AJP - Lung AJP: Gastrointestinal and Liver Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L18-L24, 2007. First published August 4, 2006; doi:10.1152/ajplung.00076.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L18    most recent
00076.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bulau, P.
Right arrow Articles by Eickelberg, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bulau, P.
Right arrow Articles by Eickelberg, O.

EDITORIAL FOCUS

Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA

Patrick Bulau,1 Dariusz Zakrzewicz,1 Kamila Kitowska,1 James Leiper,2 Andreas Gunther,1 Friedrich Grimminger,1 and Oliver Eickelberg1

1University of Giessen Lung Center, University of Giessen School of Medicine, Giessen, Germany; and 2Centre for Clinical Pharmacology and Therapeutics, British Heart Foundation Laboratories, Division of Medicine, University College London, London, United Kingdom

Submitted 4 March 2006 ; accepted in final form 25 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). Three forms of methylarginine have been identified in eukaryotes: monomethylarginine (L-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. L-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase isoforms. SDMA is eliminated almost entirely by renal excretion, whereas L-NMMA and ADMA are further metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To explore the interplay between methylarginine synthesis and degradation in vivo, we determined PRMT expression and DDAH activity in mouse lung, heart, liver, and kidney homogenates. In addition, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting for the assessment of tissue-specific patterns of arginine methylation. The salient findings of the present investigation can be summarized as follows: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation; 2) pulmonary ADMA degradation was undertaken by DDAH1; 3) bronchoalveolar lavage fluid and serum exhibited almost identical ADMA/SDMA ratios, and 4) kidney and liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. Together, these observations suggest that methylarginine metabolism by the pulmonary system significantly contributes to circulating ADMA and SDMA levels.

protein arginine methyltransferases; asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase

THREE FORMS OF METHYLARGININE have been identified in eukaryotes: NG-monomethylarginine (L-NMMA), NGNG- (asymmetric) dimethylarginine (ADMA), and NGN'G- (symmetric) dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. Free methylarginine is generated by proteolysis of posttranslationally methylated tissue proteins. L-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase (NOS) isoforms (29, 36) and thus modulates the biological effects of nitric oxide (NO), particularly in the cardiovascular system (19, 20, 36). Several studies have suggested that ADMA plasma levels are a diagnostic marker of endothelial dysfunction in cardiovascular diseases (4, 24, 38).

Methylation of the terminal nitrogen atom(s) of arginine is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMT). PRMT activity was initially identified by Paik and Kim (28), and PRMTs are now divided into two groups, type I or type II, according to substrate and product specificity. Type I and II PRMTs both form L-NMMA, possibly as an intermediary species before the formation of the dimethylated species, but differ in that type I enzymes produce ADMA, whereas type II enzymes produce SDMA (8, 21). Nine PRMT genes have been cloned to date and are termed PRMT1 to PRMT9. In vitro methyltransferase activity has been demonstrated for all PRMT gene products except PRMT2 (9, 15, 17).

Up to now, the biological impact of protein arginine methylation remains to be determined, but this process is thought to be involved in the regulation of RNA export, control of transcription, DNA repair, protein localization, protein-protein interaction, signal transduction, and recycling of receptors (17, 21, 35). In vivo substrates for type I PRMTs include histones and RNA-binding proteins including heterogeneous nuclear ribonuclear protein (hnRNP) A1, fibrillarin, and nucleolin. Methylation of RNA-binding proteins occurs in all cells. The hnRNPs are a group of proteins that regulate mRNA maturation, stability, and export to the cytoplasm, and their activity is regulated by methylation. Approximately 65% of total cellular ADMA occurs in hnRNPs (3). Large-scale proteomic approaches have revealed a potentially broad range of substrate proteins for PRMT methylation (5, 27), suggesting a significant role for arginine methylation in cellular processes. Synthesis and degradation of methylated proteins is closely coupled to protein synthesis and degradation (23). Thus it is assumed that upon proteolysis of these PRMT substrates, significant amounts of free methylarginine are released in the cytoplasm. Intracellular ADMA and SDMA levels may therefore be determined by PRMT activity and protein turnover as direct methylation of free arginines has thus far not be demonstrated.

To date, three pathways for the in vivo elimination of methylarginine have been identified: 1) renal excretion (11), 2) degradation of dimethylarginine by pyruvate aminotransferase (25), and 3) specific metabolic conversion of ADMA and L-NMMA to citrulline and dimethylamine or monomethylamine. The enzyme responsible for this specific pathway was purified from rat and human tissues and is called NG,NG-dimethylarginine dimethylaminohydrolase (DDAH) (13, 16). Subsequent studies have shown that >90% of ADMA is metabolized by DDAH. DDAH activity was found in kidney, pancreas, liver, and brain, and this correlates well with the high-protein expression levels as revealed by immunoblotting analyses in rat tissues (14). A study of DDAH expression in the rat kidney also revealed colocalization of DDAH with NOS at several anatomical sites (34). Thus the substrate specificity of DDAH and its distribution in NO-generating systems supported the idea that regulation of intracellular ADMA levels by DDAH might in turn regulate NOS activity.

To date, the interplay between methylarginine synthesis and degradation in vivo has not been described. Thus we set out to determine PRMT and DDAH activity in lung, heart, liver, and kidney homogenates. To this end, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting and ADMA degradation assays, for the assessment of tissue-specific patterns of arginine methylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sample preparation and protein hydrolysis. Tissues from C57/bl6 mice were surgically excised after thoracotomy and immediately homogenized in liquid nitrogen followed by addition of ice-cold cell lysis buffer [20 mM Tris·Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100, 2 mM Na3VO4]. Homogenized tissue was incubated for 1 h on ice and centrifuged for 15 min at 16,000 g. The resulting supernatant was stored at –20°C. Proteins were then precipitated by mixing 100 µl of tissue extract with an equal volume of 20% (vol/vol) trichloroacetic acid for 20 min. After centrifugation at 16,000 g for 12 min, the supernatants were aspirated, and the protein pellets were washed with 100 µl of ice-cold acetone for 60 min at –20°C. The suspension was centrifuged (16,000 g for 12 min), and the resulting protein pellet was dissolved in 100 µl of distilled water. Protein concentrations of crude extracts and precipitated proteins were determined by Quick Start Bradford Dye Reagent using a SmartSpecTM 3000 spectrophotometer (both Bio-Rad Laboratories, Munich, Germany). Before protein hydrolysis, 20 µl of each sample was combined with 10 µl of L-homoarginine (4 pmol/µl) as an internal standard. Total hydrolysis of precipitated protein fractions was achieved by gas-phase hydrolysis with 6 M HCl (constant boiling, sequencing grade; Pierce, Bonn, Germany) at 110°C for 16 h. Samples were dried by use of a vacuum centrifuge and stored at –20°C until further analyzed.

Human serum and bronchoalveolar lavage. This study was approved by the local ethics committee, and informed consent was obtained from each individual before entering into the study and subsequent bronchoscopy. Flexible fiberoptic bronchoscopy was performed in a standardized manner as previously described (10). The examined group consisted of seven healthy volunteers (age: 37 ± 2 yr; sex, M/F: 4/3) who had never smoked and had no history of cardiac or pulmonary disease. They also showed normal chest X-rays and pulmonary function tests.

Isolation of basic amino acids and derivatization. Tissue extracts, serum, bronchoalveolar lavage (BAL) fluid, and amino acid hydrolysates were subjected to crude fractionation on Oasis MCX solid-phase extraction (SPE) cartridges (30 mg, 1 ml; Waters, Eschborn, Germany). For tissue extracts, 30 µl of each sample was combined with 10 µl of L-homoarginine (4 pmol/µl) as an internal standard and adjusted to a final volume of 1 ml with PBS (PAA Laboratories, Pasching, Austria). Amino acid hydrolysates were directly dissolved in 1 ml of PBS. All conditioning, washing, and elution steps were performed on a vacuum manifold with a capacity for 24 columns (Waters) at a flow rate of ~0.5 ml/min. The SPE cartridges were conditioned with 2 ml of methanol/water/ammonia (50:45:5, vol/vol/vol) followed by 2 ml of PBS before sample application. Samples were passed through SPE cartridges, and contaminating components were removed with 2 ml of 0.1 M HCl followed by 2 ml of methanol. Basic compounds were eluted with 1 ml of methanol/water/ammonia (50:45:5, vol/vol/vol). Samples were dried in a vacuum centrifuge and stored at –20°C until further analyzed. Eluates were redissolved in 230 µl of distilled water and centrifuged at 14,000 g for 2 min to remove particulates before derivatization for HPLC.

Derivatization and chromatographic separation. Ortho-phthaldialdehyde (OPA) reagent was freshly prepared in potassium borate buffer (both Grom, Rottenburg-Hailfingen, Germany) according to the manufacturer's instructions. Samples (115 µl) were combined with 50 µl of OPA reagent, immediately transferred to the auto sampler, and injected after exactly 2 min.

