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 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.
Bosch
R R
RR
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Smeets
R L
RL
Sleutels
F
F
Patel
A M
AM
Emst-de Vries
S E
SE
Joep
J
J
de Pont
H H
HH
Willems
P H
PH
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism*; D015536:Down-Regulation; D004789:Enzyme Activation; D020404:Glycerophospholipids / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D051381:Rats; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012844:Sincalide / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D020404:Glycerophospholipids; D010617:Phenanthridines; D047428:Protein Kinase Inhibitors; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D011994:Recombinant Proteins; C051521:phosphatidylethanol; D019284:Thapsigargin; C016299:chelerythrine; D011493:Protein Kinase C; D010739:Phospholipase D; D012844:Sincalide; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium