HYDROLYSIS OF CELL-SURFACE INOSITOL PHOSPHOLIPID LEADS TO THE DELAYEDSTIMULATION OF PHOSPHATIDYLINOSITOL SYNTHESIS IN BOVINE AORTIC ENDOTHELIAL-CELLS

In order to address the issue of how inositol phospholipid synthesis i s controlled in a resting cell we looked for enhanced [H-3]phosphatidy linositol (PtdIns) labelling in response to the hydrolysis of cell sur face PtdIns. Bacillus thuringiensis PtdIns-PLC when added to intact bo vine aortic endothelial (BAE) cells rapidly hydrolysed 9.1 +/- 1% of t he total cellular PtdIns. This result suggests that BAE cells have a c ell surface pool of PtdIns. Hydrolysis of cell surface PtdIns, in cont rast to the agonist-stimulated hydrolysis of inner leaflet PtdIns, did not lead to a rapid (minutes) stimulation of PtdIns resynthesis. Prol onged incubation of BAE cells with PtdIns-PLC led to further hydrolysi s of PtdIns (up to 20% of total cellular PtdIns). This second phase of PtdIns-PLC induced hydrolysis was inhibited by the addition of brefel din A suggesting that it was dependent on vesicular traffic to the pla sma membrane from the endoplasmic reticulum. Furthermore, the above re sult suggests that prolonged incubation of intact cells with PtdIns-PL C leads to the slow depletion of intracellular PtdIns stores. This sec ond phase of PtdIns-PLC induced hydrolysis was associated with PtdIns resynthesis since prolonged incubation with PtdIns-PLC, but not B. cer eus PtdCho-PLC (which does not hydrolyse PtdIns), led to enhanced PtdI ns labelling. The results indicate that extracellular PtdIns-PLC induc ed PtdIns resynthesis may occur due to PtdIns-PLC induced intracellula r PtdIns depletion.