Abstract
BackgroundPABA/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 FindingsWe 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 Ca2+-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca2+ release and causing auto-regulation of eNOS through S-glutathionylation.Conclusions/SignificanceThe 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 Ca2+ 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.
Highlights
Endogenous nitrogen monoxide (NO) is a potent signaling molecule influencing numerous physiological functions
HL60 cells as compare to that of the short- and long-lived standard NO generators Figure 1 shows the intracellular generation of NO in HL60 cells exposed to either PABA/NO or diethylenetriamine/nitric oxide adduct (DETA/NO)
The kinetics of NO release after PABA/NO are shown in Fig. 1, panel A
Summary
Endogenous NO is a potent signaling molecule influencing numerous physiological functions. Cellular levels of NO are controlled by several isoforms of nitric oxide synthase (NOS): neuronal (nNOS, NOS1), inducible (iNOS, NOS2), and endothelial (eNOS, NOS3). NNOS and eNOS, are constitutively expressed and primarily isolated from neurons and endothelial cells, respectively. NO generation by these enzymes is controlled by the elevation of intracellular Ca2+ and the consequent activation of calmodulin (CaM). High levels of intracellular NO are toxic and provide a translational opportunity to induce cytotoxicity in tumor cells [2]. This led to the development of a class of anticancer agents selectively activated in tumors by glutathione S-transferase pi (GSTP) to liberate toxic levels of NO [3]. Its mechanism of action and effect on cells is not yet fully understood
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