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Circulation. 1997;96:689-697

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*(L)-ARGININE
*MONOCROTALINE
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*Pulmonary Hypertension

(Circulation. 1997;96:689-697.)
© 1997 American Heart Association, Inc.

Articles

Prolonged Administration of L-Arginine Ameliorates Chronic Pulmonary Hypertension and Pulmonary Vascular Remodeling in Rats

Yoshihide Mitani, MD; Kazuo Maruyama, MD, PhD; ; Minoru Sakurai, MD, PhD

From the Department of Pediatrics (Y.M., M.S.) and Anesthesiology (K.M.), Mie University School of Medicine, Tsu, Mie, Japan.

Correspondence to Yoshihide Mitani, MD, Department of Pediatrics, Mie University School of Medicine, 2-174 Edobashi, Tsu City, Mie Pref, Japan 514.


*    Abstract
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Background Endothelium-dependent nitric oxide–mediated vasodilation is impaired in rats with pulmonary hypertension (PH) induced by chronic hypoxia or by monocrotaline injection. We therefore investigated whether the prolonged administration of the nitric oxide precursor L-arginine would alleviate PH in both rat models.

Methods and Results Fifty-nine rats were exposed to hypobaric hypoxia (380 mm Hg, 10 days) or room air and injected intraperitoneally with L-arginine (500 mg/kg), D-arginine (500 mg/kg), or saline once daily from day -3 to day 10. An additional 38 rats injected subcutaneously with monocrotaline (60 mg/kg) or saline were treated similarly with L-arginine or saline from day -3 to day 17. At the end of the experiment, awake mean pulmonary arterial pressure was determined. The heart was dissected to weigh the right ventricle, and the lungs were obtained for vascular morphometric analysis. Hypoxic rats developed PH (30.8±0.7 versus 19.2±0.4 mm Hg in controls; P<.05) and right ventricular hypertrophy. Their pulmonary arterial wall thickness and the proportion of muscular arteries in the peripheral arteries increased. L-Arginine but not D-arginine reduced PH (24.8±0.7 mm Hg; P<.05), right ventricular hypertrophy, and pulmonary vascular disease. Monocrotaline rats developed PH (34.9±2.1 versus 18.8±1.2 mm Hg in controls; P<.05), right ventricular hypertrophy, and pulmonary vascular disease. Again, L-arginine reduced PH (24.3±1.7 mm Hg; P<.05), right ventricular hypertrophy, and pulmonary vascular disease.

Conclusions We conclude that L-arginine ameliorated the changes associated with PH in rats, perhaps by modifying the endogenous nitric oxide production.


Key Words: endothelium-derived factors • hypoxia • pulmonary heart disease • physiology • hypertension, pulmonary


*    Introduction
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Structural and functional alterations in the pulmonary vascular endothelium have been observed in experimental models1 2 3 4 5 as well as in patients with pulmonary hypertension.6 Pulmonary hypertension associated with pulmonary endothelial injury is accompanied by various pathological changes, including muscularization of the normally nonmuscular peripheral arteries, medial hypertrophy of proximal muscular arteries, and an increase in intercellular connective tissue.1 2 6 The mechanisms underlying these alterations, however, are poorly understood.

The endothelium plays an important role in the control of vascular tone and vascular remodeling by releasing endothelium-derived constricting and relaxing factors.7 NO is one endothelium-derived relaxing factor that is derived from the metabolism of L-arginine.7 This endogenous vasodilator also inhibits platelet adherence and aggregation, smooth muscle proliferation, and endothelial cell–leukocyte interactions, all of which are key events in various vascular diseases.7 We and others have demonstrated that endothelium-dependent, NO-mediated relaxation of pulmonary arteries is impaired in rats exposed to chronic hypoxia4 or injected with MCT,5 as well as in patients with pulmonary hypertension.8 9 Inhaled NO, which reaches the pulmonary vessels through an abluminal route, leads to selective pulmonary vasodilation in patients with pulmonary hypertension.10 11 Similarly, long-term NO inhalation prevents chronic pulmonary hypertension and reduces pulmonary vascular remodeling in rats exposed to chronic hypoxia.12 13 Intravenous L-arginine recently was shown to acutely reduce the PAP of patients with pulmonary hypertension by increasing the endogenous production of NO.14

In the present study, we investigated whether the prolonged administration of L-arginine would lessen chronic pulmonary hypertension and pulmonary vascular remodeling in rats exposed to chronic hypoxia or injected with MCT. In addition, we analyzed the effects of D-arginine, an L-arginine enantiomer, to evaluate the stereospecificity of this effect.


*    Methods
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 *Methods
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Study Design, Hemodynamic Studies, and Tissue Preparation
Pulmonary hypertension was induced by chronic hypoxia or MCT injection in S-D rats, as described previously.15 16 For chronic hypoxia–induced pulmonary hypertension, 59 S-D rats weighing 250 to 310 g (CLEA Japan Inc, Osaka, Japan) were randomly assigned to one of five groups: rats exposed to hypobaric hypoxia for 10 days (air at 380 mm Hg) and injected intraperitoneally with L-arginine (Nacalai Tesque Inc) (Hypoxia/LA), rats exposed to hypobaric hypoxia and injected intraperitoneally with D-arginine (Nacalai Tesque Inc) (Hypoxia/DA), rats exposed to hypobaric hypoxia and injected intraperitoneally with an equal volume of 0.9% saline vehicle (Hypoxia/V), rats exposed to ambient air and injected intraperitoneally with L-arginine (Air/LA), and rats exposed to ambient air and injected intraperitoneally with an equal volume of saline vehicle (Air/V). The animals, injected with L-arginine or D-arginine, received 500 mg·kg-1·d-1 of the compound suspended in 10 mL/kg saline. Injection of the compound began 3 days before the hypoxic exposure period and continued until the last day of hypoxic exposure. Food and water were provided ad libitum throughout the experiment. The rats were removed from the hypobaric chamber once daily for 15 minutes for the injection.

After the period of hypoxic exposure, the rats were anesthetized with pentobarbital sodium (33 mg/kg IP). A pulmonary artery catheter of silicone elastomer tubing (0.31 mm ID and 0.64 mm OD) was inserted through the right external jugular vein into the pulmonary artery by a closed-chest technique as described previously.17 PAP was monitored with a physiological transducer (Uniflow, Baxter), an amplifier system (AP-620G, Nihon Koden), and a monitor (Polygraph system, Nihon Koden). The catheter was passed under the skin and exteriorized at the back of the animal's neck. At 48 hours after catheterization, PAP was recorded in ambient air while the rat was fully conscious. After the hemodynamic measurement was performed in ambient air, an additional limited study was performed to determine PAP responses to acute hypoxia in each treatment group. PAP of the rats was monitored in a chamber flooded with 10% O2 and 90% N2 for 15 minutes.

For MCT induction of pulmonary hypertension, 38 S-D rats weighing 250 to 310 g (CLEA Japan Inc, Osaka, Japan) were randomly assigned to one of four groups: rats injected subcutaneously with 60 mg/kg MCT (Sigma Chemical Co) and intraperitoneally with L-arginine (500 mg·kg-1·d-1 in 10 mL/kg saline) (MCT/LA), rats injected subcutaneously with MCT and intraperitoneally with an equal volume of 0.9% saline vehicle (MCT/V), rats injected subcutaneously with an equal volume of saline and intraperitoneally with L-arginine (Sal/LA), and rats injected subcutaneously with saline and intraperitoneally with saline vehicle (Sal/V). MCT solutions were prepared from the crystalline compound and dissolved in pH 7.0 buffer.15 L-Arginine injection began 3 days before the injection of MCT and continued until day 17. On day 17, the rats were catheterized, and PAP was measured 48 hours later while the rats were fully conscious. To determine whether L-arginine was effective during the early or late period of MCT-induced pulmonary hypertension, we injected an additional 30 rats with MCT (60 mg/kg SC) and administered L-arginine (500 mg·kg-1·d-1) or an equal volume of saline intraperitoneally from day 9 to 17. On day 17, the rats were catheterized, and PAP was measured 48 hours later similarly.

After hemodynamic measurements were completed, a lung tissue was prepared for vascular morphometry as previously reported in detail.15 16 Briefly, after a rat was mechanically ventilated under pentobarbital sodium anesthesia, the lung was perfused through a pulmonary artery cannula with a hot (60°C) mixture of radiopaque barium and gelatin at 1000 mm H2O pressure for 5 minutes. Then the isolated lung was distended and fixed by perfusion through the tracheal tube with 10% formalin at 360 mm H2O pressure for 72 hours. A 10x10x5-mm tissue block, obtained from the midsection of the left lung, was processed for light microscopy by paraffin embedding. Sections were stained by the elastic van Gieson method. The RV was dissected from the LV+S and weighed separately. The ratios of RV/(LV+S) and of RV/BW were calculated.

Morphometric Analysis of Pulmonary Arteries
Light-microscopic slides were analyzed blindly without knowledge of the treatment groups. All barium-filled arteries >15 µm external diameter were assessed at 400x magnification. Each artery was first categorized according to its accompanying airway (ie, a terminal bronchiole, respiratory bronchiole, alveolar duct, or alveolar wall). The structural type of each artery was determined as muscular (ie, with a complete medial coat of muscle), partially muscular (ie, with only a crescent of muscle), or nonmuscular (ie, no apparent muscle). The percentage of muscular and partially muscular arteries at the alveolar wall and alveolar duct levels was determined. For all muscular arteries with an external diameter of 50 to 100 µm or 101 to 200 µm, the wall thickness of the media (ie, distance between external and internal elastic laminae) was measured along the shortest curvature and expressed as %MWT, that is, the percentage of the external diameter.

Systemic Blood Pressure
Systemic blood pressure was determined in control rats that did not receive MCT but were injected intraperitoneally with L-arginine (500 mg·kg-1·d-1) (n=6) or an equivalent volume of saline (n=5) for 21 days. Twenty-four hours after the last injection, systemic blood pressure was measured by use of a tail cuff attached to a blood pressure analyzer (UR-5000, Ueda).

Plasma Arginine Level
To determine the plasma level of arginine, nine additional weight-matched male S-D rats were injected intraperitoneally with L-arginine (500 mg/kg) (n=4) or an equal volume of saline (n=5) once daily for 10 days. On day 8, the rats were anesthetized with sodium pentobarbital (33 mg/kg), and central venous catheters were inserted via the right jugular vein for blood sampling. On day 10, blood samples (0.5 mL) were obtained just before as well as 30 minutes, 3 hours, 6 hours, 24 hours, and 48 hours after L-arginine injection. The blood was immediately centrifuged, and the plasma was stored at -80°C for arginine measurement by use of an automated amino acid analyzer (880-PU, Nihon Bunko).

Statistical Analysis
Data are presented as mean±SE. Differences between the treatment groups in BW and hemodynamic and morphological parameters were determined by a one-way ANOVA followed by Scheffé's F test. The effects of study groups and time points on plasma arginine levels were also determined similarly. The unpaired t test was used to evaluate the differences between study groups in blood pressure. A level of P<.05 was accepted as statistically significant.


*    Results
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Weight Gain
Hypoxic Rats
Initial BWs were similar in all five experimental groups. The hypoxic rats lost weight during the first 6 days of hypoxic exposure but regained some of it afterward (Fig 1Down, top). In contrast, the rats kept in ambient air gained weight steadily. On day 6 and the last day, hypoxic rats had significantly lower BWs than the control rats. Neither L-arginine nor D-arginine injection significantly affected BW in hypoxia-exposed or ambient animals.



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  		Figure 1. Effects of chronic hypoxia (top) and MCT injection (bottom) on the BW of rats. In both groups, final BWs of rats with pulmonary hypertension were lower than in control rats. Neither L-arginine (LA) nor D-arginine (DA) affected the BW of rats with pulmonary hypertension or of control rats. V indicates vehicle. Values are mean±SE. *P<.05 vs Air/V or MCT/V; {dagger}P<.05 vs Air/LA or MCT/LA.

MCT Rats
The animals in the four experimental groups had similar initial BWs. All rats gained weight steadily, but on days 6, 12, and 19, the MCT rats had significantly lower BWs than the control rats (Fig 1Up, bottom). L-Arginine treatment had no significant effect on BW in either group.

Plasma Arginine Level
Plasma levels of arginine were assessed after 10 days of L-arginine or saline injection. In the arginine-treated animals, the levels of L-arginine after the last injection rose from 317.2±54.1 to 1321.0±134.7 µmol/L within 30 minutes (P<.05 versus saline control) but returned to 316.6±52.9 µmol/L after 3 hours, 297.6±42.7 µmol/L after 6 hours, 274.6±67.0 µmol/L after 24 hours, and 230.1±84.9 µmol/L after 48 hours. Plasma levels of arginine before and >=3 hours after the last injection tended to be higher in the arginine-treated rats than in the saline-treated rats (244.1±37.8 µmol/L), but these differences were not statistically significant.

Systemic Blood Pressure
L-rginine treatment did not affect systemic blood pressure in the rats. Blood pressure was 119.3±2.3 mm Hg in rats injected with L-arginine compared with 121.3±3.6 mm Hg in rats injected with saline.

Hemodynamic Assessments and RV Hypertrophy
Hypoxic Rats
PAP was determined 48 hours after the last injection of L-arginine, when the plasma levels of arginine had returned to normal, to exclude any direct effects of L-arginine on PAP. The mean PAP was similar in Air/V rats (19.2±0.4 mm Hg) and Air/LA rats (18.1±0.7 mm Hg) (P=NS; Fig 2Down, top). Hypoxia/V rats exhibited significant pulmonary hypertension, with a PAP of 30.8±0.7 mm Hg (P<.05 versus Air/V). L-Arginine significantly reduced PAP in the hypoxic rats (24.8±0.7 mm Hg; P<.05 versus Hypoxia/V), whereas D-arginine had no effect (28.6±0.5 mm Hg; P=NS versus Hypoxia/V). Under conditions of acute hypoxia, the changes of PAP, expressed both as an absolute value and as a percent increase compared with the ambient-air value, were similar in the five treatment groups. Thus, acute hypoxia increased PAP from the ambient-air value by 6.6±1.7 mm Hg (33.8±7.8%) in Air/V rats (n=4), 3.6±1.4 mm Hg (21.0±7.7%) in Air/LA rats (n=6), 10.7±1.2 mm Hg (35.1±4.1%) in Hypoxia/V rats (n=10), 7.8±1.6 mm Hg (29.1±5.6%) in Hypoxia/LA rats (n=8), and 7.1±1.7 mm Hg (24.5±5.8%) in Hypoxia/DA rats (n=8).



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  		Figure 2. Effects of administering L-arginine (LA) or D-arginine (DA) for 14 days on mean PAP (top) and RV/(LV+S) ratio (bottom) in rats exposed to chronic hypoxia or ambient air. L-Arginine significantly but partially reduced mean PAP and RV/(LV+S) ratio in the hypoxic but not the control rats. D-Arginine had no effect. V indicates vehicle. Values are mean±SE. *P<.05 vs Air/V; {dagger}P<.05 vs Air/LA; {ddagger}P<.05 vs Hypoxia/V. IVS indicates interventricular septum.

The RV/(LV+S) and RV/BW ratios did not differ significantly between the Air/V and Air/LA groups. The RV/(LV+S) ratios were 0.27±0.01 in Air/V rats versus 0.27±0.01 in Air/LA rats (P=NS; Fig 2Up, bottom), and the respective RV/BW ratios were 0.47±0.03 versus 0.49±0.02 (P=NS). Hypoxia/V rats exhibited significant RV hypertrophy, reflected by an increased RV/(LV+S) ratio (0.37±0.01; P<.05 versus Air/V) and an increased RV/BW ratio (0.75±0.02; P<.05 versus Air/V). L-Arginine treatment reduced the extent of RV hypertrophy in the hypoxic rats, as indicated by significantly lower RV/(LV+S) and RV/BW ratios (0.31±0.01 and 0.62±0.02, respectively; P<.05 versus Hypoxia/V). Animals in the Hypoxia/DA group, in contrast, had significant RV hypertrophy, as indicated by an RV/(LV+S) ratio of 0.35±0.01 and an RV/BW ratio of 0.70±0.03, similar to the Hypoxia/V groups.

MCT Rats
The mean PAP was similar in the control Sal/V rats or Sal/LA rats (18.8±1.0 and 19.6±1.2 mm Hg, respectively; P=NS). The MCT/V rats exhibited significant pulmonary hypertension (34.8±2.0 mm Hg; P<.05 versus Sal/V), which was reduced by L-arginine treatment (24.3±1.7 mm Hg; P<.05 versus MCT/V) (Fig 3Down, top).



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  		Figure 3. Effects of administering L-arginine for 21 days on mean PAP (top) and RV/(LV+S) ratio (bottom) in rats injected with MCT or saline. L-Arginine (LA) significantly and completely reduced mean PAP and RV/(LV+S) ratio in rats injected with MCT but not in those injected with saline. V indicates vehicle. Values are mean±SE. *P<.05 vs Sal/V; {dagger}P<.05 vs Sal/LA; {ddagger}P<.05 vs MCT/V. IVS indicates interventricular septum.

The RV/(LV+S) and RV/BW ratios were similar in Sal/V and Sal/LA rats. Thus, the RV/(LV+S) ratio was 0.26±0.01 in Sal/V and 0.25±0.02 in Sal/LA animals (P=NS; Fig 3Up, bottom), and the RV/BW ratios were 0.52±0.04 and 0.43±0.02, respectively (P=NS). MCT/V rats had significant RV hypertrophy, as indicated by a higher RV/(LV+S) ratio (0.35±0.01; P<.05 versus Sal/V). The RV/BW ratio also tended to be higher in MCT/V (0.64±0.02) than in Sal/V animals, but this difference was not statistically significant. L-Arginine treatment reduced RV hypertrophy, as indicated by a lower RV/(LV+S) ratio in the MCT/LA animals (0.29±0.01; P<.05 versus MCT/V).

Morphometric Analysis of Pulmonary Arteries
Hypoxic Rats
Treatment with L-arginine did not affect the muscularization of pulmonary arteries in the control animals at the alveolar wall or alveolar duct levels (Fig 4Down). At the level of the alveolar wall, the percentages of muscularized arteries in Air/V and Air/LA animals were 5.7±3.4% and 9.9±3.9%, respectively (P=NS). At the alveolar duct level, the respective values were 4.7±2.0% and 10.1±5.0% (P=NS). In Hypoxia/V animals, an increased percentage of normally nonmuscular arteries were muscularized (74.6±2.9% at the alveolar wall level and 74.2±3.6% at the alveolar duct level; P<.05 versus Air/V). This effect was attenuated by L-arginine treatment, with muscularization levels in the Hypoxia/LA animals of 21.7±2.9% at the alveolar wall level (P<.05 versus Hypoxia/V) and 28.3±2.1% at the alveolar duct level (P<.05 versus Hypoxia/V). Treatment with D-arginine, however, had no effect on muscularization (84.1±3.3% at the alveolar wall level and 81.6±2.9% at the alveolar duct level; P=NS versus Hypoxia/V).



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  		Figure 4. Effects of administering L-arginine (LA) or D-arginine (DA) for 14 days on the percentage of muscularized arteries (% Muscularization) in normally nonmuscular peripheral pulmonary arteries at the alveolar duct and alveolar wall levels in rats exposed to chronic hypoxia or ambient air. L-Arginine significantly reduced percent muscularization partially at the alveolar duct level and completely at the alveolar wall level in hypoxic rats but not in control rats. D-Arginine had no effects. V indicates vehicle. Values are mean±SE. *P<.05 vs Air/V; {dagger}P<.05 vs Air/LA; {ddagger}P<.05 vs Hypoxia/V.

We also analyzed the %MWT for two groups of arteries, those with external diameters of 50 to 100 µm and those with external diameters of 101 to 200 µm. In the control rats, %MWT was unaffected by arginine treatment. The 50- to 100-µm arteries had a %MWT of 3.3±0.1% and 4.3±0.8% in the Air/V and Air/LA rats, respectively (P=NS; Fig 5Down). For the 101- to 200-µm arteries, the corresponding values were 3.2±0.2% and 3.7±0.3% (P=NS). In Hypoxia/V rats, the %MWT increased significantly to 6.4±0.5% in the 50- to 100-µm arteries and 6.7±0.7% in 101- to 200-µm arteries (P<.05 versus Air/V). Hypoxia/LA rats, in contrast, had a significantly decreased %MWT in the 101- to 200-µm arteries (3.8±0.2%; P<.05 versus Hypoxia/V). For the 50- to 100-µm arteries, the %MWT was also decreased in Hypoxia/LA rats (4.4±0.2%) compared with Hypoxia/V rats, but the differences were not statistically significant. In Hypoxia/DA rats, the %MWT was similar to Hypoxia/V animals (7.8±1.0% for 50- to 100-µm arteries and 7.2±1.0% for 101- to 200-µm arteries).



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  		Figure 5. Effects of administering L-arginine (LA) or D-arginine (DA) for 14 days on the %MWT of muscular pulmonary arteries with external diameters of 50 to 100 µm and 101 to 200 µm in rats exposed to chronic hypoxia or ambient air. In hypoxic rats, L-arginine significantly and completely reduced %MWT in the 101- to 200-µm arteries but not in 50- to 100-µm arteries. D-Arginine had no effects. L-Arginine did not affect %MWT of any arteries in control rats. V indicates vehicle. Values are mean±SE. *P<.05 vs Air/V; {dagger}P<.05 vs Air/LA; {ddagger}P<.05 vs Hypoxia/V.

MCT Rats
The Sal/V and Sal/LA rats exhibited similar degrees of muscularization at the alveolar wall level (5.3±1.2% and 3.1±1.3%, respectively) and at the alveolar duct level (4.4±0.9% and 4.8±0.6%, respectively; Fig 6Down). In the MCT/V animals, the degree of muscularization increased significantly to 83.1±3.9% at the alveolar wall level and 83.6±3.9% at the alveolar duct level (P<.05 versus Sal/V). L-Arginine treatment reduced the muscularization to 22.2±4.3% at the alveolar wall level and 28.2±4.4% at the alveolar duct level (P<.05 versus MCT/V).



