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Am. J. Respir. Cell Mol. Biol., Volume 19, Number 2, August 1998 308-315

L-Arginine Uptake and Metabolism by Lung Macrophages and Neutrophils Following Intratracheal Instillation of Silica In Vivo

Ralph M. Schapira, John H. Wiessner, James F. Morrisey, Urias A. Almagro, and Leif D. Nelin

Division of Pulmonary and Critical Care Medicine, and Departments of Pathology and Pediatrics, Medical College of Wisconsin; and Sections of Pulmonary and Critical Care Medicine, and Pathology and Laboratory Medicine, and Research Service, Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Nitric oxide (NO) has been associated with lung inflammation following exposure to silica. L-arginine can be converted to NO and L-citrulline by nitric oxide synthase (NOS), or into urea and L-ornithine by arginase. We tested the hypothesis that after instillation of silica into rat lungs in vivo, lung inflammatory cells increase L-arginine metabolism by both NOS and arginase, which is associated with an increase in L-arginine uptake. We isolated lung inflammatory cells 3 d after silica or saline (control) exposure. The uptake of [3H]L-arginine at 24 h by cells from silica-exposed lungs (73.9 ± 4.8%) was significantly greater than uptake by control cells (24.7 ± 2.2%; P < 0.05) and was a saturable process. The greater [3H]L-arginine uptake by cells from silica-exposed lungs was associated with greater NO and urea production than by control cells. The uptake of [3H]L-arginine by cells from control or silica-exposed lungs was blocked in a dose-dependent manner by L-ornithine (an inhibitor of L-arginine transport) and by Nomega -nitro-L-arginine methyl ester (L-NAME) (an NOS inhibitor), but not by L-valine (an arginase inhibitor). The production of NO by cells from silica-exposed lungs was completely blocked by L-NAME. The addition of L-arginine to media resulted in dose-dependent production of NO and urea. The results show that lung inflammatory cells increase L-arginine uptake and metabolism by both NOS and arginase following in vivo silica exposure. The increase in L-arginine uptake may represent a mechanism to maintain an intracellular supply of this amino acid. NO can react to generate peroxynitrite, a potential mediator of lung injury following silica exposure.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Silica is a naturally occurring mineral oxide dust that exists as a crystal structure of silicon dioxide (SiO2) (1, 2). Silicosis, one of the most prevalent chronic occupational lung diseases in the world, results from the inhalation of silica (3, 4). The inflammatory reaction of the lungs following silica exposure is characterized by an influx of alveolar macrophages (AM) and neutrophils (5, 6). Recently, interest in the pulmonary toxicity of mineral oxide dusts has focused on nitrogen oxides (NOx), and particularly on the role of the nitrogen-based free radical, nitric oxide (NO) (7, 8). The enzyme nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to L-citrulline and NO (9). In addition to its well-described roles in mediating vasodilator tone and as a neurotransmitter, NO may play an important role in both acute and chronic inflammation (9, 10).

Studies suggest that NO may be involved in the inflammatory response in the lungs following exposure to mineral oxide dusts. Exposure of rat AM to asbestos fibers in vitro has been associated with NO production (8). Following the intratracheal instillation of silica into the lungs of rats, lung inflammatory cells isolated by bronchoalveolar lavage showed increased NOS mRNA expression and NO production (7). Furthermore, NO production by lung inflammatory cells following silica instillation is reduced by the systemic administration of steroids (11).

L-arginine is transported across the plasma membrane into cells by a group of amino acid transporters, including systems y+, B0,+, and b0,+. Macrophages have been found to transport L-arginine mainly by system y+ (12). L-ornithine is also transported by system y+, and therefore L-ornithine is used as a competitive inhibitor of L-arginine transport (12). The intracellular metabolism of L-arginine involves both NOS, forming NO and L-citrulline, and arginase, forming urea and L-ornithine (Figure 1). Various types of inflammatory stimuli have been shown to increase both NOS and arginase activity in macrophages (13, 14). In the present study, we tested the hypothesis that rat lung inflammatory cells isolated after the instillation of silica in vivo would demonstrate greater L-arginine uptake and metabolism than inflammatory cells isolated after instillation of saline.


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  		Figure 1.   The L-arginine enzymatic metabolic pathways of interest in these studies. Compounds in italics are competitive inhibitors of the indicated enzymes.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Animals and Instillation of Silica or Saline

Rats were dosed with either silica or saline vehicle as previously described (5, 6). Briefly, Sprague-Dawley rats weighing 351 to 400 g were anesthetized with 30 mg/kg body weight of intraperitoneal pentobarbital, and were intratracheally instilled with either 0.3 ml (50 mg) of silica in 0.9% sterile saline or 0.3 ml of sterile 0.9% saline (control). Following instillation, the rats were returned to the vivarium for recovery. Food and water were available ad libitum. The silica used in the studies was minusil-5 (Pennsylvania Glass and Sand, Pittsburgh, PA).

Isolation and Culture of Lung Inflammatory Cells

Three days after instillation of either silica or sterile 0.9% saline, the rats were killed with intraperitoneal pentobarbital, the lungs and trachea removed en bloc, and the lungs lavaged with ice-cold, sterile, 0.9% phosphate-buffered saline (PBS) (60 ml/rat). Lavage fluid was centrifuged at 400 × g for 10 min, after which the cell pellet was resuspended in cold PBS and the centrifugation repeated. After the second wash, the cells pellet was resuspended in L-arginine-free RPMI 1640 (GIBCO, Grand Island, NY). An aliquot of the cell suspension was used to determine the cell count and differential with a hemocytometer, with 80 µl of 1:3 (vol/vol) trypan blue added. The cells were then washed in RPMI 1640 medium and 106 cells were placed in each of the wells of a 12-well culture plate (4-cm2 wells). The plates were placed in an incubator at 37°C in 95% room air/5% CO2, and the cells were allowed to adhere for 1 h. The wells were then washed with RPMI 1640 medium at 37°C to remove unattached cells, and final culture was done with L-arginine-free RPMI 1640.

Experimental Protocols

In the first series of experiments, inflammatory cells isolated from saline- and silica-exposed lungs were cultured in medium to determine [3H]L-arginine uptake and the metabolism of [3H]L-arginine to [3H]L-ornithine and [3H]L-citrulline, as well as to NO, under five experimental conditions (Figure 1):

  1. No additions to the medium to determine basal 24-h [3H]L-arginine uptake and metabolism to NO (n = 12 silica- and n = 12 saline-exposed rats). In a subset of these experiments (n = 5 silica- and n = 5 saline-exposed rats), [3H]L-arginine uptake was measured at 2 h, 4 h, 8 h, 12 h, and 24 h of culture.
  2. Addition of excess nonradioactive L-arginine to the medium in concentrations of 0.1 µM to 1,000 µM, to determine whether the [3H]L-arginine uptake process was saturable (n = 3 silica- and n = 3 saline-exposed rats per concentration of nonradioactive L-arginine).
  3. Addition of the L-arginine-transport competitive inhibitor L-ornithine to the medium in concentrations of 2 mM (n = 4 silica- and n = 4 saline-exposed rats), 5 mM (n = 3 silica- and n = 3 saline-exposed rats), 20 mM (n = 2 silica- and n = 2 saline-exposed rats), and 40 mM (n = 4 silica- and n = 4 saline-exposed rats) to determine whether the [3H]L-arginine uptake process could be competitively blocked.
  4. Addition of the NOS competitive inhibitor Nomega -nitro- L-arginine methyl ester (L-NAME) to the medium in concentrations of 2 mM (n = 4 silica- and n = 4 saline-exposed rats), 5 mM (n = 3 silica- and n = 3 saline- exposed rats), 20 mM (n = 2 silica- and n = 2 saline- exposed rats), and 40 mM (n = 2 silica- and n = 2 saline-exposed rats) to determine whether L-NAME had an effect on the [3H]L-arginine uptake process.
  5. Addition of the arginase competitive inhibitor L-valine to the medium in concentrations of 2 mM (n = 4 silica- and n = 4 saline-exposed rats), 5 mM (n = 3 silica- and n = 3 saline-exposed rats), 20 mM (n = 2 silica- and n = 2 saline-exposed rats), and 40 mM (n = 3 silica- and n = 3 saline-exposed rats) to determine whether L-valine had an effect on the [3H]L-arginine uptake process (15).

