Mitochondrial nitric oxide synthase: a masterpiece of metabolic adaptation, cell growth, transformation, and death.

Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)-dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome.

BACKGROUND: In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO(2) and H(2)O or to drive glucose storage to glycogen. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4-8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (-25 to -60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-(14)C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37+/-3 vs 10+/-1 micromolO(2)/h.g tissue and 13+/-1 vs 7.2+/-1 micromol (3)H(2)O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half. CONCLUSIONS/SIGNIFICANCE: These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.

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.

Hypoxia induces complex I inhibition and ultrastructural damage by increasing mitochondrial nitric oxide in developing CNS.

NO-mediated toxicity contributes to neuronal damage after hypoxia; however, the molecular mechanisms involved are still a matter of controversy. Since mitochondria play a key role in signalling neuronal death, we aimed to determine the role of nitrative stress in hypoxia-induced mitochondrial damage. Therefore, we analysed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia-reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. A transient but substantial increase in mtNOS content and activity was observed at 0-2 h posthypoxia, resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations in nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-nitroindazole 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage by increasing mitochondrial NO in the developing brain.

The biological significance of mtNOS modulation.

In the last years, nitric oxide synthases (NOS) have been localized in mitochondria. At this site, NO yield directly regulates the activity of cytochrome oxidase, O(2) uptake and the production of reactive oxygen species. Recent studies showed that translocated neuronal nitric oxide synthase (nNOS) is posttranslationally modified including phosphorylation at Ser 1412 (in mice) and myristoylation in an internal residue. Different studies confirm that modified nNOS alpha is the main modulable isoform in mitochondria. Modulation of mtNOS was observed in different situations, like adaptation to reduced O(2) availability and hypoxia, adaptation to low environmental temperature, and processes linked to life and death by effects on kinases and transcription factors. We present here evidence about the role of mtNOS in the analyzed conditions.

HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism.

This study investigated whether inducible heme oxygenase-1[corrected] (HO-1) [corrected] is targeted to mitochondria and its putative effects on oxidative metabolism in rat liver. Western blot and immune-electron microscopy in whole purified and fractionated organelles showed basal expression of HO-1 protein in both microsomes and mitochondria (inner membrane), accompanied by a parallel HO activity. Inducers of HO-1 increased HO-1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase, supporting that both enzymes are in the same compartmentalization. Induction of mitochondrial HO-1 was associated with a decrease of mitochondrial heme content and selective reduction of protein expression of cytochrome oxidase (COX) subunit I, which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability; these changes resulted in decreased COX spectrum and activity. Mitochondrial HO-1 induction was also associated with down-regulation of mitochondrial-targeted NO synthase expression and activity, resulting in a reduction of NO-dependent mitochondrial oxidant yield; inhibition of HO-1 activity reverted these effects. In conclusion, we demonstrated for the first time localization of HO-1 protein in mitochondria. It is surmised that mitochondrial HO-1 has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis, in which substantially increased mitochondrial NO and oxidant production have been implicated.

Mitochondrial nitric oxide synthase drives redox signals for proliferation and quiescence in rat liver development.

Mitochondrial nitric oxide synthase (mtNOS) is a fine regulator of oxygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cycle progression. From this perspective, we examined liver mtNOS modulation and mitochondrial redox changes in developing rats from embryonic days 17-19 and postnatal day 2 (proliferating hepatocyte phenotype) through postnatal days 15-90 (quiescent phenotype). mtNOS expression and activity were almost undetectable in fetal liver, and progressively increased after birth by tenfold up to adult stage. NO-dependent mitochondrial hydrogen peroxide (H(2)O(2)) production and Mn-superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolic H(2)O(2) concentration ([H(2)O(2)](ss)) and cell fluorescence. mtNOS-dependent [H(2)O(2)](ss) was a good predictor of extracellular signal-regulated kinase (ERK)/p38 activity ratio, cyclin D1, and tissue proliferation. At low 10(-11)-10(-12) M [H(2)O(2)](ss), proliferating phenotypes had high cyclin D1 and phospho-ERK1/2 and low phospho-p38 mitogen-activated protein kinase, while at 10(-9) M [H(2)O(2)](ss), quiescent phenotypes had the opposite pattern. Accordingly, leading postnatal day 2-isolated hepatocytes to embryo or adult redox conditions with H(2)O(2) or NO-H(2)O(2) scavengers, or with ERK inhibitor U0126, p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio, cyclin D1 expression, and [(3)H] thymidine incorporation in the cells. Accordingly, p38 inhibitor SB202190 or N-acetyl-cysteine prevented H(2)O(2) inhibitory effects on proliferation. In conclusion, the results suggest that a synchronized increase of mtNOS and derived H(2)O(2) operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence.