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Am J Physiol Lung Cell Mol Physiol 292: L1480-L1487, 2007. First published February 23, 2007; doi:10.1152/ajplung.00360.2006
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Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

Akihito Sasaki,1 Shouzaburoh Doi,1 Shuki Mizutani,1 and Hiroshi Azuma2

1Department of Pediatrics and Developmental Biology, and 2Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, Kanda, Chiyoda-ku, Tokyo, Japan

Submitted 14 September 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares L-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.

L-arginine-nitric oxide pathway; dimethylarginine dimethylaminohydrolase

PULMONARY HYPERTENSION (PH) is a fatal disease characterized by intimal and medial hyperplasia and adventitial fibrosis in peripheral pulmonary arteries. Impairment of endogenous release of nitric oxide (NO), which causes vasodilation (38) and inhibition of both platelet aggregation (3) and vascular smooth muscle cell proliferation (17), has been proposed to be important in developing the PH (1, 13). Actually, under clinical settings, NO level in lung tissue is lower in patients with PH compared with healthy controls (22), and NO inhalation is a specific pulmonary vasodilator therapy (39). In addition, it has been reported that short-term oral administration of L-arginine improves hemodynamics and exercise capacity in patients with precapillary PH (35).

NO production, on one hand, depends on the nitric oxide synthase (NOS) activity and protein expression. There is a report describing that the NOS activity was decreased in the rabbit corpus cavernosum after ischemia (29). In contrast, whether or not impaired NO production in PH is due to decreased expression of NOS protein has been controversial. According to Tyler et al. (44), expression of endothelial NOS (eNOS) protein was decreased in lung of monocrotaline (MCT)-induced PH rats, whereas eNOS immunostaining in pulmonary arteries was increased. Giaid and Saleh (18) have demonstrated the reduced eNOS expression in the lungs of patients with PH, whereas eNOS protein expression was enhanced in plexiform lesion in patients with PH (27). On the other hand, NO production absolutely depends on the availability of L-arginine to NOS, since NOS shares L-arginine as a common substrate with arginase (11, 34). In this regard, the L-arginine catabolism via the arginase pathway can act as an endogenous negative control system to regulate overall NO production. Recent studies have demonstrated the increases in arginase activity and arginase II expression in patients with PH (46). Furthermore, the elevated endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) may be another important factor in impaired NO production (4, 45). These methylarginines competitively inhibit the NOS activity and impair NO production in endothelial cells. An elevated level of ADMA has been found in multiple disorders where NOS dysfunction has been implicated such as hypercholesterolemia (9), renal failure (45), hypertension (43), and hyperglycemia (28). MMA and ADMA are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and methylamines (37). Therefore, the impaired DDAH activity results in the accumulation of endogenous NOS inhibitors (25, 26), thereby impairing NO production. Recently, it has been reported that dysregulation of DDAH activity is implicated in PH (2, 32, 40). Considering the mechanisms that cause PH, it appears important to analyze the pathogenesis of PH by comprehensively evaluating the alterations in L-arginine metabolism.

