Introduction

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Lung surfactant is a complex composed predominately of phospholipids and apoproteins that is produced and secreted by the alveolar type II cell. Phosphatidylcholine (PC) comprises approximately 60% of the phospholipid in lung surfactant and is the predominant surface-active phospholipid. Approximately 60% of the PC present in lung surfactant is in the form of disaturated PC (DSPC), of which the major component is dipalmitoyl PC (DPPC). The principal pathway for de novo synthesis of DSPC in the lung involves production of cytidine diphosphocholine (CDP-choline), formed from choline by the sequential action of choline kinase (EC 2.7.1.32) and cytidine triphosphate (CTP):phosphocholine cytidylyltransferase (CT) (EC 2.7.7.15). Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) subsequently catalyzes the formation of DPPC from dipalmitoylglycerol and CDP-choline. Precursors of dipalmitoylglycerol are glucose, contributing the glycerol backbone, and palmitic acid (PA) derived either from de novo synthesis or uptake of free fatty acids. Very low-density lipoproteins (VLDL) in the presence of lipoprotein lipase (LPL) are a potent source of long-chain fatty acids. Both the lipolytic activity of LPL and LPL-induced VLDL catabolism by type II cell lipoprotein receptors may be of importance (1).

Control of the de novo production of PC may thus reside at a number of synthetic steps. As reviewed by Van Golde and colleagues (2) and Possmayer (3), several indirect lines of evidence suggest that CT is of critical importance in regulation of DPPC synthesis. These include pulse- label experiments and determination of precursor and product pool sizes (4, 5), and correlation between CT activity and DPPC synthesis during fetal development (6) or after exposure to various hormones (7).

Relatively little is known of lung CT, although understanding of this enzyme may lead to strategies for manipulating production of endogenous PC. In contrast, rat and human liver CT complementary DNAs (cDNAs) have been cloned, sequenced, and expressed in COS cells (8, 9). The proposed catalytic domains are 100% identical, and the predicted and observed molecular weights of the expressed protein are approximately 42 kD. The murine CT gene has been sequenced, but little is yet known of the regulation of expression (10). Mechanisms by which CT activity is regulated may include: reversible phosphorylation (11); translocation from cytosol to microsomal membranes (12); enhanced gene expression (13); regulation by palmitate or other fatty acids (14), cholesterol (15), or VLDL (1); and inhibition by sphingosine (16).

To test directly the hypothesis that CT is both a rate-limiting and regulatory enzyme in the biosynthesis of PC, we used rat liver CT (rCT) cDNA to transfect rat alveolar type II cells in primary culture or relevant cell lines (A549 or H441) derived from alveolar epithelium. CT expression and activity were thereby increased. DSPC synthesis was increased in transfected rat alveolar type II cells under basal conditions and was further increased after exposure to dexamethasone. DSPC synthesis was not increased in the cell lines tested. These data directly confirm the hypothesis that, in mature rat alveolar type II cells, CT is rate-limiting and regulatory in the de novo production of PC.

	Materials and Methods

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Cell Culture

Cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured at 37°C in 5% CO₂/95% air. A549 cells were maintained in F12K media and H441 cells were maintained in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin (100 µg/ml-100 mg/ ml). Primary rat type II cells were isolated by the method of Dobbs and associates (17) and cultured in six-well plates (Falcon Primaria plates; Becton Dickinson, Franklin Lakes, NJ). At the time of isolation, cells were 85% type II cells as assessed by polychrome/tannic acid staining to highlight lamellar bodies; after 3 d cell purity was at least 95%, as contaminating macrophages were removed during media changes. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) free of phenol red with 10% FBS and penicillin-streptomycin at 37°C in 10% CO₂/90% air.