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  		Figure 6. Effects of administering L-arginine (LA) for 21 days on the percentage of muscularized arteries (% Muscularization) in normally nonmuscular peripheral pulmonary arteries at the alveolar duct and alveolar wall levels in rats injected with MCT or saline. L-Arginine partially but significantly reduced percent muscularization at the alveolar duct and the alveolar wall levels in MCT rats but not in control rats. D-Arginine had no effect. V indicates vehicle. Values are mean±SE. *P<.05 vs Sal/V; {dagger}P<.05 vs Sal/LA; {ddagger}P<.05 vs MCT/V.

The %MWT of muscular arteries was similar among the Sal/V and Sal/LA control animals: 3.8±0.3% and 3.8±0.1%, respectively, for the 50- to 100-µm arteries, and 3.9±0.3% and 3.7±0.3% for the 101- to 200-µm arteries (Fig 7Down). In the MCT/V rats, the %MWT was significantly increased compared with the Sal/V rats (6.8±0.4% for the 50- to 100-µm arteries and 6.7±0.8% for 101- to 200-µm arteries; P<.05). L-Arginine reversed this increase: the %MWT in the MCT/LA rats was 3.9±0.4% for 50- to 100-µm arteries and 3.8±0.3% for 100- to 200-µm arteries (P<.05).



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  		Figure 7. Effects of administering L-arginine (LA) for 21 days on %MWT of muscular pulmonary arteries with external diameters of 50 to 100 µm and 101 to 200 µm in rats injected with MCT or saline. L-Arginine significantly and completely reduced %MWT in both categories of arteries in MCT rats but not in control rats. V indicates vehicle. Values are mean±SE. *P<.05 vs Sal/V; {dagger}P<.05 vs Sal/LA; {ddagger}P<.05 vs MCT/V.

Effects of L-Arginine on the Late Stage of MCT-Induced Pulmonary Hypertension
L-Arginine supplementation from day 9 to 17 after MCT injection did not affect final BW and hematocrit values (TableDown). Moreover, the mean PAP and RV hypertrophy, expressed as RV/(LV+S) and RV/BW, were not reduced by L-arginine treatment in the late stage. Whereas %MCT in the 50- to 100-µm and 101- to 200-µm arteries did not change after late-stage L-arginine treatment, the percent muscularization at the alveolar duct and alveolar wall levels was slightly but significantly reduced (P<.05).


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  		Table 1. BW, Hemodynamic Measurements, and Morphometric Pulmonary Parameters After Late-Stage L-Arginine Treatment


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we have demonstrated that prolonged administration of the NO precursor L-arginine alleviated pulmonary hypertension, RV hypertrophy, and pulmonary vascular remodeling in rats with chronic hypoxia–induced or MCT-induced pulmonary hypertension. D-Arginine, an enantiomer of L-arginine, did not inhibit these pulmonary hypertensive changes. These findings suggest that L-arginine supplementation exerts its effects by modifying endogenous NO production.

To induce pulmonary hypertension in rats by chronic hypoxia, we exposed the animals to 10 days' hypobaric hypoxia of 380 mm Hg, which is equivalent to living at an altitude of 5500 m.18 19 This experimental period was selected because the degree of RV hypertrophy and medial thickening reached a plateau after a 10-day treatment.18 19 Moreover, this experimental protocol in rats is equivalent to keeping beagles at an altitude of 3100 m for 12 months20 or to humans living at altitudes >3000 m.21 To investigate the effects of L-arginine treatment, we administered 500 mg/kg of the drug by once-daily intraperitoneal injection. We chose this dose because preliminary experiments using 200 mg·kg-1·d-1 L-arginine for 10 days (Hypoxia/V, n=5; Hypoxia/LA, n=8) found no significant inhibition of RV hypertrophy in hypoxic rats (data not shown). L-Arginine at 500 mg·kg-1·d-1 was well tolerated by the animals and did not affect their BW gain. This dose resulted in a transient fivefold increase in plasma arginine levels to >1 mmol/L, a concentration that has been shown to restore endothelium-dependent relaxation of perfused lungs from hypoxic rats.22 Although in some previous studies, L-arginine had been administered in the drinking water,23 24 we chose intraperitoneal injection to ensure the administration of constant L-arginine amounts throughout the experiment. Chronically hypoxic rats drink little during the first several days of hypoxic exposure and therefore do not receive sufficient L-arginine from the drinking water. Although the once-daily administration of L-arginine increased plasma levels only transiently, its effects were physiologically significant. For example, salt-sensitive hypertension in Dahl/Rapp rats is abrogated by the intraperitoneal administration of L-arginine (250 mg·kg-1·d-1) for 2 weeks.25 Similarly, the endothelium-dependent nephrotoxicity induced by cyclosporin A is improved after 15 days of intraperitoneal L-arginine administration (200 mg·kg-1·d-1).26 To further investigate the mechanism underlying the effects of L-arginine and to determine whether it is effective during the early or late stage of MCT-induced pulmonary hypertension, we performed an additional series of experiments during which the drug was administered from day 9 after the injection of MCT. This time was chosen because a previous study2 had demonstrated that PAP increased only after day 8, whereas the muscularization of peripheral small arteries occurred earlier.

