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1 Department of Medical Physiology, Microcirculation Research Institute, Texas A&M University Health Science Center, College Station, Texas 77843-1114; and 2 Department of Chemical Engineering, University of California, Los Angeles, California 90095-1592
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In macrophages and many other cell types, L-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability of L-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor, L-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 µg/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels of L-arginine (0.01-0.8 mM) produced a concomitant increase in NO production of activated macrophages. L-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by L-norvaline was inversely related to the extracellular level of L-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular L-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellular L-arginine, respectively. When the L-arginine concentration exceeded 0.5 mM, the L-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited.
lipopolysaccharide; nitric oxide synthase; septic shock; inflammation
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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IN MAMMALIAN CELLS,
L-arginine is used as a
substrate by nitric oxide synthase (NOS), arginase, arginine-glycine
transaminase, kyotorphine synthase, and arginine decarboxylase
(24). Among these enzymes, NOS and arginase are presumed
to play the most significant roles in the metabolism of
L-arginine in cytotoxic macrophages (7, 11, 15, 36).
L-Arginine is metabolized by NOS
to form nitric oxide (NO) and citrulline and by arginase to form
ornithine and urea. NO, an important regulator and mediator in many
physiological and pathophysiological events, is produced by the
oxidation of one of the guanidino nitrogens of
L-arginine by a family of NOS
isoforms. Although some NOS isoforms are constitutively expressed and
Ca2+ dependent, the
NOS expressed in macrophages [inducible NOS (iNOS)] is
independent of Ca2+ and can be
induced by certain cytokines, such as tumor necrosis factor-
,
interleukin-1
, and interferon-
, and by microbes or microbial
membrane components such as lipopolysaccharide (LPS) and lipid A (34,
37). On the induction of iNOS, NO is produced continuously at a high
rate in the presence of adequate
L-arginine supply. This high NO
production has been implicated in several cytostatic and cytotoxic
actions mediated by macrophages, including the host tumoricidal and
antimicrobial effects (11, 13, 22, 25).
Besides iNOS, the other major L-arginine-consuming enzyme, arginase, was also found to have a high activity in activated macrophages (11, 15, 19, 21, 30, 32, 36). Although arginase has been shown to be induced by LPS in macrophages (30, 32, 33, 36), its physiological function remains to be elucidated. Because arginase and NOS both use L-arginine as a substrate, we hypothesized that NO production may be regulated by arginase through competition for the intracellular L-arginine pool. In addition, it has been shown that NO production from activated macrophages is dependent on the extracellular supply of L-arginine (15). It is possible that the extracellular level of L-arginine might affect the modulatory role of arginase for NO production. This issue is important because macrophages are known to be one of the chief defense instruments of mammalian hosts, and many of the cytotoxic effects exerted by macrophages have been shown to involve sustained NO production by iNOS (13, 22, 25). The purposes of the present study were therefore to determine whether NO production in LPS-activated macrophages is modulated by arginase and to investigate whether arginase-regulated NO production is influenced by the extracellular level of L-arginine. In this regard, the role of arginase in modulating NO production was investigated in the absence and presence of the arginase inhibitor L-norvaline at various L-arginine concentrations.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Activation of macrophages. The mouse macrophage J774A.1 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in either 60-mm culture dishes or 12-well culture plates with Dulbecco's modified Eagle's medium (DMEM) containing 0.4 mM L-arginine and supplemented with 10% fetal bovine serum (FBS; Summit Biotechnology, Ft. Collins, CO), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C under a humidified 7% CO2 atmosphere. Medium was changed daily and cells were passaged by trypsinization in Dulbecco's phosphate-buffered saline (DPBS) containing 0.25% trypsin (GIBCO, Grand Island, NY) and 0.2% EDTA after confluence. Experiments were performed two days after cells reached confluence.
To allow the L-arginine level in the medium to be precisely controlled, we used RPMI-1640 (GIBCO) select amine kit to prepare specific culture medium. Before each experiment, DMEM was replaced with modified RPMI-1640 medium containing 0.1 mM L-arginine, 200 µM glutamine, 4% FBS, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Two hours after the medium was changed, 1 µg/ml LPS (from Escherichia coli 0.111:B4) was added to activate macrophages, and cells were incubated for 22 h. The medium was then changed to the modified RPMI-1640 containing LPS and various concentrations of L-arginine and L-norvaline as specified in each experiment. In another series of experiments, D-norvaline, D-arginine, and the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) were used to examine the specificity of L-norvaline- and L-arginine-related NO production. After 12 h of incubation, culture medium and cells were sampled for NO measurements and enzyme assays (i.e., NOS and arginase), respectively. In some experiments, the cell lysate was incubated with L-norvaline (10 mM) for 1 h to determine the direct effect of L-norvaline on arginase and NOS activities. Cellular protein levels were quantified by bicinchoninic acid protein assay (Pierce, Rockford, IL) and were used as a basis to normalize the enzyme activity and NO production.NO measurement. It has been shown that the primary decomposition product of NO in hemoglobin-free solution is nitrite (17). Our preliminary studies confirmed this finding and found that >95% of total NO released from activated macrophages was converted to nitrite in the culture medium. Therefore, in the present study, the production of NO was evaluated by measuring nitrite using a chemiluminescence NO analyzer (Sievers Instruments, Boulder, CO). Basically, at the end of each respective treatment, the supernatant of cell culture medium was collected for NO analysis. The collected sample (100 µl) was injected into a reflux chamber containing glacial acetic acid and 1% potassium iodide at room temperature. Under these conditions, nitrite is quantitatively converted to NO. The NO gas was then purged into the chemiluminescence NO analyzer and quantitated by reference to NaNO2 standards.
Arginase assay.
After each specified treatment, cells were rinsed with ice-cold DPBS
twice, scraped into 3 ml of DPBS, and then centrifuged at 1,200 revolutions/min (250 g) for 5 min. Cell pellets were resuspended in 200 µl lysis buffer containing 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.5), 0.1 mM EDTA, 0.1 mM ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1 mM dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml aprotinin,
and 0.1 mM phenylmethylsulfonyl fluoride. Finally, cells were lysed by
three freeze-and-thaw cycles. The activity of arginase was determined
from the urea production using the method described by Corraliza et al.
(8). In brief, cell lysate (50 µl) was added into 50 µl of
Tris · HCl (50 mM; pH 7.5) containing 10 mM
MnCl2. Macrophage arginase was
then activated by heating the mixture at 55-60°C for 10 min.
The hydrolysis reaction of
L-arginine by arginase was
carried out by incubating the lysate with 50 µl of
L-arginine (0.5 M; pH 9.7) at
37°C for 1 h and was stopped by adding 400 µl of the acid
solution mixture (1 H2SO4:3 H3PO4:7
H2O).
-Isonitrosopropiophenone (25 µl, 9%; dissolved in 100%
ethanol) was then added to the mixture, followed by heating at 100°C for 45 min. After the mixture was incubated in
the dark for 10 min at room temperature, the urea concentration was
determined spectrophotometrically by the absorbance at 550 nm measured
with a microplate reader (Molecular Devices, Menlo Park, CA).
The rate of urea production was used as an index for arginase
activity.
NOS assay. Cell lysate was prepared as described in Arginase assay. Cell lysate (25 µl) was mixed with 100 µl lysis buffer and the reaction mixture containing (final concentrations) 100 mM L-arginine, 1.25 mM NADPH, 0.5 µM FAD, and 1 µM tetrahydrobiopterin. The mixture was incubated at 37°C for 1 h, and 100 µl of the sample were then collected for nitrite measurement as described in NO measurement.
Materials. All chemicals and drugs, except where otherwise noted, were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis. Results were presented as means ± SE. Data analysis was performed by one-way analysis of variance followed by Fisher's protected least-significant differences test using StatView 4 (Abacus Concepts, Berkeley, CA). Differences were considered to be statistically significant when the P value was <0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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L-Arginine dependence of NO production from activated macrophages. Our hypothesis states that the competition of arginase with NOS for their common substrate is one of the mechanisms that influences NO production. This hypothesis is predicated on the assumption that L-arginine is a limiting factor for NO production, so that the competition from arginase will be effective. We therefore began by investigating the effect of extracellular L-arginine supply on NO production in LPS-activated macrophages. In the control experiment, when macrophages were not stimulated by LPS, only a very small amount of NO was produced in the presence of L-arginine (0.1 mM) for 12 h (Fig. 1A). This basal release of NO from nonactivated macrophages was independent of the extracellular level of L-arginine (0.1-0.5 mM; data not shown). In the activated macrophage, NO production was significantly increased by 12-fold at 0.1 mM L-arginine; however, this increase was not observed when the same level of D-arginine was used instead of L-arginine in the culture medium (Fig. 1A). In addition, the NOS inhibitor L-NMMA (0.01 mM) also markedly reduced NO production from activated macrophages cultured with 0.1 mM L-arginine. In contrast to release of NO from resting macrophages, the amount of NO released from activated macrophages was correlated with the increased extracellular level of L-arginine (0-0.5 mM) (Fig. 1B). There was no further increase in NO production from activated macrophages when L-arginine concentration in the medium was increased beyond 0.5 mM. Collectively, these results demonstrated that the NO produced by LPS-activated macrophages was specifically derived from L-arginine in a concentration-dependent manner.
