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Bronchopulmonary dysplasia (BPD) is the chronic inflammatory lung disease associated with prematurity and is a major cause of morbidity for mechanically ventilated premature infants(1). Although the pathogenesis of BPD remains unclear, exposure of immature lungs to prolonged periods of high inspired oxygen is widely accepted as an important contributor to the development of BPD through both the effects of oxygen toxicity and the promotion of inflammation(1,2). Recent reports suggest a role for NO produced by activation of the various isoforms of NOS in mediating the inflammatory response of the lungs to hyperoxia and implicate a possible role for NO in the development or prevention of BPD(3–8).

NO is an important component of cellular communication in a wide range of biological systems including regulation of the pulmonary circulation, smooth muscle contraction, and host defense/inflammatory mechanisms(9). NO is produced from arginine and oxygen by constitutive [brain (b) and endothelial or epithelial (e) NOS; type I and III] and inducible iNOS (type II), with its major physiologic effect mediated via the production of cGMP after guanylyl cyclase stimulation(9). All three isoforms of NOS seem to be located within human, rat, and ovine lung including the pulmonary epithelium and nerves (type I, bNOS), pulmonary epithelium and macrophages (type II, iNOS), and pulmonary endothelium (type III, eNOS)(10,11). Although there are no existing pulmonary data describing levels of bNOS and iNOS protein after oxygen exposure, hyperoxic exposure is accompanied by elevations in pulmonary neutrophils and macrophages, which are sources for NO(1,10). Hyperoxic exposure of ovine fetal pulmonary artery endothelial cells for 2 d has been shown to elevate pulmonary vascular eNOS protein and mRNA levels(12). Nonetheless, the role of NO produced by these enzymes in mediating chronic hyperoxic damage within the lung is still unclear.

The purpose of the present study, therefore, was to investigate the role of chronic hyperoxic exposure on levels of the different NOS isozymes within the lungs of 21-d-old rats. We also sought to investigate the effects of hyperoxia on NO activity by measuring cGMP levels in the presence and absence of selective NOS blockade. We hypothesized that hyperoxic exposure would increase NO production and that blockade of endogenous NO would further aggravate hyperoxic lung injury. We, therefore, additionally examined whether selective NOS inhibition altered the pathologic response of immature rat lungs to prolonged hyperoxic exposure.

METHODS

Animal preparation. The present study used 21-d-old Sprague-Dawley rat pups exposed to hyperoxic or normoxic conditions for 8 d as approved by the Institutional Review Board of the Animal Resource Center. Rat pups from multiple litters were arbitrarily assigned to hyperoxic or normoxic groups. The hyperoxic groups were placed in a Plexiglas chamber (38 L) with continuous flow of oxygen (∼ 12 L/min), and oxygen concentration (>95%) in the chamber was monitored 4 times daily with a Minion III (Echidyne) oxygen monitor. The normoxic pups were housed in an open cage. Both groups were given rat chow and water ad libitum, and weights were obtained initially and when the rats were killed. A total of 34 rats were arbitrarily assigned to six groups of five to six pups as follows: 1) normoxia/normal saline; 2) normoxia/NOS inhibition with AG; 3) normoxia/NOS inhibition with L-NNA; 4) hyperoxia/normal saline; 5) hyperoxia/NOS inhibition with AG; and 6) hyperoxia/NOS inhibition with L-NNA. Twice a day for 8 d the rats were injected intraperitoneally with either 200 mg/kg of AG(13), 75 mg/kg of L-NNA(14), or normal saline in the same volume (0.5 mL). No mortality was noted over the 8 d in any group. After 8 d of exposure, the animals were killed by an overdose of sodium pentobarbital (90 mg/kg intraperitoneally); their chests were opened and the lungs were removed, immediately frozen in liquid nitrogen, and stored at -80°C for protein isolation. In an additional series of control experiments, eight 21-d-old rat pups were exposed to normoxia in an open cage and eight others were exposed to normoxia in the Plexiglas chamber (12 L/min flow but in the absence of hyperoxia).

Western blot analysis for NOS protein. Frozen lung tissues were obtained from four normoxia/normal saline and four hyperoxia/normal saline rats. Tissues were homogenized in 50 mM Tris(hydroxymethyl)aminomethane (Tris)-HCl buffer with 0.1 mM ethylenediaminetetraacetate (EDTA) and 0.1 mM ethyleneglycol-bis(β-aminoethyl-ether)-N,N′-tetraacetic acid (EGTA) at a pH of 7.4 plus the following protease inhibitors (in mM): 1 pepstatin, 0.1 leupeptin, 1 aprotinin, and 1 phenymethyl-sulfonyl fluoride. Homogenates were centrifuged at 14 000 × g at 4°C for 30 min. Protein content in the supernatant was determined by Bio-Rad (Bradford) method. Two hundred micrograms of protein from the supernatant were electrophoresed in 7% sodium dodecyl sulfate (SDS)-polyacrylamide gel and electro-transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) (20 mM Tris, pH 7.6, and 150 mM NaCl) and immunoblotted with primary antibody (1:2000 of either bNOS, iNOS, or eNOS from Transduction Laboratories, Lexington, KY) for 3 h.

For normalizing protein loading, the expression level of smooth muscle β-actin was also examined by Western blot analysis using an MAB from Sigma Chemical Co. (St. Louis, MO)(15). The membrane was then washed 3 times with TBS plus 1% Tween 20 and incubated with either 1:3000 goat anti-rabbit IgG or goat anti-mouse IgG labeled with peroxidase in TBS plus 5% skim milk for 1 h. After the final wash, the immunoreactive bands were detected by enhanced chemiluminescence (Amersham Corp.), and the signals were quantitated by a densitometric scanner (USB Specialty Biochemicals). Multiple exposures were performed to obtain optimal conditions and the most reliable images for both iNOS and eNOS expression.

RIA for cGMP analysis. Frozen lung tissues were taken from four animals in each of the six groups as follows: normoxia/normal saline, normoxia/AG, normoxia/L-NNA, hyperoxia/normal saline, hyperoxia/AG, and hyperoxia/L-NNA-exposed rat pups. One volume of tissue was mixed with 10 volumes of 1.07 N perchloric acid, homogenized, and centrifuged at 14 000 × g for 10 min, and the supernatant was collected for cGMP assay. cGMP assays were performed in duplicate with a commercially available kit (Immunotech, Westbrook, ME) by use of a rabbit polyclonal antibody.

Lung weights. After the rats were killed, the left upper lobe of lung was removed, dried with paper towels, and weighed. The lung weight (left upper lobe) versus body weight ratio was then calculated in 30 rat pups.

Lung histology. After the rats were killed, 12 rat lungs (two from each of the six groups) were inflated with 10% formalin with 1% glutaraldehyde at a pressure of 20 cm H2O for 15 min. The lungs were then stored for 3 d in formalin. Fixed lungs were embedded in paraffin, and 6-µm sections were placed on microscopic glass slides and stained with hematoxylin and eosin. All lung slides were reviewed by a pathologist blinded to the original treatment. For electron microscopy, formalin-fixed lungs were further processed according to a routine protocol including additional fixation in 0.1 M cocodylate-buffered 3.75% glutaraldehyde and postfixation in cocodylate-buffered 1% osmium tetroxide. Tissues were dehydrated in graded alcohols and embedded in Spear epoxy resin. Sections were cut at a thickness of 60 to 80 nm, stained in uranyl acetate and lead citrate, and examined by use of a Philips 400T electron microscope.

Analysis of data. Data are presented as mean ± SEM unless otherwise stated. Comparison of iNOS and eNOS expression between normoxic and hyperoxic lung tissues (both n = 4) was normalized to the expression of smooth muscle β-actin, and statistical analysis was performed via unpaired t test. cGMP content in each normoxic, hyperoxic, and NOS-blockaded group was averaged from four animals and expressed in pmol/mg protein. Statistical analysis of cGMP content between normoxic and hyperoxic groups in the absence of blockade was by unpaired t test; comparison of the effect of NOS blockers during normoxia and hyperoxia was by 1-way ANOVA with post hoc comparison by use of Newman Keuls. Body weight, lung weight (left upper lobe), and lung weight/body weight ratios between the entire normoxia- and hyperoxia-exposed groups were analyzed by unpaired t test. A p value <0.05 was considered statistically significant.

RESULTS

iNOS and eNOS expression. Western blot analysis for iNOS and eNOS revealed a major band with a molecular weight of 130-140 kD in rat lung parenchyma. To control for the variations in protein loading, an identical set of gels was prepared and tested for the expression of smooth muscle β-actin by Western blot analysis (Fig. 1). After 8 d of hyperoxic exposure, densitometric scanning showed a 5-fold and 2-fold increase in iNOS/β-actin and eNOS/β-actin, respectively. The ratio of iNOS/β-actin during normoxia and hyperoxia was 0.07 ± 0.02 and 0.36 ± 0.06, respectively, p < 0.01. The ratio of eNOS/β-actin during normoxia and hyperoxia was 0.15 ± 0.04 and 0.34 ± 0.06, respectively, p < 0.05. Under normoxic conditions, the presence or absence of flow (as used during hyperoxic exposure) did not affect ratios of either iNOS/β-actin or eNOS/β-actin. bNOS was not detected in either normoxia- or hyperoxia-exposed rat lungs.

Figure 1
figure 1

Western blot analysis of iNOS and eNOS protein in four normoxic lungs (lanes 1-4) and four hyperoxic lungs (lanes 5-8). An identical set of gels was used to detect β-actin protein for normalization. There was a striking (5-fold) increase in iNOS/β-actin ratio and a more modest (2-fold) increase in eNOS/β-actin ratio in response to hyperoxic exposure.

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cGMP measurements. RIA showed a significant increase in cGMP levels in the lungs of hyperoxia-exposed versus normoxia-exposed rats (287 ± 15 versus 106 ± 9 pmol/mg protein, p < 0.001) (Fig. 2). There was a significant effect of NOS blockade with either AG or L-NNA on cGMP levels in the hyperoxic rats. The inhibitory effect on cGMP production was greater in the animals treated with AG (287 ± 15 versus 83 ± 9 pmol/mg protein, p < 0.001) than in those treated with L-NNA (287 ± 15 versus 172 ± 27 pmol/mg protein, p < 0.01). In contrast, NOS blockade by AG or L-NNA did not significantly affect cGMP levels in the normoxic rat pups (p < 0.05 in both groups).

Figure 2
figure 2

cGMP levels in lung homogenates from normoxia-exposed animals and from hyperoxia-exposed animals in the absence and presence of NOS blockade with L-NNA or AG. The increase in cGMP levels in the hyperoxic animals was attenuated by blockade with L-NNA or AG. These blockers had no effect on cGMP levels in the normoxic rat pups. Each column represents mean data from five or six animals.

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Lung/body weight ratios. Because body and lung weights were not affected by NOS blockade during normoxia or hyperoxia (Table 1), data were combined for all normoxic and all hyperoxic rat pups. Initial mean body weights were comparable between groups. Body weights were significantly decreased in the hyperoxia-exposed group versus the normoxic controls (p < 0.001, Table 1). Lung weights (left upper lobe) were significantly elevated in the hyperoxic lungs compared with the normoxic controls (p < 0.05, Table 1). Consistent with the above results, the lung weight/body weight ratios were significantly elevated in the hyperoxic group versus the normoxic controls (p < 0.05, Table 1).

Table 1 Comparison of body weight, lung weight, and lung weight/body weight ratio between groups
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Light and electron microscopy. Lungs from rats treated with hyperoxia showed interstitial and alveolar edema and areas of interstitial fibrin deposits. These changes were patchy but involved the majority of the lung (Fig. 3, low-power light microscopy). Ultrastructural analysis of these lungs confirmed the presence of alveolar edema but did not identify definite epithelial or endothelial injury. Areas of edema contained a proteinaceous-like material of medium electron density along with inflammatory cells including plasma cells, neutrophils, and macrophages. Interstitial fibroblasts extending long tapering and thread-like processes were also identified. In addition, acute inflammatory cells were observed in the peri-bronchiolar spaces and within alveolar spaces. The airways, including the smooth muscle and bronchiolar epithelium, and the pulmonary vasculature showed no significant pathology based on microscopic changes. There were no qualitative differences noted by either light or electron microscopy between the hyperoxic animal groups treated with and without the NOS inhibitors. Normoxic animals treated with and without the NOS inhibitors showed no significant pulmonary pathology by light microscopy.

