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.     

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. II. Its relation to net phosphatidylcholine biosynthesis

The effect of a single dose (50 mg/kg body weight) of 3-methylcholanthrene on de novo phosphatidylcholine biosynthetic activities in rat liver was studied both in a cell-free system and with slice experiments. 3-Methylcholanthrene caused a significant depression of either [methyl-14C]choline or [2-(3)H]glycerol incorporation into phosphatidylcholine when the precursor was incubated with liver slices. At the same time, there occurred a significant accumulation of radioactivity in either cholinephosphate or diacylglycerol molecule from [14C]choline or [3H]glycerol, respectively, suggesting that 3-methylcholanthrene could cause an inhibitory effect on hepatic phosphatidylcholine synthesis at the cholinephosphotransferase or/and cholinephosphate cytidylyltransferase step. Subsequent studies, where the activities of the three enzymes involved in de novo phosphatidylcholine synthesis were compared between control and 3-methylcholanthrene-pretreated rat liver subcellular fractions, demonstrated that the cholinephosphotransferase step could be the site of inhibition by 3-methylcholanthrene. On the other hand, 3-methylcholanthrene caused a significant induction of choline kinase activity in a time-dependent manner and, at the same time, the cholinephosphate pool size in liver cytosol was enlarged 2-3-fold when compared to the respective control. The overall results suggested strongly that 3-methylcholanthrene causes the counteractive effects on the de novo phosphatidylcholine biosynthesis, induction of choline kinase activity and inhibition of cholinephosphotransferase activity, both of which could participate in a concomitant increase in cholinephosphate pool size in rat liver.     

Phosphatidylglycerol stimulates cholinephosphate cytidylyltransferase activity and phosphatidylcholine synthesis in type II pneumocytes

Phosphatidylcholine synthesis in type II pneumocytes is stimulated by inclusion of phosphatidylglycerol and other phospholipids in the culture medium (Gilfillan, A.M., Chu, A.J. and Rooney, S.A. (1984) Biochim. Biophys. Acta 794, 269-273). We have now examined the effect of phosphatidylglycerol in the medium on enzymes of de novo phosphatidylcholine synthesis in adult rat type II cells. Activities of choline kinase, cholinephosphate cytidylyltransferase and cholinephosphotransferase in homogenates of whole lung and type II cells were generally similar. Phosphatidate phosphatase activity in type II cells, however, was only 16% that in whole lung. Addition of phosphatidylglycerol (10 microM) to the culture medium had no effect on choline kinase, cholinephosphotransferase or phosphatidate phosphatase activities in type II cells but it increased the activity of cholinephosphate cytidylyltransferase by 56%. Since it is known that cholinephosphate cytidylyltransferase is stimulated in vitro by addition of phospholipids to the assay mixture, we also measured its activity in the presence of sufficient phosphatidylglycerol (1.1 mM) to maximally stimulate in vitro. Even under these conditions cholinephosphate cytidylyltransferase activity in type II cells cultured in the presence of phosphatidylglycerol was 32% greater than in control cells. These data show that the stimulatory effect of phospholipid in the culture medium on phosphatidylcholine synthesis in type II cells is mediated by increased cholinephosphate cytidylyltransferase activity. The mechanism of increased cytidylyltransferase activity remains to be elucidated but it is not due to direct in vitro activation by the phospholipid.     

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.     

Effect of 1-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes

We previously reported that addition of phosphatidylglycerol to the culture medium stimulates phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in type II pneumocytes. In view of the known biological effects of diacylglycerols and since phosphatidylglycerol could be metabolized to diacylglycerol, we now examined the effects of diacylglycerols on the same parameters. The rate of choline incorporation into phosphatidylcholine was increased 30-60% by 10 microM phosphatidylglycerol, diolein, mixed diacylglycerols and 1-oleoyl-2-acetylglycerol (OAG). The effects of phosphatidylglycerol and OAG were not additive, suggesting a similar mechanism of action. The diacylglycerols and phosphatidylglycerol increased the activity of cholinephosphate cytidylyltransferase in type II cell sonicates by 35-50%, but had no effect on the activities of choline kinase, cholinephosphotransferase or 1-acylglycerophosphocholine acyltransferase. Again, the effects of OAG and phosphatidylglycerol on cytidylyltransferase were not additive. It is known that addition of lipids to the assay mixture increases the activity of cholinephosphate cytidylyltransferase in vitro and inclusion of the above lipids (1.1 mM) in the in vitro assay mixture increased cytidylyltransferase activity in type II cell sonicates. In addition, the stimulatory effects of OAG and of diolein, as well as of phosphatidylglycerol as reported previously, in the culture medium on cytidylyltransferase activity in type II cells were diminished or abolished when the assay was carried out in the presence of sufficient amounts of the same lipids to stimulate maximally the activity in vitro. These data show that lipids in the culture medium stimulate phosphatidylcholine biosynthesis in type II cells by direct activation of cholinephosphate cytidylyltransferase.     

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.     

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.     

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50 nM of lovastatin for 60 h, lovastatin was found to decrease the incorporation of [³H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [³H]sphingomyelin by lovastatin. The increase of [³H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: II. Effects of sterol biosynthesis inhibitors on cell division

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity — 25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9–16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G₂M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.     

Evidence for a regulatory role of CTP : choline phosphate cytidylyltransferase in the synthesis of phosphatidylcholine in fetal lung following premature birth

The sequence of reactions which function to incorporate choline into phosphatidylcholine was investigated in lung from fetuses following premature delivery. The rate of [methyl-14C]choline incorporation by rat lung slices into phosphatidylcholine increases following premature delivery at both 20 and 21 days gestation. The increase in choline incorporation is primarily due to an increased specific activity of phosphorylcholine resulting from a decreased pool size of phosphorylcholine. The decrease in the concentration of phosphorylcholine following premature delivery is apparently caused by an increased activity of cytidylyltransferase which leads to an increase in the conversion of phosphorylcholine to phosphatidylcholine. The total activity of choline kinase, cytidylyltransferase, cholinephosphotransferase and phosphatidate phosphohydrolase did not change significantly. However, the cytidylyltransferase activity in the microsome fraction increased following premature delivery at 20 and 21 days gestation. The amount of cytidylyltransferase in the H form in the cytosol fraction increased following premature delivery at 21 days gestation but not at 20 days gestation. The results are interpreted to indicate that the active form of cytidylyltransferase in lung cells is the membrane-bound enzyme and this form increases following birth resulting in an increased synthesis 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.     

Coordinate increases in the enzyme activities responsible for phosphatidylglycerol synthesis and CTP:cholinephosphate cytidylyltransferase activity in developing rat lung

The enzymes responsible for the biosynthesis of phosphatidylglycerol, CTP:phosphatidate cytidylyltransferase, CDP-diacylglycerol: glycerophosphate phosphatidyltransferase and phosphatidylglycerophosphate phosphatase demonstrated a coordinate increase in activity in fetal rat lung at term when the demand for pulmonary surfactant increases. The activity of CTP:cholinephosphate cytidylyltransferase, the enzyme responsible for CDP-choline production also increased in the perinatal period. The activity of cholinephosphate cytidylyltransferase in fetal and neonatal cytosol was stimulated by the addition of phosphatidylglycerol but no effect was noted with cytosol from adult lung. These results are consistent with the suggestion that the activity of cholinephosphate cytidylyltransferase, a potential rate-determining enzyme in pulmonary phosphatidylcholine synthesis, may be regulated in the perinatal period both through an activation by phosphatidylglycerol and by an increase in total enzyme units.     

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.     

Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.     

Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

A previous study showing that ethanol (ETOH) blocked [³H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [³H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 μM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing /reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.     

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

The capacity of the sarcoplasmic reticulum for phospholipid synthesis: a developmental study

The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes wee undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was 8-fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.     

Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase alpha translocation to the nuclear envelope

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.