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
| Autor: | Emst-de Vries Se, Peter H.G.M. Willems, R. R. Bosch, de Pont Hh, A. M. P. Patel, Sleutels F, R.L.L. Smeets, Joep J |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
medicine.medical_specialty
Thapsigargin Phospholipase D Chinese hamster ovary cell Cell Biology Biology Biochemistry Molecular biology enzymes and coenzymes (carbohydrates) chemistry.chemical_compound Endocrinology Chelerythrine chemistry Internal medicine Tetradecanoylphorbol Acetate medicine lipids (amino acids peptides and proteins) Phosphatidylethanol Cholecystokinin A receptor Molecular Biology Protein kinase C |
| Zdroj: | Biochemical Journal. 337:263-268 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj3370263 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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