Introduction

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Peroxiredoxin (Prx) is a 25-kD enzyme, initially identified in yeast, that reduces hydrogen peroxide (H₂O₂) by transfer of electrons provided by thioredoxin (Trx) (1). Yeast Prx exists as a homodimer and contains two essential cysteine residues, Cys47 and Cys170, in each subunit (1). The Cys47-SH group is the primary site of oxidation by H₂O₂, and the oxidized Cys47 rapidly reacts with Cys170-SH of the other subunit to form an intermolecular disulfide. This disulfide is subsequently reduced by Trx. Mammalian Prx can be divided into three distinct groupsPrx I, Prx II, and Prx IIIon the basis of their amino acid sequence and immunologic properties (2). Recently, a fourth member of the family, Prx IV, a secretory form, has been identified (3). Recombinant proteins from each group are able to reduce peroxides with the use of electrons from Trx. Until recently, catalase and glutathione peroxidase have been viewed as the major enzymes responsible for removal of cytotoxic H₂O₂. However, Prx has recently been shown to play a role in removal of H₂O₂ (2). In rat hepatocytes, catalase is largely or entirely localized to peroxisomes, whereas glutathione peroxidase is present in various intracellular compartments, including mitochondria (42%), nuclei (26%), cytosol (21%), peroxisomes (7%), and lysosomes (4%) (4). On the other hand, Prx III appears to be localized in the mitochondria, whereas Prx I and Prx II are cytosolic proteins. The sum of the three Prx enzymes in cultured cells amounts to 2 to 8 µg/mg soluble protein (2). Additionally, all three Prx enzymes are abundant in various rat tissues and in some cultured cells (2).

The biochemical adaptation of the lung to air or oxygen breathing at birth is incompletely understood. The sudden exposure of the air-blood interface of the lung to increased oxygen tension must pose an acute oxidative stress compared with the relatively anaerobic fetal environment. Indeed, the pulmonary epithelium is usually exposed to the highest oxygen tension present in the organism. In several mammalian species there is a gradual increase in the lung activities of multiple antioxidant enzymes during the final 15 to 30% of gestation (5, 6). It has been suggested that in the premature newborn, failure to elevate the activities of these antioxidant enzymes can increase the lung damage caused by hyperoxia during treatment of the respiratory distress syndrome (6). We have demonstrated earlier that two major proteins of the Trx system, Trx and Trx reductase, were upregulated by oxygen in premature newborn baboons (7). In addition, Trx messenger RNA (mRNA), but not Trx reductase mRNA, was upregulated after term birth. Trx along with Trx reductase and Prx form a system similar to the glutathione system. Because Prxs are important antioxidant proteins that reduce H₂O₂ to molecular oxygen and H₂O using electrons from Trx, we hypothesized that in hyperoxia Prx would be upregulated as a defensive mechanism during the fetal/neonatal transition. Therefore, in this investigation we sought to determine the regulation of Prx gene expression in a premature newborn baboon model of chronic lung disease (bronchopulmonary dysplasia [BPD]).

One cell-signaling protein that frequently shows increased activity in response to changes in ambient oxygen tension or to oxidative stress is protein kinase (PK) C (8, 9). PKC, a phospholipid-dependent protein serine/threonine kinase, plays an important role in the regulation of numerous cellular processes, including transcription, cell growth, and modulation of membrane structure (reviewed in ref. 10). The prevalence of this enzyme family in signaling is exemplified by the diverse transduction mechanisms that result in formation of PKC's activator diacylglycerol (reviewed in ref. 11).

Herein, we report that the expression of Prx I mRNA was upregulated in the newborn baboon lung in response to oxygen. Studies in fetal lung cultures demonstrated that ambient oxygen tension was crucial in regulating Prx I gene expression. Additionally, oxygen-induced Prx I gene expression was inhibited by specific PKC inhibitors, indicating a role for PKC in hyperoxia-mediated induction of Prx I gene expression.

