Phospholipid scramblase 3 controls mitochondrial structure, function, and apoptotic response

Phospholipid scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for phospholipid translocation between two lipid compartments. To study PLS3 function in mitochondria, we disrupted its conserved calcium-binding motif yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity. Mitochondrial analysis revealed that PLS3(F258V)-expressing cells have decreased mitochondrial mass shown by lower cytochrome c and cardiolipin (CL) content, poor mitochondrial respiration, and reduced oxygen consumption and intracellular ATP; whereas wild-type PLS3-transfected cells exhibit increased mitochondrial mass and enhanced respiration. Electron microscopic examination revealed that the mitochondria in PLS3(F258V)-expressing cells have densely packed cristae and are fewer in number and larger than those in control cells. The abnormal mitochondrial metabolism and structure in PLS3(F258V)-expressing cells were associated with decreased sensitivity to UV- and tBid-induced apoptosis and diminished translocation of CL to the mitochondrial outer membrane. In contrast, wild-type PLS3-transfected cells displayed increased sensitivity to apoptosis and enhanced CL translocation. These studies identify PLS3 as a critical regulator of mitochondrial structure and respiration, and CL transport in apoptosis.     

Nitric oxide and mitochondrial respiration.

Nitric oxide (NO) and its derivative peroxynitrite (ONOO-) inhibit mitochondrial respiration by distinct mechanisms. Low (nanomolar) concentrations of NO specifically inhibit cytochrome oxidase in competition with oxygen, and this inhibition is fully reversible when NO is removed. Higher concentrations of NO can inhibit the other respiratory chain complexes, probably by nitrosylating or oxidising protein thiols and removing iron from the iron-sulphur centres. Peroxynitrite causes irreversible inhibition of mitochondrial respiration and damage to a variety of mitochondrial components via oxidising reactions. Thus peroxynitrite inhibits or damages mitochondrial complexes I, II, IV and V, aconitase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, superoxide dismutase, and induces mitochondrial swelling, depolarisation, calcium release and permeability transition. The NO inhibition of cytochrome oxidase may be involved in the physiological regulation of respiration rate, as indicated by the finding that isolated cells producing NO can regulate cellular respiration by this means, and the finding that inhibition of NO synthase in vivo causes a stimulation of tissue and whole body oxygen consumption. The recent finding that mitochondria may contain a NO synthase and can produce significant amounts of NO to regulate their own respiration also suggests this regulation may be important for physiological regulation of energy metabolism. However, definitive evidence that NO regulation of mitochondrial respiration occurs in vivo is still missing, and interpretation is complicated by the fact that NO appears to affect tissue respiration by cGMP-dependent mechanisms. The NO inhibition of cytochrome oxidase may also be involved in the cytotoxicity of NO, and may cause increased oxygen radical production by mitochondria, which may in turn lead to the generation of peroxynitrite. Mitochondrial damage by peroxynitrite may mediate the cytotoxicity of NO, and may be involved in a variety of pathologies.     

Yeast flavohemoglobin protects against nitrosative stress and controls ferric reductase activity

The role of Saccharomyces cerevisiae flavohemoglobin (Yhb1) is controversial and far from understood. This study compares the effects of nitrosative and oxidative challenge on the yeast mutant lacking the YHB1 gene. Growth of the mutant was impaired by nitrosoglutathione and peroxynitrite, whereas increased sensitivity to reactive oxygen species was not observed. Increased levels of intracellular NO(*) after incubation with NO(*) donors were found in the mutants cells as compared to the wild-type cells. Deletion of the YHB1 gene was found to augment the reduction of Fe(3+) by yeast cells which suggests that flavohemoglobin participates in regulation of the activity of plasma membrane ferric reductase(s).     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Redox metabolism in malaria: from genes,through biochemistry and pathology,to drugs

Abstract

The role of Saccharomyces cerevisiae flavohemoglobin (Yhb1) is controversial and far from understood. This study compares the effects of nitrosative and oxidative challenge on the yeast mutant lacking the YHB1 gene. Growth of the mutant was impaired by nitrosoglutathione and peroxynitrite, whereas increased sensitivity to reactive oxygen species was not observed. Increased levels of intracellular NO^? after incubation with NO^? donors were found in the mutants cells as compared to the wild-type cells. Deletion of the YHB1 gene was found to augment the reduction of Fe³⁺ by yeast cells which suggests that flavohemoglobin participates in regulation of the activity of plasma membrane ferric reductase(s).     

A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.     

Visualization of myoglobin-facilitated mitochondrial O(2) delivery in a single isolated cardiomyocyte

The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats. Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution. Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation. Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered. Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions. Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells. Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.     

Decreased mitochondrial OGG1 expression is linked to mitochondrial defects and delayed hepatoma cell growth

Many solid tumor cells exhibit mitochondrial respiratory impairment; however, the mechanisms of such impairment in cancer development remain unclear. Here, we demonstrate that SNU human hepatoma cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial-targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang-L cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, our results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells.     

