1Vascular Physiology Group and Department of Pediatrics, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; and 2Center for Developmental Pharmacology and Toxicology, Columbus Children's Research Institute, Department of Pediatrics, The Ohio State University, Columbus, Ohio 43205
Submitted 18 June 2003 ; accepted in final form 18 February 2004
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ABSTRACT |
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lipopolysaccharide; tumor necrosis factor-; nitric oxide synthase; L-arginine; L-valine
Given that NOS and AR share a common substrate, inhibition of one enzyme may augment the activity of the other enzyme. We have previously found that in bPAEC urea production is 20100 times greater than NO production (19). We hypothesized that inhibiting AR activity in bPAEC, particularly in L/T-treated bPAEC, will increase NO production by increasing L-Arg bioavailability to NOS. If this hypothesis is correct, then this would also demonstrate that NOS and AR compete for a common intracellular pool of L-Arg. To test this hypothesis, we determined the AR isoform(s) induced by L/T treatment in bPAEC. We measured NO and urea production by bPAEC incubated with various concentrations of the nonspecific AR inhibitor L-valine (L-Val). We then measured NO and urea production by bPAEC incubated with either no additives, L-Val, L-Arg, or both L-Val and L-Arg. Finally, since we have previously demonstrated that urea production by bPAEC depends on extracellular L-Arg (19), we excluded a nonspecific effect of L-Val on L-Arg uptake by measuring [3H]L-Arg uptake in the presence of L-Val.
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METHODS |
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bPAEC were cultured as previously described (19). Briefly, bPAEC were obtained from Clonetics (San Diego, CA). Upon arrival bPAEC were placed in T-25 flasks with 5 ml of endothelial growth media (EGM, Clonetics), which contains 250 µM L-Arg. When the bPAEC were 8090% confluent, the bPAEC were passaged with trypsin-EDTA followed by trypsin-neutralizing solution. The bPAEC were centrifuged at 1,200 g for 5 min, and the bPAEC pellet was resuspended in EGM. Nine milliliters of EGM were placed in a T-75 flask, and then 1 ml of the resuspended bPAEC pellet was added, and the T-75 flask was returned to the incubator at 37°C in 5% CO2-balance air. bPAEC between passages 3 and 8 were used for these studies.
On the day of study, the bPAEC were washed three times with 4 ml of HEPES balanced salt solution (HBSS, Clonetics). Then 5 ml of EGM were placed on the bPAEC (control), and the bPAEC were returned to the incubator at 37°C in 5% CO2-balance air for 24 h. In the L/T-treated bPAEC, 0.5 µg/ml LPS (Sigma Chemical, St. Louis, MO) and 0.5 ng/ml TNF- (Sigma Chemical) were included in the EGM as previously described (18). After 24 h, the medium was removed, stored in 1-ml aliquots, and frozen at 70°C. The bPAEC were washed three times with 4 ml of HBSS and treated with either lysis buffer for protein extraction or TRIzol (Life Technologies) for RNA isolation.
bPAEC Protein Isolation
Protein was isolated from the bPAEC as previously described (19). Briefly, bPAEC were washed with HBSS, and 750 µl of lysis buffer (0.2 M NaOH, 0.2% SDS) were added to each flask. Thirty minutes before use, the following protease inhibitors were added to each milliliter of lysis buffer: 0.2 µl aprotinin [10 mg/ml double distilled (dd) H2O], 0.5 µl leupeptin (10 mg/ml ddH2O), 0.14 µl pepstatin A (5 mg/ml methanol), and 5 µl of phenylmethylsulfonyl fluoride (34.8 mg/ml methanol). This was sterile filtered in a syringe and added to each T-75 flask of bPAEC. The bPAEC were scraped and placed in sterile centrifuge tubes on ice. The supernatant was stored in 1-ml tubes at 70°C for Western blot analysis. Total protein concentration was determined by the Bradford method with a commercially available assay (Bio-Rad, Hercules, CA).
