AJP - Lung Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 294: L498-L504, 2008. First published January 11, 2008; doi:10.1152/ajplung.00242.2007
1040-0605/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/L498    most recent
00242.2007v1
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 (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belik, J.
Right arrow Articles by Grasemann, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belik, J.
Right arrow Articles by Grasemann, H.

Developmental changes in arginase expression and activity in the lung

Jaques Belik, Darakhshanda Shehnaz, Jingyi Pan, and Hartmut Grasemann

Department of Pediatrics, Physiology and Experimental Medicine, The Hospital for Sick Children Research Institute, and University of Toronto, Toronto, Ontario, Canada

Submitted 22 June 2007 ; accepted in final form 8 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arginases compete with nitric oxide (NO) synthases for L-arginine as common substrate. Pulmonary vascular and airway diseases in which arginase activity is increased are associated with decreased NO production and reduced smooth muscle relaxation. The developmental patterns of arginase activity and type I and II isoforms expression in the lung have not been previously evaluated. Hypothesizing that lung arginase activity is developmentally regulated and highest in the fetus, we measured the expression of both arginase isoforms and total arginase activity in fetal, newborn, and adult rat lung, pulmonary artery, and bronchial tissue. In addition, intrapulmonary arterial muscle force generation was evaluated in the absence and presence of the arginase inhibitor N{omega}-hydroxy-nor-L-arginine (nor-NOHA). Arginase II content, as well as total arginase activity, was highest in fetal rat lung, bronchi, and pulmonary arterial tissue and decreased with age (P < 0.05), and its lung cell expression was developmentally regulated. In the presence of nor-NOHA, pulmonary arterial force generation was significantly reduced in fetus and newborn (P < 0.01). No significant change in force generation was noted in bronchial tissue following arginase inhibition. In conclusion, arginase II is regulated developmentally, and both expression and activity are maximal during fetal life. We speculate that the maintenance of a high pulmonary vascular resistance and decreased lung NO production prenatally may, in part, be dependent on increased arginase expression and/or activity.

pulmonary vascular resistance; airway resistance; nitric oxide

GIVEN THE HIGH FETAL PULMONARY vascular resistance, pulmonary blood flow prenatally is less than 10% of the total cardiac output. Pulmonary vascular resistance rapidly decreases at birth and reaches the low physiological level of adults at the end of the neonatal period (12). The factors accounting for maintenance of high pulmonary vascular resistance prenatally and its rapid decrease after birth are not completely understood. Pulmonary vascular nitric oxide (NO) availability and its relaxant effect on smooth muscle are considered to play an important role in the transition from fetal to neonatal circulation (1).

NO is produced by nitric oxide synthases (NOS). In the lung, three NOS isoforms are present. Endothelial NOS (eNOS) is expressed in pulmonary vascular endothelium and bronchial epithelium, whereas neuronal (nNOS) and inducible (iNOS) NOS are the predominant isoforms in vascular and airway tissue smooth muscle, bronchial epithelial cells, and macrophages (3).

The amino acid L-arginine is a substrate for both NOSs and arginases. One role of arginases is deamination of L-arginine in the liver by catalyzing the last step of the urea cycle and converting ammonia to the less toxic urea. There are two known isoforms of arginase. Arginase I is located in the cytosol and strongly expressed in liver, whereas arginase II is confined to the mitochondrial matrix and mainly expressed in kidney. The role of arginase I in the liver is well-defined, where it catalyzes the deamination of L-arginine to produce ornithine and urea. The role of extrahepatic arginases is not very clear, however, ornithine, the downstream product of arginase activity, is known to be further metabolized into polyamines that are involved in tissue repair and growth, as well as proline, the precursor of collagen formation (21).

Both isoforms of arginase are expressed in lung, and increased arginase activity has been reported in obstructive airway diseases such as asthma (3, 34), cystic fibrosis (13–15), as well as pulmonary hypertension (22–25, 30, 33). High arginase activity in the pathophysiology of these diseases is believed to result in decreased availability of L-arginine for constitutive NOSs and consequently low NO production.

