Lam Rebecca S, Shumilina Ekaterina, Matzner Nicole, Zemtsova Irina M, Sobiesiak Malgorzata, Lang Camelia, Felder Edward, Dietl Paul, Huber Stephan M, Lang Florian
Department of Physiology, University of Tübingen, Germany.
Cell Physiol Biochem. 2008;22(1-4):169-76. doi: 10.1159/000149794. Epub 2008 Jul 25.
Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation.
抗原刺激肥大细胞免疫球蛋白E受体（FcepsilonRI）会导致钙离子内流增加，随后肥大细胞脱颗粒并释放炎症介质。钙离子进一步激活钙激活钾通道，进而为钙离子内流提供电驱动力。由于此前已表明磷脂酰肌醇（PI）-3激酶是肥大细胞激活和脱颗粒所必需的，我们探究了肥大细胞钙离子及钙激活钾通道是否对PI3激酶活性敏感。在用PI3激酶抑制剂LY-294002（10μM）和渥曼青霉素（100 nM）处理或未处理的小鼠骨髓来源肥大细胞中，进行了全细胞膜片钳实验以及用于测定胞质钙离子浓度的Fura-2荧光测量。PI3激酶抑制后，抗原刺激的钙离子内流显著减少，但细胞内钙库释放钙离子不受影响。TRPV阻滞剂钌红（10μM）进一步抑制了钙离子内流。在用毒胡萝卜素耗尽钙库后重新添加钙离子时，LY-294002再次降低了钙离子内流，表明其抑制了钙库操纵性通道（SOCs）。此外，PI3激酶抑制消除了IgE刺激的钙激活钾通道，但不影响离子霉素诱导的刺激。这些观察结果揭示了PI3激酶对钙离子内流和钙激活钾通道的依赖性调节，而这又参与触发肥大细胞脱颗粒。
