Involvement of phospholipase D and protein kinase C in phorbol ester and fatty acid stimulated turnover of phosphatidylcholine and phosphatidylethanolamine in neural cells

Hydrolysis of phosphatidylcholine (PtdCho) can provide lipid second messengers involved in sustained signal transduction. Four neural-derived cell lines (C6 rat glioma; N1E-115 mouse and SK-N-MC and SK-N-SH human neuroblastoma) express different protein kinase C (PKC) isoforms and differentially respond to 4β-12-O-tetradecanoylphorbol-13-acetate (β-TPA)-stimulation of PtdCho synthesis. We examined involvement of PLD and PKC in the hydrolysis and resynthesis of PtdCho and phosphatidylethanolamine stimulated by β-TPA, bryostatin (a non-phorbol PKC activator) and oleic acid (18:1n - 9) in the four cell lines. β-TPA or bryostatin produced similar enhancement of [³H]Cho incorporation, loss of stimulated synthesis after down regulation of PKC, and activation of PLD. In C6 cells, staurosporine (STS) and bis-indolylmaleimide (BIM) only partially inhibited basal and β-TPA-stimulated PLD activity measured as choline or ethanolamine release; phosphatidylbutanol formation after prelabeling with [9,10-³H]18:1n - 9, [9,10-³H]myristic acid (14:0), [1-¹⁴C]eicosapentaenoic acid (20:5n - 3) or 1-O-[alkyl-1',2-³H]-sn-glyceryl-3-phosphorylcholine gave similar results. STS at > 200 nM activated PLD in the presence or absence of β-TPA. In SK-N-SH cells where PtdCho synthesis was stimulated by β-TPA or bryostatin, no effect of these agents on PLD was observed. 18:1n - 9 stimulated PtdCho synthesis anti, to a lesser extent, hydrolysis by PLD both with and without β-TPA present. Fatty acids had no effect on PKC activities and down regulation of PKC with β-TPA enhanced fatty acid stimulation of PtdCho synthesis. Thus, activation of PLD hydrolysis preceding resynthesis is involved in the stimulatory effects of β-TPA on PtdCho synthesis in some but not all of these neural derived cells. Further, PLD hydrolysis of PtdCho and PtdEtn appear to have differing aspects of regulation. Fatty acid regulation of PtdCho synthesis occurs independent of PKC activation. Accordingly, regulation of membrane phospholipid degradation and resynthesis in association with lipid second messenger generation can involve a complex interplay of PLD, PKC, and fatty acids.