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Am J Physiol Lung Cell Mol Physiol 290: L298-L306, 2006. First published September 9, 2005; doi:10.1152/ajplung.00140.2005
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Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells

Kate P. Stanley,1 Louis G. Chicoine,1,3 Tamara L. Young,3 Kristina M. Reber,3 C. Richard Lyons,2 Yusen Liu,3 and Leif D. Nelin1,3

1Vascular Physiology Group and 2Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; and 3Centers for Developmental Pharmacology and Toxicology, Gene Therapy, and Cell and Vascular Biology, Columbus Children's Research Institute, Department of Pediatrics, The Ohio State University, Columbus, Ohio

Submitted 29 March 2005 ; accepted in final form 4 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide (NO) is a vasodilator produced from L-arginine (L-Arg) by NO synthase (NOS). Gene therapy for hypertensive disorders has been proposed using the inducible isoform of NOS (iNOS). L-Arg also can be metabolized to urea and L-ornithine (L-Orn) by arginase, and L-Orn can be metabolized to proline and/or polyamines, which are vital for cellular proliferation. To determine the effect of iNOS gene transfer on arginase, we transfected bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for iNOS (AdiNOS). As expected, NO production in AdiNOS bPAEC was substantially greater than in control bPAEC. Although urea production was significantly less in the AdiNOS bPAEC than in the control bPAEC, despite similar levels of arginase I protein, AdiNOS transfection of bPAEC had no effect on the uptake of L-Arg. Inhibiting NO production with N{omega}-nitro-L-arginine methyl ester increased urea production, and inhibiting urea production with L-valine increased nitrite production, in AdiNOS bPAEC. The addition of L-Arg to the medium increased urea production by AdiNOS bPAEC in a concentration-dependent manner. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. This competition for substrate resulted in impaired proliferation in the AdiNOS-transfected bPAEC. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders may not only be beneficial through NO-mediated pulmonary vasodilation but also may decrease vascular remodeling by limiting L-Orn production by native arginase.

nitric oxide; urea; pulmonary hypertension; pulmonary vascular remodeling

NITRIC OXIDE (NO) is a potent pulmonary vasodilator (26). NO is also a highly reactive molecule and binds readily with hemoglobin to form the nonvasoactive oxidative products nitrite and nitrate (26, 36). These properties of NO have led to the use of inhaled NO in neonates with hypoxic respiratory failure, wherein it has been shown to decrease the need for extracorporeal membrane oxygenation (12, 16, 33, 38). However, a subgroup of patients are unresponsive to inhaled NO therapy (12, 16, 33, 38), and others develop life-threatening "rebound" pulmonary hypertension (1, 31). Therefore, there is interest (10, 18, 36) in developing gene therapy with NO synthase (NOS), the enzyme that produces NO, to treat pulmonary hypertension. In humans there are at least three isoforms of NOS: endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed, and their activities depend on Ca2+, whereas inducible NOS (iNOS) expression is upregulated by various stimuli, including inflammatory cytokines, and its activity does not depend on changes in intracellular Ca2+ concentrations. Although all three NOS isoforms synthesize NO and L-citrulline (L-Cit) from L-arginine (L-Arg), iNOS may be the isoform of choice for gene therapy, because NO production by iNOS is relatively high volume and does not depend on Ca2+ (7, 10, 18, 36).

L-Arg also can be metabolized to L-ornithine (L-Orn) and urea by arginase, and there are two isoforms of arginase (arginase I and arginase II). L-Orn then can be metabolized by ornithine carboxyl transferase and/or ornithine amino transferase to ultimately produce proline and/or polyamines (19, 24, 41). Proline and polyamines are vital to cell proliferation (3, 37, 39), necessary for the pulmonary vascular remodeling seen in many forms of pulmonary hypertension. We previously have found that in bPAEC treated with lipopolysaccharide and tumor necrosis factor-{alpha}, inhibiting arginase results in increased NO production (11), suggesting that iNOS and arginase compete for a common intracellular pool of L-Arg. The cellular bioavailability of L-Arg to NOS and/or arginase is determined by the balance among the uptake of extracellular L-Arg, the intracellular metabolism of L-Arg, and the intracellular production of L-Arg (5, 15, 19). Under certain conditions, NO production depends on the uptake of extracellular L-Arg (22, 28, 30, 34). If overexpressing iNOS in endothelial cells results in an increase in L-Arg uptake by the cells, then L-Arg bioavailability to both iNOS and arginase may be maintained during this high-NO output state. Therefore, we set out to determine the effects of iNOS gene therapy on L-Arg uptake and urea production in bovine pulmonary arterial endothelial cells (bPAEC). The working hypothesis is that the increased utilization of L-Arg by transfected iNOS results in decreased L-Arg bioavailability to arginase, and thereby decreased urea and L-Orn production. A decrease in L-Orn production as an effect of iNOS gene therapy in pulmonary hypertensive disorders characterized by vascular remodeling may be particularly beneficial, allowing for pulmonary vasodilation and preventing further vascular remodeling.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Pulmonary arterial endothelial cell culture. bPAEC were obtained from Clonetics (San Diego, CA) and cultured as previously described (28). Briefly, bPAEC were placed in a T25 flask containing 4 ml of endothelial growth medium (EGM; Clonetics) and incubated at 37°C in 5% CO2-95% air. Once the bPAEC were 80–90% confluent, the EGM was removed from the flask, and the bPAEC were washed with HEPES balanced salt solution (HBSS; Clonetics) and passaged with trypsin-EDTA and trypsin-neutralizing solution. bPAEC between passages 3 and 8 were used for the following studies.

