(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), ()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.
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,
200.
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-, 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.
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.