The bioactivity of neuronal-derived nitric oxide in aging and neurodegeneration: Switching signaling to degeneration.

The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical scientists since it was promoted from atmospheric air pollutant to biological ubiquitous signaling molecule. Its unique physical chemical properties expand beyond its radical nature to include fast diffusion in aqueous and lipid environments and selective reactivity in a biological setting determined by bioavailability and reaction rate constants with biomolecules. In the brain, •NO is recognized as a key player in numerous physiological processes ranging from neurotransmission/neuromodulation to neurovascular coupling and immune response. Furthermore, changes in its bioactivity are central to the molecular pathways associated with brain aging and neurodegeneration. The understanding of •NO bioactivity in the brain, however, requires the knowledge of its concentration dynamics with high spatial and temporal resolution upon stimulation of its synthesis. Here we revise our current understanding of the role of neuronal-derived •NO in brain physiology, aging and degeneration, focused on changes in the extracellular concentration dynamics of this free radical and the regulation of bioenergetic metabolism and neurovascular coupling.

Tobacco Smoke-Induced Hepatic Injury with Steatosis, Inflammation, and Impairments in Insulin and Insulin-Like Growth Factor Signaling.

BACKGROUND: Alcoholic liver disease (ALD) is associated with impairments in hepatic insulin and insulin-like growth factor (IGF) signaling through cell growth, survival, and metabolic pathways. Since not all heavy drinkers develop ALD, co-factors may be important. Epidemiologic data indicate that most heavy drinkers smoke tobacco and experimental data revealed that low-level nitrosamine exposures, including those from tobacco, can cause steatohepatitis with hepatic insulin/IGF resistance and exacerbate ALD. We hypothesize that cigarette smoke (CS) exposures also cause liver injury with impaired hepatic insulin/IGF signaling, and thereby contribute to ALD. METHODS: Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 (CS4) or 8 (CS8) weeks, or CS for 8 weeks with 2 weeks recovery (CS8+R). RESULTS: CS exposures caused progressive liver injury with disruption of the normal hepatic chord architecture, lobular inflammation, apoptosis or necrosis, micro-steatosis, sinusoidal dilatation, and nuclear pleomorphism. Histopathological liver injury scores increased significantly from A8 to CS4 and then further to CS8 (P<0.0001). The mean histological grade was also higher in CS8+R relative to A8 (P<0.0001) but lower than in CS4, reflecting partial resolution of injury by CS withdrawal. CS exposures impaired insulin and IGF-1 signaling through IRS-1, Akt, GSK-3ß, and PRAS40. Livers from CS8+R mice had normalized or elevated levels of insulin receptor, pYpY-Insulin-R, 312S-IRS-1, 473S-Akt, S9-GSK-3ß, and pT246-PRAS40 relative to A8, CS4, or CS8, reflecting partial recovery. CONCLUSION: CS-mediated liver injury and steatohepatitis with impairments in insulin/IGF signalling are reminiscent of the findings in ALD. Therefore, CS exposures (either first or second-hand) may serve as a co-factor in ALD. The persistence of several abnormalities following CS exposure cessation suggests that some aspects of CS-mediated hepatic metabolic dysfunction are not readily reversible.

Selective oestrogen receptor modulators differentially potentiate brain mitochondrial function.

The mitochondrial energy-transducing capacity of the brain is important for long-term neurological health and is influenced by endocrine hormone responsiveness. The present study aimed to determine the role of oestrogen receptor (ER) subtypes in regulating mitochondrial function using selective agonists for ERα (propylpyrazoletriol; PPT) and ERß (diarylpropionitrile; DPN). Ovariectomised female rats were treated with 17ß-oestradiol (E(2) ), PPT, DPN or vehicle control. Both ER selective agonists significantly increased the mitochondrial respiratory control ratio and cytochrome oxidase (COX) activity relative to vehicle. Western blots of purified whole brain mitochondria detected ERα and, to a greater extent, ERß localisation. Pre-treatment with DPN, an ERß agonist, significantly increased ERß association with mitochondria. In the hippocampus, DPN activated mitochondrial DNA-encoded COX I expression, whereas PPT was ineffective, indicating that mechanistically ERß, and not ERα, activated mitochondrial transcriptional machinery. Both selective ER agonists increased protein expression of nuclear DNA-encoded COX IV, suggesting that activation of ERß or ERα is sufficient. Selective ER agonists up-regulated a panel of bioenergetic enzymes and antioxidant defence proteins. Up-regulated proteins included pyruvate dehydrogenase, ATP synthase, manganese superoxide dismutase and peroxiredoxin V. In vitro, whole cell metabolism was assessed in live primary cultured hippocampal neurones and mixed glia. The results of analyses conducted in vitro were consistent with data obtained in vivo. Furthermore, lipid peroxides, accumulated as a result of hormone deprivation, were significantly reduced by E(2) , PPT and DPN. These findings suggest that the activation of both ERα and ERß is differentially required to potentiate mitochondrial function in brain. As active components in hormone therapy, synthetically designed oestrogens as well as natural phyto-oestrogen cocktails can be tailored to improve brain mitochondrial endpoints.

Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation.

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

Kinetic regulation of an alkaline p-nitrophenylphosphate phosphatase from Halobacterium salinarum in low water system by Mn2+ and monovalent cations.

Reversed micelles were used as a cytoplasmic model to study the effect of the multi-ionic equilibria on kinetics of extreme halophilic enzymes. The enzymatic system used was an alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium). This enzyme was solubilised in reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The p-nitrophenylphosphate phosphatase is a good system to study the regulation of the enzymatic activity, because it utilises manganese, water and potassium or sodium as cofactors and reacts with p-nitrophenylphosphate. Kinetic behaviour was determined by the ratio between [Mn2+] and [Na+] or [K+]. When the [Mn2+] increased and [Na+] or [K+] decreased, the kinetics showed cooperative behaviour. Rabin's model describes the kinetic behaviour of the p-nitrophenylphosphate phosphatase in reversed micelles.

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium.

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture.

We have re-examined the lysosomal hypothesis of oxidative-stress-induced apoptosis using a new technique for exposing cells in culture to a low steady-state concentration of H(2)O(2). This steady-state technique mimics the situation in vivo better than the bolus-administration method. A key aspect of H(2)O(2)-induced apoptosis is that the apoptosis is evident only after several hours, although cells may become committed within a few minutes of exposure to this particular reactive oxygen species. In the present work, we were able to show, for the first time, several correlative links between the triggering effect of H(2)O(2) and the later onset of apoptosis: (i) a short (15 min) exposure to H(2)O(2) caused almost immediate, albeit limited, lysosomal rupture; (ii) early lysosomal damage, and later apoptosis, showed a similar dose-related response to H(2)O(2); (iii) both events were inhibited by pre-treatment with iron chelators, including desferrioxamine. This compound is known to be taken up by endocytosis only and thus to become localized in the lysosomal compartment. After exposure to oxidative stress, when cells were again in standard culture conditions, a time-dependent continuous increase in lysosomal rupture was observed, resulting in a considerably lowered number of intact lysosomes in apoptotic cells, whereas non-apoptotic cells from the same batch of oxidative-stress-exposed cells showed mainly intact lysosomes. Taken together, our results reinforce earlier findings and strongly suggest that lysosomal rupture is an early upstream initiating event, and a consequence of intralysosomal iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state.

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space.

It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the intermembrane space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the intermembrane space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium trioxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the intermembrane space. The generation of superoxide anion towards the intermembrane space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.

Mitochondrial free radical generation, oxidative stress, and aging.

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.

Regulation of mitochondrial respiration by oxygen and nitric oxide.

Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2-) and peroxynitrite (ONOO-). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. No exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

Estimation of H2O2 gradients across biomembranes.

When cells are exposed to an external source of H2O2, the rapid enzymatic consumption of H2O2 inside the cell provides the driving force for the formation of the gradient across the plasma and other subcellular membranes. By using the concepts of enzyme latency, the following gradients - formed after a few seconds following the exposure to H2O2 - were estimated in Jurkat T-cells: [H2O2](cytosol)/[H2O2](peroxisomes)=3; [H2O2](extracellular)/[H2O2](cytosol)=7. The procedure presented in this work can easily be applied to other cell lines and provides a quantitative framework to interpret the data obtained when cells are exposed to an external source of H2O2.

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage.

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

p21WAF1 regulates anchorage-independent growth of HCT116 colon carcinoma cells via E-cadherin expression.

The cyclin-dependent kinase inhibitor p21WAF1 has been characterized as an important effector of the tumor suppressor p53 and has been linked to various growth-regulatory processes. To identify a potential role of p21 in anchorage-dependent growth control, we analyzed a pair of HCT116 human colon carcinoma cell lines that differed only in their p21 status. We found that during suspension culture, HCT116 cells (which contain wildtype p53 and p21) continued to proliferate and formed compact multicellular spheroids (MCSs). In contrast, HCT116 cells engineered to lack functional p21 (HCTp21-/-) were unable to form MCSs in suspension culture, ceased proliferation, and eventually died through apoptosis. The parental HCT116 cells underwent the same fate when treated with hyaluronidase, indicating that cell-cell contact might be required for survival in suspension culture. We established that E-cadherin was induced in HCT116 but not in HCTp21-/- cells and accounted for the formation of MCSs. Forced expression of E-cadherin or p21 in HCTp21-/- cells restored the ability to form MCSs and to grow independently of anchorage. Moreover, HCTp21-/- cells exhibited a severely reduced transformed phenotype and demonstrated greatly enhanced chemosensitivity in suspension culture. Thus, our results link an important regulator of the cell cycle machinery to the expression of a cell-cell adhesion molecule involved in tumor formation. Because our results indicate that loss of p21 severely impairs the ability of HCT cells to grow independently of anchorage, it may not be coincidental that inactivating mutations of this gene are very rarely found in tumor cells.

Mitochondrial production of hydrogen peroxide regulation by nitric oxide and the role of ubisemiquinone.

Mitochondria are considered the major cellular site for hydrogen peroxide production, a process that is kinetically controlled by the availability of oxygen and nitric oxide to cytochrome oxidase and of ADP to F1-ATPase. The multisite regulation of mitochondrial respiration and energy-transducing pathways support a critical regulatory role of mitochondrion in cell signaling pathways. The cellular steady-state levels of hydrogen peroxide and the role of mitochondria in maintaining these levels are reviewed.

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.