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Am. J. Respir. Cell Mol. Biol., Volume 18, Number 5, May 1998 712-720

Adaptation of Rat Type II Pneumocytes to NO2: Effects of NO2 Application Mode on Phosphatidylcholine Metabolism

Bernd Müller, Carola Seifart, Peter von Wichert, and Peter J. Barth

Laboratory of Respiratory Cell Biology, Department of Internal Medicine, and Institute of Pathology, Philipps University of Marburg, Marburg, Germany


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies have shown that nitrogen dioxide (NO2) inhalation affects the extracellular surfactant as well as the structure and function of type II pneumocytes. Since in these studies there were great variabilities in oxidant concentration, duration of exposure, and mode of NO2 application, we evaluated the influence of the NO2 application mode on the phospholipid metabolism of type II pneumocytes. Rats were exposed to identical NO2 body doses (720 ppm × h), which were applied continuously (10 ppm for 3 d), intermittently (10 ppm for 8 h per day, for 9 d), and repeatedly (10 ppm for 3 d, 28 d rest, and then 10 ppm for 3 d). Immediately after exposure, type II cells were isolated and evaluated for cell yield, vitality, phosphatidylcholine (PC) synthesis, and secretion. Type II pneumocyte cell yield from animals that had been continuously exposed to NO2 was significantly increased, whereas intermittently and repeatedly treated rats exhibited cell yields that were nonsignificantly enhanced. Vitality of the isolated type II pneumocytes was not affected by the NO2 exposure modes. Continuous application of 720 ppm × h NO2 resulted in increased activity of the cytidine-5-diphosphate (CDP)-choline pathway. After continuous NO2 application, specific activity of choline kinase, cytidine triphosphate (CTP):cholinephosphate cytidylyltransferase, uptake of choline, and pool sizes of CDP-choline and PC were significantly increased over those of controls. Intermittent application of this NO2 body dose also provoked an increase in PC synthesis, but this increase was less prominent than after continuous exposure. After repeated exposure, the synthesis parameters were comparable to those for cells from control animals. Whereas PC synthesis in type II cells was obviously stimulated by NO2, the secretory activity of the cells was reduced. Continuous exposure reduced this activity most, whereas intermittent exposure nonsignificantly reduced this activity as compared with that of controls. The repeated application of NO2 produced no differences. We conclude that type II pneumocytes adapt to NO2 atmospheres depending on the mode of its application, at least for the metabolism of PC and its secretion from isolated type II pneumocytes. Further studies are necessary to determine whether additional metabolic activities will also adapt to NO2 atmospheres, and if these observations are specific for NO2 or represent effects generally due to oxidants.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is well known that type II pneumocytes synthesize and secrete the pulmonary surfactant that lines the alveolar surface. Numerous studies have shown that this phospholipid-protein rich material is a prerequisite for normal lung function (1). Inhalation of environmental oxidant gases results in pulmonary injury that depends on the depth of penetration of the gas into the respiratory tract and on its reactivity (5). Among all atmospheric pollutants, nitrogen dioxide (NO2) is one of the principal oxidants present in the urban environment and is known to generate free radicals that are sufficiently stable in ambient air (6). Its toxicology has attracted additional interest since the application of nitrogen oxide (NO) for pulmonary vasodilatation, which under certain circumstances produces measurable amounts of NO2 (9).

Numerous studies, done mainly on small rodents, have evaluated the toxicology of NO2 and show the type II cell to be the progenitor cell of the type I pneumocyte that is affected first by oxidants (5, 10, 11). In addition, it was shown that NO2 affects the structure and function of type II pneumocytes as well as of extracellular surfactant (12). Since there is great variability in the use of oxidant atmospheres, duration of exposure, and time point of analysis, numerous and sometimes seemingly conflicting data have been obtained. Besides the different exposure models, this may also be related to the animals themselves, because it is known that various species show different susceptibilities to this oxidant (13).

Although many NO2-induced effects have been reported, so far no information exists about whether animals have the potential to adapt to this oxidant. We report here for the first time that identical body doses of NO2 (720 ppm × h) applied in different modes to rats (continuously, at 10 ppm for 3 d; intermittently at 10 ppm for 8 h per day, for 9 d; and repeatedly at 10 ppm for 3 d, 28 d rest, and reapplication at 10 ppm for 3 d) result in different metabolic activities of type II cells.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals and Exposure Experiments

Experiments were done on male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) with an initial body weight of 160 to 170 g. They were kept in cages (4 animals per cage) with free access to food and water. The animals were either exposed to normal air as controls or to NO2 atmospheres for different periods.

For NO2 exposure, the cages containing the animals were placed into gas-tight chambers and exposed to 10 ppm NO2 atmospheres according to the exposure modes described earlier, as follows: (1) continuous: 10 ppm for 3 d; (2) intermittent: 10 ppm for 8 h per day, 9 d; and (3) repeated: 10 ppm for 3 d and 28 d rest, and then 10 ppm for 3 d. During rest periods the animals breathed normal air. With these protocols during the exposure period, the animals received an NO2 body dose of 720 ppm × h. The atmospheric NO2 concentration was 10 ± 0.4 ppm (mean ± SD) as measured within the exposure chambers with an NO2-sensitive electrochemical element (ECS 102-1; MPSensor System, Munich, Germany).

Type II Cell Isolation

Immediately after the animals' exposure to NO2, type II pneumocytes were isolated according to the procedure described elsewhere (14). In brief, after bronchoalveolar lavage (BAL), the lungs were washed with the solutions described by Dobbs and colleagues (14) before elastase solution was instilled. Digestion with elastase was allowed to take place at 37°C for 20 min before the large airways were removed. In the presence of deoxyribonuclease I (DNAse I) (250 µg/ml), the lungs were minced with scissors and the elastase reaction was then stopped by addition of 5 ml fetal bovine serum (FBS) per lung (Gibco-BRL, Eggenstein, Germany). The final cell suspension was filtered several times through nylon gauze and washed by gentle centrifugation. The cell pellet was resuspended in Dulbecco's modified Eagles' medium (DMEM) and finally transferred to rat immunoglobulin G (IgG)-coated bacteriologic Petri dishes to a density of 30 × 106 cells. After a 1 h incubation in a 10% CO2-air incubator, the macrophages were adherent to the plastic dishes. The unattached type II pneumocytes were removed, centrifuged, and prepared as cytospin preparations, which were then stained with hematoxylin and eosin (H&E) and used for secretion experiments.

Vitality

After cell isolation, type II cell vitality was determined by trypan blue dye exclusion (15). Samples of every exposure condition were evaluated per microscopic field in triplicate, and an average value was determined. Vitalities are expressed as percentages of cells excluding trypan blue dye. Exposure-group vitalities are expressed as mean ± SD.

Cell Fractionation

For determination of enzyme activities, only freshly isolated type II pneumocytes were used. To obtain the cytosol and the microsome compartment, cells were fractionated (16). In brief, isolated type II cells were homogenized in 150 mM NaCl/5 mM Tris/HCl, pH 7.4, for determination of choline kinase, or in 150 mM NaCl/6.6 mM ethylenediamine tetraacetic acid (EDTA)/20 mM Tris/HCl, pH 7.4, for determination of cytidylyltransferase. Homogenization was done with 60 strokes of a tight-fitting pestle in a Dounce glass/glass homogenizer. The homogenate was centrifuged for 5 min at 10,000 × g, after which the resulting pellet was discarded. The supernatant was recentrifuged for 8 min in a Beckman Airfuge (Beckman, Munich, Germany) at 130,000 × g. The supernatant was designated "cytosol fraction" and the pellet "microsomal fraction."

Synthesis of Phosphatidylcholine in Freshly Isolated Type II Cells

Phosphatidylcholine synthesizing enzymes. To test the effects of NO2 exposure modes on phosphatidylcholine (PC) synthesis, the choline kinase (EC 2.7.1.3.2) and cytidine triphosphate (CTP):cholinephosphate cytidylyltransferase (EC 2.7.7.15) of the cytidine-5-diphosphate (CDP)-choline pathway were analyzed in freshly isolated type II cells.

Choline kinase activity was measured according to the phosphocholine formation method (16, 17). In brief, enzyme activity was determined by conversion of [Me-14C]choline to [14C]phosphocholine. The assay buffer contained 50 mM NaCl, 50 mM adenosine triphosphate (ATP), 67 mM Tris-HCl (pH 8.5), 100 mM MgCl2, 5 mM [Me-14C]choline, and 11 to 40 µg of cytosolic protein in a final test volume of 60 µl. Reaction was allowed to proceed for 10 min at 37°C and was then stopped by boiling the reaction mixture. Choline and phosphocholine were separated by paper chromatography, and the radioactivity in the phosphocholine was calculated by liquid-scintillation counting.

CTP:cholinephosphate cytidylyltransferase was assayed by formation of [14C]CDP-choline from [Me-14C]choline (18). In type II cell cytosol and microsomes, the enzyme was measured with and without 1 mM phosphatidylglycerol as activator; the final test volume was 100 µl and the protein content ranged from 10 to 37 µg for the cytosol fraction and from 8 to 11 µg for the microsome fraction. The incubation (10 min at 37°C) was terminated by boiling. CDP-choline and phosphocholine were separated by thin-layer chromatography, and the radioactivity in the CDP-choline was determined (19).

Choline uptake into isolated type II cells. Freshly isolated type II cells were plated at a density of 5 × 105 cells/ plate in DMEM with 10% FCS, containing 1 µCi 14C-choline, and were cultured for 4 h. After washing the cells (4 × 106) with Krebs-Ringer buffer (KRB), they were counted for radioactivity and their choline uptake was calculated.

Pool-size analysis of the choline-containing intermediate metabolic products. Determination of the pool sizes of CDP-choline and PC was done according to a procedure described elsewhere (20). Freshly isolated type II pneumocytes were homogenized in cold methanol/water (1:1, vol/vol) and extracted for phospholipids (21). After thin layer chromatography, the PC band was quantified for phospholipids (22). The aqueous phase, containing among other substances the choline and CDP-choline, was separated via anion-exchange chromatography on AG1-X18 with a linear gradient (0 to 0.03 M) ammonium bicarbonate solution. Quantification of the CDP-choline fraction was done with an enzymatic procedure (23).

Secretion of PC from Isolated Type II Cells

Secretion studies were performed after a 22-h primary culture period in which isolated type II pneumocytes synthesized PC from a [14C]choline precursor (Amersham Buchler, Braunschweig, Germany) (14). After this period the cells were washed, and serum-free medium was added without radioactivity. After a 30-min equilibration interval, secretagogues were added, followed by a 3-h secretion period. The medium containing the secreted material was then removed and the radioactivity of the extracted phospholipids was calculated as the percentage of total radioactivity from medium plus cells. The secretagogues used were 12- O-tetradecanoylphorbol-12,13-acetate (TPA, 10-7 M) and terbutaline (Terb, 10-6 M).

Other Methods

Protein was measured with the Bio-Rad (Munich, Germany) reagent (24), and DNA and phospholipid were calculated according to standard procedures (22, 25). Lactate dehydrogenase (LDH) activity was measured in cells and culture media by conversion of beta -nicotinamide adenine dinucleotide (beta -NAD) (26).

Statistical Analysis

For each sample from each exposure condition, assays were done in triplicate, and an average value was determined. Average values for each sample were then used for analysis. Results are expressed as mean ± SD. Statistical analysis was done with one-way analysis of variance (ANOVA) and consecutive Scheffé's tests. Values of P < 0.05 were considered significant.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Yield and Vitality

Isolation of type II pneumocytes showed an increased yield of cells for those experimental groups that were exposed to NO2. The data for the cell yield had to be adjusted to body weight (BW), since during the experiments animals gained body weight to different extents, depending on age and mode of NO2 application. Although continuous exposure provoked a significant (P =< 0.01) increase in cell yield (21.7 ± 4.3 × 106/100 g BW, n = 12) compared with the control group (13.3 ± 4.1 × 106/100 g BW, n = 12), intermittent and repeated exposure also increased the yield, although not to a significantly different degree from that of controls (intermittent: 14.8 ± 4.6 × 106/100 g BW, n = 10; repeated: 12.5 ± 3.7 × 106/100 g BW, n = 8).

Vitality of freshly isolated type II pneumocytes was determined by trypan blue dye exclusion and was found to be decreased in cells from the rats exposed under different conditions. The difference from cells of control animals was, however, not significant for the repeated-exposure group (Figure 1). Microscopic inspection of the isolated type II cell preparations showed a maximum of only 5% of cells that differed from the characteristic appearance of type II pneumocytes.