Adenoviral transfection. Adenovirus, serotype 5, containing either the cDNA for Escherichia coli {beta}-galactosidase and a CMV promoter (Ad{beta}-gal) or the cDNA for murine iNOS and a CMV promoter (AdiNOS) were derived and prepared as previously described (7, 10). Three groups of bPAEC were prepared in the following manner: bPAEC were transfected with 1) AdiNOS, 2) Ad{beta}-gal as a control for nonspecific gene transfer effects, and/or 3) vehicle (EGM containing no virus). A cell count was performed on a single flask of bPAEC before the transfection. The amount of virus placed on each flask was determined by multiplying the number of bPAEC per T75 flask by the multiplicity of infection (MOI), divided by the number of plaque-forming units per milliliter of virus. For each flask, the required amount of viral stock was added to 4 ml of EGM. bPAEC were incubated at 37°C in 5% CO2-95% air for 4 h. The medium was then removed, and the bPAEC were washed three times with HBSS. The medium (4 ml) to be used in the specific experimental protocols was placed on the bPAEC, and the bPAEC were incubated at 37°C in 5% CO2-95% air. After a 24-h incubation period, the medium was removed and frozen at –70°C in 1-ml aliquots for nitrite, citrulline, and/or urea analysis. The bPAEC were harvested in cell lysis buffer for protein isolation.

Nitrite assay. NO production in the cell medium was measured as previously described (11, 28, 34). When exposed to oxygen, NO is rapidly oxidized to nitrite (NO2–). NO2– in the cell medium was measured using a chemiluminescence nitric oxide analyzer (model 270B; Sievers Instruments, Boulder, CO). Briefly, a reaction chamber contained 4 ml of glacial acetic acid and 50 mg of sodium iodide. Each sample (100 µl) was injected into the reaction chamber to reduce the NO2– to NO gas, and the NO gas was carried into the analyzer by a constant flow of N2 gas. The amount of NO2– in each sample was determined using a standard curve prepared with sodium nitrite.

Urea and citrulline assay. Urea and citrulline production in the cell medium was measured using a colorimetric assay as previously described (11, 27, 28). Briefly, 500 µl of sample were added to 3 ml of a solution containing 33 µg/ml thiosemicarbazide, 1.67 mg/ml diacetyl monoxime, and 0.25 mg/ml FeCl3 in 25% H2SO4 and 20% H3PO4. In a second set of samples, 0.8 unit of urease was added and incubated for 1 h before addition to the same solution as above. The samples were vortexed and boiled at 100°C for 5 min. After cooling to room temperature, absorbance was measured at 530 nm. The concentration of citrulline was determined by comparing the absorbance in the samples containing urease to a standard curve prepared from citrulline. Urea concentrations within the samples were determined by the differences in absorbance between samples with and without urease and compared with a standard curve prepared from urea.

bPAEC protein isolation. Protein was isolated from bPAEC as previously described (11, 28). The bPAEC were washed with HBSS, and 750 µl of lysis buffer (0.2 M NaOH, 0.2% SDS) were added to each flask. The following protease inhibitors were added to each milliliter of lysis buffer before use: 2 µg of aprotinin, 5 µg of leupeptin, 0.7 µg of pepstatin, and 174 µg of phenylmethylsulfonyl fluoride. The bPAEC were scraped, and 100-µl aliquots were stored at –70°C. Total protein concentration was determined using the Bradford method with a commercially available assay (Bio-Rad, Hercules, CA).

Western blot analysis. Western blot analysis was performed as previously described (10, 27, 28). To detect iNOS, eNOS, or arginase I protein, we resolved samples (100 µg for iNOS, 20 µg for eNOS, and 40 µg for arginase I) using SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked overnight at 4°C with 5% nonfat milk and 3% bovine serum albumin (Sigma Chemical, St Louis, MO) in a Tris-buffered saline solution (TBS) containing 10 mM Tris·HCl, 50 mM NaCl (pH 7.5), and 0.1% Tween 20. Blots were incubated for 4 h at room temperature with a mouse monoclonal antibody against human iNOS (1:1,000), human eNOS (1:2,500), or arginase I (1:1,000; all obtained from Transduction Laboratories, San Diego, CA). Immunochemical labeling was achieved by incubation for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000; Bio-Rad). Chemiluminescence detection was performed per kit instructions (Amersham, Piscataway, NJ) using chemiluminescence-sensitive film and quantitated using densitometric analysis (Sigma Gel; Jandel Scientific, San Rafael, CA). We were unable to directly examine arginase II protein expression, because currently there is no commercially available antibody against bovine arginase II.

L-[3H]arginine uptake. L-[3H]Arg uptake was measured as previously described (11, 28). After the 24-h incubation in EGM, bPAEC were washed with warm HBSS. bPAEC were then incubated at room temperature for 15 min with either 4 ml of EGM containing 1 µCi/ml L-[3H]Arg, to measure total L-[3H]Arg uptake, or 4 ml of EGM with 1 µCi/ml L-[3H]Arg and 10 mM nonlabeled L-Arg, to measure nonspecific L-[3H]Arg uptake. After incubation, the flasks were washed with ice-cold HBSS. The cells were lysed with 4 ml of lysis buffer containing 0.2 M NaOH and 0.2% SDS and incubated overnight on a rocker at room temperature. To determine the amount of L-[3H]Arg in the bPAEC, 100-µl samples of the lysate were aliquoted into a scintillation counting cocktail in duplicate and then placed in a scintillation counter. The specific uptake of L-[3H]Arg was determined by subtracting the nonspecific L-[3H]Arg uptake from the total L-[3H]Arg uptake. Total protein concentration per T75 flask was determined using the Bradford method (Bio-Rad). We have found in preliminary experiments that the uptake of L-[3H]Arg is linear over this time period in bPAEC. In a second set of experiments, bPAEC were incubated with 1 µCi/ml L-[3H]Arg and 1, 10, 100, or 1,000 µM unlabeled (cold) L-Arg for 15 min. The cells were lysed, and 100-µl samples of cell lysate were aliquoted into scintillation counting cocktail in duplicate and placed in a scintillation counter as described above. Total protein was determined using the Bradford method (Bio-Rad).

Proliferation assay. The proliferation of bPAEC was determined in six-well plates. Briefly, 80–90% confluent bPAEC in T75 flasks were transfected with either AdiNOS or vehicle for 4 h as described in Adenoviral transfection. The bPAEC were then washed three times with HBSS and incubated in 5% CO2-95% air for 24 h to allow for stable expression of iNOS. The bPAEC were then passaged onto six-well plates. Each well was seeded with 1.8 x 104 cells and then incubated in 5% CO2-95% air. At 24, 48, and 72 h, the EGM was collected, and the cells were washed and trypsinized. Trypan blue was used, and the numbers of viable and nonviable cells were counted using a hemacytometer. To ensure iNOS overexpression in the AdiNOS-transfected bPAEC, we isolated protein from vehicle and AdiNOS-transfected bPAEC at 48 and 72 h for Western blot analysis of iNOS protein levels.

Statistical analysis. All data are expressed as means ± SE. NO2–, citrulline, and urea production are expressed as either nanomoles per milligram of protein or as percentages of vehicle-transfected production. L-[3H]Arg uptake is expressed as picomoles per milligram of protein. One-way ANOVA with a Newman-Keuls post hoc test was used to compare groups. Differences were considered significant when P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of AdiNOS on iNOS expression, NO production, and eNOS expression. To verify that the AdiNOS-transfected bPAEC had increased iNOS protein levels and NO production, we incubated vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC for 24 h, and then the bPAEC were lysed for protein determination and Western blot analysis for iNOS as described in METHODs. Only bPAEC transfected with AdiNOS had detectable iNOS protein bands (Fig. 1A). The lack of a detectable endogenous iNOS protein in the Ad{beta}-gal-transfected bPAEC indicates that the adenoviral construct alone had little effect on endogenous iNOS expression in cultured bPAEC.



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  		Fig. 1. Transfection of bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for inducible NO synthase (AdiNOS) resulted in transgene expression and increased NO production. A: Western blot for iNOS and {beta}-actin proteins from bPAEC transfected with vehicle (n = 3), adenovirus containing cDNA for Escherichia coli {beta}-galactosidase (Ad{beta}-gal; n = 3), and AdiNOS (n = 3). Blots were stripped and reprobed for {beta}-actin protein bands to confirm equal protein loading in all 9 lanes. B: NO2– and citrulline production increased in AdiNOS-transfected bPAEC (n = 6) compared with either vehicle-treated (n = 5) or Ad{beta}-gal-transfected (n = 5) bPAEC. Addition of 10 mM N{omega}-nitro-L-arginine methyl ester (L-NAME; n = 6) to the medium inhibited NO2– and citrulline production in AdiNOS-transfected bPAEC. *P < 0.01, different from vehicle. #P < 0.01, different from AdiNOS alone.