bPAEC RNA Isolation
RNA was isolated from the bPAEC as previously described (19). Briefly, 1 ml of TRIzol (Life Technologies) was added to the flask containing the bPAEC and incubated for 5 min at room temperature. Chloroform (0.2 ml) was added, and the tubes were shaken for 15 s and then incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was transferred to a fresh 15-ml tube. Isopropyl alcohol (0.5 ml) was added, and the mixture was incubated at room temperature for 10 min and then centrifuged at 12,000 g for 15 min at 4°C. The supernatant was discarded, and the pellet was washed with 75% ethanol and centrifuged at 7,500 g for 5 min at 4°C. The supernatant was discarded, and the pellet was partially dried, dissolved in RNAse-free water, and stored at 70°C.
Nitrite Assay
The EGM samples were assayed in duplicate for nitrite (NO2) with a chemiluminescence NO analyzer (model 270B; Sievers Instruments, Boulder, CO) as previously described (19, 23). Briefly, 100 µl of sample were placed in a reaction chamber containing a mixture of NaI in glacial acetic acid to reduce NO2 to NO. The NO gas was carried into the NO analyzer by a constant flow of He gas. The analyzer was calibrated with an NaNO2 standard curve.
Nitrite/Nitrate Assay
EGM samples were assayed in duplicate for nitrite/nitrate (NOX) using a chemiluminescence NO analyzer (model 280, Sievers Instruments) as previously described (18). Briefly, 100 µl of sample were injected into a reaction chamber containing a mixture of vanadium (III) chloride in 2 M HCl heated to 90°C to reduce NOX to NO gas. The NO gas was carried into the analyzer by a constant flow of He gas. The analyzer was calibrated with an NaNO3 standard curve.
Urea Assay
The EGM samples were assayed in duplicate for urea colorimeterically as previously described (18, 19, 23). Briefly, 100 µl of sample were 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] or the same reagents with 0.5 units of urease added. After 1 h at 37°C, the mixtures were vortexed and then boiled at 100°C for 5 min. The mixtures were cooled to room temperature, and the difference in absorbance (530 nm) with and without urease was determined and compared with a urea standard curve.
[3H]L-Arg Uptake
After a 24-h incubation in either EGM or L/T, the bPAEC were washed three times with HBSS, and [3H]L-Arg uptake was measured as previously described (19). Briefly, 4 ml of HBSS with 1 µCi/ml of [3H]L-Arg were placed on the bPAEC in the T-75 flask. Two 100-µl samples of the [3H]L-Arg/HBSS were placed in scintillation counting cocktail and placed in a scintillation counter. After 10 min, the [3H]L-Arg was removed, and the bPAEC were washed three times with ice-cold HBSS. Lysis buffer (300 µl) was added to the bPAEC in the T-75 flask and incubated at room temperature overnight. Then 100-µl samples of lysed bPAEC sample were placed in the scintillation counting cocktail and placed in a scintillation counter.
Western Blotting
The lysed bPAEC were assayed for ARI protein by Western blot analysis as previously described (18, 19). Aliquots of cell lysate were diluted 1:1 with SDS sample buffer, heated to 80°C for 15 min, and then centrifuged at 10,000 g at room temperature for 2 min. Aliquots of the supernatant were used for SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and blocked overnight in phosphate-buffered saline with 0.1% Tween (PBS-T) containing 5% nonfat dried milk and 3% albumin. The membranes were then incubated with the primary antibody ARI (1:1,000; Transduction Laboratories) for 4 h and then washed with PBS-T with 1% nonfat dried milk. The membranes were then incubated with the biotinylated IgG secondary antibody (1:5,000; Vector Laboratories) for 1 h, washed, and then incubated with streptavidin-horseradish peroxidase conjugate (1:1,500; Bio-Rad) for 30 min. The bands for ARI were visualized by chemiluminescence (Amersham ECL) and quantified by densitometry (Sigma Gel, Jandel Scientific). Authentic ARI (Transduction Laboratories) was used as a positive control. To control for protein loading, the blots were then stripped with a stripping buffer (each 100 ml contained 6.25 ml 1 M Tris·HCl, pH 6.8, 20 ml 10% SDS, 0.7 ml 2-mercaptoethanol, and 73 ml ddH2O). The blots were reprobed for -actin (1:10,000; Abcam) as described above.
RT-PCR
RT-PCR was performed as previously described (18, 19). Briefly, 2 µg of total RNA were reversed transcribed in 2.5 µM dT16 (Applied Biosystems), 20 units AMV-RT, 1 mM dNTP, 1x buffer (Promega), and balance RNase-free water, with a total volume of 40 µl. The samples were incubated in a PCR-iCycler (Bio-Rad) at 42°C for 60 min, 95°C for 5 min, and stored at 20°C. Multiplex PCR for the expression of the ARII gene was internally standardized by direct comparison to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in the same reaction. PCR reactions (total volume of 50 µl) contained 5 µl of RT product, 1 mM MgCl2, 1.25 units AmpliTaqGold (Applied Biosystems), 0.2 mM dNTP (Promega), and 15 µM forward (5'-TTGTGTTGATCTGGGTTGATGC-3') and reverse (5'-TGCCTTCTCGATAGGTCAGTCC-3') primers for ARII. The mixed samples were heated to 94°C for 4 min and then cycled as follows: 94°C for 1 min, 53°C for 1 min, and 72°C for 2 min for 17 cycles, and then 15 µM of forward (5'-GAAGACTGTGGATGGCCCCTCC-3') and reverse (5'-GTTGAGGGCAATGCCAGCCCC-3') primers for GAPDH were added to each sample and allowed to run for an additional 18 cycles. The PCR products were visualized and sized by 2.0% agarose gel electrophoresis and poststained with Syber Gold (Molecular Probes) for 30 min. The gels were scanned with a MultiGenius Bio Imaging System (Syngene), and band density analysis was performed on a personal computer with SigmaGel (Jandel Scientific) software. The PCR product sizes were the expected 422 and 356 bp for ARII and GAPDH, respectively. Preliminary PCR reactions run at various total cycle numbers between 20 and 45 demonstrated that 35 total cycles was well within the linear range for each reaction product.
Experimental Protocols
Time-dependent NO and urea production. We have previously shown that L/T treatment of bPAEC increased both NO and urea production (19). Therefore in the first set of experiments, we determined the 24-h time course of NO2 and urea production in bPAEC. Immediately after placing 5 ml of EGM or EGM containing L/T on bPAEC, we removed a 0.5-ml sample of the medium and placed the control (n = 6) and L/T (n = 6) bPAEC in an incubator. At 2, 4, 6, 8, and 24 h, 0.5-ml samples of medium were removed; after the 24-h sample was removed, protein was isolated as described above. The samples were assayed for NO2 and urea production.
NO2 vs. NOX in medium. To determine the concentrations of NO2 and NOX in the medium from bPAEC we did the following study. Immediately after placing 5 ml of EGM containing L/T on bPAEC (n = 3), we obtained a 0.5-ml sample of medium and placed the bPAEC in an incubator. At 6 and 24 h, 0.5-ml samples of medium were removed. The samples were assayed for NO2 and NOX concentrations as described above.
Effect of L/T on ARI and ARII expression. In these experiments, the effect of L/T treatment on ARI protein expression (n = 3 in each group) and ARII mRNA expression (n = 8 in each group) was determined. Control and L/T bPAEC were incubated for 24 h and washed, and either the protein was extracted or the RNA was extracted as described above. We used a commercially available antibody against ARI (Transduction Laboratories). There is no commercially available antibody directed against ARII at this time; therefore, we determined changes in ARII mRNA expression.
Concentration-dependent effect of L-Val on NO production by bPAEC. In these experiments the effect of increasing concentrations of L-Val on NO and urea production was determined in control (n = 4 for each concentration) and L/T-treated bPAEC (n = 4 for each concentration). Control and L/T bPAEC were incubated for 24 h in EGM containing 1 mM L-Arg and 3, 10, 30, or 100 mM L-Val added to the medium. We chose a concentration of 1 mM L-Arg to approximate plasma L-Arg concentration (6, 13). The L-Val concentrations were chosen given that L-Val is a competitive inhibitor of AR. The medium was collected and assayed for NO2 and urea production.
To exclude an effect of L-Val on the L/T-induced expression of NOS, we studied control bPAEC and L/T bPAEC after a 2-h incubation, a time point chosen to eliminate L/T-induced increases in NO2 or urea production. BPAEC were incubated for 2 h in EGM (n = 3), L/T (n = 3), or L/T containing 30 mM L-Val (n = 3). The medium was collected and assayed for NO2 and urea production, and protein was isolated as described above.
Effect of L-Val, L-Arg, or both L-Arg and L-Val on NO production by bPAEC. In these experiments the effect of vehicle, 30 mM L-Val, 10 mM L-Arg, or 30 mM L-Val + 10 mM L-Arg on NO and urea production was determined in control (n = 10 for each treatment) and L/T (n = 10 for each treatment) bPAEC. bPAEC were incubated for 24 h as described above with vehicle, 30 mM L-Val, 10 mM L-Arg, or both 30 mM L-Val and 10 mM L-Arg added to the medium. The medium was collected and assayed for NO2 and urea production. These experiments were repeated (n = 5 for each treatment and group) with another competitive inhibitor of AR, L-norvaline.
Effect of L-Val on L-Arg uptake by bPAEC. We have previously shown that L/T increases L-Arg uptake in bPAEC and that both urea and NO production depend on L-Arg uptake (19). Thus to determine whether L-Val had any effect on L-Arg uptake, we did the following studies. First, the effect of inhibiting L-Arg uptake on urea production was determined. Two sets of L/T bPAEC (n = 4 for each L-Arg concentration) were incubated for 24 h with 0.3, 1, 3, or 10 mM L-Arg added to the medium. In one set, 30 mM L-leucine (a competitive inhibitor of transporter-dependent L-Arg uptake) was included in the medium. In the second set, the vehicle for L-leucine was included in the medium. The medium was collected and assayed for urea production. In a separate set of experiments after a 24-h incubation with L/T, the uptake of [3H]L-Arg was measured as described above, in the presence of vehicle, 30 mM L-Val, 10 mM L-Arg, or both 30 mM L-Val and 10 mM L-Arg (n = 3 for each group) added to the medium.
Statistical Analysis
Values are means ± SE. One-way analysis of variance was used to compare the densitometry data between control and L/T bPAEC and to compare the effect of the additives on either NO2 or urea production. Significant differences were identified by a Newman-Keuls post hoc test. Differences were considered significant when P < 0.05.
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RESULTS |
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There was a time-dependent increase in NO2 production in both control and L/T-treated bPAEC (Fig. 1A). There was no difference in NO2 production in control or L/T-treated bPAEC at 2 h of incubation; however, by 4 h of incubation with L/T, there was greater NO2 production than in control bPAEC. Although, the 24-h time course of NO2 production was not exactly linear, there was continued NO2 production throughout the 24-h incubation, such that a straight line could be fit to the data (Fig. 1A, r = 0.991 for control and r = 0.989 for L/T bPAEC). The slope of the regression line approximated the NO2 production rate in these bPAEC, and the NO2 production rate was 0.19 nmol·mg protein1·h1 in control bPAEC and
0.32 nmol·mg protein1·h1 in L/T (different from control, P < 0.05). There were no significant differences in the y-intercepts (0.27 for control bPAEC and 0.44 for L/T bPAEC). There was also a time-dependent increase in urea production in both control and L/T-treated bPAEC (Fig. 1B). There was no difference in urea production in control or L/T-treated bPAEC at 2 or 4 h of incubation; however, by 6 h of incubation with L/T there was greater urea production than in control bPAEC. Although the 24-h time course of urea production may not be exactly linear, there was continued urea production throughout the 24-h incubation, such that a straight line could be fit to the data (Fig. 1B, r = 0.99 for control and r = 0.99 for L/T bPAEC). The slope of the regression line approximated the urea production rate in these bPAEC, and the urea production rate was
21 nmol·mg protein1·h1 in control bPAEC and
46 nmol·mg protein1·h1 in L/T (different from control, P < 0.02). There were no significant differences in the y-intercepts (7 for control bPAEC and 5 for L/T bPAEC). Because the increase in NO2 production occurred at
4 h and was maintained for 24 h, and the increase in urea production occurred at
6 h and was maintained for 24 h, therefore, 24-hincubations were employed in the remainder of the experiments.
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Because NO can be oxidized to NO2 and nitrate (NO3), we measured both NO2 and NOX (nitrites and nitrates) concentrations in bPAEC treated with L/T and incubated for 0, 6, and 24 h. We found that both NO2 and NOX concentrations increased with time (Fig. 2). The increase in NO2 concentration was 1.33 ± 0.09 µM, or about sevenfold. However, at time 0 there was a large NOX concentration (3.08 ± 0.60 µM) in the medium employed in these studies, and therefore although the increase in NOX concentration was 3.39 ± 0.85 µM, it was only an approximate doubling of the concentration at time 0. The NO2 concentration was 25% of the total NOX measured at 24 h. In these bPAEC NO2 represented a significant portion of total oxidation products of NO and was a more sensitive measure of NO production. Therefore, to improve the sensitivity of our measurements for NO production, we measured NO2 production (nmol/24 h) in the remainder of the experiments.
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Consistent with our previous study (19), the treatment of bPAEC with L/T resulted in significantly greater NO2 production than in control bPAEC (2.05 ± 0.13 nmol/24-h L/T vs. 1.60 ± 0.09 nmol/24-h control, n = 10, P < 0.01) and a significantly greater urea production than in control bPAEC (161 ± 30 nmol/24-h L/T vs. 43 ± 20 nmol/24-h control, n = 10, P < 0.01). The greater urea production in the L/T bPAEC was associated with a significantly greater ARI protein expression in L/T-treated bPAEC than in control bPAEC (Fig. 3). The expression of ARII mRNA was also significantly greater in L/T-treated bPAEC than in control bPAEC (Fig. 4). Thus L/T increased the expression of both ARI and ARII in these bPAEC.
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As expected, the addition of L-Val to the incubation medium of control bPAEC resulted in inhibition of urea production, with 10, 30, and 100 mM L-Val resulting in undetectable urea production (Fig. 5A). The inhibition of urea production in the control bPAEC was associated with a concentration-dependent increase in NO2 production (Fig. 5A). The increase in NO2 production was seen even though urea production was undetectable; this is probably due to the chemiluminescence NO2 assay being more sensitive than the colorimetric urea assay. In L/T-treated bPAEC, the urea production with 3 mM L-Val added to the incubation medium was significantly greater than in control bPAEC (142 ± 31 nmol/24-h L/T vs. 9.5 ± 8.0 nmol/24-h control, P < 0.05). In the L/T-treated bPAEC, L-Val caused a concentration-dependent inhibition of urea production with 100 mM L-Val resulting in undetectable urea production (Fig. 5B). This inhibition of urea production in L/T-treated bPAEC was associated with a concentration-dependent increase in NO2 production (Fig. 5B).
