Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Type II pneumocytes in alveoli of the mature lung synthesize and secrete lung surfactant, a mixture of lipids and proteins that serves to lower the surface tension of the alveolar air-liquid interface and thereby stabilize alveoli at low lung volumes. Phosphatidylcholine (PC) comprises up to 80% of lung surfactant, and of this the majority is dipalmitoylphosphatidylcholine (DPPC) (1). The cytidine diphosphocholine (CDP-choline) pathway is the major biosynthetic pathway for DPPC in alveolar type II cells, and CTP:phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in this pathway (2).

Little is known of the mechanisms that regulate production of DPPC by type II pneumocytes. In the setting of acute lung injury, alveolar levels of DPPC are decreased, most likely as a result of decreased production (3). In several models of such injury, damage is prevented or decreased by administration of surfactant or surfactant lipids (4). Thus, understanding mechanisms that regulate surfactant lipid synthesis and secretion might lead to means of controlling endogenous surfactant production and consequently to strategies designed to modify the development of acute lung injury.

Our prior studies showed that rat type II cells transfected with rat CT (rCT) cDNA had increased expression of CT, increased CT activity, and increased rates of DSPC synthesis (2). Study of the role of CT in type II cells in vivo is required, however, to determine the biologic relevance of the in vitro observations. Although we have explored the use of an adenoviral vector to deliver CT cDNA, we found that the amount of virus required for gene transfer resulted in unacceptable pulmonary inflammation. Consequently, to study the role of CT in control of DSPC biosynthesis in vivo, we have developed a transgenic mouse model in which rCT is expressed specifically in type II pneumocytes. This report documents the enhanced expression and activity of rCT in the lungs and type II pneumocytes of mice that overexpress CT, and the effect on DSPC biosynthesis and secretion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Phosphatidylcholine, phosphocholine, oleic acid, DNase I, and fibronectin were purchased from Sigma Chemical Co. (St. Louis, MO). Protease inhibitor cocktail (Complete, EDTA-free) was from Boehringer Mannheim (Indianapolis, IN). Dispase was from Fisher Scientific (Tustin, CA). Fetal bovine serum was from HyClone (Logan, UT). Rat serum was from Biocell (Rancho Dominguez, CA). Anti-mouse CD16/32 and anti-mouse CD45 were from BD PharMingen (San Diego, CA). Radioactive compounds were from NEN Life Science Products (Boston, MA).

Production and Characterization of CT Transgenic Mice

Plasmid pAX-rCT (a gift of Dr. R. Cornell, Simon Fraser University, Burnaby, BC, Canada) containing wild-type rat CT cDNA, described as CT-2 by Walkey and coworkers [7]) and plasmid p3.7-tpA (a gift of Dr. J. Whitsett, Cincinnati Children's Hospital, Cincinnati, OH, and comprised of the human SP-C promoter region nucleotides -3683 to +18, followed by a multiple cloning site, SV40 small t-intron, and polyadenylation signal) (8) were propagated in DH5 Escherichia coli. Rat CT cDNA was released from pAX-rCT with Sal I and inserted into the Sal I site of p3.7-tpA. The rCT cDNA was aligned in the 5' to 3' orientation with the SP-C promoter creating a SP-C-rCT chimeric transgene construct. The SP-C promoter has been previously used in transgenic mouse models to achieve high levels of transgene expression restricted to pulmonary epithelial cells (9). The SP-C-rCT sequences were isolated for microinjection into CB6 F₂ embryos at the single-cell stage, which were subsequently implanted into pseudopregnant BD F₁ surrogate mothers, generating four rCT transgenic founder mice (rCT-25, -33, -40, and -42). Tail-clip DNA was isolated using a Dneasy Tissue Kit (Qiagen, Valencia, CA). rCT mice were identified by polymerase chain reaction (PCR) of tail-clip DNA using the following primer sequences: 5'-GCTCTAGAA GCTTCGATGGATGCACAGAGTTCAGCT-3' (corresponding to the rCT amino terminus) and 5'-CAGACTGTGAGGACTG AGGG-3' (corresponding to unique downstream sequences in the chimeric transgene). The PCR program was 94°C for 4 min followed by 36 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min. PCR products were separated electrophoretically on 1% agarose gels, stained with ethidium bromide, and visualized with UV light. A PCR product of the expected size (1.5 kb) was observed with rCT mice, whereas no PCR products were detected with CB6 wild-type mice (Figure 1). Mice from founder 40 consistently gave lower intensity bands compared with mice from the other founders. The original rCT founder mice were backcrossed with wild-type mice to produce rCT transgenic and wild-type progeny. Individual founder lines were propagated by interbreeding rCT mice. There were no obvious phenotypic differences between rCT and wild-type mice. Mice were raised on regular grain-based rodent chow with free access to water, and were typically studied at 6-12 wk of age. Littermates that were rCT() by PCR were used as experimental controls. All experimental procedures involving mice were approved by the Department of Veterans Affairs Subcommittee on Animal Studies in compliance with federal regulations pertaining to the care and use of laboratory animals.

View larger version (94K): [in this window] [in a new window]   Figure 1.   Detection of rCT transgene. Purified p3.7S-tpA-rCT plasmid DNA (0.3 pg, lane 1) and tail-clip DNA (25 ng) from individual wild-type mice (lanes 2 and 7) and transgenic mice from founder lines rCT-25 (lanes 3 and 4), rCT-33 (lanes 5 and 6), rCT-40 (lanes 8 and 9), and rCT-42 (lanes 10 and 11) were used as templates for PCR as described in MATERIALS AND METHODS. The expected 1.5-kb PCR product was observed with plasmid DNA and with DNA from transgenic, but not wild-type mice. The left lane is a 1-kb DNA ladder (New England Biolabs, Beverly, MA).

Lung CT Gene Expression

Age-matched mice of either sex were given intraperitoneal pentobarbital to achieve deep anesthesia and the aorta was severed to exsanguinate each animal. The lungs were quickly removed and placed in RNA Stabilization Reagent (Qiagen). Approximately 100 mg of tissue was then placed directly into 1 ml of RNAwiz RNA Isolation Reagent (Ambion, Austin, TX) and the tissue was homogenized 2-3 min with a hand-held, dual-speed electric grinder (Model 850; Dremel, Racine, WI). Total RNA was isolated from homogenized lung tissue according to the RNAwiz protocol. Total RNA yields varied, but were between 50-100 µg for all samples. Northern analysis of samples was performed using the NorthernMax system (Ambion) according to the instructions provided. Five micrograms of total RNA from each sample was separated electrophoretically on a formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized under low stringency conditions with a ³²P-labeled CT probe that was a 611-bp PCR product generated from the SP-C-rCT construct. Hybridization with a ³²P-labeled mouse -actin probe was also performed as a control to evaluate variability in RNA loading between samples. Autoradiographs were exposed for 3 h.

Lung CT Expression and DSPC Content

Age-matched mice of either sex were killed with intraperitoneal pentobarbital. To perform alveolar lavage, the chest of the animal was opened and a 20-gauge blunt needle was tied into the proximal trachea. Five 1-ml aliquots of 150 mM NaCl were sequentially instilled into the lungs and withdrawn by syringe three times for each aliquot. Recovered lavage fluid was pooled, the volume was measured, and the fluid was stored at 70°C until analyzed. DSPC was isolated from lavage fluid by extracting the fluid with chloroform/methanol (10) and then treating the lipid extract with OsO₄ followed by silica column chromatography (11). Isolated DSPC was quantified by phosphorus assay (12). After lavage, lungs were removed from each animal and the tissue was homogenized in 0.5 ml of 150 mM NaCl containing protease inhibitors. An aliquot (100 µl) was centrifuged (10 min, 2,000 × g) and the supernatant was stored at 70°C for analysis of CT enzyme activity, total protein concentration, and presence of immunoreactive CT protein. The remainder of the homogenate was adjusted to a final volume of 4.5 ml with 150 mM NaCl and subjected to DSPC isolation and measurement as described above. CT activity was measured as previously described (2). Activity assays were performed in the presence or absence of 0.5 mM PC/oleic acid (1:1 molar ratio) phospholipid vesicles. Total protein concentration was measured using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin as standard. Western blotting was performed as described (2) using anti-rat CT serum (a gift of Dr. C. Kent, University of Michigan, Ann Arbor, MI).

DSPC Precursor Incorporation In Vivo

Age-matched mice of either sex were injected intraperitoneally with 8 µl/g body weight [³H]choline chloride (0.1 µCi/g) and [¹⁴C]palmitic acid (0.2 µCi/g). At 3, 8, 24, and 48 h after injection, groups of 3-5 wild-type and transgenic mice were killed with intraperitoneal pentobarbital. Alveolar lavage was performed on each animal and the lung tissue was homogenized as described above. DSPC was isolated from lavage fluid and tissue homogenate as described above, and the radioactivity of each isotope (³H and ¹⁴C) incorporated into DSPC was measured.

Isolation and Culture of Alveolar Type II Cells

Primary alveolar type II cells were isolated from mice by a modification of the method of Corti and associates (13). Briefly, mice were killed with intraperitoneal pentobarbital and the lungs were perfused with 150 mM NaCl. The lungs were filled with Dispase solution via the trachea followed by low-melt agarose (1%, 0.45 ml, 45°C), and then covered with crushed ice for 2 min. The lungs were removed to 2 ml of dispase, incubated for 45 min at room temperature, and then held on ice until the next step. The lungs were transferred to 7 ml of Dulbecco's modified Eagle's medium (DMEM) with 0.01% Dnase I in a 60-mm Petri dish. After teasing apart the digested tissue, the resulting cell suspension was filtered sequentially through 100-, 40-, and 20-µm nylon mesh. Cells were collected by centrifugation (8 min, 130 × g) and resuspended in 10 ml of DMEM containing 10% fetal bovine serum and penicillin/streptomycin (P/S; 100 µg/ml and 100 mg/ml, respectively). Contaminating cell types were removed by incubating cell suspensions for 2 h at 37°C, 5% CO₂ in 100-mm tissue culture plates coated with a mixture of anti-CD16/32 and anti-CD45 (0.65 µg and 1.5 µg, respectively, per plate), and then overnight at 37°C, 5% CO₂ in uncoated plates. Nonadherent cells from groups of three mice were pooled, collected by centrifugation, and resuspended in complete media (DMEM containing 5% rat serum and P/S) at a concentration of 1.5 × 10⁶ cells/ml. Cell suspensions were added to six-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) (2 ml/well) previously coated with fibronectin, and cultured overnight at 37°C, 10% CO₂. During this time type II cells became firmly attached to the wells.

CT Expression in Type II Cells

After 24-48 h in culture, cells were washed twice with Dulbecco's phosphate-buffered saline (PBS) and collected by scraping into 100 µl of 50 mM imidazole, 150 mM KCl, 2 mM EDTA, pH7.4. The cell suspension was sonicated with three 5-s pulses using an ultrasonic cell disrupter (Heat Systems Ultrasonics, Plainview, NY) at 60% peak output with a microtip attachment. Cell sonicates were stored at 70°C until analyzed for CT enzyme activity, total protein concentration, and immunoreactive CT protein by Western blotting.

DSPC Synthesis and Secretion by Type II Cells

After 24-48 h in culture, cells were washed once with PBS and then incubated overnight in complete media containing [³H]choline (1 µCi/well). Subsequently, cells were washed twice with PBS and cultured for an additional 3 h in serum-free media (1 ml/well). At the time of harvest, conditioned media were collected and cells were scraped into 1 ml of distilled water. Cell suspensions were sonicated and a small aliquot of each sonicate was reserved for protein assay. DSPC was isolated from conditioned media and cell sonicates as described above, and the radioactivity incorporated into DSPC was measured. The amount of newly synthesized DSPC (expressed as nmol [³H]DSPC/mg cell protein) was determined after calculating specific activity of the labeling media.

Analysis of Choline Metabolites in Type II Cells

After 24 h in culture, the medium was changed and the cells were labeled for an additional 24 h with [³H]choline (2.4 µCi/well). Cells were washed three times with PBS and then scrape-harvested into 0.4 ml of ice-cold distilled water. Cell suspensions were sonicated and a small aliquot was taken for protein assay. Hydrophilic (choline, phosphocholine, CDP-choline, glycerophospho-choline) and hydrophobic (PC, lyso-PC) metabolites were recovered by chloroform/methanol extraction (10), separated by thin-layer chromatography (TLC) as described by Walkey and colleagues (7), and counted.

Statistics

Data are presented as mean ± SEM. Comparisons between groups were performed using an unpaired Student's t Test (two-tailed) or two-way ANOVA with post hoc comparison of groups using the Student-Newman-Keuls test. Differences were considered significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Lung CT Gene Expression

Expression of the CT transcript in lung tissue was evaluated by Northern blotting using a 611-bp probe derived from the coding region of rCT cDNA. Because of the extensive homology between rat and mouse CT cDNA sequences, it appeared likely that the probe would hybridize with both the endogenous (mouse) and transgenic (rat) CT transcripts. Indeed, a diffuse band of the expected size (~ 1.6 kb) was observed with total RNA from both wild-type and transgenic mice. Expression of the CT gene was quite variable between founder lines. Compared with wild type, the greatest expression was observed in rCT-25 and rCT-42 mice, and minimal expression was observed in rCT-33 and rCT-40 mice (Figure 2). Control hybridization with a -actin probe indicated that lanes were loaded with approximately equivalent amounts of RNA (Figure 2). Based on these findings, mice from founder lines rCT-25 and rCT-42 were targeted for further studies.

View larger version (79K): [in this window] [in a new window]   Figure 2.   Lung CT gene expression. A Northern blot of total lung RNA (5 µg/lane) from wild-type and transgenic mice was prepared and hybridized using CT and -actin probes as described in MATERIALS AND METHODS. Samples from individual wild-type mice (lanes 5 and 10) and transgenic mice from founder lines rCT-33 (lanes 1 and 2), rCT-25 (lanes 3 and 4), rCT-40 (lanes 6 and 7), and rCT-42 (lanes 8 and 9) are shown.

Lung CT Expression and DSPC Content

To determine the effects of enhanced lung CT gene expression on CT protein levels, lung tissue homogenates were prepared from transgenic and wild-type mice and analyzed for immunoreactive CT protein and CT enzyme activity. Increased amounts of immunoreactive CT protein were observed by Western blotting in lung homogenates from rCT-25 and rCT-42 mice (Figure 3A), with approximately 3- and 5-fold amounts relative to wild type, respectively, based on densitometric scanning. CT enzyme activity was increased approximately 4-fold in rCT-25 mice (19.7 ± 2.3 nmol/min/mg) and over 6-fold in rCT-42 mice (28.8 ± 5.1 nmol/min/mg) compared with wild type (4.6 ± 0.3 nmol/min/mg) when assays were conducted in the presence of phospholipid vesicles (Figure 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3.   Lung CT protein expression. (A) Lung homogenates from wild-type (WT) and transgenic (25, 42) mice were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10 µg protein per lane), and immunoreactive CT protein was identified by Western blotting. A representative blot from one mouse in each group is shown. (B) CT enzyme activity was measured in lung tissue homogenates from wild-type (WT, n = 9) and transgenic (25, n = 7; 42, n = 10) mice. ***P < 0.001 compared with wild type.

To determine whether the transgenic enzyme is physiologically inactive due to loose binding of known lipid inhibitors, CT activity in lung homogenates from separate groups of animals was measured in the presence and absence of lipid vesicles. As shown in Table 1, CT activity was increased significantly to the same degree (~ 4-fold) in the presence or absence of lipid activator in both the transgenic (rCT-42) and wild-type mice. Thus, the overexpressed transgenic enzyme appears able to be activated in a fashion similar to that of the wild-type enzyme.

To determine the effect of increased CT enzyme activity on lung DSPC content, DSPC from alveolar lavage fluid and lung tissue was isolated and measured (Figure 4). Total DSPC (alveolar plus tissue) was increased 25% (P = 0.028) in rCT-42 compared with wild-type mice (54.3 ± 2.8 versus 43.5 ± 3.3 µmol/kg). Alveolar and tissue pools of DSPC were both elevated in rCT-42 compared with wild-type mice (13.3 ± 1.6 versus 9.8 ± 1.1 and 41.0 ± 2.1 versus 33.7 ± 2.5 µmol/kg, respectively). Mice from founder line rCT-25 also showed increased amounts of alveolar, tissue, and total lung DSPC, but the differences compared with wild-type mice were not statistically significant (Figure 4).

