1 Department of Pediatrics, University of Manitoba, Winnipeg, Manitoba R3A 1S1; Departments of 2 Medicine and 3 Pediatrics, Duke University Medical Center, Durham, North Carolina 27710; and 4 Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Extracellular superoxide dismutase
(EC-SOD), which scavenges extracellular superoxide
(O
antioxidant; nitric oxide; lungs; neonatal; oxygen tension
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INTRODUCTION |
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THE NEONATAL LUNG
ADAPTS to postnatal conditions by decreasing the pulmonary
vascular resistance to facilitate pulmonary blood flow and increasing
antioxidant defenses to protect against the rise in alveolar oxygen
tension associated with room air breathing. Multiple antioxidant
enzymes are developmentally regulated in the neonatal lung in
preparation for birth, including catalase, glutathione peroxidase,
Cu,Zn superoxide dismutase (SOD), and extracellular SOD (EC-SOD)
(2, 4, 9, 13, 14, 25). The isoforms of the SODs, which
include mitochondrial Mn SOD, cytosolic Cu,Zn SOD, and EC-SOD, each
have a specific cellular distribution and catalyze the dismutation of
O
EC-SOD is highly expressed in the lung and is produced by alveolar type II cells, airway epithelial cells, and vascular endothelial cells (12, 26, 27, 32). We have previously shown that EC-SOD is located inside the cell in the prenatal rabbit lung and that active EC-SOD is secreted after birth (25). Although EC-SOD has been shown to protect against hyperoxic lung injury in animal models (3, 6, 11), the factors that regulate the secretion of active EC-SOD or the consequence of alterations in secretion of EC-SOD in the immature lung are not known.
The secretion of active EC-SOD in the neonatal lung over the first week of life correlates with the adaptation to room air breathing. On the basis of this observation, we speculated that changes in oxygen tension in the prenatal period regulate the postnatal secretion of EC-SOD in the lung. It is well known that maternal factors, including maternal infection or placental insufficiency, which limit oxygen delivery to the fetus, disrupt alveolarization and lung vascular growth and alter the health outcome of the newborn infant (33). In this study, we hypothesized that maternal exposure to hypoxia in late gestation would delay the secretion of active EC-SOD in the lung. We tested this hypothesis by treating pregnant rabbits in late gestation to 36 h of hypobaric hypoxia (15,000 ft) and measuring EC-SOD expression and activity in the lungs of preterm, term, and 1-wk-old kits.
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MATERIALS AND METHODS |
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Animal model and environmental exposures. Animal studies were performed in the Center for Hyperbaric Medicine and Environmental Physiology at Duke University Medical Center. Protocols were developed and approved by the Institutional Animal Care and Use Committee. Timed pregnant New Zealand White rabbits were obtained at 24 days of gestation (Robinson's Animal Farm) and housed in the vivarium. All animals were given food and water ad libitum and were maintained on a regular 12-h light/dark cycle.
Pregnant animals were maintained in room air throughout pregnancy (control) or exposed to hypobaric hypoxia for 36 h in late gestation to produce transient prenatal hypoxia. Pregnant rabbits at day 26 of gestation were exposed to 15,000 ft in an altitude chamber [0.5 atmospheres (atm)] for 36 h. Pregnant rabbits consumed food and drank well during the exposures. Use of hypobaric hypoxia at this altitude produces a reproducible decrease in oxygen tension with no measurable changes in lung function resulting from the change in atmospheric pressure (7). Lung tissue from the kits of control or hypoxia-exposed pregnant rabbits was harvested immediately after environmental exposure, at term, and at 1 wk of age. We delivered preterm rabbits by cesarean section after euthanizing the pregnant rabbits with 50 mg/kg iv pentobarbital sodium on day 28 of gestation immediately after environmental exposure (saccular stage of lung development, normal gestation 30 ± 1 days). Kits were euthanized with intraperitoneal pentobarbital sodium (100 mg/kg). Term lungs were harvested within 12 h of birth, and 1-wk-old lungs were harvested at the beginning of day 8 of life. Lungs were flushed with saline to remove blood and were either flash-frozen in liquid nitrogen for Western blot analysis, EC-SOD activity assays, and mRNA analysis, or inflation-fixed at 20 cmH2O × 10 min in 4% paraformaldehyde. Fixed tissue was immersed for 24 h in 4% paraformaldehyde followed by 70% ethanol for immunohistochemistry.RNA isolation and RT-PCR. Total RNA was isolated from rabbit lung by TRIzol reagent (Life Technologies, Gaithersburg, MD) based on the Chomczynski and Sacchi (8) method. Briefly, tissue was homogenized in TRIzol reagent containing phenol and guanidine isothiocyanate, followed by chloroform extraction and isopropanol precipitation. Samples were quantified by absorption spectrophotometry, and RNA integrity was confirmed using nondenaturing agarose gel electrophoresis. RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI) at 37°C for 30 min to remove contaminating DNA, followed by 20 mM EGTA for 10 min at 65°C to inactivate the enzyme.
