[Nobel Prize in medicine 1998: resurrection of an inorganic gas into a molecule of high biological significance].
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History, 20th Century↗
Biomedical subjects
J J Poderoso
Publications and source records attributed to J J Poderoso.
Production of nitric oxide by mitochondria.
The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.
Animals↗
Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart.
Isolated rat heart perfused with 1.5-7.5 microM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2 uptake from 3.2 +/- 0.3 to 1.6 +/- 0.1 (7.5 microM NO, n = 18, P < 0.05) and to 1.2 +/- 0.1 microM O2.min-1.g tissue-1 (10 microM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent (r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 microM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2 uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 microM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2 uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.
Animals↗
[Shock: concepts for a definition].
The shock syndrome has been classically considered as a consequence of both decreased tissue perfusion and O2 supply; however, in some types of shock like septic or traumatic ones, regional blood flows may be increased. A decade ago, mitochondrial alterations consistent with uncoupling of oxidative phosphorylation were reported in either endotoxemic or hemorrhagic experimental shock or in humans. Recently, the discovery of nitric oxide (NO) and its increase in the shock state, has opened new perspectives in the understanding of this problem. Nitric oxide produces vasodilatation and, at the same time, increases the mitochondrial production of O2 active species like superoxide anion. Both radicals react to form a strong oxidant that is able to nitrate the phenolic rings of proteins: peroxynitrite. This effect leads to the impairment of the activities of different mitochondrial enzymes like succinate dehydrogenase and ATPase and the mitochondrial function and finally, to decreased energy levels and to multiorgan failure. The increase in NO release is due to the effects of circulating peptides and of increased adhesion of neutrophils to the endothelium and to the positive effects of inflammatory mediators like TNF-alpha and cytokines on inducible NOS (iNOS) expression in endothelium and tissues. It is suggested that the shock state is the consequence of an imbalance between NO and O2 and their metabolites.
Adenosine Triphosphatases↗
Effects of respiratory burst inhibitors on nitric oxide production by human neutrophils.
Human neutrophils (PMN) activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O2.-) and its dismutation product, hydrogen peroxide (H2O2). To assess whether .NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 micrograms/ml) decreased H2O2 yield without significantly changing .NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 micrograms/ml) completely abolished H2O2 release by fMLP-activated neutrophils; conversely, .NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased .NO production and increased O2.- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H2O2 and .NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O2.- and H2O2 generation and simultaneously maintains .NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O2.- and .NO production.
Adenylyl Cyclases↗
Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles.
Nitric oxide (.NO) released by S-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective .NO concentration of 0.1 microM, while succinate- and NADH-cytochrome-c reductase activities were half-maximally inhibited at 0.3 microM .NO. Submitochondrial particles treated with .NO (either from GSNO or from a pure solution) showed increased O(-)(2) and H202 production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with .NO also showed increased H2O2 production. Cytochrome spectra and decreased enzymatic activities in the presence of .NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone-cytochrome b region of the respiratory chain, the latter leading to the increased O2- production. Electrochemical detection showed that the buildup of a .NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of .NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial O2- reacts with .NO to form peroxynitrate and, by removing .NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of .NO, inhibition of electron transfer, .NO-induced O2- production, and ONOO- formation participate in the regulatory control of mitochondrial oxygen uptake.
Animals↗
Neutrophil function, nitric oxide, and blood oxidative stress in Parkinson's disease.
We studied nitrogen radical nitric oxide (.NO) release and reactive oxygen species (ROS) production by isolated neutrophils after phorbol myristate acetate (PMA) stimulation in 12 newly diagnosed and nine treated Parkinson's disease (PD) patients and 10 age-matched healthy controls. Neutrophils of both groups of PD patients had an elevated PMA-activated release of .NO [61 and 57%, respectively, higher than that of controls (p < 0.05)]. In contrast, H2O2 release was only significantly increased by 56% in chronically treated patients. In agreement, the maximum rate of luminol-dependent chemiluminescence, which partly represents O2- H2O2- .NO interactions, was increased only in the treated group. When other blood markers of oxidative stress were compared, only erythrocyte catalase activity was decreased in both PD patient series by 33 and 39%, respectively (p < 0.05), whereas plasma antioxidant capacity and erythrocyte superoxide dismutase activity levels were decreased only in treated PD patients. This study suggests that neutrophils express a primary alteration of .NO release in PD patients, whereas H2O2 and oxidative-stress parameters are more probably related to the evolution of PD or to effects of treatment with L-dopa.
Antiparkinson Agents↗
Oxidative stress in rodent closed duodenal loop pancreatitis.
CONCLUSION: Production of excited oxygen species is earlier in the liver than in the pancreas and could contribute to damage in a reflux model. Treatment with SOD could attenuate 59% light emission in pancreas, but did not modify serum enzyme levels or pancreatic edema, resulting as an insufficient isolated therapy. Unexpectedly, it was found an increased plasma antioxidant capacity that was related to total bilirubin levels, and declined at late stages probably denoting other circulating antioxidant consumption. BACKGROUND: Oxidative stress has been shown to play a role in different models of acute pancreatitis, although it has not been studied in the severe necrohemorrhagic model produced by closed duodenal loop pancreatitis. METHODS: We studied Sprague Dawley female rats in two groups: a closed duodenal loop pancreatitis group and a control, sham-operated group. In order to evidence the oxygen excited species production, in situ spontaneous chemiluminescence from living and naturally perfused pancreas and liver was measured at 0, 0.5, 1.5, 3, 6, 12, and 24 h after the duodenal ligature. Blood pancreatic amylase and aminotransferases levels were determined as expression of tissue damage in pancreas and liver. At the same time, plasma antioxidant capacity was measured by the peroxyl radical trapping capability of plasma samples compared to that of Trolox (synthetic analog of vitamin E), and results are expressed as Trolox equivalence. Bovine superoxide dismutase (SOD) was administered to attenuate oxygen free radicals activity at the beginning of the peroxidation chain and also as a therapeutic tool. RESULTS: The experimental procedure induced a severe pancreatitis, as evidenced by pancreatic enzymes that rose markedly in the early hours of disease and remained heightened throughout the experiment. The results show early light emission from the liver at 3 h and peak levels at 12 h, whereas in the pancreas, luminescence increased at 6 h and doubled later at 12 h, both returning to control levels at 24 h.
Acute Disease↗
Measurement of nitric oxide and hydrogen peroxide production from human neutrophils.
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Cells, Cultured↗
Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence.
Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO-) was shown to react with luminol to yield CL in a cell-free system. We evaluated the role of the L-arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorphonuclear (PMN) leukocytes using the NOS inhibitors NG-monomethyl-L-arginine (L-NMMA) and N-iminoethyl-L-ornithine (L-NIO). Nitric oxide (.NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O2.-) production was measured. Luminol CL was notably diminished by L-NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and L-NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O2.- production was significantly decreased by L-NMMA; moreover, luminol-dependent CL but not O2.- production was attenuated by L-NIO. These data suggest that products of catalytic activity of both .NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO- would be an unrecognized mediator in this phenomenon.
Animals↗
Isoproterenol-dependent decrease in oxygen uptake and respiratory enzyme activities in rat myocardial tissue and mitochondria.
OBJECTIVE: Myocardial damage induced by isoproterenol is believed to be secondary to increased oxygen demands on the heart. Our objective was to test an additional primary action of isoproterenol on tissue and mitochondrial oxidative metabolism and to compare these effects with the effects of other adrenergic agents in the presence of adrenergic inhibitors. DESIGN: Prospective, dose-response study. SETTING: Research laboratory at a university hospital. SUBJECTS: Fifty Sprague-Dawley female rats (200 to 350 g), slightly anesthetized with ether and divided into several groups. INTERVENTIONS: In 26 rats, the heart was removed, cut into fine slices (0.5-mm thickness), and placed in an ice-cold buffer. In 22 animals, the hearts were perfused in the Langendorff manner and chopped and processed for mitochondrial studies. MEASUREMENTS AND MAIN RESULTS: We determined the following: a) the direct "in vitro" effects of isoproterenol and related catecholamines on normal oxygen uptake using myocardial slices; b) rat heart oxygen consumption and mitochondrial oxygen uptake from isolated organs, perfused with isoproterenol; c) measurements of enzyme activities in submitochondrial particles from the same perfused hearts; and d) the direct effects of isoproterenol on normal mitochondria isolated from normal nonperfused hearts. The oxygen uptake was determined polarographically with a Clark-type electrode and enzymatic activities were assayed by spectrophotometric reduction of cytochrome c at 550 nm with different mitochondrial substrates. Isoproterenol (0.01 to 100 nM) decreased the oxygen uptake by the heart slices in a dose-dependent manner. In comparison, epinephrine or norepinephrine per se did not change the parameter. However, with the addition of alpha-adrenergic receptor inhibitors, oxygen uptake decreased to values similar to those values obtained with isoproterenol. Also, mitochondria isolated from hearts perfused with isoproterenol had decreased state 3 respiratory rates (by 50%) and decreased respiratory control ratios (by 30%), without changes in adenosine 5'-diphosphate/oxygen ratios. The respiratory chain enzyme activities were also lowered. CONCLUSIONS: The data suggest that while isoproterenol increases "in vivo" oxygen uptake by the working rat heart, isoproterenol can simultaneously decrease maximal adenosine 5'-diphosphate-induced mitochondrial oxygen uptake and in vitro myocardial tissue oxygen uptake, probably by modifying the mitochondrial respiratory enzymes. This action could be counteracted by alpha-adrenergic agonist effects.
