| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Endocrinology, Dokkyo University School of Medicine, Tochigi 321-0293, Japan; and Department of Pharmacology, Cornell University Medical College, New York, New York 10021
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Immunostimulants trigger vascular smooth muscle cells (VSMC)
to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is
considered to be mediated via the
y+ system, we show here that rat
VSMC in culture express the cat-1 gene
transcript as well as an alternatively spliced transcript of the
cat-2 gene. An RT-PCR cloning sequence
strategy was used to identify a 141-base nucleotide sequence encoding
the low-affinity domain of alternatively spliced
CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of
CAT-2B in VSMC activated with
lipopolysaccharide (LPS) in combination with interferon-
(IFN). With
this sequence as a probe, Northern analyses showed that
CAT-1 mRNA and
CAT-2B mRNA are constitutively present
in VSMC, and the expression of both mRNAs was rapidly stimulated by
treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels
within 6 h after LPS-IFN. CAT-2A mRNA
was not detectable in unstimulated or stimulated VSMC. Arginine
transporter activity significantly increased 4-10 h after LPS-IFN.
iNOS activity was reduced to almost zero in the absence of
extracellular arginine uptake via system
y+. Induction of arginine
transport seems to be a prerequisite to the enhanced synthesis of NO in
VSMC. Moreover, this work demonstrates tissue expression of
CAT mRNAs with use of a model of LPS
injection in rats. RT-PCR shows that the expression of
CAT-1 and
CAT-2B mRNA in the lung, heart, and
kidney is increased by LPS administration to rats, whereas
CAT-2A mRNA is abundantly expressed in
the liver independent of LPS treatment. These findings suggest that
together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with
membrane arginine transport.
nitric oxide synthase; arginine; inducible isoform of nitric oxide synthase
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
IMMUNOSTIMULANTS TRIGGER vascular smooth muscle cells (VSMC) to express the inducible isoform of nitric oxide (NO) synthase (iNOS) (3, 5, 15). Induction of iNOS and overproduction of NO in VSMC have been implicated in the genesis of septic and cytokine-induced circulatory shock (23, 36). Elucidation of the factors that control iNOS activity should provide information for the design of therapeutics that effectively limit pathophysiological NO overproduction. The activity of iNOS appears to be regulated mainly at the transcriptional level (41). However, regulation of arginine availability can also determine the cellular rate of NO production, since arginine is the only physiological substrate for the NO synthase reaction. Recent reports indicate that iNOS activity is strictly dependent on the presence of extracellular L-arginine. The cytokine-stimulated production of NO by VSMC and the NO-mediated vascular hyporeactivity after endotoxin exposure can be reversed by removing L-arginine from the extracellular environment (3, 34). These findings indicate that the transport of L-arginine into VSMC may be an important regulatory mechanism for determining the rate of VSMC NO production.
The transport of cationic amino acids by VSMC appears to be mediated by the system y+ carrier (14, 26, 27). Recently, genes encoding the proteins responsible for the activity of the murine system y+ carrier have been cloned and designated CAT-1, CAT-2A, and CAT-2B (1, 8-10, 22, 29, 30). CAT-1 was initially identified as an ectropic retrovirus receptor in murine fibroblasts (1) and was subsequently shown to be a basic amino acid transporter in Xenopus oocytes (24, 37). CAT-2B was first detected in activated thymocytes (29) and has recently been cloned from lipopolysaccharide (LPS)-treated macrophages (10). CAT-1 and CAT-2B are low-capacity transporters that have a high affinity for cationic amino acids. In contrast, CAT-2A is an alternate splice variant of CAT-2B that was cloned from murine liver and possesses low affinity but high transport capacity (9, 22).
In the present study, to elucidate the molecular mechanism of
L-arginine transport in
immunostimulated VSMC, we characterized the expression of
CAT mRNAs in these cells. Rat VSMC
express mRNAs for CAT-1 as well as
CAT-2B, both of which are upregulated
by LPS in combination with interferon- (IFN). Here we report the partial cloning of a cDNA for CAT-2A
and CAT-2B from rat VSMC. These
isoforms result from mutually exclusive alternative splicing of the
transcript in a tissue-specific manner. Using a model of LPS injection
in rats, we also investigated the tissue distribution of
CAT mRNAs and alteration of their
expression during endotoxic shock. We show that
CAT-1 and
CAT-2B mRNAs are increased by
endotoxin treatment of cardiovascular tissues such as lung, heart, and
kidney, whereas CAT-2A mRNA, which is
highly expressed in the liver, is unaffected by LPS administration.
These data suggest that an immunostimulant-elicited increase in
arginine transport activity plays a key role in NO formation by VSMC
and that arginine transport is stimulated by endotoxin in rat tissues
during sepsis.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Cell culture, animal treatment, and extraction of RNA.
VSMC were isolated by elastase and collagenase digestion of thoracic
aortas from male Wistar rats (6). Cultures were fed twice weekly with
DMEM containing 10% fetal bovine serum and antibiotics (100 µg/ml
piperacillin and 100 µg/ml streptocymin). Cells in passages 10-15 were seeded and
grown to confluence in 96-well plates for nitrite assay and in
75-cm2 culture flasks for
preparation of cell lysate and RNA. The cells exhibited a classical
VSMC phenotype, with hill-and-valley morphology, and stained positively
for smooth muscle 
-actin with a monoclonal antibody (DAKO, Via Real,
CA). Male Wistar rats (250-300 g) were injected intravenously with
LPS (Escherichia coli serotype
0111:B4, 10 mg/kg; Sigma, St. Louis, MO) or saline (1 ml/kg) in the
endotoxin and control groups, respectively. The animals were killed 3 h later by exsanguination, and various organs were removed, immediately frozen in liquid nitrogen, and stored at 
70°C until RNA
extraction. The guanidinium isothiocyanate-acid phenol method (7) was
used to extract total RNA from the VSMC and rat tissues.
Nitrite measurement. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of VSMC, as previously described (18). Nitrite was measured by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid) to 100-µl samples of cell culture medium. Absorbance at 550 nm was determined with a microplate reader. Nitrite concentrations were calculated by comparison with the absorbance of standard solutions of sodium nitrite prepared in cell culture medium.
Arginine transport measurement. After incubation in the presence of LPS-IFN, the cells were washed with HEPES-buffered saline (HBS) and further incubated in HBS at 37°C for 10 min. The reaction was started by changing the medium to HBS containing L-[3H]arginine (2.15 TBq/mmol). The concentration of L-[3H]arginine was 100 nM, and the reaction time was 2 min. The reaction was stopped by washing the cells three times with ice-cold HBS. The cells were then lysed in 0.32 N NaOH with 1% SDS, and the radioactivity incorporated was determined by a liquid scintillation counter. The protein concentration of the cell lysate was measured using the Lowry method, with BSA as a standard (28).
Determination of arginine concentration in cells and medium.
After 12- and 24-h incubation periods, the medium was collected,
centrifuged at 10,000 g for 5 min, and
stored at 80°C for subsequent analysis by HPLC. Cells were
rinsed with PBS (Ca2+ and
Mg2+ free) and lysed with
methanol, and cell lysates were stored at 80°C for HPLC
analysis. The deproteinized samples were injected into a Wakosil 5C18
column (Wako, Tokyo, Japan) attached to an HPLC system of Waters
chromatography (Millipore, Milford, MA), and the concentrations of
arginine and other amino acids in the samples were determined as
previously described (21). Intracellular amino acid concentrations are
expressed in millimolar, where the volume is the intracellular water
space (0.5 ± 0.05 pl, n = 3) measured as described previously (25).
CAT mRNA expression.
RT-PCR was performed using a standard method (25). cDNA was synthesized
from total RNA from LPS-IFN-activated VSMC by avian myeloblastosis
virus RT with random 9 mers as primers and then amplified by PCR with
primers derived from the published sequences of rat
CAT-1 and murine
CAT-2A. The forward
5'-GCCATCGTCATCTCCTTCCTG-3' [corresponding to sense
bp 272-292 of CAT-1 (23)]
and reverse 5'-CCCTCCCTCACCGTATTTCAC-3'
[corresponding to sense bp 782-802 of
CAT-1 (23)] primers were used to
detect the presence of a 531-bp CAT-1
transcript, whereas the forward 5'-AACGTGCTTTTATGCCTTTGT-3' [corresponding to sense bp 795-815 of
CAT-2A (13)] and reverse 5'-GGTGACCTGGGACTCGCTCTT-3' [corresponding to sense
bp 1387-1407 of CAT-2A
(13)] primers common to CAT-2A
and CAT-2B were used to detect
CAT-2A and/or
CAT-2B transcript(s). The PCR products were subcloned into pCR2.1 plasmids (Invitrogen, San Diego, CA) and
sequenced to ensure that they corresponded to the expected 613-bp
CAT-2A and 616-bp
CAT-2B transcripts. From this
sequencing result of rat partial cDNA for
CAT-2A and
CAT-2B, second primers that
distinguish CAT-2A from
CAT-2B were made to detect the
presence of 115-bp CAT-2A and 121-bp
CAT-2B transcripts; the primers are 5'-CCTTACCCCGCATTCTGTTTG-3' [forward
(P2A-F)] and
5'-AAATGACCCCTGCAGTCATCG-3' [reverse
(P2A-R)] for 115-bp
CAT-2A and
5'-CCCAATGCCTCGTGTAATCTA-3' [forward
(P2B-F)] and
5'-TGCCACTGCACCCGATGACAA-3' [reverse
(P2B-R)] for 121-bp
CAT-2B. The PCR products for
CAT-1, 115-bp
CAT-2A, and 121-bp
CAT-2B were labeled with
d-[-32P]CTP by
random priming and used as probes for Northern blot analysis of VSMC
mRNA. Blotting procedures were performed as described elsewhere (19).
