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1.  Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: Implications for identifying molybdopterin nitrite reductases 
Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite ( NO2-) to •NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated •NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki = 1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed •NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5–100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki = 13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to •NO (EC50 = 64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50 = 60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50 = 4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50 = 613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO2- reductase activity of AO and XOR.
PMCID: PMC4260934  PMID: 24406683
Aldehyde oxidase; Nitrite; Xanthine oxidoreductase; Raloxifene; Nitric oxide; Febuxostat
2.  [No title available] 
PMCID: PMC3917719  PMID: 24333562
3.  Nitric Oxide is Less Effective at Inhibiting Neointimal Hyperplasia in Spontaneously Hypertensive Rats 
Exogenous administration of nitric oxide (NO) markedly decreases neointimal hyperplasia following arterial injury in several animal models. However, the effect of NO on neointimal hyperplasia in hypertension remains unknown. Here, we employ the spontaneously hypertensive rat (SHR) strain, inbred from Wistar Kyoto (WKY) rats, and the carotid artery balloon injury model to assess the effects of NO on neointimal hyperplasia development. Two weeks after arterial injury, we showed that both rat strains developed similar levels of neointimal hyperplasia, but local administration of NO was less effective at inhibiting neointimal hyperplasia in the SHR compared to WKY rats (58% vs. 79%, P<0.001). Interestingly, local administration of NO did not affect systemic blood pressure in either rat strain. Compared to WKY, the SHR displayed more proliferation in the media and adventitia following balloon injury, as measured by BrdU incorporation. The SHR also showed more inflammation in the adventitia after injury, as well as more vasa vasorum, than WKY rats. NO treatment reduced the vasa vasorum in the SHR but not WKY rats. Finally, while NO decreased both injury-induced proliferation and inflammation in the SHR, it did not return these parameters to levels seen in WKY rats. We conclude that NO is less effective at inhibiting neointimal hyperplasia in the SHR than WKY rats. This may be due to increased scavenging of NO in the SHR, leading to diminished bioavailability of NO. These data will help to develop novel NO-based therapies that will be equally effective in both normotensive and hypertensive patient populations.
PMCID: PMC3877576  PMID: 24149190
balloon injury; endothelial dysfunction; hypertension; neointimal hyperplasia; nitric oxide; SHR
4.  Chemical Foundations of Hydrogen Sulfide Biology 
Following nitric oxide (nitrogen monoxide) and carbon monoxide, hydrogen sulfide (or its newer systematic name sulfane, H2S) became the third small molecule that can be both toxic and beneficial depending on the concentration. In spite of its impressive therapeutic potential, the underlying mechanisms for its beneficial effects remain unclear. Any novel mechanism has to obey fundamental chemical principles. H2S chemistry was studied long before its biological relevance was discovered, however, with a few exceptions, these past works have received relatively little attention in the path of exploring the mechanistic conundrum of H2S biological functions. This review calls attention to the basic physical and chemical properties of H2S, focuses on the chemistry between H2S and its three potential biological targets: oxidants, metals and thiol derivatives, discusses the applications of these basics into H2S biology and methodology, and introduces the standard terminology to this youthful field.
