Phospholipase D hydrolysis of plasmalogen and diacyl ethanolamine phosphoglycerides by protein kinase C dependent and independent mechanisms.
Ethanolamine phosphoglycerides (EPG) are potential sources of lipid second messengers in signal transduction pathways. We investigated EPG turnover, including both 1-alkenyl-2-acyl- (plasmalogen) and diacyl-classes, in response to stimulation of protein kinase C (PKC) by phorbol ester (4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA)) in cultured C6 rat glioma cells. Release of ethanolamine to the medium from EPG prelabeled with [14C]ethanolamine indicated that initial (< 60 min) TPA-stimulated hydrolysis of EPG was predominantly by phospholipase D (PLD). Effects of TPA on PLD activity specifically with EPG was confirmed using trans-phosphatidylation by incubating cells prelabeled with [14C]eicosapentaenoic acid (20:5n-3) with 100 nM TPA and 1% butanol. Analysis of acid-labile phosphatidylbutanol and remaining EPG showed utilization of both plasmalogen and non-plasmalogen EPG. Staurosporine (STS) inhibited PKC at 200-500 nM but stimulated PLD activity 2-fold at > or = 1 microM. However, STS did not eliminate all TPA-stimulated PLD activity, even when PKC was > 98% inhibited. Bis-indolylmaleimide (BIM) fully inhibited PKC activity but had no independent effects on PLD and did not completely inhibit TPA- or bryostatin-stimulated PLD activity. Down-regulation of PKC by chronic exposure to TPA eliminated stimulation of PLD by TPA but not by STS. Thus, PLD hydrolysis of both plasmalogen and diacyl-EPG is a source of potential lipid second messengers in C6 glioma cells. PLD is stimulated by activation of PKC and by PKC-independent action of STS. Further, the possibility that TPA may also elicit responses through a mechanism independent of PKC activity is suggested.
Van Iderstine
S C
SC
Atlantic Research Centre, Department of Biochemistry, Dalhousie University, Halifax, NS Canada.
Byers
D M
DM
Ridgway
N D
ND
Cook
H W
HW
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005910:Glioma; D006868:Hydrolysis; D007700:Kinetics; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010955:Plasmalogens / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D010714:Phosphatidylethanolamines; D010955:Plasmalogens; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate