Synthesis of Phosphatidylcholine and Phosphatidylglycerol by Alveolar Type I1 Cells in Primary Culture*

Saturated phosphatidylcholine and phosphatidyl- glycerol are important components of pulmonary surface active material. We studied the synthesis of these two phospholipid classes by alveolar type I1 cells in primary culture. During a 20-h incubation, type I1 cells incorporated a high percentage of glycerol, acetate, and palmitate into phosphatidylcholine (61.2, 76.4, and 76.8% of lipid radioactivity, respectively) and into phosphatidylglycerol (16.7, 5.8, and 6.6%). Acetate was incorporated principally by de nouo synthesis of fatty acids rather than by chain elongation. We studied the pathways for synthesis of saturated phosphatidylcho- line and phosphatidylglycerol with type I1 cells that had been in culture for 1 day. Palmitate was incorpo- rated nearly equally into positions 1 and 2 of saturated phosphatidylglycerol, but predominantly (72%) into position 2 of saturated phosphatidylcholine. These data imply that saturated phosphatidylcholine is synthesized at least in part by acylation of 1-acyl-2-lysophos- phatidylcholine. Alveolar type I1 cells also incorpo- rated a mixture of saturated l-[9,10-3H]palmitoyl-2-ly-sophosphatidylcholine and 1-acyl-2-lysophosphatidyl- [1,2-14C]choline

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material. We studied the synthesis of these two phospholipid classes by alveolar type I1 cells in primary culture. During a 20-h incubation, type I1 cells incorporated a high percentage of glycerol, acetate, and palmitate into phosphatidylcholine (61.2, 76.4, and 76.8% of lipid radioactivity, respectively) and into phosphatidylglycerol (16.7, 5.8, and 6.6%). Acetate was incorporated principally by de nouo synthesis of fatty acids rather than by chain elongation. We studied the pathways for synthesis of saturated phosphatidylcholine and phosphatidylglycerol with type I1 cells that had been in culture for 1 day. Palmitate was incorporated nearly equally into positions 1 and 2 of saturated phosphatidylglycerol, but predominantly (72%) into position 2 of saturated phosphatidylcholine. These data imply that saturated phosphatidylcholine is synthesized at least in part by acylation of 1-acyl-2-lysophosphatidylcholine. Alveolar type I1 cells also incorporated a mixture of saturated l-[9,10-3H]palmitoyl-2-lysophosphatidylcholine and 1-acyl-2-lysophosphatidyl-[1,2-14C]choline from the medium by direct acylation rather than by transacylation. As the duration of culture increased beyond l day, type I1 cells incorporated a lower percentage of palmitate into phosphatidylglycerol and saturated phosphatidylcholine.
Pulmonary surface active material, which is synthesized and secreted by the alveolar type I1 cell, lowers the surface tension at the air-liquid interface within alveoli and thereby prevents alveoli from collapsing at low transpulmonary pressures (1-3). Two classes of phospholipids, phosphatidylglycerol and dipalmitoyl phosphatidylcholine, are found in unusually high concentrations in surface active material and in alveolar type I1 cells from adult animals. Dipalmitoyl phosphatidylcholine is thought to account for the stability and low surface tension of the surface film (1, 2). The quantitative contribution of different pathways for the synthesis of dipalmitoyl phosphatidylcholine by type I1 cells is not established. Studies on the synthesis of saturated phosphatidylcholine in whole lung and in subcellular fractions of whole lung have been reviewed recently (4-6). Most investigators agree that the serial methylation of phosphatidylethanolamine is not quantitatively important. However, the relative importance of three alternative synthetic pathways (de nouo synthesis from saturated diglyceride, deacylation-reacylation ( 7 ) , and deacylation-transacylation (8)), remains uncertain, partly because it is difficult to interpret biochemical studies performed on a tissue composed of many different cell types. The lung contains more than 40 different kinds of cells (9); only 15% of the cells of an adult rat lung are type I1 cells (10). It is therefore important to study phospholipid synthesis in isolated type I1 cells. We previously developed a method of purifying type I1 cells by differential adherence in primary culture (11,12). In this report, we show that type I1 cells in primary culture synthesize saturated phosphatidylcholine and phosphatidylglycerol and provide evidence that the deacylation-reacylation pathway is important in synthesizing saturated phosphatidylcholine in vitro.