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  		Figure 1.   Cell yield and vitality of freshly isolated type II pneumocytes from rats after exposure to a 720 ppm × h body dose of NO2 applied with different modes (i.e., continuous: 10 ppm for 3 d; intermittent: 10 ppm 8 h per day for 9 d; repeated: 10 ppm for 3 d and 28 d rest, 10 ppm for 3 d). The open bars represent the vitality of the freshly isolated cells; the solid bars represent the cell yield per 100 g body weight. Significant increase of cell yield was observed after continuous NO2 exposure; **P < 0.01. Viability was comparable for all groups.

Enzyme Analyses

Exposure to NO2 atmospheres significantly enhanced the activities of the choline kinase in the continuous (7.6 ± 0.8 nmol × mg-1min-1, n = 6) and intermittent (6.9 ± 0.9 nmol × mg-1min-1, n = 6) exposure groups compared with normal air-breathing controls (5.4 ± 0.6 nmol × mg-1min-1, n = 10). Animals that were repeatedly exposed to NO2 showed kinase activities that were comparable to those of normal controls (5.5 ± 0.8 nmol × mg-1min-1, n = 6) (Figure 2).


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  		Figure 2.   Choline kinase activity (nmol × mg-1 min-1) of freshly isolated type II pneumocytes after exposure to a 720 ppm × h body dose of NO2 applied in different modes. Both continuous and intermittent exposure elevated the specific enzyme activity; **P < 0.01 and *P < 0.05, respectively. After repeated exposure, the enzyme activity was unchanged as compared with the control.

The CTP:cholinephosphate cytidylyltransferase, which catalyzes the rate-limiting step in the CDP-choline pathway, showed changes in activity similar to those of the choline kinase for the different NO2 application modes. In the microsome fraction, the enzyme activities were comparable for all modes of NO2 application (range: 2.04 to 2.30 nmol × mg-1min-1). Evaluation of the cytosol fraction in the presence of phosphatidylglycerol (PG) as activator also showed comparable values for all experimental groups and for the control (range 2.14 to 2.39 nmol × mg-1min-1) (data not shown). Evaluation of the cytosol fraction without PG exhibited for the continuous-exposure group an increase in CTP:cholinephosphate cytidylyltransferase specific activity that is significantly twice as high as for the control group (0.97 ± 0.23 nmol × mg-1min-1, n = 6 versus 0.53 ± 0.25 nmol × mg-1min-1, n = 10). Although there was a clear increase in the activity of this enzyme in the intermittent and the repeated exposure groups (0.70 ± 0.23 nmol × mg-1min-1, n = 5 and 0.80 ± 0.42 nmol × mg-1min-1, n = 5), the increase was not significantly different from the control value (Figure 3).


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  		Figure 3.   CTP:cholinephosphate cytidylyltransferase activity (nmol × mg-1min-1) of freshly isolated type II pneumocytes after exposure to a 720 ppm × h body dose of NO2 applied in different modes. Whereas the microsomal fraction of this enzyme (solid bars) exhibited no change in enzyme activity after the different NO2 application modes, the activity of the enzyme was significantly elevated. *P < 0.05 in the cytosomal fraction (open bars) after continuous exposure.

In addition to evaluating the activities of phospholipid-synthesizing enzymes, we also evaluated uptake of phospholipid precursor choline and formation of the intermediate metabolic product CDP-choline, as well as the final PC pool size. To allow a comparison on an absolute level, the data are adjusted to µg DNA. A significant increase was found in choline uptake after continuous inhalation of NO2 atmospheres as compared with the control group (422.5 ± 50.3 pmol × µg DNA-1, n = 8, versus 319.6 ± 42.5 pmol × µg DNA-1, n = 6). The choline precursor uptake was also significantly enhanced with the intermittent NO2 inhalation mode (392.4 ± 33.0 pmol × µg DNA-1, n = 6), whereas the repeated exposure group showed no difference from the control (349.6 ± 73.7 pmol × µg DNA-1, n = 6) (Figure 4).


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  		Figure 4.   Choline uptake of isolated type II cells (nmol/µg DNA) after exposure to a 720 ppm × h body dose of NO2 applied in different modes. Continuous as well as intermittent exposure modes resulted in a significant elevation of the choline precursor uptake; **P < 0.01 for the continuous exposure and *P < 0.05 for the intermittent exposure. No change of uptake was observed after repeated exposure.

