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Biomedical subjects

D E Vance

Publications and source records attributed to D E Vance.

At least 127 records · Page 7Linked to original sources
PubMed1986

The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells.

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.

Acyl Coenzyme A↗
PubMed1985

Evaluation of possible mechanisms of phorbol ester stimulation of phosphatidylcholine synthesis in HeLa cells.

Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12,13-dibutyrylphorbol acetate, markedly stimulate the synthesis of phosphatidylcholine (PC) and the activity of CTP:phosphocholine cytidylyltransferase (CT) in cultured HeLa cells. Two possible mechanisms whereby the phorbol esters stimulated PC biosynthesis were investigated. One consideration was that phorbol esters may induce the release of fatty acyl chains from endogenous complex lipids; increased fatty acids or fatty acyl-CoAs could cause the translocation of CT from cytosol to microsomes and thereby increase the activity of the rate-limiting enzyme in PC synthesis. In HeLa cells prelabeled with [3H]oleate or [3H]arachidonate, phorbol ester treatment increased the redistribution of arachidonate in phospholipids and neutral lipids and release of label to the medium, but there was little effect on the cellular fatty acid pools with either of the labeled fatty acids or of the phorbol esters. A second possibility was that protein kinase C (PKC), a receptor for phorbol esters, might be involved in activation of CT activity. TPA stimulated the phosphorylation of cytosolic proteins of HeLa cells more than twofold during a 10- or 60-min incubation with 32Pi. However, an approximate sixfold purified preparation of PKC from rat brain did not stimulate the activity of partially purified (12- to 15-fold) CT; a slight inhibition, dependent on ATP but independent of Ca2+ and diolein, was observed. Our results suggest that intracellular release of free fatty acids or direct phosphorylation of CT by PKC probably do not account for the observed levels of stimulation by phorbol esters. Other possible mechanisms are discussed.

Choline-Phosphate Cytidylyltransferase↗
PubMed1985

The role of phosphatidylcholine biosynthesis in the secretion of lipoproteins from hepatocytes.

An investigation of the role of phospholipids in lipoprotein assembly and secretion is important since phospholipids, particularly phosphatidylcholine, are prominent components of all plasma lipoproteins. The fatty acid composition of phosphatidylcholine is virtually identical in human very low (VLDL), low, and high density lipoproteins, which supports the idea that phosphatidylcholine exchanges freely among plasma lipoproteins. However, the fatty acid composition of phosphatidylcholine from cultured rat hepatocytes is different from that in the secreted lipoproteins. In addition, the composition of molecular species of phosphatidylcholine is quite different in the rat liver, plasma, and red cells. Phosphatidylcholine is made in liver by two alternate pathways, by the CDP-choline pathway and by the methylation of phosphatidylethanolamine. Regulation of phosphatidylcholine biosynthesis by the CDP-choline pathway in rat liver is well established. In most instances, the rate of phosphatidylcholine synthesis is governed by the activity of CTP:phosphocholine cytidylyltransferase, which is present in the cytosol and also associated with microsomes. The cytosolic enzyme is inactive but can be reversibly translocated to the microsomes, where it is active. Translocation of this enzyme to the microsomes can be achieved either by a dephosphorylation reaction or by the presence of fatty acids in the cytosol. Once synthesized, how is phosphatidylcholine assembled into lipoprotein particles? The sequence of assembly of phospholipids into VLDL has been investigated in several studies. In a pulse-chase experiment, there was an initial labelling (within 15 min of the pulse) of phospholipids in secreted VLDL, which probably reflected the rapid movement of the phospholipids from their site of synthesis (the endoplasmic reticulum) to the Golgi. There appears to be a rapid exchange of phospholipid between the Golgi membranes and contents. There was also a delayed labelling (after 30 min) of the phospholipids and triacylglycerols (from [3H]glycerol) and the apoproteins (from [3H]leucine) in the secreted VLDL. This lag was attributed to the time taken for the nascent VLDL particles to move from the lumen of the endoplasmic reticulum to the Golgi and into the medium. Is phosphatidylcholine biosynthesis is required for lipoprotein secretion? This question was investigated in rats maintained on a choline-deficient diet for 10 days. The total amount of plasma phosphatidylcholine decreased by approximately 40% and the rats developed fatty livers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Membrane-bound CTP:phosphocholine cytidylyltransferase regulates the rate of phosphatidylcholine synthesis in HeLa cells treated with unsaturated fatty acids.

