Nitric Oxide In Living Cells Research Articles

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

A dual-response triphenylamine-based fluorescent probe for selective sensing of copper(II) and nitric oxide in live cells

The development of bifunctional fluorescent probes for rapid and sensitive detection of copper(II) and nitric oxide (NO) in vivo is of significant importance to better understand their roles in human health. In this paper, a novel dual-response triphenylamine-based fluorescent probe, BQ, was developed for the detection of Cu2+ and NO in live cells. The sensing mechanism of the probe towards Cu2+can be attributed to the photoinduced electron transfer (PET) process of BQ-Cu2+, resulting in the remarkable quenching of the fluorescence emission. Meanwhile, the addition of NO can make the fluorescent wavelength of the probe red shift due to the intramolecular charge transfer (ICT) effect of BQ-NO, and the sensing mechanisms of BQ towards Cu2+ and NO have been further confirmed by 1H NMR and the high resolution mass spectrometry (HR-MS) analysis and the density functional theory (DFT) theoretical computation. Furthermore, BQ has the obvious advantages of high sensitivity, rapid fluorescent response, and reliability at physiological pH towards Cu2+ and NO. It is noteworthy that BQ can be used as a sensor for the detection of Cu2+ and NO in live RAW 264.7 cells by fluorescence imaging.

A Dual-Response Triphenylamine-Based Fluorescent Probe for Selective Sensing of Copper(Ii) and Nitric Oxide in Live Cells

A Dual-Response Triphenylamine-Based Fluorescent Probe for Selective Sensing of Copper(Ii) and Nitric Oxide in Live Cells

Simultaneous imaging of hypochlorous acid and nitric oxide in live cells based on a dual-channel fluorescent probe

Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) are inevitably produced during normal human metabolism. Various ROS and RNS together form tangled networks that play important roles in many physiological and pathological processes. Here we used 1,8-naphthalene diamine as a reactive group to develop a fluorescent probe, N-[2-(6-phenylethynyl)quinolinylmethyl]-1,8-diamino naphthalene (QBN), for HOCl and NO. QBN showed a “turn-on” fluorescent response at 464 nm to HOCl in the range of 0–75 μM with rapid responding time (10 s) and detection limit (0.11 ± 0.03 μM). Furthermore, a “turn-on” fluorescent responses at 512 nm to NO in the range of 0–40 μM with responding time (20 s) and detection limit (25.7 ± 3.4 nM) was found. The response mechanisms of QBN to HOCl and NO were discussed based on mass analysis of the different products. The dual-channel probe was then successfully applied for simultaneous imaging of both exogenous and endogenous HOCl and NO in live cells.

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Selective and sensitive fluorescence imaging reveals microenvironment-dependent behavior of NO modulators in the endothelial system

Nitric oxide (NO) is a second messenger playing crucial roles in the signaling of a variety of cellular functions. Due to its pathophysiological significance, various NO modulators have been developed to explore NO pathways and some have been used as therapies. These modulators are often used directly to observe pharmacological effects in cell lines, but their actual effect on intracellular NO level is seldom analyzed. Herein, facilitated by a selective and sensitive fluorescence probe, we observed that some NO modulators displayed unexpected behaviors with both NO scavenger carboxy-PTIO and endothelial nitric oxide synthase (eNOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME) failing to decrease intracellular free NO level in EA. hy926 cells while NO donor diethylamine-NONOate (DEA·NONOate) and eNOS activator calcimycin (A23187) failing to increase free NO level in human umbilical vein endothelial cell line (HUV-EC-C), although the reagents were confirmed to work normally in the primary human umbilical vein endothelial cells (primary HUVECs) and RAW 264.7 macrophage cells. Further research suggested that these unusual behaviors might be attributed to the cellular microenvironments including both the NO synthase (NOS) level and the endogenous glutathione (GSH) level. Genetically manipulating eNOS level in both cells restores the expected response, while decreasing GSH level restores the ability of DEA·NONOate to increase NO level in HUV-EC-C. These results reveal that the cellular microenvironment has a profound impact on pharmacological effect. Our study suggests GSH as a reservoir for NO in live cells and highlights the value of chemical probes as valuable tools to reveal microenvironment-dependent pharmacological effects.

Rapid and sensitive detection of nitric oxide by a BODIPY-based fluorescent probe in live cells: glutathione effects.

