MGD)2 complex was assessed by cryogenic X-band ESR
spectroscopy. In frozen state, this paramagnetic complex exhibits an
anisotropic triplet signal with axial symmetry at g
= 2.035, g
= 2.02 (24). The mononitros-iron complex
with diethyldithiocarbamate (NOFe(DETC)2) (12 µM; dissolved in Me2SO) served as a standard.
ESR spectra were recorded at 77 K on a BRUKER ESR 300E at a microwave
frequency of 9.47 GHz, a modulation frequency of 100 kHz, a modulation
amplitude of 0.5 millitesla, microwave power of 20 mW, and a time
constant of 655 ms. Each spectrum was collected over 160 s with
1024 points resolution.
Caspase-3-like Activity--
HUVEC transfected with pcDNA3.1
plasmid were lysed in 200 µl of buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin, 2 mM dithiothreitol, 10 mM Tris/HCl,
pH 8) for 15 min at 4 °C. After centrifugation (20,000 × g for 10 min), caspase-3-like activity of the supernatant was detected by measuring the proteolytic cleavage of the fluorogenic substrate 7-amino-4-coumarin (AMC)-DEVD and AMC as a standard using
excitation and emission wavelengths of 380 and 460 nm, respectively. Protein content was analyzed, and enzyme activity was calculated as mol
AMC released × mg protein
1 × s
1
(11).
Caspase-3-induced Cell Death--
To assess cell death
induction, HUVEC were cotransfected as described previously (22) and
incubated for 6-18 h. The transfected cells were identified by
-galactosidase staining. Viable versus dead stained cells
were counted by two blinded investigators, and results were expressed
as dead/viable cells × 100. In addition, potential differences in
cell death rate due to necrosis were excluded by measuring LDH release
to indicate that death induction was apoptotic in nature.
Statistical Analysis--
Data are expressed as the means ± S.E. from at least three independent experiments. Statistical
analysis was performed by one-way analysis of variance (variance: LSD test).
 |
RESULTS AND DISCUSSION |
S-Nitrosation of Caspase-3 by Exogenous NO Donors in Vivo--
To
demonstrate the in vivo occurrence of
caspase-3-S-nitrosation by exogenous NO donors, a vector
encoding the Myc-tagged p17 subunit of caspase-3
(pcDNA3.1-p17wt-Myc) was transiently transfected into COS-7 cells.
Cells were incubated for 36 h to allow for protein expression and
subsequently treated with Cys-NO (1 mM) for 10 min. Then
p17wt-Myc was isolated by immunoprecipitation using an anti-Myc
antibody and protein A/G-agarose beads. To remove contaminating
S-nitrosothiols and other low molecular mass NO donors, the
p17-Myc-anti-Myc immunocomplexes attached to the beads were extensively
washed. NO bound to the free thiol group (Cys-163) available on p17 was
then detected by a newly developed procedure that makes use of the
catalytic decomposition of S-nitrosothiols by
dithiocarbamates and subsequent spin trapping of released NO by a
water-soluble dithiocarbamate-iron complex (Fe(MGD)2)
(cf. "Experimental Procedures"). The beads were removed
by centrifugation, and the supernatant containing spin trapped NO was
assessed by cryogenic ESR spectroscopy (27). The efficiency of this
procedure was verified using bovine S-nitroso-albumin as a
standard. The recovery of NO from 100 to 1000 nM
S-nitroso-albumin kept overnight in immunoprecipitation
buffer amounted to 50 ± 5% (n = 3), and the
lower limit for ESR detection (signal to noise = 2:1) was 30 nM NOFe(MGD)2 complex (data not shown). As
shown in Fig. 1A, ESR spectra
typical for NOFe(MGD)2 were clearly visible in samples prepared from immunoprecipitates of Cys-NO exposed p17-Myc-transfected COS cells (middle trace). For comparison, the ESR signal of
a standard is shown in Fig. 1A (bottom trace).
This signal was absent in samples prepared from immunoprecipitates of
control cells not exposed to Cys-NO (Fig. 1A, top
trace). The concentration of spin trapped NO present in the ESR
probe of Cys-NO-exposed cells was 150 ± 20 nM.
