Bromoenol lactone, an inhibitor of Group V1A calcium-independent phospholipase A2 inhibits antigen-stimulated mast cell exocytosis without blocking Ca2+ influx.

Calcium-independent phospholipase A2 (iPLA2beta) has recently been suggested to regulate Ca2+ entry by activating store-operated Ca2+ 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), Ca2+ 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 iPLA2beta antibody, we demonstrate that the 84kDa iPLA2beta is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA2beta 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 Ca2+ ionophore A23187, which passively transports Ca2+ down a concentration gradient and also in permeabilised mast cells where Ca2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen- or thapsigargin-stimulated Ca2+ signalling, both the release from internal stores and sustained elevation due to Ca2+ influx. These results cast doubt on the proposed mechanism involving iPLA2beta required for Ca2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA2beta activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis.

Reconstitution system based on cytosol-depleted cells to study the regulation of phospholipase D.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membrane-associated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes--PLD1 and PLD2--have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5'-[gamma-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.

Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2+, degranulation, and membrane ruffling in the cultured rat basophilic leukemia-2H3 mast cell line.

Mast cells are key regulators in allergy and inflammation, and release histamine, cytokines, and other proinflammatory mediators. In the classical view, IgE acts merely to prime mast cells, attaching to FcepsilonRs but not evoking any cell signaling response until cross-linked by the presence of a multivalent allergen. However, several recent studies have reported that IgE alone can promote cell survival and cytokine production in the absence of cross-linking by allergen. In this study we demonstrate that acute addition of monomeric IgE elicits a wide spectrum of responses in the rat basophilic leukemia-2H3 mast cell line, including activation of phospholipases Cgamma and D, a rise in cytosol Ca(2+), NFAT translocation, degranulation, and membrane ruffling within minutes. Calcium transients persist for hours as long as IgE is present resulting in the maintained translocation of the transcription factor NFAT to the nucleus. Removal of IgE reverses the signaling processes. Our results indicate that, far from simply preparing the cells for a response to allergen, monomeric IgE can stimulate signaling pathways that lead to degranulation, membrane ruffling, and NFAT translocation. The mechanism of activation is likely to be via aggregation of the FcepsilonR1 because activation by IgE can be inhibited with monovalent hapten.

Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid.

We have examined the specificity of oleate as an activator of phospholipase D2 (PLD2) and whether it can be used to study PLD2 localization and its involvement in cell function. Oleate stimulates PLD activity in intact RBL-2H3 mast cells. Comparing PLD1- with PLD2-overexpressing cells, oleate enhanced PLD activity only in PLD2-overexpressing cells. Membranes were also sensitive to oleate and when membranes prepared from PLD1- and PLD2-overexpressing cells were examined, oleate further increased PLD activity only in membranes from PLD2-overexpressing cells. Overexpressed green fluorescent protein (GFP)-PLD2 fusion protein was localized at the plasma membrane and GFP-PLD1 was found in an intracellular vesicular compartment. Oleate was used to examine whether overexpressed PLD2 co-localized with endogenous PLD2. RBL-2H3 mast cell homogenates were fractionated on a linear sucrose gradient and analysed for both oleate-stimulated activity and ADP ribosylation factor 1-stimulated PLD1 activity. The oleate-stimulated activity co-localized with markers of the plasma membrane including the beta-subunit of the FcepsilonRI and linker for activation of T cells. Fractionation of homogenates from PLD2-overexpressing cells demonstrated that the overexpressed PLD2 fractionated in an identical location to the endogenous oleate-stimulated activity and this activity was greatly enhanced in comparison with control membranes. Examination of membranes prepared from COS-7, Jurkat and HL60 cells indicated a relationship between oleate-stimulated PLD2 activity and PLD2 immunoreactivity. We examined whether oleate could be used to activate secretion and membrane ruffling in adherent RBL-2H3 mast cells. Oleate did not stimulate secretion but did stimulate membrane ruffling, which was short-lived. We conclude that oleic acid is a selective activator of PLD2 and can be used for localization studies, but its use as an activator of PLD2 in intact cells to study function is limited due to toxicity.