1 Division of Pulmonary and Critical Care, Department of Medicine, Duke University Medical Center, Durham 27710; and 2 Environmental Protection Agency, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Heme oxygenase (HO)-1 is an oxygen-dependent enzyme that may regulate vascular tone and cell proliferation through the production of carbon monoxide (CO). We tested the hypothesis that HO-1 is upregulated in the lung in chronic hypoxia by exposing male Sprague-Dawley rats to 17,000 feet (395 Torr) for 0, 1, 3, 7, 14, or 21 days. After exposure, blood gases, carboxyhemoglobin (COHb) levels, and hematocrit were measured, and the lungs were either inflation fixed for immunohistochemistry or frozen for later measurement of HO enzyme activity, Western blot for HO-1 protein, and RT-PCR for HO-1 mRNA. The heart was excised and weighed, and the right-to-left heart weight ratio was determined. During hypoxia, the hematocrit increased progressively, reaching significantly higher values than the control value after 3 days. COHb levels increased above the control value after 1 day of hypoxia and increased progressively between 14 and 21 days, whereas arterial PO2 and arterial PCO2 did not vary significantly. HO-1 protein determined by Western blot increased for the first 7 days and declined thereafter; however, enzyme activity was elevated only after 1 day. Changes in HO-1 during hypoxia were localized by immunohistochemistry to inflammatory cells (early) and newly muscularized arterioles (later). Lung HO-1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was increased after 1 and 21 days. The data indicate that lung HO-1 protein and activity are upregulated only during early chronic hypoxia, whereas persistent COHb elevations indicate high endogenous CO production rates at nonpulmonary sites. If CO has antiproliferative properties, the lack of HO enzyme activity in the lung may be permissive for pulmonary vascular proliferation in hypoxia.
pulmonary hypertension; hypoxic pulmonary vasoconstriction; carbon monoxide; adaptation
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INTRODUCTION |
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CHRONIC HYPOXIA produces well-known adaptive changes in the lungs and pulmonary vasculature, heart, and other systems (26). Acute hypoxia reversibly constricts the pulmonary arteries and dilates systemic arteries, whereas chronic hypoxia leads to structural changes in the pulmonary arteries including smooth muscle proliferation and hypertrophy in arterial vessels that are not muscularized under normal circumstances (12, 26, 27, 30). Reversible pulmonary hypertension gradually becomes irreversible, accompanied by right heart hypertrophy and failure. These responses to chronic hypoxia occur clinically in disease states such as interstitial and chronic obstructive pulmonary disease and cyanotic heart diseases. In contrast to patients with hypoxic diseases, both sojourners and natives of high altitudes are "adapted" to hypoxia in ways that preserve exercise capacity (11). The molecular and cellular mechanisms and biological advantages of most of the adaptive and pathological responses are unknown. Molecular changes include upregulation of both mitogenic and antiproliferative mediators, and the ultimate vascular changes are likely determined by the balance between these responses (17).
Heme oxygenase (HO) is an enzyme that degrades heme to biliverdin, with the release of iron and carbon monoxide (CO) (29). The inducible isoform of this enzyme, HO-1, responds to hypoxia and may influence the vascular response through CO production (28). Like nitric oxide (NO), CO vasodilates by activating guanylyl cyclase to produce cGMP, resulting in smooth muscle relaxation (15, 16, 23, 32). In vascular smooth muscle cells, hypoxia increases HO-1 gene expression through activation of hypoxia-inducible factor-1 (HIF-1) (10). This specific nuclear activating factor increases gene transcription of several other hypoxia-responsive proteins including erythropoietin, vascular endothelial growth factor, NO synthase, and several glycolytic enzymes (7, 18, 20, 24). In contrast, HO-1 induction by lipopolysaccharide and NO donors is stimulated through nuclear binding activator protein-1 (1, 8). Control of the HO-1 gene by HIF-1 suggests that this enzyme may have a unique function in hypoxia other than responding to increased heme turnover.
Hypoxia increases HO-1 protein, activity, and mRNA and cGMP in cultured vascular smooth muscle cells (15) and increases HO-1 mRNA in rat lungs after short-term hypoxia (120 min) (10). The duration, extent, and distribution of the HO-1 response in lung cells and the pulmonary vasculature in hypoxia are not known. One role proposed for HO-1 in hypoxia is to oppose hypoxic vasoconstriction by endogenous CO production (10, 15). In cultured vascular smooth muscle cells, CO also decreases cellular proliferation (14). Thus HO-1 also could modulate the vascular proliferative response and development of pulmonary hypertension in chronic hypoxia.
We tested the hypothesis that HO-1 gene expression and protein are upregulated in the lungs of rats during chronic hypoxia. After rats were exposed to hypobaric hypoxia for 1, 3, 7, 14, or 21 days, HO-1 protein, mRNA, and activity were measured, and the distribution of HO-1 in the lung was localized by immunohistochemistry. These findings were correlated with physiological responses to hypoxia, including hematocrit, right-to-left heart weight ratio, arterial blood gases, and smooth muscle hypertrophy in pulmonary arterial vessels.
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METHODS |
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Materials. Unless indicated otherwise, all materials were obtained from Sigma (St. Louis, MO).
Hypoxic exposure in rats. Rats were exposed in an altitude chamber to 17,000 feet (395 Torr) for 1, 3, 7, 14, or 21 days (n = 8 animals/group). The protocol was approved by the Duke University (Durham, NC) Animal Care and Use Committee. The chamber was returned briefly to sea level once a day to clean the cages and replenish the food and water supply for the animals. At the end of the exposure, the animals were anesthetized with pentobarbital sodium (50 mg/kg ip), and an arterial catheter was placed to draw blood samples at the altitude to which the rats were exposed. Arterial blood gases, pH, carboxyhemoglobin (COHb), and hematocrit were measured with a calibrated blood gas analyzer (Instrumentation Laboratories model 1640) and CO-oximeter (Instrumentation Laboratories model 480).
The lungs were flushed with 0.9% NaCl through the right ventricle,
excised from the hilar structures, snap-frozen in liquid nitrogen, and
stored at 80°C. Frozen lungs were later used to quantitate
HO-1 protein by Western blot, HO activity, and HO-1 mRNA by RT-PCR. The
heart was excised, and the right ventricle-to-left ventricle plus
septum ratio was determined from the wet weights as a measure of right
heart hypertrophy. Lungs from several rats in each group were
simultaneously perfusion fixed at 100 cmH2O and inflation
fixed at 20 cmH2O pressure with 4% paraformaldehyde to
localize HO-1 by immunohistochemistry.
Western blot for HO-1. Tissue was homogenized on ice in cold
lysis buffer [150 mM NaCl, 50 mM Tris, pH 7.6, 1% SDS (Bio-Rad, Carpenteria, CA), 3% Nonidet P-40, 5 mM EDTA, 1 mM MgCl2,
2 mM 1,3-dichloroisocoumarin, 2 mM 1,10-phenanthroline, and 0.5 mM E-64]. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant was decanted, and an aliquot was stored at
20°C for the measurement of protein concentration. The
remaining supernatant was mixed with an equal volume of double-strength
Laemmli sample buffer [250 mM Tris · HCl
(Bio-Rad), pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and
2%
-mercaptoethanol], divided into aliquots, and stored at
80°C.
Electrophoresis was performed on 12% polyacrylamide gels under reducing conditions with a minigel system (Hoefer Scientific Instruments, San Francisco, CA). All lanes were loaded with 15 µg of protein, and electrophoresis was performed for 1.5 h under a constant current of 30 mA. The proteins were electrotransferred with a TE series Transphor unit at 100 V (Hoefer Scientific) to a polyvinylidene fluoride membrane (Millipore, Amersham Life Sciences, Cleveland, OH) and blocked overnight at 4°C in Tris-buffered saline (TBS) with 1% Tween 20 (TBS-T) containing 5% nonfat dry milk. The following day, the membranes were washed six times over 30 min in TBS-T at room temperature. Western blots were performed with a rabbit polyclonal antibody against rat HO-1 (StressGen, Vancouver, BC). Incubation with the primary antibody was performed for 1 h at room temperature at a dilution of 1:1,000 in TBS-T with 5% milk. After multiple washes in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson Laboratories) at a 1:15,000 dilution in TBS-T with 5% milk. The membranes were then washed in TBS-T, and the signal was detected on Biomax film (Eastman Kodak, Rochester, NY) with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). HO-1 was quantitated by comparison of signal strength intensity with a BioImage densitometer (BioImage, Ann Arbor, MI).
HO activity assay. HO activity was measured as previously
reported (3), modified from the method of Tenhunen et al. (29). Briefly, the lungs were homogenized on ice in one volume of 100 mM
phosphate buffer with 2 mM MgCl2. The homogenates were
sonicated and centrifuged. HO activity in the supernatant was
measured with 2 mg of liver cytosol (source of biliverdin
reductase), 20 mM hemin, 0.8 mM NADPH, 2 mM glucose 6-phosphate, and
0.0016 U/µl of glucose-6-phosphate dehydrogenase. An NADPH-free
reaction mixture was a background sample. Bilirubin was extracted with
chloroform and quantitated spectrophotometrically based on the change
in optical density at 464 nm minus that at 530 nm, with an extinction coefficient of 40 mM1 · cm
1.
Liver cytosol was prepared by hand-douncing rat liver in four volumes of 100 mM phosphate buffer with 2 mM MgCl2 and centrifuging the homogenate at 105,000 g for 27 min at 4°C. The supernatant (cytosol), a rich source of biliverdin reductase, was used in the HO enzyme activity assay.
Immunohistochemistry. Fixed lungs were removed from paraformaldehyde after 24 h and placed in 70% ethanol until embedded in paraffin. Sections of 10 µm thickness were cut for light microscopy. Before being labeled, the tissue sections were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The sections were blocked in a solution of 5% nonfat dry milk, 1% BSA, 5% goat serum in 0.01 M PBS, and 0.1% Triton X-100 before incubation overnight at 4°C with a monoclonal antibody to HO-1 (StressGen) in 1% milk and 1% BSA in 0.01 M PBS and 0.1% Triton X-100 (1:200 dilution). The sections were washed three times with PBS with 0.1% Triton X-100 for 5 min each and incubated for 1 h with secondary antibody (biotinylated goat anti-mouse IgG; Jackson Laboratories) at a dilution of 1:1,000 in 1% milk, 0.01 M PBS, and 0.1% Triton X-100 at room temperature. The signal was detected with peroxidase-conjugated avidin and diaminobenzidine. The slides were counterstained with 1% hematoxylin. For negative controls, sections were processed as above except that the primary incubation was performed with nonimmune rabbit serum (Jackson Laboratories) instead of primary antibodies.
RT-PCR. Lung tissue was homogenized (1 g/5 ml) with 4 M guanidine thiocyanate (Boehringer Mannheim, Indianapolis, IN), 50 mM sodium citrate, 0.5% sarkosyl, and 0.01 M dithiothreitol. RNA was pelleted by ultracentrifugation through cesium chloride (Boehringer Mannheim) and 0.1 M EDTA. One hundred nanograms of total RNA were reverse transcribed (Moloney murine leukemia virus reverse transcriptase, Life Technologies), and the resultant cDNA was amplified for 27 and 37 cycles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and HO-1, respectively, in separate reactions with gene-specific primers. Oligonucleotide sequences were synthesized with an Applied Biosystems 391 DNA synthesizer (Foster City, CA) based on sequences published in the GenBank DNA database. The sense and antisense sequences were 5'-CCATGGAGAAGGCTGGGG-3' and 5'-CAAATTGTCATGGATGACC-3', respectively, for GAPDH and 5'-ATTGGAGGCTGGAGCTATTCTG-3' and 5'-CCTTCGGTGCAGCTCCTCAG-5', respectively, for HO. Amplification products were separated on 2% denaturing agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. The resulting negative (type 55 film, Polaroid, Cambridge, MA) was quantitated with a BioImage (Ann Arbor, MI) densitometer. For each experimental condition, the integrated optical density of the HO DNA band was divided by that of the GAPDH DNA band (as a reference gene) to correct for variation in the amount of amplifiable cDNA in each sample.
Statistical analysis. Experimental data from each group are
expressed as means ± SE. Statistical analyses were performed with two-way analysis of variance (factorial design) with a post hoc comparison test (Fisher's exact test) with commercially available software (Statview 4.0, Calabasas, CA). A P value of 0.05 was accepted as significant.
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RESULTS |
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Physiological effects of chronic hypoxia in rats. After rats
were exposed to an altitude of 17,000 feet (395 Torr) for
0, 1, 3, 7, 14, or 21 days, hematocrit and COHb were determined by arterial blood gas analysis (Fig. 1). The
hematocrit increased progressively after exposure to hypoxia (Fig.
1A) and reached significance over control values after 3 days
(P 0.05). Hematocrit also continued to increase at the later
time points, reaching a plateau at 60% by 21 days.
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COHb levels (expressed as percent of total hemoglobin) in arterial
blood were measured as a marker of endogenous CO production. With the
assumption of a constant PO2 and
ventilation, COHb provides an indirect assessment of heme turnover. As
shown in Fig. 1B, COHb levels were highest after 1 and 21 days
of hypoxia. The increases in COHb levels were significant after 1, 7, 14, and 21 days of hypoxia (P 0.05). Arterial blood gas
measurements indicated that the arterial
PO2
(PaO2) and arterial
PCO2 (PaCO2) both declined at altitude
(normal values for the rat are PaO2 of
90 Torr and PaCO2 of 40 Torr) but did
not vary significantly over the 21 days of the experiments
except that PaCO2 normalized after 21 days of hypoxia (Table 1). Thus the
elevated COHb could not be attributed to hypoxia or differences in
ventilation.
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Figure 2 shows the right-to-left heart
weight ratios of each group expressed as means ± SD compared with the
control values. After 7 days of hypoxia, the right-to-left heart weight
ratios were significantly increased compared with the control value
(P 0.05). Right-to-left heart weight ratios increased
further after 14 and 21 days. These data indicate that progressive
right ventricular hypertrophy developed over time after the exposure to
altitude in these studies.
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HO-1 expression in the lung after hypoxia. HO-1 protein was
quantitated in the lungs of control and hypoxia-exposed rats by Western
blot analysis and by measurement of HO-1 activity. Figure 3 shows a Western blot for
HO-1 in the lungs of a control rat and a representative experimental
animal exposed to 1, 3, 7, 14, or 21 days of hypoxia. Rat spleen
homogenate was used as a positive control for the Western blots. The
blot demonstrates that HO-1, expressed constitutively at low levels in
control rat lungs, increases after 1, 3, and 7 days. After 14 and 21 days, however, HO-1 expression had declined toward the control values.
The changes in HO-1 in the lung after hypoxia were quantitated by
densitometry to compare the relative intensity of the bands (Fig.
4). The data in Fig. 4 are expressed as a
ratio of the mean intensity for four animals from each group normalized
to the lowest control value. After 1 day of hypoxia, HO-1 in the lung
was increased twofold over the control value (P 0.05). After
3 and 7 days, HO-1 was lower than in the 1-day exposed group but
remained elevated over the control value. After 14 and 21 days,
however, HO-1 protein was not significantly increased over the control
values, although it did remain elevated in a few individual animals.
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HO enzyme activity was measured in the lungs from control rats and from
rats after 1, 3, 7, 14, or 21 days of hypoxia (n = 6/group).
Spleen tissue from a control animal was used as a positive control for
the HO activity assay. Figure 5 shows HO
activity expressed as picomoles of bilirubin formed per milligram of
protein per hour. After 1 day of hypoxia, HO activity in the lung
increased significantly compared with the control value (P 0.05). After 3, 7, 14, and 21 days of hypoxia, HO activity in the lung
was not significantly different than the control value despite the presence of more immunoreactive protein on days 3 and
7.
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HO-1 distribution in the lung after hypoxia was localized by
immunohistochemistry (Fig. 6). Figure 6
demonstrates light microscopy immunohistochemistry at low and high
power for control lungs and lungs after 1 and 21 days of hypoxia. In
control rat lungs, HO-1 staining was primarily detected in alveolar
macrophages (Fig. 6, A and D). This contrasts with the
more uniform staining pattern in inflammatory cells and the alveolar
region of the lung after 1 day of hypoxia (Fig. 6, B and
E). After 1 day of hypoxia, increased numbers of inflammatory
cells were present in the lung and the macrophages stained intensely
for HO-1. Although diffuse staining for HO-1 was present in the
alveolar region, minimal staining for HO-1 was found in pulmonary
vessels (data not shown). After 3 days of hypoxia, HO-1 staining was
still present in the epithelium as well as in the macrophages, and
after 7 days of hypoxia, HO-1 was distributed similarly in the
bronchial and alveolar epithelial regions and in inflammatory cells
(data not shown). After 14 and 21 days, the small pulmonary arteries
began to show smooth muscle hypertrophy in the vascular wall. By 21 days, HO-1 staining was present in the thickened smooth muscle layer in
many small pulmonary arteries (Fig. 6, C and F). The
alveolar regions of the lung in rats after 21 days of hypoxia had
minimal staining, similar to that in control lungs, whereas macrophages
maintained heavy staining for HO-1.
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HO-1 mRNA content of the lung after hypoxia was quantitated by RT-PCR.
Figure 7 shows the mRNA data normalized to
the GAPDH content of lungs from each group. GAPDH mRNA content remained stable across the experiment with respect to time. Like HO-1 protein, HO-1 mRNA was present constitutively in control rat lungs. After 1 day
of hypoxia, HO-1 mRNA increased threefold over the control value
(P 0.05). After 3, 7, and 14 days, however, HO-1 mRNA was
similar to the control value. After 21 days, HO-1 mRNA again increased
significantly above the control value, indicating the onset of a second
response in HO-1 gene expression after hypoxia (P
0.05).
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DISCUSSION |
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Over the first 3 wk of chronic hypoxia, the young adult rat develops polycythemia, right ventricular hypertrophy, and pulmonary vascular remodeling. In our study of HO-1 expression in the lung during this period, we found that HO-1 protein, mRNA, and activity were increased significantly in the first 24 h of hypoxia. HO-1 protein remained elevated for 3-7 days. Over the next 2 wk, however, HO-1 protein levels decreased and HO activity in the lung remained flat. After 14 and 21 days of hypoxia, HO-1 protein and total HO activity were quantitatively similar to the control value, but the enzyme distribution had shifted to the hypertrophied small arteries. Meanwhile, COHb levels increased on day 1, stabilized, and then increased further by 21 days of hypoxia. This pattern of response at later time points indicates greater endogenous CO production by HO activity in other tissues of the body.
We studied HO-1 expression during chronic hypoxia because the enzyme
may have roles in both acute and chronic responses of the lung to
hypoxia. First, the activation of HO-1 through HIF-1 (10) suggests that
the enzyme is part of the specific coordinated response to hypoxia in
the lung. HIF-1 controls the expression of a number of proteins
that seem to be important in the response to hypoxia, and mice
deficient in HIF-1- have delayed polycythemia, right ventricular
hypertrophy, and pulmonary vascular remodeling after hypoxic exposure
(34). Second, HO is the source of endogenous CO, which has vasorelaxant
properties and may inhibit vascular smooth muscle cell proliferation.
Finally, NO, another major vasorelaxant molecule that signals through
guanylyl cyclase (GC), has an affinity for GC many times higher than
that of CO; however, in hypoxia, NO may not be produced as readily
because of reduced activity of NO synthase (9, 21, 33).
Because HO requires molecular oxygen, its Michaelis-Menten constant for oxygen is an important predictor of the behavior of the enzyme during hypoxia. Because the enzyme has a Michaelis-Menten constant for oxygen of only 8 Torr (4), this could provide a role for endogenous CO that otherwise might overlap in vascular signaling function with NO through GC.
