Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of mastoparan

Treating Chlamydomonas moewusii cells with non-permeabilizing concentrations of mastoparan (1-5 mu M) increased inositol 1,4,5-trisphosphate (InsP(3)) levels up to 20-fold in a dose-dependent manner and rapidly induced deflagellation and mating-structure activation, two well-defmed Ca2+ responses. When metabolism of the phospholipid precursors was monitored in P-32(i)-labelled cells, as much as 70% of the radioactivity in phosphatidylinositol bisphosphate (PtdInsP(2)) was lost within 20 s. Thereafter, the P-32-label in PtdInsP(2) increased to twice the control level within 10 min. A similar pattern of P-32-labelling was also exhibited by PtdInsP. An HPLC-headgroup analysis revealed that only PtdIns4P and PtdIns(4,5)P-2 were involved and not the D3-phosphorylated isomers. Correlated with the increased polyphosphoinositide (PPI) turnover, there was a massive (5- to 10-fold) increase in P-32-labelled phosphatidic acid (PtdOH) and, slightly later, an increase in its metabolic product, diacylglycerol pyrophosphate (DGPP), reflecting the phosphorylation of the resulting diacylglycerol (DAG) and PtdOH, respectively. Mastoparan-treatment of P-32-labelled cells in the presence of 0.2% n-butanol increased the formation of radioactive phosphatidylbutanol (PtdBut), a specific reporter of phospholipase D (PLD) activity. This means that mastoparan activates both phospholipase C (PLC) and PLD, and thus both pathways could contribute to the increase in PtdOH. To distinguish between them, a differential labelling strategy was applied based on the fact that P-32(i)-label is slowly incorporated into structural phospholipids but rapidly incorporated into ATP. Since PLD hydrolyses a structural lipid, radioactivity only appears slowly in PtdOH(PLD) (and PtdBut). In contrast, PtdOH(PLC) is synthesised by phosphorylation of DAG, and therefore should rapidly incorporate radioactivity. In practice, PtdOH formed on addition of mastoparan was rapidly labelled, reflecting the specific radioactivity of the [P-32]ATP pool. Based on the production of [P-32]PtdBut, we estimate that about 5-17% of the PtdOH was generated through the PLD pathway, while the majority originated from PLC activity. Together, this is the first demonstration (i) that PLC activation is correlated with increases in Ca2+, InsP(3), PtdOH and DGPP, at the cost of PtdInsP and PtdInsP(2), all in one and the same cell, (ii) of the characteristics of stimulated and unstimulated PPI turnover, (iii) that stimulated turnover affects the D-4 PPI and not the 3-isomers, (iv) that PLC and PLD are activated at the same time, (v) of a simple labelling method to discriminate between the two in terms of PtdOH production.