Department of Sports Medicine, Medical Clinic, University of Heidelberg, 69115 Heidelberg, Germany
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ABSTRACT |
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In
oxygen-sensitive excitable cells, responses to hypoxia are initiated by
membrane depolarization due to closing of the K channels that is
thought to be mediated by a decrease in reactive oxygen species (ROS).
Because the mechanisms of hypoxic inhibition of ion transport of
alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C,
and Clerici C. Am J Physiol Lung Cell Mol Physiol 271:
L70-L78, 1996; Mairbäurl H, Wodopia R, Eckes S, Schulz S,
and Bärtsch P. Am J Physiol Lung Cell Mol Physiol 273: L797-L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as 86Rb and
22Na uptake in A549 cells exposed to normoxia, hyperoxia,
or hypoxia together with ROS donors and scavengers.
H2O2 < 1 mM did not affect transport,
whereas 1 mM H2O2 activated
22Na uptake (+200%) but inhibited 86Rb uptake
(30%). Also hyperoxia, aminotriazole plus menadione, and
diethyldithiocarbamate inhibited 86Rb uptake.
N-acetyl-L-cysteine, diphenyleneiodonium, and
tetramethylpiperidine-N-oxyl, used to reduce ROS, inhibited
86Rb uptake, thus mimicking the hypoxic effects, whereas
deferoxamine, superoxide dismutase, and catalase were ineffective.
Also, hypoxic effects on ion transport were not prevented in the
presence of H2O2, diethyldithiocarbamate, and
N-acetyl-L-cysteine. These results indicate that
ion transport of A549 cells is significantly affected by decreasing or
increasing cellular ROS levels and that it is possible that certain
species of ROS might mediate the hypoxic effects on ion transport of
alveolar epithelial cells.
cation transport; sodium/potassium pump; sodium/potassium/2 chloride cotransport; hyperoxia; reactive oxygen species; superoxide anion; A549 cells; oxygen sensing; pulmonary edema
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INTRODUCTION |
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REACTIVE OXYGEN SPECIES (ROS) appear to mediate
specific responses to changes in PO2
in oxygen-sensitive tissues such as carotid body cells, pulmonary
vascular smooth muscle, and erythropoietin (EPO)-producing hepatocytes.
On the other hand, ROS are involved in the degradation of biological
material, and elevated levels of ROS can cause significant cell damage,
apoptosis, and cell death (14). In these oxygen-sensing cells,
H2O2 seems the most suitable of all ROS to
serve as an intracellular messenger. In comparison to other ROS such as
superoxide anion or hydroxyl radical, H2O2 is
characterized by high stability and free membrane permeability.
Production of H2O2 proportional to changes in
PO2 could be demonstrated in HepG2
hepatocytes (11). Acker et al. (1) showed a decreasing nerve
discharge of rat carotid bodies by increasing concentrations of
H2O2, thus mimicking hyperoxia, whereas hypoxic
stimulation was suppressed during the period of H2O2 addition. In pulmonary vascular smooth
muscle, Archer et al. (2) showed a
PO2-dependent ROS production and subsequent changes in vascular tone that appeared to depend on ROS
concentration in isolated rat lungs. Fandrey et al. (11) demonstrated
inhibition of hypoxia-induced EPO production by
H2O2 in HepG2 hepatocytes. Moreover,
H2O2-induced inhibition of EPO production could
be antagonized by incubation with catalase (31), and a relationship
between the oxygen dependence of EPO expression induced by
hypoxia-inducible factor-1 and ROS has been discussed (31, 35).
Molecular analysis of EPO expression showed great similarities with
other known hypoxia-responsive genes (31), thus supporting the
hypothesis of a highly conserved oxygen sensor. However, a link between
ROS and the regulation of ion transport of alveolar epithelial cells in
hypoxia has not yet been demonstrated.
Both hypoxia and hyperoxia were shown to affect ion transport of alveolar epithelial cells (for a review, see Ref. 6). Alveolar epithelial cells respond to hypoxia with decreased activity of the Na/K pump, Na/K/2Cl cotransport (NKCC), and other Na-transporting systems (22, 27, 28), which was discussed as one mechanism crucial for developing and aggravating hypoxia-induced pulmonary edema (30). Whereas acute hyperoxia appears to inhibit the Na/K pump, prolonged exposure activates and thus helps clear hyperoxia-induced edema (6, 25, 26). Alterations of transport might also occur after a respiratory burst of macrophages and other cells of the immune system that liberate ROS for host defense.
