The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.     

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.     

Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).     

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.     

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.     

The coordination of sterol and phospholipid synthesis in cultured myogenic cells: Effect of cholesterol synthesis inhibition on the synthesis of phosphatidylcholine

The coordination of biosynthesis of cholesterol and phosphatidylcholine has been investigated in a myoblast cell line, L₆, grown in lipid-depleted medium. The addition of 25-hydroxycholesterol or compactin to this medium inhibits cholesterol synthesis by over 95%. The rate of [³H]choline incorporation into phosphatidylcholine begins to decline after 6 h and eventually falls to 45% of control. Measurements of choline flux through the CDPcholine pathway and of the pool sizes of choline-containing intermediates indicate that the formation of CDPcholine is the rate-limiting step in phosphatidylcholine synthesis in L₆. The rate of CDPcholine synthesis was measured in vivo by pulse-chase experiments. Culturing cells with 25-hydroxycholesterol or compactin results in an inhibition of this step, which parallels the inhibition of incorporation of [³H]choline into phosphatidylcholine. The specific activities of the enzymes of phosphatidylcholine synthesis were assayed under optimal substrate conditions. Growth in the presence of sterol-synthesis inhibitors for 24 h has a significant, but variable, effect on the activity of microsomal and cytosolic cholinephosphate cytidylyltransferase. Inhibition is seen in approximately one-half of the preparations and ranges up to 60%. The degree of inhibition of the enzyme in vitro correlates with an elevation of cytosolic triacylglycerol and phospholipid levels, and is not eliminated by the inclusion of excess stimulatory phospholipids in the assay. The pool sizes of the substrates, cholinephosphate and CTP, are unaffected by cholesterol synthesis inhibition. In contrast to the effects on cholinephosphate cytidylyltransferase, the microsomal enzymes glycerol-3-phosphate acyltransferase and choline phosphotransferase are stimulated 2-fold or more. Choline kinase specific activity was inhibited 2-fold after 24 h of treatment with 25-hydroxycholesterol; however, no effect on this step was observed in vivo. These results indicate that the coordination of cholesterol and phosphatidylcholine synthesis involves regulation at the cytidylyltransferase-catalyzed step.     

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.     

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.     

Diacylglycerol synthesized in vitro from sn-glycerol 3-phosphate and the endogenous diacylglycerol are different substrate pools for the biosynthesis of phosphatidylcholine in rat lung microsomes

In microsomes of rat lung, labeled diacylglycerol was synthesized from sn-[3H]glycerol 3-phosphate, which had been added, and from the endogenous free fatty acids. In these microsomes containing biosynthesized [3H]diacylglycerol as well as endogenous nonlabeled diacylglycerol, the synthesis of phosphatidylcholine was measured from added [14C]CDPcholine. The incorporation of [methyl-14C]choline and of [3H]diacylglycerol into phosphatidylcholine showed an entirely different progress in the time-course of incubation. The 14C label of phosphatidylcholine increased continuously, whereas the 3H label remained constant after 2 min up to the end of the incubation period of 20 min. From this result we concluded that the diacylglycerols, synthesized in vitro from glycerol 3-phosphate over an incubation period of 20 min, constitute a separate substrate pool for the biosynthesis of phosphatidylcholine, and are not mixed with the endogenous diacylglycerol pool.     

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.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

Further evidence for the existence of different diacylglycerol pools of the phosphatidylcholine synthesis in microsomes

Endogenous diacylglycerol and diacylglycerol, synthesized in vitro by glycerol 3-phosphate acylation, are not mixed and represent different substrate pools for the biosynthesis of phosphatidylcholine in microsomes of rat muscle, liver and lung. Freshly isolated lung microsomes contain 12-18 nmol diacylglycerol per mg protein, and incubation with CDPcholine showed a biphasic curve for the synthesis of phosphatidylcholine as lung microsomes enriched in diacylglycerol through the glycerol phosphate pathway. With respect to the synthesis of phosphatidylcholine, a part of this endogenous diacylglycerol (0.4-0.8 nmol/mg) was comparable with diacylglycerol de novo formed in vitro by glycerol 3-phosphate acylation. An increase in the relative proportion of de novo-formed diacylglycerol in the total amount of diacylglycerol caused an increase in phosphatidylcholine synthesis by nearly the same factor. The apparent Km of the de novo-formed diacylglycerol substrate for the choline phosphotransferase was 10-times higher than the pool size of this diacylglycerol substrate in freshly isolated lung microsomes. The results supported the idea that the availability of this substrate type may be rte limiting for the de novo synthesis of phosphatidylcholine. As shown by use of the proteolytic technique measuring the mannose-6-phosphatase as lumenal control activity, the phosphatidylcholine synthesis from de novo-formed diacylglycerol and endogenous as well as exogenous diacylglycerol seems to be located on the cytoplasmic leaflet of the microsomal vesicles isolated from rat lung.     

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.     

Microsomal membranes contain phosphatidylcholine that equilibrates across the bilayer, and phosphatidylcholine that does not

Results of experiments using phosphatidylcholine transfer protein and phospholipase C as probes indicate that there are at least two pools of phosphatidylcholine in rat liver microsomes. One of these is preferentially labelled with [14C]choline and does not equilibrate across the bilayer. The second pool is labelled with [3H]glycerol and does equilibrate across the bilayer. Our observations also confirm that phosphatidylcholine exchange protein does not modify the distribution of phospholipids or cause randomization of the inner and outer leaflet pools of phosphatidylcholine when these are differentially labelled by [14C]choline.     

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids.     

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.     

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.