Abstract
Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material. We studied the synthesis of these two phospholipid classes by alveolar type II cells in primary culture. During a 20-h incubation, type II 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 novo synthesis of fatty acids rather than by chain elongation. We studied the pathways for synthesis of saturated phosphatidylcholine and phosphatidylglycerol with type II 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 II cells also incorporated a mixture of saturated 1-[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 1 day, type II cells incorporated a lower percentage of palmitate into phosphatidylglycerol and saturated phosphatidylcholine.
Highlights
Rial-We tested the ability of type I1 cells in primary culture to synthesize the lipids of surface active materibayl incubating the cells for 20 hwith radioactiveacetate,palmitate,or glycerol
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 d e novo synthesisratherthan by chain elongationW. e determined whether acetatewas incorporated into fattyacids by de novo synthesioscrhain elongationby incubating ceUs with
Incorporation ofpalmitate into totallipid and disatfatty acids only by de novo synthesis and if palmitate were urated phosphatidylcholine (DSPC).Day 0 designates the day on the sole product, 12.5%of the radioactivitywould be found in the carboxyl carbon
Summary
Extraction a n d Separation-After the incubation period, medium was removed and thecells were washedthree timeswith ME medium/. KC1 to which we added sodium acetate (1111~)or glycerol (1mM) for the experimentsin which radioactive acetate orglycerol was used as precursor.Individualphospholipidswere separated by two-dimensional thin layer chromatography(TLC)on Silica Gel G plates impregnated with boric acid [17]. To isolate boththesaturatedandtheunsaturated 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: N ammonium hydroxide: water (75:25:1:2).For analysesrequiring onlythe isolation of saturated phosphatidylcholine The reaction products were separated by TLC, localized by iodine vapor, and scraped into counting vials for determination of radioactivity [18] With the exception of diethyl ether, were redistilled before use
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