Data supporting a direct role of CO in regulating vascular tone are limited, and little is known about its role in the pulmonary vasculature. In adult dogs, ventilation with CO inhibits hypoxic pulmonary vasoconstriction (HPV) (13); however, low concentrations of CO do not prevent HPV in isolated perfused rat lungs (2). Less is known about the role of CO in modulating the response to chronic hypoxia. A possible role for CO is suggested by a study (19) of neonatal rats showing that exposure to low inspired CO prevents development of HPV. In vitro data also suggest that CO has antiproliferative effects in vascular smooth muscle (14).
During severe alveolar hypoxia, HO-1 protein, activity, and mRNA in the rat lung increase acutely within 24 h, but the responses are not maintained over the next few weeks as pulmonary vascular hypertrophy and right heart enlargement develop. A second increase in HO-1 mRNA occurs after 21 days of hypoxia that appears to herald a second induction of HO-1 after substantial pulmonary hypertension has developed. The early peak in HO-1 correlates with an increased number of macrophages that stain very intensely for HO-1, and the early rise in HO activity may reflect this inflammatory response. The presence of HO-1 in the vasculature at the later time points suggests a compensatory response to the cardiovascular effects of hypoxia. This very interesting observation raises the possibility that the enzyme has different functions in different cell types in the lung.
An important reason for HO induction in hypoxia could be related to increased red blood cell turnover because the heme substrate induces the enzyme (5). However, in the lung, the HO-1 increase preceded the onset of erythrocytosis, and this response was not maintained as the hemoglobin concentration increased. This observation provides further indirect evidence that HO-1 induction in the lung is a specific response to hypoxia. The progressive increase in COHb over 21 days of hypoxia could reflect increased HO activity in response to increased heme turnover at sites other than the lung.
Although activation of HO-1 through HIF-1 suggests that the response is specific to hypoxia, the HO-1 increase in hypoxia could also represent an oxidant stress response if hypoxia results in reactive oxygen species production. This effect has been reported in vitro in some studies (25), whereas others (31) have shown decreased reactive oxygen species production with decreasing oxygen concentrations. After 3 days of hypoxia in rats, however, antioxidant enzymes including superoxide dismutase, catalase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase increase in the lung (6, 25). In addition, HO-1 has potential antioxidant properties including catabolism of free heme, altered iron handling, and production of antioxidant substances including biliverdin and CO (5, 22). A more complete understanding of the factors influencing gene transcription for HO-1 in chronic hypoxia may help delineate the function of the enzyme at different times.
HO enzyme activity in the lung increases in accordance with the immunoreactive protein after 1 day of hypoxia. However, HO-1 activity is similar to the control value at all later time points despite persistently increased immunoreactive protein at 3 and 7 days. Because the activity assay is performed ex vivo, it provides optimal conditions and substrates for the reaction, indicating that the enzyme in the lung may be inactivated in vivo during hypoxia. Several interesting explanations for this finding can be proposed, but the mechanism and its significance will require further study. One possibility is that the enzyme is modified in a redox-sensitive manner during hypoxia. Also, posttranslational modifications of new HO-1 enzyme produced during hypoxia or decreased degradation of inactive enzyme could result in an increase in nonfunctional enzyme. Finally, CO itself could inactivate the enzyme as has been demonstrated in vitro (29). These considerations raise the question of whether maintaining HO-1 enzyme activity could prevent or modify pulmonary vascular and right heart remodeling. This possibly could be further investigated by determining the duration of the HIF-1 response and other factors that regulate HO-1 production and degradation.
In summary, HO-1 mRNA and protein are induced by acute hypoxia in the rat lung in vivo. The immunoreactive protein remains elevated for at least 1 wk, but it is likely enzymatically inactive. HO-1 mRNA increases again after 3 wk of hypoxia, which may precede another increase in protein. These HO-related events correlate with compensatory changes in the cardiopulmonary system. Thus regulation of HO-1 expression may be controlled specifically in hypoxia to influence adaptation through its substrate or one or more of its products.
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ACKNOWLEDGEMENTS |
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Funding for this project was provided by National Heart, Lung, and Blood Institute Grant P01-HL-42444-09.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. S. Carraway, Division of Pulmonary and Critical Care, PO Box 3221, Duke Univ. Medical Center, Durham, NC 27710.
Received 18 May 1999; accepted in final form 26 October 1999.
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