	Materials and Methods

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Animal Studies

All animal care procedures were performed according to the National Research Council's Guide for the Care and Use of Laboratory Animals. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Southwest Foundation for Biomedical Research, San Antonio, Texas. Fetal baboons of varying gestational ages (± 2 d) were delivered by hysterotomy. Gestational ages were determined by timed matings as previously described (7), with confirmation by ultrasound at intervals during pregnancy.

In other studies pertinent to the effect of variable oxygen tension in vivo and to the development of BPD after hyaline membrane disease, treatment groups were delivered at either 140 ± 2 or 125 ± 2 d of gestation and immediately placed on positive pressure ventilation. Animals of 140 d gestation were given either continuous 100% oxygen or an inspired oxygen tension as needed (pro re nada [prn]) to maintain arterial oxygen pressure at 40 to 50 torr. Within 10 d, those 140-d animals given 100% oxygen developed lung histopathologic lesions which closely resemble human BPD, whereas the prn animals did not develop these lesions, allowing near-normal lung development (12, 13). More premature animals of 125 d gestation received immediate resuscitation with artificial surfactant, positive pressure ventilation, and prn oxygen. These animals also developed BPD despite receiving lesser (prn) inspired concentrations of oxygen (13). In either case, all animals received state-of-the-art care in a neonatal intensive care unit for up to 17 d. After treatment, animals were killed by intravenous pentobarbital administration. The lungs were perfused via the pulmonary artery with phosphate-buffered saline (PBS) (37°C), and distal lung tissue was dissected free from major airways and central structures and processed immediately as for fetal tissue.

Fetal Lung Culture

Fetal lung explant cultures were carried out as described previously (7). Briefly, distal fetal lung tissue (gestational ages 125 to 175 d) was dissected into 1-mm³ pieces, with careful attention to removal of any attached airways. Explant pieces were placed at four or five per well in 3.5-cm-diameter wells (six-well plates) that had been precoated with 2 ml Waymouth's A157 medium. Explants were then placed in a 1% oxygen, 5% CO₂, and 94% nitrogen-containing certified gas mixture in a plastic modular exposure chamber (Billups-Rothenberg, La Jolla, CA) and allowed to equilibrate for 16 h (37°C). After this period of equilibration, the medium was changed and the explants were exposed for an additional period of 24 h or greater to otherwise identical conditions but with variable oxygen tension.

Cell Culture

A549 cells were obtained from ATCC (Manassas, VA) and were cultured in F12K Kaigan's modified medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U each/ml).

Isolation of Lung Total RNA and Northern Analysis of Prx mRNA

Lung tissue was homogenized in guanidinium isothiocyanate (GITC) with a polytron homogenizer, followed by centrifugation of the lysate in cesium chloride (147,000 g, 20 to 25°C) in a Beckman ultracentrifuge using a modified method of Sambrook and colleagues (14). Total RNA was quantitated spectrophotometrically. A total of 20 µg of RNA was resolved by electrophoresis in 1% agarose/2.5 M formaldehyde gel in a buffer containing 20 mM 3-(N-Morpholino)propanesulfonic acid and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.4). RNA was transferred to nitrocellulose and blots were prehybridized for 2 to 12 h in 50% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate (pH 7.0), 5× Denhardt's solution, 50 µg/ml salmon-sperm DNA, and 0.1% sodium dodecyl sulfate (SDS) at 42°C. Blots were hybridized with complementary DNA (cDNA) for human Prx I (15) and, in some cases, for 28S ribosomal RNA (rRNA) labeled to a specific activity of 2 to 7 × 10⁷ counts per min using ³²P- cytidine triphosphate (ICN, Costa Mesa, CA) in hybridization solution at 42°C overnight, and then washed in 0.3 M sodium chloride, 0.03 M sodium citrate, and 0.1% SDS at 42°C. Autoradiographs were made by exposing blots to X-ray film (Kodak, Rochester, NY) at 70°C with intensifying screens. In some autoradiographs, densitometry was performed with Image 135 software (NIH, Bethesda, MD). Some of the blots were exposed to phosphor screens (Molecular Dynamics, Uppsala, Sweden) and densitometry was performed on phosphor screens by Macintosh computer using MD Image Quant version 3.3 software.

