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Biomedical subjects

D E Vance

Publications and source records attributed to D E Vance.

At least 145 records · Page 8Linked to original sources
PubMed1982

Prolonged effects of cyclic AMP analogues of phosphatidylcholine biosynthesis in cultured rat hepatocytes.

The short- and long-term effects of cyclic AMP analogues and phosphodiesterase inhibitors on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes were investigated. All the compounds tested produced an inhibition of phosphatidylcholine biosynthesis for up to 6 h after addition to the hepatocyte medium. The reduced rate of lipid synthesis was a function of the concentration of cyclic AMP analogue and was independent of the concentration of choline in the medium. The proportion of choline oxidized to betaine was relatively unaffected. Choline was incorporated into hepatocytes by saturable and non-saturable mechanisms. Although the various cAMP analogues had different effects on choline uptake, chlorophenylthio-cAMP reduced uptake of choline by 28% for cells treated for 1.5-15 h. This analogue lowered the Vmax of the saturable component of choline transport by 3.6-fold. Prolonged incubation of the hepatocytes with cAMP analogues resulted in a reversal of the inhibition of phosphatidylcholine synthesis. After 15 h all the compounds tested stimulated the relative incorporation of [methyl-3H]choline into phosphatidylcholine. For hepatocytes incubated with chlorophenylthio-cAMP for 14-16 h, there was a 2.8-fold stimulation of the rate of phosphatidylcholine synthesis. The enzymes responsible for the conversion of choline into phosphatidylcholine were examined at various times after addition of the chlorophenylthio-cAMP to the hepatocyte medium. The reduced synthesis of phosphatidylcholine strongly correlated with inhibition of CTP:phosphocholine cytidylyltransferase activity. After 12 h of treatment with the analogue, the relative inhibition of the cytidylyltransferase activity was reversed.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Diethylstilbestrol treatment increases the amount of choline kinase in rooster liver.

Studies have been performed on the mechanism by which diethylstilbestrol stimulates the activity of choline kinase in livers from cockerels. The enzyme was purified 700-900-fold by affinity chromatography. The increased enzyme activity could not be accounted for by diethylstilbestrol alteration of the kinetic constants of the enzyme. Rabbit antibody was raised to the purified enzyme. Titration studies with antiserum demonstrated a 2-fold increase in the amount of choline kinase in diethylstilbestrol-treated cytosol, which correlated with a 2-fold elevation of the activity of the enzyme. We conclude that diethylstilbestrol stimulates the activities of choline kinase in cockerel liver by corresponding increase in the amount of enzyme.

Animals↗
PubMed1981

Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters.

It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.

Animals↗
PubMed1981

Diethylstilbestrol treatment modulates the enzymatic activities of phosphatidylcholine biosynthesis in rooster liver.

The effect of diethylstilbestrol injection on the activities of phosphatidylcholine biosynthetic enzymes in rooster liver has been determined. Choline kinase activity was stimulated within 4 h after the first hormone injection. By the third day enzyme activity reached 5.47 nmol . min-1 . mg-1 protein compared to control values (1.83 nmol . min-1 . mg-1 protein) which were unchanged during the the experiment. CTP : phosphocholine cytidylyltransferase activity was unaffected until Day 3 when its activity was 50% that of control values. When assayed in the presence of exogenous phospholipid, no significant change was noted in cytidylyltransferase activity. The activity of CDPcholine : 1,2-diacylglycerol phosphocholinetransferase was not altered by the hormone injections. The activity of phosphatidylethanolamine-N-methyltransferase gradually increased so that by Day 3, the enzyme activity was elevated 2-fold (0.12 to 0.24 nmol methyl group transferred per mg microsomal protein). These results are consistent with earlier in vivo studies (Vigo, C. and Vance, D.E. (1981) Eur. J. Biochem., in the press) that indicated a stimulation of phosphatidylcholine biosynthesis via CDPcholine during the first 2 days of diethylstilbestrol injection and inhibition on the third day.

Animals↗
PubMed1981

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes.

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).

Animals↗
PubMed1981

Semliki Forest virus does not inhibit phosphatidylcholine biosynthesis in BHK-21 cells.