The maturation-dependent changes in arginase expression and activity in the lung as well as their physiological implications have not been previously evaluated and constitute the main goal of this study. Reflecting the rapid decrease in pulmonary vascular resistance after birth, we hypothesized that arginase expression and activity in lung tissue are highest prenatally. Accordingly, we evaluated rat fetal, early neonatal, and adult lung tissue, as well as the specific contribution of pulmonary arterial vs. airways tissue arginase activity and their age-dependent functional changes.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Timed-pregnant and nonpregnant young adult (2–4 mo old, 250–300 g) as well as 3-day-old newborn Sprague-Dawley rats (Charles River, Ontario, Canada) were studied. All procedures were conducted according to criteria established by the Canadian Council on Animal Care and approved by the Hospital for Sick Children Research Institute Animal Care Review Committee.

Preparation of tissue extracts. Tissue extracts were made in the lysis buffer consisting of 50 mM HEPES buffered with sodium hydroxide to pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 10% glycerol, 1.5 mM magnesium chloride, 2 mM EDTA, 1 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin. Lung tissue was homogenized in a rotor-stator-type homogenizer whereas pulmonary artery and bronchi were frozen in liquid nitrogen and then grounded with mortar and pestle before ice-cold lysis buffer was added. After 1 h on ice, the homogenates were centrifuged at 14,000 rpm for 20 min. The supernatants as extracts were transferred to fresh tubes. Total protein concentration was measured according to the Bradford method (7); extracts were diluted to a final concentration of 4 mg/ml. Given the small size, fetal and newborn tissue was pooled such that each sample reflected tissue derived from three separate animals, respectively.

Measurement of arginase activity. Arginase activity was measured according to the method described by Corraliza et al. (9). In brief, the extracts were incubated with equal volumes of 10 mM manganese chloride in 25 mM Tris·HCl, pH 7.4, at 56°C for 10 min to activate the enzyme. Then 50 µl of the activated extracts were transferred to the boil-proof Eppendorf tubes containing 50 µl of 20–250 mM of L-arginine, pH 9.7, with or without N{omega}-hydroxy-nor-L-arginine (nor-NOHA; Calbiochem, San Diego, CA), the competitive inhibitor of arginase activity. These assay mixtures were incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by adding 800 µl of the acid mix comprised of sulfuric acid, phosphoric acid, and water in a ratio of 1:3:7. Fifty microliters of 9% {alpha}-isonitrosopropiophenone (ISPF) dissolved in ethanol was added, and the tubes were incubated in a boiling water bath for 45 min. The color was developed by keeping the tubes in the dark at room temperature for 10 min. Aliquots of 200 µl were transferred to a 96-well plate, and absorbance at 540 nm was measured in VersaMax microplate reader (Plate Technologies, Sunnyvale, CA). Each assay was run in triplicate, and the activity was completely inhibited with nor-NOHA confirming that the urea produced was an outcome of the arginase action.

Immunoblotting for arginase I, arginase II, and eNOS. The tissue extracts were digested with Laemmli sample loading buffer at 100°C for 5 min and electrophoresed on 10% SDS-PAGE gel. Proteins separated on the gel were electrotransferred to nitrocellulose membrane (Amersham Biosciences, Mississauga, Ontario, Canada) at room temperature for 1 h at a constant voltage of 100 V. The membranes were blocked by rocking with 5% nonfat dry milk in 20 mM Tris·HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The Trans-Blots were then incubated at 4°C overnight on a rocking platform with commercially available polyclonal antibodies raised against arginase I or II (Santa Cruz Biotechnology) or eNOS (Transductions Laboratories, Lexington, KY) at dilutions of 1:500, 1:1,000, and 1:5,000 in TBS-T containing 5% milk, respectively. After washing with TBS-T for 30 min, the Trans-Blots were incubated with IgG conjugated with horseradish peroxidase (1:20,000 dilution in TBS-T containing 5% milk) at room temperature for 60 min and washed with TBS-T for 40 min at room temperature. The blots were subsequently treated with chemiluminescence reagent (ECL; PerkinElmer, Shelton, CT) and exposed to Kodak Scientific Imaging Systems film. Membrane blots were stripped and exposed to β-actin (1:40,000 dilution). The bands for arginase I, arginase II, and eNOS on imaging film were quantitated by measuring density on a chemiluminescence imaging instrument with a built-in program for analysis (FluorChem FC2; Alpha Innotech, San Leandro, CA), and results were expressed as a ratio to the corresponding β-actin densities.

