Properties of freshly isolated type II alveolar epithelial cells

Properties of freshly isolated type II alveolar epithelial cells

Biochimicu el BiphvsicaActa. 762 (1983)398-404 398 Elsevier BBA 11153 PROPERTIES OF FRESHLY ISOLATED TYPE I! ALVEOLAR EPITHELIAL CELLS JACOB N. FI...

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Biochimicu el Bi<>phvsicaActa. 762 (1983)398-404



BBA 11153


" Dicision of Neonatolog)', Department of Pediatrics and ~' Department of Radiation Bioloyv and Biophysics. Unit:ersitv of Rochester School of Medicine, Rochester, N Y 14642 (U.S.A.) (Received August 25th, 1982) (Revised manuscript received January 12th, 1983)

Key words": Phospholipid ~vnthesis," Secretion," 7~Loe II cell," (Rabbit lung)

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors IU-14Clglucose, [methyl-3Hlcholine and 13H]palmitate into cellular phosphatidylcholine at rates 2-10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-iysophosphatidylcholine acyitransferase, an enzyme believed to be involved in pulmona~' surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type lI cells. These observations support the utility of freshly isolated ~ p e II cells as a model system for the study of the functions of the alveolar epithelium.

Introduction The type II alveolar epithelial cell is the principal site of synthesis of the surface-active material (pulmonary surfactant) found in the lung [1 3]. Recent studies of specific biochemical events in surfactant synthesis have focused on the properties of these type II cells. Generally, studies have utilized type II cells prepared by proteolytic digestion of the alveolar epithelium followed by a purification step, which may include density gradient centrifugation [4-6], elutriation [7], laser flow cytometry [8] and differential adherence in primary culture [9,10]. Studies of metabolism in such cells have been equivocal because cell damage is inherent in the Abbreviation: Hepes, ethanesulfonic acid.


0167-4889/83/$03.00 '-'i 1983 Elsevier Science Publishers B.V.

isolation methodology. The choice of proteolytic conditions for isolation may affect measured metabolic activity. Evidence exists for detrimental effects of trypsin on cellular metabolism in general and on enzymes of phospholipid synthesis in type II cells in particular [4]. This phenomenon has prompted investigators to culture isolated cells for 18 24 h before attempting metabolic studies. The rationale is to allow repair of injury and stabilization of metabolism. However, type II cells are not stable in culture [5,11]. Furthermore, the effects of nutritional and other factors (e.g., fetal bovine serum) in the culture environment on type II cell function are not known. We have recently developed methods for highyield isolation of type II cells from adult rabbit using low concentrations of a protease mixture [12]. Using this method, enzymes of phospholipid synthesis were not inactivated, and other studies

399 have indicated minimal cell damage [12]. This report continues the characterization of these cells with additional enzyme measurements, evaluating synthesis of phospholipids from labelled precursors and surfactant secretion. The high activities of enzymes of phospholipid synthesis, the rapid rate of substrate utilization in the synthesis of phosphatidylcholine, and the intact secretory response all support the suitability of freshly isolated type II cells as an appropriate model system to study surfactant synthesis. Methods and Materials Cell isolation Type II cells were isolated from adult New Zealand white male rabbits by instillation of protease solution as described previously [12]. Briefly, animals were killed by intravenous pentabarbital and the lung ventilated and perfused with 0.15 M NaCI containing 3 mM EDTA until free of blood. The lungs were then excised and lavaged with Ca2+-free balanced salt solution. After extensive washing the lungs were inflated to total lung volume with Joklik modified minimum essential medium (Gibco, Grand Island, NY) (pH 7.6, with 10 mM Hepes) containing 0.025 m g / m l trypsin (Type IX, Sigma, St. Louis, MO), 1.3 units/ml elastase (type ES, Millipore, Inc., Worthington Div., Freehold, N J) and 10 /,g/ml DNAase I (Sigma, St. Louis, MO). The lungs were held in a sterile siliconized beaker at 37°C for 30 rain to release the cells. The reaction was stopped by the addition of cold (4°C) Joklik solution containing 50 ffg/ml DNAase, 10% fetal bovine serum and 250 ffg/ml trypsin inhibitor (type I, Sigma, St. Louis, MO). The major bronchi and vessels were removed and the remaining tissue minced in a sterile petri dish. The minced lung and all fluid retained by the lung were combined and stirred for 10 rain, The resulting suspension was filtered through nylon gauze (HC 160, HC 41, H D 14, Tetko, Inc., Elmsford, NY) and the cells washed free of protease. Type II cells were isolated from this mixed cell population by centrifigation on a discontinuous gradient of Ficoll (type 400, Sigma) in Joklik media. Cell counts and viability were determined by hemocytometer counts and trypan blue-exclusion, respectively. Purity was monitored

by use of modified Papanicolau staining procedure [13] on air-dried smears and occasional transmission electron microscopy as described previously [121. Incubation of type I I cells with radioactive precursors and secretory responsiveness Metabolic studies were carried out by incubating freshly isolated type II cells with radioactively labelled substrates at 37°C in 12 x 75 mm polypropylene culture tubes. The incubation medium (0.5 ml) consisted of Krebs-Ringer phosphate buffer (or Eagle's minimum essential medium) at pH 7.4 supplemented with other components as specific in the legends to the figures and tables. The incubation was stopped by adding 0.3 ml of cold (4°C) 0.15 M NaC1 and transferring the cells and media to a tube containing 3 ml chloroform/methanol (1:2, v/v). Extraction of the lipids from this mixture was performed by the method of Bligh and Dyer [14]. Secretion by type I1 cells was measured after incubation of cells with labelled choline or palmitare under the conditions outlined above, After cells had been labelled, isproterenol was added and incubation continued an additional hour. Incubation was terminated as above except that, prior to addition of the chloroform/methanol, the tubes were centrifuged at 300 x g for 10 rain to separate ceils and media. The cells were suspended in 0.8 ml of 0.15 M NaC1 and extracted by the method of Bligh and Dyer [14]. Cell-free medium was treated in a similar way. Subcellular fractionation and enzyme assays Freshly isolated cells were resuspended in 2 mM Tris-HC1 (pH 7.4)/1 mM EDTA, pH 7.4, buffer and homogenized by a single 10-s sonication using a microprobe at 20 W maintained at 4°C. This was immediately followed by the addition of an equal volume of 0.66 M sucrose, giving a final concentration of 0.33 M. This homogenate was subjected to differential centrifugation as described previously [12]. The following subcellular fractions were produced: cell-free homogenate, the supernatant after centrifugation at 300 x g for 10 min; fraction 1, the pellet after centrifugation at 1 6 0 0 x g (10 rain); fraction 2. pellet after centrifugation at 8000 x g for 10 rain; fraction 3, pellet after centrifugation at 16000 x g for 10 min:


fraction 4, pellet after centrifugation at 150000 × g for 60 min; fraction 5, supernatant after centrifugation at 150 000 × g for 60 min. CDP choline: 1,2-diacylglycerol choline phosphotransferase was assayed essentially as described previously [12]. sn-Glycerol-3-phosphate : acyl coenzyme A acyltransferase was assayed as described by Garcia et al. [15]. Lysophosphatidylcholine : acyl coenzyme A acyltransferase activity was determined by measuring the incorporation of [14C]palmitoyl coenzyme A into phosphatidylcholine in the presence of 1-acyl-2lysophosphatidylcholine (palmitoyl form) similar to the procedure described by Oldenborg and Van Golde [16]. The assay contained 10 mM Tris-HC1 buffer, pH 7.4, 50 p.M lysophosphatidylcholine, 100 mM palmitoyl coenzyme A and 0.7 m g / m l fatty acid-free bovine serum albumin in a total volume of 0.2 ml. The properties of this enzyme and the development of this assay have been described earlier [17]. All enzyme assays were porportioned to time of incubation and added protein in the range used (2 40 /~g per assay). Specific activites were expressed as nmol of produ c t / m i n per mg of added protein.

Analytical procedures The incorporation of radioactivity into phosphatidylcholine or neutral lipids was determined by subjecting an aliquot of the total lipid extract to chromatography on LK5D plates (Whatman, Inc.) with chloroform/methanol/7 M ammonium hydroxide (65:35:5) as the eluent. The phosphatidylcholine spots were scraped from the plate and placed directly in scintillation vials for counting. In this system, neutral lipids (except free fatty acids) migrate with the solvent front, free fatty acids behind. The spots were scraped and counted. In order to discriminate between labelling of the glycerol and fatty acid moieties of phosphatidylcholine, an aliquot of the phosphatidylcholine (isolated by chromatography) was subjected to mild alkaline hydrolysis as described previously [18]. Protein measurements were performed by the method of Lowry et al. [19] using crystalline bovine serum albumin as the standard.

Materials Radioactively labelled precursors



strates: [U-~4C]glucose (360 mCi/mmol), [methylH]choline chloride (80 Ci/mol), [9,10 -3 H]palmitic acid (11.8 Ci/mmol), [1-14C]oleic acid (57 m C i / mmol), [U-E4C]glycerol 3-phosphate (144 m C i / retool), [14C]cytidine diphosphocholine (52.5 mCi/mmol), and [1-14C]palmitoyl coenzyme A were purchased from New England Nuclear, Boston, Massachusetts. Fatty acid-free bovine serum albumin was from Miles Laboratories (Elkhart, IN). Unlabelled precursors and substrates were purchased from Sigma Chemical Co. (St. Louis, MO). Results

Incorporation of precursors into phospholipids The incorporation of labelled glucose into phosphatidylcholine by freshly isolated type II cells is shown in Fig. 1. Incorporation into total lipid (data not shown) and phosphatidylcholine was saturated at 0.25 mM glucose. At 5.5 mM glucose, incorporation into phosphatidylcholine was linear with time for 4h (Fig. 1). Under the incubation conditions employed in Fig. 1 (5.5 mM glucose plus 0.2 mM choline chloride) the rate of incorporation of [U- 14C]glucose into phosphatidylcholine was 1.08 + 0.08 (n = 10) nmol/10 ~' cells per h at 37°C. This value is approximately 50% of the radioactively labelled glucose found in the total lipid extract. Alkaline hydrolysis of labelled phosphatidylcholine showed that 50% of the incorporated label was recovered in the fatty acyl moiety. The effect of changing incubation conditions on the utilization of labelled glucose by fresh type II cells was examined. Choline chloride stimulates phosphatidylcholine synthesis from labelled glucose (5.5 mM glucose alone, 0.69 nmol/10 ~ cells: glucose plus 0.2 M choline chloride, 1.08 + 0.08). This is due primarily, to a 40% increase in incorporation of glucose into phosphatidylcholine fatty acids. Other investigators have shown [6] that exogenously added fatty acids influence [U-14C] glucose incorporation by cultured type II ceils. Our results confirm this observation. Addition of 1 mM palmitic acid (complexed to bovine serum albumin at a molar ratio of 2 : 1) to the incubation media resulted in a 30% reduction of glucose incorporation into phosphatidylcholine (to 0.70 _+ 0.03 nmol/106 cells). As shown in Table I, incor-


i ~.~








Cells (2.10 6) were incubated in 1.0 ml of media containing 0.2 m M choline chloride and 5.5 m M [U-J4C]glucose (1 m C i / r n tool). The concentration of added palmitate was 1.0 m M (complexed to bovine serum albumin at a 2 : 1 molar ratio). Fatty acids were isolated by alkaline hydrolysis. Results are mean_+ S.D. of duplicate assays.



o.4 0.2





Glucose incorporation (nmol/106 cells per h)


0 t.) ,.j




Phosphatidylcholine Total lipid Phosphatidylcholine fatty acids Total lipid fatty acids




/~Jl t





Control (n = 6)

Palmitate (n = 4)

