Katrina M Miranda

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• Basudhar, D., Cheng, R. C., Bharadwaj, G., Ridnour, L. A., Wink, D. A., & Miranda, K. M. (2015). Chemotherapeutic potential of diazeniumdiolate-based aspirin prodrugs in breast Cancer. Free radical biology & medicine.
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  Diazeniumdiolate-based aspirin prodrugs have previously been shown to retain the anti-inflammatory properties of aspirin while protecting against the common side effect of stomach ulceration. Initial analysis of two new prodrugs of aspirin that also release either nitroxyl (HNO) or nitric oxide (NO) demonstrated increased cytotoxicity toward human lung carcinoma cells compared to either aspirin or the parent nitrogen oxide donor. In addition, cytotoxicity was significantly lower in endothelial cells, suggesting cancer-specific sensitivity. To assess the chemotherapeutic potential of these new prodrugs in breast cancer, we studied their effect both in cultured cells and in a nude mouse model. Both prodrugs reduced growth of breast adenocarcinoma cells more effectively than the parent compounds while not being appreciably cytotoxic in a related non-tumorigenic cell line (MCF-10A). The HNO donor also was more cytotoxic than the related NO donor. The basis for the observed specificity was investigated in terms of impact on metabolism, DNA damage and repair, apoptosis, angiogenesis and metastasis. The results suggest a significant pharmacological potential for treatment of breast cancer.
• Jorolan, J. H., Buttitta, L. A., Cheah, C., & Miranda, K. M. (2015). Comparison of the chemical reactivity of synthetic peroxynitrite with that of the autoxidation products of nitroxyl or its anion. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society, 44, 39-46.
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  Donors of nitroxyl (HNO) exhibit pharmacological properties that are potentially favorable for treatment of a variety of diseases. To fully evaluate the pharmacological utility of HNO, it is therefore important to understand its chemistry, particularly involvement in deleterious biological reactions. Of particular note is the cytotoxic species formed from HNO autoxidation that is capable of inducing double strand DNA breaks. The identity of this species remains elusive, but a conceivable product is peroxynitrous acid. However, chemical comparison studies have demonstrated that HNO autoxidation leads to a unique reactive nitrogen oxide species to that of synthetic peroxynitrite. Here, we extend the analysis to include a new preparation of peroxynitrite formed via autoxidation of nitroxyl anion (NO(-)). Both peroxynitrite preparations exhibited similar chemical profiles, although autoxidation of NO(-) provided a more reliable sample of peroxynitrite. Furthermore, the observed dissimilarities to the HNO donor Angeli's salt substantiate that HNO autoxidation produces a unique intermediate from peroxynitrite.
• Bharadwaj, G., Benini, P. G., Basudhar, D., Ramos-Colon, C. N., Johnson, G. M., Larriva, M. M., Keefer, L. K., Andrei, D., & Miranda, K. M. (2014). Analysis of the HNO and NO donating properties of alicyclic amine diazeniumdiolates. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society, 42, 70-8.
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  Nitroxyl (HNO) donors have been shown to elicit a variety of pharmacological responses, ranging from tumoricidal effects to treatment of heart failure. Isopropylamine-based diazeniumdiolates have been shown to produce HNO on decomposition under physiological conditions. Herein, we report the synthesis and HNO release profiles of primary alicyclic amine-based diazeniumdiolates. These compounds extend the range of known diazeniumdiolate-based HNO donors. Acetoxymethyl ester-protected diazeniumdiolates were also synthesized to improve purification and cellular uptake. The acetoxymethyl derivative of cyclopentylamine diazeniumdiolate not only showed higher cytotoxicity toward cancer cells as compared to the parent anion but was also effective in combination with tamoxifen for targeting estrogen receptor α-negative breast cancer cells.
• Cheng, R. Y., Basudhar, D., Ridnour, L. A., Heinecke, J. L., Kesarwala, A. H., Glynn, S., Switzer, C. H., Ambs, S., Miranda, K. M., & Wink, D. A. (2014). Gene expression profiles of NO- and HNO-donor treated breast cancer cells: insights into tumor response and resistance pathways. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society, 43, 17-28.
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  Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.
• Ford, P., Pereira, J., & Miranda, K. (2013). Mechanisms of nitric oxide reactions mediated by biologically relevant metal centers. Structure and Bonding: Special Issue on Nitrosyl Complexes in Inorganic Chemistry, Biochemistry and Medicine.
• Roveda, A. C., Aguiar, H. d., Miranda, K. M., Tadini, C. C., & Franco, D. W. (2014). Light-triggered and cysteine-mediated nitric oxide release from a biodegradable starch-based film. JOURNAL OF MATERIALS CHEMISTRY B, 2(41), 7232-7242.
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  A new nitric oxide-releasing material produced with cassava starch is described. The ruthenium nitrosyl complex trans-[Ru(NH3)(4)(isn)NO](BF4)(3) (RuNOisn; isn = isonicotinamide) is able to release NO upon either photolysis or chemical reduction. Impregnating this complex under mild conditions into cassava starch (CS) films produced a NO-delivery platform (CSx-RuNOisn). Spectroscopic analysis of CSx-RuNOisn indicates that the coordination sphere of RuNOisn remains intact during film production. Exposure of CSx-RuNOisn to long wave UV-light (lambda(irr) = 355 nm) leads to NO release and formation of the paramagnetic photoproduct trans-[RuIII(NH3)(4)isn(H2O)](3+) in the CS film. Reaction of this aquaruthenium(III) complex with aqueous nitrite regenerates RuNOisn in the film. Delivery of NO upon photolysis of CSx-RuNOisn was verified by trapping with oxymyoglobin. Moreover, NO release upon chemical reduction was carried out using L-cysteine as a reductant. Cysteine-mediated NO delivery from CSx-RuNOisn persisted for more than 7 h, during which physiologically relevant NO concentrations were liberated. These results suggest that CSx-RuNOisn is a promising candidate for use in biological applications.
• Staurengo-Ferrari, L., Zarpelon, A., Longhi-Balbinot, D., Marchesi, M., Cunha, T., Cunha, T., Alves-Filho, J., Cunha, F., Ferriera, S., Casagrande, R., Miranda, K., & Verri, W. (2014). Nitroxyl inhibits overt pain-like behavior in mice: role of cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Pharmacological Reports.
• Wink, D., & Miranda, K. (2014). Persulfides and the cellular thiol landscape. Proceedings of the National Academy of Science USA.
• Basudhar, D., Bharadwaj, G., Cheng, R. Y., Jain, S., Shi, S., Heinecke, J. L., Holland, R. J., Ridnour, L. A., Caceres, V. M., Spadari-Bratfisch, R. C., Paolocci, N., Velázquez-Martínez, C. A., Wink, D. A., & Miranda, K. M. (2013). Synthesis and chemical and biological comparison of nitroxyl- and nitric oxide-releasing diazeniumdiolate-based aspirin derivatives. Journal of Medicinal Chemistry, 56(20), 7804-7820.
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  PMID: 24102516;Abstract: Structural modifications of nonsteroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but they may increase the risk of myocardial infarction with chronic use. The fact that nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction, and enhances contractility led us to synthesize a diazeniumdiolate-based HNO-releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward nonsmall cell lung carcinoma cells (A549), but they were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening on murine ventricular myocytes compared to control. Together, these anti-inflammatory, antineoplasic, and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer, or heart failure. © 2013 American Chemical Society.
• Heinrich, T. A., S., R., Miranda, K. M., Switzer, C. H., Wink, D. A., & Fukuto, J. M. (2013). Biological nitric oxide signalling: Chemistry and terminology. British Journal of Pharmacology, 169(7), 1417-1429.
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  PMID: 23617570;PMCID: PMC3724101;Abstract: Biological nitrogen oxide signalling and stress is an area of extreme clinical, pharmacological, toxicological, biochemical and chemical research interest. The utility of nitric oxide and derived species as signalling agents is due to their novel and vast chemical interactions with a variety of biological targets. Herein, the chemistry associated with the interaction of the biologically relevant nitrogen oxide species with fundamental biochemical targets is discussed. Specifically, the chemical interactions of nitrogen oxides with nucleophiles (e.g. thiols), metals (e.g. hemeproteins) and paramagnetic species (e.g. dioxygen and superoxide) are addressed. Importantly, the terms associated with the mechanisms by which NO (and derived species) react with their respective biological targets have been defined by numerous past chemical studies. Thus, in order to assist researchers in referring to chemical processes associated with nitrogen oxide biology, the vernacular associated with these chemical interactions is addressed. © 2013 The British Pharmacological Society.
• Johnson, G., Chozinski, T., Salmon, D., Moghaddam, A., Chen, H., & Miranda, K. (2013). Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent. Free Radical Biology and Medicine, 63, 476-484.
• Miranda, K., Basudhar, D., Bharadwaj, G., Cheng, R. Y., Jain, S., Shi, S., Heinecke, J. L., Holland, R. J., Ridnour, L. A., Caceres, V. M., Spadari-Bratfisch, R. C., Paolocci, N., Velázquez-Martínez, C. A., Wink, D. A., & Miranda, K. M. (2013). Synthesis and Chemical and Biological Comparison of Nitroxyl- and Nitric Oxide-Releasing Diazeniumdiolate-Based Aspirin Derivatives. Journal of medicinal chemistry.
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  Structural modifications of nonsteroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but they may increase the risk of myocardial infarction with chronic use. The fact that nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction, and enhances contractility led us to synthesize a diazeniumdiolate-based HNO-releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward nonsmall cell lung carcinoma cells (A549), but they were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening on murine ventricular myocytes compared to control. Together, these anti-inflammatory, antineoplasic, and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer, or heart failure.
• Miranda, K., Johnson, G. M., Chozinski, T. J., Salmon, D. J., Moghaddam, A. D., Chen, H. C., & Miranda, K. M. (2013). Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent. Free radical biology & medicine, 63.
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  Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.
• Zarpelon, A. C., Souza, G. R., Cunha, T. M., Schivo, I. R., Marchesi, M., Casagrande, R., Pinge-Filho, P., Cunha, F. Q., Ferreira, S. H., Miranda, K. M., & Verri Jr., W. A. (2013). The nitroxyl donor, Angeli's salt, inhibits inflammatory hyperalgesia in rats. Neuropharmacology, 71, 1-9.
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  PMID: 23541720;PMCID: PMC3666861;Abstract: Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO-) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na2N2O 3). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 μg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1β and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K+ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO+ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K+ channel signaling pathway in vivo. © 2013 Elsevier Ltd. All rights reserved.
• Flores-Santana, W., Salmon, D. J., Donzelli, S., Switzer, C. H., Basudhar, D., Ridnour, L., Cheng, R., Glynn, S. A., Paolocci, N., Fukuto, J. M., Miranda, K. M., & Wink, D. A. (2011). The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems. Antioxidants and Redox Signaling, 14(9), 1659-1674.
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  PMID: 21235346;PMCID: PMC3070000;Abstract: The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review. © 2011 Mary Ann Liebert, Inc.
• Miranda, K., Salmon, D. J., Torres de Holding, C. L., Thomas, L., Peterson, K. V., Goodman, G. P., Saavedra, J. E., Srinivasan, A., Davies, K. M., Keefer, L. K., & Miranda, K. M. (2011). HNO and NO release from a primary amine-based diazeniumdiolate as a function of pH. Inorganic chemistry, 50(8).
