ABSTRACT
Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.
Subject(s)
Dynamins , Energy Metabolism , Hexokinase , Mitochondria , Mitochondrial Dynamics , Mitochondrial Proteins , Hexokinase/metabolism , Hexokinase/genetics , Humans , Mitochondria/metabolism , Mitochondria/genetics , Mitochondria/enzymology , Dynamins/metabolism , Dynamins/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Animals , Adenosine Triphosphate/metabolism , Stress, Physiological , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Citric Acid Cycle , Glucose-6-Phosphate/metabolism , Mice , HeLa Cells , HEK293 Cells , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , MutationABSTRACT
Neuronal activity is accompanied by a net outflow of potassium ions (K+) from the intra- to the extracellular space. While extracellular [K+] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K+ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K+] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K+] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K+] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K+]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K+] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.
Subject(s)
Biosensing Techniques , Neurons , Potassium , Animals , Potassium/metabolism , Neurons/metabolism , Mice , Biosensing Techniques/methods , Action Potentials , Cells, Cultured , Fluorescence Resonance Energy Transfer/methods , Optogenetics/methodsABSTRACT
The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.
Subject(s)
Biosensing Techniques , Endothelial Cells , Hydrogen Peroxide , Nitric Oxide Synthase Type III , Nitric Oxide , Oxidative Stress , Oxygen , Hydrogen Peroxide/metabolism , Nitric Oxide/metabolism , Humans , Oxygen/metabolism , Endothelial Cells/metabolism , Endothelial Cells/drug effects , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type III/genetics , Biosensing Techniques/methods , NG-Nitroarginine Methyl Ester/pharmacologyABSTRACT
When a cell sustains damage, it liberates cytosolic ATP, which can serve as an injury signal, affecting neighboring cells. This study presents a methodological approach that employs in vitro axotomy and in vivo laser ablation to simulate cellular injury. Specially tailored biosensors are employed to monitor ATP dynamics and calcium transients in injured cells and their surroundings. To simultaneously visualize extracellular and cytosolic ATP, we developed bicistronic constructs featuring GRABATP1.0 and MaLionR biosensors alongside the calcium sensor RCaMP, enabling multiparametric imaging. In addition to transducing primary neuron cultures, we developed another method where we cocultured dorsal root ganglion neurons together with specialized "sniffer" cell lines expressing the bicistronic biosensors. Exploiting these approaches, we successfully demonstrated the release of ATP from the injured neurons and its extracellular diffusion in response to cellular injury in vitro and in vivo. Axotomy triggered intracellular calcium mobilization not only in the injured neuron but also in the intact neighboring cells, providing new insights into ATP's role as an injury signal. The tools developed in this study have demonstrated remarkable efficiency in unraveling the intricacies of ATP-mediated injury signaling.
Subject(s)
Biosensing Techniques , Calcium , Rats , Animals , Calcium/metabolism , Rats, Sprague-Dawley , Neurons/metabolism , Adenosine TriphosphateABSTRACT
Trauma, vascular events, or neurodegenerative processes can lead to axonal injury and eventual transection (axotomy). Neurons can survive axotomy, yet the underlying mechanisms are not fully understood. Excessive water entry into injured neurons poses a particular risk due to swelling and subsequent death. Using in vitro and in vivo neurotrauma model systems based on laser transection and surgical nerve cut, we demonstrated that axotomy triggers actomyosin contraction coupled with calpain activity. As a consequence, neurons shrink acutely to force water out through aquaporin channels preventing swelling and bursting. Inhibiting shrinkage increased the probability of neuronal cell death by about 3-fold. These studies reveal a previously unrecognized cytoprotective response mechanism to neurotrauma and offer a fresh perspective on pathophysiological processes in the nervous system.
ABSTRACT
Chemogenetic Operation of iNTRacellular prOton Levels (pH-Control) is a novel substrate-based enzymatic method that enables precise spatiotemporal control of ultralocal acidification in cultured cell lines and primary neurons. The genetically encoded biosensor SypHer3s showed that pH-Control effectively acidifies cytosolic, mitochondrial, and nuclear pH exclusively in the presence of ß-chloro-d-alanine in living cells in a concentration-dependent manner. The pH-Control approach is promising for investigating the ultralocal pH imbalance associated with many diseases.
Subject(s)
Protons , Hydrogen-Ion Concentration , Cell Line , Homeostasis , Cytosol/metabolismABSTRACT
Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N'-bis (2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.
Subject(s)
Biosensing Techniques , Metalloproteins , Iron , Nitric Oxide , Edetic Acid , Ferric Oxide, Saccharated , Salts , EthylenediaminesABSTRACT
BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.
Subject(s)
CD27 Ligand , Endothelial Cells , Nitric Oxide , CD27 Ligand/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Humans , Hydrogen Peroxide/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolismABSTRACT
Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
Subject(s)
Biosensing Techniques , Metalloproteins , Endothelial Cells/metabolism , HEK293 Cells , Humans , Iron/metabolism , Metalloproteins/metabolism , Nitric Oxide/metabolism , Oxygen/metabolismABSTRACT
Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redox-dependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors.
Subject(s)
Biosensing Techniques , Hydrogen Peroxide , Animals , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Oxidative Stress , Signal TransductionABSTRACT
The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.
Subject(s)
Cardiomyopathy, Dilated , Heart Failure , Alanine/pharmacology , Amino Acids/metabolism , Amino Acids/pharmacology , Amino Acids/therapeutic use , Animals , Cardiomyopathy, Dilated/metabolism , Dependovirus/metabolism , Disease Models, Animal , Heart Failure/genetics , Heart Failure/metabolism , Hydrogen Peroxide/metabolism , Myocytes, Cardiac/metabolism , Oxidative Stress , Rats , TranscriptomeABSTRACT
Dimethyl disulfide (DMDS) has a specific unpleasant odour and is profoundly toxic with an odour threshold of around 7-12 ppb. In this study, the removal of DMDS was investigated by adsorption on activated carbon cloth (ACC) in the gas phase. Kinetics and isotherm studies were performed. Adsorption kinetics followed by GC-MS and the data were processed using different models. When correlation coefficients (R2) of linear regression analysis are analyzed, it is seen that the concordance of experimental data to the pseudo-second-order equation is quite good. Isotherm data have been examined using Freundlich, Temkin and Langmuir models. The regression coefficient (R2) of data to fit the Langmuir model is 0.9993, which means that the fit is very good. The monolayer adsorption capacity (qm) of DMDS has been calculated as 118 mL.g-1 according to the Langmuir model.
ABSTRACT
Chemogenetics refers to experimental systems that dynamically regulate the activity of a recombinant protein by providing or withholding the protein's specific biochemical stimulus. Chemogenetic tools permit precise dynamic control of specific signaling molecules to delineate the roles of those molecules in physiology and disease. Yeast d-amino acid oxidase (DAAO) enables chemogenetic manipulation of intracellular redox balance by generating hydrogen peroxide only in the presence of d-amino acids. Advances in biosensors have allowed the precise quantitation of these signaling molecules. The combination of chemogenetic approaches with biosensor methodologies has opened up new lines of investigation, allowing the analysis of intracellular redox pathways that modulate physiological and pathological cell responses. We anticipate that newly developed transgenic chemogenetic models will permit dynamic modulation of cellularredox balance in diverse cells and tissues and will facilitate the identification and validation of novel therapeutic targets involved in both physiological redox pathways and pathological oxidative stress.
Subject(s)
Hydrogen Peroxide , Oxidative Stress , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Signal TransductionABSTRACT
A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H2O2 to living cells or tissues. However, the addition of H2O2 to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of H2O2 on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids. Much has been learned about the intracellular transport of H2O2 through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of H2O2 under hypoxic conditions imaged with the novel ultrasensitive H2O2 sensor (HyPer7). Moreover, this approach was suitable for generating H2O2 in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of d-amino acids differentially affects mDAAO-dependent intracellular H2O2 generation. When paired with the hydrogen sulfide (H2S) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust H2S signals. We also show that chemogenetic H2O2 generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision.
Subject(s)
Hydrogen Peroxide , Oxidants , Amino Acids , Cells, Cultured , Oxidation-ReductionABSTRACT
Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method's potential as a practicable and informative multiparametric live-cell imaging technique.
Subject(s)
Biosensing Techniques , Coculture Techniques , Endothelial Cells/metabolism , Hydrogen Peroxide/metabolism , MitochondriaABSTRACT
The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).
Subject(s)
Biological Phenomena , Cartilage, Articular , Sirtuins , Aged , Animals , Cartilage, Articular/metabolism , Chondrocytes , Homeostasis , Humans , Hydrogen Peroxide/metabolism , Mice , Oxidation-Reduction , Oxidative Stress , Sirtuins/genetics , Sirtuins/metabolismABSTRACT
Hydrogen peroxide (H2O2) modulates critical phosphorylation pathways in vascular endothelial cells, many of which affect endothelial nitric oxide synthase (eNOS) signal transduction. Both intracellular and extracellular sources of H2O2 have been implicated in eNOS regulation, yet the specific endothelial pathways remain incompletely understood. Here we exploited chemogenetic approaches and live-cell imaging methods to both generate and detect H2O2 in different subcellular compartments (cytosol, nucleus, and caveolae) of cultured EA.hy926 human endothelial cells. We developed novel recombinant constructs encoding differentially-targeted yeast d-amino acid oxidase (DAAO), which generates H2O2 only when its d-amino acid substrate is provided. DAAO was expressed as a fusion protein with the new H2O2 biosensor HyPer7.2, which allowed us to quantitate intracellular H2O2 levels by ratiometric imaging in living endothelial cells following the activation of DAAO by d-alanine. The addition of extracellular H2O2 to the HyPer-DAAO-transfected cells led to increases in H2O2 throughout different regions of the cell, as measured using the differentially-targeted HyPer biosensor for H2O2. The sensor response to extracellular H2O2 was more rapid than that quantitated following the addition of d-alanine to transfected cells to activate differentially-targeted DAAO. The maximal intracellular levels of H2O2 observed in response to the addition of extracellular H2O2 vs. intracellular (DAAO-generated) H2O2 were quantitatively similar. Despite these similarities in the measured levels of intracellular H2O2, we observed a remarkable quantitative difference in the activation of endothelial phosphorylation pathways between chemogenetically-generated intracellular H2O2 and the phosphorylation responses elicited by the addition of extracellular H2O2 to the cells. Addition of extracellular H2O2 had only a nominal effect on phosphorylation of eNOS, kinase Akt or AMP-activated protein kinase (AMPK). By contrast, intracellular H2O2 generation by DAAO caused striking increases in the phosphorylation of these same key signaling proteins. We also found that the AMPK inhibitor Compound C completely blocked nuclear H2O2-promoted eNOS phosphorylation. However, Compound C had no effect on eNOS phosphorylation following H2O2 generation from cytosol- or caveolae-targeted DAAO. We conclude that H2O2 generated in the cell nucleus activates AMPK, leading to eNOS phosphorylation; in contrast, AMPK activation by cytosol- or caveolae-derived H2O2 does not promote eNOS phosphorylation via AMPK. These findings indicate that H2O2 generated in different subcellular compartments differentially modulates endothelial cell phosphorylation pathways, and suggest that dynamic subcellular localization of oxidants may modulate signaling responses in endothelial cells.