Abstract
Rationale: Airway nitric oxide is reduced in cystic fibrosis airways. Asymmetric dimethylarginine is an endogenous nitric oxide synthase inhibitor that may contribute to nitric oxide deficiency in cystic fibrosis.
Objectives: To test the hypothesis that asymmetric dimethylarginine is increased in cystic fibrosis and contributes to nitric oxide deficiency and airway obstruction.
Methods: The concentrations of asymmetric dimethylarginine, symmetric dimethylarginine, and l-arginine were measured in sputum of clinically stable patients with cystic fibrosis, in patients with cystic fibrosis before and after treatment for a pulmonary exacerbation, and in healthy control subjects, using liquid chromatography-tandem mass spectrometry.
Measurements and Main Results: Asymmetric dimethylarginine was increased in cystic fibrosis compared with control sputum, and the l-arginine/asymmetric dimethylarginine ratio was decreased. Symmetric dimethylarginine exceeded asymmetric dimethylarginine concentrations in control sputum, but this ratio was reversed in cystic fibrosis. Treatment for pulmonary exacerbation resulted in a decrease in sputum asymmetric dimethylarginine and an improved l-arginine/asymmetric dimethylarginine ratio. The treatment-related decrease in asymmetric dimethylarginine correlated significantly with an increase in sputum nitric oxide metabolites and improvement in pulmonary function. The activity of the asymmetric dimethylarginine-metabolizing enzyme, dimethylarginine dimethylaminohydrolase, was higher in cystic fibrosis sputum before rather than after treatment, suggesting that the accumulation of asymmetric dimethylarginine is caused by increased production, not decreased degradation, of asymmetric dimethylarginine.
Conclusions: Asymmetric dimethylarginine is increased in cystic fibrosis airways and may contribute to airway obstruction in patients with cystic fibrosis by reducing nitric oxide formation.
Nitric oxide (NO) production is decreased in cystic fibrosis airways. Whether endogenous inhibitors of nitric oxide synthases contribute to NO deficiency and to related pathophysiology in patients with cystic fibrosis is unknown.
The endogenous competitive nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is increased in cystic fibrosis airways. Therapeutic interventions resulting in a decrease in ADMA could potentially lead to increased airway nitric oxide production and improved pulmonary function.
Arginine residues in proteins are methylated by methyltransferases (protein arginine methyltransferases), and are likely important in lung function under normal and disease conditions (6, 7). Methylated arginine derivates, including asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are products of the hydrolysis of these methylated proteins during protein degradation (8). ADMA and SDMA are found in equimolar concentrations in human plasma. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to form dimethylamine and l-citrulline, whereas SDMA is excreted renally. ADMA functions as a competitive inhibitor of NOS and as such leads to reduced NO production and a shift of l-arginine to the arginase pathway. SDMA does not inhibit NOS but competes with l-arginine transport into the cell and therefore may limit the availability of l-arginine for intracellular NO production (7). Accumulation of ADMA has been shown to play a role in the pathophysiology of a number of vascular diseases characterized by increased vascular smooth muscle constriction (9–13). Little is known about ADMA in lung diseases, but data from animals and in vitro experiments suggest that ADMA modulates inducible NOS expression in lung epithelial cells in inflammatory airway diseases, such as asthma (14). In addition, subcutaneous infusion of ADMA into otherwise naive mice resulted in reduced NO production but increased deposition of collagen as a downstream product of increased arginase activity (15). Similarly, treatment of cultured primary mouse lung fibroblasts with ADMA stimulated arginase activity and collagen formation in vitro (15).
We here explore whether the methylated arginine derivates ADMA and SDMA are present in the sputum of patients with CF. Some of the results of these studies have been previously reported in the form of an abstract (16).
The study was approved by the Institutional Research Ethics Board and written informed consent was obtained in all cases. Thirty-four patients with CF and 11 healthy control subjects were included. The diagnosis of CF had been confirmed by repeated sweat tests (chloride > 60 mmol/L) and CFTR gene mutation analysis in all patients. Clinically stable patients with CF were recruited consecutively during routine outpatient visits. The 16 (10 boys) stable patients were free of evidence of a pulmonary exacerbation and had no significant drop in pulmonary function from previous baseline. The mean (range) age was 12.7 (7–17) years and the FEV1 was 80.4 (47–117)% of predicted values (17). Sputum from patients with CF with a pulmonary exacerbation was obtained within the first 2 days of hospital admission and after 14 days of intravenous antibiotic treatment. These 18 (7 boys) patients were 12.9 (7–17) years old and had a mean (range) FEV1 of 52.5 (36–69)% of predicted values before treatment. Patients with CF were compared with 11 (5 male) healthy control subjects, 15.5 (14–22) years of age, who were nonsmokers without a history of asthma or other chronic respiratory diseases, and free of a respiratory tract infection for at least 2 weeks before recruitment.
Sputum samples from patients with CF were collected after either spontaneous expectoration or induction with inhaled hypertonic saline as described (18). The pre- and posttreatment samples were obtained by the same technique in each patient, respectively. Nine patients underwent sputum induction before and after antibiotic treatment and the remaining nine patients produced sputum spontaneously. ADMA and SDMA levels were similar after induction or spontaneous expectoration. Sputum production in all healthy control subjects was induced with hypertonic saline. All samples were processed within 1 hour.
To determine free extracellular as well as intracellular concentrations of ADMA, SDMA, and l-arginine in sputum, supernatants of cell suspensions and sputum homogenates were used, respectively. The nonliquid phase of the sputum was processed by adding 0.1% dithiothreitol in Dulbecco's phosphate-buffered saline (4:1, vol:wt), and the clear supernatant of the cell suspension was separated from the cells by centrifugation (19). Sputum homogenates were prepared in 0.1% Triton X-100 and added protease inhibitors, similar to a previous report (4). Processed samples were aliquoted and stored at −80°C for subsequent analyses. Analyses were performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Briefly, the sample were deproteinized, butylated, separated by high-performance liquid chromatography, and subjected to tandem mass spectrometry. Concentrations were measured by comparison with standard curves prepared and processed in a similar manner as the samples.
DDAH enzyme activity was measured in sputum homogenates, in a similar manner as previously reported (20). In brief, sputum homogenate was incubated with 0.5 mM ADMA (Sigma-Aldrich, Oakville, ON, Canada) at pH 6.5, for 1 hour, and the reaction was stopped by adding sulfosalicylic acid (3%), which inhibits DDAH activity. ADMA concentrations were measured by LC-MS/MS and DDAH activity was expressed as ADMA consumption (nmol/g/h) (20).
Sputum NO metabolite (NOx) levels were measured, as described previously (21).
All results are expressed as the mean ± standard error of the mean (SEM). Binary comparisons were made by two-tailed Student t test or Wilcoxon, where appropriate. The paired t test was used to compare patients with CF before and after treatment for a pulmonary exacerbation. Correlations were determined by Spearman's test. P values less than 0.05 were considered significant. All statistical analyses were conducted with GraphPad Prism 4.0c (Graphpad Software Inc., La Jolla, CA).
Measurable concentrations of ADMA were found in all sputum samples. Extracellular ADMA concentrations in sputum were significantly higher in patients with stable CF (1.53 ± 0.45 μmol/L; P = 0.01) and in patients with a pulmonary exacerbation (2.58 ± 0.57 μmol/L; P = 0.002) compared with control subjects (0.04 ± 0.01 μmol/L). ADMA decreased significantly in patients who were treated for a CF pulmonary exacerbation (0.64 ± 0.14 μmol/L; P < 0.01, paired t test) but remained increased compared with control subjects (P = 0.002) (Figure 1).
Figure 1. Asymmetric dimethylarginine (ADMA) sputum concentrations in healthy control subjects (HC), patients with stable cystic fibrosis (CF stable), and patients with CF with a pulmonary exacerbation before (CF pre) and after (CF post) treatment with intravenous antibiotics (ABx). Results are expressed as means ± SEM. ADMA sputum concentrations were significantly higher in patients with CF compared with control subjects and significantly decreased in patients treated with antibiotics.
[More] [Minimize]The absolute concentrations of extracellular SDMA in CF sputum, when compared with healthy control subjects (0.12 ± 0.01 μmol/L), was higher in both stable patients (0.28 ± 0.05 μmol/L; P = 0.02) and patients presenting with a pulmonary exacerbation (0.47 ± 0.08 μmol/L; P = 0.004) but decreased with antibiotic treatment (0.19 ± 0.04 μmol/L; P < 0.01, paired t test) to levels comparable to control subjects (P = 0.14) (Figure 2).