Thus we designed the present experiments to investigate whether the ability to produce NO, eNOS activity and protein expression, endogenous NOS inhibitors in endothelial cells, arginase activity, and DDAH activity and protein expression were implicated in MCT-induced PH rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. Male Sprague-Dawley rats (170–230 g body wt) were used for all experiments. They received a single subcutaneous injection of saline (control) or MCT (60 mg/kg; Sigma, St. Louis, MO), which was known to cause severe PH in rats. We performed all experiments on day 24 after MCT injection. After the rats were anesthetized with sodium pentobarbital (30 mg/kg ip), right ventricular systolic pressure (RVP) was directly measured by the subcutaneous puncture with the tip of a needle connected to a pressure transducer into the right ventricle (RV). After measurement of RVP, the RV was dissected from the left ventricle (LV) and interventricular septum (IVS) and weighed for evaluating the extent of RV hypertrophy as the weight ratio of RV/(LV+IVS). The extrapulmonary arteries were carefully isolated and cleaned of their connective tissue in ice-cold modified Krebs solution. The composition of the modified Krebs solution was in mM 118.0 NaCl, 4.7 KCl, 1.2 MgSO4·7H2O, 2.5 CaCl2·2H2O, 1.2 KH2PO4, 25.0 NaHCO3, and 10.0 glucose (pH 7.4). The freshly isolated pulmonary artery specimens were used for isometric tension experiments and measurement of cGMP production. Remaining pulmonary arteries were frozen at –80°C until used for enzyme activity assays. According to the method described previously (6), pulmonary artery endothelial cells (PAECs) were collected by gently rubbing the luminal surface with each buffer specified for enzyme activity assays and Western blot analyses. To confirm the endothelial origin of the collected cells, they were stained using von Willebrand factor antibody (rabbit polyclonal antibody for human von Willebrand factor; Dako, Kyoto, Japan) and the labeled streptavidin-biotin staining reagent [LSAB kit/horseradish peroxidase (HRP); Dako] according to the method described previously (6). The population of von Willebrand factor-positive cells was determined as >98% of the collected cells. In addition, scanning electron microscopy revealed that the internal elastic lamina remained intact, and few endothelial cells were detectable after the collection by gently rubbing the luminal surface.

The present experimental protocol complied with and was approved by the Animal Welfare Regulation of Tokyo Medical and Dental University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society.

Isometric tension measurements. Extrapulmonary arteries were cut into transverse strips (~2 mm in width and 5 mm in length) and submerged in 10-ml organ chambers filled with Krebs solution maintained at 37 ± 0.5°C and continuously bubbled with 95% O2 and 5% CO2. One end of each strip was connected to a force-displacement transducer (TB-612T; Nihon-Kohden Kogyo, Tokyo, Japan) to record the changes in isometric tension on a pen-writing oscillograph (R64; Rika Denki Kogyo, Tokyo, Japan). The strip length was adjusted several times until a stable resting tension of 1 g was attained. In the preliminary experiments, we compared the contraction caused by 10–5 M phenylephrine (PE) and relaxation in response to 10–6 M acetylcholine (ACh) during the PE contraction under the basal resting tension of 0.5, 1, and 1.5 g. We determined that the reproducibility, magnitude, and stability of the responses were optimal under the basal resting tension of 1 g. Before beginning the experiments, strips were allowed to equilibrate for at least 60 min in the bathing solution, and during this period, the bathing solution was replaced every 20 min with fresh solution. All experiments were performed in the presence of guanethidine (10–5 M) as an inhibitor of adrenergic neurotransmission and indomethacin (10–6 M) as an inhibitor of cyclooxygenase. Endothelium-dependent relaxation (EDR) in response to ACh was measured during the contraction caused by 10–5 M PE. After the 60-min washout period, the strips were precontracted again with 10–5 M PE, and then endothelium-independent relaxation (EIR) to sodium nitroprusside (SNP) was examined in the presence of 10–4 M NG-nitro-L-arginine (NOARG) as an authentic NOS inhibitor. ACh (10–9 M to 3 x 10–5 M) and SNP (10–10 M to 3 x 10–6 M) were applied into organ chamber in a cumulative fashion at increasing concentrations of 0.5 log unit. ACh- or SNP-induced maximum relaxation was respectively represented as a percentage of their dilatory effect against the PE (10–5 M)-induced contraction.

Measurement of cGMP. The cGMP level was determined according to the method described previously (36). Briefly, extrapulmonary arteries weighing ~10 mg with intact endothelium were preincubated in modified Krebs solution under the 1-g resting tension for 20 min at 37°C in the organ chamber. After they were washed with fresh Krebs solution, tissues were subjected to further 30-min incubation. Preparations were then rapidly transferred into 10% trichloroacetic acid (TCA) and snap-frozen with liquid nitrogen to stop the reaction. In contrast, norepinephrine (NE; 10–6 M) and ACh (10–5 M) were added immediately and 15 min after transferring the specimens into the fresh modified Krebs solution, respectively. All experiments were performed in the presence of 10–5 M 3-isobutyl-1-methylxanthine (IBMX) as a nonselective inhibitor of phosphodiesterases. The net production of cGMP was calculated as the difference between the production in the presence of 10–4 M NOARG plus NE plus ACh and that in the presence of NE plus ACh. The basal cGMP level was taken as the value without any agonist and antagonist except for IBMX.

Measurement of NOS activity. NOS activity in PAECs was measured by determining the conversion of L-[14C(U)]arginine (specific activity: 11,581 MBq/mmol) to L-[14C(U)]citrulline as described previously (29). In brief, PAECs were collected into the buffer, which was composed of 50 mM Tris·HCl, 10 mM CHAPS, 2 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µM pepstatin A, and 2 µM leupeptin (pH 7.4). The collected PAECs were sonicated at 50 W, 28 kHz for 15 s (3 times at 1-min intervals) and centrifuged at 10,000 g for 20 min at 4°C to separate the supernatant, in which protein concentration was determined using bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL). Incubation mixtures consisted of 100 µl of the supernatant and 20 µl of the buffer described above containing 1 mM NADPH, 4 µM FAD, 4 µM flavin mononucleotide, 10 µM tetrahydrobiopterin (BH4), 1 mg/l calmodulin, 2.5 mM CaCl2, and 0.1 µCi/ml L-[14C(U)]arginine. The reaction mixture was incubated at 37°C for 1 h in a shaking water bath. The incubation was terminated by keeping the tubes on ice for 5 min. Samples were then applied to a 1-ml column of Dowex AG 50W-X8 (Na+ form) to remove unmetabolized L-[14C(U)]arginine. The columns were then washed with 1.5 ml of distilled water, and L-[14C(U)]citrulline was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB 2750TR/LL; Packard Instrument, Meriden, CT). NOS activity was expressed as pmol L-citrulline·mg protein–1·60 min–1. The activity was also measured in the presence of 10–4 M NOARG or 30 µM aminoguanidine as a selective inhibitor for inducible NOS (iNOS). Moreover, calcium-independent NOS activity was measured in the calcium-free incubation medium with 20 mM EDTA. The net activity was expressed as the difference between activities in the absence and presence of NOARG.

Measurement of arginase activity. The arginase activity in PAECs was measured in the same preparations as those obtained for the determination of NOS activity. Arginase activity was measured by determining the conversion of L-[guanido-14C]arginine to [14C]urea according to the method described previously (42). Aliquots of PAEC extracts (10 µl) were incubated in a final volume of 100-µl buffer containing 9 mM Tris·HCl, 0.08 µCi/ml of L-[guanido-14C]arginine (specific activity: 1,776 MBq/mmol), and 1 mM MnCl2, pH 9.6, for 2 h at 37°C. Reactions were terminated by the addition of 400 µl of ice-cold stop buffer containing 250 mM sodium acetate and 100 mM urea, pH 4.5. Samples were passed through a column containing 1.5 ml of Dowex 50W-X8 resin to remove unmetabolized L-[guanido-14C]arginine. The columns were then washed with 1.5 ml of distilled water, and [14C]urea was quantified in the flow-through fraction using a liquid scintillation counter. The enzyme activity was determined in the presence or absence of 20 µM NG-hydroxy-L-arginine (NOHA) as an inhibitor of arginase. Results were given as the net activity calculated from the difference in the activities between the presence and absence of NOHA.

Contents of L-arginine and methylarginines. Contents of MMA, ADMA, symmetric dimethylarginine (SDMA), and L-arginine in PAECs were determined by means of HPLC as reported previously (6, 29). PAECs were collected from the pulmonary artery specimens by gently rubbing the luminal surface according to the method described previously (6). The collected PAECs were sonicated at 50 W, 28 kHz for 15 s (3 times at 1-min intervals) in ice-cold 5 mM HEPES buffer (pH 7.4) and centrifuged at 10,000 g for 20 min at 4°C to separate the supernatant, which was lyophilized with the aid of the centrifugal vaporizer (CVE-100D; Eyela, Tokyo, Japan). Twenty-microliter aliquots of the reconstituted solution with HEPES buffer were assayed for DNA by a fluorometric method. After addition of TCA in a final concentration of 5%, PAECs were centrifuged at 1,600 g for 15 min, and the supernatant was obtained, of which a 100-µl aliquot was applied for HPLC to determine L-arginine and methylarginines. The minimum concentration of methylarginines that can be detected was 5 pM. The intracellular concentrations of MMA, ADMA, SDMA, and L-arginine were determined according to the method described previously (5, 19).

Measurement of dimethylarginine dimethylaminohydrolase activity. DDAH activity was measured by determining the conversion of [3H]MMA [NG-monomethyl-L-arginine(2,3,4-3H)] to L-[3H]citrulline. The PAECs were collected in the buffer, which was composed of 100 mM sodium phosphate buffer (pH 6.5), 1 mM PMSF, 2 mM 2-mercaptoethanol, 1 µM pepstatin A, and 2 µM leupeptin. The collected PAECs were sonicated at 50 W, 28 kHz for 15 s (3 times at 1-min intervals) and centrifuged at 10,000 g for 20 min at 4°C to separate the supernatant, in which protein concentration was determined using BCA protein assay reagent kit (Pierce). Incubation mixtures consisted of 90 µl of the supernatant and 10 µl of the buffer described above containing 20 mM EDTA, 0.1 µM MMA, and 0.01 µCi/ml of [3H]MMA. [3H]MMA (specific activity: 2.00 TBq/mmol) was manufactured by Daiichi Pure Chemicals (Ibaraki, Japan). Radiochemical purity of [3H]MMA, which had been determined by DuPont NEN Analytical Service (Boston, MA) with the aid of HPLC, was 97.6%. The reaction mixture was incubated at 37°C for 2 h in a shaking water bath. The incubation was terminated by keeping the tubes on ice for 5 min. Samples were applied to a 1-ml column of Dowex AG 50W-X8 (Na+ form) to remove unmetabolized [3H]MMA. The columns were then washed with 1.5 ml of distilled water, and L-[3H]citrulline was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB 2750TR/LL, Packard Instrument). DDAH activity was expressed as pmol L-citrulline·mg protein–1·120 min–1.

Western blot analysis. The PAECs were collected in lysis buffer containing 50 mM Tris, pH 7.5, 300 mM NaCl, 1% Triton X-100 according to the method described previously (6). The PAEC samples (10 µg of protein for NOS, 15 µg of protein for DDAH) were subjected to 10% SDS polyacrylamide gel and blotted onto polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL) with a wet transfer unit (Bio-Rad, Hercules, CA). The membranes were incubated with monoclonal antibody against anti-eNOS and anti-iNOS (BD Biosciences, San Jose, CA), polyclonal antibody against anti-DDAH1 (Orbigen, San Diego, CA) and anti-DDAH2 (Abcam, Cambridge, UK), followed by respective HRP-conjugated secondary antibodies. Mouse macrophages stimulated with IFN-{gamma}/LPS (BD Biosciences) were used for positive control for protein expression of iNOS. The bands were visualized using an enhanced ECL detection kit (Amersham) and by densitometry. Equal amount of protein loading was confirmed by blotting membranes with a monoclonal antibody against anti-beta-actin (Sigma).

Data analysis. Cumulative concentration-response curves to ACh or SNP were statistically analyzed by two-way repeated measures ANOVA. All other data are presented as means ± SE. For comparison between two groups, unpaired Student's t-test was used. Significance was accepted at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MCT-induced PH and RVP. Twenty-four days after MCT injection, the RVP was determined to be 28 ± 2.6 mmHg in the control (n = 12) and 55 ± 2.4 mmHg in the MCT group (n = 12; P < 0.05). In addition, RV/(LV+IVS) weight ratio as a marker of RV hypertrophy was 0.22 ± 0.004 in the control and 0.43 ± 0.02 (P < 0.0001) in the MCT group (Fig. 1, A and B).


Figure 1
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  		Fig. 1. The monocrotaline (MCT) group showed increased right ventricular systolic pressure (RVP) (A) and weight ratio of right ventricle (RV) to left ventricle (LV) and interventricular septum (IVS) (LV+IVS), which represent pulmonary hypertension (PH; n = 12) (B). C: comparison of the endothelium-dependent relaxation (EDR) in the pulmonary artery (PA) specimens of control and MCT group. The EDR was obtained from the concentration-response curves for acetylcholine (ACh; 10–9 to 3 x 10–5 M) during the contraction caused by 10–5 M phenylephrine (PE; n = 4). D: comparison of the endothelium-independent relaxation (EIR) in the PA specimens of control and MCT group. The EIR was obtained from the concentration-response curves for sodium nitroprusside (SNP; 10–10 to 3 x 10–6 M) during the contraction caused by 10–5 M PE (n = 4). Open circle indicates control group, and closed circle indicates MCT group. Results are expressed as a percentage of the PE contraction. Results are given as means ± SE. *P < 0.05; #P < 0.0001.

 
Isometric tension measurement. The ACh-induced EDR in isolated pulmonary artery specimens was significantly (P < 0.05) impaired in the MCT group (Fig. 1C). The SNP-induced EIR was not different between MCT and control groups (Fig. 1D).

cGMP production. The cGMP production as a marker of NO production was examined in pulmonary artery specimens with endothelial cells (20). Both the basal and the net production of cGMP were significantly decreased in MCT group (the basal production was 0.23 ± 0.05 vs. 1.36 ± 0.17 pmol/mg protein; P < 0.001; the net production was 0.18 ± 0.05 vs. 0.48 ± 0.09 pmol/mg protein; P < 0.05) (Fig. 2 ).


Figure 2
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  		Fig. 2. Basal and net production of cGMP as a marker of nitric oxide (NO) production in MCT and control groups. The net production of cGMP was calculated as the difference between the production in the presence of 10–4 M NG-nitro-L-arginine (NOARG) plus norepinephrine (NE) plus ACh and that in the presence of NE plus ACh. The basal cGMP level was taken as the value without any agonist and antagonist except for IBMX. Results are given as means ± SE (n = 4). *P < 0.05; **P < 0.001.

 
NOS activity and protein expression. Under the present experimental conditions, the NOS activity was almost abolished in calcium-free medium containing 20 mM EDTA or in the presence of 100 µM NOARG as a nonselective inhibitor of NOS. However, the enzyme activity remained unaffected by 30 µM aminoguanidine as an inhibitor of iNOS (590.2 ± 41.0 vs. 597.0 ± 62.2 pmol/mg protein in control group; 395.0 ± 38.0 vs. 409.0 ± 21.5 pmol/mg protein in MCT group; NS). These results indicate that the NOS activity derived from PAECs dominantly represented the constitutive but not iNOS. This was supported by the findings that iNOS protein expression was not detected by Western blotting in both groups. NOS activity was significantly (P < 0.05) decreased in PAECs of MCT group (409.0 ± 21.5 vs. 597.0 ± 62.2 pmol/mg protein; Fig. 3). The eNOS expression in PAECs was significantly (P < 0.05) decreased in MCT group (Fig. 4, A and B).


Figure 3
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  		Fig. 3. NO synthase (NOS) activity in pulmonary artery endothelial cells (PAECs) of MCT and control groups. Results are given as means ± SE (n = 4). *P < 0.05.

 

Figure 4
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  		Fig. 4. A: endothelial NOS (eNOS) protein expression in PAECs of MCT and control groups. Results are given as means ± SE (n = 4). B: eNOS level was shown as ratio to beta-actin level as an internal standard. *P < 0.05. C: inducible NOS (iNOS) expression was not detected in PAECs of either group.

 
Arginase activity. Arginase activity was significantly (P < 0.01) higher in PAECs of MCT group (889.0 ± 59.1 vs. 416.0 ± 49.9 pmol urea/mg protein; Fig. 5).


Figure 5
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  		Fig. 5. Arginase activity in PAECs of MCT and control groups. Results are given as means ± SE (n = 4). ##P < 0.01.