Although a variety of systems exist that affect formation of ROS inside the cell, it appears that ROS concentration also depends on available oxygen. This is the basis for the assumption of a ubiquitous, ROS-dependent cellular oxygen-sensing mechanism. The present study was performed to investigate the role of ROS in regulating alveolar epithelial cell ion transport and to test whether changes in ROS are involved in mediating the hypoxic inhibition of ion transport (21, 22, 28). Effects of modulating cellular ROS by pro- and antioxidative substances on ion transport were measured on A549 cells, a human lung-derived carcinoma cell line with conservation of many functions of alveolar type II cells, in particular the hypoxic inhibition of ion transport (22). The results indicate that cellular ROS depletion modulates cation transport of A549 cells similar to hypoxia.
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MATERIALS AND METHODS |
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Reagents, oxidants and antioxidants. All reagents were of analytic grade, and stock solutions were prepared from deionized water. Phosphate-buffered saline (PBS), Ham's F-12 medium, penicillin-streptomycin, FCS, and HEPES were from GIBCO BRL (Life Technologies). H2O2 (30% stock solution), menadione sodium bisulfite (Men), and 3-amino-1,2,4-triazole (ATZ; aminotriazole), Cu/Zn superoxide dismutase (SOD; from bovine liver), catalase (from bovine liver), diphenyleneiodonium chloride (DPI), N-acetyl-L-cysteine (NAC), and deferoxamine (DFO; desferrioxamine mesylate), tetramethylpiperidine-N-oxyl (TEMPO), and diethyldithiocarbamate (DETC) were from Sigma.
A549 cells. A549 cells, a human pulmonary carcinoma cell line with many characteristics of alveolar type II cells (20), were from American Type Culture Collection. The cells were cultured on untreated 24-well plates (Costar) in Ham's F-12 medium supplemented with 7% FCS, 100 U/ml penicillin-0.1 mg/ml streptomycin, 10 mM HEPES, and 10 mM sodium bicarbonate. Confluence was reached 3-4 days after the cells were seeded, and the experiments were carried out on 2- to 8-day-old confluent monolayers passaged 3-12 times after purchase from American Type Culture Collection.
Hypoxia was applied by an initial change to a gas-equilibrated culture medium. In most experiments, oxidants and antioxidants were added at this point. Culture plates then were transferred to a CO2-O2-controlled incubator (NUNC) adjusted to 3% O2, 5% CO2 and 92% N2 at 37°C as described previously (22). For exposure to hyperoxia, gas mixtures containing 30 or 40% O2, 5% CO2 and balance N2 were used. Control cells were treated similarly under the usual tissue conditions (5% CO2 and 37°C) in a tissue culture incubator (Heraeus).
Flux measurements. The activity of ion transport pathways was
determined by unidirectional tracer uptake measurements (22). After
exposure to normoxia or hypoxia, the cells were washed two times with
washing medium [150 mM NaCl and 2 mM HEPES, pH 7.4, at room
temperature (RT)] and incubated for 15 min at RT with the flux
medium equilibrated with the respective CO2-free gas. The
flux medium contained (in mM) 140 NaCl, 20 HEPES, 10 glucose, 5 KCl, 1 NaH2PO4, 1 MgCl2, and 0.2 CaCl2, pH 7.4, at RT. Fluxes were started by adding flux
medium containing 86Rb (as a tracer for K) or
22Na at final activities of 0.5-2 µCi/ml and the
respective inhibitors. Ouabain (100 µM) and bumetanide (50 µM) were
added to quantify the activity of the Na/K pump and NKCC,
respectively. Equivalent volumes of the solvent were added to the
control cells. Tracer uptake was stopped, and contaminant tracer was
removed with five washes with ice-cold washing medium. The cells were
lysed with 0.1 M NaOH. Radioactivity of the lysate was determined in a
-counter (model TR 2100, Canberra Packard). The protein
concentration in the lysate was measured photometrically with a test
kit from Bio-Rad, with human serum albumin and globulin standards in
saline diluted with 0.1 M NaOH.
Photometric measurement of H2O2 concentration. To measure the capacity of A549 cells to metabolize H2O2, its concentration was measured photometrically according to the method previously described (11). Briefly, one volume of supernatant was mixed with four volumes of 2,2'-azino-di(3-ethylbenzthiazoline-sulfonate) reagent (100 mg/l) in 100 mM NaCl and 50 mM NaH2PO4, pH 4.4. Horseradish peroxidase (Boehringer Mannheim) was added to start the reaction. Absorption was measured after 30 min of incubation at RT, and H2O2 concentration was calculated from known standards. The lower detection limit of this assay was ~4 µM H2O2.
Measurements of ATP and ADP. ATP and ADP concentrations were measured as described by Weicker et al. (33). Briefly, the cells were washed with ice-cold PBS, and protein was denatured by adding ice-cold 0.6 M perchloric acid onto the cell layer. The cells were scrubbed from the tissue culture plate and vortexed, and the precipitate was sedimented by a 3-min centrifugation at 10,000 g in a microfuge. The pH of the clear supernatant was neutralized before measurement with HPLC with an ultraviolet detection system.
Statistics. All measurements were repeated on several batches
of cells from different passages, and fluxes were determined in
triplicate. Results are means ± SD. The level of significance was
P 0.05. Graphs were designed with SigmaPlot version 2.01 (Jandel Scientific).
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RESULTS |
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H2O2 elimination by A549 cells. Taking
into consideration the potential cytotoxicity of oxidants and
especially of H2O2, it was important to
evaluate the capability of A549 cells to handle a
H2O2 charge. Due to the limited sensitivity of
the assay used, neither basal levels of H2O2 in
A549 cells nor PO2-dependent changes
in H2O2 could be detected. Elimination of
H2O2 was tested after known amounts of
H2O2 were added to the cells kept in culture medium. Figure 1A shows the high
capacity of A549 cells to eliminate within 2 min
H2O2 that amounted to ~40% of the
H2O2 added initially. The
H2O2 concentration fell almost instantly when
NAC (10 mM) was present. The rate of H2O2
degradation was not affected by hypoxia (Fig. 1B). The catalase
inhibitor ATZ did not significantly influence
H2O2 elimination (data not shown).
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Effects of oxidants and hyperoxia on ion uptake of A549 cells.
Cellular levels of ROS were increased by the addition of
H2O2 to test whether elevated ROS
concentrations would abolish the decrease in ion uptake by A549 cells
in hypoxia (22). It has to be noted that incubation with
H2O2 at a concentration of 1 mM caused cell
damage, indicated by floating cells (only ~50-75% of the cells
remained adherent to tissue culture plates as quantified by measuring
the protein content per culture well). This cytotoxic effect was quite
variable because it was not observed in ~40% of all experiments. It
was less pronounced at a H2O2 concentration of 100 µM (85% adherent cells) but was not observed at 10 µM. Occurrence of lysis was not related to passage number, the age of
the cells, or the feeding cycle. However, changes in transport activity
were found regardless of whether cell damage was observed or not.
Figure 2 shows that total 22Na
uptake of adherent cells was affected by H2O2
in a dose- and time-dependent manner: a 1-h incubation with 1 mM
H2O2 caused a sixfold stimulation of
22Na uptake; at a concentration of 100 µM, activation was
about twofold, whereas no significant effect was seen at lower
concentrations. After a 4-h incubation,
H2O2 affected 22Na uptake only when
applied at 1 mM. Total 86Rb uptake was inhibited by
H2O2 when applied at concentrations > 100 µM (Fig. 3A). This was due to an
inhibition of the Na/K pump (Fig. 3B). NKCC was
not affected, whereas ouabain- and bumetanide-insensitive (OBI)
86Rb uptake was increased significantly (Fig. 3B).
Concentrations of 100 and 10 µM H2O2 did not
affect 86Rb uptake components.
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To test whether H2O2 actually simulates the
effects of an increased level of oxygen, the cells were also exposed to
hyperoxia. Four hours of exposure to 30% O2 had no effect
on either component of 86Rb uptake, whereas exposure to
40% O2 inhibited total 86Rb uptake by ~28%,
which was mainly due to inhibition of the Na/K pump. The slight
decrease in NKCC was not significant. In contrast to
H2O2, hyperoxia did not affect OBI
86Rb uptake (Fig. 4A)
nor did it affect total 22Na uptake (Fig. 4B).
