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(Received for publication, October 2, 1995) From the
Nitric oxide (NO) is synthesized from arginine by nitric oxide
synthase (NOS), and citrulline which is generated can be recycled to
arginine by argininosuccinate synthetase (AS) and argininosuccinate
lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS),
and expression of the inducible isoform of NOS (iNOS), AS, and AL was
analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the
LPS treatment but was induced by LPS in the lung, heart, liver, and
spleen, and less strongly in the skeletal muscle and testis. AS mRNA
was induced in the lung and spleen, and AL mRNA was weakly induced in
these tissues. AS and AL mRNAs were abundant in the control liver and
remained unchanged after the treatment. Kinetic studies showed that
iNOS mRNA increased rapidly in both spleen and lung, reached a maximum
2-5 h after the treatment, and decreased thereafter. On the other
hand, AS mRNA increased more slowly and reached a maximum in 6-12
h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in
the spleen and lung increased slowly and remained high up to 24 h. In
immunoblot analysis, increase of iNOS protein was evident in the lung,
liver, and spleen, and there was an increase of AS protein in the lung
and spleen. In immunohistochemical analysis, macrophages in the spleen
that were negative for iNOS and AS before LPS treatment were strongly
positive for both iNOS and AS after this treatment. As iNOS, AS, and AL
were coinduced in rat tissues and cells, citrulline-arginine recycling
seems to be important in NO synthesis under the conditions of
stimulation.
Nitric oxide (NO) is a major messenger molecule regulating blood
vessel dilatation and immune function and functions as a
neurotransmitter in the brain and peripheral nervous system (see (1, 2, 3) for reviews). NO is synthesized
from arginine by nitric oxide synthase (NOS), (
Figure 1:
RNA blot analysis for iNOS (A), AS (B), and AL (C) in LPS-treated rat
tissues. Total RANs (1.0 µg in A and 5.0 µg in B and C) from brain (Br), lung (Lu),
heart (He), liver (Li), spleen (Sp),
skeletal muscle (Mu), testis (Te), and kidney (Ki) of control(-) and LPS-treated (6 h) (+) rats
were electrophoresed in formaldehyde-containing 1% agarose gels and
transferred to nylon membranes. The filters were hybridized using as
probes digoxigenin-labeled antisense RNA for iNOS (A), AS (B), or AL (C) according to the protocol supplied by
Boehringer Mannheim. Detection was made using the DIG chemiluminescence
detection kit (Boehringer Mannheim). The positions of 28 S (4.6 kb) and
18 S (1.9 kb) rRNAs are shown on the left. Integrity of the
mRNAs was verified by the apparently identical intensities of 28 S and
18 S rRNA bands following the ethidium bromide staining after
electrophoresis.
AS and AL mRNAs were
measured in the lung, spleen, and liver of the same rats in which iNOS
mRNA was markedly induced. AS mRNA of about 1.5 kb was detected in the
lung before the LPS treatment and increased by the treatment (Fig. 1B). AS mRNA was present at a lower level in the
spleen than in the lung before the treatment, and increased markedly by
the treatment. In the liver where AS is involved in urea synthesis, AS
mRNA was much more abundant and was not induced by LPS. AL mRNA of
about 2.0 kb was also detected in the lung and spleen before the LPS
treatment, and increased weakly in these tissues after the treatment (Fig. 1C). It was much more abundant in the liver where
AL is also involved in urea synthesis and remained unchanged by the
treatment.
Figure 2:
Time course of induction of iNOS (a), AS (b), and AL (c) mRNAs in the spleen. A, total RNA was isolated from the rat spleen at indicated
times after the LPS injection. Two rats at 0 and 24 h and 3 rats at 2,
6, and 12 h were used. RNA blot analysis was performed as described in
the legend for Fig. 1. The chemiluminograms for iNOS (a), AS (b), and AL (c) mRNAs are shown. One
lane for AL mRNA at 6 h is missing. d shows ethidium bromide
staining of 28 S and 18 S rRNAs. B, the chemiluminograms were
quantified and the results are represented by mean ± S.D. (solid bar; n = 3) or mean ± range (dotted bar; n = 2). Maximal values are set at
100.