Animals↗
Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils.
Nitric oxide (.NO) release, oxygen uptake and hydrogen peroxide (H2O2) production elicited by increasing phorbol 12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 micrograms PMA/ml (.NO) and 0.001-0.01 micrograms PMA/ml (H2O2). At saturated PMA concentrations, .NO production, oxygen uptake and H2O2 release were 0.56 +/- 0.04, 3.32 +/- 0.52 and 1.19 +/- 0.17 nmol.min-1.10(6) cells-1. .NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.001-0.01 micrograms PMA/ml. Preincubation with NG-monomethyl-L-arginine (L-NMMA) decreased O2 uptake and .NO release but increased H2O2 production, while superoxide dismutase (SOD) increased .NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that .NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of .NO and O2.- release favour the formation of other oxidants like peroxynitrite.
Humans↗
Decreased production of nitric oxide by human neutrophils during septic multiple organ dysfunction syndrome. Comparison with endotoxin and cytokine effects on normal cells.
The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O-2) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O-2 by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-gamma), or tumor necrosis factor (TNF-alpha) alone or in combination. Results showed that PMN release of both NO and O-2 was reduced in septic samples; in contrast, an association of LPS, IFN-gamma, and TNF-alpha promoted maximal NO release by normal cells (40-50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O-2 release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.
Adult↗
Oxidative stress in muscle and liver of rats with septic syndrome.
Sepsis, as infection associated to systemic manifestations, was produced in rats by cecal ligation and double perforation. Sham-operated rats were used as controls. The spontaneous chemiluminescence of rat adductor muscle and liver were measured at 6, 12, 24, and 30 h after the surgical procedure. Muscle chemiluminescence showed a maximal increase of about twofold (control emission 10 +/- 1 cps/cm2) after 6-12 h of sepsis, while liver chemiluminescence increased by about 80% (control emission: 11 +/- 1 cps/cm2) after 24 h of sepsis. The activities of muscle antioxidant enzymes were found maximally diminished after 12 h of sepsis: 46% decrease for Mn-superoxide dismutase, 83% decrease for catalase, and 55% decrease for glutathione peroxidase. In liver, only catalase activity showed a 52% decrease after 24 h of sepsis. State 3 oxygen uptake of muscle mitochondria with either malate-glutamate or succinate as substrates was 40% decreased after 12 h of sepsis in both cases. State 4 oxygen uptake of muscle mitochondria was not affected. The rate of H2O2 production of muscle mitochondria after 12 h of sepsis with either malate-glutamate or succinate as substrates was increased about 2.5 times but was not affected when assayed in the presence of as rotenone and antimycin. The oxygen uptake of liver mitochondria isolated from septic rats did not show differences as compared with those of control rats after 6 to 24 h of sepsis. Oxidative stress appears to occur in skeletal muscle early at the onset of the septic syndrome, with inhibition of active mitochondrial respiration and inactivation of antioxidant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Liver oxygen uptake dependence and mitochondrial function in septic rats.
Defective oxygen consumption and a pathological dependence of oxygen uptake on O2 supply have been considered important events in sepsis. To relate these features with tissue and mitochondrial metabolism, we studied oxygen uptake in whole isolated and perfused rat liver at two O2 supply levels, in the same liver slices, and in isolated liver mitochondria. Experimental sepsis in rats was induced by cecal ligation and double-gauge puncture. The results showed that liver and tissue slices from septic animals had a 60% greater O2 uptake than that of controls and that, during sepsis, liver O2 uptake was markedly dependent on O2 supply. Concomitantly, mitochondrial O2 uptake was nearly 30% greater with malate-glutamate as substrate, but not with succinate; lowering O2 concentration in the medium did not alter the enhanced function. In submitochondrial, only NADH-dehydrogenase activity was 100% higher in septic samples. At least, in some tissues, O2 dependence is a function of O2 availability, sensitized by increased mitochondrial O2 uptake related to changes in respiratory enzymes.