 
Concentrations of L-arginine and methylarginines. The concentrations of L-arginine in PAECs tended to decrease in MCT group but were not significantly different (147.8 ± 21.4 vs. 214.3 ± 4.1 µM; Table 1). The concentrations of MMA and ADMA in PAECs were significantly increased in MCT group (0.63 ± 0.04 vs. 0.45 ± 0.03 µM for MMA; P < 0.05; and 2.52 ± 0.14 vs. 1.55 ± 0.03 µM for ADMA; P < 0.001). The concentrations of SDMA remained unchanged in two groups (0.39 ± 0.02 vs. 0.32 ± 0.04 µM; NS; Table 1).


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  		Table 1. Concentrations of L-arginine and methylarginines in PAECs of control and MCT groups

 
The IC50 values of authentic MMA and ADMA for NOS, which had been prepared from endothelial cells of rabbit thoracic aorta, were determined to be 2.7 ± 0.2 (n = 5) and 15.4 ± 1.0 µM (n = 5), respectively.

DDAH activity and protein expression. DDAH activity was significantly (P < 0.0005) decreased in PAECs of MCT group (5,258 ± 487 vs. 8,948 ± 223 pmol L-citrulline/mg protein; Fig. 6). The DDAH1 protein expression in PAECs was significantly (P < 0.005) reduced in MCT-induced PH rats (Fig. 7, A and B). In contrast, there was no significant change in DDAH2 protein expression between two groups (Fig. 7, C and D).


Figure 6
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  		Fig. 6. Dimethylarginine dimethylaminohydrolase (DDAH) activity in PAECs of MCT and control groups. Results are given as means ± SE (n = 4). ***P < 0.0005.

 

Figure 7
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  		Fig. 7. DDAH1 (A) and DDAH2 (C) protein expression in PAECs of MCT and control groups. Quantitative data of DDAH1 and DDAH2 signals were shown as ratios to beta-actin level as an internal standard (B and D). Results are given as means ± SE (n = 4). ###P < 0.005.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, the RVP and RV/(LV+IVS) weight ratio were significantly increased on day 24 following the administration of MCT, indicating that PH was apparently induced by MCT in the rat under the present experimental conditions. Furthermore, we demonstrated that the EDR and cGMP production as a marker of NO production in pulmonary artery specimens were significantly impaired in MCT group. These alterations were associated with the decreases in NOS activity and protein expression, accelerated arginase activity, accumulation of endogenous NOS inhibitors, and the impaired DDAH activity and decreased DDAH1 protein expression in endothelial cells.

The major vasomotor alterations in PH are vasoconstriction, endothelial and smooth muscle cell proliferation, and thrombosis, which are closely related to homeostatic imbalances between vasodilators and vasoconstrictors, growth inhibitors and mitogenic factors, and antithrombotic and prothrombotic determinants (16). These alterations are probably resulting from pulmonary endothelial cell dysfunction. We showed that there was no difference in EIR of pulmonary artery in response to SNP between MCT and control groups. SNP is well known as a NO donor to produce cGMP through activation of soluble guanylyl cyclase. Thus it is suggested that the decreased cGMP production in MCT group would be due to the impaired NO production in PAECs, not due to impaired guanylyl cyclase activity in smooth muscle cells. We recognized that only cGMP experiments were difficult to interpret for differences in the activity of guanylyl cyclase between MCT and control groups. However, the concentration-response curves for SNP as a NO donor and an activator of guanylyl cyclase remained unaffected between MCT and control groups, leading us to speculate that guanylyl cyclase is not impaired in smooth muscle cells of two groups.

Another possible explanation for the decreased cGMP production observed in the MCT group would be an increased phosphodiesterase activity. The reason is that phosphodiesterase type 5 is the predominant phosphodiesterase isoform in the lung that metabolizes cGMP (41) and has been shown to be upregulated in conditions associated with PH (7, 12, 14, 21). In the present experiments, however, the phosphodiesterase activity was not determined and thus the involvement of the enzyme in decreasing cGMP production in MCT group remains to be investigated.