Total RNA (1 µg) extracted from the lungs was reverse transcribed in a final volume of 25 µl containing 0.4 units of avian myeloblastosis virus (AMV) RT (Promega), 1× AMV RT buffer [50 mM Tris · HCl, (pH 8.3), 50 mM KCl, 10 mM MgCl, 0.5 mM spermidine, and 10 mM dithiothreitol], 0.01 µg oligo(dT), 0.8 mM 2'-deoxynucleoside 5'-triphosphate (dNTP) (Life Technologies), and 1.6 units RNasin ribonuclease inhibitor. The samples were incubated at 42°C for 90 min and enzyme inactivated at 95°C for 10 min. The synthesized single-stranded cDNA was stored atWestern blot analysis.
Lungs from kits of different ages with and without exposure to prenatal
hypoxia were examined by Western blot analysis for expression of EC-SOD
protein. Tissue was homogenized in lysis buffer (50 mM Tris, pH 7.6, 3% Igepal, 150 mM NaCl, 1 mM MgCl2, 5 mM EDTA, pH 7.6)
with 1:20 protease inhibitor [2 mM 1,10-phenanthroline, 2 mM
3,4-diisocoumarin, 0.4 mM
trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane] on ice. Homogenates were centrifuged at 10,000 g for 20 min,
and supernatants were used for protein assay (BCA protein assay kit, Sigma). Twenty micrograms of protein were loaded onto a 12%
polyacrylamide gel for electrophoresis and electrophoretically
transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Each
membrane was blocked in 3% milk-Tris-buffered saline-Tween 20 (TBST)
overnight at 4°C. The membranes were incubated with a mouse IgG
antibody against a 20-amino acid EC-SOD peptide (1:1,000 in 3%
milk-TBST) (25) for 1 h at room temperature, followed
by a secondary goat anti-mouse IgG antibody conjugated to horseradish
peroxidase (1:20,000 in 3% milk-TBST; BD Transduction Laboratories,
San Diego, CA) for 1 h at room temperature. The blots were
developed with enhanced chemiluminescence (Amersham Pharmacia Biotech,
Piscataway, NJ). The blots were stripped with 0.1 M glycine (pH 2.9)
and reprobed using a monoclonal mouse IgG antibody against -actin
(1:5,000 in 3% milk-TBST), followed by a secondary goat anti-mouse IgG antibody conjugated to horseradish peroxidase (1:20,000 in 3% milk-TBST; BD Transduction Laboratories) to confirm equal protein loading.
Immunohistochemistry. Paraffin-embedded tissue sections (4 µm) were immunostained to assess localization of EC-SOD protein in airways and vessels. Sections were deparaffinized, and endogenous peroxidase activity was inhibited using graded ethanol and 3% hydrogen peroxide. Sections were subsequently blocked with 5% normal goat serum, 1% BSA, and 3% milk in PBS for 1 h at room temperature. Slides were incubated with primary mouse IgG against EC-SOD (25) overnight at 4°C. The secondary antibody incubation was performed with a biotinylated goat anti-mouse IgG antibody 1:20 in 1% BSA-PBS for 1 h at room temperature, followed by labeling with peroxidase-conjugated streptavidin in 1% BSA-PBS for 1 h (Biogenex Link and Label Kit; Biogenex, San Ramon, CA). Negative controls were performed with normal mouse serum. Slides were washed with PBS and Tris · Cl and developed with 3,3-diaminobenzine. Slides were counterstained with hematoxylin, rinsed, and dehydrated with graded alcohol. Sections were examined by light microscopy and photographed at ×180.
EC-SOD activity measurement. EC-SOD activity was measured after separation from intracellular SOD (Cu,Zn SOD and Mn SOD). Lung tissue was homogenized in 10 vol of ice-cold buffer (50 mM potassium phosphate, pH 7.4, with 0.3 M KBr, 0.05 mM phenylmethylsulfonyl fluoride, and 3 mM diethylenetriaminepentaacetic acid) and then passed over a concavalin A sepharose column as described (22). EC-SOD activity was measured by inhibition of cytochrome c reduction at pH 10.0 as described by Crapo et al. (10).
Reagents. Reagents, unless specified, were obtained from Sigma.
Data analysis. Data are expressed as means ± SE as indicated. Comparisons were made by an unpaired t-test for RT-PCR or Western blot data or two-way analysis of variance for activity data using Statview software (SAS Institute, Cary, NC).
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RESULTS |
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Effects of prenatal hypoxia on EC-SOD mRNA expression.
To evaluate the effects of prenatal oxygen tension on EC-SOD gene
expression, EC-SOD mRNA transcription was examined by semiquantitative RT-PCR in control preterm, term, and 1-wk-old rabbit lungs and compared
with EC-SOD mRNA transcripts after prenatal exposure to
hypoxia. Experiments were performed in triplicate. Figure
1 shows a representative EC-SOD
mRNA signal along with corresponding 18S rRNA extracted from
lung samples from preterm, term, and 1-wk-old rabbits with and without
prenatal exposure to hypoxia. OD of the signals expressed as a ratio of
EC-SOD to 18S rRNA were compared by unpaired t-test at each
age group. In control animals, EC-SOD mRNA transcripts in the lung
decreased by 60-70% from term to 1 wk of age (ratio of EC-SOD/18s
rRNA: 1.19 ± 0.12 in term control lungs vs. 0.36 ± 0.15 in
1-wk control lungs, P < 0.05 by unpaired t-test). Prenatal exposure to hypoxia significantly
decreased EC-SOD mRNA transcripts in term animal lung (ratio of
EC-SOD/18s rRNA 0.52 ± 0.15, P < 0.05 vs. term
control lung by unpaired t-test). Prenatal hypoxia induced
subtle but not significant decreases in EC-SOD mRNA signals in the
lungs from preterm and 1-wk-old kits.
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Effects of prenatal hypoxia on EC-SOD protein expression.
EC-SOD protein was constitutively expressed in preterm and term
rabbits. Interestingly, protein expression appeared to be age
dependent, because the EC-SOD signal on Western blot analysis tended to
decrease in control lung tissue from preterm to 1 wk (P = 0.06 by unpaired t-test, n = 4). Prenatal
hypoxia significantly decreased EC-SOD immunoreactive protein in lungs
from preterm, term, and 1-wk-old kits. Densitometry showed that with
hypoxia, EC-SOD protein levels decreased by 33% in preterm lungs, 45%
in term lungs, and 29% in 1-wk-old lungs compared with age-matched control lungs (P < 0.05, n = 4 for
each except n = 3 for 1-wk hypoxia; Fig.
2). One week after birth, prenatal
hypoxia did not change EC-SOD protein expression in the lung. -actin
levels, used to confirm equal loading of protein samples, were similar in all animals (not illustrated).
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Effects of prenatal hypoxia on EC-SOD protein activity.
EC-SOD activity increases with age in developing rabbit lung
(25). In control animals, EC-SOD activity tended to
increase (1.8-fold) 1 wk after birth compared with that of preterm and term kits (n = 5 for each). Prenatal hypoxia sharply
lowered EC-SOD activity by one-half in the 1-wk-old rabbit lung,
compared with age-matched controls (n = 4, P < 0.05 by ANOVA). EC-SOD activity was not decreased
in preterm lungs after prenatal hypoxia (n = 5, P = 0.4; Fig. 4). Low
EC-SOD activity in term lungs (average 0.15 U/mg protein for
n = 2) is similar to levels in the preterm (0.14 ± 0.04, n = 5) and 1-wk lungs (0.15 ± 0.07, n = 4), but we are unable to provide statistical
analysis with control term lungs due to small sample size.
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DISCUSSION |
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We present new information on the developmental regulation of the important extracellular antioxidant enzyme EC-SOD in the immature rabbit lung and report the interesting finding that prenatal hypoxia disrupts the normal developmental secretion of active EC-SOD in the lung. This study contributes new data to our previous observations that secretion of active EC-SOD into the extracellular compartment is developmentally regulated in the rabbit lung (25).
In control rabbit lungs, EC-SOD mRNA expression significantly decreased during the first week of life. This pattern is consistent with other antioxidant enzymes, which are upregulated in the immediate prenatal period and subsequently decrease to adult levels (2, 4, 9, 13, 14). Whereas EC-SOD mRNA transcripts were constitutively expressed in preterm and term lungs, the message significantly decreased approximately threefold in the 1-wk-old animals (Fig. 1). The changes in mRNA transcription were followed by a similar trend toward less protein expression at 1 wk of age. As noted in our previous report, EC-SOD protein in rabbit lung consists of primarily uncleaved EC-SOD with intact heparin binding domain (25).
The model of maternal exposure to hypobaric hypoxia at 0.5 atm was selected to decrease oxygenation in the fetus by decreasing umbilical venous oxygen saturations. A previous study showed similar levels of hypobaric hypoxia (4,300 m) in pregnant ewes' decreased fetal umbilical venous O2 saturation from 74-61% to 45-28% (20). Human maternal hypoxemia (PAO2 < 60 mmHg) reduces umbilical venous PvO2 from 32 to 26 mmHg to reduce fetal oxygen content (36). In our study, although the maternal exposures to hypobaric hypoxia were limited to 36 h, the changes in EC-SOD expression in the lungs of the kits were evident for at least 1 wk.