Preparation of Alveolar Type I I Cells
Alveolar type I1 cells were prepared from specific pathogen-free Sprague-Dawley male rats that weighed from 180 to 300 g (11,12).
Type I1 cells were partially purified by dissociation of intact excised lung with crystalline trypsin and centrifugation of the resulting cell suspension over a discontinuous density gradient made with albumin.
A higher concentration of trypsin (3 mg/ml) was used for the experiments in Table I and Figs. 1 and 2 and a lower concentration of trypsin (0.1 mg/ml) was used for all other experiments (12). The dispersed cells were further purified by differential adherence (11,12). Cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum, and antibiotics. For experiments in Table I and Figs. 1 and 2, we used 10 pg of gentamicin/ml and in all other experiments we used 50 pg of gentamicin/ml and 100 units of penicillin G/ml. Cells were incubated for 3 h in a T-75 culture flask, during which time few cells (mostly macrophages) attached to the plastic. The nonadherent cells were removed and, in most experiments, were placed in 35-mm tissue culture dishes with 2.5 ml of medium and 2.5 X 10" cells/dish; during the next 20 h of culture, type I1 cells adhered to the plastic. Nonadherent cells (mostly lymphocytes and nonviable cells), were removed and discarded. After the differential adherence, approximately 10" cells remained attached to each culture dish. Cells were quantitated by DNA determination (13); by this method, there is 8.3 pg of DNA/106 type 11 cells.' The percentage of type I1 cells was determined from differential counts of cells stained with the modified Papanicolaou stain (14). Cells prepared with 3 mg/ml of trypsin and purified in culture yielded 83 -t 4% type I1 cells (mean -C S.D.; n = 6) and cells prepared with 0.1 mg/ml of trypsin and purified in culture yielded 94 * 4% type I1 cells ( n = 12). The total yield of purified adherent type I1 cells from six rat lungs dissociated with 0.1 mg/ml of trypsin was 10 to 15 x 10" cells.

Incorporation ofAcetate, Palmitate, and Glycerol into Lipids
During the period of attachment of type I1 cells, the cells were incubated with radioactive acetate, palmitate, or glycerol as described in the legend to Table I. ' E. Geppert, unpubliihed data.

Metabolic Incubations
After 23 h in culture, the adherent cells were washed three times with phosphate-buffered saline and then incubated with minimal essential medium, Hanks' salts, 25 mM Hepes,' pH 7.4. This medium is designated ME medium/Hepes. Fraction V bovine serum albumin and appropriate radioactive substrates were added to the ME medium/Hepes; the final concentrations of added substances are stated in the legends to the figures and tables.

Lipid Analyses
Extraction a n d Separation-After the incubation period, medium was removed and the cells were washed three times with ME medium/ Hepes. The cells were extracted six times with ethanol (15), lipids (0.6 to 1.0 mg) isolated from dog lung were added as carriers, and the ethanol was evaporated under a stream of nitrogen. The residue was extracted with ch1oroform:methanol (2:1), and the solution was partitioned into organic and aqueous phases by the method of Folch et al. (16). The aqueous phase was formed by the addition of 100 mM KC1 to which we added sodium acetate (1 111~) or glycerol (1 mM) for the experiments in which radioactive acetate or glycerol was used as precursor. Individual phospholipids were separated by two-dimensional thin layer chromatography (TLC) on Silica Gel G plates impregnated with boric acid (17). The spots were identified by brief exposure to iodine vapor; phosphatidylcholine and phosphatidylglycerol were eluted from the silica with ch1oroform:methanol:water: acetic acid (50:502:1) followed by ch1oroform:methanol:water (30:60: 5) (18).
To isolate both the saturated and the unsaturated species of phosphatidylcholines (Tables I1 and V), we reacted the total phosphatidylcholine fraction with osmium tetroxide in carbon tetrachloride (19) and separated the saturated species from the unsaturated species by TLC on Silica Gel G plates impregnated with boric acid with a solvent of chloroform:methanol:14 N ammonium hydroxide: water (75:25:1:2). For analyses requiring only the isolation of saturated phosphatidylcholine (the phospholipase AP degradation in Table I1 and the incorporation of palmitate in Figs. 1 and 2), we took an aliquot of the total phosphatidylcholine fraction, added carrier lipids from dog lung, reacted the mixture with osmium tetroxide, and isolated with saturated species by column chromatography on neutral alumina (19).
To resolve the individual species of phosphatidylcholine and phosphatidylglycerol (Tables 111 and IV), we converted the phospholipids to diglyceride acetates and separated the major classes of diglyceride acetates by argentation TLC. Phosphatidylcholine and phosphatidylglycerol were converted to diglycerides with phospholipase C and the diglycerides were acetylated with acetic anhydride (15). The diglyceride acetates were purified by TLC and the acetates were separated by TLC on Silica Gel G plates impregnated with silver nitrate (20). The diglyceride acetates were localized with 2,7-dichlorofluorescein and were eluted with ch1oroform:methanol (9:l). The eluates were washed successively with methanol:l% ammonium hydroxide, methanol:0.15 M NaCI, and methanol:water, all 1:l by volume to remove any eluted 2,7-dichlorofluorescein and silver nitrate. The recovery of diglyceride acetates from the TLC plates ranged from 85 to 97%. We measured radioactivity by liquid scintillation counting in a dioxane: naphtha1ene:water system (21). Counts were corrected for quenching and for spillover of the I4C into the "H channel with the external standard channels ratio and quench curves generated in our laboratory.
Degradation of Saturated Phosphatidylcholine with Phospholipase A2-An aliquot of saturated phosphatidylcholine and 60 pg of egg phosphatidylcholine were evaporated to dryness and dissolved in 2 ml of diethyl ether:ethanol, 191 by volume (18.22). Fifty microliters of phospholipase AB (Crotalus adamanteus, 1 mg/ml in 5 mM Hepes and 5 mM CaCI2, pH 7.4) was added, and the reaction mixture was incubated for 4 h a t 37OC. The reaction mixture was evaporated to dryness and redissolved in ch1oroform:methanol (1:l). Oleic acid, egg phosphatidylcholine, and lysophosphatidylcholine were then added as carrier lipids. The reaction products were separated by TLC, localized by iodine vapor, and scraped into counting vials for determination of radioactivity (18). In these experiments (Table 11). from 93 to 98% of the phosphatidylcholine was hydrolyzed and recovery of total radioactivity was 89 to 96%. In analyzing the data, we assumed that the radioactivity found in the fatty acid fraction came exclusively from position 2 of phosphatidylcholine and the radioactivity in the The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; ME medium, minimal essential medium. lysophosphatidylcholine fraction came from position 1.
Degradation of Saturated Diglyceride Acetates with Pancreatic Lipase-Analyses were performed according to the method of Renkonen (23). The reaction mixture consisted of an aliquot of diglyceride acetate, 200 pg of triolein, 425 p1 of 1 M Tris (pH 8.0, prepared in water saturated with diethyl ether), 25 pl of 238 CaCI2, 10 p1 of 1% sodium deoxycholate, and 25 p1 of pancreatic lipase (5 to 8 units dissolved in 1 M Tris, pH 8.0). These reagents were incubated for 10 min a t 40°C. The reaction was stopped by the addition of 200 pl of 6 N HCl. We added 1 ml of methanol and extracted the reaction products three times with diethyl ether. The diethyl ether was removed and backwashed with water. The reaction products were extracted with ether and separated by TLC in a solvent system of hexane:diethyl ether:acetic acid (50501). We found that the separation of diglyceride acetates, diglycerides, fatty acids, monoglyceride acetates, and monoglycerides was improved by spraying the Silica Gel G plate briefly with water before applying the samples. For each analysis, the amount of radioactivity in saturated diglyceride acetates derived from phosphatidylcholine ranged from 1650 to 5900 cpm for I4C and from 1940 to 6380 cpm for "H. For saturated diglyceride acetates derived from phosphatidylglycerol, the amount of radioactivity ranged from 580 to 1890 cpm for I4C and from 740 to 3400 cpm for ,'H. The percentage of hydrolysis of diglyceride acetates ranged from 25 to 43%. The recovery of total radioactivity in these analyses ranged from 97 to 100%. Monoglyceride acetates contained nearly twice as much radioactivity as monoglycerides. Fatty acids liberated by pancreatic lipase during these limited hydrolyses were assumed to come from position 1, whereas the monoglycerides and monoglyceride acetates were assumed to contain long chain fatty acids only in position 2 (23).
