Effect of nitric oxide donors on oxygen-dependent cytotoxic responses mediated by neutrophils

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.     

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Mitochondrial nitric oxide in the signaling of cell integrated responses

Mitochondria are the specialized organelles for energy metabolism, but, as a typical example of system biology, they also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or oppositely promote cell arrest and programmed cell death by a limited number of oxidative or nitrosative reactions. These reactions are influenced by matrix nitric oxide (NO) steady-state concentration, either from local production or by gas diffusion to mitochondria from the canonical sources. Likewise, in a range of 30–200 nM, NO turns mitochondrial O₂ utilization down by binding to cytochrome oxidase and elicits a burst of superoxide anion and hydrogen peroxide that diffuses outside mitochondria. Depending on NO levels and antioxidant defenses, more or less H₂O₂ accumulates in cytosol and nucleus, and the resulting redox grading contributes to dual activation of proliferating and proapoptotic cascades, like ERK1/2 or p38 MAPK. Moreover, these sequential activating pathways participate in rat liver and brain development and in thyroid modulation of mitochondrial metabolism and contribute to hypothyroid phenotype through complex I nitration. On the contrary, lack of NO disrupts pathways like S-nitrosylation or H₂O₂ production and likewise is a gateway to disease in amyotrophic lateral sclerosis with superoxide dismutase 1 mutations or to cancer proliferation. peroxynitrite; hydrogen peroxide; mitochondrial nitric oxide synthase; mitogen-activated protein kinase     

Progesterone prevents mitochondrial dysfunction in the spinal cord of wobbler mice

In the Wobbler mouse, a mutation of the Vps54 protein increases oxidative stress in spinal motoneurons, associated to toxic levels of nitric oxide and hyperactivity of nitric oxide synthase (NOS). Progesterone neuroprotection has been reported for several CNS diseases, including the Wobbler mouse neurodegeneration. In the present study, we analyzed progesterone effects on mitochondrial-associated parameters of symptomatic Wobbler mice. The activities of mitochondrial respiratory chain complexes I, II-III and IV and protein levels of mitochondrial and cytosolic NOS were determined in cervical and lumbar cords from control, Wobbler and Wobbler mice receiving a progesterone implant for 18 days. We found a significant reduction of complex I and II-III activities in mitochondria and increased protein levels of mitochondrial, but not cytosolic nNOS, in the cervical cord of Wobbler mice. Progesterone treatment prevented the reduction of complex I in the cervical region and the increased level of mitochondrial nNOS. Wobbler motoneurons also showed accumulation of amyloid precursor protein immunoreactivity and decreased activity and immunostaining of MnSOD. Progesterone treatment avoided these abnormalities. Therefore, administration of progesterone to clinically afflicted Wobblers (i) prevented the abnormal increase of mitochondrial nNOS and normalized respiratory complex I; (ii) decreased amyloid precursor protein accumulation, a sign of axonal degeneration, and (iii) increased superoxide dismutation. Thus, progesterone neuroprotection decreases mitochondriopathy of Wobbler mouse cervical spinal cord.     

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

A New Paradigm for MAPK: Structural Interactions of hERK1 with Mitochondria in HeLa Cells

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.     

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.