Tamara Zaobornyj

Oxygen dependence of mitochondrial nitric oxide synthase activity

The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.

Strategic localization of heart mitochondrial NOS: a review of the evidence

Heart mitochondria play a central role in cell energy provision and in signaling. Nitric oxide (NO) is a free radical with primary regulatory functions in the heart and involved in a broad array of key processes in cardiac metabolism. Specific NO synthase (NOS) isoforms are confined to distinct locations in cardiomyocytes. The present article reviews the chemical reactions through which NO interacts with biomolecules and exerts some of its crucial roles. Specifically, the article discusses the reactions of NO with mitochondrial targets and the subcellular localization of NOS within the myocardium and analyzes the available data about heart mitochondrial NOS activity and identity. The article also describes the regulation of heart mtNOS by the distinctive mitochondrial environment by showing the effects of Ca(2+), O(2), l-arginine, mitochondrial transmembrane potential, and the metabolic states on heart mitochondrial NO production. The article depicts the effects of NO on heart function and highlights the relevance of NO production within mitochondria. Finally, the evidence on the functional implications of heart mitochondrial NOS is delineated with emphasis on chronic hypoxia and ischemia-reperfusion studies.

Kidney mitochondrial nitric oxide synthase.

Nitric oxide synthase activity was recognized in rat renal cortex mitochondria (mtNOS) with nitric oxide (NO) production rates of 0.14-0.78 nmol/min/mg of protein. Rat pretreatment with enalapril (30 mg/kg/day i.p., up to 15 days) increased NO production in kidney, liver, and heart mitochondria. In kidney, mtNOS activity and mtNOS protein, measured by western blot densitometry, were 5 and 2.3 times increased, respectively. Electron paramagnetic resonance analysis with the probe N-methyl-D-glucamine dithiocarbamate/FeSO(4) detected NO production in mitochondria isolated from enalapril-treated rats, but not in control untreated animals. Polyclonal antibodies anti-iNOS and anti-nNOS detected kidney mtNOS in western blots and inhibited mtNOS biochemical activity. The enzymatic activity of kidney mtNOS generates intramitochondrial NO concentrations that regulate mitochondrial functions: state 3 respiration was decreased by 12-28%, and state 4 hydrogen peroxide production was increased 12-35%.

Functional activity of mitochondrial nitric oxide synthase.

The functional activity of mitochondrial nitric oxide synthase (mtNOS) is determined by inhibiting O2 uptake and by enhancing H2O2 production. The effect of mtNOS activity on mitochondrial O2 uptake is assayed in state 3 respiration in two limit conditions of intramitochondrial NO: at its maximal and minimal levels. The first condition is achieved by supplementation with L-arginine and superoxide dismutase (SOD), and the second by addition of an NOS inhibitor and oxyhemoglobin. The difference between state 3 O2 uptake in both conditions constitutes the mtNOS functional activity in the inhibition of cytochrome oxidase activity. The functional activity of mtNOS in enhancing mitochondrial H2O2 generation in state 4 is given by the NO inhibition of ubiquinol-cytochrome c reductase activity. Simple determinations with the oxygen electrode or the measurement of mitochondrial H2O2 production can be used to assay the effects of physiological and pharmacological treatments on mtNOS activity.

Role of matrix metalloproteinase‐2 in the cardioprotective effect of ischaemic postconditioning

The activation of matrix metalloproteinases (MMPs) contributes to myocardial injury at the onset of reperfusion; however, their role in ischaemic postconditioning is unknown. The aim of the present study was to examine the effects of ischaemic postconditioning on MMP activity in isolated rabbit hearts. The isolated rabbit hearts were subjected to 30 min of global ischaemia followed by 180 min of reperfusion (I/R group; n= 8). In the ischaemic postconditioning group (n= 8), a postconditioning protocol was performed (2 cycles of 30 s reperfusion–ischaemia). In other experiments, we added doxycycline, an MMP inhibitor, at 25 (n= 7) or 50 μmol l−1 (n= 8) during the first 2 min of reperfusion. Coronary effluent and left ventricular tissue were collected during pre‐ischaemic conditions and at different times during the reperfusion period to measure MMP–2 activity and cardiac protein nitration. We evaluated ventricular function and infarct size. In the I/R group, infarct size was 32.1 ± 5.2%; Postcon reduced infarct size to 9.5 ± 3.8% (P < 0.05) and inhibited MMP–2 activity during reperfusion. The administration of doxycycline at 50 μmol l−1 inhibited MMP–2 activity and cardiac protein nitration and reduced the infarct size to 9.7 ± 2.8% (P < 0.05). A lower dose of doxycycline (25 μmol l−1) failed to inhibit MMP–2 activity and did not modify the infarct size. Our results strongly suggest that ischaemic postconditioning may exert part of its cardioprotective effects through the inhibition of MMP–2 activity.

Polyphenols and Red Wine as Antioxidants against Peroxynitrite and other Oxidants

The antioxidant capacity of polyphenols (+)-catechin, (-)-epicatechin and myricetin, and of different types of red wines (Cabernet Sauvignon, Malbec and blended wine) was evaluated by three assays. (a) NADH oxidation by peroxynitrite (ONOO-): the ONOO- scavenging activity was higher for myricetin (IC50=35 microM) than for (+)-catechin (IC50=275 microM) and (-)-epicatechin (IC50=313 microM). (b) Peroxynitrite initiated chemiluminescence in rat liver homogenate: (-)-epicatechin (IC50=7.0 microM) and (+)-catechin (IC50=13 microM) were more potent than myricetin (IC50=20 microM) in inhibiting the chemiluminescence signal. (c) Lucigenin chemiluminescence in aortic rings: (-)-epicatechin (IC50=15 microM) and (+)-catechin (IC50=18 microM) showed higher antioxidant capacity than myricetin (IC50=32 microM). All the assayed red wines were able to scavenge the oxidants and free radical species that generate the signal in each assay. Cabernet Sauvignon was the red wine with the highest antioxidant capacity in comparison with Malbec and blended wine. It is concluded that the use of sensitive biological systems (as the aortic ring chemiluminescence) provides important information in addition to the results from chemical (NADH oxidation by peroxynitrite) and biochemical (homogenate chemiluminescence) assays and offers advances in the physiological role of polyphenols.

Mitochondrial nitric oxide metabolism during rat heart adaptation to high altitude: effect of sildenafil, l-NAME, and l-arginine treatments

Rats submitted to high altitude (Cerro de Pasco, Perú, 4,340 m, Po(2) = 12.2 kPa) for up to 84 days showed a physiological adaptive response with decreased body weight gain (15%), increased right ventricle weight (100%), and increased hematocrit (40%) compared with sea level animals. These classical parameters of adaptation to high altitude were accompanied by an increase in heart mitochondrial enzymes: complexes I-III activity by 34% and mitochondrial nitric oxide synthase (mtNOS) activity and expression by >75%. The hyperbolic increase for mtNOS activity during adaptation to high altitude was similar to the observed pattern for hematocrit. Hematocrit and mtNOS activity mean values correlated linearly (r(2) = 0.75, P <or= 0.05). Chronic treatment for 28 days with sildenafil (50 mg*kg(-1).day(-1)) decreased the response of mtNOS to high altitude by 25%. Conversely, N(G)-nitro-l-arginine methyl ester treatment (8.3 mg*kg(-1)*day(-1)) increased such response by 40%, whereas l-arginine treatment (106 mg*kg(-1)*day(-1)) had no effect. Nitric oxide (NO) production by mtNOS accounts for approximately 49% of total cellular NO production in sea level rats and for approximately 54% in rats exposed to high altitude for 84 days. It is concluded that mtNOS is a substantial source of cardiac NO, a factor in the adaptive response to sustained heart hypoxia that is susceptible to be modified by pharmacological treatments.

Nitric oxide and superoxide anion production during heparin-induced capacitation in cryopreserved bovine spermatozoa.

The aim of this work was to quantify NO,O(2)(-) and ONOO(-) production during heparin-induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin-dependent capacitation, O(2) uptake, and NO production. Conversely, O(2)(-) production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O(2) consumption (5.9 ± 0.6 nmol/min × 10(7) cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10(7) cells), and a five-fold increase in O(2)(-) production (1.3 ± 0.07 nmol/min × 10(7) cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O(2)(-) generation and the second-order rate constant of the reaction between these species. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O(2) uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O(2)(-) production by a membrane-bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.

Mitochondrial contribution to the molecular mechanism of heart acclimatization to chronic hypoxia: role of nitric oxide.

A remarkable number of adaptive responses; including changes in the cardiovascular, respiratory and hematologic systems; takes place during acclimatization to natural or simulated high altitude. This adaptation to chronic hypoxia confers the heart an improved tolerance to all major deleterious consequences of acute O2 deprivation, not only reducing infarct size but also alleviating post-ischemic contractile dysfunction and ventricular arrhythmias. There is growing evidence about the involvement of mitochondria and NO in the establishment of cardioprotection. This review focuses on evidence about the putative role of different effectors of heart acclimatization to chronic hypoxia. Along with classical parameters, we consider NO, specially that generated by mtNOS, mitochondrial respiratory chain, mitoK(ATP) channels, reactive oxygen species and control of gene expression by HIF-1.

Polyphenols and Red Wine as Peroxynitrite Scavengers

Abstract: A novel chemiluminescent assay for evaluating peroxynitrite (ONOO−)‐scavenging capacity was developed. The experimental protocol ensures sensitivity and reproducibility of measurements. The addition of 0‐500 μM ONOO− to rat liver homogenate generated a luminous signal that was analyzed by chemiluminescence in a LKB Wallac liquid scintillation counter. The obtained optimal conditions were: 1‐2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 220 μM ONOO− at 30°C. As polyphenols we used (+)‐catechin, (−)‐epicatechin, and myricetin. The most efficient of the compounds tested was myricetin with an IC50 of 20 μM. The effectiveness of this method was verified by evaluating the antioxidant ability of three red wine samples to decrease peroxynitrite‐initiated chemiluminescence. The ONOO−‐scavenging activity of wines measured by this assay was related to the phenolic level of the samples. The quickness and reliability of this assay makes it particularly suitable for a large‐scale screening of watery food extracts.