Ion channel modulators mediated alterations in NO-induced free radical generation and neutrophil membrane potential

Patel, Satyananda; Vemula, Jayaraj; Konikkat, Salini; Barthwal, Manoj Kumar; Dikshit, Madhu

doi:10.1080/10715760902887276

Cited by 16 publications

(13 citation statements)

References 43 publications

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“…1A and 1B). We found that at same concentrations, SNP was less effective than SNAP, in tune with reported slow release of NO by SNP (22). Generation of ROS was also confirmed by using an antioxidant, NAC (Fig.…”

Section: Discussionmentioning

confidence: 50%

“…In this study, we have used commonly utilized NO donors, SNP and SNAP to explore reactive species modulation (14,15,17,19,21,22). Addition of NO donor, SNP or SNAP to the neutrophil suspension augmented ROS generation in a concentration-and time-dependent manner, as measured by DCF (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Neutrophils have also been coupled commonly with inflammatory conditions, which are mostly associated with the accumulation of NO and its metabolites. Various reports demonstrated NOmediated biphasic regulation (14,16,17), stimulation (15,22), and even inhibition (18,20) of PMNs reactive species formation. These outcomes could be due to the differences in probes used to detect ROS/RNS, which exhibit different reactivity, specificity, and selectivity, and some probes even interact with NO (25,26).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, NO suppressed NADPH oxidase-dependent superoxide production by S-nitrosylation of p47 subunit in human endothelial cells (21). We recently reported augmentation in the generation of ROS even with micromolar concentrations of NO (22). These differences in the interpretation were due to the use of different probes being used for the assessment of reactive species formation.…”

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Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

et al. 2010

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Previous reports from this laboratory and others demonstrated NO-mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/ methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO-mediated enzymatic reactive species formation by using multiple probes, human neutrophils/ HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM-1 mM) to PMNs suspension, exhibited a concentration-and time-dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO-nitrone adduct, dityrosine and rhodamine-123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL-60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO-dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7-NI). However, 7-NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO-mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO-mediated modulation of reactive oxygen species in particular in various pathological situations. ' 2010 International Society for Advancement of Cytometry

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Section: Discussionmentioning

confidence: 50%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

et al. 2010

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“…Interestingly, NO was later identified to modulate diverse signaling cascades in neutrophil to regulate various functions such as adhesion, chemotaxis, phagocytosis, respiratory burst, apoptosis, and PMN-mediated bacterial killing or tissue damage (18). Previous studies from this laboratory have also demonstrated NO-mediated modulation of neutrophil free radical generation (19)(20)(21)(22).…”

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

et al. 2010

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Previous studies from this laboratory have demonstrated importance of neutrophilderived 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. ' 2010 International Society for Advancement of Cytometry Key terms nitric oxide; NOS; neutrophils; neutrophil precursor cells; maturation NEUTROPHILS (PMNs) are orchestrating cells of the innate immune system (1,2), circulating through the body and extravasating to the sites of infection and injury, to perform important roles in the host defense. This process involves recruitment of lysosomes and various types of granules to release proteolytic enzymes, antimicrobial peptides, and free radical formation (1,2). PMN development in the bone marrow has classically been divided into six stages myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), band cells (BCs), and segmented neutrophil on the basis of cell size, nuclear morphology, and granule content (3,4). Production of these cells is continuous to provide the continual demand for the tissues and mostly to maintain the circulating pool in the blood.Nitric oxide (NO), a versatile signaling molecule, mediates immune response, vasodilation, neurotransmission, proliferation, and apoptosis (5-8). NO is synthesized by a class of NADPH-dependent N...

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Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential

et al. 2011

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Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and assessment of NO-mediated free radical formation by using a consistent NO donor, DETA-NONOate. NO-mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO-mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO-or PMA-mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1b and IL-8, while with THP-1 cells, release of IL-1b, IL-8, and TNFa was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines. ' 2011 International Society for Advancement of Cytometry Key terms nitric oxide; neutrophil extracellular traps; NADPH-oxidase; myeloperoxidase; inflammatory cytokine; mitochondrial and nuclear DNA

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Ion channel modulators mediated alterations in NO-induced free radical generation and neutrophil membrane potential

Cited by 16 publications

References 43 publications

Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

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

Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential

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