The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.     

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.     

Hormonal regulation of phosphatidylcholine synthesis by reversible modulation of cytidylyltransferase.

The effect of both lipolytic and antilipolytic hormones on the turnover of phosphatidylcholine in freshly isolated rat adipocytes was investigated. Treatment of adipocytes with agonists such as glucagon or isoprenaline that stimulate lipolysis through a cyclic AMP-dependent mechanism caused an increase in the incorporation of [Me-3H]choline into phosphatidylcholine. Pulse-chase studies indicated that the stimulation was due to an increase in the conversion of choline into phosphatidylcholine, which was both time- and dose-dependent. The stimulatory effect of isoprenaline was inhibited in a dose-dependent manner by oxytocin or insulin. Oxytocin inhibited the incorporation of [Me-3H]choline into phosphatidylcholine in both the presence and the absence of isoprenaline, whereas in the absence of isoprenaline insulin increased the incorporation of [Me-3H]choline into phosphatidylcholine. The effects of isoprenaline, oxytocin and insulin on the incorporation of [3H]choline into phosphatidylcholine were paralleled by changes in the activity of CTP:phosphocholine cytidylyltransferase.     

Role of a specific choline pool in sphingomyelin synthesis in rat heart.

Sphingomyelin synthesis was studied in slices of rat heart by using [Me-14C]choline, [1,2-14C]ethanolamine, S-adenosyl-L-[14C]methionine and [32P]Pi as as precursors. In the presence of both [Me-14C]choline and [32P]Pi the ratio of the specific radioactivities of 14C and 32P in phosphatidylcholine was greater than in sphingomyelin at all the times studied. This suggested that synthesis of phosphatidylcholine and sphingomyelin de novo did not involve the utilization of a common pool of cytidine diphosphate choline. In addition, studies with [1,2-14C]ethanolamine and S-adenosyl-L-[14C]methionine indicated that a quantitatively significant pool of choline, derived from these precursors, was selectively utilized for sphingomyelin formation. This pool was not represented by phosphatidylcholine formed by methylation of phosphatidylethanolamine or by other pathways.     

Arterio-venous differences of choline and choline lipids across the brain of rat and rabbit.

The concentration of unesterified choline in the plasma in the jugular vein of the rat (0.85 nmol/ml) was found to be three times that of the arterial supply to the brain (0.25 nmol/ml), indicating a higher efflux than uptake of unesterified choline by the brain. No such difference was found for the rabbit and no arterio-venous difference for phosphatidylcholine or lysophosphatidylcholine was observed in either species. No arterio-venous difference was found for choline in blood cells. The infusion of [Me-3H]choline into the circulation of the rat or rabbit indicated an uptake of radioactive choline by the brain and an efflux of non-radioactive choline. In the rabbit such an infusion produced a steady rise in the labelling of phosphatidylcholine and lysophosphatidylcholine in the plasma. When [14C2]ethanolamine was injected intraperitoneally into the rat there was a labelling of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in the plasma and cells of blood from the jugular vein and the arterial supply, as well as in the brain tissue. However, no labelling of unesterified choline in these tissues could be detected. Unesterified choline was shown to be liberated into the plasma when whole blood from the rat or man, but not the rabbit, was incubated for short periods at 30 degrees C.     

Rapid transport of phosphatidylcholine occurring simultaneously with protein transport in the frog sciatic nerve

1. Either l-[4,5-(3)H]leucine or [Me-(3)H]choline, or both l-[U-(14)C]leucine and [Me-(3)H]-choline, were injected into the ninth dorsal root ganglion of the frog, and peripheral transport of labelled proteins and/or phospholipids, mostly phosphatidylcholine, was studied by analysis of consecutive segments of the sciatic nerve. 2. At 25 degrees C, approx. 5% of the (3)H-labelled protein was transported at the rate of 152mm/day. The rate was temperature-dependent with the Q(10) value of 2.6. The flow was completely blocked by the local application of colchicine, but was unaffected by cytochalasin D. 3. [Me-(3)H]-Choline was incorporated into phosphatidylcholine at a comparatively slow rate, but was transported in the nerve at a rate equivalent to that for (3)H-labelled proteins. 4. The simultaneous transport of phosphatidylcholine and the protein was further supported in the double-labelling experiments by an identical transport rate of (3)H-labelled phosphatidylcholine and (14)C-labelled proteins, by their identical temperature dependence, by simultaneous blockade with colchicine, and also by the parallel distribution of the two labels in subcellular fractions. Specific radioactivities on a protein basis of both (3)H and (14)C labels were highest in microsomal subfractions enriched with Na(+)-plus-K(+)-stimulated adenosine triphosphatase and acetylcholinesterase. It is suggested that (3)H-labelled phosphatidylcholine and (14)C-labelled proteins transported in the nerve reside in the same structural entity, most probably a membrane component.     