Figure 3
figure 3

A representative hyperoxia-exposed lung showing acute lung injury with intra-alveolar fibrin and scattered acute inflammation surround a bronchiole (B). The bronchiolar epithelium is unremarkable. (Hematoxylin and eosin, ×200).

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DISCUSSION

Exposure to high inspired oxygen contributes to the development of BPD via both free radical effects on endothelial and epithelial cell barriers that lead to pulmonary edema and triggering mechanisms that lead to activation and accumulation of inflammatory cells, as confirmed in this study(1). NO has been implicated as an important component of oxygen-induced pulmonary changes because it can act as an antioxidant by combining with the oxygen radical superoxide, can promote oxygen membrane damage through the formation of peroxynitrite, and can propagate the inflammatory response of the lung(16,17). Our data suggest that prolonged hyperoxic exposure does elevate both NOS levels and NO activity within the immature rat lung. Nonetheless, blockade of excess NO production neither promoted nor attenuated the pulmonary parenchymal damage induced by hyperoxia.

There is considerable evidence for high levels of NOS during the perinatal period, and this is consistent with a significant role for NO in regulation of pulmonary vascular resistance during early postnatal life in multiple mammalian species(18–22). Endothelial NOS protein peaks at term gestation in rat lung with a slow decline during the first weeks of postnatal life culminating in very low levels in adult tissue(19,22). A similar maturational pattern has been observed in guanylyl cyclase expression in pulmonary vascular and bronchial smooth muscle as well as in alveolar epithelial cells of neonatal versus adult rats(23). Previous data in maturing rats have not shown a dramatic increase in iNOS immunoreactivity at term, although iNOS distribution did become cytoplasmically diffuse postnatally(21). In the current study, we chose to study rat pups whose hyperoxic exposure began at 21 d to avoid the marked changes in eNOS that normally occur during the immediate perinatal period. Our inability to detect bNOS in rat lung at 28 d is in contrast with prior studies in which immunoreactive bNOS was observed before and within 7 d of birth(19,21). This may reflect suppression of bNOS expression during later postnatal life. Our various findings, therefore, may not be applicable to normoxia- or hyperoxia-exposed neonatal rat pups, and future studies should use such animals before weaning.

We found both iNOS and eNOS were present in lung parenchyma of rat pups at 21-29 d of age, with a significant elevation produced by hyperoxia. Increased eNOS within the lung during oxygen exposure is consistent with the data of North et al.(12) showing a 2.7-fold increase in eNOS protein in pulmonary endothelial cells after 48 h of hyperoxic exposure. This was not confirmed in hyperoxia-exposed rats who showed a decrease in eNOS accompanied by a small increase of NO in bronchoalveolar lavage fluid(24). Elevation of iNOS suggests an inflammatory process produced by hyperoxic toxicity and mediated by alveolar macrophages or neutrophils; however, the current data provide no evidence for the source of the increased NOS protein. Although an increase in iNOS has not been previously reported under hyperoxic conditions, this is consistent with the expression of iNOS protein in macrophages and rat lung after endotoxin stimulation(25,26). Although the present study is unable to elucidate the mechanism whereby NOS elevation occurs, the increase in both enzymes within the lung parenchyma in response to hyperoxic conditions suggests that NO may be involved in the response of the lung to excessive oxygen exposure. Future studies will need to address the effect of NO on cytokine production and the interactions between NO and reactive oxygen species in the hyperoxia exposure rat pup model.

Because NO stimulates guanylyl cyclase to produce cGMP, measurement of cGMP levels should be an indicator of NO activity(9). Our finding of elevated cGMP levels within the lungs exposed to hyperoxia suggests increased NO production from elevated NOS isozymes. The attenuation of these levels by selective blockade of either iNOS or eNOS suggests that both enzymes contribute to this increased NO production. We acknowledge that cGMP is only an indirect marker for NO activity. Preliminary data from our laboratory, however, indicate no evidence for an alteration of phosphodiesterase (degrading enzyme) activity in cultured human lung epithelial cells exposed to hyperoxia. Nonetheless, future studies might consider measurement of nitrate and nitrite as alternative markers of NO activity.

