Biological Functions Of Nitric Oxide Research Articles

Ratiometric fluorescent biosensor for detection and real-time imaging of nitric oxide in mitochondria of living cells

Nitric oxide (NO), a ubiquitous gaseous messenger, plays critical roles in various pathological and physiological progresses. The abnormal levels of NO in organisms are closely related to a large number of maladies. Mitochondria are the main area that produce NO in mammalian cells. Thus, detecting and real-time imaging of NO in mitochondria is of great significance for exploring the biological functions of NO. Herein, a ratiometric fluorescent biosensor (Mito-GNP-pNO520) is developed for sensitive and selective detection and real-time imaging of NO in mitochondria of living cells. The detection is achieved through the fluorescence off-on response of Mito-GNP-pNO520 toward NO. This biosensor shows excellent characteristics, such as high sensitivity toward NO with a low detection limit of 0.25 nM, exclusive selectivity to NO without interference from other substances, good biological stability and low cytotoxicity. More importantly, the biosensor is specifically located in mitochondria, enabling the detection and real-time imaging of endogenous and exogenous NO in mitochondria of living cells. Therefore, our biosensor offers a new approach for dynamic detecting and real-time imaging of NO in subcellular organelles, providing an opportunity to explore new biological effects of NO.

Roles of Protein S-Nitrosylation in Endothelial Homeostasis and Dysfunction.

Significance: Nitric oxide (NO) plays several distinct roles in endothelial homeostasis. Except for activating the guanylyl cyclase enzyme-dependent cyclic guanosine monophosphate signaling pathway, NO can bind reactive cysteine residues in target proteins, a process known as S-nitrosylation (SNO). SNO is proposed to explain the multiple biological functions of NO in the endothelium. Investigating the targets and mechanism of protein SNO in endothelial cells (ECs) can provide new strategies for treating endothelial dysfunction-related diseases. Recent Advances: In response to different environments, proteomics has identified multiple SNO targets in ECs. Functional studies confirm that SNO regulates NO bioavailability, inflammation, permeability, oxidative stress, mitochondrial function, and insulin sensitivity in ECs. It also influences EC proliferation, migration, apoptosis, and transdifferentiation. Critical Issues: Single-cell transcriptomic analysis of ECs isolated from different mouse tissues showed heterogeneous gene signatures. However, litter research focuses on the heterogeneous properties of SNO proteins in ECs derived from different tissues. Although metabolism reprogramming plays a vital role in endothelial functions, little is known about how protein SNO regulates metabolism reprogramming in ECs. Future Directions: Precisely deciphering the effects of protein SNO in ECs isolated from different tissues under different conditions is necessary to further characterize the relationship between protein SNO and endothelial dysfunction-related diseases. In addition, identifying SNO targets that can influence endothelial metabolic reprogramming and the underlying mechanism can offer new views on the crosstalk between metabolism and post-translational protein modification. Antioxid. Redox Signal. 40, 186-205.

Selective visualization of nitric oxide in living cells and tissue slices using a mitochondria-targetable fluorescent probe

Nitric oxide (NO) is an important messaging molecule which plays a vital role in many biological systems. Because the endogenous NO is synthesized mainly in the mitochondria of mammalian cells, the detection of NO in mitochondria is of great importance for studying the biological functions of NO. Here, a novel fluorescence turn-on probe (MTNO) for monitoring the mitochondrial NO is designed and synthesized. The fluorescence intensity of MTNO exhibits a ∼430-fold enhancement in response to NO with a detection limit as low as 14 nM. MTNO also shows good mitochondrial localization, pH insensitivity and high selectivity to NO. With the aid of MTNO, the imaging of exogenous and endogenous NO in living cells and tissue slices is achieved through two-photon microscopy.

Blood–Brain Barrier Permeable Photoacoustic Probe for High-Resolution Imaging of Nitric Oxide in the Living Mouse Brain

Alternations in the brain nitric oxide (NO) homeostasis are associated with a variety of neurodegeneration diseases; therefore, high-resolution imaging of NO in the brain is essential for understanding pathophysiological processes. However, currently available NO probes are unsuitable for this purpose due to their poor ability to cross the blood-brain barrier (BBB) or to image in deep tissues with spatial resolution. Herein, we developed a photoacoustic (PA) probe with BBB crossing ability to overcome this obstacle. The probe shows a highly selective ratiometric response toward NO, which enables the probe to image NO with micron resolution in the whole brain of living mice. Using three-dimensional PA imaging, we demonstrated that the probe could be used to visualize the detailed NO distribution in varying depth cross-sections (0-8 mm) of the living Parkinson's disease (PD) mouse brain. We also investigated the therapeutic properties of natural polyphenols in the PD mouse brain using the probe as an imaging agent and suggested the potential of the probe for screening therapeutic agents. This study provides a promising imaging agent for imaging of NO in the mouse brain with high resolution. We anticipate that these findings may open up new possibilities for understanding the biological functions of NO in the brain and the development of new imaging agents for the diagnosis and treatment of brain diseases.

Biological Functions of Nitric Oxide in the Brain and Brain Stem

Nitric oxide (NO) is a small biological arbitrator and signaling molecule that has numerous significant biological roles in our body. Most of the neurons produce NO by neuronal nitric oxide synthase (nNOS). NO has been involved in the regulation of neurogenesis, synaptic plasticity, learning, and memory. Also, it contributes to the regulation of circulation and synapses, cerebral map formation, and neuropeptides. In the current review, we focused on previous research that has demonstrated structural aspects, subcellular localization, and some factors that adjust nNOS function. Furthermore, we have characterized the effect of nNOS in the brain in some physiological situations, particularly long-term potentiation and depression (LTP and LTD) and neural plasticity during development . Moreover, the effect of NO on neuropeptidergic neurons, including orexin, in reward systems was reviewed. Also, this study has focused on the NO involvement in brain circulation, the excitability of neurons, and the homeostatic balance of excitatory and inhibitory signaling in the brain.

An N-nitrosation reaction-based fluorescent probe for detecting nitric oxide in living cells and inflammatory zebrafish

Nitric oxide (NO), an essential biological messenger molecule, participates in various physiological and pathological processes. The sensitive and specific detection of NO is of great significance for understanding the biological function of NO. Here, we synthesized a fluorescent probe (Rho-NO) for highly selective detection of NO both in vitro and in vivo. The high selectivity of Rho-NO is attributed to the fact that NO is easily replaced by electron donor amino group to form N-nitrosation products, causing rhodamine spiro ring open and fluorescence emit. Rho-NO showed a good linear response to NO (0–100 µM) with a low detection limit (0.06 µM). Importantly, it exhibited excellent specificity for NO detection in human serum and was also applied for imaging NO in living cells and inflammatory model of zebrafish. This work proves the potential of Rho-NO in pathological research and disease diagnosis.

A tough nitric oxide-eluting hydrogel coating suppresses neointimal hyperplasia on vascular stent

Vascular stent is viewed as one of the greatest advancements in interventional cardiology. However, current approved stents suffer from in-stent restenosis associated with neointimal hyperplasia or stent thrombosis. Herein, we develop a nitric oxide-eluting (NOE) hydrogel coating for vascular stents inspired by the biological functions of nitric oxide for cardiovascular system. Our NOE hydrogel is mechanically tough and could selectively facilitate the adhesion of endothelial cells. Besides, it is non-thrombotic and capable of inhibiting smooth muscle cells. Transcriptome analysis unravels the NOE hydrogel could modulate the inflammatory response and induce the relaxation of smooth muscle cells. In vivo study further demonstrates vascular stents coated with it promote rapid restoration of native endothelium, and persistently suppress inflammation and neointimal hyperplasia in both leporine and swine models. We expect such NOE hydrogel will open an avenue to the surface engineering of vascular implants for better clinical outcomes.

Biological Mechanisms of S-Nitrosothiol Formation and Degradation: How Is Specificity of S-Nitrosylation Achieved?

The modification of protein cysteine residues underlies some of the diverse biological functions of nitric oxide (NO) in physiology and disease. The formation of stable nitrosothiols occurs under biologically relevant conditions and time scales. However, the factors that determine the selective nature of this modification remain poorly understood, making it difficult to predict thiol targets and thus construct informatics networks. In this review, the biological chemistry of NO will be considered within the context of nitrosothiol formation and degradation whilst considering how specificity is achieved in this important post-translational modification. Since nitrosothiol formation requires a formal one-electron oxidation, a classification of reaction mechanisms is proposed regarding which species undergoes electron abstraction: NO, thiol or S-NO radical intermediate. Relevant kinetic, thermodynamic and mechanistic considerations will be examined and the impact of sources of NO and the chemical nature of potential reaction targets is also discussed.

Dual channel mitochondria-targeted fluorescent probe for detection of nitric oxide in living cells and zebrafish

A novel dual channel mitochondria-targeted fluorescent probe has been developed to detect nitric oxide (NO), an important and short-life reactive nitrogen species (RNS) which mainly generates in the mitochondria. The new probe Mi-NO has exhibited dual emission property as well as unique spectrum responses towards NO. Upon the addition of NO, the fluorescence of Mi-NO quenched significantly. Molecular docking calculations suggest a potential molecular target for Mi-NO in the mitochondria protein adenine nucleotide translocator (ANT). High colocalization coefficient also suggested that Mi-NO can effectively target mitochondria. Moreover, Mi-NO has been successfully employed in imaging NO in living cells and zebrafish, affording a potential for studying the biological functions of NO in living systems.

A Novel Mechanism for Nitrosative Stress Tolerance Dependent on GTP Cyclohydrolase II Activity Involved in Riboflavin Synthesis of Yeast

The biological functions of nitric oxide (NO) depend on its concentration, and excessive levels of NO induce various harmful situations known as nitrosative stress. Therefore, organisms possess many kinds of strategies to regulate the intracellular NO concentration and/or to detoxify excess NO. Here, we used genetic screening to identify a novel nitrosative stress tolerance gene, RIB1, encoding GTP cyclohydrolase II (GTPCH2), which catalyzes the first step in riboflavin biosynthesis. Our further analyses demonstrated that the GTPCH2 enzymatic activity of Rib1 is essential for RIB1-dependent nitrosative stress tolerance, but that riboflavin itself is not required for this tolerance. Furthermore, the reaction mixture of a recombinant purified Rib1 was shown to quench NO or its derivatives, even though formate or pyrophosphate, which are byproducts of the Rib1 reaction, did not, suggesting that the reaction product of Rib1, 2,5-diamino-6-(5-phospo-d-ribosylamino)-pyrimidin-4(3 H)-one (DARP), scavenges NO or its derivatives. Finally, it was revealed that 2,4,5-triamino-1H-pyrimidin-6-one, which is identical to a pyrimidine moiety of DARP, scavenged NO or its derivatives, suggesting that DARP reacts with N2O3 generated via its pyrimidine moiety.

Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells.

Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high-NO concentration environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl- o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry technology allowed the characterization of the proteome under NO-generated conditions. Moreover, we demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.

Acid-Promoted D-A-D Type Far-Red Fluorescent Probe with High Photostability for Lysosomal Nitric Oxide Imaging.

To accurately monitor the variations of lysosomal nitric oxide (NO) under physiological condition remains a great challenge for understanding the biological function of NO. Herein, we developed a new chemotype probe, namely, MBTD, for acid-promoted and far-red fluorescence imaging of lysosomal NO in vitro and ex vivo. MBTD was rationally designed by incorporating o-phenylenediamino (OPD) moiety into the donor-acceptor-donor (D-A-D) type fluorophore based on a dual intramolecular charge transfer (ICT) mechanism. Compared to previously reported OPD-based NO probes, MBTD displays several distinct advantages including large stroke shift, huge on-off ratio with minimal autofluorescence, and high NO specificity. Particularly, MBTD exhibits an acid-promoted response to NO with high acid tolerance, which greatly improves the spatial resolution to lysosomal NO by excluding the background noise from other nonacidic organelles. Furthermore, MBTD displayed much longer-lived and more stable fluorescence emission in comparison with the commercialized NO probe. MBTD was employed for ratiometric examination of the exogenous or endogenous NO of macrophages. More importantly, MBTD was able to detect the variation of lysosomal NO level in an acute liver injury mouse model ex vivo, implying the potential of MBTD for real-time monitoring the therapeutic efficacy of drug candidates for the treatment of acute liver injury. MBTD as a novel type of NO probe might open a new avenue for precisely sensing lysosomal NO-related pathological and therapeutic process.

Detection of S-Nitrosated Nuclear Proteins in Pathogen-Treated Arabidopsis Cell Cultures Using Biotin Switch Technique.

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.

