Molecular, biochemical characterization and localization of neuronal nitric oxide synthase in human neutrophil

Abstract

Nitric oxide (NO) is a potent free radical with diverse roles in regulating several important functions of neutrophils (PMNs), including chemotaxis, adhesion, aggregation, apoptosis, bacterial killing, however the expression status, biochemical characterization and localization study remains the least defined in human PMNs. The present study was attempted to characterize expression of NOS isoforms and NO generation potential in healthy human PMNs. Real time PCR using NOS primers have identified neuronal NOS (nNOS) as higher copy number (45 fold, *p<0.001) than inducible NOS (iNOS). Identification of nNOS PDZ domain (Exon‐2, N‐terminal) was done by RT‐PCR using exon‐2 specific primer and Western blotting using N‐terminal specific antibody. Localization of nNOS was explored by electron and confocal microscopy. Biochemical study involving NOS activity (L‐Arginine to L‐Citruline conversion) exhibited higher calcium dependent activity (0.21±0.05 pmol/30 min./107cells) than calcium independent (0.039±.007 pmol/30 min/107) which positively correlated with higher expression of nNOS. Immunocytochemical studies exhibited distribution of nNOS isoform primarily in the cytosol, granules, mitochondria and nucleus. The results thus suggest constitutive expression of PDZ domain containing nNOS localized mainly to cytoplasm contributes to calcium dependent activity and NO generation in human PMNs.