scholarly journals Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail

1997 ◽  
Vol 327 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Bo LIU ◽  
Shigeru NAKASHIMA ◽  
Takahito ADACHI ◽  
Yuzuru ITO ◽  
Tomoko TAKANO ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Mrna Levels ◽  
Primary Role ◽  
Ovary Cells ◽  
Protein Kinase Cα ◽  
Paf Receptor

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Cα (PKCα) to membrane was similar to that of DG formation. In WT-H cells, PKCα was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCα was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCα. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCα, which might play an important role in the expression of c-fos and c-jun.

Activation of Phospholipase D in Chinese Hamster Ovary Cells Expressing Platelet-Activating Factor Receptor1

1994 ◽  
Vol 116 (4) ◽  
pp. 882-891 ◽  
Author(s):  
Bo Liu ◽  
Shigeru Nakashima ◽  
Hiroyuki Kanoh ◽  
Tomoko Takano ◽  
Takao Shimizu ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Ovary Cells

scholarly journals Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells

1998 ◽  
Vol 329 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Richard HEAL ◽  
John McGIVAN
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Asparagine Synthetase ◽  
Mrna Levels ◽  
Amino Acid Deprivation ◽  
Ovary Cells ◽  
Protein Levels

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and α-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.

scholarly journals Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor

1999 ◽  
Vol 337 (2) ◽  
pp. 263-268 ◽  
Author(s):  
Remko R. BOSCH ◽  
Rolf L. L. SMEETS ◽  
Frank SLEUTELS ◽  
Anjana M. P. PATEL ◽  
Sjenet E. Van EMST-De VRIES ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Kinase C ◽  
Peptide Amide ◽  
Sustained Increase

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26–33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 µM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 µM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

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