The choline-depleted type II pneumonocyte. A model for investigating the synthesis of surfactant lipids.

When type II pneumonocytes from adult rats were maintained in a medium that lacked choline, the incorporation of [14C]glycerol into phosphatidylcholine was not greatly diminished during the period that the cells displayed characteristics of type II pneumonocytes. Cells that were maintained in choline-free medium that contained choline oxidase and catalase, however, became depleted of choline and subsequent synthesis of phosphatidylcholine by these cells was responsive to choline in the extracellular medium. Incorporation of [14C]glycerol into phosphatidylcholine by choline-depleted cells was stimulated maximally (approx. 6-fold) by extracellular choline at a concentration (0.05 mM) that also supported the greatest incorporation into phosphatidylglycerol. The incorporation of [14C]glycerol into other glycerophospholipids by choline-depleted cells was not increased by extracellular choline. When cells were incubated in the presence of [3H]cytidine, the choline-dependent stimulation of the synthesis of phosphatidylcholine and phosphatidylglycerol was accompanied by an increased recovery of [3H]CMP. This increased recovery of [3H]CMP reflected an increase in the intracellular amount of CMP from 48 +/- 9 to 76 +/- 16 pmol/10(6) cells. Choline-depleted cells that were exposed to [3H]choline contained [3H]CDP-choline as the principal water-soluble choline derivative. As the extracellular concentration of choline was increase, however, the amount of 3H in phosphocholine greatly exceeded that in all other water-soluble derivatives. Choline-depletion of cells resulted in an increase in the specific activity of CTP:phosphocholine cytidylyltransferase in cell homogenates (from 0.40 +/- 0.15 to 1.31 +/- 0.20 nmol X min-1 X mg of protein-1). These data are indicative that the biosynthesis of phosphatidylcholine is integrated with that of phosphatidylglycerol and are consistent with the proposed involvement of CMP in this integration. The choline-depleted type II pneumonocyte provides a new model for investigating the regulation of CTP:phosphocholine cytidylyltransferase activity.     

The influence of myo-inositol on phosphatidylglycerol synthesis by rat type II pneumonocytes.

Type II pneumonocytes isolated from adult rat lung were incubated in a serum-free medium containing [14C]glycerol and the incorporation of 14C into glycerophospholipids was measured. After 24 h, more than 80% of the 14C incorporated into total lipids or into phosphatidylcholine and approx. 90% of the 14C incorporated into phosphatidylglycerol after 24 h was recovered in the glycerophosphoester moieties of these molecules. Supplementation of the incubation medium with foetal-bovine serum (10%, v/v) did not alter the incorporation of [14C]glycerol by type II pneumonocytes after 24 h into either a total lipid extract or phosphatidylcholine. In the presence of foetal-bovine serum, however, the incorporation of 14C into phosphatidylglycerol was decreased and the incorporation of 14C into phosphatidylinositol was increased. In the absence of foetal-bovine serum, the incorporation of 14C into phosphatidylglycerol was decreased progressively as the concentration of myo-inositol in the incubation medium was increased. The range of concentration (0.04-0.50 mM) over which myo-inositol had the greatest influence on [14C]glycerol incorporation into phosphatidylglycerol by type II pneumonocytes in vitro encompassed the concentration range measured in foetal-rat serum late in gestation. At 4 days before birth, the concentration of myo-inositol in foetal-rat serum was 0.36 mM and decreased to 0.23 mM 1 day before birth. The concentration of myo-inositol in adult rat serum increased from 0.03 mM to 0.06 mM during pregnancy. Isolated rat type II pneumonocytes were found to take up myo-inositol by a saturable process. A half-maximal rate of myo-inositol uptake occurred at a concentration of myo-inositol of 0.29 mM. The results of this investigation are consistent with the hypothesis that late in gestation there is a decreasing availability of myo-inositol to the foetal lungs and that this favours the biosynthesis of phosphatidylglycerol for surfactant at the expense of phosphatidylinositol biosynthesis.     

Altered lipid synthesis in type II pneumonocytes exposed to lung surfactant.

When type II pneumonocytes were exposed to purified lung surfactant that contained 1-palmitoyl-2-[3H]palmitoyl-glycero-3-phosphocholine, radiolabelled surfactant was apparently taken up by the cells since it could not be removed by either repeated washing or exchange with non-radiolabelled surfactant, but was released when the cells were lysed. After 4 h of exposure to [3H]surfactant, more than half of the 3H within cells remained in disaturated phosphatidylcholine. Incorporation of [3H]choline, [14C]palmitate and [14C]acetate into glycerophospholipids was decreased in type II cells exposed to surfactant and this inhibition, like surfactant uptake, was half-maximal when the extracellular concentration of surfactant was approx. 0.1 mumol of lipid P/ml. Inhibition of incorporation of radiolabelled precursors by surfactant occurred rapidly and reversibly and was not due solely to dilution of the specific radioactivity of intracellular precursors. Activity of dihydroxyacetone-phosphate acyltransferase, but not glycerol-3-phosphate acyltransferase, was decreased in type II cells exposed to surfactant and this was reflected by a decrease in the 14C/3H ratio of total lipids synthesized when cells incubated with [U-14C]glycerol and [2-3H]glycerol were exposed to surfactant. Phosphatidylcholine, phosphatidylglycerol and cholesterol, either individually or mixed in the molar ratio found in surfactant, did not mimic purified surfactant in the inhibition of glycerophospholipid synthesis. In contrast, an apoprotein fraction isolated from surfactant inhibited greatly the incorporation of [3H]choline into lipids and this inhibitory activity was labile to heat and to trypsin. It is concluded that the apparent uptake of surfactant by type II cells in vitro is accompanied by an inhibition of glycerophospholipid synthesis via a mechanism that involves a surfactant apoprotein.     

Comparison of the enzyme activities of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in freshly isolated type II pneumocytes and whole lung from the adult rat

We compared the activities of enzymes of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in whole lung tissue and freshly isolated type II pneumocytes from adult rats. The activities of 1-acylglycerophosphocholine acyltransferase and CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase were 2.9- and 4.4-fold higher, respectively, in type II cell sonicates than in whole lung homogenates. There was little difference between the type II cells and whole lung in the activities of choline kinase, choline-phosphate cytidyltransferase, cholinephosphotransferase, phosphatidate phosphatase, phosphatidate cytidylytransferase or CDPdiacylglycerol-inositol 3-phosphatidyltransferase. Since the type II cell is the source of pulmonary surfactant, and disaturated phosphatidylcholine and phosphatidylglycerol are major components of surfactant, it is of interest that this cell is enriched in the activities of enzymes exclusively involved in the synthesis of these lipids. In view of possible proteolytic damage during isolation we compared freshly isolated type II cells with those cultured for 1 day. The rates of incorporation of [methyl-3H]choline and [2-3H]glycerol into phospholipids, L-[U-14C]phenylalanine into protein and [methyl-3H]thymidine into DNA were the same in the freshly isolated and cultured cells. The composition of the phospholipids synthesized from [2-3H]glycerol and sodium [1-14C]acetate were also the same. The freshly isolated cells were at least 90% pure and did not release significant amounts of lactate dehydrogenase. Since use of freshly isolated cells avoids cell loss during culture they provide an attractive alternative, particularly in studies requiring large amounts of material.     

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Regulation by cytidine nucleotides of the acylation of sn-[14C]glycerol 3-phosphate. Regional and subcellular distribution of the enzymes responsible for phosphatidic acid synthesis de novo in the central nervous system of the rat

1. The regional and subcellular distribution of the incorporation of sn-[(14)C]glycerol 3-phosphate into rat brain lipids in vitro was investigated and compared with the relative specific activity of various chemical and enzyme markers. The similarity between the subcellular distribution of this incorporation and of NADPH-cytochrome c reductase activity indicated that the synthesis of phosphatidic acid via this route correlated with the presence of endoplasmic reticulum. 2. Experiments in which various amounts of the microsomal fraction were added to fixed amounts of nuclear, myelin, nerve-ending and mitochondrial preparations clearly demonstrated that the endoplasmic-reticulum contamination of these fractions was entirely responsible for the incorporation of sn-[(14)C]glycerol 3-phosphate. 3. The presence of CMP or CTP inhibited the incorporation of sn-[(14)C]glycerol 3-phosphate into the whole homogenate. Similar effects were observed with individual fractions, except for the mitochondria. With the mitochondrial fraction the effect of these cytidine nucleotides varied with the preparation, stimulating in some preparations and inhibiting with other preparations. The presence of CDP-choline stimulated the incorporation into the whole homogenate and to a lesser extent into the subcellular fractions. 4. These results indicate that the various organelles of the central nervous system are more dependent on endoplasmic reticulum for the production of glycerolipids de novo than has previously been appreciated.     

Role of microtubules in surfactant secretion

In the isolated perfused rat lung and cultured type II cells, surfactant secretion and cellular adenosine 3',5'-cyclic monophosphate (cAMP) content was stimulated by beta-adrenergic agonists. Isoproterenol-induced surfactant secretion was inhibited by the antimicrotubule agents colchicine and vinblastine. Incorporation of [3H]glycerol into disaturated phosphatidylcholine was augmented by beta-adrenergic agents but was not significantly different from the enhanced incorporation rate when colchicine was present. This suggests that the augmented incorporation of [3H]glycerol into disaturated phosphatidylcholine was a secondary response to storage depletion rather than direct cAMP stimulation. beta-Adrenergic agents shifted the equilibrium in the isolated perfused rat lung and cultured type II cells to favor microtubules. The stimulatory effect of 1.0 microM isoproterenol on tubulin polymerization was observed as early as 1 min and was augmented 2.8-fold at a half-maximal stimulation of 4 nM in cultured type II cells. Cytochalasin B, an antimicrofilament agent, potentiated the isoproterenol-induced secretion. These results suggest that an intact microtubule-microfilament system may be obligatory for enhanced surfactant secretion and that beta-adrenergic agents not only induce surfactant release but also tubulin polymerization.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphatidylglycerol of rat lung. Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant.

The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant.     

Phosphatidyglycerol in rat lung. II. Comparison of occurrence, composition, and metabolism in surfactant and residual lung fractions.

A comparison of the occurrence, fatty acid composition, and metabolism of phosphatidyglycerol and phosphatidylcholine in the surfactant and residual fraction of rat lung has been carried out. The surfactant and residual fractions were separated by discontinuous sucrose density gradient centrifugation. The surfactant fraction was found to contain 69 percent phosphatidylcholine and 7 percent phosphatidylglycerol. The residual fraction contained 46 percent phosphatidylcholine and 3 percent phosphatidylglycerol. Phosphatidylcholine and phosphatidylglycerol were found to contain 85 and 79 percent palmitate in the surfactant fraction and 67 and 68 percent in the residual fraction, respectively. Isolated rat lungs were perfused with medium containing [U-14C]glucose, [9,10-3H]palmitate, and [1-14C]acetate and the incorporation into palmitate isolated from the alpha and beta position of phosphatidylcholine and phosphatidylglycerol was determined. Each radioactive substrate was found to be incorporated into palmitate of phosphatidylcholine equally at the alpha and beta position of the surfactant fraction. In the residual fraction the specific activity of the beta position palmitate was found to be twice that of the alpha position. The incorporation of [9,10-3H]palmitate and [1-14C]acetate into palmitate at the alpha and beta positions of phosphatidylglycerol was similar in both the surfactant and residual fractions. In each case palmitate at the alpha position had approximately twice the specific activity of that at the beta position. The incorporation of [U-14C]glucose into phosphatidylglycerol of the surfactant fraction was, however, greater in palmitate at the beta position than at the alpha. The results show that phosphatidylglycerol is associated with the lung surfactant fraction and suggest that palmitate esterified to the alpha and beta positions of phosphatidylglycerol and phosphatidylcholine occurs at different rates and is dependent upon the precursor source of palmitate.     

Biosynthesis of murein lipoprotein in Escherichia coli: effects of 3,4-dihydroxybutyl-1-phosphonate.

The effects of 3,4-dihydroxybutyl-1-phosphonate, a four-carbon analog of sn-glycerol 3-phosphate, on the biosynthesis of the glyceryl moiety in murein lipoprotein of Escherichia coli were studied. The compound at a concentration of 55 microM strong inhibits in the incorporation of [2-3H]glycerol radioactivity into lipoprotein by virtue of its inhibition of the synthesis of phosphatidylglycerol. On the other hand, the incorporation of prelabeled [2-3H]glycerol radioactivity into lipoprotein was only partially inhbited by 3,4-dihydroxybutyl-1-phosphonate even at a much higher concentration (1 mM). These data were consistent with the postulated pathway for the biosynthesis of the lipid moiety in lipoportein: cysteine-lipoprotein + phosphatidylglycerol leads to glycerylcystein-lipoprotein + phosphatidic acid.     

Glycerolipid metabolism in lung from ventilated and unventilated rabbits

The incorporation of [14C]-glycerol 3-phosphate and [3H]-palmitate into phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and triacylglycerols by lung microsomes from ventilated and unventilated rabbits was measured. Unventilated lung microsomes showed an impairment of the "de novo" synthesis of phosphatidic acid and, therefore, a general decrease of glycerolipids synthesized from glycerol 3-phosphate. The incorporation of [3H]-palmitate into phosphatidic acid was considerably lower than the incorporation of [14C]-glycerol 3-phosphate by lung microsomes from both ventilated and unventilated rabbits, and the 3H/14C molar ratio did not change during incubation time. These observations suggest the preferential utilization of endogenous fatty acids by acyltransferases involved in the formation of phosphatidic acid. The activities of the enzymes implicated in the synthesis of phosphatidylcholine from lysophosphatidylcholine remained unchanged in lung from both ventilated and unventilated rabbits.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.