PMCID: PMC3843984  PMID: 23850631
hydrogen sulfide; sulfane; sulfhydration; oxidants; metal; transsulfuration
5.  Agmatine Induced NO Dependent Rat Mesenteric Artery Relaxation and its Impairment in Salt-Sensitive Hypertension 
L-arginine and its decarboxylated product, agmatine are important mediators of NO production and vascular relaxation. However, the underlying mechanisms of their action are not understood. We have investigated the role of arginine and agmatine in resistance vessel relaxation of Sprague-Dawley (SD) and Dahl salt-sensitive hypertensive rats. Second or 3rd-order mesenteric arterioles were cannulated in an organ chamber, pressurized and equilibrated before perfusing intraluminally with agonists. The vessel diameters were measured after mounting on the stage of a microscope fitted with a video camera. The gene expression in Dahl rat vessel homogenates was ascertained by real-time PCR. L-arginine initiated relaxations (EC50, 5.8 ± 0.7 mM; n = 9) were inhibited by arginine decarboxylase (ADC) inhibitor, difluoromethylarginine (DFMA) (EC50, 18.3 ± 1.3 mM; n = 5) suggesting that arginine-induced vessel relaxation was mediated by agmatine formation. Agmatine relaxed the SD rat vessels at significantly lower concentrations (EC50, 138.7 ± 12.1 μM; n = 22), which was compromised by L-NAME (L-NG-Nitroarginine methyl ester, an eNOS inhibitor), RX821002 (α-2 AR antagonist) and pertussis toxin (G-protein inhibitor). The agmatine-mediated vessel relaxation from high salt Dahl rats was abolished as compared to that from normal salt rats (EC50, 143.9 ± 23.4 μM; n = 5). The α-2A AR, α-2B AR and eNOS mRNA expression was downregulated in mesenteric arterioles of high-salt treated Dahl hypertensive rats. These findings demonstrate that agmatine facilitated the relaxation via activation of α-2 adrenergic G-protein coupled receptor and NO synthesis, and this pathway is compromised in salt-sensitive hypertension.
PMCID: PMC3844099  PMID: 23994446
agmatine; arginine decarboxylase; L-arginine; nitric oxide; α-2 adreno receptor; hypertension
6.  Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase 
We introduce a strategy for generating mixtures of nitric oxide (NO) and nitroxyl (HNO) at tunable rates in physiological media. The approach involves converting a spontaneously HNO/NO-generating ion to a caged (prodrug) form that is essentially stable in neutral media, but that can be activated for HNO/NO release by adding an enzyme capable of efficiently opening the cage to regenerate the ion. By judiciously choosing the enzyme, substrate, and reaction conditions, unwanted scavenging of the HNO and NO by the protein can be minimized and the catalytic efficiency of the enzyme can be maintained. We illustrate this approach with a proof-of-concept study wherein the prodrug is Gal-IPA/NO, a diazeniumdiolate of structure iPrHN-N(O)=NOR, with R = β-D-galactosyl. E. coli-derived β-D-galactosidase at concentrations of 1.9 to 15 nM hydrolyzed 56 μM substrate with half-lives of 140 to 19 min, respectively, producing the IPA/NO anion (iPrHN-N(O)=NO−, half-life ~3 min), which in turn spontaneously hydrolyzed to mixtures of HNO with NO. Using saturating substrate concentrations furnished IPA/NO generation rates that were directly proportional to enzyme concentration. Consistent with these data, the enzyme/substrate combination applied to ventricular myocytes isolated from wild-type mouse hearts resulted not only in a significant positive inotropic effect, but also rescued the cells from the negative inotropy, hypercontractions, and occasional cell death seen with the enzyme alone. This mechanism represents an alternate approach for achieving controlled fluxes of NO/HNO to investigate their biological actions.
PMCID: PMC3881966  PMID: 24126017
IPA/NO; Prodrug; Nitroxyl; Diazeniumdiolate; Gal-IPA/NO; β-Galactosidase
Organic nitrates (ORNs) are commonly used anti-ischemic and anti-anginal agents, which serve as an exogenous source of the potent vasodilator nitric oxide (NO). Recently, both mitochondrial aldehyde dehydrogenase-2 (ALDH2) and cytosolic aldehyde dehydrogenase-1a1 (ALDH1A1) have been shown to exhibit the ability to selectively bioactivate various ORNs in vitro. The objective of the present research was to examine the potential role of ALDH3A1, another major cytosolic isoform of ALDH, in the in vitro bioactivation of various ORNs, and to estimate the enzyme kinetic parameters toward ORNs through mechanistic modeling. The extent of bioactivation was assayed by exposing recombinant ALDH3A1 to various concentrations of ORNs, and measuring the concentration-time profiles of released NO via a NO-specific electrode. Metabolite formation kinetics was monitored for nitroglycerin (NTG) using LC/MS/MS. Our results showed that ALDH3A1 mRNA and protein were highly expressed in C57BL/6 mouse aortic, cardiac, and hepatic tissues, and it was able to release NO from several ORNs, including NTG, isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN), and nicorandil with similar Vmax (0.175 – 0.503 nmol/min/mg of ALDH3A1), and Km values of 4.01, 46.5, 818 and 5.75 × 103 μM respectively. However, activation of isosorbide-5-mononitrate (IS-5-MN) by ALDH3A1 was undetectable in vitro. ALDH3A1 was also shown to denitrate NTG, producing primarily glyceryl 1, 2-dinitrate (1, 2-GDN) in preference to glyceryl 1, 3-dinitrate (1, 3-GDN). Therefore, ALDH3A1 may contribute to the bioactivation of ORNs in vivo.
PMCID: PMC3895916  PMID: 24126018
Organic nitrates; Aldehyde dehydrogenase 3A1; Nitric oxide; Kinetics
8.  Hydrogen Sulfide Chemical Biology: Pathophysiological roles and detection 
Hydrogen sulfide (H2S) is the most recent endogenous gasotransmitter that has been reported to serve many physiological and pathological functions in different tissues. Studies over the past decade have revealed that H2S can be synthesized through numerous pathways and its bioavailability regulated through its conversion into different biochemical forms. H2S exerts its biological effects in various manners including redox regulation of protein and small molecular weight thiols, polysulfides, thiosulfate/sulfite, iron-sulfur cluster proteins, and anti-oxidant properties that affect multiple cellular and molecular responses. However, precise measurement of H2S bioavailability and its associated biochemical and pathophysiological roles remains less well understood. In this review, we discuss recent understanding of H2S chemical biology, its relationship to tissue pathophysiological responses and possible therapeutic uses.
PMCID: PMC4077051  PMID: 23850632
sulfide; cysteine; nitric oxide; cardiovascular; oxidative stress
9.  Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry 
Nitric Oxide  2014;42:32-39.
Graphic Abstract
Graphic entry for the Table of Contents (TOC).
•A DFT-derived barrier for nitrite linkage isomerism on heme center is reported.•EPR spectra of nitrite adducts show evidence for linkage isomerism.•The electronic structure of Fe(III)-nitrite heme is conformation-dependent.•Certain conformations are inducive to EPR silence.•Fe(II)-nitrite is undetectable on stopped-flow time scales.
The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.
PMCID: PMC4256065  PMID: 25172022
Hemoglobin; Myoglobin; Nitrite; DFT; EPR
10.  Sulfide-Inhibition of Mitochondrial Respiration at Very Low Oxygen Concentrations 
Our aim was to study the capacity of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the capacity of the pathway metabolizing and eliminating sulfide, which is linked to the mitochondrial respiratory chain and therefore operates under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide’s inhibition of cellular respiration would be dependent of the oxygen concentration in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5 – 1 × 106 cells in 2 ml of continuously stirred respiration medium at 37°C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73 ± 0.05 μM, 3.1 ± 0.2 μM, and 6.2 ± 0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3; 3.5] and 11.7 [6.2;21.2] min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6[15.5;28.1] min (coupled) and 35.9[27.4;59.2] min (uncoupled), as well as 42.4 [27.5;42.4] min (coupled) and 51.5 [46.4;51.7] min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results suggest that elimination of sulfide in these cells is limited by oxygen availability when approaching the anoxic condition. This property may contribute to the physiological role of sulfide as an oxygen sensor.
PMCID: PMC4231205  PMID: 24963794
11.  H2S during circulatory shock: Some unresolved questions 
Numerous papers have been published on the role of H2S during circulatory shock. Consequently, knowledge about vascular sulfide concentrations may assume major importance, in particular in the context of “acute on chronic disease”, i.e., during circulatory shock in animals with pre-existing chronic disease. This review addresses the questions i) of the “real” sulfide levels during circulatory shock, and, ii) to which extent injury and pre-existing co-morbidity may affect the expression of H2S producing enzymes under these conditions. In the literature there is a huge range on sulfide blood levels during circulatory shock, in part as a result of the different analytical methods used, but also due to the variable of the models and species studied. Clearly, some of the very high levels reported should be questioned in the context of the well-known H2S toxicity. As long as “real” sulfide levels during circulatory shock are unknown and/or undetectable “on line” due to the lack of appropriate techniques, it appears to be premature to correlate the measured blood levels of hydrogen sulfide with the severity of shock or the H2S therapy-related biological outcomes. The available data on the tissue expression of the H2S-releasing enzymes during circulatory shock suggest that a “constitutive” CSE expression may play a crucial role of for the maintenance of organ function, at least in the kidney. The data also indicate that increased CBS and CSE expression, in particular in the lung and the liver, represents an adaptive response to stress states.