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  The growing evidence that nitroxyl (HNO) has a rich pharmacological potential that differs from that of nitric oxide (NO) has intensified interest in HNO donors. Recently, the diazeniumdiolate (NONOate) based on isopropylamine (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO)]) was demonstrated to function under physiological conditions as an organic analogue to the commonly used HNO donor Angeli's salt (Na(2)N(2)O(3)). The decomposition mechanism of Angeli's salt is dependent on pH, with transition from an HNO to an NO donor occurring abruptly near pH 3. Here, pH is shown to also affect product formation from IPA/NO. Chemical analysis of HNO and NO production led to refinement of an earlier, quantum mechanically based prediction of the pH-dependent decomposition mechanisms of primary amine NONOates such as IPA/NO. Under basic conditions, the amine proton of IPA/NO is able to initiate decomposition to HNO by tautomerization to the nitroso nitrogen (N(2)). At lower pH, protonation activates a competing pathway to NO production. At pH 8, the donor properties of IPA/NO and Angeli's salt are demonstrated to be comparable, suggesting that at or above this pH, IPA/NO is primarily an HNO donor. Below pH 5, NO is the major product, while IPA/NO functions as a dual donor of HNO and NO at intermediate pH. This pH-dependent variability in product formation may prove useful in examination of the chemistry of NO and HNO. Furthermore, primary amine NONOates may serve as a tunable class of nitrogen oxide donor.
• Salmon, D. J., L., C., Thomas, L., Peterson, K. V., Goodman, G. P., Saavedra, J. E., Srinivasan, A., Davies, K. M., Keefer, L. K., & Miranda, K. M. (2011). HNO and NO release from a primary amine-based diazeniumdiolate as a function of pH. Inorganic Chemistry, 50(8), 3262-3270.
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  PMID: 21405089;PMCID: PMC3075328;Abstract: The growing evidence that nitroxyl (HNO) has a rich pharmacological potential that differs from that of nitric oxide (NO) has intensified interest in HNO donors. Recently, the diazeniumdiolate (NONOate) based on isopropylamine (IPA/NO; Na[(CH 3) 2CHNH(N(O)NO)]) was demonstrated to function under physiological conditions as an organic analogue to the commonly used HNO donor Angeli's salt (Na 2N 2O 3). The decomposition mechanism of Angeli's salt is dependent on pH, with transition from an HNO to an NO donor occurring abruptly near pH 3. Here, pH is shown to also affect product formation from IPA/NO. Chemical analysis of HNO and NO production led to refinement of an earlier, quantum mechanically based prediction of the pH-dependent decomposition mechanisms of primary amine NONOates such as IPA/NO. Under basic conditions, the amine proton of IPA/NO is able to initiate decomposition to HNO by tautomerization to the nitroso nitrogen (N 2). At lower pH, protonation activates a competing pathway to NO production. At pH 8, the donor properties of IPA/NO and Angeli's salt are demonstrated to be comparable, suggesting that at or above this pH, IPA/NO is primarily an HNO donor. Below pH 5, NO is the major product, while IPA/NO functions as a dual donor of HNO and NO at intermediate pH. This pH-dependent variability in product formation may prove useful in examination of the chemistry of NO and HNO. Furthermore, primary amine NONOates may serve as a tunable class of nitrogen oxide donor. © 2011 American Chemical Society. © 2011 American Chemical Society. © 2011 American Chemical Society.
• Andrei, D., Salmon, D. J., Donzelli, S., Wahab, A., Klose, J. R., Citro, M. L., Saavedra, J. E., Wink, D. A., Miranda, K. M., & Keefer, L. K. (2010). Dual mechanisms of HNO generation by a nitroxyl prodrug of the diazeniumdiolate (NONOate) class. Journal of the American Chemical Society, 132(46), 16526-16532.
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  PMID: 21033665;PMCID: PMC2984372;Abstract: Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)=NO-Na+ (1, IPA/NO) with BrCH2OAc produced a stable derivative of structure iPrHN-N(O)=NO-CH2OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH3-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies. © 2010 American Chemical Society.
• Jourd'heuil, D., Lancaster Jr., J. R., Fukuto, J., Roberts, D. D., Miranda, K. M., Mayer, B., Grisham, M. B., & Wink, D. A. (2010). The bell-shaped curve for peroxynitrite-mediated oxidation and nitration of NO/O₂^.- is alive and well. Journal of Biological Chemistry, 285(35), le15.
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  PMID: 20729216;PMCID: PMC2930750;
• Kumars, M. R., Fukuto, J. M., Miranda, K. M., & Farmer, P. J. (2010). Reactions of HNO with heme proteins: New routes to HNO-heme complexes and insight into physiological effects. Inorganic Chemistry, 49(14), 6283-6292.
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  PMID: 20666387;PMCID: PMC2912650;Abstract: The formation and interconversion of nitrogen oxides has been of interest in numerous contexts for decades. Early studies focused on gas-phase reactions, particularly with regard to industrial and atmospheric environments, and on nitrogen fixation. Additionally, investigation of the coordination chemistry of nitric oxide (NO) with hemoglobin dates back nearly a century. With the discovery in the early 1980s that NO is blosynthesized as a molecular signaling agent, the literature has been focused on the biological effects of nitrogen oxides, but the original concerns remain relevant. For instance, hemoglobin has long been known to react with nitrite, but this reductase activity has recently been considered to be important to produce NO under hypoxic conditions. The association of nitrosyl hydride (HNO; also commonly referred to as nitroxyl) with heme proteins can also produce NO by reductive nitrosylation. Furthermore, HNO is considered to be an intermediate in bacterial denitrification, but conclusive identification has been elusive. The authors of this article have approached the bioinorganic chemistry of HNO from different perspectives, which have converged because heme proteins are important biological targets of HNO. © 2010 American Chemical Society.
• Flores-Santana, W., Switzer, C., Ridnour, L. A., Basudhar, D., Mancardi, D., Donzelli, S., Thomas, D. D., Miranda, K. M., Fukuto, J. M., & Wink, D. A. (2009). Comparing the chemical biology of NO and HNO. Archives of Pharmacal Research, 32(8), 1139-1153.
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  PMID: 19727606;Abstract: For the past couple of decades nitric oxide (NO) and nitroxyl (HNO) have been extensively studied due to the important role they play in many physiological and/or pharmacological processes. Many researchers have reported important signaling pathways as well as mechanisms of action of these species, showing direct and indirect effects depending on the environment. Both NO and HNO can react with, among others, metals, proteins, thiols and heme proteins via unique and distinct chemistry leading to improvement of some clinical conditions. Understanding the basic chemistry of NO and HNO and distinguishing their mechanisms of action as well as methods of detection are crucial for understanding the current and potential clinical applications. In this review, we summarize some of the most important findings regarding NO and HNO chemistry, revealing some of the possible mechanisms of their beneficial actions. © 2009 The Pharmaceutical Society of Korea.
• Jackson, M. I., Han, T. H., Serbulea, L., Dutton, A., Ford, E., Miranda, K. M., Houk, K. N., Wink, D. A., & Fukuto, J. M. (2009). Kinetic feasibility of nitroxyl reduction by physiological reductants and biological implications. Free Radical Biology and Medicine, 47(8), 1130-1139.
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  PMID: 19577638;Abstract: Nitroxyl (HNO), the one-electron reduced and protonated congener of nitric oxide (NO), is a chemically unique species with potentially important biological activity. Although HNO-based pharmaceuticals are currently being considered for the treatment of chronic heart failure or stroke/transplant-derived ischemia, the chemical events leading to therapeutic responses are not established. The interaction of HNO with oxidants results in the well-documented conversion to NO, but HNO is expected to be readily reduced as well. Recent thermodynamic calculations predict that reduction of HNO is biologically accessible. Herein, kinetic analysis suggests that the reactions of HNO with several mechanistically distinct reductants are also biologically feasible. Product analysis verified that the reductants had in fact been oxidized and that in several instances HNO had been converted to hydroxylamine. Moreover, a theoretical analysis suggests that in the reaction of HNO with thiol reductants, the pathway producing sulfinamide is significantly more favorable than that leading to disulfide. Additionally, simultaneous production of HNO and NO yielded a biphasic oxidative capacity. © 2009 Elsevier Inc. All rights reserved.
• Miller, T. W., Cherney, M. M., Lee, A. J., Francolean, N. E., Farmer, P. J., King, S. B., Hobbs, A. J., Miranda, K. M., Burstyn, J. N., & Fukuto, J. M. (2009). The effects of nitroxyl (HNO) on soluble guanylate cyclase activity. Interactions at ferrous heme and cysteine thiols. Journal of Biological Chemistry, 284(33), 21788-21796.
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  PMID: 19531488;PMCID: PMC2755905;Abstract: It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Switzer, C. H., Flores-Santana, W., Mancardi, D., Donzelli, S., Basudhar, D., Ridnour, L. A., Miranda, K. M., Fukuto, J. M., Paolocci, N., & Wink, D. A. (2009). The emergence of nitroxyl (HNO) as a pharmacological agent. Biochimica et Biophysica Acta - Bioenergetics, 1787(7), 835-840.
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  PMID: 19426703;PMCID: PMC2761033;Abstract: Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one-electron oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment before ischemia-reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.
• Donzelli, S., Espey, M. G., Flores-Santana, W., Switzer, C. H., Yeh, G. C., Huang, J., Stuehr, D. J., King, S. B., Miranda, K. M., & Wink, D. A. (2008). Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine. Free Radical Biology and Medicine, 45(5), 578-584.
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  PMID: 18503778;PMCID: PMC2562766;Abstract: The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H2O2 to oxidize hydroxylamine (NH2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH2 was observed upon oxidation of NH2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH2OH, the highest yields of GS(O)NH2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.
• Novais, Z., Sérgio, M., Molin, J. C., Lunardi, C. N., Miranda, K. M., Bendhack, L. M., Ford, P. C., & Santana, R. (2008). The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO₂)L(bpy)₂]⁺ complex. Dalton Transactions, 4282-4287.
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  PMID: 18682867;Abstract: The synthesis of [Ru(NO2)L(bpy)2]+ (bpy = 2,2′-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO)L(bpy)2](PF6) 3 to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2)L(bpy)2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around -0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at -0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS+ and spectroelectrochemical experiment where cis-[Ru(bpy) 2L(H2O)]2+ was obtained as a product of the reduction of cis-[RuII(NO2)L(bpy)2] +. The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 μmol L-1 phenylephrine responded with relaxation in the presence of cis-[RuII(NO2)L(bpy)2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO 2)L(bpy)2]+ was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO2)L(bpy)2]+ inside the cell. The maximum vasorelaxation was achieved with 1 × 10-5 mol L-1 of cis-[RuII(NO2)L(bpy)2] + complex. © 2008 The Royal Society of Chemistry.
• Omsland, A., Miranda, K. M., Friedman, R. L., & Boitano, S. (2008). Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses. FEMS Microbiology Letters, 284(1), 92-101.
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  PMID: 18462394;PMCID: PMC2517120;Abstract: Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-μM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
• Väänänen, A. J., Salmenperä, P., Hukkanen, M., Miranda, K. M., Harjula, A., Rauhala, P., & Kankuri, E. (2008). Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the presence of endogenous nitric oxide. Free Radical Biology and Medicine, 45(6), 749-755.
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  PMID: 18572022;Abstract: Nitrosation of enzyme regulatory cysteines is one of the key posttranslational modification mechanisms of enzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such as cathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO production could provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally, endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteines prior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-γ, on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-γ induce iNOS with generation of nitrate up to 9 μM in the media after a 24-h stimulation, while native RAW 264.7 macrophages neither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0-316 μM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOS activity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, where papain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 μM) in the presence of the NO donor DEA/NO (0-316 μM). This clearly showed that a high molar excess of DEA/NO (EC50 406 μM) is needed to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide first evidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteases for modification by HNO. © 2008 Elsevier B.V. All rights reserved.
• Paolocci, N., Jackson, M. I., Lopez, B. E., Miranda, K., Tocchetti, C. G., Wink, D. A., Hobbs, A. J., & Fukuto, J. M. (2007). The pharmacology of nitroxyl (HNO) and its therapeutic potential: Not just the janus face of NO¹1This review is dedicated to the career of Prof. Herbert T. Nagasawa, a pioneer in the field of HNO chemistry, biochemistry and pharmacology.. Pharmacology and Therapeutics, 113(2), 442-458.
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  PMID: 17222913;PMCID: PMC3501193;Abstract: Nitroxyl (HNO), the 1-electron reduced and protonated congener of nitric oxide (NO), has received recent attention as a potential pharmacological agent for the treatment of heart failure and as a preconditioning agent for the mitigation of ischemia-reperfusion injury. Interest in the pharmacology and biology of HNO has prompted examination, or in some instances reexamination, of many of its chemical properties. Such studies have provided insight into the chemical basis for the biological effects of HNO, although the biochemical mechanisms for many of these effects remain to be established. In this review, a brief description of the biologically relevant chemistry of HNO is given, followed by a more detailed discussion of the pharmacology and potential toxicology of HNO. © 2006 Elsevier Inc. All rights reserved.
• Donzelli, S., Espey, M. G., Thomas, D. D., Mancardi, D., Tocchetti, C. G., Ridnour, L. A., Paolocci, N., King, S. B., Miranda, K. M., Lazzarino, G., Fukuto, J. M., & Wink, D. A. (2006). Discriminating formation of HNO from other reactive nitrogen oxide species. Free Radical Biology and Medicine, 40(6), 1056-1066.
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  PMID: 16540401;Abstract: Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GS(O)NH 2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GS(O)NH2 was not observed with other nitrogen oxides (NO, N 2O3, NO2, or ONOO-), indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GS(O)NH2. Detection of GS(O)NH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.
• Donzelli, S., Switzer, C. H., Thomas, D. D., Ridnour, L. A., Espey, M. G., Isenberg, J. S., Tocchetti, C. G., King, S. B., Lazzarino, G., Miranda, K. M., Roberts, D. D., Feelisch, M., & Wink, D. A. (2006). The activation of metabolites of nitric oxide synthase by metals is both redox and oxygen dependent: A new feature of nitrogen oxide signaling. Antioxidants and Redox Signaling, 8(7-8), 1363-1371.
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  PMID: 16910783;Abstract: Nitrite (NO2-), NG-hydroxy-L-arginine (NOHA), and hydroxylamine (NH2OH) are products of nitric oxide synthase (NOS) activity and can also be formed by secondary reactions of nitric oxide (NO). These compounds are commonly considered to be rather stable and as such to be dosimeters of NO biosynthesis. However, each can be converted via metal-catalyzed reactions into either NO or other reactive nitrogen oxide species (RNOS), such as nitrogen dioxide (NO2) and nitroxyl (HNO), which have biologic activities distinct from those of the parent molecules. Consequently, certain aspects of tissue regulation controlled by RNOS may be dictated to a significant extent by metal-dependent reactions, thereby offering unique advantages for cellular and tissue regulation. For instance, because many metal-catalyzed reactions depend on the redox and oxygen status of the cellular environment, such reactions could serve as redox indicators. Formation of RNOS by metal-mediated pathways would confine the chemistry of these species to specific cellular sites. Additionally, such mechanisms would be independent both of NO and NOS, thus increasing the lifetime of RNOS that react with NO. Thus metal-mediated conversion of nitrite, NOHA, and NH2OH into biologically active agents may provide a unique signaling mechanism. In this review, we discuss the biochemistry of such reactions in the context of their pharmacologic and biologic implications. © Mary Ann Liebert, Inc.
• Dutton, A. S., Suhrada, C. P., Miranda, K. M., Wink, D. A., Fukuto, J. M., & Houk, K. N. (2006). Mechanism of pH-dependent decomposition of monoalkylamine diazeniumdiolates to form HNO and NO, deduced from the model compound methylamine diazeniumdiolate, density functional theory, and CBS-QB3 calculations. Inorganic Chemistry, 45(6), 2448-2456.
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  PMID: 16529464;PMCID: PMC3164114;Abstract: Isopropylamine diazeniumdiolate, IPA/NO, the product of the reaction of isopropylamine and nitric oxide, NO, decomposes in a pH-dependent manner to afford nitroxyl, HNO, in the pH range of 13 to above 5, and NO below pH 7. Theoretical studies using B3LYP/6-311+G(d) density functional theory, the polarizable continuum and conductor-like polarizable continuum solvation models, and the high-accuracy CBS-QB3 method on the simplified model compound methylamine diazeniumdiolate predict a mechanism involving HNO production via decomposition of the unstable tautomer MeNN+(O-)NHO -. The production of NO at lower pH is predicted to result from fragmentation of the amide/NO adduct upon protonation of the amine nitrogen. © 2006 American Chemical Society.
• Katori, T., Donzelli, S., Tocchetti, C. G., Miranda, K. M., Cormaci, G., Thomas, D. D., Ketner, E. A., Lee, M. J., Mancardi, D., Wink, D. A., Kass, D. A., & Paolocci, N. (2006). Peroxynitrite and myocardial contractility: In vivo versus in vitro effects. Free Radical Biology and Medicine, 41(10), 1606-1618.
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  PMID: 17045928;Abstract: Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish β-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted β-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function. © 2006 Elsevier Inc. All rights reserved.
• Espey, M., Xavier, S., Thomas, D., Miranda, K., & Wink, D. (2005). Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 99(6), 3481-3486.
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  3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 muM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC50 approximate to 20 muM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC50 > 250 muM). The more physiologically relevant conditions of either peroxynitrite infusion (1 muM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO2) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO2 can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of NO2 may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.
• Fukuto, J. M., Switzer, C. H., Miranda, K. M., & Wink, D. A. (2005). Nitroxyl (HNO): Chemistry, biochemistry, and pharmacology. Annual Review of Pharmacology and Toxicology, 45, 335-355.
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  PMID: 15822180;Abstract: Recent discoveries of novel and potentially important biological activity have spurred interest in the chemistry and biochemistry of nitroxyl (HNO). It has become clear that, among all the nitrogen oxides, HNO is unique in its chemistry and biology. Currently, the intimate chemical details of the biological actions of HNO are not well understood. Moreover, many of the previously accepted chemical properties of HNO have been recently revised, thus requiring reevaluation of possible mechanisms of biological action. Herein, we review these developments in HNO chemistry and biology.
• Miranda, K. M. (2005). The chemistry of nitroxyl (HNO) and implications in biology. Coordination Chemistry Reviews, 249(3-4 SPEC. ISS.), 433-455.
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  Abstract: Over the past century, HNO research has evolved from fundamental physical examinations to elucidation of interactions in atmospheric, industrial and bacterial processes. Most recently, the HNO literature has been primarily concerned with the pharmacological effects and potential physiological functions of HNO in mammalian systems. The chemistry of HNO is inordinately complicated for a triatomic molecule. Further, the rapid self-consumption of HNO through dehydrative dimerization impedes detection and necessitates in situ production. This review provides a detailed discussion of the most common donors of HNO and of the current understanding of the aqueous chemistry of HNO and the synthesis, consumption and reactivity of HNO in a cellular environment, as ascertained with these donors. Additionally, the consequences of the molecular interactions of HNO on physiology are described, and a comparison is made to NO in terms of cellular signaling and pharmacological potential. © 2004 Elsevier B.V. All rights reserved.
• Miranda, K. M., Dutton, A. S., Ridnour, L. A., Foreman, C. A., Ford, E., Paolocci, N., Katori, T., Tocchetti, C. G., Mancardi, D., Thomas, D. D., Espey, M. G., Houk, K. N., Fukuto, J. M., & Wink, D. A. (2005). Mechanism of aerobic decomposition of Angeli's salt (Sodium Trioxodinitrate) at physiological pH. Journal of the American Chemical Society, 127(2), 722-731.
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  PMID: 15643898;Abstract: The recent determination that Angeli's salt may have clinical application as a nitrogen oxide donor for treatment of cardiovascular diseases such as heart failure has led to renewed interest in the mechanism and products of thermal decomposition of Angeli's salt under physiological conditions. In this report, several mechanisms are evaluated experimentally and by quantum mechanical calculations to determine whether HNO is in fact released from Angeli's salt in neutral, aerobic solution. The mechanism of product autoxidation is also considered.
• Miranda, K. M., Katori, T., L., C., Thomas, L., Ridnour, L. A., McLendon, W. J., Cologna, S. M., Dutton, A. S., Champion, H. C., Mancardi, D., Tocchetti, C. G., Saavedra, J. E., Keefer, L. K., Houk, K. N., Fukuto, J. M., Kass, D. A., Paolocci, N., & Wink, D. A. (2005). Comparison of the NO and HNO donating properties of diazeniumdiolates: Primary amine adducts release HNO in vivo. Journal of Medicinal Chemistry, 48(26), 8220-8228.
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  PMID: 16366603;Abstract: Diazeniumdiolates, more commonly referred to as NONOates, have been extremely useful in the investigation of the biological effects of nitric oxide (NO) and related nitrogen oxides. The NONOate Angeli's salt (Na 2N2O3) releases nitroxyl (HNO) under physiological conditions and exhibits unique cardiovascular features (i.e., positive inotropy/lusitropy) that may have relevance for pharmacological treatment of heart failure. In the search for new, organic-based compounds that release HNO, we examined isopropylamine NONOate (IPA/NO; Na[(CH 3)2-CHNH(N(O)NO]), which is an adduct of NO and a primary amine. The chemical and pharmacological properties of IPA/NO were compared to those of Angeli's salt and a NO-producing NONOate, DEA/NO (Na[Et 2NN(O)NO]), which is a secondary amine adduct. Under physiological conditions IPA/NO exhibited all the markers of HNO production (e.g., reductive nitrosylation, thiol reactivity, positive inotropy). These data suggest that primary amine NONOates may be useful as HNO donors in complement to the existing series of secondary amine NONOates, which are well-characterized NO donors. © 2005 American Chemical Society.
• Miranda, K. M., Nagasawa, H. T., & Toscano, J. P. (2005). Donors of HNO. Current Topics in Medicinal Chemistry, 5(7), 649-664.
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  PMID: 16101426;Abstract: Recent comparisons of the pharmacological effects of nitric oxide (NO) and nitroxyl (HNO) donors have demonstrated that the responses to these redox-related nitrogen oxides are nearly universally dissimilar. These analyses have suggested the existence of mutually exclusive signaling pathways as a result of discrete chemical interactions of HNO and NO with a variety of critical biomolecules. Although the mechanisms of action are currently unresolved, the pharmacological responses to HNO are promising for clinical treatment of cardiovascular diseases such as heart failure, myocardial infarction and stroke. This review provides a detailed discussion of the most commonly utilized donors of HNO as well as a guideline for the characterization of novel donors. © 2005 Bentham Science Publishers Ltd.
• Miranda, K. M., Ridnour, L., Esprey, M., Citrin, D., Thomas, D., Mancardi, D., Donzelli, S., Wink, D. A., Katori, T., Tocchetti, C. G., Ferlito, M., Paolocci, N., & Fukuto, J. M. (2005). Comparison of the chemical biology of NO and HNO: An inorganic perspective. Progress in Inorganic Chemistry, 54, 349-384.
• Miranda, K., Espey, M., Yamada, K., Krishna, M., Ludwick, N., Kim, S., Jourd'heuil, D., Grisham, M., Feelisch, M., Fukuto, J., & Wink, D. (2005). Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion. JOURNAL OF BIOLOGICAL CHEMISTRY, 276(3), 1720-1727.
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  The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O-2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O-2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O-2(-) this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.
• Paolocci, N., Saavedra, W., Miranda, K., Martignani, C., Isoda, T., Hare, J., Espey, M., Fukuto, J., Feelisch, M., Wink, D., & Kass, D. (2005). Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 98(18), 10463-10468.
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  Nitroxyl anion (NO-) is the one-electron reduction product of nitric oxide (NO.) and is enzymatically generated by NO synthase in vitro. The physiologic activity and mechanism of action of NO- in vivo remains unknown. The NO- generator Angeli's salt (AS, Na2N2O3) was administered to conscious chronically instrumented dogs, and pressure-dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac preload volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of N-acetyl-L-cysteine. Cardiac inotropic signaling by NO- was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP-(8-37) prevented this effect but not systemic vasodilation. Thus, NO- is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking NO- to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, NO- may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.
• Mancardi, D., Ridnour, L. A., Thomas, D. D., Katori, T., Tocchetti, C. G., Espey, M. G., Miranda, K. M., Paolocci, N., & Wink, D. A. (2004). The chemical dynamics of NO and reactive nitrogen oxides: A practical guide. Current Molecular Medicine, 4(7), 723-740.
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  PMID: 15579020;Abstract: Nitric oxide has emerged as one of the most important and diverse players in physiology. This small diatomic radical stunned researchers because of its existence and unique biological properties in human physiology. Over the last two decades it was found that NO often has fickle behavior in pathophysiological mechanisms. Where benefiting the host in one case yet inducing and augmenting injury in another. This has lead to confusion in is NO good or bad? Much of the answers to this dichotomy lies in the chemistry of NO and its related nitrogen oxide species. To help understand the complex chemistry with perspective to biology, a discussion on the chemical biology of NO is useful. The chemical biology defines the relevant chemical reaction of NO and nitrogen monoxide in the context of the biological conditions. We discuss in this article the chemistry of nitrogen oxide with different types of biological motifs. Reaction of NO with metal complexes and radicals require low concentration, where formation of reactive nitrogen oxide species require considerably higher amounts and generally are isolated to specific microenvironments in vivo. Though many reactive nitrogen oxide species are formed from chemical reactions with NO, there are several which appear to not require NO to be present, HNO and NO2. These two species have unique physiological effects and represent additional complexity to this biological picture. From this discussion, a picture can be formed concerning the possible chemical dynamics, which can be plausible in different biological mechanisms. © 2004 Bentham Science Publishers Ltd.
• Pagliaro, P., Mancardi, D., Rastaldo, R., Penna, C., Gattullo, D., Miranda, K., Feelisch, M., Wink, D., Kass, D., & Paolocci, N. (2004). Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning. FREE RADICAL BIOLOGY AND MEDICINE, 34(1), 33-43.
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  Nitric oxide (NO) donors mimic the early phase of ischemic preconditioning (IPC). The effects of nitroxyl (HNO/NO-), the one-electron reduction product of NO, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated whether HNO/NO-, produced by decomposition of Angeli's salt (AS; Na2N2O3), has a cardioprotective effect in isolated perfused rat hearts. Effects were examined after intracoronary perfusion (19 min) of either AS (1 muM), the NO donor diethylamine/NO (DEA/NO, 0.5 muM), vehicle (100 nM NaOH) or buffer, followed by global ischemia (30 min) and reperfusion (30 min or 120 min in a subset of hearts). IPC was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each intervention was assessed by changes in myocardial contractility as well as lactate dehydrogenase (LDH) release and infarct size. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly improved with IPC and pre-exposure to AS, as opposed to control or DEA/NO-treated hearts. Infarct size and LDH release were also significantly reduced in IPC and AS groups, whereas DEA/NO was less effective in limiting necrosis. Co-infusion in the triggering phase of AS and the nitroxyl scavenger, N-acetyl-L-cysteine (4 mM) completely reversed the beneficial effects of AS, both at 30 and 120 min reperfusion. Our data show that HNO/NO- affords myocardial protection to a degree similar to IPC and greater than NO, suggesting that reactive nitrogen oxide species are not only necessary but also sufficient to trigger myocardial protection against reperfusion through species-dependent, pro-oxidative, and/or nitrosative stress-related mechanisms. (C) 2002 Elsevier Science Inc.
• Ridnour, L. A., Thomas, D. D., Mancardi, D., Donzelli, S., Paolocci, N., Pagliaro, P., Miranda, K. M., Krishna, M., Fukuto, J., Grisham, M. B., Mitchell, J. B., Espey, M. G., & Wink, D. A. (2004). Antioxidant properties of nitric oxide in cellular physiological and pathophysiological mechanisms. The implications of biological balance between ^•NO and oxidative stress. Current Medicinal Chemistry: Anti-Inflammatory and Anti-Allergy Agents, 3(3), 181-188.
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  Abstract: The function of nitric oxide (•NO) in pathophysiology remains confounding as both protective and cytotoxic effects of •NO have been demonstrated in many disease processes. Nitric oxide chemistry culminating in the generation of oxidative as well as nitrosative intermediates have generally been proposed as mediators of pathophysiology and have overshadowed the antioxidant capabilities of •NO. However, the counteracting role of •NO in providing a balance under conditions of oxidative and nitrosative stress has been underappreciated. The purpose of this review is the discussion of the role of •NO as an antioxidant and interceptor of more potent reactive intermediates in normal physiology and disease. © 2004 Bentham Science Publishers Ltd.
• Ridnour, L. A., Thomas, D. D., Mancardi, D., Espey, M. G., Miranda, K. M., Paolocci, N., Feelisch, M., Fukuto, J., & Wink, D. A. (2004). The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting perspective on stressful biological situations. Biological Chemistry, 385(1), 1-10.
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  PMID: 14977040;Abstract: This review addresses many of the chemical aspects of nitrosative stress mediated by N2O3. From a cellular perspective, N2O3 and the resulting reactive nitrogen oxide species target specific motifs such as thiols, lysine active sites, and zinc fingers and is dependant upon both the rates of production as well as consumption of NO and must be taken into account in order to access the nitrosative environment. Since production and consumption are integral parts of N2O3 generation, we predict that nitrosative stress occurs under specific conditions, such as chronic inflammation. In contrast to conditions of stress, nitrosative chemistry may also provide cellular protection through the regulation of critical signaling pathways. Therefore, a careful evaluation of the chemistry of nitrosation based upon specific experimental conditions may provide a better understanding of how the subtle balance between oxidative and nitrosative stress may be involved in the etiology and control of various disease processes. Copyright © by Walter de Gruyter.
• Espey, M., Miranda, K., Thomas, D., & Wink, D. (2003). Distinction between nitrosating mechanisms within human cells and aqueous solution. JOURNAL OF BIOLOGICAL CHEMISTRY, 276(32), 30085-30091.
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  The quintessential nitrosating species produced during NO autoxidation is N2O3. Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt2NN(O)NO (DEA/NO), hum an tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N2O3 through the intermediacy of NO2. This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO2 was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.
• Espey, M., Miranda, K., Thomas, D., Xavier, S., Citrin, D., Vitek, M., Wink, D., Chiueh, C., Hong, J., & Leong, S. (2003). A chemical perspective on the interplay between NO, reactive oxygen species, and reactive nitrogen oxide species. NITRIC OXIDE: NOVEL ACTIONS, DELETERIOUS EFFECTS AND CLINICAL POTENTIAL, 962, 195-206.
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  Nitric oxide (nitrogen monoxide, NO) plays a veritable cornucopia of regulatory roles in normal physiology. In contrast, NO has also been implicated in the etiology and sequela of numerous neurodegenerative diseases that involve reactive oxygen species (ROS) and nitrogen oxide species (RNOS). In this setting, NO is often viewed solely as pathogenic; however, the chemistry of NO can also be a significant factor in lessening injury mediated by both ROS and RNOS. The relationship between NO and oxidation, nitrosation, and nitration reactions is summarized. The salient factors that determine whether NO promotes, abates, or interconnects these chemistries are emphasized. From this perspective of NO chemistry, the type, magnitude, location, and duration of either ROS or RNOS reactions may be predicted.
• Hofseth, L. J., Saito, S., Hussain, S. P., Espey, M. G., Miranda, K. M., Araki, Y., Jhappan, C., Higashimoto, Y., Peijun, H. e., Linke, S. P., Quezado, M. M., Zurer, I., Rotter, V., Wink, D. A., Appella, E., & Harris, C. C. (2003). Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. Proceedings of the National Academy of Sciences of the United States of America, 100(1), 143-148.
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  PMID: 12518062;PMCID: PMC140909;Abstract: Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 post-translational modifications, leading to an increase in p53 transcriptional targets and a G2/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21WAF1 was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation.
• Hofseth, L., Saito, S., Hussain, S., Espey, M., Miranda, K., Araki, Y., Jhappan, C., Higashimoto, Y., He, P., Linke, S., Quezado, M., Zurer, ., Rotter, ., Wink, D., Appella, E., & Harris, C. (2003). Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(1), 143-148.
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  Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a G(2)/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21(WAF1) was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation.
• Lorkovic, I., Miranda, K., Lee, B., Bernhard, S., Schoonover, ., & Ford, P. (2003). Flash photolysis studies of the ruthenium(II) porphyrins Ru(P)(NO)(ONO). Multiple pathways involving reactions of intermediates with nitric oxide. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 120(45), 11674-11683.
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  Described are the spectra and kinetics of transients formed by laser flash photolysis of the ruthenium nitrosyl nitrito complexes Ru(P)(NO)(ONO), P = TPP (meso-tetraphenylporphyrin), OEP (octaethylporphyrin), TmTP (tetra(m-tolyl)porphyrin), and FTTP (tetra(m-trifluoromethylphenyl)porphyrin) in benzene solutions. Two transient decay processes are seen on the time frame (
• Miranda, K. M., Nims, R. W., Thomas, D. D., Espey, M. G., Citrin, D., Bartberger, M. D., Paolocci, N., Fukuto, J. M., Feelisch, M., & Wink, D. A. (2003). Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins: A chemical discussion of the differential biological effects of these redox related products of NOS. Journal of Inorganic Biochemistry, 93(1-2), 52-60.
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  PMID: 12538052;Abstract: Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO-) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.
• Miranda, K. M., Paolocci, N., Katori, T., Thomas, D. D., Ford, E., Bartberger, M. D., Espey, M. G., Kass, D. A., Feelisch, M., Fukuto, J. M., & Wink, D. A. (2003). A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system. Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9196-9201.
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  PMID: 12865500;PMCID: PMC170895;Abstract: The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O2/O2- couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.