The formation of CDP-choline is the prerequisite for the synthesis of PC, and was found to be higher for all NO2 application modes than for normal air-breathing animals. As with the results for CTP:cholinephosphate cytidylyltransferase activity, the pool size of CDP-choline was also significantly increased for the continuous-exposure group (542.2 ± 119.6 pmol × µg DNA-1, n = 6; control: 304.3 ± 102.4 pmol × µg DNA-1, n = 6). Intermittent (402.7 ± 98.8 pmol × µg DNA-1, n = 6) and repeated NO2 application (419.5 ± 95.1 pmol × µg DNA-1, n = 6) also enhanced the CDP-choline pool size, although the difference was not significantly different from the control value (Figure 5).


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  		Figure 5.   CDP-choline pool size in isolated type II cells after different modes of NO2 application. The intermediate metabolic product showed an increased pool size only after the continuous NO2 application mode; **P < 0.01.

The pool sizes of the final PC product showed the same pattern as the intermediate metabolic product CDP-choline. Measurements revealed the largest PC pool size in type II cells after continuous inhalation of the oxidant atmosphere (14.5 ± 2.2 nmol × µg DNA-1, n = 6, versus control: 9.8 ± 0.9 nmol × µg DNA-1, n = 6). The pneumocytes in the intermittent- and repeated-exposure groups also had a larger PC pool size than in the control group (intermittent: 12.3 ± 1.7 nmol × µg DNA-1, n = 6; repeated: 12.0 ± 1.3 nmol × µg DNA-1, n = 6); however the difference was not significant as compared with the control value (Figure 6).


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  		Figure 6.   Formation of phosphatidylcholine (PC) in isolated type II cells after different modes of NO2 application. All NO2 application modes provoked a significant increase in the PC pool size as compared with cells from control; **P < 0.01, *P < 0.05.

Plating Efficiency and LDH Activity in Primary Culture of Type II Pneumocytes

After overnight culture of type II cells (22 h), plating efficiency was comparable for all cultures from the differently NO2-exposed animals (range: 82 to 85%). Determinations of LDH activities in the culture media were made at the end of the secretion experiments, and clearly showed that they constituted less than 3% of total cellular LDH activity, thus representing no cell degeneration.

Secretion Studies

Besides surfactant synthesis, the other important metabolic function of type II cells is surfactant secretion. Since basal surfactant secretion in primary culture of type II pneumocytes is very low, TPA (10-7 M) and Terb (10-6 M) were used as secretagogues in order to produce clearer effects. Secretion rates were corrected for the basal secretory activity, and for type II pneumocytes from controls showed a secretory activity of 10.2 ± 2.5% (n = 14). The continuous-exposure mode significantly decreased the TPA-modulated secretory activity to 5.8 ± 1.7% (n = 8). A reduced secretory activity was also found for the intermittent- (7.9 ± 2.4%, n = 8) and repeated-exposure (7.9 ± 1.1%, n = 8) treatments. Treatment with the beta -agonist Terb increased the secretion rate to 3.35 ± 1.6% (n = 14) for cells from the control group. This secretion rate was also determined for cells from the repeated-exposure group (3.0 ± 0.9%, n = 8), whereas for cells after intermittent inhalation treatment, a slightly decreased secretory activity was determined (2.8 ± 1.0%, n = 8). The most prominent effect was again observed after continuous NO2 exposure, after which the isolated type II cells exhibited significantly reduced secretory activity (1.7 ± 0.4%, n = 8) (Figure 7).


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  		Figure 7.   Stimulated secretion of phosphatidylcholine (PC) from isolated type II cells in primary culture after different modes of NO2 application. Both terbutalin (10-5 M) (open bars) and TPA (10-7 M) (solid bars) significantly reduced the secretory activity of the cells from the continuous-exposure group; **P < 0.01. Cells from the intermittent- and repeated-exposure groups did not show significant differences in secretion activity as compared with cells from controls.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Toxicology of NO2 has gained much interest because this oxidant is a major anthropogenic air pollutant derived from manufacturing and traffic in the outdoor environment and from cigarette smoke in the indoor environment (5). The toxicology of NO2 has also attracted additional interest because of the application of nitrogen oxide for pulmonary vasodilatation, which under certain circumstances, produces measurable amounts of NO2 (9). Inhalation of NO2 is known to cause alterations of lung structure and function, depending on its atmospheric concentration and the duration of its inhalation (5, 12, 27, 28). One prominent structural observation is the degeneration of type I pneumocytes and their replacement by type II pneumocytes (10, 11). However, it is not known whether exposure to identical body doses of NO2 applied with different modes (continuous, intermittent, repeated) has differing effects on the phospholipid metabolism of type II cells, representing adaptive reactions to NO2 atmospheres.