Adenosine Diphosphate↗
[Nitric oxide, nervous system, and Parkinson disease].
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Central Nervous System↗
Active core rewarming in neurologic, hypothermic patients: effects on oxygen-related variables.
OBJECTIVES: To determine in hypothermic patients if a) the decrease in oxygen consumption (VO2) is exclusively dependent on the decrease in metabolic rate, or b) as a consequence of the greater hemoglobin affinity for oxygen, hypothermic tissues have impaired oxygen extraction. DESIGN: Clinical, prospective study; sequential measurements of oxygen-related variables during active core rewarming. SETTING: Intensive care unit of a university hospital. PATIENTS: Twelve patients (44 +/- 16 yrs of age) admitted to the intensive care unit with a core temperature of < 34 degrees C due to severe neurologic damage. INTERVENTIONS: Rewarming (with heated enemas, gastric infusions, and heated blankets) to increase body temperature at a rate of approximately 1 degree C/hr. Measurements of oxygen-related variables were performed at a baseline of 31.0 +/- 1.1 degrees C, and repeated at each 1 degree C increase to reach a core temperature of approximately 35 degrees C. MEASUREMENTS AND MAIN RESULTS: Oxygen-related variables of rewarmed patients were allocated into two groups, above or below the observed mean core temperature of 33.1 degrees C recorded for all measurements (n = 45). Comparison of the low core temperature group (31.1 +/- 1.4 degrees C; n = 20) with the high core temperature group (34.7 +/- 0.9 degrees C; n = 25) showed that the group with the lower core temperatures had a significant increase in VO2 index (67 +/- 22 vs. 103 +/- 38 mL/min/m2 [p < .001]), oxygen delivery index (183 +/- 73 vs. 290 +/- 123 mL/min/m2 [p < .001]), and the PO2 value at which hemoglobin was half-saturated with oxygen ([P50] 23 +/- 5.7 vs. 27.7 +/- 5.7 torr [3.0 +/- 0.7 vs. 3.6 +/- 0.7 kPa] [p < .02]). An increase in metabolic acidosis could be observed in the lower temperature group: arterial pH 7.47 +/- 0.15 vs. 7.34 +/- 0.13 (p < .01); base deficit -3.7 +/- 6.7 vs. -8.2 +/- 4.9 mEq/L (p < .02). The oxygen extraction ratio remained unchanged: 0.39 +/- 0.10 vs. 0.38 +/- 0.10 (NS). CONCLUSIONS: These data show that VO2 was reduced to half of normal values during hypothermia. Active core rewarming produced an average 4.5% increase in VO2 per 1 degree C that was characterized by the wide variation observed in this metabolic response between different patients and for individual cases. Despite the rightward shift of P50 observed during rewarming (mainly due to the Bohr effect), no change was reflected on the oxygen extraction ratio.
Acidosis, Lactic↗
Oxidative stress in skeletal muscle during sepsis in rats.
Skeletal muscle is a target organ during sepsis; nevertheless, there is no evidence of a possible free radical overproduction with tissue damage in this situation. We studied Sprague Dawley female rats in two groups: a septic group with cecal ligation and double cecal perforation and a control group that was sham operated. Hind limb adductor muscles spontaneous chemiluminescence was measured at 2, 4, 6, 12, 24, and 30 hr after the surgical procedure as the expression of oxygen excited species generation. Muscle samples were also taken and activity of the principal antioxidant enzymes--superoxide dismutase (SOD), catalase, and glutathione peroxidase--as well as myeloperoxidase, an index of neutrophil infiltration was determined. CPK seric assays at 12 and 24 hr were used to reflect muscle injury and revealed high levels. Previously administered bovine superoxide dismutase was employed to prevent or attenuate oxidative stress. The results showed that light emission by rat skeletal muscle doubled from 4 to 12 hr of sepsis and could be attenuated with SOD pretreatment. Observed changes may be attributed to the production of oxygen free radicals that do not depend on local neutrophil infiltration. The detoxifying antioxidant enzyme activities in skeletal muscle were diminished (Mn SOD 46% at 6 hr, catalase 83% at 12 hr glutathione peroxidase 55% at 12 hr), which would also facilitate muscle septic damage.
Animals↗