Considering the recovery of NO from S-nitrosothiol after the
washing and trapping procedure, it can be estimated that 300 nM NO would account for the total amount of NO initially released from the immunprecipitates, equivalent to 150 pmol NO in 0.5 ml of sample volume. As a control, one fraction of the immunoprecipitates was analyzed by Western blot with an antibody against Myc to demonstrate equal expression of Myc-tagged wild type p17
(p17wt-Myc, data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
S-Nitrosation of p17 in
vivo: ESR spectra and Western blot of p17-Myc
immunoprecipitates from COS-7 cells overexpressing p17 in the presence
or absence of exogenous NO. A, Myc-tagged
p17-overexpressing COS-7 cells were incubated in the presence or
absence of Cys-NO for 10 min (1 mM, middle and
top trace, respectively). Anti-Myc immunprecipitates of cell
lysates were assessed for the incorporation of NO by ESR
spectrophotometry. Bottom trace, ESR signal of a standard.
B, COS-7 cells were transfected with Myc-tagged p17 wild
type (wt) or p17 mutant, where the essential Cys-163 residue
was replaced by alanine (C163A). Transfected cells were incubated for
1 h with SNP (50 µM), and anti-Myc
immunoprecipitates were analyzed by ESR. Inset, anti-Myc
Western blot analysis of the immunoprecipitates demonstrates the
expression of p17-Myc in COS-7 cells.
|
|
To test whether S-nitrosation still occurs under exposure to
low concentrations of NO donors, which have been shown to efficiently inhibit apoptotic cell death, COS-7 cells expressing the Myc-tagged p17
subunit were incubated for 1 h with 50 µM SNP. As
shown in Fig. 1B, a significant NO release could be detected
by ESR in immunoprecipitated Myc-tagged p17. Calculated NO in the
immunoprecipitate derived from SNP-treated COS-7 cells expressing
p17wt-Myc was 70 nM. Moreover, the S-nitrosation
of caspase-3 was detectable for at least 12 h after stimulation
with 50 µM SNP (data not shown).
Caspase-3 Is S-Nitrosated at the Essential Cys-163--
The
essential cysteine residue of caspase-3 is located in the active center
of the p17 subunit of caspase-3 at position Cys-163. To specifically
assign the S-nitrosation of caspase-3 to the thiol group of
Cys-163, Cys-163 was mutated into alanine by site-directed mutagenesis
(p17mt-Myc). COS-7 cells expressing p17mt-Myc underwent the same
incubation procedure with SNP as control cells transfected with p17
wild type (50 µM SNP, 1 h). As shown in Fig.
1B, mutation of Cys-163 completely prevented
S-nitrosation of the p17 subunit, thus excluding that other
cysteine residues within the molecular structure of p17 may serve as
acceptor amino acids for the S-nitrosation. Again, Western
blot analysis confirmed that similar amounts of p17 protein were
immunoprecipitated (Fig. 1B, inset).
Caspase-3-like Activity in Caspase-3-overexpressing HUVEC Is
Inhibited by NO Donors in Vivo--
To explore the functional impact
of caspase-3-S-nitrosation by exogenous NO in
vivo, HUVEC were transiently transfected with a plasmid containing
full-length caspase-3 (pcDNA3.1Casp-3) or with the control vector
(pcDNA3.1). During protein expression, cells were treated with
exogenous NO donated by SNP (50 µM) or PAPA NONOate (50 µM) or were kept in the absence of an NO donor as a
control. As shown in Fig. 2, the increase
in caspase-3-like activity induced by caspase-3 overexpression is
significantly reduced by 75 and 70% in the presence of SNP and PAPA
NONOate, respectively (vector, 100%; *, p < 0.001).