PMCID: PMC4229245  PMID: 24650697
H2S; NaSH; Na2S; GYY4137; cystathionine-γ-lyase; cystathione-β-synthase
12.  AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], a mitochondrially targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro 
The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. AP39 (30 – 300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.
PMCID: PMC4225488  PMID: 24755204
mitochondria; bioenergetics; DNA repair; oxidative stress; cytoprotection
13.  Tocopherol Supplementation Reduces NO Production and Pulmonary Inflammatory Response to Bleomycin 
Bleomycin causes acute lung injury through production of reactive species and initiation of inflammation. Previous work has shown alteration to the production of reactive oxygen species results in attenuation of injury. Vitamin E, in particular, γ-tocopherol, isoform, has the potential to scavenge reactive oxygen and nitrogen species. This study examines the utility of dietary supplementation with tocopherols in reducing bleomycin-mediated acute lung injury. Male C57BL6/J mice were intratracheally instilled with PBS or 2 units/kg bleomycin. Animals were analyzed 3 and 8 days post instillation at the cellular, tissue, and organ levels. Results showed successful delivery of tocopherols to the lung via dietary supplementation. Also, increases in reactive oxygen and nitrogen species due to bleomycin are normalized in those mice fed tocopherol diet. Injury was not prevented but inflammation progression was altered, in particular macrophage activation and function. Inflammatory scores based on histology demonstrate limited progression of inflammation in those mice treated with bleomycin and fed tocopherol diet compared to control diet. Upregulation of enzymes and cytokines involved in pro-inflammation were limited by tocopherol supplementation. Day 3 functional changes in elastance in response to bleomycin are prevented, however, 8 days post injury the effect of the tocopherol diet is lost. The effect of tocopherol supplementation upon the inflammatory process is demonstrated by a shift in the phenotype of macrophage activation. The effect of these changes on resolution and the progression of pulmonary fibrosis has yet to be elucidated.
PMCID: PMC3769481  PMID: 23669183
Lung Mechanics; Macrophage Phenotype; Macrophage Activation; Reactive Oxygen; Nitrogen Species
14.  Proteomic analysis of the NOS2 interactome in human airway epithelial cells 
The cytokine-inducible isoform of nitric oxide synthase (NOS2) is constitutively expressed in human respiratory epithelia and is upregulated in inflammatory lung disease. Here, we sought to better define the protein interactions that may be important for NOS2 activity and stability, as well as to identify potential targets of NOS2-derived NO, in the respiratory epithelium. We overexpressed Flag-tagged, catalytically-inactive NOS2 in A549 cells and used mass spectrometry to qualitatively identify NOS2 co-immunoprecipitating proteins. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify the coordinate effects of cytokine stimulation on NOS2-protein interactions. Multi-protein networks dominated the NOS2 interactome, and cytokine-inducible interactions with allosteric activators and with the ubiquitin-proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination. The ubiquitin ligase scaffolding protein, FBXO45, was identified as a novel, direct NOS2 interactor. Similar to the SPRY domain-containing SOCS box (SPSB) proteins, FBXO45 requires Asn27 in the 23DINNN27 motif of NOS2 for its interaction. However, FBXO45 is unique from the SPSBs in that it recruits a distinct E3 ligase complex containing MYCBP2 and SKP1. Collectively, these findings demonstrate the general utility of interaction proteomics for defining new aspects of NOS2 physiology.
PMCID: PMC3769490  PMID: 23438482
nitric oxide synthase; nitric oxide; B30.2/SPRY domain; ubiquitination; proteomics
15.  Xanthine Oxidoreductase-Catalyzed Reduction of Nitrite to Nitric Oxide: Insights Regarding Where, When and How 
Numerous inflammatory disorders are associated with elevated levels of xanthine oxidoreductase (XOR) and allied enhancement of reactive species formation contributory to systemic pathology. Despite a long standing association between increased XOR activity and negative clinical outcomes, recent reports describe a paradigm shift where XOR mediates beneficial actions by catalyzing the reduction of NO2− to •NO. While provocative, these observations contradict reports of improved outcomes in similar models upon XOR inhibition as well as reports revealing strict anoxia as a requisite for XOR-mediated •NO formation. To garner a more clear understanding of conditions necessary for in vivo XOR-catalyzed •NO production, this review critically analyzes the impact of O2 tension, pH, substrate concentrations, glycoaminoglycan docking and inhibition strategies on the nitrite reductase activity of XOR and reveals a hypoxic milieu where this process may be operative. As such, information herein serves to link recent reports in which XOR activity has been identified as mediating the beneficial outcomes resulting from nitrite supplementation to a microenviromental setting where XOR can serve as substantial source of •NO.
PMCID: PMC3706534  PMID: 23454592
nitrite; xanthine oxidoreductase; nitric oxide; oxygen tension; inflammation; hypoxia
16.  A nanoparticle delivery vehicle for S-nitroso-N-acetyl cysteine: Sustained vascular response 
Interest in the development of nitric oxide (NO) based therapeutics has grown exponentially owing to its well elucidated and established biological functions. In line with this surge, S-nitroso thiol (RSNO) therapeutics are also receiving more attention in recent years both as potential stable sources of NO as well as for their ability to serve as S-nitrosating agents; S-nitrosation of protein thiols is implicated in many physiological processes. We describe two hydrogel based RSNO containing nanoparticle platforms. In one platform the SNO groups are covalently attached to the particles (SNO-np) and the other contains S-nitroso-N-acetyl cysteine encapsulated within the particles (NAC-SNO-np). Both platforms function as vehicles for sustained activity as trans-S-nitrosating agents. NAC-SNO-np exhibited higher efficiency for generating GSNO from GSH and maintained higher levels of GSNO concentration for longer time (24 h) as compared to SNO-np as well as a previously characterized nitric oxide releasing platform, NO-np (nitric oxide releasing nanoparticles). In vivo, intravenous infusion of the NAC-SNO-np and NO-np resulted in sustained decreases in mean arterial pressure, though NAC-SNO-np induced longer vasodilatory effects as compared to the NO-np. Serum chemistries following infusion demonstrated no toxicity in both treatment groups. Together, these data suggest that the NAC-SNO-np represents a novel means to both study the biologic effects of nitrosothiols and effectively capitalize on its therapeutic potential.
PMCID: PMC4156139  PMID: 22705913
Nitric Oxide; RSNO; S-nitroso-N-acetyl-cysteine; S-Nitrosoglutathione; Vasodilation; Nanotechnology
17.  Contribution of iNOS/sGC/PKG pathway, COX-2, CYP4A1, and gp91phox to the protective effect of 5,14-HEDGE, a 20-HETE mimetic, against vasodilation, hypotension, tachycardia, and inflammation in a rat model of septic shock☆ 
We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), prevents vascular hyporeactivity, hypotension, tachycardia, and inflammation in rats treated with lipopolysaccharide (LPS) and mortality in endotoxemic mice. These changes were attributed to decreased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO, cyclooxygenase (COX)-2-derived vasodilator prostanoids, and proinflammatory mediators associated with increased cyctochrome P450 (CYP) 4A1-derived 20-HETE and CYP2C23-dependent antiinflammatory mediator formation. The aim of this study was to determine whether decreased expression and activity of iNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG), COX-2, gp91phox (NOX2; a superoxide generating NOX enzyme), and peroxynitrite production associated