The unique mitochondrial form and function of Antarctic channichthyid icefishes

Antarctic icefishes of the family Channichthyidae are the only vertebrate animals that as adults do not express the circulating oxygen-binding protein hemoglobin (Hb). Six of the 16 family members also lack the intracellular oxygen-binding protein myoglobin (Mb) in the ventricle of their hearts and all lack Mb in oxidative skeletal muscle. The loss of Hb has led to substantial remodeling in the cardiovascular system of icefishes to facilitate adequate oxygenation of tissues. One of the more curious adaptations to the loss of Hb and Mb is an increase in mitochondrial density in cardiac myocytes and oxidative skeletal muscle fibers. The proliferation of mitochondria in the aerobic musculature of icefishes does not arise through a canonical pathway of mitochondrial biogenesis. Rather, the biosynthesis of mitochondrial phospholipids is up-regulated independently of the synthesis of proteins and mitochondrial DNA, and newly-synthesized phospholipids are targeted primarily to the outer-mitochondrial membrane. Consequently, icefish mitochondria have a higher lipid-to-protein ratio compared to those from red-blooded species. Elevated levels of nitric oxide in the blood plasma of icefishes, compared to red-blooded notothenioids, may mediate alterations in mitochondrial density and architecture. Modifications in mitochondrial structure minimally impact state III respiration rates but may significantly enhance intracellular diffusion of oxygen. The rate of oxygen diffusion is greater within the hydrocarbon core of membrane lipids compared to the aqueous cytosol and impeded only by proteins within the lipid bilayer. Thus, the proliferation of icefish's mitochondrial membranes provides an optimal conduit for the intracellular diffusion of oxygen and compensates for the loss of Hb and Mb. Currently little is known about how mitochondrial phospholipid synthesis is regulated and integrated into mitochondrial biogenesis. The unique architecture of the oxidative muscle cells of icefishes highlights the need for further studies in this area.     

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.     

Dietary inorganic nitrate improves mitochondrial efficiency in humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.     

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.     

Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration

Nitric oxide (NO) is a widespread biological messenger that has many physiological and pathophysiological roles. Most of the physiological actions of NO are mediated through the activation of sGC (soluble guanylate cyclase) and the subsequent production of cGMP. NO also binds to the binuclear centre of COX (cytochrome c oxidase) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Although sGC is more sensitive to endogenous NO than COX at atmospheric oxygen tension, the more relevant question is which enzyme is more sensitive at physiological oxygen concentration. Using a system in which NO is generated inside the cells in a finely controlled manner, we determined cGMP accumulation by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry at 30 microM oxygen. In the present paper, we report that the NO EC50 of sGC was approx. 2.9 nM, whereas that required to achieve IC50 of respiration was 141 nM (the basal oxygen consumption in the absence of NO was 14+/-0.8 pmol of O2/s per 10(6) cells). In accordance with this, the NO-cGMP signalling transduction pathway was activated at lower NO concentrations than the AMPKs (AMP-activated protein kinase) pathway. We conclude that sGC is approx. 50-fold more sensitive than cellular respiration to endogenous NO under our experimental conditions. The implications of these results for cell physiology are discussed.     

Nitric oxide and carotid body chemoreception.

Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensory responses to hypoxia. It is believed that NO modulates carotid chemoreception by several mechanisms, which include the control of carotid body vascular tone and oxygen delivery and reduction of the excitability of chemoreceptor cells and petrosal sensory neurons. In addition to the well-known inhibitory effect, we found that NO has a dual (dose-dependent) effect on carotid chemoreception depending on the oxygen pressure level. During hypoxia, NO is primarily an inhibitory modulator of carotid chemoreception, while in normoxia NO increased the chemosensory activity. This excitatory effect produced by NO is likely mediated by an impairment of mitochondrial electron transport and oxidative phosphorylation, which increases the chemosensory activity. The recent findings that mitochondria contain an isoform of NO synthase, which produces significant amounts of NO for regulating their own respiration, suggest that NO may be important for the regulation of mitochondrial energy metabolism and oxygen sensing in the CB.     

Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Nitric oxide inhibition of mitochondrial respiration and its role in cell death

Nitric oxide (NO) or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial respiration in two different ways: (i) an acute, potent, and reversible inhibition of cytochrome oxidase by NO in competition with oxygen; and, (ii) irreversible inhibition of multiple sites by RNS. NO inhibition of respiration may impinge on cell death in several ways. Inhibition of respiration can cause necrosis and inhibit apoptosis due to ATP depletion, if glycolysis is also inhibited or is insufficient to compensate. Inhibition of neuronal respiration can result in excitotoxic death of neurons due to induced release of glutamate and activation of NMDA-type glutamate receptors. Inhibition of respiration may cause apoptosis in some cells, while inhibiting apoptosis in other cells, by mechanisms that are not clear. However, NO can induce (and inhibit) cell death by a variety of mechanisms unrelated to respiratory inhibition.