Figure 2. Symmetric dimethylarginine (SDMA) sputum concentrations in healthy control subjects (HC), patients with stable cystic fibrosis (CF stable), and patients with CF with a pulmonary exacerbation before (CF pre) and after (CF post) treatment with intravenous antibiotics (ABx). Results are expressed as means ± SEM. SDMA sputum concentrations were significantly higher in patients with CF compared with control subjects and significantly decreased, to levels similar to those of healthy control subjects, in patients treated with antibiotics.
[More] [Minimize]SDMA has previously been described to be present in equimolar concentrations to ADMA in serum and bronchoalveolar lavage fluid of healthy individuals (6). However, sputum concentrations of extracellular ADMA in healthy control subjects were three times lower than those found for SDMA (P < 0.0001). In contrast, free ADMA concentrations exceeded those for SDMA in CF sputum. The ADMA/SDMA ratio in sputum supernatant was 0.31 ± 0.04 in control subjects, but was increased to 6.03 ± 0.71 in stable CF (P < 0.0001, t test) (Figure 3). The ADMA/SDMA ratio in sputum supernatants of patients with CF presenting with a pulmonary exacerbation was decreased after treatment with antibiotics (4.92 ± 0.35 vs. 3.74 ± 0.49; P = 0.02, paired t test) but remained approximately 10-fold higher than in control subjects (Figure 3).
Figure 3. The ratio of asymmetric dimethylarginine (ADMA) to symmetric dimethylarginine (SDMA) in the sputum of healthy control subjects (HC), patients with stable cystic fibrosis (CF stable), and patients with CF with a pulmonary exacerbation before (CF pre) and after (CF post) treatment with intravenous antibiotics (ABx). Results are expressed as means ± SEM. The ratio of ADMA to SDMA was significantly higher in patients with CF compared with control subjects and decreased in patients treated with antibiotics.
[More] [Minimize]The ADMA/SDMA ratio in sputum lysates was similar in control subjects and treated patients with CF (Table 1), suggesting that increased free methylarginine in CF is largely derived from lysed cells in the CF sputum.
Supernatant | Lysate | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ADMA (μmol/L) | SDMA (μmol/L) | ADMA/SDMA | ADMA (μmol/L) | SDMA (μmol/L) | ADMA/SDMA | |||||
| Control subjects | 0.04 ± 0.01* | 0.12 ± 0.01 | 0.30 ± 0.04* | 0.10 ± 0.04 | 0.03 ± 0.01* | 2.20 ± 0.10 | ||||
| CF stable | 1.53 ± 0.45 | 0.28 ± 0.05 | 4.45 ± 0.53* | 1.06 ± 0. 35* | 0.16 ± 0.04* | 6.03 ± 0.71 | ||||
| CF pre | 2.07 ± 0.44 | 0.39 ± 0.06 | 5.15 ± 0.41 | 2.01 ± 0.41 | 0.43 ± 0.05 | 4.38 ± 0.55 | ||||
| CF post | 0.55 ± 0.10 | 0.18 ± 0.03 | 3.86 ± 0.80 | 0.49 ± 0.12 | 0.22 ± 0.03 | 2.09 ± 0.37 | ||||
Because ADMA acts as a competitive inhibitor for NOS, the ratio of l-arginine to ADMA (i.e., the ratio of NOS substrate to inhibitor) can be used as an indicator of l-arginine availability for functional impairment of NOS, as previously described for cardiovascular diseases (22). The l-arginine/ADMA ratio was higher in control than in stable CF sputum samples (mean ± SD: 628 ± 74 vs. 394 ± 68; P = 0.03) and was further reduced in patients with CF with a pulmonary exacerbation (195 ± 31; P < 0.001). Treatment of pulmonary exacerbations resulted in an increase in the ratio to values similar to stable CF; however, the ratio remained significantly decreased compared with control subjects (312 ± 48; P < 0.001) (Figure 4).
Figure 4. The ratio of l-arginine to asymmetric dimethylarginine (ADMA) (i.e., ratio of nitric oxide synthase substrate to inhibitor) in the sputum of healthy control subjects (HC), patients with stable cystic fibrosis (CF stable), and patients with CF with a pulmonary exacerbation before (CF pre) and after (CF post) treatment with intravenous antibiotics (ABx). Results are expressed as means ± SEM. The ratio of l-arginine to ADMA was significantly lower in patients with CF compared with control subjects and increased in patients treated with antibiotics.
