Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

Background PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood. Methodology/Principal Findings We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca 2+ release and causing auto-regulation of eNOS through S-glutathionylation. Conclusions/Significance The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca 2+ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.