The impaired NO production in PAECs of MCT group was in line with the previous reports (22). However, whether this simply resulted from reduced expression of NOS protein has been controversial (18, 27). Furthermore, there were few reports describing the decreased NOS activity in pulmonary artery of PH. In the current study, the NOS activity in PAECs was significantly reduced in MCT group, which was accompanied by the decreased expression of eNOS, suggesting that the impaired NO production in pulmonary artery was at least partly due to the decreased NOS activity, which would result from the decreased eNOS protein expression.

Arginase metabolizes L-arginine to urea and L-ornithine and shares L-arginine as a common substrate with NOS (34). Thus the accelerated arginase activity decreases the L-arginine availability to NOS, leading to the impairment of the NO production (46). The arginase activity in PAECs was significantly accelerated in MCT group. Thus it is suggested that the impaired NO production in MCT group would be partly due to the decreased L-arginine availability resulting from the accelerated arginase activity. However, in this study, L-arginine as a substrate for NOS was only slightly decreased in PAECs. These results are possibly related to the "arginine paradox," which means that the application of excess L-arginine increases the NO production, although the intracellular L-arginine concentration well exceeded the Km value of NOS (8). The proposed explanations for this phenomenon are the existence of arginine pools and the colocalization of arginine transporters and NOS in caveolae (30). Thus, if arginase colocalizes with NOS in caveolae, accelerated arginase activity might deprive the adjacent NOS of available L-arginine without significantly decreasing the total levels of L-arginine in PAECs. However, further experiments should be performed to examine the colocalization of arginase and NOS.

Endothelial concentrations of MMA and ADMA as endogenous NOS inhibitors were significantly increased in MCT group. It is important to consider whether concentrations of accumulated MMA and ADMA are sufficient to inhibit NOS. The inhibition of eNOS activity by MMA and ADMA at determined concentrations of 0.63 and 2.52 µM (Table 1) was assessed to be 24.2 ± 0.8% and 18.2 ± 1.1%, respectively (unpublished observations, which were obtained from the regression line for IC50 experiments of authentic MMA and ADMA). These values were significantly (P < 0.001) greater than corresponding values in the control. Thus the increased concentrations of MMA and ADMA in MCT group seem to be sufficient to inhibit NO production. Meanwhile, it seems plausible that the ratio of intracellular L-arginine to methylarginines also contributes to NO production in endothelial cells. In the present experiments, the significantly decreased L-arginine-to-methylarginines ratio in endothelial cells (slightly decreased L-arginine and significantly increased MMA and ADMA in MCT group) was associated with the impaired cGMP production as a marker of NO production, suggesting that the accumulation of endogenous NOS inhibitors in pulmonary endothelial cells would, at least partly, result in the impaired NO production. Moreover, the probable localization of endogenous NOS inhibitors at the vicinity of NOS in endothelial cells suggests that they may exert their actual inhibitory effect on NOS (29). Recently, several studies have suggested that the plasma concentration of ADMA provide a marker of risk for endothelial dysfunction and cardiovascular disease (9, 33, 45, 47). Furthermore, there have been reports describing that the contents of MMA as well as ADMA were increased in the corpus cavernosum after bilateral iliac artery ischemia (29) and in endothelial cells of human uterine arteries with intimal hyperplasia (25). It has also been reported that the inhibitory effect of MMA on NOS was more potent than that of ADMA (29). Taken together, it is suggested that not only ADMA but also MMA possibly play an important role in impairing NO production for one of the causes of PH.

DDAH is an enzyme that metabolizes MMA and ADMA to L-citrulline and monomethylamine or dimethylamine. Thus the impaired DDAH activity results in the accumulation of these endogenous NOS inhibitors (24). The DDAH activity and protein expression in PAECs were significantly decreased in MCT group, leading us to assume that the accumulation would be due to the decreased metabolism of MMA and ADMA by DDAH. This possibility seems to be partly supported by the findings that the level of SDMA, which is not a substrate for DDAH (37), remained unchanged in the control and MCT groups. On the other hand, it could be argued that the accumulation of endogenous NOS inhibitors is due to an acceleration of the transmembrane transport into endothelial cells. There is a report describing that L-arginine as well as methylarginines including SDMA enter cells through the cationic amino acid transporter known as system y+ (10). The enhanced transport would result in the increased intracellular contents of not only MMA and ADMA but also SDMA. However, we showed that the accumulated MMA and ADMA were associated with unchanged SDMA concentration. Thus the enhanced transmembrane transport is possibly not related to the accumulation of endogenous NOS inhibitors in endothelial cells of MCT group.