with increased expression of COX-1 and CYP4A1 and 20-HETE formation in renal and cardiovascular tissues of rats contributes to the effect of 5,14-HEDGE to prevent vasodilation, hypotension, tachycardia, and inflammation in response to systemic administration of LPS. Mean arterial pressure fell by 28 mmHg and heart rate rose by 47 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of iNOS and COX-2 associated with a decrease in COX-1 and CYP4A1 mRNA and protein expression. Increased NOS activity, iNOS-heat shock protein 90 complex formation (an index for iNOS activity), protein expression of phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), gp91phox, p47phox (NOXO2; organizer subunit of gp91phox), and nitrotyrosine (an index for peroxynitrite production) as well as cGMP (an index for sGC activity), 6-keto-PGF1α (a stable metabolite PGI2) and PGE2 levels (indexes for COX activity), and nitrotyrosine levels by LPS were also associated with decreased CYP hydroxylase activity as measured by 20-HETE formation from arachidonic acid in renal microsomes of LPS-treated rats. These effects of LPS, except iNOS mRNA and COX-1 protein expression, were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid (30 mg/kg, s.c.; 1 h after LPS) reversed the effects of 5,14-HEDGE, except iNOS and COX-1 mRNA and protein expression as well as expression of CYP4A1 mRNA. These results suggest that increased CYP4A1 expression and 20-HETE formation associated with suppression of iNOS/sGC/PKG pathway, COX-2, and gp91phox participate in the protective effect of 5,14-HEDGE against vasodilation, hypotension, tachycardia, and inflammation in the rat model of septic shock.
PMCID: PMC3759626  PMID: 23684565
Endotoxin; Hypotension; iNOS/sGC/PKG pathway; COX-2; CYP4A1; gp91phox/NOX2
18.  Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells 
The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5- ceramide-labelled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluoresence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl- rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the presence of the proteasomal inhibitor MG132. The present data complete the remarkable insight that the structural integrity of three closely juxtaposed cytoplasmic organelles - Golgi apparatus, endoplasmic reticulum and mitochondria -is dependent on nitric oxide.
PMCID: PMC3759657  PMID: 23770576
Nitric oxide scavenging (c-PTIO); pulmonary arterial endothelial cells; organellar remodeling; endoplasmic reticulum; Golgi apparatus; mitochondria; reticulon-4b/Nogo-A; atlastin 3; ubiquitination; MG132
19.  Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons 
Surges of nitric oxide compromise mitochondrial respiration primarily by competitive inhibition of oxygen binding to cytochrome c oxidase (complex IV) and are particularly injurious in neurons, which rely on oxidative phosphorylation for all their energy needs. Here, we show that transgenic overexpression of the neuronal globin protein, neuroglobin, helps diminish protein nitration, preserve mitochondrial function and sustain ATP content of primary cortical neurons challenged by extended nitric oxide exposure. Specifically, in transgenic neurons, elevated neuroglobin curtailed nitric oxide-induced alterations in mitochondrial oxygen consumption rates, including baseline oxygen consumption, consumption coupled with ATP synthesis, proton leak and spare respiratory capacity. Concomitantly, activation of genes involved in sensing and responding to oxidative/nitrosative stress, including the early-immediate c-Fos gene and the phase II antioxidant enzyme, heme oxygenase-1, was diminished in neuroglobin-overexpressing compared to wild-type neurons. Taken together, these differences reflect a lesser insult produced by similar concentrations of nitric oxide in neuroglobin-overexpressing compared to wild-type neurons, suggesting that abundant neuroglobin buffers nitric oxide and raises the threshold of nitric oxide-mediated injury in neurons.
PMCID: PMC3816961  PMID: 23587847
neuroglobin transgene; nitric oxide; primary neurons; mitochondrial respiration; ATP synthesis; bioenergetics
20.  PEX7 and EBP50 Target iNOS to the Peroxisome in Hepatocytes 
iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8 hours of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals.
