Production of Adrenomedullin in Macrophage Cell Line and
Peritoneal Macrophage*
Atsushi
Kubo
§,
Naoto
Minamino
¶,
Yoshitaka
Isumi
,
Takeshi
Katafuchi
,
Kenji
Kangawa
,
Kazuhiro
Dohi§, and
Hisayuki
Matsuo
From the
National Cardiovascular Center Research
Institute, Fujishirodai, Suita, Osaka 565-8565 and the
§ First Department of Internal Medicine, Nara Medical
University, Shijo, Kashihara, Nara 634-0813, Japan
 |
ABSTRACT |
We demonstrate that adrenomedullin (AM) is
produced and secreted from cultured murine monocyte/macrophage cell
line (RAW 264.7) as well as mouse peritoneal macrophage. Immunoreactive
(IR) AM secreted from RAW 264.7 cells was chromatographically
identified to be native AM. To elucidate the regulation mechanism of AM
production in macrophage, we examined the effects of various substances
inducing differentiation or activation of monocyte/macrophage. Phorbol ester (TPA), retinoic acid (RA), lipopolysaccharide (LPS), and interferon-
(IFN-
) increased AM production 1.5-7-fold in RAW 264.7 cells in a dose- as well as time-dependent manner. By
LPS stimulation, the AM mRNA level in RAW 264.7 cells was augmented up to 7-fold after 14 h incubation. RA exerted a synergistic
effect when administered with TPA, LPS, or IFN-
, whereas IFN-
completely suppressed AM production in RAW 264.7 cells stimulated with
LPS. Dexamethasone, hydrocortisone, estradiol, and transforming growth factor-
dose-dependently suppressed AM production in RAW
264.7 cells. AM production was also investigated in mouse peritoneal macrophage. Primary mouse macrophage secreted IR-AM at a rate similar
to that of RAW 264.7 cells, and its production was enhanced 9-fold by
LPS stimulation. AM was found to increase basal secretion of tumor
necrosis factor
(TNF-
) from RAW 264.7 cells, whereas AM
suppressed the secretion of TNF-
and interleukin-6 from that stimulated with LPS. Thus, macrophage should be recognized as one of
the major sources of AM circulating in the blood. Especially in cases
of sepsis and inflammation, AM production in macrophage is augmented,
and the secreted AM is deduced to function as a modulator of cytokine
production.
 |
INTRODUCTION |
Adrenomedullin (AM)1 is
a potent vasorelaxant peptide originally isolated from extracts of
human pheochromocytoma by monitoring the elevating activity of platelet
cAMP (1). AM shows slight homology with calcitonin gene-related peptide
(CGRP) and has a potent and long-lasting depressor effect when injected
intravenously into anesthetized rats (1, 2). We have shown that
cultured endothelial cells (ECs) and vascular smooth muscle cells
(VSMCs) produce and secrete AM into culture medium (3, 4). The
production and secretion of AM in VSMC and EC were augmented by
interleukin-1 (IL-1), tumor necrosis factor
(TNF-
), and
lipopolysaccharide (LPS) (5, 6), which are known to be major factors
inducing septic shock (7-9). In the in vivo study,
intravenous administration of LPS into rats actually elevated plasma AM
concentration 20-fold and augmented AM gene expression in blood
vessels, lung, and intestine (10). Plasma AM levels were also
remarkably increased in patients with septic shock compared with those
in healthy volunteers (11, 12). These data suggest the possibility that
AM contributes to induction of refractory hypotension in septic
shock.
On the other hand, macrophages are activated by exposure to stimuli of
foreign bodies such as LPS and then start to produce and secrete
various cytokines, such as IL-1 and TNF-
. These data suggest that
macrophage is presumed to be another candidate for AM-producing cells
in sepsis in addition to VSMC and EC. In fact, Miller et al.
(13) have shown the expression of the AM gene in various tumor cell
lines, including histiocytic lymphoma cell line (U937), by using the
reverse transcriptase-PCR method. Recent reports have also demonstrated
that AM or its mRNA is significantly detected in the alveolar
macrophages and endometrial macrophages (14, 15). However, the
secretion of AM from macrophage has not yet been studied. In this
study, we demonstrate that AM is produced and secreted from cultured
murine macrophage cell line (RAW 264.7) as well as murine peritoneal
macrophage.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human AM-(40-52) and its N-Tyr derivative were
synthesized by peptide synthesizer 431A (Applied Biosystems, Foster
City, CA). The following materials were used: rat interferon-
(IFN-
) (Life Technologies, Inc.), murine recombinant IL-1
(Intergen, Purchase, NY), mouse recombinant TNF-
(Boehringer
Mannheim, Mannheim, Germany), dexamethasone, human recombinant
transforming growth factor-
1 (TGF-
),
12-O-tetradecanoyl phorbol-13-acetate (TPA), forskolin, hydrocortisone (Wako Pure Chemical, Osaka, Japan), 17-estradiol, hypoxanthine (Nacalai Tesque, Kyoto, Japan), Escherichia
coli LPS (serotype O26: B6) (Parsel+Lorei, Frankfurt, Germany),
all-trans-retinoic acid (RA), 8-bromoadenosine 3',5'-cyclic
monophosphate (8-Br-cAMP), low density lipoprotein (LDL) (Sigma),
acetylated LDL (ac-LDL) (Biomedical Technologies Inc., Stoughton, MA),
and recombinant human macrophage colony-stimulating factor (R & D
Systems, Minneapolis, MN). TPA and dexamethasone were first dissolved
in ethanol and then diluted with an incubation medium (Dulbecco's
modified Eagle's medium (DMEM) (Nikken BioMedical Laboratory, Kyoto,
Japan) containing 0.1% bovine serum albumin (BSA)). RA was dissolved
in dimethyl sulfoxide and diluted with the incubation medium. LPS was
dissolved in 0.9% NaCl solution and diluted with the incubation
medium. The other lyophilized substances were dissolved according to
the producer's manuals and diluted with the incubation medium.
Oxidized LDL (ox-LDL) prepared by the EC method was kindly donated by
Dr. Shimokado of this institute. Peptides of rat AM, rat CGRP, rat vasoactive intestinal polypeptide, human AM-(22-52) and human CGRP-(8-37) were obtained from Peptide Institute (Osaka, Japan).
