(Circulation. 2000;102:1302.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Nitric Oxide Provokes Tumor Necrosis Factor-
Expression in Adult Feline Myocardium Through a cGMP-Dependent Pathway
From the Winters Center for Heart Failure Research, Cardiology Section, Department of Medicine, Veterans Administration Medical Center and Baylor College of Medicine, Houston, Tex.
Correspondence to Douglas L. Mann, MD, Cardiology Research (151C), VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030. E-mail dmann{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-
(TNF-) and nitric oxide (NO) in the failing heart is
unknown.
Methods and Results—To determine whether NO was sufficient to
provoke TNF- biosynthesis, we examined the effects of an NO donor,
S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff
hearts. SNAP (1 µmol/L) treatment resulted in a time- and
dose-dependent increase in myocardial TNF- mRNA and protein
biosynthesis in adult cat hearts. The effects of SNAP were completely
abrogated by a NO quenching agent,
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide
(C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic
mobility shift assays demonstrated that SNAP treatment led to the rapid
induction of nuclear factor kappa-beta (NF-
B) but not AP-1.
The importance of the cGMP pathway in terms of mediating NO-induced
TNF- biosynthesis was shown by studies that demonstrated that
8-bromo-cGMP mimicked the effects of SNAP and that the effects
of SNAP could be completely abrogated using a cGMP
antagonist, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one
(ODQ), or protein kinase G antagonist
(Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the
site-specific phosphorylation (serine 32) and
degradation of IB in isolated cardiac myocytes. Finally, protein
kinase G was sufficient to directly phosphorylate
IB on serine 32, a critical step in the activation of
NF-B.
Conclusions—These studies show that NO provokes TNF-
biosynthesis through a cGMP-dependent pathway, which suggests that the
coincident expression of TNF- and NO may foster self-sustaining
positive autocrine/paracrine feedback inflammatory circuits within the
failing heart.
Key Words: nitric oxide • tumor necrosis factor • cyclic GMP • NF-B • heart failure, congestive
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous studies have shown that tumor necrosis factor-
(TNF-) is expressed de novo within the
myocardium in response to tissue injury.1 In
most instances, the expression of TNF- mRNA and protein within the
myocardium is self-limited1 ; that is, after
the removal of the inciting injury, both the TNF- gene and protein
expression are no longer evident within the
myocardium.1 Nonetheless, in certain settings,
such as chronic heart failure, TNF- mRNA and protein are
persistently expressed in the myocardium.2
However, the mechanisms that are responsible for persistent TNF-
expression within the myocardium are not known. On the
basis of the observation that TNF- and the inducible form of nitric
oxide synthase (iNOS) are coexpressed in failing human
hearts3 and of experimental studies showing that TNF-
upregulates iNOS in cardiac myocytes4 and that nitric
oxide (NO) donors are sufficient to increase TNF- production
in various nonmyocyte cell types,5 we considered
the possibility that TNF- and NO might participate in
self-sustaining positive autocrine/paracrine feedback circuits within
the myocardium. Accordingly, to begin to address this
question, in the present study, we systematically examined the
effects of NO on TNF- mRNA and protein biosynthesis in the adult
heart. The results of this simple experimental study constitute an
initial demonstration that NO is a sufficient stimulus to provoke
TNF- expression within the adult mammalian myocardium.
Moreover, the results of this study show that NO provokes TNF-
biosynthesis through a novel cGMP-dependent pathway. |
Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Myocardial TNF-
Biosynthesis Ex VivoMyocardial TNF-
biosynthesis was assessed ex vivo, under
endotoxin-free conditions, using a modified Langendorff perfusion
apparatus, as detailed previously.1 The heart
was perfused with a recirculating Krebs-Henseleit bicarbonate
buffer containing S-nitroso-N-acetyl penicillamine (SNAP) or diluent
(control hearts). In preliminary control experiments using
10-4 to 10-9 mol/L SNAP,
we determined that 10-6 mol/L SNAP produced
stable nitrite concentrations of 2.8 to 3.1 µmol/L for at least
180 minutes. Insofar as this range of nitrite concentrations
corresponded to the nitrite levels that have been observed after the
breakdown of pathophysiologically relevant
concentrations of NO6 and that they did not produce
detectable quantities of peroxynitrite in isolated cardiac myocytes, we
used 1 µmol/L SNAP for the majority of the experiments described
herein.
