Constanza Lisdero

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)

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.

Peroxynitrite-mediated mitochondrial dysfunction.

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.

The Mitochondrial Interplay of Ubiquinol and Nitric Oxide in Endotoxemia

Publisher Summary This chapter focuses on role of ubiquinol (UQ) and nitric oxide in endotoxemia. UQ is endogenously synthesized in every organ in a specific pathway branched from cholesterol biosynthesis. Organs have different ubiquinol content depending on the activity of the biosynthetic pathway. In addition to its role as electron carrier in the mitochondrial respiratory chain, UQ exhibits prooxidant and antioxidant properties. The chapter provides the methodological bases that support the relation between UQ content, mitochondrial NO steady-state concentration, and peroxynitrite-mediated mitochondrial damage in representative organs. The chapter also focuses on the contribution of mtNOS to peroxynitrite formation in mitochondria. The chapter presents an experimental model, elaborating endotoxemic animals and sample preparation, and homogenate and mitochondrial preparations. Sources of NO in endotoxemia are also analyzed in the chapter. The chapter discusses the measurement of NOS expression and activity and analyzes the role of ubiquinol in endotoxemia. The endotoxemic mitochondrial damage is also analyzed in the chapter. The chapter elaborates the ubiquinone content and NO-induced hydrogen peroxide production by mitochondria

Activation of a thioesterase specific for very-long-chain fatty acids by adrenergic agonists in perfused hearts.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.

β-Adrenergic stimulation controls the expression of a thioesterase specific for very-long-chain fatty acids in perfused hearts

Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobilization of this compound have been proposed, including a novel mechanism that involves the release of arachidonic acid from arachidonoyl-CoA by a thioesterase with substrate specificity for very-long-chain fatty acids. In rat heart, the acyl-CoA thioesterase activity can be regulated by a mechanism that involves beta-adrenoceptors. In this paper we demonstrate that beta-adrenergic agonists also regulate the acyl-CoA thioesterase mRNA levels. Isoproterenol (10(-7)M)-a concentration known to exert physiological responses-increases in a time-dependent manner the acyl-CoA thioesterase mRNA levels, an effect blocked by a specific beta-adrenoceptor antagonist. In addition, our results show that cAMP is involved in this process. The acyl-CoA thioesterase mRNA levels are also increased by fasting, but not by di(2-ethylhexyl)phthalate, a peroxisome proliferator. These results may suggest the existence of a beta-adrenoceptor-activated regulatory pathway for arachidonic acid release in cardiac tissue.