Premium
Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies
Author(s) -
Saini R.,
Patel S.,
Saluja R.,
Sahasrabuddhe A. A.,
Singh M. P.,
Habib S.,
Bajpai V. K.,
Dikshit M.
Publication year - 2006
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.0605320
Subject(s) - nitric oxide synthase , immunogold labelling , citrulline , biology , microbiology and biotechnology , endothelial nos , cytoplasm , nitric oxide , subcellular localization , immunocytochemistry , immunoprecipitation , blot , biochemistry , arginine , antibody , immunology , enos , endocrinology , amino acid , gene
Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin‐1, a prominent NOS‐interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L‐[ 3 H] arginine and 4,5‐diaminofluorescein diacetate (DAF‐2DA). Reverse transcriptase‐polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L‐[ 3 H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin‐1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin‐1 in rat PMNs.