Main

In the lungs of neonatal animals, oxidative stress such as hyperoxia can cause acute and chronic injury. Oxidative stress regulates the expression of critical antioxidant enzymes(1) and other genes such as HO-1, an isoenzyme of HO (EC 1.14.99.3), the rate-limiting enzyme in mammalian heme catabolism(2, 3). HO is an inducible stress protein which may serve in limiting oxidative damage by removing intracellular heme, a prooxidant compound(4), producing bilirubin, an antioxidant molecule(5, 6), and increasing ferritin synthesis to further sequester redox active iron(7).

Several investigators have demonstrated transcriptional regulation of HO-1 in various models of oxidant stress(2, 8, 9). Treatment of adult rats with diethylmaleate, a glutathione-depleting agent, was shown to result in a 6-fold increase in brain HO-1 mRNA(8). Exposure of human umbilical vein endothelial cells to 95% oxygen resulted in a 7-fold induction of HO-1 mRNA at 48 h. In addition, pretreatment of the cells with L-buthionine-(S,R)-sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase, further increased HO-1 mRNA levels after hyperoxic exposure(2). In further corroboration with transcriptonal regulation of HO-1 in oxidant stress, a sequence upstream of the mRNA cap site was indentified, which responds to oxidant agents such as hydrogen peroxide, resulting in 3-5-fold increases in expression of a reporter construct of the gene(10). Furthermore, HO-1 mRNA was transcribed at an increased rate with various oxidant stresses, such as hydrogen peroxide, but no change in mRNA stability was noted(11). However, others have observed that cycloheximide increased HO-1 mRNA half-life 5-fold after heme induction, suggesting that a protein may play a key role in HO-1 mRNA regulation and thus alluding to possible posttranscriptional mechanisms of HO-1 regulation(12).

In neonatal rats, other lung enzymes such as superoxide dismutase and catalase are regulated posttranscriptionally in hyperoxia(13, 14). For example, perinatal catalase mRNA half-life is increased 2-fold in hyperoxia without changes in catalase mRNA rate of transcription, in contrast to adult lung catalase(15). Although it is well known that HO-1 is transcriptionally regulated by oxidant stress in adult models, it is not known whether neonatal lung HO-1 is regulated in hyperoxia and, if so, what type of regulation occurs. Therefore, we attempted to answer these questions by examining lung HO-1 mRNA, protein, and activity in neonatal rats during hyperoxic exposure.

METHODS

Animals. Litters of Wistar rat pups and their dams were obtained from Simonsen (Gilroy, CA). The animals were kept in a 12-h light-dark cycle and allowed to feed ad libitum until the time of experimentation. Animals were newly born (<12 h old) when experiments began.

Experimental design. Neonatal rats and their mothers were placed in a flow-through, Plexiglas cylindrical exposure chamber (Vreman Scientifics, Los Altos, CA) that had ports for gas entry and exit, and access to nesting materials, food, and water. Either air or hyperoxic atmospheres(>95% O2) were provided by commercial cylinders (Liquid Carbonic, Chicago, IL). Rats were kept in a 12-h light-dark cycle throughout the exposures, and pups were taken out daily for 10-15 min to allow for bedding changes and chamber cleaning. At this time, dams that had been in one atmosphere (i.e. air or hyperoxia) were changed to the other (air to hyperoxia and vice versa) every 24 h and allowed to nurture the new litter to obviate the effects of hyperoxia on the dams. Some of the pups were removed daily for sample collection, but litter size remained constant between experimental and control groups on each day to control for nutritional status of the pups.

Tissue preparation. The neonatal rats were killed by decapitation. Blood was drained from the body and collected. The lungs of each animal were rinsed in cold 0.1 M KPO4 buffer to remove any excess blood and processed immediately.

Preparation of lung microsomes. Some experiments were reproduced in microsomes because HO is concentrated in this cellular compartment(9). Lungs were removed, homogenized in 0.1 M KPO4 buffer (pH 7.4), and centrifuged at 13,000 × g. The 13,000 × g supernatant was centrifuged at 100,000 ×g for 60 min, and the microsomal pellet resuspended in 0.01 M Na/K phosphate buffer (pH 7.4) containing 1 mM EDTA, 1.1 mM phenylmethylsulfonyl fluoride, 100μg/mL leupeptin, and 20 μg/mL soybean trypsin inhibitor to prevent protein degradation, and 20% glycerol(16). The suspended microsomes were snap frozen in liquid nitrogen and stored at -80°C until assays could be performed.

Tissue heme oxygenase activity. Assays were conducted with the room lights off. Twenty microliters of lung 13,000 × g supernatants or lung microsomes were reacted with NADPH and hemin in a septum-sealed, amber-colored vial at 37 °C. Vials were purged with CO-free air and allowed to incubate for 15 min in the dark. The reaction was stopped with dry ice (-78 °C), and CO generation in the vial gas headspace was analyzed by gas chromatography as per the methods of Vreman et al.(18). This assay is conducted under saturating conditions, therefore the presence of heme does not interfere with the determination of CO(17).

Lipid peroxidation. Thiobarbituric reactive substance in lung supernatants were determined by the methods of Asakawa et al.(18) and read at 532 nm with a spectrophotometer(Shimadzu, Columbia, MD). In addition, serum samples were assessed for the presence of lipid hydroperoxides with a commercially available kit (Determiner LPO; Kamiya Chemicals, Thousand Oaks, CA). This colorimetric assay detects the Hb-catalyzed reaction of hydroperoxides with a methylene blue derivative(19).

Tissue protein content. Lung 13,000 × g supernatants or microsomes were analyzed for protein by the method of Lowryet al.(20).

Preparation of HO-1 antisera. Antisera was prepared by Berkeley Antibodies, Inc. (Berkeley, CA). Polyclonal rabbit anti-rat HO-1 antibodies were raised against a 30-kD soluble HO-1 protein expressed inEscherichia coli using rat liver cDNA(21)(gift of A. Wilks, University of California, San Francisco, CA). Antisera was precleared by incubating at 4 °C with normal rat serum for 2 h, followed by 10% Staphylococcus aureus protein A-Sepharose CL-4B beads (Sigma Chemical Co., St. Louis, MO) for an additional 2 h. After centrifugation at 6,000 × g for 5 min, the supernatant was recovered and used as a source of HO-1 antibody for immunoprecipitation and Western analyses. This antibody does not cross-react with HO-2 in all tissues tested.

Western blot analysis. Fifty-microgram aliquots of 13,000× g supernatant or 20 μg of lung microsomes were electrophoresed on a 12% polyacrylamide gel according to the method of Laemmli(22). Proteins were transferred for 2 h to polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) with a Bio-Rad Mini Trans-Blot apparatus according to the method of Towbin et al.(23). Membranes were blocked overnight at 25 °C in 10% nonfat milk, washed briefly with TBS containing 0.1% Tween 20, incubated overnight with 1:200 dilution of rabbit anti-rat HO-1 IgG, and washed with TBS. Antigen-antibody complexes were visualized with the alkaline phosphatase chemiluminescent system according to the manufacturer's instructions(Bio-Rad). Microsomes prepared from adult rat spleen were used as a positive control for identification of the HO-1 protein. Blots were subsequently washed in TBS with 0.1% Tween overnight and reincubated for 2 h with a 1:600 dilution of rabbit anti-rat HO-2 IgG, and antigen antibody complexes were visualized as described above. Quantification was performed by densitometry (Applied Biosystems, Santa Clara, CA). Equal loading of the samples was verified by Coomassie Blue staining.

Plasmid and probe preparation. The plasmid pBKRHO1 was constructed in pBluescript II SK(-) using a rat HO-1 cDNA fragment prepared by reverse transcription-PCR that corresponded to HO-1 mucleotides -33 to +833 as reported by Shibahara et al.(24). Poly(A+) RNA was isolated from rat spleen (Poly(A) Quik mRNA isolation kit, Stratagene, La Jolla, CA) and used for first strand cDNA synthesis(Superscript™ II Kit, Life Technologies, Inc., Gaithersburg, MD) using an oligo(dT)15 primer (Promega Corp., Madison, WI). HO-1-specific oligonucleotides were obtained from Eppendorf (Madison, WI): the 5′-sense oligonucleotide (5′GCAAGCTTAGCGGAGCCAGCCTGAA-3′) corresponded to residues -33 to -16 and contained 6 additional nucleotides coding for a HindIII restriction site; the 3′-antisense oligonucleotide (5′-GGCTCGAGGAAACTGAGTGTGAGGA-3′) corresponded to residues 814-833 and contained 8 additional nucleotides coding for anXho I restriction site. After 2 × 30 cycles of amplification(GeneAmp PCR Reagent Kit, Perkin-Elmer Corp.), the PCR product was phenol-chloroform-extracted and ethanol-precipitated. The resuspended cDNA was digested with HindIII and XhoI, gel purified, and cloned into pBlue-script II SK(-). The resulting plasmid (pBKRHO1) was used to transform E. coli DH 5∂ (Life Technologies, Inc.). Transformants were screened by restriction digestion and the nucleotide sequence of the insert (RHO-1 cDNA) was confirmed by the dideoxynucleotide chain termination method (PAN Facility, Beckman Center, Stanford University). The housekeeping genes GAPD cDNA(25) and β-actin(26) (American Type Culture Collection, Rockville, MD) were prepared as EcoRI digests by standard methods(27). Labeled probes were prepared by the random primer method(28) using [32P]dCTP.

Northern hybridization. RNA was isolated by the guanidinium thiocyanate:phenol extraction method of Chomczynski and Sacchi(29) and quantitated spectrophotometrically at 260 nm. With each gel, 2-5 μg of RNA derived from adult rat spleen were denatured and electrophoresed as a positive control, because the spleen is a tissue with high HO-1 expression. As to the neonatal lung samples, 20 μg of RNA were denatured, electrophoresed on a 1.2% agarose gel containing 1.1 M formaldehyde, transferred to a positively charged nylon membrane (Hybond N+, Amersham Corp., Arlington Heights, IL) and immobilized by UV irradiation (UV-Stratalinker, Stratagene). Membranes were prehybridized for 4-6 h at 42 °C in buffer, containing 50% formamide, 5 × SSPE, 5× Denhardt's solution, 1% SDS, 10% dextran sulfate, and 50 μg/mL denatured salmon sperm DNA, and subsequently hybridized for 18-24 h at 42°C in buffer containing 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 1% SDS, 10% dextran sulfate, and 32P-labeled probe(5-10 × 108 cpm/μg, 1-5 × 106 cpm/mL). Membranes were washed twice at 25 °C for 5 min in 2 × SSPE, 0.1% SDS, and twice at 50 °C for 20 min in 0.2 × SSPE, 0.1% SDS, dried briefly, and exposed to Kodak X-Omat film with an intensifying screen at -80 °C. For reprobing, membranes were stripped according to the manufacturers protocol using boiling 0.5% SDS. HO-1, GAPD, and β-actin mRNA quatifications were performed by densitometry (PDI, Santa Clara, CA), and statistical analysis was performed on the ratio of HO-1 to GAPD or β-actin normalized to the value of the preexposure control (D0) from the same membrane. Because the two ratios did not differ significantly, data were expressed as the normalized ratio of HO-1 to GAPD only.

Immunoprecipitation of HO-1 protein. Methods used were generally as described by Harlow and Lane(30). Briefly, lung 13,000 × g supernatants from air- and hyperoxia-exposed animals were incubated at 4 °C with HO-1 antibody at 1:300 dilution for 1 h. A sufficient quantity of S. aureus protein A-Sepharose CL-4B beads was added (10% final concentration), and the mixture was incubated for 1 h at 4 °C. After centrifugation at 10,000 × g for 3 min, the supernatant was removed and analyzed for HO activity as described above.

Statistical analysis. For comparison between treatment groups, the null hypothesis that there was no difference between treatment means was tested by a single factor analysis of variance for multiple groups or unpairedt test for two groups (Statview 4.02; Abacus Concepts, Inc., Berkeley, CA). Statistical significance (p < 0.05) between and within groups was determined by means of the Fischer method of multiple comparisons.

RESULTS

HO-1 mRNA. In the lungs of neonatal rats, no significant change in levels of steady state mRNA of HO-1 was observed in the lungs of animals exposed to hyperoxia compared with air-exposed rat pups on any of the days of the exposures (see Fig. 1,A andB). Furthermore, earlier sampling at 12 h did not reveal any significant changes in lung HO-1 mRNA levels in air or hyperoxia when normalized to D0 values (1.23 ± 0.15 versus 1.28 ± 0.29, respectively; p = 0.91;n = 4 in each group).

Figure 1
figure 1

(A) Detection of neonatal lung HO-1 mRNA transcripts with hyperoxic exposure. Total RNA was isolated in neonatal rat lungs exposed to air or hyperoxia and subjected to Northern blot analysis using 32P-labeled HO-1 cDNA as described in “Methods.”Upper panel, HO-1; middle panel, GAPD (reference transcript); lower panel, β-actin (reference transcript).Lane C, 2 μg of RNA from adult rat spleen (positive control for HO-1); lane 1, neonatal rat lung RNA, preexposure (d0);lane 2, d 1 of air exposure (d1air); lane 3, d 1 of O2 exposure (d1O2); lane 4, d 2 of air exposure(d2air); lane 5: d 2 of O2 exposure(d2O2); lane 6, d 3 of air exposure (d3air);lane 7, d 3 of O2 (d3O2). All lanes other than lane C (positive control) contained 20 μg of RNA. (B) Densitometric quantitation of lung HO-1 to GAPD mRNA for air or hyperoxia exposed neonatal rats and normalization to preexposure (D0) values derived from the same membrane. Values are expressed as mean ± SEM,n = 10 in each group.

Full size image

HO-1 protein. In the lungs of rat pups, steady state levels of immunoreactive HO-1 protein decreased after d 0 in both air and hyperoxia. On d 2, HO-1 protein was two times greater (p < 0.05) in the lungs of rats exposed to hyperoxia compared with air-exposed animals. On d 3, lung HO-1 protein levels were 1.5-fold higher in hyperoxia-exposed rats compared with air-exposed rats, but this difference did not achieve statistical significance (p = 0.19) (Fig. 2B). A similar pattern was observed in the lung microsome samples (Fig. 2A).

Figure 2
figure 2

(A) Detection of immunoreactive HO-1 protein in the lungs of neonatal rats exposed to air or hyperoxia by Western analysis with chemiluminescence. Lane C: positive control microsome from adult rat spleen. Lane 1, d0; lane 2, d1air;lane 3, d1O2; lane 4, d2air;lane 5, d2O2; lane 6, d3air;lane 7, d3O2. Equal amounts (20 μg) of lung microsomal protein were loaded in each lane. (B) Quantitation of immunoreactive HO-1 protein in the 13,000 × g supernatants of lungs from neonatal rats exposed to air or hyperoxia. Values are expressed as mean densitometric units ± SE; *p < 0.05vs air-exposed, n = 6 in each group.

Full size image

Total HO activity. Activity was expressed per mg of protein of the 13,00 × g supernatant. No differences in lung 13,000× g total HO activity were observed between air- and hyperoxia-exposed rat pups on d 0-2. However, hyperoxic exposure was associated with increased total HO activity [1.8-fold (p < 0.05)] on d 3 (Fig. 3). These data were further confirmed in the lung microsome samples expressing HO activity as per mg of the microsomal fraction (1.9-fold increase; p < 0.05; n = 4 in each group).

Figure 3
figure 3

Determination of HO-1 activity by gas chromatography in the 13,000 × g supernatant of lungs from neonatal rats exposed to hyperoxia. Samples were reacted with hemin in the presence (total) and absence (blank) of NADPH. Values for HO activity were derived by calculating the CO content of the total sample minus that of the blank and values were expressed as mean ± SE: *p < 0.05 vs air-exposed, n = 8 in each group.

Full size image

Lipid peroxidation. In hyperoxia, an increase in lung lipid peroxidation was observed on d 2 preceding the increase in total HO activity(0.42 ± 0.16 nmol/mg of protein in air versus 1.13 ± 0.21 nmol/mg protein in hyperoxia; p < 0.05, n = 6). Increased lipid peroxidation on d 2 was substantiated by significantly elevated lipid hydroperoxides in the serum of the hyperoxia-exposed rat pups compared with air-exposed controls (49.56 ± 4.8 in air versus 72.41 ± 5.4 nmol/mL in hyperoxia; p < 0.05, n = 4).

Effect of immunoprecipitation of lung HO-1 protein. Because total HO activity is the sum of the enzymatic activity of both isoforms of HO, we attempted to remove HO-1 protein through immunoprecipitation, and thus eliminate HO-1-derived activity from rat lung samples. The percentage of HO-1-related activity was calculated as follows: [(total HO activity - residual HO activity after immunoprecipitation)/ total HO activity] × 100. After 3 d of exposure, the percentage of HO-1-related activity was significantly greater in hyperoxia-exposed animals compared with those air-exposed (Fig. 4A). The efficacy of immunoprecipitation was verified in five 13,000 × g supernatant samples and in three microsome samples by comparing the HO-1 antibody-antigen complex signal on Western analysis pre- and postimmunoprecipitation. The average reduction in signal intensity postimmunoprecipitation in the 13,000 × g supernatants was 96 ± 3% of the preimmunoprecipitation signal. Microsomal samples revealed a similar pattern with immunoprecipitation (94%± 3%) (Fig. 4B).