Immunohistochemistry. Fetal, newborn, and adult lungs were removed immediately after killing, inflated (20 cmH20 airway pressure), and maintained in 4% paraformaldehyde in a 0.2 M sodium phosphate buffer (pH 7.4). The tissue was postfixed at 4°C for 4–20 h, dehydrated in a graded series of ethanols, and cleared in xylene. The fixed tissue was embedded in molten paraffin in a heating incubator at 54°C for 2–3 days. After tissues were embedded, the paraffin block containing the tissue was trimmed and mounted on a rotary microtome (model 45; Lipshaw, Detroit, MI) equipped with a standard knife holder and a forward-moving block. The tissue was oriented to allow for the cutting of transverse sections (6-µm thickness). Sections were mounted on gelatin-subbed microscope slides and immersed in xylene for 15 min to remove wax before staining. The dewaxed sections were rehydrated through a graded series of ethanols, stained with Gill hematoxylin for 2 min for nuclear staining, and rinsed with distilled water. After they were washed with distilled water, the sections were rehydrated in ethanol, equilibrated in xylene, and positioned under a glass cover with Entellan.

Tissues for all immunolabeling were immersed in methanol containing 0.3% H2O2 for 20–45 min, microwaved for 20 min in 10 mM sodium citrate buffer, rinsed with PBS, and blocked with Dako serum-free protein (Dako, Mississauga, Ontario, Canada) for 30 min. All tissues were incubated overnight with goat anti-arginase I or II (1:100 dilution; Santa Cruz Biotechnology) at 4°C. After three 5-min PBS washes, tissues were incubated with the horse anti-goat IgG biotinylated secondary antibody (1:200 dilution; Vector Laboratories, Burlington, Ontario, Canada) for 1–2 h at room temperature. Tissues treated with antibodies were further incubated in a 1% avidin-biotinylated-peroxidase complex (1:50 ABC; kit PK-4000, Vectastain, Vector Laboratories) for 30 min. Antibody labeling was then revealed by 3,3-diaminobenzidine tetrahydrochloride (DAB) for 10 min at room temperature. Controls were provided by primary antibody omission.

Organ bath studies. Third or fourth (adult) generation left lung intralobar pulmonary artery ring segments or their adjacent bronchus (average diameter, 80–100 µm; length, 2 mm) were dissected free and mounted in a wire myograph (Danish Myo Technology A/S, Aarhus, Denmark). Isometric changes were digitized and recorded online (Myodaq, Danish Myo Technology A/S). Tissues were bathed in Krebs-Henseleit buffer (NaCl, 115 mM; NaHCO3, 25 mM; NaHPO4, 1.38 mM; KCl, 2.51 mM; MgSO4 7H2O, 2.46 mM; CaCl2, 1.91 mM; and dextrose, 5.56 mM) bubbled with air-6% CO2 and maintained at 37°C. After 1 h of equilibration, the optimal tissue resting tension was determined by repeated stimulation with 128 mM KCl until maximum active tension was reached. All subsequent force measurements were obtained at optimal resting tension.

Pulmonary vascular muscle force generation was evaluated by stimulating with the thromboxane A2-mimetic U-46619, whereas the muscarinic receptor stimulant acetylcholine was used in bronchial tissue. Contractile responses were normalized to the tissue cross-sectional area as (width x diameter) x 2 and expressed as mN/mm2.

The arginases inhibitor nor-NOHA (Calbiochem) was added to the muscle bath 20 min before the initiation of the dose-responses at a 10–5 M concentration. This concentration has been shown by others to effectively inhibit vascular and airway tissue arginases activity. Relaxation was induced with the endothelium-dependent agonist acetylcholine in the presence and absence of the nonspecific NOS inhibitor nitro-L-arginine methyl ester (L-NAME; 10–4 M) following precontraction with U-46619 at age-specific concentrations equivalent to the concentration giving 75% maximal response (EC75).

Drugs. Unless otherwise indicated, all drugs were obtained from Sigma-Aldrich, Ontario, Canada.

Data analysis. Data were evaluated by one- or two-way ANOVA with multiple comparisons obtained by the Tukey-Kramer test when appropriate. Statistical significance was accepted at P < 0.05. All statistical analysis was performed with the Number Cruncher Statistical System (NCSS, Kaysville, UT). Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tissue arginase activity was measured in total lung, intrapulmonary arteries and their adjacent bronchi (Fig. 1). Significantly higher arginase activity was found in all fetal compared with adult tissues. Arginase activity in pulmonary artery was also significantly higher in newborn compared with adult, whereas no differences in arginase activity were seen between newborn and adult rats in whole lung tissue or bronchi (Fig. 1).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Arginase activity for fetal, newborn, and adult lung and pulmonary arterial and bronchial tissue (n = 3 for each tissue and age). *P < 0.05 and **P < 0.01 compared with adult values. {dagger}P < 0.05 compared with newborn values by one-way ANOVA and Tukey-Kramer multiple comparisons test.

 
Both arginase isoforms were identified in rat lung tissues throughout development by Western blots, and their expression was significantly different in fetal compared with postnatal lung (P < 0.01; Fig. 2). Whereas the expression of arginase type II was highest in fetal whole lung homogenates, the type I content was significantly lower in fetal compared with newborn and adult lungs (Fig. 2). eNOS was also expressed in lungs of all age groups with a tendency toward higher levels of expression in the fetus and newborn compared with adult (Fig. 2).


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 2. Arginase isoform types I and II and endothelial nitric oxide synthase (eNOS) expression normalized to β-actin in fetal (n = 3), newborn (n = 3), and adult (n = 3) whole lung. **P < 0.01 compared with the respective newborn and adult tissue by one-way ANOVA and Tukey-Kramer multiple comparisons test.

 
To comparatively evaluate tissue-specific expression of the arginase isoforms in pulmonary artery and airway, we further extracted protein from intrapulmonary 3rd to 4th generation arteries and bronchi. As shown in Fig. 3, arginase I expression in adult tissue was at the limits of antibody sensitivity. A further characterization of the maturational pattern of arginase I in the small quantities of bronchial and pulmonary arterial tissue of fetal and neonatal animals was therefore not feasible. In contrast, arginase type II expression could be ascertained in bronchial but not pulmonary arterial adult tissue (Fig. 3). As such, we further evaluated the developmental expression pattern of arginase type II in bronchial tissue (Fig. 4). Its expression was significantly greater (P < 0.01) in the fetus compared with newborn and adult in a pattern similar to the expression of this isoenzyme in whole lung homogenate.


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. Representative Western blots of adult pulmonary arterial, bronchial, and lung tissue (200 µg per protein loading) for arginases I and II and β-actin. Note the faint arginase type I bands present for the pulmonary arterial and bronchial tissue as well as for arginase II in pulmonary arteries.

 

Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. Arginase II expression in fetal (n = 3), newborn (n = 3), and adult (n = 3) bronchial tissue. **P < 0.01 compared with the respective newborn and adult tissue by one-way ANOVA.

 
To determine the cellular localization of arginases in the lung, immunohistochemical staining for both isoforms was obtained. As shown in Fig. 5, arginases I and II are both expressed in the rat lung at all three ages studied. Arginase I appeared to be localized predominately in pulmonary artery endothelium and airway epithelium. The type II isoform was mostly expressed in the muscle and adventitial layers of the pulmonary arteries and airways (Fig. 5).


Figure 5
View larger version (87K):
[in this window]
[in a new window]

  		Fig. 5. Fetal, newborn, and adult rat lung immunohistochemistry for arginases I and II. Brown staining (arrows) identify arginase expression in vascular and airway tissue. See text for methodological details. Magnification, x200.

 
To further evaluate the functional significance of arginase expression and its activity with regard to developmental changes, we studied pulmonary arterial and bronchial smooth muscle contraction in the presence of the arginase inhibitor nor-NOHA. The rationale for this set of experiments relates to the expected increase in availability of L-arginine for endogenous NO production following arginase inhibition that would result in reduced force generation in response to agonist stimulation. Figure 6 illustrates the pulmonary arterial muscle force that developed in response to the thromboxane A2 analog U-46619 in the absence and presence of the arginases inhibitor nor-NOHA. In the presence of arginase inhibitor, both fetal and newborn pulmonary arteries generated significantly lower force for all molar concentrations tested (P < 0.01, respectively). In contrast, except for the 10–7 M U-46619 concentration, nor-NOHA had no effect on force generation in adult pulmonary arteries. Similarly, incubation with nor-NOHA resulted in significant relaxation of U-46619-contracted vessels to levels comparable with the age-matched control acetylcholine-mediated values in the fetus and newborn but not adult arteries (Fig. 7). Experiments conducted in the presence of L-NAME (10–4 M) completely eliminated the nor-NOHA-induced relaxation in newborn pulmonary arteries (Fig. 7). These data suggest that arginase inhibition led to an increase in NO production in the fetal and newborn arteries of a magnitude comparable with the acetylcholine-induced eNOS-mediated response.


