INTRODUCTION

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NITRIC OXIDE (NO) is an inorganic, highly reactive, and rapidly diffusible gas that was discovered to be the endothelium-derived relaxation factor (13, 18). Endogenous NO is produced by many cell types and is important in regulating normal basal vasomotor tone. In the lung, NO produced by the pulmonary endothelium is responsible for the fall of pulmonary vascular resistance soon after the birth of the newborn infant (1). High vascular pressure persists when this response fails to occur, resulting in persistent pulmonary hypertension of the newborn. Exogenously administered NO is also a potent vasodilator (5, 7, 12, 16, 20, 22). Its mechanism of action is primarily through the activation of guanylate cyclase, which increases the production of guanosine 3',5'-cyclic monophosphate and results in smooth muscle relaxation (24). A dynamic cycle exists in which hemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated, and the NO group is released during arterial-venous transit. Changes in oxygen (O₂) tension might also regulate NO delivery because NO release is facilitated by deoxygenation (14).

Various early reports (16, 20) showed that inhalation of NO together with high inspired concentrations of O₂ significantly improved oxygenation in infants with persistent pulmonary hypertension of the newborn and in some cases reduced the need for extracorporeal membrane oxygenation. NO has also been reported to improve oxygenation in premature infants with respiratory distress syndrome and in full-term infants with congenital heart disease and diaphragmatic hernia (5, 12). Clinical use of NO in the United States is currently limited to experimental protocols for treating extremely sick patients with primary diseases of the lungs or heart. These patients are invariably receiving high concentrations of O₂ (hyperoxia) to maintain adequate oxygenation. Hyperoxia alone is cytotoxic (9). In most instances, NO is added to the existing flow of O₂. Recent evidence suggests that the combined use of NO+hyperoxia has adverse effects on lung surfactant (10, 11, 19). However, there is a paucity of studies addressing whether the combination of inhaled NO+hyperoxia can injure cells or organs. Here we report that this combination produces synergistic cytotoxicity in cultured alveolar epithelial and pulmonary microvascular endothelial cells.

	METHODS

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Cell culture and cytotoxicity assays. Human alveolar epithelial A549 cells (American Type Culture Collection CCL-185) were grown in Ham's F-12-K medium (GIBCO BRL, Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Human microvascular endothelial lung cells (HMVEC-L) (Clonetics, San Diego, CA) were grown in endothelial growth medium (Microvascular, BulletKit, Clonetics). Cells were maintained at 37°C in 95% room air-5% CO₂ in a humidified chamber. A549 cells were seeded at 6 × 10⁴ cells/ml, and HMVEC-L were seeded at 4.8 × 10⁴ cells/ml. Cells adhered overnight in 35-mm culture dishes. The next day, the dishes were divided into four groups, and cells were grown in 95% room air-5% CO₂ (control), room air+NO, 95% O₂-5% CO₂ (hyperoxia), and hyperoxia+NO. NO was generated in culture medium using one of two NO donors: S-nitroso-N-acetylpenicillamine (SNAP; Biomolecular Research Laboratories, Plymouth Meeting, PA) or 2,2'-(hydroxynitrosohydrazono)-bis-ethanamine (DETA NONOate; Cayman Chemical, Ann Arbor, MI). NO levels generated in culture medium were measured using the Iso-NO meter (World Precision Instruments, Sarasota, FL). Media, NO donors, and gasses were refreshed once each day. The total cell count and number of live cells were determined daily with a hemacytometer; live cells were distinguished by the exclusion of trypan blue dye.

Assays of apoptosis. Cells were grown on glass coverslips and treated as described above for 3 days. The protocol utilized for the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay was as described previously (15). Fluorescent TUNEL reagents were obtained from Boehringer Mannheim. Cells were dual labeled with 4 µg/ml of the DNA-binding dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Boehringer Mannheim, Indianapolis, IN) for 5 min at room temperature. With the use of a Nikon Optiphot microscope with epifluorescence and an ultraviolet-2A filter (Nikon Instruments, Melville, NY), apoptotic nuclei were identified as smaller and brighter objects than their normal counterparts (15).

Western blot analysis. Cells were washed three times with phosphate-buffered saline (PBS) and lysed in 300 µl of lysis buffer [10 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride, pH 6.8, 10 µg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluoride] per dish. Cellular proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4-20% acrylamide gradient gels (BioRad Laboratories, Hercules, CA) under reducing conditions (17). Proteins were transferred to Immobilon polyvinylidene fluoride membranes (Millipore, Bedford, MA) at 24 V for 30 min using an electrophoretic blotting apparatus (Idea Scientific, Minneapolis, MN). Nonspecific binding to the membrane was blocked by incubation with 5% nonfat dry milk overnight at 4°C. Nitrotyrosine-containing proteins were immunodetected using 1 µg/ml of rabbit polyclonal anti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) in PBS and 0.1% Tween 20. A goat anti-rabbit secondary antibody [immunoglobulin G (IgG)] conjugated to alkaline phosphatase (Boehringer Mannheim) was used to detect the primary antibody and was visualized by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphate buffer (100 mM Tris · HCl, pH 9.5, and 50 mM MgCl₂).

Immunocytochemistry. Cells cultured on coverslips were rinsed with ice-cold PBS and fixed and permeabilized for 30 min in 2% paraformaldehyde and 0.1% Triton X-100. Cells were incubated for 1 h at 4°C with 3 µg/ml of the same rabbit polyclonal anti-nitrotyrosine antibody. Primary antibody was detected with 240 µg/ml of goat anti-rabbit IgG conjugated with rhodamine (Organon Teknika, Durham, NC). The cellular localization of the antigen was visualized using a G-2A filter (Nikon) attached to a fluorescent microscope.

	RESULTS

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To compare the cytotoxicity of combined NO+hyperoxia with either agent alone, cells were divided into four groups: room air (control), room air+NO, hyperoxia (95% O₂-5% CO₂), and a combination of hyperoxia+NO. A series of preliminary experiments was performed to identify conditions under which the NO donors would not be overtly cytotoxic to A549 cells or HMVEC-L. On the basis of these experiments, we determined that the highest concentration that caused virtually no cell death over the time course of 1 wk was 2 mM SNAP or 0.5 mM DETA NONOate (data not shown). In the culture medium used, 2 mM SNAP yielded peak levels of NO (4.4 µM) after 2 h, and NO levels gradually declined to baseline over the next 10 h (Fig. 1). As expected, control untreated cells increased in number during the entire experiment when cultured in room air alone. Figure 2 shows that there was no appreciable cell death in SNAP alone during the experiment, although the cell population did not increase. Similarly, cell death was minimal in the hyperoxia group until day 4. Unlike these relatively modest cytotoxic effects, cells exposed to the combination of NO+hyperoxia died rapidly (Fig. 2). Cell death in large numbers was evident as early as day 2, and the remaining cells died soon thereafter. This cytotoxicity was clearly synergistic, with the death rate exceeding any additive effect. To determine whether the synergistic cytotoxicity was due specifically to NO+hyperoxia and not to a nonspecific artifact of the NO donor SNAP, the experiment was repeated using a different NO donor, DETA NONOate. A concentration of 0.5 mM was chosen on the basis of several preliminary experiments that showed no cytotoxicity at this concentration over a period of 6 days. NO release from 0.5 mM DETA NONOate was slower but more sustained than with 2 mM SNAP (Fig. 1). Figure 3 shows that the NO donor alone had few cytotoxic effects but was synergistic when used in combination with hyperoxia. This observation confirms the earlier results with SNAP and supports the conclusion that synergistic cytotoxicity was due to hyperoxia+NO and not a by-product of either NO donor. To determine whether cell death synergy is limited to these transformed cells or occurs in any primary lung cells, the experiments were repeated using primary HMVEC-L. Figure 4 shows that synergistic cytotoxicity was evident in these primary cells, suggesting that the effects of O₂+NO might be widespread. HMVEC-L cultured under these conditions were relatively resistant to both NO and hyperoxia, and the synergistic death did not occur until day 6.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Production of nitric oxide (NO) during cell culture in 2 mM S-nitroso-N-acetylpenicillamine (SNAP) or 0.5 mM 2,2'-(hydroxynitrosohydrazono)-bis-ethanamine (DETA NONOate). SNAP or DETA NONOate were added to culture media as described in METHODS, and NO levels were monitored over a 24-h time period. Results are of a typical experiment reproduced at least 3 times.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Synergistic cytotoxicity of NO from 2 mM SNAP+hyperoxia in cultured human alveolar epithelial A549 cells. Cells were cultured in 95% room air-5% CO2 (control) or 95% O2-5% CO2 (hyperoxia) in presence or absence of 2 mM SNAP as described in METHODS. Cells (total and live) were counted each day. Values are means ± SD.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Synergistic cytotoxicity of NO from 0.5 mM DETA NONOate+hyperoxia in cultured A549 cells. Cells were cultured in 95% room air-5% CO2 (control) or 95% O2-5% CO2 (hyperoxia) in presence or absence of 0.5 mM DETA NONOate as described in METHODS. Cells (total and live) were counted each day. Values are means ± SD.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Synergistic cytotoxicity of NO from 2 mM SNAP+hyperoxia in cultured human microvascular endothelial lung cells (HMVEC-L). Cells were cultured in 95% room air-5% CO2 (control) or 95% O2-5% CO2 (hyperoxia) in presence or absence of 2 mM SNAP as described in METHODS. Cells (total and live) were counted each day. Values are means ± SD.

To determine whether combined NO+hyperoxia causes cell death via apoptosis, two different assays of apoptosis were utilized. DAPI is a DNA-binding dye that is fluorescent when bound to DNA and excited by ultraviolet light. Apoptotic nuclei are clearly distinguished from normal nuclei on the basis of their intense fluorescence and much smaller size (15). Figure 5 shows that apoptosis was not evident after culture in NO alone, hyperoxia alone, or combined NO+hyperoxia. As a positive control, cells were exposed to a level of H₂O₂ that is known to induce widespread apoptosis in these cells (15). Figure 5 shows widespread apoptosis under these conditions. To independently confirm these observations, apoptosis was also studied with the TUNEL assay, which labels 3'-COOH ends of DNA in chromatin, which result from endonucleolytic cleavage occurring during apoptosis (15). Figure 5 shows that cells cultured in NO+hyperoxia, in NO alone, and in hyperoxia alone were all TUNEL negative, whereas cells exposed to H₂O₂ (positive control) clearly demonstrate apoptosis. These data confirm the observation that hyperoxia does not cause epithelial cell apoptosis (15) and indicate that combined NO+hyperoxia also causes cell death via nonapoptosis.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   Nonapoptotic cell death from NO+hyperoxia. A549 cells were cultured for 3 days on coverslips in room air in absence (A and F) or presence (B and G) of 2 mM SNAP and in hyperoxia in absence (C and H) or presence (D and I) of 2 mM SNAP for 3 days as described in METHODS. E and J: a typical field of cells exposed to 5 mM H2O2 for 3 h (positive control for apoptosis). Cells in A-E were terminal deoxynucleotidyltransferase-mediated dUTP nick end labeled. Cells in F-J were stained with DNA-binding dye 4',6-diamidino-2-phenylindole dihydrochloride. Only H2O2-treated cells showed apoptosis.

Hyperoxia is known to cause intracellular accumulation of reactive O₂-derived intermediates including the superoxide anion () (23). Superoxide can react rapidly with NO to form peroxynitrite (ONOO), an extremely reactive and toxic molecule (3). Although ONOO is too reactive to measure directly, one of its reaction products, nitrotyrosine, which forms when ONOO donates a nitro group to tyrosine residues in cellular proteins, is stable and detectable in tissues and cells. To determine whether the synergistic cytotoxicity of NO+hyperoxia is associated with increased nitrotyrosine, immunocytochemistry was performed using anti-nitrotyrosine antibodies. Nitrotyrosine was barely detected by immunofluorescence in cells grown in room air alone or in hyperoxia (Fig. 6). Cells cultured in NO showed diffuse immunofluorescence, suggesting the accumulation of nitrotyrosine. However, cells grown in combined NO+hyperoxia showed a more intense signal, perhaps because the intracellular immunofluorescence pattern became eccentric (Fig. 6). Cells incubated with secondary antibodies alone showed no signal (not shown).

View larger version (64K): [in this window] [in a new window]   View larger version (94K): [in this window] [in a new window]   View larger version (63K): [in this window] [in a new window]   View larger version (84K): [in this window] [in a new window]   View larger version (68K): [in this window] [in a new window]   View larger version (84K): [in this window] [in a new window]   View larger version (64K): [in this window] [in a new window]   View larger version (67K): [in this window] [in a new window]   Fig. 6.   Nitrotyrosine immunofluorescence in cultured A549 cells. Cells on coverslips were grown for 3 days and fixed for immunofluorescence as described in METHODS and were incubated with anti-nitrotyrosine antibody. A and B: room-air controls. C and D: hyperoxia. E and F: NO (SNAP) alone. G and H: NO+hyperoxia. Cells were visualized by fluorescence (A, C, E, G) and phase-contrast (B, D, F, H) microscopy. Only cells exposed to NO alone (E) or NO+hyperoxia (G) showed immunofluorescence. Note eccentric fluorescence in G.

To gain a further understanding of the nature of the proteins modified by ONOO, cellular proteins were size fractionated on polyacrylamide gels and assayed on Western blots using anti-nitrotyrosine antibodies. Figure 7 shows that immunoreactive proteins were barely detected in either control cells cultured in room air or cells exposed to hyperoxia alone. In contrast, cells exposed to combined NO+hyperoxia showed a notable increase in the levels of two protein bands, migrating at 25 and 35 kDa. Interestingly, cells cultured in NO alone also showed immunoreactive proteins at 25 and 35 kDa. The specificity of this result was confirmed with control assays by incubating Western blots with nonimmune rabbit IgG or with anti-nitrotyrosine antibodies preabsorbed with excess (10 mM) nitrotyrosine (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Western blot for nitrotyrosine-containing proteins. Cells were cultured for 3 days, and Western blots were prepared as described in METHODS. Blots were incubated with anti-nitrotyrosine antibody. Two proteins, at 25 and 35 kDa, were specifically enriched after treatment with NO alone or NO+hyperoxia. These specific bands were not detected when blots were incubated with nonimmune rabbit IgG or with anti-nitrotyrosine antibodies preabsorbed with excess (10 mM) nitrotyrosine (data not shown).

	DISCUSSION

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Inhaled NO therapy is currently being studied in patients also receiving concurrent treatment with O₂ and mechanical ventilation, yet there is a paucity of data from animal or cell culture studies on the combined effects of these two agents. We have performed a set of experiments in cultured alveolar epithelial and lung microvascular endothelial cells that demonstrates that the combination of NO+hyperoxia is much more cytotoxic than either agent alone. These observations are important because they demonstrate that the beneficial effects of inhaled NO therapy have the potential to be accompanied by deleterious side effects when combined with hyperoxia. Although there is some evidence that endogenous NO or lower doses of exogenous (10 parts/million) NO improved alveolar liquid clearance, higher doses of exogenous NO (100 parts/million) increased vascular permeability to protein, and survival of rats exposed to hyperoxia was not improved by inhaled NO (8). Together with reports of lung injury and surfactant dysfunction in animals receiving concurrent inhaled NO+hyperoxia (10, 11, 19), these observations suggest that there is a potential for damage to lung epithelium and endothelium from this therapy. It may therefore be important to minimize exposure to these agents when administered clinically.

The precise relationship between these cell culture experiments and the experimental use of NO in patients is not yet clear, and the full range of NO doses and O₂ levels is yet unexplored. The levels used in these experiments are probably high relative to those experienced by cells in intact lungs. Moreover, the effects of NO+hyperoxia on the multiple cell types of intact lungs are likely to differ in many ways from the effects we observed in homogeneous cell culture. Because NO and ONOO are highly reactive, we postulated that the cells would die via apoptosis, as is typically reported to occur in response to oxidative cellular damage (2, 4, 6, 21, 23). However, the mode of cell death by combined NO+hyperoxia was not apoptosis, as shown by nuclear size, DNA staining intensity, and TUNEL assay. The capacity of these cells to undergo typical oxidant-induced apoptosis is well described (15) and was evident in positive control experiments when the cells were exposed to H₂O₂. The mode of cell death from combined NO+hyperoxia may be similar to the mode of death from hyperoxia alone (15), although the time course of cell death was much more rapid when exogenous NO was present. However, the observed nonapoptotic cell death was substantially slower in combined NO+hyperoxia relative to oxidant-induced apoptosis (2 or more days vs. 4 h), consistent with the nuclear morphology and TUNEL assays showing the absence of apoptosis.

The observation that intense immunofluorescence with anti-nitrotyrosine antibody was correlated with severe cytotoxicity suggested that ONOO may have a role in mediating the cytotoxic effects shown here. The Western blot analysis further suggested that the specific nitration of two proteins with molecular masses of 25 and 35 kDa may be relevant. However, nitration of bands of the same size also occurred in the cells grown in NO alone, indicating that nitration of these proteins is not sufficient for synergistic cell death. Moreover, on the basis of the fact that ONOO formation requires superoxide (10), this observation suggests that sufficient superoxide is produced in these cells to generate some ONOO, even when cultured in room air. The observation that nitrotyrosine immunofluorescence appeared notably enhanced is difficult to understand in the absence of a large increase in the abundance of nitrotyrosine-containing proteins on Western blots. Perhaps ONOO-modified proteins become translocated in cells in NO+hyperoxia, resulting in eccentric fluorescence. Alternatively, changes in conformation (perhaps increased denaturation) may underlie the enhanced cellular immunofluorescence. Further work is needed to identify these proteins and to determine whether they have a role in cytotoxicity.