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L-Norvaline enhances NO production from activated macrophages. Because L-arginine was required for NO production in activated macrophages, we investigated whether inhibition of the arginase pathway would enhance the amount of NO released. L-Norvaline was used as an arginase inhibitor, because it does not affect NOS activity (see below). Figure 2 shows that NO production from activated macrophages at 0.05 mM L-arginine was elevated in the presence of L-norvaline (5-20 mM). This effect was dose dependent up to 10 mM of L-norvaline. Concentrations >10 mM did not further enhance NO production. In contrast to L-norvaline, its stereoisomer D-norvaline (10 mM) had no effect on NO production (Fig. 2).
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L-Norvaline inhibits macrophage arginase but not NOS. For further characterization of the effect of L-norvaline on NO production, it is essential to evaluate the specificity of L-norvaline by demonstrating that 1) L-norvaline does inhibit arginase activity at the enzyme kinetic level and 2) L-norvaline has no effect on NOS activity either at the enzyme kinetic or gene expression levels. To probe the inhibitory effect at the enzyme kinetic level, we cultured cells without L-norvaline, and then the cell lysate was used for enzyme assays in the presence and absence of L-norvaline (10 mM). It was found that arginase activity (i.e., urea production) was significantly inhibited by L-norvaline in the cell lysate (Fig. 4A), indicating that arginase activity can be directly inhibited by L-norvaline. To examine the effect of L-norvaline on arginase gene expression level, we initially cultured cells in the presence of L-norvaline (10 mM) for 12 h. After the L-norvaline in the cell culture was washed out, the cell lysate was then prepared and assayed for arginase activity in the presence and absence of L-norvaline (10 mM). Figure 4B shows that arginase activity was inhibited only when the cell lysate was treated with L-norvaline, indicating that L-norvaline did not alter arginase gene expression. These results demonstrate that L-norvaline does inhibit arginase activity and that the inhibition is at the enzyme kinetic level.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The biological importance of NO synthesized from L-arginine has been well documented (22, 25). In particular, the rate of NO production has been shown to be regulated at several levels: transcription initiation, mRNA stability, phosphorylation, intracellular Ca2+ concentration, and availability of tetrahydrobiopterin (23). In the present study, we investigated whether L-arginine availability modulated by arginase can serve as another control mechanism for NO production. Our results indicate that arginase is capable of diminishing NO production by reducing L-arginine availability to NOS. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited. To our knowledge, the present study is the first to directly demonstrate the role of arginase in the modulation of NO production in activated macrophages. To provide a perspective for our observations and conclusions, we will address the specificity of L-norvaline in the inhibition of arginase. In addition, the possible physiological and pathophysiological role of arginase in modulation of NO production will be discussed.
Specificity of L-norvaline in arginase inhibition. L-Norvaline, a nonmetabolizable analog of L-valine, has been reported to be one of the most potent arginase inhibitors in various preparations (29). This inhibition appears to be enantiomer dependent inasmuch as its stereoisomer, D-norvaline, had no effect on NO production from activated macrophages (Fig. 2). Directly, L-norvaline inhibits arginase simply because of its structural similarity to ornithine (14, 16, 31). Ornithine is one of the products from the arginase pathway, and it has been shown to inhibit arginase (4, 16, 26, 28). Indirectly, L-norvaline was found to inhibit arginase activity through its inhibition of ornithine transcarbamylase, which converts ornithine to citrulline in the urea cycle (3, 29). The inhibition of ornithine transcarbamylase by L-norvaline may lead to the accumulation of ornithine, which in turn inhibits arginase. Furthermore, L-norvaline has also been shown to inhibit argininosuccinate synthase (35), which catalyzes the endogenous synthesis of L-arginine from recycled citrulline (23, 38). The inhibition of argininosuccinate synthase by L-norvaline would decrease endogenous L-arginine synthesis and thus reduce NO production. However, the net effect of L-norvaline observed in the present study was an increase in NO production (Fig. 2). If inhibition of argininosuccinate synthase by L-norvaline indeed occurred in our preparation, the role of arginase in the modulation of NO production might have been actually underestimated in the present study. However, it has been shown that the contribution of endogenous synthesis of L-arginine for NO production is relatively small in the activated macrophages (15, 38); thus it is unlikely that the inhibition of argininosuccinate synthase by L-norvaline played an important role in our experimental settings.