Quantification of amino acids was performed on an HPLC system consisting of an ASI-100 auto sampler, a model P680 gradient pump, a model RF-2000 fluorescence detector, and a data acquisition system (Chromeleon, version 6.60; all Dionex, Idstein, Germany). Separation was carried out according to the method described by Teerlink et al. (33) with slight modifications. Fluorescent amino acid derivatives were separated on a SunFire C18 column (4.6 x 150 mm; 3.5-µm particle size; 100 Å pore size) with a µBondapak C18 guard column at 30°C and a flow rate of 1.1 ml/min (all columns were from Waters). After sample injection (125 µl), separation was performed under isocratic conditions with 8.8% (vol/vol) acetonitrile in 25 mM potassium phosphate buffer (pH 6.8) as solvent. The isocratic conditions were maintained for 24 min. To elute strongly bound compounds, the column was flushed with acetonitrile/water (50:50, vol/vol) for 5 min and reequilibrated under isocratic conditions for 15 min before the next injection. Fluorescent derivatives were detected at excitation and emission wavelengths of 330 and 450 nm, respectively. L-Arginine, ADMA, and SDMA were quantified by two separation steps. For the detection of ADMA and SDMA, the gain of the detector was switched to a hundred-fold higher sensitivity.

Calibration was performed as described previously (6). Briefly, six combined standards spanning the range 1.5 to 450 pmol (60 nM to 18 µM) for L-Arg, 0.15 to 45 pmol (6 nM to 1.8 µM) for L-NMMA and ADMA, and 0.09 pmol to 9 pmol (3.6 nM to 0.36 µM) for SDMA (all Sigma, St. Louis, MO) were used for calibration. Standard solutions were combined with 15 pmol of the internal standard L-homoarginine and subjected to SPE, derivatization, and chromatography as described above. Calibration curves were calculated by plotting the ratio of the peak area of analyte (arginine, L-NMMA, ADMA or SDMA) to the area of the internal standard (L-homoarginine) vs. analyte quantity. Since L-homoarginine is highly abundant in crude liver extract, quantification of methylarginine was performed by external calibration. Statistical analysis was performed using Student's t-test.

Measurement of ADMA degradation. For tissue extracts and serum, 25 µl of each sample was combined with 6 µl of combined ADMA/SDMA solution (500 pmol/µl each) and adjusted to a final volume of 0.5 ml with 0.1 M sodium phosphate buffer (pH 6.5) (26). After incubation for 2 h at 37°C, samples were directly subjected to crude fractionation on Oasis MCX cartridges, HPLC separation, and fluorescence detection as described above. As assay blank, ADMA and SDMA from the crude tissue extracts were directly quantified.

Determination of NO concentration. The NO concentrations of tissue extracts were determined using QuantiChrom Nitric Oxide Assay Kit (BioAssay Systems, Hayward, CA) according to the manufacturer's instructions.

Western blot analysis. Equal amounts of protein extracts (30 µg) were separated on 10% SDS-PAGE gels and transferred to PVDF-PLUS membranes (Osmonics, Moers, Germany). Western blots were performed with antibodies against PRMT1 (at a dilution of 1:2,000; Upstate, Dundee, UK), PRMT2 (1:1,000; Abcam, Cambridge, UK), PRMT3 (1:2,000; Upstate), PRMT4 (1:1,000; Upstate), PRMT5 (1:2,000; Upstate), PRMT6 (1:500; Imgenex, San Diego, CA), PRMT7 (1:1,000; Upstate), and DDAH1/2 [1:1,000 (22)]. After incubation with the respective secondary antibodies, specific bands were visualized by autoradiography using enhanced chemiluminescence according to the manufacturer's instructions (LUMIGEN; Amersham, Buckinghamshire, UK). The specific antibodies did not exhibit any cross-reactivity with other PRMT isoforms.

Densitometric analysis of autoradiographies was performed using a GS-800 Calibrated Densitometer and the 1-D analysis software Quantity One (both Bio-Rad Laboratories).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Methylarginine and NO content of mouse lung, heart, liver, and kidney. We first sought to establish whether different organs exhibited distinct methylation characteristics. As such, we measured the concentration of free cellular and protein-incorporated methylarginine in crude tissue extracts and protein hydrolysates, respectively, derived from mouse lung, heart, liver, and kidney. As depicted in Fig. 1A, we detected similar free ADMA and SDMA levels in lung (ADMA: 0.010 ± 0.004 nmol/mg protein; SDMA: 0.0017 ± 0.0005 nmol/mg protein) and heart (ADMA: 0.014 ± 0.004 nmol/mg protein; SDMA: 0.0026 ± 0.0005 nmol/mg protein) crude tissue extracts, whereas kidney (ADMA: 0.181 ± 0.04 nmol/mg protein; SDMA: 0.033 ± 0.012 nmol/mg protein) and liver (ADMA: 0.088 ± 0.02 nmol/mg protein; SDMA: 0.011 ± 0.003 nmol/mg protein) exhibited significantly higher concentrations for both dimethylarginines. As expected, crude kidney extracts exhibited the highest levels of free ADMA and SDMA (n = 6, P < 0.001), supporting the idea that the kidneys provide the main route for clearance of both methylarginines. Surprisingly, liver extracts displayed four- to eightfold higher levels of free cellular ADMA and SDMA compared with lung and heart (n = 6, P < 0.001). Because of the low cellular arginine concentration, liver homogenates exhibited a dramatically increased arginine/ADMA ratio of ~2, which is significantly higher than the ratios observed in lung, heart, and kidney.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Concentration of free cellular arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) (A) and nitric oxide (NO; B) in crude lung, heart, kidney, and liver tissue extracts. Values are represented as means ± SD (n = 6). **P < 0.001 kidney vs. all tissues.

