Functional and molecular characterization of NOS isoforms in rat neutrophil precursor cells

Previous studies from this laboratory have demonstrated importance of neutrophil-derived nitric oxide (NO) in free radical generation, characterized nitric oxide synthase (NOS) isoforms, and have reported subcellular distribution of NOS in rat peripheral neutrophils. Maximum number of neutrophils are added per day to the circulation from bone marrow, thus neutrophils might add substantial amount of NO in the bone marrow. NO generating ability and NOS isoforms characteristics in bone marrow neutrophil precursor cells is, however, still unexplored. This study was, therefore, undertaken to investigate NO generation ability and the molecular/biochemical characteristics of NOS isoforms in neutrophil precursor cells. The neutrophil precursors were separated on Percoll density gradient and characterized by Giemsa staining, CD markers, and by their size and granularity at various stages of maturation as Bands 1, 2, and 3. Mature neutrophils were efficient in free radical generation and phagocytosis, whereas immature cells had more mitochondria and myeloperoxidase. Amount of NO augmented from immature to mature neutrophils as assessed by fluorescent probe DAF-2DA and Griess reagent. Measurement of NOS enzyme activity further confirmed the functional status of NOS in these cells. NOS isoforms were differentially expressed during neutrophil maturation as confirmed by enzyme activity, Western blotting, flowcytometry, and RT-PCR. Expression of nNOS was predominantly stable in all the stages of neutrophil maturation. iNOS expression was, however, consistently augmented during maturation, whereas eNOS expression was downregulated with neutrophil maturation. Furthermore, all NOS isoforms proteins were distributed in cytosol as well as nucleus as assessed by confocal microscopy. This study for the first time report biochemical and molecular characteristics of NOS isoforms in rat neutrophil precursor cells.