Quantitation of Prx Activity

Prx activity was determined as described by Cha and Kim (16) with the following modifications. The reaction mixture contained 100 µM H₂O₂, 250 µM nicotinamide adenine dinucleotide phosphate (NADPH), 20 µM Escherichia coli Trx-S₂, and 2 µg bovine Trx reductase in N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid-NaOH buffer, pH 7.0. The reaction was initiated by addition of lung homogenates. The total reaction volume was 150 µl. The oxidation of NADPH was followed by monitoring the absorbance at 340 nm. Prx activity was expressed as nanomoles of NADPH oxidized/min/mg protein.

PKC Assay

PKC activity was determined using the PKC assay kit of Life Technologies, Inc. (Rockville, MD), on the basis of the measurement of the phosphorylation of acetylated myelin basic protein as described by Yasuda and associates (17). Briefly, 5 × 10⁶ cells were washed in PBS followed by scraping of the monolayers in 0.5 ml of buffer A (20 mM Tris [pH 7.5], 0.5 mM EDTA, 0.5 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid [EGTA], 0.5% Triton X-100, and 25 µg/ml each of aprotinin and leupeptin). Cells were homogenized with 10 to 15 strokes of a precooled Dounce homogenizer with a B-type pestle at 4°C. After incubation of the cell homogenate with buffer A for 30 min, the lysate was centrifuged and the supernatant was applied to a diethylaminoethyl (DEAE) ion exchange column (Bio-Rad, Hercules, CA). Partially purified PKC was eluted with 5 ml of buffer C (20 mM Tris [pH 7.5], 0.5 mM EDTA, 0.5 mM EGTA, 10 mM 2-mercaptoethanol, and 0.2 M NaCl). The DEAE eluate (25 µl) was assayed for PKC activity using 50 µM acetylated myelin basic protein as a substrate. Activity of PKC was expressed as pmol/min/5 × 10⁶ cells.

Statistical Analysis

Mean values were calculated by averaging data from each experimental group, and the standard error of the mean of each group was calculated. Means of two groups were compared by two-tailed unpaired t test, and those from more than two groups were compared by one-way analysis of variance (18). P < 0.05 was considered significant.

	Results

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Effect of Fetal Gestational Age and Term Birth on the Expression of Baboon Lung Prx mRNA

To determine whether Prx I is developmentally regulated in baboons, we compared the mRNA levels of fetal baboons at 125, 140, 160, and 175 d of gestation by Northern blotting. As shown in Figure 1A, there was no significant change in the expression of Prx I mRNA in fetal baboon lung during the various gestational ages, demonstrating a lack of developmental regulation of Prx I. In addition, there were no changes in the levels of Prx I mRNA during the final third of gestation, in contrast to other antioxidant enzymes (5, 6). We further determined the Prx expression in response to term birth. As shown in Figure 1B, there was significant increase in Prx I mRNA at Day 2 after term birth (P < 0.005). However, at Day 3 Prx I mRNA levels came back to the 175-d level, indicating that sudden exposure to ambient oxygen only transiently increased Prx I mRNA expression. In contrast, Prx II mRNA expression did not change significantly in response to term birth (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 1.   (A) Effect of gestational age on expression of baboon lung Prx I mRNA. Lung tissue was obtained from fetal baboons of different gestational ages delivered by Caesarian section as described in MATERIALS AND METHODS. Tissue was homogenized in GITC and processed for total RNA, and Prx I mRNA was determined by hybridizing total RNA with cDNA for Prx I or 28S rRNA. Densitometry was performed on the autoradiographs, and the density of Prx I mRNA was normalized to 28S rRNA. The ratio of Prx I density to 28S rRNA density was plotted against the gestational age. (B) Effect of air-breathing on Prx I gene expression. Lung tissue was collected from fetal or term-gestation, spontaneously delivered infant baboons, and processed for total RNA isolation and Northern blotting for Prx I as described for A. (GC, gestational controls). *Significant difference from all other groups (P < 0.05).