Cell Culture--
RAW 264.7 macrophages (an Abelson leukemia
virus-transformed macrophage cell line of Balb/c mouse origin) (16)
were obtained from American Type Culture Collection (Rockville, MD) and
were cultured in DMEM containing 10% fetal calf serum (FCS) at
37 °C in a humidified atmosphere containing 5% CO2.
Preparation of Conditioned Medium--
RAW 264.7 cells, grown to
confluence in a 6-well plate, were washed twice with DMEM, replaced
with 1 ml of DMEM containing 0.1% BSA and stimulants, and incubated
for 4-14 h at 37 °C (n = 6). To evaluate
effects of co-administration of more than two substances, all
substances were generally administered at the same time, except that
suppressors were added 1 h before administration of stimulants.
Viability of RAW 264.7 cells after 14 h incubation was more than
97% by estimating with trypan blue staining. Culture medium (1 ml) was
acidified with acetic acid (final concentration, 0.5 M),
Triton X-100 added (final concentration, 0.002%), heated at 100 °C
for 10 min to inactivate protease, and lyophilized. The lyophilizates
were dissolved in radioimmunoassay (RIA) buffer and submitted to RIA
for AM.
RIA for AM--
Details of preparation and characterization of
antiserum 172CI-7 against human AM-(40-52), which recognizes the
C-terminal amide structure common to human, rat, and mouse AM, have
been reported by Sakata et al. (17). Monoiodinated
N-Tyr-AM-(40-52) isolated by reverse phase HPLC was used as a tracer
(3).
Characterization of IR-AM in Culture Medium of RAW 264.7 Cells--
Culture medium (100 ml) of RAW 264.7 cells was acidified
with acetic acid (final concentration, 1 M) and boiled for
10 min. After cooling, the acidified medium was loaded onto Sep-Pak
C18 ENV cartridges (Waters Chromatography Division,
Millipore Corp., Millford, MA) and washed with 0.1% trifluoroacetic
acid, and absorbed materials were eluted with 60% CH3CN
containing 0.1% trifluoroacetic acid. The eluate was evaporated and
subjected to gel filtration on Sephadex G-50 column (fine, 1.5 × 100 cm, Amersham Pharmacia Biotech, Sweden). Fractions containing IR-AM
were pooled and separated by reverse phase HPLC on a µ-Bondasphere 5 µ C18 column (300 Å, 3.9 × 150 mm, Millipore
Corp.) using a linear gradient elution of CH3CN from 10 to
60% in 0.1% trifluoroacetic acid over 60 min.
RNA Blot Analysis--
RAW 264.7 cells, grown to confluence in a
10-cm dish, were washed twice with DMEM and incubated with stimulants
in the incubation medium for 4-14 h. Total RNA (10 µg), extracted by
the acid guanidinium thiocyanate/phenol/chloroform method, was
denatured, electrophoresed, and then transferred to Zeta probe membrane
(Bio-Rad) (3). Hybridization and washing of the membrane were carried
out as reported (3). EcoRI-BglI cDNA fragment
of rat AM (nucleotide 153-422), EcoRI-BamHI
cDNA fragment of rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (nucleotide 492-799), cDNA fragments of mouse TNF-
(nucleotide 169-651) and mouse IL-6 (nucleotide 123-431) were used as
probes (8, 18, 19). Band intensity was estimated by BioImage analyzer
(BAS 5000, Fuji Photofilm, Tokyo, Japan). AM, TNF-
, and IL-6
mRNA band intensities were compared after correcting band intensity
using GAPDH mRNA band as an internal standard.
Determination of TNF-
, IL-6, and IL-1
Concentrations--
TNF-
, IL-6, and IL-1
concentrations were
measured with a solid-phase sandwich enzyme-linked immunoadsorbent
assay (ELISA) specific for murine TNF-
, IL-6, and IL-1
(Genzyme,
Cambridge, MA) after 4 h incubation of the cells in the presence
or absence of AM (10
6 M), because the
previous study reported that the TNF-
bioactivity in RAW 264.7 cells
increased rapidly and plateaued after 4 h of LPS stimulation
(20).
Quantification of AM, TNF-
, and IL-6 mRNA by Real Time
Quantitative PCR--
RAW 264.7 cells, grown to confluence in a 6-well
dish, were washed twice with DMEM and incubated with stimulants in the
incubation medium for 0-8 h. Total RNA (2 µg), extracted by the RNA
zol B (Tel-Test Inc., Friendwood, TX), was reverse-transcribed into cDNA with SuperScript reverse transcriptase (Life Technologies, Inc.). Mouse AM cDNA, TNF-
cDNA, IL-6 cDNA, and GAPDH
cDNA were each amplified with respective pairs of the
following oligonucleotides: TCCTGGACGAGCAGAACACAAC (base
1704-1725, sense primer) and TGGTTCATGCTCTGGCGGTA (base 1797-1816,
antisense primer) for AM cDNA (21); GTGATCGGTCCCCAAAGG (base
326-343) and GGGTCTGGGCCATAGAACTG (base 394-414) for TNF-
cDNA
(18); TGAAGTTCCTCTCTGCAAGAGACT (base 33-56) and TAGGGAAGGCCGTGGTTGT (110-128) for IL-6 cDNA (19); ACATGTTCCAGTATGACTCCACTCAC (base 174-199) and TCTCGCTCCTGGAAGATGGT (base 282-301) for GAPDH cDNA (22). To measure mouse AM, TNF-
, IL-6, and GAPDH mRNA levels, a
novel quantitative PCR method, real time quantitative PCR (Prism 7700 Sequence Detector, Applied Biosystems), was performed as reported
previously (23, 24). We used the following oligonucleotide probes
labeled with 6-carboxyfluorescein as reporter fluorescence and
6-carboxytetramethylrhodamine as quencher fluorescence:
ACAAGCCAGCAATCAGAGCGAAGC (bases 1735-1758) for AM (21);
ATGAGAAGTTCCCAAATGGCCTCCCT (bases 346-369) for TNF-
(18);
TTGCCTTCTTGGGACTGATGCTGGT (bases 66-90) for IL-6 (19); and
AACGGCACAGTCAAGGCCGAGAAT (bases 209-233) for GAPDH (22). AM, TNF-
,
and IL-6 mRNA levels were compared after correcting them by GAPDH
mRNA level as an internal standard. Known amounts of cDNA
fragments of mouse AM (bases 1704-1816), mouse TNF-
(bases
326-414), mouse IL-6 (bases 33-128), and mouse GAPDH (bases 174-301)
were used as standards. The data were averages of three individual
experiments.