The rate-limiting reaction in phosphatidylcholine synthesis by alveolar type II cells isolated from fetal rat lung

The rate-limiting reaction in the formation of phosphatidylcholine by type II cells isolated from fetal rat lung was examined. Studies on the uptake of [Me-3H]choline and its incorporation into its metabolites indicated that in these cells the choline phosphate pool was much larger than both the choline and CDPcholine pools. Chemical measurements of the pool sizes showed that the choline phosphate pool was indeed much larger than the intracellular choline and CDPcholine pools. Pulse-chase studies with [Me-3H]choline revealed that labelled choline taken up by the cells was rapidly phosphorylated to choline phosphate and that the radioactivity lost from choline phosphate during the chase period appeared in phosphatidylcholine. Little change was observed in the labelling of CDPcholine during the chase period. These results indicate that cholinephosphate cytidylyltransferase catalyzes a rate-limiting reaction in phosphatidylcholine formation by fetal rat lung type II cells.     

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.     

Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters.

It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.     

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.     

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro

We investigated the intracellular metabolic fate of [Me-14C]choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5:4:1). With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of water-soluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine. With [Me-14C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents. It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.     

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.     

Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids?

1. The use of ;marker' enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH-cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-(14)C]choline or phosphoryl[Me-(14)C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [(32)P]P(i) precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-(14)C]choline but the synaptic vesicles and probably the intraterminal ;endoplasmic reticulum' appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-(14)C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca(2+). Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH-cytochrome c reductase. 5. On the intraperitoneal injection of [(32)P]P(i) into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little (32)P incorporation up to 10h.     

Early Stimulation of Phosphatidylcholine Biosynthesis During Wallerian Degeneration of Rat Sciatic Nerve

Phospholipid metabolism was studied in rat sciatic nerve during Wallerian degeneration induced by crush injury. Portions of crushed sciatic nerve, incubated with labeled substrates, showed significantly higher phosphatidylcholine synthesis than normal nerve, prior to any measurable alterations of phospholipid composition. Maximum synthesis occurred 3 days after crush injury, at which time the metabolism of other phospholipids was unchanged. After a rapid decrease in biosynthetic activity, a second phase of enhanced phosphatidylcholine synthesis occurred, beginning 6 days after crush injury. Increased incorporation of [33P]phosphate, [2-3H]glycerol, and [Me-14C]choline indicated stimulation of de novo synthesis of phosphatidylcholine 3 days after injury. Neither base exchange reactions nor sequential methylation of ethanolamine phospholipids contributed significantly to phosphatidylcholine synthesis. Assay of certain key enzymes under optimal conditions in subcellular fractions of sciatic nerve revealed higher activities of cholinephosphate cytidyltransferase, choline phosphotransferase, and acyl-CoA:lysophosphatidylcholine acyltransferase in injured nerve, while choline kinase activity remained unchanged. This indicates that stimulation of phosphatidylcholine synthesis occurs via the cytidine nucleotide pathway, as well as by increased acylation of lysophosphatidylcholine. Although the cause of stimulated phosphatidylcholine synthesis remains unexplained, it is possible that trace amounts of lysophospholipids or other metabolites produced by injury-enhanced phospholipase activity may be responsible.     

Phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum.

1. The anaerobic rumen protozoon Entodinium caudatum was incubated either intact or with various radioactive precursors of phospholipids after ultrasonication. 2. Pulse-chase experiments showed a rapid turnover of phosphatidylinositol and much slower turnovers of phosphatidylethanolamine and phosphatidylcholine. 3. E. caudatum imbibed choline very rapidly; this was immediately and exclusively converted into phosphatidylcholine which was shown by radioautography after 10 min to be distributed throughout the cell membranes. 4. Phosphatidylcholine was synthesized through a phosphorylcholine-CDP-choline pathway, the methylation or base-exchange pathways not being present. 5. Under suitable conditions [Me-14C]choline can be substantially (50-60%) converted into CDP-choline by sonicated E. caudatum and this provides an excellent method of preparing this biosynthetic intermediary. 6. [2-14C]Ethanolamine was taken up much less readily than choline. The former was incorporated into phosphatidylethanolamine by the CDP-ethanolamine pathway. 7. Doubly labelled [32P]phosphatidyl[2-3H]ethanolamine was converted into ceramide phosphorylethanolamine and N-(1-carboxyethyl)phosphatidyl-ethanolamine, without change in the isotopic ratio. Ceramide phosphoryl [2-14C]-ethanolamine was converted into phsophatidylethanolamine. 8. Palmitic acid, oleic acid and linoleic acid were taken by E. caudatum cells and incorporated into phospholipids. By contrast, although stearic acid was taken up it was hardly incorporated into phospholipids.     

