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: | R R, Bosch, R L, Smeets, F, Sleutels, A M, Patel, S E, Emst-de Vries, J, Joep, H H, de Pont, P H, Willems |
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| Rok vydání: | 1999 |
| Předmět: |
Benzophenanthridines
Brain Down-Regulation CHO Cells Glycerophospholipids Recombinant Proteins Sincalide Phenanthridines Rats Receptor Cholecystokinin A Enzyme Activation enzymes and coenzymes (carbohydrates) Alkaloids Cytosol Cricetinae Phospholipase D Animals Tetradecanoylphorbol Acetate Thapsigargin lipids (amino acids peptides and proteins) Calcium Receptors Cholecystokinin Protein Kinase Inhibitors Protein Kinase C Research Article |
| Zdroj: | The Biochemical journal. 337 |
| ISSN: | 0264-6021 |
| 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 microM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM 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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