Alexandre Samouilov

Enzyme-independent formation of nitric oxide in biological tissues

The gaseous free radical nitric oxide (NO˙) is an important regulator of a variety of biological functions and also has a role in the pathogenesis of cellular injury. It has been generally accepted that NO˙ is solely generated in biological tissues by specific nitric oxide synthases, NOSs, which metabolize arginine to citrulline with the formation of NO˙. We report that NO˙ can also be generated in the ischaemic heart by direct reduction of nitrite to NO˙ under the acidotic and highly reduced conditions that occur. This NO˙ formation is not blocked by NOS inhibitors, and with long periods of ischaemia progressing to necrosis, this mechanism of NO˙ formation predominates. We observe that enzyme-independent NO˙ generation results in myocardial injury with a loss of contractile function. The existence of this enzyme independent mechanism of NO˙ formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Non-enzymatic nitric oxide synthesis in biological systems

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions, the magnitude of enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Characterization of the Magnitude and Kinetics of Xanthine Oxidase-catalyzed Nitrite Reduction EVALUATION OF ITS ROLE IN NITRIC OXIDE GENERATION IN ANOXIC TISSUES

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.

Evidence for the Pathophysiological Role of Endogenous Methylarginines in Regulation of Endothelial NO Production and Vascular Function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated l-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-l-arginine (l-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and l-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for l-arginine is 3.14 μm with a Vmax of 0.14 μmol mg–1 min–1, whereas Ki values of 0.9 μm and 1.1 μm were determined for ADMA and l-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 μm level of l-arginine, MA levels of >10μm were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 μm. Similar effects were observed in isolated vessels where 5 μm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and l-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. l-Arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, ∼4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.

Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of 10 microM. Similarly, nitrite (10 microM-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO.

Mechanisms of nitrite reduction to nitric oxide in the heart and vessel wall

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, over the last 15 years, nitrite-mediated NO production has been shown to be an important mechanism of NO formation in the heart and cardiovascular system. Now numerous studies have demonstrated that nitrite can be an important source rather than simply a product of NO in mammalian cells and tissues and can be a potential vasodilator drug for cardiovascular diseases. There are a variety of mechanisms of nitrite reduction to NO and it is now appreciated that this process, while enhanced under hypoxic conditions, also occurs under normoxia. Several methods, including electron paramagnetic resonance, chemiluminescence NO analyzer, and NO electrode have been utilized to measure, quantitate, and image nitrite-mediated NO formation. Results reveal that nitrite-dependent NO generation plays critical physiological and pathological roles, and is controlled by oxygen tension, pH, reducing substrates and nitrite levels. In this manuscript, we review the mechanisms of nitrite-mediated NO formation and the effects of oxygen on this process with a focus on how this occurs in the heart and vessels.

Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems.

While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.

In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real‐time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L‐band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony‐stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony‐stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony‐stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double‐resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med, 2011.

Development of a hybrid EPR/NMR coimaging system

Electron paramagnetic resonance imaging (EPRI) is a powerful technique that enables spatial mapping of free radicals or other paramagnetic compounds; however, it does not in itself provide anatomic visualization of the body. Proton magnetic resonance imaging (MRI) is well suited to provide anatomical visualization. A hybrid EPR/NMR coimaging instrument was constructed that utilizes the complementary capabilities of both techniques, superimposing EPR and proton‐MR images to provide the distribution of paramagnetic species in the body. A common magnet and field gradient system is utilized along with a dual EPR and proton‐NMR resonator assembly, enabling coimaging without the need to move the sample. EPRI is performed at ∼1.2 GHz/∼40 mT and proton MRI is performed at 16.18 MHz/∼380 mT; hence the method is suitable for whole‐body coimaging of living mice. The gradient system used is calibrated and controlled in such a manner that the spatial geometry of the two acquired images is matched, enabling their superposition without additional postprocessing or marker registration. The performance of the system was tested in a series of phantoms and in vivo applications by mapping the location of a paramagnetic probe in the gastrointestinal (GI) tract of mice. This hybrid EPR/NMR coimaging instrument enables imaging of paramagnetic molecules along with their anatomic localization in the body. Magn Reson Med 58:156–166, 2007.