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 6. Fetal (n = 4), newborn (n = 16), and adult (n = 6) pulmonary arterial dose-response to the thromboxane A2 analog U-46619 in the absence (control) and presence of the arginase inhibitor N{omega}-hydroxy-nor-L-arginine (nor-NOHA; 10–5 M). **P < 0.01 and *P < 0.05 compared with control values by ANOVA.

 

Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 7. Acetylcholine-induced relaxation of fetal (n = 4), newborn [n = 16 for control and nor-NOHA, n = 4 for nor-NOHA + nitro-L-arginine methyl ester (L-NAME)], and adult (n = 6) pulmonary arteries precontracted with thromboxane A2 analog U-46619 (EC75; concentration giving the 75% maximal response) in the absence (control) and presence of the arginase inhibitor nor-NOHA (10–5 M) and NOS inhibitor L-NAME (10–4 M). Relaxation is expressed as percentage of respective control postcontraction values. **P < 0.01 compared with control values by ANOVA.

 
Similar measurements were obtained for the same generation bronchial tissue. In contrast to the pulmonary arterial tissue, arginase inhibition had no effect on the bronchial smooth muscle acetylcholine-induced force generation at any age (Fig. 8).


Figure 8
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 8. Fetal (n = 8), newborn (n = 4), and adult (n = 12) bronchial muscle dose-response to acetylcholine in the absence (control) and presence of the arginase inhibitor nor-NOHA (10–5 M). No statistical significant effect of nor-NOHA was noted.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Arginases have been shown to control smooth muscle contraction by modulating NO production through substrate limitation for NOS. In the lung, there is evidence that increased arginase activity plays a role in both vascular and airway diseases. In the present study, we documented for the first time that the pattern of arginase expression and activity in the healthy lung changes with maturation. Total lung arginase activity and tissue expression of arginase type II were maximal in fetal compared with postnatal values. In line with these observations, functional studies showed that inhibition of arginase activity resulted in a greater reduction in muscle tone of fetal and neonatal pulmonary artery compared with adult. In addition, as judged by the reduction in force, arginase inhibition resulted in an increase in fetal and newborn pulmonary arteries NO production comparable to the postacetylcholine-induced eNOS-dependent response.

Changes in arginase expression and/or activity have been reported in a number of human systemic vascular diseases. These include increased arginase activity and/or expression in diabetic patients with erectile dysfunction (16), systemic hypertension (10), atherosclerosis (19), and preeclampsia (27). In the adult lung, increased arginase type II expression in pulmonary artery endothelial cells together with higher arginase activity in serum was documented in subjects with pulmonary arterial hypertension (33). Serum arginase activity has been also shown to be increased in sickle cell disease-related secondary pulmonary hypertension (25) and thalassemia (24). In the newborn rat, we (5) have previously reported that chronic hyperoxia induces pulmonary hypertension. Hyperoxia is known to upregulate arginase expression in lung (29) and possibly contributed to decreased NO production that we reported in this newborn model of pulmonary hypertension (5).

Arginase I expression is upregulated in older rats and may account for the endothelial dysfunction observed later in life (6). Yet the pulmonary vascular age-dependent arginase expression changes from fetal to adult life had not been evaluated. In this study, we documented a significantly higher lung arginase activity in the fetal compared with adult tissue, suggesting that arginase may be important in the regulation of smooth muscle tone in early life.

In keeping with the maturational pattern of lung arginase activity and expression in the present study, we showed that arginase inhibition had a significantly greater functional effect on fetal compared with adult pulmonary arteries. In the presence of the arginase inhibitor nor-NOHA, fetal pulmonary arteries showed less agonist-induced force indicative of an increase of L-arginine availability for NOS and consequently higher vascular NO production that resulted in a lower smooth muscle tone. Arginase inhibition led to relaxation of precontracted fetal pulmonary arteries of comparable magnitude as obtained following eNOS stimulation (acetylcholine-induced). Physiologically, these findings correlate with the observed reduction in pulmonary vascular tone during the transition from fetal to postnatal life, suggesting that the higher lung arginase activity prenatally is one of the contributing regulatory factors to this phenomenon.

