Analysis of hormone‐stimulated phosphatidylinositol synthesis

Agonist‐stimulated phosphoinositide turnover is accompanied by compensatory resynthesis of these lipids. Several lines of evidence suggest that resynthesis of phosphatidylinositol (PtdIns) involves phosphorylation of diacylglycerol (DG) (salvage pathway) rather than acylation of glycerol phosphate (de novo pathway), although a contribution from the de novo pathway has not been ruled out. To determine the relative contribution of the de novo and salvage pathways in stimulated PtdIns resynthesis, an inhibitor of de novo synthesis (Triacsin C) was incubated simultaneously with the hormone agonist. Results indicate that at early times (90 min), hormone‐stimulated PtdIns synthesis proceeds predominantly via the salvage pathway, although some de novo synthesis is also taking place. At later times (24 h), stimulated synthesis is solely via the de novo pathway. Increasing cellular DG content by either adding exogenous DG or treating cells with bacterial phospholipase C (bPLC) results in deacylation of the DG rather than phosphorylation; however, inhibition of this deacylation fails to stimulate phosphorylation by DG kinase (DGK), suggesting channeling of the DG substrate between PLC and DG kinase. Receptor activation is not required for activation of DGK, since treatment with a calcium ionophore induces the same Triacsin C‐insensitive PtdIns synthesis. Depletion of the polyphosphoinositide pools by treatment with wortmannin prevents both hormone and A23187‐induced polyphosphoinositide hydrolysis; however, A23187 is still able to induce hydrolysis of PtdIns and subsequent compensatory resynthesis. The inability of R59949 to inhibit either hormone‐induced or ionophore‐induced PtdIns resynthesis suggests that the α isoform is not involved; however, its possible that the channeling phenomenon prevents the inhibitor from gaining access to the diacylglycerol kinase enzyme. Further study will be required to determine which isoform catalyzes hormone‐induced resynthesis of PtdIns. © 2002 Wiley‐Liss, Inc.