(Received for publication, November 13, 1995; and in revised form, January 30, 1996)
From the
The production of nitric oxide (NO) from L-arginine by
nitric oxide synthase (NOS) in cytokine-stimulated vascular smooth
muscle cells (VSMC) is thought to play an important role in the
pathophysiology of several vascular disease states including septic
shock. This study examines the relationship between cytokine-stimulated
NO production and L-arginine transport in cultured VSMC.
Cultured VSMC from rat aorta were stimulated with interleukin-1,
tumor necrosis factor-
, and/or angiotensin II (Ang II); and the
accumulation of nitrite, a stable product of NO metabolism, in the
culture media and the rates of net L-arginine uptake were
measured. Interleukin-1
and tumor necrosis factor-
, alone or
in combination, stimulated both the uptake of L-arginine and
the accumulation of nitrite in the culture media in a dose-dependent
manner. Inhibition of NOS activity by substituted analogues of L-arginine had no effect on cytokine-stimulated L-arginine transport. Ang II in the presence of cytokines
up-regulated L-arginine transport while inhibiting nitrite
accumulation. Two forms of the L-arginine transporter, cat-1b and cat-2, are expressed in VSMC. Northern
analysis revealed that the cytokine-stimulated increase in L-arginine transport coincided with increased levels of cat-2 mRNA. In contrast, cat-1b does not appear to be
regulated by cytokines at the mRNA level, although significant
increases in response to Ang II were observed. These results show that,
while cytokines can stimulate both NOS activity and L-arginine
uptake, NO production is not required to signal the increase in L-arginine transport. Furthermore, Ang II and cytokine
stimulation of L-arginine uptake involves the differential
regulation of the cationic amino acid transporter (cat) genes.
Nitric oxide (NO) ()is an important regulator of
vascular tone(1) , and several studies have shown that excess
production of NO may contribute to the hypotension and vascular
hyporesponsiveness observed in septic shock. The activation of
inflammatory cells during infection leads to the release of cytokines
such as tumor necrosis factor-
(TNF-
), interleukin-1
(IL-1
), and
-interferon. These cytokines, along with
bacterial toxins such as lipopolysaccharides (LPS), can stimulate the
expression of the inducible isoform of nitric oxide synthase (iNOS) in
most vascular cells, including tissue macrophages, endothelial cells,
and vascular smooth muscle cells
(VSMC)(2, 3, 4, 5) . Expression of
iNOS leads to the sustained production of NO as measured by the
accumulation of its stable end product, nitrite. Recently, treatment of
sepsis patients with NOS inhibitors was shown to produce a widespread
increase in vascular tone and a rise in blood pressure(6) .
This observation, along with the finding that mice lacking a functional inos gene have an increased survival rate over wild-type
animals during LPS-induced septic shock(7) , strongly suggests
that the overproduction of NO is an important mechanism in the vascular
dysfunction and mortality associated with sepsis.
Because NO
production requires free L-arginine as a substrate, the
availability of L-arginine provides a potential regulatory
site for NO production. Cationic amino acids are transported across
mammalian cell membranes by a small number of well defined transport
systems (y, b
,
y
L)(8, 9, 10) . In VSMC,
transport (influx and efflux) of cationic amino acids appears to be
mediated by the Na
-independent System
y
(11, 12) . Genes encoding three
murine System y
transporters (cat-1, cat-2,
and cat-2a) have been
cloned(10, 13, 14, 15, 16, 17, 19) and shown to infer System y
transport
activity when expressed in Xenopus oocytes(10, 13, 14, 17, 19) .
Recently, genes for two rat homologues for cat-1 have been
reported. The first was detected and cloned from a rat hepatoma genomic
library (20) . Subsequently, we have reported the partial
cloning of a cDNA for a second gene (cat-1b) from
VSMC(12) . These cells also express a cat-2 homologue.
In murine macrophages LPS- and -interferon-induced NO
production is absolutely dependent on the availability of extracellular L-arginine. When murine macrophages were treated with LPS,
System y
transport activity was shown to be increased
more than 10-fold(21, 22) . The induction of L-arginine transport was sensitive to the protein synthesis
inhibitor cycloheximide, implying that the LPS-stimulated activity of
System y
in these cells requires the de novo protein synthesis(23) . The aim of the present work was to
determine whether the cytokine stimulation of NO production in VSMC was
associated with changes in the System y
transport
activity and with specific changes in the expression of cat genes.
