[Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules]

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine to the granules caused an increase in DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines on DPI synthesis in the rat mast cell granules was dose-dependent and maximal effects were observed at 1 mM spermine and 10 mM spermidine respectively. When the effect of 1 mM spermine on 32P incorporation into DPI in rat mast cell granules was investigated serially, 32P incorporation into DPI in rat mast cell granules incubated with spermine for 15 min was enhanced significantly (p less than 0.05) compared with that in the granules in the absence of spermine.