scholarly journals Basal and phorbol-ester-stimulated turnover of phosphatidylcholine in HeLa cells involve different pathways

1992 ◽  
Vol 288 (3) ◽  
pp. 983-985 ◽  
Author(s):  
C S T Hii ◽  
Y S Edwards ◽  
A W Murray
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Hela Cells ◽  
Rapid Accumulation

HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.

scholarly journals Epidermal-growth-factor-induced production of phosphatidylalcohols by HeLa cells and A431 cells through activation of phospholipase D

1992 ◽  
Vol 287 (1) ◽  
pp. 51-57 ◽  
Author(s):  
M Kaszkin ◽  
L Seidler ◽  
R Kast ◽  
V Kinzel
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Hela Cells ◽  
Total Phospholipid ◽  
Primary Alcohol ◽  
A431 Cells ◽  
Intact Cells ◽  
Epidermal Growth

In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.

Phorbol Esters Stimulate Phospholipase D Activity in C-6 Glioma Cells

1989 ◽  
Vol 16 (3) ◽  
pp. 257-262
Author(s):  
Lena Gustavsson ◽  
Christofer Lundqvist ◽  
Christer Ailing
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Culture Medium ◽  
Phorbol Esters ◽  
Glioma Cells

The effects of phorbol esters on phospholipase D activity were studied in C-6 glioma cells. The cell lipids were prelabelled with [3H]-glycerol or [14C]-arachidonic acid. Phosphatidylethanol was formed during stimulation with 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), when ethanol was present in the culture medium. After 30 minutes of stimulation, phosphatidylethanol constituted 2.6% of the [3H]-glycerol-labelled lipids. Stimulating the cells with TPA in the absence of ethanol caused a significant increase in labelled phosphatidic acid. This increase was inhibited by ethanol. The present findings demonstrate that TPA stimulates phospholipase D activity in cultured C-6 glioma cells.

scholarly journals A shift in the optimum pH of phospholipase D produced by activating long-chain anions

1969 ◽  
Vol 112 (5) ◽  
pp. 795-799 ◽  
Author(s):  
R. H. Quarles ◽  
R. M. C. Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Activity Profile ◽  
Ph Optimum ◽  
Surface Ph ◽  
Electrophoretic Mobilities ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Long Chain

1. The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) towards ultrasonically treated phosphatidylcholine or large phosphatidylcholine particles activated with ether was maximal near pH5, and there was little activity above pH6. 2. When the enzyme was activated by the addition of phosphatidic acid to large phosphatidylcholine particles the pH optimum was shifted to pH6·5 irrespective of the amount of activator added. 3. When the enzyme was activated with low concentrations of dodecyl sulphate the pH optimum was 5·5 with little activity above pH6. With higher concentrations of dodecyl sulphate the pH–activity profile was shifted upwards towards a pH optimum of 6·5–6·6, the magnitude of the shift depending on the extent of the hydrolysis. 4. The shifts in the pH–activity profiles cannot be correlated with changes in the ‘surface pH’ of the substrate particles calculated from the measurement of their ζ-potentials (electrophoretic mobilities).

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

scholarly journals Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate

2000 ◽  
Vol 22 (2) ◽  
pp. 147-154 ◽  
Author(s):  
Teun Munnik ◽  
Harold J. G. Meijer ◽  
Bas ter Riet ◽  
Heribert Hirt ◽  
Wolfgang Frank ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Hyperosmotic Stress
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