Thapsigargin-induced mast cell degranulation requires PI3Kγ, but not GPCR signaling.

Abstract

<p>(A) Granule release of wild type and p110γ^−/− BMMCs was determined detecting β-hexosaminidase (β-Hex) release into extracellular media. BMMC stimulation with IgE/antigen was initiated with the antigen (Ag, DNP-HSA at 10 ng/ml; 100 ng/ml IgE overnight). Alternatively, BMMCs were stimulated by the addition of thapsigargin (1 µM). Where indicated, BMMCs were preincubated for 15 min with 100 nM wortmannin. Released β-Hex was quantified 20 min after stimulation, and is expressed as mean ± standard error of the mean (SEM) (<i>n</i> = 3; <i>p</i>-values in all figures are * or &: <i>p</i><0.05, **: <i>p</i><0.005; ***: <i>p</i><0.0005; * depict here comparison with respective wild type control; & refer to comparison of untreated versus treated samples). (B) Granule release was assessed as above, but ADA (10 units/ml) was added to BMMC suspensions 1 min before stimulation where depicted. (C) Wild type or p110γ^−/− BMMCs were stimulated with adenosine (Ade; 1 µM) or thapsigargin (1 µM) for 2 min, and phosphorylation of PKB/Akt on Thr308 (pPKB), total PKB and p110γ was analyzed by Western blotting. BMMCs were incubated in starving medium (2% FCS, without IL-3) for 3 h before stimulation, and pretreated with ADA where indicated. (D) Heterotrimeric Gα_i proteins were inactivated by preincubation of wild type and p110γ^−/− BMMCs with 100 ng/ml <i>P</i>Tx for 4 h, before thapsigargin (Tg) or adenosine was added as in (C).</p