Formation of phosphatidylinositol-4-phosphate in human peripheral blood eosinophils

To evaluate the presence of phosphatidylinositol-4-phosphate in peripheral blood eosinophils, venous blood was drawn from normal healthy volunteers. The eosinophils were isolated on a Percoll gradient and were incubated with [gamma32P]ATP in the presence of Mg2+. After stopping the reaction, lipid extraction was performed with acidic medium and phospholipids were separated by thin-layer chromatography on 1% (w/v) oxalic acid- and potassium oxalate-impregnated silica gel plates. Considerable amounts of radioactivity were found to be incorporated into phosphatidylinositol-4-phosphate on both plates. This reaction requires ATP and Mg2+, and maximal response is seen at 10 microM ATP and 20 mM Mg2+. The reaction is dependent upon the time and temperature of the assay system. No significant superoxide anion generation from the eosinophils incubated with ATP at the concentrations used in the study was observed. These results suggest the possible presence of phosphatidylinositol kinase which catalyses the formation of phosphatidylinositol-4-phosphate from endogenous phosphatidylinositol in human peripheral blood eosinophils.