 
In contrast, arginine residues in protein hydrolysates of the lung exhibited a fourfold higher degree of asymmetrical and a twofold higher degree of symmetrical dimethylation (ADMA: 4.23 ± 2.19 nmol/mg protein; SDMA: 0.37 ± 0.17 nmol/mg protein) compared with heart (ADMA: 1.11 ± 0.53 nmol/mg protein; SDMA: 0.22 ± 0.07 nmol/mg protein), kidney (ADMA: 1.41 ± 0.26 nmol/mg protein; SDMA: 0.27 ± 0.03 nmol/mg protein), or liver (ADMA: 1.05 ± 0.42 nmol/mg protein; SDMA: 0.21 ± 0.07 nmol/mg protein) hydrolysates (n = 6, P < 0.05), which exhibited almost identical levels of protein-incorporated ADMA and SDMA (Fig. 2). In conclusion, we found that, in the lung, 1.4% of all protein-incorporated arginine residues were asymmetrically dimethylated, whereas all other tissues represented levels of 0.3–0.6%. We were unable to detect any monomethylation of arginine residues in either hydrolysates or crude tissue lysates.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. Concentration of protein-incorporated arginine, ADMA, and SDMA in lung, heart, kidney, and liver protein hydrolysates. Values are represented as means ± SD (n = 6). *P < 0.05 lung vs. all tissues.

 
In addition, we measured the concentration of NO in crude tissue extracts. As depicted in Fig. 1B, we detected similar NO levels in lung (0.66 ± 0.23 nnmol/mg protein) and heart (0.81 ± 0.13 nnmol/mg protein) crude tissue extracts, whereas kidney extracts (0.32 ± 0.20 nnmol/mg protein) exhibited lower concentrations and liver extracts (1.10 ± 0.86 nnmol/mg) higher concentrations for NO.

Protein expression of PRMT1–7. To determine whether the increased concentration of protein-incorporated ADMA observed in the mouse lung correlated with increased expression of type I PRMTs, immunoblot analysis was performed on crude tissue homogenates. The expression of PRMT isoforms in selected tissues is illustrated in Fig. 3. Densitometric analysis revealed that mouse lungs expressed significantly higher levels of PRMT1, 2, and 6 compared with heart, kidney, and liver (n = 3, P < 0.05).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 3. Expression of protein arginine methyltransferases (PRMT) 1–7 in mouse lung, heart, kidney, and liver. Tissue extracts (30 µg of protein) were subjected to immunoblot analysis using antibodies against PRMT1–7.

 
Methylarginine content of serum and BAL fluid. The increased type I PRMT expression and activity observed in the mouse lung may result in significant levels of free cellular ADMA after protein breakdown, leading to a release of ADMA in the BAL fluid. To determine whether this was the case, we quantified ADMA in mouse and human BAL fluid by HPLC analysis (Table 1). Both ADMA and SDMA were detected in mouse and human BAL fluid. Interestingly, the BAL fluid of mouse and human exhibited an ADMA/SDMA ratio similar to the ratio observed in the respective sera (see Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Comparison of free arginine methylation in serum and BAL fluid from mice and human

 
DDAH protein expression and ADMA degradation. Western blot analysis of tissue homogenates indicated a tissue-specific expression of DDAH isoforms (Fig. 4A). Densitometry revealed that DDAH1 expression in kidney and liver was significantly higher compared with lung. DDAH1 was not detected in heart lysates. In contrast, DDAH2 was equally expressed in liver, lung, and heart but significantly less in kidney lysates (Fig. 4B). To determine whether the different patterns in DDAH protein expression were correlated with enzymatic activity, in vitro ADMA degradation was measured in crude tissue lysates (Fig. 5). Kidney lysates exhibited the highest activity compared with liver and lung. Moreover, degradation activity was found to be significantly higher in liver vs. lung. ADMA degradation was not detected in heart lysates and serum. Significant degradation of SDMA was not detected in any of the tissues investigated.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 4. Expression of dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 in mouse lung, heart, kidney, and liver. A: tissue extracts (30 µg of protein) were subjected to immunoblot analysis with antibodies against DDAH1 and 2. B: relative quantification of DDAH expression by densitometry. Values are represented as means ± SD in optical density (n = 3). *P < 0.05. N.D., not detected.