Myocardial TNF mRNA and Protein Biosynthesis
Starting after the addition of SNAP (time 0), a 500 mg sample of
myocardium was excised from the suspended heart (carefully
sparing the large epicardial vessels) every 30 minutes for a total of
180 minutes. This sample was frozen in liquid nitrogen and stored at
-70°C. Total RNA was extracted using the guanidinium
thiocyanate/phenol method. For the detection of TNF- mRNA, we
developed a feline cDNA probe that was subsequently used for
ribonuclease protection assays (see Data Supplement, which can be found
at www.circulationaha.org). To detect TNF- protein levels, 0.5 mL of
recirculating buffer was collected every 30 minutes for a total of 180
minutes. TNF- protein levels were determined by ELISA using a
commercially available kit that recognizes feline TNF- (Human
Ultrasensitive Cytoscreen, Biosource).
Cellular Source for Myocardial TNF-
Biosynthesis
To determine whether NO was sufficient to induce TNF- in
cultured myocytes, we treated cultured cardiac myocytes with SNAP or
diluent. The methods for isolating adult feline cardiac myocytes and
the characteristics and purity of the cell culture system have been
detailed previously.7 Total RNA was extracted 0, 1, and 6
hours after the addition of 1 µmol/L SNAP or diluent, and RNase
protection assays were performed. Cytosolic TNF- protein was
determined at time 0 and at 1 and 6 hours after the addition of 1
µmol/L SNAP or diluent. Myocyte cultures were lysed with 200 µL of
0.05% Triton X-100 and harvested. A 100-µL aliquot of the cell
lysate was used to determine total protein (Bicinchoninic Acid
assay, Pierce), and the remaining cell lysate was used to measure
TNF- protein levels (ELISA).
Transcription Factors for TNF-
To determine whether SNAP-induced myocardial TNF-
biosynthesis was mediated, at least in part, by the activation of
NF-B or AP-1, we performed electrophoretic mobility shift assays.
Freshly isolated cat hearts were treated with 1 µmol/L SNAP or
diluent for a total of 180 minutes. Starting at time 0 and every 30
minutes thereafter, we obtained myocardial biopsies (
500 mg) from
the SNAP-treated hearts. All myocardial samples were frozen and stored
at -70°C. Electrophoretic mobility shift assays (EMSAs) were
performed by incubating 10 µg of nuclear extracts from the
SNAP-treated hearts with 8 fmol/L (20 000 cpm) of the
double-stranded consensus sequence for either nuclear factor kappa-beta
(NF-B) (5'-AGTTGA-GGGGACTTTCCCAGGC-3') or
AP-1 (5'-CGCTTGATGACTC-AGCCGGAA-3')
(Data Supplement available at www.circulationaha.org). The
specificity of binding was determined in "cold" competition
experiments using 25x and 50x molar excess of the respective
unlabeled oligonucleotides. To determine the components
of the DNA-protein binding complexes, we performed supershift assays by
incubating the nuclear extracts with 2 µg of anti-human polyclonal
antibodies directed against the various components of NF-B,
including p50, p52, p65 (Rel A), Rel B, and cRel, for 15 minutes at
room temperature before the addition of the labeled
oligonucleotide consensus sequences.
Role of the cGMP Pathway in TNF- Biosynthesis and
NF-B Activation
Myocardial cGMP Levels and Protein Kinase G Activity
In preliminary control experiments, we determined that
SNAP (1 µmol/L) stimulation of Krebs-Henseleit
bicarbonate–perfused hearts led to a 14-fold increase in cGMP levels
within 2 minutes and a 2-fold increase in protein kinase G (PKG)
levels, whereas no increase occurred in either cGMP or PKG in
diluent-treated hearts (Data Supplement available at
www.circulationaha.org).
Inhibition and Activation of cGMP
To determine whether the cGMP pathway was important in terms of
SNAP-induced TNF- biosynthesis, we examined the effects of the
activation and inhibition of the cGMP/PKG pathway. Buffer-perfused
hearts were pretreated for 60 minutes with either
10-5 mol/L
1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a specific
inhibitor of guanylyl cyclase, or with Rp-8-Br-cGMPS
(25 µmol/L), a specific inhibitor of PKG, before
stimulating the hearts with SNAP. Next, we stimulated freshly isolated
hearts for 180 minutes with 10-5 mol/L
8-Br-cGMP, a biologically active cGMP analog. As an additional control
experiment, we also stimulated a separate group of hearts for 180
minutes with 10-7 mol/L atrial
natriuretic factor, a peptide that activates cGMP
through an NO-independent pathway. We measured TNF- mRNA by
ribonuclease protection assay, protein biosynthesis by ELISA, and
NF-B activation by EMSA.
Mechanism for cGMP-Induced NF-B Activation
To determine whether SNAP stimulation and cGMP stimulation
phosphorylated IB on serine 32 (Ser32), we
stimulated 35S-methionine/cysteine-labeled
myocyte cultures with 1 µmol/L SNAP or
10-5 mol/L 8-Br-cGMP and incubated the cell
lysates with either a C-21 antibody that recognizes the terminal 21
amino acid residues of the carboxyl terminus of IB or a
phosphospecific antibody that recognizes IB only when it is
phosphorylated on Ser32 (Data Supplement available at
www.circulationaha.org). The IB protein-antibody complex was
immunoprecipitated using protein A-agarose beads, and the gels were
then fixed, soaked in a fluorographic solution, dried, and exposed.
To determine whether PKG was sufficient to directly
phosphorylate IB on Ser32, we used a simple cell-free
in vitro assay system (Data Supplement). For these studies, an
IB-glutathione-S-transferase (GST) protein was incubated
with 1000 U of PKG for 10 minutes, and the samples were incubated
overnight with the phosphospecific Ser32 antibody. Immune complexes
were precipitated and analyzed by SDS-PAGE, and the gels
were vacuum-dried and exposed to x-ray film at -70°C.
Statistical Analysis
Data are expressed as mean±SEM. One-way ANOVA was used to
test for differences between group means. When appropriate, post hoc
multiple comparisons were performed to test for differences between
control and experimental groups (Dunnett’s test) or between
experimental groups (Newman-Keuls test). Two-way ANOVA was used to
evaluate overall differences in the means between different groups as a
function of time. Significant differences were said to exist at
P<0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Myocardial TNF-
Biosynthesis Ex VivoMyocardial TNF-
mRNA BiosynthesisThe salient finding shown by Figure 1A
is that treatment with SNAP (1
µmol/L) resulted in a time-dependent increase in TNF- mRNA
biosynthesis. As shown, TNF- mRNA expression was detectable within
30 minutes, and it increased 7-fold by 180 minutes. Similar
qualitative findings were observed in 2 additional SNAP-treated hearts.
In contrast, TNF- mRNA was not detectable in the hearts (n=3) that
were treated with diluent alone, as we reported
previously.1
|
) and SNAP-treated (1 µmol/L)
hearts (
). Inset in B shows results of group data after stimulation
with diluent or 10-9 to 10-4 mol/L SNAP (n=3
to 4 hearts per concentration). *P<0.05,
**P<0.01 compared with diluent-treated hearts.
Myocardial TNF- Protein Biosynthesis
Figure 1B
shows that TNF- protein synthesis was
detectable as early as 90 minutes after SNAP (1 µmol/L)
treatment, and it increased 19-fold by 180 minutes. In contrast,
TNF- protein levels were not detectable in the diluent-treated
hearts. The inset of Figure 1B shows that the SNAP-induced
(10-9 to 10-4 mol/L)
increase in TNF- protein biosynthesis was concentration-dependent
(P<0.001 by ANOVA). Post hoc multiple comparison testing
showed that there were significant differences (P<0.05)
between these and control values for SNAP concentrations
10-9 mol/L.
Two additional control studies were performed to be certain that
the SNAP-induced TNF- biosynthesis was not a nonspecific effect of
the NO donor chosen. First, we pretreated hearts (n=3) for 60 minutes
with the NO-scavenger
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide
(C-PTIO) (10-4 mol/L) and then treated them
with 1 µmol/L SNAP for 180 minutes. Second, we stimulated
another group of hearts (n=3) for 180 minutes with 5 µmol/L
sodium nitroprusside, a structurally dissimilar NO donor. As shown in
Figure 2A, pretreatment with C-PTIO
completely abrogated TNF- mRNA expression, whereas sodium
nitroprusside treatment induced TNF- mRNA synthesis. Figure
2B shows that C-PTIO prevented SNAP-induced TNF- protein
synthesis, whereas sodium nitroprusside provoked TNF- protein
synthesis.
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Cellular Source for Myocardial TNF- Biosynthesis
To determine whether SNAP stimulation was sufficient to
provoke TNF- biosynthesis in adult cardiac myocytes, we examined
TNF- mRNA and protein biosynthesis in diluent and SNAP-treated adult
cardiac myocytes cultures. Figure 3A
shows that TNF- mRNA levels were barely detectable in the
diluent-treated cultures, whereas TNF- mRNA levels were increased in
a time-dependent manner in the SNAP-treated (1 µmol/L) myocyte
cultures. Similar qualitative results were observed in 2 additional
primary myocyte isolations. Figure 3B shows that TNF- protein
levels were negligible in diluent-stimulated myocyte cultures (n=6
cultures per time point), whereas an 11- and 16-fold increase occurred
at 1 and 6 hours, respectively, in TNF- protein synthesis in the
SNAP-treated (1 µmol/L) myocyte cultures (n=6 cultures per time
point). Two-way ANOVA indicated that a statistically significant
difference existed in the effect of SNAP on myocyte TNF- synthesis
when compared with diluent treatment (P<0.001).
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Transcription Factors for TNF-
To determine whether SNAP-induced TNF- biosynthesis was
related to the activation of NF-B and/or AP-1, we performed EMSAs in
SNAP-treated hearts. Figure 4A shows the
temporal increase in NF-B–DNA complexes in SNAP-treated (1
µmol/L) hearts. As shown, the NF-B binding activity was increased
at as early as 30 minutes, which is consistent with the rapid
onset of TNF- mRNA biosynthesis in the SNAP-treated hearts. After
180 minutes of SNAP stimulation, 3 separate binding complexes were
detectable by EMSA; they were denoted C1 (slowest moving), C2, and C3
(fastest moving). Similar findings were obtained in 3 additional
experiments. The specificity of the binding to the radiolabeled
oligonucleotides was demonstrated by cold chase
experiments, in which the C1 through C3 complexes decreased in
intensity and eventually disappeared when a 25- and 50-fold molar
excess of unlabeled oligonucleotide was added,
respectively, to the nuclear extracts. To determine the specific
components that comprised the NF-B/Rel complexes, we also performed
supershift assays using antibodies to the various known components of
NF-B and Rel (p50, p52, p65, RelB, and cRel). These studies showed
that NF-B protein complexes 2 and 3 were comprised of p50 and p65
proteins. In contrast to the findings observed with NF-B, we did not
observe any evidence of AP-1-DNA binding activity in nuclear extracts
from the SNAP-treated hearts. As shown (Figure 4C),
angiotensin II (positive control) was sufficient to
activate AP-1.
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Role of the cGMP Pathway in TNF- Biosynthesis and NF-B
Activation
Inhibition and Activation of cGMP
We next examined the effects of inhibiting and activating the
cGMP/PKG pathway in our model system. For the series of studies that
follow, TNF- mRNA and protein levels are presented in
Figures 5A and 5B, respectively, and the
corresponding NF-B gel shift assays are presented in Figure
5C. When hearts were pretreated for 60 minutes with either ODQ
(10-5 mol/L), an inhibitor of
guanylyl cyclase, or Rp-8-Br-cGMPS (2.5x10-5
mol/L), an inhibitor of PKG, before stimulating them with
1 µmol/L SNAP for 180 minutes, TNF- mRNA/protein biosynthesis
and NF-B activation were completely abrogated. Treating hearts with
10-5 mol/L 8-Br-cGMP or
10-7 mol/L atrial natriuretic factor
resulted in TNF- mRNA/protein biosynthesis and NF-B activation.
Similar qualitative results were obtained for at least 3 hearts in each
experimental group. Finally, we showed that stimulating the cells with
10-6 to 10-2 mol/L
8-Br-cGMP led to a dose-dependent increase in TNF- biosynthesis in
isolated myocytes, whereas stimulation with 10-6
to 10-2 mol/L 8-Br-cAMP had no discernible
effect on TNF- biosynthesis (Data Supplement).
