Abstract
Rationale: Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that competes with l-arginine for binding to NOS. It has been suggested that ADMA contributes to inflammation, collagen deposition, nitrosative stress, and lung function in murine models.
Objectives: To test the hypothesis that ADMA is increased in asthma and that NOS inhibition by ADMA contributes to airways obstruction.
Methods: We assessed alterations of l-arginine, ADMA, and symmetric dimethylarginine (SDMA) levels in a murine model of allergic airways inflammation using LC-tandem mass spectrometry. Based on the levels of ADMA observed in the murine model, we further tested the direct effects of nebulized inhaled ADMA on airways responsiveness in naive control mice. We also assessed alterations of l-arginine, ADMA, and SDMA in humans in adult lung specimens and sputum samples from pediatric patients with asthma.
Measurements and Main Results: ADMA was increased in lungs from the murine model of allergic airways inflammation. Exogenous administration of ADMA to naive mice, at doses consistent with the levels observed in the allergically inflamed lungs, resulted in augmentation of the airways responsiveness to methacholine. ADMA levels were also increased in human asthma lungs and sputum samples.
Conclusions: ADMA levels are increased in asthma and contribute to NOS-related pathophysiology.
Reduced availability of substrate for the nitric oxide synthase (NOS) isozymes has recently been shown to be important in asthma airways. In addition to limitations in l-arginine bioavailability, accumulation of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) may also play a role in the pathogenesis of asthma.
ADMA is increased in asthma and contributes to NOS-related pathophysiology. Measurements of ADMA and l-arginine in sputum may be useful noninvasive measures of NOS function in asthma and other respiratory diseases.
The biosynthesis of nitric oxide (NO) from the semiessential amino acid l-arginine by the NO synthase (NOS) family of isoenzymes is important in the maintenance of airway tone (1–3). Asthma is a chronic inflammatory disease characterized by variable and reversible obstruction of airways. Expired NO levels are elevated in asthma and are closely related to the increased expression of inducible NOS (iNOS) in asthma airway epithelium (4, 5). However, recent studies suggest that a reduction in the local bioavailability of l-arginine and reduced NO production from constitutive NOS in airway smooth muscle contributes to the airways hyperresponsiveness characteristic of asthma (6, 7).
Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are produced by methylation of arginine residues in proteins by protein methyl transferases and are liberated during proteolysis (8). Although ADMA is an important endogenous inhibitor of NOS, SDMA has been suggested to impair cellular uptake of l-arginine (9), which could contribute to a reduction in intracellular substrate availability. ADMA has been shown to be important in a number of vascular diseases characterized by increased smooth muscle constriction (10–14). It has recently been suggested that ADMA levels are increased in a mouse model of allergic airways inflammation (15). Thus, it is conceivable that ADMA competes with l-arginine for binding to NOS in asthma airways, which would result in decreased NO production. The balance between l-arginine metabolism and the release of endogenous inhibitors of these respective enzymes may be important to understand the underlying pathologic mechanisms of asthma. However, it remains unknown as to whether ADMA levels are altered in human asthma and whether ADMA contributes to asthma pathophysiology.
The aims of this study are (1) to determine whether ADMA levels are altered in human asthma; (2) to determine physiologic importance of ADMA in a murine model of allergic airways inflammation; and (3) to determine concentrations of SDMA, a methylation product of protein arginine residues that is thought to compete with l-arginine for transport into the cell, and its potential role in asthma. Some of the results related to the patient population and animal data included in this study have been previously reported in the form of abstracts at the Annual Meeting of the American Thoracic Society (16, 17).
Additional experimental details are available in the online supplement.
This study was approved by the University of Toronto Faculty Advisory Committee on Animal Services and was conducted in accordance with the guidelines of the Canadian Council on Animal Care. This acute ovalbumin (OVA)-sensitization and -challenge model of allergic airways inflammation has been described previously (18–21). Twenty-four hours after the final aerosol challenge, mice underwent ventilator-based measurement of respiratory function and responsiveness to nebulized methacholine using the flexiVent (SciReq Inc., Montréal PQ, Canada) (22, 23). After the functional end points were obtained, the mice were killed and the lungs isolated and frozen for subsequent mass spectrometric analysis.
To further test the functional consequences of increased ADMA levels on airways tone and methacholine responsiveness in naive mice, we administered either phosphate-buffered saline (PBS) or ADMA (20–200 nmol per gram body weight in PBS) intratracheally via nebulizer after the mice were intubated for pulmonary function testing. Methacholine responsiveness was assessed after a 15-minute equilibration period. NO metabolite (NOx−) concentrations were measured from lung homogenates, as previously published (24).
ADMA, SDMA, and l-arginine levels were determined in human lung specimen homogenates prepared from deceased control subjects (nonpulmonary disease) and subjects with asthma (n = 6 and 5 for control and asthma groups, respectively; National Disease Research Interchange, Philadelphia, PA) (19).
Sputum samples were collected from 17 pediatric patients with moderate, atopic asthma who presented with current symptoms as part of a standard protocol in the asthma clinic (16, 25). Written informed consent was obtained from each patient or their parents. The study was approved by the Institutional Review Board at the Hospital for Sick Children. The median age was 12 years (range, 8–16 yr), and there were 3 boys and 14 girls. Nine of the subjects exhibited sputum eosinophilia (≥ 2.5% of total cell counts). All patients were treated with inhaled corticosteroids. All but two were on a fixed combination of inhaled corticosteroids with long-acting β-agonists, and six were on additional montelukast. The mean FEV1 for all patients was 79.7 ± 3.1% (range, 56–109%) of predicted values (16). Patients were compared with 12 healthy control subjects (median age, 18.5 yr [range, 15–22 yr]; seven males and five females). Sputum samples were processed within 1 hour and stored at −80°C for subsequent analyses, as recently described (26). The fraction of exhaled NO (FeNO) was measured in these subjects according to published consensus guidelines for the measurement of FeNO in children (27).
The concentrations of ADMA, SDMA, and l-arginine were measured in homogenates of mouse and human lung tissue and sputum samples by running them against standard curves with their respective internal standards, prepared in their own matrices. Both samples and standards were butylated before analysis on the liquid chromatography–mass spectrometry (see the online supplement). Dimethylarginine dimethylaminohydrolase activity in lung homogenates was measured in a similar manner to that described previously (28), and modified by us to indicate the conversion of the stable isotopomer of ADMA (M+7) to citrulline (M+7).
All data are expressed as the mean ± SEM. All binary comparisons were made with two-tailed Student t test. Correlations between the biochemical parameters and functional end points (i.e., respiratory system or airways resistance in the mouse model and FeNO in the human subjects with asthma) were determined by Spearman test. P values less than 0.05 were considered significant. All statistical analyses were conducted using GraphPad Prism 4.0c (Graphpad Software Inc., La Jolla, CA).
ADMA levels were significantly increased in lung homogenates of OVA-sensitized and -challenged (OVA/OVA) mice compared with PBS-challenged (OVA/PBS) control animals (4.1-fold; 2.1 ± 0.5 vs. 0.5 ± 0.05 nmol per milligram protein for the OVA/OVA lungs compared with OVA/PBS control lungs; P < 0.01) (Figure 1A). l-Arginine levels were reduced by 47% in the OVA-model lungs (47 ± 6.5 vs. 88.6 ± 6.8 nmol per milligram protein for the OVA/OVA compared with OVA/PBS control lungs; P < 0.01). The l-arginine:ADMA ratios, representing substrate over the endogenous NOS inhibitor as an indicator of impaired NOS activity, exhibited an 81% reduction in OVA/OVA lungs compared with control lungs (32 ± 5 vs. 175 ± 5 for the OVA/OVA compared with OVA/PBS control lungs; P < 0.0001) (Figure 1B), suggestive of NOS impairment in diseased lungs. SDMA levels were also significantly increased in the OVA-model lungs, compared with control lungs (187 ± 27 vs. 17 ± 6 pmol per milligram protein for the OVA/OVA lungs compared with OVA/PBS control lungs; P < 0.0001) (Figure 1C).