Plasmid Preparation

rCT cDNA (9) in pAX-142 plasmid (a generous gift of Dr. R. Cornell, Simon Fraser University, Burnaby, BC, Canada) and pAX-142 without insert were propagated in DH5-P3 Escherichia coli. The pCMV expression vector containing the lacZ insert (Clontech Laboratories, Palo Alto, CA) was propagated in DH5 E. coli. Plasmid preparations were isolated using a Qiagen Plasmid Maxi Kit (Qiagen, Inc., Santa Clarita, CA). The rCT cDNA insert was recovered by restriction with Sal1 and was demonstrated to be of the expected size (1.26 kb) by gel electrophoresis. Negative control observations were made using pAX-142 without insert and using nontransfected cells.

Cell Transfection

A549 or H441 cells were split the day before transfection and plated at a density of 0.5 or 1.0 × 10⁶ cells/60-mm dish. Cells were then transfected for 12 h using 5 µg Lipofectin (Bethesda Research Laboratories, Gaithersburg, MD) and 10 µg pAX-rCT DNA (unless otherwise stated) and incubated for a subsequent 60 h. These conditions were chosen after performing preliminary experiments in which transfection conditions for A549 cells were varied by altering the ratio of Lipofectin to plasmid DNA. Using 5 µg Lipofectin and 0 to 20 µg pAX-rCT DNA, maximal CT-specific activity was detected when 10 or more µg plasmid DNA was transfected (data not shown). In addition, the time course of CT specific activity expression was investigated by incubating cells for 12 h with 5 µg Lipofectin/10 µg plasmid DNA and harvesting and fractionating cells at various times up to 72 h after transfection (Figure 1). CT activity of both cytosolic and particulate fractions at times 36 h or more after transfection were significantly higher than control activities. Consequently, observations were subsequently made on cells cultured for 60 h after transfection.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Time course of CT activity in A549 cells after transfection. A549 cells were transfected with pAX-rCT as described. Cells were harvested and fractionated at the times indicated, and CT activity was measured in cytosolic and particulate fractions. CT activities 36 h or more after transfection were significantly greater than control. Results are the means of three experiments.

Rat type II cells at a density of approximately 1.5 × 10⁶/ 35-mm plate were transfected immediately after isolation for 8 to 10 h using 5 µg Lipofectin and 2 µg cDNA and harvested 60 h later. For staining to disclose -galactosidase activity, cells were incubated for 48 h after transfection and stained in situ with X-gal (18).

Cell Fractionation

At the time of harvest, cells were washed once with iced phosphate-buffered saline (PBS: 150 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄ · 7 H₂O, and 1.4 mM KH₂PO₄, pH 7.4), collected by scraping into 1 ml PBS, centrifuged (250 × g for 10 min), and resuspended in 100 µl homogenizing buffer (50 mM imidazole, 150 mM KCl, and 2 mM ethylenediaminetetraacetic acid [EDTA], pH 7.4). The suspension was sonicated with three 5-s pulses using an ultrasonic cell disrupter (60% peak output) with microtip (Heat Systems-Ultrasonics, Inc., Plainview, NY) and centrifuged at 100,000 × g for 60 min. Supernatants, representing the cytosolic fraction, were collected and the pellet, representing the particulate membrane or particulate fraction, was resuspended in homogenizing buffer in a volume equal to that of the supernatant. All steps were performed at 4°C. Cell-fraction protein was determined by the method of Bradford using the Pierce protein assay kit (Pierce, Inc., Rockford, IL) with bovine serum albumin (BSA) as a standard.

Immunoblot Detection of Protein

Cytosol or particulate fraction proteins (30 µg) were separated electrophoretically on 12% (wt/vol) sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Inc., Bedford, MA). After incubation with 5% blotting grade blocker (Bio-Rad, Hercules, CA) in buffer A (24 mM Tris and 0.5 mM NaCl, pH 7.4) for 2 h at 24°C, the membrane was then incubated in buffer B (buffer A plus 3% blocker, 0.1% Tween-20, and 0.1% BSA) with anti-rat CT serum (1:5,000) (a generous gift of Dr. C. Kent, University of Michigan, Ann Arbor, MI) for 18 h at 24°C. After washing three times with 0.1% Tween-20, the membrane was incubated with donkey antirabbit immunoglobulin G-horseradish peroxidase (1:10,000) in buffer B for 90 min, then washed twice with 0.3% Tween-20 in buffer A and twice with 0.1% Tween-20 in buffer A. The membrane was soaked in Amersham ECL reagent (Amersham, Inc., Arlington Heights, IL) for 2 min and exposed to X-ray film for up to 5 min.

Type II cells were studied under both resting and stimulated conditions. PA or dexamethasone was used as stimulant, as PA is reported to increase the membrane-bound CT activity (19, 20), and glucocorticoids stimulate CT activity through effects mediated by fatty acids (16). Type II cells were transfected and cultured as described with additions of 100 µm PA or 0.25 µm dexamethasone. PA was dissolved in ethanol which was added (1:1,000 vol/vol) to culture media. Additions were made immediately or 40 h after the transfection period. At 60 h after the transfection period all cells, including control cells, were harvested. Thus, in all experiments, type II cells were cultured for identical lengths of time. Protein (10 µg) from each fraction was separated electrophoretically and CT protein was detected by immunoblot.

Enzyme Activity

CT activity was assayed using a modification of the method of Weinhold and coworkers (21). Each fraction was assayed in a reaction mixture of 100 µl containing 20 to 60 µg sample protein, 1.6 mM phosphoryl-[¹⁴CH₃]choline (1,390 dpm/nmol), 3.0 mM CTP, 12.0 mM MgCl₂, 2.0 mM EDTA, 60 to 100 mM NaCl, 0.5 mM PC-oleic acid (1:1 molar ratio), and 65 mM Tris, pH 7.5. The reaction proceeded at 37°C for 30 min and was terminated by addition of 100 µl iced stop solution (150 mM phosphocholine and 10% vol/ vol trichloroacetic acid). Charcoal (500 µl of a 6% suspension in H₂O) was added to the mixture. After 30 min incubation the mixture was centrifuged (250 × g for 2 min), the supernatant was removed, and the charcoal was washed four times with 1.0 ml H₂O. The charcoal was then extracted three times with 1.0 ml extraction solution (EtOH: NH₄OH:H₂O, 190:11:118 by volume). The pooled extracts were added to 10 ml scintillation solution (EcoScint; National Diagnostics, Atlanta, GA) and counted. For each assay batch, duplicate blank samples were assayed and recovery was determined by assay of samples containing a known amount of [¹⁴CH₃]choline. Recoveries were in the range of 60 to 80%. All data were corrected for background and recovery.

Cell Production of DSPC

A549 or H441 cells were washed three times with PBS and then loaded with methyl-[³H]choline by incubation with 5 µCi [³H]choline (82 Ci/mmol) for 1 h in 2 ml F12K (containing 100 nmol choline/ml) or RPMI 1640 media (containing 22 nmol choline/ml), respectively. Media contained 0.1% fatty acid-free BSA. Alternatively, rat type II cells that had been washed three times with PBS were loaded with [³H]choline by incubation with 2.5 µCi [³H]choline for 3 h in 1 ml DMEM (containing 28.5 nmol choline/ml) and 0.1% fatty acid-free BSA. Subsequently, cells were washed three times with PBS and cultured for various lengths of time in the appropriate media. At the time of harvest, media were aspirated and cells were scraped into 1 ml distilled water. Cells were sonicated, and aliquots were reserved for protein assay. Total lipid from cell sonicates was extracted (22). DSPC was isolated as described by Mason and associates (23) and radioactivity was counted as described. Production of DSPC was determined after calculating specific activity of the labeling media and is expressed as nmol DSPC/mg cell protein.

Data Analysis

Values are presented as means ± standard error of the mean. Differences among multiple groups were assessed using one- or two-way analysis of variance (ANOVA) as appropriate, with post hoc testing using Student-Neuman- Keuls test or Bonferroni t test. Differences between specified groups were assessed using Student's t test. Values of P < 0.05 are described as significant.

	Results

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Transfection Efficiency

The efficiency of transfection was assessed semiquantitatively by visual assessment of cells stained with X-gal to detect -galactosidase activity, and quantitatively by determination of CT specific activity. X-gal staining of alveolar type II cells revealed that 19 ± 8% (n = 3 observations) of cells exhibited blue staining, consistent with transfection with pCMV.