 

Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 5. A: HPLC chromatograms of a combined standard containing 15 pmol of ADMA and SDMA (top) and from crude lung extract incubated with the identical amount of combined standard (bottom). Chromatographic conditions and enzymatic activity assay are described in detail in MATERIALS AND METHODS. B: enzymatic degradation of 0.75 nmol of ADMA in 25 µl of lung, heart, kidney, and liver tissue crude extracts and serum. Enzymatic activity is defined as the degradation of ADMA in 2 h at 37°C. Activity is expressed as means ± SD (n = 3) in nmol/mg protein. *P < 0.05 kidney and liver vs. lung.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Arginine methylation of proteins is catalyzed by the action of PRMTs. There is potentially a broad range of target proteins for both types of PRMTs, and the enzymes and their substrates are widely distributed throughout the body (5, 37). In the cardiovascular system, the expression of PRMT has only been reported for type I PRMTs in the heart, smooth muscle cells, and endothelial cells (35). Initially, we characterized the expression of PRMTs in lung, heart, liver, and kidney. We found that mouse lungs expressed significantly higher levels of PRMT1, 2, and 6 compared with the heart, kidney, or liver. To elucidate whether the increased pulmonary expression of type I PRMTs correlated with increased asymmetrical dimethylation of lung proteins, protein hydrolysis of tissue proteins and HPLC-analysis were performed. In lung protein hydrolysates, we found that arginine residues exhibited an almost fourfold higher degree of asymmetrical and twofold higher degree of symmetrical dimethylation compared with arginine residues from heart, kidney, and liver tissue.

Free cellular methylarginine levels are dependent on PRMT activity, the rate of protein degradation, the rates of ADMA metabolism by DDAHs, degradation of ADMA and SDMA by pyruvate aminotransferase, and the rates of active methylarginine uptake and release. We found similar free cellular ADMA and SDMA levels in the lung and heart, whereas kidney and liver exhibited significantly higher concentrations for both dimethylarginines. Kidney lysates exhibited the highest levels of both dimethylarginines. Because kidney proteins did not display a higher degree of protein-incorporated methylarginine, the increased ADMA and SDMA levels may be a result of active renal methylarginine uptake, as suggested in previously published studies (1, 18, 36). Kidney homogenates exhibited significantly higher DDAH1 expression and ADMA degradation activity compared with liver, lung, or heart homogenate, suggesting that the kidney provides the main route for clearance and metabolic conversion of circulating methylarginines. Moreover, kidney lysates displayed the highest capacity for ADMA degradation, albeit DDAH2 expression was significantly lower than that observed in the liver, lung, or heart. Furthermore, no significant degradation of SDMA (as a consequence of pyruvate aminotransferase activity) was observed. These findings suggest that renal metabolism of ADMA is due to the metabolic action of DDAH1 and not DDAH2 and pyruvate aminotransferase.

This is the first report on the direct characterization of methylarginine metabolism in the liver. Liver lysates displayed four- to eightfold higher levels of free cellular ADMA and SDMA compared with the lung and heart. Because liver proteins did not exhibit a higher degree of protein-incorporated methylarginine, the increased ADMA and SDMA levels may be a result of active hepatic methylarginine uptake by the y+ transporter, as suggested in previously published studies (31, 32). Liver lysates also exhibited significantly higher DDAH1 expression and ADMA degradation activity than did lysates from lung and heart tissue. A previously published study on the characterization of ADMA clearance in rat plasma after nephrectomy has suggested that ADMA does not require the kidney for its elimination from the plasma (7). Moreover, transplanted liver graft is capable of clearing circulating ADMA in human patients (32). Thus our data suggest that the liver provides an alternative route for the clearance and metabolic conversion of circulating ADMA.

Together, kidney and liver tissues exhibited a high capacity for ADMA degradation, supporting the idea that both organs provide complementary routes for clearance and metabolic conversion of circulating ADMA.

ADMA is a competitive inhibitor of all NOS isoforms and thus modulates the formation of NO, particularly in the cardiovascular system. We found similar NO levels in lung and heart tissue extracts, whereas kidney extracts exhibited lower, and liver extracts exhibited higher, concentrations of NO. Taking the tissue-specific ADMA levels into account, a connection between NO formation and free cellular ADMA levels was not evident. Thus the increased methylarginine levels in the kidney and liver do not contribute to the regulation of NOS activity but rather reflect their function of metabolic conversion of circulating methylarginine.

Compared with the heart, pulmonary expression of PRMTs was significantly increased and correlated with enhanced asymmetrical and symmetrical dimethylation of proteins in the lung. In contrast, we found similar levels of free cellular ADMA and SDMA in lung and heart. Lung homogenates exhibited DDAH1 expression and ADMA degradation activity, which was not detected at all in heart lysates, suggesting that the lung is capable of metabolic conversion of free cellular methylarginine. Heart lysates did not display a capacity for ADMA degradation, although their DDAH2 expression was slightly higher than those of lung lysates. Therefore, relevant pulmonary degradation of ADMA is a result of DDAH1 activity, whereas no contribution of DDAH2 is evident. Furthermore, ADMA and also SDMA were detected in mouse and human BAL fluid. The BAL fluid of mouse and human exhibited an ADMA/SDMA ratio is similar to the ratio observed in the respective sera, suggesting that the similar levels are a result of diffusion through paracellular spaces. Circulating ADMA levels are raised in patients with pulmonary hypertension and in experimental models of pulmonary hypertension (2, 12, 22, 30). In the rat model of chronic hypoxia-induced pulmonary hypertension, the effect was caused by a decreased expression and activity of DDAH1 (22). Thus the overall pulmonary ADMA output reflects a balance of PRMT activity, rates of protein turnover, intracellular DDAH1 activity, and active extrusion from the cell.

The relative contribution of each component remains to be determined, but the salient findings of the present investigation can be summarized as: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation of the lung proteome; 2) pulmonary ADMA degradation was undertaken by DDAH1; and 3) BAL fluid and serum exhibited almost identical ADMA/SDMA ratios. Together, these observations suggest that methylarginine metabolism by the lung significantly contributes to circulating ADMA and SDMA levels.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Deutsche Forschungsgemeinschaft Grant DFG-SFB547 (to F. Grimminger and O. Eickelberg) and the Alexander von Humboldt Foundation (Sofja Kovalevskaja Award) (to O. Eickelberg). D. Zakrzewicz and K. Kitowska were supported by predoctoral fellowships of the International Graduate Program Molecular Biology and Medicine of the Lung.