The blots were quantitated for radioactivity with a BAS2000 image
analyzer (Fuji Photo Film, Tokyo, Japan). In addition, expression of
the CAT mRNAs in rat tissues was
evaluated by RT-PCR with use of primers specific for
CAT-1 and
CAT-2. The identity of the PCR
products was confirmed by direct sequencing.
Statistical analysis. Values are means ± SD. Multiple comparisons were evaluated by ANOVA followed by Fisher's protected least significant difference test. Student's unpaired t-test was used for comparisons between two experiments. P < 0.05 was considered significant.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Exposure of rat VSMC to LPS in culture triggers the production of
nitrite, an accumulating oxidation product of NO. Nitrite accumulation
is not observed in untreated cells but appears 6-8 h after
addition of LPS to the culture medium. Although IFN alone does not
elicit nitrite production, it has a potent synergistic effect on
LPS-induced nitrite accumulation. Figure 1
shows the time course of nitrite production in VSMC treated with a
combination of LPS (30 µg/ml) and IFN (100 U/ml) (LPS-IFN). Nitrite
accumulation 24 h after stimulation was 960 ± 24 nmol/mg cell
protein. On Northern blot analysis of iNOS mRNA, a dominant transcript
of ~4.6 kb is recognized in LPS-IFN-stimulated VSMC by means of a
probe for iNOS. The transcript is barely detectable at 2 h, becomes
evident at 4 h, peaks at 8 h, and is sustained at slightly lower levels at 24 h after stimulation (Fig. 1,
inset).
|
Nitrite formation was examined in the culture media containing various
concentrations of L-arginine in
the VSMC activated with LPS-IFN for 24 h. The rate of nitrite formation
was saturable to arginine concentration within an apparent
Michaelis-Menten constant
(Km) of 40 µM
and almost saturated at 100 µM arginine (Fig.
2A). The
transport of L-arginine into
immunostimulant-activated VSMC has been shown to be mediated by the
cationic amino acid transport system
y+ (12-15, 26, 27). The
dependency of NO formation on
L-arginine transport was studied
by incubating VSMC for 24 h with LPS-IFN with dialyzed serum, amino
acid-depleted medium containing
L-arginine (0.1 mM), and
increasing concentrations of
L-lysine (0-10 mM), which
shares this transport system and competitively inhibits the transport
of arginine (38-40). Under these conditions, the production of
nitrite was inhibited in a concentration-dependent manner by lysine
(Fig. 2B) but not by serine, which
does not share the y+ system (data
not shown). This inhibition by lysine was bypassed by the presence of
higher concentrations of arginine (1 mM; Fig. 2B).
|
Changes in initial arginine uptake were investigated in VSMC cultured
with LPS-IFN. As shown in Fig.
3A, the
rate of uptake was significantly augmented in VSMC at 4-10 h after
LPS-IFN, and the rate declined thereafter. Treatment of VSMC with
cycloheximide (5 µg/ml) abolished the increase in arginine uptake
without reducing uptake below control levels (data not shown). We
measured changes in the intracellular concentration of arginine when
VSMC were activated with LPS-IFN. At 12 and 24 h after LPS-IFN, the
intracellular and extracellular arginine concentrations were
determined. The intracellular arginine concentration was higher in
LPS-IFN-stimulated cells, and the intracellular-to-extracellular
distribution ratio of arginine was much higher in these stimulated
cells (Fig. 3B). The intracellular
concentration of arginine decreased when lysine was present in the
medium (data not shown). We also measured intracellular concentrations
of cationic amino acids other than arginine in VSMC (Table
1). After 12 h in culture the total
concentration of cationic amino acids (arginine + lysine + ornithine)
was 0.54 mM in unstimulated cells and 1.53 mM in LPS-IFN-treated cells. After 24 h the concentration of cationic amino acids in the
LPS-IFN-treated cells was further increased to 2.19 mM.
|
|
cDNA prepared by RT from LPS-IFN-activated rat VSMC RNA served as a
template for PCR. The products obtained were for a 531-bp CAT-1 and the mutually exclusive
alternatively spliced isoforms of
CAT-2 (high-affinity isoform
CAT-2B or low-affinity isoform CAT-2A). As shown in Fig.