Schmidt Decarboxylation Reaction-We saponified the lipids synthesized by type I1 cells from [l-'4C]acetate. The fatty acids were extracted, purified by TLC, and decarboxylated according to a modification of the Schmidt procedure (24). In each decarboxylation procedure, we ran [l-'4C]palmitate as a control; the percentage of radioactivity recovered as CO1 ranged from 75 to 88%. We calculated the percentage of radioactivity in position C-1 of the fatty acids synthesized from [ l-14C]acetate by the formula:  (19), combined, and hydrolyzed by phospholipase A2 (C. adamanteus). We separated lysophosphatidylcholine by TLC 607:14), eluted the lysophosphatidylcholine, and repurified it by the with a solvent system of ch1oroform:methanol:water:acetic acid (100: same method; the final purity was 99%. The labeled lysophosphatidylcholine was bound to albumin just prior to each experiment.

Other Methods
Palmitate was bound to defatted bovine serum albumin according to the method of Spector et al. (25).

Materials
Materials and animals for isolating type I1 cells were obtained from the same suppliers as described previously (13 grade and, with the exception of diethyl ether, were redistilled before use.

RESULTS
Synthesis of the Phospholipids of Surface Active Material-We tested the ability of type I1 cells in primary culture to synthesize the lipids of surface active material by incubating the cells for 20 h with radioactive acetate, palmitate, or glycerol. The results, which have been published in preliminary form in recent symposia (12, 26), are shown in Table I. The pattern of distribution of radioactivity in the various phospholipid classes is noteworthy for two reasons. First, the types of lipids synthesized from these precursors are very similar to the phospholipid composition of surface active material and alveolar type I1 cells from rats (12). Second, the pattern of distribution was the same when either acetate or palmitate was used as the precursor, a result which suggests that acetate is incorporated into fatty acids by de novo synthesis rather than by chain elongation. We determined whether acetate was incorporated into fatty acids by de novo synthesis or chain elongation by incubating ceUs with [1-'4C]   days become larger and the cellular cytoplasmic inclusions become smaller and less distinct (12, 27), we wanted to determine whether type I1 cells kept in culture retained their ability to produce the lipid components of surface active material. We maintained cells in culture for 1,2,3, and 6 days and then incubated the cells for 2 h with [ l-'4C]palmitate. The results are shown in Figs. 1 and 2. Incorporation of palmitate into total lipid and into saturated phosphatidyIcholine/pg of DNA increased with time in culture (Fig. 1). This is consistent with the observation that type I1 cells become larger. However, because the percentage of palmitate incorporated into saturated phosphatidylcholine and phosphatidylglycerol decreased with the duration of time in culture (Fig. 2), we concluded that as time in culture increased, there was relatively less synthesis of the lipids of surface active material and more synthesis of other cellular phospholipids. We therefore performed the rest of the studies of lipid biosynthesis on Day 1 of culture.
Studies of the Synthesis of Saturated Phosphatidylcholine and Saturated Phosphatidylglycerol-We wanted to determine the relative importance of various pathways for synthesizing saturated phosphatidylcholine, i.e. de novo synthesis (via saturated phosphatidic acid and saturated diglyceride), deacylation-reacylation, and deacylation-transacylation (4).
