2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Lakshmi, Vijaya M.; Zenser, Terry V.

doi:10.1016/j.lfs.2006.10.010

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…The NO donor SNP [33], [34] served as NO positive control and applied to analyze its effects on the examined parameters. The NO scavenger cPTIO [35] oxidizes NO to nitrite [36] and was therefore used as a negative control to study the effects of diminished NO content.…”

Section: Methodsmentioning

confidence: 99%

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

et al. 2013

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BackgroundRed blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.Methods/FindingsBlood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.Conclusion/SignificanceThis study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.

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Section: Methodsmentioning

confidence: 99%

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

et al. 2013

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“…While testing whether the activity involves NO synthase by applying a NOSͲinhibitor is straightforward (see below), untangling which species accounts for the fluorescence increase can be challenging. [39]. This may also occur with DAFs, leading to false-negative results by enhancing rather than quenching fluorescence.…”

Section: Caveatsmentioning

confidence: 99%

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Cortese‐Krott

Rodriguez-Mateos

Kuhnle

et al. 2012

Free Radical Biology and Medicine

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Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4ͲaminoͲ5Ͳ methylaminoͲ2',7'Ͳdifluorofluorescein (DAFͲFM), has gained increasing popularity in recent years due to its improved NOͲsensitivity, pHͲstability, and resistance to photoͲbleaching compared to the firstͲ generation compound, DAFͲ2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAFͲFMͲT and DAFͲ2ͲT, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAFͲFMͲT formation using an array of fluorescenceͲbased methods (laserͲscanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversedͲphase HPLC and LCͲ MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

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“…is an oxidant [48,50] and has a similar structure to MTS and MTT [46,51]. Thus, at high concentrations cPTIO competes with MTS and MTT for electrons, leading to reduced transformations of MTS and MTT to formazan, leading to decrease of absorbance values.…”

Section: Journal Of Adenocarcinoma Issn 2572-309xmentioning

confidence: 99%

In Vitro Evaluation of Hepg2 Cell Proliferation Altered by Reactive Oxygen and Nitrogen Species

Li¹

2016

J Adenocarcinoma

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This study was to investigate cell proliferation regulated by reactive oxygen and nitrogen species and their scavengers. Earlier conclusions are paradoxical on roles that reactive oxygen and nitrogen species and their scavengers enhance or inhibit cancer cell proliferations. This study employed the MTS assay to evaluate proliferations of HepG2 cells treated with various concentrations of two different nitric oxide donors, hydrogen peroxide and their scavengers for 24 hours. The MTS assay revealed that low concentrations of SNP, SNAP and H 2 O 2 can significantly enhance HepG2 cell proliferation, and high concentrations of cPTIO significantly inhibited HepG2 cell proliferation. Additionally, the assay also indicated that 100 µM H 2 O 2 , 100 µM cPTIO or 100 U/mL catalase did not have evident synergisms with NSP or SNAP. However, due to reactions of the chemicals with reagents of kits of the MTS assay, the data were not inferred that low concentrations of NO and H 2 O 2 enhance proliferation, whereas high concentrations inhibit proliferation, and whether there are synergistic effects with the combination of SNP (or SNAP) and H 2 O 2 , cPTIO or catalase.

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2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Cited by 8 publications

References 25 publications

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

In Vitro Evaluation of Hepg2 Cell Proliferation Altered by Reactive Oxygen and Nitrogen Species

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