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ATZ and Men were applied to increase ROS generation by inhibiting
catalase and affecting electron shuttling between different electron
acceptors, respectively. The cells were incubated with 50 mM ATZ and
0.6 mM Men over 2 h before exposure to hypoxia to establish their effects (11). DETC was used to increase cellular superoxide anion levels by inhibiting SOD. Figure
5A shows that ATZ and Men together
inhibited 86Rb uptake in normoxic cells. This effect can
mainly be attributed to the inhibition of NKCC. Also, DETC inhibited
total 86Rb uptake by ~40%, which is brought about by a
20% inhibition of the Na/K pump and a 60% inhibition of NKCC
(Fig. 5B). Pretreatment with DETC for at least 30 min was
required to detect significant inhibition (time course not shown).
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Effects of antioxidants on ion uptake by A549 cells. Application of antioxidants to normoxic cells should lower the cellular ROS levels thought to mimic a decrease in ROS as in hypoxia. SOD and catalase added to A549 cells had no effect on 86Rb uptake regardless of the concentration applied and duration of treatment (data not shown).
NAC and DFO were added at different concentrations to the culture
medium, and the cells were exposed for 1 and 4 h before the flux
measurement. NAC caused a dose-dependent inhibition of total
86Rb uptake (Fig. 6A)
that was complete at ~10 mM NAC and was independent of the duration
of the treatment. However, at this high concentration of NAC, some cell
damage was observed as indicated by floating cells. NAC (2 mM) also
caused a slight inhibition of total 22Na uptake (Fig.
4B). DFO had no significant effect on 86Rb
uptake (Fig. 6B).
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DPI reduces superoxide anion production and ROS generation by
inhibition of NAD(P)H oxidase and other flavine-containing enzymes. TEMPO has been used as a ROS scavenger that appears to be more selective for the superoxide anion (29). Figure
7A shows that 10 µM DPI inhibited
total 86Rb uptake, which was independent of the duration of
treatment (10 min to 4 h; data not shown). One micromolar DPI was
ineffective; a greater variability of results was observed at 100 µM
(data not shown). The inhibition of total 86Rb uptake by 10 µM DPI was caused by inhibition of the Na/K pump. NKCC and OBI
86Rb uptake did not change significantly. Because DPI might
also affect mitochondrial NADPH oxidases, it might exert its action by
energy depletion. Figure 8 shows that DPI
did not affect cellular ATP and ADP concentrations. Figure 7B
shows that TEMPO (3 mM) inhibited total 86Rb uptake by
~20% due to inhibition of the Na/K pump and NKCC.
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Combined effects of oxidants, NAC, and hypoxia. To establish a
role for ROS in sensing cellular oxygen levels and their involvement in
the modulation of ion transport by hypoxia, oxidants and antioxidants were applied to cells exposed to normoxia and hypoxia because clamping
intracellular ROS levels should render transport
insensitive to hypoxia if ROS are involved in oxygen-dependent
transport modulation. Figure 9A
indicates that H2O2 stimulated 22Na
uptake in normoxic and hypoxic A549 cells, but the effect was smaller
in hypoxia. In both oxygenation states, 2 mM NAC had no effect on
22Na uptake. Figure 9B shows that 1 mM
H2O2 inhibited 86Rb uptake in
normoxia and hypoxia but did not prevent the hypoxia-induced transport
inhibition. Also, in the presence of NAC, hypoxic inhibition of
86Rb uptake was detectable. Figure 9C shows that
the SOD inhibitor DETC reduced transport activity in normoxia. However,
in the presence of DETC, no significant inhibition of transport by
hypoxia could be seen.
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DISCUSSION |
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Our results indicate that ROS and their scavengers as well as hyperoxia and hypoxia have profound effects on ion transport of A549 cells but that different transport systems are affected in different ways: hypoxia and scavengers of ROS inhibit Na and Rb uptake, whereas hyperoxia and increased levels of ROS activate Na uptake but inhibit Rb uptake, particularly the Na/K pump. Increasing cellular ROS levels, however, did not prevent transport inhibition by hypoxia.
These results contrast to some extent with the hypothesis of a sensor detecting hypoxia by a decrease in cellular ROS levels, which might also be involved in the modulation of cation transport of alveolar epithelial cells by hypoxia, analogous to results obtained on peripheral chemoreceptor glomus cells, pulmonary artery smooth muscle, and EPO-producing hepatocytes (24, 31, 34). This hypothesis predicts that varying the cellular levels of ROS by the addition of ROS scavengers and oxidants would mimic the respective effects of hypoxia and hyperoxia on ion transport (6). Addition of oxidants should then prevent hypoxic transport inhibition, and transport should be independent of PO2 at any preset concentration of ROS adjusted by adding and scavenging ROS.
If the above-discussed arguments held true, a direct relationship might be expected between ROS concentration and cellular ion transport. ROS depletion should inhibit transport, whereas increased ROS levels should activate ion transport of A549 cells. Lung epithelial cells are constantly exposed to high levels of ROS from inhaled oxidants or formation by phagocytic and inflam-matory cells inside the lung as well as by alveolar epithelial cells (32). These cells require an effective protection system consisting of enzymes eliminating ROS (10) and ion transport pathways that adjust cellular solute and water contents on oxidative cell damage. Increased levels of ROS have been shown to modulate ion transport of lung alveolar cells (3, 7, 13, 15, 16, 25). Acute exposure to increased ROS concentrations induced by severe hyperoxia and addition of H2O2 was shown to inhibit Na-K-ATPase (7, 13), whereas long-term hyperoxia activates ion transport (4, 13). A brief pulse of externally added ROS was sufficient for long-term transport modulation despite rapid H2O2 degradation (13). Whereas the initial inhibition of the Na/K pump by increased ROS might be a consequence of oxidative cell damage as well as of direct damage of the transport protein and/or its regulating systems, stimulation of the Na/K pump on prolonged ROS exposure by increasing its expression might represent a mechanism to reduce edema formation caused by oxidative lung damage (6, 23). In contrast to the above-mentioned prediction, this time-dependent diversification of the response of the Na/K pump to increased levels of ROS indicates that no direct relationship exists between the actual ROS concentration and transport activity. Our results indicate that short-term exposure of cells to increased ROS affects different transporters in different ways. Although 22Na uptake was activated by H2O2 but not by short-term hyperoxia, the Na/K pump (and, to some extent, also NKCC) were inhibited by 40% O2, whereas ATZ plus Men and DETC affected mainly NKCC (Figs. 4 and 5). Clerici et al. (7) also found inhibition with 2.5 mM H2O2 of the Na/K pump and Na-coupled transport of phosphate and amino acids of alveolar type II cells in primary culture. The authors did not comment on total Na uptake. The mechanisms involved in ROS activation of total 22Na uptake and inhibition of the Na/K pump and NKCC are not understood. It appears that increased ROS might modulate ion transport by alveolar epithelial cells not by action on a common signaling pathway but by transporter-specific effects because some transporters are more sensitive to oxidant stress than others. In alveolar epithelial cells, Ca-dependent mechanisms seem not to be involved in mediating oxidant-induced transport inhibition. In contrast to other cell types, which respond to micromolar concentrations of H2O2 with a pronounced increase in intercellular Ca (Cai) (9, 19), primary cultured rat AII cells as well as A549 cells do not increase Cai unless concentrations of H2O2 > 2 mM are applied (results not shown). In the latter case, the increase in Cai is certainly a consequence of H2O2-induced cell damage.
Scavengers of ROS are well known to prevent oxidative tissue injury. However, the effects of decreased ROS levels on ion transport have not been studied in great detail. Our results indicate a pronounced decrease in ion transport by alveolar epithelial cells when cellular ROS levels were decreased by the addition of ROS scavengers (TEMPO and NAC) and an inhibitor of ROS formation (DPI; Figs. 6, 7, and 9). This result is consistent with the notion that hypoxia causes transport inhibition by decreasing the concentration of ROS. It is, however, not in line with results on B cells where oxygen sensitivity was found to be independent of NAD(P)H oxidase, the enzyme thought to be mainly responsible for formation of the superoxide radical from oxygen (36). Inhibitors of NAD(P)H oxidase, such as DPI, were also shown to affect mitochondrial function by inhibition of flavine-containing enzymes, which might impair ATP synthesis (8, 12). Our results on cellular ATP and ADP concentrations in cells treated with low doses of DPI show no indications for an impairment of mitochondrial function (Fig. 8). Energy depletion can therefore be ruled out as a reason for transport inhibition by DPI.
Recently, Rafii et al. (29) reported activation of Na transport and
epithelial Na-channel expression when fetal lung distal epithelial
cells cultured in 3% O2 were exposed to increased
PO2 (normoxia). This effect was
prevented only by ROS scavengers that appear to be more specific in
binding and neutralizing the superoxide radical rather than peroxides
(29). The similarity between hypoxia- and scavenger-related changes in
transcription factor nuclear factor-B led to the conclusion that in
this case, an oxygen-induced increase in cellular ROS levels might
mediate upregulation of the epithelial Na channel. Our results contrast
with this finding because we could not demonstrate an upregulation of
Na transport with hyperoxia but only with H2O2.
Do changes in ROS mediate transport inhibition by hypoxia? The inhibition of transport by free radical scavengers in normoxic cells is consistent with the notion of an ROS-dependent oxygen-sensing mechanism that measures the degree of hypoxia as the decrease in cellular levels of ROS. This is in line with findings on EPO-producing hepatocytes (11). It is contrasted by recent results (5) on a hypoxia-induced increase in dichlorofluorescin fluorescence, which was interpreted to show an increase in mitochondrial ROS formation in hypoxia. With the methods applied, we were unable to detect changes in ROS of A549 cells during hypoxia. Our results taken together indicate no direct relationship between ROS concentration and transport activity. Although transport inhibition by depletion of cellular ROS supports the model, the lack of H2O2 and DETC to prevent hypoxia-induced transport inhibition contrasts with the notion that a ROS-dependent oxygen sensor mediates inhibition of ion transport in hypoxia. It might be, however, that the high capacity of A549 cells to remove added H2O2 effectively still allows hypoxia to exert its effects. This argument is weakened because the H2O2-induced inhibition of 86Rb transport was stable over several hours despite the rapid removal of H2O2 by A549 cells. Also, in hypoxic hepatocytes, an initial pulse of H2O2 was sufficient to prevent the hypoxia-induced EPO formation (11). In those experiments, a constantly elevated level of ROS could only be maintained in the presence of inhibitors of catalase (11). It appears, therefore, that hypoxic effects can occur even though cellular ROS levels are increased, which makes their involvement in an oxygen-sensing process unlikely unless the signal consists of a (transient) change in ROS rather than absolute values. The fact that hypoxic inhibition of transport is also seen in the presence of NAC (Fig. 9), when cellular ROS concentrations are already low, further supports this notion.
Cellular concentrations of ROS vary due to alterations in ambient oxygen and changes in metabolic activity. They indicate, therefore, the metabolic state of a cell. On the basis of this assumption, ROS were discussed as "sensors" for altered oxygen availability in various cell types that show oxygen-dependent changes in cell functions. The alveolar epithelium, however, is constantly exposed to high levels of oxidants from, e.g., the high oxygen content of inspired air, pollutants like NO2, and a respiratory burst of activated neutrophils and macrophages. Alveolar epithelial cells were even shown to be capable of initiating respiratory bursts themselves due to a highly active NADPH oxidase (18, 32). As a protective mechanism, they also have high-capacity ROS eliminating pathways such as extracellular catalase (10, 17). It is, therefore, questionable whether in cells that have a high capacity to both produce and eliminate ROS, an oxygen-sensing system based on changes in ROS would be very sensitive unless the oxygen sensor consists of a distinct ROS species that is not directly affected by cellular ROS detoxification systems and that we were unable to modulate with the above-described maneuvers.
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ACKNOWLEDGEMENTS |
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We thank Christiane Herth and Sonja Engelhardt for excellent technical assistance.
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FOOTNOTES |
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The project was supported by Grant MA 1503/11-1 from the German Research Foundation (Deutsche Forschungsgemeinschaft).
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: H. Mairbäurl, Medizinische Klinik und Poliklinik, Innere Medizin VII, Sportmedizin, Universität Heidelberg, Hospitalstrasse 3, Geb. 4100, 69115 Heidelberg, Germany (E-mail: heimo_mairbaeurl{at}med.uni-heidelberg.de).
Received 20 July 1999; accepted in final form 5 November 1999.
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