Experimental Lung Research
ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type II Alveolar Cell from Fetal Rabbit Lungs J. E. Scott To cite this article: J. E. Scott (1992) Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type II Alveolar Cell from Fetal Rabbit Lungs, Experimental Lung Research, 18:4, 563-580, DOI: 10.3109/01902149209064346 To link to this article: http://dx.doi.org/10.3109/01902149209064346
Published online: 02 Jul 2009.
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Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type I1 Alveolar Cell from Fetal Rabbit Lungs
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J. E. Scott
ABSTRACT: Evidence indicates that pulmonary pool sizes of choline and related intermediates available f . r synthesis of phosphatidylcholine, the major component of the surfactant, change during gestation. Furthermore, recent data suggest that the type 11 lung cells that produce the suvfactant potentially can reutilize components of this material. However, the relationship of the de novo synthetic mechanism to the secretion and reutilization of phosphatidylcholine has not been established. This is particularly true in the case of the fetal lung where, although alterations i n precursor pool sizes, including choline, have been demonstrated, few or no data are available concerning how phosphatidylcholine synthesis affects or is affected by secretion and reutilization of this phospholipid by fetal type 11 cells. The present study was undertaken to determine the effect of availability of choline on de novo synthesis of phosphatidylcholine by isolated fetal rabbit type 11 cells during the differentiation process. In addition, differentiating type 11 alveolar cells were used to examine the hypothesis that these cells incorporate phospholipid from the extracellular environment and the quantity and/or composition of this phospholipid differently affects cellular secretion or de novo phosphatidylcholine synthesis. Assuming that the cells did not discriminate between radioactive and nonradioactive choline, elevation of extracellular choline increased the synthesis of cellular phosphatidylcholine and disaturated phosphatidylcholine in a dose-dependent manner to 0.08 mM choline. Cells induced to differentiated by exposure to fibroblast-conditioned medium synthesized more total and disaturated phosphatidylcholine at all extracellular choline concentrations. Incubation of the fetal type 11 cells with dipalmitoylphosphatidylcholine or l-palmitoyl-2oleoylphosphatidylcholine signlficantly depressed the incorporation of (3H/choline into
From the Departments of Oral Biology and Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada. Address all cowespondence to Dv. J. E. Scott, Department of Oral Biology, Faculty of Dentistry, University of Manttoba, 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E OW3, Canada. Received 6 June 1990; accepted 20 November 1991.
Experimental Lung Research 18:563-580 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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cellular phosphatidylcholine after 24 or 48 h, but not necessarily at both times. Dipalmitoylphosphatidylcholine depressed the secretion of PH]choline-labeled phosphatidylcholine after incubation for 24 h. 1-Palmitoyl-2-oleoylphosphatidylcholine stimulated the secretion of tritium-labeled phosphatidylcholine at a concentration of 25 ,ug/mL after 48 h. Comparison of the phospholipid efect by incubating the cells with 50 ng of I4C-labeled phospholipid for 24 h showed that I-palmitoyl-2-oleoylphosphatidylcholine significantly reduced the synthesis of 'H-labeled phosphatidylcholine compared to dipalmitoylphosstimulated secrephatidylcholine. I n contrast, 1-palmitoyl-2-oleoylphosphatidylcholine tion of 'H-labeled phosphatidylcholine compared with the disaturated moiety. B e d g e r entiation state of the cells altered the magnitude of the cellular secretion response but not the character. Finally, sign2ficantly less [14C'1-palmitoyl-2-oleoylphosphatidylcholinewas associated with the non-trypsin-dissociable fraction of the cellular phospholipid pool than was the 14C-labeled disaturated moiety Altering the dgerentiation state of the cells sign 2ficantly increased the cell-associated fraction of the disaturated phospholipid; the amount of unsaturated phospholip~associated with the type 11 cells was not altered. These results indicate that the level of synthesis of phosphatidylcholine in differentiating type 11alveolar cells is at least to some degree dependent on the availability of choline. I n addition, the extracellular phospholipid environment may influence both the rate of synthesis and secretion of phosphatidylcholine by these cells.
INTRODUCTION The pulmonary surfactant is a complex mixture of phospholipids and proteins that acts primarily at the air-liquid interface of the lung to reduced surface tension within the alveolus, thereby preventing collapse of the lung at maximum expiration. In the developing lung, surfactant synthesis is initiated prior to term within the type I1 alveolar cells. In type I1 cells, phosphatidylcholine, the main constituent of the surfactant, is produced by a process of dephosphorylation of phosphatidic acid to diacylglycerol, which is converted enzymatically to phosphatidylcholine by cholinephosphotransferase (see reviews by Rooney [l] and Possmayer [2]). The high levels of the disaturated form of phosphatidylcholine that characterize the surfactant appear to be supplied by two mechanisms. The first is a direct synthetic route, whereby the cholinephosphotransferase adds the cholinephosphate moiety of CDPcholine to dipalmitoylglycerol [3]. The second is the reacylation route, whereby 1-palmitoyl-2-unsaturated phosphatidylcholine is deacylated by a phospholipase A, and reacylated with palmitoyl CoA [l, 41. In either case, the evidence suggests that the limiting step in PC synthesis is the production of CDPcholine [2]. However, changes that occur in pool sizes of choline and its associated anabolic products toward the end of gestation [5] suggest that the entire mechanism by which phosphatidylcholine synthesis is controlled undergoes a fundamental adjustment in the developing lung. Indeed, as surfactant-related PC synthesis is initiated in the fetal rabbit lung, the pool size of choline doubles while the pool sizes of other cholinecontaining intermediates decline [5]. This is associated with a doubling of
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phosphatidylcholine content prior to birth. However, it is not clear what relation this change in pool size bears to phosphatidylcholine synthesis specifically within the developing surfactant-producing type I1 cells. Therefore, as a component of the present study, radioactive choline incorporation into total and disaturated phosphatidylcholine was examined as a function of the concentration and availability of extracellular choline in isolated fetal rabbit I1 cells. In addition to the two de novo pathways for phosphatidylcholine synthesis, recent evidence suggests that the type I1 alveolar cells may reutilize components of previously secreted surfactant, presumably there by maintaining intracellular tissue stores [6-111. In fact, this latter mechanism would appear to constitute a major metabolic route considering that surfactant phosphatidylcholine apparently is reutilized with a high efficiency at least by 3-day-old rabbit lung [9, 101. Other evidence indicates that this process occurs by a net uptake mechanism, since isolated rat type I1 cells are observed to incorporate liposomal-encapsulated radioactive sucrose intact [ 12, 131. Nevertheless, the potential for phospholipid reincorporation and reutilization has not been explored in type I1 cells of the preterm lung when the pool size of disaturated phospholipid is small [14] and one might expect a mechanism for conservation of secreted surfactant material to be developing. Using isolated differentiating type I1 alveolar cells, we have established previously that the P-adrenergic agonist isoxsuprine stimulates secretion and synthesis of phosphatidylcholine de novo from radioactively labeled choline [ 151. Furthermore, we observed a paradoxical decline in cell-associated radioactively labeled phospholipid when the incubation was extended past 24 h. Since evidence from the investigations cited above [6-131 indicates that secreted phospholipid material may be reutilized by lung and lung cells, the possibility exists that our previous observations may be due in part to the influence of reincorporated phospholipid on the de novo synthetic or secretory rate of phosphatidylcholine in fetal type I1 alveolar cells. Therefore, to test the hypothesis that availability of extracellular choline influences fetal type I1 alveolar cell phosphatidylcholine synthetic rate and that this rate may be affected by phospholipid reutilization and P-adrenergic-based secretion processes, the incorporation of radioactive precursor into cellular phosphatidylcholine was measured in isolated differentiating fetal rabbit type I1 cells as the concentration of extracellular choline was increased, as well as under varying conditions of chemical form and concentration of added extracellular phosphatidylcholine. In addition, in some experiments radioactively labeled dipalmitoylphosphatidylcholine or l-palmitoyl-2oleoylphosphatidylcholine was incubated with fetal alveolar type I1 cells to confirm that they take up phospholipid from the external environment and to determine if degree of saturation affects labeling of the cellular phospholipid pool.
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MATERIALS AND METHODS Animals and Materials New Zealand White timed-pregnant rabbits were purchased from Blue Farm Rabbitry (St. Adolphe, MB). Culture materials were from Gibco Laboratories (Burlington, Ont.) unless otherwise specified. Radioactives including [rnethylJH]choline chloride (specific activity, 58 mCi/mmol), L-a[dipalmityl-l-'4C]dipalmitoylphosphatidylcholine(specific activity, 112.0 mCi/mmol), and ~-cr-palmitoyl-2-[l-'4C]oleoylphosphatidylcholine(specific activity, 57 mCi/mmol) were from New England Nuclear (Lachine, PQ). Other chemicals were from Sigma Chemical Co. (St. Louis, MO).
