Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.