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Figure 3


Fig. 3. Phospholipase C causes the depletion of plasma membrane PtdIns(4,5)P2, and the release of Ca2+ from intracellular stores. (A,B) PLC inhibitors prevent mast cell degranulation. RPMCs were pre-incubated with U73-122 and/or U73-343 for 5 minutes (A), or Et-18-OMe (or EtOH, as vehicle control) for 20 minutes (B), at the indicated concentration before stimulation with 48/80 at 25°C. After 10 minutes, the medium was assayed for released ß-hexosaminidase (ß-hex) activity. Data are means of triplicates ± s.e.m. (C) PLC inhibitors block depletion of PtdIns(4,5)P2. Mast cells were untreated (ctrl) or incubated with 5 µM BAPTA/AM or as described in A and B. Subsequently, degranulation was evoked with 48/80 for 30 seconds. Cells were fixed and stained with 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Bar, 10 µm. (D) PLC inhibitors block Ca2+ signalling. RPMCs were loaded for 20 minutes with Fluo3/AM, pre-incubated with the indicated compound as in A-C or for 20 minutes with 5 µM BAPTA/AM, and activated with 48/80. Normalised fluorescence intensity traces are shown from a single representative cell for each condition.





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