   ACKNOWLEDGMENTS
 
We are indebted to Drs. Werner Seeger and Rory E. Morty for critical reading of the manuscript; Günter Lochnit for performing protein hydrolysis; and all members of the Eickelberg Laboratory for valuable discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Bulau, Univ. of Giessen Lung Center, Univ. of Giessen School of Medicine, Aulweg 123, D-35329 Giessen, Germany (e-mail: Patrick.Bulau{at}uglc.de)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Al Banchaabouchi M, Marescau B, Possemiers I, D'Hooge R, Levillain O, De Deyn PP. NG,NG-dimethylarginine and NG,NG-dimethylarginine in renal insufficiency. Pflügers Arch 439: 524–531, 2000.[CrossRef][Web of Science][Medline]
  2. Arrigoni FI, Vallance P, Haworth SG, Leiper JM. Metabolism of asymmetric dimethylarginines is regulated in the lung developmentally and with pulmonary hypertension induced by hypobaric hypoxia. Circulation 107: 1195–1201, 2003.
  3. Boffa LC, Karn J, Vidali G, Allfrey VG. Distribution of NG,NG-dimethylarginine in nuclear protein fractions. Biochem Biophys Res Commun 74: 969–976, 1977.[CrossRef][Web of Science][Medline]
  4. Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 98: 1842–1847, 1998.
  5. Boisvert FM, Cote J, Boulanger MC, Richard S. A proteomic analysis of arginine-methylated protein complexes. Mol Cell Proteomics 2: 1319–1330, 2003.[Abstract/Free Full Text]
  6. Bulau P, Zakrzewicz D, Kitowska K, Leiper J, Eickelberg O. Quantitative assessment of arginine methylation in free versus protein-incorporated amino acids in vitro and in vivo using protein hydrolysis and high-performance liquid chromatography. Biotechniques 40: 305–310, 2006.[Web of Science][Medline]
  7. Carello KA, Whitesall SE, Lloyd MC, Billecke SS, D'Alecy LG. Asymmetrical dimethylarginine plasma clearance persists after acute total nephrectomy in rats. Am J Physiol Heart Circ Physiol 290: H209–H216, 2006.[Abstract/Free Full Text]
  8. Clarke S. Protein methylation. Curr Opin Cell Biol 5: 977–983, 1993.[CrossRef][Medline]
  9. Cook JR, Lee JH, Yang ZH, Krause CD, Herth N, Hoffmann R, Pestka S. FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues. Biochem Biophys Res Commun 342: 472–481, 2006.[CrossRef][Web of Science][Medline]
  10. Gunther A, Siebert C, Schmidt R, Ziegler S, Grimminger F, Yabut M, Temmesfeld B, Walmrath D, Morr H, Seeger W. Surfactant alterations in severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema. Am J Respir Crit Care Med 153: 176–184, 1996.[Abstract]
  11. Kakimoto Y, Akazawa S. Isolation and identification of N-G,N-G- and N-G,N'-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. J Biol Chem 245: 5751–5758, 1970.[Abstract/Free Full Text]
  12. Kielstein JT, Bode-Boger SM, Hesse G, Martens-Lobenhoffer J, Takacs A, Fliser D, Hoeper MM. Asymmetrical dimethylarginine in idiopathic pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol 25: 1414–1418, 2005.[Abstract/Free Full Text]
  13. Kimoto M, Miyatake S, Sasagawa T, Yamashita H, Okita M, Oka T, Ogawa T, Tsuji H. Purification, cDNA cloning and expression of human NG,NG-dimethylarginine dimethylaminohydrolase. Eur J Biochem 258: 863–868, 1998.[Web of Science][Medline]
  14. Kimoto M, Tsuji H, Ogawa T, Sasaoka K. Detection of NG,NG-dimethylarginine dimethylaminohydrolase in the nitric oxide-generating systems of rats using monoclonal antibody. Arch Biochem Biophys 300: 657–662, 1993.[CrossRef][Web of Science][Medline]
  15. Lee J, Sayegh J, Daniel J, Clarke S, Bedford MT. PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280: 32890–32896, 2005.[Abstract/Free Full Text]
  16. Leiper JM, Santa Maria J, Chubb A, MacAllister RJ, Charles IG, Whitley GS, Vallance P. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem J 343: 209–214, 1999.
  17. Leiper JM, Vallance P. The synthesis and metabolism of asymmetric dimethylarginine (ADMA). Eur J Clin Pharmacol 62, Suppl 13: 33–38, 2006.[CrossRef]
  18. MacAllister RJ, Rambausek MH, Vallance P, Williams D, Hoffmann KH, Ritz E. Concentration of dimethyl-L-arginine in the plasma of patients with end-stage renal failure. Nephrol Dial Transplant 11: 2449–2452, 1996.[Abstract/Free Full Text]
  19. MacAllister RJ, Whitley GS, Vallance P. Effects of guanidino and uremic compounds on nitric oxide pathways. Kidney Int 45: 737–742, 1994.[Web of Science][Medline]
  20. Machado RF, Londhe Nerkar MV, Dweik RA, Hammel J, Janocha A, Pyle J, Laskowski D, Jennings C, Arroliga AC, Erzurum SC. Nitric oxide and pulmonary arterial pressures in pulmonary hypertension. Free Radic Biol Med 37: 1010–1017, 2004.[CrossRef][Web of Science][Medline]
  21. McBride AE, Silver PA. State of the arg: protein methylation at arginine comes of age. Cell 106: 5–8, 2001.[CrossRef][Web of Science][Medline]
  22. Millatt LJ, Whitley GS, Li D, Leiper JM, Siragy HM, Carey RM, Johns RA. Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension. Circulation 108: 1493–1498, 2003.
  23. Miyake M, Kakimoto Y. Synthesis and degradation of methylated proteins of mouse organs: correlation with protein synthesis and degradation. Metabolism 25: 885–896, 1976.[CrossRef][Web of Science][Medline]
  24. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Imaizumi T. Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 99: 1141–1146, 1999.
  25. Ogawa T, Kimoto M, Sasaoka K. Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2. J Biol Chem 265: 20938–20945, 1990.[Abstract/Free Full Text]
  26. Ogawa T, Kimoto M, Sasaoka K. Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. J Biol Chem 264: 10205–10209, 1989.[Abstract/Free Full Text]
  27. Ong SE, Mittler G, Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat Methods 1: 119–126, 2004.[CrossRef][Web of Science][Medline]
  28. Paik WK, Kim S. Protein methylase I. Purification and properties of the enzyme. J Biol Chem 243: 2108–2114, 1968.[Abstract/Free Full Text]
  29. Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664–666, 1988.[CrossRef][Medline]
  30. Pullamsetti S, Kiss L, Ghofrani HA, Voswinckel R, Haredza P, Klepetko W, Aigner C, Fink L, Muyal JP, Weissmann N, Grimminger F, Seeger W, Schermuly RT. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J 19: 1175–1177, 2005.[Abstract/Free Full Text]
  31. Siroen MP, van der Sijp JR, Teerlink T, van Schaik C, Nijveldt RJ, van Leeuwen PA. The human liver clears both asymmetric and symmetric dimethylarginine. Hepatology 41: 559–565, 2005.[CrossRef][Web of Science][Medline]
  32. Siroen MP, Warle MC, Teerlink T, Nijveldt RJ, Kuipers EJ, Metselaar HJ, Tilanus HW, Kuik DJ, van der Sijp JR, Meijer S, van der Hoven B, van Leeuwen PA. The transplanted liver graft is capable of clearing asymmetric dimethylarginine. Liver Transpl 10: 1524–1530, 2004.[CrossRef][Web of Science][Medline]
  33. Teerlink T, Nijveldt RJ, de Jong S, van Leeuwen PA. Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography. Anal Biochem 303: 131–137, 2002.[CrossRef][Web of Science][Medline]
  34. Tojo A, Welch WJ, Bremer V, Kimoto M, Kimura K, Omata M, Ogawa T, Vallance P, Wilcox CS. Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. Kidney Int 52: 1593–1601, 1997.[Web of Science][Medline]
  35. Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 24: 1023–1030, 2004.[Abstract/Free Full Text]
  36. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339: 572–575, 1992.[CrossRef][Web of Science][Medline]
  37. Wada K, Inoue K, Hagiwara M. Identification of methylated proteins by protein arginine N-methyltransferase 1, PRMT1, with a new expression cloning strategy. Biochim Biophys Acta 1591: 1–10, 2002.[Medline]
  38. Zoccali C, Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L, Cataliotti A, Bellanuova I, Fermo I, Frolich J, Boger R. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 358: 2113–2117, 2001.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
J. Li, A. Wilson, X. Gao, R. Kuruba, Y. Liu, S. Poloyac, B. Pitt, W. Xie, and S. Li
Coordinated Regulation of Dimethylarginine Dimethylaminohydrolase-1 and Cationic Amino Acid Transporter-1 by Farnesoid X Receptor in Mouse Liver and Kidney and Its Implication in the Control of Blood Levels of Asymmetric Dimethylarginine
J. Pharmacol. Exp. Ther., October 1, 2009; 331(1): 234 - 243.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. M. Wells, M. C. Buford, C. T. Migliaccio, and A. Holian
Elevated Asymmetric Dimethylarginine Alters Lung Function and Induces Collagen Deposition in Mice
Am. J. Respir. Cell Mol. Biol., February 1, 2009; 40(2): 179 - 188.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
B. Wojciak-Stothard, B. Torondel, L. Zhao, T. Renne, and J. M. Leiper
Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function
Mol. Biol. Cell, January 1, 2009; 20(1): 33 - 42.
[Abstract] [Full Text] [PDF]