View larger version (27K): [in this window] [in a new window]   Figure 4.   Lung DSPC content. Amounts of DSPC recovered by alveolar lavage (alveolar), in lung tissue after alveolar lavage (tissue), and in alveolar lavage plus lung tissue (total) were determined in wild-type (closed bar, n = 8) and transgenic rCT-25 (open bar, n = 8) and rCT-42 (hatched bar, n = 7) mice. *P < 0.05 compared with wild type.

DSPC Precursor Incorporation In Vivo

Because lung DSPC pool sizes were increased in transgenic mice, weight-adjusted doses [³H]choline and [¹⁴C]palmitate were injected into mice intraperitoneally, and net incorporation into lung DSPC, secretion of labeled DSPC into alveoli, and any loss of labeled DSPC at 48 h after precursor injection were determined (Figure 5). The labeling patterns over time were similar for transgenic and wild-type mice. There was a gradual loss of labeled DSPC from lung tissue over 48 h, and maximal appearance of labeled DSPC in the alveoli at 24 h. Total labeled DSPC was decreased at 48 h, which is consistent with catabolism of lung DSPC. Recovery of DSPC labeled with either [³H]choline (Figure 5A) or [¹⁴C]palmitate (Figure 5B) followed similar patterns.

View larger version (34K): [in this window] [in a new window]   Figure 5.   Incorporation of [3H]choline (A) and [14C]palmitate (B) into DSPC in vivo. The amounts of radiolabeled DSPC (dpm/g body weight) recovered by alveolar lavage (top), in lung tissue after alveolar lavage (middle), and in alveolar lavage plus lung tissue (bottom) were determined in wild-type (closed bars), rCT-25 (open bars), and rCT-42 (shaded bars) mice.

CT Expression in Type II Cells

To verify that CT expression and activity were augmented in alveolar type II cells, we performed studies on primary cultures of type II cells isolated from transgenic and wild-type mice. Enhanced levels of immunoreactive CT protein were observed in type II cell extracts from transgenic mice (Figure 6A). CT activity was increased 3-fold in rCT-25 cells (8.8 ± 1.1 nmol/min/mg) and 6-fold in rCT-42 cells (18.0 ± 3.3 nmol/min/mg) compared with cells from wild-type mice (2.9 ± 0.6 nmol/min/mg) (Figure 6B). The relative increases in CT activity in type II cells from transgenic mice were similar to those observed in whole lung (Figure 3).

View larger version (25K): [in this window] [in a new window]   Figure 6.   CT protein expression in alveolar type II cells. Cells were isolated, cultured in duplicate wells (3 × 106 cells/ well) for 24-48 h, and then harvested as described in Materials and Methods. (A) Cell extracts from wild-type (WT) and transgenic (25, 42) mice were subjected to SDS-PAGE (7.5 µg protein per lane) and immunoreactive CT protein was identified by Western blotting. A representative blot from one mouse in each group is shown. Nonspecific staining of several bands was consistently observed in cell extracts from both transgenic and wild-type mice. (B) CT activity was measured in cell extracts from wild-type (WT) and transgenic (25, 42) mice. Data represent results from four experiments. ***P < 0.001 compared with wild type.

DSPC Synthesis and Secretion by Type II Cells

To determine whether enhanced CT expression by type II cells would lead to increased DSPC synthesis and secretion, cells from transgenic and wild-type mice were cultured in the presence of [³H]choline, and incorporation of the label into newly synthesized DSPC was measured in cell extracts and conditioned media. Incorporation of [³H]choline into total DSPC (media plus cells) was increased 18% (P = 0.024) in rCT-42 compared with wild-type cells (33.6 ± 1.2 versus 28.4 ± 1.7 nmol/mg) (Figure 7A). Conditioned media and cellular pools of [³H] DSPC from rCT-42 cells were both elevated compared with wild type (13.3 ± 1.6 versus 9.0 ± 1.0 and 41.0 ± 2.1 versus 34.8 ± 1.5 µmol/kg, respectively). Cells from rCT-25 mice also produced increased amounts of [³H]DSPC, but the differences between rCT-25 and wild-type pools were not statistically significant (Figure 7A). These data are in good agreement with DSPC measurements in lung tissue and alveolar lavage fluid (Figure 4). [³H]DSPC secretion by rCT-42 cells was increased 56% (P = 0.006) compared with wild type (6.7 ± 0.5 versus 4.3 ± 0.5%) (Figure 7B). In contrast, [³H]DSPC secretion by rCT-25 cells was not significantly different compared with wild type (Figure 7B).