Preparation of Mouse Peritoneal
Macrophages--
Thioglycolate-elicited macrophages were collected by
peritoneal lavage with pyrogen-free saline at 7 days after
intraperitoneally injecting 2 ml of sterile thioglycolate to 6-week-old
C3H/He mice (Charles River Japan, Yokohama, Japan).
Thioglycolate-elicited macrophages were cultured in 10-cm dishes at
5 × 105 cells/dish in RPMI 1640 medium (Life
Technologies, Inc.) supplemented with 10% FCS. After 1 h
incubation to allow for adherence of macrophages, the dishes were
washed to remove nonadherent cells. Macrophage monolayers were then
cultured in RPMI 1640 medium containing 10% FCS with TPA, RA, LPS, or
IFN-
for 24 h (n = 5).
Intracellular cAMP Measurement--
RAW 264.7 cells and mouse
peritoneal macrophages, grown to confluence in 24-well plates, were
washed thoroughly with DMEM. The media were then replaced with
Hepes-buffered DMEM (pH 7.4, 25 mM Hepes, 0.01% BSA, and
0.5 mM isobutylmethylxanthine) containing various
concentrations of rat AM, rat CGRP, and their antagonists, and the
cells were incubated precisely for 10 min. The reaction was terminated
by aspirating the media followed by addition of 0.2 M HCl
and 5 mM EDTA. After scraping the cells, the cell
suspension was sonicated and centrifuged. From the resulting extracts,
the aliquot (100 µl) was lyophilized, and its cAMP content was
measured by a specific RIA as reported previously (n = 3) (25).
Statistical Analysis--
Statistical analysis of the results
were performed by one-way analysis of variance, followed by a multiple
comparison test (Fisher's test). All data were expressed as means ± S.E. A level of p < 0.05 was considered to be
statistically significant.
 |
RESULTS |
Production and Secretion of IR-AM from RAW 264.7 Cells and Its
Chromatographic Identification--
We measured IR-AM content in
culture medium of RAW 264.7 cells after incubation for 4, 8, or 14 h by using RIA specific to AM. As shown in Fig.
1, IR-AM was accumulated in the medium
almost linearly up to 14 h without any stimulation. Intracellular
IR-AM content of RAW 264.7 cells was also measured after extraction and
condensation. The intracellular level of IR-AM was constant and much
lower (less than 10%) than that in culture medium after 14 h
incubation (data not shown). This result suggests that IR-AM synthesized in RAW 264.7 cells is not stored in the cells but is
secreted constitutively into the medium after synthesis. Thus, we
measured IR-AM content in the culture medium to evaluate IR-AM production in RAW 264.7 cells.

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Fig. 1.
Time-dependent AM production in
RAW 264.7 cells stimulated with TPA, RA, LPS, and IFN- . RAW
264.7 cells were stimulated with TPA, RA, LPS, and IFN- for 4, 8, and 14 h at concentrations listed below. After stimulation, IR-AM
concentration in culture medium was measured by RIA specific for AM.
Closed circles, TPA (10 7 M);
closed triangle, RA (10 5 M);
open circles, LPS (100 ng/ml); open triangles,
IFN- (100 units/ml); closed squares, control (without
stimulation). Each point represents the mean ± S.E. of six
separate dishes. *, p < 0.05.
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|
To identify IR-AM secreted into culture medium from RAW 264.7 cells, we
characterized IR-AM by gel filtration and reverse phase HPLC. The
culture medium was collected after stimulation with 10
7
M TPA for 14 h, since the secretion rate of IR-AM from
RAW 264.7 cells was low without any stimulation. In Sephadex G-50 gel
filtration, more than 70% of total IR-AM in the culture medium was
eluted at a molecular weight region corresponding to 6000 (Fig.
2a). Fractions 33-35 of 6000 IR-AM were pooled, and a portion of the pooled fractions was subjected
to reverse phase HPLC. More than 70% of IR-AM was eluted at a
retention time almost identical to that of native rat AM (Fig.
2b). Based on these data, we conclude that IR-AM secreted
from RAW 264.7 cells is chromatographically composed of a single
component that has properties very close to those of rat AM of 50 residues. In fact, the amino acid sequence of mouse AM has recently
been determined which has only three homologous replacements in the
50-residue peptide compared with rat AM (20).

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Fig. 2.
Characterization of IR-AM secreted from RAW
264.7 cells. a, Sephadex G-50 gel filtration of IR-AM from
culture medium of RAW 264.7 cells stimulated with TPA. Sample: 100 ml
eq of culture medium. Column: 1.5 × 100 cm. Solvent: 2 M acetic acid. Fraction size: 4.0 ml/tube. Flow rate: 5.0 ml/h. b, reverse phase HPLC of IR-AM from culture medium of
RAW 264.7 cells stimulated with TPA. Sample: 2/3 of fractions 33-35
(a). Column: µ-Bondasphere 5 µ C18 column
(300 Å, 3.9 × 150 mm). Flow rate: 1.0 ml/min. Fraction size: 0.5 ml/tube. Solvent system: Linear gradient elution from 10 to 60%
CH3CN in 0.1% trifluoroacetic acid over 60 min.
Arrows indicate elution positions of BSA (1),
human AM-(1-52) (2), human AM-(22-52) (3),
human AM-(40-52) (4), NaCl (5), and rat
AM-(1-50) (6).
|
|
Regulation of IR-AM Production in RAW 264.7 Cells--
We first
examined various substances that induced monocyte-macrophage
differentiation or activation (26). Among them, TPA, RA, LPS, and
IFN-
significantly increased IR-AM secretion from RAW 264.7 cells
compared with the control (Table I). In
contrast, TNF-
and IL-1
, which augmented AM production in rat
VSMCs and ECs (5, 6), showed no apparent effect on AM production in RAW
264.7 cells.
