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Salvador Moncada

Publications and source records attributed to Salvador Moncada.

At least 19 recordsLinked to original sources
PubMed2006

Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide.

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 microM L-arginine at 70 microM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0+/-0.8 and 24.8+/-0.8 microM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

Arginine↗
PubMed2006

TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents.

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Adenosine Triphosphate↗
PubMed2006

Inhibition of cellular respiration by endogenously produced carbon monoxide.

Endogenously produced nitric oxide (NO) interacts with mitochondrial cytochrome c oxidase, leading to inhibition of cellular respiration. This interaction has been shown to have important physiological and pathophysiological consequences. Exogenous carbon monoxide (CO) is also known to inhibit cytochrome c oxidase in vitro; however, it is not clear whether endogenously produced CO can inhibit cellular respiration and, if so, what the significance of this might be. In this study, we show that exogenous CO inhibits respiration in a moderate but persistent manner in HEK293 cells under ambient (21%) oxygen concentrations (K(i) = 1.44 microM). This effect of CO was increased (K(i) = 0.35 microM) by incubation in hypoxic conditions (1% oxygen). Endogenous CO, generated by HEK293 cells transfected with the inducible isoform of haem oxygenase (haem oxygenase-1; HO-1), also inhibited cellular respiration moderately (by 12%) and this was accompanied by inhibition (23%) of cytochrome c oxidase activity. When the cells were incubated in hypoxic conditions during HO-1 induction, the inhibitory effect of CO on cell respiration was markedly increased to 70%. Furthermore, endogenously produced CO was found to be responsible for the respiratory inhibition that occurs in RAW264.7 cells activated in hypoxic conditions with lipopolysaccharide and interferon-gamma, in the presence of N-(iminoethyl)-L-ornithine to prevent the synthesis of NO. Our results indicate that CO contributes significantly to the respiratory inhibition in activated cells, particularly under hypoxic conditions. Inhibition of cell respiration by endogenous CO through its interaction with cytochrome c oxidase might have an important role in inflammatory and hypoxic conditions.

Animals↗
PubMed2006

Mitochondria as signaling organelles in the vascular endothelium.

Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O(2)) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O(2) concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the alpha-subunit of hypoxia-inducible factor 1. At higher O(2) concentrations (1-3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O(2) or O(2)-derived species as signaling molecules. Diversion of O(2) away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells.

Adenylate Kinase↗
PubMed2006

Nitric oxide is a factor in the stabilization of hypoxia-inducible factor-1alpha in cancer: role of free radical formation.

Widespread expression of the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) was observed in samples of human oral squamous cell carcinoma. In all the cases, this was accompanied by a widespread distribution of nitric oxide (NO) synthases (NOS). Furthermore, in three human cell lines derived from human oral squamous cell carcinoma, the accumulation of HIF-1alpha was prevented either by inhibition of NOS activity with the nonspecific NOS inhibitor N(G)-monomethyl-L-arginine or by the antioxidants N-acetyl-L-cysteine and ascorbic acid. We suggest that, in certain forms of cancer, NO might be responsible for the accumulation of HIF-1alpha by a mechanism dependent on free radicals.

Carcinoma, Squamous Cell↗
PubMed2006

Nitric oxide, cell bioenergetics and neurodegeneration.

Following stimulation of NMDA receptors, neurons transiently synthesize nitric oxide (NO) in a calcium/calmodulin-dependent manner through the activation of neuronal NO synthase. Nitric oxide acts as a messenger, activating soluble guanylyl cyclase and participating in the transduction signalling pathways involving cyclic GMP. Nitric oxide also binds to cytochrome c oxidase, and is able to inhibit cell respiration in a process that is reversible and in competition with oxygen. This action can also lead to the release of superoxide anion from the mitochondrial respiratory chain. Here, we discuss recent evidence that this mitochondrial interaction represents a molecular switch for cell signalling pathways involved in the control of physiological functions. These include superoxide- or oxygen-dependent modulation of gene transcription, calcium-dependent cell signalling responses, changes in the mitochondrial membrane potential or AMP-activated protein kinase-dependent control of glycolysis. In pathophysiological conditions, such as brain ischaemia or neurological disorders, NO is formed excessively by NMDA receptor over-activation in neurons, or by inducible NO synthase from neighbouring glia (microglial cells and astrocytes). Elevated NO concentrations can then interact with superoxide anion, generated by the mitochondria or by other mechanisms, leading to the formation of the powerful oxidant species peroxynitrite. During pathological conditions activation of the NAD(+)-consuming enzyme poly(APD-ribose) polymerase-1 (PARP-1) is also a likely mechanism for NO-mediated energy failure and neurotoxicity. Activation of PARP-1 is, however, a repair process, which in milder forms of oxidative stress protects neurons from death. Thus, whilst NO plays a physiological role in neuronal cell signalling, its over-production may cause neuronal energy compromise leading to neurodegeneration.