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Effect of L-Val, L-Arg, or both L-Arg and L-Val on NO Production by bPAEC
In control bPAEC, as expected, the addition of 30 mM L-Val to the incubation medium resulted in significantly less urea production than in vehicle-treated bPAEC (Fig. 6A). The decrease in urea production was associated with a significantly greater NO2 production (Fig. 6B). The addition of 10 mM L-Arg to the incubation medium resulted in significantly greater urea and NO2 production than in vehicle-treated bPAEC (Fig. 6, A and B). The addition of both 30 mM L-Val and 10 mM L-Arg resulted in significantly less urea production than the addition of 10 mM L-Arg alone (Fig. 6A), whereas the NO2 production was significantly more than with the addition of 30 mM L-Val alone (Fig. 6B). In the L/T-treated bPAEC a similar pattern of change in urea (Fig. 7A) and NO2 production (Fig. 7B) was seen, although the NO2 and urea production were greater than in control bPAEC. Substituting L-norvaline for L-Val in these experiments led to a similar pattern of response as described above for both control and L/T-treated bPAEC (data not shown).
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Figure 8 demonstrates the effect of inhibiting L-Arg uptake on urea production in L/T-treated bPAEC. In L/T-treated bPAEC, the addition of L-Arg led to a concentration-dependent increase in urea production. Including 30 mM L-leucine in the medium resulted in a lower urea production at each L-Arg concentration than in L/T bPAEC without L-leucine included in the medium (Fig. 8). The effect of L-Val on NO2 and urea production in L/T-treated bPAEC was not due to inhibition of L-Arg uptake as demonstrated in Fig. 9. There was no significant difference in [3H]L-Arg uptake in L/T bPAEC with either vehicle or 30 mM L-Val added to the medium. However, as expected, addition of 10 mM nonlabeled L-Arg nearly completely inhibited [3H]L-Arg uptake, and this was true when 10 mM unlabeled L-Arg and 30 mM L-Val were added together (Fig. 9).
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DISCUSSION |
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Treatment with L/T resulted in an increase in NO2 production starting at 4 h and an increase in urea production starting at
6 h. Therefore, it appears that both NOS and AR were upregulated in these bPAEC relatively rapidly. Furthermore, the bPAEC continued to produce both NO and urea throughout the 24-h incubation period, as shown by the reasonable fit of the NO2 and urea production vs. time data by linear regression. This suggests that the necessary cofactors and substrates were available to NOS throughout the 24-h incubation period in both control and L/T-treated bPAEC. Therefore, the changes in NO2 and urea production found with inhibition of AR (L-Val), addition of substrate (L-Arg), or inhibition of L-Arg uptake (L-leucine) most likely reflect enzymatic interactions rather than nonspecific effects.
NO can be oxidized to NO2, and NO2 can be further oxidized to NO3. It is of interest to note that the NO2 concentration was 25% of the total NOX concentration in the medium after 24 h. This finding is consistent with a report by Ignarro et al. (11), wherein they found that, although in an oxygenated buffer solution the principal oxidation product of NO was NO2, in the unpurified cytosolic fractions from rat cerebellum the molar ratio of NO2/NO3 was
0.25. Furthermore, in the bPAEC in this study, there was a sizable NOX concentration in the medium at time 0,
48% of the NOX concentration at 24 h, whereas the concentration of NO2 in the medium from the bPAEC at time 0 was negligible, only
12% of the 24-h medium NO2 concentration. Thus to improve sensitivity we employed measurement of the NO2 concentration as a marker of NO production rather than measurement of the NOX concentration.
Treatment with L/T increased urea production, ARI protein expression, and ARII mRNA expression in bPAEC in our study. These findings suggest that L/T treatment increased urea production by increasing the expression of AR and are in agreement with previous studies. For example in rat aortic endothelial cells, LPS and IFN- treatment increased urea production, had no effect on ARI mRNA expression, and increased ARII activity (1). Similarly, hyperoxic exposure increased AR activities and expression in the lungs of rats (21). In macrophages, treatment with LPS resulted in increased ARI and ARII protein expression (5, 13, 17). LPS treatment of mice increased AR activities, protein, and mRNA expression in the lungs (22). The role of increased urea production in the bPAEC is unclear. If bPAEC in culture are representative of in vivo conditions, then the increased AR activity and expression may be involved in the formation of polyamines and/or L-proline from L-ornithine, which are important in tissue healing after injury (10, 12a, 12b). Consistent with this concept is the finding in murine macrophages that T helper (Th) 2 cytokines (IL-4 and IL-10) are potent inducers of AR, whereas a Th1 cytokine (IFN-
) is a potent inducer of iNOS (16). Similarly, in mouse peritoneal exudate cells, the growth factor transforming growth factor-
attenuated IFN-
-induced increased NOS activity and resulted in increased AR activity (24). Thus together these studies suggest that AR induction leading to increased urea production may be associated with tissue repair in some conditions.
We found that L/T treatment increased both urea and NO production. It has been suggested that the co-induction of AR with iNOS may limit L-Arg bioavailability to iNOS and thereby serve to decrease NO production (3, 4, 25, 27, 28). On the other hand, Buga et al. (1) found in rat aortic endothelial cells that when NO production was increased 20-fold by LPS/IFN-
treatment, AR activity was inhibited. The authors found that this effect was due to the intermediate in NO production from L-Arg, N
-hydroxy-L-Arg. The Ki for N
-hydroxy-L-Arg inhibition of AR was
10 µM. Similarly, Waddington et al. (26) found in macrophages that N
-hydroxy-L-Arg inhibited AR activity at concentrations of 20 and 200 µM but not at a concentration of 2 µM. Because the levels of NO produced in our culture media were
16 µM and because treatment with L-Val decreased urea production and increased NO production even in the L/T-treated bPAEC, it is unlikely that the levels of N
-hydroxy-L-Arg produced would be sufficient to inhibit urea production. Therefore, in situations where both AR and iNOS are induced, the degree of iNOS induction may determine whether AR activity will limit L-Arg bioavailablity to iNOS or whether enough N
-hydroxy-L-Arg will be produced to inhibit AR and thereby increase L-Arg bioavailablity to iNOS. Further studies are needed to examine the cellular mechanisms involved in iNOS and AR induction and the interrelationship between iNOS and AR activities during inflammation.
In this study we found that inhibiting AR increased NO production. This finding is consistent with a recent study in coronary microvascular endothelial cells, wherein NO production in response to serotonin or adenosine was significantly enhanced by inhibition of AR (28). In LPS-activated macrophages, inhibition of AR with L-norvaline decreased urea production and increased NO production, and L-norvaline resulted in a concentration-dependent increase in NO production (3), which is similar to what we found in bPAEC in this study. In macrophages, treatment with LPS and IL-13 induced both ARI and iNOS protein expression and increased urea and NO production; furthermore, treatment of these macrophages with L-norvaline increased NO production (4). Together these results demonstrate that inhibiting AR activity in a wide variety of cell types increases NO production.
There is no evidence that a product of AR activity inhibits NOS; therefore, the effect of AR inhibition on NO production suggests that AR and NOS compete for intracellular L-Arg. Considering that AR is the major L-Arg metabolic pathway in bPAEC, a decrease in AR activity might be expected to result in an increase in the intracellular concentration of L-Arg. However, when one considers that the L-Arg Km for the various NOS isoforms is 310 µM and that the intracellular concentration of L-Arg in cultured endothelial cells is
100800 µM (6, 13), it seems unlikely that the changes in L-Arg concentration from inhibition of AR would contribute significantly to L-Arg bioavailability to NOS. However, we (19, 23) and others (1, 3, 7) have previously shown that, despite an L-Arg Km of
310 µM for the various NOS isoforms, increasing the extracellular concentration of L-Arg increases NO production. This phenomenon has been termed the L-Arg paradox (15). Thus a reasonable interpretation of our findings in light of these studies is that despite the L-Arg Km for isolated NOS isoforms, AR and NOS do indeed compete for their common substrate L-Arg. Further support for the concept that AR and NOS compete for intracellular L-Arg comes from experiments wherein AR is overexpressed in cells. For example in HEK-293 cells with stable overexpression of nNOS, transfection with ARI decreased NO production, and the addition of 1 mM L-Arg to the culture medium restored NO production (20). Furthermore, transfection of coronary venular endothelial cells with either ARI or ARII increased urea production and decreased NO production (12a). Similarly, transfection of J774A.1 cells with ARI increased LPS-induced urea formation and decreased LPS-induced NO production compared with nontransfected cells, and NO production increased when the transfected ARI was inhibited with L-norvaline (2). Together these studies suggest that AR and NOS compete for intracellular L-Arg, such that increasing AR activity leads to a decrease in NO production, whereas decreasing AR activity leads to an increase in NO production. Thus changes in AR activities may be involved in the cellular regulation of NO production.