• Pagliaro, P., Mancardi, D., Rastaldo, R., Penna, C., Gattullo, D., Miranda, K. M., Feelisch, M., Wink, D. A., Kass, D. A., & Paolocci, N. (2003). Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning. Free Radical Biology and Medicine, 34(1), 33-43.
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  PMID: 12498977;Abstract: Nitric oxide (NO) donors mimic the early phase of ischemic preconditioning (IPC). The effects of nitroxyl (HNO/NO-), the one-electron reduction product of NO, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated whether HNO/NO-, produced by decomposition of Angeli's salt (AS; Na2N2O3), has a cardioprotective effect in isolated perfused rat hearts. Effects were examined after intracoronary perfusion (19 min) of either AS (1 μM), the NO donor diethylamine/NO (DEA/NO, 0.5 μM), vehicle (100 nM NaOH) or buffer, followed by global ischemia (30 min) and reperfusion (30 min or 120 min in a subset of hearts). IPC was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each intervention was assessed by changes in myocardial contractility as well as lactate dehydrogenase (LDH) release and infarct size. Postischemic contractility, as indexed by developed pressure and dP/dtmax, was similarly improved with IPC and pre-exposure to AS, as opposed to control or DEA/NO-treated hearts. Infarct size and LDH release were also significantly reduced in IPC and AS groups, whereas DEA/NO was less effective in limiting necrosis. Co-infusion in the triggering phase of AS and the nitroxyl scavenger, N-acetyl-L-cysteine (4 mM) completely reversed the beneficial effects of AS, both at 30 and 120 min reperfusion. Our data show that HNO/NO- affords myocardial protection to a degree similar to IPC and greater than NO, suggesting that reactive nitrogen oxide species are not only necessary but also sufficient to trigger myocardial protection against reperfusion through species-dependent, pro-oxidative, and/or nitrosative stress-related mechanisms. © 2002 Elsevier Science Inc.
• Paolocci, N., Katori, T., Champion, H. C., E., M., Miranda, K. M., Fukuto, J. M., Wink, D. A., & Kass, D. A. (2003). Positive inotropic and lusitropic effects of HNO/NO^- in failing hearts: Independence from β-adrenergic signaling. Proceedings of the National Academy of Sciences of the United States of America, 100(9), 5537-5542.
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  PMID: 12704230;PMCID: PMC154380;Abstract: Nitroxyl anion (HNO/NO-), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO- augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts β-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO- generated by Angelis' salt (AS) was infused (10 μg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)/NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed β-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted β-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO- cardiotropic action. Thus, HNO/NO- has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to β-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO- donors for the treatment of heart failure.
• Sidorkina, O., Espey, M. G., Miranda, K. M., Wink, D. A., & Laval, J. (2003). Inhibition of poly(ADP-ribose) polymerase (PARP) by nitric oxide and reactive nitrogen oxide species. Free Radical Biology and Medicine, 35(11), 1431-1438.
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  PMID: 14642390;Abstract: The poly(ADP-ribose) polymerase (PARP) family of nuclear enzymes is involved in the detection and signaling of single strand breaks induced either directly by ionizing radiation or indirectly by the sequential action of various DNA repair proteins. Therefore, PARP plays an important role in maintaining genome stability. Because PARP proteins contain two zinc finger motifs, these enzymes can be targets for reactive nitrogen oxide intermediates (RNOS) generated as a result of nitric oxide (NO) biosynthesis in an aerobic environment. The effects of RNOS on the activity of purified PARP were examined using donor compounds. Both NO and nitroxyl (HNO) donors were found to be inhibitory in a similar time and concentration manner, indicating that PARP activity can be modified under both nitrosative and oxidative conditions. Moreover, these RNOS donors elicited comparable PARP inhibition in Sf21 insect cell extract and intact human MCF-7 cancer cells. The concentrations of donor required for 90% inhibition of PARP activity produce RNOS at a similar magnitude to those generated in the cellular microenvironment of activated leukocytes, suggesting that cellular scavenging of RNOS may not be protective against PARP modification and that inhibition of PARP may be significant under inflammatory conditions. © 2003 Elsevier Inc.
• Thomas, D. D., Miranda, K. M., Colton, C. A., Citrin, D., Espey, M. G., & Wink, D. A. (2003). Heme proteins and nitric oxide (NO): The neglected, eloquent chemistry in NO redox signaling and regulation. Antioxidants and Redox Signaling, 5(3), 307-317.
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  PMID: 12880485;Abstract: The role of nitric oxide (NO) in cellular physiology and signaling has been an important aspect in biomedical science over the last decade. As NO is a small uncharged radical, the chemistry of NO within the redox environment of the cell dictates the majority of its biological effects. The mechanisms that have received the most attention from a biological perspective involve reactions with oxygen and superoxide, despite the rich literature of metal-NO chemistry. However, NO and its related species participate in important chemistry with metalloproteins. In addition to the well known direct interactions of NO with heme proteins such as soluble guanylate cyclase and oxyhemoglobin, there is much important, but often underappreciated, chemistry between other nitrogen oxides and heme/metal proteins. Here the basic chemistry of nitrosylation and the interactions of NO and other nitrogen oxides with metal-oxo species such as found in peroxidases and monoxygenases are discussed.
• Wink, D. A., Miranda, K. M., Katori, T., Mancardi, D., Thomas, D. D., Ridnour, L., Espey, M. G., Feelisch, M., Colton, C. A., Fukuto, J. M., Pagliaro, P., Kass, D. A., & Paolocci, N. (2003). Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: A novel redox paradigm. American Journal of Physiology - Heart and Circulatory Physiology, 285(6 54-6), H2264-H2276.
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  PMID: 12855429;Abstract: Endogenous formation of nitric oxide (NO) and related nitrogen oxides in the vascular system is critical to regulation of multiple physiological functions. An imbalance in the production or availability of these species can result in progression of disease. Nitrogen oxide research in the cardiovascular system has primarily focused on the effects of NO and higher oxidation products. However, nitroxyl (HNO), the one-electron-reduction product of NO, has recently been shown to have unique and potentially beneficial pharmacological properties. HNO and NO often induce discrete biological responses, providing an interesting redox system. This article discusses the emerging aspects of HNO chemistry and attempts to provide a framework for the distinct effects of NO and HNO in vivo.
• Bartberger, M. D., Liu, W., Ford, E., Miranda, K. M., Switzer, C., Fukuto, J. M., Farmer, P. J., Wink, D. A., & Houk, K. N. (2002). The reduction potential of nitric oxide (NO) and its importance to NO biochemistry. Proceedings of the National Academy of Sciences of the United States of America, 99(17), 10958-10963.
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  PMID: 12177417;PMCID: PMC123192;Abstract: A potential of about-0.8 (±0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (3NO-) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to 1NO- is highly unfavorable, with a predicted reduction potential of about -1.7 (±0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and -0.35 V for the NO/3NO-and NO/1NO- couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of 3NO- has been reevaluated as 11.6 (±3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, 1NO-, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O2 under reductive conditions is discussed.
• Espey, M. G., Miranda, K. M., Thomas, D. D., & Wink, D. A. (2002). Ingress and reactive chemistry of nitroxyl-derived species within human cells. Free Radical Biology and Medicine, 33(6), 827-834.
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  PMID: 12208370;Abstract: The mechanisms that control the biological signaling and toxicological properties of the nitrogen oxide species nitroxyl (HNO) are largely unknown. The ingress and intracellular reactivity of nitroxyl-derived species were examined using Angeli's salt (AS), which decomposes initially to HNO and nitrite at physiologic pH. Exposure of 4,5-diaminofluorescein (DAF) to AS resulted in fluorescent product formation only in the presence of molecular oxygen. Kinetic analysis and the lack of signal from a nitric oxide (NO)-sensitive electrode suggested that these processes did not involve conversion of HNO to NO. On an equimolar basis, bolus peroxynitrite (ONOO-) exposure generated only 15% of fluorescent product formation observed from AS decomposition. Moreover, infusion of synthetic ONOO- at a rate comparable to AS decomposition resulted in only 4% of the signal. Quenching of AS-mediated product formation within intact human MCF-7 breast carcinoma cells containing DAF by addition of urate to buffer suggested involvement of an oxidized intermediate formed from reaction between HNO and oxygen. Conversely, intact cells competitively sequestered the HNO-derived species from reaction with DAF in solution. These data show this intermediate to be a long-lived diffusible species. Relative product yield from intracellular DAF was decreased 5- to 8-fold when cells were lysed immediately prior to AS addition, consistent with the partitioning of HNO and/or derived species into the cellular membrane, thereby shielding these reactive intermediates from either hydrolysis or cytoplasmic scavenger pools. These findings establish that oxygen-derived species of nitroxyl can readily penetrate and engage the intracellular milieu of cells and suggest this process to be independent of NO and ONOO- intermediacy. The substantial facilitation of oxygen-dependent nitroxyl chemistry by intact lipid bilayers supports a focusing role for the membrane in modulation of cellular constituents proteins by this unique species.
• Espey, M. G., Miranda, K. M., Thomas, D. D., Xavier, S., Citrin, D., Vitek, M. P., & Wink, D. A. (2002). A chemical perspective on the interplay between NO, reactive oxygen species, and Reactive Nitrogen Oxide Species. Annals of the New York Academy of Sciences, 962, 195-206.
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  PMID: 12076975;Abstract: Nitric oxide (nitrogen monoxide, NO) plays a veritable cornucopia of regulatory roles in normal physiology. In contrast, NO has also been implicated in the etiology and sequela of numerous neurodegenerative diseases that involve reactive oxygen species (ROS) and nitrogen oxide species (RNOS). In this setting, NO is often viewed solely as pathogenic; however, the chemistry of NO can also be a significant factor in lessening injury mediated by both ROS and RNOS. The relationship between NO and oxidation, nitrosation, and nitration reactions is summarized. The salient factors that determine whether NO promotes, abates, or interconnects these chemistries are emphasized. From this perspective of NO chemistry, the type, magnitude, location, and duration of either ROS or RNOS reactions may be predicted.
• Espey, M. G., Thomas, D. D., Miranda, K. M., & Wink, D. A. (2002). Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. Proceedings of the National Academy of Sciences of the United States of America, 99(17), 11127-11132.
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  PMID: 12177414;PMCID: PMC123221;Abstract: The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide (O2-) simultaneously generated at nanomolar to low micromolar per minute rates by spermine/NO decomposition and xanthine oxidase-catalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of O2- to NO approached equimolar, then decreased precipitously as O2- exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the N2O3 scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (NO+ addition). The latter mechanism predominated with higher rates of NO formation relative to O2-. The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the O2- + NO pathway in vivo. The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by O2- toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of O2- production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.
• Espey, M. G., Xavier, S., Thomas, D. D., Miranda, K. M., & Wink, D. A. (2002). Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 99(6), 3481-3486.
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  PMID: 11904413;PMCID: PMC122549;Abstract: 3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 μM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC50 ≈ 20 μM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC50 > 250 μM). The more physiologically relevant conditions of either peroxynitrite infusion (1 μM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxidecatalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO2) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO2 can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidasecatalyzed formation of NO2 may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.
• Miranda, K. M., K-i, Y., Espey, M. G., Thomas, D. D., Degraff, W., Mitchell, J. B., Krishna, M. C., Colton, C. A., & Wink, D. A. (2002). Further evidence for distinct reactive intermediates from nitroxyl and peroxynitrite: Effects of buffer composition on the chemistry of Angeli's salt and synthetic peroxynitrite. Archives of Biochemistry and Biophysics, 401(2), 134-144.