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Cell Isolation Undifferentiated type I1 alveolar cells were isolated as described previously with modifications as described below [16]. O n the 24th gestational day (day of breeding designated day O), does were killed by an IV overdose of Euthanyl (240 mg/mL sodium pentobarbitol). The fetuses were removed, immediately decapitated, and placed in sterile ice-cold Hanks balanced salt solution (HBSS). Fetal lungs were removed, chopped on a Sorval tissue chopper (Sorval Instruments, Newtown, CT), and immersed in a solution of trypsin/EDTA (0.05°/~/0.010/~)with 75 mg deoxyribonuclease I in calcium- and magnesium-free HBSS in water-jacketed trypsinization flasks (Wheaton, Millville, NJ). The suspension was stirred on a magnetic plate for 45 min at a constant temperature of 37OC. The action of the trypsin was stopped by addition of 50 mL of minimum essential medium with 10% carbon-stripped fetal bovine serum (v/v), prepared as described by Tanswell et al. [7]. After trypsinization, the material was filtered through three layers of Nitex gauze and centrifuged at 250g for 15 min to pellet the cells. Cells were resuspended in minimum essential medium and plated into 80-cm2flasks, which were incubated at 37OC in an atmosphere of 5% CO, and 95% air to allow fibroblasts to adhere. After 1.5 h the unattached cells were collected. The attached fibroblasts were grown to confluence in minimum essential medium supplemented with 10% fetal bovine serum (v/v) and used to prepare conditioned medium (CM) by incubation with serumfree minimum essential medium for 24 h, as described previously [18]. The CM was used to induce differentiation [19]. The unattached cells were layered on a discontinuous metrizamide gradient (10 mL of 0.10 M over 10 mL of 0.22 M metrizamide) and centrifuged at 2OOg for 20 min. Cells that collected at the interface of the gradient were aliquoted into 2-cm2 wells in 24 multiwell plates (deep well, uncoated, Falcon Plastics, Canlab, Winnipeg, MB). Cells were grown for 3 days at 37°C in minimum essential medium with 10% carbon-stripped fetal bovine serum (v/v) in a humidified atmo-
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sphere of 5% CO,. Purity estimates indicate that greater than 90% of these cells develop lamellar bodies [16]. To examine the cells, after 2-3 days in culture, portions were exposed to CM for 24 h, while others were incubated in standard medium indicated above. Cells were fixed in 1.5% glutaraldehyde in phosphate-buffered saline, as described by Mason et al. [20], to visualize lamellar bodies, and postfixed with 1.0% OsO, in the same buffer. The cell monolayers were treated for 18 h with 1.0% tannic acid in phosphate-buffered saline adjusted to p H 6.8, washed in distilled water, dehydrated in a graded ethanol series, and stained with the blue and red stains as described [20]. Monolayers were cut from bottom of flasks and coverslipped using an aqueous-based mounting medium.
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Treatment and Analyses All experiments were conducted in serum-free minimum essential medium. To characterize the incorporation of radioactive precursor ([3H]choline) into phosphatidylcholine (PC) by these cells, 3 days after isolation, the immature type I1 alveolar cells were incubated with minimum essential medium (control) or minimum essential medium plus CM (20°/0, v/v) to induce differentiation, under increasing concentrations of extracellular choline. Concurrently, 0.5 pCi of [-‘H]choline was added to the incubation. After 24 h, the cells were removed by trypsin and extracted into ch1oroform:methanol (1:2), according to High and Dyer 1211. The incorporation of radioactive label into phosphatidylcholine was determined by thin-layer chromatography of an aliquot of the organic solvent extract in a solvent system of ch1oroform:methanol:acetic acid:water (75:25:1:3) adapted from that described by Skipski and Barclay [22]. The position of phosphatidylcholine was established by running authentic internal standards with alternate samples and visualizing with iodine vapor. Appropriate spots were scraped into scintillation vials and radioactivity was estimated on a Beckman LS5801 scintillation counter with a counting efficiency of 65% for tritium. Quench compensation and disintegrations per minute (DPM) were determined by the method of H# (Beckman Instruments, Palo Alto, CA). Disaturated phosphatidylcholine (DSPC) was isolated by the method of Mason et al. [23] after reaction of aliquots of the samples with osmium tetroxide. Column eluent was collected in scintillation vials, the solvent was allowed to evaporate, and the radioactivity was determined as above. To examine the effect of phosphatidylcholines on synthesis and secretion by these cells, experiments were performed as follows. Cells were prelabeled for 24 h with 0.5 pCi of [-‘H]choline in serum-free minimum essential medium. The medium was removed and fresh serum-free medium with 0.5 pCi of VHIcholine was added with or without CM. Concurrently, an aliquot of phospholipid suspension in 50 mM Tris (pH 7.2) or only 50 mM Tris (control) was added to the cell cultures such that the final concentration was 0, 25,
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or 100 pg phospholipid/mL. The phospholipids used were dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine. Each had been prepared by drying under N, and sonicating (2 x 30 s) into 50 mM Tris (pH 7.2). To most cell monolayers, isoxsuprine was added to a final concentration of 10 pM to induce secretion. Some monolayers were maintained without isoxsuprine to measure the baseline levels of synthesis and secretion of radioactive phospholipid. We have shown previously that this 0adrenergic agonist at a concentration of 10 pM induces secretion in these cells [16]. After 24 or 48 h, the medium was collected by carefully aspirating into individual tubes through Whatman GF/C filters to remove any cellular debris. The cell monolayer was washed with balanced salt solution and the cells were released by trypsin and extracted into ch1oroform:methanol as described above. Radioactivity in phosphatidylcholine within the medium and the cells was determined as described above. To examine the potential of fetal rabbit alveolar type I1 cells to incorporate phospholipid, on the third day after isolation the cells were prelabeled with 0.5 pCi/ml of ['Hlcholine for 24 h. Cells were transferred to fresh medium with additional fresh ['Hlcholine and small amounts (50 &well) of either dipalmitoyl-~dipalmitoyl-l-'4C]phosphatidylcholine or l-palmitoyl2-[l-'4C]oleoylphosphatidylcholine. Both phospholipids were prepared as described in 50 mM Tris and an identical aliquot of Tris was added to the control cultures. Portions of the cultures were exposed to CM (20%, v/v) and the volumes of other additions adjusted accordingly. After 24 h the medium and cells were collected, extracted, and assayed by thin layer chromatography as described above to determine the incorporation of 14Cor 'H into total phosphatidylcholine. In addition, since some phospholipid may be bound to the external cell membrane and not incorporated into the cellular pool, trypsin-resistant uptake of dipalmitoyl-[dipalmitoyl-'4C]pho~phatidylcholine or l-palmitoyl-2['4C]oleoylphosphatidylcholinewas assessed after 24 h as described by Rice et al. [24] with minor modifications. In all calculations of incorporation of ['4C]phospholipid into cells, the trypsin-releasable uptake was subtracted from the total phospholipid uptake to obtain a value representing incorporation of the phospholipid into the cell. Where necessary, dual label scintillation counting was done with window settings of 0-400 for tritium and 400-700 for I4C. Under these conditions, radioactivity is determined with an efficiency of 50% for isotope 1 ('H) and 80% for isotope 2 ("C) with a ratio of 'H/I4C > 50/1 and < I % spill between channels.
Statistical Analyses Statistical comparisons were done using Duncan's new multiple range test for multiple comparisons after determining the validity of such a compari-
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son by analysis of variance [25]. A probability of P for significant differences.
< .05 was used
to test
RESULTS
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The typical appearance of the fetal type I1 alveolar cells is shown in Fig. 1, Exposure to CM for 24 h induced the appearance of opaque inclusion bodies that were very prominent within the cytoplasm of these cells. Few were
Figure 1 Mason’s tannic acid-based stain [20]for lamellar bodies shows that in differentiating fetal cells not exposed to C M very few, possibly precursor-type inclusions are visible (d,arrows). In contrast, after exposure to C M for 24 h lamellar bodies are easily discernible in virtually all the cells (b). Cells appeared identical after a total of 48 h exposure to CM. (250 X )
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observed without prior exposure to CM. Cells appeared identical after exposure of the cells to CM for an additional 24 h. The incorporation of [3H]choline into total and disaturated phosphatidylcholine by fetal type I1 cells as a function of the extracellular choline concentration is shown in Fig. 2. After 24 h, at choline concentrations of 0.007mM as supplied by the stock minimum essential medium (indicated by the arrow in the figure), taking isotope dilution into account, the cells synthesized a total of 350 pmol/well of phosphatidylcholine, of which slightly less than 45% was disaturated. As extracellular choline levels were raised, the synthesis of total cellular phosphatidylcholine increased and peaked at approximately 1050 pmol in 0.08mM choline. Concurrently, the formation of DSPC increased in parallel with that of total phosphatidylcholine such that at a choline concentration of 0.08 mM the proportion of radioactive label in either phospholipid was similar to that observed at lower choline concentrations. In cultures that were exposed to 20% CM (v/v) at an extracellular choline concentration of 0.007 mM, the incorporation of radioactive precursor into total phosphatidylcholine was greater than that observed in cell cultures without CM. Similarly, the DSPC labeling level was higher than that in those cell monolayers not exposed to the CM. At higher choline concentrations associated with a peak level of phospholipid formation, the response to CM was augmented such that those cell monolayers incubated
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m M Choline Figure 2 Incorporation of [3H]choline into total phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC) over 24 h by fetal rabbit type I1 cells in vitro with or without 20% (v/v) of conditioned medium (CM). The arrow indicates the approximate concentration of choline in serum-free medium. Each point represents the mean f SEM for three separate preparations with a minimum of eight samples per preparation. Lines are best-fit approximations fit by computer.
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with 0.08 mM choline synthesized approximately 1950 pmol total phosphatidylcholine per well, of which 50% was disaturated. Figure 3a shows the effect of dipalmitoylphosphatidylcholine (DPPC) on the incorporation of [‘Hlcholine into phosphatidylcholine by fetal rabbit type I1 alveolar cells in vitro. DPPC reduced the incorporation of [‘Hlcholine into phosphatidylcholine in a dose-dependent manner. This reduction was marked particularly after 48 h in that either 25 or 100 pg DPPC per well significantly reduced (P < .05,P < .01, respectively) the incorporation of [3H]choline into phosphatidylcholine. In those monolayer cultures incubated with 20% CM, the level of incorporation of fH]choline into phosphatidylcholine was elevated compared with those cultures not exposed to CM. DPPC significantly reduced (P < .Ol) the incorporation of VHIcholine into phosphatidylcholine, but only in those cultures exposed to the phospholipid for 48 h at a concentration of 100 pg/well. The corresponding secretion of tritium-labeled phosphatidylcholine by S y n t h e s i s of [ 3 H ] - l a b c l l e d PC
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Figure 3 (a) Incorporation of [3H]choline into cellular phosphatidylcholine by fetal type I1 cells in the presence of DPPC. Cells were prelabeled with [3H]choline for 24 h and transferred to fresh medium with or without 20% conditioned medium (CM) and differing amounts of D P P C (0,25, or 100 &well) with 10 p M isoxsuprine for 24 or 48 h. Each bar represents the mean t SEM of four separate experiments with a minimum of six observations per experiment. C , cultures not treated with isoxsuprine; ‘), , significantly different ( P < .05, P < 01, respectively) from cultures not exposed to phospholipid (0 &well) as determined by Duncan’s multiple range test; PC, phosphatidylcholine. (b) Secretion of [3H]choline-labeled phosphatidylcholine by fetal type I1 cells in the presence of DPPC. Cells were treated as described in (a), the medium was collected after 24 or 48 h and filtered, and the radioactivity in SEM of four separate experiments phosphatidylcholine was determined. Each bar represents the mean with a minimum of six observations per experiment. C , cultures not treated with isoxsuprine; ”, significantly different (P < .05) from cultures not exposed t o phospholipid (0 pg/well) as determined by Duncan’s multiple range test; CM, conditioned medium; PC, phosphatidylcholine.
