Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate

Sugimoto, Kenkichi; Fujii, Sachiko; Takemasa, Tohru; Yamashita, Kazuo

doi:10.1007/s004180000151

Cited by 49 publications

(17 citation statements)

References 24 publications

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“…In these experiments, 500 mM H 2 O 2 was applied in instantaneous ROS dynamics observations lasting for 5 min (to collect fluorescence signal with enough intensity changes in confocal microscope), while 0e500 H 2 O 2 was applied in others studies lasting for several hours. (Bindokas et al, 1996); DAF/AM can also specifically react with intracellular NO (Sugimoto et al, 2000). Unfortunately, there is currently no specific probe for H 2 O 2 in living cells.…”

Section: Methodsmentioning

confidence: 99%

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress‐induced mitochondrial dysfunction

Lü

Gong

2009

Cell Biology International

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Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H(2)O(2)), superoxide radical (O(2)(*)(-)), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H(2)O(2) significantly increased the production of intracellular H(2)O(2), O(2)(*)(-), and NO (P<0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.

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Section: Methodsmentioning

confidence: 99%

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress‐induced mitochondrial dysfunction

Lü

Gong

2009

Cell Biology International

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“…The animals were then anesthetized in seawater containing 2% ethanol, dissected to expose the ventral surface of the light organ, and viewed with a Zeiss 510 laser scanning confocal microscope (LSM). In experiments for the detection of NO in fixed tissues (Sugimoto et al ., 2000), hatchlings were fixed as described (see above, The detection of NOS activity by the NADPH‐diaphorase reaction ), rinsed with mPBS, stained with DAF, and viewed by LSM. Physiological analyses of V. fischeri have shown that it neither produces NO nor uses it as a terminal electron acceptor (E.G.…”

Section: Methodsmentioning

confidence: 99%

NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association

et al. 2004

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SummaryDuring colonization of the Euprymna scolopes light organ, symbiotic Vibrio fischeri cells aggregate in mucus secreted by a superficial ciliated host epithelium near the sites of eventual inoculation. Once aggregated, symbiont cells migrate through ducts into epithelium-lined crypts, where they form a persistent association with the host. In this study, we provide evidence that nitric oxide synthase (NOS) and its product nitric oxide (NO) are active during the colonization of host tissues by V. fischeri. NADPHdiaphorase staining and immunocytochemistry detected NOS, and the fluorochrome diaminofluorescein (DAF) detected its product NO in high concentrations in the epithelia of the superficial ciliated fields, ducts, and crypt antechambers. In addition, both NOS and NO were detected in vesicles within the secreted mucus where the symbionts aggregate. In the presence of NO scavengers, cells of a non-symbiotic Vibrio species formed unusually large aggregates outside of the light organ, but these bacteria did not colonize host tissues. In contrast, V. fischeri effectively colonized the crypts and irreversibly attenuated the NOS and NO signals in the ducts and crypt antechambers. These data provide evidence that NO production, a defense response of animal cells to bacterial pathogens, plays a role in the interactions between a host and its beneficial bacterial partner during the initiation of symbiotic colonization.

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“…Intracellular NO was examined in HUVECs loaded with NOsensitive fluorescent dye diaminofluorescein-2 diacetate (DAF2DA; 5 lmol/L), with or without acetylcholine (ACh) treatment (1 lmol/L, 20 minutes). 19 In transfected HUVECs, NO was detected by using 4-amino-5-methylmino-2 0 ,7 0 -difluorofluorescein (DAF-FM) diacetate. Nuclei were stained with 4 0 ,6-diamidino-2-phenylindole (DAPI; 1 lmol/L).…”

Section: Confocal Fluorescence Microscopymentioning

confidence: 99%

Glutathionylation Mediates Angiotensin II–Induced eNOS Uncoupling, Amplifying NADPH Oxidase‐Dependent Endothelial Dysfunction

Galougahi

Liu

Gentile

et al. 2014

JAHA

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BackgroundGlutathionylation of endothelial nitric oxide synthase (eNOS) “uncouples” the enzyme, switching its function from nitric oxide (NO) to O2•− generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)‐induced endothelial dysfunction.Methods and ResultsAng II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2•− generation. Ang II‐induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin‐1. Moreover, Ang II‐induced increase in O2•− and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds‐tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II‐induced impairment of endothelium‐dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2•−, improved endothelium‐dependent vasorelaxation and reduced blood pressure.ConclusionsUncoupling of eNOS by glutathionylation is a key mediator of Ang II‐induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II‐induced O2•− generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation‐dependent eNOS uncoupling.

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Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate

Cited by 49 publications

References 24 publications

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress‐induced mitochondrial dysfunction

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress‐induced mitochondrial dysfunction

NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association

Glutathionylation Mediates Angiotensin II–Induced eNOS Uncoupling, Amplifying NADPH Oxidase‐Dependent Endothelial Dysfunction

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