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  PMID: 12054463;Abstract: The nitroxyl (HNO) donor Angeli's salt (Na2N2O3; AS) is cytotoxic in vitro, inducing double strand DNA breaks and base oxidation, yet may have pharmacological application in the treatment of cardiovascular disease. The chemical profiles of AS and synthetic peroxynitrite (ONOO-) in aerobic solution were recently compared, and AS was found to form a distinct reactive intermediate. However, similarities in the chemical behavior of the reactive nitrogen oxide species (RNOS) were apparent under certain conditions. Buffer composition was found to have a significant and unexpected impact on the observed chemistry of RNOS, and varied buffer conditions were utilized to further distinguish the chemical profiles elicited by the RNOS donors AS and synthetic ONOO-. Addition of HEPES to the assay buffer significantly quenched oxidation of dihydrorhodamine (DHR), hydroxylation of benzoic acid (BA), and DNA damage by both AS and ONOO-, and oxidation and nitration of hydroxyphenylacetic acid by ONOO-. Additionally, H2O2 was produced in a concentration-dependent manner from the interaction of HEPES with both the donor intermediates. Interestingly, clonogenic survival was not affected by HEPES, indicating that H2O2 is not a contributing factor to in vitro cytotoxicity of AS. Variation in RNOS reactivity was dramatic with significantly higher relative affinity for the AS intermediate toward DHR, BA, DNA, and HEPES and increased production of H2O2. Further, AS reacted to a significantly greater extent with the unprotonated amine form of HEPES while the interaction of ONOO- with HEPES was pH-independent. Addition of bicarbonate only altered ONOO- chemistry. This study emphasizes the importance of buffer composition on chemical outcome and thus on interpretation and provides further evidence that ONOO- is not an intermediate formed between the reaction of O2 and HNO produced by AS. © 2002 Elsevier Science (USA). All rights reserved.
• Miranda, K., Dutton, A., Ridnour, L., Foreman, C., Ford, E., Paolocci, N., Katori, T., Tocchetti, C., Mancardi, D., Thomas, D., Espey, M., Houk, K., Fukuto, J., & Wink, D. (2002). Mechanism of aerobic decomposition of Angeli's salt (sodium trioxodinitrate) at physiological pH. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 127(2), 722-731.
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  The recent determination that Angeli's salt may have clinical application as a nitrogen oxide donor for treatment of cardiovascular diseases such as heart failure has led to renewed interest in the mechanism and products of thermal decomposition of Angeli's salt under physiological conditions. In this report, several mechanisms are evaluated experimentally and by quantum mechanical calculations to determine whether HNO is in fact released from Angeli's salt in neutral, aerobic solution. The mechanism of product autoxidation is also considered.
• Miranda, K., Katori, T., de Holding, C., Thomas, L., Ridnour, L., MeLendon, W., Cologna, S., Dutton, A., Champion, H., Mancardi, D., Tocchetti, C., Saavedra, J., Keefer, L., Houk, K., Fukuto, J., Kass, D., Paolocci, N., & Wink, D. (2002). Comparison of the NO and HNO donating properties of diazeniumdiolates: Primary amine adducts release HNO in vivo. JOURNAL OF MEDICINAL CHEMISTRY, 48(26), 8220-8228.
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  Diazeniumdiolates, more commonly referred to as NONOates, have been extremely useful in the investigation of the biological effects of nitric oxide (NO) and related nitrogen oxides. The NONOate Angell's salt (Na2N2O3) releases nitroxyl (HNO) under physiological conditions and exhibits unique cardiovascular features (i.e., positive inotropy/lusitropy) that may have relevance for pharmacological treatment of heart failure. In the search for new, organic-based compounds that release HNO, we examined isopropylamine NONOate (IPA/NO; Na[(CH3)(2)CHNH(N(O)NO]), which is an adduct of NO and a primary amine. The chemical and pharmacological properties of IPA/NO were compared to those of Angeli's salt and a NO-producing NONOate, DEA/NO (Na[Et2NN(O)NO]), which is a secondary amine adduct. Under physiological conditions IPA/NO exhibited all the markers of HNO production (e.g., reductive nitrosylation, thiol reactivity, positive inotropy). These data suggest that primary amine NONOates may be useful as HNO donors in complement to the existing series of secondary amine NONOates, which are well-characterized NO donors.
• Miranda, K., Miranda, K., Bu, X., Bu, X., Lorkovic, ., Lorkovic, ., Ford, P., & Ford, P. (2002). Synthesis and structural characterization of several ruthenium porphyrin nitrosyl complexes. INORGANIC CHEMISTRY, 36(21), 4838-4848.
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  The synthesis, X-ray crystal structures, and some spectroscopic and chemical properties of the nitrosylruthenium(II) porphyrin complexes Ru(TPP)(NO)(ONO), Ru(TPP)(NO)(OH), Ru(OEP)(NO)(ONO), and Ru(OEP)(NO)(OH) (TPP = tetraphenylporphyrinato dianion; OEP = octaethylporphyrinato dianion) derived from the analogous Ru(II) carbonyl complexes are reported. Also described are experiments which quantitatively demonstrate that N2O is formed as a product of the synthesis scheme and that NO serves as the principal oxidant in the transformation of N(II) to N(III). The two TPP complexes are isostructural and consist of columns of molecules stacked along the c axis. The two OEP complexes are also isostructural and can be considered as layers of OEP complexes stacked along the b axis with solvent molecules situated at the cavities between layers. The nitrite ions are coordinated in a unidentate fashion through the oxygen atom. Crystal data for Ru(TPP)(NO)(ONO) (1): M = 789.79, space group I4/m (no. 87), a = 13.6529(6) Angstrom, c = 9.7904(5) Angstrom, V = 1825.0(2) Angstrom(3), Z = 2, rho = 1.437 g cm(-3), purple bipyramid, 2 theta(max) = 50.0 degrees, R(F) = 4.87% for 86 parameters and 838 reflections with I > 2 sigma(I). Crystal data for Ru(TPP)(NO)(OH) (2): M = 760.79, space group I4/m (No. 87), a = 13.5423(4) Angstrom, c = 9.7150(4) Angstrom, V = 1781.7(1) Angstrom(3), Z = 2, rho = 1.418 g cm(-3), dark red plate, 2 theta(max) = 50.0 degrees, R(F) = 3.92% for 83 parameters and 811 reflections with I > 2 sigma(I). Crystals data for Ru(OEP)(NO)(OH). C2H5OH (4): M = 726.91, space group P2(1) (No. 4), a = 10.8474(7) Angstrom, b = 21.002(1) Angstrom, c = 8.3646(5) Angstrom, beta = 103.571(1)degrees, V = 1852.4(2) Angstrom(3), Z = 2, rho = 1.303 g cm(-3), brown plate, 2 theta(max) = 45.0 degrees, R(F) = 6.74% for 421 parameters and 3527 reflections with I > 2 sigma(I).
• Miranda, K., Nims, R., Thomasa, D., Espey, M., Citrin, D., Bartberger, M., Paolocci, N., Fukuto, J., Feelisch, M., & Wink, D. (2002). Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins - A chemical discussion of the differential biological effects of these redox related products of NOS. JOURNAL OF INORGANIC BIOCHEMISTRY, 93(1-2), 52-60.
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  Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO-) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO. Published by Elsevier Science Inc.
• Thomas, D. D., Espey, M. G., Vitek, M. P., Miranda, K. M., & Wink, D. A. (2002). Protein nitration is mediated by heme and free metals through Fenton-type chemistry: An alternative to the NO/O₂^- reaction. Proceedings of the National Academy of Sciences of the United States of America, 99(20), 12691-12696.
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  PMID: 12226478;PMCID: PMC130522;Abstract: The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONOO-) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide (O2-), from spermine/NO (2.7 μM/min) and xanthine oxidase (1-28 μM O2-/min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of O2- concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONOO- might not be obligatory to this process. Hemin-mediated NT formation required only the presence of NO2- and H2O2, which are stable end-products of NO and O2- degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONOO- can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and NO2- subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an H2O2 source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation.
• Thomas, D. D., Miranda, K. M., Espey, M. G., Citrin, D., Jourd'Heuil, D., Paolocci, N., Hewett, S. J., Colton, C. A., Grisham, M. B., Feelisch, M., & Wink, D. A. (2002). Guide for the use of nitric oxide (NO) donors as probes of the chemistry of NO and related redox species in biological systems. Methods in Enzymology, 359, 84-105.
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  PMID: 12481562;
• Bartberger, M., Liu, W., Ford, E., Miranda, K., Switzer, C., Fukuto, J., Farmer, P., Wink, D., & Houk, K. (2001). The reduction potential of nitric oxide (NO) and its importance to NO biochemistry. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 99(17), 10958-10963.
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  A potential of about -0.8 (+/-0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion ((NO-)-N-3) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to (NO-)-N-1 is highly unfavorable, with a predicted reduction potential of about -1.7 (+/-0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +039 V and -0.35 V for the NO/(NO-)-N-3 and NO/(NO-)-N-1 couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of (NO-)-N-3 has been reevaluated as 11.6 ( 3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, (NO-)-N-1, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O-2 under reductive conditions is discussed.
• Colton, C. A., Gbadegesin, M., Wink, D. A., Miranda, K. M., Espey, M. G., & Vicini, S. (2001). Nitroxyl anion regulation of the NMDA receptor. Journal of Neurochemistry, 78(5), 1126-1134.
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  PMID: 11553686;Abstract: Nitric oxide (NO) is an important regulator of NMDA channel function in the CNS. Recent findings suggest that nitroxyl anion (NO-) may also be generated by nitric oxide synthase, which catalyzes production of NO. Using recombinant NMDA receptors (NMDA-r) transfected into human embryonic kidney cells, our data demonstrate that the nitroxyl anion donor, Angeli's salt (AS; Na2N2O3) dramatically blocked glycine-independent desensitization in NMDA-r containing NR1-NR2A subunits. AS did not affect glycine-dependent desensitization, calcium dependent inactivation or glutamate affinity for the NMDA-r. This effect could be mimicked by treatment with DPTA, a metal chelator and was not evident under hypoxic conditions. In contrast, receptors containing the NR1-NR2B subunits demonstrated an approximate 25% reduction in whole cell currents in the presence of AS with no apparent change in desensitization. Our data suggest that the regulation of NMDA-r function by nitroxyl anion is distinctly different from NO and may result in different cellular outcomes compared with NO.
• Espey, M. G., Miranda, K. M., Thomas, D. D., & Wink, D. A. (2001). Distinction between Nitrosating Mechanisms within Human Cells and Aqueous Solution. Journal of Biological Chemistry, 276(32), 30085-30091.
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  PMID: 11404354;Abstract: The quintessential nitrosating species produced during NO autoxidation is N2O3. Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt 2NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N2O3 through the intermediacy of NO2. This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO2 was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.
• Espey, M., Miranda, K., Pluta, R., & Wink, D. (2001). Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals. JOURNAL OF BIOLOGICAL CHEMISTRY, 275(15), 11341-11347.
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  Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1 beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells S-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O-2 reaction leading to N2O3 formation. These data mimicked the pattern observed with a 5 mu M concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl) -N-(n-propyl) aminoldiazen-1-ium- 1,2-diolate (PAPA/NO), The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N2O3 and elicit nitrosative stress.
• Jourd'heuil, D., Miranda, K., Kim, S., Espey, M., Vodovotz, Y., Laroux, S., Mai, C., Miles, A., Grisham, M., & Wink, D. (2001). The oxidative and nitrosative chemistry of the nitric oxide superoxide reaction in the presence of bicarbonate. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 365(1), 92-100.
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  The primary product of the interaction between nitric oxide (NO) and superoxide (O-2(-)) is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or O-2(-) can further react with ONOO- to form species which mediate nitrosation, Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and O-2(-). Since ONOO- reacts not only with NO and O-2(-) but also with CO2, the effects of bicarbonate (HCO3-) on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and O-2(-) were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of HCO3- did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or O-2(-). However, an increase in the rate of DHR oxidation by ONOO- in the presence of HCO3- suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or O-2(-) with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with O-2(-) and NO. (C) 1999 Academic Press.
• Miranda, K. M., Espey, M. G., & Wink, D. A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide - Biology and Chemistry, 5(1), 62-71.
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  PMID: 11178938;Abstract: Numerous methods are available for measurement of nitrate (NO3-). However, these assays can either be time consuming or require specialized equipment (e.g., nitrate reductase, chemiluminescent detector). We have developed a method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format. The principle of this assay is reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction. This assay is sensitive to 0.5 μM NO3- and is useful in a variety of fluids including cell culture media, serum, and plasma. S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents. However, these compounds are generally minor constituents of biological fluids relative to the concentration of nitrate/nitrite. This report introduces a new, convenient assay for the stable oxidation products of nitrogen oxide chemistry in biological samples. © 2001 Academic Press.
• Miranda, K. M., Espey, M. G., Yamada, K., Krishna, M., Ludwick, N., Kim, S., Jourd'heuil, D., Grisham, M. B., Feelisch, M., Fukuto, J. M., & Wink, D. A. (2001). Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion. Journal of Biological Chemistry, 276(3), 1720-1727.
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  PMID: 11042174;Abstract: The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2/- and NO3/- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3/- or by oxidation of an HN2O3/- molecule to form NO2/-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2/-, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.
• Ogawa, R., Pacelli, R., Espey, M. G., Miranda, K. M., Friedman, N., Kim, S., Cox, G., Mitchell, J. B., Wink, D. A., & Russo, A. (2001). Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: Insights into Listeriocidal activity of oxidative and nitrosative stress. Free Radical Biology and Medicine, 30(3), 268-276.
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  PMID: 11165873;Abstract: The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6) (N[N(O)NO]C3H7)) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-γ (INF-γ) + tumor necrosis factor-α (TNF-α or INF-γ + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections. © 2001 Elsevier Science Inc.
• Paolocci, N., Saavedra, W. F., Miranda, K. M., Martignani, C., Isoda, T., Hare, J. M., Espey, M. G., Fukuto, J. M., Feelisch, M., Winkt, D. A., & Kass, D. A. (2001). Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling. Proceedings of the National Academy of Sciences of the United States of America, 98(18), 10463-10468.
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  PMID: 11517312;PMCID: PMC56983;Abstract: Nitroxyl anion (NO-) is the one-electron reduction product of nitric oxide (NO') and is enzymatically generated by NO synthase in vitro. The physiologic activity and mechanism of action of NO- in vivo remains unknown. The NO- generator Angeli's salt (AS, Na2N2O3) was administered to conscious chronically instrumented dogs, and pressure-dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac preload volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of N-acetyl-L-cysteine. Cardiac inotropic signaling by NO- was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP-(8-37) prevented this effect but not systemic vasodilation. Thus, NO- is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking NO- to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, NO- may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.
• Thomas, D., Espey, M., Vitek, M., Miranda, K., & Wink, D. (2001). Protein nitration is mediated by heme and free metals through Fenton-type chemistry: An alternative to the NO/O-2(-) reaction. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 99(20), 12691-12696.
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  The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONOO-) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide (O-2(-)), from spermine/NO (2.7 muM/min) and xanthine oxidase (1-28 muM O-2(-)/min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of O-2(-) concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONOO- might not be obligatory to this process. Hemin-mediated NT formation required only the presence of NO2- and H2O2, which are stable end-products of NO and O-2(-) degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONOO- can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and NO2- subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an H2O2 source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation.
• Wink, D. A., Miranda, K. M., & Espey, M. G. (2001). Cytotoxicity related to oxidative and nitrosative stress by nitric oxide. Experimental Biology and Medicine, 226(7), 621-623.
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  PMID: 11444095;
• Wink, D. A., Miranda, K. M., Espey, M. G., Pluta, R. M., Hewett, S. J., Colton, C., Vitek, M., Feelisch, M., & Grisham, M. B. (2001). Mechanisms of the antioxidant effects of nitric oxide. Antioxidants and Redox Signaling, 3(2), 203-213.
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  PMID: 11396476;Abstract: The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.
• Espey, M. G., Miranda, K. M., Feelisch, M., Fukuto, J., Grisham, M. B., Vitek, M. P., & Wink, D. A. (2000). Mechanisms of cell death governed by the balance between nitrosative and oxidative stress. Annals of the New York Academy of Sciences, 899, 209-221.
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  PMID: 10863541;Abstract: Many cellular functions in physiology are regulated by the direct interaction of NO with target biomolecules. In many pathophysiologic and toxicologic mechanisms, NO first reacts with oxygen, superoxide or other nitrogen oxides to subsequently elicit indirect effects. The balance between nitrosative stress and oxidative stress within a specific biological compartment can determine whether the presence of NO will be ultimately deleterious or beneficial. Nitrosative stress can be defined primarily through reactions mediated by N2O3, a reactive nitrogen oxide species generated by high fluxes of NO in an aerobic environment. In contrast, oxidative stress is mediated primarily by superoxide and peroxides. In addition to reactive oxygen species, several reactive nitrogen oxide species such as peroxynitrite, nitroxyl, and nitrogen dioxide can also impose oxidative stress to a cell. We here describe how the mechanisms of cell death are interwoven in the balance between the different chemical intermediates involved in nitrosative and oxidative stress.
• Espey, M. G., Miranda, K. M., Pluta, R. M., & Wink, D. A. (2000). Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals. Journal of Biological Chemistry, 275(15), 11341-11347.
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  PMID: 10753947;Abstract: Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-γ and either tumor necrosis factor-α or interleukin-1β resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-γ and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O2 reaction leading to N2O3 formation. These data mimicked the pattern observed with a 5 μM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N2O3 and elicit nitrosative stress.
• Miranda, K. M., Espey, M. G., & Wink, D. A. (2000). A discussion of the chemistry of oxidative and nitrosative stress in cytotoxicity. Journal of Inorganic Biochemistry, 79(1-4), 237-240.
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  PMID: 10830872;Abstract: Nitric oxide (NO) has been shown to be a key bioregulatory agent in a wide variety of biological processes, yet cytotoxic properties have been reported as well. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. We have investigated the effects of NO on a variety of toxic agents and correlated how its chemistry might pertain to the observed biology. The results generate a scheme termed the chemical biology of NO in which the pertinent reactions can be categorized into direct and indirect effects. The former involves the direct reaction of NO with its biological targets generally at low fluxes of NO. Indirect effects are reactions mediated by reactive nitrogen oxide species, such as those generated from the NO/O2 and NO/O2- reactions, which can lead to cellular damage via nitrosation or oxidation of biological components. This report discusses several examples of cytotoxicity involved with these stresses. (C) 2000 Elsevier Science Inc.
• Miranda, K., Paolocci, N., Katori, T., Thomas, D., Ford, E., Bartberger, M., Espey, M., Kass, D., Feelisch, M., Fukuto, J., & Wink, D. (2000). A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(16), 9196-9201.
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  The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O-2/O-2(-) couple, HNO is a weak reductant the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O-2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.
• Paolocci, N., Katori, T., Champion, H., St John, M., Miranda, K., Fukuto, J., Wink, D., & Kass, D. (2000). Positive inotropic and lusitropic effects of HNO/NO- in failing hearts: Independence from beta-adrenergic signaling. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(9), 5537-5542.
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  Nitroxyl anion (HNO/NO-), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO- augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts beta-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO- generated by Angelis' salt (AS) was infused (10 mug/kg per min, im.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)/NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed beta-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted beta-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO- cardiotropic action. Thus, HNO/NO- has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to beta-adrenergic stimulation and stimulates CGRIP release. This suggests potential of HNO/NO- donors for the treatment of heart failure.
• Wink, D. A., Miranda, K. M., & Espey, M. G. (2000). Effects of oxidative and nitrosative stress in cytotoxicity. Seminars in Perinatology, 24(1), 20-23.
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  PMID: 10709853;Abstract: Nitric oxide is a key bioregulatory agent in a wide variety of biological processes, yet it also can have cytotoxic properties. This dichotomy raises the question of how this potentially toxic species can be involved in so many fundamental physiological processes. This articles discusses how the chemistry of nitric oxide might pertain to its observed biology as it relates to oxidative and nitrosative stress in different mechanisms of cytotoxicity.
• Zhou, X., Espey, M. G., Chen, J. X., Hofseth, L. J., Miranda, K. M., Hussain, S. P., Wink, D. A., & Harris, C. C. (2000). Inhibitory effects of nitric oxide and nitrosative stress on dopamine- β-hydroxylase. Journal of Biological Chemistry, 275(28), 21241-21246.
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  PMID: 10887204;Abstract: Dopamine-β-hydroxylase (DβH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the β-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase- dependent mechanism. In this report, we investigated the relationship between NO and DβH enzymatic activity. In the initial in vitro experiments, the activity of purified DβH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC50 of 1 mM. The inclusion of either azide or GSH partially restored DβH activity, suggesting the involvement of the reactive nitrogen oxide species, N2O3. Treatment of human neuroblastoma cells (SK-N- MC) with diethylamine/NO decreased cellular DβH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Coculture of the SK-N-MC cells with interferon-γ and lipopolysaccharide- activated macrophages, which release NO, also reduced the DβH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N2O3, can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation.
• Espey, M., Miranda, K., Feelisch, M., Fukuto, J., Grisham, M., Vitek, M., Wink, D., & Chiueh, C. (1999). Mechanisms of cell death governed by the balance between nitrosative and oxidative stress. REACTIVE OXYGEN SPECIES: FROM RADIATION TO MOLECULAR BIOLOGY, 899, 209-221.
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  Many cellular functions in physiology are regulated by the direct interaction of NO with target biomolecules, In many pathophysiologic and toxicologic mechanisms, NO first reacts with oxygen, superoxide or other nitrogen oxides to subsequently elicit indirect effects. The balance between nitrosative stress and oxidative stress within a specific biological compartment can determine whether the presence of NO will be ultimately deleterious or beneficial, Nitrosative stress can be defined primarily through reactions mediated by N(2)O(3), a reactive nitrogen oxide species generated by high fluxes of NO in an aerobic environment. In contrast, oxidative stress is mediated primarily by superoxide and peroxides, In addition to reactive oxygen species, several reactive nitrogen oxide species such as peroxynitrite, nitroxyl, and nitrogen dioxide can also impose oxidative stress to a cell. We here describe how the mechanisms of cell death are interwoven in the balance between the different chemical intermediates involved in nitrosative and oxidative stress.
• Jourd'heuil, D., Miranda, K. M., Kim, S. M., Espey, M. G., Vodovotz, Y., Laroux, S., Mai, C. T., Miles, A. M., Grisham, M. B., & Wink, D. A. (1999). The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate. Archives of Biochemistry and Biophysics, 365(1), 92-100.