CT Protein

Enzyme protein was detected by immunoblot in both A549 and H441 cells after transfection with pAX-rCT (Figure 2). In contrast, rCT protein in both cytosolic and particulate fractions of control-transfected A549 and H441 cells was beneath the detection limits of the assay method. Type II cell CT protein was also detected in cells transfected with pAX-rCT and in control cells exposed to dexamethasone (Figure 3). Qualitatively, more CT protein appeared to be associated with the particulate fraction after exposure for 20 h to palmitate or dexamethasone.

View larger version (33K): [in this window] [in a new window]   Figure 2.   Immunodetection of CT protein in transfected A549 and H441 cells. A549 and H441 cells were transfected as described, harvested after 60 h, and fractionated; and 30 µg protein of each fraction was separated electrophoretically under reducing conditions, blotted, and immunostained as described. Results from A549 cells are in lanes 1-4; those from H441 cells are in lanes 5-8. Fractions from cells transfected with pAX-rCT are in lanes 3, 4, 7, and 8; and those from cells transfected with pAX-142 (negative control) are in lanes 1, 2, 5, and 6. Cytosolic fractions are in lanes 1, 3, 5, and 7. Particulate fractions are in lanes 2, 4, 6, and 8. Results are representative of two experiments. Only in cells transfected with pAX-rCT was CT protein detected.

View larger version (80K): [in this window] [in a new window]   Figure 3.   Immunodetection of CT protein in stimulated or resting transfected rat type II cells. Rat type II cells were transfected and cultured for a subsequent 60-h period. (A) Results from exposure of cells to 100 µM PA for 20 h. (B) Results from exposure of cells to 0.25 µM dexamethasone for 20 h. C and P denote cytosolic and particulate fractions, respectively. PA and DX denote exposure to PA or dexamethasone, respectively. Control and rCT denote transfection with pAX-142 or pAX-rCT, respectively. Both total CT protein and the fraction present in the particulate compartment are increased after exposure to PA or dexamethasone. CT protein is detected in control-transfected cells exposed to dexamethasone. Results are representative of two experiments.

CT Activity

To determine the location of CT activity in cells transfected with pAX-rCT or control plasmid, A549 or H441 cells were transfected as described and, after 60 h, harvested and fractionated. CT activity was measured in cytosolic and particulate fractions and found to be augmented significantly (P < 0.001) in both fractions in both cell lines for cells transfected with pAX-rCT (Figure 4). The ratio of activity in the particulate fraction to that in the cytosolic fraction was not significantly altered in cells transfected with pAX-rCT compared with control-transfected cells.

View larger version (29K): [in this window] [in a new window]   Figure 4.   CT activity in cytosolic and particulate fractions of A549 and H441 cells. A549 cells or H441 cells were transfected with pAX-142 (pAX) or pAX-rCT (rCT) as described, harvested after 60 h, and fractionated. CT activity was measured as described. The CT activity of both cytosolic and particulate fractions from both cell lines was significantly augmented as a result of transfection with pAX-rCT. Results are the means of three experiments.

CT activity was studied in both stimulated and unstimulated rat type II cells. Study of the time course of the increase in CT activity after transfection (Figure 5) confirmed that activity was significantly increased relative to control cells after 36 h or more and appeared to be maximal after 60 h. CT activity of nontransfected cells or of pAX-transfected cells did not increase during the incubation period. CT activity in all subsequent studies of type II cells was measured at the 60-h time point.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Time course of CT activity in type II cells after transfection. Type II cells were transfected with pAX-142 (pAX) or pAX-rCT (rCT) as described, harvested, and fractionated at the times indicated after transfection; and CT activity was measured in cytosolic and particulate fractions. Control (nontransfected) type II cells (con) were cultured for equivalent lengths of time. CT activities 36 h or more after transfection with rCT were significantly greater than control values. Results are the means of three experiments.

