title: Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase.
abstract: 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.
  
  
  RESULTS
  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 Ca2+-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca2+ release and causing auto-regulation of eNOS through S-glutathionylation.
  
  
  CONCLUSIONS
  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 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.
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina, United States of America.
authors.forename: Yefim
authors.initials: Y
authors.lastname: Manevich
authors.forename: Danyelle M
authors.initials: DM
authors.lastname: Townsend
authors.forename: Steven
authors.initials: S
authors.lastname: Hutchens
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Tew
mesh_terms: D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D001391:Azo Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D018922:HL-60 Cells; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D062366:para-Aminobenzoates
chemical_list: D001391:Azo Compounds; D008565:Membrane Proteins; D020030:Nitric Oxide Donors; C485370:O(2)-(2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate; D013438:Sulfhydryl Compounds; D013449:Sulfonamides; C452539:diazeniumdiolate; D062366:para-Aminobenzoates; D009569:Nitric Oxide; C017967:W 7; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005978:Glutathione; D002118:Calcium; D010129:4-Aminobenzoic Acid