The influence of fatty acids on phosphatidylcholine biosynthesis in HeLa cell cultures was investigated. Oleate and other unsaturated fatty acids stimulated the incorporation of phospho[Me-3H]choline into phosphatidylcholine from 5 to 20-fold, while saturated fatty acids were without effect. Stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) by 1 mM oleate was evident within 15 min and could be reversed within 40 min after removal of the oleate-supplemented cell medium. Cytidylyltransferase activity was 11-fold higher in homogenates from cells exposed to oleate. Treatment of the HeLa cells with oleate produced almost complete translocation of the cytidylyltransferase from the cytosol to the microsomal fraction. Additional support for conversion of the cytidylyltransferase to a membrane-bound form in oleate-treated cells was obtained in studies with digitonin. Exposure of control cells to digitonin for 2 min caused the release of 60% of the total cytidylyltransferase into the medium, while oleate-treated cells leaked only 5% of the enzyme in the presence of digitonin. Finally, oleate (50 microM) was shown to promote complete aggregation of cytosolic cytidylyltransferase with microsomes from HeLa cells and 22-fold stimulation of the enzyme activity. It appears that the rate of phosphatidylcholine biosynthesis is governed by the amount of cytidylyltransferase bound to endoplasmic reticulum in HeLa cells.

Choline↗
PubMed1984

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.

Chlorpromazine↗
PubMed1984

Phorbol esters stimulate phosphatidylcholine biosynthesis by translocation of CTP:phosphocholine cytidylyltransferase from cytosol to microsomes.

Previous studies have demonstrated that 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates phosphatidylcholine biosynthesis in HeLa cells. The stimulation was apparently caused by an acceleration of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) (Paddon, H.B. and Vance, D.E. (1980) Biochim. Biophys. Acta 620, 636-640). We now provide evidence that the enzyme activation is due to a translocation of the cytidylyltransferase from the cytosol to the microsomes. The rate of phospho[Me-3H]choline conversion into phosphatidylcholine was approx. 3-fold faster in HeLa cells treated with 100 nM TPA. This stimulation correlated with a 2.3-fold activation (P less than 0.05) of cytidylyltransferase in homogenates from treated cells. There was a 1.7-fold increase in the enzyme associated with microsomes (P less than 0.05) and a corresponding decrease in enzyme recovered from cytosol (P less than 0.01). The total amount of enzyme recovered from the homogenates was unchanged. Further evidence for TPA causing an increased association of cytidylyltransferase with cellular membranes was obtained when cells were treated with digitonin. The release of cytidylyltransferase into the medium was inhibited by 4-fold from cells previously treated with TPA. Similar results on phospho[Me-3H]choline incorporation into phosphatidylcholine were found with cells incubated with phorbol-12,13-dibutyrate, a water-soluble tumor-promoting agent.

Biological Transport↗
PubMed1984

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes.

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.

Animals↗
PubMed1984

Evidence that the enzymes involved in the methylation of phosphatidylethanolamine are on the external side of the microsomal vesicles.

The topology of the phosphatidylethanolamine (PE)-N-methyltransferase(s) on rat liver microsomes has been studied. The activity for the conversion of PE to phosphatidylcholine decreased by 50% after 2 min of exposure of the microsomes to trypsin and was virtually eliminated with 15 min. When exogenous monomethyl-PE or dimethyl-PE were incubated with microsomes, the formation of dimethyl-PE and phosphatidylcholine were also eliminated as a result of trypsin digestion. During the experiments the microsomes remained intact, since the latency of the mannose-6-phosphate phosphohydrolase remained approx. 90%. It is concluded that the active site(s) of the enzyme(s), or portions of the enzyme(s) indispensable to its activity, are present at the cytosolic side of the microsomes.

Animals↗
PubMed1984

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes.

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.

Animals↗
PubMed1984

Glucagon inhibits phosphatidylcholine biosynthesis via the CDP-choline and transmethylation pathways in cultured rat hepatocytes.

The short-term influence of insulin and glucagon on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes was investigated. Under conditions in which insulin (100 nM) stimulated [3H]acetate incorporation into fatty acid almost twofold, synthesis of phosphatidylcholine via CDP-choline and from phosphatidylethanolamine were unaffected. By contrast, glucagon (100 nM), even in the presence of insulin (100 nM), reduced the rate of phosphatidylcholine formation from [Me-3H]phosphocholine by approximately 25% (p less than 0.05) within 1 h. Similarly, [3H]phosphatidylethanolamine incorporation into phosphatidylcholine was inhibited in cells exposed to glucagon. Insulin and glucagon had little or no effect on [Me-3H]choline uptake by the hepatocytes. No changes in the activities of the phosphatidylcholine biosynthetic enzymes in cytosol and microsomes from glucagon-treated cells could be detected.

Animals↗
PubMed1983

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.

Animals↗
PubMed1983

Pitfalls and problems in studies on the methylation of phosphatidylethanolamine.

