From the Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Königinstrasse 16, 80539 Munich, Germany
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ABSTRACT |
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Atrial natriuretic peptide (ANP), a
cardiovascular hormone, has been shown to inhibit synthesis of nitric
oxide in lipopolysaccharide (LPS)-activated mouse bone marrow-derived
macrophages via activation of its guanylate cyclase-coupled receptor.
The goal of the present study was to elucidate the potential sites of
inducible nitric-oxide synthase (iNOS) regulation affected by ANP and
revealed the following. 1) ANP and dibutyryl-cGMP did not inhibit
catalytic iNOS activity measured by the conversion rate of
L-[3H]arginine to
L-[3H]citrulline in homogenates of
LPS-treated cells. 2) Pretreatment of cells with ANP
dose-dependently reduced the LPS-induced
L-[3H]citrulline production that has been
shown to be due to reduced iNOS protein levels detected by Western
blot. 3) ANP does not alter the ratio of catalytically active iNOS
dimer versus inactive iNOS monomer considered to be a major
post-translational regulatory mechanism for the enzyme. 4) Macrophages
exposed to ANP display decreased LPS-induced iNOS mRNA levels. 5)
Importantly, two basic mechanisms seem to be responsible for this
observation, i.e. ANP specifically induced acceleration of
iNOS mRNA decay and ANP reduced binding activity of NF-B, the
transcription factor predominantly responsible for LPS-induced iNOS
expression in murine macrophages. Moreover, 6) ANP acts via an
autocrine mechanism since recently ANP was shown to be secreted by
LPS-activated macrophages, and we demonstrated here that LPS-induced NO
synthesis was increased after blocking the binding of endogenous ANP by
a receptor antagonist. These observations suggest ANP as a new
autocrine macrophage factor regulating NO synthesis both
transcriptionally and post-transcriptionally. ANP may help to balance
NO production of activated macrophages and thus may allow successful
immune response without adverse effects on host cells.
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INTRODUCTION |
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Atrial natriuretic peptide (ANP)1 is a 28-amino acid
polypeptide secreted mainly by the heart
atria in response to atrial stretch. The main and best studied actions
of ANP are geared toward the regulation of volume pressure homeostasis
(for review see Refs. 1 and 2). There are two biochemically and
functionally distinct classes of natriuretic peptide receptors (NPR).
Clearance receptors (NPR-C) are by far the most abundant class of NPR.
Besides their well established role in removing ANP from the
circulation, the NPR-C elicit biological functions by interacting with
G-proteins (2, 3). The guanylate cyclase-coupled receptors (NPR-A) are
signaling receptors that mediate all known cardiovascular and renal
effects of ANP via cGMP (2, 4). The functions of the natriuretic
peptides, however, are not restricted to the regulation of volume
homeostasis as suggested earlier by demonstration of ANP and its
receptors in diverse tissues besides the cardiovascular and renal
system (5). ANP was suggested to play a role in the immune system
because thymus (6-8) and macrophages (9, 10) are sites of synthesis of
the natriuretic peptide and its receptors. In the course of functional
investigations concerning ANP in the immune system, the peptide was
found to inhibit maturation and differentiation of fetal thymus (11) as
well as proliferation of thymocytes of adult animals (8). Recently, ANP
was shown to reduce nitrite accumulation in lipopolysaccharide
(LPS)-activated murine macrophages (10). Thus, the peptide might
interfere with the synthesis of a mediator that plays an important role
in inflammation and host defense response (12). The enzyme nitric-oxide
synthase (NOS), which catalyzes the synthesis of NO from
L-arginine, exists in three different isoforms that differ
in their tissue distribution, calcium dependence, and in the regulation
of their expression (13, 14). The two constitutive isoforms expressed
in neurons (neuronal nitric-oxide synthase; NOS I) and endothelial
cells (endothelial nitric-oxide synthase; NOS III) are
calcium/calmodulin-dependent. They are mainly involved in
neurotransmission and vascular regulation, respectively (15). A major
function of NO derived from the inducible NO synthase (iNOS; NOS II) is
target cell cytotoxicity. Target cells may include tumor cells as well
as bacteria, viral particles, and other microorganisms (12). However,
NO produced by iNOS of macrophages (as well as other cells) also has
the potential for adverse activities depending on its concentration and
site of release. These include the induction of severe hypotension and
cardiovascular shock and cytotoxicity toward host cells such as
vascular cells, lymphocytes, or even macrophages themselves (12).