Because transfection efficiency in HUVEC was 30% on average, the
increase in caspase-3-like activity is likely to be even higher in
those cells that were actually subjected to transfection by caspase-3
compared with the mean value of all cells.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Inhibition of caspase-3-like activity in
caspase-3-overexpressing HUVEC by coincubation with exogenous NO
donors. HUVEC transiently transfected with either a plasmid
containing the full-length caspase-3 insert or vector alone were
incubated in the presence or absence of the NO donors SNP and PAPA
NONOate (50 µM) for 12 h. DEVD-directed cleavage of
the AMC-coupled substrate was detected by spectrofluorometry. Vector,
100%. The data are the means ± S.D. *, p < 0.001 versus caspase-3.
|
|
Apoptosis Induction in HUVEC by Caspase-3 Overexpression Is
Suppressed by Exogenous NO--
To test whether NO also affects
apoptotic cell death induced by caspase-3 overexpression, HUVEC were
cotransfected with pcDNA3.1-lacZ and pcDNA3.1-caspase-3, and
cell death was detected at different time points (Fig.
3, A-C). A maximal increase
in cell death rate of positively stained cells was observed after
12 h (Fig. 3A). Dead cells were characterized by
membrane blebs and/or a general cell shrinkage (Fig. 3C). No
significant differences in LDH release as an indicator for cell
necrosis were observed, when measured at time points in analogy with
the determination of cell death (Fig. 3B). This indicates
that cell death observed following transfection of HUVEC with
full-length caspase-3 is likely due to apoptosis rather than cell
necrosis. To assess the sensitivity to exogenous NO of the key
apoptosis execution step driven by caspase-3, HUVEC overexpressing
caspase-3 were incubated in the presence of the NO donor SNP (50 µM). As displayed in Fig. 3A, the induction of cell death by caspase-3 was abolished by NO treatment (*,
p < 0.001). Similar results were obtained when PAPA
NONOate or S-nitrosopenicillamine were used as NO donors
(Fig. 3D). In contrast, LDH activity was not affected by
coincubation with NO donors (Fig. 3B), suggesting that
exogenous NO at concentrations used here has no impact on necrotic cell
death.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Antiapoptotic effect of exogenous NO in HUVEC
transiently transfected with caspase-3. HUVEC were cotransfected
with full-length caspase-3 or pcDNA3.1 vector together with
pcDNA3.1 Gal and incubated for the indicated periods of time in
the presence or absence of SNP (50 µM). A,
cell death induction in transfected HUVEC identified by light
microscopy given as percentage of transfected cells. The data are the
means ± S.D. (n = 3). *, p < 0.001 for caspase-3 versus caspase-3 + SNP at the indicated
times. B, LDH release from HUVEC culture during incubation
for 6, 12, and 18 h post-transfection with or without NO donors.
The data are calculated as percentages of intracellular LDH content
(means ± S.D., n = 3). C, light
microscopy of HUVEC transfected according to the above protocol. Blue
staining indicates transfected cells. Arrows indicate
apoptotic cells that are characterized by a general cell shrinkage
leading to a dark, spheric appearance, and/or by cytoplasmic membrane
blebbing. D, effect of 12 h of incubation with
different NO donors on caspase-3-induced cell death rate in HUVEC. The
data are the means ± S.D. (n = 3). *,
p < 0.001 versus caspase-3 overexpression
in the absence of an NO donor.
|
|
Taken together the present results demonstrate that exogenous NO donors
induce S-nitrosation of caspase-3 in intact cells. S-Nitrosation was still detected to a considerable extent,
even when cells were exposed to low concentrations of NO donors, which resemble native endothelial NO production (11). In addition to the
short term effect of exogenous NO on cellular caspase-3, when the NO
donor was applied 1 h before protein isolation,
S-nitrosation of caspase-3 was also detectable after 12 h of incubation of cells in the presence of an NO donor (data not
shown). These data support the observed long lasting inhibition of
caspases following the addition of exogenous NO (11).
The observed modification of cellular caspase-3 by NO leading to the
inhibition of the key signal to regulate apoptosis might be of major
importance in endothelial physiology where NO has been described as a
major protective factor (28, 29). Disturbances in endothelial cell
integrity are known to be involved in the early pathophysiological
changes leading to atherosclerosis, and increased endothelial apoptosis
was suggested to contribute to the initial stages of atherogenesis and
plaque erosion (30, 31) that eventually can cause heart attack and
stroke. One may hypothesize that the vasoprotective potencies of NO
could at least partially be based on its apoptosis-suppressive effects
in the vascular endothelium.
