Nitric Oxide positively regulates Ag (I)-induced Ca(2+) influx and mast cell activation: role of a Nitric Oxide Synthase-independent pathway

Abstract

NO is generated by NOS activity and known to act as a negative regulator of mast cell activation. We reported previously that Ag (I) directly evokes mast cell degranulation and LTC(4) release via Ca(2+) influx through thiol-sensitive, store-independent channels. Here, we report that NO generated independently of NOS activity mediates the store-independent Ca(2+) influx. Exposure of mast cells to Ag (I) resulted in increased intracellular NO levels and NO(2)(-)/NO(3)(-) contents in the extracellular fluid. The NO increase was blocked by NO scavenger Hb and DTT but not by NOS inhibitors such as amino-BH(4) and L-NAME. This NO production occurred independently of the Src family kinase and PI3K activities, both of which were necessary for antigen-induced, NOS-dependent NO production. Hb and DTT reduced Ag (I)-induced beta-hexosaminidase release and LTC(4) release, whereas the NO scavengers and NOS inhibitors augmented antigen-induced mediator release. Moreover, Hb and DTT, but not the NOS inhibitors, abolished the Ag (I)-induced Ca(2+) influx, and none of the drugs blocked CRAC channel activity. Finally, Ag (I)-induced Ca(2+) influx was distinct from LTCC activity in terms of its sensitivities to wortmannin and LTCC antagonists and the effects of Ca(v)1.2 LTCC gene silencing. These data show that NOS-independent NO regulates mast cell activation positively via a unique store-independent Ca(2+) influx pathway. The present findings suggest multiple sources and functions of NO in mast cell biology.