PMCID: PMC3642215  PMID: 23474170
Inflammation; Sepsis; Inducible Nitric Oxide Synthase; Peroxisome; Liver; Subcellular Localization
21.  Nitroxyl accelerates the oxidation of oxyhemoglobin by nitrite 
Angeli’s salt (Na2N2O3) decomposes into nitroxyl (HNO) and nitrite (NO2−), compounds of physiological and therapeutic interest for their impact on biological signaling both through nitric oxide and nitric oxide independent pathways. Both nitrite and HNO oxidize oxygenated hemoglobin to methemoglobin. Earlier work has shown that HNO catalyzes the reduction of nitrite by deoxygenated hemoglobin. In this work, we have shown that HNO accelerates the oxidation of oxygenated hemoglobin by NO2−. We have demonstrated this HNO mediated acceleration of the nitrite/oxygenated hemoglobin reaction with oxygenated hemoglobin being in excess to HNO and nitrite (as would be found under physiological conditions) by monitoring the formation of methemoglobin in the presence of Angeli’s salt with and without added NO2−. In addition, this acceleration has been demonstrated using the HNO donor 4- nitrosotetrahydro-2H-pyran-4-yl pivalate, a water-soluble acyloxy nitroso compound that does not release NO2− but generates HNO in the presence of esterase. This HNO donor was used both with and without NO2− and acceleration of the NO2− induced formation of methemoglobin was observed. We found that the acceleration was not substantially affected by catalase, superoxide dismutase, c-PTIO, or IHP, suggesting that it is not due to formation of extramolecular peroxide, NO2 or H2O2, or to modulation of allosteric properties. In addition, we found that the acceleration is not likely to be related to HNO binding to free reduced hemoglobin, as we found HNO binding to reduced hemoglobin to be much weaker than has previously been proposed. We suggest that the mechanism of the acceleration involves local propagation of autocatalysis in the nitrite-oxygenated Hb reaction. This acceleration of the nitrite oxyhemoglobin reaction could affect studies aimed at understanding physiological roles of HNO and perhaps nitrite and use of these agents in therapeutics such as hemolytic anemias, heart failure, and ischemia reperfusion injury.
PMCID: PMC3652897  PMID: 23545404
Hemoglobin; kinetics; nitrite; nitroxyl; therapeutics
22.  Effects of the nitric oxide donor JS-K on the blood-tumor barrier and on orthotopic U87 rat gliomas assessed by MRI 
Nitric oxide (NO) released from NO donors can be cytotoxic in tumor cells and can enhance the transport of drugs into brain tumors by altering blood-tumor barrier permeability. The NO donor JS-K [O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] releases NO upon enzymatic activation selectively in cells overexpressing glutathione-S-transferases (GSTs) such as gliomas. Thus, JS-K-dependent NO effects - especially on cell viability and vascular permeability - were investigated in U87 glioma cells in vitro and in an orthotopic U87 xenograft model in vivo by magnetic resonance imaging (MRI). In vitro experiments showed dose-dependent antiproliferative and cytotoxic effects in U87 cells. In addition, treatment of U87 cells with JS-K resulted in a dose-dependent activation of soluble guanylate cyclase and intracellular accumulation of cyclic guanosine monophosphate (cGMP) which was irreversibly inhibited by the selective inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo(4,3a)quinoxaline-1-one). Using dynamic contrast enhanced MRI (DCE-MRI) as a minimally invasive technique, we demonstrated for the first time a significant increase in the DCE-MRI read-out initial area under the concentration curve (iAUC60) indicating an acute increase in blood-tumor barrier permeability after i.v. treatment with JS-K. Repeated MR imaging of animals with intracranial U87 gliomas under treatment with JS-K (3.5 μmol/kg JS-K 3×/week) and of untreated controls on day 12 and 19 after tumor inoculation revealed no significant changes in tumor growth, edema formation or tumor perfusion. Immunohistochemical workup of the brains showed a significant antiproliferative effect of JS-K in the gliomas. Taken together, in vitro and in vivo data suggest that JS-K has antiproliferative effects in U87 gliomas and opens the blood-tumor barrier by activation of the NO/cGMP signaling pathway. This might be a novel approach to facilitate entry of therapeutic drugs into brain tumors. DCE-MRI is a non-invasive, repeatable imaging modality to monitor biological effects of NO donors and other experimental therapeutics in intracranial tumor models.
PMCID: PMC3617040  PMID: 23370169
Nitric oxide; JS-K; blood-brain barrier; blood-tumor barrier; DCE-MRI; U87 glioma; cGMP
Plasma hemoglobin (Hb) scavenges endothelium-derived nitric oxide (NO), producing systemic and pulmonary vasoconstriction in many species. We hypothesized that i.v. administration of murine cell-free Hb would produce pulmonary vasoconstriction and enhance hypoxic pulmonary vasoconstriction (HPV) in mice.
To assess the impact of plasma Hb on basal pulmonary vascular tone in anesthetized mice we measured left lung pulmonary vascular resistance (LPVRI) before and after infusion of Hb at thoracotomy. To confirm the findings obtained at thoracotomy, measurements of right ventricular systolic pressure (RVSP) and systemic arterial pressure (SAP) were obtained in closed-chest wild-type mice. To elucidate whether pretreatment with Hb augments HPV we assessed the increase in LPVRI before and during regional lung hypoxia produced by left mainstem bronchial occlusion (LMBO) in wild-type mice pretreated with Hb.
Infusion of Hb increased SAP but did not change pulmonary arterial pressure (PAP), left lung pulmonary arterial flow (QLPA) or LPVRI in either wild-type or diabetic mice with endothelial dysfunction. Scavenging of NO by plasma Hb did not alter HPV in wild-type mice. Inhibition of NO synthase with L-NAME did not change the basal LPVRI, but augmented HPV during LMBO.
Our data suggest that scavenging of NO by plasma Hb does not alter pulmonary vascular tone in mice. Therefore, generation of NO in the pulmonary circulation is unlikely to be responsible for the low basal pulmonary vascular tone of mice.
PMCID: PMC3764648  PMID: 23313572
hemoglobin; endothelium; hypoxia; vasoconstriction; nitric oxide
24.  Quinoid radio-toxin (QRT) induced metabolic changes in mice: An ex vivo and in vivo EPR investigation 
Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from γ-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2 mg QRT. A dose of 2 mg QRT was fatal to mice within 45–60 min of treatment. Nitrosyl hemoglobin complexes α-(Fe2+–NO)α-(Fe2+)β-(Fe2+)2 were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO2 in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with γ-irradiation.
PMCID: PMC3955719  PMID: 18230367
Radio-toxins; Nitric oxide; Nitrosyl hemoglobin complexes; Blood; Tissue; EPR
25.  Kinetic analysis of DAF-FM activation by NO: Toward calibration of a NO-sensitive fluorescent dye 
Nitric oxide (NO) research in biomedicine has been hampered by the absence of a method that will allow quantitative measurement of NO in biological tissues with high sensitivity and selectivity, and with adequate spatial and temporal resolution. 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) is a NO sensitive fluorescence probe that has been used widely for qualitative assessment of cellular NO production. However, calibration of the fluorescent signal and quantification of NO concentration in cells and tissues using fluorescent probes, have provided significant challenge. In this study we utilize a combination of mathematical modeling and experimentation to elucidate the kinetics of NO/DAF-FM reaction in solution. Modeling and experiments suggest that the slope of fluorescent intensity (FI) can be related to NO concentration according to the equation: ddt[FI]=2αk1[NO]2[O2][DAF-FM]k[NO]+[DAF-FM] where α is a proportionality coefficient that relates FI to unit concentration of activated DAF-FM, k1 is the NO oxidation rate constant, and k was estimated to be 4.3 ± 0.6. The FI slope exhibits saturation kinetics with DAF-FM concentration. Interestingly, the effective half-maximum constant (EC50) increases proportionally to NO concentration. This result is not in agreement with the proposition that N2O3 is the NO oxidation byproduct that activates DAF-FM. Kinetic analysis suggests that the reactive intermediate should exhibit NO-dependent consumption and thus NO2• is a more likely candidate. The derived rate law can be used for the calibration of DAF-FM fluorescence and the quantification of NO concentration in biological tissues.
PMCID: PMC3544979  PMID: 23063986
nitric oxide; 4-amino-5-methylamino-2′,7′-difluorofluorescein; reaction kinetics

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