Nitric Oxide Synthase - Nitric Oxide Involvement in the Human Neutrophil Free Radical Generation: Role of iNOS and Rac2 Interaction
Authors: Jyoti Anupam , Singh Abhishek , Keshari Ravi , Kumar Sachin , Saluja Rohit , Dubey Megha , Verma Anupam , Bajpai Virendra , Barthwal M K , Tripathi Anil K , Dikshit Madhu 
Published: October 2019

Abstract
Abstract 1036

Aims:
Previous reports from this lab demonstrated importance of nitric oxide (NO) in augmenting neutrophils (PMNs) free radical generation. To explore further, present study investigated interaction of nitric oxide synthase (NOS) with Rac2 in resting and PMA activated human PMNs, and also nitration and nitrosylation of BSA following phagocytosis of coated beads.

Results:
Rac2 and iNOS interaction was evident in the cytosolic fraction of resting cells, which was augmented significantly in the membrane fraction of PMA treated PMNs. Nitration/nitrosylation of BSA, implicated functional importance of iNOS, and interaction of NO with NADPH-oxidase (NOX) and myeloperoxidase (MPO) derived species, as it was reduced significantly by the inhibitors of NOX (VAS), MPO (ABAH) and iNOS (1400W). Moreover, studies to assess the molecular characteristics and sub-cellular distribution of iNOS using standard methodologies, demonstrated iNOS transcript (RT-PCR, Real time PCR) and protein (immunoprecipitation and WB) in PMNs. iNOS protein was distributed in the granules, mitochondria, cytosol, nucleus and plasma membrane (immuno-electron microscopy and confocal microscopy) of human PMNs.
Molecular characteristics, sub-cellular distribution of iNOS and its interaction with Rac2 are herein shown for the first time in human neutrophils. This study also elucidates the functional importance of iNOS-Rac2 interaction and highlighted the role of iNOS in nitration and nitrosylation of BSA following phagocytosis.

Conclusions:
Altogether these results suggest the functional importance of iNOS-Rac2 interaction in the nitration and S-nitrosylation of phagocytosed pathogens.
Association of iNOS with Rac2 and its migration to the membrane in activated PMNs (A) Total cell lysate of human PMNs (1 × 108 cells) were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-iNOS (Lane 1) or anti-nNOS antibody (Lane 4). Lanes 2 and 5 – IP with rabbit IgG (negative control for anti-Rac2). iNOS recombinant protein (Lane 3) and rat brain homogenate protein (Lane 6) were used as positive controls for iNOS (130 kDa) and nNOS (160 kDa) respectively. Lanes 7 and 8 were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2. Inducible NOS and Rac2 interaction in the cytosol and membrane fractions of resting, PMA and NSC23766 with PMA stimulated cells. Both cytosol and membrane fractions were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2 (B) and anti-iNOS (C). The data represent 5 independent experiments, and are presented as Mean ± SEM (For Rac2, *** p<0.001 for control cytosol vs membrane fraction in control PMNs, * p<0.01 for cytosol vs membrane fraction in PMA treated PMNs or cytosol vs membrane fraction in NSC23766 with PMA treated PMNs, @p<0.01 for PMA vs control in cytosolic fraction and @@p<0.01 for PMA vs control in membrane fraction or NSC23766 PMA vs control in membrane fraction). (For iNOS, *** p<0.01 for cytosol vs membrane fraction in control PMNs or cytosol vs membrane fraction in NSC23766 with PMA treated PMNs, * p<0.01 for cytosol vs membrane fraction in PMA treated PMNs @p<0.05 for PMA cytosolic vs control cytosolic fraction or in PMA membrane vs control membrane fraction).

Disclosures:
No relevant conflicts of interest to declare.