Figure 3:
Time
course of induction of iNOS (a), AS (b), and AL (c) mRNAs in the lung. Total RNA was isolated from the rat
lung and RNA blot analysis was performed as described in the legend for Fig. 2.
Figure 4:
Immunoblot analysis of iNOS (A)
and AS (B) proteins in LPS-treated rat tissues. Rats were
treated with LPS for 12 h. Tissue extracts (10 µg of protein) of
the lung (Lu), liver (Li), spleen (Sp), and
kidney (Ki) of control and LPS-treated rats were subjected to
SDS-5% (A) or 10% (B) polyacrylamide gel
electrophoresis. Proteins were electrotransferred to nitrocellulose
membranes, and the membranes were immunoblotted with mouse monoclonal
antibody against mouse iNOS (0.25 µg of IgG/ml) (A) or
rabbit anti-rat AS antiserum (diluted 5000-fold) (B).
Molecular mass markers (Rainbow protein molecular size markers,
Amersham Corp.) are myosin (200 kDa), phosphorylase b (97
kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), and carbonic
anhydrase (30 kDa).
Figure 5:
Immunostaining of the spleen of control
and LPS-treated rats with antibodies against iNOS, AS, and AL and with
a macrophage-specific antibody RM4. Spleens of control (a, c, e, and g) and an LPS-treated rat (12 h) (b, d, f, and h) were stained with
a monoclonal antibody against iNOS (10 µg of IgG/ml) (a and b), antiserum against AS (diluted 300-fold) (c and d) or AL (diluted 300-fold) (e and f), or RM4 (10 µg of IgG/ml) (g and h). Left portion of each photograph (a-h) shows red
pulp (RP), and right portion shows white pulp (WP). Original magnifications,
Cellular NO production is determined by NOS activity,
intracellular arginine concentration, and other factors. Induction of
iNOS in response to various stimuli has been reported for a variety of
cell types in vitro(33, 34, 35) and
also in rat tissues in
vivo(36, 37, 38) . Arginine can be
supplied via the blood circulation or regenerated from citrulline by
the citrulline-NO cycle that is composed of iNOS, AS, and AL. The
coinduction of iNOS and AS was noted in a murine macrophage cell line (9) and a murine aortic smooth muscle cell line(10) ,
thereby suggesting the importance of the citrulline-NO cycle, at least
in these cell types. The present study shows for the first time that
iNOS, AS, and AL are coinduced by LPS in vivo. Coinduction of
iNOS and AS was evident in the rat spleen and lung at both mRNA and
protein levels. AL mRNA was induced weakly in the spleen and lung. iNOS
mRNA was highly induced in the liver, but AS and AL mRNAs were abundant
in this tissue prior to LPS treatment and did not increase further.
This can be explained by the roles of AS and AL in urea synthesis in
the liver. iNOS mRNA was induced also in the heart, muscle and testis,
but apparently not in the brain and kidney. Ohshima et al.(36) reported that iNOS protein was induced in the liver, lung,
and spleen of the rat by administration of Propionibacterium acnes and LPS. Hom et al.(37) reported that iNOS mRNA
and protein were induced by LPS in many tissues of the rat, including
the brain and kidney. Sato et al.(38) reported that
iNOS protein was induced in the lung, liver, spleen, and peritoneal
macrophages of the LPS-treated rats. The minor discrepancies in these
studies may be due to differences in rat strain, amount of LPS, and
route of LPS administration. Kinetic studies showed that iNOS mRNA
is induced very rapidly with little time lag both in the spleen and
lung, whereas AS mRNA is induced more slowly with a time lag of about 2
h in these tissues. Induction of AL mRNA in these tissues is also slow,
albeit less marked. A rapid induction of NOS activity, followed by a
delayed induction of AS activity in the rat aortic smooth muscle cells
treated with LPS and interferon- An immunohistochemical study of iNOS in LPS-treated rats (39) showed that iNOS immunoreactivity was strongly detected in
macrophages in the heart, lung, liver, and kidney 6-9 h later.