Animals↗
PubMed2005

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Adipose Tissue↗
PubMed2005

Discrepancies between nitroglycerin and NO-releasing drugs on mitochondrial oxygen consumption, vasoactivity, and the release of NO.

It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.

Acetylcholine↗
PubMed2005

Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis.

Well-known risk factors for atherosclerosis include hypercholesterolaemia, hypertension, diabetes, and smoking. These conditions are associated with endothelial dysfunction, which itself is associated with reduced endothelial generation of nitric oxide (NO). This is an overview of the implications of NO generation in atherosclerosis and of the potential therapeutic benefit of drugs which donate NO, such as organic nitrates, nicorandil, and sydnonimines, or those which increase the availability of endogenous NO, such as statins, angiotensin-converting enzyme inhibitors, L-arginine, and tetrahydrobiopterin.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2005

Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo.

BACKGROUND: Endothelium-dependent dilatation is mediated by 3 principal vasodilators: nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). To determine the relative contribution of these factors in endothelium-dependent relaxation, we have generated mice in which the enzymes required for endothelial NO and PGI2 production, endothelial NO synthase (eNOS) and cyclooxygenase-1 (COX-1), respectively, have been disrupted (eNOS-/- and COX-1-/- mice). METHODS AND RESULTS: In female mice, the absence of eNOS and COX-1 had no effect on mean arterial blood pressure (BP), whereas BP was significantly elevated in eNOS-/-/COX-1-/- males compared with wild-type controls. Additionally, endothelium-dependent relaxation remained intact in the resistance vessels of female mice and was associated with vascular smooth muscle hyperpolarization; however, these responses were profoundly suppressed in arteries of male eNOS-/-/COX-1-/- animals. Similarly, the endothelium-dependent vasodilator bradykinin produced dose-dependent hypotension in female eNOS-/-/COX-1-/- animals in vivo but had no effect on BP in male mice. CONCLUSIONS: These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whereas NO and PGI2 are the predominant mediators in male mice. Moreover, the gender-specific prevalence of EDHF appears to underlie the protection of female eNOS-/-/COX-1-/- mice against hypertension.

6-Ketoprostaglandin F1 alpha↗
PubMed2005

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo.

1 GW274150 ([2-[(1-iminoethyl) amino]ethyl]-L-homocysteine) and GW273629 (3-[[2-[(1-iminoethyl)amino]ethyl]sulphonyl]-L-alanine) are potent, time-dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100-fold) or neuronal NOS (nNOS) (>80-fold). GW274150 and GW273629 are arginine competitive, NADPH-dependent inhibitors of human iNOS with steady state K(d) values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time-dependent manner, reaching IC(50) values of 0.2+/-0.04 and 1.3+/-0.16 microM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260-fold and 219-fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150-fold and 365-fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half-life of approximately 6 h. That of GW273629 was also biphasic in rats, producing a terminal half-life of approximately 3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (approximately 10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS-induced plasma NO(x) levels in mice with an ED(50) of 3.2+/-0.7 mg kg(-1) after 14 h intraperitoneally (i.p.) and 3.8+/-1.5 mg kg(-1) after 14 h when administered orally. GW273629 showed shorter-lived effects on plasma NO(x) and an ED(50) of 9+/-2 mg kg(-1) after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.

Animals↗
PubMed2005

Nitric oxide: orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response.

Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations, the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of "metabolic hypoxia" -- a situation in which, although oxygen is available, the cell is unable to utilise it.

Animals↗
PubMed2005

Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition.

We tested whether nitric oxide (NO) could synergize with hypoxia to induce damage to the aorta isolated from rat. We found that 4 h of mild hypoxia (5% O2) caused substantial necrosis of isolated rat aortae (measured as lactate dehydrogenase release) if inducible NO synthase (iNOS) had previously been induced by endotoxin plus interferon-gamma. Mild hypoxia caused no significant necrosis in the absence of this inflammatory activation, and inflammatory activation caused little damage at a higher oxygen levels (21% oxygen). An iNOS inhibitor (1400W) prevented the necrosis induced by inflammation plus mild hypoxia, whereas the NO donor diethylenetriamine (DETA)/NO adduct, 0.5 mM) greatly sensitized the noninflammed aorta to necrosis induced by mild hypoxia. NO inhibited aortic respiration to a greater degree at lower oxygen concentrations, consistent with NO inhibition of cytochrome oxidase in competition with oxygen. A specific inhibitor of mitochondrial respiration, myxothiazol, caused necrosis of aortae over a similar time course to NO. DETA/NO plus mild hypoxia-induced cell death was substantially reduced by a glycolytic intermediate 3-phosphoglycerate, suggesting that necrosis resulted from energy depletion secondary to respiratory inhibition. This NO-induced sensitization of aorta to mild hypoxia may be important in sepsis and other pathologies where iNOS is expressed.

Animals↗
PubMed2004

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Adenosine Triphosphate↗
PubMed2004

Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response.

Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. This action of NO has both physiological and pathophysiological consequences. Here we report that endogenously generated NO, which disrupts the respiratory chain, may cause changes in mitochondrial calcium flux. This induces cleavage of the endoplasmic reticulum (ER) stress-regulated transcription factor p90 ATF6 into an active p50 form. Cleavage depends on a calcium-dependent serine protease through a regulated intramembrane proteolysis (RIP) process. p50 ATF6 then translocates to the nucleus to upregulate expression of the ER-resident molecular chaperone, glucose-regulated protein 78 (Grp78). The increase in Grp78 provides significant cytoprotection against toxic agents, including thapsigargin, a selective ER calcium-ATPase inhibitor. Cytoprotection is abolished after treatment with cyclosporin A (CsA), which disrupts mitochondrial calcium signalling, or with the calcium chelator BAPTA-AM. The NO-mediated ER stress response is diminished in rho(0) cells devoid of mitochondrial DNA, consistent with our evidence that NO-dependent mitochondrial disruption is coupled to the ER stress response.

Base Sequence↗
PubMed2004

Hypoxia accelerates nitric oxide-dependent inhibition of mitochondrial complex I in activated macrophages.

Excess production of nitric oxide (NO) is implicated in the development of multiple organ failure, with a putative mechanism involving direct mitochondrial inhibition, predominantly affecting complex I. The persistent effects of NO on complex I may be mediated through S-nitrosylation and/or nitration. The temporal contribution of these chemical modifications to the inhibition of respiration and the influence of concurrent hypoxia have not been previously examined. We therefore addressed these questions using J774 macrophages activated by endotoxin and interferon-gamma over a 24-h period, incubated at 21% and 1% oxygen. Oxygen consumption and complex I activity fell progressively over time in the activated cells. This was largely prevented by coincubation with the nonspecific NO synthase inhibitor L-N5-(1-iminoethyl)-ornithine. Addition of glutathione ethyl ester reversed the inhibition at initial time points, suggesting an early mechanism involving nitrosylation. Thereafter, the inhibition of complex I became more persistent, coinciding with a progressive increase in mitochondrial nitration. Hypoxia accelerated the persistent inhibition of complex I, despite a reduction in the total amount of NO generated. Our results suggest that hypoxia amplified the mitochondrial inhibition induced by NO generated during inflammatory disease states.

Animals↗
PubMed2004

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.

2-Methoxyestradiol↗