Phospholipase d activities in neural cells in relation to protein kinase c isoform and substrate expression

Rat glioma (C6), mouse neuroblastoma (N1E-115) and human neuroblastoma (SK-N-MC and SK-N-SH) cells in culture differentially express isoforms of protein kinase C (PKC). We have reported correlation between PKC-α, its substrate, Myristoylated Alanine-Rich C-Kinase Substrate, and ability of the four cells to increase synthesis of phosphatidylcholine in response to 4β-12-tetradecanoylphorbol-13-acetate (TPA). To further assess mechanisms involved, we determined phospholipid turnover by PLD in response to TPA and bryostatin using several PLD assays: headgroup release from cells prelabeled with [³H]choline or [¹⁴C]ethanolamine and phosphatidylbutanol formation in cells prelabeled with [³H]14:0, [¹⁴C]₂0:5(n-3) or [¹⁴C]lyso- alkylphosphatidylcholine. In C6 cells expressing PKC-α and MARCKS, a delayed, 2-fold increase in PLD by TPAwas incompletely reversed by pretreatment with staurosporine (STS) or bis-indolylmaleimide; >1 M STS was itself stimulatory. In N1E-115 and SK-N-MC cells with little PKC-α or MARCKS, TPA did not increase PLD although 100 nM STS inhibited basal levels. In SK-N-SH cells expressing both PKC-α and MARCKS, TPA and bryostatin were only slightly (<15%) stimulatory and this was inhibited by STS. Thus, in these cell lines,stimulation of PLD did not uniformly correlate with MARCKS and PKC-α expression or activity suggesting involvement of other mechanisms mediating phorbol ester effects.