Recent articles have confused the steady state concentration of radioactivity in N-methylphosphatidylethanolamine (PME) and N,N-dimethylphosphatidylethanolamine (PDE) with the amount of these products formed during the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). This paper clarifies this problem and reports the apparent Km values for AdoMet and pH optima for the conversion of PE to PME, PDE, and PC by rat liver microsomes. We purified AdoMet and [methyl-3H]AdoMet and measured the transfer of tritium to PME, PDE, and PC as a function of time. There was an initial lag in the formation of [3H]PC followed by linear incorporation of isotope. In contrast, labeled PME and PDE reached and maintained steady state levels within 1 to 2 min. Hence, calculations of the rate of formation of PME, PDE, and PC must take into account the subsequent conversion of PME and PDE to PC. The PE N-methyltransferase was assayed at pH 6.6, 9.2, and 10.25 and the apparent Km for AdoMet for the three methylation reactions was calculated. The formation of PME was best estimated by the dpm in PME + 1/2 dpm in PDE + 1/3 dpm in PC. The synthesis of PDE from PME was estimated from 1/2 dpm in PDE and 1/3 dpm in PC, and the formation of PC from PDE estimated by 1/3 dpm in PC. The apparent Km for AdoMet at pH 10.25 for the conversion of PE to PME was 58 microM, PME to PDE was 65 microM, and PDE to PC was 96 microM. The pH optimum for each of these methylation reactions was 10.25. This high value was not due to alkaline degradation of AdoMet or denaturation of the enzyme. The apparent Km for AdoMet was also estimated for the conversion of exogenous PME to PDE (50 microM) and exogenous PDE to PC (45 microM). Since recent studies on the methylation of PE have not taken into account the conversion of newly formed PME and PDE to PC, the results and conclusions about apparent Km values for AdoMet, pH optima, and the number of enzymes involved must be re-evaluated.

Chromatography, Ion Exchange↗
PubMed1983

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes.

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.

Animals↗
PubMed1983

Trifluoperazine and other anaesthetics inhibit rat liver CTP: phosphocholine cytidylyltransferase.

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

Anesthetics, Local↗
PubMed1983

Fatty acids promote translocation of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine synthesis.

The mechanism by which fatty acids stimulate the biosynthesis of phosphatidylcholine has been studied in cultured rat hepatocytes. Long chain fatty acids (1 mM) stimulated approximately 1.9-fold [methyl-3H] choline incorporation from phosphocholine into phosphatidylcholine by isolated hepatocytes. Oleate and palmitate (4 mM) enhanced phosphatidylcholine production by 3- and 2.2-fold, respectively. Stimulation of phosphatidylcholine synthesis by oleate was evident within 30 min after addition of the fatty acid to the hepatocyte medium. The effect could be correlated with a doubling of the microsomal CTP:phosphocholine cytidylyltransferase activity. Additional evidence for a direct effect by fatty acids and their CoA derivatives on the cytidylyltransferase was obtained in vitro. Arachidonate, oleate and palmitate (0.1 mM) stimulated rat liver cytosolic cytidylyltransferase activity 4.1-, 3.5- and 3.2-fold, respectively. Activation by oleate was accompanied by a 3.6-fold reduction in the apparent Km of the cytidylyltransferase for CTP and aggregation of the enzyme to high molecular weight species. Acceleration of the cytidylyltransferase reaction by fatty acids provides a positive feed-forward mechanism for regulation of phosphatidylcholine anabolism.

Acyl Coenzyme A↗
PubMed1983

Activities of the phosphatidylcholine biosynthetic enzymes in rat liver during development.

The activities of the enzymes of rat hepatic phosphatidylcholine biosynthesis have been measured as a function of development in the rat (term, 23 days). During the last 5 days of gestation, the specific activity of choline kinase was elevated almost fivefold (p less than 0.05). After parturition, choline kinase activity was reduced to adult values by the 5th postnatal day. Over 75% of the total CTP:phosphocholine cytidylyltransferase protein in prenatal liver was detected in the cytosolic fraction. On the day of birth, most of the cytidylyltransferase translocated to the microsomes so that the microsomal specific enzyme activity was 3.3-fold higher (p less than 0.01) and the cytosolic specific enzyme activity (measured in the presence of phospholipid) was 68% lower (p less than 0.001) than the day before parturition. CDPcholine:diacylglycerol cholinephosphotransferase activity (measured in the presence of diacylglycerol) increased 130-fold (p less than 0.001) during the last 5 days of gestation. On the 10th postnatal day, cholinephosphotransferase activity was 1.7-fold higher (p less than 0.001) than immediately after birth, but declined to adult values by the 19th day. Between the 5th day prior to parturition and the 10th postnatal day, phosphatidylethanolamine N-methyltransferase activity steadily increased 16-fold (p less than 0.001). The results are in agreement with the hypothesis that the increase in phosphatidylcholine in rat liver during the perinatal period is due to an increased synthesis of CDPcholine, which is a consequence of the translocation of the cytidylyltransferase from cytosol to the endoplasmic reticulum.

Animals↗