View larger version (21K): [in this window] [in a new window]   Figure 7.   DSPC synthesis and secretion in alveolar type II cells. (A) Cells from wild-type (closed bars), rCT-25 (open bars), and rCT-42 (shaded bars) mice were isolated and cultured in duplicate wells (3 × 106 cells/well) overnight in the presence of [3H]choline. After washing, cells were cultured an additional 3 h in serum-free media. [3H]DSPC was then isolated from conditioned media and cell extracts, and measured as described in MATERIALS and METHODS. Total [3H]DSPC synthesis (media plus cells) is also presented. Data represent results from four experiments. *P < 0.05, ***P < 0.001 compared with wild type. (B) Data from A is presented as [3H]DSPC secretion, which was calculated as the percentage of [3H]DSPC in conditioned media relative to total [3H]DSPC (media plus cells). **P < 0.01 compared with wild type.

Relative Pool Sizes of Choline Metabolites in Type II Cells

Although the CT activity of transgenic rCT-42 cells increased ~ 6-fold (Figure 6B), the amount of [³H]choline incorporated into DSPC appeared to be elevated by only ~ 18% (Figure 7A). This discrepancy might be explained by enhanced metabolism of DSPC in the transgenic mice. To investigate this possibility, type II cells from rCT-42 and wild-type mice were labeled for 24 h (to presumed equilibrium) with [³H]choline, and the cell distribution of choline-labeled metabolites was determined. The total disintegrations per min (dpm)/µg protein recovered in hydrophilic plus hydrophobic metabolites from rCT-42 and wild-type mice was similar (9,081 ± 1,524 and 11,166 ± 3,106 dpm/µg, respectively; P = 0.34, n = 3). Table 2 shows that the phosphocholine pool was reduced 25% (P = 0.017) in rCT-42 transgenic cells, in keeping with acceleration of the CT-catalyzed step. However, the CDP-choline pool was not significantly increased in these cells, suggesting rapid utilization of this intermediate product by cells from both transgenic and wild-type mice. There was a trend toward increased pool sizes of PC and glycerophosphocholine (GPC), a breakdown product of PC, but the differences between wild-type and transgenic cells were not statistically significant.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The control of surfactant phospholipid pool size is poorly understood. To investigate the influence of type II pneumocyte CT activity on surfactant phospholipid synthesis and pool size, we have developed transgenic mice that overexpress the rat CT gene under control of the SP-C promoter. The tissue specificity achieved through use of this promoter is well established (14, 15). Four founder lines were generated. Gene expression, as determined by Northern analysis, was greater in two of these (rCT-25 and rCT-42). Further study of these two lines showed that immunoreactive CT protein levels (Figure 3A) and activity (Figure 3B) were increased, relative to wild-type animals, and that immunoreactive protein level and activity appeared to be correlated, with the higher values in animals from line rCT-42. Moreover, the relative difference in CT activity between wild-type and transgenic mice was the same regardless of whether the activities were measured in the presence or absence of lipid activator (Table 1), suggesting that the transgenic enzyme is physiologically active.

CT is ubiquitous in cells of higher eukaryotes, as it is a component of the biosynthetic pathway for phosphatidylcholine, which is the major cell membrane phospholipid in those animals. Two distinct genes, CCT and CCT, are described; with the latter producing two splice variants. Whereas CCT and both CCT isoforms are expressed in fetal lung, only CCT appears to be expressed in adult lung (16).

Thus, whole lung CT activity includes activity present in type II cells and also activity present in all other cells of the lung. Consequently, it is likely that the 4- to 6-fold increase in CT activity reported in Figure 3B under-represents the actual increase in type II cell activity that was present in the lungs of rCT-25 and rCT-42 mice.