Therefore, a better understanding of the physiological regulation of
iNOS is important. The major activator of iNOS in macrophages has been
shown to be bacterial lipopolysaccharide (LPS) (12, 16). Co-stimulatory
effects were demonstrated for INF- and a variety of other cytokines
such as TNF-
, IL-2, INF-
, and -
(12).
So far, little is known about what terminates the production of NO by
macrophages. However, this is of particular importance regarding the
severe pathophysiological effects of sustained NO production such as
circulatory failure and tissue damage. Again cytokines, i.e.
transforming growth factor-, IL-4, and IL-10, have been described to
suppress NO release of macrophages (12, 17). The underlying mechanisms
appeared to be different for the respective cytokines (12, 17, 18). The
observation that ANP, a circulating hormone best known for its
vasodilative effects, inhibits NO synthesis in activated macrophages is
particularly interesting since ANP concentrations are highly elevated
in septic shock (19), and moreover LPS-exposed macrophages were shown to produce increased ANP (9). Thus, ANP may be a novel autocrine substance modulating NO production. Consecutively, aim of the present
study was to clarify the basic mechanisms underlying the inhibition of
NO synthesis by ANP.
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EXPERIMENTAL PROCEDURES |
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Materials--
Mouse ANP 99-126 was purchased from Calbiochem
(Bad Soden, Germany). HS-142-1 was a gift from Dr. Matsuda, Tokyo
Research Laboratories, Tokyo, Japan. iNOS cDNA probe was provided
by Dr. Kleinert, University of Mainz, Germany; COX-2 cDNA was a
gift from Dr. Herschman, UCLA; TNF- cDNA was obtained from Dr.
Decker, University of Freiburg, Germany; IL-6 cDNA was provided by
Dr. Kremer, GSF, Munich, Germany. Monoclonal antibody against
macrophage iNOS was obtained from Transduction Laboratories (Lexington,
KY); antiserum against the macrophage antigen F4/80 was from Serotec LTD (Wiesbaden, Germany); cell culture media (RPMI 1640, DMEM), fetal
calf serum (FCS), penicillin/streptomycin, and TRIzolTM
were from Life Technologies, Inc. (Eggenstein, Germany). cGMP radioimmunoassay kit (cGMP 125I-assay system) and
[3H]L-arginine (60 Ci/mmol), ECL detection
system, and random primer labeling system (Rediprime®)
were from Amersham (Braunschweig, Germany); Dowex 50 WX8
(Na+ form) was obtained from Serva (Heidelberg, Germany);
[
-32P]UTP (800 Ci/mmol), [
-32P]ATP,
and [
-32P]dCTP (both 3000 Ci/mmol) were from Hartmann
Analytic (Braunschweig, Germany); T3/T7 RNA polymerase transcription
system was obtained from Stratagene (Heidelberg, Germany);
dexamethasone solution was ordered from Centravet (Bad Bentheim,
Germany); NF-
B and AP-2 binding oligonucleotides, SP6 polymerase,
and T4 polynucleotide kinase were obtained from Boehringer Ingelheim
Bioproducts (Heidelberg, Germany); IRF-1 binding oligonucleotide was
from Santa Cruz Biotechnology (Heidelberg, Germany). Bradford protein
assay was from Bio-Rad (Munich, Germany). All other materials were
purchased from either Sigma (Deisenhofen, Germany) or ICN Biomedicals
(Eschwege, Germany).
Cell Culture-- Mouse bone marrow macrophages (BMM) were prepared as described previously (9) and were seeded at a density of 2 × 105 cells/ml in 24-well tissue plates and grown for 5 days (5% CO2, 37 °C) in RPMI 1640 medium supplemented with 20% L-929 cell-conditioned medium, 10% heat-inactivated FCS, and penicillin (100 units/ml)/streptomycin (100 µg/ml). L-cell-conditioned medium was removed at least 12 h before experiments. BMM were found >95% pure as judged by fluorescence-activated cell sorter analysis (FACscan, Becton Dickinson, San Jose, CA) using an antiserum against the macrophage antigen F4/80 (20). Adult bovine aortic endothelial cells (ABAE, provided by Dr. Plendl, Munich) were cultivated in 24-well tissue plates in DMEM containing 10% FCS (21).
Measurement of cGMP-- Confluent BMM or ABAE (24-well tissue plates) were washed three times and pretreated with 3-isobutyl-1-methylxanthine (0.5 mM) in serum-free RPMI 1640 for 10 min at 37 °C. Various stimuli were added for 30 min. Thereafter, medium was aspirated and cGMP was extracted immediately by the addition of HCl (0.1 N). After 10 min of incubation on ice the cell extracts were transferred to fresh tubes, lyophilized, and assayed for cGMP content by radioimmunoassay using a commercially available kit.
Nitrite Accumulation-- BMM (24-well plates, 200 µl) were treated with lipopolysaccharide (LPS, E. coli, serotype 055:B5, 1 µg/ml) in the presence or absence of various concentrations of ANP 99-126 and/or HS-142-1. After 20 h the stable metabolite of NO, nitrite, was measured in the medium by the Griess reaction (22). 100 µl of cell culture supernatant was removed, and 90 µl of 1% sulfanilamide in 5% H3PO4 and 90 µl of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in H2O was added, followed by spectrophotometric measurement at 550 nm (reference wavelength 620 nm).
iNOS Enzyme Activity--
BMM (24-well plates) were either
untreated, stimulated with LPS (1 µg/ml) only, or co-incubated with
ANP 10 nM-1 µM) for 12 h, washed three
times with cold PBS, frozen immediately, and stored at 70 °C. iNOS
activity was determined by measuring the conversion of
[3H]L-arginine to
[3H]L-citrulline according to Ref. 23.
Briefly, cells were homogenized in 50 mM Tris, pH 7.6, containing EDTA (0.1 mM), EGTA (0.1 mM), and
phenylmethylsulfonyl fluoride (PMSF, 1 mM) by freezing and thawing. Homogenates of equal protein concentration (Bradford method)
(100 µl, 200 µg of protein) were incubated at 37 °C for 30 min
in the presence of L-arginine (10 µM), NADPH
(1 mM), L-valine (50 mM), FAD (4 µM), tetrahydrobiopterin (4 µM), and
[3H]L-arginine (0.2 µCi; 0.033 µM). Reactions were stopped by adding ice-cold sodium
acetate, pH 5.5 (20 mM, 1 ml), containing EDTA (2 mM) and L-citrulline (0.1 mM).
[3H]L-Citrulline was separated by ion
exchange columns (Dowex 50 WX8, Na+ form) and measured by
scintillation counting. The effect of test substances on the specific
enzyme activity was evaluated using homogenates of cells treated with
LPS for 12 h. Substances were incubated with cell homogenate for
10 min and further processed as described above. Extent of
L-[3H]citrulline formation independent of
iNOS activity was determined in each experiment by employing
homogenates of cells not exposed to LPS. The results are expressed as
percentage of iNOS-specific [3H]citrulline formation.
Western Blot Analysis--
BMM (24-well plates) were treated
with LPS (1 µg/ml) or a combination of LPS (1 µg/ml) plus ANP (1 µM) with or without HS-142-1 (100 µg/ml) for 12 h.
Cells were washed with ice-cold PBS and stored at 70 °C. Western
blot analysis was performed according to Ref. 24 except that the lysis
buffer (50 mM Tris-HCl, pH 6.8, 1% SDS, 2%
mercaptoethanol, 10% glycerol, 0.004% bromphenol blue) was
supplemented with a protease inhibitor mixture (Complete®). After
sonication lysates were either boiled for 5 min (fully denaturing conditions) or not boiled (partially denaturing conditions) to discriminate between iNOS dimer and monomer (24). Samples (60 µg of
protein) were loaded on an SDS-polyacrylamide gel (7.5%) and
electroblotted, and iNOS protein was detected using an anti-iNOS monoclonal antibody and the ECL detection system. Signal intensities were evaluated by densitometric analysis (Herolab, E.A.S.Y. plus system, Wiesloch, Germany).
Detection of iNOS mRNA--
BMM were stimulated with or
without LPS (1 µg/ml) in the presence or absence of ANP (1 µM) or dexamethasone (10 µM) for 6 h
(24-well plates). RNA was prepared using TRIzolTM reagent
and pooled from 6 wells. Northern blot was performed in principle as
described previously (6, 9). Membranes were hybridized to a
32P-labeled murine macrophage iNOS cRNA probe (2 × 106 cpm/ml). iNOS cDNA (558 base pairs) was subcloned
in a pBluescript SK(+) vector, linearized (HindIII), and
labeled with [32P]UTP (50 µCi) using a T3 RNA
polymerase transcription system. Signal intensity was evaluated by
densitometric analysis. To control for comparable amounts of intact
mRNA loaded on the gel, membranes were rehybridized with a
32P-labeled -actin probe (2 × 106
cpm/ml) as described (6).
Analysis of mRNA Stability--
BMM in 24-well plates were
treated with LPS (1 µg/ml) or a combination of LPS (1 µg/ml) plus
ANP 99-126 (1 µM) for 5 h before addition of
actinomycin D (5 µg/ml). Total RNA was prepared at the times
indicated and further processed for Northern blot hybridization as
described above. In addition to hybridization with an iNOS cRNA probe,
blots have been rehybridized with probes for TNF- (25), COX-2 (26),
and IL-6 (27), respectively. To normalize mRNA concentrations
membranes were hybridized with a
-actin probe. The iNOS,
-actin,
TNF-
, and COX-2 probes were radiolabeled by in vitro
transcription and the IL-6 DNA probe by random-primed cDNA
synthesis. The signal intensities were analyzed densitometrically, and
signal density for iNOS or the other mRNAs was divided by that of
-actin in order to correct the loading differences. mRNA decay
was evaluated based on the assumption that change of mRNA concentration at any time is a first-order process depending on the
amount of mRNA present at that time (28). Accordingly, the ratios
of the signal intensities of the respective mRNA/
-actin mRNA
at each time point were expressed as percentage of the time point 0, i.e. before addition of actinomycin D and plotted against time.
Preparation of Nuclear Extracts--
BMM were grown in 24-well
plates and stimulated with LPS (1 µg/ml) in the presence or absence
of ANP 99-126 (1 µM-10 nM) for 2 h.
Dibutyryl-cGMP (1 mM) or pyrrolidinedithiocarbamate (PDTC, 50 µM) was added 1 h prior to LPS stimulation.
Nuclear extracts were prepared as described (29). Briefly, cells were
washed with PBS, resuspended in 400 µl of hypotonic buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF), and were allowed to swell on ice for 15 min. Nonidet P-40 (10%,
25 µl) was added followed by 10 s of vigorous vortexing and
centrifugation at 12,000 × g for 30 s. The
supernatant was removed, and the nuclear pellet was extracted with 50 µl of hypertonic buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) by shaking at 4 °C for 15 min. The extract was centrifuged at 12,000 × g, and
the supernatant was frozen at 70 °C. The protein concentration was
determined by the Lowry method (30).
Electrophoretic Mobility Shift Assay (EMSA)--
Two 22-mer
double-stranded oligonucleotide probes containing a consensus binding
sequence for either NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') or IRF-1 (5'-GGAAGCGAAAATGAAATTGACT-3') were 5'-end-labeled
with [
-32P]-ATP (10 µCi) using T4 polynucleotide
kinase. 10 µg of nuclear protein were incubated (20 min at room
temperature) in a 15-µl reaction volume containing 10 mM
Tris-HCl, pH 7.5, 5 × 104 cpm radiolabeled
oligonucleotide probe, 2 µg of poly(dI-dC), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, and 0.5 mM DTT. Nucleoprotein-oligonucleotide complexes were resolved by
electrophoresis (4.5% non-denaturing polyacrylamide gel, 100 V). The
gel was autoradiographed with an intensifying screen at
70 °C
overnight. Specificity of the DNA-protein complex was confirmed by
competition with a 100-fold excess of unlabeled NF-
B and AP-2
(5'-GATCGAACTGACCGCCCGCGGCCCGT-3') binding sequences, respectively.
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RESULTS |
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Demonstration of Guanylate Cyclase-coupled NPR-A in BMM, Its Implication in ANP-induced NO Inhibition-- Previous data that have been confirmed by a representative experiment shown in Fig. 1A demonstrated that ANP dose-dependently inhibits nitrite accumulation of LPS-activated BMM. The ANP-induced NO inhibition seems to be mediated by the guanylate cyclase-coupled NPR-A receptor, since HS-142-1, an NPR-A antagonist (31), abrogated the ANP effect. In addition, expression of the mRNA coding for both of the ANP receptors (NPR-A and NPR-C) has been shown before in BMM (10). The aim here was to clarify the actual presence of functional NPR-A protein in BMM. BMM were treated with ANP, and cGMP production was determined. Intracellular cGMP levels were significantly elevated in cells exposed to ANP (10 µM-10 nM) for 30 min compared with untreated cells (Fig. 1B). HS-142-1 (100 µg/ml) was able to antagonize this increase. To exclude that elevated cGMP levels were due to enzymatic activity of soluble guanylate cyclase (sGC) we incubated the cells with sodium nitroprusside (SNP), which is able to release NO, a known activator of sGC (14, 15). SNP (100 µg/ml) did not change intracellular cGMP levels in BMM (Fig. 1C). In contrast, cGMP production of ABAE, known to express sGC (32), was dose-dependently elevated by the addition of SNP (10 and 100 µg/ml) under identical conditions. Thus, BMM release NO when activated but do not possess sGC as an important target system for NO action.
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iNOS Activity in Cell Homogenates-- Suppression of nitrite accumulation in BMM by ANP has to be discussed as the sum of potentially cumulative actions of ANP on the iNOS system and, for instance, does not allow us to assess effects of ANP on specific enzyme activity. Therefore, ANP (1 µM) was added to the homogenate of LPS (1 µg/ml)-stimulated cells, and iNOS enzyme activity was measured as conversion rate of [3H]L-arginine to [3H]L-citrulline (Fig. 2A). Incubation of cell homogenate with ANP did not result in a change of iNOS activity. The cGMP analog dibutyryl-cGMP (1 mM) had no effect either. The known specific inhibitor of NOS enzyme activity NG-monomethyl-L-arginine (3 mM) served as control and was able to inhibit specific iNOS enzyme activity up to 90%. By having excluded a direct effect of ANP on the enzyme activity, we next wanted to examine whether reduction of NO synthesis by ANP is due to factors such as decreased uptake of L-arginine or availability of other necessary substrates and cofactors for iNOS. By using an indirect approach, measurement of enzyme activity was performed with cell homogenates of ANP-pretreated cells in the presence of optimal concentrations of substrates and cofactors (i.e. NADPH, L-arginine, BH4, FAD, and FMN). ANP pretreatment of cells dose-dependently (10 µM to 10 nM) reduced iNOS activity up to 70% (1 µM) compared with the activity of only LPS-stimulated cells (Fig. 2B). Thus, ANP induces inhibition of NO synthesis most likely not by causing an intracellular shortage of iNOS substrates. [3H]L-Citrulline formation in the homogenate of ANP-exposed cells co-treated with the ANP-receptor antagonist HS-142-1 (10 µg/ml) was almost restored completely to levels of LPS treatment only (Fig. 2B).
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ANP Treatment Reduces iNOS Protein Expression-- The reduced activity of iNOS after ANP treatment reflected a decrease of iNOS protein as revealed by immunoblot with a monoclonal mouse anti-iNOS antibody (Fig. 3A). No iNOS was detected in unstimulated BMM. Upon stimulation with LPS (1 µg/ml, 12 h) a single band (130 kDa) corresponding to iNOS appeared when fully denaturing conditions were used for sample preparation. The expression of iNOS protein was significantly reduced by ANP (1 µM), whereas co-incubation with the NPR-A receptor antagonist HS-142-1 (100 µg/ml) attenuated the decrease in iNOS protein by ANP.
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iNOS Dimerization-- The next question was to determine whether ANP influenced the ratio of catalytically active iNOS dimer versus inactive monomer. Western blots were performed under partially denaturing conditions that allow detection of iNOS dimer (260 kDa) (24). Signal intensity of both dimer and monomer diminished in ANP-treated cells, but the proportion of monomer/dimer remained unchanged compared with LPS treatment only (Fig. 3B).
ANP Treatment Reduces iNOS mRNA Levels-- Northern blot analysis was performed to determine whether ANP inhibits iNOS mRNA accumulation when added simultaneously with LPS (1 µg/ml). Time course experiments showed that appearance of iNOS mRNA in BMM was maximal between 4 and 8 h and decreases after 12 h (data not shown). Subsequently mRNA was isolated from cells after 6 h of treatment. In unstimulated cells no iNOS mRNA was detectable (Fig. 4). ANP (1 µM) caused a marked reduction of LPS-induced iNOS mRNA steady-state levels. Dexamethasone (10 µM), a known inhibitor of iNOS induction (33), completely blocked iNOS mRNA accumulation.