We first incubated type I1 cells with [l-'4C]palmitate for short periods of time and determined the percentage of palmitate that was incorporated into positions l and 2 of saturated phosphatidylcholine. As shown in Table 11, the percent- Tvpe II cells were cultured in 35-mm culture dishes and were puriked by differential adherence as described in the text. They were preincubated for 15 min at 37'C in ME medium/Hepes which contained 3 mg/ml of Fraction V bovine serum albumin. The medium was removed and replaced with warm medium (1.6 ml) which consisted of ME medium/Hepes, 3 mg/ml of bovine serum albumin (34 hrnol of mixed fatty acids/liter), and albumin-bound [l-"C]-palmitate (20 pmol/liter, 0.9 &i). The calculated total fatty acid to albumin molar ratio was 1.2. After the designated periods of incubation, duplicate dishes were washed, the cells were extracted, and the lipids were processed as described in the text. In this experiment, there was 9.24 pg of DNA/culture dish (the equivalent of 1.1 x IO" cells/dish). Based on the assumptions that all fatty acids are esterified at an equal rate (28) and that the intracellular specific activity of fatty acids is the same as the specific activit.v of the fatty acids in the medium, the calculated synthesis of total phosphatid.vlcholine was 2.54 nmol/ 10" cells h-'. than into position 1. We next showed that the distribution of palmitate incorporated into positions 1 and 2 of saturated phosphatidylglycerol was quite different from the distribution in saturated phosphatidylcholine.
We incubated type II cells for 20 h with [1,'H]gIyceroI (to achieve equilibrium labeling (29) of the phospholipid backbone) and then incubated the cells for 30 min with  We isolated phosphatidylcholine and phosphatidylglycerol, converted these phospholipids to diglyceride acetates, isolated the saturated diglyceride ace-tates, and partially degraded the diglyceride acetates with pancreatic lipase to ascertain the distribution of radioactive palmitate in positions 1 and 2. The results are shown in Table  III. Palmitate was incorporated predominantly into position 2 of saturated phosphatidylcholine, whereas it was incorporated nearly equally into positions 1 and 2 of saturated phosphatidylglycerol (Table IIIA). Because the distribution of palmitate (Table IIIA) was calculated from a limited hydrolysis with pancreatic lipase as contrasted with the complete degradation of saturated phosphatidylcholine with phospholipase A2 (Table II), we also compared the '%/"H (fatty acid/backbone) ratio in monoglyceride acetates and diglyceride acetates as an internal check on the direct measurement of the "'C. The '% was derived from [ l-'%]palmitate and the ,'H from [ l,3-"H]glycerol. The comparison of the '%f'H ratio is independent of the extent of the hydrolysis of the diglyceride acetates by pancreatic lipase and requires only physical separation of the two compounds. In the conversion of diglyceride acetates to monoglyceride acetates by pancreatic lipase, the fatty acid in position 1 is removed. This analysis was complicated, however, by the fact that 10 to 19% of the "H radioactivity was found in the fatty acid portion of the phospholipids and, therefore, the exact distribution of palmitate was not calculated from these data. Nevertheless, it was instructive to compare the 'YJ/"H ratios of the diglyceride acetates with those of the monoglyceride acetates (or monoglycerides) produced by pancreatic lipase (Table IIIB). The ratio of "C/"H of monoglyceride acetate:diglyceride acetate was 0.57 for those species derived   Type II cells were isolated and purified by differential adherence in 35mm culture dishes as described in the text. The adherent cells were washed and then incubated for 15 min at 37'C in ME medium/ Hepes that contained 3 mg/ml of bovine serum albumin. The medium was removed and replaced with 1.6 ml of ME medium/Hepes which contained 30 mg/ml of defatted bovine serum albumin and a mixture of l-~9,lO-~~H~palmitoyl-2-Iysophosphatidylcho~ine and l-acyl-2-iysophosbhatidycil,2-'4Cjchol&e -(7 ti 40 PM, final concentration). The lvsoohosuhatidvlcholine contained 19.lCQ dpm of "H and 955 dpm of "C/nmoi. Aftei the cells were incubaied foi60 min, the medi&n was removed.