Despite the marked increase in whole-lung CT activity, only a modest increase in DSPC content was detected (Figure 4), and this increase was only significant in rCT-42 mice, the line that had the most marked enzyme amount and activity. Several factors may explain why greater increases in lung DSPC content were not seen. First, it is possible that the rate of DSPC synthesis is significantly enhanced, but that regulatory mechanisms increase DSPC degradation or clearance and thereby control type II cell and alveolar DSPC pool sizes. To investigate this possibility, we studied the in vivo incorporation of radiolabeled precursors into DSPC following intraperitoneal injection, a strategy that has been used previously for measurements of surfactant metabolism in mice. The results are qualitatively very similar to those reported by other investigators (17) and do not suggest increased precursor incorporation by rCT-42 mice. However, a modest increase in DSPC synthesis within type II cells may have been obscured by the global analysis of all cell types present in the whole lung.

Others have noticed a significant dissociation between CT activity and DSPC precursor incorporation. In studies of rCT expressed in COS cells, Walkey and associates found a 20-fold increase in CT activity in the microsomal fraction, but only a modest increase in DSPC precursor incorporation (7). In these cells, the phosphocholine pool size was reduced by up to 50%, consistent with increased CT activity. The choline pool was also reduced by ~ 50%, consistent with stimulation of the choline kinase reaction. However, the CDP-choline pool was found to be markedly elevated, suggesting limitation of synthesis at the cholinephosphotransferase step (7). Efforts to overcome this limitation by augmenting the amount of diacylglycerol were consistent with a rate-limiting role for cholinephosphotransferase. To explore whether similar mechanisms might be operative in rCT-42 mice, type II cells were labeled to equilibrium with [³H]choline, and the cellular distribution of [³H]choline metabolites was determined. A significant reduction in the phosphocholine pool was observed in cells from rCT-42 mice, consistent with enhanced CT activity. However, no significant differences in other metabolic pool sizes between rCT-42 and wild-type mice were detected. Thus, these experiments do not support the hypothesis that increased DSPC metabolism occurs in vivo in rCT-42 mice. Neither do they indicate limitation of conversion at metabolic steps downstream from that catalyzed by CT.

One must consider whether DSPC synthesis might be limited by availability of choline. The K_m for choline kinase (from yeast) is 0.3 mM (18), and type II cell concentrations may be in the range of 0.7 mM (19). Thus, it is unlikely that significant substrate limitation occurs. Further, in studies in which we labeled type II cells in vitro with [³H]choline, substrate limitation did not occur.

Although the metabolism of type II cells in primary culture may not fully reflect the in vivo situation, observations on type II cells recovered from mice from the rCT-25 and rCT-42 lines were consistent with findings in whole lung. Specifically, both rCT protein and activity were greatest in cells from rCT-42 mice (Figure 6). In addition, a minimal increase in incorporation of [³H]choline into DSPC was detected in cells from rCT-42 mice (Figure 7A) when DSPC in both cells and media was analyzed.

It has been proposed by others that there is a fine balance between PC synthesis and degradation, so that enhanced expression of CT results in accelerated PC synthesis that is accompanied by similarly accelerated PC degradation, with the effect that overall PC cell content remains relatively constant (20). This concept appears to hold true in certain cell lines during enforced expression of both CT genes (7, 21), and, as reviewed by Baburina and coworkers, during accelerated PC synthesis that occurs in ras-transformed cell lines and during the G₁ stage of the cell cycle (20). In type II pneumocytes, cell PC is found not only in cell membranes but also in organelles devoted to surfactant biosynthesis. Control of PC content in this compartment will also be significantlyperhaps predominantlydetermined by secretion of PC in lamellar bodies. It is thus significant that the percentage of newly synthesized DSPC that was secreted was also moderately increased in cells from the transgenic line that had the greatest CT activity and DSPC synthesis (Figure 7B).

In summary, we have shown that overexpression of CT activity in alveolar type II pneumocytes results in enhanced CT activity, both as detected in lung homogenate and in isolated type II cells. Tissue pools of DSPC were increased in mice with the greatest gene expression and rCT protein levels. Further, in isolated type II cells from these mice, we have found a modest increase in precursor incorporation into DSPC and increased DSPC secretion. There appears to be a discrepancy between the elevation of physiologically active CT we observed in transgenic rCT-42 mice and the production of DSPC. Our experiments suggest that mechanisms other than enhanced DSPC metabolism, limitation of conversion of CDP-choline to DSPC, or limitation of the choline substrate are of significance. Further study may reveal additional steps at which DSPC biosynthesis is controlled, and the extent to which DSPC synthesis and secretion may be augmented by modulating those controls.