Electron paramagnetic resonance characterization of rat neuronal nitric oxide production ex vivo

Kozlov, Andrey V.; Bini, Anna; Iannone, Anna; Zini, Isabella; Tomasi, Aldo

doi:10.1016/s0076-6879(96)68025-2

Cited by 32 publications

(33 citation statements)

References 11 publications

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“…The preparation of nitroso-heme complex standards was performed as described earlier. 34 Determination of Protein Levels of Transferrin and Hepcidin All procedures were performed at room temperature under agitation. Two ml of plasma were spotted on nitrocellulose membranes (Trans-Blot Transfer Medium, Bio-Rad) and membranes were blocked overnight with 2% BSA in PBS.…”

Section: Epr Measurementsmentioning

confidence: 99%

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

Duvigneau

Piskernik

Haindl

et al. 2008

Laboratory Investigation

100

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Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe 2 þ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-a and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

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Section: Epr Measurementsmentioning

confidence: 99%

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

Duvigneau

Piskernik

Haindl

et al. 2008

Laboratory Investigation

100

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“…2A), the EPR spectrum of HbNO cannot be observed in most cases. To solve these problems, we introduced and modified an EPR signal subtraction method (14,32).…”

Section: Discussionmentioning

confidence: 99%

“…NO gas was obtained commercially from Sumitomo Seika Chemicals (Osaka, Japan), and higher oxides such as NO 2 and NO3 (NOx) were removed by passing through a trap containing 1 M KOH. An NO-saturated aqueous solution was prepared by bubbling NO gas for 15 min through water that had been previously deoxygenated by bubbling with purified argon gas for 30 min (32). Temocapril (0.5 or 5 mg/ml) was suspended in carboxyl methyl cellulose (0.5%) and orally given to the animals.…”

Section: Methodsmentioning

confidence: 99%

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Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

Kirima¹,

Tsuchiya²,

Séi³

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

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.-The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N -nitro-Larginine methyl ester (L-NAME; 120 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] ϭ 1.6-5.5 M) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] ϭ 5.7 M). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method. nitric oxide; hemoglobin-nitric oxide adduct; electron paramagnetic resonance; temocapril NITRIC OXIDE (NO), a free radical molecule, has numerous roles in various physiological functions, such as the regulation of the cardiovascular, immune, and nervous systems. An imbalance of NO results in many diseases such as hypertension, atherosclerosis, septic and hemorrhage shock, ischemia-reperfusion injury, diabetes, and neurodegenerative disorders (18, 40,70).Because of increasing interest in the effect of NO, an accurate method is required for the measurement of this radical. Several methods for the quantitation of NO such as chemiluminescence (46), methemoglobin formation (23), and electron paramagnetic resonance (EPR) spectroscopy of nitrosyl-metal complexes (15) have been developed (3). The Griess and cGMP methods have been widely used to measure NO production in vivo (35, 62) and in vitro (34, 61), but these values do not always reflect the concentration of NO, because of their poor specificities for blood NO. EPR spectroscopy is a technique used for the detection and measurement of free radical species. EPR methods using endogenous and exogenous spin-trapping agents, which stabilize NO as a polyatomic spin adduct, have been developed (1,4,29, 41,72).In the circulation, NO exists as relatively stable hemoglobin (Hb)-NO adduct (HbNO) (48), which means that the amount of HbNO may reflect the blood NO ...

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“…The principle of the EPR signal subtraction method of HbNO is to give the EPR signal of HbNO prominence by subtraction of the NO-depleted EPR signal from the objective one (59,60). This manipulation is accomplished using the special software EPRMAIN, which was obtained from the National Institute of the Environmental Health Sciences (NIEHS) (http://epr.…”

Section: Epr Measurement and Data Processingmentioning

confidence: 99%

New Methods to Evaluate Endothelial Function: Evaluation of Endothelial Function by Hemoglobin-Nitric Oxide Complex Using Electron Paramagnetic Resonance Spectroscopy

et al. 2003

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Abstract. This minireview describes the practical use of assay systems to detect nitric oxide (NO) by electron paramagnetic resonance (EPR) spectroscopy for evaluation of endothelial functions. The iron(II)-dithiocarbamate complexes, such as iron(II)-(N-methyl-D-glucamine dithiocarbamate), are commonly used in EPR detection of NO both in vivo and in vitro. However, due to their redox activity, these complexes have some drawbacks that limit their usefulness for the detection of NO. On the other hand, the measurement of hemoglobin-NO adduct (HbNO) in whole blood by the EPR method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and an unknown radical species overlapping the same magnetic field as that of HbNO, which makes it physically impossible to measure small amounts of HbNO. Thus, to reveal the EPR spectrum of HbNO, we developed the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of the sample blood using software. Using this technique, we succeeded in measuring the steady blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril reduces abnormalities of NO dynamics in the L-NAME (N w -nitro-L-arginine-methylester)-induced endothelial dysfunction of rats using the EPR HbNO signal subtraction method.

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Electron paramagnetic resonance characterization of rat neuronal nitric oxide production ex vivo

Cited by 32 publications

References 11 publications

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

New Methods to Evaluate Endothelial Function: Evaluation of Endothelial Function by Hemoglobin-Nitric Oxide Complex Using Electron Paramagnetic Resonance Spectroscopy

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