Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

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.     

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.     

Influence of starvation on the lung: effect on glucose and palmitate utilization.

The relative utilization of [U-14C]glucose and [1-14C]palmitate was examined in lung slices of male Long Evans hooded rats fed ad libitum and starved for 72 h. Food deprivation (72-h fast) significantly decreased [U-14C]flucose oxidation and incorporation into lung lipids. Glucose incorporation into phospholipid-fatty acid (53%) was, in proportion, more markedly reduced than into phospholipid-gluceride glycerol (33%), suggesting that glucose was being conserved for the formation of alpha-glycerol phosphate. (1-14C) palmitate utilization following fasting showed a significant 40% increase in oxidation, and a significant 16% increase in phospholipids, indicating preferential utilization of fatty acids over glucose. Phospholipid fatty acid composition, surface tension measurements and volume-pressure curves were not affected by fasting. Khe data indicate that glucose and palmitate metabolism are interrelated, and that the relative utilization of these substrates is changed to maintain essential lung lipids during an altered physiologic state.     

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.     

Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro

Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.     

Utilization of disaturated and unsaturated phosphatidylcholine and diacylglycerols by cholinephosphotransferase in rat lung microsomes

1. Cholinephosphosphotransferase catalyzes the conversion of diacylglycerol and CDPcholine into phosphatidylcholine and CMP. Incubation of rat lung microsomes containing phosphatidyl[Me-14C]choline with CMP resulted in an increase in water-soluble radioactivity, suggesting that also in rat lung microsomes the cholinephosphotransferase reaction is reversible. 2. Microsomes containing 14C-labeled disaturated and 3H-labeled monoenoic phosphatidylcholine were prepared by incubation of these organelles with [1-14C]palmitate and [9,10-3H2]oleate in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine, ATP, coenzyme A and MgCl2. Incubation of these microsomes with CMP resulted in an equal formation of 14C- and 3H-labeled diacylglycerols, indicating that disaturated and monoenoic phosphatidylcholines were used without preference by the backward reaction of the cholinephosphotransferase. When in a similar experiment the phosphatidylcholine was labeled with [9,10-3H2]palmitate and [1-14C]linoleate, somewhat more 14C- than 3H-labeled diacylglycerol was formed. 3. The backward reaction was used to generate membrane-bound mixtures of [1-14C]palmitate- and [9,10-3H2]oleate- or of [9,10-3H2]palmitate- and [1-14C]linoleate-labeled diacylglycerols. When the microsomes containing diacylglycerols were incubated with CDPcholine, both 3H- and 14C-labeled diacylglycerols were used for the formation of phosphatidylcholine, indicating that there is no absolute discrimination against disaturated diacylglycerols. This observation is in line with our previous findings and indicates that also the CDPcholine pathway may contribute to dipalmitoylphosphatidylcholine synthesis in lung.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. Measurement of the incorporation of [14C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure: the conditioned medium was more effective than the steroid. These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-³H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-³H]palmitic acid, 458±18pmol of [1-¹⁴C]oleic acid and 252±15pmol of [U-¹⁴C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [³H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-¹⁴C]glucose increased by 125%. Incorporation of [1-¹⁴C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

The effect of betamethasone and fetal sex on the synthesis and maturation of lung surfactant phospholipids in rabbits

In the present study we investigated the maturation of the surfactant phospholipids and the role of fetal sex on the effect of betamethasone in male and female rabbit fetuses. Betamethasone was administered to the doe (0.2 mg/kg intramuscularly) 42 and 18 h prior to killing. The fetuses were studied at 27 and 28 days from conception. Results from the alveolar lavage show that male fetuses tended to have a lower disaturated phosphatidylcholine/sphingomyelin ratio and lower levels of phosphatidylinositol. Phosphatidylglycerol was detected in trace amounts. This was apparently due to the high extracellular levels of myo-inositol inhibiting the synthesis of surfactant phosphatidylglycerol while increasing the synthesis of surfactant phosphatidylinositol. Betamethasone increased the recovery of disaturated phosphatidylcholine and phosphatidylinositol from the lung lavage in both sexes. As studied in lung slices in vitro, the betamethasone treatment decreased the incorporation of glucose into phospholipids, including into the fatty acid moiety of disaturated phosphatidylcholine, although it had no significant effect on the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. However, the addition of palmitate increased the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. The betamethasone treatment did not increase the incorporation of [1-14C]pyruvate into disaturated phosphatidylcholine. Following betamethasone administration, the availability of fatty acids may become rate-limiting for the synthesis of surfactant phospholipids. Betamethasone increased the activities of phosphatidic acid phosphohydrolase and phosphatidate cytidyltransferase in a fraction of microsomal membranes. The present evidence suggests that the glucocorticoid-induced lung maturation and the maturation of the normal lung are associated with an increase in the activity of the enzymes which are involved in metabolizing phosphatidic acid to neutral and acidic surfactant secretion of the male fetus was not explained by possible sex-related differences in the biosynthesis of the phospholipids.     

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.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean.

1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.     

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.