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.     

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.     

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.     

Properties of freshly isolated type II alveolar epithelial cells

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

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.     

Regulation of CTP: phosphocholine cytidylyltransferase activity in type II pneumonocytes.

Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.     

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.     

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.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Digestion of phosphatidylcholines,absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphati-dylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1-[1-¹⁴C]acyl- and 1-[³H] alkyl-lysophosphatidylcholine, [1-¹⁴C]oleic acid, [2-¹⁴C]glycerol, phosphoryl[methyl-¹⁴C]choline, and [methyl-¹⁴C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A₂, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and P_i, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoalkylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine. Arch. Insect Biochem. Physiol. 36:273–293, 1997. © 1997 Wiley-Liss, Inc.     

Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes.

Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+-free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo-inositol that were found previously to be associated with this period of development.     

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

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.     

c-fos基因在内皮素-1促肺泡Ⅱ型细胞表面活性物质合成中的作用

应用反义技术探讨ｃ－ｆｏｓ基因ＥＴ－１调控肺泡Ⅱ型细胞（ＡＴⅡ）表面活性物质（ＰＳ）合成的胞内信号转导中的作用,结果显示：（１）内皮素－１（ＥＴ－１）可提高ＡＴⅡ细胞的＾３Ｈ－胆碱掺入。（２）蛋白激酶Ｃ（ＰＫＣ）激活剂ＰＭＡ可使ＡＴⅡ细胞的＾３Ｈ－胆碱掺入量增加,ＰＫＣ抑制剂Ｈ７可抑制ＥＴ－１的促ＰＳ合成效应。（３）ＥＴ－１和ＰＭＡ可显著提高Ｆｏｓ蛋白表达量,Ｈ７和ｃ－ｆｏｓ反义寡核苷酸（ＯＤＮ）     

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl-sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid were compared as precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μm) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid into diacyl-sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl-sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μm. In short-term labeling experiments, the label in disaturated diacyl-sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1-¹⁴C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl-sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phospho-[³H-methyl]choline was incorporated into total cellular diacyl-sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in ¹⁴C. These findings indicate the cells take up intact monoacyl-sn-glycero-3-phosphocholine and incorporate it into diacyl-sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl-sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Control of glucose metabolism in isolated acini of the lactating mammary gland of the rat. The ability of glycerol to mimic some of the effects of insulin.

Inhibition of glucose uptake by acetoacetate and relief of this inhibition by insulin found previously in slices of rat mammary gland [Williamson, McKeown & Ilic (1975) Biochem. J. 150. 145-152] was confirmed in acini, which represent a more homogeneous population of cells. Glycerol (1mM) behaved like insulin (50 minuits/ml) in its ability to relieve the inhibition of glucose (5 mM) utilization caused by acetoacetate (2 mM) in acini. Both glycerol and insulin reversed the increase in [citrate] and the decrease in [glycerol 3-phosphate] and the [lactate]/[pyruvate] ratio in the presence of acetoacetate. Lipogenesis from 3H2O, [3-14C] acetoacetate, [1-14C]- and [6-14C]-glucose was stimulated, whereas 14CO2 formation from [3-14C]acetoacetate was decreased. Neither insulin nor glycerol relieved the acetoacetate inhibition of glucose uptake when lipogenesis was inhibited by 5-(tetradecyloxy)-2-furoic acid. From measurements of [3-14C]acetoacetate incorporation into lipid in the various situations it is suggested that a cytosolic pathway for acetoacetate utilization may exist in rat mammary gland. In the absence of acetoacetate, glycerol inhibited glucose utilization by 60% and increased both [glycerol 3-phosphate] and the [lactate/[pyruvate] ratio. Possible ways in which glycerol may mimic the effects of insulin are discussed.     

Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.