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  PMID: 10222043;Abstract: The primary product of the interaction between nitric oxide (NO) and superoxide (O2/-) is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or O2/- can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and O2/-. Since ONOO- reacts not only with NO and O2/- but also with CO2, the effects of bicarbonate (HCO3/-) on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3- diaminonaphthalene and reduced glutathione were examined. Nitric oxide and O2/- were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of HCO3/- did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOOby either NO or O2/-. However, an increase in the rate of DHR oxidation by ONOO- in the presence of HCO3/- suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or O2/- with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with O2/- and NO.
• Ford, P. C., Bourassa, J., Miranda, K., Lee, B., Lorkovic, I., Boggs, S., Kudo, S., & Laverman, L. (1998). Photochemistry of metal nitrosyl complexes. Delivery of nitric oxide to biological targets. Coordination Chemistry Reviews, 171(1), 185-202.
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  Abstract: The discoveries that nitric oxide serves important roles in mammalian bioregulation and immunology have stimulated intense interest in the chemistry and biochemistry of NO and derivatives such as metal nitrosyl complexes. Also of interest are strategies to deliver NO to biological targets on demand. One such strategy would be to employ a precursor which displays relatively low thermal reactivity but is photochemically active to give NO. This proposition led the authors to investigate photochemical properties of metal nitrosyl complexes such as the iron-sulfur-nitrosyl Roussin cluster anions Fe2S2(NO)2-4 and Fe4S3(NO)-7 as well as metalloporphyrin nitrosyls including ferriheme complexes (with M. Hoshino of the Institute of Physical and Chemical Research, Japan) and nitrosyl nitrito complexes of ruthenium porphyrins Ru(P)(ONO)(NO). Continuous and flash photolysis studies of these compounds are reviewed here as are studies (with D.A. Wink and J.B. Mitchell of the Radiation Biology Branch of the US National Cancer Institute) using metal nitrosyl photochemistry as a vehicle for delivering NO to hypoxic cell cultures in order to sensitize γ-radiation damage. © 1998 Elsevier Science S.A.
• Lorković, I. M., Miranda, K. M., Lee, B., Bernhard, S., Schoonover, J. R., & Ford, P. C. (1998). Flash photolysis studies of the ruthenium(II) porphyrins RU(P)(NO)(ONO). Multiple pathways involving reactions of intermediates with nitric oxide. Journal of the American Chemical Society, 120(45), 11674-11683.
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  Abstract: Described are the spectra and kinetics of transients formed by laser flash photolysis of the ruthenium nitrosyl nitrito complexes Ru(P)(NO)(ONO), P= TPP (meso-tetraphenylporphyrin), OEP (octaethylporphyrin), TmTP (tetra(m- tolyl)porphyrin); and FTTP (tetra(m-trifluoromethylphenyl)porphyrin)in benzene solutions. Two transient decay processes are seen on the time frame (< 1 ms) of the flash photolysis experiment, and a residual difference spectrum, which decays to baseline on a longer time frame, is noted as well. The accumulated evidence points to the formation of two primary photoproducts, Ru(P)(ONO) (A) formed by NO photolabilization and Ru(P)(NO) (B) formed by NO2 photolabilization. Both decay by NO dependent pathways, the reaction of A with NO to re-form Ru(P)(NO)(ONO) being substantially faster (2.4-5.5 x 108 M-1 s-1 in ambient temperature benzene) than the reaction of B with NO (2.4-10 x 107 M-1 s-1). The product of the latter reaction is apparently the dinitrosyl complex Ru(P)(NO)2, which undergoes a much slower thermal reaction with excess NO to give again Ru(P)(NO)(ONO). The possibility of B being the oxo complex O=Ru(P)(No) formed by NO loss from coordinated nitrite was considered but concluded to be a minor pathway at best. Isotopic exchange reactions using either labeled complex or labeled NO in cyclohexane demonstrate photochemical exchange of NO into both the nitrosyl and nitrito complexes, and time-resolved infrared experiments are consistent with formation of a long-lived nitrosyl-containing intermediate. Flash photolysis studies of the respective nitrosyl chloro complexes Ru(TPP)(NO)Cl and Ru(OEP)(NO)Cl indicate that only a single transient species, presumably Ru(P)Cl, is formed in each case, and this decays by a single NO dependent pathway back to starting material.
• Miranda, K., Espey, M., & Wink, D. (1998). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. NITRIC OXIDE-BIOLOGY AND CHEMISTRY, 5(1), 62-71.
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  Numerous methods are available for measurement of nitrate (NO3-). However, these assays can either be time consuming or require specialized equipment (e.g., nitrate reductase, chemiluminescent detector). We have developed a method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format. The principle of this assay is reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction. This assay is sensitive to 0.5 muM NO3- and is useful in a variety of fluids including cell culture media, serum, and plasma. S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents. However, these compounds are generally minor constituents of biological fluids relative to the concentration of nitrate/nitrite. This report introduces a new, convenient assay for the stable oxidation products of nitrogen oxide chemistry in biological samples. (C) zool Academic Press.
• Espey, M., Thomas, D., Miranda, K., & Wink, D. (1997). Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 99(17), 11127-11132.
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  The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide (O-2(-)) simultaneously generated at nanomolar to low micromolar per Minute rates by spermine/NO decomposition and xanthine oxidase-catalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of O-2(-) to NO approached equimolar, then decreased precipitously as O-2(-) exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the N2O3 scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (NO+ addition). The latter mechanism predominated with higher rates of NO formation relative to O-2(-). The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the O-2(-) NO pathway in vivo. The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by O-2(-) toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of O-2(-) production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.
• Miranda, K. M., Miranda, K. M., Xianhui, B. u., Xianhui, B. u., Lorković, I., Lorković, I., Ford, P. C., & Ford, P. C. (1997). Synthesis and Structural Characterization of Several Ruthenium Porphyrin Nitrosyl Complexes. Inorganic Chemistry, 36(21), 4838-4848.
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  Abstract: The synthesis, X-ray crystal structures, and some spectroscopic and chemical properties of the nitrosylruthenium(II) porphyrin complexes Ru(TPP) (NO) (ONO), Ru(TPP) (NO) (OH), Ru(OEP) (NO) (ONO), and Ru(OEP) (NO)-(OH) (TPP = tetraphenylporphyrinato dianion; OEP = octaethylporphyrinato dianion) derived from the analogous Ru(II) carbonyl complexes are reported. Also described are experiments which quantitatively demonstrate that N2O is formed as a product of the synthesis scheme and that NO serves as the principal oxidant in the transformation of N(II) to N(III). The two TPP complexes are isostructural and consist of columns of molecules stacked along the c axis. The two OEP complexes are also isostructural and can be considered as layers of OEP complexes stacked along the b axis with solvent molecules situated at the cavities between layers. The nitrite ions are coordinated in a unidentate fashion through the oxygen atom. Crystal data for Ru(TPP) (NO) (ONO) (1): M = 789.79, space group I4/m (No. 87), a = 13.6529(6) Å, c = 9.7904(5) Å, V = 1825.0(2) Å3, Z = 2, ρ = 1.437 g cm-3, purple bipyramid, 2θmax = 50.0°, R(F) = 4.87% for 86 parameters and 838 reflections with I > 2σ(I). Crystal data for Ru(TPP) (NO) (OH) (2): M = 760.79, space group I4/m (No. 87), a = 13.5423(4) Å, c = 9.7150-(4) Å, V= 1781.7(1) Å3, Z = 2, ρ = 1 .418 g cm-3, dark red plate, 2θmax = 50.0°, R(F) = 3.92% for 83 parameters and 811 reflections with I > 2σ(I). Crystal data for Ru(OEP) (NO)(ONO)·CH2Cl2 (3): M = 794.77, space group P21 (No. 4), a = 10.7687(2) Å, b = 21.0320(2) Å, c = 8.5936(2) Å, β= 102.683(1)°, V= 1898.85(6) Å3, Z = 2, ρ = 1.390 g cm-3, black plate, 2θmax = 50.0°, R(F) = 6.23% for 453 parameters and 4702 reflections with I > 2σ(I). Crystal data for Ru(OEP) (NO) (OH)·C2H5OH (4): M = 726.91, space group P21 (No. 4), a = 10.8474-(7) Ǎ, b = 21.002(1) Å, c = 8.3646(5) Å, β= 103.571(1)°, V= 1852.4(2) Å3, Z= 2, ρ = 1.303 g cm-3, brown plate, 2θmax = 45.0°, R(F) = 6.74% for 421 parameters and 3527 reflections with I > 2σ(I).

Reviews

• Miranda, K. (2005. The chemistry of nitroxyl (HNO) and implications in biology(pp 433-455).
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  Over the past century, HNO research has evolved from fundamental physical examinations to elucidation of interactions in atmospheric, industrial and bacterial processes. Most recently, the HNO literature has been primarily concerned with the pharmacological effects and potential physiological functions of HNO in mammalian systems. The chemistry of HNO is inordinately complicated for a triatomic molecule. Further, the rapid self-consumption of HNO through dehydrative dimerization impedes detection and necessitates in situ production. This review provides a detailed discussion of the most common donors of HNO and of the current understanding of the aqueous chemistry of HNO and the synthesis, consumption and reactivity of HNO in a cellular environment, as ascertained with these donors. Additionally, the consequences of the molecular interactions of HNO on physiology are described, and a comparison is made to NO in terms of cellular signaling and pharmacological potential. (C) 2004 Elsevier B.V. All rights reserved.
• Ridnour, L., Thomas, D., Mancardi, D., Espey, M., Miranda, K., Paolocci, N., Feelisch, M., Fukuto, J., & Wink, D. (2003. The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting perspective on stressful biological situations(pp 1-10).
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  This review addresses many of the chemical aspects of nitrosative stress mediated by N2O3. From a cellular perspective, N2O3 and the resulting reactive nitrogen oxide species target specific motifs such as thiols, lysine active sites, and zinc fingers and is dependant upon both the rates of production as well as consumption of NO and must be taken into account in order to access the nitrosative environment. Since production and consumption are integral parts of N2O3 generation, we predict that nitrosative stress occurs under specific conditions, such as chronic inflammation. In contrast to conditions of stress, nitrosative chemistry may also provide cellular protection through the regulation of critical signaling pathways. Therefore, a careful evaluation of the chemistry of nitrosation based upon specific experimental conditions may provide a better understanding of how the subtle balance between oxidative and nitrosative stress may be involved in the etiology and control of various disease processes.
• Fukuto, J., Switzer, C., Miranda, K., & Wink, D. (2002. Nitroxyl (HNO): Chemistry, biochemistry, and pharmacology(pp 335-355).
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  Recent discoveries of novel and potentially important biological activity have spurred interest in the chemistry and biochemistry of nitroxyl (HNO). It has become clear that, among all the nitrogen oxides, HNO is unique in its chemistry and biology. Currently, the intimate chemical details of the biological actions of HNO are not well understood. Moreover, many of the previously accepted chemical properties of HNO have been recently revised, thus requiring reevaluation of possible mechanisms of biological action. Herein, we review these developments in HNO chemistry and biology.
• Wink, D., Miranda, K., Espey, M., Pluta, R., Hewett, S., Colton, C., Vitek, M., Feelisch, M., & Grisham, M. (2001. Mechanisms of the antioxidant effects of nitric oxide(pp 203-213).
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  The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O-2(-) that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.