Inhibitory Effects of Catecholamines and Anti-Oxidants on the Fluorescence Reaction of 4,5-Diaminofluorescein, DAF-2, a Novel Indicator of Nitric Oxide

Nagata, Naoko; Momose, Kazutaka; Ishida, Yukisato

doi:10.1093/oxfordjournals.jbchem.a022333

Cited by 87 publications

(60 citation statements)

References 13 publications

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“…Prior investigations have called attention to the quenching of DAF-2 nitrosation by naturally occurring reducing agents such as Asc and GSH [7,46,67]. Consistent with these findings, our …”

Section: Do Antioxidants Such As Gsh or Asc Affect Daf Imaging?supporting

confidence: 91%

“…This chemical modification of the dye by a secondary product of NO is associated with a lowering of the energetic charge transfer state below the ground state of the fluorophore, thus removing the quenching effect of the benzoic structure and allowing the fluorophore to emit efficiently. The high yield of fluorescence of the triazole forms of DAF and DAR make these indicators suitable for the detection of NO production in biological systems, a property that has been exploited by now in many studies using fluorometry [6][7][8][9][10][11][12][13][14][15][16][17], flow cytometry [18,19], high-pressure liquid chromatography (HPLC) [20], and fluorescence microscopy [4,14,. Furthermore, when used in conjunction with other probes, diamino fluorophores theoretically provide the option to test for co-localization of NO formation with other signaling events, such as a change in intracellular calcium concentration using Fura-2 [12,16,38], or its association with certain cell organelles by e.g.…”

Section: Introductionmentioning

confidence: 99%

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Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Rodríguez

Specian

Maloney

et al. 2005

Free Radical Biology and Medicine

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An emergent approach to the detection of nitric oxide (NO) in tissues relies on the use of fluorescence probes that are activated by products of NO autoxidation. Here we explore the performance of the widely-used NO probe 4,5-diaminofluorescein diacetate (DAF-2 DA) for the localization of sources of NO in rat aortic tissue, either from endogenous NO synthesis or from chemically or photolytically-released NO from targets of nitrosation/nitrosylation. Of importance toward understanding the performance of this probe in tissues is the finding that, with incubation conditions commonly used in the literature (10 µM DAF-2 DA), intracellular DAF-2 accumulates to concentrations that approach the millimolar range. Whereas such high probe concentrations do not interfere with NO release or signaling, they help to clarify why DAF-2 nitrosation is possible in the presence of endogenous nitrosation scavengers (e.g. ascorbate and glutathione). The gain attained with such elevated concentrations is, however, mitigated by associated high levels of background autofluorescence from the probe. This, together with tissue autofluorescence, limits the sensitivity of the probe to low-micromolar levels of accumulated DAF-2 triazole (DAF-2 T), the activated form of the probe, which is higher than the concentrations of endogenous nitrosation/nitrosylation products found in tissues. We further show that the compartmentalization of DAF-2 around elastic fibers further limits its potential to characterize the site of NO production at the subcellular level. Moreover, we find that reaction of DAF-2 with HgCl 2 and other commonly employed reagents are associated with spectral changes that may be misinterpreted as NO signals. Finally, UV illumination can lead to high levels of nitrosating species that interfere with NO detection from enzymatic sources. These findings indicate that while DAF-2 may still represent an important tool for the localization of NO synthesis, provided important pitfalls and limitations are taken into consideration, it is not suited for the detection of basally-generated nitrosation/nitrosylation products.Performance of DAF-2 for NO Imaging 3

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“…Prior investigations have called attention to the quenching of DAF-2 nitrosation by naturally occurring reducing agents such as Asc and GSH [7,46,67]. Consistent with these findings, our …”

Section: Do Antioxidants Such As Gsh or Asc Affect Daf Imaging?supporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Rodríguez

Specian

Maloney

et al. 2005

Free Radical Biology and Medicine

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“…Evidence suggests that DAF-based probes can also react with oxidized derivatives of NO, e.g., peroxynitrite [ONOO 2 (29,38)], reductants [dehydroascorbic acid, ascorbic acid, dithiothreitol, 2-mercaptoethanol, and glutathione (39,40)] divalent cations [mainly calcium, (41)], and endogenous nitroso/nitrosyl species [i.e., nitrosothiols, nitrosamines, and heme-NO products, (29)], which could account for the residual DAF-2T fluorescence present after treatment with C-PTIO.…”

Section: Resultsmentioning

confidence: 99%

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

et al. 2010

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Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds. ' International Society for Advancement of Cytometry Key termsanti-leishmanial; DAF-2DA; flow cytometry; leishmaniasis; nitric oxide LEISHMANIA are intracellular protozoan parasites that infect host mononuclear phagocytes, the resultant complex of diseases being leishmaniasis affecting 12 million people world wide in 88 countries (1). This disease is characterized by development of dermal lesions that may be cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), or mucocutaneous leishmaniasis (MCL) as also there can be systemic involvement as in the case of visceral leishmaniasis (VL), which is potentially fatal if left untreated (2).This obligate intracellular parasite resides and multiplies within macrophages, and therefore it should be able to evade the cytotoxic mechanisms innately operative within the macrophages for its survival (3). One of the predominant cytotoxic mechanisms includes production of reactive nitrogen intermediates, which the Leishmania parasite effectively decreases to ensure its survival (4,5).

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“…DAF-2 DA is a specific NO indicator that can penetrate rapidly into the cell where it is hydrolyzed to diaminofluorescin (DAF-2) by intracellular esterases (20). DAF-2 selectively traps NO, yielding fluorescent triazolofluorescein (excitation 480 nm/emission 520 nm) (25). DAF-2 DA has proven useful in detecting rapid sequential NO generation in our cardiomyocytes (21).…”

Section: Chemicals Nmentioning

confidence: 99%

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Shao

Chang

Chan

et al. 2007

American Journal of Physiology-Heart and Circulatory Physiology

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timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 Ϯ 3.4% cell death. Hypothermia induction to 25°C was most protective (14.3 Ϯ 0.6% death, P Ͻ 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 Ϯ 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 Ϯ 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N -nitro-L-arginine methyl ester (200 M) reversed these changes and abrogated hypothermia protection. In addition, the PKC⑀ inhibitor myr-PKC⑀ v1-2 (5 M) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase C⑀; nitric oxide synthase.

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Inhibitory Effects of Catecholamines and Anti-Oxidants on the Fluorescence Reaction of 4,5-Diaminofluorescein, DAF-2, a Novel Indicator of Nitric Oxide

Cited by 87 publications

References 13 publications

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

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