Effect of Oxygen Exposure on Lung Prx mRNA In Vivo

To investigate the effect of oxygen breathing on Prx gene expression, premature newborn baboons were exposed to oxygen as described in MATERIALS AND METHODS. Within 24 h after birth, Prx I mRNA increased approximately 40% in lungs of animals of 140 d gestation with respiratory distress syndrome (Figure 2A, lane 2). In those receiving 100% oxygen, Prx I mRNA levels remained significantly elevated (P < 0.05) relative to fetal values even at 6 and 10 d of age (Figure 2A, lanes 6 and 7 and Figure 2C, lanes 4 and 5). By contrast, in lungs of 140-d animals given only as-needed oxygen (24 h prn = 42% oxygen [mean]; 6 d prn = 45% oxygen [mean]; 10 d prn = 30% oxygen [mean]) Prx I mRNA levels increased about 30% (Figure 2A, lanes 3 and 4 and Figure 2C, lanes 2 and 3).

View larger version (51K): [in this window] [in a new window]   Figure 2.   Effect of birth and respiratory distress on lung Prx I mRNA. (A) Lung tissue was obtained from 140-d fetal controls or 140-d gestational premature baboons delivered by Caesarian section and exposed to various oxygen tensions. Tissue was homogenized in GITC and processed for total RNA, and Prx I mRNA was determined by hybridizing total RNA with cDNA probe for Prx I as described in MATERIALS AND METHODS. Lane 1, 140-d fetal premature baboon delivered and immediately necropsied and lung tissue processed for RNA isolation; lane 2, 140-d fetal premature baboon delivered by Caesarian section and ventilated with as-needed (prn) oxygen for 24 h in critical care unit and necropsied after this treatment; lane 3, similar treatment as in lane 2 with prn oxygen administered continuously for 6 d; lane 4, similar to treatment in lane 3 with prn oxygen administered continuously for 10 d; lane 5, similar to lane 2 but newborns received 100% oxygen continuously for 24 h; lane 6, similar to lane 5 with continuous 100% oxygen administration for 6 d; lane 7, similar to lane 6 with continuous 100% oxygen administration for 10 d. (B) Same treatments as in A. Blot was stripped and reprobed with 28S rRNA. (C) Ratio of Prx I mRNA to 28S rRNA. *Significant difference from all other groups (P < 0.05).

Effect of Oxygen on Prx mRNA Expression in Fetal Lung Explant Culture

To isolate the acute effects of hyperoxia from other early postnatal factors in respiratory distress syndrome, such as barotrauma, inflammation, and bacterial colonization, a fetal lung explant culture system was used. Exposure to 95% oxygen at Denver (approximately 80% O₂ at sea level) for 24 h caused a 4- to 5-fold increase in Prx I mRNA relative to the levels of this mRNA during continued culture in 1% oxygen (Figure 3A). Prx I mRNA was significantly increased in explants exposed to 95% oxygen compared with 1% oxygen (P < 0.05; Figure 3C). The relative increase in Prx I mRNA between 1% and 95% oxygen was fairly constant among the various gestational ages evaluated (125 to 175 d), and the capacity to increase these messages in response to hyperoxia was also retained in lung tissue from adult baboons (not shown).

View larger version (51K): [in this window] [in a new window]   Figure 3.   Effect of oxygen on Prx I mRNA expression in fetal lung explant culture. Fetal (140-d) distal lung explants were cultured as described in MATERIALS AND METHODS. Explants were exposed either to 1% or 95% oxygen for 24 h and processed for total RNA (n = 3 per condition). Total RNA was hybridized with cDNA probe for human Prx I or 28S rRNA. (A) Lanes 1-3, explants exposed to 1% oxygen; lanes 4-6, explants exposed to 95% oxygen. (B) Same treatments as in A. Blots were stripped and reprobed with 28S rRNA. (C) Ratio of Prx I mRNA to 28S rRNA. *Significant difference from all other groups (P < 0.05).

Effect of Oxygen on Prx Activity In Vivo

Prx contains catalytic cysteines that react with peroxides, and the activity of Prxs is dependent on the catalytic cysteines. Because many sulfhydryl-containing enzymes are known to be inactivated in oxidative stress, we sought to determine the activity of Prx in respiratory distress. As shown in Figure 4A, the activity of Prx was significantly increased in prn animals (P < 0.05), but was decreased relative to prn animals in premature baboons exposed to 100% oxygen. Although the activity in the lungs of 100% oxygen- exposed animals remained elevated compared with gestational control animals, there was a lack of statistical significance at the P < 0.05 level. Nevertheless, there was a trend for an increase in the Prx activity in premature baboons exposed to 100% oxygen.

View larger version (25K): [in this window] [in a new window]   Figure 4.   (A) Effect of respiratory distress on lung Prx I activity. Lung tissue was obtained from 140-d gestational controls or 140-d fetal premature baboons delivered by Caesarian section and exposed to various oxygen tensions as described in MATERIALS AND METHODS. Tissue was homogenized in Tris-HCl buffer, pH 7.4, and centrifuged in a microcentrifuge. The supernatant was assayed for Prx activity as described in MATERIALS AND METHODS (n = 3 per condition). The error bar for the middle column (140d PRN O2) is so small that it cannot be seen. Prx activity was expressed as nmol of NADPH oxidized/min/mg protein. (GC, gestational controls). *Significant difference from all other groups (P < 0.05). (B) Effect of oxygen on Prx I activity in lung explant cultures. Fetal distal lung explants were cultured as described in MATERIALS AND METHODS. Explants were exposed to either 1% or 95% oxygen for 24 h and were homogenized as described for A. Prx activity was assayed as described for A (n = 3 per condition). *Significant difference from all other groups (P < 0.05).

Effect of Oxygen on Prx Activity in Lung Explant Culture

To determine the effect of oxygen on lung Prx activity, we exposed explants of lungs of 140-d gestation fetal animals to 1% or 95% oxygen for 24 or 48 h, and Prx activity was determined as described in MATERIALS AND METHODS. There was increased Prx activity in premature fetal 140-d lung explants exposed to 95% oxygen for 24 h over that of explants exposed to 1% oxygen for 24 h (Figure 4B). However, the increased Prx activity was not statistically significant at a P < 0.05 level. When explants were further incubated in 95% oxygen for 48 h, there was a decrease in Prx activity. However, this decrease also was not statistically significant.

Effect of Actinomycin D and Cycloheximide on Prx I mRNA Increase by Oxygen in Lung Explant Culture

To determine whether the increase in Prx I mRNA after exposure to oxygen could be mediated at the transcriptional level, lung explants were incubated in 1% or 95% oxygen with actinomycin D (1 µg/mL), an inhibitor of transcription. Actinomycin D inhibited Prx I mRNA induction in lung explants exposed to hyperoxia (Figure 5). Further, cycloheximide, a protein synthesis inhibitor, lowered the Prx I mRNA elevation by more than 50%, suggesting that protein synthesis de novo is, in part, also involved in the induction of the Prx I mRNA.

View larger version (31K): [in this window] [in a new window]   Figure 5.   Effect of actinomycin D and cycloheximide on Prx I mRNA increase by oxygen in lung explants. Lung explants were treated with actinomycin D (1 µg/ml; Calbiochem) for 1 h. They were then exposed to either 1% or 95% oxygen for 24 h. The experiment was performed in duplicate. After exposure, explants were removed, rinsed in Hanks' balanced salt solution (HBSS) and homogenized in GITC. Total RNA was prepared as described in MATERIALS AND METHODS. Total RNA (20 µg) was probed with cDNA for human Prx I or 28S rRNA. Densitometry was performed on autoradiographs. Density of Prx I was normalized to the density of 28S rRNA.

Effect of Hyperoxia on PKC Activity in A549 Cells, and of Specific PKC Inhibitors Calphostin C and GF109203X on Oxygen-Induced Elevation of Prx I mRNA in Lung Explant Cultures

Oxidants such as H₂O₂ and nitric oxide (NO), as well as perturbation of oxygen tension, can cause increased activity of PKC in a number of cell types (8, 9). Because intracellular oxidants are produced in excess in response to elevated oxygen tension, we reasoned that PKC could be involved in the signaling mechanism by which hyperoxia increased the expression of Prx I. We exposed human lung epithelial-like cell line A549 to either 21% (baseline) or 95% O₂ for various time periods as shown in Figure 6A and measured the PKC catalytic activity. Total cellular PKC activity increased when A549 cells were exposed to 95% O₂ relative to 21% O₂ exposure. Significant increases in PKC activity occurred after 30, 60, 90, and 120 min of continuous hyperoxic exposure, relative to baseline (Figure 6A). Maximal activation occurred after 1 h with a slight decline occurring within 2 hours. Calphostin C is a specific inhibitor of PKC that binds to the regulatory subunit of the enzyme (19). GF109203X is a specific inhibitor of PKC that binds to the catalytic subunit of the enzyme (20). Because total cellular PKC activity was increased in A549 cells in response to oxygen, we investigated the effect of specific inhibitors of PKC on Prx I gene expression in response to hyperoxia in lung explant cultures. Lung explants were pre-exposed to calphostin C (200 ng/ml) or GF103203X (15 µM) for 1 h, followed by exposure to 1% or 95% oxygen for 24 h. As shown in Figure 6B, treatment with either inhibitor decreased oxygen-mediated Prx I mRNA induction to < 25% that of untreated explants, suggesting a PKC-dependent pathway for Prx I induction in response to oxygen.

View larger version (23K): [in this window] [in a new window]   Figure 6.   (A) Effect of hyperoxia on PKC activity in A549 cells. Lung epithelial-like A549 cells were grown in F12K medium as described in MATERIALS AND METHODS. Confluent monolayers were exposed to 95% oxygen, and after incubation, PKC was partially purified as described in MATERIALS AND METHODS. PKC activity was assayed using a PKC assay kit (GIBCO BRL, Rockville, MD) as per manufacturer's protocol. PKC activity was expressed as pmol/min/ mg protein. *Significant difference from all other groups (P < 0.05). (B) Effect of PKC inhibitors on Prx I mRNA increase in lung explant cultures. Lung explants (140-d) were pretreated with calphostin C (200 ng/ml; Calbiochem, San Diego, CA) or GF109203X (15 µM) for 1 h. They were then exposed to either 1% or 95% oxygen for 24 h. The experiment was performed in duplicate. After exposure, explants were rinsed in HBSS and homogenized in GITC. Total RNA was prepared as described, and probed with cDNA for human Prx I or 28S rRNA. Densitometry was performed on autoradiographs. Density of Prx I was normalized to the density of 28S rRNA.

	Discussion

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The data presented here indicate that the gene for Prx I, an important protein of the Trx system, is expressed at low levels in the prenatal period. By contrast, its expression is increased postnatally, primarily by ambient oxygen tension. This pattern of expression differs considerably from a number of classical antioxidant enzymes (5, 6), although it is clear that oxygen can play an important role in modulating expression of some of these in the newborn (20). At 6 d after birth in 140-d-gestation baboons, the increase in Prx I mRNA occurred to a comparable extent in both 100% oxygen-exposed animals that were destined to develop BPD (12, 13) and in those exposed only to prn oxygen that did not acquire chronic lung disease. In 100% oxygen- exposed newborns, elevation of lung Prx I mRNA persisted even after 10 d. Additionally, the Prx I mRNA expression declined in prn oxygen-treated animals as the inspired oxygen tension was weaned. Although a smaller number of newborns were studied at this time point, newborn baboons exposed to 100% or prn oxygen for only 24 h also had strong elevation of Prx I mRNA. Thus, pulmonary Prx I gene expression did not appear developmentally regulated during the final third of gestation, and upregulation of Prx I mRNA in response to oxygen was unimpaired in newborns throughout this interval.