Figure 1. Asymmetric dimethylarginine (ADMA) (A) and symmetric dimethylarginine (SDMA) (C) concentrations in lungs from the murine model of allergic airways inflammation. Ratios of ADMA (B) to l-arginine in the same tissues (***P < 0.0001 to ovalbumin [OVA]/phosphate-buffered saline [PBS]); n = 10 and 13 for lungs from OVA/PBS and OVA/OVA mice, respectively.
[More] [Minimize]Correlation comparisons were made between the arginine metabolite and functional parameters in the lung (i.e., ADMA, SDMA, l-arginine, and l-arginine:ADMA) with total respiratory system resistance (Rrs), airways Newtonian resistance (RN), and peripheral tissue damping values (G) (Figure 2). Interestingly, and consistent with our overall hypothesis that l-arginine metabolism is key to the maintenance of airways tone in disease, l-arginine levels correlated inversely, and the ADMA and SDMA levels correlated directly with all three measures of respiratory and airways tone.
Figure 2. Correlations between l-arginine metabolites in the lung and (A) baseline respiratory resistance (Rrs), (B) central airways resistance (RN), and (C) peripheral tissue damping (G). Inset Spearman r-values for the correlation (*P < 0.05, **P < 0.01, ***P < 0.0001; n = 20 and 22 for ovalbumin [OVA]/phosphate-buffered saline [PBS, open circles] and OVA/OVA [filled circles] mice, respectively). ADMA = asymmetric dimethylarginine; SDMA = symmetric dimethylarginine.
[More] [Minimize]To determine whether the increased ADMA levels in the mouse lungs were caused by decreased metabolism of ADMA to l-citrulline, we measured DDAH activities in the OVA/PBS and OVA/OVA mouse lung homogenates. DDAH activities were 71 ± 5.2 and 67.2 ± 4 pmol l-citrulline per milligram protein per hour in the OVA/OVA and OVA/PBS groups, respectively (n.s., n = 7 per group) (Figure S1).
To determine the functional effect of relevant doses of ADMA, we nebulized ADMA into the airways of naive wild-type mice (Figure 3). Based on the approximately fourfold increase of ADMA, from 0.5–2 nmol per milligram protein in the OVA/OVA mice compared with the OVA/PBS control animals, we estimated that a dose of 20–200 nmol per gram body weight ADMA delivered by nebulization into the airways would approximate the levels observed in the OVA-sensitized and -challenged mice. Although 20 nmol ADMA per gram body weight caused no change in maximum responsiveness to methacholine, 200 nmol ADMA per gram body weight resulted in significant 1.6- and 1.4-fold increases in maximum total respiratory resistance (8.5 ± 1.3 vs. 5.3 ± 0.6 cm H2O·s/ml for ADMA vs. control mice, respectively; P < 0.05) (Figure 3A) and airways Newtonian resistance to methacholine (2.2 ± 0.2 vs. 1.6 ± 0.1 cm H2O·s/ml for ADMA vs. control mice, respectively; P < 0.05) (Figure 3B), but did not significantly affect peripheral tissue damping (Figure 3C). ADMA administration by this means also resulted in significantly reduced NOx− concentrations in lung homogenates (55.8 ± 4.3 vs. 90 ± 14.6 μmol per gram of lung tissue for ADMA-treated vs. control mice, respectively; P < 0.05) (Figure 3D), suggesting that the effect of ADMA on pulmonary function was caused by inhibition of NOS.
