Discrete generations of intracellular hydrogen peroxide and superoxide in antigen-stimulated mast cells: Reciprocal regulation of store-operated Ca 2+ channel activity

Abstract

Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (FcɛRI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca 2+ channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca 2+ channel (SOC) activity is regulated by superoxide (O 2 •−) and hydrogen peroxide (H 2O 2) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O 2 •− and H 2O 2, respectively, in antigen-stimulated cells. The H 2O 2 generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca 2+, and the FcɛRI β-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O 2 •− generation was strictly dependent on extracellular Ca 2+, but negatively regulated by the SFK and PI3K activities. Inhibition of O 2 •− generation resulted in increased H 2O 2 generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca 2+ store depletion. On the contrary, inhibition of H 2O 2 generation resulted in increased intracellular O 2 •− generation and augmented SOC activity. The findings suggest that O 2 •− and H 2O 2, which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca 2+ signaling in mast cells.