From earlier studies, it is known that low NO2 concentrations or short exposure periods do not cause significant alterations in surfactant PC synthesis (29). Although the NO2 concentration used in the present study (10 ppm) appears to be high in relation to environmental conditions, there are circumstances (e.g., smoking) in which NO2 concentrations are considerably higher (6, 7). However, the 10 ppm NO2 atmosphere was chosen because earlier studies have shown that it guarantees injury of the respiratory epithelia and, after cessation, allows epithelial recovery (30).

In our experiments, evidence of an increased proliferation of type II pneumocytes was provided by the cell yields of type II pneumocytes from the differently exposed animals. The absolute cell yield was significantly greater for all experimental groups as compared with the control group (data not shown). However, because the rats gained body weight to different extents depending on the experimental protocol and the animal's age, the cell-yield data had to be corrected for body weight. With this adjustment, cell yield was significantly higher only after continuous exposure, whereas intermittent exposure produced no significant increase and repeated exposure produced only a slight decrease in cell yield compared with the control value. Type II cell vitality as determined by trypan blue dye exclusion showed comparable values for all exposure groups, and did not differ from the control value. Also, plating efficiency and LDH release did not differ between the exposure groups. Therefore, it was not necessary to adjust the data from metabolic studies to cell vitality. From this observation we conclude that in the present study, the mode of exposure obviously had no influence on vitality of isolated type II cells.

As an initial measure of the metabolic activity of isolated type II pneumocytes, we analyzed the synthesis of PC via the CDP-choline pathway. All enzyme activities and pool sizes of intermediate metabolic products measured in these experiments were found to be in the ranges reported in the literature (16, 20, 31). Acute exposure to 10 ppm NO2 applied continuously for 72 h is known to enhance the specific activity of type II cells' choline kinase (29). This effect was confirmed in our study. In addition, we found that after intermittently applied NO2, the specific activity of the choline kinase declined but was still significantly different from the control value. In the repeated-exposure experiments, however, the enzyme activity was no longer significantly different from that of the control. Although a higher lipid-synthesizing capacity has been reported after recovery from acute exposure to relatively high NO2 concentrations (35), no information was given in these reports for the activity of choline kinase.

In the CDP-choline pathway, CTP:cholinephosphate cytidylyltransferase is a very important enzyme because it is thought to regulate the rate-limiting step in the synthesis of PC (20). According to the translocation hypothesis, the active form of this enzyme is thought to be located in the microsomal fraction, at least for hepatocytes (39). In the present study, the microsomes exhibited a higher activity of CTP:cholinephosphate cytidylyltransferase than did the cytosol fraction. Exposure-mode-dependent activity of this enzyme, however, was only observed in the cytosol, which exhibited a significantly higher specific activity after continuous exposure than the control value. Intermittent and repeated exposure also produced a higher CTP:cholinephosphate cytidylyltransferase activity in the cytosol, although this was not significantly different from the control. Therefore, these data make it questionable whether the translocation hypothesis is valid for type II pneumocytes. Other authors have reached similar conclusions (39). Since the enzyme was measured under optimal conditions, which did not produce a further increase when PG was used as an activator in the cytosol fraction, this suggests that the increased activity of CTP:cholinephosphate cytidylyltransferase is due to exposure-mode-dependent activation of existing enzyme, rather than synthesis of new protein. Expression studies, however, are necessary to confirm this assumption. Although the content of either enzyme was not analyzed within the type II pneumocytes, it is hypothesized that the increase in specific activity of these phospholipid-synthesizing enzymes represents a repair or adaptational activity of type II cells. This activity clearly depends on the mode of NO2 application.

We analyzed the activities of the PC-synthesizing enzymes at the same time (i.e., directly after cell isolation) as we quantified the pool sizes of CDP-choline and PC. For CDP-choline, as well as PC, the exposure modes determined the magnitude of pool-size enhancement, which is in accord with an increased activity of choline kinase and CTP:cholinephosphate cytidylyltransferase.