4, a specific DNA sequence (138 or 141 bp)
was nested within a 613- or 616-nt sequence obtained by RT-PCR by using
the primers that flanked the region of alternative splicing. One of
these sequences encodes the
L-arginine high affinity of the
CAT-2 isoform polypeptide (CAT-2B),
which, expressed in Xenopus oocytes,
acts as cationic amino acid transport system
y+, and the other encodes CAT-2A,
which is the low-affinity liver isoform of this cationic amino acid
transport activity (13-15). The deduced amino acid sequences,
based on the rat nucleotide sequences, of the domain that differs
between the two isoforms (46 and 47 amino acids) displays 100%
identity with that of mouse CAT-2A (8) and differs by only one residue
from that of mouse CAT-2B (29) (Fig. 4). On the basis of the nucleotide
sequence differences in this domain between
CAT-2A and
CAT-2B, we made new primer sets for
PCR amplification of cDNA prepared from rat VSMC and tissue RNAs to
discriminate CAT-2A mRNA and
CAT-2B mRNA. PCR primer sets for
amplification of 115-bp CAT-2A cDNA
are P2A-F and
P2A-R, and for 121-bp
CAT-2B they are
P2B-F and
P2B-R, respectively (Fig. 4). The
identity of the PCR products 115-bp
CAT-2A and 121-bp CAT-2B, as those corresponding to rat
CAT-2A and
CAT-2B, was confirmed by direct
sequencing of the PCR products.
|
The time course of CAT mRNAs was
investigated by Northern analysis of total RNA from LPS-IFN-activated
VSMC with rat CAT cDNAs (531-bp
CAT-1 cDNA, 115-bp
CAT-2A cDNA, and 121-bp
CAT-2B cDNA) as probes. As shown in
Fig. 5, two mRNAs for
CAT-1 (7.9 and 3.4 kb) and for
CAT-2B (8.5 and 4.5 kb) were
identified in vascular smooth muscle.
CAT-1 mRNA, which was detectable in
untreated VSMC, substantially increased after the stimulation with
LPS-IFN, peaked at 2 h and then decreased below basal levels by 8 h.
The CAT-2A signal was absent or
negligible in the untreated and stimulated VSMC throughout the time
course (data not shown). CAT-2B mRNA, which was detectable in untreated VSMC, increased within 2 h after the
stimulation, peaked by 4 h, and subsequently decreased up to 24 h.
|
Figure 6 shows the effects of
dexamethasone, cycloheximide, and actinomycin D on the expression of
CAT mRNAs in VSMC. The steady-state
level of CAT-1 mRNA in VSMC stimulated
with LPS-IFN was decreased by 6 h, but it remained increased by
treatment of the cells with cycloheximide. The steady-state level of
CAT-2B mRNA in VSMC after LPS-IFN,
which returned to near basal level by 6 h, was abolished by actinomycin
D at 6 h. In contrast, CAT-2B mRNA in
VSMC treated with dexamethasone or cycloheximide was increased compared
with control (LPS-IFN only) at 6 h.
|
We next used a model of LPS injection in rats to investigate the tissue
distribution of CAT mRNAs and
alteration of their expression during endotoxic shock. As has been
shown previously, iNOS mRNA is markedly increased in the lung, heart,
liver, and kidney from LPS-treated rats but is absent from all tissues
studied in control rats (20). The
CAT-1 signal was absent or negligible in the control lung, heart, and liver, whereas a signal was detected in
the control kidney. After LPS the
CAT-1 signal became detectable in the
lung and heart and was substantially increased in the kidney; however,
it remained absent in the liver (Fig. 7).
Although the CAT-2 signal was very low
in the control lung and heart, a large signal was observed in the
control liver and a weak signal in the control kidney. After LPS the
CAT-2 signal remained elevated in the
liver and was further increased in the kidney (Fig. 7). By separate PCR
amplification with use of primers
P2A-F and
P2A-R for
CAT-2A and
P2B-F and
P2B-R for
CAT-2B, a large
CAT-2A signal in the liver was
detected independently of LPS treatment, and a modest induction by LPS
of CAT-2A mRNA was observed in the
kidney. The CAT-2B mRNA was clearly
induced in the lung, heart, and kidney by LPS treatment and also was
detectable in the liver before and after LPS (Fig. 7). Control PCR
experiments demonstrated equivalent expression of the glyceraldehyde
3-phosphate dehydrogenase gene in all samples.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Several lines of evidence argue that L-arginine transport by rat aortic VSMC is mediated by the system y+ carrier and that LPS or cytokines stimulate the rate of L-arginine transport by upregulation of the system y+ family of cationic amino acid transporters (12-14, 16, 26, 27). This study shows clearly that immunostimulants can stimulate the system y+ activity of VSMC and the mRNA concentrations of CAT-1 and CAT-2B. Moreover, this work defines the profile of tissue expression of CAT mRNAs in LPS-treated rat: expression of CAT-1 and CAT-2B mRNA in lung, heart, and kidney is increased by LPS administration, whereas CAT-2A mRNA is abundantly expressed in liver in the presence or absence of LPS treatment.
