Juan José 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 trapN-methyl-d-glucamine-dithiocarbamate-FeIIproduced a signal ascribed to the NO⋅ spin adduct (g = 2.04; a N = 12.5 G). The intensity of the signal increased with time, protein concentration, andl-Arg, and decreased with the addition of the NOS inhibitorN G-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⋅ × (min·mg protein)−1, respectively, with aK m for l-Arg of 5–7 μm. 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, O2consumption, and O2 free radical formation.

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 μg (.NO) and 0.001‐0.01 μg (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 · 106 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 μg . Preincubation with N G‐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.

Nitric oxide inhibits mitochondrial NADH : ubiquinone reductase activity through peroxynitrite formation

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

The Regulation of Mitochondrial Oxygen Uptake by Redox Reactions Involving Nitric Oxide and Ubiquinol

The reversible inhibitory effects of nitric oxide (·NO) on mitochondrial cytochrome oxidase and O2uptake are dependent on intramitochondrial ·NO utilization. This study was aimed at establishing the mitochondrial pathways for ·NO utilization that regulate O⨪2 generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO–) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO–-supplemented mitochondrial membranes were used. The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for ·NO utilization in mitochondria, which effectively compete with the binding of ·NO to cytochrome oxidase, thereby releasing this inhibition and restoring O2 uptake. The pathways for ·NO utilization are discussed in terms of the steady-state levels of ·NO and O⨪2 and estimated as a function of O2 tension. These calculations indicate that mitochondrial ·NO decays primarily by pathways involving ONOO– formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysac‐charide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady‐state concentration of 0.5 µM NO in the muscle associated with increased detection of hydrogen peroxide (H2O2). In vitro, the same NO concentration produced a marked increase in H2O2 production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O2−·). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H2O2 production associated with uncoupling and decreased phosphor‐ylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L‐NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O2−· that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility.—Boczkowski, J., Lis‐dero, C. L., Lanone, S., Samb, A., Carreras, M. C., Boveris, A., Aubier, M., Poderoso, J. J. Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J. 13, 1637–1647 (1999)

Regulation of mitochondrial respiration by oxygen and nitric oxide.

Abstract: Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1‐ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2−) and peroxynitrite (ONOO−). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. NO exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

Mitochondrial Regulation of Cell Cycle and Proliferation

Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O₂, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O₂ utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis.

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5 μM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 μM NO, n = 18, P < 0.05) and to 1.2 ± 0.1 μM O2 ⋅ min-1 ⋅ g tissue-1 (10 μM 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 μM 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 O2uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 μM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.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.

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)