The mechanism by which Semliki Forest virus inhibits the incorporation of [methyl-3H]choline into phosphatidylcholine has been investigated. Decreased labeling of the lipid was not due to altered uptake of [methyl-3H]choline. The specific activities of choline kinase and CTP:phosphocholine cytidylyltransferase were unchanged. The previously observed inhibition (Vance, D. E. & Burke, D. C. (1974) Eur. J. Biochem. 43, 327-336) of CDP-choline:1,2-diacylglycerol phosphocholinetransferase was confirmed. Since the decreased activity of the phosphocholinetransferase may not have caused the reduced labeling of phosphatidylcholine, the amounts of this lipid and its precursors were measured. We observed changes in the concentration of phosphocholine (34 +/- 12 and 120 +/- 40 nmol x g cells-1 in mock- and virus-infected cells, respectively) and CTP (116 +/- 35 and 36 +/- 13 nmol x g cells-1 in mock- and virus-infected cells, respectively). Pulse-chase studies with [methyl-3H]choline demonstrated that, initially, most of the radioactivity was in phosphocholine. As it disappeared from this compound, it appeared in phosphatidylcholine. From these results, we calculated the rate of phosphatidylcholine biosynthesis to be 0.56 and 1.23 nmol x min-1 x g cells-1 in mock- and virus-infected BHK-21 cells, respectively. We conclude that phosphatidylcholine biosynthesis is not inhibited in Semliki Forest virus infected BHK cells, but rather is stimulated 6.75 h after infection. The decreased labeling observed during pulse studies with [methyl-3H]choline is due to dilution of the labeled choline into a pool of phosphocholine which is 3.5 times larger in the infected cells.

Animals↗
PubMed1980

Tetradecanoyl-phorbol acetate stimulates phosphatidylcholine biosynthesis in HeLa cells by an increase in the rate of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase.

Studies have been performed on the mechanism by which 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates phosphatidylcholine biosynthesis in HeLa cells. The phorbol acetate did not alter the transport of choline into the cells, nor did it stimulate choline phosphorylation. When [methyl-3H]choline was added to HeLa cells for a 1 h pulse, virtually all of the radioactivity in the aqueous phase of the cell extracts became associated with phosphocholine. The addition of the phorbol ester caused an accelerated disappearance of radioactivity from phosphocholine and a concomitant increase in the incorporation into phosphatidylcholine. The radioactivity associated with CDPcholine remained low and constant throughout the experiment. These results provide strong evidence that TPA accelerates phosphatidylcholine biosynthesis in HeLa cells by stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase.

Choline↗
PubMed1980

The possible functional significance of phosphatidylethanolamine methylation.

The conversion of phosphatidylethanolamine to phosphatidylcholine was first reported in rat liver microsomes by Bremer and Greenberg. The reaction requires three successive methylations of the ethanolamine moiety by S-adenosylmethionine. The highest activity for this enzyme has been found in liver microsomes and the enzyme from rat liver was recently solubilized and partially purified. Although the activity is highest in liver, it is the minor pathway in this tissue for the synthesis of phosphatidylcholine. The enzyme has recently been identified in bovine adrenal medulla and reports exist on the activity of phosphatidylethanolamine methyltransferase in erythrocyte ghosts, reticulocyte ghosts and mammary gland membranes. In view of the relatively minor importance of the methylation pathway in phosphatidylcholine biosynthesis in liver, we were intrigued by the reports of significant physiological changes attributed to phospholipid methylation. Calculations reported here show that this enzymatic activity in reticulocytes, erythrocytes and mammary gland membranes is 0.1% of that observed in liver microsomes. Furthermore, in conditions where marked changes in microviscosity of the erythrocyte membrane were observed, only extremely small amounts of phosphatidylethanolamine were methylated. For these and other reasons, there is considerable doubt that methylation of phosphatidylethanolamine could account for the many physiological responses attributed to this activity.

Adrenal Medulla↗
PubMed1980

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.

Cell Transformation, Viral↗
PubMed1980

Evidence that Sindbis virus infects BHK-21 cells via a lysosomal route.

Chloroquine and NH4Cl, potent inhibitors of lysosomal function, decreased the production of infectious Sindbis virus particles in BHK-21 cells by 10- and 12-fold, respectively. There were no apparent toxic effects on cells exposed to these lysosomotropic agents. These chemicals did not alter the rate of cellular protein synthesis, with the exception of a reversible twofold inhibition by chloroquine. No additive effects of chloroquine and NH4Cl were observed when the cells were saturated with these weak bases, which suggests that their effect is exerted via the same mechanism, most likely as a result of an increase in lysosomal pH. The reduction in the formation of Sindbis virions was monitored by incorporation of [35S]methionine and shown to be fourfold by chloroquine or NH4Cl at 5 h postinfection but negligible at 11 h postinfection. These results strongly suggest that a productive Sindbis virus infection requires functional lysosomes. Thus, endocytosis is probably the main infectious mechanism for penetration of this virus into BHK-21 cells.

Ammonium Chloride↗