In the present study, L-arginine alleviated pulmonary vascular remodeling in rats with pulmonary hypertension but did not affect PAP and systemic blood pressure in control rats, consistent with previous results.22 23 24 25 27 28 29 For example, in a study using vascular strips, exogenous L-arginine did not enhance acetylcholine-induced endothelium-dependent relaxation in freshly prepared normal vessels but did restore endothelium-dependent relaxation in isolated bovine pulmonary arteries after prolonged tension or exposure to calcium ionophore.27 28 These data suggest that although endogenous L-arginine is sufficient to saturate NO synthase in normal vessels, intracellular L-arginine may be overutilized in the latter conditions. Similarly, endothelial cells cultured in arginine-deficient medium released little NO until exogenous L-arginine was supplemented.29 The present study also confirmed that L-arginine but not D-arginine affected pulmonary parameters. Similarly, only L-arginine restored impaired acetylcholine-induced pulmonary vasodilation in perfused lungs from chronic hypoxic rats.22 In diseases such as atherosclerosis in hypercholesterolemic rabbits,23 intimal hyperplasia after balloon injury in rabbits,24 and salt-sensitive hypertension in Dahl/Rapp rats,25 the administration of L-arginine but not D-arginine improved the endothelium-dependent relaxation of the diseased vessels. These results suggest that the prevention of pulmonary hypertensive changes by L-arginine in rats may be mediated at least in part by endothelial NO production. The changes in PAP in response to acute hypoxia did not differ between the Air/V and Hypoxia/V groups, which is consistent with a previous study.30 The effects of L-arginine on PAP responses were not confirmed. Because L-arginine levels completely returned to control levels in 48 hours after the last injection of L-arginine, a lack of its direct effect may explain their similar responses.

Studies have not consistently confirmed the effects of L-arginine on pulmonary parameters. For example, one report31 found that administration of L-arginine in drinking water for 3 weeks did not reduce pulmonary hypertension and RV hypertrophy in hypoxic rats. However, because hypoxic rats drink little for the first several days, it is questionable whether the animals received a sufficient amount of L-arginine; plasma arginine levels were not measured. Another study32 using a vascular-strip method showed that exogenous L-arginine did not restore the impaired endothelium-dependent relaxation of pulmonary arteries associated with chronic obstructive lung disease. The discrepancies between these findings and our study may be due to differences in the methods used and in the stages of the pulmonary changes analyzed. The influence of the method of L-arginine administration is emphasized by findings that in vivo pretreatment of hypoxic rats with L-arginine restores the endothelium-dependent relaxation of isolated pulmonary arteries, whereas the addition of L-arginine to an organ bath did not restore the vasodilatory response.33