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Table I
Effect of various substances on AM production in RAW 264.7 cells
RAW 264.7 cells were incubated with various substances, which induced
monocyte-macrophage differentiation or activation, as well as steroids,
and lipoproteins in the indicated concentrations for 14 h. IR-AM
concentrations secreted into culture media were measured by RIA
specific for AM. Each value represents mean ± S.E. of six
separate dishes. M-CSF, macrophage-colony stimulating factor; VIP,
vasoactive intestinal polypeptide.
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|
TPA, RA, LPS, and IFN-
dose-dependently stimulated
secretion of IR-AM from RAW 264.7 cells, and increased IR-AM
concentration in the media to 164, 190, 600, and 460% of the control
at concentrations of 10
7 and 10
5
M, 1000 ng/ml, and 100 units/ml, respectively (Fig.
3). Time-dependent effects of
these substances in concentrations inducing almost maximal stimulation
are shown in Fig. 1. AM production was augmented after 8 h
stimulation in the cases of TPA, RA, and IFN-
, whereas LPS was found
to have already enhanced AM production before 4 h stimulation.

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Fig. 3.
Dose-dependent AM production in
RAW 264.7 cells by stimulation with TPA, RA, LPS, and IFN- . RAW
264.7 cells were stimulated with TPA (a), RA (b),
LPS (c), and IFN- (d) of indicated
concentrations for 14 h. IR-AM concentration in culture medium was
measured by RIA specific for AM. Each value represents the mean ± S.E. of six separate dishes. *, p < 0.05.
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AM gene transcription was estimated by RNA blot analysis, and AM
mRNA levels in RAW 264.7 cells stimulated with LPS (100 ng/ml) were
measured after 0, 4, 8, and 14 h stimulation. LPS
time-dependently increased AM mRNA level (Fig.
4, a and c), and
this result was parallel to the peptide production level of AM in RAW
264.7 cells. Furthermore, AM mRNA level after 8 h stimulation
was elevated according to the increase of LPS concentration and was
also well correlated with the peptide production level of AM shown in
Fig. 3c (Fig. 4, b and d). AM mRNA
levels were also increased by stimulation with RA, TPA, and IFN-
to
116, 148, and 199% of the control in concentrations of
10
7 and 10
5 M and 100 units/ml
after 8 h stimulation, respectively (data not shown).

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Fig. 4.
RNA blot analysis of AM gene transcripts in
RAW 264.7 cells. Total cellular RNA was extracted from RAW 264.7 cells after stimulation with LPS and was subjected to RNA blot analysis
for AM mRNA. a, AM mRNA levels in RAW 264.7 cells
after 0, 4, 8, and 14 h stimulation with LPS (100 ng/ml).
b, AM mRNA levels in RAW 264.7 cells after 8 h
stimulation with 0, 1, 10, and 100 ng/ml LPS. c, RNA blot
analysis of AM transcripts in RAW 264.7 cells after 0, 4, 8, and
14 h stimulation with LPS (100 ng/ml). d, RNA blot
analysis of AM transcripts in RAW 264.7 cells after 8 h
stimulation with 0, 1, 10, and 100 ng/ml LPS. Lower panels of
c and d indicate RNA blot analysis of GAPDH
transcripts. AM mRNA level was estimated after correcting band
intensity using GAPDH mRNA level as an internal standard.
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Next, we examined effects of steroid hormones on IR-AM production in
RAW 264.7 cells. Dexamethasone, hydrocortisone, and estradiol reduced
IR-AM concentrations to 24, 49, and 67% of the control at the
concentrations of 10
5, 10
6, and
10
5 M, respectively (Fig.
5, a, b, and
c). We also investigated the effects of TGF-
that has
been recognized as a potential inhibitor of macrophage activation (27).
TGF-
suppressed AM production to 75% of the control in a
concentration of 10 ng/ml (Fig. 5d).

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Fig. 5.
Dose-dependent inhibition of AM
production with dexamethasone, hydrocortisone, estradiol, and TGF-
in RAW 264.7 cells. RAW 264.7 cells were incubated for 14 h
in the presence of dexamethasone (Dexa) (a),
hydrocortisone (Hyd) (b), estradiol
(c), and TGF- (d) at the indicated
concentrations. IR-AM concentration in culture medium was measured by
RIA specific for AM. Each value represents the mean ± S.E. of six
separate dishes. *, p < 0.05.
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To evaluate cooperative effects of stimulators and suppressors, IR-AM
concentration in the cultured medium of RAW 264.7 cells was measured by
co-administering substances that had significantly altered AM
production. When 10
5 M of RA was administered
with TPA (10
8 M), LPS (100 ng/ml), or IFN-
(100 units/ml), IR-AM concentration in the medium was elevated to a
level higher than that deduced from summation of the effect of each
substance (Fig. 6). Specifically, simultaneous addition of LPS and RA increased IR-AM content in the
medium to 10 times higher than that of control. Although IL-1
, TNF-
, and LPS showed additive effects on AM production in cultured rat VSMCs (5), combining these three substances did not induce any
cooperative effect on AM production in RAW 264.7 cells (data not
shown). Since combination of LPS and IFN-
has been reported to
induce synergistic effects on cytotoxic activity and nitric oxide (NO)
production in RAW 264.7 cells (28), we examined the effect of
co-administration of LPS and IFN-
. In contrast, IFN-
suppressed
IR-AM secretion stimulated with 10 ng/ml LPS even at a concentration of
1 unit/ml (Fig. 7).

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Fig. 6.
Effects of co-administration of RA with TPA,
LPS, or IFN- on AM production in RAW 264.7 cells.
10 5 M RA was administrated with TPA
(10 8 M), LPS (100 ng/ml), or IFN- (100 units/ml) to RAW 264.7 cells and the cells were incubated for 14 h. Closed bars and open bars indicate IR-AM
concentrations in the presence or absence of RA, respectively. Each
value represents the mean ± S.E. of six separate dishes.
Significant difference (p < 0.05) was observed in all
cases between the presence and absence of RA.
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Fig. 7.
Inhibitory effect of IFN- on AM production
in RAW 264.7 cells stimulated with LPS. LPS and IFN- were
simultaneously administered to RAW 264.7 cells as indicated at the
bottom of the figure. IR-AM content in the culture medium
was measured after 14 h incubation by RIA specific for AM. Each
value represents the mean ± S.E. of six separate dishes. *,
p < 0.05.
