sincalide and phosphatidylethanol

We have studied phospholipase D activation in [(32)P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of (32)P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [(32)P]phosphatidylethanol and [(32)P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of (32)P-phosphatidylalcohol, yet they decreased [(32)P]phosphatidylcholine content and stimulated the generation of [(32)P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity.

Topics: Animals; Butanols; Carbachol; Enzyme Activation; Ethanol; Glycerophospholipids; Hydrogen Peroxide; Kinetics; Pancreas; Phosphates; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Rats; Rats, Wistar; Sincalide; Tetradecanoylphorbol Acetate; Vanadates

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.

Topics: Alkaloids; Animals; Benzophenanthridines; Brain; Calcium; CHO Cells; Cricetinae; Cytosol; Down-Regulation; Enzyme Activation; Glycerophospholipids; Phenanthridines; Phospholipase D; Protein Kinase C; Protein Kinase Inhibitors; Rats; Receptor, Cholecystokinin A; Receptors, Cholecystokinin; Recombinant Proteins; Sincalide; Tetradecanoylphorbol Acetate; Thapsigargin