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.