Bromoenol lactone, an inhibitor of Group V1A calcium-independent phospholipase A 2 inhibits antigen-stimulated mast cell exocytosis without blocking Ca 2+ influx

Calcium-independent phospholipase A 2 (iPLA 2 β) has recently been suggested to regulate Ca 2+ entry by activating store-operated Ca 2+ channels. These studies have been conducted in mast cells using thapsigargin to deplete intracellular stores. In RBL 2H3 and bone marrow-derived mast cells (BMMCs), Ca 2+ entry is critical for exocytosis and therefore we have examined whether the proposed mechanism would be relevant when a physiological stimulus is applied to these cells. Using an iPLA 2 β antibody, we demonstrate that the 84 kDa iPLA 2 β is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA 2 β it was used to probe this potential mechanism. We observe inhibition of exocytosis stimulated either with antigen or with thapsigargin. However, BEL also inhibits exocytosis when stimulated using a Ca 2+ ionophore A23187, which passively transports Ca 2+ down a concentration gradient and also in permeabilised mast cells where Ca 2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen- or thapsigargin-stimulated Ca 2+ signalling, both the release from internal stores and sustained elevation due to Ca 2+ influx. These results cast doubt on the proposed mechanism involving iPLA 2 β required for Ca 2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA 2 β activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis.