An Improved Assay for Measurement of Nitric Oxide Synthase Activity in Biological Tissues

Giraldez, Roberto R.; Zweíer, Jay L.

doi:10.1006/abio.1998.2721

Cited by 29 publications

(14 citation statements)

References 25 publications

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“…We wish to acknowledge that there exist alternative pathways other than NOS by which citrulline may be generated in tissues. 22 Although we did not measure the NOS-inhibitable fraction of citrulline generation in our assay, we have recently documented an increase in the immunodetectable levels of NOS that parallels the functional data reported here (C.K.R. et al, unpublished data, 2000).…”

Section: Discussionsupporting

confidence: 74%

Enhanced NO Inactivation and Hypertension Induced by a High-Fat, Refined-Carbohydrate Diet

et al. 2000

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Abstract-We have recently demonstrated that long-term consumption of a high-fat, refined-carbohydrate (HFS) diet induces hypertension (HTN) in normal rats compared with a low-fat, complex-carbohydrate (LFCC) diet. Limited evidence suggests that high-fat or high-sugar diets cause enhanced generation of reactive oxygen species (ROS). We therefore hypothesized that by inducing oxidative stress, the HFS diet may promote nitric oxide (NO) inactivation and HTN. To test this hypothesis, female Fischer rats were placed on either the HFS or the LFCC diet starting at 2 months of age. Blood pressure, urinary NO metabolites (NO x ), and total renal NO synthase activity were monitored, and the tissue abundance of nitrotyrosine (NT), which is the stable "footprint" of NO oxidation by ROS, was determined. The HFS diet group exhibited a gradual rise in arterial blood pressure and were hypertensive by 18 months. This trend was accompanied by a marked accumulation of NT in all tested tissues, an initial rise and a subsequent fall in NO synthase activity, and a fall in urinary NO x excretion. The HFS diet-fed animals had a blunted blood pressure response to the NO synthase inhibitor N -nitro-L-arginine methyl ester (L-NAME) compared with the LFCC diet group, which showed a marked hypertensive response to L-NAME. L-NAME-induced HTN was reversible with L-arginine in the LFCC diet group; however, HTN was not corrected by L-arginine supplementation in the HFS diet group. These findings point to enhanced ROS-mediated inactivation and sequestration of NO, which may contribute to the reduction of bioactive NO and HTN in the HFS diet-fed animals. Key Words: arginine Ⅲ endothelial Ⅲ free radicals Ⅲ insulin resistance Ⅲ L-NAME Ⅲ nitric oxide H ypertension (HTN) is the most common cardiovascular disease in the United States and is a hallmark risk factor for myocardial infarction, stroke, and congestive heart failure. 1 It has been estimated that one quarter of all adults and one half of all individuals Ͼ65 years of age have HTN, and it is more common in men than women. 1 The vast majority of patients with HTN have essential HTN of unknown etiology.The vascular endothelium is responsible for the production of several vasoactive substances, 1 of the most vital of which is nitric oxide (NO), a potent vasodilator synthesized from L-arginine by the enzyme NO synthase (NOS). Several investigations have demonstrated that endotheliumdependent relaxation is impaired in patients with essential HTN. 2 This endothelial dysfunction could be the result of a decrease in biologically active NO, ultimately leading to a depressor/pressor imbalance stemming from tonic removal of NO-mediated vasodilation. 2,3 There is evidence that hyperlipidemia 4 and high-sugar diets, 5 high-fat diets, 6 or both induce oxidative stress. We have recently demonstrated increased lipid peroxidation 7 and subsequently found direct evidence of NO oxidation by reactive oxygen species (ROS) in a model of lead-induced HTN. 8 In addition, we recently reported that female and male rats ...

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Section: Discussionsupporting

confidence: 74%

Enhanced NO Inactivation and Hypertension Induced by a High-Fat, Refined-Carbohydrate Diet

et al. 2000

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“…On the other hand, the radioactive signal observed by Lacza group could also not be quenched using the NOS inhibitor, N -nitro- L -arginine methyl esther (100 μM), clearly indicating that the signal was not NOS-dependent. Attempts to inhibit arginase by use of arginase inhibitors in the MT samples during the NOS activity assays, and/or actual removal of arginase using KCl washes during MT preparation have been suggested previously [19; 37; 38; 39]. Nevertheless, most groups studying mtNOS activity (using either the 14 C or 3 H labeled L -arginine assay) do not make an effort to inhibit arginase.…”

Section: Resultsmentioning

confidence: 99%

Arginase activity in mitochondria – An interfering factor in nitric oxide synthase activity assays

Venkatakrishnan

Nakayasu

Almeida

et al. 2010

Biochemical and Biophysical Research Communications

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Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [ 14 C]-L-arginine to [ 14 C]-Lcitrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent [1]. Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [ 14 C]-L-arginine to [ 14 C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays. (Supported by ES 011982 & 2G12RR008124 to RTM & UTEP, respectively).

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“…Our experiments measuring the effect of pH on AO-mediated nitrite reduction showed that at pH 6.0 an ϳ35% increase occurred compared with pH 7.4 (Table 1). Thus, in the heart, AO-mediated NO generation could approach that of the maximal NO generation from constitutive NO synthase, estimated to be 1.5 nM/s (12,48). Of note, NO synthase requires molecular oxygen as a substrate so that its production of NO would be impaired under the hypoxic or anoxic conditions observed to trigger AO-mediated nitrite reduction.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite

Kundu

Zweíer

2009

Journal of Biological Chemistry

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111

110

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Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K m value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite. Nitric oxide (NO)3 exerts a large number of important regulatory biological functions and also plays an important role in the pathogenesis of cellular injury (1-5). NO synthesis was first discovered in macrophages, endothelial cells, and neuronal cells (1, 6 -8). A group of enzymes were identified, NO synthases, which metabolize arginine to citrulline with the formation of NO (9, 10). More recent studies have shown that in addition to NO generation from specific NO synthases, nitrite can be an important source of NO in biological tissues, especially under ischemic conditions (11-16). However, questions remain regarding the precise mechanisms involved in this nitrite reduction.Aldehyde oxidase (AO) (aldehyde:oxygen oxidoreductase; EC 1.2.3.1) is a cytosolic enzyme that plays an important role in the biotransformation of drugs and xenobiotics (17). AO belongs to the family of molybdenum-containing proteins with two iron-sulfur clusters, a flavin cofactor, and a molybdopterin cofactor (18,19). The similar molybdenum-containing enzyme xanthine oxidoreductase (XOR) has been shown previously to be a highly effective nitrite/nitrate reductase playing an important role in catalyzing NO generation from nitrite in mammalian tissues, especially under acidic conditions (14, 16, 20 -26).AO is present in highest levels in the liver but is also broadly distributed in o...

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An Improved Assay for Measurement of Nitric Oxide Synthase Activity in Biological Tissues

Cited by 29 publications

References 25 publications

Enhanced NO Inactivation and Hypertension Induced by a High-Fat, Refined-Carbohydrate Diet

Enhanced NO Inactivation and Hypertension Induced by a High-Fat, Refined-Carbohydrate Diet

Arginase activity in mitochondria – An interfering factor in nitric oxide synthase activity assays

Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite

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