Cells were transfected as described, exposed to 100 µM PA for the final 20 or 60 h (of the 60-h post-transfection incubation), harvested, and fractionated, and CT activity in the cell fractions was measured (Figure 6A). Transfection with rCT increased CT activity significantly (in both cytosolic and particulate fractions), independent of the effect of PA (two-way ANOVA). Exposure to PA resulted in significantly augmented CT activity in the particulate fraction of control-transfected (20-h exposure) compared with control-transfected unexposed cells and in the particulate fraction of rCT-transfected cells (60-h exposure) compared with rCT-transfected unexposed cells. Although the mean values for CT activity in other particulate fractions rose with PA exposure, the increase did not reach statistical significance.

View larger version (18K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 6.   CT activity in cytosolic and particulate fractions of type II cells under resting or stimulated conditions. Type II cells were transfected with pAX-142 (pAX) or pAX-rCT (rCT) and subsequently exposed for the times indicated to either 100 µM PA (A) or 0.25 µM dexamethasone (DX) (B). Nontransfected cells (Control) were cultured for equivalent lengths of time. Cells were harvested and fractionated, and CT activity was measured. Transfection with rCT increased CT activity significantly, independent of exposure to PA or DX. Results are the means of four experiments. *P < 0.05 relative to comparable pAX fraction. **P < 0.05 relative to comparable rCT fraction.

Type II cells were exposed to 0.25 µM dexamethasone for the final 20 h of the 60-h incubation period and were processed as described. Transfection with rCT increased CT activity significantly (in both cytosolic and particulate fractions), independent of the effect of dexamethasone (two-way ANOVA). Dexamethasone exposure resulted in a significant increase in CT activity in the particulate fraction of control-transfected cells (compared with unexposed control-transfected cells) and in both the cytosolic and particulate fractions of rCT-transfected cells (compared with unexposed rCT-transfected cells) (Figure 6B). In the presence of dexamethasone, CT activity increased 33% in the cytosolic fraction and 31% in the particulate fraction of rCT-transfected cells, whereas CT activity increased 55% in the particulate fraction of control-transfected cells. The 13% increase in CT activity in the cytosolic fraction of control-transfected cells with exposure to dexamethasone was not significant.

To further explore the stimulation of CT activity by exposure to dexamethasone, cells were exposed for the final 20 h of the 60-h post-transfection period to concentrations of dexamethasone ranging from 2.5 × 10¹² to 2.5 × 10⁶ M. Results, shown in Figure 7, confirm the effect of dexamethasone exposure on CT activity of type II cells. CT activity of cytosolic and particulate fractions was significantly increased after exposure of cells to concentrations greater than or equal to 10¹⁰ and 10⁸ M, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 7.   CT activity in type II cells exposed to various concentrations of dexamethasone. Type II cells transfected with pAX-142 (pAX) or pAX-rCT (rCT) were exposed for 20 h to concentrations of dexamethasone ranging from 2.5 × 1012 M to 2.5 × 106 M and whole-cell CT activity was determined. Results are the means of three experiments. *P < 0.05 relative to unexposed cells.

Cell Production of DSPC

To determine whether augmented CT activity resulted in enhanced production to DSPC, A549 or H441 cells transfected with either pAX-rCT or control plasmid were pulsed with methyl-[³H]choline as described to label the intracellular phosphocholine pool, washed, and incubated in media containing 20 µM choline chloride. At times from 0 to 90 min cells were harvested and [³H]DSPC was recovered and counted. The incorporation of the choline label into DSPC during the choline chase period occurred in both cell types transfected with either pAX-rCT or pAX-142. However, as shown in Figures 8A and 8B, there was no significant difference in the rate of incorporation between cells transfected with pAX-rCT and control-transfected cells.

View larger version (12K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 8.   Synthesis of DSPC by transfected cells. A549 cells (A), H441 cells (B), and rat type II cells (C) were transfected with pAX-rCT or pAX-142 (negative control), pulsed with 2.5 µCi/ml choline, and chased with 20 mM choline. During the chase period the rate of incorporation of label into DSPC was not significantly different between A549 or H441 cells transfected with pAX-rCT and control transfected cells. Rat type II cells containing rCT incorporated label into DSPC at a significantly greater rate than did control-transfected cells. Results are the means of three to seven experiments.