 
In a second set of studies, after transfection with an MOI of 3.0, NO2– and citrulline levels were measured in vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC after a 24-h incubation. A group of AdiNOS-transfected bPAEC treated with the competitive NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 10 mM) also was studied. Citrulline and NO2– production, as expected, were significantly greater in AdiNOS-transfected bPAEC compared with either vehicle- or Ad{beta}-gal-transfected bPAEC (Fig. 1B). Citrulline and NO2– production in AdiNOS-transfected bPAEC incubated in the presence of L-NAME were not significantly different from that in vehicle- or Ad{beta}-gal-transfected bPAEC, suggesting that the main source of the increased citrulline and NO2– production in the AdiNOS bPAEC was NOS activity. Because of the robust increase in NO2– production with an MOI of 3.0, in subsequent studies we employed an MOI of 1.0, unless otherwise noted.

To determine the effect of AdiNOS transfection on eNOS protein expression, we incubated vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC for 24 h and then lysed bPAEC for protein determination and Western blot analysis for eNOS. The changes in NO and citrulline production in the AdiNOS-transfected bPAEC are largely due to the overexpression of iNOS, because AdiNOS transfection had no apparent effect on eNOS protein expression, as demonstrated by the similar densities of eNOS protein bands in the three groups (Fig. 2).



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  		Fig. 2. AdiNOS had no effect on endothelial NO synthase (eNOS) expression in these bPAEC. Top: a representative Western blot for eNOS protein from vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC. First lane at left represents a commercially available positive control. Bottom: bar graph representing the band densities for the three groups. AU, arbitrary units.

 
Time course of NO and urea production after transfection. To determine the time course of NO production by AdiNOS-transfected bPAEC, we transfected bPAEC with either Ad{beta}-gal or AdiNOS for 4 h. The bPAEC were then washed, and EGM was placed on the cells. The EGM was sampled at 1, 2, 4, 6, and 24 h for determination of NO2– and urea production. After the 24-h sampling, the bPAEC were washed and harvested for protein determination as described above. NO production in Ad{beta}-gal-transfected bPAEC was essentially linear over the experimental time course (Fig. 3A), which is consistent with our previous findings in cultured control bPAEC (11). Conversely, the NO production increased dramatically in the AdiNOS-transfected bPAEC after 6 h (Fig. 3A). Urea production increased over time in both Ad{beta}-gal- and AdiNOS-transfected bPAEC (Fig. 3B), which is consistent with our previous findings in cultured control bPAEC (11). However, at 24 h, the urea production in the AdiNOS-transfected bPAEC was less than in the Ad{beta}-gal-transfected bPAEC (Fig. 3B). These results demonstrate that the decrease in urea production in AdiNOS-transfected bPAEC occurred only after NO production increased.



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  		Fig. 3. Transfection of bPAEC with AdiNOS resulted in an increase in NO production and a decrease in urea production, which began after 6 h. A: time course of NO production by Ad{beta}-gal- and AdiNOS-transfected bPAEC (n = 3–5 for each time point). There was a nearly linear time-dependent increase in NO production in the Ad{beta}-gal-transfected groups (y = 0.28x – 0.22, r = 0.996, P < 0.001). In the AdiNOS-transfected bPAEC, NO production started out following the same general pattern as that of Ad{beta}-gal transfection; however, after 6 h, NO production increased such that by 24 h there was a doubling of NO production in the AdiNOS-transfected bPAEC compared with the Ad{beta}-gal-transfected bPAEC. *P < 0.05, AdiNOS different from Ad{beta}-gal at 24 h. B: time course of urea production by Ad{beta}-gal- and AdiNOS-transfected bPAEC (n = 3 for each time point). There was a time-dependent increase in urea production in both the Ad{beta}-gal- and AdiNOS-transfected groups. However, in the AdiNOS-transfected group, there was decreased urea production starting after 6 h compared with the Ad{beta}-gal-transfected group such that urea production was significantly less at 24 h in the AdiNOS-transfected bPAEC than in the Ad{beta}-gal-transfected bPAEC. *P < 0.05, AdiNOS different from Ad{beta}-gal at 24 h.

 
Effects of iNOS overexpression on L-[3H]Arg uptake. To determine the effect of overexpression of iNOS on L-Arg uptake, we measured specific L-[3H]Arg uptake in vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC. AdiNOS transfection did not significantly change the specific uptake of L-Arg by bPAEC (Fig. 4A). The mean specific L-Arg uptake was 4.7 ± 0.3, 5.0 ± 0.3, and 4.1 ± 0.4 pmol/mg protein in vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC, respectively. In these studies, transfection of bPAEC with AdiNOS was confirmed by a substantially greater NO2– production in the medium of AdiNOS-transfected bPAEC compared with either the vehicle- or Ad{beta}-gal-transfected bPAEC (data not shown).



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  		Fig. 4. AdiNOS transfection had no effect on L-arginine (L-Arg) uptake by bPAEC. A: specific uptake of L-[3H]Arg was not different in AdiNOS-transfected bPAEC compared with vehicle-treated or Ad{beta}-gal-transfected bPAEC (n = 4). B: uptake of L-[3H]Arg was inhibited in a concentration-dependent manner by unlabeled (cold) L-Arg in both vehicle-treated (n = 3 for each concentration) and AdiNOS-transfected bPAEC (n = 3 for each concentration).

 
A second set of experiments was carried out in vehicle-treated and AdiNOS-transfected bPAEC in which L-[3H]Arg uptake was measured with increasing concentrations of unlabeled (cold) L-Arg added to the medium. Both vehicle-treated and AdiNOS-transfected bPAEC had similar decreases in L-[3H]Arg uptake with increasing competitive competition by cold L-Arg (Fig. 4B).

Effect of iNOS overexpression on NO and urea production. To test the hypothesis that the increase in NO production caused by overexpression of iNOS would decrease urea production, we performed three sets of studies. In the first set of studies, Ad{beta}-gal- and AdiNOS-transfected bPAEC were incubated with EGM containing 1 mM L-Arg for 24 h. NO2– production in the Ad{beta}-gal-transfected bPAEC was not significantly different from that in vehicle-treated bPAEC, whereas NO2– production was substantially greater in AdiNOS-transfected bPAEC compared with either vehicle- or Ad{beta}-gal-transfected bPAEC (Fig. 5A). Urea production was unaffected by Ad{beta}-gal transfection relative to vehicle. However, urea production was ~40% lower in the AdiNOS-transfected bPAEC compared with either vehicle- or Ad{beta}-gal-transfected bPAEC (Fig. 5A). The protein content per flask of bPAEC did not differ among the three study groups: vehicle, 5.0 ± 0.4 mg/flask; Ad{beta}-gal, 5.5 ± 0.4 mg/flask; and AdiNOS, 4.4 ± 0.3 mg/flask. Thus transfection of bPAEC with AdiNOS increased NO production and decreased urea production.



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  		Fig. 5. Transfection of bPAEC with AdiNOS decreased urea production. A: NO production (nitrite) and urea production in Ad{beta}-gal- and AdiNOS-transfected bPAEC [multiplicity of infection (MOI) = 1.0; n = 5], expressed as a percentage of values for vehicle-treated bPAEC (n = 5), represented by the dashed line at 100%. *P < 0.05, different from vehicle and Ad{beta}-gal. B: NO production (nitrite) increased and urea production decreased in a dose-dependent manner in AdiNOS-transfected bPAEC (n = 3 for each MOI group). The y-axis indicates the percentage of either NO2– or urea production compared with values for vehicle-treated bPAEC, represented by the dashed line at 100%. *P < 0.05, different from vehicle. #P < 0.05, different from preceding MOI group.

 
In a second set of studies, bPAEC were transfected as described above with 0 (vehicle), 0.1, 0.5, or 1.0 MOI AdiNOS and then washed and incubated for 24 h in EGM containing 1 mM L-Arg. The increase in NO2– production in the AdiNOS-transfected bPAEC was dependent on the viral dose used to transfect (Fig. 5B), and the decrease in urea production in the AdiNOS-transfected bPAEC also was dependent on the viral dose used to transfect (Fig. 5B).

In a third set of studies, the effects of AdiNOS transfection of bPAEC on levels of endogenous arginase I protein were examined. AdiNOS transfection of bPAEC had little effect on arginase I protein levels, as demonstrated by the similar densities of the arginase I protein bands in the three groups (Fig. 6). Thus the decreased urea production in AdiNOS-transfected bPAEC was not due to alterations in arginase I protein levels.



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  		Fig. 6. AdiNOS transfection had no effect on arginase I expression. A: a representative Western blot for arginase I from vehicle-, Ad{beta}-gal-, and AdiNOS-transfected bPAEC (top). The same blot, stripped and reprobed for {beta}-actin, is shown at bottom. B: densitometry data from the arginase I Western blots from vehicle (n = 3)-, Ad{beta}-gal (n = 3)-, and AdiNOS (n = 3)-transfected bPAEC normalized to {beta}-actin. There were no significant differences among the groups in arginase I protein level.

