Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.     

Effect of sodium chloride concentration on fluid-phase assembly and stability of the C3 convertase of the classical pathway of the complement system.

The specificity of the phospholipid head-group for feedback regulation of CTP: phosphocholine cytidylyltransferase was examined in rat hepatocytes. In choline-deficient cells there is a 2-fold increase in binding of cytidylyltransferase to cellular membranes, compared with choline-supplemented cells. Supplementation of choline-deficient cells with choline, dimethylethanolamine, monomethylethanolamine or ethanolamine resulted in an increase in the concentration of the corresponding phospholipid. Release of cytidylyltransferase into cytosol was only observed in hepatocytes supplemented with choline or dimethylethanolamine. The apparent EC50 values (concn. giving half of maximal effect) for cytidylyltransferase translocation were similar for choline and dimethylethanolamine (25 and 27 microM respectively). The maximum amount of cytidylyltransferase released into cytosol with choline supplementation (1.13 m-units/mg membrane protein) was twice that (0.62) observed with dimethylethanolamine. Supplementation of choline-deficient hepatocytes with NN'-diethylethanolamine, N-ethylethanolamine or 3-aminopropanol also did not cause release of cytidylyltransferase from cellular membranes. The translocation of cytidylyltransferase appeared to be mediated by the concentration of phosphatidylcholine in the membranes and not the ratio of phosphatidylcholine to phosphatidylethanolamine. The results provide further evidence for feedback regulation of phosphatidylcholine biosynthesis by phosphatidylcholine.     

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.     

Induction of phosphatidylcholine biosynthesis via CDPcholine pathway in lung and liver of rats following intratracheal administration of DDT and endosulfan

The induction of phosphatidylcholine (PC) biosynthesis via the CDPcholine pathway in lung and liver of rats has been shown following the intratracheal administration of 1,1,1-trichloro-2m2-bis(p-chlorophenyl) ethane (DDT) (5 mg/100 g body weight) and endosulfan (1 mg/100 g body weight) for 3 days. Controls received only the vehicle solution (groundnut oil, 0.1 m1/100 g body weight). The treatment of DDT and endosulfan significantly increased the PC contents and the incorporation of radioactive [methyl-3H]choline into PC of lung and liver microsomes. The incorporation of radioactive [methyl-14C]methionine into microsomal PC of lung and liver was not affected significantly by treatment with either of the insecticides. 1,4,5,6,7-hexachloro-5-norbornene-2,3-dimethano cyclic sulfite (endosulfan) administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal) of lung, whereas DDT increased the activity of only latter. In liver, both DDT and endosulfan administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal). However, the activity of phosphocholinetransferase was not affected in both lung and liver microsomes of rats treated with these insecticides. The PC precursor pool sizes, choline and phosphorylcholine, of lung and liver tissues were not altered by DDT and endosulfan treatments. The present results suggest that the increased level of PC and incorporation of radioactive [methyl-3H]choline into microsomal PC could be the result of increased activity of choline kinase and phosphocholine cytidylyltransferase of lung and liver of rats following intratracheal administration of DDT and endosulfan.     

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Trifluoperazine and other anaesthetics inhibit rat liver CTP: phosphocholine cytidylyltransferase

Chlorpromazine (25 microM) and trifluoperazine (25 microM) inhibited by 5-fold the activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme for phosphatidylcholine biosynthesis, in rat liver cytosol. Addition of saturating amounts of rat liver phospholipid to the enzyme assay rapidly reversed the drug-mediated inhibition. Three-fold or greater concentrations of these drugs were required to produce a 50% inhibition of the microsomal cytidylyltransferase. Incubation of rat hepatocytes with 20 microM trifluoperazine or chlorpromazine did not inhibit phosphatidylcholine biosynthesis. These results provide additional evidence for the hypothesis that the active form of cytidylyltransferase is on the endoplasmic reticulum and the enzyme in cytosol appears to be latent.     