Figure 3. Effect of exogenous nebulized asymmetric dimethylarginine (ADMA) on methacholine-responsiveness in naive female BALB/c mice. Total respiratory system resistance (A), Newtonian airways resistance (B), and peripheral tissue damping (C) were determined in mice treated with either phosphate-buffered saline (PBS) vehicle, or 20 or 200 nmol ADMA per gram body weight. *P < 0.05 to PBS vehicle control; n = 10–12 per group. (D) Nitric oxide (NO)x levels in lung homogenates from control mice and ADMA-treated naive mice (n = 4 and 7, respectively; *P < 0.05 to control mice).
[More] [Minimize]ADMA and SDMA levels were increased 1.7- and 1.8-fold, respectively, in the lungs from subjects with asthma, compared with control subjects; however, this difference did not reach statistical significance for ADMA, likely because of the small number of samples available. (ADMA, 2.9 ± 0.6 vs. 1.7 ± 0.2 nmol per milligram protein, P = 0.125; SDMA, 0.76 ± 0.20 vs. 0.41 ± 0.19 nmol per milligram protein, P < 0.05). Meanwhile, the l-arginine:ADMA ratios were significantly reduced in these asthma lung tissues compared with control lung tissues (55 ± 10 vs. 138 ± 11; P < 0.01), matching our observation in the mouse lungs. DDAH activities were unaltered in the asthma lung tissues compared with control lung tissues (31.1 ± 5.4 vs. 34.3 ± 5 pmol l-citrulline per milligram protein per hour for asthma and control subjects, respectively; n.s.; n = 5 and 6, respectively) (Figure S1).
Measurable levels of ADMA and SDMA were observed in all sputum samples (Figure 4). ADMA levels were elevated 2.4-fold in sputum from patients with asthma compared with control subjects (91.8 ± 14.6 vs. 38.6 ± 5.9 pmol per milligram protein; P < 0.001). By contrast SDMA levels were attenuated by 36% in the asthma sputum (77 ± 15 vs. 120 ± 3.4 pmol per milligram protein; P < 0.05). Sputum ADMA levels and l-arginine:ADMA ratios correlated significantly with FeNO in the patients with asthma (Spearman r-value −0.5319 and 0.500, respectively; P < 0.05) (Figure S2), also suggestive of NOS inhibition in asthma airways.
Figure 4. Asymmetric dimethylarginine (ADMA) (A) and symmetric dimethylarginine (SDMA) (B) concentrations in sputum samples obtained from pediatric patients with asthma and healthy control subjects (*P < 0.05 and ***P < 0.0001 compared with control subjects; n = 11 vs. 17 samples from control subjects and patients with asthma, respectively).
[More] [Minimize]Herein, we describe increased ADMA concentrations in lungs of mice that underwent an acute OVA model of induced allergic airways disease, similar findings in human asthma lung tissue, and increased ADMA in sputum from pediatric patients with asthma. We further provide evidence supporting the direct physiologic significance of these findings, because nebulized ADMA resulted in increased airways responsiveness to methacholine and decreased lung NOx levels in mice. The inhibitory effect of ADMA on NO production in asthma was further supported by the observation that ADMA and the l-arginine:ADMA ratio in sputum correlated inversely with FeNO in patients with asthma. Although increased ADMA has previously been observed in a mouse model of allergic airways disease (15), this is the first study to show the physiologic relevance of this for airways hyperresponsiveness and for NO production in patients with asthma.
In addition to ADMA, we also demonstrated that SDMA, which competes with l-arginine for transport into the cell (29), was augmented in lungs from human subjects with asthma and in the murine model of allergic airways inflammation. These alterations in l-arginine metabolism have significant implications in the understanding of the biochemical mechanisms underlying the airways hyperresponsiveness of asthma.
Although roles for the endogenous NOS inhibitor ADMA have been proposed in cardiovascular diseases, including pulmonary hypertension (10), little is known about the contribution of ADMA to pulmonary diseases (8, 30). A role for ADMA in asthma has only recently been hypothesized, based on findings in murine models (15, 31–34). Ahmad and coworkers (15) reported an approximately twofold increase in ADMA in lung cytosol and mitochondria from OVA-sensitized and -challenged mice, which they hypothesized was responsible for increased nitrosative stress (31) and mitochondrial dysfunction in asthma. Although the absolute ADMA concentrations in the cytosolic and mitochondrial fractions seem to be approximately 1,000-fold greater than our findings, this is likely caused by the greater protein content in our lung homogenates. Furthermore, the fold-increase in ADMA observed by Ahmad and coworkers (15) was approximately half that observed by ourselves, which may be explained by differences in the murine models used; whereas the durations of the challenge phases were similar between the studies (7 vs. 12 d), the challenge phase of the current study used a dose of OVA that was twice that used by Ahmad and coworkers (15) (6% vs. 3%). In a recent paper by Klein and coworkers (33) allergen-induced lung inflammation was potentiated in mice when circulating ADMA was elevated and ADMA increased iNOS expression in lung epithelial cells in these OVA-model animals, suggesting that ADMA may play a role in asthma through modulation of iNOS expression in airways. To determine whether the changes in ADMA observed in the allergen-induced inflamed lungs were sufficient to elicit functional changes in airways responsiveness, we acutely administered nebulized doses of ADMA into the lungs of otherwise naive mice in a dose range consistent with our observed increases in ADMA levels in OVA/OVA lung tissue, and demonstrated increased responsiveness to methacholine. Thus, ADMA at increased levels similar to those observed in the murine model of allergic airways inflammation was sufficient to increase airways responsiveness in the absence of inflammation. Interestingly, the fold increases in ADMA levels observed in our OVA-model were similar to the levels administered to mice in the recent study by Wells and coworkers (32). In their study, chronic administration of exogenous ADMA via osmotic pumps resulted in increased collagen deposition, airways remodeling, and increased methacholine responsiveness, also in the absence of inflammation, although the altered responsiveness to methacholine could have been caused by the remodeled airway. Thus, the potential mechanisms by which ADMA contributes to remodeling remains an interesting area for further investigation. Although modulation of DDAH expression or activity represents one potential mechanism underlying the increased ADMA levels in asthma, our observation that DDAH activity remained unchanged in human asthma lungs and in the OVA-model lung homogenates supports that the increased levels likely reflect increased ADMA formation, and not a decrease in enzymatic degradation. However, in a previous publication by Ahmad and coworkers (15) a decrease in DDAH2 protein expression in a similar model was found in lung cytosol using Western blot, which may suggest that alterations in specific cellular compartments and cell populations may contribute to changes in ADMA metabolism, at least at the cellular level.
To provide evidence that ADMA may also be important in patients with asthma, we measured ADMA levels in human asthma lung specimens and sputum samples, and showed increased ADMA levels in both. A direct comparison between lung tissue and sputum could not be performed, because these samples were obtained from different patient populations. Indeed, one of the limitations of this study was the small number of lung tissue specimens investigated, which is reflective of the difficulty to obtain lung specimens from this population and the fact that little information was available regarding the treatment status of the subjects before their death. However, we were able to study a sufficient number of asthma sputum specimens. Not only were ADMA levels increased in these samples when compared with healthy control subjects, but ADMA also correlated significantly with FeNO in these patients, supporting the hypothesis that NO formation in the airways is impaired as a consequence of increased ADMA. Although the relationship between sputum ADMA, FeNO, and pulmonary function remains unclear at this point, these findings may also support the use of sputum samples in investigations of ADMA and arginine metabolism in patients with asthma. However, it must be considered that the information gained from sputum samples may differ substantially from that of lung tissue samples because they represent different compartments with potential differences in cellular composition and inflammatory activity and l-arginine metabolism.