To investigate the relationship between cytokine-stimulated
NO production and L-arginine transport in VSMC, cells were
incubated for 24 h with IL-1 and TNF-
alone, or in
combination. Both cytokines stimulated nitric oxide synthase activity
as determined by the recovery of nitrite in the media and by the
initial rates of uptake of L-arginine into the cells (Fig. 1). The cytokines in combination produced results that
appeared to be additive with respect to the effects observed when used
alone. In contrast, while LPS and
-interferon both stimulated
nitrite production, no changes in the L-arginine uptake rates
could be detected (data not shown). Dose-response curves showed that
IL-1
stimulated both processes with an EC
of between
1 and 3 ng/ml (data not shown), suggesting that the same receptor
population was controlling the induction in each case. When the time
courses of IL-1
stimulation of nitrite accumulation and L-arginine uptake activity were determined, detectable
increases in nitrite concentration were observed from 6 h increasing
linearly through to 48 h. In contrast, while the L-arginine
uptake activity was enhanced from 6 h and increased through to 24 h, no
further increase could be detected (data not shown). Both lysine and
ornithine inhibited the cytokine-stimulated component of the L-arginine transport (data not shown), confirming that the
System y
activity was being stimulated.
Figure 1:
TNF- and IL-1
stimulate
nitrite accumulation and L-arginine transport in VSMC.
Quiescent VSMC were incubated for 24 h in the absence (Control) or presence of TNF-
(100 ng/ml) or IL-1
(10 ng/ml), alone or in combination. The amount of nitrite accumulated
in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described
under ``Experimental Procedures.'' Values are the means
± S.D. of three replicate determinations. Differences between
values not sharing the same letter are statistically significant (p < 0.05).
While
investigating whether NO production was required for the stimulation of L-arginine transport, inhibitors, or nitric oxide synthase, N-methyl-L-arginine and N-nitro-L-arginine methyl ester were both shown to
largely block the IL-1
/TNF-
stimulation of nitrite production
without affecting the changes in L-arginine transport (Fig. 2). In a parallel experiment, dexamethasone treatment (1
mM for 24 h) was shown to reduce the cytokine-stimulated
nitrite accumulation by over 70%, whereas there was no change in the
cytokine stimulation of L-arginine uptake (data not shown).
There was, however, a decrease in the basal L-arginine uptake
rate in the absence of cytokines suggesting that dexamethasone
inhibited the synthesis of some component in the L-arginine
transport pathway. Further evidence for the requirement of new protein
synthesis was obtained when cells were exposed to the protein synthesis
inhibitor cycloheximide, which completely blocked the
cytokine-stimulated nitrite accumulation and the increased L-arginine uptake rate. However, cycloheximide caused a
significant decrease in the L-arginine uptake rate in the
absence of cytokines (Fig. 3). These results suggest that new
protein synthesis is required not only for the cytokine-stimulated
nitrite production and increases in L-arginine uptake but is
also necessary for the maintenance of the basal L-arginine
uptake.
Figure 2:
Inhibitors of NOS decrease nitrite
accumulation but do not affect cytokine-stimulated L-arginine
uptake in VSMC. Quiescent VSMC were treated for 24 h without NOS
inhibitors (open bars), with 1 mMN-methyl-L-arginine (hatched
bars), and with 1 mMN-nitro-L-arginine
methyl ester (filled bars) in the absence (Control)
or presence of a combination of IL-1
(10 ng/ml) and TNF-
(100
ng/ml). The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental
Procedures.'' Values are the means ± S.D. of four replicate
determinations. Differences between values not sharing the same letter
are statistically significant (p <
0.05).
Figure 3:
Cytokine-stimulated increases in nitrite
accumulation and L-arginine uptake require de novo protein synthesis. Quiescent VSMC were treated for 24 h with the
inhibitor of protein translation cycloheximide (10 µg/ml, hatched bars) or without cycloheximide (open bars) in
the absence (Control) or presence of a combination of
IL-1 (10 ng/ml) and TNF-
(100 ng/ml). The amount of nitrite
accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described
under ``Experimental Procedures.'' Values are the means
± S.D. of three replicate determinations. Differences between
values not sharing the same letter are statistically significant (p < 0.05).
Recently we reported that the vasoactive peptide angiotensin
II (Ang II) could stimulate L-arginine transport in
VSMC(12) . When tested in combination with the cytokines
IL-1 and TNF-
, Ang II was shown to partially inhibit the
cytokine-induced accumulation of nitrite in the media while stimulating
the L-arginine transport in a manner that was additive
compared with that produced by the cytokines alone (Fig. 4). Ang
II alone did not affect the control levels of nitrite production, and
it appeared to delay the cytokine stimulation of nitrite production by
about 6 h with the rates of nitrite production over the final 12 h of
the experiment being similar in both the presence and absence of Ang
II. These data confirm the observation of Nakayama et al.(33) that Ang II suppresses NOS activity in these cells
and further indicate that Ang II is likely to stimulate L-arginine uptake by a mechanism that is independent of that
of the cytokines.