In identical experiments with rat type II cells, the rate of incorporation of methyl-[³H]choline into DSPC was significantly increased in rCT-transfected as compared with control-transfected cells (Figure 8C). After 90 min of choline "chase," pAX-rCT and control-transfected cells had produced 0.79 ± 0.07 and 0.48 ± 0.05 nmol/mg protein, respectively (P < 0.006, n = 7). The respective rates of incorporation over the 90-min period were 0.16 ± 0.06 and 0.03 ± 0.03 nmol/mg protein/h (P = 0.03).

Having shown that PA and dexamethasone stimulated type II cell CT activity, we determined DSPC production in rCT-transfected and control-transfected cells under resting and stimulated conditions. Cells were transfected as described and exposed to 100 µM PA or 250 nM dexamethasone for 20 h. They were then washed, exposed to methyl- [³H]choline (1 µCi/ml) for 3 h and harvested, and DSPC was isolated and counted. As shown in Figure 9, significantly more DSPC was produced by rCT-transfected cells than by control-transfected cells, independent of exposure to stimulant. Control-transfected cells appeared to produce slightly greater amounts of DSPC after exposure to PA, although this increase was not significant. On the other hand, rCT-transfected cells increased production of DSPC by approximately 10% after exposure to PA (P < 0.003) or dexamethasone (P < 0.04).

View larger version (15K): [in this window] [in a new window]   Figure 9.   Synthesis of DSPC by transfected rat type II cells. Type II cells were transfected with pAX-142 (pAX) or pAX-rCT (rCT) and subsequently exposed to either 100 µM PA or 0.25 µM dexamethasone for the times shown. Cells were harvested, and [3H]DSPC content was determined. Results are the means of eight experiments. *P < 0.05 relative to pAX. **P < 0.05 relative to rCT.

	Discussion

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This study tested the hypothesis that CT activity is rate-limiting in the synthesis by alveolar epithelial cells of DSPC, the major phospholipid component of pulmonary surfactant. Further, the ability of CT to regulate DSPC synthesis in response to stimuli known to affect DSPC synthesis was also tested. The investigational strategy was to enhance CT activity in cells by introducing the CT cDNA and to show, first, that CT protein and activity levels were augmented. Subsequently, the ability of transfected cells, either resting or stimulated, to increase DSPC synthesis was tested.

Both rat alveolar type II cells in primary culture and human lung tumor cell lines exhibiting some type II cell characteristics were studied. The results show that transfection of CT cDNA does result in increased CT protein and activity in all cells studied, but that only in rat type II cells is this increase accompanied by an increase in DSPC synthesis. Further, in type II cells, CT protein levels (Figure 3) and activity (Figure 6) increased in response to exposure to PA and dexamethasone. This increase is most evident in the particulate cell fractions. Dexamethasone, but not PA, resulted in an overall increase in the rate of type II cell DSPC synthesis.

In vitro studies of alveolar epithelial cell functions have provided substantial information regarding the biosynthesis of surfactant lipid and protein components. Immortalized cell lines (e.g., A549 and H441), derived from human lung epithelial tumors, and type II cells in primary culture have been used. The former have obvious advantages, but are limited in the extent to which they mimic type II cell functions. A549 cells synthesize DSPC in vitro, and dexamethasone and coculture with fetal lung fibroblasts stimulate this synthesis significantly (24). Although these cells develop lamellated inclusion bodies reminiscent of the lamellar bodies of type II cells, the lipid composition of A549 cells has been reported to resemble that of fibroblasts more than that of freshly isolated type II cells (25). Specifically, the phospholipid fraction of DSPC is significantly less in A549 cells than in type II cells.

