Prenatal hypoxia decreases lung extracellular superoxide dismutase expression and activity

Brenda-Louise Giles1, Hagir Suliman2, Lisa B. Mamo3, Claude A. Piantadosi2, Tim D. Oury4, and Eva Nozik-Grayck3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular superoxide dismutase (EC-SOD), which scavenges extracellular superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·), is highly regulated in the developing lung. In the prenatal rabbit, EC-SOD is predominantly intracellular and inactive, and postnatally, active EC-SOD is secreted. We hypothesized that prenatal hypoxia would delay the normal postnatal secretion of active EC-SOD in the lung. Pregnant New Zealand White rabbits were exposed to hypobaric hypoxia (15,000 ft × 36 h) to alter fetal O2 tension or were maintained in room air. Lungs were harvested from preterm (28 days), term (30 ± 1 day), and 1-wk-old kits. After prenatal hypobaric hypoxia, EC-SOD mRNA expression was significantly decreased in lungs of full-term kits, whereas EC-SOD protein decreased at all ages. Immunohistochemical staining for EC-SOD showed that hypoxia delayed secretion of the isoenzyme in the airways and pulmonary vasculature. Furthermore, pulmonary EC-SOD enzyme activity was significantly decreased in the 1-wk-old kits exposed to prenatal hypoxia. We conclude that prenatal hypoxia downregulates EC-SOD expression at both the transcriptional and posttranslational levels. Furthermore, prenatal hypoxia delays secretion of active EC-SOD enzyme. These findings have important implications for the effects of prenatal asphyxia on postnatal response to oxidant stress.

antioxidant; nitric oxide; lungs; neonatal; oxygen tension


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· (Kd ~ 1 × 109) (21, 23, 35). EC-SOD, the only known enzymatic scavenger of extracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, is also involved in the preservation of nitric oxide (NO) activity in the vasculature and airways due to its ability to compete with the rapid reaction between O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and NO (1, 28). Therefore, the regulation of EC-SOD in the neonatal lung is potentially important both to protect from extracellular oxidative stress with the change in oxygen tension and to preserve NO-dependent transition from fetal to adult circulation.

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -20°C until use.

EC-SOD was amplified by PCR using 20 pmol rabbit EC-SOD sense and antisense primers, 0.2 mM dNTP, 0.075 units Taq DNA polymerase (Qiagen, Valencia, CA), and 1× PCR buffer [25 mM KCl, 25 mM Tris · HCl (pH 8.3), 0.75 mM MgCl2]. Primers were designed by MacVector to span the intron-exon 3 coding region of rabbit EC-SOD (sense: 5' TGG ATG TTG CAA GTG ACC AG 3'; anti-sense: 5' GAC TAC CAA GCC GCT GAG TC 3') (Life Technologies). The cDNA was amplified in Hot Start reaction tubes (Molecular BioProducts, San Diego, CA) with the Hybaid OmniGene thermocycler (Hybaid, Teddington, United Kingdom) using the following conditions: 1 min at 95°C × 1 cycle; 3 min at 95°C × 1 cycle; 30 s at 95°C, 40 s at 59.4°C, and 30 s at 72°C × 35 cycles followed by a terminal extension of 7 min at 72°C × 1 cycle. EC-SOD amplicons were normalized to 18S ribosomal RNA (rRNA) as an external standard.

PCR products were analyzed by agarose gel electrophoresis (0.8%). A 315-bp and a 489-bp fragment of EC-SOD and 18S, respectively, were stained with gelStar nucleic acid gel stain (BioWhittaker Molecular Applications, Rockland, MD) and visualized under ultraviolet transillumination. Band intensity was determined by densitometry analysis via the GS-710 calibrated imaging densitometer and Quantity One software (Bio-Rad, Hercules, CA). Results were expressed as the ratio between optical density (OD) of EC-SOD and 18S amplicons to determine relative expression values.

Western 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 beta -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Extracellular superoxide dismutase (EC-SOD) mRNA expression by RT-PCR after prenatal exposure to hypobaric hypoxia. EC-SOD mRNA expression in preterm, term, and 1-wk-old rabbit lung is shown with each corresponding 18S rRNA as an internal standard. EC-SOD mRNA expression did not decrease from control levels in preterm lungs after prenatal hypoxia. EC-SOD mRNA expression in term control lungs decreased in term rabbit lung after prenatal hypoxia. EC-SOD mRNA in both control and hypoxia-exposed lung decreased at 1 wk of age. 18S rRNA was similar in all experimental groups.

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. beta -actin levels, used to confirm equal loading of protein samples, were similar in all animals (not illustrated).


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Fig. 2.   Western blot analysis of EC-SOD after prenatal exposure to hypobaric hypoxia. EC-SOD protein expression decreased in preterm, term, and 1-wk-old rabbit lung after prenatal hypoxia compared with control lungs. The corresponding densitometry analysis for each blot shows the significant decrease in protein expression in preterm (n = 4), term (n = 4), and 1-wk-old lungs (n = 3) with hypoxia compared with age-matched controls (n = 4 in each) by unpaired t-test (* P < 0.05). Data is expressed as optical density of the signal (OD/mm2).

By 1 wk of age, EC-SOD was detectable by immunostaining in both the intracellular and extracellular compartments of pulmonary blood vessels in control lungs (Fig. 3, left). We found that prenatal hypoxia strongly decreased the release of EC-SOD protein into the extracellular space of 1-wk-old rabbits lungs, particularly in the extracellular matrix surrounding small pulmonary arteries (Fig. 3, right). Strong intracellular immunostaining was evident in the airway epithelial and alveolar type II epithelial cells (not illustrated) of preterm and term animals with and without exposure to prenatal hypoxia.


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Fig. 3.   Localization of EC-SOD in pulmonary arteries at 1 wk of age after exposure to prenatal hypobaric hypoxia. Staining of the extracellular matrix surrounding small pulmonary arteries is apparent by 1 wk of age in normoxic control lungs (left). Extracellular staining is minimal in lungs of 1-wk-old kits after exposure to prenatal hypoxia (right). Micrographs were photographed at ×180.

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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Fig. 4.   EC-SOD activity levels in neonatal rabbit lung after prenatal exposure to hypobaric hypoxia. EC-SOD activities (expressed as EC-SOD activity U/mg protein) tended to increase over the first week of life (n = 5; closed bars). EC-SOD activities in 1-wk-old rabbits significantly decreased after prenatal hypoxia (n = 4; open bars) compared with control levels (* P < 0.05). Low levels of EC-SOD activity in preterm lung after prenatal hypoxia (n = 5) were not significantly different from age-matched controls.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· and peroxynitrite associated with reduced EC-SOD activity (33). In a recent study, administration of NO altered alveolar lung development in fetal lung explants (37), which suggests that changes in EC-SOD expression could influence pulmonary vascular development by a NO-dependent mechanism. In addition, EC-SOD is important in protection from oxygen toxicity, as shown by studies in adult and neonatal transgenic mice overexpressing EC-SOD, which resist pulmonary oxygen toxicity, and EC-SOD knockout mice, which are more sensitive to hyperoxia (3, 6, 11).

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.


    ACKNOWLEDGEMENTS

The authors acknowledge the assistance of Lisa Schaeffer and P. Owen Doar for excellent technical expertise.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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