Figure 4
figure 4

(A) Effect of immunoprecipitation of HO-1 protein on total HO activity in 13,000 × g supernatant of lungs from neonatal rats exposed to air hyperoxia. The percentage of HO-1-related activity was calculated as follows [(total HO activity - residual HO activity)/total HO activity] × 100. Values expressed as mean ± SE; *p < 0.05 vs native activity, n = 5 in each group. (B) Detection of immunoreactive HO-1 protein in the microsomes of lungs from neonatal rats exposed to 3 d of air or hyperoxia before and after immunoprecipitation with HO-1 antibodies. A representative of three Western analyses detected with chemiluminescence is depicted.Lane 1, air-exposed, preimmunoprecipitation; lane 2, air-exposed, postimmunoprecipitation; lane 3, oxygen exposed, preimmunoprecipitation; lane 4, oxygen-exposed, postimmunoprecipitation.

Full size image

DISCUSSION

In neonatal rats, we noted increased lung total HO activity in hyperoxia but the regulation of lung HO-1 in hyperoxia seemed to differ from previous reports. We saw little to no change in lung HO-1 mRNA but we observed higher HO-1 protein and total HO activity in the lungs of neonatal rats after hyperoxic exposure. Nonetheless, we could not conclude that the increased HO activity was due purely to HO-1, because total HO activity is the sum of both HO-1- and HO-2-derived activities. There are no established assays that allow differentiation of HO-1- and HO-2-derived activities, therefore, we attempted to remove HO-1 protein by immunoprecipitation to evaluate the contribution of HO-1 to total HO activity. After immunoprecipitation, we noted a large decrease in total HO activity in neonatal lungs after 3 d of hyperoxia (80%) whereas, after air exposure, the decrease was much smaller (25%). Furthermore, in recent preliminary work, we did not observe any changes in lung HO-2 steady state protein levels in the first few days of life and no hyperoxia-related increase in HO-2 (C. S. Lee, unpublished observations). These data suggest that the increased total HO activity noted in hyperoxia was principally due to HO activity related to HO-1 protein and may indirectly denote that only HO-1 protein is regulated in hyperoxia. Other investigators have shown that HO-1 is the isoform of HO that can be regulated in oxidant stress, whereas HO-2 does not undergo such regulation(8, 31).

Because hyperoxic exposure of 3 d duration was associated with an increase in HO-1 derived activity, we expected increased immunoreactive HO-1 protein in the lungs of hyperoxia-exposed neonatal rats. However, hyperoxic exposure was associated with a lesser decrease in lung HO-1 protein than air exposure on d 2, suggesting less degradation of HO-1 protein in hyperoxia on d 2. On d 3 of hyperoxic exposure, the lesser decrease in HO-1 protein compared with air did not reach statistical significance. Because no change in lung HO activity occurred on d 2 of hyperoxic exposure but lung HO-1 protein levels differed from those of air-exposed pups, it is possible that the total lung HO activity on d 2 was limited by the activity of CCR, the enzyme that transfers reducing equivalents to HO during heme catabolism. Previous studies have shown that hyperoxia may in fact decrease CCR activity in adult rats(32, 33); however, in 5-d-old rat pups exposed to hyperoxia, the level of CCR increased after an initial lag period(34). Furthermore, neonatal rats have relatively higher levels of CCR activity than their adult counterparts(34). Together, these data imply that it is unlikely that lung HO activity was suppressed on d 2 of hyperoxia due to limited CCR activity. The rapid changes in expression of lung HO-1 protein that we note are in agreement with a previous report of a half-life for HO-1 protein of 15 h in vitro(12). Although we cannot directly extrapolate from in vitro data, it is conceivable that in a 24-h period, HO-1 protein levels could decrease to 32.5% of their previous values; thus a significant increase at d 2 of hyperoxic exposure can be followed by a lower, nonsignificant value on d 3 if no new HO-1 protein were made. Another explanation for increased HO activity on d 3 of hyperoxic exposure without a similar increase in HO-1 protein could have been due to changes in HO-2 protein expression, but HO-2 protein did not increase on any day of hyperoxic exposure (C. S. Lee, unpublished observations).