 
Effect of NOS or arginase inhibitors on production of NO and urea. To examine whether transfected iNOS and endogenous arginase compete for L-Arg, vehicle-treated bPAEC, AdiNOS-transfected bPAEC, and AdiNOS-transfected bPAEC with 10 mM L-NAME included in the medium were incubated for 24 h. The medium was collected and analyzed for NO2– and urea. In this set of experiments, AdiNOS-transfected bPAEC had ~180% greater NO2– production and ~40% less urea production than did vehicle-treated bPAEC (Fig. 7A). When L-NAME (10 mM) was added to the medium of AdiNOS-transfected bPAEC, NO2– production was ~80% less than in AdiNOS-transfected bPAEC, resulting in an ~130% greater urea production than in AdiNOS-transfected bPAEC (Fig. 7A).



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  		Fig. 7. Transfected iNOS and arginase compete for L-Arg in bPAEC. A: inhibition of NOS in AdiNOS-transfected bPAEC increased urea production (n = 6 in each group). The effect of AdiNOS transfection in the presence of 10 mM L-NAME, a competitive NOS inhibitor, on NO2– (nitrite) and urea production is shown. The y-axis indicates the difference in either NO2– or urea production compared with values for vehicle-treated bPAEC, expressed as percentages, where no difference from vehicle is shown as 0%. *P < 0.01, different from vehicle. #P < 0.05, AdiNOS + L-NAME different from AdiNOS alone. B: inhibition of arginase in AdiNOS-transfected bPAEC by adding 30 mM L-valine to the media resulted in greater NO2– production than in either vehicle- or AdiNOS-transfected bPAEC (n = 6 in each group). The y-axis again indicates the difference in either NO2– or urea production compared with values for vehicle-treated bPAEC, expressed as percentages, where no difference from vehicle is shown as 0%. The differences in NO2– (nitrite) and urea are shown in bPAEC transfected with AdiNOS alone or with AdiNOS plus 30 mM L-valine included in the medium. *P < 0.01, different from vehicle transfected. #P < 0.05, AdiNOS + L-valine different from AdiNOS.

 
To further examine whether transfected iNOS and endogenous arginase compete for L-Arg, AdiNOS-transfected bPAEC were incubated with either 1 mM L-Arg added to the medium or 1 mM L-Arg and 30 mM L-valine (L-Val), a competitive inhibitor of arginase, added to the medium. The medium was collected and analyzed for NO2– and urea. In this set of experiments, AdiNOS-transfected bPAEC had ~160% greater NO2– production and ~42% less urea production than did vehicle-treated bPAEC (Fig. 7B). When L-Val, a competitive inhibitor of arginase, was added to the medium of AdiNOS-transfected bPAEC, urea production was ~34% less than in AdiNOS-transfected bPAEC, and this resulted in ~42% greater NO2– production than in AdiNOS-transfected bPAEC (Fig. 7B).

Effect of extracellular L-Arg on urea production. To test the hypothesis that increasing the extracellular L-Arg concentration would increase L-Arg bioavailability to arginase and therefore increase urea production in AdiNOS-transfected bPAEC, we incubated AdiNOS-transfected bPAEC in EGM containing 1, 3, 10, or 30 mM L-Arg for 24 h. Vehicle-transfected bPAEC also were incubated in 1 mM L-Arg to serve as a control. Urea production increased in AdiNOS-transfected bPAEC in a concentration-dependent manner with the addition of extracellular L-Arg (Fig. 8). Consistent with the experiments described above, urea production was significantly less in AdiNOS-transfected bPAEC incubated in 1 mM L-Arg compared with vehicle-transfected bPAEC incubated in 1 mM L-Arg (Fig. 8). Urea production increased in a concentration-dependent manner in AdiNOS-transfected bPAEC with the addition of 3, 10, and 30 mM L-Arg to the medium (Fig. 8), indicating that urea production by AdiNOS-transfected bPAEC was restored by exogenous L-Arg.



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  		Fig. 8. Increasing L-Arg bioavailability restored urea production in AdiNOS-transfected bPAEC. The addition of extracellular L-Arg to the medium increased urea production in a concentration-dependent manner in AdiNOS-transfected bPAEC (n = 3 for each bar). The y-axis indicates the percentage of urea production compared with that in vehicle-treated bPAEC, represented by the dashed line at 100%. *P < 0.05, different from vehicle. #P < 0.05, different from AdiNOS + 1 mM L-Arg.

 
Effect of iNOS overexpression on cell proliferation. To determine whether the alterations in NO and urea production caused by AdiNOS transfection had physiological effects, we determined the proliferation of AdiNOS- and vehicle-transfected bPAEC. AdiNOS-transfected or vehicle-transfected bPAEC were plated in six-well plates 24 h after transfection. At 24, 48, and 72 h, the numbers of viable and nonviable bPAEC were determined using Trypan blue exclusion. There was no difference in the number of viable bPAEC at 24 h between AdiNOS-transfected and control bPAEC, and in both groups the number of viable bPAEC increased at 48 h and again at 72 h (Fig. 9A). However, there were significantly more viable cells in the vehicle-transfected bPAEC than in the AdiNOS-transfected bPAEC at 72 h (Fig. 9A). There was no difference between vehicle- and AdiNOS-transfected bPAEC in the numbers of nonviable cells at each time point (Fig. 9B). As expected, the AdiNOS-transfected bPAEC had easily discernable iNOS bands as shown by Western blot analysis at 48 and 72 h, whereas vehicle-transfected bPAEC did not. These results indicate that AdiNOS-transfected bPAEC did not proliferate as well as vehicle-transfected bPAEC.



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  		Fig. 9. Transfection with AdiNOS impaired cell proliferation. A: viable bPAEC at 24, 48, and 72 h after transfected bPAEC were seeded onto 6-well plates (n = 3 at each time point). The numbers of viable bPAEC increased in both vehicle-transfected (control) and AdiNOS-transfected bPAEC (n = 3 at each time point) at both 48 and 72 h. The number of viable bPAEC was greater at 72 h in vehicle-transfected (control) than in AdiNOS-transfected bPAEC. *P < 0.05, different from 24 h in same group. #P < 0.01, AdiNOS different from control at same time point. B: nonviable bPAEC at 24, 48, and 72 h after transfected bPAEC were seeded onto 6-well plates (n = 3 for each time point). The numbers of nonviable bPAEC increased in both vehicle-transfected (control) and AdiNOS-transfected by 72 h. However, there was no difference in the number of nonviable bPAEC at 72 h between vehicle- and AdiNOS-transfected bPAEC. *P < 0.05, different from 24 h in same group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of this study were that in bPAEC transfected with AdiNOS: 1) L-Arg uptake was not affected; 2) NO production was increased, whereas urea production was decreased; 3) arginase I protein levels were not affected; 4) urea production was increased by inhibition of NOS with L-NAME; 5) NO production was increased by inhibition of arginase with L-Val; and 6) urea production was restored to control levels by increasing the extracellular L-Arg concentration. Together, these results support our hypothesis that in bPAEC transfected with iNOS, iNOS and arginase compete for their common substrate, L-Arg. This competition between iNOS and arginase occurred, at least in part, because L-Arg uptake did not significantly increase in AdiNOS-transfected bPAEC. Furthermore, the decreased urea production had physiological consequences, as demonstrated by impaired proliferation in the AdiNOS-transfected bPAEC.

The bioavailability of L-Arg for NO and/or urea synthesis may be affected by the recycling of L-Cit to L-Arg. It has been found that some vascular endothelial cells have a basal capacity to recycle L-Cit to L-Arg via argininosuccinate synthetase and argininosuccinate lyase (15). As expected, citrulline production increased in AdiNOS-transfected bPAEC in the current experiments. However, there does not appear to be sufficient citrulline recycling to arginine in the AdiNOS-transfected bPAEC to maintain urea production. Thus it is likely that recycling of L-Cit to L-Arg does not account for a large portion of the L-Arg bioavailability to arginase in these AdiNOS-transfected bPAEC.

Extracellular L-Arg is transported into the lung mainly via CAT-1 and CAT-2 (14, 27, 28, 30). We previously have found that the uptake of L-[3H]Arg, as well as the expression of CAT-1 and/or CAT-2 mRNA, was enhanced in rat lung exposed to silica in vivo or in bPAEC treated with lipopolysaccharide and tumor necrosis factor-{alpha}, an effect associated with increased NO production, increased urea production, and increased iNOS protein expression (27, 28). These studies suggest that inflammation-mediated increases in NO production were associated with enhanced L-Arg uptake and may represent a mechanism to maintain the intracellular supply of L-Arg to iNOS. These findings led us to ask the question, Does increased NO production alone affect CAT activity? This question was specifically addressed in the current study, and L-Arg uptake was not affected by the increased NO production caused by iNOS gene transfer in bPAEC. Thus these findings demonstrate that increased NO production per se does not upregulate L-Arg uptake.

It appears that in endothelial cells, arginase represents the major intracellular metabolic pathway for L-Arg (6, 19, 28). In the current study we found that iNOS overexpression decreased urea production by arginase, and increasing extracellular L-Arg concentrations enhanced urea production, indicating that addition of substrate could overcome the effect of AdiNOS on urea production. In a study analogous to the current investigation, Li et al. (20) overexpressed arginase in bovine coronary venular endothelial cells and found that urea production was increased and NO production was decreased compared with controls. Furthermore, Que et al. (32) demonstrated that transfection with arginase decreased NO production in 293 cells overexpressing nNOS. We also found that inhibiting arginase increased NO production in the iNOS-overexpressing bPAEC, suggesting that L-Arg metabolism by arginase affects NO production by the transfected iNOS. This finding is consistent with recent studies in bPAEC, coronary microvascular endothelial cells, and cytokine-stimulated macrophages, wherein NO production was significantly enhanced by inhibition of arginase (8, 9, 11, 35, 43). Therefore, the effects of arginase overexpression and arginase inhibition on NO production are consistent with the concept that arginase and NOS compete for a common pool of intracellular L-Arg.