Figure 4:
Ang II increases cytokine-stimulated L-arginine uptake but decreases nitrite accumulation in VSMC.
Quiescent VSMC were treated with a combination of IL-1 (10 ng/ml)
and TNF-
(100 ng/ml, filled squares), Ang II (100
nM, open circles), or a combination of IL-1
,
TNF-
, and Ang II (filled circles) for the indicated
times. Control (untreated) VSMC were co-cultured in parallel (open
squares). The amount of nitrite accumulated in the culture media (a) and the initial rate of L-arginine uptake (b) were determined as described under ``Experimental
Procedures.'' Values are the means ± S.D. of four replicate
determinations. Differences between 24-h values not sharing the same
letter are statistically significant (p <
0.05).
Previous studies have shown that cultured VSMC
express two different genes of the L-arginine transporter, cat-1b and cat-2 (12). To examine the induction of
the cat and inos genes, cells were exposed to either
Ang II or the cytokines TNF- and IL-1
for periods up to 24 h
before harvesting and extraction of total RNA for Northern analysis.
The cytokines in combination led to parallel increases in the abundance
of inos and cat-2 transcripts without any effect on
the level of cat-1b mRNA (Fig. 5). In contrast, Ang II
was able to enhance cat-1b mRNA concentrations at early time
points, 3 and 6 h, but not at longer time points, which was consistent
with our previous results(12) .
Figure 5:
Cytokines regulate inos and cat-2 mRNA levels in VSMC. Quiescent VSMC were treated with
Ang II (100 nM) or a combination of IL-1 (10 ng/ml) and
TNF-
(100 ng/ml) for the times indicated with control (untreated)
VSMC co-cultured in parallel. Total RNA was prepared and analyzed (20
µg/lane) for the expression of inos, cat-1b, and cat-2/mRNA as described under ``Experimental
Procedures.'' The integrity of the RNA was determined by
visualizing ethidium bromide-stained rRNA under ultraviolet light
following electrophoresis on a formaldehyde
gel.
This work has clearly demonstrated that the cytokines
IL-1 and TNF-
stimulate both NO production and the rate at
which L-arginine is transported into VSMC. The increase in L-arginine transport is due to up-regulation of the System
y
family of cationic amino acid transporters, a result
which is consistent with this family being the only transporter of L-arginine expressed in these
cells(11, 12, 34) . The finding that the
cytokine-stimulated increase in L-arginine transport in VSMC
was inhibited by cycloheximide, and therefore required de novo protein synthesis suggested that regulation of L-arginine
transport possibly required protein synthesis of the L-arginine transporter itself. However, the observation that
cycloheximide caused a decrease in the non-stimulated rate of L-arginine transport suggests either that the transporter has
a rapid turnover rate or that some other component was required for the
regulation of the transporter activity.
The presence of messenger
RNA for two cat genes has been demonstrated in
VSMC(12) . However, other studies have shown that the CAT-1 and
CAT-2 isoforms have identical kinetic properties and are
indistinguishable at the cellular
level(10, 13, 14, 17, 19) .
Our study using transcript-specific probes has clearly demonstrated
that IL-1 and TNF-
not only regulate the expression of inos mRNA but specifically up-regulate the expression of the cat-2 mRNA transcript. The cytokine-stimulated expression of
iNOS is at least partially mediated in other cells through the nuclear
translocation of the cytoplasmic transcription factor, nuclear factor
B(35) . Recent analysis of the murine cat-2 gene
has demonstrated the presence of multiple promoters, suggesting a
complex regulation of this gene(36) .
Our observation that
Ang II inhibits the cytokine stimulation of NO production in VSMC is
consistent with that of Nakayama et al.(33) . In our
experiment, this appears to be a result of the Ang II delaying the
cytokine effect rather than blocking it completely. In contrast, Ang II
stimulates the System y activity over and above that
observed for the cytokines, suggesting that there are multiple
signaling pathways regulating the expression of the cat genes.
The inos and cat genes are not the only genes
involved in L-arginine metabolism that are regulated by
cytokines(35) . Both the activity and the mRNA level of
argininosuccinate synthetase are coinduced with inos in VSMC
in response to cytokines(37) . Recently, the type II arginase
activity in macrophages has been shown to be induced by LPS but not by
-interferon(18) . These results suggest that there is a
complex regulation of genes encoding proteins required for L-arginine metabolism that may ultimately affect the ability
of cells to sustain NO production following chronic stimulation by
cytokines.