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fetal type I1 cells exposed to DPPC is shown in Fig. 3b. Secretion of tritiumlabeled phospholipid was reduced significantly (P < .05) after exposure of the cells to 25 or 100 pg DPPC for 24 h. This effect was overcome largely by 48 h. The effect of 1-palmitoyl-2-oleoylphosphatidylcholine on the incorporation of [)H]choline into phosphatidylcholine is shown in Fig. 4a. The radiolabeling of cellular phosphatidylcholine was reduced significantly (P < .05) after 24 h exposure to 1-palmitoyl-2-oleoylphosphatidylcholineat either 25 or 100 pg per well. Similarly, after 48 h the incorporation of ['Hlcholine was reduced significantly (P < .01) in the presence of 100 pg phospholipid per well. Conditioned medium augmented the radioactive labeling level after 48 h, but did not prevent a significant reduction (P < .05) in ['Hlcholine incorporation into cellular phosphatidylcholine when 100 pg of 1-palmitoyl-2-oleoylphosphatidylcholine was added to the incubation for
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Figure 4 (a) Incorporation of ['Hlcholine into cellular phosphatidylcholine by fetal type I1 cells in the presence of 1-palmitoyl-2-oleoylphospharidylcholine. Cells were treated as described for Fig. 3a except (0, 25, or 100 pg/well) was used in place of DPPC. After 24 that I-palmitoyl-2-oleoylphosphatidylcholine or 48 h the cells were collected. Incorporation of ['Hlcholine into cellular phosphatidylcholine was determined. Each bar represents the mean k SEM of four separate experiments with a minimum of six observations per experiment. C, cultures not treated with isoxsuprine; '*, + , significantly different (P < .05, P < .01, respectively) from cultures not exposed to phospholipid (0 pg/well) as determined by Duncan's multiple range test; CM, conditioned medium; PC, phosphatidylcholine. (b) Secretion of [3H]choline-labeled phosphatidylcholine by fetal type If cells in the presence of I-palmiroyl-2oleoylphosphatidylcholine. Cells were treated as described in (u) and the radioactivity in phosphatidylcholine in the medium was determined. Each bar represents the mean zk SEM of four separate experiments with a minimum of six observations per experiment. C, cultures not treated with isoxsuprine; + , significantly different (P < .01) from cultures not exposed to phospholipid (0pg/well) as determined by Duncan's multiple range test; CM, conditioned medium; PC, phosphatidylcholine.
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24 h. Similarly, radioactive precursor incorporation was reduced significantly in the presence of conditioned medium by exposure of the cells to 25 or 100 pg phospholipid per well for 48 h ( P < .05, P < .01, respectively). The secretion of tritium-labeled phosphatidylcholine by fetal type I1 cells in the presence of ~-palmitoyl-2-oleoylphosphatidylcholine is shown in Fig 4b. Exposure of the cells to 25 pg of ~-palmitoyl-2-oleoylphosphatidylcholine for 48 h increased significantly (P < .05) the secretion of tritium-labeled phosphatidylcholine. No other effects were observed. Since the incubations described above were conducted on separate cell preparations, a direct comparison of the effect of the two phospholipids on the cellular labeling, uptake, and secretion was not possible. To directly compare DPPC and 1-palmitoyl-2-oleoylphosphatidylcholine uptake and effect on cell labeling, isolated fetal type I1 cells were prelabeled for 24 h with [3H]choline and removed to fresh medium with ['Hlcholine and exposed concurrently t o 50 ng of either [14C]DPPC or ['4C]l-palmitoyl-2oleoylphosphatidylcholine for 24 h. Comparison of the effect of these phospholipids on fH]choline incorporation into cellular phosphatidylcholine is shown in Fig. 5a. The unsaturated phospholipid significantly depressed ( P < .01) the ['Hlcholine incorporation into phosphatidylcholine compared with DPPC. The presence of 20% conditioned medium did not alter this effect. Comparison of the effect of DPPC o r l-palmitoyl-2oleoylphosphatidylcholine on secretion of 3H-labeledphosphatidylcholine is shown in Fig. 5b. 1-Palmitoyl-2-oleoylphosphatidylcholine significantly augmented secretion compared with DPPC in the presence or absence of conditioned medium (P < .Ol). Conditioned medium increased significantly (P < . O l ) the potential of the cells to release tritium-labeled phosphatidylcholine compared with the cells incubated under serum-free conditions. Figure 6 shows the non-trypsin-dissociable I4C-labeledphosphatidylcholine incorporated over 24 h by the fetal type I1 cells expressed as a percentage of that which was initially available. The level of I4C-labeled l-palmitoyl-2oleoylphosphatidylcholine associated with the cells was significantly less ( P < .05) than that of the I4C-labeledsaturated moiety. This observation was consistent for the cells exposed to conditioned medium as well. However, this medium did augment significantly the incorporation of "C-labeled disaturated phospholipid compared with cells in serum-free medium.
DISCUSSION Type I1 alveolar cells are the source of the phospholipid components of the pulmonary surfactant. These cells secrete this material onto the alveolar surface where it adsorbs to the surface monolayer after passing through a transitional tubular myelin phase [26]. Presumably, after cycling through the monomolecular film at the air-water interface during the expansion and contraction phases of the respiratory cycle, certain of the phospholipid com-
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Figure 5 (a) Corn arison of the effect of DPPC or I-palmitoyl-2-oleoylphosphatidylcholine (50 ng/well of either phospholipid) on incorporation of [ Hlcholine into cellular phosphatidylcholine by fetal rabbit type I1 cells after 24 h. Each bar represents the mean f SEM of three separate experiments with a minimum of six observations per experiment. +, significantly different (P < .01) from corresponding cultures exposed t o DPPC determined by Duncan’s multiple range test. (b)Comparison of the effect of DPPC or 1palmiroyl-2-oleoylphosphatidylcholine on secretion of [3H]choline-labeled phosphatidylcholine by fetal rabbit type I1 cells over 24 h. Each bar represents the mean f SEM of three separate experiments with a minimum of six observations per experiment. +, significantly different (P < .01) from corresponding cultures exposed to DPPC as determined by Duncan’s multiple range test; A, significantly different (P < .01) from cultures not exposed to conditioned medium as determined by Duncan’s multiple range test.
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Figure 6 Incorporation of [14C]DPPC or [’4C]1-palmitoyl-2-oleoylphosphatidylcholine by fetal type I1 cells over 24 h. Results are expressed as a percentage of the total phospholipid available for incorporation and represent non-trypsin-dissociable radioactivity. Each sample was exposed t o an identical amount of either phospholipid. Each bar represents the mean zk SEM of three separate experiments with a minimum of six observations per experiment. A, significantly different (P < .05)from corresponding cultures exposed to DPPC as determined by Duncan’s multiple range test; 6 , significantly different (P < .01) from corresponding cultures not exposed to conditioned medium as determined by Duncan’s multiple range test.
ponents are replenished with new phospholipid [ll]. The fate of the “old” surfactant that had been displaced from the monomolecular film has not been determined. Lavage of rabbit or mouse lung yields several subpopulations of material, components of which may represent this used surfactant [27, 281. Estimates of the cellular reutilization of this material suggest that substantial quantities (> 90°/o) may be reincorporated by lung tissue of 3day-old rabbits [9, lo]. Since fetal lung synthesizes and secretes lameller body material [29], it was of interest to us to examine the potential for isolated fetal type I1 cells to incorporate phospholipid as an analogy to reutilizing surfactant components. In addition, while little is known concerning circulating choline levels or how these levels fluctuate during gestation [30-321, it has been demonstrated that the pool size of choline doubles in rabbit lung between the 25th gestational day and the adult [5]. We therefore wished to examine the dynamics of phosphatidylchohne synthesis in fetal rabbit type I1 cells as a function of availability of extracellular choline and the differentiation state of these cells. Previous observations using isolated fetal type 11 cells and a pulse-chase method suggested that under the culture conditions we normally employ, using minimum essential medium, the incorporation pathway for [3H]choline was not oper-
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ating at maximum capacity. The present results under varying extracellular choline indicate that isolated fetal type I1 cells produce more phosphatidylcholine as well as respond to the stimulus of fibroblast conditioned medium (which contains FPF [33]) to a greater extent as choline concentration is raised. Furthermore, these cells appear to respond to changes in the extracellular choline concentrations to a greater degree than other cells, which may reflect the importance of phosphatidylcholine metabolism in type I1 cells. Using cultured rat hepatocytes, Pritchard and Vance [34] observed that varying medium choline concentration from 5 to 40 pM increased phosphatidylcholine synthesis by about 50%. Comparable variations in choline concentration using fetal type I1 cells showed that under unstimulated conditions, total phosphatidylcholine synthesis at least doubled between 5 pM (or 7 pM supplied by standard culture conditions) and 40 pM. The situation was even more marked after exposure of the type I1 cells to conditioned medium. However, the relation this may bear to adjustments in vivo in the fetal lung phosphatidylcholine synthesis is not clear since circulating fetal choline levels throughout gestation have not been examined. There is some evidence that the premature human infant has significantly higher plasma choline levels than the mother [32], which therefore may correspond to the higher fetal lung choline pool sizes observed by Tokmakjian et al. [5] in the fetal rabbit and elevated phosphatidylcholine synthesis by fetal type I1 alveolar cells, as shown here. Preliminary evidence has appeared recently that lung may take up extracellular surfactant-associatedphospholipid. Jacobs et al. [&lo] and Young et al. [35] have shown that in vivo the rabbit and rat lung, respectively, have the capacity to incorporate instilled phospholipid intact and add this to the secretory pool. In addition, several studies indicate that isolated type I1 cells incorporate radioactively labeled phosphatidylcholine and may reprocess this material [12, 131. Furthermore, component parts of the surfactant may alter the reincorporation of phospholipid by these cells [8-10, 35-37]. The present study extends these observations to differentiating fetal rabbit type I1 cells and demonstrates that they can incorporate phospholipid from the extracellular pool. It also shows that these cells incorporate phosphatidylcholines of differing degrees of saturation at different rates. In addition, while both phospholipids substantially reduced the de novo synthesis of phosphatidylcholine as measured by incorporation of [3H]choline, 1palmitoyl-2-oleoylphosphatidylcholinewas more potent in this regard. Such an effect may be due to entrance of this phospholipid into the deacylation/reacylation pathway [4]. O n the other hand, portions of the disaturated phospholipid may directly enter the intracellular surfactant pool or be degraded such that it does not compete with de novo synthesized intermediates for enzyme sites. The secretion of de novo synthesized phosphatidylcholine also appeared to be affected by the presence of these phospholipids. DPPC in particular
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depressed the release of tritiated phosphatidylcholine after 24 h of incubation compared with cultures not exposed to phospholipid. Comparison of the potency of the two phospholipids with regard t o secretion showed that DPPC reduced release of tritium-labeled phosphatidylcholine to a greater degree than did the unsaturated phospholipid. This suggests that synthesis, secretion, and reutilization in these cells may function in a concerted fashion to regulate alveolar surfactant levels. Furthermore, the processes of phosphatidylcholine synthesis and secretion may be altered by the presence of phospholipid supplied by a reprocessing system. The presence of /3adrenergic stimulation in this scheme must also be considered. As observed previously [15], addition of isoxsuprine, in most cases, resulted in a modest elevation in release of radiolabeled phospholipid. Considering that the biological half-life of this drug in the adult human is in the order of 1.25 h [38], we could expect little effect of the drug after a maximum of 7.5 h given that the previous observations indicate that low concentrations in the order of 1 pM could still induce secretion [15]. The response induced by such a relatively long, unphysiological exposure, compared with what might be expected in vivo, suggests that the overall effect of this drug on secretion by the type I1 cells is not of a large magnitude. These results support the concept of a dynamic equilibrium in the surfactant system composed of mechanisms of synthesis, secretion, and reutilization. With regard to the developing lung, these mechanisms appear to become functionally competent as fetal lung cells undergo the final stages of prenatal development. Specifically, the cells acquire a greater capacity to synthesize and secrete phosphatidylcholine as substrate availability increases. It is further apparent that induction of differentiation in type I1 alveolar cells affects the capacity to incorporate phospholipid and, presumably, to reutilize secreted material. However, it is important to note that the dissimilarity between differentiated and undifferentiated cells occurred predominantly in the magnitude and not in the characteristics of the response. The results of this and other studies indicate that fetal and adult alveolar type I1 cells not only synthesize pulmonary surfactant but also reincorporate and reprocess surfactant phospholipid material. The chemical character of the phospholipid as well as the availability of precursors for de novo synthesis and, in the case of the developing fetal lung, the differentiation state of the fetal lung cells are all determinants of the dynamic surfactant system. Clearly, the pathways whereby these components are degraded and/or returned to the surfactant pool and the control of the development of these pathways requires considerably more examination. The author acknowledges the support of the Medical Research Council of Canada, the Manitoba Health Research Council, and the University of Manitoba Senate Research Committee.
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REFERENCES 1. Rooney SA: Lung surfactant. Environ Hlth Persp 55:205-226, 1984. 2. Possmayer F: Biochemistry of pulmonary surfactant during fetal development and in the perinatal period. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 295-355. 3. Miller JC, Weinhold PA: Cholinephosphotransferase in rat lung. J Biol Chem 256: 12662-12665, 1981. 4. Batenburg JJ: Biosynthesis and secretion of pulmonary surfactant. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 237-270. 5. Tokmakjian S, Haines DSM, Possmayer F: Pulmonary phosphatidylcholine biosynthesis: alterations in the pool sizes of choline and choline derivatives in rabbit fetal lung during development. Biochim Biophys Acta 663:557-568, 1981. 6. Desai R, Tetley TD, Curtis CG, Powell GM, Richards RJ: Studies on the fate of pulmonary surfactant in the lung. Biochem J 176:455-462, 1978. 7. Dobbs LG, Wright JR, Hawgood S, Gonzalez R, Venstrom K, Nellenbogen J: Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type I1 cells. Proc Natl Acad Sci 84:lOlO-1014, 1987. 8. Jacobs HC, Ikegami M, Jobe A, Barry DD, Jones S: Reutilization of surfactant phosphatidylcholine in adult rabbits. Biochim Biophys Acta 837:77-84, 1985. 9. Jacobs H, Jobe A, Ikegami M, Conaway D: The significance of reutilization of surfactant phospholipids. J Biol Chem 258:4156-4165, 1983. 10. Jacobs H, Jobe A, Ikegami M, Jones S: Surfactant phosphatidylcholine source, fluxes and turnover times in 3-day-old, 10-day-old and adult rabbits. J Biol Chem 257:1805-1810, 1982. 11. Jobe A, Jacobs HC: Catabolism of pulmonary surfactant. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 271-293. 12. Chander A, Claypool WD, Strauss JF, Fisher AB: Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am J Physiol 245:C397-C404, 1983. 13. Chander A, Reicherter J, Fisher AB: Degradation of dipalmitoyl phosphatidylcholine by isolated rat granular pneumocytes and reutilization for surfactant synthesis. J Clin Invest 79:1133-1138, 1987. 14. Clements JA, Tooley WH: Kinetics of surface-active material in the fetal lung. In Hodson CWA, ed., Development of the Lung. New York, Dekker, 1972, pp. 349-366. 15. Rasmusson M-G, Scott JE, Oulton MR, Temple S: characterization and comparison of the role of 0-agonists on in vivo and in vitro surfactant-related phospholipid synthesis and secretion by fetal rabbit lung and isolated type I1 alveolar cells. Exp Lung Res 142311-822, 1988. 16. Scott JE, Possmayer F, Harding PGR: Alveolar pre-type I1 cells from the fetal rabbit lung: isolation and characterization. Biochim Biophys Acta 753: 195-204, 1983. 17. Tanswell AK, Joneja MG, Lindsay J, Vreeken E: Differentiation-arrested rat
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fetal lung in primary monolayer cell culture, I: development of a differentiationarrested and growth-supporting culture system using carbon-stripped bovine fetal calf serum. Exp Lung Res 5:37-48, 1983. 18. Scott JE, Possmayer F, Quirie MA, Tanswell AK, Harding PGR: Alveolar pretype I1 cells from the fetal rabbit lung: Characterization of the production of disaturated phospholipid during cellular differentiation. Biochim Biophys Acta 879:292-300, 1986. 19. Smith BT: Pulmonary surfactant during fetal development and neonatal adaptation: hormonal control. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 357-382. 20. Mason RJ, Walker SR, Shields BA, Henson JE, Williams MC: Identification of rat alveolar type I1 epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131:786-788, 1985. 21. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917, 1959. 22. Skipski VP, Barclay M: Thin-layer chromatography of lipids. Methods Enzymol 14~530-598,1969. 23. Mason RJ, Nellenbogen J, Clements JA: Isolation of disaturated phosphatidylcholine with osmium tetroxide. J Lipid Res 17:281-284, 1976. 24. Rice WR, Sarin VK, Fox JL, Baatz J, Wert S, Whitsett JA: Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells. Biochim Biophys Acta 1006:237-245, 1989. 25. Ott L: An Introduction to Statistical Methods and Data Analysis. North Scituate, Duxbury, 1977, pp. 392-393. 26. Stratton CJ: Morphology of surfactant producing cells and of the alveolar lining layer. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 67-118. 27. Magoon MW, Wright JR, Baritussio A, Williams MC, Goerke J, Benson BJ, Hamilton RL, Clements JA: Subfractionation of lung surfactant: implications for metabolism and surface activity. Biochim Biophys Acta 750:18-31, 1983. 28. Gross NJ, Nardine KR: Surfactant subtypes in mice: characterization and quantitation. J Appl Physiol 66:342-349, 1989. 29. Kikkawa Y, Motoyama EK, Gluck L: Study of the lungs of fetal and newborn rabbits. Am J Pathol 52:177-209, 1968. 30. Haubrich DR, Gerber N, Pflueger AB, Zweig M: Tissue choline studied using simple chemical assay. J Neurochem 36:1409-1417, 1981. 31. Knipper M, Boekhoff I, Breer H: Isolation and reconstitution of the highaffinity choline carrier. FEBS Lett 235-237, 1989. 32. McMahon KE, Farrell PM: Measurement of free choline concentrations in maternal and neonatal blood by micropyrolysis gas chromatography. Clin Chim Acta 149:l-12, 1985. 33. Smith BT: Lung maturation in the fetal rat: acceleration by injection of fibroblast-pneumocyte factor. Science 204:1094-1095, 1979. 34. Pritchard PH, Vance DE: Choline metabolism and phosphatidylcholine biosynthesis in cultured rat, hepatocytes. Biochem J 196:261-267, 1981. 35. Young SL, Wright JR, Clements JA: Cellular uptake and processing of surfactant lipids and apoprotein SP-A by rat lung. J Appl Physiol66:1336-1342, 1989.
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36. Rice WR, Ross GF, Singleton FM, Dingle S, Whitsett JA: Surfactant-associated protein inhibits phospholipid secretion from type I1 cells. J Appl Physiol 63:692-698, 1987. 37. Wright JR, Wager RE, Hawgood S, Dobbs L, Clements JA: Surfactant apoprotein M, = 26,000-36,000 enhances uptake of liposomes by type I1 cells. J Biol Chem 262:2888-2894, 1987. 38. Avery GS: Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics. London, Churchill Livingstone, 1976, p. 896.
ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type II Alveolar Cell from Fetal Rabbit Lungs J. E. Scott To cite this article: J. E. Scott (1992) Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type II Alveolar Cell from Fetal Rabbit Lungs, Experimental Lung Research, 18:4, 563-580, DOI: 10.3109/01902149209064346 To link to this article: http://dx.doi.org/10.3109/01902149209064346
Published online: 02 Jul 2009.
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Phosphatidylcholine Synthesis, Secretion, and Reutilization During Differentiation of the Surfactant-Producing Type I1 Alveolar Cell from Fetal Rabbit Lungs
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J. E. Scott
ABSTRACT: Evidence indicates that pulmonary pool sizes of choline and related intermediates available f . r synthesis of phosphatidylcholine, the major component of the surfactant, change during gestation. Furthermore, recent data suggest that the type 11 lung cells that produce the suvfactant potentially can reutilize components of this material. However, the relationship of the de novo synthetic mechanism to the secretion and reutilization of phosphatidylcholine has not been established. This is particularly true in the case of the fetal lung where, although alterations i n precursor pool sizes, including choline, have been demonstrated, few or no data are available concerning how phosphatidylcholine synthesis affects or is affected by secretion and reutilization of this phospholipid by fetal type 11 cells. The present study was undertaken to determine the effect of availability of choline on de novo synthesis of phosphatidylcholine by isolated fetal rabbit type 11 cells during the differentiation process. In addition, differentiating type 11 alveolar cells were used to examine the hypothesis that these cells incorporate phospholipid from the extracellular environment and the quantity and/or composition of this phospholipid differently affects cellular secretion or de novo phosphatidylcholine synthesis. Assuming that the cells did not discriminate between radioactive and nonradioactive choline, elevation of extracellular choline increased the synthesis of cellular phosphatidylcholine and disaturated phosphatidylcholine in a dose-dependent manner to 0.08 mM choline. Cells induced to differentiated by exposure to fibroblast-conditioned medium synthesized more total and disaturated phosphatidylcholine at all extracellular choline concentrations. Incubation of the fetal type 11 cells with dipalmitoylphosphatidylcholine or l-palmitoyl-2oleoylphosphatidylcholine signlficantly depressed the incorporation of (3H/choline into
From the Departments of Oral Biology and Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada. Address all cowespondence to Dv. J. E. Scott, Department of Oral Biology, Faculty of Dentistry, University of Manttoba, 780 Bannatyne Avenue, Winnipeg, Manitoba, R3E OW3, Canada. Received 6 June 1990; accepted 20 November 1991.
Experimental Lung Research 18:563-580 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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cellular phosphatidylcholine after 24 or 48 h, but not necessarily at both times. Dipalmitoylphosphatidylcholine depressed the secretion of PH]choline-labeled phosphatidylcholine after incubation for 24 h. 1-Palmitoyl-2-oleoylphosphatidylcholine stimulated the secretion of tritium-labeled phosphatidylcholine at a concentration of 25 ,ug/mL after 48 h. Comparison of the phospholipid efect by incubating the cells with 50 ng of I4C-labeled phospholipid for 24 h showed that I-palmitoyl-2-oleoylphosphatidylcholine significantly reduced the synthesis of 'H-labeled phosphatidylcholine compared to dipalmitoylphosstimulated secrephatidylcholine. I n contrast, 1-palmitoyl-2-oleoylphosphatidylcholine tion of 'H-labeled phosphatidylcholine compared with the disaturated moiety. B e d g e r entiation state of the cells altered the magnitude of the cellular secretion response but not the character. Finally, sign2ficantly less [14C'1-palmitoyl-2-oleoylphosphatidylcholinewas associated with the non-trypsin-dissociable fraction of the cellular phospholipid pool than was the 14C-labeled disaturated moiety Altering the dgerentiation state of the cells sign 2ficantly increased the cell-associated fraction of the disaturated phospholipid; the amount of unsaturated phospholip~associated with the type 11 cells was not altered. These results indicate that the level of synthesis of phosphatidylcholine in differentiating type 11alveolar cells is at least to some degree dependent on the availability of choline. I n addition, the extracellular phospholipid environment may influence both the rate of synthesis and secretion of phosphatidylcholine by these cells.
INTRODUCTION The pulmonary surfactant is a complex mixture of phospholipids and proteins that acts primarily at the air-liquid interface of the lung to reduced surface tension within the alveolus, thereby preventing collapse of the lung at maximum expiration. In the developing lung, surfactant synthesis is initiated prior to term within the type I1 alveolar cells. In type I1 cells, phosphatidylcholine, the main constituent of the surfactant, is produced by a process of dephosphorylation of phosphatidic acid to diacylglycerol, which is converted enzymatically to phosphatidylcholine by cholinephosphotransferase (see reviews by Rooney [l] and Possmayer [2]). The high levels of the disaturated form of phosphatidylcholine that characterize the surfactant appear to be supplied by two mechanisms. The first is a direct synthetic route, whereby the cholinephosphotransferase adds the cholinephosphate moiety of CDPcholine to dipalmitoylglycerol [3]. The second is the reacylation route, whereby 1-palmitoyl-2-unsaturated phosphatidylcholine is deacylated by a phospholipase A, and reacylated with palmitoyl CoA [l, 41. In either case, the evidence suggests that the limiting step in PC synthesis is the production of CDPcholine [2]. However, changes that occur in pool sizes of choline and its associated anabolic products toward the end of gestation [5] suggest that the entire mechanism by which phosphatidylcholine synthesis is controlled undergoes a fundamental adjustment in the developing lung. Indeed, as surfactant-related PC synthesis is initiated in the fetal rabbit lung, the pool size of choline doubles while the pool sizes of other cholinecontaining intermediates decline [5]. This is associated with a doubling of
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phosphatidylcholine content prior to birth. However, it is not clear what relation this change in pool size bears to phosphatidylcholine synthesis specifically within the developing surfactant-producing type I1 cells. Therefore, as a component of the present study, radioactive choline incorporation into total and disaturated phosphatidylcholine was examined as a function of the concentration and availability of extracellular choline in isolated fetal rabbit I1 cells. In addition to the two de novo pathways for phosphatidylcholine synthesis, recent evidence suggests that the type I1 alveolar cells may reutilize components of previously secreted surfactant, presumably there by maintaining intracellular tissue stores [6-111. In fact, this latter mechanism would appear to constitute a major metabolic route considering that surfactant phosphatidylcholine apparently is reutilized with a high efficiency at least by 3-day-old rabbit lung [9, 101. Other evidence indicates that this process occurs by a net uptake mechanism, since isolated rat type I1 cells are observed to incorporate liposomal-encapsulated radioactive sucrose intact [ 12, 131. Nevertheless, the potential for phospholipid reincorporation and reutilization has not been explored in type I1 cells of the preterm lung when the pool size of disaturated phospholipid is small [14] and one might expect a mechanism for conservation of secreted surfactant material to be developing. Using isolated differentiating type I1 alveolar cells, we have established previously that the P-adrenergic agonist isoxsuprine stimulates secretion and synthesis of phosphatidylcholine de novo from radioactively labeled choline [ 151. Furthermore, we observed a paradoxical decline in cell-associated radioactively labeled phospholipid when the incubation was extended past 24 h. Since evidence from the investigations cited above [6-131 indicates that secreted phospholipid material may be reutilized by lung and lung cells, the possibility exists that our previous observations may be due in part to the influence of reincorporated phospholipid on the de novo synthetic or secretory rate of phosphatidylcholine in fetal type I1 alveolar cells. Therefore, to test the hypothesis that availability of extracellular choline influences fetal type I1 alveolar cell phosphatidylcholine synthetic rate and that this rate may be affected by phospholipid reutilization and P-adrenergic-based secretion processes, the incorporation of radioactive precursor into cellular phosphatidylcholine was measured in isolated differentiating fetal rabbit type I1 cells as the concentration of extracellular choline was increased, as well as under varying conditions of chemical form and concentration of added extracellular phosphatidylcholine. In addition, in some experiments radioactively labeled dipalmitoylphosphatidylcholine or l-palmitoyl-2oleoylphosphatidylcholine was incubated with fetal alveolar type I1 cells to confirm that they take up phospholipid from the external environment and to determine if degree of saturation affects labeling of the cellular phospholipid pool.
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MATERIALS AND METHODS Animals and Materials New Zealand White timed-pregnant rabbits were purchased from Blue Farm Rabbitry (St. Adolphe, MB). Culture materials were from Gibco Laboratories (Burlington, Ont.) unless otherwise specified. Radioactives including [rnethylJH]choline chloride (specific activity, 58 mCi/mmol), L-a[dipalmityl-l-'4C]dipalmitoylphosphatidylcholine(specific activity, 112.0 mCi/mmol), and ~-cr-palmitoyl-2-[l-'4C]oleoylphosphatidylcholine(specific activity, 57 mCi/mmol) were from New England Nuclear (Lachine, PQ). Other chemicals were from Sigma Chemical Co. (St. Louis, MO).
