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Verónica D'Annunzio,
Martín Donato,
Andrea Fellet,
Bruno Buchholz,
Valeria G Antico Arciuch,
María C Carreras,
Laura B Valdez, Tamara Zaobornyj,
Celina Morales,
Alberto Boveris,
Juan J Poderoso,
Ana M Balaszczuk,
Ricardo J Gelpi
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ABSTRACT: Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.
Molecular and Cellular Biochemistry 08/2011; 359(1-2):169-76. · 2.06 Impact Factor
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ABSTRACT: Isolated rabbit hearts were exposed to ischemia (I; 15 min) and reperfusion (R; 5-30 min) in a model of stunned myocardium. I/R decreased left-ventricle O(2) consumption (46%) and malate-glutamate-supported mitochondrial state 3 respiration (32%). Activity of complex I was 28% lower after I/R. The pattern observed for the decline in complex I activity was also observed for the reduction in mitochondrial nitric oxide synthase (mtNOS) biochemical (28%) and functional (50%) activities, in accordance with the reported physical and functional interactions between complex I and mtNOS. Malate-glutamate-supported state 4 H(2)O(2) production was increased by 78% after I/R. Rabbit heart Mn-SOD concentration in the mitochondrial matrix (7.4±0.7 μM) was not modified by I/R. Mitochondrial phospholipid oxidation products were increased by 42%, whereas protein oxidation was only slightly increased. I/R produced a marked (70%) enhancement in tyrosine nitration of the mitochondrial proteins. Adenosine attenuated postischemic ventricular dysfunction and protected the heart from the declines in O(2) consumption and in complex I and mtNOS activities and from the enhancement of mitochondrial phospholipid oxidation. Rabbit myocardial stunning is associated with a condition of dysfunctional mitochondria named "complex I syndrome." The beneficial effect of adenosine could be attributed to a better regulation of intracellular cardiomyocyte Ca(2+) concentration.
Free radical biology & medicine 06/2011; 51(6):1203-12. · 5.42 Impact Factor
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Martín Donato,
Verónica D'Annunzio,
Bruno Buchholz,
Verónica Miksztowicz,
Cristina Lorenzo Carrión,
Laura B Valdez, Tamara Zaobornyj,
Laura Schreier,
Regina Wikinski,
Alberto Boveris,
Gabriela Berg,
Ricardo J Gelpi
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ABSTRACT: 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 micromol 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 micromol 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 micromol 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.
Experimental physiology 10/2009; 95(2):274-81. · 3.17 Impact Factor
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ABSTRACT: 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.
AJP Heart and Circulatory Physiology 04/2009; 296(6):H1741-7. · 3.71 Impact Factor
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ABSTRACT: 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.
Frontiers in Bioscience 02/2007; 12:1247-59. · 3.52 Impact Factor
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ABSTRACT: The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.
Biochimica et Biophysica Acta 04/2006; 1757(3):166-72. · 4.66 Impact Factor
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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.
Annals of the New York Academy of Sciences 01/2006; 957(1):271 - 273. · 3.15 Impact Factor
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Journal of Applied Physiology 01/2006; 99(6):2453-4. · 3.75 Impact Factor
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ABSTRACT: Mitochondrial nitric oxide (NO) production was assayed in rats submitted to hypobaric hypoxia and in normoxic controls (53.8 and 101.3 kPa air pressure, respectively). Heart mitochondria from young normoxic animals produced 0.62 and 0.37 nmol NO.min(-1).mg protein(-1) in metabolic states 4 and 3, respectively. This production accounts for a release to the cytosol of 29 nmol NO.min(-1).g heart(-1) and for 55% of the NO generation. The mitochondrial NO synthase (mtNOS) activity measured in submitochondrial membranes at pH 7.4 was 0.69 nmol NO.min(-1).mg protein(-1). Rats exposed to hypobaric hypoxia for 2-18 mo showed 20-60% increased left ventricle mtNOS activity compared with their normoxic siblings. Left ventricle NADH-cytochrome-c reductase and cytochrome oxidase activities decreased by 36 and 12%, respectively, from 2 to 18 mo of age, but they were not affected by hypoxia. mtNOS upregulation in hypoxia was associated with a retardation of the decline in the mechanical activity of papillary muscle upon aging and an improved recovery after anoxia-reoxygenation. The correlation of left ventricle mtNOS activity with papillary muscle contractility (determined as developed tension, maximal rates of contraction and relaxation) showed an optimal mtNOS activity (0.69 nmol.min(-1).mg protein(-1)). Heart mtNOS activity is regulated by O(2) in the inspired air and seems to play a role in NO-mediated signaling and myocardial contractility.
Journal of Applied Physiology 07/2005; 98(6):2370-5. · 3.75 Impact Factor
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ABSTRACT: Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO.min(-1).g heart(-1) (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.
AJP Heart and Circulatory Physiology 07/2005; 288(6):H2568-73. · 3.71 Impact Factor
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ABSTRACT: 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.
Methods in Enzymology 02/2005; 396:444-55. · 2.04 Impact Factor
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ABSTRACT: 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.
Biological research 02/2004; 37(2):279-86. · 1.03 Impact Factor
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ABSTRACT: 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%.
Antioxidants and Redox Signaling 07/2003; 5(3):265-71. · 8.46 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 07/2003; 305(3):771-5. · 2.48 Impact Factor
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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 microM 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 microM ONOO(-) at 30 degrees C. As polyphenols we used (+)-catechin, (-)-epicatechin, and myricetin. The most efficient of the compounds tested was myricetin with an IC(50) of 20 microM. 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.
Annals of the New York Academy of Sciences 06/2002; 957:271-3. · 3.15 Impact Factor
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ABSTRACT: Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.
Biochimica et Biophysica Acta 1757(5-6):535-42. · 4.66 Impact Factor
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ABSTRACT: The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.
Molecular Aspects of Medicine 25(1-2):49-59. · 9.97 Impact Factor
