title: Interaction of inducible nitric oxide synthase with rac2 regulates reactive oxygen and nitrogen species generation in the human neutrophil phagosomes: implication in microbial killing.
abstract: OBJECTIVE
  Present study explores importance of inducible nitric oxide synthase (iNOS) and its interaction with Rac2 in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, protein-nitration and in microbial killing by neutrophils.
  
  
  RESULTS
  The iNOS transcript and protein were constitutively present in human as well as in mice neutrophils. iNOS protein was found in cytosol, granules containing elastase and gelatinase, and in other subcellular organelles in resting human neutrophils. After phagocytosis of bovine serum albumin (BSA) coated beads, both human and mice neutrophils showed significant elevation in superoxide radicals, nitric oxide (NO), ROS/RNS and consequent BSA nitration. These responses were significantly reduced in presence of iNOS, NADPH oxidase (NOX), myeloperoxidase or Rac inhibitors, as well as in iNOS, Nox2 and Rac2 silenced human or iNOS-knockout mice neutrophils. Complex formed on interaction of iNOS with Rac2 coprecipitated with anti-Rac2, predominantly in cytosol in resting human neutrophils, while iNOS-Rac2 complex translocated to phagosomes after phagocytosis. This was accompanied by generation of superoxide radicals, NO, ROS/RNS and consequent BSA-nitration. Importance of Rac2 in iNOS mediated NO formation and microbial killing was confirmed by pretreatment of mice with Rac inhibitor, NSC23766 that significantly abrogated NO release and microbial killing in vivo.
  
  
  METHODS
  Present study highlights previously undefined role of Rac2-iNOS interaction, in translocation of iNOS to phagosomal compartment and consequent NO, superoxide radicals, ROS/RNS generation, BSA nitration and microbial killing.
  
  
  CONCLUSIONS
  Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. These studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils.
authors.affiliation: 1 Pharmacology Division, CSIR-Central Drug Research Institute , Lucknow, Uttar Pradesh, India .
authors.forename: Anupam
authors.initials: A
authors.lastname: Jyoti
authors.forename: Abhishek K
authors.initials: AK
authors.lastname: Singh
authors.forename: Megha
authors.initials: M
authors.lastname: Dubey
authors.forename: Sachin
authors.initials: S
authors.lastname: Kumar
authors.forename: Rohit
authors.initials: R
authors.lastname: Saluja
authors.forename: Ravi Shankar
authors.initials: RS
authors.lastname: Keshari
authors.forename: Anupam
authors.initials: A
authors.lastname: Verma
authors.forename: Tulika
authors.initials: T
authors.lastname: Chandra
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Kumar
authors.forename: Virendra Kumar
authors.initials: VK
authors.lastname: Bajpai
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Barthwal
authors.forename: Madhu
authors.initials: M
authors.lastname: Dikshit
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000254:growth & development; D006801:Humans; D051379:Mice; D009504:Neutrophils / Q000378:metabolism* / Q000382:microbiology; D009569:Nitric Oxide / Q000097:blood; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism; D060066:Protein Interaction Maps; D026361:Reactive Nitrogen Species / Q000097:blood; D017382:Reactive Oxygen Species / Q000097:blood; D013481:Superoxides / Q000097:blood; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D013481:Superoxides; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein