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 me ssengers involved in sustained signal transduction. Four neural-derive d cell lines (C6 rat glioma; N1E-115 mouse and SK-N-MC and SK-N-SH hum an neuroblastoma) express different protein kinase C (PKC) isoforms an d differentially respond to 4 beta-12-O-tetradecanoylphorbol-13-acetat e (beta-TPA)-stimulation of PtdCho synthesis. We examined involvement of PLD and PKC in the hydrolysis and resynthesis of PtdCho and phospha tidylethanolamine stimulated by beta-TPA, bryostatin (a non-phorbol PK C activator) and oleic acid (18:1n-9) in the four cell lines. beta-TPA or bryostatin produced similar enhancement of [H-3]Cho incorporation, loss of stimulated synthesis after down regulation of PKC, and activa tion of PLD, In C6 cells? staurosporine (STS) and bis-indolylmaleimide (BIM) only partially inhibited basal and beta TPA-stimulated PLD acti vity measured as choline or ethanolarnine release; phosphatidylbutanol formation after prelabeling with [9,10-H-3]18:1n-9, [9,10-H-3]myristi c acid (14:0), [1-C-14]eicosapentaenoic acid (20:5n-3) or O-[alkyl-1', 2-H-3]-sn-glyceryl-3-phosphorylcholine gave similar results. STS at > 200 nM activated PLD in the presence or absence of beta-TPA. In SK-N-S H cells where PtdCho synthesis was stimulated by beta-TPA or bryostati n, no effect of these agents on PLD was observed. 18:1n-9 stimulated P tdCho synthesis and, to a lesser extent, hydrolysis by PLD both with a nd without beta-TPA present. Fatty acids had no effect on PKC activiti es and down regulation of PKC with beta-TPA enhanced fatty acid stimul ation of PtdCho synthesis. Thus, activation of PLD hydrolysis precedin g resynthesis is involved in the stimulatory effects of beta-TPA on Pt dCho synthesis in some but not all of these neural derived cells. Furt her, PLD hydrolysis of PtdCho and PtdEtn appear to have differing aspe cts of regulation. Fatty acid regulation of PtdCho synthesis occurs in dependent of PKC activation. Accordingly, regulation of membrane phosp holipid degradation and resynthesis in association with lipid second m essenger generation can involve a complex interplay of PLD, PKC, and f atty acids. (C) 1998 Elsevier Science B.V.