Utilization of High-Density Lipoprotein Sphingomyelin by the Developing and Mature Brain in the Rat

Utilization of very long chain saturated fatty acids by brain was studied by injecting 20-day-old and adult rats with high-density lipoprotein containing [stearic or lignoceric acid-14C, (methyl-3H)choline]sphingomyelin. Labeling was followed for 24 h. Very small amounts of 14C were recovered in the brain of all rats, and there was no preferential uptake of lignoceric acid. Approximately 20% of the entrapped 14C was located in the form of unchanged sphingomyelin 24 h after injection. This result shows that the rat brain utilizes very little very long chain fatty acids (greater than or equal to 20 C atoms) from high-density lipoprotein sphingomyelin, even during the myelinating period. The [3H]choline moiety from sphingomyelin was recovered in brain phosphatidylcholine in a higher proportion in comparison with the 14C uptake. The brain 3H increased throughout the studied period in all experiments, but was much higher in the myelinating brain than in the mature brain. From the radioactivity distribution in liver and plasma lipids, it is clear that the choline 3H in the brain originates from either double-labeled phosphatidylcholine of lipoproteins or tritiated lysophosphatidylcholine bound to albumin, both synthesized by the liver.     

The incorporation of choline into disaturated phosphatidylcholine in the microsomal, lamellar body, and surfactant fractions of hamster lung tissue

The specific activity of disaturated phosphatidylcholine in microsomes and lamellar bodies prepared from hamster lung tissue and in surfactant obtained by lung lavage was determined at various times following the intraperitoneal administration of [Me-3H]choline. The highest specific activity of disaturated phosphatidylcholine in the lung microsomes was attained 1 h after the administration of [3H]choline; thereafter, the specific activity declined. The specific activity of disaturated phosphatidylcholine in lamellar bodies increased steadily for 12 h after [3H]choline administration. The specific activity in lamellar bodies ater 12 h exceeded the maximum specific activity achieved in the microsomal fraction (p less than 0.005). The specific activity of the disaturated phosphatidylcholine in the alveolar lavage increased after an initial lag period of approximately 3 h, attaining the same specific activity as that of the lamellar bodies at the 12-h time point. The reported results are discussed in relation to the biosynthesis, storage, and secretion of the disaturated phosphatidylcholine associated with the lipoprotein, surfactant.     

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 roles of phospholipase D and a GTP-binding protein in guanosine 5''-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.     

Time-course of utilization of [stearic or lignoceric acid]sphingomyelin from high-density lipoprotein by rat tissues

Utilization of stearic and lignoceric acids supplied by high-density lipoprotein (HDL) sphingomyelin to different tissues was followed for 24 h after rats were injected with HDL containing [[1-14C]stearic (18:0) or [1-14C]lignoceric (24:0) acid [Me-3H]choline]sphingomyelin. Both isotopes reached a maximum in tissue lipids 3-12 h after injection and were recovered mainly in the liver (30%) and small intestine (3%), whereas the other tissues contained approx. 1% or less of the injected dose. All the tissues were able to take up some intact sphingomyelin from HDL and hydrolyze it. In the lung and erythrocytes, the 3H:14C ratio of sphingomyelin remained unchanged throughout the studied period, while an increase in the isotopic ratio was observed in the kidney due to the 3H choline moiety re-used for synthesis of new sphingomyelin. Conversely, the isotopic ratio of sphingomyelin decreased in the liver, indicating a saving of the 14C-labelled fatty acids, especially 24:0. Furthermore, [24:0]ceramide in the liver remained at a high level (6% of the injected dose), whereas [18:0]ceramide decreased to 1%. When the tissues were examined 24 h after injection, the proportion of the 14C linked to sphingomyelin in the total 14C was always higher for both kinds of sphingomyelin than the molar proportion of sphingomyelin in the whole of lipid classes. However, in the majority of the extra-hepatic tissues, more [14C]18:0 than [14C]24:0 was recovered in sphingomyelin, and more 14C radioactivity from 18:0 than from 24:0 was redistributed in the other lipids. The choline moiety from both kinds of sphingomyelin was re-used to synthesize phosphatidylcholine, especially in the liver (up to 20% of the injected dose). All these results show that utilization of sphingomyelin from HDL by tissues normally occurs in vivo and that this phenomenon should be taken into account in the study of the phospholipid turnover of cell membranes. They also show that metabolism of sphingomyelin from HDL in the liver and other tissues is dependent on the sphingomyelin acyl moiety.