Several other potential mechanisms exist for reducing pulmonary vascular disease in addition to the vasodilatory effect of enhanced endogenous NO production. For example, one may speculate that the effects of L-arginine are related to the direct effects of NO on smooth muscle cell proliferation and/or hypertrophy. NO has been shown to inhibit hypoxia-induced expression of endothelin 1 and PDGF B-chain in endothelial cells.34 The role of endothelin has been supported by findings that an endothelin antagonist ameliorated pulmonary hypertension and vascular remodeling in hypoxic rats.35 Furthermore, increased expression of PDGF A- and B-chain genes was found in lungs in the same model.36 Because both of these substances are potent vasoconstrictors and mitogens, NO may reduce smooth muscle cell proliferation by interfering with endothelial production of these substances. In the present study, L-arginine reduced both hypoxia-induced and MCT-induced pulmonary vascular remodeling. Inhaled NO, in contrast, reduced pulmonary hypertension and pulmonary vascular remodeling in chronically hypoxic rats12 but not in rats with MCT-induced pulmonary hypertension, even though acutely inhaled NO reduced PAP in these animals.37 Therefore, the inhibitory effects of inhaled NO on pulmonary hypertensive changes may depend on the mechanism or causes that underlie pulmonary hypertension. Little is known about the different biological effects that inhaled NO and systemically administered L-arginine have on pulmonary vascular changes. In hypoxic rats, hypoxic pulmonary vasoconstriction precedes the pulmonary vascular changes,17 whereas in MCT-treated rats, endothelial cell injury caused by MCT precedes pulmonary hypertension and pulmonary vascular remodeling.2 Pulmonary vascular disease in hypoxic rats may be alleviated by reducing the vasoconstriction that precedes pulmonary vascular remodeling. In MCT-injected rats, in contrast, a platelet-activating factor antagonist,38 a serotonin synthesis inhibitor,39 and an IL-1 antagonist40 have been reported to prevent pulmonary hypertension. These observations, together with the findings of leukocyte infiltration in the lungs of rats injected with MCT,41 suggest that platelets and leukocytes contribute to MCT-induced pulmonary hypertension in rats. Prolonged oral L-arginine administration inhibits platelet aggregation in humans.42 Furthermore, NO inhibits the IL-1{alpha}–stimulated expression of proinflammatory cytokines (IL-6, IL-8) and cellular adhesion molecules (VCAM-1, E-selectin, and ICAM-1) by inhibiting a transcription factor, nuclear factor-{kappa}B.43 Finally, the antiatherogenic effects of L-arginine in hypercholesterolemic rabbits were associated with the inhibition of monocyte-endothelium interactions via increased endothelial NO production.44 Together, these observations suggest that NO may alleviate MCT-induced pulmonary hypertension by inhibiting platelet function and leukocyte adhesion.

The present study also demonstrated that L-arginine specifically affects MCT-induced pulmonary hypertension during the early induction of vascular disease. This finding is interesting, as it is consistent with findings that an elastase inhibitor prevents muscularization of small arteries in the early period of MCT-induced pulmonary hypertension.15 NO inhibits neutrophil superoxide anion via a direct effect on NADPH oxidase.45 Because NO avidly scavenges superoxide anion, it can also prevent superoxide anion from forming hydrogen peroxide.46 Such reactive oxygen species inactivate an endogenous elastase inhibitor, {alpha}1-protease inhibitor.47 Reactive oxygen species also induce early response genes such as c-fos, which is involved in activation of a transcription factor, AP-1, required for the production of collagenase and other matrix metalloproteinases.48 These findings suggest that L-arginine exerts its effects via the inhibition of elastolytic or other proteolytic activities in the lung. It remains to be determined, however, whether L-arginine supplementation can inhibit vascular elastolysis in MCT-induced pulmonary hypertension or whether increased intracellular NO levels can transcriptionally regulate the expression of vascular elastase.

The modulation of endothelial NO production may be a new therapeutic concept in the treatment of pulmonary hypertension. Recent studies have demonstrated the upregulation of endothelial NO production by ACE inhibitors,49 estrogen,50 and fish oil51 as well as L-arginine, even in clinical cases of atherosclerosis, systemic hypertension, and heart failure.49 The possible role of modulating endothelial NO production in the treatment of pulmonary hypertension therefore warrants further investigation.


*    Selected Abbreviations and Acronyms
 
Air/LA = rats exposed to ambient air and injected with L-arginine
Air/V = rats exposed to ambient air and injected with an equal volume of saline vehicle
BW = body weight
Hypoxia/DA = rats exposed to hypobaric hypoxia and injected with D-arginine
Hypoxia/LA = rats exposed to hypobaric hypoxia and injected with L-arginine
Hypoxia/V = rats exposed to hypobaric hypoxia and injected with an equal volume of 0.9% saline vehicle
IL = interleukin
LV+S = left ventricle plus septum
MCT = monocrotaline
MCT/LA = rats injected with monocrotaline and L-arginine
MCT/V = rats injected with monocrotaline and an equal volume of 0.9% saline vehicle
NO = nitric oxide
PAP = pulmonary arterial pressure
PDGF = platelet-derived growth factor
%MWT = percent medial wall thickness
RV = right ventricle, right ventricular
Sal/LA = rats injected with an equal volume of saline and L-arginine
Sal/V = rats injected with saline and saline vehicle
S-D = Sprague-Dawley


*    Acknowledgments
 
This work was supported in part by grants-in-aid for scientific research (No. 07771228) from the Japanese Ministry of Education, Science and Culture and grants for pediatric research (Nos. 5C-02 and 8C-2) from the Japanese Ministry of Health and Welfare.

Received October 14, 1996; revision received December 17, 1996; accepted January 9, 1997.


*    References
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