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ANP Accelerates iNOS mRNA Decay--
The ANP-induced reduction
of iNOS mRNA could be due to changes in either transcription or in
mRNA stability. The effect of ANP on stability of iNOS mRNA was
assessed by experiments employing the transcription inhibitor
actinomycin D. BMM were stimulated with LPS (1 µg/ml) in the presence
or absence of ANP (1 µM) for 5 h. Thereafter,
actinomycin D was added (5 µg/ml), and total RNA was extracted at the
indicated times and examined by Northern blot analysis. In order to
evaluate the specificity of the ANP effect for iNOS mRNA blots were
hybridized with probes for COX-2, TNF-, and IL-6 mRNA,
respectively (Fig. 5A). These
mRNAs were chosen for their similarity to the iNOS mRNA,
i.e. they are inducible and contain AUUUA sequences in their
3'-untranslated region, known to be important for mRNA degradation
(34, 35). To assess for equal loading of RNA and to normalize the
amount of RNA in each sample, blots were rehybridized with a probe for
-actin (Fig. 5A). Autoradiographs were subjected to
densitometry and evaluated as described under "Experimental
Procedures." The signal density ratio of iNOS/
-actin mRNA at
each time expressed as percentage of that of the control (0 h) was
plotted against time (Fig. 5B). It showed that the half-life
of iNOS mRNA in ANP-treated macrophages was considerably decreased
(1.9 versus 1.1 h untreated versus ANP-treated cells). Evaluation of the signal intensities of the other
mRNAs based on the autoradiographs shown in Fig. 5A,
revealed no reduction of mRNA stability by ANP for TNF-
and IL-6
mRNA, respectively (data not shown). COX-2 mRNA decay, however,
was increased by ANP similar to iNOS mRNA (Fig. 5C).
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ANP Inhibits NF-B Binding Activity--
As reduction of iNOS
mRNA stability may not be the only mechanism by which ANP reduces
NO synthesis, we determined its effect on binding activity of NF-
B,
the transcription factor known to be essential for iNOS induction in
mouse macrophages (36). NF-
B binding activity of nuclear extracts
was assessed by EMSA after stimulation of cells with LPS (1 µg/ml)
for 2 h (Fig. 6). Formation of the
specific DNA probe-NF-
B complex was dose-dependently
reduced when nuclear extracts of cells co-incubated with ANP (10 nM-1 µM) were employed. The cGMP analog
dibutyryl-cGMP (1 mM) could mimic the effect. The known
inhibitor of NF-
B binding activity, PDTC (50 µM), was
used as a control (36). Binding specificity was determined by addition
of a 100-fold excess of unlabeled NF-
B or AP-2 binding sequence. For
further control, binding activity of IRF-1, a transcription factor
involved in INF-
-induced iNOS expression (37), was tested. No
IRF-1·DNA complex formation could be demonstrated in LPS-treated BMM
(data not shown).
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Autocrine Mechanism of ANP-induced NO Inhbition-- By learning that ANP is able to interfere with iNOS expression, the question of its functional significance arises. It has previously been shown that ANP synthesis and secretion is markedly increased in LPS-activated BMM (9, 38), and thus, our working hypothesis was that ANP inhibits NO production via an autocrine mechanism.
We investigated whether blocking the NPR-A activation through endogenous ANP with the specific antagonist HS-142-1 would have an effect on LPS-induced NO secretion measured by nitrite accumulation. As shown in Fig. 7 treatment of BMM with HS-142-1 alone did not induce NO production as compared with untreated cells (Co). LPS (1 µg/ml) induced a marked NO production (100%) that was elevated dose-dependently by co-treating the cells with HS-142-1 (100 and 10 µg/ml).
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DISCUSSION |
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This paper focuses on the regulation of iNOS expression by ANP in
LPS-activated macrophages. ANP inhibits NO synthesis via the guanylate
cyclase-coupled NPR-A receptor. Evidence is provided that inhibition by
ANP is regulated at the transcriptional and post-transcriptional level
as follows. 1) ANP did not affect the catalytic activity of iNOS. 2)
Stability of iNOS mRNA was decreased in ANP-treated cells. 3) ANP
inhibited binding activity of NF-B, the predominant transcription
factor for iNOS induction in LPS-activated murine macrophages (36).
Furthermore, cGMP is suggested to be the second messenger of the ANP
effect based on the following data. 1) Exposure of BMM to ANP results
in increased cGMP production. The increase of cGMP originated from
activation of the particulate guanylate cyclase (NPR-A) rather than the
soluble guanylate cyclase, which is not present in BMM (Ref. 39 and
present data). 2) A specific NPR-A receptor antagonist (HS-142-1) (31)
was shown to abrogate, whereas stable cGMP analogs could mimic the
effects of ANP on various levels of iNOS regulation.
