The kinetics of phosphoinositide hydrolysis in rat basophilic leukemia (RBL-2H3) cells varies with the type of IgE receptor cross-linking agent used.

We have re-examined, by high pressure liquid chromatographic (HPLC) procedures, the hydrolysis of 3H-labeled inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells. Previous studies showed no clear correlation between the release of any particular inositol metabolite and the calcium signal in these cells. Paradoxically no responses were observed when the cells were stimulated with the antigen, aggregated ovalbumin, in the absence of external Ca2+. We report here that in the absence of external Ca2+ aggregation of the IgE receptor by agents other than aggregated ovalbumin causes the release of small amounts of [3H]inositol phosphates and a small increase in levels of cytosol Ca2+ (approximately 25 nM). The response, however, varied with the type of stimulant used. Within seconds after addition of 24 mol of dinitrophenol conjugated with 1 mol of bovine serum albumin to cells primed with dinitrophenol-specific IgE there was a small burst in release of [3H]inositol 1,4,5-triphosphate, [3H]inositol 1,3,4,5-tetrakisphosphate, and [3H]inositol 1,3,4-trisphosphate which was followed by a gradual rise in inositol 1,3,4-trisphosphate, inositol bisphosphate, and inositol monophosphate. Eventually, all inositol phosphates reached different steady state levels which were maintained for at least 40 min. In contrast, the initial response to oligomeric IgE, which aggregates receptors at a relatively slow rate, was muted although the subsequent development of the response was the same. The levels of inositol pentakisphosphate and hexakisphosphate remained unchanged. These and other studies with cell extracts support the conclusion that inositol 1,4,5-trisphosphate, a putative messenger for release of intracellular Ca2+, was converted to inositol 1,3,4,5-tetrakisphosphate and thence to inositol 1,3,4-trisphosphate. Both trisphosphate metabolites were dephosphorylated in sequential fashion by phosphatase enzymes in the cytosolic and membrane fractions. However, the appearance of several isomers of inositol monophosphates and bisphosphates suggested that degradation proceeded through multiple pathways in the intact cell.