One of the key issues in understanding the potential role of NOS in health and disease is the “arginine paradox” (35), by which NOS activity is impaired in vivo, despite the presence of substrate at levels 15- to 30-fold greater than the KM values for the NOS isozymes (i.e., in the low micromolar levels). This has been suggested to be moderated, in part, through inhibition of the NOS isozymes by endogenous ADMA. Bode-Boger and coworkers (35) recently proposed the calculation of the l-arginine:ADMA ratio as a novel index of this paradox, which is reflective of imbalances in NOS activity caused by the accumulation of ADMA. As such, a “normal” l-arginine:ADMA ratio is in the range of 132–225 (35), which is consistent with our mean values in the control (OVA/PBS) mouse lungs. Thus, when ADMA levels accumulate in the tissue, the ratio would decrease and reflect reduced substrate availability and NOS activity (the average l-arginine:ADMA ratio in our OVA/OVA lungs was 32 ± 5). We achieved similar findings in the human lung specimens (i.e., a high l-arginine:ADMA ratio in control subjects, which was reduced in asthma). Our findings may help explain why inhalation of l-arginine resulted in a greater increase in FeNO in patients with asthma compared with healthy controls (36) and why chronic l-arginine supplementation, as recently studied in a clinical trial (37) and in a murine model of allergic airways inflammation (38), may result in long-term functional improvements. These investigations suggest that measurement of the l-arginine:ADMA ratio in sputum could be potentially useful in monitoring therapeutic interventions in patients with asthma that are directed toward increasing the bioavailability of l-arginine for the NOS isozymes (i.e., supplemental l-arginine and potentially arginase inhibition), because arginase expression and activity is increased in asthma (7, 19, 39) and contributes to the reduction in bioavailability of l-arginine (7) in asthma.
This is the first report to demonstrate a significant correlation between quantitative measures of l-arginine metabolism (i.e., l-arginine, ADMA, and SDMA levels and calculations reflective of impaired l-arginine availability for NOS) and functional outcomes (i.e., total respiratory and airways resistance) in mice. Thus, as the correlation between the l-arginine:ADMA ratio and the airways resistance was negative, the more NOS activity seems to be impaired because of increased ADMA, which was supported by the correlation between ADMA and FeNO in the pediatric subjects with asthma. These data support that ADMA is important in asthma.
SDMA is also produced by proteolytic degradation of methylarginine residues in proteins, and has been reported to competitively block l-arginine uptake into cells (8, 29). We report for the first time that SDMA levels are augmented in asthma both in humans and in the murine model. Interestingly, SDMA levels were not increased but decreased in sputum samples from pediatric patients with asthma, which may reflect differences in SDMA metabolism or transport between the compartments (i.e., airway epithelial and inflammatory cells vs. parenchymal and smooth muscle cells). Although the reasons for increased SDMA levels in lung tissue but decreased levels in sputum cannot be explained at this point, these findings may suggest a role for SDMA in regulating l-arginine uptake in airway epithelial cells. These findings also suggest that impaired l-arginine uptake in the lung via the cationic amino acid transporters (29) might also contribute toward the limited bioavailability for epithelial iNOS, resulting in decreased NO production in asthma airway because of uncoupling of NOS activity and the production of peroxynitrite. This is the first observation of altered levels of SDMA in asthma and further mechanistic investigations are necessary to understand the potential role for SDMA and l-arginine metabolism in asthma.
Our findings suggest that methylated arginine metabolites inhibit NO formation in asthma airways and are physiologically important for the development of airways hyperresponsiveness. Because sputum samples can be obtained conveniently, sputum may eventually be used to monitor l-arginine bioavailability by measuring l-arginine and ADMA and may also be helpful to investigate the response to therapeutics targeting the l-arginine metabolism in asthma.
M.L.N. was supported by the AllerGen NCE, an Ontario Graduate Scholarship in Science and Technology, and the Frederick Banting and Charles Best Canada Graduate Scholarships – Doctoral Research Award.
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Supported by the Ontario Thoracic Society.
Author contributions: Conception and design, J.A.S., P.P., P.S., and H.G. Analysis and interpretation, J.A.S., M.L.N., M.R., H.H., P.P., and H.G. Drafting the manuscript for important intellectual content, J.A.S., P.P., P.S., J.B., and H.G.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201011-1810OC on June 30, 2011
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