Determination and Bioimaging Method for Nitric Oxide in Biological Specimens by Diaminofluorescein Fluorometry

Itoh, Yoshinori; Hai, Fu; Hoshi, Hanae; Oka, Michiko; Noda, Kumiko; Ukai, Yojiro; Kojima, Hirotatsu; Nagano, Tetsuo; Toda, Noboru

doi:10.1006/abio.2000.4859

Cited by 188 publications

(142 citation statements)

References 19 publications

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“…Urchin Eggs-DAF-FM is said to be a more faithful indicator of NO above pH 5.8 (28) and has been used to image NO production in several cell types, including rat neuronal cells and smooth muscle cells (37,38). We confirmed the specificity of DAF-FM in sea urchin eggs at fertilization (see "Experimental Procedures").…”

Section: Levels Of No As Measured With Daf-fm Rise After the Initiasupporting

confidence: 65%

The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs

Leckie

Empson

Becchetti

et al. 2003

Journal of Biological Chemistry

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Both the inositol 1,4,5-trisphosphate (InsP 3 ) and ryanodine receptor pathways contribute to the Ca 2؉ transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP 3 receptor pathway has a primary role in causing Ca 2؉ release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca 2؉ at fertilization and establish that NO levels rise after, not before, the Ca 2؉ wave is initiated and that this rise is Ca 2؉ -dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca 2؉ transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca 2؉ wave to regulate the duration of the fertilization Ca 2؉ transient.

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Section: Levels Of No As Measured With Daf-fm Rise After the Initiasupporting

confidence: 65%

The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs

Leckie

Empson

Becchetti

et al. 2003

Journal of Biological Chemistry

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“…Changes in intracellular NO concentration were monitored using DAF-FM, a non-fluorescent compound that is irreversibly nitrosated by an NO-dependent mechanism to a fluorescent triazole (Itoh et al, 2000). DAF-FM intensity also exhibits no pH dependence over the physiologic range (Kojima et al, 1999).…”

Section: Methodsmentioning

confidence: 99%

“…Because ⌬⌿ m dissipates immediately after the onset of the NMDA-induced [Ca 2ϩ ] i increase, we used the onset of this [Ca 2ϩ ] i increase as a proxy for the onset of ⌬⌿ m dissipation. To estimate NMDAinduced changes in NO production, we simultaneously measured time-dependent changes in fura-2 and DAF-FM fluorescence, a pH-independent, NO-specific indicator (Itoh et al, 2000;Kojima et al, 2001). We used linear regression to quantify the slope of the DAF-FM fluorescence increase before and during the first 4 min of NMDA stimulation, beginning with the onset of the [Ca 2ϩ ] i rise.…”

Section: No Production Increases During Nmda Receptor Activation In Pmentioning

confidence: 99%

Mitochondrial Nitric Oxide Mediates Decreased Vulnerability of Hippocampal Neurons from Immature Animals to NMDA

Marks¹,

Boriboun²,

Wang³

2005

J. Neurosci.

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“…A number of studies have reported on the nitrosation of DAF-2 for varying concentrations of NO donors, either in buffer solutions [9,10,20,21,30] using fluorometry or in cells/tissues [26,29] using fluorescence microscopy. …”

Section: Comparative Nitrosation Of Daf-2 In Buffer Solutions and Tismentioning

confidence: 99%

“…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%

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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Determination and Bioimaging Method for Nitric Oxide in Biological Specimens by Diaminofluorescein Fluorometry

Cited by 188 publications

References 19 publications

The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs

The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs

Mitochondrial Nitric Oxide Mediates Decreased Vulnerability of Hippocampal Neurons from Immature Animals to NMDA

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

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