Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Expression of
endothelial nitric oxide synthase (eNOS) in transfected U-937 cells
upregulates phorbol 12-myristate 13-acetate (PMA)-induced tumor
necrosis factor- (TNF-
) production through a superoxide
(O
regulation, their possible role in
eNOS-derived O
(5.8 ± 1.0 fold; P = 0.01) and
increase the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. Although
S-nitroso-N-acetylpenicillamine, a nitric oxide
(NO) donor, also increased TNF-
production, NO exposure led to
phosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-
production by eNOS transfection was associated with increases in
activated p42/44 MAPK (P = 0.001), whereas levels of
phosphorylated p38 MAPK were unaffected. Furthermore, cotransfection
with Cu/Zn superoxide dismutase, which blocks TNF-
upregulation by
eNOS, also abolished the effects on p42/44 MAPK. Expression of
Gln361eNOS, a mutant that produces O
upregulation by eNOS (P = 0.02).
Thus O
production
via a mechanism that involves p42/44 MAPK activation.
signal transduction; tumor necrosis factor-; reactive oxygen
species; endothelial nitric oxide synthase
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INTRODUCTION |
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REACTIVE OXYGEN
SPECIES (ROS) can function as important and specific signal
transduction intermediates that regulate cell growth, differentiation,
proliferation, and apoptosis (17, 20, 27, 28).
Although there are many cellular sources of ROS, the molecular
identities and precise origins of ROS involved in signal transduction
events have largely remained obscure (13, 37). Several
investigations have shown that endothelial nitric oxide synthase
(eNOS), a NADPH oxidase, is capable of releasing oxygen intermediates
in cell-free systems deficient in tetrahydrobiopterin (BH4)
and Ca2+ (32, 38). This finding raises the
provocative possibility of a dual role for this enzyme in both nitric
oxide (NO) and ROS signal transduction. Notably, increased eNOS
expression in endothelial cells has been associated with elevated ROS
production (4, 9, 24). In vivo, the infusion of
BH4 in hypercholesterolemic patients has been shown to
restore endothelium-dependent vascular responses (26),
supporting the concept that eNOS dysfunction, with impaired NO
production and increased O
Despite interest in the NADPH oxidase activity of eNOS and its
potential importance in atherogenesis (24, 26), a number of technical obstacles have hindered the direct demonstration in intact
cells of eNOS-derived O
Using this intact cell model, we recently demonstrated that eNOS
expression upregulates tumor necrosis factor- (TNF-
) promoter activity and TNF-
production through a ROS-dependent signaling pathway (35). Signaling by eNOS-derived
O
-methyl-L-arginine
(L-NMA), a NOS inhibitor, or by an eNOS mutation that
disrupts NO formation but leaves NADPH oxidase activity intact. Finally, using a sensitive aconitase assay, eNOS upregulation of
TNF-
production was directly linked to O
through a
mechanism dependent on O
A possible role for mitogen-activated protein kinases (MAPK) in
eNOS-derived O responses (2, 20,
31). The MAPK family is a conserved network of signal transduction enzymes that are activated by phosphorylation and can be
translocated to the nucleus (7). Upon activation, MAPK phosphorylate serine and threonine residues in target proteins, typically other kinases and transcription factors. By utilizing task-related signaling complexes, and through both kinase-dependent and
kinase-independent functions, relatively few MAPK components can be
differentially activated, thereby resulting in a much larger number of
pathway-specific signaling events (7, 23). Here we
investigate the relationship between ROS-dependent signal transduction initiated by eNOS and MAPK activation in PMA-differentiated U-937 cells. Furthermore, the ROS and NO specificity of MAPK responses is
examined in these cells.