1.08 _+0.10 2.48 _+0.10

0.70 _+0.03 2.44 ± 0.10

0.54 _+0.04 1.22_+0.07

0.08 _+0.04 0.64±0.16



Fig. IA. Effect of glucose concentration on [U-14C]glucose incorporation into phosphatidylcholine, Freshly isolated type II cells were incubated for 60 rain at 37°C in Krebs-Ringer phosphate buffer plus 0.1 m M choline chloride with the indicated glucose concentration. The specific activity was constant (1 p, C i / m o l ) in all incubations. Each point represents the m e a n ± S.D. of duplicate determinations in four separate experiments. B. Effect of time on glucose incorporation into phosphatidylcholine. Type II cells were incubated in KrebsRinger phosphate buffer containing 5.5 m M glucose (1 p, C i / m o l ) and 0.1 m M choline chloride for the indicated times. Each point represents the mean_4_-S.D, of duplicate determinations in four separate cell preparations.

poration into total lipids was unaffected during these short-term incubations. Alkaline hydrolysis of phosphatidylcholine and the total lipid extract showed that the addition of exogenous palm±tic acid significantly decreased [U-14C]glucose incorporation into fatty acyl groups of both total lipid and phosphatidylcholine. The incorporation of glucose into the glyceride moiety of phosphatidylcholine was increased by addition of palmitate such that the overall rate of glucose into phosphatidylcholine was only reduced by 30%. The incorporation of exogenous fatty acid into phosphatidylcholine is shown in Table II. [~ H]Palmitate and [ 1-14C]oleate were incorporated

at a constant rate from zero time up to at least 2 h (data not shown). Saturating concentrations, determined in the presence of 5.5 mM glucose and 0.2 mM choline chloride, were 0.1 mM for both fatty acids. In the absence of added glucose, the incorporation of palmitate into phosphatidylchoT A B L E II FATTY ACID DYLCHOLINE




Cells were incubated in media containing 0.2 m M choline chloride and 5,5 m M glucose. Labelled fatty acids were added as a complex with bovine serum albumin (molar ratio, 2: 1). Results are mean_+S.D, of triplicate assays of four separate preparations of cells. Fatty acid added (mM) C16:0


0.1 0.5 1.0

0.1 0.5 1.0

Rate of incorporation (nmol/106 cells per h)

2,15±0.15 2.25_+0.15 2.15_+0.10 0.1 0.5 1.0 + 0.1 + 0.5 + 1.0

1.1 +--0.06 1.0 -+0.05 1.0 -+0.08

1.0 4-0.15 1.15_+0.10 1.05 _+0.09 0.35___0.04 0.30-+0.03 0.30_+0.01

402 line by type II cells was reduced 25%. As can be seen in Table I1, palmitate was incorporated into phosphatidylcholine at twice the rate of oleate (2.2 n m o l / h per 10 ~ cells vs. 1.06 n m o l / h per 10 ~ cells). Incorporation of exogenous palmitate into phosphatidylglycerol showed the same time-course and concentration relationship, with the observed rate approximately one-sixth that of phosphatidylcholine (0.42 n m o l / h per 10 ~ cells, n = 3). In normal in vivo situations, exogenous fatty acids are rarely present as pure c o m p o n e n t s but are usually mixtures derived from serum lipoproteins. To simulate these conditions and study the relationship between fatty acid precursors under more physiologic conditions, palmitate and oleate were added simultaneously and incorporation rates of each followed independently (each being labelled by a different radioisotope). Table I I shows the rates of incorporation of fatty acids alone or added as mixtures into phosphatidylcholine. At equimolar concentrations, palmitic acid was preferentially utilized over oleate. The ratio of incorporation rates varied from 2 : 1 for the individual fatty acids to 3 : 1 in the mixtures. The incorporation of [rnelhyl-3H]choline into phosphatidylcholine was linear from 30 rain to at least 2 h of incubation. The concentration curve, in the presence of 5,5 mM glucose, showed choline incorporation into phosphatidylcholine to be saturated above 0.025 raM. The rate of incorporation of [methyl- 3H]choline into phosphatidylcholine in the presence of 5.5 m M glucose was 0.750 n m o l / 1 0 ~' cells per h. The addition of exogenous fatty acid (complexed to bovine serum albumin) increased [metltvl-3H]choline incorporation into phosphatidylcholine by 25% to 1.0 n m o l / 1 0 ~ cells per h. Exogenous palmitate or oleate were equally effective in this stimulation.

