Laura B. Valdez

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.

Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.

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.

Free radical chemistry in biological systems.

Mitochondria are an active source of the free radical superoxide (O2-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O2 uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O2. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O2 uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O2- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.

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 Radical Production by Human Mononuclear Leukocytes

Human mononuclear cells (90% lymphocytes, 9% monocytes, and 1% polymorphonuclear leukocytes) produced spontaneously in resting state 0.11+/-0.01 nmol of nitric oxide (NO)/min/10(6) cells and 0.25+/-0.02 nmol of superoxide anion (O2-)/min/10(6) cells, as primary products. When these cells were stimulated with phorbol 12-myristate 13-acetate (PMA), the NO and O2- production increased by 82% and 204% to 0.25+/-0.02 nmol of NO/min/10(6) cells and 0.76+/-0.12 nmol of O2-/min/10(6) cells, respectively. Oxygen uptake reasonably accounted for the sum of the rates of NO and hydrogen peroxide (H2O2), the latter calculated as 0.5 O2- production, in nonstimulated and in PMA-stimulated cells. H2O2 and peroxynitrite formation were detected and measured as secondary products of the primary products O2- and NO. An original assay to determine H2O2 steady-state concentration and production rates is described. The determined production rates of the involved reactive species are in good agreement with known chemical equations. It is apparent that NO and O2- production by human mononuclear cells may constitute the basis of intercellular signaling and cell toxicity.

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.