Two distinct isoforms of DDAH (DDAH1 and DDAH2) have been identified. These two isoforms have distinct tissue distributions with similar enzyme activity, and both of them have been identified in the cardiovascular system (23). In the current study, we demonstrated that protein expression of DDAH1 in PAECs was significantly downregulated in MCT group, whereas the protein expression of DDAH2 remained unchanged, suggesting that the downregulation of DDAH1 is at least in part responsible for the decreased overall DDAH activity in MCT group. This finding is in line with a previous report describing that the protein expression of DDAH1 was decreased in the lungs of the hypoxia-induced PH rats (32). On the other hand, the protein expression of DDAH2 but not DDAH1 was downregulated in the piglet PH model (2). The different expression profiles of DDAH1 and DDAH2 in PH may be due to the different species used as experimental animals.

According to Pullamsetti et al. (40), ADMA and SDMA levels were increased in lung homogenate from both MCT-treated rats and patients with PH by dot-blot analysis. They also showed that in immunohistochemical staining, immunoreactivity to dimethylated arginines was observed in the alveolar epithelium and arterial endothelium and that alveolar macrophages and dimethylated arginine immunoreactivity were preferentially increased in pulmonary endothelium of lung specimen in PH. In contrast, as described above, our HPLC data revealed that MMA and ADMA levels were significantly increased in PAECs of MCT-treated rats with no change in SDMA level, and we demonstrated that the accumulation of MMA and ADMA in endothelial cells possibly resulted from the attenuated DDAH activity due to the decreased DDAH1 protein expression. In addition, the level of SDMA, which is not a substrate for DDAH, remained unaltered between the control and the MCT groups, supporting the accumulation of MMA and ADMA in endothelial cells with the attenuated DDAH activity.

In conclusion, the impairment of cGMP production as a marker of NO production in PAECs of MCT-induced PH rats is possibly due to the blunted eNOS activity resulting from the downregulation of eNOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors (Fig. 8). To our knowledge, this is the first report that comprehensively assessed L-arginine metabolism and NO production in PAECs of MCT-induced PH rats. The results obtained in the current study may provide a rationale for the development of novel therapeutic targets other than L-arginine supplementation (31, 35) in treating the pulmonary arterial hypertension such as the inhibition of accelerated arginase activity and the decrease in accumulated endogenous NOS inhibitors by enhancing the DDAH activity.


Figure 8
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  		Fig. 8. Schematic overview of suggested mechanisms for causing PH by MCT in the rat. Accumulated endogenous NOS inhibitors, attenuated NOS, and DDAH activities are involved in MCT-induced PH, leading to the decreased ability of PAECs to produce NO. The attenuated NOS and DDAH activities are at least partly due to the decreased NOS and DDAH1 protein expression, respectively. Enhanced arginase activity is also involved in causing PH. ADMA, asymmetric dimethylarginine; MMA, monomethylarginine.

 
Study limitation. We recognized that the results of the study were obtained from experiments using conduit arteries as opposed to resistance vessels. However, we designed the present experiment to investigate the mechanisms in impairing NO production in PAECs. Therefore, we collected endothelial cells from conduit artery because resistance vessels were too small to collect only endothelial cells. On the other hand, the role of pulmonary resistance vessel is very important in causing PH. Further investigation using lung tissue containing pulmonary resistance vessels should be performed.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Azuma, Dept. of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental Univ., 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-0062, Japan (e-mail: azuma.bsr{at}tmd.ac.jp)

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.


   REFERENCES
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 RESULTS
 DISCUSSION
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