Intriguingly, transient prenatal exposure to hypoxia decreased EC-SOD mRNA transcription and protein expression for up to 1 wk of life. The significant decrease in both EC-SOD mRNA and protein in term lungs indicates that the regulation of transcription and translation was coordinated in these lungs after prenatal hypoxia. The lack of change in mRNA levels in the 1-wk-old rabbit after hypoxia likely reflects the lower baseline expression of EC-SOD mRNA.
The factors that regulate expression of EC-SOD mRNA or protein are unknown. Hypoxia is known to affect fetal development, matrix production, and secretion of signaling molecules (18, 29). It is possible that hypoxia represses EC-SOD expression in part by decreasing stability of the mRNA transcripts and enhancing the RNA decay (17). On the other hand, hypoxia may induce or repress the expression of a specific factor that would recognize nucleotide consensus sequence in the EC-SOD gene promoter. The formation of complexes with such a factor could regulate EC-SOD gene expression during prenatal hypoxia. Such a mechanism has been implicated in the regulation of Mn SOD in rabbit airway epithelial cells, as well as in mouse, rat, and A549 cells (17, 30). Moreover, EC-SOD regulation may occur via corticosteroid-responsive element in the promoter of EC-SOD gene, which may also suppress the isoenzyme gene expression. Notably, sustained hypoxemia results in an upregulation of adrenocorticotropic hormone expression in association with a sustained increase in plasma cortisol (15, 16).
Our data strongly imply that the mechanism(s) that regulates EC-SOD enzyme activity is distinct from those that regulate gene and protein expression. Although the amount of immunoreactive EC-SOD protein in control rabbit lung tended to decrease by 1 wk of age, the activity of the isoenzyme increased with levels 1.8-fold higher than in preterm and term rabbit lung, revealing discordance between enzyme activity and protein expression. These results are reminiscent of our earlier study in which adult rabbit lung contained similar amounts of EC-SOD immunoreactive protein compared with neonatal lung but as much as fivefold more enzyme activity (25). The exact mechanisms responsible for this discordance between EC-SOD protein expression and its enzymatic activity are unclear, but ample evidence supports the importance of posttranslational regulatory mechanisms in several other systems (5, 24, 34).
One potentially important site for posttranslational regulation is the secretion of active EC-SOD into the extracellular compartment. In developing rabbit lungs, the increase in EC-SOD activity correlates with the secretion of EC-SOD protein into the extracellular space. We found that both EC-SOD activity levels and the secretion of EC-SOD in 1-wk-old rabbit lungs were decreased by prenatal exposure to hypoxia. This important and novel finding supports our hypothesis that EC-SOD activity is regulated by posttranslational modification of the protein and that hypoxia interrupts this process by delaying its secretion into the extracellular compartment.
The diminished secretion of EC-SOD in hypoxic 1-wk-old rabbits could very well be due to protein modification by oxidants of targets, including tyrosine residues or carbonyl molecules. Although we did not measure these oxidative markers in this study, previous work has shown increases in nitrosylated and carbonylated proteins in human fetal tissue after intrauterine hypoxia (31). Because in vitro studies show that CO2 influences radical production, it is possible that the hypocarbic alkalosis produced in response to hypoxia alters free radical reactions and nitrosylation of proteins in this model (19). Future studies will be essential to understand the regulation of EC-SOD and its physiological relevance.
In utero, hypoxia may occur during maternal hypoxia or with placental
insufficiency. A reduction in EC-SOD activity in the lung could place
an infant at risk for disruption of normal postnatal development of the
pulmonary vasculature or pulmonary oxidative damage after birth,
especially with supplemental O2 therapy. Abnormal development of the vasculature is associated with perinatal hypoxia and
increased pulmonary vascular resistance, which may be mediated by
increased concentrations of O
In summary, we have shown that maternal exposure to hypobaric hypoxia in late gestation decreases EC-SOD expression and activity in the neonatal lung that are sustained over the first week of life. These are the first data that show that EC-SOD expression can be altered by prenatal conditions and provide important rationale for understanding the regulation of EC-SOD in the lung and prenatal conditions that influence postnatal susceptibility to lung diseases.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the assistance of Lisa Schaeffer and P. Owen Doar for excellent technical expertise.
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FOOTNOTES |
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This work was supported by the Duke Neonatal Perinatal Research Institute and National Heart, Lung, and Blood Institute Grant HL-63700-01 (T. D. Oury).
These data were presented in part at the American Thoracic Society International Conference, Toronto 2000 and San Francisco 2001.
Address for reprint requests and other correspondence: E. Nozik-Grayck, Box 3046, Dept. of Pediatrics, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: grayc001{at}mc.duke.edu).
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.
April 19, 2002;10.1152/ajplung.00018.2002
Received 15 January 2002; accepted in final form 12 April 2002.
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