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Cell Isolation Undifferentiated type I1 alveolar cells were isolated as described previously with modifications as described below [16]. O n the 24th gestational day (day of breeding designated day O), does were killed by an IV overdose of Euthanyl (240 mg/mL sodium pentobarbitol). The fetuses were removed, immediately decapitated, and placed in sterile ice-cold Hanks balanced salt solution (HBSS). Fetal lungs were removed, chopped on a Sorval tissue chopper (Sorval Instruments, Newtown, CT), and immersed in a solution of trypsin/EDTA (0.05°/~/0.010/~)with 75 mg deoxyribonuclease I in calcium- and magnesium-free HBSS in water-jacketed trypsinization flasks (Wheaton, Millville, NJ). The suspension was stirred on a magnetic plate for 45 min at a constant temperature of 37OC. The action of the trypsin was stopped by addition of 50 mL of minimum essential medium with 10% carbon-stripped fetal bovine serum (v/v), prepared as described by Tanswell et al. [7]. After trypsinization, the material was filtered through three layers of Nitex gauze and centrifuged at 250g for 15 min to pellet the cells. Cells were resuspended in minimum essential medium and plated into 80-cm2flasks, which were incubated at 37OC in an atmosphere of 5% CO, and 95% air to allow fibroblasts to adhere. After 1.5 h the unattached cells were collected. The attached fibroblasts were grown to confluence in minimum essential medium supplemented with 10% fetal bovine serum (v/v) and used to prepare conditioned medium (CM) by incubation with serumfree minimum essential medium for 24 h, as described previously [18]. The CM was used to induce differentiation [19]. The unattached cells were layered on a discontinuous metrizamide gradient (10 mL of 0.10 M over 10 mL of 0.22 M metrizamide) and centrifuged at 2OOg for 20 min. Cells that collected at the interface of the gradient were aliquoted into 2-cm2 wells in 24 multiwell plates (deep well, uncoated, Falcon Plastics, Canlab, Winnipeg, MB). Cells were grown for 3 days at 37°C in minimum essential medium with 10% carbon-stripped fetal bovine serum (v/v) in a humidified atmo-
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sphere of 5% CO,. Purity estimates indicate that greater than 90% of these cells develop lamellar bodies [16]. To examine the cells, after 2-3 days in culture, portions were exposed to CM for 24 h, while others were incubated in standard medium indicated above. Cells were fixed in 1.5% glutaraldehyde in phosphate-buffered saline, as described by Mason et al. [20], to visualize lamellar bodies, and postfixed with 1.0% OsO, in the same buffer. The cell monolayers were treated for 18 h with 1.0% tannic acid in phosphate-buffered saline adjusted to p H 6.8, washed in distilled water, dehydrated in a graded ethanol series, and stained with the blue and red stains as described [20]. Monolayers were cut from bottom of flasks and coverslipped using an aqueous-based mounting medium.
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Treatment and Analyses All experiments were conducted in serum-free minimum essential medium. To characterize the incorporation of radioactive precursor ([3H]choline) into phosphatidylcholine (PC) by these cells, 3 days after isolation, the immature type I1 alveolar cells were incubated with minimum essential medium (control) or minimum essential medium plus CM (20°/0, v/v) to induce differentiation, under increasing concentrations of extracellular choline. Concurrently, 0.5 pCi of [-‘H]choline was added to the incubation. After 24 h, the cells were removed by trypsin and extracted into ch1oroform:methanol (1:2), according to High and Dyer 1211. The incorporation of radioactive label into phosphatidylcholine was determined by thin-layer chromatography of an aliquot of the organic solvent extract in a solvent system of ch1oroform:methanol:acetic acid:water (75:25:1:3) adapted from that described by Skipski and Barclay [22]. The position of phosphatidylcholine was established by running authentic internal standards with alternate samples and visualizing with iodine vapor. Appropriate spots were scraped into scintillation vials and radioactivity was estimated on a Beckman LS5801 scintillation counter with a counting efficiency of 65% for tritium. Quench compensation and disintegrations per minute (DPM) were determined by the method of H# (Beckman Instruments, Palo Alto, CA). Disaturated phosphatidylcholine (DSPC) was isolated by the method of Mason et al. [23] after reaction of aliquots of the samples with osmium tetroxide. Column eluent was collected in scintillation vials, the solvent was allowed to evaporate, and the radioactivity was determined as above. To examine the effect of phosphatidylcholines on synthesis and secretion by these cells, experiments were performed as follows. Cells were prelabeled for 24 h with 0.5 pCi of [-‘H]choline in serum-free minimum essential medium. The medium was removed and fresh serum-free medium with 0.5 pCi of VHIcholine was added with or without CM. Concurrently, an aliquot of phospholipid suspension in 50 mM Tris (pH 7.2) or only 50 mM Tris (control) was added to the cell cultures such that the final concentration was 0, 25,
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or 100 pg phospholipid/mL. The phospholipids used were dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine. Each had been prepared by drying under N, and sonicating (2 x 30 s) into 50 mM Tris (pH 7.2). To most cell monolayers, isoxsuprine was added to a final concentration of 10 pM to induce secretion. Some monolayers were maintained without isoxsuprine to measure the baseline levels of synthesis and secretion of radioactive phospholipid. We have shown previously that this 0adrenergic agonist at a concentration of 10 pM induces secretion in these cells [16]. After 24 or 48 h, the medium was collected by carefully aspirating into individual tubes through Whatman GF/C filters to remove any cellular debris. The cell monolayer was washed with balanced salt solution and the cells were released by trypsin and extracted into ch1oroform:methanol as described above. Radioactivity in phosphatidylcholine within the medium and the cells was determined as described above. To examine the potential of fetal rabbit alveolar type I1 cells to incorporate phospholipid, on the third day after isolation the cells were prelabeled with 0.5 pCi/ml of ['Hlcholine for 24 h. Cells were transferred to fresh medium with additional fresh ['Hlcholine and small amounts (50 &well) of either dipalmitoyl-~dipalmitoyl-l-'4C]phosphatidylcholine or l-palmitoyl2-[l-'4C]oleoylphosphatidylcholine. Both phospholipids were prepared as described in 50 mM Tris and an identical aliquot of Tris was added to the control cultures. Portions of the cultures were exposed to CM (20%, v/v) and the volumes of other additions adjusted accordingly. After 24 h the medium and cells were collected, extracted, and assayed by thin layer chromatography as described above to determine the incorporation of 14Cor 'H into total phosphatidylcholine. In addition, since some phospholipid may be bound to the external cell membrane and not incorporated into the cellular pool, trypsin-resistant uptake of dipalmitoyl-[dipalmitoyl-'4C]pho~phatidylcholine or l-palmitoyl-2['4C]oleoylphosphatidylcholinewas assessed after 24 h as described by Rice et al. [24] with minor modifications. In all calculations of incorporation of ['4C]phospholipid into cells, the trypsin-releasable uptake was subtracted from the total phospholipid uptake to obtain a value representing incorporation of the phospholipid into the cell. Where necessary, dual label scintillation counting was done with window settings of 0-400 for tritium and 400-700 for I4C. Under these conditions, radioactivity is determined with an efficiency of 50% for isotope 1 ('H) and 80% for isotope 2 ("C) with a ratio of 'H/I4C > 50/1 and < I % spill between channels.
Statistical Analyses Statistical comparisons were done using Duncan's new multiple range test for multiple comparisons after determining the validity of such a compari-
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son by analysis of variance [25]. A probability of P for significant differences.
< .05 was used
to test
RESULTS
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The typical appearance of the fetal type I1 alveolar cells is shown in Fig. 1, Exposure to CM for 24 h induced the appearance of opaque inclusion bodies that were very prominent within the cytoplasm of these cells. Few were
Figure 1 Mason’s tannic acid-based stain [20]for lamellar bodies shows that in differentiating fetal cells not exposed to C M very few, possibly precursor-type inclusions are visible (d,arrows). In contrast, after exposure to C M for 24 h lamellar bodies are easily discernible in virtually all the cells (b). Cells appeared identical after a total of 48 h exposure to CM. (250 X )
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observed without prior exposure to CM. Cells appeared identical after exposure of the cells to CM for an additional 24 h. The incorporation of [3H]choline into total and disaturated phosphatidylcholine by fetal type I1 cells as a function of the extracellular choline concentration is shown in Fig. 2. After 24 h, at choline concentrations of 0.007mM as supplied by the stock minimum essential medium (indicated by the arrow in the figure), taking isotope dilution into account, the cells synthesized a total of 350 pmol/well of phosphatidylcholine, of which slightly less than 45% was disaturated. As extracellular choline levels were raised, the synthesis of total cellular phosphatidylcholine increased and peaked at approximately 1050 pmol in 0.08mM choline. Concurrently, the formation of DSPC increased in parallel with that of total phosphatidylcholine such that at a choline concentration of 0.08 mM the proportion of radioactive label in either phospholipid was similar to that observed at lower choline concentrations. In cultures that were exposed to 20% CM (v/v) at an extracellular choline concentration of 0.007 mM, the incorporation of radioactive precursor into total phosphatidylcholine was greater than that observed in cell cultures without CM. Similarly, the DSPC labeling level was higher than that in those cell monolayers not exposed to the CM. At higher choline concentrations associated with a peak level of phospholipid formation, the response to CM was augmented such that those cell monolayers incubated
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m M Choline Figure 2 Incorporation of [3H]choline into total phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC) over 24 h by fetal rabbit type I1 cells in vitro with or without 20% (v/v) of conditioned medium (CM). The arrow indicates the approximate concentration of choline in serum-free medium. Each point represents the mean f SEM for three separate preparations with a minimum of eight samples per preparation. Lines are best-fit approximations fit by computer.
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with 0.08 mM choline synthesized approximately 1950 pmol total phosphatidylcholine per well, of which 50% was disaturated. Figure 3a shows the effect of dipalmitoylphosphatidylcholine (DPPC) on the incorporation of [‘Hlcholine into phosphatidylcholine by fetal rabbit type I1 alveolar cells in vitro. DPPC reduced the incorporation of [‘Hlcholine into phosphatidylcholine in a dose-dependent manner. This reduction was marked particularly after 48 h in that either 25 or 100 pg DPPC per well significantly reduced (P < .05,P < .01, respectively) the incorporation of [3H]choline into phosphatidylcholine. In those monolayer cultures incubated with 20% CM, the level of incorporation of fH]choline into phosphatidylcholine was elevated compared with those cultures not exposed to CM. DPPC significantly reduced (P < .Ol) the incorporation of VHIcholine into phosphatidylcholine, but only in those cultures exposed to the phospholipid for 48 h at a concentration of 100 pg/well. The corresponding secretion of tritium-labeled phosphatidylcholine by S y n t h e s i s of [ 3 H ] - l a b c l l e d PC
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Figure 3 (a) Incorporation of [3H]choline into cellular phosphatidylcholine by fetal type I1 cells in the presence of DPPC. Cells were prelabeled with [3H]choline for 24 h and transferred to fresh medium with or without 20% conditioned medium (CM) and differing amounts of D P P C (0,25, or 100 &well) with 10 p M isoxsuprine for 24 or 48 h. Each bar represents the mean t SEM of four separate experiments with a minimum of six observations per experiment. C , cultures not treated with isoxsuprine; ‘), , significantly different ( P < .05, P < 01, respectively) from cultures not exposed to phospholipid (0 &well) as determined by Duncan’s multiple range test; PC, phosphatidylcholine. (b) Secretion of [3H]choline-labeled phosphatidylcholine by fetal type I1 cells in the presence of DPPC. Cells were treated as described in (a), the medium was collected after 24 or 48 h and filtered, and the radioactivity in SEM of four separate experiments phosphatidylcholine was determined. Each bar represents the mean with a minimum of six observations per experiment. C , cultures not treated with isoxsuprine; ”, significantly different (P < .05) from cultures not exposed t o phospholipid (0 pg/well) as determined by Duncan’s multiple range test; CM, conditioned medium; PC, phosphatidylcholine.
