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The reversible inhibitory effects of nitric oxide (⅐NO) on mitochondrial cytochrome oxidase and O 2 uptake are dependent on intramitochondrial ⅐NO utilization. This study was aimed at establishing the mitochondrial pathways for In the early 1970s, it was recognized that isolated respiring mitochondria produce hydrogen peroxide (H 2 O 2 ) at rates that depend on the redox state of the components of the respiratory chain and, consequently, on the mitochondrial metabolic state and the presence of inhibitors (1, 2). Mitochondrial production of H 2 O 2 accounts for about 1% of the O 2 uptake under physiological conditions, according to evidence obtained from perfused rat liver and heart (3). Mitochondrial H 2 O 2 is produced through the manganese-superoxide dismutase-catalyzed disproportionation of O 2. (4 -6), which is vectorially generated into the mitochondrial matrix during ubisemiquinone autoxidation (4, 7, 8) and NADH-dehydrogenase activity (9 value of 0.5-1.0 ϫ 10 -10 M can be estimated (3, 10). Nitric oxide (⅐NO) produced by the endothelium elicits cellular physiological effects within a wide concentration range (10 -9 to 10 -5 M). The effects of ⅐NO on mitochondria-inhibition of cytochrome c oxidase (11-19), impairment of electron flow at the cytochrome bc 1 region (17), and oxidation of ubiquinol (20, 21)-require progressively increasing concentrations of this species. ⅐NO regulates O 2 uptake and promotes H 2 O 2 release by mitochondria (17,22) (an effect also demonstrated in the isolated beating rat heart (23)); the increase in mitochondrial H 2 O 2 formation may be understood as an antimycin-like effect of ⅐NO accomplished by its effective binding to the cytochrome bc 1 segment (17).The ⅐NO influx in the mitochondrial compartment is expected to affect the steady-state levels of O 2 . due to the diffusion-controlled reaction between these species (24, 25) to yield peroxynitrite (ONOO -) (26). Three recently recognized facts add complexity to the mitochondrial interactions between O 2 .and ⅐NO: first, ⅐NO inhibits succinate-cytochrome c reductase activity and increases O 2 . production in submitochondrial particles, isolated mitochondria, and perfused rat heart (17, 23). Second, membrane-bound mitochondrial NOS 1 generates ⅐NO at rates that are similar to the rates of mitochondrial O 2 . production (27-29). Third, ⅐NO can be reduced to the nitroxyl anion (NO -) by one-electron transfers from three reduced components of the mitochondrial respiratory chain: ubiquinol, cytochrome c, and cytochrome c oxidase (20,30,31).The fine metabolic control of the intramitochondrial steadystate concentrations of ⅐NO-performed through a series of oxidative and reductive reactions involving O 2 . , ubiquinol, the cytochrome bc 1 segment, and cytochrome c oxidase-is relevant to mitochondrial physiology with further implications for cell energy production. This study is aimed at establishing the mitochondrial pathways for ⅐NO utilization that regulate O 2 .generation via reductive and oxidative reactions involving ubiquinol...

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 lipopolysaccharide (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 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating 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 O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.

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.

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Pharmacological evidence supports a role of a transient decreased endogenous nitric oxide (NO) synthesis in ovalbumin (OVA)-induced early airway hyperresponsiveness in guinea pigs. However, no data are available regarding the expression and activity of the constitutive NO synthases (cNOS; NOS1 and NOS3, nNOS and eNOS, respectively) in this model. Therefore, we evaluated cNOS activity (conversion of L-[3H]arginine to L-[3H]citrulline in the presence of Ca2+ and calmodulin), nitrate and nitrite (NOx) concentration (modified Griess method), and NOS1 and NOS3 protein expression (Western blot) in lung homogenates and in the tracheal smooth muscle from OVA-immunized and multiple aerosol-challenged guinea pigs (six challenges, once daily). The expression and activity of the inducible NOS isoform (NOS2), the levels of exhaled NO, and the in vivo airway reactivity were also determined. Constitutive NOS activity and NO(x) concentration were significantly lower 6 h after the last OVA challenge as compared with saline exposure, being similar at 24 h. Expression of NOS1 paralleled cNOS activity, which was reduced 6, but not 24 h after OVA challenge. The decrease in NOS1 expression was accompanied by a significant decrease in the amounts of exhaled NO and by a maximal airway hyperresponsiveness to histamine. The levels of NOS3 were not modified at the two time points evaluated, and no NOS2 expression and activity were found at any time point. Similar modifications were observed in the tracheal smooth muscle. We conclude that OVA stimulation in immunized guinea pigs induced a transient reduction in NOS1 protein expression and activity in the respiratory system, which probably participates in airway hyperresponsiveness.

Peroxynitrite anion (ONOO^–) is a potent biological oxidant produced by the near diffusion-limited reaction of superoxide and nitric oxide. Peroxynitrite has been implicated in diverse forms of free radical-induced tissue injury. Experimental evidence showed that exogenous and endogenous peroxynitrite causes alterations of the structure and function of mitochondrial proteins, leading to mitochondrial dysfunction and cellular or organ injury. These data are discussed along with its physiopathological implications.

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