Another immunohistochemical study (38) revealed that
inflammatory cells in many tissues, hepatocytes, and endothelial cells
of the aorta of rats were positive for iNOS around 6 h after LPS
injection, and that macrophages were positive in the liver and spleen
after 12 h. The present immunohistochemical study shows that the iNOS
and AS are colocalized in the spleen macrophages of LPS-treated rats.
All these results suggest strongly that the citrulline-NO cycle
functions in vivo in macrophages of the spleen and perhaps in
other tissues and cell types. Further immunohistochemical and in
situ hybridization analyses are under way.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2658-2662
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
RNA BLOT, IMMUNOBLOT, AND IMMUNOHISTOCHEMICAL ANALYSES (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)generating
citrulline as another product. Cellular NO production is absolutely
dependent on availability of arginine. This amino acid can be obtained
from exogenous sources via the blood circulation, from intracellular
protein degradation, or by endogenous synthesis of arginine. Major
sites of arginine synthesis in ureotelic animals are the liver, where
arginine generated in the urea cycle (ornithine cycle) is rapidly
converted to urea and ornithine by arginase, and the kidney, where
arginine is synthesized from citrulline and released into the blood
circulation (see (4) for a review). However, other tissues and
cell types also contain low levels of argininosuccinate synthetase (AS)
and argininosuccinate lyase (AL), which together synthesize arginine
from citrulline(5, 6, 7, 8) .
Therefore, arginine can be generated from citrulline which is produced
as a co-product of the NOS reaction, forming a cycle which could be
termed the ``citrulline-NO cycle'' (9) or
``arginine-citrulline cycle''(10) . Vascular
endothelial cells can convert citrulline to arginine(11) , and
this conversion is increased when cells are stimulated to produce
NO(12) . Cytokine-activated macrophages, which produce a large
amount of NO, have an increased capacity to produce arginine from
citrulline(13) . Furthermore, the inducible isoform of NOS
(iNOS) and AS are coinduced in a murine macrophage cell line (9) and cultured vascular smooth muscle cells (10) . AS (14, 15, 16) and AL (17, 18, 19, 20) have been purified
and characterized. cDNAs and genomic clones for AS (21, 22, 23) and AL (24, 25, 26, 27) were isolated, and
the promoters of the AS gene (28) and the AL gene (29) were characterized. To better understand the role of these
citrulline-NO cycle enzymes in NO synthesis in vivo, we
examined expression of the enzymes in lipopolysaccharide (LPS)-treated
rats using RNA blots, immunoblots and immunohistochemical analyses. We
report here that iNOS, AS, and AL are coinduced by LPS in the spleen
and lung of rats. Immunohistochemical analysis revealed induction of
both iNOS and AS in macrophages of the spleen after LPS treatment.
Materials
A monoclonal antibody against mouse
iNOS was obtained from Transduction Laboratories, Lexington, KY.
Antisera against rat AS and AL were as reported elsewhere(8) .