Reciprocal regulation of arginases and NOS has been previously reported where inhibition of arginase resulted in increased NOS activity (6). This cannot be supported by our findings of eNOS expression in lung tissue. The developmental pattern of eNOS expression is thought to be such that it peaks after the neonatal period as shown in porcine lung (2) and also rat liver (28). In the present study, we documented that eNOS expression in rat lung was similar across ages with a tendency toward higher values in the fetus and newborn compared with adult lung. We did not, however, observe an effect of arginase inhibition on bronchial smooth muscle force generation suggesting that L-arginine availability is not a limiting factor for the maintenance of a low airway resistance. In contrast, in adult guinea pig trachea (17), arginase inhibition was found to enhance noncholinergic nerve-mediated airway relaxation. Species- and age-related differences may explain the discrepant results. In addition, we have not evaluated the electrical field stimulation-induced relaxant potential of the rat bronchial muscle as done in a study by Maarsing et al. (17). Under physiological conditions, the airway resistance is low and does not significantly change during maturation. However, increased airway arginase activity has been documented in diseases such as asthma (18, 26) and cystic fibrosis (13–15) and may contribute to airflow obstruction in these patients. In this study, we further demonstrated that the expression of arginase I in airway epithelium is likely not developmentally regulated (Fig. 5).

It is currently not known which arginase isoform is the predominant contributor to total arginase activity in the lung. Studies in transgenic mice suggest that arginase I is essential for detoxification, whereas these animals can compensate for arginase II deficiency. Arginase I-deficient mice exhibited a severe debilitated phenotype with these animals dying from high ammonia levels during the newborn period (20). Arginase II knockout mice showed no apparent phenotypic change, possibly because of compensatory upregulation of arginase I (31). In the present study, we documented that both isoforms were expressed in the rat lung in a cell type- and maturation-specific pattern. Arginase I predominates in airway epithelium and pulmonary arterial endothelium, whereas the type II isoform is mostly present in the medial and adventitial layers of both anatomical structures (Fig. 5).

Evidence for compensatory regulation of the two arginase isoforms was also found based on the observed significant increase in arginase II in the presence of decreased arginase I expression the fetal lung. Previously, Berkowitz et al. (6) had shown that both arginase isotypes were expressed in the rat aortic endothelium. Arginase I had been found to be constitutively expressed in endothelial cells (35), whereas arginase II appeared to be inducible in response to cytokines (8). White et al. (32) have demonstrated that arginase type I knockdown could enhance NOS activity in adult rat thoracic aortic tissue, and Zhang et al. (35) had shown in the porcine coronary artery that arginase type I is responsible for modulating the endothelial-dependent relaxation. Bachetti et al. (4) demonstrated that both isoforms were expressed in human umbilical vein endothelial cells (HUVEC) with a predominance of the type I isoform. In contrast and highlighting the discrepancies with the use of presently available antibodies, Ming et al. (19) showed that arginase type II is most abundant in HUVEC. Future studies will therefore be needed to define the role of the arginase isoforms in maturation of the human lung tissues.

Aside from regulating vascular tone, increased arginase activity may also alter vascular resistance by contributing to tissue remodeling in chronic conditions. In fact, ornithine, the product of arginase activity, is the precursor of proline and the polyamines, which promote collagen production and cell proliferation, respectively. The view that arginase contributes to pulmonary vascular remodeling is supported by studies demonstrating that upregulated L-arginine transport and metabolism by TGF-β1 resulted in increased polyamine and L-proline formation in vascular smooth muscle cells (11).

In summary, we showed for the first time that expression and activity of arginases in the rat lung follow a developmental pattern. The pulmonary arterial tissue arginase is highest in the fetus, and its in vitro inhibition markedly reduces the agonist-stimulated muscle contraction, indicating increased vascular NO production. These findings suggest that arginase is involved in the maintenance of a high pulmonary vascular resistance during fetal life.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the Canadian Institutes of Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Belik, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8 Canada (e-mail: jaques.belik{at}sickkids.ca)