We sought to investigate the role played by these NOS isozymes in mediating the pulmonary responses of rats to oxygen. Both a protective role and a pathologic role have been implicated for NO within the lung exposed to hyperoxia. Elevated nitrotyrosine levels in rat lungs exposed to hyperoxia suggest that toxic levels of the oxidant peroxynitrite are formed from NO under these conditions(3). Also, exposure of hyperoxic lungs to the NO precursor L-arginine or to inhaled NO promotes pulmonary lung damage, surfactant inactivation, and pulmonary inflammation(6,27,28) as well as cytotoxicity in cultured alveolar epithelial and endothelial cells(29). In contrast, blockade of NO production with L-NAME has been shown to aggravate pulmonary oxygen toxicity and/or mortality in newborn(5) and adult rats(30,31). Further, low-dose inhaled NO attenuated oxygen-mediated lung endothelial injury and decreased pulmonary edema and neutrophil accumulation(4,8,32). Adult rats exposed to high-dose inhaled NO exhibited improved survival after high oxygen exposure, although the mechanism for the protective effect of NO was unclear(33). To investigate this controversy in the immature rat, we performed pathologic examination on young rat lungs exposed to chronic hyperoxia during selective inhibition of the NOS isozymes. We hypothesized that blockade of endogenous NO production would aggravate hyperoxic lung injury in rat pups.

The hyperoxia-exposed rat pups weighed significantly less than the normoxic rats, which is consistent with the data of Hershenson et al.(34). Nonetheless, the hyperoxic lungs weighed significantly more than their normoxic controls, and there was no effect of NOS blockade on body or lung weights in rat pups exposed either to normoxia or to hyperoxia. The failure of blockade to influence lung weight changes suggests that iNOS and eNOS did not affect the alterations in lung fluid or protein flux induced by hyperoxia, although more subtle changes may have been seen if more animals had been studied and endothelial permeability or alveolar fluid clearance had actually been measured. We also did not observe any increase in mortality as a result of NOS blockade. These results are in contrast to a previous study that used NG-nitro-L-arginine-methyl ester (L-NAME) administered to maternal rats during late gestation and early lactation(5). Subsequent hyperoxic exposure of the rat pups was associated with increased mortality in the L-NAME-treated animals(5). Apart from obvious differences in timing and route of L-NAME administration, our results may differ from this earlier study because of the selective nature of L-NNA and AG compared with L-NAME. Because NO is a major component of many physiologic systems, nonselective blockade may be expected to have a greater impact on a stressed immature animal than would selective blockade. Alternatively, these findings may indicate that a protective role for NO is more critical during earlier stages of development, as in the first days of postnatal life before weaning.

Histologic examination of the lungs showed consistent acute lung injury due to oxygen exposure when compared with normoxic controls. Ultrastructural analysis showed interstitial fibrin and platelet aggregates within the interstitial capillaries, suggestive of early endothelial injury as has been previously reported(4,8,30). Inhaled NO has been shown to prevent microvascular injury, endothelial dysfunction, and pulmonary neutrophil accumulation after ischemia and reperfusion(4,35–37). In the current study, blockade of endogenous NO formed from eNOS and iNOS did not seem to change hyperoxic damage within the immature lung parenchyma and alveolar structures. Before these findings can be fully accepted, however, more subtle histologic changes need to be quantified in future rigorous morphometric analyses of structural changes in the developing lung.

The present study shows that there is a marked up-regulation of iNOS, a modest up-regulation of eNOS, and an increased level of NO activity as measured by cGMP within the young rat lung exposed to chronic hyperoxia. We speculate that iNOS elevations are partly caused by neutrophil and alveolar macrophage migration into the lung due to oxygen-induced pulmonary inflammation whereas eNOS up-regulation is because of an oxygen-induced overexpression of eNOS within vascular endothelium and airway epithelium. Blockade of NO production by either isozyme, however, does not aggravate oxygen-induced pulmonary damage at this stage of development in our qualitative pathologic studies. Characterization of the mechanisms whereby hyperoxia induces up-regulation of NOS will require identification of the source of NO, the effect of NO on the lung inflammatory response, and the interaction between NO and reactive oxygen species. The biologic consequences of the excess NO activity remain to be determined.