Preparation of a monoclonal antibody RM4 against rat macrophages will
be published elsewhere. (
)Animals and LPS Treatment
Specific
pathogen-free male Wistar rats (5-6 weeks of age) were injected
intraperitoneally with Escherichia coli LPS (serotype 0127:68,
Sigma) at 1.0 mg/kg body weight, and then killed following
anesthetization with ether.RNA Blot Analysis
Total RNA from rat tissues was
prepared by the guanidium thiocyanate-phenol-chloroform extraction
procedure (30) . After electrophoresis in
formaldehyde-containing agarose gels, RNAs were transferred to nylon
membranes (Boehringer Mannheim). Hybridization was performed using as
probes digoxigenin-labeled rat iNOS antisense RNA ((31) ;
nucleotides 2344-3026), rat AS antisense RNA(8) , or rat
AL antisense RNA(8) . The iNOS antisense RNA was synthesized
using iNOS cDNA that was cloned using reverse transcription-polymerase
chain reaction and subcloned into pcDNAII, as templates and the DIG-RNA
labeling kit (Boehringer Mannheim). Chemiluminescence signals derived
from hybridized probes were detected on x-ray films using DIG
luminescence detection kits (Boehringer) and quantified by
densitometry.Immunoblot Analysis
Rat tissues were excised and
homogenized in nine volumes of 20 mM potassium HEPES buffer
(pH 7.4) containing 1 mM dithiothreitol, 50 µM antipain, 50 µM leupeptin, 50 µM chymostatin, and 50 µM pepstatin. The homogenates
were centrifuged at 25,000 
g for 30 min at 4 °C,
and the supernatants were used as tissue extracts. The tissue extracts
were subjected to SDS-polyacrylamide gel electrophoresis, and proteins
were electrotransferred to nitrocellulose membranes. Immunodetection
was performed using an ECL kit (Amersham) according to the protocol
supplied by Amersham. Chemiluminescence signals on x-ray films were
quantified by densitometry.Immunohistochemical Staining
The excised rat
tissues were embedded in OCT Compound® (Miles, Elkhart, IN) and
frozen in liquid nitrogen. Five-µm sections were cut, air-dried,
washed in phosphate-buffered saline, and fixed with 2%
periodate-lysine-paraformaldehyde for 10 min. After inhibition of
endogenous peroxidase activity by the method of Isobe et al.(32) , the specimens were incubated for 2 h with one of
the primary antibodies diluted with phosphate-buffered saline
containing 0.5% bovine serum albumin (Fraction V). After washing in
phosphate-buffered saline, they were covered with sheep anti-mouse or
donkey anti-rabbit Ig[F(ab`)] conjugated with
peroxidase (Amersham) (diluted 100-fold) for 1 h. Peroxidase activity
was visualized cytochemically using 3,3`-diaminobenzidine as substrate.
For control, tissue slides were incubated with non-immunized mouse or
rabbit serum instead of primary antibody, and afterward processed by
the procedure described above. Sections were slightly counterstained
with hematoxylin.Other Methods
Protein was determined with the
protein assay reagent (Bio-Rad) using bovine serum albumin as a
standard.
Induction of iNOS, AS, and AL mRNAs in LPS-treated Rat
Tissues
Rats were injected intraperitoneally with bacterial LPS,
and iNOS mRNA in various tissues was measured by RNA blot analysis 6 h
after the treatment. iNOS mRNA was not detected in any tissue before
the treatment (Fig. 1A). However, the mRNA of about 4.5
kb was induced most strongly in the lung and spleen, followed by the
heart and liver, and less strongly in skeletal muscle and testis, but
apparently not in the brain and kidney.
Kinetics of Induction of iNOS, AS, and AL mRNAs in the
Spleen
mRNAs for iNOS, AS, and AL in the spleen were examined at
various times after the LPS treatment (Fig. 2). iNOS mRNA
increased to a near-maximum 2 h after the treatment, reached a maximum
at 6 h, decreased thereafter, and returned to a hardly detectable level
at 24 h. On the other hand, AS mRNA started to increase after 2 h with
a time lag, reached maximum at 6 h on the average (about 10-fold
increase), increased further or decreased at 12 h depending on the
animal, and returned to control levels at 24 h. AL mRNA increased with
kinetics similar to that for AS mRNA, although the increase was less
marked (about 2.5-fold); partly induced levels were retained for 24 h.
These results show that iNOS, AS, and AL are coinduced by LPS in the
rat spleen. iNOS mRNA was induced rapidly and strongly, whereas AS and
AL mRNAs were induced somewhat more slowly and AL mRNA was weakly
induced. Induction varied from one animal to another, but the three
mRNAs behaved similarly in each animal. For example, animal 3 at 6 h (6 h lanes) and animal 1 at 12 h (12 h lanes) were
relatively high for all mRNAs.
