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.     

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.     

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.     

Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase.

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.     

Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells.

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.     

Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

The effect of lidocaine on [³H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3·2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0·0005) or 55 per cent (n = 10; p < 0·0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [³H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until ³H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.     

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.