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.

Nitric Oxide Produced in Response to Engagement of β2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

“…Activation of Src tyrosine kinases is the earliest event that occurs during engagement of ␤2 integrins (2), and these tyrosine kinases have been shown to up-regulate the activity of PLC␥2 (leading to activation of PKC and mobilization of calcium) and PI 3-kinase (leading to the generation of phosphatidylinositol 3,4,5-trisphosphate). Although we don't know yet the link that exists between these signaling molecules and activation of iNOS, it might be that they affect the relocalization of iNOS to a specific compartment (68) and/or its interaction with binding partners. We also found that the effect of NO on the regulation of Rap GTPases is mediated through activation of guanylate cyclase, production of cGMP, and activation of cGKI.…”

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

“…Specifically, rat neutrophils constitutively express nNOS and generate higher levels of NO; whereas human neutrophils express low level of enzyme at mRNA and protein level. The previous reports from this laboratory have shown the presence of nNOS and iNOS and their subcellular localization but failed to detect eNOS in peripheral rat PMNs (29,30).…”

“…Control samples were processed similarly as mentioned earlier, omitting the primary antibodies (29).…”

“…The mixture was passed through Dowex 50WX (T-400) columns. Radioactivity in the eluent was measured using beta scintillation counter (LKB Wallace 1409, Liquid Scintillation counter) as described by Saini et al (29). NO synthesis in the neutrophil lysate has been reported as pmol of L-[ …”

“…A total of 5 3 10 6 cells were suspended in protein extraction buffer [0.1 M NaCl, 0.01 M Tris HCl pH 7.4, 0.001 M EDTA pH 7.4, 1 lg/ml aprotinin, 100 lg/ml phenylmethylsulfonyl fluoride (PMSF), Pepstatin 20 lg/ml, Sodium orthovenadate (Na 3 VO 4 ) 2 mM, DFP 1 mM] for 30 min on ice (29). Centrifuged at 13,000 rpm, 20 min, 48C, Protein concentrations were measured by BCA protein assay reagent kit (Pierce).…”