Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTα spanning residues 1–236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the β-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.     

Characterization of a lipid activated CTP:phosphocholine cytidylyltransferase from Drosophila melanogaster

CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V(max) value of 1352+/-159 nmol CDP-choline/min/mg, a K(m) value of 0.50+/-0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72+/-0.10 mM for CTP. The 6 x -His-tagged enzyme has similar properties with a V(max) value of 2254+/-253 nmol CDP-choline/min/mg, a K(m) value of 0.63+/-0.13 mM for phosphocholine and a K' for CTP equal to 0.81+/-0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.     

Identification and characterization of the nuclear isoform of Drosophila melanogaster CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.     

The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization.

Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.     

CTP:phosphocholine cytidylyltransferase: Function,regulation, and structure of an amphitropic enzyme required for membrane biogenesis

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-limiting and regulated step in the CDP-choline pathway for the synthesis of phosphatidylcholine (PC) and PC-derived lipids. Control of CCT activity is multi-layered, and includes direct regulation by reversible membrane binding involving a built-in lipid compositional sensor. Thus CCT contributes to phospholipid compositional homeostasis. CCT also modifies the curvature of its target membrane. Knowledge of CCT structure and regulation of its catalytic function are relatively advanced compared to many lipid metabolic enzymes, and are reviewed in detail. Recently the genetic origins of two human developmental and lipogenesis disorders have been traced to mutations in the gene for CCTα.     

A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0--22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphocholine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 mumol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.     

Purification and kinetic characterization of CTP:phosphocholine cytidylyltransferase from Saccharomyces cerevisiae

CTP:phosphocholine cytidylyltransferase (CCT) regulates the biosynthesis of phosphatidylcholine in mammalian cells. In order to understand the mechanism by which this enzyme controls phosphatidylcholine synthesis, we have initiated studies of CCT from the model genetic system, the yeast Saccharomyces cerevisiae. The yeast CCT gene was isolated from genomic DNA using the polymerase chain reaction and was found to encode tyrosine at position 192 instead of histidine, as originally reported. Levels of expression of yeast CCT activity in Escherichia coli or in the yeast, Pichia pastoris, were somewhat low. Expression of yeast CCT in a baculovirus system as a 6x-His-tag fusion protein was higher and was used to purify yeast CCT by a procedure that included delipidation. Kinetic characterization revealed that yeast CCT was activated approximately 20-fold by 20 microM phosphatidylcholine:oleate vesicles, a level 5-fold lower than that necessary for maximal activation of rat CCT. The k(cat) value was 31.3 s(-1) in the presence of lipid and 1.5 s(-1) in the absence of lipid. The K(m) values for the substrates CTP and phosphocholine did not change significantly upon activation by lipids; K(m) values in the presence of lipid were 0.80 mM for phosphocholine and 1.4 mM for CTP while K(m) values in the absence of lipid were 1.2 mM for phosphocholine and 0.8 mM for CTP. Activation of yeast CCT, therefore, appears to be due to an increase in the k(cat) value upon lipid binding.     

Identification of lysine 122 and arginine 196 as important functional residues of rat CTP:phosphocholine cytidylyltransferase alpha

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) contains a central region that functions as a catalytic domain, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the subsequent synthesis of phosphatidylcholine. We have investigated the catalytic role of lysine 122 and arginine 196 of rat CCTalpha using site-directed mutagenesis and a baculovirus expression system. Arginine 196 is part of the highly conserved RTEGIST motif, while lysine 122 has not previously been identified by protein sequence alignment as a candidate catalytic amino acid. Removing the side chain of lysine 122 compromises the catalytic ability of CCTalpha, decreasing the apparent V(max) value in mutant enzymes Lys122Ala and Lys122Arg to 0.30 and 0.09% of the wild-type value, respectively. The decrease in V(max) is accompanied by dramatic 471- and 80-fold increases in the apparent K(m) value for phosphocholine but no greater than 3-fold increases in the apparent Hill constant (K*) value for CTP. Mutation of arginine 196 to lysine results in an enzyme that retains 24% of the wild-type V(max) value with a modest 5-fold increase in the K(m) value for phosphocholine. However, the Arg196Lys mutant enzyme exhibits a 23-fold increase in the K* value for CTP. These data suggest lysine 122 and arginine 196 of rat CTP:phosphocholine cytidylyltransferase are functionally important amino acids, perhaps at or near the active site involved in forming contacts with the substrates phosphocholine and CTP, respectively.     