Although inhibition of arginase by L-norvaline has been generally characterized in the perfused liver or purified arginase (3, 14, 16, 31), it is not known whether L-norvaline can exert its inhibitory function at the gene expression level in addition to the enzyme kinetic level. It is also unclear whether L-norvaline has any effect on the activity and/or gene expression of NOS. This is a critical question because our data interpretation relies on its specific action. In the arginase enzyme assay, we demonstrated that L-norvaline (10 mM) inhibits arginase activity by 50% (Fig. 4A), indicating that L-norvaline has a direct inhibitory effect on arginase function. When cell lysate from macrophages pretreated with L-norvaline for 12 h was analyzed, inhibition of arginase activity was only observed when L-norvaline was added to the cell lysate (Fig. 4B). These results strongly suggest that the inhibitory action of L-norvaline is not at the gene expression level because preincubation of the cells with L-norvaline for 12 h (to allow the inhibitory process to act at the gene expression level) did not reduce arginase activity. These results indicate that arginase is directly inhibited by L-norvaline at the enzyme kinetic level. Most importantly, L-norvaline has no effect on iNOS either at the enzyme kinetic or gene expression level, as shown in Fig. 5. Therefore, it appears that the enhanced NO production by L-norvaline is a result of reduced arginase activity rather than the activation of iNOS. In contrast to our finding that the activity of iNOS was not altered by L-norvaline, Hrabák et al. (14) compared the inhibitory effect of various amino acids and derivatives on arginase and found that macrophage NOS activity was increased ~40% by L-norvaline. This discrepancy could be explained by the low concentration of L-arginine used by Hrabák et al. in their enzyme assay. In our NOS assay, a very high concentration of L-arginine (100 mM) was used in order to saturate arginase [the value of the Michaelis-Menten constant, Km, in mouse macrophage was reported to be 10 mM, with a range of 4-45 mM, in various animal tissues (18)]. The high concentration of L-arginine allowed us to study only the effect of L-norvaline on NOS activity and to eliminate indirect effects caused by changes in arginase activity. In contrast, Hrabák et al. used 4-8 µM L-arginine for the measurement of NOS activity in activated macrophages obtained from dephosphorylated casein-treated animals (14). Although the agent used by Hrabák et al. for cell activation was different from ours (LPS), it is very likely that the increased amount of L-arginine available to NOS during arginase inhibition, i.e., by L-norvaline, might have caused the apparent increase in NOS activity in their study. Therefore, the study of Hrabák et al. may indirectly support our hypothesis that arginase can play a modulatory role in NO production from activated macrophages.Modulation of NO production by arginase. A previous study of NO production in activated macrophages has suggested that both the NO synthesis pathway and the arginase pathway rely on the extracellular supply of L-arginine and that 90% of L-arginine is consumed by arginase, while 10% of the L-arginine routes to the NO synthesis pathway (15). This view is supported by our data showing that NO production from activated macrophages is determined by the level of L-arginine in the culture medium (Fig. 1). When comparing the activities of arginase (Fig. 4) and NOS (Fig. 5) in terms of urea and NO production, respectively, we found that arginase has a higher activity in metabolizing L-arginine. Therefore, the changes in arginase activity may subsequently alter L-arginine availability for NOS and thus influence NO production. Nevertheless, this hypothesis is feasible only under the conditions that arginine availability is limiting for NO production. Indeed, the NO production from LPS-activated macrophages was dependent on the replenishment of intracellular L-arginine from the extracellular medium up to 0.5 mM (Fig. 1), suggesting that the intracellular L-arginine is limiting for NO production within this range. This observation is important because the normal plasma L-arginine concentration is usually at the range of 0.1-0.3 mM and does not exceed 0.5 mM in various animal species (1, 10, 12, 24). Therefore, we speculated that arginase might play an important role in the modulation of NO production from activated macrophages in vivo.
When the competition between arginase and NOS was studied at different concentrations of L-arginine, L-norvaline caused a 28% enhancement in NO production at the normal physiological level of L-arginine (e.g., 0.1 mM) (Fig. 3). When L-arginine concentration was reduced to 0.05 mM, the NO production was further increased to 55% (Fig. 3). These results suggest that the competition between arginase and NOS is more pronounced when L-arginine availability is compromised, that is, the influence of arginase on NO synthesis is enhanced when the L-arginine level is reduced. It has been shown that the interstitial L-arginine concentration was markedly reduced by at least 30-50% in wound, during sepsis, and in other inflammatory sites (1, 10). This low level of L-arginine would make arginase even more relevant to the regulation of NO production from NOS. Interestingly, when the L-arginine level was increased to
0.5 mM, inhibition of arginase did not further enhance
NO production (Fig. 3). This phenomenon can be explained by the
saturation of iNOS for NO production at the higher levels of
L-arginine (0.5 mM).
Because NO production in the activated macrophage was correlated with
the increased level of
L-arginine and reached maximum
(saturated) at 0.5 mM (Fig. 1), it is expected that further supply of
L-arginine (i.e., inhibition of
arginase by L-norvaline) would
not further increase NO production. It appears that the competition
between arginase and NOS can be overcome if
L-arginine availability is high
enough for NOS to fully exert its function. Similar to its effect at
high levels of
L-arginine, the effect of
L-norvaline was not observed
when the L-arginine level was
extremely low (i.e., 0.01 mM). This may be explained by the fact that
the source of L-arginine for
both arginase and NOS in macrophages is mainly exogenous (Fig. 1) (15)
and that the relatively high
L-arginine affinity for NOS vs.
arginase apparently reduces the
L-arginine available to arginase
and thus subsequently reduces the contribution of arginase to NO
production. The modulation of NO production by arginase can be further
supported by the in vivo studies showing that NO production in
LPS-treated rats was significantly reduced when plasma
L-arginine was depleted by
administration of arginase to the animals (6, 12). Furthermore, the
effect of arginase could be reversed by infusion of
L-arginine (12). Collectively,
these in vivo findings are consistent with our contention that arginase
does have an influence on NO production by possibly controlling the
substrate availability.
Pathophysiological significance of modulation of NO release by arginase. High NO throughput has been shown to be involved in many pathophysiological conditions, such as inflammation and sepsis. In addition, the exclusive extracellular L-arginine requirement in this NO production process makes manipulation of substrate availability for NOS an attractive target for therapeutic intervention. For example, regulation of the L-arginine level by its transport (2) and by administration of arginase (6, 12) has been shown to be beneficial in experimental models of septic shock. Because a concomitant change in arginase activity, in addition to NOS, has been found under some pathophysiological conditions, it is conceivable that the alteration of arginase activity may subsequently influence the cellular function by altering the production of NO. For example, a high arginase activity in the infiltrating macrophages was found to be associated with cancer development (9, 20, 27, 32). Although the metabolism of L-arginine to NO is important for the macrophage antitumoral ability, the latter could be compromised by enhancing the arginase activity. In fact, the metabolism of L-arginine by arginase into ornithine and subsequently toward polyamines may actually favor tumor cell proliferation (5, 20, 27). Therefore, L-arginine metabolism in macrophages within a tumor could promote inhibition or growth of the tumor, depending on whether the NOS or arginase pathway is prevailing. For an understanding of the dual capacity of macrophages, the elucidation of the molecular nature of arginase and the regulatory behavior of macrophage arginase is considerably important. New clues for understanding the disparate roles of arginase have been provided by recent studies (5, 21, 27, 36), but many of the features of arginase still remain to be investigated. Although providing the ornithine substrate for polyamines and proline synthesis has been suggested to be one of the main functions of arginase, here we provide evidence that arginase can compete with NOS for their common substrate, L-arginine, and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-55524 and by a Whitaker Foundation Biomedical Engineering Grant.
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FOOTNOTES |
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Address for reprint requests: L. Kuo, Dept. of Medical Physiology, Microcirculation Research Institute, Texas A&M Univ. Health Science Center, College Station, TX 77843-1114.
Received 21 May 1997; accepted in final form 26 September 1997.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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)], NO production
from macrophages was minimal. In contrast, NO released from
LPS-activated macrophage [LPS(+)] was significantly
elevated in presence of 0.1 mM
L-Arg but not
D-arginine
(D-Arg). In presence of the NOS
inhibitor NG-monomethyl-L-arginine
(L-NMMA) with
L-Arg, the increased NO
production was significantly attenuated. Values are means ± SE;
n = 3 independent experiments.
* Significantly different from LPS(
significantly different from LPS(+) with 0.1 mM
L-Arg
(P < 0.05).
B: NO production from LPS-treated
macrophages was increased with increasing extracellular level of
L-Arg up to 0.5 mM. Values are
means ± SE; n = 3 independent
experiments. * Significantly different from conditions without
L-Arg
(P < 0.05).