[More] [Minimize]The increase in l-arginine/ADMA ratio after treatment for exacerbation was paralleled by a significant increase in sputum NO metabolite (NOx) concentrations (432 ± 84 vs. 894 ± 144 μmol/L; P < 0.001). In contrast, l-arginine sputum concentrations decreased in patients with CF treated with antibiotics (354 ± 58 vs. 135 ± 23 μmol/L; P = 0.001), suggesting that the increase in NO production during treatment for pulmonary exacerbation is not a direct consequence of higher substrate concentrations, but rather is due to increased l-arginine availability for NOS.
To explore whether accumulation of ADMA in CF sputum could be related to impaired degradation of ADMA, we measured the activity of its metabolizing enzyme, dimethylarginine dimethylaminohydrolase (DDAH), in sputum derived from 12 patients with CF before and after antibiotic treatment for a pulmonary exacerbation. DDAH activity was significantly higher before compared with after treatment (264 ± 104 vs. 92 ± 61 nmol/g/h; P = 0.024, Wilcoxon), suggesting that increased ADMA concentrations in CF are not the result of reduced degradation (Figure 5). DDAH activity in CF sputum was not compared with control subjects as sputum from healthy individuals does not contain enough cells to produce lysate sufficient for the DDAH activity assay used.
Figure 5. Dimethylarginine dimethylaminohydrolase (DDAH) activity, measured as asymmetric dimethylarginine (ADMA) consumption over time in the sputum of patients with cystic fibrosis before (pre) and after (post) treatment for a pulmonary exacerbation. Results are expressed as means ± SEM. DDAH activity significantly decreased with treatment (P = 0.024).
[More] [Minimize]Treatment of pulmonary exacerbations for 14 days resulted in a significant increase in FEV1 (52.5 ± 2.5 vs. 65.8 ± 3.2% predicted; P = 0.0004, Wilcoxon). Changes in sputum ADMA concentrations during antibiotic treatment showed a significant correlation (Spearman) with improvement in FEV1 (R = −0.66, P = 0.003), as well as with changes in sputum NOx levels (R = −0.63, P = 0.006) (Figure 6). Similar correlations were seen for sputum SDMA (data not shown).
Figure 6. Correlations between changes in sputum asymmetric dimethylarginine (ΔADMA) concentrations during treatment of a pulmonary exacerbation with (A) changes in nitric oxide metabolite (ΔNOx) levels in sputum as well as (B) changes in pulmonary function (ΔFEV1). Each symbol represents one individual. Reductions in ADMA concentration were significantly correlated with increases in NOx (R = −0.63, P = 0.006) and improvements in FEV1 (R = –0.66, P = 0.003).
[More] [Minimize]We here describe for the first time that the endogenous NOS inhibitor ADMA is present in airway secretions of patients with CF. ADMA sputum concentrations are increased in CF compared with healthy control subjects and are the highest in patients with CF presenting with a pulmonary exacerbation. Although antibiotic therapy resulted in a significant decrease, ADMA after treatment remained significantly higher in CF than in control sputum. The ratio of extracellular l-arginine to ADMA (i.e., the ratio of NOS substrate to inhibitor), which can be used as an index of substrate availability and functional impairment of NOS at a given l-arginine concentration (22), was lower in patients with CF than in control subjects, but improved after treatment for exacerbation, as did airway NO production, reflected by an increase in sputum NOx concentrations. The change in extracellular sputum ADMA concentrations during treatment for a pulmonary exacerbation correlated significantly with the increase in NOx and the improvement in FEV1 with treatment, suggesting that ADMA contributes to CF airway obstruction by reducing airway NO production.
Methylated arginine derivates such as ADMA and SDMA are released during protein degradation and are products of protein arginine methyltransferases (7). ADMA is a competitive inhibitor of NOS. The formation of NO in airways is decreased in CF and we have previously shown that reduced l-arginine availability due to an increase in arginase activity contributes to NO deficiency in patients with CF (4, 5). The finding of increased concentrations of ADMA further adds to the notion that reduced l-arginine bioavailability is an important contributor to NO deficiency in CF, and considers the functional impairment of NOS in the CF airway. The observation that NOx increased with treatment for a pulmonary exacerbation matches previous reports of an increase in exhaled NO in patients with CF during antibiotic treatment (23), and taken together these findings suggest that an increase in airway NO is not only related to a decrease in arginase activity but also to a decrease in sputum ADMA concentrations and an increase in the extracellular l-arginine/ADMA ratio in sputum.
ADMA, by inhibiting NOS, increases the availability of l-arginine for arginases and increases arginase activity. The balance between NOS and arginase is thought to be critical for maintaining lung mechanics and structure (15). NO is a mediator for bronchodilation and increased arginase in airways results in reduced NO formation, airway obstruction, and airway hyperreactivity (24, 25). Data from patients with CF suggest that an imbalance between the l-arginine–metabolizing enzymes exists in CF and is physiologically important. In patients with CF airway NO is low, arginase activity in sputum is high, fractional exhaled NO (FeNO) and sputum NOx correlate directly with pulmonary function, and changes in sputum arginase activity after antibiotic treatment for pulmonary exacerbation correlate with changes in FeNO and pulmonary function (4). Our findings that patients with the highest changes in sputum ADMA during treatment for a pulmonary exacerbation also had the highest increase in sputum NOx and the greatest improvement in FEV1 further suggest that ADMA contributes to NO deficiency and airway obstruction in CF.
Downstream products of arginase activity include the polyamines and collagen, molecules that may contribute to airway remodeling and fibrosis. This was supported by a study showing that treatment with ADMA induces collagen deposition of the lung and alters lung function in otherwise naive mice (15). Thus increased ADMA may not only result in NO deficiency but also contribute to remodeling of the CF lung. The contribution of increased ADMA to morphological changes of the CF lung needs to be addressed in future studies.
Methylated arginine derivates are eliminated by renal excretion and hepatic metabolism (26, 27). In addition, ADMA, but not SDMA, is subject to enzymatic degradation by DDAH (to form dimethylamine and citrulline) (28). CF sputum is rich in protein and the increase in ADMA in CF sputum is likely related to increased protein turnover. Alternatively, accumulation of ADMA could result from decreased enzymatic degradation. However, when looking at DDAH activity in lysates of CF sputum samples from patients presenting with pulmonary exacerbation, where ADMA levels were the highest, we did not find a decrease in DDAH activity. In fact, DDAH activity was higher in these samples than in the samples obtained after 14 days of antibiotic treatment, where ADMA had decreased.
As decreased airway NO contributes to CF lung disease, therapeutic interventions aiming to improve NO production are currently being explored. An increase in substrate availability for NOS can be achieved by l-arginine supplementation, and a single inhalation of nebulized l-arginine was shown to result in increased airway NO and improved pulmonary function in patients with CF (29). However, l-arginine is also substrate for arginase, and a sustained increase in airway l-arginine concentrations may potentially promote lung fibrosis via downstream products of arginase activity, such as polyamines. Therefore, ways to increase l-arginine availability for NOS other than substrate supplementation need to be explored. Selective arginase inhibitors are currently in preclinical testing (30), and have been demonstrated to improve NO-mediated airway hyperresponsiveness (24) and may help to improve the l-arginine/ADMA ratio without promoting remodeling. Improvement in airway NO production in CF airways may also be achieved with statins (or HMG-CoA reductase inhibitors), as treatment with mevastatin not only restores expression of NOS2 in models of CF epithelium (31) but also has the potential to lower protein expression and activity of arginase-1, as shown in mouse models of allergic asthma (32, 33). Other potential treatments to improve the l-arginine/ADMA ratio could include interventions aiming to prevent ADMA production, for instance, by inhibition of protein arginine methyltransferases (34), or the accumulation of ADMA. One of the drugs that could help reduce ADMA in CF airways is the hormone melatonin. In vivo experiments in rats have shown that treatment with melatonin can prevent increases in hepatic ADMA by blocking H2O2-induced down-regulation of DDAH2 expression and activity (35), to increase hepatic DDAH2 expression and activity and subsequently decrease ADMA content in both liver and kidney in bile duct ligation–induced oxidative stress and kidney injury (36), and to prevent a decrease in renal DDAH activity in spontaneous hypertensive rats, which impedes the development of hypertension by decreasing ADMA and restoring the l-arginine/ADMA ratio in plasma (37).
Further studies evaluating these potential interventions in animal models are needed to define the best interventional strategy that should be moved into human trials.
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