Endothelial nitric oxide synthase is essential for nitric oxide generation, L-type Ca2+ channel activation and survival in RBL-2H3 mast cells

Abstract

Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Cav1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcɛRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcɛRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Cav1.2 LTCC activation. FcɛRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Cav1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Cav1.2 LTCC activation but not CRAC channel activation. Similar to Cav1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcɛRI engagement.