Phosphorylation of lipids in rat primary glial cells and immortalized astrocytes (DITNC)

Phosphatidylinositol 4,5-bisphosphate, a substrate in the signal transduction pathway involving phospholipase C, is synthesized in cell membranes by phosphorylation of phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI-4-P). Incubation with [gamma-32P]adenosine triphosphate of membranes isolated from primary glial cells in culture and from an immortalized astrocyte cell line (DITNC) and subsequent separation of the lipids by high-performance thin-layer chromatography revealed a number of labeled lipid bands. Further analysis of the deacylated products by high-performance liquid chromatography indicated the presence of two PI-4-P bands and a band corresponding to lysoPIP (PIP, PI phosphate). The two PI-4-P bands were more prominent in the samples from astrocyte cell membranes than from synaptic plasma membranes and plasma membranes. Analysis of the fatty acid composition of PI by gas-liquid chromatography revealed that both 20:3n-9 and 20:4n-6 were present in PI of cultured cell membranes, whereas the brain membranes contained exclusively 20:4n-6. The two PI-4-P bands in these membranes can be attributed to the presence of different molecular species of PI. Assignment of the fraction corresponding to lysoPIP was consistent with the observation that an increase in label in this lipid band occurred upon incubation of DITNC cell supernatant with lysoPI (1-stearoyl PI). This suggests that endogenous lysoPI present in the cell membranes can be phosphorylated to form lysoPIP.