Some information about the influence of cyclic nucleotides on iNOS
expression are already available in the literature. In fact,
natriuretic peptides via increased cGMP have been shown to augment
IL-1-induced iNOS expression in vascular smooth muscle cells (40), and
an interaction of cyclic GMP elevating agents with the cyclic AMP
pathway has been suggested in this context (41). On the other hand,
8-Br-cGMP was recently shown to inhibit iNOS expression and NO
production in LPS-activated J774 macrophages (42). These data together
with our results argue that the effect of cGMP on iNOS seems to be
cell- and probably stimulus-dependent. A similar phenomenon
has been reported for transforming growth factor-, i.e.
it inhibits iNOS synthesis in macrophages but induces iNOS in 3T3
fibroblasts (18, 43).
We found that ANP does not directly interact with the iNOS protein to reduce NO synthesis. This is in accordance with our previous observation that exogenous ANP is basically not taken up by BMM (9). A receptor-mediated effect on the catalytic site of iNOS could be excluded as well, since the cGMP analog did not affect the enzyme activity. However, iNOS activity was significantly reduced in the homogenate of ANP-exposed cells. Several mechanisms could account for this observation: ANP may stimulate substrate-degrading pathways such as arginase. This is unlikely since arginase activity has been inhibited in our experiments by addition of L-valine (44). Furthermore, ANP might activate the induction of yet unidentified iNOS inhibitors. The Western blot analysis showing a strikingly decreased amount of iNOS protein, however, draws attention to sites of regulation other than enzyme activity which might be affected by ANP.
In this regard a major post-translational mechanism of iNOS regulation has been demonstrated to be intracellular dimerization of the iNOS molecule (24). iNOS is catalytically active only in the dimeric form. Dimer formation seems to be dependent on the availability of tetrahydrobiopterin, heme, and L-arginine (24). Interestingly, NO regulates its own synthesis by limiting intracellular assembly of iNOS by preventing heme insertion and decreasing heme availability (45). We addressed this rarely investigated regulatory mechanism of iNOS and could not provide evidence that ANP alters the extent of iNOS dimerization.
Effects of ANP on other regulatory mechanisms at the protein level such as translational activity or protein stability certainly cannot be ruled out since the corresponding experiments have not been performed in this study. However, based on the data obtained by Northern blot analysis, the decreased amount of iNOS protein is most likely a consequence of the significantly lower concentration of iNOS mRNA observed in ANP-exposed cells.
Levels of mRNA are controlled via transcriptional activity and via
mRNA stability. mRNA degradation seems to be an important regulatory mechanism for iNOS expression (12, 17, 18) as the iNOS gene
contains AUUUA sequences within its 3'-untranslated region known to be
responsible for the instability of mRNA (for review see Ref. 28).
This sequence represents a binding motif for short-lived RNase that
attaches to this site and is responsible for the extreme lability of
the corresponding mRNAs (46). Importantly, ANP reduces stability of
iNOS mRNA in BMM. Half-life of iNOS mRNA in ANP-exposed cells
was reduced around 50% as compared with only LPS-activated cells. In
order to elucidate whether ANP specifically affects the iNOS mRNA
stability, we examined three other inducible mRNAs known to possess
AUUUA sequences in their 3'-untranslated region. Interestingly, ANP did
not accelerate the decay of TNF- and IL-6 mRNA. However, the
stability of COX-2 mRNA was decreased by ANP. ANP may interact with
AUUUA binding proteins or mRNases which then specifically interact with
AUUUA sequences of iNOS and COX-2 mRNA, but neither with those of
TNF-
nor IL-6 mRNA (28). However, the nature as well as the
effects of AUUUA binding proteins seem to be complex, and little
information exists as yet on the features of mRNases (28, 46).
We hypothesize that ANP beside affecting post-transcriptional processes
decreases the transcription of iNOS mRNA. ANP has been shown to
reduce the binding activity of NF-B, the transcription factor
essential for iNOS induction in murine macrophages (36). Since iNOS was
induced solely with LPS in the present study and LPS is known to induce
NF-
B, transcription factors that are involved in iNOS induction by
cytokines such as INF-
or TNF-
were assumed not to considerably
contribute to iNOS induction in our cell system. In fact, we tested for
activation of the transcription factor IRF-1 (37), mediating
IFN-
-induced iNOS activation, and did not obtain positive results.
However, iNOS is not the only gene induced by LPS in macrophages via
activation of NF-
B. Other important mediators of macrophage
functions such as TNF-
or INF-
possess NF-
B sites in their
genes (47) and are induced by LPS. The fact that ANP is able to
interfere with such a common target of gene transcription as NF-
B
leads to the speculation that ANP may interfere with the synthesis of
these cytokines.
TNF- as well as INF-
have been shown to influence iNOS expression
(48, 49). Thus, the question arises whether the effect of ANP on the
regulation of iNOS is a direct one or indirectly mediated by
influencing the synthesis or activity of these cytokines. Experiments
employing corresponding antibodies against these cytokines have to be
performed in order to clarify this question.
It is important to note that ANP has recently been shown to interfere with other cell signaling systems that control gene transcription, i.e. the mitogen-activated protein kinase (MAPK) route (50, 51). ANP inhibits MAPK by inducing mitogen-activated protein kinase phosphatase-1 in glomerular mesangial cells (52). Interestingly, ANP was demonstrated to inhibit MAPK in astrocytes through the clearance receptor, i.e. independent of cGMP (51). Moreover, ANP abrogates endothelin-3-induced stimulation of early growth-related protein-1 transcription and basic fibroblast growth factor transactivation in these cells (52).
Finally, we would like to propose that ANP regulates NO synthesis in an autocrine circuit. The peptide was demonstrated earlier to be a constituent of macrophages, and its synthesis and secretion is increased upon exposure of cells to LPS (9, 38). We showed that blocking the NPR-A receptor by the specific receptor antagonist HS-142-1 in LPS-stimulated BMM resulted in an increase of NO release, which is explained by the blockade of the NO inhibitory action of BMM-endogenous ANP. These observations draw attention to the potential physiological significance of ANP-induced NO inhibition; ANP is supposed to be an autocrine macrophage mediator that terminates or at least reduces the production of NO by iNOS. Thus, ANP might be an important endogenous factor for deactivating macrophages and conserving their homeostasis in inflammation since NO has been shown to be autotoxic for macrophages (53). Modulation of NO synthesis by ANP may have broad implications in situations such as endotoxic shock, where, in fact, increased ANP plasma levels have been reported (19). Furthermore, in host defense ANP may guarantee optimal NO concentration for antimicrobial activity of macrophages without adversely affecting host cells (12).
In conclusion, we could demonstrate a novel mechanism of action for the
cardiovascular hormone ANP. By inhibition of NF-B activation via
cGMP and destabilization of iNOS mRNA, ANP specifically interacts
with NO production of LPS-activated macrophages.
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ACKNOWLEDGEMENTS |
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We thank Dr. Matsuda (Tokyo, Japan) for
kindly providing the receptor antagonist HS-142-1. We also thank Dr.
Herschman (UCLA) for giving us the COX-2 probe; Dr. Kleinert
(Mainz, Germany) for the iNOS cDNA probe; Dr. Decker (Freiburg,
Germany) for the TNF- cDNA; and Dr. Kremer (GSF Munich, Germany)
for the IL-6 probe. Dr. J. Plendl (Munich, Germany) is thanked for
supply of the bovine aortic endothelial cells. We thank Dr. H. Ammer
(Munich, Germany) for help performing the Western blots. The excellent
technical support of U. Rueberg is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Vo 376/8-1.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.: 89-2180-6349;
Fax: 89-342316; E-mail: vollmar{at}pharmtox.vetmed.uni-muenchen.de.
1
The abbreviations used are: ANP, atrial
natriuretic peptide; AP-2, activating protein-2; BMM, bone
marrow-derived macrophages; ABAE, adult bovine aortic endothelial
cells; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EMSA,
electrophoretic mobility shift assay; FCS, fetal calf serum; IL,
interleukin; INF, interferon; iNOS, inducible nitric-oxide synthase;
IRF-1, interferon responding factor-1; LPS, lipopolysaccharide; MAPK,
mitogen-activated protein kinase; NF-B, nuclear factor
B; nNOS,
neuronal nitric-oxide synthase; NO, nitric oxide; NOS, nitric-oxide
synthase; NPR, natriuretic peptide receptor; PBS,
phosphate-buffered saline; PDTC, pyrrolidinedithiocarbamate; PMSF,
phenylmethylsulfonyl fluoride; SNP, sodium nitroprusside; TNF-
,
tumor necrosis factor-
.
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REFERENCES |
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