The cationic amino acid transport system y+ has been well studied in mammalian cells (8, 30, 38-40). Physiological transport system y+ activity is encoded by the cationic amino acid transporter genes (cat-1 and cat-2), with cDNA clones denoted as CAT-1, CAT-2A, and CAT-2B (1, 7-9, 22, 29, 30). Three different polypeptides encoded by these genes each catalyze sodium-independent cationic amino acid uptake. CAT-2A and CAT-2B are distinct proteins encoded by two mature mRNAs originating from one primary transcript of the cat-2 gene (10, 22). These isoforms result from the mutually exclusive alternative splicing of the transcript in a tissue-specific manner. In this study we used PCR primers for rat CAT-2 that flanked the region of alternative splicing and found that the rat and mouse sequences differ by only one base within this alternatively spliced region of CAT-2A or CAT-2B. This results in a single amino acid conservative substitution of "I" in the rat for "V" in the mouse predicted the 47-amino acid polypeptide sequence of CAT-2B, whereas the rat and mouse predicted sequences are 100% identical within this 46-amino acid region of CAT-2A (10, 22). The optimal alignment between the alternatively spliced regions of rat CAT-2 amino acid sequence shows only 50% identity.
In mouse lymphocytes, expression of CAT-2B results in high-affinity (Km ~ 40-100 µM) system y+ uptake. The CAT-2A transporter isoform has a much lower substrate affinity (Km ~ 5 mM) and is expressed predominantly in liver. In the rat VSMC we could detect mRNAs for CAT-1 and CAT-2B, but not for CAT-2A, by Northern blot analysis. Using RT-PCR, we were able to detect the presence of CAT-2A mRNA in rat VSMC, the product of which was not derived from genomic DNA but was confirmed by sequencing to be the product derived from CAT-2A mRNA. Indeed, the CAT-2A signal was detectable at lower levels even by Northern blot analysis when VSMC were treated with LPS-IFN in the presence of cycloheximide. Nonetheless, CAT-1 and CAT-2B are the isoforms expected to take up arginine from the medium (DMEM), in which the arginine concentration routinely used is 400 µM. Because rat plasma L-arginine concentrations are ~100 µM or lower under septic shock conditions (11, 33), CAT-1 and CAT-2B are most likely to be the physiologically relevant isoforms in most tissues, including lung, heart, and kidney, with a negligible contribution by CAT-2A, except in liver (9, 10, 22).
We reported earlier that, along with iNOS, immunostimulants synergistically induce VSMC to express argininosuccinate synthase (AS) mRNA and activity (17). With constitutively expressed argininosuccinate lyase (AL), AS confers on cells an "arginine-citrulline cycle" (17, 32), which can sustain NO production via continuous regeneration of the iNOS substrate L-arginine from the iNOS coproduct L-citrulline. This allows for NO synthesis from citrulline even in the absence of extracellular arginine. We also showed the coinduction of AS/AL and iNOS in vivo in tissues of LPS-treated rats most clearly in the kidney, where arginine is regenerated from citrulline by use of AS and AL (20). We found the apparent Km to be 40 µM L-arginine for NO biosynthesis from extracellular substrate L-arginine and that nitrite production was a hyperbolic function of extracellular L-arginine concentration, such that upregulated nitrite production was reduced to almost zero when extracellular L-arginine was 0 mM (Fig. 2). Thus extracellular L-arginine concentration is a rate-limiting factor for NO synthase activity in VSMC exposed to immunostimulants under normal conditions where intracellular citrulline concentration is not high enough to support arginine supply via the arginine-citrulline cycle (17, 32). This dependency on extracellular L-arginine concentration could be determined by the arginine transport y+ system activity. Indeed, increasing concentrations of L-lysine dose dependently inhibited NO synthesis. It has been shown that the activity of iNOS was not directly inhibited by lysine in a murine macrophage cell line and in rat peritoneal macrophages (4, 35). The induction of CAT-1 and CAT-2B mRNAs and arginine transport activity in LPS-IFN-treated VSMC preceded iNOS mRNA induction and NO biosynthesis. Thus upregulated arginine transport activity may play a more important role in supporting high-output NO synthesis by VSMC, especially in the earlier stages before the recycling system from citrulline to arginine by AS and AL becomes effective. It is likely that arginine must be concentrated from the extracellular fluid into the cells for continuous production of NO. Under these conditions, intracellular concentrations of other cationic amino acids (lysine and ornithine) in VSMC also are increased. Transport of arginine via system y+ is subject to transstimulation; i.e., the influx of arginine is significantly increased by the presence of competing substrates in the cells (38). Therefore, the increase in the arginine transport activity in LPS-IFN-treated cells might be due, in part, to transstimulation.