Whereas continuous NO2 application significantly increased the pool size of both metabolites, the continuous- and repeated-exposure modes produced significantly higher values as compared with control cells. It is possible that NO2-exposed type II cells do not necessarily produce increased PC only for surfactant phospholipid production, but also for incorporation into membrane phospholipids. Since in type II cells from the differently NO2-exposed animals the pool sizes of PC were much larger than the pool sizes of CDP-choline, the rate-limiting role of CTP:cholinephosphate cytidylyltransferase also seems obvious for type II cells from the differently NO2-exposed animals.

For determination of phospholipid precursor uptake, a 3-h incubation period is known to guarantee an isotopic equilibrium in the precursor pool (16). Choline incorporation into type II pneumocytes followed the activity of choline kinase in the differently applied NO2-exposed groups in our study. Whereas cells from continuously and intermittently exposed animals showed a higher choline incorporation, cells from animals subjected to repeated NO2 exposure exhibited the same uptake as control cells.

The second metabolic function of type II pneumocytes is to secrete phospholipids into the alveolar lumen. This activity was tested on isolated type II cells and found to be affected differently by the different NO2 application modes. However, this was detected only after Terb and TPA stimulation, with no loss of NO2-induced alteration during primary culture. The possibility that the basal secretion was different from the control could not be excluded, but despite the very low basal secretory activity, differences could not be detected with the methods used (29). As we have reported earlier, phospholipid secretion from isolated type II cells was significantly decreased after continuous NO2 exposure (43). In addition to this, the results from this study provide evidence that intermittent and repeated exposure does not significantly change the cells' stimulated phospholipid-secretory activity.

The graduation of enzyme activities, choline incorporation, pool sizes of intermediate metabolic products, and secretion activity suggest different reactions of type II cells to the different modes of NO2 application used in the present study. It is known that type II pneumocytes react to acute NO2 exposure by enhancing their proliferation. At this acute stage in lung injury by NO2, the capacity to produce surfactant phospholipids is greater because of the increase in type II cells and/or the reported increase in specific activity of phospholipid-synthesizing enzymes (10, 11, 29, 38). We conclude that the early phase of acute alveolar epithelial damage is followed in vivo by an adaptive or recovery phase in which type II cells proliferate.

After intermittent and repeated exposure to NO2, the metabolic activity of the isolated type II cells in our study seemed to be reduced. It is obvious that all oxidant-induced signs of lung injury are paralleled by repair processes that are less prominent than with acute injury (44). It is also well known that there are overall antioxidative protective systems that can defend lung cells against cytotoxic substances and free-radical-induced damage (47). Additionally, during intermittent and repeated modes of in vivo exposure, antioxidative mechanisms may be developed to prevent type II pneumocytes from direct effects of the oxidant (50). For example, studies of type II cells from hyperoxic lungs have shown increased PC synthesis after exposure to 60% oxygen but reduced PC synthesis after 90% oxygen (51). However, there are also adaptation reactions to oxidants that fail to produce an increase in antioxidant systems (54).

The present study leads to the general suggestion that type II pneumocytes have the potential to react differently to different modes of NO2 application. We conclude that the mode of NO2 application is a dominant determinant of the reaction of the type II pneumocyte. Preliminary analyses of BAL specimens have shown that lung lavage protein also depends on the NO2 application mode, thus indicating its influence on alveolar barrier function (unpublished results). Additional studies will be needed to elucidate whether these suggested adaptive reactions are accompanied by detoxifying processes such as the induced expression of antioxidative enzymes and/or increases in their enzymatic activities. This may provide further insight into mechanisms of NO2 toxicology and the adaptation of lung cells to NO2 exposure.

    Footnotes

Address correspondence to: Bernd Müller, Laboratory of Respiratory Cell Biology, Department of Internal Medicine, Philipps University of Marburg, 35033 Marburg, Germany. E-mail: bmueller{at}mailer.uni-marburg.de

(Received in original form July 16, 1997 and in revised form October 13, 1997).

Acknowledgments: The authors are indebted to Ms. F. Koerner and Mrs. C. Skurk for their valuable technical assistance. The study was funded by the VERUM foundation.

Abbreviations NO2, nitrogen dioxide; PC, phosphatidylcholine; PG, phosphatidylglycerol; RA, room air; Terb, terbutalin; TPA, 12-O-tetradecanoylphorbol-12,13-acetate.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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