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fetal type I1 cells exposed to DPPC is shown in Fig. 3b. Secretion of tritiumlabeled phospholipid was reduced significantly (P < .05) after exposure of the cells to 25 or 100 pg DPPC for 24 h. This effect was overcome largely by 48 h. The effect of 1-palmitoyl-2-oleoylphosphatidylcholine on the incorporation of [)H]choline into phosphatidylcholine is shown in Fig. 4a. The radiolabeling of cellular phosphatidylcholine was reduced significantly (P < .05) after 24 h exposure to 1-palmitoyl-2-oleoylphosphatidylcholineat either 25 or 100 pg per well. Similarly, after 48 h the incorporation of ['Hlcholine was reduced significantly (P < .01) in the presence of 100 pg phospholipid per well. Conditioned medium augmented the radioactive labeling level after 48 h, but did not prevent a significant reduction (P < .05) in ['Hlcholine incorporation into cellular phosphatidylcholine when 100 pg of 1-palmitoyl-2-oleoylphosphatidylcholine was added to the incubation for
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Figure 4 (a) Incorporation of ['Hlcholine into cellular phosphatidylcholine by fetal type I1 cells in the presence of 1-palmitoyl-2-oleoylphospharidylcholine. Cells were treated as described for Fig. 3a except (0, 25, or 100 pg/well) was used in place of DPPC. After 24 that I-palmitoyl-2-oleoylphosphatidylcholine or 48 h the cells were collected. Incorporation of ['Hlcholine into cellular phosphatidylcholine was determined. Each bar represents the mean k SEM of four separate experiments with a minimum of six observations per experiment. C, cultures not treated with isoxsuprine; '*, + , significantly different (P < .05, P < .01, respectively) from cultures not exposed to phospholipid (0 pg/well) as determined by Duncan's multiple range test; CM, conditioned medium; PC, phosphatidylcholine. (b) Secretion of [3H]choline-labeled phosphatidylcholine by fetal type If cells in the presence of I-palmiroyl-2oleoylphosphatidylcholine. Cells were treated as described in (u) and the radioactivity in phosphatidylcholine in the medium was determined. Each bar represents the mean zk SEM of four separate experiments with a minimum of six observations per experiment. C, cultures not treated with isoxsuprine; + , significantly different (P < .01) from cultures not exposed to phospholipid (0pg/well) as determined by Duncan's multiple range test; CM, conditioned medium; PC, phosphatidylcholine.
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24 h. Similarly, radioactive precursor incorporation was reduced significantly in the presence of conditioned medium by exposure of the cells to 25 or 100 pg phospholipid per well for 48 h ( P < .05, P < .01, respectively). The secretion of tritium-labeled phosphatidylcholine by fetal type I1 cells in the presence of ~-palmitoyl-2-oleoylphosphatidylcholine is shown in Fig 4b. Exposure of the cells to 25 pg of ~-palmitoyl-2-oleoylphosphatidylcholine for 48 h increased significantly (P < .05) the secretion of tritium-labeled phosphatidylcholine. No other effects were observed. Since the incubations described above were conducted on separate cell preparations, a direct comparison of the effect of the two phospholipids on the cellular labeling, uptake, and secretion was not possible. To directly compare DPPC and 1-palmitoyl-2-oleoylphosphatidylcholine uptake and effect on cell labeling, isolated fetal type I1 cells were prelabeled for 24 h with [3H]choline and removed to fresh medium with ['Hlcholine and exposed concurrently t o 50 ng of either [14C]DPPC or ['4C]l-palmitoyl-2oleoylphosphatidylcholine for 24 h. Comparison of the effect of these phospholipids on fH]choline incorporation into cellular phosphatidylcholine is shown in Fig. 5a. The unsaturated phospholipid significantly depressed ( P < .01) the ['Hlcholine incorporation into phosphatidylcholine compared with DPPC. The presence of 20% conditioned medium did not alter this effect. Comparison of the effect of DPPC o r l-palmitoyl-2oleoylphosphatidylcholine on secretion of 3H-labeledphosphatidylcholine is shown in Fig. 5b. 1-Palmitoyl-2-oleoylphosphatidylcholine significantly augmented secretion compared with DPPC in the presence or absence of conditioned medium (P < .Ol). Conditioned medium increased significantly (P < . O l ) the potential of the cells to release tritium-labeled phosphatidylcholine compared with the cells incubated under serum-free conditions. Figure 6 shows the non-trypsin-dissociable I4C-labeledphosphatidylcholine incorporated over 24 h by the fetal type I1 cells expressed as a percentage of that which was initially available. The level of I4C-labeled l-palmitoyl-2oleoylphosphatidylcholine associated with the cells was significantly less ( P < .05) than that of the I4C-labeledsaturated moiety. This observation was consistent for the cells exposed to conditioned medium as well. However, this medium did augment significantly the incorporation of "C-labeled disaturated phospholipid compared with cells in serum-free medium.
DISCUSSION Type I1 alveolar cells are the source of the phospholipid components of the pulmonary surfactant. These cells secrete this material onto the alveolar surface where it adsorbs to the surface monolayer after passing through a transitional tubular myelin phase [26]. Presumably, after cycling through the monomolecular film at the air-water interface during the expansion and contraction phases of the respiratory cycle, certain of the phospholipid com-
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Figure 5 (a) Corn arison of the effect of DPPC or I-palmitoyl-2-oleoylphosphatidylcholine (50 ng/well of either phospholipid) on incorporation of [ Hlcholine into cellular phosphatidylcholine by fetal rabbit type I1 cells after 24 h. Each bar represents the mean f SEM of three separate experiments with a minimum of six observations per experiment. +, significantly different (P < .01) from corresponding cultures exposed t o DPPC determined by Duncan’s multiple range test. (b)Comparison of the effect of DPPC or 1palmiroyl-2-oleoylphosphatidylcholine on secretion of [3H]choline-labeled phosphatidylcholine by fetal rabbit type I1 cells over 24 h. Each bar represents the mean f SEM of three separate experiments with a minimum of six observations per experiment. +, significantly different (P < .01) from corresponding cultures exposed to DPPC as determined by Duncan’s multiple range test; A, significantly different (P < .01) from cultures not exposed to conditioned medium as determined by Duncan’s multiple range test.
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Figure 6 Incorporation of [14C]DPPC or [’4C]1-palmitoyl-2-oleoylphosphatidylcholine by fetal type I1 cells over 24 h. Results are expressed as a percentage of the total phospholipid available for incorporation and represent non-trypsin-dissociable radioactivity. Each sample was exposed t o an identical amount of either phospholipid. Each bar represents the mean zk SEM of three separate experiments with a minimum of six observations per experiment. A, significantly different (P < .05)from corresponding cultures exposed to DPPC as determined by Duncan’s multiple range test; 6 , significantly different (P < .01) from corresponding cultures not exposed to conditioned medium as determined by Duncan’s multiple range test.