Kinetics of Induction of iNOS, AS, and AL mRNAs in the
Lung
Time course of induction of iNOS, AS and AL mRNAs in the
lung after the LPS treatment is shown in Fig. 3. iNOS mRNA
reached a maximum at 2 h and remained at near-maximal levels at 6 h,
then decreased slowly, and was still detectable at 24 h. Thus, the mRNA
in the lung increased similarly as that in the spleen, and decreased
more slowly that in the spleen. AS mRNA decreased somewhat 2 h after
the treatment, increased at 6-12 h by about 2-fold on the
average, and then decreased. The induction of AS mRNA was again slower
than that of iNOS mRNA and was much less marked than that of AS mRNA in
the spleen. AL mRNA in the lung increased gradually up to 12 h by about
2-fold and remained at high levels at 24 h. The profile of AL mRNA
induction in the lung resembled that in the spleen.
Induction of iNOS and AS Proteins by LPS
Induction
of iNOS and AS proteins in LPS-treated rat tissues was examined using
immunoblot analysis. iNOS protein of about 150 kDa that was
undetectable before the LPS treatment, was induced 12 h after the
treatment in the lung, liver, and spleen, but apparently not in the
kidney (Fig. 4A). These results parallel findings for
iNOS mRNA. On the other hand, AS protein of 46 kDa was detected in the
lung before the LPS treatment, and increased 12 h after the treatment (Fig. 4B). It was present in a lower amount in the
spleen than in the lung before the LPS treatment and increased markedly
by the treatment. Again, these results parallel those of AS mRNA.
Immunohistochemical Detection of Spleen Macrophages
Positive for iNOS and AS
To identify cells positive for iNOS and
AS, immunohistochemical analysis of the spleen, in which both enzymes
were markedly induced, was performed (Fig. 5). iNOS
immunoreactivity was nil in the spleen prior to LPS treatment. However,
a strong iNOS immunoreactivity was found in macrophages in the red pulp
of the spleen 12 h after the LPS treatment. AS immunoreactivity was
absent before the LPS treatment, and after the treatment, macrophages
in the red pulp became strongly positive for AS immunoreactivity.
Distributions and staining patterns of iNOS-positive macrophages and
AS-positive macrophages were similar. Distributions of these iNOS- and
AS-positive cells were similar to macrophages positive with a
macrophage-specific antibody RM4. However, staining patterns of iNOS-
and AS-positive cells differed from that of RM4-positive cells, perhaps
because iNOS and AS are cytosolic enzymes, whereas the antibody RM4
preferentially stains lysosomes in macrophages. These
results suggest strongly that iNOS and AS are colocalized in all
macrophages in the spleen. No change was found in the distribution of
RM4-positive cells before and after LPS treatment. These results
suggest that iNOS and AS are induced in macrophages that are already
present before the treatment. On the other hand, AL immunoreactivity
was not found before and after the LPS treatment. This apparent lack of
AL immunoreactivity in macrophages may be due to a small amount of the
AL protein and a low induction of the protein (about 2.5-fold induction
at the mRNA level).
200.
, was also reported(10) .
This means that the expression of the iNOS gene and those of the AS and
AL genes are partly coordinated and partly dissociated, possibly
because of different roles of these enzymes; iNOS is involved only in
NO synthesis, whereas AS and AL are responsible to synthesis of
arginine, a precursor for synthesis of proteins, polyamines, and
creatine phosphate in addition to NO. Studies on regulatory elements of
the iNOS, AS, and AL genes and trans-acting factors will need to be
done.
))
We thank S. Yano (First Department of Pharmacology,
Kumamoto University) for the iNOS cDNA clone, K. Iwase and K. Akagi of
this department (Molecular Genetics) for the AS and AL cDNA clones, and
K. Iyama (Division of Developmental Neurology, Kumamoto University) for
advice on histological techniques. We also thank M. Ohara for comments
on the manuscript and Y. Kusano for secretarial services.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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