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.     

On the insensitivity of sheep to the almost complete microbial destruction of dietary choline before alimentary-tract absorption.

1. Injection of [Me-14C]choline into sheep indicated that the small amount of phosphatidylcholine present in abomasal digesta was largely (69%) of non-dietary or ruminal origin. 2. Long-term feeding of [Me-3H]choline to sheep produced insignificant labelling of plasma phosphatidylcholine, indicating that more than 99% of the choline body pool was of non-dietary origin. 3. In contrast, when rats were fed with [Me-3H]choline for similar periods, 18-54% of the tissue phosphatidylcholine was derived from dietary choline. 4. The loss of [14C]choline and 32P from the plasma phosphatidylcholine after a single injection of these isotopes indicated a markedly slower turnover of choline in the sheep compared with the rat. This observation, coupled with a lack of liver glycerophosphocholine diesterase, provides an explanation for the insensitivity of the sheep to an almost complete microbial destruction of dietary choline before alimentary-tract absorption.     

Ethanolamine inhibits choline uptake in the isolated hamster heart

The effect of exogenous ethanolamine on phosphatidylcholine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [Me-3H]choline in the presence of 0.05-0.5 mM ethanolamine. Incorporation of label into phosphatidylcholine was decreased 26-63% at 0.1-0.5 mM ethanolamine. Similar decreases in the labelling of the metabolites of the CDP-choline pathway were observed at these ethanolamine concentrations. The observed decrease in phosphatidylcholine labelling at 0.1-0.5 mM ethanolamine was attributed to an inhibition of labelled choline uptake by ethanolamine. The inhibitory role of ethanolamine to choline uptake was examined by comparison to hemicholinium-3. Both compounds inhibited choline uptake in a competitive manner. Intracellular choline, phosphocholine and CDP-choline concentrations were not altered under all experimental conditions. It can be concluded that exogenous ethanolamine has no immediate effect on the rate of phosphatidylcholine biosynthesis in the isolated hamster heart. The reduced labelling of phosphatidylcholine in the presence of ethanolamine is a direct result of the reduction of labelled choline taken up by the heart.     

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.     

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.     

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.     

Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm.

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.     

A deazaadenosine-insensitive methylation of phosphatidylethanolamine is involved in lipoprotein secretion

We have examined the effect of inhibitors of methylation of phosphatidylethanolamine on lipoprotein secretion from cultured rat hepatocytes. The incorporation of [1-3H]ethanolamine into phosphatidylcholine of hepatocytes and secreted lipoproteins was inhibited by greater than 90% by the methylation inhibitors 3-deazaadenosine and Neplanocin. In addition, these compounds strongly inhibited the incorporation of [3-3H]serine into the choline moiety of phosphatidylcholine of the hepatocytes, but had no effect on incorporation of [3-3H]serine into secreted phosphatidylcholine. The results suggest that a pool of phosphatidylcholine targeted for lipoprotein secretion originates from phosphatidylethanolamine made from serine and this methylation reaction has the unique property of being insensitive to 3-deazaadenosine.     

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.     

Regulation of ethanolamine and choline phosphatide biosynthesis in isolated rat intestinal villus cells

The effects of ethanolamine, choline, and different fatty acids on phospholipid synthesis via the CDP-ester pathways were studied in isolated rat intestinal villus cells. The incorporation of [14C]glucose into phosphatidylethanolamine was stimulated severalfold by the addition of ethanolamine and long-chained unsaturated fatty acids, while the addition of lauric acid inhibited the incorporation of radioactivity into phosphatidylethanolamine. At concentrations of ethanolamine higher than 0.2 mM, phosphoethanolamine accumulated, but the concentrations of CDP-ethanolamine and the incorporation of radioactivity into phospatidylethanolamine did not increase further. The incorporation of [14C]glucose into phosphatidylcholine responded in a way similar to that of phosphatidylethanolamine, except that a 10-fold higher concentration of choline was required for maximal stimulation. CCC inhibited the incorporation of choline into phosphatidylcholine. In contrast with hepatocytes, villus cells did not form phosphatidylcholine via phospholipid N-methylation. The data indicate that, in intestinal villus cells, the cytidylyltransferase reactions are rate limiting in the synthesis of phosphatidylethanolamine and probably also of phosphatidylcholine. The availability of diacylglycerol and its fatty acid composition may also significantly affect the rate of phospholipid synthesis.     

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 base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

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.     

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.     

2-hydroxyethylhydrazine as a potent inhibitor of phospholipid methylation in yeast

The effect of 2-hydroxyethylhydrazine on the phosphatidylethanolamine methylation pathway in yeast was studied. 2-Hydroxyethylhydrazine inhibited the growth of cells. The concentration required for 50% inhibition was 66 microM. The growth rate decreased by 2-hydroxyethylhydrazine was restored by the addition of a low concentration of choline. Incorporation of radioactivity from L-[3-14C]serine, L-[methyl-14C]methionine and S-adenosyl-L-[methyl-14C]methionine into phosphatidylcholine was markedly reduced by 2-hydroxyethylhydrazine. The restoration of growth by choline was not due to the reversal of the inhibition, but to the formation of phosphatidylcholine via the CDPcholine pathway. Thus, the site of action of 2-hydroxyethylhydrazine in vivo was the phosphatidylethanolamine methylation pathway. Experiments with methylation mutants indicated that all three steps of methylation were sensitive to 2-hydroxyethylhydrazine. 2-Hydroxyethylhydrazine was shown to inhibit the methyltransferase after it had become chemically or metabolically transformed in cells. 2-Hydroxyethylhydrazine-resistant mutants were obtained and were found to have a defect in choline transport activity. Genetic data indicated that the uptake of 2-hydroxyethylhydrazine into cells is mediated by the choline transport system.     

Studies on the origin of choline in the brain of the rat

1. Labelled precursors of choline, namely ethanolamine, dimethylaminoethanol and methionine and also labelled choline itself were injected intraperitoneally into the adult female rat and the incorporation into lipids and water-soluble fractions was traced in liver, blood and brain. 2. No significant free choline was detected and no labelling of the phosphorylcholine of blood. There was, however, considerable labelling of the phosphorylcholine of brain and liver. 3. After intracerebral injection, [1,2-(14)C]dimethylaminoethanol was rapidly phosphorylated and converted into phosphatidyldimethylaminoethanol, presumably by the cytidine pathway. 4. In view of the pattern of labelling and the amount of phosphatidylcholine in the tissues examined, it seems highly likely that choline is transported to the brain by the blood in a lipid-bound form.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

Base exchange reactions of the phospholipids in rat brain particles

A particulate fraction from rat brain catalyzes the incorporation of [(14)C]choline, [(14)C]ethanolamine, and l-[(14)C]serine into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, respectively. The reaction appears to be energy-independent since Mg(2+), CTP, ATP, and NaF have no stimulatory action. The incorporation is inhibited by EDTA and activated by Ca(2+). The pH optimum for the incorporation of choline is 9.5, for ethanolamine it is 9.0, and for l-serine it is 8.5. Tris, bicine, and imidazole buffers are inhibitory. The incorporations are inhibited by a variety of structurally related alcohols and are stimulated by isoserine (alpha-hydroxy,beta-aminopropionic acid).     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.