The cells were washed and extracted, and the lipids were processed as described in the text. was in position 2; in contrast, the ratio was 0.83 for the species derived from saturated phosphatidylcholine, suggesting that there was more palmitate in position 2 than in position 1.
The 20-h incubation with [1,3-"H]glycerol should allow sufficient time for equilibrium labeling of the various species of phosphatidylcholine and phosphatidylglycerol (29). We analyzed the distribution of radioactivity in 'H in different species of diglyceride acetates derived from phosphatidylcholine and phosphatidylglycerol (Table IV). The actual percentages of the different species may be slightly different from the distribution of radioactivity shown in Table IV, since 10 to 19% of the radioactivity in the diglyceride acetates was in the fatty acid portion of the molecules. However, since both the total and the saturated diglyceride acetates had the same percentage of "H in the fatty acid and glycerol portions of the molecules, it is unlikely that the percentages of saturated species are overestimated.
We did not determine the chemical composition of the different species because we did not have a sufficient number of cells and we had to add carrier lipids for our analyses.
To test for the presence of transacylase pathway in type II cells, we incubated cells for 1 h with a mixture of l-[9,10-"H]palmitoyl-2Jysophosphatidylcholine and I-acyl-2-lysophosphatidyl-[ l,2-'4C]choline. The results are shown in Table  V. Lysophosphatidylcholine was readily incorporated into phosphatidylcholine, but there was no change in the "H/"'C ratio (palmitate/choline) of the phosphatidylcholine, suggesting that exogenous lysophosphatidylcholine was incorporated by direct acylation rather than by transacylation.

DISCUSSION
There ia now substantial evidence (5,12,14,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) that type II cells can make and secrete the lipid components of surface active material. What is not known is what factors regulate lipid synthesis and by what pathways the saturated species of phospholipids are synthesized. There are four main pathways that have been considered possible for the synthesis of saturated phosphatidylcholine.

transacylation.
A detailed discussion of the evidence in SUPport of or against each pathway can be found in recent reviews (4-6). We will focus on the results of this report and of others who used isolated type II cells. Kikkawa et uZ. (14) showed that serial methylation of phosphatidylethanolamine is not a quantitatively important reaction in the synthesis of phosphatidylcholine by isolated type II cells. We have tried to compare the relative contribution of the three other possible routes of synthesis.
Our data support previous observations of others (20,39) which indicate that the deacylation-reacylation pathway is quantitatively important. The assumptions and limitations inherent in our data should be considered before the data are discussed, First, the cell isolation procedure may have altered metabolic pathways in our cells. We dissociated lungs with trypsin; Finkelstein and Mavis (40) reported that trypsin can alter certain enzymes of lipid synthesis in type II cells. Second, we performed most of our studies after 1 day of culture. We found both morphologic and biochemical evidence of change during short term culture and it is possible that the cells we studied after 1 day in culture are different from freshly isolated type II cells (12). Third, because the fatty acids used in culture medium can affect which species of phospholipid are synthesized ~'a Ctro (35,41,42), we chose to perform the long (20-h) incubations (Table I) in the presence of 10% fetal calf serum and the shorter incubations in the presence of the mixed fatty acids found in bovine serum albumin.
Fourth, we used methods that isolate total saturated species of phosphatidylcholine and phosphatidylglycerol.
2) Palmitate is incorporated equally into positions 1 and 2 of saturated phosphatidylglycerol. 3) Type II cells readily acylate lysophosphatidylcholine to form saturated phosphatidylcholine.