An increase in cytoplasmic CTP accelerates the reaction catalyzed by CTP:phosphocholine cytidylyltransferase in poliovirus-infected HeLa cells

Poliovirus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (Vance, D.E., Trip, E.M., and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069). The mechanism for the virus effect has been investigated. An assay for the cytidylyltransferase which mimics the physiological conditions within the cell was developed. The enzyme activity was not changed at 3 h but was stimulated more than 2-fold at 4 and 5 h after infection with poliovirus. Enzyme activity was stimulated by addition of CTP to the assay. At 0.10 mM CTP the difference in activities from poliovirus- and mock-infected cells was abolished. Mg2+ inhibited the cytidylyltransferase activities and eliminated the differences between the two activities at a concentration of 0.05 mM. However, the endogenous amount of Mg2+ in the postmitochondrial supernatants was the same for infected and mock-infected cells. The addition of CDP-choline or PPi inhibited the cytidylyltransferase activity but had no effect on the relative differences in activities from infected and mock-infected cells. Measurement of CTP in the postmitochondrial fraction showed no differences at 3 h but was elevated 2- to 3-fold in poliovirus-infected cells at 4 and 5 h. It appears that the cytidylyltransferase reaction is faster in poliovirus-infected HeLa cells because of an increase of CTP in the cytoplasmic compartment. Moreover, it appears that the concentration of CTP in the cytoplasm can determine the rate of phosphatidylcholine biosynthesis in HeLa cells.     

The glutathione-binding site in glutathione S-transferases. Investigation of the cysteinyl, glycyl and gamma-glutamyl domains.

In hamster heart, the majority of the phosphatidylcholine is synthesized via the CDP-choline pathway, and the rate-limiting step of this pathway is catalysed by CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15). We have shown previously [Choy (1982) J. Biol. Chem. 257, 10928-10933] that, in the myopathic heart, the level of cardiac CTP was diminished during the development of the disease. In order to maintain the level of CDP-choline, and consequently the rate of phosphatidylcholine biosynthesis, cardiac cytidylyltransferase activity was increased. However, it was not clear if the same compensatory mechanism would occur when the cardiac CTP level was decreased rapidly. In this study, hypoxia of the hamster heart was produced by perfusion with buffer saturated with 95% N2. The heart was pulse-labelled with radioactive choline and then chased with non-radioactive choline for various periods under hypoxic conditions. There was a severe decrease in ATP and CTP levels within 60 min of hypoxic perfusion, with a corresponding fall in the rate of phosphatidylcholine biosynthesis. Analysis of the choline-containing metabolites revealed that the lowered ATP level did not affect the phosphorylation of choline to phosphocholine, but the lower CTP level resulted in the decreased conversion of phosphocholine to CDP-choline. Determination of enzyme activities revealed that hypoxic treatment resulted in the enhanced translocation of cytidylyltransferase from the cytosolic to the microsomal form. This enhanced translocation was probably caused by the accumulation of fatty acids in the heart during hypoxia. We postulate that the enhancement of translocation of the cytidylyltransferase to the microsomal form (a more active form) is a mechanism by which the heart can compensate for the decrease in CTP level during hypoxia in order to maintain phosphatidylcholine biosynthesis.     

The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung.

The reaction catalyzed by CTP:phosphocholine cytidylyltransferase in the reverse direction, i.e. the formation of CTP and phosphocholine from CDP-choline and pyrophosphate, is slightly faster than the reaction in the forward direction. The reverse reaction is optimal at 2 mM pyrophosphate and 6 mM Mg2+, in both fetal and adult preparations. The apparent substrate Km values for phosphocholine, CDP-choline, and pyrophosphate are similar in the fetal and adult forms of the enzyme. The enzyme activity is separated into two forms by gel filtration. The enzyme from adult lung exists as a high molecular weight species, ranging in size from 5 X 10(6) to 50 X 10(6). The enzyme from fetal lung exists as a 190,000 molecular weight species and is totally dependent upon added anionic phospholipid for activity in both the forward and reverse direction. The addition of phosphatidylglycerol gives maximal activity, while phosphatidylinositol or cardiolipin produce about 60 to 70% of the maximal activity. Enzyme activation is accompanied by an aggregation of the enzyme. A sonicated preparation of phosphatidylglycerol is a more efficient activator than a preparation mixed on a Vortex mixer (KA = 30 micronM) and also converts a larger proportion of enzyme from fetal lung into a high molecular weight species. The enzyme from adult lung can be dissociated into a form in fetal lung. The dissociated species can be converted back to a high molecular weight form in the presence of phosphatidylglycerol.     

Regulation of CTP:phosphocholine cytidylyltransferase by amphitropism and relocalization

Phosphatidylcholine (PC) synthesis in animal cells is generally controlled by cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (CCT). This enzyme is amphitropic, that is, it can interconvert between a soluble inactive form and a membrane-bound active form. The membrane-binding domain of CCT is a long amphipathic alpha helix that responds to changes in the physical properties of PC-deficient membranes. Binding of this domain to membranes activates CCT by relieving an inhibitory constraint in the catalytic domain. This leads to stimulation of PC synthesis and maintenance of membrane PC content. Surprisingly, the major isoform, CCT alpha, is localized in the nucleus of many cells. Recently, a new level of its regulation has emerged with the discovery that signals that stimulate PC synthesis recruit CCT alpha from an inactive nuclear reservoir to a functional site on the endoplasmic reticulum.     

Phosphatidylcholine metabolism in ischemic and hypoxic hearts

The rates of phosphatidylcholine biosynthesis in the isolated hamster hearts under ischemic and hypoxic conditions were examined. Global ischemia was produced by perfusion of the heart with a reduced flow, whereas hypoxia was produced by perfusion with a N₂-saturated buffer. A 51% reduction in the biosynthesis of phosphatidylcholine was observed in the ischemic heart. The reduction was caused by a severe decrease in ATP level which resulted in a diminished conversion of choline into phosphocholine. A 22% reduction in the biosynthetic rate of phosphatidylcholine was also detected in the hypoxic heart. The reduction was caused by a diminished level of CTP which resulted in a decreased conversion of phosphocholine to CDP-choline. No compensatory mechanism was triggered during ischemia, but the CTP: phosphocholine cytidylyltransferase activity was enhanced in the hypoxic heart. Our results demonstrate the possible rate-limiting role of choline kinase and reconfirm the regulatory role of the cytidylyltransferase in the biosynthesis of phosphatidylcholine. (Mol Cell Biochem116: 53–58, 1992)     

Caspase processing and nuclear export of CTP:phosphocholine cytidylyltransferase alpha during farnesol-induced apoptosis

CTP:phosphocholine cytidylyltransferase alpha (CCT alpha) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway, the primary route for synthesis of phosphatidylcholine (PtdCho) in eukaryotic cells. Induction of apoptosis by farnesol (FOH) and other cytotoxic drugs has been shown to alter PtdCho synthesis via the CDP-choline pathway. Here we report that FOH-induced apoptosis in CHO cells caused a dose-dependent activation of CCT alpha and inhibition of the final step in the pathway, resulting in a biphasic effect on PtdCho synthesis. Activation of CCT alpha was accompanied by enzyme translocation to the nuclear envelope within 30 min of FOH addition to cells. Following translocation to membranes, CCT alpha was exported from the nucleus and underwent caspase-mediated proteolysis that coincided with poly(ADP-ribose) polymerase cleavage. Site-directed mutagenesis and in vivo and in vitro expression studies mapped a caspase 6 and/or 8 cleavage site to TEED(28 downward arrow)G, the final residue in the CCT alpha nuclear localization signal. Nuclear export of CCT alpha appeared to be an active process in FOH-treated CHO cells that was independent of caspase removal of the nuclear localization signal. Caspase cleavage of CCT alpha occurred during UV or chelerythrine-induced apoptosis; however, nuclear membrane translocation and nuclear export were not evident under these conditions. Thus, caspase cleavage of CCT alpha was a late feature of several apoptotic programs that occurred in the nucleus or at the nuclear envelope. Activation and nuclear export of CCT alpha were early events in FOH-induced apoptosis that contributed to altered PtdCho synthesis and, in conjunction with caspase cleavage, excluded CCT alpha from the nucleus.     

CTP:phosphocholine cytidylyltransferase: insights into regulatory mechanisms and novel functions

A key regulatory enzyme in phosphatidylcholine biosynthesis, CTP:cholinephosphate cytidylyltransferase (CCT), catalyzes the formation of CDP-choline. This review discusses the essential features of CCT and addresses intriguing new insights into the catalytic and regulatory properties of this complex enzyme. Characterization of a lipid-binding segment in rat CCT is described and the role of lipids in CCT activation is discussed. An analysis of the phosphorylation domain is presented and possible physiological rationales for reversible phosphorylation of CCT are discussed. The nuclear localization of CCT is examined in the context of multiple CCT isoforms, as is recent evidence establishing a potential link between CCT activity and vesicular transport.     

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.     

Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells

The production and characterization of an antibody to rat liver CTP:phosphocholine cytidylyltransferase is described. This antibody quantitatively precipitated cytidylyltransferase from both rat liver and HeLa cell cytosol. Following affinity purification, the antibody was used to demonstrate, for the first time, the phosphorylation of cytidylyltransferase in vivo. Following the immunoprecipitation of cytidylyltransferase from HeLa cells, acid hydrolysis, and thin layer electrophoresis of the amino acids, only [32P]phosphoserine was detected. The phosphorylation state of cytidylyltransferase in HeLa cells was examined following treatment with phorbol ester for 1 h. In agreement with previous studies, the incorporation of [3H]choline into phosphatidylcholine via the CDP-choline pathway was stimulated 5-fold in cultures of HeLa cells following treatment with phorbol ester for 1 h. However, no appreciable translocation of cytidylyltransferase was detected, despite the utilization of two different methods of cell lysis. Furthermore, the inclusion of phosphatase inhibitors and chelators of divalent cations in the homogenization buffers had no effect on the observed distribution or activity of the enzyme. Immunoprecipitated cytidylyltransferase was phosphorylated to the same extent, and on serine residues only, in both control and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated cells. Measurement of the pool sizes of the aqueous intermediates of the CDP-choline pathway, following TPA treatment, revealed a modest decrease in the phosphocholine pool only, consistent with an activation of cytidylyltransferase.     

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.     

CDP-choline significantly restores phosphatidylcholine levels by differentially affecting phospholipase A2 and CTP: phosphocholine cytidylyltransferase after stroke

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTalpha protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 +/- 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTalpha after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.