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We also examined effects of glucocorticoids and TGF-
on AM
production in RAW 264.7 cells stimulated with LPS, since these substances have been reported to suppress production of NO and cytokines, such as TNF-
, under stimulation with LPS (29, 30). When
dexamethasone and hydrocortisone were each administered simultaneously with LPS, these substances elicited slight inhibitory effects on AM
secretion from RAW 264.7 cells (data not shown). However, 1 h
pretreatment of the cells with dexamethasone or hydrocortisone strongly
inhibited IR-AM production stimulated with LPS (100 ng/ml) (Fig.
8). These results indicate that
glucocorticoids are potent inhibitors of AM production in RAW 264.7 cells, overcoming the effect of LPS. TGF-
(10 ng/ml) also suppressed
AM production to 60% that stimulated with LPS (100 ng/ml) (data not
shown).

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Fig. 8.
Inhibitory effects of dexamethasone and
hydrocortisone on AM production in RAW 264.7 cells stimulated with
LPS. Dexamethasone (Dexa) (a) and
hydrocortisone (Hyd) (b) of indicated
concentrations were preincubated for 1 h before LPS stimulation.
After preincubation, the medium was replaced, and RAW 264.7 cells were
incubated for 14 h with dexamethasone (a) and
hydrocortisone (b) in the presence of LPS (100 ng/ml). IR-AM
content in the culture medium was measured after 14 h incubation
by RIA specific for AM. Each value represents the mean ± S.E. of
six separate dishes. *, p < 0.05.
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We examined the effect of the substances that elevate intracellular
cAMP concentrations, such as forskolin, 8-Br-cAMP, and vasoactive
intestinal polypeptide, on AM production in RAW 264.7 cells, since we
previously showed that these substances suppressed AM production in
VSMCs (31). However, these substances elicited no significant effect on
AM production in RAW 264.7 cells (Table I).
Finally, effects of lipoproteins, such as LDL, ac-LDL, and ox-LDL, on
AM production in RAW 264.7 cells were estimated, since macrophages were
well known to have an important role in cholesterol deposition in
atherosclerosis (32). LDL, ac-LDL, and ox-LDL significantly increased
AM production in RAW 264.7 cells to 141, 123, and 181% of the control,
respectively (Table I).
AM Modulates Secretion and Gene Transcription of TNF-
and IL-6
in RAW 264.7 Cells--
To elucidate physiological functions of AM
secreted from macrophages and their cell lines, we examined the effects
of AM on production of inflammatory cytokine, TNF-
, IL-1
, and
IL-6, in RAW 264.7 cells, since these cytokines were known to play
important roles in the pathophysiology of sepsis. AM increased TNF-
concentration in the culture medium of RAW 264.7 cells to about 160%
in the absence of LPS stimulation (Fig.
9a). LPS (1 ng/ml) markedly
enhanced TNF-
production about 120-fold in RAW 264.7 cells. On the
contrary, AM significantly suppressed TNF-
secretion from RAW 264.7 cells stimulated with LPS maximally to about 50%, and the inhibitory effect was observed even in high concentrations of LPS (Fig.
9b). RNA blot analysis demonstrated that LPS
dose-dependently increased TNF-
mRNA level in RAW
264.7 cells after 4 h stimulation. AM also suppressed TNF-
mRNA level in RAW 264.7 cells stimulated with 1, 10, 100, and 1000 ng/ml LPS to 69, 80, 62, and 70%, respectively (Fig.
10a).

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Fig. 9.
Effects of AM on TNF- secretion from RAW
264.7 cells. a, TNF- concentration in the culture medium
was measured by ELISA after 4 h incubation of RAW 264.7 cells with
AM (10 6 M). b, TNF-
concentration was measured by ELISA after 4 h incubation of RAW
264.7 cells with 1, 10, 100 and 1000 ng/ml LPS in the presence
(open bar) or absence (closed bar) of AM
(10 6 M). Each value represents the mean ± S.E. of four separate dishes. *, p < 0.05.
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Fig. 10.
Quantification of TNF- mRNA level in
RAW 264.7 cells. a, TNF- mRNA levels in RAW 264.7 cells were evaluated by RNA blot analysis after 4 h stimulation of
with 1, 10, 100, 1000 ng/ml LPS in presence (open bar) or
absence (closed bar) of AM (10 6
M). b, TNF- mRNA levels in RAW 264.7 cells after 0, 1, 2, 4, and 8 h stimulation with LPS (100 ng/ml)
were measured by real time quantitative PCR in the presence (open
bars) or absence (closed bars) of AM (10 6
M) (n = 3). c, TNF- mRNA
levels in RAW 264.7 cells after 4 h stimulation with 0, 1, 10, and
100 ng/ml LPS were measured by real time quantitative PCR in the
presence (open bars) or absence (closed
bars) of AM (10 6 M) (n = 3). TNF- mRNA level was estimated after correcting it using
GAPDH mRNA level as an internal standard.
|
|
Basal secretion levels of IL-1
and IL-6 were below detection limits
(10 and 5 pg/ml, respectively). However, LPS enhanced IL-6 production
(27.6 ± 0.57 pg/ml) at a concentration of 100 ng/ml, and AM
slightly but significantly reduced IL-6 concentration (22.8 ± 1.78 pg/ml, p < 0.05) in a manner similar to that of
TNF-
. RNA blot analysis also demonstrated that AM suppressed IL-6
mRNA levels to about 85% in the presence of 100 ng/ml LPS (data
not shown). On the other hand, IL-1
production in RAW 264.7 cells was not significantly altered by the addition of AM.
Measurement of Mouse AM mRNA and TNF-
mRNA Levels By
Using Real Time Quantitative PCR--
To evaluate whether real time
quantitative PCR is usable for quantifying the amount of mRNA, we
first measured mouse AM levels in RAW 264.7 cells stimulated with LPS.
As shown in Fig. 11a, the fluorescent emission data were collected during the PCR amplification, and threshold cycles were calculated by determining the point at which
the fluorescence intensity exceeded the level 10 times higher than the
standard deviation of the base lines. As the larger amounts of the
standard were applied, the significant emission of fluorescence was
detected at the earlier phase of threshold cycle in the amplification
plot. The threshold cycle decreased linearly with increasing quantity
of mouse AM cDNA standard (r = 0.998, p < 0.0001), indicating that the amount of mRNA
was correctly evaluated in a wide range by this method (Fig.
11b). By using the real time quantitative PCR, we
demonstrated that LPS time- and dose-dependently increased
AM mRNA levels in RAW 264.7 cells in profiles similar to those
measured by RNA blot analysis (Fig. 11, c and d).
Thus, the real time quantitative PCR was shown to be usable for
determining the amounts of mRNA instead of RNA blot analysis.

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|
Fig. 11.
Quantification of AM mRNA level in RAW
264.7 cells by real time quantitative PCR. a, amplification
plots of AM mRNA levels measured by real time quantitative PCR.
Standard samples of 0.001, 0.01, 0.1, 1, 10, 100, and 1000 fg were
amplified, and the Rn (fluorescent emission) was plotted
against the cycle number. As the larger amounts of the standard sample
were applied, the significant emission of fluorescence was detected at
the earlier phase of threshold cycle in the amplification plot.
Threshold cycle (Ct) was calculated by determining the point
at which the fluorescence exceeded 10 times the standard deviation of
the base line. b, standard curve for AM mRNA
levels measured by real time quantitative PCR. The amounts of standard
samples were plotted versus threshold cycles in duplicates.
c, AM mRNA levels were evaluated by real time
quantitative PCR after 0, 1, 2, 4, and 8 h stimulation with LPS
(100 ng/ml) (n = 3). d, AM mRNA levels were
measured by real time quantitative PCR after 4 h stimulation with
0, 1, 10, and 100 ng/ml LPS (n = 3). AM mRNA level was
compared after correcting it using GAPDH mRNA level as an internal
standard (c and d). *, p < 0.05.
|
|
Next, TNF-
mRNA level in RAW 264.7 cells was evaluated by real
time quantitative PCR. TNF-
mRNA level was
time-dependently increased up to 8 h by stimulation
with LPS (100 ng/ml), and AM (10
6 M)
suppressed TNF-
mRNA levels maximally to 77% at 4 h after LPS stimulation (Fig. 10b). In addition, LPS
dose-dependently increased TNF-
mRNA levels, and AM
(10
6 M) suppressed TNF-
mRNA levels
maximally to 66% at a dose of 10 ng/ml after 4 h of stimulation
(Fig. 10c).
Production of IR-AM in Murine Peritoneal Macrophages--
Based on
data obtained from macrophage cell line, RAW 264.7, we next assessed
whether AM was produced and secreted from primary mouse macrophages.
Mouse primary macrophages secreted IR-AM without any stimulation at a
rate (0.2 ± 0.04 fmol/105 cells/24 h) about 15% that
of RAW 264.7 cells (Table II). Peritoneal exudate macrophages were treated with TPA (10
7
M), RA (10
6 M), LPS (100 ng/ml),
and IFN-
(100 units/ml) for 24 h. Among them, LPS increased
IR-AM content in the culture medium about 7-fold compared with that of
control, but the others did not alter IR-AM concentrations (Table II).
When 1000 ng/ml LPS was added to primary macrophages, IR-AM
concentration was increased at least 9-fold over 24 h. To examine
synergistic effects, TPA, RA, and IFN-
were co-administered with 100 ng/ml LPS and incubated for 24 h (Table II). As in the case of
that observed in RAW 264.7 cells, LPS dose-dependently
augmented AM production, and co-administration of LPS and RA
synergistically enhanced their stimulatory effects on IR-AM secretion
from mouse primary macrophage. With regard to the combination of LPS
and IFN-
, IFN-
also inhibited IR-AM secretion stimulated with LPS
in a manner similar to that of RAW 264.7 cells. However, TPA inhibited
IR-AM secretion stimulated with LPS in contrast to the results obtained
in RAW 264.7 cells. Based on these results, mouse primary macrophages
were shown to have the ability to produce and secrete AM. Regulation of
AM production in primary macrophage was deduced to be similar to that
of RAW 264.7 cells, as observed in the case of LPS, but was not
completely identical to that of the established cell line.
View this table:
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|
Table II
Production of AM in mouse peritoneal macrophages
Mouse primary macrophages, elicited by the thioglycolate, were
stimulated with TPA, RA, LPS, or IFN- of indicated concentrations
for 24 h. Furthermore, 100 ng/ml LPS was co-administered to
primary macrophage with TPA, RA, and IFN- of indicated
concentrations and cultured for 24 h. IR-AM concentrations in
cultured medium were measured by RIA specific for AM. Each value
represents mean ± S.E. of five separate dishes.
|
|
Intracellular cAMP Production in RAW 264.7 Cells and Mouse
Peritoneal Macrophages--
Since AM was shown to modulate production
of cytokines in RAW 264.7 cells, we examined the expression and
properties of AM receptors on this macrophage cell line and mouse
peritoneal macrophages. AM (10
6 M) did not
increase cAMP production in RAW 264.7 cells (data not shown). On the
other hand, AM (10
6 M) augmented the
intracellular cAMP level in mouse peritoneal macrophages 6.7-fold
compared with the basal level. Rat CGRP dose-dependently elevated the intracellular cAMP level with an ED50 value of
7.6 × 10
10 M, and AM antagonist, human
AM-(22-52) did not affect the ED50 value. In the presence
of 10
6 M CGRP antagonist, human CGRP-(8-37),
the dose-response curve of the cAMP production was markedly shifted to
the high concentration side, and the ED50 value was 145 times higher than that without CGRP antagonist. In contrast, the
ED50 value of rat AM in the cAMP production assay was
4.3 × 10
7 M, being 570 times higher
than that of rat CGRP. Furthermore, the dose-response curve of AM was
not shifted with human AM-(22-52) but with human CGRP-(8-37). Based
on these results, the receptors expressed on mouse peritoneal
macrophages are found to be specific for CGRP, and AM is not deduced to
significantly stimulate the cAMP production under the physiological
conditions.
 |
DISCUSSION |
In this study, we demonstrated that the macrophage cell line RAW
264.7 produced and secreted IR-AM that was identified as native AM by
chromatographic procedures (Fig. 2). Although the production level of
AM in RAW 264.7 cells was lower than that of cultured rat ECs and VSMCs
(4, 5), we found substances that stimulated differentiation and
activation of monocyte/macrophage potently augmented AM production
(Table I). As shown in Fig. 3, the secretion rate of AM from RAW 264.7 cells stimulated with LPS or IFN-
was comparable to that of cultured
VSMCs, indicating that monocyte/macrophage system was another candidate
for secreting AM into the bloodstream.
