scholarly journals Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the ‘de novo’ pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake

1993 ◽  
Vol 293 (3) ◽  
pp. 739-744 ◽  
Author(s):  
H Tronchère ◽  
F Tercé ◽  
M Record ◽  
H Chap
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Phorbol Ester ◽  
De Novo ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Choline Uptake ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Choline Incorporation ◽  
Unique Effect

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the ‘de novo’ pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the ‘de novo’ pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).

scholarly journals The Major Sites of Cellular Phospholipid Synthesis and Molecular Determinants of Fatty Acid and Lipid Head Group Specificity

2002 ◽  
Vol 13 (9) ◽  
pp. 3148-3161 ◽  
Author(s):  
Annette L. Henneberry ◽  
Marcia M. Wright ◽  
Christopher R. McMaster
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
De Novo ◽  
Brefeldin A ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Fatty Acyl ◽  
Head Group ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Lipid Head Group

Phosphatidylcholine and phosphatidylethanolamine are the two main phospholipids in eukaryotic cells comprising ∼50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized almost exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway being cell type dependent. Two human genes, CEPT1 and CPT1, code for the total compliment of activities that directly synthesize phosphatidylcholine and phosphatidylethanolamine through the CDP-alcohol pathways. CEPT1 transfers a phosphobase from either CDP-choline or CDP-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPT1 synthesizes phosphatidylcholine exclusively. We show through immunofluorescence that brefeldin A treatment relocalizes CPT1, but not CEPT1, implying CPT1 is found in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was found in the Golgi and CEPT1 was found in both the endoplasmic reticulum and nuclear membranes. The rate-limiting step for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase α, which is found in the nucleus in most cell types. CTP:phosphocholine cytidylyltransferase α is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase α to nuclear located CEPT1 is the mechanism by which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In addition, a series of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acid residues as structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214–228 allowed for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned.

scholarly journals Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells

1996 ◽  
Vol 320 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Christina DECKER ◽  
Maria Jesus MIRO OBRADORS ◽  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Site Of Action

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

scholarly journals Antagonism of phorbol-ester-stimulated phosphatidylcholine biosynthesis by the phospholipid analogue hexadecylphosphocholine

1993 ◽  
Vol 291 (2) ◽  
pp. 561-567 ◽  
Author(s):  
T Wieder ◽  
C C Geilen ◽  
W Reutter
Keyword(s):  
Cell Lines ◽  
Hela Cells ◽  
Mdck Cells ◽  
Fold Increase ◽  
Choline Uptake ◽  
Choline Incorporation ◽  
Contrast Enhanced ◽  
Phosphatidylcholine Biosynthesis ◽  
Membrane Bound ◽  
Stimulation Of

The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase activity in PMA-treated MDCK cells. In contrast, enhanced PtdCho biosynthesis in HeLa cells is achieved by a 2-fold increase in particulate cytidylyltransferase activity after PMA stimulation. Translocation of cytidylyltransferase from the cytosol to membranes is therefore important in HeLa cells. Nevertheless, in both cell lines, the main target of HePC seems to be the translocation process. In MDCK cells, addition of 50 microM HePC decreases membrane-bound cytidylyltransferase activity by about 45%, compared with control cells and PMA-treated cells. In HeLa cells, PMA-induced translocation of cytidylyltransferase to membranes is totally abolished by HePC.

scholarly journals Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells

1992 ◽  
Vol 282 (2) ◽  
pp. 333-338 ◽  
Author(s):  
F Tercé ◽  
M Record ◽  
H Tronchère ◽  
G Ribbes ◽  
H Chap
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Cyclic Amp ◽  
Subcellular Fractionation ◽  
Oleic Acid Content ◽  
Noticeable Increase ◽  
Cell Incubation ◽  
Membrane Bound

Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.7.15), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

Abstract 269: 5-aminoimidazole-4-carboxamide-3-ribonucleoside (AICAR) Decreases Soluble Fms-like Tyrosine Kinase-1 (sFlt-1) and Attenuates Endoplasmic Reticulum Stress in Rat Placental Villi Explants

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Christopher T Banek ◽  
Haley E Gillham ◽  
Sarah M Johnson ◽  
Hans C Dreyer ◽  
Jeffrey S Gilbert
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
De Novo ◽  
Vegf Receptor ◽  
Sprague Dawley ◽  
Hypoxic Conditions ◽  
Ampk Phosphorylation ◽  
Angiogenic Balance ◽  
Placental Villi ◽  
Placental Ischemia

Preeclampsia, defined by the onset of de novo hypertension and proteinuria near the 20th week of gestation, is a major contributor to maternal and fetal morbidity and mortality worldwide. Preeclampsia is often preceded by placental ischemia and an imbalance in circulating angiogenic factors (e.g. VEGF - vascular endothelial growth factor, sFlt-1 - soluble VEGF receptor 1). Recent studies also report increased expression of endoplasmic reticulum (ER) stress products in preeclamptic placentas. Our laboratory recently reported 5-aminoimidazole-4-carboxamide-3-ribonuceloside (AICAR) reduces blood pressure and improves angiogenic balance (increased VEGF, decreased sFlt-1) in rats with placental ischemia-induced hypertension, but the mechanism is unclear. We hypothesized AICAR would decrease sFlt-1, increase AMPK phosphorylation, and decrease ER stress in hypoxic placental villous explants. On day 19 of pregnancy, placentas were isolated from four Sprague-Dawley rats and immediately dissected in ice-cold phosphate-buffered saline. Explants were cultured for 12 hours in physiologic normoxic (8% O2) and hypoxic (1.5% O2) conditions. All experiments were performed in triplicate. VEGF secretion was unaffected by AICAR treatment in both normoxic and hypoxic conditions. AICAR decreased sFlt -1 secretion in hypoxic villi (2147±116 vs. *1411±67, P<0.05). Additionally, AMPK activation ratio was increased with AICAR, and was hypoxic-dependent (8%: 2.9±0.3; 8%+A: 3.3±0.1; 1.5%: 3.5±0.1; 1.5%+A: *4.5±0.01;*P<.05). Moreover, markers of ER stress were increased with hypoxia, and decreased with AICAR treatment (BiP 8%: 1.2±0.2; 8%+A: 1.0±0.0; 1.5%: *8.3±0.7; 1.5%+A: 1.9±0.0.3;*P<.05), (CHOP 8%: 0.5±0.0; 8%+A: 0.3±0.1; 1.5%: *1.7±0.1; 1.5%+A: 0.7±0.1;*P<.05). ATF4 was not changed with hypoxia or AICAR treatment. The present data show that AICAR stimulates AMPK phosphorylation and decreases ER stress response proteins in hypoxic placental villi. Further, the present data support the hypothesis that AICAR restores angiogenic balance by decreasing sFlt-1 rather than increasing VEGF secretion from placental villi. These findings suggest AICAR may improve placental function during pregnancies complicated by placental-ischemia.

Sign in / Sign up

Export Citation Format

Share Document