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
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abman SH, Chatfield BA, Hall SL, McMurtry IF. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol Heart Circ Physiol 259: H1921–H1927, 1990.[Abstract/Free Full Text]
  2. Arrigoni FI, Hislop AA, Pollock JS, Haworth SG, Mitchell JA. Birth upregulates nitric oxide synthase activity in the porcine lung. Life Sci 70: 1609–1620, 2002.[CrossRef][Web of Science][Medline]
  3. Ashutosh K. Nitric oxide and asthma: a review. Curr Opin Pulm Med 6: 21–25, 2000.[CrossRef][Medline]
  4. Bachetti T, Comini L, Francolini G, Bastianon D, Valetti B, Cadei M, Grigolato P, Suzuki H, Finazzi D, Albertini A, Curello S, Ferrari R. Arginase pathway in human endothelial cells in pathophysiological conditions. J Mol Cell Cardiol 37: 515–523, 2004.[CrossRef][Web of Science][Medline]
  5. Belik J, Jankov RP, Pan J, Yi M, Chaudhry I, Tanswell AK. Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane. J Appl Physiol 96: 725–730, 2004.[Abstract/Free Full Text]
  6. Berkowitz DE, White R, Li D, Minhas KM, Cernetich A, Kim S, Burke S, Shoukas AA, Nyhan D, Champion HC, Hare JM. Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels. Circulation 108: 2000–2006, 2003.[Abstract/Free Full Text]
  7. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
  8. Chicoine LG, Paffett ML, Young TL, Nelin LD. Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells. Am J Physiol Lung Cell Mol Physiol 287: L60–L68, 2004.[Abstract/Free Full Text]
  9. Corraliza IM, Soler G, Eichmann K, Modolell M. Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages. Biochem Biophys Res Commun 206: 667–673, 1995.[CrossRef][Web of Science][Medline]
  10. Demougeot C, Prigent-Tessier A, Marie C, Berthelot A. Arginase inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats. J Hypertens 23: 971–978, 2005.[Web of Science][Medline]
  11. Durante W, Liao L, Reyna SV, Peyton KJ, Schafer AI. Transforming growth factor-beta(1) stimulates L-arginine transport and metabolism in vascular smooth muscle cells: role in polyamine and collagen synthesis. Circulation 103: 1121–1127, 2001.[Abstract/Free Full Text]
  12. Fineman JR, Soifer SJ, Heymann MA. Regulation of pulmonary vascular tone in the perinatal period. Annu Rev Physiol 57: 115–134, 1995.[CrossRef][Web of Science][Medline]
  13. Grasemann H, Kurtz F, Ratjen F. Inhaled L-arginine improves exhaled nitric oxide and pulmonary function in patients with cystic fibrosis. Am J Respir Crit Care Med 174: 208–212, 2006.[Abstract/Free Full Text]
  14. Grasemann H, Schwiertz R, Grasemann C, Vester U, Racke K, Ratjen F. Decreased systemic bioavailability of L-arginine in patients with cystic fibrosis. Respir Res 7: 87, 2006.[CrossRef][Medline]
  15. Grasemann H, Schwiertz R, Matthiesen S, Racke K, Ratjen F. Increased arginase activity in cystic fibrosis airways. Am J Respir Crit Care Med 172: 1523–1528, 2005.[Abstract/Free Full Text]
  16. Jiang M, Ding Y, Su Y, Hu X, Li J, Zhang Z. Arginase-flotillin interaction brings arginase to red blood cell membrane. FEBS Lett 580: 6561–6564, 2006.[CrossRef][Web of Science][Medline]
  17. Maarsingh H, Tio MA, Zaagsma J, Meurs H. Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation. Respir Res 6: 23, 2005.[CrossRef][Medline]
  18. Meurs H, McKay S, Maarsingh H, Hamer MA, Macic L, Molendijk N, Zaagsma J. Increased arginase activity underlies allergen-induced deficiency of cNOS-derived nitric oxide and airway hyperresponsiveness. Br J Pharmacol 136: 391–398, 2002.[CrossRef][Web of Science][Medline]
  19. Ming XF, Barandier C, Viswambharan H, Kwak BR, Mach F, Mazzolai L, Hayoz D, Ruffieux J, Rusconi S, Montani JP, Yang Z. Thrombin stimulates human endothelial arginase enzymatic activity via RhoA/ROCK pathway: implications for atherosclerotic endothelial dysfunction. Circulation 110: 3708–3714, 2004.[Abstract/Free Full Text]
  20. Modolell M, Corraliza IM, Link F, Soler G, Eichmann K. Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. Eur J Immunol 25: 1101–1104, 1995.[Web of Science][Medline]
  21. Mori M, Gotoh T. Arginine metabolic enzymes, nitric oxide and infection. J Nutr 134: 2820S–2825S, 2004.[Abstract/Free Full Text]
  22. Morris CR. New Strategies for the Treatment of Pulmonary Hypertension in Sickle Cell Disease: The Rationale for Arginine Therapy. Treat Respir Med 5: 31–45, 2006.[CrossRef][Medline]