In contrast to upregulation of mRNA, data obtained from fetal lung explants cultured in different oxygen concentrations indicated that there was no increase in Prx activity in response to hyperoxia after 48 h in the lung tissue from animals of 140 d gestation. In contrast, there was a 40% decrease in Prx activity relative to 1% O₂ after 48 h of exposure to hyperoxia in lung explant cultures. One potential mechanism for this decline in activity is that the Cys47 of Prx could react with H₂O₂, causing its oxidation during prolonged exposure to hyperoxia. The regeneration of this sulfhydryl group may not occur in hyperoxia due to the oxidation of Trx itself, which normally regenerates the Cys47 of Prx (1). Besides facilitating evaluation of the effects of acute exposures, the explant system also allowed the isolation of the effect of oxygen tension from other factors, such as bacterial or fungal colonization or infection, inflammation, exogenous cytokines, and barotrauma, which may be present in the intubated, artificially ventilated newborn. Data obtained from 140-d premature newborns in vivo after 6 d of exposure to hyperoxia indicated that, by this time point, Prx activity still had not increased in response to elevated oxygen tension. However, 140-d prematures exposed to prn oxygen demonstrated a 2.6-fold increase in Prx activity. Hence, this increase in Prx activity may offer protection against oxygen-mediated injury to prn animals. On the other hand, the failure of 100% oxygen-exposed animals to significantly increase lung Prx activity may contribute to the subsequent development of lung injury (BPD).

Because oxidants such as H₂O₂ and NO, as well as perturbation of oxygen tension, can cause increased activity of PKC in a number of cell types (8, 9, 21), and because intracellular oxidants are produced in excess in response to elevated oxygen tension, we reasoned that PKC could be involved in the signaling mechanism by which hyperoxia increased expression of Prx I. We used a human lung epithelial-like cell line (A549) to further explore the mechanism of Prx I gene activation by hyperoxia. We found A549 cells to demonstrate many of the same changes in intermediary metabolism in response to hyperoxia as we and others have observed in lungs and/or lung cells. These include the inactivation of aconitase (21), increased glucose utilization (22) and decreased respiration (21), induction of Trx (7) and of hexokinase II gene expression (23), and many other effects. Because access to fetal lung tissue from primates is limited, we used A549 cells to determine the effect of oxygen on PKC activity. In addition, 100% oxygen-exposed animals are now only infrequently studied at the BPD center in San Antonio, Texas. Therefore, we examined the effect of hyperoxia on PKC activity at multiple early time points during exposure to hyperoxia. PKC catalytic activity was increased in 95% oxygen exposure of A549 cells. This study provided an additional mechanistic understanding of our findings in inhibitor studies with lung explants. Calphostin C is a specific inhibitor of PKC that binds to the regulatory subunit of the enzyme (18). GF109203X is a specific inhibitor of PKC that binds to the catalytic site and thereby causes its inhibition (19). When 140-d lung explants were pretreated with calphostin C (1 µM) or GF109203X (15 µM) for 1 h, followed by continuous exposure to 1 or 95% oxygen, significant inhibition of the oxygen-dependent increase in Prx mRNA was observed (Figure 6). The extent of inhibition was about 90% when Prx mRNA was normalized to 28S RNA. These studies indicate that activation of PKC may play a significant role in signaling of Prx I expression induced by elevated oxygen tension.

Elevation of Prx I expression in the lung may provide important protection against oxidative stress during fetal adaptation to aerobic life. Prx I can protect a number of different cell types, including endothelial and epithelial cells, against injury by H₂O₂. Because prn-exposed premature infant baboons have less lung injury and improved survival compared with 100% oxygen-exposed animals, the response of prn animals to increased Prx activity may contribute to their more successful adaptation.