Home page
 JPEN J Parenter Enteral NutrHome page
M. C. Richir, R. H. Bouwman, T. Teerlink, M. P.C. Siroen, T. P.G.M. de Vries, and P. A.M. van Leeuwen
The Prominent Role of the Liver in the Elimination of Asymmetric Dimethylarginine (ADMA) and the Consequences of Impaired Hepatic Function
JPEN J Parenter Enteral Nutr, November 1, 2008; 32(6): 613 - 621.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. Lara, S. B. Khatri, Z. Wang, S. A. A. Comhair, W. Xu, R. A. Dweik, M. Bodine, B. S. Levison, J. Hammel, E. Bleecker, et al.
Alterations of the Arginine Metabolome in Asthma
Am. J. Respir. Crit. Care Med., October 1, 2008; 178(7): 673 - 681.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
M. R. Wilkins, J. Wharton, F. Grimminger, and H. A. Ghofrani
Phosphodiesterase inhibitors for the treatment of pulmonary hypertension
Eur. Respir. J., July 1, 2008; 32(1): 198 - 209.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
K. Kitowska, D. Zakrzewicz, M. Konigshoff, I. Chrobak, F. Grimminger, W. Seeger, P. Bulau, and O. Eickelberg
Functional role and species-specific contribution of arginases in pulmonary fibrosis
Am J Physiol Lung Cell Mol Physiol, January 1, 2008; 294(1): L34 - L45.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. M. Wells and A. Holian
Asymmetric Dimethylarginine Induces Oxidative and Nitrosative Stress in Murine Lung Epithelial Cells
Am. J. Respir. Cell Mol. Biol., May 1, 2007; 36(5): 520 - 528.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. A. Dweik
The lung in the balance: arginine, methylated arginines, and nitric oxide
Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L15 - L17.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L18    most recent
00076.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bulau, P.
Right arrow Articles by Eickelberg, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bulau, P.
Right arrow Articles by Eickelberg, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.