Hydrogen sulfide inhibits development of atherosclerosis through up-regulating protein S-nitrosylation.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule that serves many important regulatory roles in physiological and pathophysiological conditions. H2S exerts an anti-atherosclerotic effect through mediating the biological functions of nitric oxide (NO). However, its mechanism of action is unclear. The purpose of this study is to explore the effect mechanism of H2S on the development of atherosclerosis with regard to protein S-nitrosylation. A total of 45 male apoE-/- mice were randomly divided into three groups. Atherosclerosis was induced by Western diet (21% fat and 0.15% cholesterol) with/without administration of a H2S donor (NaHS) or an endogenous cystathionine γ-lyase inhibitor (d, l-propargylglycine) for 12 weeks. After 12 weeks, plasma lipid and plasma NO levels were measured. Aortic gross lesion area and histopathological features of aortic lesion were determined. Additionally, the level of S-nitrosylated proteins in vascular smooth muscle cells (VSMCs) was detected using immunofluorescence in aorta. Rat VSMCs were performed in an in vitro experiment. Inducible nitric oxide synthase (iNOS) protein expression, NO generation, protein S-nitrosylation, and cell proliferation and migration were measured. We found that H2S significantly reduced the aortic atherosclerotic lesion area (P=0.006) and inhibited lipid and macrophage accumulation (P=0.004, P=0.002) and VSMC proliferation (P=0.019) in apoE-/- mice. H2S could up-regulate levels of plasma NO and protein S-nitrosylation in aorta VSMCs. However, d, l- propargylglycine had the opposite effect, increasing the lesion area and the content of lipids and macrophages in the lesions of apoE-/- mice and down-regulating plasma NO levels and protein S-nitrosylation in aorta VSMCs. In vitro experiments, H2S could significantly reverse the reduction of iNOS expression and NO generation induced by oxidized low-density lipoprotein in VSMCs. Moreover, H2S could increase the protein S-nitrosylation level of VSMCs in a dose-dependent manner, and the effect could be inhibited by iNOS inhibitors. In addition, proliferation and migration of VSMCs could be inhibited by H2S in a dose-dependent manner, which could be blocked by an iNOS inhibitor or protein S-nitrosylation removal agent. Our data suggest that H2S could inhibit the development of atherosclerosis by up-regulating plasma NO and protein S-nitrosylation, thereby inhibiting the proliferation and migration of VSMCs.

Abstract 4328: Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells

Abstract Background: Nitric oxide (NO) is an important signaling molecule in the immune, nervous and cardiovascular systems. NO involves in the regulation of cellular behavior as well as cytotoxic events. Studies demonstrated that resveratrol (RES) (a polyphenolic compound in the skin of red fruits) at high concentration (≥50μM) induces NO production in endothelial F-2 cells and in human umbilical vein endothelial cells. However, the impact of RES on the biological functions of NO are still poorly understood. Therefore, the goal of this study was to investigate the impact of RES on NO-mediated oxidative stress at mitochondria level in mouse prostate cancer (PCa) cells. Experimental Procedures: The transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (mirrors the pathogenesis of human prostate cancer) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 IU/ml, streptomycin 100 μg/ml, 0.005mg/ml bovine insulin, and 10nM Dehydroisoandrosterone in 5% CO2 at 370C. Cells were harvested by trypsinization after 80% confluency (at 4-5 pass), counted (105/ml), and incubated with RES (50uM), RES (50uM) + Deta-NONOate (750uM), Deta-NONOate (750uM), RES (50uM) +L-NMMA (750uM), and L-NMMA (750uM). Deta-NONOate was used as nitric oxide donor while L-NMMA was used as nitric oxide inhibitor. Thereafter, cell viability, NO-estimation in culture supernatant, mitochondrial membrane potential (MMP), total ROS-production in mitochondria and cytoplasm, and apoptotic assay (Annexin-V and caspase-3/7) were carried out. Results and Conclusion: PCa cells incubated with RES showed decreased cell viability, decreased NO production in culture supernatant, decreased MMP, and decreased level of ROS in mitochondria. However, PS-externalization, and total ROS in cytoplasm were found high after normalization with control cells. Further, treatment with Deta-NONOate results in decreased cell viability, decreased total ROS in the cytoplasm, and decreased MMP, while mitochondrial ROS and NO production in culture supernatant were observed highly significant as compared control cells. Furthermore, cells were also exposed to the combination of RES and NO which suggest that RES reduced NO-mediated cell killing and reactive oxygen species generation in TRAMP cells. However, no significant changes were observed after L-NMMA exposure to TRAMP cells. The result demonstrates that RES protect cells from NO-mediated cell killing and induced NO-independent apoptosis in PCa cells. This suggests that role of RES as a potential therapeutic agent to control cancer. However, further investigation is needed to explore potential mechanism of action of RES in controlling cancer proliferation. Citation Format: Sanjay Kumar, James Stokes, Karyn Scissum Gunn, Selvarangan Ponnazhagan, Upender Manne, Manoj K. Mishra. Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4328.

Biodegradable poly(lactic-co-glycolic acid) microspheres loaded with S-nitroso-N-acetyl-D-penicillamine for controlled nitric oxide delivery

Nitric oxide (NO) is a fascinating and important endogenous free-radical gas with potent antimicrobial, vasodilating, smooth muscle relaxant, and growth factor stimulating effects. However, its wider biomedical applicability is hindered by its cumbersome administration, since NO is unstable especially in biological environments. In this work, to ultimately develop site-specific controlled release vehicles for NO, the NO donor S-nitroso-N-acetyl-D-penicillamine (SNAP) was encapsulated within poly(lactic-co-glycolic acid) 50:50 (PLGA) microspheres by using a solid-in-oil-in-water emulsion solvent evaporation method. The highest payload was 0.56(±0.01) μmol SNAP/mg microspheres. The in vitro release kinetics of the donor were controlled by the bioerosion of the PLGA microspheres. By using an uncapped PLGA (Mw=24,000–38,000) SNAP was slowly released for over 10days, whereas by using the ester capped PLGA (Mw=38,000–54,000) the release lasted for over 4weeks. The presence of copper ions and/or ascorbate in solution was necessary to efficiently decompose the released NO donor and obtain sustained NO release. It was also demonstrated that light can be used to induce rapid NO release from the microspheres over several hours. SNAP exhibited excellent storage stability when encapsulated in the PLGA microspheres. These new microsphere formulations may be useful for site-specific administration and treatment of pathologies associated with dysfunction in endogenous NO production, e.g. treatment of diabetic wounds, or in diseases involving other biological functions of NO including vasodilation, antimicrobial, anticancer, and neurotransmission.

Protein Control of S-Nitrosothiol Reactivity: Interplay of Antagonistic Resonance Structures

There is currently great interest in S-nitrosothiols (RSNOs) because formation of protein-based RSNOs-protein S-nitrosation-has been recently recognized as a major pathway of the biological function of nitric oxide, NO. Despite the growing number of S-nitrosated proteins identified in vivo, enzymatic processes that control reactions of biological RSNOs are still not well understood. In this article, we use a range of models to computationally demonstrate that specific interactions of RSNOs with charged and polar residues in proteins can result in dramatic modification of RSNO structure, stability, and reactivity. This unprecedented sensitivity of the -SNO group toward interactions with charged species is related to their unusual electronic structure that can be elegantly expressed in terms of antagonistic resonance structures. We propose a 'ligand effect map' (LEM) approach as an efficient way to estimate the environment effects on the -SNO groups in proteins without performing electronic structure calculations. Furthermore, the calculated (15)N NMR signatures of these specific interactions suggest that (15)N NMR spectroscopy can be an effective technique to identify and study these interactions experimentally. Overall, the results of this study suggest that RSNO reactions in vivo should be tightly controlled by the protein environment via modulation of the RSNO electronic structure.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca(2+) signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

Real-time imaging of nitric oxide production in living cells with 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3′,4′-diaminophenyl)-difluoroboradiaza- s-indacence by invert fluorescence microscope

Although the importance of nitric oxide (NO) as a signalling molecule in many biological processes is becoming increasingly evident, many proposed and potential biological functions of NO still remain unclear. Bioimaging is a good technique to visualize observation of nitric oxide in biological samples. In this report, a fluorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3′,4′-diaminophenyl)-difluoroboradiaza- s-indacence (TMDCDABODIPY), has been first applied to real-time image NO produced in PC12 cells, Sf9 cells and human vascular endothelial cells at the presence of l-arginine with inverted fluorescence microscope. NO production in the cells is successfully captured and imaged with fine temporal and spatial resolution. The results prove that the probe combined with inverted fluorescence microscope can be developed into a sensitive and selective method for further study of NO release from cells.