Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies

Abstract

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-[³H] 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-[³H] 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.