In a separate, second series of experiments, lung inflammatory cells were cultured with the addition of nonradioactive L-arginine to the medium (without [3H]L-arginine) in concentrations of 0.1 µM to 1,000 µM to determine a dose response at 24 h for the metabolism of L-arginine to urea (n = 3 silica- and n = 3 saline-exposed rats) and NO (n = 4 silica- and n = 4 saline-exposed rats).

[3H]L-arginine Uptake by Lung Inflammatory Cells

The cells were washed with L-arginine-free RPMI 1640 and were then placed in triplicate in 1 ml L-arginine-free RPMI 1640, containing 1 µCi of [3H]L-arginine (L-[2,3,4,5- [3H]arginine monohydrochloride (specific activity: 59 Ci/ mmol; Amersham International, Amersham, UK); the final concentration of [3H]L-arginine was 17 nM. The plates were placed in an incubator at 37°C in 95% room air/5% CO2. After 24 h, two 100-µl aliquots of supernatant were removed from each well and placed directly into 5 ml of scintillation-counting cocktail, after which the mixture was placed in a liquid-scintillation counter. To determine the amount of [3H]L-arginine in the medium, two 100-µl aliquots of the medium were placed directly into scintillation-counting cocktail, and the mixture was placed in a liquid-scintillation counter. The uptake of [3H]L-arginine was determined from the amount of [3H]L-arginine added, the volume of medium at 24 h, and the corrected counts/min at 24 h. The amount of [3H]L-arginine uptake by the inflammatory cells was determined from the ratio of [3H] in L-arginine to that in its metabolites, L-ornithine and L-citrulline, as determined by thin-layer chromatography (TLC) as subsequently described.

[3H]L-arginine Metabolism by Lung Inflammatory Cells

The metabolism of L-[2,3,4,5-3H]arginine monohydrochloride to L-ornithine and L-citrulline can be quantitated by measuring the appearance of [3H] in L-ornithine and L-citrulline through TLC. A sample of medium from each [3H]L-arginine-uptake experiment (40 µl) was streaked directly on a TLC plate (Silica Gel 60 F254; EM Science, Darmstadt, Germany), which was exposed to a mobile phase consisting of chloroform/methanol/ammonium hydroxide/ water (9:9:4:1 by volume). The Rf values with this system are 0.38 for L-arginine, 0.63 for L-ornithine, and 0.88 for L-citrulline. The TLC plates were scraped in 1-cm bands into liquid-scintillation vials, to which 10 ml of scintillation fluid was added, and the counts were measured in the liquid-scintillation counter.

Nitrite Production by Lung Inflammatory Cells

Nitrite (NO2-) is the stable breakdown product of NO in aerobic aqueous solutions such as culture media (16). The measurement of NO2- is used to quantify NO production in culture media of inflammatory cells (16). We measured NO2- in inflammatory cell culture supernatants from media of silica- and saline-exposed lungs in duplicate at 24 h of culture, using a chemiluminescence NO analyzer (Model 270B; Sievers, Boulder, CO). For this measurement, 100 µl of supernatant was injected into a reaction chamber containing a mixture of NaI in acetic acid to reduce NO2- to NO, allowing the liberation of the NO from the aqueous mixture. The NO was carried into the analyzer with a constant flow of N2. The analyzer was calibrated with a NaNO2 standard curve. In the first series of experiments, the supernatants from the studies of uptake of [3H]L-arginine (described previously) were assayed for NO2-. For the separate, second series of experiments, inflammatory cells were incubated with 0.1 µM to 1,000 µM nonradioactive L-arginine added to media (without [3H]L-arginine) to determine the dose-response effect of L-arginine on NO2- production at 24 h, and NO2- from the media was assayed in an identical manner.

Urea Production by Lung Inflammatory Cells

In another set of experiments, done to determine the dose-response effect of L-arginine on urea production, inflammatory cells from silica- and saline-exposed lungs were incubated with the addition of 0.1 µM to 1,000 µM nonradioactive L-arginine to media (without [3H]L-arginine), and urea in the media was measured in duplicate at 24 h of culture, using a colorimetric assay (19). For this measurement, a 100 µl sample of medium was added to 3 ml of chromogenic reagent (5 mg thiosemicarbazide, 250 mg diacetyl monoxime, and 37.5 mg FeCl3 in 150 ml 25% (vol/vol) H2SO4 and 20% (vol/vol) H3PO4. The absorbance (530 nm) was determined with a spectrophotometer and compared with a standard curve prepared with urea (Sigma Chemicals, St. Louis, MO)

Statistics

All data are expressed as means ± SEM. One-way analysis of variance (ANOVA) was used to compare differences between groups. A Newman-Keuls post hoc test was used to identify significant differences between groups. Differences were considered significant when P < 0.05.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Inflammation Following Intratracheal Instillation of Silica or Saline

Silica exposure resulted in greater lung inflammation, as reflected by differential analysis of inflammatory cell populations from lung lavage fluid, which revealed significantly more neutrophils from silica-exposed lungs (n = 12 silica- and n = 12 saline-exposed rats) (Table 1) (P < 0.01). In addition, following instillation of silica, lung inflammation was manifested by an accumulation of inflammatory cells within the airways and alveolar spaces, as demonstrated histopathologically (n = 5) (Figure 2). Saline-exposed lungs were normal (n = 5).

                              
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TABLE 1
Lung lavage fluid characteristics


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  		Figure 2.   Photomicrographs of rat lungs exposed to either saline or silica in vivo for 3 d. (top panel ) Saline-exposed lungs showing normal histologic findings. (Original magnification: ×160.) (bottom panel ) Silica-exposed lungs showing numerous confluent nodules consisting of inflammatory cells. (Original magnification ×160).

Uptake of [3H]L-arginine by Lung Inflammatory Cells

Table 2 reveals that there was significantly more basal [3H]L-arginine uptake at 24 h by inflammatory cells from silica-exposed lungs than by cells from saline-exposed lungs (P < 0.02). Figure 3 shows that the [3H]L-arginine uptake was essentially linear for 24 h for cells from saline-exposed lungs and for 12 h for cells from silica-exposed lungs. The uptake of [3H]L-arginine by inflammatory cells from silica-exposed lungs was a saturable process (Figure 4). The inflammatory cells from silica-exposed lungs had an increased [3H]L-arginine uptake, and the saturation curve was shifted to the right as compared with that for cells from saline-exposed lungs. This suggests that the [3H]L-arginine uptake involved a cellular transport process.

                              
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TABLE 2
[3H]L-arginine uptake and metabolism at 24 h of culture


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  		Figure 3.   [3H]L-arginine uptake by lung inflammatory cells from saline- and silica-exposed lungs over 24 h. [3H]L-arginine uptake was essentially linear for 24 h for cells from saline-exposed lungs and for 12 h for cells from silica-exposed lungs. *Silica-exposed significantly different from saline-exposed at same time point (P < 0.05).


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  		Figure 4.   Inhibition of [3H]L-arginine uptake by lung inflammatory cells from saline- and silica-exposed lungs by increasing concentrations of nonradioactive L-arginine. The y axis is the percent of basal control [3H]L-arginine uptake (i.e., the uptake by saline-exposed cells without nonradioactive L-arginine added to the medium). The x axis is the concentration of nonradioactive L-arginine added to the medium. *Significantly different from same exposure at 0.1 µM nonradioactive L-arginine (P < 0.05); +silica-exposed significantly different from saline-exposed at same concentration of nonradioactive L-arginine (P < 0.05).