In a more general point of view, the in vivo inhibition of
caspase-3 by NO reported here could represent a universal mechanism by
which cell death rates of a whole variety of tissues are fine tuned by
NO via controlling the execution step of the caspase cascade, where the
main apoptosis signal machinery converges. In such a model, the
susceptibility of the cell to death signals transmitted by caspases is
prone to an additional regulatory influence dependent on the cellular
and exogenous NO levels. In view of the ambivalent capabilities of NO
to act either in a proapoptotic or in an antiapoptotic fashion
depending on cell type and NO dosage (5), a complex spectrum of
NO-mediated control of apoptosis is conceivable. Thus, corresponding to
the activation status of the cellular NO synthases and to the cytosolic
redox balance of the individual cell type in a certain physiological
scenario, NO may either function as an apoptosis inhibitor to stabilize tissue integrity by the above mechanism or, at higher concentrations, may exert toxic effects by direct degradation of DNA (32, 33).
 |
ACKNOWLEDGEMENT |
We thank S. Ficus for technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Sonderforschungsbereich 553).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.
¶
To whom correspondence should be addressed. Tel.:
49-69-6301-7440; Fax: 49-69-6301-7113; E-mail:
Dimmeler{at}em.uni-frankfurt.de.
 |
ABBREVIATIONS |
The abbreviations used are:
HUVEC, human
umbilical vein endothelial cells;
BCS, bathocuproine-disulfonic acid;
DTPA, diethylene-triamine-pentaacetic acid;
MGD, N-methyl-D-glucamine-dithiocarbamate;
ESR, electron spin resonance;
AMC, 7-amino-4-coumarin;
SNP, sodium
nitroprusside;
Cys-NO, S-nitroso-L-cysteine.
 |
REFERENCES |
-
White, E.
(1996)
Genes Dev.
10,
1-15[CrossRef][Medline]
[Order article via Infotrieve]
-
Nagata, S.
(1997)
Cell
88,
355-365[Medline]
[Order article via Infotrieve]
-
Enari, M.,
Sakahira, H.,
Yokoyama, H.,
Okawa, K.,
Iwamatsu, A.,
and Nagata, S.
(1998)
Nature
391,
43-50[CrossRef][Medline]
[Order article via Infotrieve]
-
Nicholson, D. W.,
Ali, A.,
Thornberry, N. A.,
Vaillancourt, J. P.,
Ding, C. K.,
Gallant, M.,
Gareau, Y.,
Griffin, P. R.,
Labelle, M.,
Lazebnik, Y. A.,
et al..
(1995)
Nature
376,
37-43[CrossRef][Medline]
[Order article via Infotrieve]
-
Dimmeler, S.,
and Zeiher, A. M.
(1997)
Nitric Oxide
1,
275-281[CrossRef][Medline]
[Order article via Infotrieve]
-
Nicotera, P.,
Brune, B.,
and Bagetta, G.
(1997)
Trends Pharmacol. Sci.
18,
189-190[CrossRef][Medline]
[Order article via Infotrieve]
-
Albina, J.,
Cui, S.,
Mateo, R.,
and Reichner, J.
(1993)
J. Immunol.
150,
5080-5085[Abstract/Free Full Text]
-
Messmer, U.,
and Brüne, B.
(1996)
Biochem. J.
319,
299-305[Medline]
[Order article via Infotrieve]
-
Mannick, J. B.,
Asano, K.,
Izumi, K.,
Kieff, E.,
and Stamler, J. S.
(1994)
Cell
79,
1137-1146[Medline]
[Order article via Infotrieve]
-
Mannick, J. B.,
Miao, X. Q.,
and Stamler, J. S.
(1997)
J. Biol. Chem.
272,
24125-24128[Abstract/Free Full Text]
-
Dimmeler, S.,
Haendeler, J.,
Nehls, M.,
and Zeiher, A. M.
(1997)
J. Exp. Med.
185,
601-608[Abstract/Free Full Text]
-
Dimmeler, S.,
Haendeler, J.,
Sause, A.,
and Zeiher, A. M.
(1998)
Cell Growth Differ.
9,
415-422[Abstract]
-
Haendeler, J.,
Weiland, U.,
Zeiher, A. M.,
and Dimmeler, S.