Extracellular L-Arg is involved in both NO and urea production, since increasing the extracellular L-Arg concentration increased both NO and urea production, and inhibition of L-Arg uptake decreased urea production. An increase in urea production when the extracellular L-Arg concentration is increased from 0.3 to 10 mM is consistent with the L-Arg Km for AR of 13 mM (14). Although the increase in bPAEC NO production with the addition of 10 mM L-Arg to the medium is consistent with our previous findings (19) and has been described by others (1, 7) in endothelial cells, it is more difficult to explain on a purely biochemical basis, since as discussed above the L-Arg Km for the NOS isoforms is
310 µM. It has been suggested that the L-Arg paradox may involve the association of the cationic amino acid transporter-1 (CAT-1) with eNOS in the cell membrane, such that eNOS preferentially utilizes extracellular L-Arg (15). However, in this case, one would expect that inhibiting intracellular ARI would have little effect on NO production. Thus although association of eNOS and CAT-1 is involved in the L-Arg paradox, a significant portion of NO production in these endothelial cells also utilizes the same L-Arg pool as AR. Both ARI and iNOS are found in the cytosol, and they may occupy a common intracellular compartment (12a). Therefore, the effect of L-Val on NO production may represent an effect on this common intracellular compartment. However, further studies will need to be done to determine exactly which isoforms and which intracellular compartments utilize a common pool of L-Arg.
L-Arg uptake by endothelial cells has been found to be primarily by the CAT family of amino acid transporters (6, 14). In cultured endothelial cells 70% of uptake occurs via CAT, whereas
30% occurs via the Na+-dependent system b0,+ amino acid transporters, also known as the broad-scope amino acid transport (BAT) proteins (6, 14). BAT proteins transport not only cationic amino acids like L-Arg but also neutral amino acids like L-Val (6). Thus to determine whether some of the effect of the addition of 30 mM L-Val to the medium was due to attenuation of L-Arg transport, we measured the uptake of [3H]L-Arg. L-Val had no significant effect on the uptake of [3H]L-Arg, suggesting that L-Val was taken up by bPAEC predominantly through mechanisms different from those responsible for L-Arg uptake. As expected, the addition of 10 mM unlabeled L-Arg nearly completely inhibited the uptake of [3H]L-Arg, and the small amount of [3H]L-Arg uptake in the presence of 10 mM unlabeled L-Arg represents nontransporter-dependent uptake (19), demonstrating that the majority of L-Arg is transported into bPAEC via a saturable process most likely representing CAT and/or BAT activities (6, 14, 19).
In conclusion, we found that treatment of bPAEC with L/T resulted in increased expression of ARI protein and ARII mRNA, as well as increased urea and NO production. Inhibition of AR with L-Val or L-norvaline led to a concentration-dependent increase in NO production. Both urea and NO production were increased by extracellular L-Arg, and the addition of both L-Arg and L-Val led to the greatest increase in NO production. L-Val is transported into bPAEC by a mechanism different from that which accounts for L-Arg uptake. Together these data demonstrate that AR and NOS compete for their common substrate, L-Arg, in bPAEC even under control conditions. We speculate that AR may represent a therapeutic target for the manipulation of NO production, particularly in cases of NO overproduction such as seen in inflammatory lung diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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