TPA and IFN-
increased IR-AM concentration in the culture medium of
RAW 264.7 cells (Figs. 1 and 3). TPA, a potent activator of protein
kinase C, induces the differentiation of this cell line into macrophage
and stimulates production of IL-1
and TNF-
(7, 33). IFN-
is
also known to be a potent inducer of differentiation of
monocyte/macrophage and to enhance production of NO, TNF-
, and
IL-1
(28, 34, 35). RA increased AM production in RAW 264.7 cells
(Figs. 1 and 3) and stimulated AM production synergistically when
administered with TPA, IFN-
, or LPS (Fig. 6). RA has been reported
to activate macrophage and potentiate phagocytosis and IL-1 activity in
RAW 264.7 cells (36). This result suggests that RA activates
macrophages to conditions where they can respond more potently to any
stimulation and produce AM at higher rates. AM production is thought to
be increased by stimulators that induce differentiation or activation
of macrophages. On the other hand, mouse peritoneal macrophages failed
to increase AM production by stimulation with TPA, RA, and IFN-
. RAW
264.7 cells were established from peritoneal macrophages, but the cells
have been transformed and immortalized by infection of Abelson leukemia
virus (16). Although peritoneal macrophages and RAW 264.7 cells share
many biological features, these cells are reported to elicit different responses in NO synthesis when stimulated with phorbol ester (37). We
also observed in this study that AM increased cAMP production in
peritoneal macrophages but not in RAW 264.7 cells. These findings lead
us to deduce that the differences in results are due to different properties of established cell lines and primary culture.
Glucocorticoids have been shown to enhance AM production, and estradiol
increased it in rat EC and VSMC EC (6, 38), but these reagents were
found to be potent suppressors of AM production in RAW 264.7 cells
(Fig. 5). Glucocorticoids have been shown to antagonize differentiation
of macrophage (39) and are potent anti-inflammatory agents that inhibit
production and release of NO and cytokines, such as IL-1, IL-6, and
TNF-
, from macrophages (29, 40-42). These data indicate that gene
transcription and production of AM in the macrophage cell line are
regulated in a manner similar to NO synthase and inflammatory cytokines
but distinct from AM in EC and VSMC (4-6, 38). In the vascular wall,
glucocorticoids are known to increase vascular tone, but AM secreted
from EC and VSMC contributes to reduction of vascular tone (43, 44). In
the monocyte/macrophage system, AM is shown to be produced and secreted
from RAW 264.7 cells and to participate in the regulation of TNF-
and IL-6 production. Taken together, we deduce that the intracellular
signal integration mechanism regulating transcription of AM gene must
be different in each cell line in order to exert its divergent effects
after secretion from the cells. TGF-
is recognized as a potential
inhibitor for activation of macrophage as well as for production of NO
and TNF-
(27, 30) and also suppresses AM production in RAW 264.7 cells. Based on these results, AM production in RAW 264.7 cells is
thought to be increased or decreased mainly by the substances that
stimulate or suppress differentiation and activation of
macrophages.
We have reported that production and secretion of AM from VSMC and EC
are augmented by LPS stimulation (5, 6) and that intravenous
administration of LPS into rats increases plasma AM concentration
20-fold and augments AM gene transcription in almost all tissues
including blood vessels (10). Furthermore, an average concentration of
plasma AM in patients with septic shock has recently been reported to
be 10-45 times higher than that of healthy volunteers (11, 12). Based
on these data, we have attributed high plasma AM concentrations in
LPS-injected rats and in patients with septic shock to the elevated
production of AM in ECs and VSMCs. As shown in the present study, LPS
is shown to be the most potent stimulator of AM production and gene
transcription in RAW 264.7 cells (Figs. 1, 3, 4, and 11). Even in
primary mouse macrophages, LPS in a concentration of 1000 ng/ml also
increases AM production about 9-fold compared with that of control. We
also found in our recent studies that human leukemia cell line and
peripheral blood monocytes produce AM according to their
differentiation into macrophages and that LPS stimulation augments AM
production in monocyte-derived macrophages (45). Regulation of AM
production in RAW 264.7 cells is found to be similar to that of
inflammatory cytokines, such as IL-1, IL-6, and TNF-
, which
contribute to the pathophysiology of sepsis (7-9). These data indicate
that macrophage is another candidate for secreting circulatory AM in
the case of sepsis.
As shown in Fig. 8, glucocorticoids strongly suppressed AM production
in RAW 264.7 cells even in the presence of LPS. In the in
vivo experiments, glucocorticoids have been reported to induce tolerance to LPS and reduce mortality in animals with experimentally induced septic shock (46). Glucocorticoids suppress production of
inflammatory cytokines and NO, which results in reduction of vasodilation (40-42). This suppressive effect on production of inflammatory cytokines and NO is thought to be one of the reasons why
glucocorticoid induces tolerance to LPS and prevents septic shock.
Thus, our results raise the possibility that AM produced in macrophage
is an additional modulator in the pathogenesis of sepsis. Pretreatment
of macrophages with TGF-
has been reported to attenuate their
ability to produce TNF-
, NO, and prostaglandin E2 in
response to LPS (30, 47). AM production in RAW 264.7 cells was also
suppressed by TGF-
(Fig. 5d). In this study, macrophage and macrophage cell line were shown to actively produce AM after LPS
stimulation, whereas AM production was suppressed by glucocorticoids and TGF-
. These results suggest that AM plays a significant role in
the pathogenesis of sepsis and inflammation in addition to its function
as a vasodilator.
Our previous study has shown that IFN-
suppresses AM production in
rat VSMCs (31). However, IFN-
stimulates AM secretion in RAW 264.7 cells when administered alone (Fig. 3). AM gene has many
IFN-
-responsive elements in the promoter region (48). McDowell
et al. (49) reported that RAW 264.7 cells, but not less
mature WEHI-3 cells, were able to utilize transfected
IFN-
-responsive elements and induce rapid transcription by IFN-
stimulation. These facts indicate that the IFN-
-responsive element
is utilized differently depending on the cells and that in RAW 264.7 cells IFN-
induces gene transcription of AM via IFN-
-responsive
elements. On the contrary, IFN-
strongly inhibits AM secretion from
RAW 264.7 cells and mouse peritoneal macrophages stimulated with LPS (Fig. 7 and Table II). In the case of RAW 264.7 cells stimulated with
LPS, IFN-
was reported to induce hyporesponsiveness to LPS and
reduce the production of TNF-
(50). Another report also showed that
IFN-
strongly suppressed the expression of IL-1
gene stimulated
with LPS (51). These data suggest that IFN-
acts as inhibitory
cytokine under the chronically inflammatory conditions stimulated with
LPS. Since the effects of IFN-
are different in each cell type and
dependent on the physiological condition of the cells, discordant
responses of IFN-
are likely to be derived from differences in
transcriptional regulation between VSMCs and RAW 264.7 cells as well as
between the presence and absence of LPS.
In order to identify physiological functions of AM secreted from
macrophages, we examined whether AM modulates secretion of TNF-
,
IL-1
, and IL-6, the cytokines mainly produced in macrophages and RAW
264.7 cells (7-9). As shown in Fig. 9, AM increased a basal secretion
level of TNF-
from RAW 264.7 cells, whereas AM significantly
suppressed TNF-
secretion when the cells were stimulated with LPS.
AM was also shown to suppress secretion of IL-6 stimulated with LPS in
this study. RNA blot analysis and real time quantitative PCR analysis
showed that AM lowered TNF-
and IL-6 mRNA levels in RAW 264.7 cells stimulated with LPS (Fig. 10), suggesting that AM inhibits
cytokine production at the step of gene transcription. Kamoi et
al. (52) reported that AM inhibited secretion of cytokine-induced neutrophil chemoattractant, belonging to IL-8 superfamily, in rat
alveolar macrophages stimulated with LPS. These results indicate that
AM secreted from macrophages not only dilates blood vessels but also
modulates production of inflammatory cytokines, TNF-
, IL-6, and
cytokine-induced neutrophil chemoattractant.
To assess the properties of AM receptors expressed on RAW 264.7 cells
and mouse peritoneal macrophages, we measured intracellular cAMP
concentrations in these cells. However, AM did not significantly increase the intracellular cAMP concentration in RAW 264.7 cells. In
the case of mouse peritoneal macrophages, only 10
6
M AM increased intracellular cAMP concentration, and
receptors expressed on primary macrophages were found to be specific
for CGRP. These results suggest that the suppressive effect of AM on
the production and gene transcription of TNF-
and IL-6 was not
mediated by the cAMP signaling pathway. As the alternative signaling
pathway, Shimekake et al. (53) have reported that AM
increases the intracellular free calcium concentration in the case of
bovine aortic endothelial cells. Thus, we deduce that another
intracellular signaling pathway than that of cAMP exists in RAW 264.7 cells for AM-induced modulation of cytokine production.
Lipoproteins, such as LDL, ac-LDL, ox-LDL, are found to be factors that
stimulate AM production in RAW 264.7 cells. Ox-LDL has been reported to
modulate the expression of macrophage colony-stimulating factor
mRNA, platelet-derived growth factor-B mRNA, and TNF-
mRNA in mouse peritoneal macrophages, and many lines of evidence have demonstrated that these lipoproteins play crucial roles in the
development and progression of atherosclerosis (32, 54, 55). Activated
macrophages ingest plasma lipoproteins and are then converted into foam
cells being stuffed with cholesteryl ester. The resulting foam cells
are known to participate in the formation of atherosclerotic plaque.
Kohno and co-workers (56) have shown that AM inhibits proliferation of
VSMCs with the synthetic phenotype that are also major components of
atherosclerotic plaque. In this study, AM production in RAW 264.7 cells
is shown to be stimulated with lipoproteins. Based on these data, AM is
deduced to participate in the development and progression of
atherosclerosis as an inhibitory factor.
In conclusion, this study demonstrate that the macrophage cell line and
primary macrophage produce and secrete AM. AM production is dynamically
regulated by LPS, steroids, and lipoproteins, as well as RA, TPA, and
IFN-
which modulate differentiation and activation of macrophages.
AM suppresses the secretion and gene transcription of TNF-
and IL-6
under stimulation with LPS. These results suggest that AM secreted from
macrophages participates in the pathogenesis of sepsis, inflammation,
and atherosclerosis.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. K. Kitamura and Prof.
T. Eto of Miyazaki Medical College for discussion and donation of
antiserum against AM and cAMP; Dr. K. Shimokado of this institute for
donation of ox-LDL; and M. Nakatani and M. Higuchi of this institute
for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by the Special Coordination
Fund for the Promotion of Science and Technology from the Science and
Technology Agency (Encouragement System of Center of Excellence), by
research grants from the Ministry of Health and Welfare, the Ministry
of Education, Science and Culture, and the Human Science Foundation of
Japan.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: National
Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel.: 81-6-833-5012 (ext. 2600); Fax:
81-6-872-7485; E-mail: minamino{at}ri.ncvc.go.jp.
1
The abbreviations used are: AM, adrenomedullin;
IR, immunoreactive; CGRP, calcitonin gene-related peptide; EC,
endothelial cell; VSMC, vascular smooth muscle cell; ELISA,
enzyme-linked immunoadsorbent assay; NO, nitric oxide; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; TPA,
12-O-tetradecanoyl phorbol-13-acetate; RA, retinoic acid;
LPS, lipopolysaccharide; IFN-
, interferon-
; TNF-
, tumor
necrosis factor
; IL-1, interleukin-1; IL-6, interleukin-6; TGF-
,
transforming growth factor-
1; LDL, low density
lipoprotein; ox-LDL, oxidized LDL; ac-LDL, acetylated LDL; DMEM,
Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; FCS,
fetal calf serum; HPLC, high pressure liquid chromatography; PCR,
polymerase chain reaction; RIA, radioimmunoassay; 8-Br-cAMP,
8-bromoadenosine 3',5'-cyclic monophosphate.
 |
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