Nitric oxide (NO) is an important signaling molecule involved in various physiological and pathological processes. The effects of NO depend on its concentration, and the spatial and temporal constraints of the cell microenvironment. Meanwhile, NO can react with some biomolecules such as biothiols, leading to a short biological lifetime. Thus, it is very crucial to establish a real-time visualization method for monitoring NO levels. In this work, we have developed a fluorescent probe, RBA, for NO, with a 3-extended BODIPY as a fluorophore and a secondary amine as the active site. The probe RBA can quickly sense NO (∼10 s) in aerobic solutions to generate a fluorescent N-nitrosamine (RBA-NO, Φf = 0.87) due to blocking of the photoinduced electron transfer (PET) process from the secondary amine to the BODIPY core. This sensing reaction displays high sensitivity (LOD = 10 nM) and high selectivity for NO over relevant analytes except some reducing reagents including biothiols, and a remarkable interference effect is observed ascribed to a competitive reaction with biothiols. Furthermore, the exo- and endogenous detection of NO in live cells and zebra fish was achieved, and it was demonstrated that glutathione (GSH) weakens drastically the fluorescence response by cell-imaging experiments. These results imply that the colorimetric and fluorescence response of the chemosensor for NO depends on the levels of both NO and GSH in environments.

A rhodamine-based fast and selective fluorescent probe for monitoring exogenous and endogenous nitric oxide in live cells.

Nitric oxide, an important cellular messenger molecule, plays a wide range of physiological roles in neurotransmission, vascular relaxation, platelet activation, and immune response. In this work, we synthesized a selective and sensitive cationic fluorescent probe (ROPD) for monitoring exogenous and endogenous nitric oxide in live cells by grafting a NO-trapper o-phenylenediamine (OPD), via one of the amino groups of OPD, onto a rhodamine fluorophore at the C-3 position. The probe exhibited high selectivity and sensitivity towards NO with fast response in the water phase over a wide pH range. At the same time, the probe ROPD is cell-membrane permeable and can be used for the real-time visualization of exogenous NO in both L929 and HeLa cells, and endogenous NO in stressed RAW 264.7 cells.

Ratiometric SERS imaging and selective biosensing of nitric oxide in live cells based on trisoctahedral gold nanostructures.

Herein, a ratiometric SERS probe was created for monitoring nitric oxide (NO) by designing a novel molecule, 3,4-diaminobenzene-thiol, and immobilizing this molecule onto trisoctahedral gold nanostructures with superior SERS capability. The established probe possessed good selectivity and biocompatibility, high sensitivity and accuracy, thus enabling imaging and biosensing of NO in live cells.

An N-nitrosation reactivity-based two-photon fluorescent probe for the specific in situ detection of nitric oxide.

In situ fluorescence imaging of nitric oxide (NO) is a powerful tool for studying the critical roles of NO in biological events. However, the selective imaging of NO is still a challenge because most currently available fluorescent probes rely on the o-phenylenediamine (OPD) recognition site, which reacts with both NO and some abundant reactive carbonyl species (RCS) (such as dehydroascorbic acid and methylglyoxal) and some reactive oxygen/nitrogen species (ROS/RNS). To address this problem, a new fluorescent probe, NCNO, based on the N-nitrosation of aromatic secondary amine was designed to bypass the RCS, ROS, and RNS interference. As was expected, the probe NCNO could recognize NO with pronounced selectivity and sensitivity among ROS, RNS, and RCS. The probe was validated by detecting NO in live cells and deep tissues owing to its two-photon excitation and red-light emission. It was, hence, applied to monitor NO in ischemia reperfusion injury (IRI) in mice kidneys by two-photon microscopy for the first time, and the results vividly revealed the profile of NO generation in situ during the renal IRI process.

286 - Novel Fluorescent Probes for Imaging and Quantitative Analysis of Intracellular Nitric Oxide

Nitric oxide (NO) is an important biological regulator involved in number of physiological and pathological processes. Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO can be rapidly oxidized, thus there is relatively low concentration of NO existing in vivo. Although a variety of fluorescent sensors have been developed to detect NO, their poor photostability, short emission wavelength and low selectivity have limited the applications of these existing reactive oxygen species (ROS) and reactive nitrogen species (RNS) probes in live cells. To address this unmet need, three novel cell-permeable NO probes Nitrixyte Orange, Nitrixyte Red and Nitrixyte NIR were developed for detecting free NO in live cells. Compared to the commonly used diaminofluorescein (DAF-2), Nitrixyte probes have 1) higher selectivity toward NO than other ROS and RNS species; 2) improved sensitivity in detecting endogenous levels of NO; 3) enhanced cell permeability and 4) better photostability. Importantly, long emission wavelength of Nitrixyte Red and Nitrixyte NIR minimize autofluorescence of cells and facilitate multicolor imaging. Taking advantages of Nitrixyte probes, fluorescence imaging was successfully applied to monitor the exogenous NO in various adherent cell lines such as HeLa and CHO-K1, and to visualize the endogenously produced NO in Raw 264.7 macrophage cell line. Fluorescence microplate reader was also successfully used to quantitatively measure NO level in above cell lines. Significantly, all these novel probes were suited for monitoring intracellular NO in suspension cell lines such as T lymphocyte Jurkat cells using flow cytometer, to provide quantitative real-time measurements of NO in individual live cells. These novel fluorescent NO probes provide valuable tools for life science research and medical diagnostic applications.