After short incubations with radioactive palmitate, position 2 of saturated phosphatidylcholine has approximately three times the radioactivity of position 1 (Tables II and III). The most likely explanation for this observation is that palmitate is incorporated at least in. part by a deacylation-reacylation mechanism. One would expect that palmitate would be incorporated equally into positions I and 2 by de nouo synthesis or by the deacylation-transacylation pathway. It is, however, also possible to imagine that either of these two pathways could produce asymmetric incorporation, for example if there were a large pool of l-acyl-2-lysophosphatidic acid or if there were different pools of acceptor and donor lysophosphatidylcholine for the transacylase (1ysolecithin:lysolecithin acyltransferase) (45). Palmitate is also incorporated predominantly into position 2 of saturated phosphatidylcholine in Phosphatidylglycerol Synthesis urethane adenoma (20), in whole lung (18,20), and in surface active material (46). We have assumed that saturated phosphatidic acid and saturated diglyceride, the precursor molecules for de novo synthesis, have palmitate equally distributed in positions 1 and 2. Because we isolated too few type I1 cells to measure these intermediates directly, we tested this assumption indirectly by determining the distribution in saturated phosphatidylglycerol, reasoning that it might reflect the distribution in saturated phosphatidic acid. The rationale for this approach is that 1) in lung, phosphatidylglycerol is thought to be synthesized de nouo (47); 2 ) type I1 cells readily synthesize saturated phosphatidylglycerol; and 3) the de novo synthesis of phosphatidylcholine and phosphatidylglycerol probably occurs on the endoplasmic reticulum (47,48), implying that the same pool of phosphatidic acid might be used for the de nouo synthesis of both phosphatidylcholine and phosphatidylglycerol. By this reasoning, the finding that radioactive palmitate was equally distributed in positions 1 and 2 of saturated phosphatidylglycerol (Table IIIA) suggests that, during the incubation, saturated phosphatidic acid also had palmitate equally distributed in positions 1 and 2. Other workers have shown that palmitate is incorporated equally into positions 1 and 2 of saturated diglyceride (by lung in uivo (49)) and saturated phosphatidic acid (by urethane adenoma in ritro (50)). Since saturated phosphatidylcholine had three times as much radioactive palmitate in position 2 as in position 1, it seems likely that saturated phosphatidylcholine was made to a large extent by a mechanism other than de novo synthesis.
We used a mixture of saturated radiolabeled lysophosphatidylcholines to evaluate the synthesis of saturated phosphatidylcholine by the transacylation pathway with intact type I1 cells. Although we did not determine the fatty acid composition of the radioactive saturated lysophosphatidylcholine, it should be the same as that of position 1 of saturated phosphatidylcholine in type I1 cells, namely 9% myristate, 87% palmitate, and 3% stearate. We, like Smith and Kikkawa (34), found that lysophosphatidylcholine was readily incorporated into saturated phosphatidylcholine. However, because there was no increase in the .'H/l4C (palmitate/choline) ratio of the saturated phosphatidylcholine synthesized from the mixture of labeled saturated lysophosphatidylcholines, we did not demonstrate transacylation. Our results are in contrast to the work of Akino et al. (51,52) and Hallman and Raivio (53) who used doubly labeled lysophosphatidylcholine to demonstrate the presence of transacylation for the synthesis of saturated phosphatidylcholine in whole lung and in lung slices. There are several explanations for these differences. First, pulmonary cells other than type I1 cells may have accounted for the results in experiments with whole lung (39). Second, our procedure for preparing type I1 cells might result in a loss of 1ysolecithin:lysolecithin acyltransferase activity (40). Third, we used intact cells in our experiments; lysophosphatidylcholine may have been acylated in the cellular plasma membrane (54) and therefore may not have entered the cells to be available to 1ysolecithin:lysolecithin acyltransferase which is located in the cytosol (45,55).
Recently, Batenburg et al. (39) measured the activity of lysolecithin acyltransferase and 1ysolecithin:lysolecithin acyltransferase in sonicates of type I1 cells and whole lung. They found that the specific activity of lysolecithin acyltransferase was much greater in type I1 cells than in whole lung and that the enzyme from type I1 cells showed a preference for palmitoyl coenzyme A over oleoyl coenzyme A. In contrast, the specific activity of 1ysolecithin:lysolecithin acyltransferase was the same in type II cells and in whole lung.
Data with intact lung, adenomas of type I1 cells, and isolated type I1 cells suggest that the deacylation-reacylation pathway is important, but clearly additional studies of the enzymes involved and direct measurement of the intracellular intermediates are needed before one will be able to describe quantitatively the pathways and regulation of the synthesis of saturated phosphatidylcholine in type I1 cells.