Regulation of phosphatidylcholine biosynthesis in mammalian cells. II. Effects of phospholipase C treatment on the activity and subcellular distribution of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary and LM cell lines

CTP:phosphocholine cytidylyltransferase was located in both the cytosolic and particulate fractions from Chinese hamster ovary cells. The activity of the cytosolic form of the enzyme was greatly enhanced by incubation with sonicated preparations of several different lipids, although incubations with either phosphatidylcholine or 1,2-sn-diolein did not increase activity. The activation of the cytidylyltransferase in Chinese hamster ovary cells treated with phospholipase C from Clostridium perfringens occurred with a concomitant shift in the subcellular distribution of the enzyme from cytosolic to particulate fractions. This shift was rapid and did not require protein synthesis. Removal of phospholipase C from the cell cultures resulted in a return to basal levels of incorporation of [3H]choline into phosphatidylcholine, a decrease in the activity of cytidylyltransferase, and a loss of the membrane-bound form of the enzyme. Similar experiments with LM cells, which are resistant to exogenous phospholipase C, showed no change in subcellular distribution of cytidylyltransferase, suggesting that the activation of CTP:phosphocholine cytidylyltransferase required a change in membrane phospholipid composition. The results presented are discussed in terms of a mechanism of regulation of phosphatidylcholine production involving monitoring of membrane phospholipid composition.     

Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.     

Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells.

The mechanism of the inhibition of phosphatidylcholine biosynthesis by the phospholipid analogue, hexadecylphosphocholine, was investigated in Madin-Darby canine kidney cells. In the presence of 50 mumol/liter hexadecylphosphocholine, there was a translocation of CTP:choline-phosphate cytidylyltransferase (EC 22.7.7.15) activity from the membranes to the cytosol of the cells. Since we recently demonstrated that hexadecylphosphocholine also inhibits protein kinase C in vitro, [methyl-3H]choline labeling experiments were repeated with phorbol ester-desensitized cells. In these cells the same inhibitory effect of hexadecylphosphocholine was measured. As a consequence of inhibition, the [methyl-3H]choline incorporation into the phosphocholine pool was increased time-dependently. In addition, there was no evidence for a difference between the choline uptake of control and hexadecylphosphocholine-treated cells. Likewise, the amount of diacylglycerol, a known activator of the translocation process, was not reduced. Finally, we showed that the inhibitory effect of hexadecylphosphocholine on CTP:choline-phosphate cytidylyltransferase translocation cannot be explained by the detergent properties of this phospholipid analogue. Therefore, we suggest a direct inhibitory effect of hexadecylphosphocholine on the translocation of CTP:choline-phosphate cytidylyltransferase.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) suggested that phospholipase A was activated in these cells. These results indicate that the ras oncogene product directly or indirectly causes an increased turnover of phosphatidylcholine in C3H10T1/2 cells.     

Immunological studies on CTP:phosphocholine cytidylyltransferase from the livers of normal and choline-deficient rats

Chickens were immunized with the purified low-molecular-weight form of CTP:phosphocholine cytidylyltransferase from rat liver cytosol. The antiserum was obtained and fractionated to yield immunoglobulin. The antibodies specifically inhibited the enzymatic activity of the partially purified low-molecular-weight form of the enzyme from pH 6.0 to 8.5. Antibodies against the low-molecular-weight form of the enzyme cross-reacted with the high-molecular-weight form of the enzyme from cytosol as well as with the cytidylyltransferase associated with the microsomal fraction. The antibodies were used for the immunochemical determination of the amount of cytosolic phosphocholine cytidylyltransferase in the livers of normal and choline-deficient rats. The amount of enzyme in rat liver cytosol was not changed for at least 18 days of choline deficiency. The decrease in specific activity of the enzyme in choline-deficiency may be caused by factors other than adaptive changes in the level of enzyme.     

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.     

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.     

c-Ha-ras oncogene expression increases choline uptake,CTP:prosphocholine cytidylyltransferase activity and phosphatidylcholine biosynthesis in the immortalized human keratinocyte cell line HaCaT

The effect of c-Ha-ras transfection on phosphatidylcholine biosynthesis of the keratinocyte cell line HaCaT was investigated. It was shown that ras-transfection caused a 3-fold increase of choline incorporation into phosphatidylcholine. By investigating the mechanisms underlying this phenomenon, two targets were obtained. First, the choline uptake was elevated by 2-fold in ras-transfected HaCaT cells as compared with untransfected HaCaT cells, and second, the activity of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, was increased by 43%. Stimulation of HaCaT cells and ras-transfected HaCaT cells with oleate revealed that the increased activity of cytidylyltransferase might be due to a higher level of enzyme. In these experiments, a 75% increase of the specific activity of fully stimulated, membrane-bound cytidylyltransferase was found in ras-transfected HaCaT cells. Choline kinase which has been previously descrived as a target of ras-transfection in fibroblasts was unaffected.     

Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis

Choline kinase alpha (CK-alpha) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka(+/-)) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by approximately 30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka(+/-) mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka(+/-) mice. Triacylglycerol and cholesterol ester were elevated approximately 2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka(+/-) and wild-type (Chka(+/+)) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.     

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.