Phosphatidylcholine metabolism in cultured cells: catabolism via glycerophosphocholine

The catabolism of phosphatidylcholine (PtdCho) has been studied in cultured murine neuroblastoma (N1E-115), C6 glioma, rat brain primary glia, and human fibroblast cells. Cells were pulse labelled for 96 h with [methyl-3H]choline followed by a chase for up to 24 h in medium containing 4 mM choline. Measurement of the radioactivity and mass of choline-containing compounds in these cells indicated that the major degradative pathway is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----choline. At all times during the chase, PtdCho, sphingomyelin and lysoPtdCho comprised 72-92% of the cell-associated radioactivity; the remaining 10-30% was water-soluble and was chiefly GroPCho (30-80%) in all cell lines. In fibroblasts, however, phosphocholine (PCho) was also a major labelled water-soluble component (33-54%). The specific activity of GroPCho closely parallelled that of PtdCho in fibroblasts, but decreased faster than PtdCho in C6 and N1E-115 cells. We postulate that this may be due to distinct pools of PtdCho in the cell with differing rates of turnover. The changes in specific activity of PCho suggest that the major portion is formed by synthesis rather than as a degradative product. However, the inability to reduce the specific activity of this fraction to that of the intracellular choline suggests that a portion may be derived from either PtdCho or GroPCho.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Phosphatidylcholine catabolism in the MCF-7 cell cycle.

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.     

Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells

Eukaryotic cells control the levels of their major membrane lipid, phosphatidylcholine (PtdCho), by balancing synthesis with degradation via deacylation to glycerophosphocholine (GroPCho). Here we present evidence that in both yeast and mammalian cells this deacylation is catalyzed by neuropathy target esterase (NTE), a protein originally identified by its reaction with organophosphates, which cause nerve axon degeneration. YML059c, a Saccharomyces cerevisiae protein with sequence homology to NTE, had similar catalytic properties to the mammalian enzyme in assays of microsome preparations and, like NTE, was localized to the endoplasmic reticulum. Yeast lacking YML059c were viable under all conditions examined but, unlike the wild-type strain, did not convert PtdCho to GroPCho. Despite the absence of the deacylation pathway, the net rate of [(14)C]choline incorporation into PtdCho in YML059c-null yeast was not greater than that in the wild type; this was because, in the null strain diminished net uptake of extracellular choline and decreased formation of the rate-limiting intermediate, CDP-choline, resulted in a reduced rate of PtdCho synthesis. In [(14)C]choline labeling experiments with cultured mammalian cell lines, production of [(14)C]GroPCho was enhanced by overexpression of catalytically active NTE and was diminished by reduction of endogenous NTE activity mediated either by RNA interference or organophosphate treatment. We conclude that NTE and its homologues play a central role in membrane lipid homeostasis.     

Effects of choline deficiency and methotrexate treatment upon rat liver

Choline deficiency and treatment with methotrexate (MTX) both are associated with fatty infiltration of the liver. Choline, methionine, and folate metabolism are interrelated and converge at the regeneration of methionine from homocysteine. MTX perturbs folate metabolism, and it is possible that it also influences choline metabolism. We fed rats a choline deficient diet for 2 weeks and/or treated them with methotrexate (MTX; 0.1 mg/kg daily). Choline deficiency lowered hepatic concentrations of choline (to 43% control), phosphocholine (PCho; to 18% control), glycerophosphocholine (GroPCho; to 46% control), betaine (to 30% control), phosphatidylcholine (PtdCho; to 62% control), methionine (to 80% control), and S-adenosylmethionine (AdoMet; to 57% control), while S-adenosylhomocysteine (AdoHcy) and triacylglycerol concentrations increased (to 126% and 319% control, respectively). MTX treatment alone lowered hepatic concentrations of PCho (to 48% control), GroPCho (to 69% control), betaine (to 55% control), and AdoMet (to 75% control). The addition of MTX treatment to choline deficiency resulted in a larger decrease in AdoMet concentrations (to 75% control) and larger increases in AdoHcy and triacylglycerol concentrations (to 150% and 500% control, respectively) than was observed in choline deficiency alone. Livers from MTX-treated animals used radiolabeled choline to make the same metabolites as did livers from controls (most of the label was converted to PCho and betaine). In choline deficient animals, most of the labeled choline was converted to PtdCho. Therefore, MTX depleted hepatic PCho, GroPCho, and betaine by a mechanism that was different from that of choline deficiency. MTX increased the extent of fatty infiltration of the liver in choline deficient rats, and choline deficiency and MTX treatment damaged hepatocytes as measured by leakage of alanine aminotransferase activity. Our data are consistent with the hypothesis that the fatty infiltration of the liver associated with MTX treatment occurs because of a disturbance in choline metabolism.     

Glycerophosphocholine catabolism as a new route for choline formation for phosphatidylcholine synthesis by the Kennedy pathway

In eukaryotes, neuropathy target esterase (Nte1p in yeast) deacylates phosphatidylcholine derived exclusively from the CDP-choline pathway to produce glycerophosphocholine (GroPCho) and release two fatty acids. The metabolic fate of GroPCho in eukaryotic cells is currently not known. Saccharomyces cerevisiae contains two open reading frames predicted to contain glycerophosphodiester phosphodiesterase domains, YPL110c and YPL206c. Pulse-chase experiments were conducted to monitor GroPCho metabolic fate under conditions known to alter CDP-choline pathway flux and consequently produce different rates of formation of GroPCho. From this analysis, it was revealed that GroPCho was metabolized to choline, with this choline serving as substrate for renewed synthesis of phosphatidylcholine. YPL110c played the major role in this metabolic pathway. To extend and confirm the metabolic studies, the ability of the ypl110cDelta and ypl206cDelta strains to utilize exogenous GroPCho or glycerophosphoinositol as the sole source of phosphate was analyzed. Consistent with our metabolic profiling, the ypl206cDelta strain grew on both substrates with a similar rate to wild type, whereas the ypl110cDelta strain grew very poorly on GroPCho and with moderately reduced growth on glycerophosphoinositol.     

Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells

PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis.     

Neuropathy target esterase and phospholipid deacylation

Certain organophosphates react with the active site serine residue of neuropathy target esterase (NTE) and cause axonal degeneration and paralysis. Cloning of NTE revealed the presence of homologues in eukaryotes from yeast to man and that the protein has both a catalytic and a regulatory domain. The latter contains sequences similar to the regulatory subunit of protein kinase A, suggesting that NTE may bind cyclic AMP. NTE is tethered via an amino-terminal transmembrane segment to the cytoplasmic face of the endoplasmic reticulum. Unlike wild-type yeast, mutants lacking NTE activity cannot deacylate CDP-choline pathway-synthesized phosphatidylcholine (PtdCho) to glycerophosphocholine (GroPCho) and fatty acids. In cultured mammalian cells, GroPCho levels rise and fall, respectively, in response to experimental over-expression, and inhibition, of NTE. A complex of PtdCho and Sec14p, a yeast phospholipid-binding protein, both inhibits the rate-limiting step in PtdCho synthesis and enhances deacylation of PtdCho by NTE. While yeast can maintain PtdCho homeostasis in the absence of NTE, certain post-mitotic metazoan cells may not be able to, and some NTE-null animals have deleterious phenotypes. NTE is not required for cell division in the early mammalian embryo or in larval and pupal forms of Drosophila, but is essential for placenta formation and survival of neurons in the adult. In vertebrates, the relative importance of NTE and calcium-independent phospholipase A2 for homeostatic PtdCho deacylation in particular cell types, possible interactions of NTE with Sec14p homologues and cyclic AMP, and whether deranged phospholipid metabolism underlies organophosphate-induced neuropathy are areas which require further investigation.     

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.     

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.     

Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis

The C3H/10T1/2 Cl8 HAbetaC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAbetaC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of alpha/betaPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAbetaC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAbetaC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [(14)C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between alpha/betaPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.     

Choline derived from the phosphatidylcholine specific phospholipase D is not directly available for the CDP choline pathway in phorbol ester- treated C3H10T1/2 C18 fibroblasts

We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [³H]/[¹⁴C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.     

Vasoactive intestinal polypeptide stimulates cyclic AMP production in mouse N1E-115 neuroblastoma cells: Modulation by a protein kinase C activator and ionomycin

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca²⁺ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4β-phorbol 12-myristate 13-acetate reduced VIP-but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca²⁺ potentiates this signaling pathway.     

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.     

Levels of choline intermediates in the visual system structures and in peripheral nerve of the rat: Comparison with neural tissues of a lower vertebrate (Mustelus canis) and an invertebrate (Loligo pealei)

The levels of choline intermediate endogenous pools in structures of the visual system (retina, optic nerve, lateral geniculate body, superior colliculus) and in sciatic nerve of adult (4-month-old) and young (30-day-old) rats were measured. The amounts were also obtained from retina, optic nerve, optic tectum and cranio-spinal nerves of a primitive elasmobranch, the smooth dogfish Mustelus canis, and from related nervous structures (retina, optic lobe, fin nerve, stellar nerve and stellate ganglia) of a marine invertebrate, the squid Loligo pealei. In all regions of rat nervous system, the pool size of CDP-choline was much smaller than that of free choline, whereas GroPCho was present in a relatively higher content. The pool sizes of choline intermediates in 30- and 120-day-old rats were nearly the same. In nervous system regions of the dogfish and squid, the values followed the same general trend as observed for rat. Squid nervous tissues had the lowest choline and GroPCho contents. The rat retina showed the lowest glycerophosphorylcholine phosphodiesterase activity. The chemical studies described here confirm the basic similarity in the pattern of choline intermediate pool sizes among animal species widely different in phylogenetic position. The data highly reinforce the idea that the precursor role of choline and catabolic pathways for the maintenance of the PtdCho membraneous pool seem highly conserved during evolution.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

L-alpha-glycerylphosphorylcholine inhibits the transfer function of phosphatidylinositol transfer protein alpha

Phosphatidylinositol transfer protein alpha (PITP-alpha) is a bifunctional phospholipid transfer protein that is highly selective for phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho). Polar lipid metabolites, including L-alpha-glycerylphosphorylcholine (GroPCho), increasingly have been linked to changes in cellular function and to disease. In this study, polar lipid metabolites of PtdIns and PtdCho were tested for their ability to influence PITP-alpha activity. GroPCho inhibited the ability of PITP-alpha to transfer PtdIns or PtdCho between liposomes. The IC(50) of both processes was dependent on membrane composition. D-myo-inositol 1-phosphate and glycerylphosphorylinositol modestly enhanced PITP-alpha-mediated phospholipid transfer. Choline, phosphorylcholine (PCho), CDP-choline, glyceryl-3-phosphate, myo-inositol and D-myo-inositol 1,4,5-trisphosphate had little effect. Membrane surface charge was a strong determinant of the GroPCho inhibition with the inhibition being greatest for highly anionic membranes. GroPCho was shown to enhance the binding of PITP-alpha to anionic vesicles. In membranes of low surface charge, phosphatidylethanolamine (PtdEtn) was a determinant enabling the GroPCho inhibition. Anionic charge and PtdEtn content appeared to increase the strength of PITP-alpha-membrane interactions. The GroPCho-enhanced PITP-alpha-membrane binding was sufficient to cause inhibition, but not sufficient to account for the extent of inhibition observed. Processes associated with strengthened PITP-alpha-membrane binding in the presence of GroPCho appeared to impair the phospholipid insertion/extraction process.     

Kinetics of the in vivo labeling of the acyl groups of rabbit lung phosphatidylcholine and disaturated phosphatidylcholine.

The kinetics of labeling of lung phosphatidylcholine and disaturated phosphatidylcholine were studied for periods from 0.75--120 min following intravenous injection of radiolabeled palmitic acid and choline into 3-day-old rabbits. The labeled palmitic acid was cleared rapidly from plasma, and rapidly appeared with identical incorporation kinetics in both phosphatidylcholine and disaturated phosphatidylcholine. The 2-acyl positions of both phosphatidylcholine and disaturated phosphatidylcholine were labeled preferentially soon after [14C]palmitic acid injection. The specific activities of palmitic acid in the 2-acyl positions of phosphatidylcholine and disaturated phosphatidylcholine 0.75 min after injection of labeled palmitic acid were 3.4 and 1.9 times, respectively, the specific activities of palmitic acid in the 1-acyl positions. By 120 min the label had randomized between the 1-acyl and 2-acyl positions, and the kinetics of that randomization were defined for both phosphatidylcholine and disaturated phosphatidylcholine. Choline did not pulse label lung phosphatidylcholine or disaturated phosphatidylcholine. The choline label appeared with equal specific activities in both phosphatidylcholine and disaturated phosphatidylcholine. Thus no analysis of the de novo synthesized product via the CDP-choline pathway was possible.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.