ponents are replenished with new phospholipid [ll]. The fate of the “old” surfactant that had been displaced from the monomolecular film has not been determined. Lavage of rabbit or mouse lung yields several subpopulations of material, components of which may represent this used surfactant [27, 281. Estimates of the cellular reutilization of this material suggest that substantial quantities (> 90°/o) may be reincorporated by lung tissue of 3day-old rabbits [9, lo]. Since fetal lung synthesizes and secretes lameller body material [29], it was of interest to us to examine the potential for isolated fetal type I1 cells to incorporate phospholipid as an analogy to reutilizing surfactant components. In addition, while little is known concerning circulating choline levels or how these levels fluctuate during gestation [30-321, it has been demonstrated that the pool size of choline doubles in rabbit lung between the 25th gestational day and the adult [5]. We therefore wished to examine the dynamics of phosphatidylchohne synthesis in fetal rabbit type I1 cells as a function of availability of extracellular choline and the differentiation state of these cells. Previous observations using isolated fetal type 11 cells and a pulse-chase method suggested that under the culture conditions we normally employ, using minimum essential medium, the incorporation pathway for [3H]choline was not oper-
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ating at maximum capacity. The present results under varying extracellular choline indicate that isolated fetal type I1 cells produce more phosphatidylcholine as well as respond to the stimulus of fibroblast conditioned medium (which contains FPF [33]) to a greater extent as choline concentration is raised. Furthermore, these cells appear to respond to changes in the extracellular choline concentrations to a greater degree than other cells, which may reflect the importance of phosphatidylcholine metabolism in type I1 cells. Using cultured rat hepatocytes, Pritchard and Vance [34] observed that varying medium choline concentration from 5 to 40 pM increased phosphatidylcholine synthesis by about 50%. Comparable variations in choline concentration using fetal type I1 cells showed that under unstimulated conditions, total phosphatidylcholine synthesis at least doubled between 5 pM (or 7 pM supplied by standard culture conditions) and 40 pM. The situation was even more marked after exposure of the type I1 cells to conditioned medium. However, the relation this may bear to adjustments in vivo in the fetal lung phosphatidylcholine synthesis is not clear since circulating fetal choline levels throughout gestation have not been examined. There is some evidence that the premature human infant has significantly higher plasma choline levels than the mother [32], which therefore may correspond to the higher fetal lung choline pool sizes observed by Tokmakjian et al. [5] in the fetal rabbit and elevated phosphatidylcholine synthesis by fetal type I1 alveolar cells, as shown here. Preliminary evidence has appeared recently that lung may take up extracellular surfactant-associatedphospholipid. Jacobs et al. [&lo] and Young et al. [35] have shown that in vivo the rabbit and rat lung, respectively, have the capacity to incorporate instilled phospholipid intact and add this to the secretory pool. In addition, several studies indicate that isolated type I1 cells incorporate radioactively labeled phosphatidylcholine and may reprocess this material [12, 131. Furthermore, component parts of the surfactant may alter the reincorporation of phospholipid by these cells [8-10, 35-37]. The present study extends these observations to differentiating fetal rabbit type I1 cells and demonstrates that they can incorporate phospholipid from the extracellular pool. It also shows that these cells incorporate phosphatidylcholines of differing degrees of saturation at different rates. In addition, while both phospholipids substantially reduced the de novo synthesis of phosphatidylcholine as measured by incorporation of [3H]choline, 1palmitoyl-2-oleoylphosphatidylcholinewas more potent in this regard. Such an effect may be due to entrance of this phospholipid into the deacylation/reacylation pathway [4]. O n the other hand, portions of the disaturated phospholipid may directly enter the intracellular surfactant pool or be degraded such that it does not compete with de novo synthesized intermediates for enzyme sites. The secretion of de novo synthesized phosphatidylcholine also appeared to be affected by the presence of these phospholipids. DPPC in particular
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depressed the release of tritiated phosphatidylcholine after 24 h of incubation compared with cultures not exposed to phospholipid. Comparison of the potency of the two phospholipids with regard t o secretion showed that DPPC reduced release of tritium-labeled phosphatidylcholine to a greater degree than did the unsaturated phospholipid. This suggests that synthesis, secretion, and reutilization in these cells may function in a concerted fashion to regulate alveolar surfactant levels. Furthermore, the processes of phosphatidylcholine synthesis and secretion may be altered by the presence of phospholipid supplied by a reprocessing system. The presence of /3adrenergic stimulation in this scheme must also be considered. As observed previously [15], addition of isoxsuprine, in most cases, resulted in a modest elevation in release of radiolabeled phospholipid. Considering that the biological half-life of this drug in the adult human is in the order of 1.25 h [38], we could expect little effect of the drug after a maximum of 7.5 h given that the previous observations indicate that low concentrations in the order of 1 pM could still induce secretion [15]. The response induced by such a relatively long, unphysiological exposure, compared with what might be expected in vivo, suggests that the overall effect of this drug on secretion by the type I1 cells is not of a large magnitude. These results support the concept of a dynamic equilibrium in the surfactant system composed of mechanisms of synthesis, secretion, and reutilization. With regard to the developing lung, these mechanisms appear to become functionally competent as fetal lung cells undergo the final stages of prenatal development. Specifically, the cells acquire a greater capacity to synthesize and secrete phosphatidylcholine as substrate availability increases. It is further apparent that induction of differentiation in type I1 alveolar cells affects the capacity to incorporate phospholipid and, presumably, to reutilize secreted material. However, it is important to note that the dissimilarity between differentiated and undifferentiated cells occurred predominantly in the magnitude and not in the characteristics of the response. The results of this and other studies indicate that fetal and adult alveolar type I1 cells not only synthesize pulmonary surfactant but also reincorporate and reprocess surfactant phospholipid material. The chemical character of the phospholipid as well as the availability of precursors for de novo synthesis and, in the case of the developing fetal lung, the differentiation state of the fetal lung cells are all determinants of the dynamic surfactant system. Clearly, the pathways whereby these components are degraded and/or returned to the surfactant pool and the control of the development of these pathways requires considerably more examination. The author acknowledges the support of the Medical Research Council of Canada, the Manitoba Health Research Council, and the University of Manitoba Senate Research Committee.
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REFERENCES 1. Rooney SA: Lung surfactant. Environ Hlth Persp 55:205-226, 1984. 2. Possmayer F: Biochemistry of pulmonary surfactant during fetal development and in the perinatal period. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 295-355. 3. Miller JC, Weinhold PA: Cholinephosphotransferase in rat lung. J Biol Chem 256: 12662-12665, 1981. 4. Batenburg JJ: Biosynthesis and secretion of pulmonary surfactant. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 237-270. 5. Tokmakjian S, Haines DSM, Possmayer F: Pulmonary phosphatidylcholine biosynthesis: alterations in the pool sizes of choline and choline derivatives in rabbit fetal lung during development. Biochim Biophys Acta 663:557-568, 1981. 6. Desai R, Tetley TD, Curtis CG, Powell GM, Richards RJ: Studies on the fate of pulmonary surfactant in the lung. Biochem J 176:455-462, 1978. 7. Dobbs LG, Wright JR, Hawgood S, Gonzalez R, Venstrom K, Nellenbogen J: Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type I1 cells. Proc Natl Acad Sci 84:lOlO-1014, 1987. 8. Jacobs HC, Ikegami M, Jobe A, Barry DD, Jones S: Reutilization of surfactant phosphatidylcholine in adult rabbits. Biochim Biophys Acta 837:77-84, 1985. 9. Jacobs H, Jobe A, Ikegami M, Conaway D: The significance of reutilization of surfactant phospholipids. J Biol Chem 258:4156-4165, 1983. 10. Jacobs H, Jobe A, Ikegami M, Jones S: Surfactant phosphatidylcholine source, fluxes and turnover times in 3-day-old, 10-day-old and adult rabbits. J Biol Chem 257:1805-1810, 1982. 11. Jobe A, Jacobs HC: Catabolism of pulmonary surfactant. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 271-293. 12. Chander A, Claypool WD, Strauss JF, Fisher AB: Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am J Physiol 245:C397-C404, 1983. 13. Chander A, Reicherter J, Fisher AB: Degradation of dipalmitoyl phosphatidylcholine by isolated rat granular pneumocytes and reutilization for surfactant synthesis. J Clin Invest 79:1133-1138, 1987. 14. Clements JA, Tooley WH: Kinetics of surface-active material in the fetal lung. In Hodson CWA, ed., Development of the Lung. New York, Dekker, 1972, pp. 349-366. 15. Rasmusson M-G, Scott JE, Oulton MR, Temple S: characterization and comparison of the role of 0-agonists on in vivo and in vitro surfactant-related phospholipid synthesis and secretion by fetal rabbit lung and isolated type I1 alveolar cells. Exp Lung Res 142311-822, 1988. 16. Scott JE, Possmayer F, Harding PGR: Alveolar pre-type I1 cells from the fetal rabbit lung: isolation and characterization. Biochim Biophys Acta 753: 195-204, 1983. 17. Tanswell AK, Joneja MG, Lindsay J, Vreeken E: Differentiation-arrested rat
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fetal lung in primary monolayer cell culture, I: development of a differentiationarrested and growth-supporting culture system using carbon-stripped bovine fetal calf serum. Exp Lung Res 5:37-48, 1983. 18. Scott JE, Possmayer F, Quirie MA, Tanswell AK, Harding PGR: Alveolar pretype I1 cells from the fetal rabbit lung: Characterization of the production of disaturated phospholipid during cellular differentiation. Biochim Biophys Acta 879:292-300, 1986. 19. Smith BT: Pulmonary surfactant during fetal development and neonatal adaptation: hormonal control. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 357-382. 20. Mason RJ, Walker SR, Shields BA, Henson JE, Williams MC: Identification of rat alveolar type I1 epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131:786-788, 1985. 21. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917, 1959. 22. Skipski VP, Barclay M: Thin-layer chromatography of lipids. Methods Enzymol 14~530-598,1969. 23. Mason RJ, Nellenbogen J, Clements JA: Isolation of disaturated phosphatidylcholine with osmium tetroxide. J Lipid Res 17:281-284, 1976. 24. Rice WR, Sarin VK, Fox JL, Baatz J, Wert S, Whitsett JA: Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells. Biochim Biophys Acta 1006:237-245, 1989. 25. Ott L: An Introduction to Statistical Methods and Data Analysis. North Scituate, Duxbury, 1977, pp. 392-393. 26. Stratton CJ: Morphology of surfactant producing cells and of the alveolar lining layer. In Robertson B, Van Golde LMG, Batenburg JJ, eds., Pulmonary Surfactant. Amsterdam, Elsevier, 1985, pp. 67-118. 27. Magoon MW, Wright JR, Baritussio A, Williams MC, Goerke J, Benson BJ, Hamilton RL, Clements JA: Subfractionation of lung surfactant: implications for metabolism and surface activity. Biochim Biophys Acta 750:18-31, 1983. 28. Gross NJ, Nardine KR: Surfactant subtypes in mice: characterization and quantitation. J Appl Physiol 66:342-349, 1989. 29. Kikkawa Y, Motoyama EK, Gluck L: Study of the lungs of fetal and newborn rabbits. Am J Pathol 52:177-209, 1968. 30. Haubrich DR, Gerber N, Pflueger AB, Zweig M: Tissue choline studied using simple chemical assay. J Neurochem 36:1409-1417, 1981. 31. Knipper M, Boekhoff I, Breer H: Isolation and reconstitution of the highaffinity choline carrier. FEBS Lett 235-237, 1989. 32. McMahon KE, Farrell PM: Measurement of free choline concentrations in maternal and neonatal blood by micropyrolysis gas chromatography. Clin Chim Acta 149:l-12, 1985. 33. Smith BT: Lung maturation in the fetal rat: acceleration by injection of fibroblast-pneumocyte factor. Science 204:1094-1095, 1979. 34. Pritchard PH, Vance DE: Choline metabolism and phosphatidylcholine biosynthesis in cultured rat, hepatocytes. Biochem J 196:261-267, 1981. 35. Young SL, Wright JR, Clements JA: Cellular uptake and processing of surfactant lipids and apoprotein SP-A by rat lung. J Appl Physiol66:1336-1342, 1989.
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36. Rice WR, Ross GF, Singleton FM, Dingle S, Whitsett JA: Surfactant-associated protein inhibits phospholipid secretion from type I1 cells. J Appl Physiol 63:692-698, 1987. 37. Wright JR, Wager RE, Hawgood S, Dobbs L, Clements JA: Surfactant apoprotein M, = 26,000-36,000 enhances uptake of liposomes by type I1 cells. J Biol Chem 262:2888-2894, 1987. 38. Avery GS: Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics. London, Churchill Livingstone, 1976, p. 896.