  23. Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC, Sachdev V, Hazen SL, Vichinsky EP, Morris SM Jr, Gladwin MT. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. JAMA 294: 81–90, 2005.[Abstract/Free Full Text]
  24. Morris CR, Kuypers FA, Kato GJ, Lavrisha L, Larkin S, Singer T, Vichinsky EP. Hemolysis-associated pulmonary hypertension in thalassemia. Ann NY Acad Sci 1054: 481–485, 2005.[CrossRef][Web of Science][Medline]
  25. Morris CR, Morris SM Jr, Hagar W, Van WJ, Claster S, Kepka-Lenhart D, Machado L, Kuypers FA, Vichinsky EP. Arginine therapy: a new treatment for pulmonary hypertension in sickle cell disease? Am J Respir Crit Care Med 168: 63–69, 2003.[Abstract/Free Full Text]
  26. Morris CR, Poljakovic M, Lavrisha L, Machado L, Kuypers FA, Morris SM Jr. Decreased arginine bioavailability and increased serum arginase activity in asthma. Am J Respir Crit Care Med 170: 148–153, 2004.[Abstract/Free Full Text]
  27. Noris M, Todeschini M, Cassis P, Pasta F, Cappellini A, Bonazzola S, Macconi D, Maucci R, Porrati F, Benigni A, Picciolo C, Remuzzi G. L-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. Hypertension 43: 614–622, 2004.[Abstract/Free Full Text]
  28. Nowicki MJ, Shi D, Cai Z, Bishop PR, May WL. Developmental expression of endothelial nitric oxide synthase (eNOS) in the rat liver. Pediatr Res 54: 732–738, 2003.[CrossRef][Web of Science][Medline]
  29. Que LG, Kantrow SP, Jenkinson CP, Piantadosi CA, Huang YC. Induction of arginase isoforms in the lung during hyperoxia. Am J Physiol Lung Cell Mol Physiol 275: L96–L102, 1998.[Abstract/Free Full Text]
  30. Sasaki A, Doi S, Mizutani S, Azuma H. Roles of accumulated endogenous NOS inhibitors, enhanced arginase activity, and attenuated NOS activity in endothelial cells for pulmonary hypertension in rats. Am J Physiol Lung Cell Mol Physiol 292: L1480–L1487, 2007.[Abstract/Free Full Text]
  31. Shi O, Morris SM Jr, Zoghbi H, Porter CW, O'Brien WE. Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene. Mol Cell Biol 21: 811–813, 2001.[Abstract/Free Full Text]
  32. White AR, Ryoo S, Li D, Champion HC, Steppan J, Wang D, Nyhan D, Shoukas AA, Hare JM, Berkowitz DE. Knockdown of arginase I restores NO signaling in the vasculature of old rats. Hypertension 47: 245–251, 2006.[Abstract/Free Full Text]
  33. Xu W, Kaneko FT, Zheng S, Comhair SA, Janocha AJ, Goggans T, Thunnissen FB, Farver C, Hazen SL, Jennings C, Dweik RA, Arroliga AC, Erzurum SC. Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J 18: 1746–1748, 2004.[Abstract/Free Full Text]
  34. Yang M, Rangasamy D, Matthaei KI, Frew AJ, Zimmmermann N, Mahalingam S, Webb DC, Tremethick DJ, Thompson PJ, Hogan SP, Rothenberg ME, Cowden WB, Foster PS. Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness. J Immunol 177: 5595–5603, 2006.[Abstract/Free Full Text]
  35. Zhang C, Hein TW, Wang W, Chang CI, Kuo L. Constitutive expression of arginase in microvascular endothelial cells counteracts nitric oxide-mediated vasodilatory function. FASEB J 15: 1264–1266, 2001.[Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
B. Chen, A. E. Calvert, H. Cui, and L. D. Nelin
Hypoxia promotes human pulmonary artery smooth muscle cell proliferation through induction of arginase
Am J Physiol Lung Cell Mol Physiol, December 1, 2009; 297(6): L1151 - L1159.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. Belik, D. Stevens, J. Pan, D. Shehnaz, C. Ibrahim, C. Kantores, J. Ivanovska, H. Grasemann, and R. P. Jankov
Chronic hypercapnia downregulates arginase expression and activity and increases pulmonary arterial smooth muscle relaxation in the newborn rat
Am J Physiol Lung Cell Mol Physiol, October 1, 2009; 297(4): L777 - L784.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. L. North, N. Khanna, P. A. Marsden, H. Grasemann, and J. A. Scott
Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma
Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L911 - L920.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/L498    most recent
00242.2007v1
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 (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belik, J.
Right arrow Articles by Grasemann, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belik, J.
Right arrow Articles by Grasemann, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.