(1997)
Nitric Oxide
1,
282-293[CrossRef][Medline]
[Order article via Infotrieve]
-
Kim, Y.-M.,
de Vera, M. E.,
Watkins, S. C.,
and Billiar, T. R.
(1997)
J. Biol. Chem.
272,
1402-1411[Abstract/Free Full Text]
-
Stamler, J. S.,
Simon, D. I.,
Osborne, J. A.,
Mullins, M. E.,
Jaraki, O.,
Michel, T.,
Singel, D. J.,
and Loscalzo, J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
444-448[Abstract]
-
Stamler, J. S.
(1994)
Cell
78,
931-936[Medline]
[Order article via Infotrieve]
-
Hausladen, A.,
Privalle, C. T.,
Keng, T.,
De Angelo, J.,
and Stamler, J. S.
(1996)
Cell
86,
719-729[Medline]
[Order article via Infotrieve]
-
Li, J.,
Billiar, T. R.,
Talanian, R. V.,
and Kim, Y. M.
(1997)
Biochem. Biophys. Res. Commun.
240,
419-424[CrossRef][Medline]
[Order article via Infotrieve]
-
Shen, Y. H.,
Wang, X. L.,
and Wilcken, D. E.
(1998)
FEBS Lett.
433,
125-131[CrossRef][Medline]
[Order article via Infotrieve]
-
Estevez, A. G.,
Spear, N.,
Thompson, J. A.,
Cornwell, T. L.,
Radi, R.,
Barbeito, L.,
and Beckman, J. S.
(1998)
J. Neurosci.
18,
3708-3714[Abstract/Free Full Text]
-
Hebestreit, H.,
Dibbert, B.,
Balatti, I.,
Braun, D.,
Schapowal, A.,
Blaser, K.,
and Simon, H.-U.
(1998)
J. Exp. Med.
187,
415-425[Abstract/Free Full Text]
-
Dimmeler, S.,
Assmus, B.,
Hermann, C.,
Haendeler, J.,
and Zeiher, A. M.
(1998)
Circ. Res.
83,
334-342[Abstract/Free Full Text]
-
Arnelle, D. R.,
Day, B.,
and Stamler, J. S.
(1997)
Nitric Oxide
1,
56-64[CrossRef][Medline]
[Order article via Infotrieve]
-
Lai, C. S.,
and Komarov, A. M.
(1994)
FEBS Lett.
345,
120-124[CrossRef][Medline]
[Order article via Infotrieve]
-
Tsuchiya, K.,
Takasugi, M.,
Minakuchi, K.,
and Fukuzawa, K.
(1996)
Free Radical Biol. Med.
21,
733-737[CrossRef][Medline]
[Order article via Infotrieve]
-
Mikoyan, V. D.,
Kubrina, L. N.,
Serezhenkov, V. A.,
Stukan, R. A.,
and Vanin, A. F.
(1997)
Biochim. Biophys. Acta
1336,
225-234[Medline]
[Order article via Infotrieve]
-
Mülsch, A.,
Mordvintcev, P.,
Bassenge, E.,
Jung, F.,
Clement, B.,
and Busse, R.
(1995)
Circulation
92,
1876-1882[Abstract/Free Full Text]
-
Moncada, S.,
and Higgs, A.
(1993)
N. Engl. J. Med.
329,
2002-2012[Free Full Text]
-
Moroi, M.,
Zhang, L.,
Yasuda, T.,
Virmani, R.,
Gold, H. K.,
Fishman, M. C.,
and Huang, P. L.
(1998)
J. Clin. Invest.
101,
1225-1232[Abstract/Free Full Text]
-
Geng, Y. J.,
and Libby, P.
(1995)
Am. J. Pathol.
147,
251-266[Abstract]
-
Haunstetter, A.,
and Izumo, S.
(1998)
Circ. Res.
82,
1111-1129[Free Full Text]
-
Burney, S.,
Tamir, S.,
Gal, A.,
and Tannenbaum, S. R.
(1997)
Nitric Oxide
1,
130-144[CrossRef][Medline]
[Order article via Infotrieve]
-
Nguyen, T.,
Brunson, D.,
Crespi, C. L.,
Penman, B. W.,
Wishnok, J. S.,
and Tannenbaum, S. R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3030-3034[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.