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

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.