Further evidence for the involvement of a transport process in [3H]L-arginine uptake comes from the inhibition of [3H]L-arginine uptake by L-ornithine (Figure 5). The inhibition curve was shifted to the right for cells from silica-exposed lungs compared with cells from saline-exposed lungs. Similarly, [3H]L-arginine uptake was inhibited by L-NAME, with a shift to the right for the cells from silica-exposed lungs compared with cells from saline-exposed lungs (Figure 6). The addition of L-valine to the medium had no effect on [3H]L-arginine uptake by cells from either saline- or silica-exposed lungs.


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  		Figure 5.   Inhibition of [3H]L-arginine uptake by lung inflammatory cells from and saline- and silica-exposed lungs by four different concentrations of L-ornithine. The y axis is the percent of basal [3H]L-arginine uptake (i.e., the uptake without L-ornithine added to the medium).


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  		Figure 6.   Inhibition of [3H]L-arginine uptake by lung inflammatory cells from saline- and silica-exposed lungs by four different concentrations of L-NAME. The y axis is the percent of basal [3H]L-arginine uptake (i.e., the uptake without L-NAME added to the medium).

[3H]L-arginine Metabolism by Lung Inflammatory Cells

There was conversion of [3H]L-arginine to both [3H]L-citrulline and [3H]L-ornithine by the inflammatory cells from both saline- and silica-exposed lungs (Table 2). Table 2 reveals that there was significantly more [3H] appearing as [3H]L-ornithine and [3H]L-citrulline in media from inflammatory cells of silica-exposed lungs at 24 h than in media from cells of saline-exposed lungs, in accord with an increase in L-arginine metabolism by NOS and arginase (P < 0.02).

NO2- Production by Lung Inflammatory Cells

Inflammatory cells from saline-exposed lungs showed little basal NO2- production at 24 h, whereas inflammatory cells from silica-exposed lungs showed significantly greater production of NO2- at 24 h (P < 0.02) (Table 2). The addition of 2, 5, or 20 mM L-ornithine had no effect on NO2- production at 24 h by cells from silica-exposed lungs. The addition of 40 mM L-ornithine decreased NO2- production by 67%. The addition of 2, 5, 20, or 40 mM L-NAME completely blocked NO2- production by cells from silica-exposed lungs. The addition of 2, 5, 20, or 40 mM L-valine to the medium had no effect on NO2- production at 24 h by cells from silica-exposed lungs. The addition of L-arginine to culture media increased NO2- production in a dose-dependent manner by inflammatory cells from both saline- and silica-exposed lungs. At each of the three highest concentrations of L-arginine added to media, cells from silica-exposed, compared with saline-exposed lungs, produced significantly greater amounts of nitrite (P < 0.05) (Figure 7).


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  		Figure 7.   Nitrite production in medium by lung inflammatory cells from saline- and silica-exposed lungs cultured in increasing amounts of nonradioactive L-arginine. The addition of increasing amounts of L-arginine resulted in a dose-dependent increase in production of nitrite. *Significantly different from saline-exposed at same concentration of L-arginine (P < 0.05).

Urea Production by Lung Inflammatory Cells

The addition of L-arginine to culture media increased urea production in a dose-dependent manner by inflammatory cells from both saline- and silica-exposed lungs. At each of the three highest concentrations of L-arginine added to media, cells from silica-exposed as compared with saline-exposed lungs produced significantly greater amounts of urea (P < 0.05) (Figure 8).


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  		Figure 8.   Urea production in medium by lung inflammatory cells from saline- and silica-exposed lungs cultured in increasing amounts of nonradioactive L-arginine. The addition of increasing amounts of L-arginine resulted in a dose-dependent increase in production of urea. *Significantly different from saline-exposed at same concentration of L-arginine (P < 0.05).

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In the present study we found that the uptake of extracellular L-arginine was significantly greater by inflammatory cells isolated from lungs exposed to silica in vivo than by inflammatory cells isolated from lungs of control rats. The difference in L-arginine uptake by the silica-exposed inflammatory cells corresponded to greater L-arginine metabolism by both arginase and NOS by the cells from silica-exposed lungs than by the cells from control rats. To the best of our knowledge, this is the first demonstration linking L-arginine uptake with L-arginine metabolism following the exposure of lungs to a mineral oxide dust in vivo. These findings suggest that L-arginine uptake and metabolism is increased in lung inflammatory cells following silica exposure.

The role of L-arginine uptake and metabolism in lung injury following exposure to silica or silicate mineral oxide dusts remains unclear. However, numerous mechanisms have been proposed to account for the development of lung inflammation and fibrosis following exposure to silica. These include the release of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1 (IL-1), and prostaglandins such as prostaglandin E2 (PGE2) (20). In addition, it is believed that silica may cause pulmonary toxicity through the production of highly reactive oxygen-based free radicals (5, 21, 22). NO is a nitrogen-based free radical that is formed from L-arginine by the closely related group of NOS enzymes (23). There is evidence that NO exerts proinflammatory effects including vasodilatation, edema, and cytotoxicity, and can contribute to tissue injury (24, 25). Despite the emphasis on NO as a mediator of deleterious effects in different tissues, it has been suggested that NO also has beneficial properties, such as inhibition of leukocyte-endothelial cell adhesion and inhibition of platelet adhesion and vascular thrombosis (26, 27).

A link between NO and lung injury from silica and silicate mineral oxide dusts has been suggested by recent studies. Rat AM exposed to asbestos in vitro generate NO (8). Following in vivo exposure to silica, rat lung inflammatory cells demonstrate NOS mRNA expression and NO production (7). It has been suggested that the role of NO formation in response to silica is related to the ability of NO to combine rapidly with O2- to form peroxynitrite (ONOO-), a potent oxidant capable of oxidizing an array of biomolecules (28, 29). Numerous mechanisms have been proposed to explain the oxidative ability of ONOO-, including direct oxidation by ONOO- or through the formation of peroxynitrous acid (ONOOH) (26, 28, 29). In addition, it has been suggested that oxidation by ONOO- could be explained by the decomposition of ONOO- to the cytotoxic hydroxyl radical (OH·) (30). OH· formation has been demonstrated in rat lungs following silica exposure in vivo (5). However, recent evidence suggests that the formation of OH· from ONOO- may not be biologically relevant (29). The production of NO in the lungs by lung inflammatory cells following silica exposure might represent a pathway leading to ONOO- production and lung injury. Following silica instillation in vivo, rat lung tissue and lung inflammatory cells demonstrate peroxynitrite-dependent chemiluminescence, which is inhibited by steroids (11).

In the present study, inflammatory cells exposed to silica in vivo showed an increase in L-arginine metabolism not only by NOS, but also by arginase. The role of arginase in inflammation is not clearly understood. It has been postulated that in macrophages the arginase pathway may play a regulatory role in NO synthesis by competing for substrate and thus limiting the availability of L-arginine to NOS (13, 33, 34). On the other hand, an in vitro study found that Nomega -hydroxyl-L-arginine, an intermediate in the five-electron oxidation of L-arginine to L-citrulline and NO, was a potent inhibitor of bovine liver arginase, leading the investigators to speculate that the inhibition might decrease competition between the two enzymes for L-arginine and thus increase the availability of L-arginine to NOS (35).

The increase in both NOS and arginase activity by lung inflammatory cells following silica exposure is consistent with finding in studies of L-arginine metabolism by other biologic systems. For example, coronary artery endothelial cells of diabetic rats have impaired L-arginine metabolism, involving both the NOS and arginase pathways, compared with endothelium from nondiabetic rats (15). Arginase and NOS activity of mesangial cells and macrophages isolated from glomeruli increase in a rat model of acute glomerulonephritis (19). The treatment of murine AM with lipopolysaccharide (LPS) induces both arginase and NOS activity (36). In addition, temporal variations in the metabolism of L-arginine by NOS and arginase of peritoneal macrophages may play a role during the phases of wound healing (37). Thus, it appears that the cellular metabolism of L-arginine by the competing NOS and arginase pathways can be altered under different conditions.

