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

Abstract

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

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

“…Methods that merely detect changes in fluorescence intensity including fluorimetry, microscopy, and flow cytometry cannot distinguish between specific (both NOͲ and DAFͲ dependent) and unspecific (DAFͲdependent but NOͲindependent) fluorescence signals. The latter could also be derived by formation of fluorescent adducts of DAFs with ascorbate and dehydroascorbate [28] or reaction with HgCl 2, as reported [23]. While principally producing comparable results, these assays differ somewhat in selectivity; a sideͲbyͲside comparison of fluorescenceͲbased techniques, as presented here later on, offers valuable insight into techniqueͲ specific assay characteristics.…”

“…Indeed, Jourd'heuil observed an increase in the NOͲdependent fluorescence signal of DAFͲ2 in the presence of oxidants [29]. Since high concentrations of antioxidants present in cells are able to scavenge nitrosating intermediates and/or reduce the oxidized probe, some of us argued that DAFͲ2 derivatives must be present at high, millimolar concentrations in order to allow detection of nitrosative chemistry inside cells [23].…”

“…These compounds have become the most popular and widely used fluorescent probes for the measurement of NO production in mammalian cells [15], tissues [22,23], as well as invertebrates and plants [22,24]. NOͲ dependent fluorescent product formation in cells and tissues has been mainly evaluated using fluorescence microscopy, flow cytometry and fluorimetry [15,22,23], but the specificity of this approach has been questioned repeatedly [20,25,26]. In fact, these techniques are not able to distinguish the NOͲspecific fluorescence signal produced by the triazole derivates from unspecific fluorescent products formed by reaction with other biological cell constituents within cells [22,23,27Ͳ 29].…”

“…Although additional controls have been included by some investigators to exclude that changes in e.g., ascorbate may have contributed to the measured changes in DAFͲrelated fluorescence, not all possibilities for false positives may be known to date. Other uncertainties relate to the cellular redox status, which may also affect the intensity of the fluorescence signal that is observed; together with a high background due to preferential accumulation of DAFs within certain compartments and autofluorescence of cells and tissues [23]. This may make it difficult to detect basal NO production, and in some cases prevent investigators from picking up a signal altogether.…”

Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

“…One issue encountered with NO fluorescence imaging in biological samples using the DAF/DAR ratiometric method is the intracellular sequestration of the fluorescent dyes. With the incubation conditions commonly used such as 10 μM DAF-2 DA and 10 μM DAR-4M AM, the intracellular concentration of these dyes can reach the millimolar range (Rodriguez et al, 2005). We observed strong fluorescence in PC12 cells.…”