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METHODS |
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Reagents. Anti-eNOS antibodies were purchased from Transduction Labs and Research Diagnostics. The enhanced chemiluminescence Western blotting detection system was obtained from Amersham Pharmacia Biotech. Polyvinylidene difluoride (PVDF) membranes and 4-20% Tris-glycine gels were obtained from Novex. Phospho-specific antibody against phospho-p38 MAPK was obtained from Promega or Santa Cruz Biotechnology. Phospho-specific antibody against phospho-p42/44 MAPK and phospho-nonspecific antibodies against p38 MAPK and p42/44 MAPK were all obtained from New England Biolabs. S-nitroso-N-acetylpenicillamine (SNAP), hygromycin B, and PD-98059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one] were purchased from Calbiochem. Phenazine methosulfate, N-formyl-methionyl-leucyl-phenylalanine (fMLP), tert-butyl hydroperoxide, and hydrogen peroxide (H2O2) were obtained from Sigma Aldrich.
Expression vectors. Construction of the plasmids used in this investigation has been described previously (35, 40). Briefly, human eNOS expression vector was constructed by insertion of human eNOS cDNA (4 kb) into the pCEP4 eukaryotic expression vector (Invitrogen). A mutant eNOS cDNA that results in a Glu-to-Gln substitution at position 361 (5) was cut from pGEM-3Z with EcoRI and ligated into pBluescript SK(+). A HindIII/NotI fragment containing the entire open reading frame of eNOS was then excised from pBluescript SK(+) and ligated into pCEP4 to create Gln361eNOS. NADPH binding site deletion mutants of eNOS, d(NADPH)eNOS and d(NADPH)Gln361eNOS, were constructed by digesting eNOS and Gln361eNOS cDNA with XhoI, thus removing 98 amino acids from the COOH-terminal end of eNOS that contain the NADPH-adenine binding site sequence. Human Cu/Zn SOD cDNA [American Type Culture Collection (ATCC)] was excised from pSP64 vector at HindIII/BamHI sites and ligated into pCEP4. For these pCEP4 constructs, transcription of the inserted sequence of interest is driven by the cytomegalovirus immediate early enhancer/promoter. The pCEP4 plasmid contains a hygromycin B-resistance gene that allows selection. All of the expression vectors were partially sequenced to confirm the correct sequence and orientation.
Transfection, selection, and eNOS expression. U-937 cells (ATCC) were grown in RPMI 1640 complete medium containing HEPES (25 mM), endotoxin-free fetal calf serum (10%), L-glutamine (2 mM), and antibiotics at 37°C in a humidified incubator with 5% CO2. Empty pCEP4 (control vector) or vector constructs containing human eNOS, Gln361eNOS, d(NADPH)eNOS, d(NADPH)Gln361eNOS, and human Cu/Zn SOD were transfected into cells by electroporation, as previously described (35, 40). Briefly, U-937 cells (5 × 106) were suspended in 260 µl of RPMI 1640 complete medium. Cells were electroporated (240 V, 960 µF) in the presence of plasmid DNA (20 µg) with an Electro Cell Manipulator 600 (BTX). After electroporation, cells were grown in RPMI complete medium for 3 days and then selected with hygromycin B (275 U/ml) for at least 2 wk. The expression and activity of eNOS in transfected cells was measured using a L-[14C]arginine to L-[14C]citrulline conversion assay, as described previously (39, 40). In addition, expression of eNOS and mutant eNOS protein was demonstrated by Western blot with the use of antibody directed at either the COOH-terminal (Transduction Labs) or NH2-terminal (Research Diagnostics) portion of eNOS.
TNF- production.
Untransfected naive or transfected U-937 cells (1 × 107 cells each), as indicated by experiment, were suspended
in RPMI 1640 complete medium (100 ml) containing PMA (100 nM) for
48 h to induce differentiation and TNF-
production. After
differentiation, adhered cells were removed by incubation with 1 mM
EDTA in Hanks' balanced salt solution without Ca2+ or
Mg2+. Cells were then washed, resuspended in RPMI 1640 complete medium, counted, tested for viability by trypan blue exclusion
(
90% viable for all experiments), and plated into 24-well plates at
5 × 105 cells/ml. For some experiments, as indicated,
these final incubations occurred in the absence or presence of
increasing concentrations of one of the following: a redox cycling
agent (phenazine methosulfate), a membrane-permeant oxidant
(tert-butyl hydroperoxide), authentic H2O2, a NO donor (SNAP), or a p42/44 MAPK
inhibitor (PD-98059). Cell-free supernatants were collected after
22 h of incubation at 37°C in 5% CO2 and then
assayed for TNF-
production with an enzyme-linked immunosorbent
assay, according to the manufacturer's protocol (R&D Systems).
Ferricytochrome c assay for O6 M fMLP. The reaction was stopped by placing the tubes
in a cold water bath. Tubes were then centrifuged at 12,000 g for 10 min, the supernatant was then transferred to
cuvettes, and the absorbance was read at 550 nm in a spectrophotometer
(Molecular Devices). Blanks were prepared by using reaction mixtures
without cells, and these measurements were subtracted as background.
MAPK phosphorylation. Again, U-937 cells, either untransfected or transfected, were PMA (100 nM) differentiated for 48 h and washed with PBS. For the oxidant stress and NO donor experiments, respectively, these PMA-differentiated cells (~1 × 107 cells/flask) were then incubated in RPMI 1640 complete medium in the presence of phenazine methosulfate or fMLP for 30 min, tert-butyl hydroperoxide or H2O2 for 10 min, or SNAP for 15 min. For each agent, MAPK phosphorylation-time curves (data not shown) had been performed, and optimal incubation times were selected for these studies. In all experiments, cells for MAPK phosphorylation determination were removed from plates with a cell scraper and then lysed with ice-cold lysis buffer containing 50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail (Roche Molecular Biochemicals). Solubilized proteins were centrifuged at 10,000 g in a microcentrifuge at 4°C for 10 min, and supernatant protein concentration was quantified by bicinchoninic acid assay (Pierce). These protein preparations (20 µg) were then separated on 4-20% Tris-glycine gels by SDS-PAGE and transferred to PVDF membranes at 30 V for 2 h. Membranes were blocked overnight with PBS containing 5% nonfat dry milk and 0.05% Tween 20 at 4°C. The blots were incubated for 1 h with primary antibody. After incubation with secondary antibody, phosphorylated and nonphosphorylated forms of MAPK were detected by enhanced chemiluminescence. Results were arbitrarily quantitated by using laser densitometry and expressed as relative change from control values.
MAPK activity. Lysates of U-937 cells (500 µg total protein) were incubated with rabbit anti-p42/44 antibody overnight at 4°C and then incubated with protein A-Sepharose beads (20 µg) for 1.5 h at 4°C with gentle rocking to immunoprecipitate the protein. The beads were washed three times with 500 µl of kinase buffer (25 mM Tris, pH 7.5, 5 mM glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2), and then an in vitro kinase assay was performed using the transcription factor Elk-1 as a substrate. Phosphorylated Elk-1 protein was then detected by Western blot with the use of an anti-phospho-Elk-1 antibody according to the manufacturer's instructions (New England Biolabs).
Statistics. Data are shown as means ± SE, and differences were considered significant where two-tailed P < 0.05. Paired comparisons were made using t-tests. Escalating dose experiments were analyzed by computing nonparametric Sen-Theil estimates of dose-response slopes and then performing one-sample t-tests to determine whether these derived slopes were different from 0. Experiments involving multiple comparisons were subjected to an initial analysis of variance (ANOVA) followed by post hoc Fisher's least significant difference tests of all individual comparisons. To determine if the response to PD-98059, a p42/44 MAPK inhibitor, was different in eNOS compared with control vector transfectants, a repeated-measures ANOVA was computed, with the comparison evaluated from the transfectant/dose interaction term.
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RESULTS |
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Effects of a redox-cycling agent, phenazine methosulfate, on
TNF- production and MAPK phosphorylation.
In a previous study we found that eNOS expression increased TNF-
production and promoter activity in PMA-differentiated U-937 cells
(35). L-NMA, an inhibitor of eNOS that blocks
NO production but not the NADPH oxidase activity of the enzyme, had no
effect on TNF-
upregulation by eNOS. Phenazine methosulfate, a
redox-cycling agent that has been used to generate intracellular
O
upregulation (35). To determine intermediate signaling
steps in the regulation of TNF-
by eNOS-derived O
production (P = 0.01) in
PMA-differentiated U-937 cells (Fig.
1A). This
effect on TNF-
regulation was associated with a concomitant,
dose-dependent change in the phosphorylation state of p42/44 MAPK (Fig.
1B). In contrast to p42/44 MAPK, the phosphorylation state
of p38 MAPK in PMA-differentiated U-937 cells, was not altered by
phenazine methosulfate exposure (Fig. 1C). These data
closely resemble the effects of eNOS-derived O
production by O
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Effect of fMLP-stimulated respiratory burst on MAPK
phosphorylation.
To determine the possible effects of enzymatically generated
O6 M; P = 0.01). Conversely, in these
PMA-differentiated U-937 cells, p38 MAPK was not significantly
activated (Fig. 2C; P = 0.37). Notably, cell
activation by fMLP is a complex event that involves multiple signal
transduction pathways (11, 22). Determining in
PMA-differentiated U-937 cells whether fMLP-induced p42/44 MAPK
activation is O
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Effect of tert-butyl hydroperoxide and H2O2
on TNF- production and MAPK phosphorylation.
We have previously associated eNOS production of O
upregulation in U-937 cells (35).
Because O
production and MAPK activation in PMA-differentiated U-937
cells. TNF-
production was not upregulated by
tert-butyl-hydroperoxide (Fig.
3A;
P = 0.057 for a downregulatory effect). However,
tert-butyl-hydroperoxide markedly increased p38 MAPK
phosphorylation (Fig. 3C; P = 0.02) and had a
modest effect on p42/44 MAPK phosphorylation (Fig. 3B;
P = 0.052). Interestingly, H2O2 also
strongly increased p38 MAPK phosphorylation (Fig.
4C; P = 0.002) while showing only a minor effect on p42/44
MAPK activation that did not reach statistical significance (Fig.
4B; P = 0.09). However, in contrast to
tert-butyl hydroperoxide, H2O2
significantly upregulated TNF-
(Fig. 4A; P =
0.01). Notably, the pattern of MAPK phosphorylation produced by either
tert-butyl hydroperoxide or H2O2
differed from that seen with phenazine methosulfate or with eNOS
expression (see Effect of eNOS expression on p42/44 MAPK and p38
MAPK activation: role of superoxide).
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Effect of a NO donor, SNAP, on TNF- production and MAPK
phosphorylation.
We have shown previously that inducible NOS (iNOS) and NO donors can
upregulate TNF-
production in PMA-differentiated U-937 cells through
an unusual cAMP-dependent mechanism (34, 40). Because the
main function of eNOS is to produce NO, MAPK activation by a NO donor,
SNAP, was investigated to determine whether its MAPK pathway effects
could be discerned from those associated with O
production (P = 0.001)
in PMA-differentiated U-937 cells (Fig.
5A).
Although SNAP had no effect on the phosphorylation state of p42/44 MAPK
(Fig. 5B), it intensely increased the amount of
phosphorylated p38 MAPK in a dose-dependent manner (Fig.
5C). Total p42/44 MAPK and p38 MAPK were not changed by NO
exposure. These results suggest that eNOS-derived NO and eNOS-derived
O
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Effect of eNOS expression on p42/44 MAPK and p38 MAPK activation:
role of superoxide.
In our previous investigation, eNOS upregulation of TNF- production
was abolished by Cu/Zn SOD cotransfection (35), so we
examined here the effect of SOD on MAPK phosphorylation induced by eNOS
expression. U-937 transfectants expressing eNOS demonstrated increased
intracellular levels of phosphorylated p42/44 MAPK compared with
control vector transfected cells (Fig.
6A; P = 0.001).
Furthermore, this increment in p42/44 MAPK phosphorylation was
substantially abolished by coexpression of Cu/Zn SOD (compared with
control vector, P = 0.5). Total p42/44 MAPK was not altered
by any transfections. As shown in Fig. 6B, eNOS expression,
as well as eNOS/SOD cotransfection, had no effect on p38
phosphorylation (P = 0.5), suggesting that p38 MAPK plays no
significant role in eNOS-induced TNF-
upregulation. Figure
6C shows that eNOS and SOD were expressed in the
transfectants used in these experiments. These data, combined with the
experimental results obtained with the use of various oxidants and a NO
donor, suggest that p42/44 MAPK activation in eNOS-transfected U-937 cells is dependent on the release of superoxide.
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Effect of mutating the L-arginine binding site or
deleting the NADPH binding site of eNOS on p42/44 MAPK and p38 MAPK
phosphorylation.
Previous work has shown that the mutant enzyme Gln361eNOS
is unable to produce NO but retains its NADPH oxidase activity and the
ability to upregulate TNF- (5, 35), whereas eNOS
mutants lacking an adenosine NADPH binding site, d(NADPH)eNOS and
d(NADPH)Gln361 eNOS, have neither NADPH oxidase activity
nor the ability to upregulate TNF-
(35). Consistent
with these previous results, we found (Fig.
7A) that both eNOS and
Gln361eNOS similarly increased the phosphorylation state of
p42/44 MAPK (P = 0.01), but NADPH binding site deletion
mutants of eNOS had no effect on p42/44 MAPK phosphorylation compared
with control vector-transfected cells (P = 0.54). In
contrast, neither eNOS nor any of its mutant forms affected p38 MAPK
phosphorylation (Fig. 7B). These results further suggest
that superoxide generated by the NADPH oxidase activity of eNOS is
essential to its ability to increase the phosphorylation state of
p42/44 MAPK and, as previously shown (35), to upregulate
TNF-
production.
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Effect of eNOS expression on p42/44 MAPK activity and effect of
PD-98059, a p42/44 MAPK pathway inhibitor, on eNOS-induced TNF-
upregulation.
To confirm that p42/44 MAPK phosphorylation reflects changes in
enzymatic activity, we performed an assay for p42/44 MAPK activity. A
specific downstream target of p42/44 MAPK, the transcription factor
Elk-1, served as a substrate for measuring p42/44 MAPK activity.
Similar to our results obtained by examining p42/44 MAPK
phosphorylation, eNOS expression compared with control vector transfection (Fig. 8A)
resulted in the upregulation of p42/44 MAPK activity (P
= 0.001). Finally, we used PD-98059, an inhibitor of p42/44 MAPK
phosphorylation, to test whether the p42/44 MAPK pathway participates
in TNF-
upregulation by eNOS. As shown in Fig. 8B,
PD-98059 dose-dependently blocked the eNOS-dependent upregulation of
TNF-
(P = 0.02 for interaction between eNOS expression and the effect of PD-98059). The inhibitory effect of PD-98059 was
significantly greater in eNOS cells than in control vector transfectants, suggesting that TNF-
upregulation in
PMA-differentiated U-937 cells is mediated by activation of the p42/44
MAPK pathway.
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DISCUSSION |
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Recently, we demonstrated in PMA-differentiated U-937 cells that
eNOS could regulate TNF- transcription and production through a
signal transduction pathway dependent on the release of
O
. Signaling by eNOS
through this pathway is blocked by Cu/Zn SOD coexpression and by
deletion of the NADPH binding site of eNOS, but not by a
L-arginine binding site mutation that only abolishes NO
production. tert-Butyl hydroperoxide and
H2O2 produce patterns of MAPK activation
dissimilar to that seen with phenazine methosulfate or eNOS expression.
Importantly, NO, the primary product of eNOS that has its own
upregulatory effects on TNF-
(30, 34, 40), was shown to
activate p38 MAPK, not p42/44 MAPK.
