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

Abstract

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.

Rat pancreatic acinar cells express a cytosolic phospholipase D1b isoform that is not regulated by cholecystokinin

“…A relatively weak membrane association may also mean that high-level PKC activation, as obtained with PMA, is required for PLD activation. Such a model is compatible with our findings that low-level PKC activation by either CCK or the thapsigargin-induced increase in cytosolic Ca 2+ is sufficient to activate PLD in CHO cells [22], but not in acinar cells (this study).…”

“…We have shown previously that the presence of PKC is a prerequisite for CCK-, thapsigargin-and PMAinduced PLD activation in CHO-CCK A cells [22]. In the same study we demonstrated that the extent to which CCK activates this PKC-dependent PLD depends largely, if not entirely, on the magnitude and duration of the hormone-induced increase in cytosolic Ca 2+ .…”

“…This conclusion is based on the following observations: (a) rat pancreatic acinar cells and CHO-CCK A cells expressed the same PLD isoform; (b) in the acinar cells, however, this isoform, PLD1b, occurred predominantly in the cytosol, whereas in CHO cells it was located mainly in the cell membrane; (c) CCK did not increase PLD activity in pancreatic acinar cells, whereas it stimulates this activity readily in CHO-CCK A cells [22]; (d) the combination of PMA and GTPγS did not increase PLD activity in a broken cell preparation of pancreatic acinar cells, whereas it stimulated this activity in a similar preparation of CHO cells markedly.…”

“…Similarly, this combination of drugs did not activate PLD significantly following homologous reconstitution of the acinar membrane and cytosol fraction. We have shown previously that PMA markedly stimulates PLD activity in a homogenate of Chinese hamster ovary (CHO) cells [22]. Preliminary experiments have revealed that GTPγS also stimulates PLD activity markedly in a CHO cell homogenate and that the effects of PMA and GTPγS are more than additive (data not shown).…”

“…After 1 h, the acini were washed and the labelling was continued for another hour. CHO cells expressing the rat acinar CCK A receptor (CHO-CCK A cells) were labelled with [ 3 H]myristic acid as described previously [21,22]. Briefly, CHO-CCK A cells were grown to confluence in the presence of [ 3 H]myristic acid (2 µCi/ml) in a humidified atmosphere of 5% CO 2 for 24 h at 37°C.…”