The lack of a significant increase in lung HO-1 protein in hyperoxia on d 3 was also corroborated by a similar lack of a statistically significant increase in steady state mRNA levels on all days of hyperoxic exposure and also at 12 h. Hyperoxic exposure has been reported to result in a 7-fold increase in HO-1 mRNA levels in human umbilical endothelial cells in culture by 48 h(2) and a 6-15-fold increase in adult rat lung HO-1 mRNA levels within 56 h(35), but no data exist on the effect of hyperoxia in neonatal tissues in vivo. Although early 3-10-fold induction of hepatic HO-1 is noted in neonatal rats injected with CoCl2 on d 0-5 of life(36), the neonatal lung may be more refractory to HO-1 mRNA induction than is the liver. Additionally, the response of neonatal lung tissue to oxidative stress may differ from that of the liver due to the need for rapid regulation of lung antioxidant capacity. This may explain the bypassing of transcriptional regulation of HO-1 in hyperoxia. Perhaps the differences in HO-1 protein, on d 2, in air- and hyperoxia-exposed lungs, may be due to more efficient translation of HO-1 mRNA. Another possible explanation for the lack of change in steady state levels of HO-1 mRNA in hyperoxia could be that changes in mRNA would have preceded our sampling (i.e. before 12 h) and rapidly returned to baseline as occurs in in vitro models of HO-1 induction(38, 39).

In this study, we observed that basal HO-1 protein levels were highest on the 1st d of life then declined, as previously observed in the neonatal liver(40). Furthermore, recent observations by our group have shown that lung HO activity is 4-fold higher in neonates compared with adults(41). This developmental regulation of HO-1 in the lung and liver may be due to increased heme levels in the neonatal tissues from a more rapid red cell turnover(40). The higher levels of HO activity and HO-1 protein in early neonatal life may explain the relative refractoriness to induction of lung HO-1 in the neonatal animals, because HO-1 expression is already at maximal levels. Lung HO-1 mRNA levels increase in adults rats in response to hyperoxia, although induction of lung HO-1 mRNA occurred late i.e. at 56-72 h of hyperoxia)(35). It may be that neonatal rats have even later hyperoxic induction of HO-1 than adult rats becasue they are relatively more tolerant to hyperoxia(1). In fact, several investigators have suggested that induction of HO-1 is a generalized response to oxidative stress(37) and may result from the sensing of oxidatively damaged proteins(8, 11). We have noted that oxygen-resistant cells that overexpress HO do not induce HO-1 mRNA after 72 h(42). Similarly, Applegate et al.(43) have shown that UVA-resistant dermal fibroblasts do not induce HO-1 mRNA as readily as more UVA-sensitive keratinocytes after exposure to UVA, a form of oxidative stress. Therefore, despite the increases in lung lipid peroxidation noted on d 2 of hyperoxic exposure, a known signal for HO-1 mRNA induction(44), neonatal rat lungs may not have incurred a sufficient threshold of oxidative damage to result in hyperoxic induction of lung HO-1 mRNA.

The overall response of neonatal lung HO-1 to hyperoxia differs from that of other classical antioxidants, because it occurs later (changes in activity beyond 48 h) and does not involve changes in mRNA levels at least by 72 h. Copper-zinc superoxide dismutase mRNA levels are seen to change in neonatal rats after 48 h of hyperoxic exposure(13). Catalase mRNA steady state levels also change in the neonatal rat lung in response to hyperoxia within 48 h of exposure(15). Nonetheless, as with other antioxidants(13), maturational differences in the regulation of HO-1 are observed.

Because HO-1 protein did not increase significantly from d 2 to 3 of hyperoxic exposure but total HO activity did, we wondered whether existing HO-1 protein could have been modified or activated in hyperoxia on d 3 in neonatal rat lungs. Therefore, the degree of activity of HO-1 protein in air and hyperoxia on d 3 was estimated by calculating the percentage values of HO-1-related activity obtained form immunoprecipitation with HO-1 antibodies(80% HO-1 in hyperoxia, 25% HO-1 in air) and estimating the relative activity of HO-1 protein using the following equation: [(total HO activity × percentage of HO-1 related activity)/HO-1 protein densitometry values]. From these calculations we speculate that on d 3 of exposure, HO-1 may be rendered more active in hyperoxia (1.223) than in air (0.483). The mechanism by which HO-1 may be activated in hyperoxia needs to be explored.

In summary, in newly born rats exposed to hyperoxia, we have shown increased lung HO-1 expression but, in contrast with adult models, have shown little to no differences in lung HO-1 mRNA levels. We speculate that neonatal lung HO-1 mRNA is regulated posttranscriptionally in hyperoxia.