Arginase activity can be inhibited by an intermediate in the L-Arg-NO pathway, NG-hydroxy-L-arginine (NOHA), during high-output NO synthesis (6, 13). For example, Buga et al. (6) demonstrated that when cytokine-induced NO production was increased ~20-fold in rat aortic endothelial cells, intracellular levels of NOHA increased and arginase activity was inhibited. Furthermore, inhibition of NOS decreased levels of NOHA and increased urea production. The IC50 value for NOHA inhibition of arginase has been found to be 10–40 µM (6, 13). The concentration of NO produced in the present experiments by the AdiNOS-transfected bPAEC was ~1–3 µM. Thus it is unlikely, given the concentrations of NO found in the current experiments, that the concentrations of NOHA achieved would be high enough to result in arginase inhibition. This conclusion also is consistent with a recent study by Waddington et al. (40), wherein NOHA inhibited arginase activity in macrophages at NO concentrations of 20 and 200 µM but not at an NO concentration of 2 µM. Thus it is unlikely that the decrease in urea production in AdiNOS-transfected bPAEC was due to inhibition of arginase by NOHA.

Arginase activity may have negative consequences in pulmonary hypertension. Arginase may limit NO production by competition for L-Arg and may increase vascular cell proliferation by producing L-Orn. In this regard, arginase activities may contribute to both vascular constriction and vascular remodeling. For example, Ming et al. (23) found that in atherosclerotic aortic rings from apoE knockout mice, there was significant vasodilation when arginase was inhibited. In terms of cellular proliferation, Ignarro et al. (17) found that vascular smooth muscle cells transfected with arginase I exhibited enhanced cellular proliferation. Bachetti et al. (2) found that human umbilical vein endothelial cells produced polyamines and that polyamine production was decreased when arginase was inhibited; furthermore, the percentage of cells positive for Ki-67, a nuclear protein indicative of cells undergoing active division, was decreased by arginase inhibition. Loyaga-Rendon et al. (21) found that arginase activity was approximately two times greater in endothelial cells from patients with intimal hyperplasia of the uterine arteries than in patients without intimal hyperplasia. In a recent study, endothelial cells from humans with primary pulmonary hypertension were found to express higher levels of arginase protein, greater arginase activity, and lower levels of NO production than endothelial cells from normal controls, despite similar levels of eNOS protein between the two groups (42). Thus, given the potential negative role of arginase in pulmonary hypertension, gene therapy using NOS may be beneficial not only by increasing NO production, and thereby causing vasodilation, but also by limiting L-Orn production, and thereby decreasing cellular proliferation and vascular remodeling. Our findings that bPAEC proliferation was decreased in the AdiNOS-transfected cells is consistent with this concept.

In conclusion, gene transfection with iNOS decreased the production of urea in bPAEC, most likely by limiting the supply of L-Arg available to arginase. The decrease in urea production was overcome by providing excess L-Arg. However, it is important to remember that the added L-Arg concentrations used in this cell culture study were much higher than levels normally found in human plasma (~100 µM) (25), and very large doses of L-Arg (30 g intravenously in adults, 500 mg/kg in pediatric patients) given over 30 min are required to produce transient plasma concentrations in the millimolar range in humans (4, 29). However, the finding that urea production was augmented by added L-Arg, together with the fact that inhibiting arginase activity increased NO production, strongly supports the concept that AdiNOS transfection increases L-Arg utilization by iNOS and thereby limits the bioavailability of L-Arg to arginase. The change in bioavailability of L-Arg to arginase arose, at least in part, because adenovirus transfection of the iNOS gene in these bPAEC had no significant effect on the uptake of extracellular L-Arg. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. Furthermore, the substrate competition-induced decrease in urea production in AdiNOS-transfected bPAEC was associated with impaired cellular proliferation. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders not only may be beneficial through pulmonary vasodilation, via an effect on vascular smooth muscle cell soluble guanylate cyclase, but also may decrease vascular remodeling by limiting L-Orn production by native arginase.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Forest Pharmaceuticals Advancing Newborn Medicine Fellowship Grant (to K. P. Stanley), National Heart, Lung, and Blood Institute Grant HL-04050 (to L. G. Chicoine), and a grant-in-aid from the American Heart Association, Desert Mountain Affiliate (to L. D. Nelin).


   ACKNOWLEDGMENTS
 
We thank Heather E. Nash and Kelly M. Billings for excellent technical assistance. We also thank Stephen E. Welty, MD, for editorial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. D. Nelin, Center for Developmental Pharmacology and Toxicology, Columbus Children's Research Institute, 700 Children's Drive, Columbus, OH 43205 (e-mail: nelinl{at}pediatrics.ohio-state.edu)

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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