In the present study, the instillation of silica into rat lungs in vivo was associated with an inflammatory response composed of AM and neutrophils, which was consistent with findings in similar studies (5, 7, 38). Although our study did not specifically compare the relative uptake and metabolism of L-arginine by AM and neutrophils following silica instillation, a previous study found that after silica exposure, both rat AM and neutrophils show increased steady-state levels of mRNA for inducible NOS (iNOS) (7). Our results demonstrate that the lung inflammatory cells from silica-exposed lungs had significantly greater uptake of [3H]L-arginine and greater NO production than did cells from saline-exposed lungs. This observation is consistent with the finding that L-arginine transport into cells is increased when inflammatory cells such as macrophages are activated, providing a supply of L-arginine to the NOS and arginase metabolic pathways (12, 39). Furthermore, a link has been demonstrated in brain astrocytes between NOS activity and genetic regulation of the L-arginine transporter system y+ when these cells are activated by LPS/interferon-gamma (40).

In our studies, the uptake of [3H]L-arginine was blocked by nonradioactive L-arginine and L-ornithine. The blockage of [3H]L-arginine uptake demonstrates that the uptake of [3H]L-arginine by the inflammatory cells is effected by an amino acid transport system that is saturable. Indeed, the transport of L-arginine into cells has been found to be blocked by certain amino acids, such as L-ornithine (12). We also found that L-NAME decreased both NO production and [3H]L-arginine uptake by lung inflammatory cells isolated from silica-exposed lungs. The effect of L-NAME on [3H]L-arginine uptake suggests that L-NAME is transported by the same transporter as L-arginine or that NOS activity is involved in the regulation of L-arginine uptake. However, it has been shown that in endothelial cells, L-NAME has no effect on L-arginine uptake, and that L-NAME is taken up by the neutral amino acid transporter, system L (41, 42). Although further study is needed, the effect of L-NAME on [3H]L-arginine uptake suggests that either inflammatory cells differ from endothelial cells in their uptake mechanisms for L-arginine, or that NOS activity by lung inflammatory cells influences the systems that transport L-arginine (40).

In summary, we have established that rat-lung inflammatory cells isolated after the intratracheal instillation of silica in vivo show greater uptake of L-arginine than do cells isolated from saline-exposed lungs. In addition, inflammatory cells from silica-exposed lungs show greater metabolism of L-arginine by both the arginase and NOS metabolic pathways than do cells from saline-exposed lungs. The increase in uptake by inflammatory cells following silica exposure may augment the L-arginine supply to intracellular metabolic pathways. NO produced as a result of L-arginine metabolism by the NOS pathway has the ability to form ONOO-, which may be responsible for lung injury following silica exposure.

    Footnotes

Address correspondence to: Ralph M. Schapira, M.D., Section of Pulmonary/Critical Care Medicine (CC-111-E), Clement J. Zablocki Veterans Affairs Medical Center, 5000 W. National Avenue, Milwaukee, WI 53295-1000. E-mail: rschap{at}post.its.mcw.edu

(Received in original form October 16, 1996 and in revised form November 24, 1997).

Acknowledgments: The authors thank Andrew J. Ghio, M.D., for technical advice and for providing the silica for this investigation. The authors also thank Carol J. Thomas and Linda Rehorst-Paea for performing the nitrite and urea assays and Helen Roddy for manuscript preparation. This work was supported by Department of Veterans Affairs Merit Review No. 7731-05, Cancer Center, Medical College of Wisconsin Grant No. 3-35914 and by the American Lung Association of Wisconsin. This work was done during the tenure of a clinician-scientist award from the American Heart Association to Dr. L. Nelin.

Abbreviations AM, alveolar macrophage; ANOVA, analysis of variance; IC50, concentration that inhibits 50%; IL-1, interleukin-1; L-NAME, Nomega -nitro-L-arginine methyl ester; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthase; NOx, nitrogen oxides; O2-, superoxide anion; OH-, hydroxyl radical; ONOO-, peroxynitrite anion; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; TLC, thin-layer chromatography; TNF-alpha , tumor necrosis factor-alpha .

    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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