We found that CT in both pAX-rCT and control-transfected cells was distributed between the cytosolic and particulate compartments, with approximately 35% of the activity found in the former in both A549 and H441 cells. This finding is similar to prior descriptions of CT distribution in A549 cells (21) and rat type II cells (26). Finally, H441 cells express surfactant apoproteins in a regulated fashion (27, 28), suggesting an origin from type II cells. Thus, although A549 and H441 cells may share some characteristics of type II cells, significant differences exist.

Studies using isolated type II cells are challenging. Cell morphology, lipid synthesis and secretion, and apoprotein expression change during the first few days of culture (29). We noted that culturing cells on Primaria plates resulted in moderate preservation of phenotypic features, as observed by phase microscopy, during 4 d of culture. However, cell functions undoubtedly changed during the time of culture. Nevertheless, in contrast to findings in A549 or H441 cells, augmented type II cell CT activity was accompanied by a modestly increased ability of cells to synthesize DSPC. We interpret this result to mean that the level of intracellular CT activity was responsible for the increase in DSPC synthesis observed. We believe it likely that type II cells in vivo, as opposed to those in culture for 3 or more d, might exhibit a greater increase in production of DSPC in response to augmented cell CT activity. That augmented DSPC synthesis was seen only in type II cells indicates that properties specific to the type II cell are necessary for allowing increased DSPC synthesis. The identification of these properties remains to be determined.

Although exposure to PA for 20 h did increase CT activity in the particulate fraction of type II cells (Figure 6A), this increase was significant only in control-transfected cells, was modest, and was not accompanied by a significant increase in DSPC production. These findings may be compared with those of Chander and Fisher (20), who also found that exposure to PA increased CT activity in the microsomal fraction of type II cells. Studying cells cultured for only 27 h, these investigators were able to demonstrate an accompanying increase in DSPC production. It is possible that this capacity was not maintained by cells cultured as described in the present investigation. Exposure of control or rCT-transfected cells to dexamethasone did result, in both instances, in an increase in both the cytosolic and particulate levels of CT mass (Figure 3) and activity (Figure 6B). A significant increase in DSPC synthesis was detected only in rCT-transfected cells. Previous investigators have described a similar effect of corticosteroids. Post and colleagues found a significant (29%) increase in incorporation of choline into DSPC in the presence of 10⁵ M cortisol in mature rat type II cells cultured for less than 30 h (30), whereas Viscardi and associates described a significant cyclohexamide-inhibitable increase in CT activity of cytosolic and microsomal fractions of fetal rat lung cells after exposure to dexamethasone that was accompanied by increased rate of DSPC synthesis (31). The effect of dexamethasone is uncertain; neither in the experiments of Viscardi and coworkers nor in ours did the effect appear to be associated with significant redistribution of the enzyme from cytosolic to microsomal compartments. That we observed a significant effect of dexamethasone at concentrations as low as 2.5 × 10¹⁰ M suggests genomic, or nuclear receptor-dependent, action. Effects mediated through products of contaminating fibroblasts may occur in fetal rat lung cell culture. The relative purity of the cells in the experiments described here make indirect effects of dexamethasone exposure less likely.

In summary, augmentation of type II cell CT activity by transfection of CT cDNA appears to be an effective mechanism of increasing cell production of DSPC. Native and recombinant CT appear to have similar biologic activities after exposure of cells to PA or dexamethasone. The results support the hypothesis that CT is a rate-limiting and regulatory enzyme in the biosynthesis of DSPC.

Our observations raise questions related to additional effects that enhanced synthesis of DSPC may have on the type II cell. Specifically, what are the effects on synthesis of other phospholipids, on synthesis of surfactant apoproteins, and on the function of the surfactant produced by these cells?

These observations also raise interesting therapeutic possibilities. Recent reports suggest that exogenous lung surfactant may be of value in the treatment of acute lung injury (ALI) (32, 33). In animal models of ALI induced by hyperoxia, sepsis, or ischemia, pretreatment with exogenous surfactant is protective. This report suggests that in vivo transfection of alveolar epithelial type II cells might be a strategy for stimulation of endogenous production of surfactant phospholipid. This strategy thus might provide a mechanism for protection of the lungs of patients at risk of developing ALI.