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 se cond messengers in signal transduction pathways. We investigated EPG t urnover, including both 1-alkenyl-2 acyl- (plasmalogen) and diacyl-cla sses, 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 pr elabeled with [C-14]ethanolamine indicated that initial (< 60 min) TPA -stimulated hydrolysis of EPG was predominantly by phospholipase D (PL D). Effects of TPA on PLD activity specifically with EPG was confirmed using trans-phosphatidylation by incubating cells prelabeled with [C- 14]eicosapentaenoic acid (20:5n-3) with 100 nM TPA and 1% butanol. Ana lysis of acid-labile phosphatidylbutanol and remaining EPG showed util ization of both plasmalogen and non-plasmalogen EPG. Staurosporine (ST S) inhibited PKC at 200-500 nM but stimulated PLD activity 2-fold at g reater than or equal to 1 mu M. However, STS did not eliminate all TPA -stimulated PLD activity, even when PKC was > 98% inhibited. Bis-indol ylmaleimide (BIM) fully inhibited PKC activity but had no independent effects on PLD and did not completely inhibit TPA- or bryostatin-stimu lated PLD activity. Down-regulation of PKC by chronic exposure to TPA eliminated stimulation of PLD by TPA but not by STS. Thus, PLD hydroly sis 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.