Secreto O, response of isolated cells The effects of a fl-adrenergic agonist (isoproterenol) on the release of labelled phosphatidylcholine from the freshly isolated cells were examined. The results show that these cells respond to the two concentrations of isoproterenol used: at 10-3 M isoproterenol, cells incubated with choline or palmitate secreted to 8.9_+ 2.1 and 8.2 + 1.7% cellular phosphatidylcholine, respectively. Data for 10 4 M isoproterenol were 3 . 4 + 1.5 and 3.5_+

TABLE Ill ENZYMES OF PHOSPHOLIPID SYNTHESIS IN ISOLATED MICROSOMAL FRACTIONS Data are expressed as nmol of product formed/min per mg of added protein. Assay conditions are as discussed in Materials and Methods. Data are the result of triplicate assays on at least five different preparations of microsomes and interassay variation was less than 10%. Source of Microsome

Whole rabbit lung Alveolar macrophage Unpurified dispersed lung cells Type II cells

Glycerol3-phosphate acyltransferase


Lysophosphatidylcholine acyltransterase







6.2 8.9

13. I 31.2

214 308

1.1%, respectively (mean + S.E. of four replicates from two separate preparations). Release of lactate dehydrogenase was not altered by this treatment (not shown).

Enzyme content of isolated cells The specific activity and subcellular location of three key enzymes in p u l m o n a r y surfactant phospholipid synthesis were measured in subcellular fractions prepared from whole lung, alveolar macrophages, unpurified dispersed lung cells, and purified lung cells. Glycerol-3-phosphate acyltransferase, choline phosphotransferase and lysophosphatidylcholine acyltransferase were localized primarily in the endoplasmic reticulum, following very closely the distribution of the microsomal marker, N A D P H - c y t o c h r o m e c reductase. Table llI shows the activity of these enzymes m the microsomal fraction prepared from the various cell populations available. In all cases the type I1 cell had the the greatest activity, being enriched 1.5-5-fold over that of whole lung tissue. Discussion In our previous study [12] we showed that by using a defined protease mixture of elastase and

403 trypsin large numbers of type lI cells could be isolated from rabbit lung with minimal proteolytic digestion. Measurement of enzymes of phospholipid biosynthesis suggested that these cells were capable of high rates of phosphatidylcholine biosynthesis. The present study extends the characterization of these cells by examining the ability of freshly isolated rabbit type II cells to utilize key metabolic precursors to synthesize phosphatidylcholine, the major phospholipid component of pulmonary surfactant, and determining that the secretory ability of these cells remained intact. Smith and Kikkawa [20,21] carried out extensive metabolic studies on freshly isolated rabbit type II cells prepared by digestion with 1.0 m g / m l crude trypsin. Although they stated isolation conditions were without effect on phospholipid synthesis [22], the rates of choline (0.135 nmol/106 cells per h), glucose (0.102 nmol/106 cells per h) and palmitate (0.647 nmol/106 cells per h) are 2-10-fold lower than those observed in the isolated rabbit cells in the present study. It is possible that some of the differences in the observed rate of substrate utilization arise from differences in experimental design. The experiments of Smith and Kikkawa [20,21] were performed by incubating cell populations in Hanks' balanced salt solution to which the precursor to be studied was added. As a consequence, all incubations contained the substrate of interest plus glucose. No consideration was given to the role of substrate interactions in the optimum utilization of precursors by freshly isolated type II cells. Our data clearly show the importance of such interactions. Choline had a significant effect on glucose utilization by type II cells, primarily by increasing fatty acids synthesized from glucose, in phosphatidylcholine. Our data have also shown glucose effects on fatty acid incorporation and an effect of exogenous fatty acid on both glucose and choline incorporation. However, the data do not completely account for the observed differences, and the possibility of extensive intracellular damage by the high concentrations of protease used in their isolation procedure must be considered. Comprehensive studies on type II cell phospholipid synthesis were carried out by Batenburg et al. [6] using cells from rat lung. The method used involved incubation for 18-21 h in culture

prior to metabolic studies. In general, the pattern of results in the present study is consistent with those of Batenburg et al. Effects of exogenous fatty acids on glucose utilization, rates of utilization of glucose, choline and palmitate were similar to results of the present study. The principal difference between these two studies is the effect of exogenous fatty acid (principally palmitate) on [methyl-)H]choline incorporation. In their study, Batenburg et al. [6] reported a 2-fold stimulation of choline utilization by exogenous palmitate, whereas in our study only a modest 25% increase was observed. This suggests that de novo synthesis of fatty acids from glucose is insufficient to supply the biosynthetic needs of the cell in the case of the cultured rat cell, but not for freshly isolated rabbit cells. Whether this may be due to down-regulation of fatty acid synthesis in culture or stimulation in the freshly isolated cell is unclear and requires further work. It is also possible that differences in observed patterns of substrate incorporation may be due either to species differences (rat vs. rabbits), the effects of cell culture nutritional factors, dedifferentiation in culture or subtle damage caused by cell isolation. The observation that freshly isolated cells are capable of responding to a/3-adrenergic agonist by secretion is the first report of such effects in a freshly isolated cell. Previous reports [23,24] have demonstrated effects in cultured cells. Our results are consistent with our finding of detectable /3adrenergic receptors on the surface of freshly isolated cells [22]. The extent of secretion observed in this study is similar to that reported for cultured rat type II cells [23]. The enzyme data of Table Ill clearly shows an enrichment of membrane-bound enzymes of phospholipid synthesis in the isolated type II cell. Lysophosphatidylcholine acyltransferase showed the greatest enrichment. As a key enzyme in the phosphatidylcholine-remodeling pathway [1], which may be responsible for the synthesis of dipalmitoyl phosphatidylcholine in type lI cell, such enrichment is not unexpected. Our results confirm those of Batenburg et al. [25] and Funkhouser er al. [26], who found higher activity of the reacylation pathway in cultured type II cells. This is the first report which compares acyla-

404 tion of glycerol 3-phosphate, the first step in the synthesis of the glycerolipid moiety, to the acylation of l y s o p h o s p h at id y l c h o l in e , the putative last step in d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e synthesis. In freshly isolated type II cells reacylation activity is 35-fold higher than acylation. In whole lung or alveolar m a c r o p h a g e s the ratio is only 1 1-12. T h e relative i m p o r t a n c e of these activities in d e t e r m i n ing the biosynthesis of p h o s p h o l i p i d s is currently u n d e r active investigation. In conclusion, the present studies have shown that freshly isolated cells when p r e p a r e d by defined p r o t e o l y t i c c o n d i t i o n s can be useful system for studying various aspects of p u l m o n a r y surfactant biology. Th e active precursor utilization and intact ,B-adrenergic receptors should p e r m i t studies on the interaction b e t w e e n stimulation of secretion a nd biosynthesis. In addition, the p o te n t ia l for studies of short t e r m in vitro and, m o r e directly, in vivo dietary or h o r m o n a l m a n i p u l a t i o n w i th o u t recourse to cell culture could be significant. U s i n g the freshly isolated cell m a y simplify e x t r a p o l a t i o n to in vivo conditions. U s i n g these freshly isolated ceils it should be possible to study the effects of cell culture on type II cell function, and thus establish c o n d i t i o n s which w o u ld m a i n t a i n p h e n o typic expression of differentiated function in vitro.

Acknowledgements T h e au t h o rs gratefully a c k n o w l e d g e the expert technical assistance of C h r i s ti n a M. K r a m e r and A n i t a B. Parkhurst. T h e investigators were supp o r t e d in part by G r a n t C A 27791 from the U.S. Public H e a l t h Service and by the A m e r i c a n H e a r t Association.

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