ROS, once thought of as metabolic by-products and toxins, are now
recognized as essential signal transduction intermediates in the
regulation of cell proliferation, differentiation, and apoptosis (13, 17, 20, 27, 28, 37). Although ROS
signaling pathways have been associated previously with MAPK activation in many cell types, the enzymatic sources and the molecular identity of
the ROS involved in these regulatory events have often remained obscure
(13, 37). The importance of MAPK activation to
ROS-dependent signaling under a variety of experimental conditions
(15, 18, 27, 28) and the ability of MAPK to regulate
TNF- responses (2, 20, 31) suggested that these
signaling cascades might be differentially and specifically regulated
by eNOS in our PMA-differentiated U-937 cell model system. We found
that eNOS-produced O
For many ROS signaling events, H2O2 is
considered a more attractive intermediate than its more toxic
precursor, O. Collectively, these results suggest in
PMA-differentiated U-937 cells that eNOS-derived O
upregulation. However,
H2O2 is well known to be a potent signal transduction intermediate in many experimental systems and, as shown
here, clearly affects MAPK pathways and TNF-
production in
PMA-differentiated U-937 cells. Therefore, the conversion of eNOS-derived O
or in other cell types. As
stated by Manna et al. (20), activations of
redox-dependent signaling pathways are complex events that likely
reflect the total cellular balance of opposing prooxidant and
antioxidant factors rather than the presence of a single reactive
intermediate. This concept suggests that the coregulation of eNOS and
SOD in endothelial cells, as described by Dimmeler et al.
(12), could act as a counterregulatory mechanism that
serves to maximize the vasodilatory and antiadherence properties of
eNOS-derived NO while suppressing the prooxidant potential of eNOS expression.
Not only did we find that eNOS expression and O upregulation. Conversely, even though NO exposure was
associated with phosphorylation of p38 MAPK, SB-203580
[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], a p38 MAPK inhibitor, had no effect on NO-induced upregulation of
TNF-
in concentrations up to 20 µM (experiment not shown). In
previous investigations, we showed that TNF-
upregulation by NO and
iNOS expression occurs through a cAMP-dependent signaling pathway
(34). NO inhibits basal adenylate cyclase activity and lowers intracellular cAMP concentrations, which in turn decreases cAMP-dependent protein kinase (PKA) activity. Notably, NO-induced upregulation of TNF-
is mimicked by PKA inhibitors and abolished by
cell-permeable cAMP analogs (34). This effect of NO on
TNF-
has been associated with decreased binding of Sp1 to the
TNF-
promoter, which results in upregulation of TNF-
promoter
activity (33). Therefore, although NO exposure leads to
the phosphorylation of p38 MAPK, it appears that this effect may be
tangential to TNF-
upregulation. Interestingly, PKA, whose activity
is decreased by NO through its cAMP-lowering effects (33,
34), is known in some cells to phosphorylate Raf-1, thereby
blocking its interaction with Ras and subsequent activation of MAPK
(8, 14). The binding of Raf-1 with Ras, which
arguably might be increased by NO inhibition of the cAMP/PKA pathway,
leads to MAPK kinase activation. Thus the effects of NO on cAMP could
independently lead to both TNF-
upregulation and p38 MAPK phosphorylation.
In summary, this investigation has identified p42/44 MAPK kinase as a
downstream target of eNOS-derived O
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ACKNOWLEDGEMENTS |
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We thank Kenneth D. Bloch for providing human eNOS cDNA and Pei-Feng Chen for providing human Gln361eNOS cDNA.
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FOOTNOTES |
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This work was supported by intramural National Institutes of Health funds.
Present address of S. Wang: The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850.
Address for reprint requests and other correspondence: R. L. Danner, Critical Care Medicine Dept., National Institutes of Health, Bldg. 10, Rm. 7D43, 10 Center Dr. MSC 1662, Bethesda, MD 20892-1662 (E-mail: rdanner{at}nih.gov).
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.
Received 3 October 2000; accepted in final form 28 March 2001.
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