It is now well established that high-output NO synthesis by immunostimulant-activated cells coincides with the induction of arginine transport activity in cultured cells. Our study shows that expression of mRNAs for CAT-1 and CAT-2B resulting in high-affinity system y+ uptake is induced in response to immunostimulants, providing a molecular basis for this in VSMC. We also show the concomitant induction of CAT-1 and CAT-2B mRNA and iNOS mRNA in various tissues of LPS-treated rats. These findings suggest that together CAT-1 and CAT-2B play a special function in providing substrate for high-output NO synthesis in vitro as well as in vivo. Several important questions await answers: 1) iNOS of VSMC has been well characterized, and the reported value of Km for arginine is 5-10 µM. The intracellular concentration of arginine shown here was much higher than this, even in VSMC incubated with lysine, yet under these conditions NO synthesis was considerably inhibited. How can this discrepancy be explained? 2) What is the advantage for a cell to express CAT-1 and CAT-2B, both coding functionally active transporters in the VSMC and the other tissues? 3) What are the relative contributions of activation of transporter(s) and the recycling pathway of citrulline to arginine to sustaining NO synthesis?
Inhibiting arginine availability affords a potential therapeutic opportunity for limiting NO production in pathophysiological conditions arising from NO excess. The challenge will be to develop specific modulators of arginine availability to the cells that can be targeted to specific cell types.
| |
ACKNOWLEDGEMENTS |
|---|
The authors are grateful to Dr. Kazumi Akimoto (Laboratory of Molecular and Cellular Biology, Dokkyo University School of Medicine) for technical assistance.
| |
FOOTNOTES |
|---|
This work was supported in part by a grant from the Japan Private School Promotion Foundation. S. S. Gross is supported by National Heart, Lung, and Blood Institute Grants HL-46403 and HL-50656.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Hattori, Dept. of Endocrinology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan.
Received 14 September 1998; accepted in final form 1 February 1999.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Albritton, L. M.,
L. Tseng,
D. Scadden,
and
J. M. Cunningham.
A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.
Cell
57:
659-666,
1989[Medline].
2.
Aulak, K. S.,
J. Liu,
J. Wu,
S. L. Hyatt,
M. Puppi,
S. J. Henning,
and
M. Hatzoglou.
Molecular sites of regulation of expression of the rat cationic amino acid transporter gene.
J. Biol. Chem.
271:
29799-29806,
1996
3.
Beasley, D.,
J. H. Schwartz,
and
B. M. Brenner.
Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells.
J. Clin. Invest.
87:
602-608,
1991.
4.
Bogle, R. G.,
A. R. Baydoun,
J. D. Pearson,
S. Moncada,
and
G. E. Mann.
L-Arginine transport is increased in macrophages generating nitric oxide.
Biochem. J.
284:
15-18,
1992.
5.
Busse, R.,
and
A. Mulsch.
Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells.
FEBS Lett.
275:
87-90,
1990[Medline].
6.
Chamley-Campbell, J.,
G. R. Campbell,
and
R. Ross.
The smooth muscle cell in culture.
Physiol. Rev.
59:
1-61,
1979
7.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
8.
Closs, E. I.
CATs, a family of three distinct mammalian cationic amino acid transporters.
Amino Acids
11:
193-208,
1996.
9.
Closs, E. I.,
L. M. Albritton,
J. W. Kim,
and
J. M. Cunningham.
Identification of a low affinity, high capacity transporter of cationic amino acids in mouse liver.
J. Biol. Chem.
268:
7538-7544,
1993
10.
Closs, E. I.,
C. R. Lyon,
C. Kelly,
and
J. M. Cunningham.
Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins.
J. Biol. Chem.
268:
20796-20800,
1993
11.
Deshmukh, D. R.,
V. S. Ghole,
B. Marescau,
and
P. P. De Deyn.
Effect of endotoxemia on plasma and tissue levels of nitric oxide metabolites and guanidino compounds.
Arch. Physiol. Biochem.
105:
32-37,
1997[Medline].
12.
Durante, W.,
L. Liao,
I. Iftikhar,
K. Cheng,
and
A. I. Schafer.
Platelet-derived growth factor regulates vascular smooth muscle cell proliferation by inducing cationic amino acid transporter gene expression.
J. Biol. Chem.
271:
11838-11843,
1996
13.
Durante, W.,
L. Liao,
I. Iftikhar,
W. O'Brien,
and
A. Schafer.
Differential regulation of L-arginine transport and nitric oxide production by vascular smooth muscle and endothelium.
Circ. Res.
78:
1075-1082,
1996
14.
Durante, W.,
L. Liao,
and
A. I. Schafer.
Differential regulation of L-arginine transport and inducible NOS in cultured vascular smooth muscle cells.
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H1158-H1164,
1995
15.
Durante, W.,
V. B. Schini,
T. Scott-Burden,
D. C. Junquero,
M. H. Kroll,
P. M. Vanhoutte,
and
A. I. Schafer.
Platelet inhibition by an L-arginine-derived substance released by IL-1
-treated vascular smooth muscle cells.
Am. J. Physiol.
261 (Heart Circ. Physiol. 30):
H2024-H2030,
1991
16.
Gill, D. J.,
B. C. Low,
and
M. R. Grigor.
Interleukin-1 and tumor necrosis factor- stimulate the cat-2 gene of the L-arginine transporter in cultured vascular smooth muscle cells.
J. Biol. Chem.
271:
11280-11283,
1996
17.
Hattori, Y.,
E. B. Campbell,
and
S. S. Gross.
Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration of arginine for nitric oxide synthesis.
J. Biol. Chem.
269:
9405-9408,
1994
18.
Hattori, Y.,
and
S. S. Gross.
GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase.
Biochem. Biophys. Res. Commun.
195:
435-441,
1993[Medline].
19.
Hattori, Y.,
K. Kasai,
K. Akimoto,
and
C. Thiemermann.
Induction of NO synthesis by lipoteichoic acid from Staphylococcus aureus in J774 macrophages: involvement of a CD14-dependent pathway.
Biochem. Biophys. Res. Commun.
233:
375-379,
1997[Medline].
20.
Hattori, Y.,
S. Shimoda,
and
S. S. Gross.
Effect of lipopolysaccharide treatment in vivo on tissue expression of argininosuccinate synthetase and argininosuccinate lyase mRNAs: relationship to nitric oxide synthase.
Biochem. Biophys. Res. Commun.
215:
148-153,
1995[Medline].
21.
Ikeda, M.,
K. Sorimachi,
K. Akimoto,
and
Y. Yasumura.
Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride.
J. Chromatogr.
621:
133-138,
1993[Medline].
22.
Kavanaugh, M. P.,
H. Wang,
Z. Zhang,
W. Zhang,
Y. N. Wu,
E. Dechant,
R. A. North,
and
D. Kabat.
Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family.
J. Biol. Chem.
269:
15445-15450,
1994
23.
Kilbourn, R. G.,
and
O. W. Griffith.
Overproduction of nitric oxide in cytokine-mediated and septic shock.
J. Natl. Cancer Inst.
84:
827-831,
1992
24.
Kim, J. W.,
E. I. Closs,
L. M. Albritton,
and
J. M. Cunningham.
Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.
Nature
352:
725-728,
1991[Medline].
25.
Kletzien, R. F.,
M. W. Pariza,
J. E. Becker,
and
V. R. Potter.
A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.
Anal. Biochem.
68:
537-544,
1975[Medline].
26.
Low, B. C.,
and
M. R. Grigor.
Angiotensin II stimulates system y+ and cationic amino acid transporter gene expression in cultured vascular smooth muscle cells.
J. Biol. Chem.
270:
27557-27583,
1995.
27.
Low, B. C.,
I. K. Ross,
and
M. R. Grigor.
Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells.
J. Cell. Physiol.
156:
626-634,
1993[Medline].
28.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
29.
MacLeod, C. L.,
K. Finley,
D. Kakuda,
C. A. Kozak,
and
M. F. Wilkinson.
Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor.
Mol. Cell. Biol.
10:
3663-3674,
1990
30.
MacLeod, C. L.,
and
D. K. Kakuda.
Regulation of CAT: cationic amino acid transporter gene expression.
Amino Acids
11:
171-191,
1996.
31.
Nunokawa, Y.,
N. Ishida,
and
S. Tanaka.
Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
191:
89-94,
1993[Medline].
32.
Nussler, A. K.,
T. R. Billiar,
Z. Z. Liu,
and
S. M. Morris, Jr.
Coinduction of nitric oxide synthase and argininosuccinate synthetase in a murine macrophage cell line. Implications for regulation of nitric oxide production.
J. Biol. Chem.
269:
1257-1261,
1994
33.
Sax, H. C.,
P. O. Hasselgren,
M. A. Talamini,
L. L. Edwards,
and
J. E. Fischer.
Amino acid uptake in isolated, perfused liver: effect of trauma and sepsis.
J. Surg. Res.
45:
50-55,
1988[Medline].
34.
Schott, C. A.,
G. A. Gray,
and
J. C. Stoclet.
Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine.
Br. J. Pharmacol.
108:
38-43,
1993[Medline].
35.
Shibazaki, T.,
M. Fujiwara,
H. Sato,
K. Fujiwara,
K. Abe,
and
S. Bannai.
Relevance of the arginine transport activity to the nitric oxide synthesis in mouse peritoneal macrophages stimulated with bacterial lipopolysaccharide.
Biochim. Biophys. Acta
1311:
150-154,
1996[Medline].
36.
Thiemermann, C.,
and
J. Vane.
Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo.
Eur. J. Pharmacol.
182:
591-595,
1990[Medline].
37.
Wang, H.,
M. P. Kavanaugh,
R. A. North,
D. Kabat,
H. Wang,
M. P. Kavanaugh,
R. A. North,
and
D. Kabat.
Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter.
Nature
352:
729-731,
1991[Medline].
38.
White, M. F.,
G. C. Gazzola,
and
H. N. Christensen.
Cationic amino acid transport into cultured animal cells. I. Influx into cultured human fibroblasts.
J. Biol. Chem.
257:
4443-4449,
1982
39.
White, M. F.,
and
H. N. Christensen.
The two-way flux of cationic amino acids across the plasma membrane of mammalian cells is largely explained by a single transport system.
J. Biol. Chem.
257:
4450-4457,
1982
40.
White, M. F.
The transport of cationic amino acids across the plasma membrane of mammalian cells.
Biochim. Biophys. Acta
822:
355-374,
1985[Medline].
41.
Xie, Q.-W.,
R. Whisnant,
and
C. Nathan.
Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon- and bacterial lipopolysaccharide.
J. Exp. Med.
177:
1779-1784,
1993
This article has been cited by other articles:
|
|
 |
|
  K. A. Munger, R. C. Blantz, and M. J. Lortie Acute renal response to LPS: impaired arginine production and inducible nitric oxide synthase activity Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2006; 291(3): R684 - R691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Stanley, L. G. Chicoine, T. L. Young, K. M. Reber, C. R. Lyons, Y. Liu, and L. D. Nelin Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L298 - L306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Schafer, T. Wallerath, E. I. Closs, C. Schmidt, P. M. Schwarz, U. Forstermann, and H.-A. Lehr Dexamethasone suppresses eNOS and CAT-1 and induces oxidative stress in mouse resistance arterioles Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H436 - H444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Lortie, J. Satriano, F. B. Gabbai, S. Thareau, S. Khang, A. Deng, D. P. Pizzo, S. C. Thomson, R. C. Blantz, and K. A. Munger Production of arginine by the kidney is impaired in a model of sepsis: early events following LPS Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2004; 287(6): R1434 - R1440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Schwartz, I. F. Schwartz, E. Gnessin, Y. Wollman, T. Chernichovsky, M. Blum, and A. Iaina Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats Am J Physiol Renal Physiol, April 1, 2003; 284(4): F788 - F795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Mann, D. L. Yudilevich, and L. Sobrevia Regulation of Amino Acid and Glucose Transporters in Endothelial and Smooth Muscle Cells Physiol Rev, January 1, 2003; 83(1): 183 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Calatayud, E. Garcia-Zaragoza, C. Hernandez, E. Quintana, V. Felipo, J. V. Esplugues, and M. D. Barrachina Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying Am J Physiol Gastrointest Liver Physiol, December 1, 2002; 283(6): G1360 - G1367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. O. Scumpia, P. J. Sarcia, V. G. DeMarco, B. R. Stevens, and J. W. Skimming Hypothermia attenuates iNOS, CAT-1, CAT-2, and nitric oxide expression in lungs of endotoxemic rats Am J Physiol Lung Cell Mol Physiol, December 1, 2002; 283(6): L1231 - L1238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D. Nelin, H. E. Nash, and L. G. Chicoine Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1232 - L1239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Fernandez, I. Yaman, R. Mishra, W. C. Merrick, M. D. Snider, W. H. Lamers, and M. Hatzoglou Internal Ribosome Entry Site-mediated Translation of a Mammalian mRNA Is Regulated by Amino Acid Availability J. Biol. Chem., April 6, 2001; 276(15): 12285 - 12291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fernandez, I. Yaman, W. C. Merrick, A. Koromilas, R. C. Wek, R. Sood, J. Hensold, and M. Hatzoglou Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2alpha Phosphorylation and Translation of a Small Upstream Open Reading Frame J. Biol. Chem., January 11, 2002; 277(3): 2050 - 2058. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) or 1 mM (
)
arginine supplemented with lysine
(B). After 24 h, nitrite
accumulation in culture medium was measured. Values are means ± SD
of 6 determinations from 2 independent experiments. When not shown,
error bars are contained within symbols.
* P < 0.05;
** P < 0.01 vs. control (no
lysine).