Quinoline-Based Two-Photon Fluorescent Probe for Nitric Oxide in Live Cells and Tissues

A two-photon fluorescent probe (QNO) for nitric oxide is reported. The probe is designed with a photoinduced electron transfer (PeT) mechanism and shows 12-fold fluorescence enhancement toward NO. Adopting a quinoline derivative as the fluorophore, QNO has a large two-photon action cross section value of 52 GM and long-wavelength emission. It also features high selectivity, low cytotoxicity, and pH insensitivity. By utilizing two-photon microscopy (TPM), QNO can detect NO in live cells and live tissues at a depth of 90-180 μm.

Targetable Fluorescent Probe for Monitoring Exogenous and Endogenous NO in Mitochondria of Living Cells

Nitric oxide (NO) is a ubiquitous cellular messenger molecule in the cardiovascular, nervous, and immune systems. Mitochondrion is the main area where endogenous NO is synthesized by inducible NOS enzymes in mammalian cells. Thus, real-time monitoring NO in mitochondria is very meaningful for NO chemical biology. Although a variety of fluorescent probes for NO have been successfully developed, they are not suited for detecting mitochondrial NO because none of them can specifically localize in mitochondria. Herein, Mito-Rh-NO, the first mitochondria-targetable "turn-on" fluorescent probe for NO, has been developed through attaching a triphenylphosphonium to a rhodamine spirolactam. The characteristics of this probe are as following: (1) Mito-Rh-NO exhibits high sensitivity toward NO. In solution, Mito-Rh-NO responds to NO by significant fluorescence enhancement up to 60-fold, and its NO detection limit is as low as 4.0 nM. (2) The NO sensing of Mito-Rh-NO is highly selective, which will not interfere with the other reactive oxygen and nitrogen species. (3) Mito-Rh-NO has a low cytotoxic effect: after being treated with 10 μM Mito-Rh-NO for 24 h, the survival rate is higher than 90%. (4) Mito-Rh-NO specifically localizing in mitochondria: colocalization experiment of Mito-Rh-NO and Rh 123, a typical mitotracker, shows the merged fluorescent microcopy image with a high Pearson's colocalization coefficient 0.92 and overlap coefficient 0.99. (5) Mito-Rh-NO demonstrates high applicability for real-time monitoring of mitochondrial NO in live cells. Both the exogenous NO released by the donor NOC13 and endogenous NO generated in cells under stimulation have been visualized under confocal microscopy.

A fluorescent ratiometric nanosensor for detecting NO in aqueous media and imaging exogenous and endogenous NO in live cells.

As an endogenously generated signal-inducing agent in the immune, cardiovascular and nervous systems, nitric oxide (NO) is involved in various biological processes and a large number of pathologies. Hence, sensitive and reliable detection of NO in biological samples and inside living cells has been of great interest. Herein, we reported a carbon-dot-based fluorescent ratiometric nanosensor for NO detection in water and imaging in living cells. Ratiometric sensing is realized through the fluorescence resonance energy transfer (FRET) process, and the carbon dots (CDs) serve as both the energy donor and the anchoring site for the NO probe moiety, which turns into the energy acceptor in the presence of NO. This nanosensor is highly selective for NO, and is operable in totally aqueous media with a very low detection limit of 3 nM. Due to the biocompatible nature and small size of the carbon dots, the nanosensor exhibits very low cytotoxicity and can easily permeate through the cell membrane. The nanosensor is capable of tracking the exogenous NO levels in several cell lines such as Hela, L929 and Raw 264.7 cells, and it can be also used to visualize the endogenously produced NO in the Raw 264.7 macrophage cell line.

Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.

Extracellular DNA Affects NO Content in Human Endothelial Cells

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17β-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein.

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.