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Mechanism for cGMP-Induced NF-B Activation
Insofar as our studies suggested that the cGMP/PKG pathway was
sufficient to activate NF-B, we next sought to determine
whether PKG was sufficient to activate NF-B through an
IB-dependent mechanism. As shown in Figure 6A, both SNAP (1 µmol/L) and
8-Br-cGMP (10-5 mol/L) were sufficient to lead
to the rapid (<30 minute) degradation (60% decrease) in the level
of IB protein in cultured adult cardiac myocytes; they also led
to the phosphorylation of IB on Ser32, which is
consistent with the rapid degradation of the
phosphorylated IB protein reported in
nonmyocyte cell types.8 To confirm these
observations in cultured cells, we next asked whether PKG, a
serine/threonine kinase,9 was sufficient to
phosphorylate IB on Ser32 in a simple cell-free assay
system. The important finding shown by lane 2 in Figure 6B is
that PKG was sufficient to phosphorylate IB at the
critical Ser32 residue, whereas phosphorylation of
IB was not detectable in the absence of either PKG (lane 1) or
the IB-GST fusion protein (lane 3). Similar results were obtained
in 3 additional experiments.
|
-P32]ATP.
I |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The major conclusion to be drawn from this study is that pathophysiologically relevant concentrations of NO are sufficient to provoke TNF-
biosynthesis in the adult
mammalian heart. Three distinct but mutually complementary lines of
evidence support this statement. First, treatment of isolated
buffer-perfused adult cat hearts with concentrations of SNAP that are
known to generate pathophysiologically relevant
concentrations of NO6 resulted in a robust increase in
TNF- mRNA (Figure 1A) and protein biosynthesis (Figure
1B). Consistent with previous observations from this
laboratory, neither TNF- mRNA nor protein biosynthesis were observed
in diluent-treated hearts.1 The fact that we could
completely inhibit the effects of SNAP-induced TNF- mRNA (Figure
2A) and protein biosynthesis (Figure 2B) with an NO
scavenger (C-PTIO) and mimic the effects of SNAP with a structurally
dissimilar NO donor (sodium nitroprusside) suggests that the effects of
SNAP were related to the generation of NO and not to a nonspecific
effect of the NO donor. Second, these findings were evident at the
level of the intact ventricle and in the isolated cardiac myocyte
itself. That is, stimulation of relatively pure (>98%) cultures of
isolated adult cardiac myocytes with SNAP resulted in an 11- and
16-fold increase in TNF- biosynthesis at 1 and 6 hours, respectively
(Figure 3). Third, SNAP stimulation resulted in the rapid (30
minutes) activation of NF-B, an important transcription factor for
TNF- (Figure 4). Importantly, the time course for activation
of NF-B was entirely consistent with the rapid de novo
expression of TNF- mRNA that we observed after SNAP stimulation.
Moreover, our findings suggest that the SNAP-induced NF-B/Rel
complexes were comprised of p50/p65 heterodimers.
A second important finding of this study was that NO
provoked TNF- biosynthesis through a cGMP-dependent pathway. In
preliminary control experiments (Data Supplement) we confirmed that
SNAP stimulation of isolated hearts resulted in a rapid 14-fold
increase in myocardial cGMP and a 2-fold increase in myocardial PKG
activity. Importantly, we were able to completely abrogate TNF- mRNA
(Figure 5A) and protein biosynthesis (Figure 5B), as well
as NF-B activation (Figure 5C), when we used specific
inhibitors to block NO-induced guanylyl cyclase or PKG
activity. To provide more direct evidence for the role of the cGMP
pathway, 3 additional studies were performed. First, we could mimic the
effects of SNAP on TNF- mRNA and protein biosynthesis and NF-B
activation (Figure 5) using 8-Br-cGMP, a biologically active
cGMP analog. Second, when we stimulated hearts with atrial
natriuretic factor, a peptide that stimulates cGMP through
an NO-independent pathway, we could provoke TNF- mRNA and protein
biosynthesis and NF-B activation (Figure 5). Third, in
additional control experiments (Data Supplement), we determined that
the SNAP-induced TNF- biosynthesis was not secondary to cross-talk
and/or activation of cAMP, as has been described in rat aortic smooth
muscle cells.10 To determine the mechanism for the
observed cGMP/PKG-mediated activation of NF-B, we demonstrated that
the stimulation of isolated cardiac myocytes with SNAP or 8-Br-cGMP led
to the degradation of IB and to the
phosphorylation of IB at the critical Ser32
residue (Figure 6A). Insofar as the above studies suggested that
PKG might be acting as an IB kinase, we further demonstrated that
PKG was sufficient to phosphorylate IB on its Ser32
residue in a simple, cell-free, in vitro kinase assay. Thus, in
summary, the above results suggest that NO provokes TNF-
biosynthesis through a pathway that involves the activation of cGMP,
PKG, and NF-B.
Although the findings in the present study are
consistent with earlier reports that have implicated NO and/or
cGMP in TNF- biosynthesis,5 11 they seem to disagree
with previous reports which suggest that NO decreases NF-B-DNA
binding12 and suppresses TNF-
biosynthesis.13 14 There are several possible explanations
for the observed differences. For example, the cell types and the
nature and doses of the NO-donors used in the aforementioned studies
were different from those used in the present study. Furthermore,
apart from these more obvious methodological differences, there is,
perhaps, a more intriguing explanation. That is, in all of the studies
in which NO was shown to decrease NF-B activation and/or TNF-
expression, the cells had been previously stimulated with agents that
activate NF-B before being treated with NO. Thus, in
settings in which NF-B is already activated, the primary
effect of NO might be inhibitory, either by increasing the
NF-B–induced transcription of inhibitory IB proteins
or, alternatively, by stabilizing IB.13 However, in the
absence of prior NF-B activation, the primary effect of NO might be
stimulatory, as was observed in the present study. Indeed, this
point of view is consonant with the recent observation in
endothelial cells that low concentrations of NO are
sufficient to activate NF-B, whereas higher concentrations
of NO inhibit NF-B activation.15
Conclusions
The present study constitutes the initial demonstration that
NO provokes TNF- biosynthesis through the cGMP pathway. Although the
exact biological significance of these findings is not known, the
results of this study may provide new insight into one mechanism for
the progressive cardiac decompensation that occurs after sustained
myocardial injury. That is, given that NO provokes myocardial TNF-
biosynthesis and that TNF- is sufficient to induce iNOS expression,
the coincident expression of TNF- and NO in the heart might foster
self-sustaining positive autocrine/paracrine feedback inflammatory
circuits within the myocardium that lead to the
inappropriate overexpression of these 2 inflammatory mediators.
Additionally, these studies suggest the intriguing possibility that
cross-talk may exist between 2 signal transduction pathways that have,
heretofore, been considered functionally distinct in the heart (ie, the
adrenergic system and the cytokine system). Indeed, although it
has long been recognized that TNF- can induce the expression of iNOS
in certain cell types, what is less well appreciated is that
catecholamines stabilize iNOS mRNA in cardiac
myocytes.16 Thus, both cytokines and adrenergic
mediators may converge on and amplify the expression of NO in certain
disease states. Whether either of these mechanisms provides a
satisfactory explanation for the sustained expression of TNF- and
iNOS in the failing human heart will require further study.
|
Acknowledgments |
|---|
The authors gratefully acknowledge the secretarial assistance of Jana Grana, as well as the spirited technical assistance of Dorellyn Lee-Jackson and Stacey Walker. The authors also thank Dr Andrew I. Schafer for his past and present support and guidance. This research was supported by research funds from the Department of Veterans Affairs and the National Institues of Health (grants P50 HL-O6H, RO1 HL58081-01, and RO1 HL61543-01).
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Footnotes |
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Guest Editor for this article was Wilson S. Colucci, MD, Boston Medical Center, Boston, Mass.
Supplementary material for this article can be found Online at www.circulationaha.org
Received February 18, 2000; revision received April 7, 2000; accepted April 10, 2000.
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 S. Gupta, R. Maitra, D. Young, A. Gupta, and S. Sen Silencing the myotrophin gene by RNA interference leads to the regression of cardiac hypertrophy Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H627 - H636. [Abstract] [Full Text] [PDF]
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 B. Das, S. Gupta, A. Vasanji, Z. Xu, S. Misra, and S. Sen Nuclear Co-translocation of Myotrophin and p65 Stimulates Myocyte Growth: REGULATION BY MYOTROPHIN HAIRPIN LOOPS J. Biol. Chem., October 10, 2008; 283(41): 27947 - 27956. [Abstract] [Full Text] [PDF]
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 A. Steensberg, C. Keller, T. Hillig, C. Frosig, J. F. P. Wojtaszewski, B. K. Pedersen, H. Pilegaard, and M. Sander Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle FASEB J, September 1, 2007; 21(11): 2683 - 2694. [Abstract] [Full Text] [PDF]
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 H.-F. Cheng, M.-Z. Zhang, and R. C. Harris Nitric oxide stimulates cyclooxygenase-2 in cultured cTAL cells through a p38-dependent pathway Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1391 - F1397. [Abstract] [Full Text] [PDF]
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 G. F. Tomaselli and D. P. Zipes What Causes Sudden Death in Heart Failure? Circ. Res., October 15, 2004; 95(8): 754 - 763. [Abstract] [Full Text] [PDF]
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 K. Sekiguchi, X. Li, M. Coker, M. Flesch, P. M Barger, N. Sivasubramanian, and D. L Mann Cross-regulation between the renin-angiotensin system and inflammatory mediators in cardiac hypertrophy and failure Cardiovasc Res, August 15, 2004; 63(3): 433 - 442. [Abstract] [Full Text] [PDF]
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R. B. Pilz and D. E. Casteel Regulation of Gene Expression by Cyclic GMP Circ. Res., November 28, 2003; 93(11): 1034 - 1046. [Abstract] [Full Text] [PDF]
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L. Connelly, A. T. Jacobs, M. Palacios-Callender, S. Moncada, and A. J. Hobbs Macrophage Endothelial Nitric-oxide Synthase Autoregulates Cellular Activation and Pro-inflammatory Protein Expression J. Biol. Chem., July 11, 2003; 278(29): 26480 - 26487. [Abstract] [Full Text] [PDF]
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T. Peng, X. Lu, M. Lei, and Q. Feng Endothelial Nitric-oxide Synthase Enhances Lipopolysaccharide-stimulated Tumor Necrosis Factor-alpha Expression via cAMP-mediated p38 MAPK Pathway in Cardiomyocytes J. Biol. Chem., February 28, 2003; 278(10): 8099 - 8105. [Abstract] [Full Text] [PDF]
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 D. L. Brutsaert Cardiac Endothelial-Myocardial Signaling: Its Role in Cardiac Growth, Contractile Performance, and Rhythmicity Physiol Rev, January 1, 2003; 83(1): 59 - 115. [Abstract] [Full Text] [PDF]
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D. L. Mann Inflammatory Mediators and the Failing Heart: Past, Present, and the Foreseeable Future Circ. Res., November 29, 2002; 91(11): 988 - 998. [Abstract] [Full Text] [PDF]
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H. Funakoshi, T. Kubota, N. Kawamura, Y. Machida, A. M. Feldman, H. Tsutsui, H. Shimokawa, and A. Takeshita Disruption of Inducible Nitric Oxide Synthase Improves {beta}-Adrenergic Inotropic Responsiveness but Not the Survival of Mice With Cytokine-Induced Cardiomyopathy Circ. Res., May 17, 2002; 90(9): 959 - 965. [Abstract] [Full Text] [PDF]
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M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]
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G. Wright, I. S. Singh, J. D. Hasday, I. K. Farrance, G. Hall, A. S. Cross, and T. B. Rogers Endotoxin stress-response in cardiomyocytes: NF-kappa B activation and tumor necrosis factor-alpha expression Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H872 - H879. [Abstract] [Full Text] [PDF]
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 G. Valen, Z.-q. Yan, and G.o. K. Hansson Nuclear factor kappa-B and the heart J. Am. Coll. Cardiol., August 1, 2001; 38(2): 307 - 314. [Abstract] [Full Text] [PDF]
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M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]
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 D. K. Kalra, X. Zhu, M. K. Ramchandani, G. Lawrie, M. J. Reardon, D. Lee-Jackson, W. L. Winters, N. Sivasubramanian, D. L. Mann, and W. A. Zoghbi Increased Myocardial Gene Expression of Tumor Necrosis Factor-{alpha} and Nitric Oxide Synthase-2: A Potential Mechanism for Depressed Myocardial Function in Hibernating Myocardium in Humans Circulation, April 2, 2002; 105(13): 1537 - 1540. [Abstract] [Full Text] [PDF]
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D. Kalra, N. Sivasubramanian, and D. L. Mann Angiotensin II Induces Tumor Necrosis Factor Biosynthesis in the Adult Mammalian Heart Through a Protein Kinase C-Dependent Pathway Circulation, May 7, 2002; 105(18): 2198 - 2205. [Abstract] [Full Text] [PDF]
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G. Baumgarten, P. Knuefermann, D. Kalra, F. Gao, G. E. Taffet, L. Michael, P. J. Blackshear, E. Carballo, N. Sivasubramanian, and D. L. Mann Load-Dependent and -Independent Regulation of Proinflammatory Cytokine and Cytokine Receptor Gene Expression in the Adult Mammalian Heart Circulation, May 7, 2002; 105(18): 2192 - 2197. [Abstract] [Full Text] [PDF]
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